Reactogenicity of BNT162b2 mRNA COVID-19 Vaccine in a Young Working Age Population: A Survey among Medical School Residents, within a Mass Vaccination Campaign, in a Regional Reference Teaching Hospital in Italy.

Vaccinations are a key prevention measure in fighting the COVID-19 pandemic. The BNT162b2 mRNA vaccine (BioNTech/Pfizer), the first to receive authorization, was widely used in the mass vaccination campaign in Italy. Healthcare workers were identified as a priority group for vaccination, but few studies have assessed its reactogenicity among the young working age population. An online survey was conducted to investigate the adverse reactions occurring in the 7 days following the first and second vaccination doses amongst resident doctors of the University of Genoa, employed at the IRCCS Ospedale Policlinico San Martino of Genoa, between 11 January and 16 March 2021. A total of 512 resident physicians were invited to participate in the study (female = 53.2%; mean age = 28.9 years), of whom 296 (female = 53.4%, mean age = 28.9 years) and 275 (female = 55.3%, mean age = 29.1 years) completed the survey after their first and second vaccination doses, respectively. In the 7 days following the first dose, most common adverse reactions were local pain (96.3%), fatigue (42.6%), headache (33.8%), arthromyalgia (28.0%), and 5.1% reported fever, while following the second dose, participants reported local pain (93.5%), fatigue (74.9%), headache (57.5%), arthromyalgia (58.2%), and fever (30.9%), with a higher prevalence among females. Systemic (but not local) reactions increased following the second vaccination, reaching severe intensity in 9.8% of participants and causing three or more events of moderate intensity in 23.7% of participants. Adverse reactions preventing regular daily activities could cause absenteeism among workers. These results can be useful to inform populations of young individuals, set expectations, and improve adherence to vaccination campaigns.

IκB kinase-ε-mediated phosphorylation triggers IRF-1 degradation in breast cancer cells.

Interferon Regulatory Factors (IRFs) are key regulators of immunity, cell survival and apoptosis. IRF transcriptional activity and subcellular localization are tightly regulated by posttranscriptional modifications including phosphorylation. The IκB kinase family member IKK-ε is essential in regulating antiviral innate immunity mediated by IRFs but is now also recognized as an oncoprotein amplified and overexpressed in breast cancer cell lines and patient-derived tumors. In the present study, we report that the tumor suppressor IRF-1 is a specific target of IKK-ε in breast cancer cells. IKK-ε-mediated phosphorylation of IRF-1 dramatically decreases IRF-1 protein stability, accelerating IRF-1 degradation and quenching IRF-1 transcriptional activity. Chemical inhibition of IKK-ε activity, fully restores IRF-1 levels and function and positively correlates with inhibition of cell growth and proliferation of breast cancer cells. By using a breast cancer cell line stably expressing a dominant negative version of IRF-1 we were able to demonstrate that IKK-ε preferentially exerts its oncogenic potential in breast cancer through the regulation of IRF-1 and point to the IKK-ε-mediated phosphorylation of IRF-1 as a therapeutic target to overcome IKK-ε-mediated tumorigenesis.

A model of the three-dimensional structure of human interferon responsive factor 1 and its modifications upon phosphorylation or phosphorylation-mimicking mutations.

Interferon responsive factor 1 (IRF-1) is a pleiotropic transcription factor, possessing non-redundant biological activities that depend on its interaction with different protein partners and multiple post-translational modifications including phosphorylation. In particular, a 5'-SXXXSXS-3' motif of the protein represents the target of the IκB-related kinases, TANK-binding kinase (TBK)-1 and inhibitor of nuclear factor kappa-B kinase (IKK)-ε. Here, a 3D model of human IRF-1 was determined by using multi-template comparative modeling and molecular dynamics approaches. Models obtained through either phosphorylation or aspartate mutation of residues 215, 219 and 221 were also calculated and compared to the wild type. Calculations indicated that each of these modifications mainly induces a rigidification of the protein structure and only slightly changes in electrostatics and hydrophobicity of IRF-1 surface, resulting in the impairment of the capacity of IRF-1 containing as partate mutations (S221D and S215D/S219D/S221D) to synergize with tumour necrosis factor (TNF)-α stimulation in inducing interferon (IFN) promoter-mediated reporter gene activation. Therefore, these changes are qualitatively correlated to the amount of negative charge located on the 215-221 segments of IRF-1 by phosphorylation or aspartate mutation. Hypotheses on the structural mechanism that governs the phosphorylation-related damping of IRF-1 activity were also drawn. Communicated by Ramaswamy H. Sarma.

