Expression of CD4 on human peripheral blood neutrophils

AbstractCD4, the primary receptor for entry of HIV, is known to be expressed on T cells and monocytes/macrophages; healthy natural killer (NK) lymphocytes; in vitro human herpesvirus 6 (HHV6)–infected CD8+, NK, and γδ T lymphocytes; CD34+ progenitor cells; and a subset of eosinophils and basophils. We here report the unconventional expression of CD4 at the surface of peripheral blood neutrophils derived from 4 of 51 (7.8%) HIV-1–infected and 3 of 25 (12%) uninfected donors, with similar frequency within the 2 groups. The percentage of CD4+ neutrophils ranged from 39% to 97% of the total neutrophil population. Both surface and cytoplasmic forms of CD4 were present in neutrophils. Quantitative RNA polymerase chain reaction (PCR) revealed that neutrophils contain levels of CD4 mRNA comparable to those of peripheral blood mononuclear cells derived from the same donor. The conformation of CD4 expressed at the surface of neutrophils was similar to that of CD4 expressed on T lymphocytes as determined by the binding of monoclonal antibodies specific for conformational epitopes and the binding of recombinant HIV-1 gp120. Thus, our data provide evidence that neutrophils express endogenous CD4 and bind HIV. Owing to their abundance in peripheral blood, CD4+ neutrophils may influence significantly the biodistribution of HIV delivering it to sites of inflammation or to additional tissue reservoirs

Arqus Openness Position Paper

The Openness Position Paper published by the Arqus European University Alliance emphasises that Arqus institutions, in line with the policies, roadmaps and strategies of the EU and a wide range of stakeholders, are striving jointly to make further progress towards realising Open Science. The Position Paper identifies and acknowledges aims and values of Open Science and relates them to values, principles, and standards shared by the Arqus Alliance, followed by a vision for a future with Open Science. In the interest of a nuanced picture, the Position Paper discusses not only desired effects, but also possible areas of tension related to Open Science. It presents a wide range of specific aims and recommendations for each of the eleven elements of Open Science defined by the Arqus Openness Task Force: Governance Publications (including Open Access) Data (including research data management, FAIR and Open Data) Infrastructures (including support staff, Open Science software and tools, repositories, Open Labs) Methods (including source code, preregistration, materials, workflows, protocols, lab notes) Awareness and training (including education of early-stage researchers) Evaluation (including Open Metrics, research assessment, Open Peer Review, rewards and incentives) Communication (including multilingualism) Citizen Science Open Education Open Innovation The Position Paper concludes with an annex that highlights the progress already made in the implementation and support of Open Science practices at Arqus institutions.Cofunded by the Erasmus+Programme of the European Unio

Valutazione delle risposte immunitarie umorali e cellulari di tipo B in soggetti vaccinati con Gardasil o Cervarix

