Effects of the age of vaccination on the humoral responses to a human papillomavirus vaccine.

Adult vaccination programs are receiving increasing attention however, little is known regarding the impact of age on the maintenance of the immune response. We investigated this issue in the context of a human papillomavirus (HPV) vaccination program collecting real-world data on the durability of humoral immunity in 315 female subjects stratified according to vaccination age (adolescents and adults) and sampled at early or late time points after the last vaccine dose. HPV-specific IgGs, but not memory B cells, were induced and maintained at higher levels in subjects vaccinated during adolescence. Nonetheless, antibody functions waned over time to a similar degree in adolescents and adults. To shed light on this phenomena, we analyzed quantitative and qualitative properties of lymphocytes. Similar biochemical features were observed between B-cell subsets from individuals belonging to the two age groups. Long term humoral responses toward vaccines administered at an earlier age were comparably maintained between adolescents and adults. The percentages of naïve B and CD4+ T cells were significantly higher in adolescents, and the latter directly correlated with IgG titers against 3 out of 4 HPV types. Our results indicate that age-specific HPV vaccine responsiveness is mostly due to quantitative differences of immune cell precursors rather than qualitative defects in B cells. In addition, our results indicate that adults also have a good humoral immunogenic profile, suggesting that their inclusion in catch-up programmes is desirable.

HPV-Specific Systemic Antibody Responses and Memory B Cells are Independently Maintained up to 6 Years and in a Vaccine-Specific Manner Following Immunization with Cervarix and Gardasil in Adolescent and Young Adult Women in Vaccination Programs in Italy.

: Human papillomavirus (HPV) persistent infections are associated with cervical cancer and other HPV-related diseases and tumors. Thus, the characterization of long lasting immunity to currently available HPV vaccines is important. A total of 149 female subjects vaccinated with Cervarix or Gardasil participated to the study and they were stratified according to age (10-12-year-old and 16-20-year-old). Humoral immune responses (IgG and neutralizing antibody titers, antibody avidity) and circulating memory B cells were analyzed after an average of 4-6 years from the third immunization. The humoral responses against HPV-16 and HPV-18 (and HPV-6 and HPV-11 for Gardasil) were high in both age groups and vaccines up to six years from the third dose. However, Cervarix induced significantly higher and more persistent antibody responses, while the two vaccines were rather equivalent in inducing memory B cells against HPV-16 and HPV-18. Moreover, the percentage of subjects with vaccine-specific memory B cells was even superior among Gardasil vaccinees and, conversely, Cervarix vaccinated individuals with circulating antibodies, but undetectable memory B cells were found. Finally, a higher proportion of Cervarix-vaccinated subjects displayed cross-neutralizing responses against non-vaccine types HPV-31 and HPV-45. Gardasil and Cervarix may, thus, differently affect long-lasting humoral immunity from both the quantitative and qualitative point of view.

Development of Lentiviral Vectors Simultaneously Expressing Multiple siRNAs Against CCR5, vif and tat/rev Genes for an HIV-1 Gene Therapy Approach.

Gene therapy holds considerable promise for the functional cure of HIV-1 infection and, in this context, RNA interference (RNAi)-based approaches represent powerful strategies. Stable expression of small interfering RNAs (siRNAs) targeting HIV genes or cellular cofactors has the potential to render HIV-1 susceptible cells resistant to infection. To inhibit different steps of virus life cycle, self-inactivating lentiviral vectors expressing multiple siRNAs targeting the CCR5 cellular gene as well as vif and tat/rev viral transcripts, under the control of different RNA polymerase III promoters (U6, 7SK, H1) were developed. The use of a single RNA polymerase III promoter driving the expression of a sequence giving rise to three siRNAs directed against the selected targets (e-shRNA) was also investigated. Luciferase assay and inhibition of HIV-1 replication in human Jurkat T-cell line were adopted to select the best combination of promoter/siRNA. The efficacy of selected developed combinatorial vectors in interfering with viral replication was evaluated in human primary CD4(+) T lymphocytes. We identified two effective anti-HIV combinatorial vectors that conferred protection against R5- and X4- tropic viruses. Overall, our results showed that the antiviral effect is influenced by different factors, including the promoter used to express the RNAi molecules and the selected cassette combination. These findings contribute to gain further insights in the design of RNAi-based gene therapy approaches against HIV-1 for clinical application.

Dihydrotanshinone-I interferes with the RNA-binding activity of HuR affecting its post-transcriptional function.

Post-transcriptional regulation is an essential determinant of gene expression programs in physiological and pathological conditions. HuR is a RNA-binding protein that orchestrates the stabilization and translation of mRNAs, critical in inflammation and tumor progression, including tumor necrosis factor-alpha (TNF). We identified the low molecular weight compound 15,16-dihydrotanshinone-I (DHTS), well known in traditional Chinese medicine practice, through a validated high throughput screening on a set of anti-inflammatory agents for its ability to prevent HuR:RNA complex formation. We found that DHTS interferes with the association step between HuR and the RNA with an equilibrium dissociation constant in the nanomolar range in vitro (Ki = 3.74 ± 1.63 nM). In breast cancer cell lines, short term exposure to DHTS influences mRNA stability and translational efficiency of TNF in a HuR-dependent manner and also other functional readouts of its post-transcriptional control, such as the stability of selected pre-mRNAs. Importantly, we show that migration and sensitivity of breast cancer cells to DHTS are modulated by HuR expression, indicating that HuR is among the preferential intracellular targets of DHTS. Here, we disclose a previously unrecognized molecular mechanism exerted by DHTS, opening new perspectives to therapeutically target the HuR mediated, post-transcriptional control in inflammation and cancer cells.