Role of interferon regulatory factor 1 in governing Treg depletion, Th1 polarization, inflammasome activation and antitumor efficacy of cyclophosphamide.

The antitumor effectiveness of cyclophosphamide (CTX) and other chemotherapeutics was shown to rely not only on direct cytotoxicity but also on immunogenic tumor cell death and systemic immunomodulatory mechanisms, including regulatory T cell (Treg) depletion, Th1 cell polarization, type I interferon (IFN) and proinflammatory cytokine production. IFN regulatory factor (IRF)-1 is a transcriptional regulator of IFNs and IFN-inducible genes, involved in the control of Th1 and Treg differentiation and in sterile inflammation. Aim of this study was to explore the role of IRF-1 in CTX-induced antitumor effects and related immune activities. This study shows for the first time that IRF-1 is important for the antitumor efficacy of CTX in mice. Moreover, experiments in tumor-bearing C57BL/6 mice showed that Irf1 gene expression in the spleen was transiently increased following CTX administration and correlated with the induction of Th1 cell expansion and of Il12p40 gene expression, which is the main Th1-driving cytokine. At the same time, CTX administration reduced both Foxp3 expression and Treg cell percentages. These effects were abrogated in Irf1-/- mice. Further experiments showed that the gene and/or protein expression of caspase-1, iNOS, IL-1ß, IL-6 and CXCL10 and the levels of nitric oxide were modulated following CTX in an IRF-1-direct- or -indirect-dependent manner, and highlighted the importance of caspase-1 in driving the sterile inflammatory response to CTX. Our data identify IRF-1 as important for the antitumor efficacy of CTX and for the regulation of many immunomodulatory activities of CTX, such as Th1 polarization, Treg depletion and inflammation.

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. IMPORTANCE: Current therapies have dramatically reduced morbidity and mortality associated with HIV infection and have converted infection from a fatal pathology to a chronic disease that is manageable via antiretroviral therapy. Nevertheless, HIV-1 infection remains a challenge, and the identification of useful cellular targets for therapeutic intervention remains a major goal. The cellular transcription factor IRF-1 impacts various physiological functions, including the immune response to viral infection. In this study, we have identified a unique mechanism by which HIV-1 evades IRF-1-mediated host immune responses and show that the viral protein Tat accelerates IRF-1 proteasome-mediated degradation and inactivates IRF-1 function. Restoration of IRF-1 functionality may thus be regarded as a potential strategy to reinstate both a direct antiviral response and a more broadly acting immune regulatory circuit.

IFN Regulatory Factors and Antiviral Innate Immunity: How Viruses Can Get Better.

The interferon regulatory factor (IRF) family consists of transcriptional regulators that exert multifaceted and versatile functions in multiple biological processes. Their crucial role as central mediators in the establishment and execution of host immunity in response to pathogen-derived signals downstream pattern recognition receptors (PRRs) makes IRFs a hallmark of the host antiviral response. They function as hub molecules at the crossroad of different signaling pathways for the induction of interferon (IFN) and inflammatory cytokines, as well as of antiviral and immunomodulatory genes even in an IFN-independent manner. By regulating the development and activity of immune cells, IRFs also function as a bridge between innate and adaptive responses. As such, IRFs represent attractive and compulsive targets in viral strategies to subvert antiviral signaling. In this study, we discuss current knowledge on the wide array of strategies put in place by pathogenic viruses to evade, subvert, and/or hijack these essential components of host antiviral immunity.