The family of human papillomaviruses comprises over 120 different types that infect cutaneous and mucosal tissues, and among them high-risk genotypes (HPV16, 18, 31, 33, 45, 51, 58 and others) are strongly associated with different cancer in the genital tract in men and women. Low-risk genotypes (HPV6, 11, 40, 43 and others) are found in genital epithelial lesions but rarely detected in malignancies. Cervical cancer is the third most common cancer in women world-wide associated with persistent infection of sexually transmitted high-risk HPV genotypes. In particular, HPV16 and 18 cause more than 70% of invasive cervix cancer in women. Immunocompetent women are able to clear high-risk HPV genotypes infections in 12-18 months. This is accompanied or closely followed by seroconversion against the major coat protein L1. The antibody titres developed during natural infection are low and don’t protect against HPV reinfection, moreover, not all women seroconvert. Approximately 10% of women fail to clear HPV infection resulting in long-term persistent infection that leads to progressive disease. In 2006-2007 two prophylactic vaccines were licenced based on virus-like particles technology: a bivalent HPV16/18 L1 VLP vaccine (Cervarix, GSK) and a tetravalent HPV 6/11/16/18 L1 VLP vaccine (Gardasil, MSD). Both vaccines showed almost 100% efficacy against CIN 2/3 against vaccine-related HPV types in naïve women. The efficacy is considerably lower against HPV types not included in vaccine formulation, and also in women with evidence of previous or current infections of vaccine-related genotypes. Furthermore, both vaccines are safe, and induce high titres of type-specific neutralizing antibodies against both linear and conformational epitopes on capsid protein L1 (4 years follow up of phase III clinical trials) preventing both high risk HPV16 and 18 infection and lesion development in the cervix. In addition, the quadrivalent vaccine is protective against occurrence of external genital warts. Despite this success, several key issues are still open. In fact, reports from phase III studies suggest that the two HPV vaccines may induce different antigen-specific immune responses in terms of intensity and persistence. The generation of memory B-cells and their responses to recall antigens are crucial factors for the long-term efficacy of vaccine induced humoral protection and up to now standardized assays are not commercially available to measure HPV immunity. Moreover, the efficacy in pre- and early adolescents, the primary targets for vaccination, has not been demonstrated. Furthermore, at present, the majority of data available on the two HPV vaccines comes from studies performed by the manufacturers. In this contest, an independent study was designed by enrolling HPV vaccinated women in Veneto and Emilia Romagna Regions to a) set up standardized B-cell elispot assays to measure the frequency of memory B-cells specific to HPV6, 11, 16 and 18 VLPs; b) screen a cohort of HPV vaccinees stratified by age (12 years old vs 20-45 years old) and by time after the 3rd dose of vaccine (1-6 months vs 4 years); c) compare the immune responses of Cervarix and Gardasil HPV vaccines. This study demonstrates that Gardasil induces high and sustained number of memory B-cells against the HPV types included in the vaccine formulation. With regard to the frequency of memory B-cells the vaccine was not influenced by the age of vaccine administration and was similar among the age groups at 1-6 months and 4 years after vaccination. Furthermore, Gardasil induces high antigen-specific IgG titres in both age groups that decrease significantly 4 years after vaccination but remains still detectable. However, the IgG titres were significantly lower in the 20-45 years old group compared to the 12 years old group both both 1-6 months and 4 years after vaccination, and the percentage of vaccinees whom IgG levels were still detectable were significantly lower in the 20-45 years old group compared to the 12 years old group 4 years after the vaccination. Cervarix induces higher B-cell responses (both frequency of memory B-cells and antigen-specific IgG titres) compared to Gardasil in 12 years old vaccinees, tested 1-6 months after vaccination. Evalutation of immune responses in 12-years old Cervarix recipients 4 years after vaccination as well as in 20-45 years old (both 1-6 months and 4 years after vaccination) is in progress.Il carcinoma della cervice uterina rappresenta la seconda causa di morte per tumore tra le giovani donne fra i 15 e i 44 anni, dopo il tumore al seno. Si stimano infatti a livello mondiale 530.000 nuovi casi di tumore