Early IFN type I response: Learning from microbial evasion strategies.

Type I interferon (IFN) comprises a class of cytokines first discovered more than 50 years ago and initially characterized for their ability to interfere with viral replication and restrict locally viral propagation. As such, their induction downstream of germ-line encoded pattern recognition receptors (PRRs) upon recognition of pathogen-associated molecular patterns (PAMPs) is a hallmark of the host antiviral response. The acknowledgment that several PAMPs, not just of viral origin, may induce IFN, pinpoints at these molecules as a first line of host defense against a number of invading pathogens. Acting in both autocrine and paracrine manner, IFN interferes with viral replication by inducing hundreds of different IFN-stimulated genes with both direct anti-pathogenic as well as immunomodulatory activities, therefore functioning as a bridge between innate and adaptive immunity. On the other hand an inverse interference to escape the IFN system is largely exploited by pathogens through a number of tactics and tricks aimed at evading, inhibiting or manipulating the IFN pathway, that result in progression of infection or establishment of chronic disease. In this review we discuss the interplay between the IFN system and some selected clinically important and challenging viruses and bacteria, highlighting the wide array of pathogen-triggered molecular mechanisms involved in evasion strategies.

Type I IFN--a blunt spear in fighting HIV-1 infection.

For more than 50 years, Type I Interferon (IFN) has been recognized as critical in controlling viral infections. IFN is produced downstream germ-line encoded pattern recognition receptors (PRRs) upon engagement by pathogen-associated molecular patterns (PAMPs). As a result, hundreds of different interferon-stimulated genes (ISGs) are rapidly induced, acting in both autocrine and paracrine manner to build a barrier against viral replication and spread. ISGs encode proteins with direct antiviral and immunomodulatory activities affecting both innate and adaptive immune responses. During infection with viruses, as HIV-1, that can establish a persistent infection, IFN although produced, is not able to block the initial infection and a chronic IFN-mediated immune activation/inflammation becomes a pathogenic mechanism of disease progression. This review will briefly summarize when and how IFN is produced during HIV-1 infection and the way this innate immune response is manipulated by the virus to its own advantage to drive chronic immune activation and progression to AIDS.

Influenza vaccination among healthcare workers in Italy.

Influenza vaccination is a fundamental tool for the prevention of influenza in healthcare settings and its administration to healthcare workers (HCWs) is recommended in more than 40 countries including United States of America and many countries of the European Union. Despite these recommendations, the compliance of HCWs to influenza vaccination is largely inadequate in Italy. Since 2005/06 season, a comprehensive multifaceted intervention project aimed at increasing the seasonal influenza vaccination coverage rates among HCWs was performed at the IRCCS AOU San Martino IST teaching hospital in Genoa, Italy, the regional tertiary adult acute-care reference center with a 1300 bed capacity. Despite almost a decade of efforts, the vaccination coverage rates registered at our hospital steadily remain unsatisfactory and very distant by the minimum objective of 75% defined by the Italian Ministry of Health. During the last influenza season (2013/14), vaccination coverage rates by occupation type resulted 30% among physicians, 11% among nurses and 9% among other clinical personnel.   Further efforts are necessary to prevent the transmission of influenza to patient and novel strategies need to be identified and implemented in order to increase the compliance of HCWs, particularly nurses, with the seasonal influenza vaccination.

HIV-1 latency: an update of molecular mechanisms and therapeutic strategies.

The major obstacle towards HIV-1 eradication is the life-long persistence of the virus in reservoirs of latently infected cells. In these cells the proviral DNA is integrated in the host's genome but it does not actively replicate, becoming invisible to the host immune system and unaffected by existing antiviral drugs. Rebound of viremia and recovery of systemic infection that follows interruption of therapy, necessitates life-long treatments with problems of compliance, toxicity, and untenable costs, especially in developing countries where the infection hits worst. Extensive research efforts have led to the proposal and preliminary testing of several anti-latency compounds, however, overall, eradication strategies have had, so far, limited clinical success while posing several risks for patients. This review will briefly summarize the more recent advances in the elucidation of mechanisms that regulates the establishment/maintenance of latency and therapeutic strategies currently under evaluation in order to eradicate HIV persistence.

IκB kinase ε targets interferon regulatory factor 1 in activated T lymphocytes.

IκB kinase ε (IKK-ε) has an essential role as a regulator of innate immunity, functioning downstream of pattern recognition receptors to modulate NF-κB and interferon (IFN) signaling. In the present study, we investigated IKK-ε 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-ε 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-ß transcription. We demonstrated that IKK-ε 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-κB RelA subunit and interfered with PCAF-mediated acetylation of NF-κB RelA. These results demonstrate that TCR/CD28 stimulation of primary T cells stimulates IKK-ε activation, which in turn contributes to suppression of IFN-ß production.

Therapeutics for HIV-1 reactivation from latency.

Intensive combined antiretroviral therapy successfully suppresses HIV-1 replication and AIDS disease progression making infection manageable, but it is unable to eradicate the virus that persists in long-lived, drug-insensitive and immune system-insensitive reservoirs thus asking for life-long treatments with problems of compliance, resistance, toxicity and cost. These limitations and recent insights into latency mechanisms have fueled a renewed effort in finding a cure for HIV-1 infection. Proposed eradication strategies involve reactivation of the latent reservoir upon induction of viral transcription followed by the elimination of reactivated virus-producing cells by viral cytopathic effect or host immune response. Several molecules identified by mechanism-directed approaches or in large-scale screenings have been proposed as latency reversing agents. Some of them have already entered clinical testing in humans but with mixed or unsatisfactory results.

The development of immune-modulating compounds to disrupt HIV latency.

Antiretroviral therapy (ART) has proved highly effective in suppressing HIV-1 replication and disease progression. Nevertheless, ART has failed to eliminate the virus from infected individuals. The main obstacle to HIV-1 eradication is the persistence of cellular viral reservoirs. Therefore, the "shock-and-kill" strategy was proposed consisting of inducing HIV-1 escape from latency, in the presence of ART. This is followed by the elimination of reactivated, virus-producing cells. Immune modulators, including protein kinase C (PKC) activators, anti-leukemic drugs and histone deacetylase inhibitors (HDACis) have all demonstrated efficacy in the reactivation of latent virus replication. This review will focus on the potential use of these small molecules in the "shock and kill" strategy, the molecular basis for their action and the potential advantages of their immune-modulating activities.

HIV-1, interferon and the interferon regulatory factor system: an interplay between induction, antiviral responses and viral evasion.

Thirty years after the first isolation of the etiological agent of AIDS, the virus HIV-1 is still a major threat worldwide with millions of individuals currently infected. Although current combination therapies allow viral replication to be controlled, HIV-1 is not eradicated and persists in drug- and immune system-insensitive reservoirs and a cure is still lacking. Pathogens such as HIV-1 that cause chronic infections are able to adapt to the host in a manner that ensures long term residence and survival, via the evolution of numerous mechanisms that evade various aspects of the innate and adaptive immune response. One such mechanism is targeted to members of the interferon (IFN) regulatory factor (IRF) family of proteins. These transcription factors regulate a variety of biological processes including interferon induction, immune cell activation and downstream pattern recognition receptors (PRRs). HIV-1 renders IRFs harmless and hijacks them to its own advantage in order to facilitate its replication and evasion of immune responses. Type I interferon (IFN), the canonical antiviral innate response, can be induced in both acute and chronic HIV-1 infection in vivo, but in the majority of individuals this initial response is not protective and can contribute to disease progression. Type I IFN expression is largely inhibited in T cells and macrophages in order to successfully establish productive infection, whereas sustained IFN production by plasmacytoid dendritic cells is considered an important source of chronic immune activation, a hallmark to AIDS progression.

Adherence to international and national recommendations for the prevention of surgical site infections in Italy: results from an observational prospective study in elective surgery.