cervicale all’anno e circa 275.000 decessi. Lo sviluppo del tumore alla cervice uterina è imputabile all’infezione persistente, sessualmente trasmessa, di alcuni genotipi ad alto rischio di Papillomavirus (HPV); in particolare più del 70% di tutti i tumori cervicali è correlato con la presenza di HPV16 e 18 mentre il rimanente 25% è legato all’infezione causata da altri genotipi di HPV come 31, 33, 45 e 58. Le donne immunocompetenti sono in grado di eliminare spontaneamente le infezioni dei genotipi ad alto rischio in 12-18 mesi. La seroconversione che deriva dall’infezione non avviene in tutte le donne e, laddove succede, i titoli degli anticorpi neutralizzanti sono molto bassi e non proteggono da successive reinfezioni. Inoltre, il 10% delle donne non è in grado di eliminare il virus e l’infezione persistente a livello della mucosa cervicale, è il punto di partenza di una serie di eventi molecolari che portano allo sviluppo di lesioni neoplastiche. Nel 2006-2007 sono stati approvati e commercializzati due vaccini profilattici anti-HPV costituiti dalle proteine L1 del capside virale, assemblate a formare degli pseudovirioni (VLP) tridimensionalmente identici alle particelle virali native ma non infettivi: Cervarix (GSK) contenente le VLP di HPV16 e 18 e Gardasil (Merck) contenente le VLP di HPV6, 11, 16 e 18, quindi protettivo anche nei confronti di infezioni genitali di tipo benigno come i condilomi. Gli studi clinici hanno dimostrato che entrambi i vaccini sono sicuri, capaci di indurre elevati livelli di anticorpi neutralizzanti contro la proteina L1 ed efficaci nel proteggere dall’infezione da parte dei genotipi di HPV presenti nel vaccino. Tuttavia, i vaccini sono in commercio solo da pochi anni e sono necessari ulteriori studi per correlare l’efficacia di protezione con l’entità delle risposte immunitarie indotte. Per studiare l’immunità a lungo termine, la determinazione dei titoli anticorpali (neutralizzanti e non) è uno strumento utile ma non sufficiente, ed è necessario quindi quantificare i linfociti B memoria antigene-specifici. Inoltre, sono ancora pochi i dati disponibili in letteratura sull’induzione delle risposte immunitarie in funzione dell’età di somministrazione del vaccino e soprattutto la maggior parte dei dati di efficacia e di risposte immunitarie indotte da Gardasil e Cervarix provengono dalle aziende che commercializzano i vaccini. In questo contesto, lo studio indipendente da noi eseguito aveva tre obiettivi: 1) sviluppare e standardizzare una metodica di B-cell Elispot per la quantificazione dei linfociti B memoria HPV genotipo-specifici; 2) quantificare i linfociti B memoria e i titoli anticorpali, specifici per ciascun antigene vaccinale, in una popolazione di adolescenti (12 anni) e di donne (20-45 anni) vaccinate con Gardasil o Cervarix, arruolate 1-6 mesi o 4 anni dopo la vaccinazione; 3) comparare l’immunogenicità dei due vaccini anti-HPV. In particolare, ad oggi sono state arruolate 283 volontarie di cui per il vaccino Gardasil n=121 per il gruppo 12 anni, n=112 per il gruppo 20-45 anni e per il vaccino Cervarix n=60 per il gruppo 12 anni. L’arruolamento dei soggetti vaccinati con Cervarix è ancora in corso e lo studio verrà completato nei prossimi 6 mesi. I risultati dello studio hanno dimostrato che le frequenze dei linfociti B memoria HPV genotipo-specifiche indotte dal Gardasil sono elevate 1-6 mesi dopo la vaccinazione in entrambe le coorti di età e, nonostante diminuiscano nel corso del tempo, dopo 4 anni continuano ad essere significativamente elevate indipendentemente dall’età di somministrazione del vaccino. Al contrario, i titoli anticorpali sono influenzati dall’età di somministrazione del vaccino. Infatti, i titoli anticorpali misurati nella coorte di adolescenti sono sempre significativamente superiori rispetto a quelli misurati nella corte delle donne, sia 1-6 mesi sia 4 anni dopo la vaccinazione. In aggiunta, la percentuale di individui i cui titoli anticorpali non sono più misurabili, dopo 4 anni dalla vaccinazione, è significativamente superiore nelle donne rispetto alle adolescenti. Relativamente alla comparazione delle risposte immunitarie indotte dai due vaccini anti-HPV, i risultati preliminari ottenuti in un gruppo di adolescenti vaccinate con Cervarix dimostrano che 1-6 mesi dopo la vaccinazione il Cervarix induce titoli anticorpali e frequenze di linfociti B memoria anti-HPV16 e 18 significativamente superiori rispetto ad adolescenti vaccinate con Gardasil. I risultati ottenuti potranno essere un utile ausilio per il Ministero della Salute per tracciare le linee guida di prevenzione primaria del tumore alla cervice uterina