BACKGROUND: An observational prospective study of the perioperative procedures for prevention of surgical site infections (SSIs) was carried out in a tertiary referral teaching hospital in Liguria, Italy, to evaluate their adherence to international and national standards. METHODS: A 1-month survey was performed in all surgical departments, monitored by turns by trained survey teams. Data regarding presurgical patient preparation and intraoperative infection control practices were collected. RESULTS: A total of 717 elective interventions were actively monitored in 703 patients who underwent surgery. Hair-shaving was performed mainly using a razor (92%) by the nurses (72.8%) on the day before the operation (83.5%). All of the patients showered, either with a common detergent (87%) or with an antiseptic solution (13%). Antimicrobial prophylaxis was administered properly in 75.7% of the patients at induction of anaesthesia; however, according to current Italian guidelines, inappropriate prophylaxis was provided in 55.2% patients. Appropriate antisepsis of the incision area was done in 97.4% of the operations, and nearly 90% of the interventions lasted less than the respective 75th percentile. The doors of the operating theatres were mostly open during the duration of the operation in 36.3% of the cases. CONCLUSIONS: This review of infection control policies identified significant opportunities for improving the safety and the quality of routine surgical practice.

IRF-8 controls melanoma progression by regulating the cross talk between cancer and immune cells within the tumor microenvironment.

The transcription factor interferon regulatory factor-8 (IRF-8) is crucial for myeloid cell development and immune response and also acts as a tumor suppressor gene. Here, we analyzed the role of IRF-8 in the cross talk between melanoma cells and tumor-infiltrating leukocytes. B16-F10 melanoma cells transplanted into IRF-8-deficient (IRF-8(-/-)) mice grow more rapidly, leading to higher numbers of lung metastasis, with respect to control animals. These events correlated with reduced dendritic cell and T cell infiltration, accumulation of myeloid-derived suppressor cells and a chemokine/chemokine receptor expression profile within the tumor microenvironment supporting tumor growth, angiogenesis, and metastasis. Noticeably, primary tumors developing in IRF-8(-/-) mice displayed a clear-cut inhibition of IRF-8 expression in melanoma cells. Injection of the demethylating agent 5-aza-2'-deoxycytidine into melanoma-bearing IRF-8(-/-) animals induced intratumoral IRF-8 expression and resulted in the re-establishment of a chemokine/ chemokine receptor pattern favoring leukocyte infiltration and melanoma growth arrest. Importantly, intrinsic IRF-8 expression was progressively down-modulated during melanoma growth in mice and in human metastatic melanoma cells with respect to primary tumors. Lastly, IRF-8 expression in melanoma cells was directly modulated by soluble factors, among which interleukin-27 (IL-27), released by immune cells from tumor-bearing mice. Collectively, these results underscore a key role of IRF-8 in the cross talk between melanoma and immune cells, thus revealing its critical function within the tumor microenvironment in regulating melanoma progression and invasiveness.

An integrated approach identifies IFN-regulated microRNAs and targeted mRNAs modulated by different HCV replicon clones.

BACKGROUND: Infections with hepatitis C virus (HCV) progress to chronic phase in 80% of patients. To date, the effect produced by HCV on the expression of microRNAs (miRs) involved in the interferon-ß (IFN-ß) antiviral pathway has not been explored in details. Thus, we compared the expression profile of 24 selected miRs in IFN-ß-treated Huh-7 cells and in three different clones of Huh-7 cells carrying a self-replicating HCV RNA which express all viral proteins (HCV replicon system). METHODS: The expression profile of 24 selected miRs in IFN-ß-treated Huh-7 cells and in HCV replicon 21-5 clone with respect to Huh-7 parental cells was analysed by real-time PCR. To exclude clone specific variations, the level of 16 out of 24 miRs, found to be modulated in 21-5 clone, was evaluated in two other HCV replicon clones, 22-6 and 21-7. Prediction of target genes of 3 miRs, confirmed in all HCV clones, was performed by means of miRGator program. The gene dataset obtained from microarray analysis of HCV clones was farther used to validate target prediction. RESULTS: The expression profile revealed that 16 out of 24 miRs were modulated in HCV replicon clone 21-5. Analysis in HCV replicon clones 22-6 and 21-7 indicated that 3 out of 16 miRs, (miR-128a, miR-196a and miR-142-3p) were modulated in a concerted fashion in all three HCV clones. Microarray analysis revealed that 37 out of 1981 genes, predicted targets of the 3 miRs, showed an inverse expression relationship with the corresponding miR in HCV clones, as expected for true targets. Classification of the 37 genes by Panther System indicated that the dataset contains genes involved in biological processes that sustain HCV replication and/or in pathways potentially implicated in the control of antiviral response by HCV infection. CONCLUSIONS: The present findings reveal that 3 IFN-ß-regulated miRs and 37 genes, which are likely their functional targets, were commonly modulated by HCV in three replicon clones. The future use of miR inhibitors or mimics and/or siRNAs might be useful for the development of diagnostic and therapeutic strategies aimed at the recovering of protective innate responses in HCV infections.

The dominant negative ß isoform of the glucocorticoid receptor is uniquely expressed in erythroid cells expanded from polycythemia vera patients.

Glucocorticoid receptor (GR) agonists increase erythropoiesis in vivo and in vitro. To clarify the effect of the dominant negative GRß isoform (unable to bind STAT-5) on erythropoiesis, erythroblast (EB) expansion cultures of mononuclear cells from 18 healthy (nondiseased) donors (NDs) and 16 patients with polycythemia vera (PV) were studied. GRß was expressed in all PV EBs but only in EBs from 1 ND. The A3669G polymorphism, which stabilizes GRß mRNA, had greater frequency in PV (55%; n = 22; P = .0028) and myelofibrosis (35%; n = 20) patients than in NDs (9%; n = 22) or patients with essential thrombocythemia (6%; n = 15). Dexamethasone stimulation of ND cultures increased the number of immature EBs characterized by low GATA1 and ß-globin expression, but PV cultures generated great numbers of immature EBs with low levels of GATA1 and ß-globin irrespective of dexamethasone stimulation. In ND EBs, STAT-5 was not phosphorylated after dexamethasone and erythropoietin treatment and did not form transcriptionally active complexes with GRα, whereas in PV EBs, STAT-5 was constitutively phosphorylated, but the formation of GR/STAT-5 complexes was prevented by expression of GRß. These data indicate that GRß expression and the presence of A3669G likely contribute to development of erythrocytosis in PV and provide a potential target for identification of novel therapeutic agents.

Human papillomavirus type 16 E5 protein induces expression of beta interferon through interferon regulatory factor 1 in human keratinocytes.

Crucial steps in high-risk human papillomavirus (HR-HPV)-related carcinogenesis are the integration of HR-HPV into the host genome and loss of viral episomes. The mechanisms that promote cervical neoplastic progression are, however, not clearly understood. During HR-HPV infection, the HPV E5 protein is expressed in precancerous stages but not after viral integration. Given that it has been reported that loss of HPV16 episomes and cervical tumor progression are associated with increased expression of antiviral genes that are inducible by type I interferon (IFN), we asked whether E5, expressed in early phases of cervical carcinogenesis, affects IFN-ß signaling. We show that the HPV type 16 (HPV16) E5 protein expression per se stimulates IFN-ß expression. This stimulation is specifically mediated by the induction of interferon regulatory factor 1 (IRF-1) which, in turn, induces transcriptional activation of IRF-1-targeted interferon-stimulated genes (ISGs) as double-stranded RNA-dependent protein kinase R (PKR) and caspase 8. Our data show a new and unexpected role for HR-HPV E5 protein and indicate that HPV16 E5 may contribute to the mechanisms responsible for cervical carcinogenesis in part via stimulation of IFN-ß and an IFN signature, with IRF-1 playing a pivotal role. HPV16 E5 and IRF-1 may thus serve as potential therapeutic targets in HPV-associated premalignant lesions.