1,25-Dihydroxyvitamin D3 Upregulates Functional CXCR4 Human Immunodeficiency Virus Type 1 Coreceptors in U937 Minus Clones: NF-κB-Independent Enhancement of Viral Replication

U937 cell clones which sustain efficient or poor replication of human immunodeficiency virus type 1 (HIV-1) (referred to herein as plus clones and minus clones, respectively) have been previously described. 1,25-Dihydroxyvitamin D3 (vitamin D3) potently induced HIV-1 replication and proviral DNA accumulation in minus clones but not in plus clones. Vitamin D3 did not induce NF-κB activation but selectively upregulated CXCR4 expression in minus clones. The CXCR4 ligand stromal-cell derived factor-1 induced Ca(2+) fluxes and inhibited both constitutive and vitamin D3-enhanced HIV replication in minus clones

EIF2A-dependent translational arrest protects leukemia cells from the energetic stress induced by NAMPT inhibition

Cell-cycle analysis andClick-iTdetection of RNA and Protein synthesis. A) Cell-cycle analysis with PI staining of the nuclei after 48 h of treatment. Overnight serum starvation is shown as a positive control of induced cell cycle synchronization in G0/G1 phase. Cell phase analysis was done with ModFit LT 3.2 software by using the Sync Wizard model (30000 cells/sample in biological duplicate). B) Jurkat cells were treated for 48 h with or without (Mock) the indicated concentration of FK866 or for 3 h with 5 μM Actinomycin D, an RNA synthesis blocking agent, then subjected to Click-it biochemistry and flow-cytometry analyses including 7-AAD to identify living cells. C) Jurkat cells were treated for 48 h with or without (Mock) the indicated concentration of FK866 or for 3 h with 350 μM Cycloheximide, as a positive control for protein synthesis inhibition, then stained as in B. In B and C. Experiments were carried out on two biological replicates (50000 events/sample). (PDF 1562 kb

Characterization of a new CDC73 missense mutation that impairs Parafibromin expression and nucleolar localization.

Mutations of the Cell Division Cycle 73 (CDC73) tumor suppressor gene (previously known as HRPT2), encoding for parafibromin, are associated with the Hyperparathyroidism-Jaw Tumor (HPT-JT) syndrome, an autosomal dominant disease whose clinical manifestations are mainly parathyroid tumors and, less frequently, ossifying fibromas of the jaws, uterine and renal tumors. Most mutations of CDC73 are nonsense or frameshift, while missense mutations are rare and generally affect the N-terminal domain of parafibromin, a region that is still poorly characterized. The aim of this study was to characterize a novel somatic CDC73 missense mutation (Ile60Asn) identified in the mandibular tumor of a HPT-JT patient carrying a germline CDC73 inactivating mutation. Immunostaining of the tumor showed reduced nuclear parafibromin immunoreactivity. Western blotting and confocal microscopy of transfected cells demonstrated that the Ile60Asn mutant parafibromin was less expressed than the wild-type protein and exhibited impaired nucleolar localization. Treatment of transfected cells with translation and proteasome inhibitors demonstrated a decreased stability of the Ile60An mutant, partially due to an increase in proteasomal degradation. Overexpression of the Ile60Asn mutant led to increased cell proliferation and to accumulation in the G2/M phase of cell cycle. Moreover, mutant parafibromin lost the ability to down-regulate c-myc expression. In conclusion, our study shows that a missense mutation in the N-terminus of parafibromin, identified in an ossifying fibroma from a HPT-JT patient, stimulated cell proliferation and impaired parafibromin expression and nucleolar localization, suggesting a relevant role of the N-terminal domain for parafibromin function