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

BACKGROUND: Nicotinamide phosphoribosyltransferase (NAMPT), the rate-limiting enzyme in NAD(+) biosynthesis from nicotinamide, is one of the major factors regulating cancer cells metabolism and is considered a promising target for treating cancer. The prototypical NAMPT inhibitor FK866 effectively lowers NAD(+) levels in cancer cells, reducing the activity of NAD(+)-dependent enzymes, lowering intracellular ATP, and promoting cell death. RESULTS: We show that FK866 induces a translational arrest in leukemia cells through inhibition of MTOR/4EBP1 signaling and of the initiation factors EIF4E and EIF2A. Specifically, treatment with FK866 is shown to induce 5'AMP-activated protein kinase (AMPK) activation, which, together with EIF2A phosphorylation, is responsible for the inhibition of protein synthesis. Notably, such an effect was also observed in patients' derived primary leukemia cells including T-cell Acute Lymphoblastic Leukemia. Jurkat cells in which AMPK or LKB1 expression was silenced or in which a non-phosphorylatable EIF2A mutant was ectopically expressed showed enhanced sensitivity to the NAMPT inhibitor, confirming a key role for the LKB1-AMPK-EIF2A axis in cell fate determination in response to energetic stress via NAD(+) depletion. CONCLUSIONS: We identified EIF2A phosphorylation as a novel early molecular event occurring in response to NAMPT inhibition and mediating protein synthesis arrest. In addition, our data suggest that tumors exhibiting an impaired LBK1- AMPK- EIF2A response may be especially susceptible to NAMPT inhibitors and thus become an elective indication for this type of agents.

Targeting the multifaceted HuR protein, benefits and caveats.

The RNA-binding protein (RBP) HuR is one of the most widely studied regulators of the eukaryotic posttranscriptional gene expression and it plays a physiological role in mediating the cellular response to apoptotic, proliferating and survival stimuli. Following physiological or stress stimuli, HuR protein binds to Adenylate-Urydinilate rich elements (AREs) generally contained in the 3'UTR of transcripts, then it shuttles from the nucleus to the cytoplasm and regulates the half-life and/or translation of cargo mRNAs. Derangements in sub-cellular localization and expression of HuR have been associated with the pathophysiology of many diseases and this protein has been proposed as a potential drug target. Recent findings also re-evaluated HuR as a splicing and polyadenylation factor, expanding its spectrum of functional activity up to the maturation of pre-mRNAs. In this review, we generate a comprehensive picture of HuR functionality to discuss the implications of considering HuR as pharmacological target and the detrimental or positive impact that can be expected upon its modulation. Firstly, we focus on the recent findings about the mechanistic role of HuR in the nucleus and in the regulation of long non coding RNAs; then we describe the animal models and the clinical association and significance in cancer; finally, we have reviewed the pharmacological tools that influence HuR's post-transcriptional control and the efforts made to identify specific HuR inhibitors.

Neutralizing and cross-neutralizing antibody titres induced by bivalent and quadrivalent human papillomavirus vaccines in the target population of organized vaccination programmes.

Aim of this investigator-initiated study was to evaluate and compare the titres of neutralizing and cross-neutralizing antibodies (NAbs) induced by the bivalent (Cervarix(®)) and quadrivalent (Gardasil(®)) HPV vaccines in a cohort of girls aged 11-13 years from organized vaccination programmes. To this aim, HPV16 and HPV18 NAbs were measured by pseudovirion-based neutralization assays in serum collected at 1-6 months after the third vaccine dose in 107 girls vaccinated with Cervarix(®) and 126 vaccinated with Gardasil(®), while HPV31 and HPV45 cross-NAbs were tested in the first 50 consecutive girls of both vaccine groups. The results of this study demonstrated that all vaccinated girls developed HPV16 and HPV18 NAbs, with the exception of two Gardasil(®) vaccinees with undetectable HPV18 NAbs. Geometric mean titres (GMTs) of both HPV16 and HPV18 NAbs were significantly higher in Cervarix(®) than in Gardasil(®) vaccinees [HPV16 NAb GMT 22,136 (95% CI, 18,811-26,073) vs 5092 (4230-6151), respectively; P<0.0001; HPV18 NAb GMT 11,962 (9536-14,363) vs 1804 (1574-2110), respectively; P<0.0001]. Cross-NAbs to HPV31 and HPV45 were detected more frequently Cervarix(®) (HPV31 NAb positivity rates 92.7% and 36%, respectively; P<0.05) than in Gardasil(®) vaccinees (HPV45 NAb positivity rates 56% and 6%, respectively; P<0.0001). The titres of cross-NAbs against HPV31 and HPV45 were also significantly higher in Cervarix(®) than in Gardasil(®) vaccinees [HPV31 NAb GMT 157.2 (95% CI, 92-269) vs 13.0 (6.5-25.8), respectively; P<0.0001; HPV45 NAb GMT 4.7 (2.1-10.2) vs 1.3 (0.3-3.1), respectively; P<0.01]. In conclusion, in adolescent girls vaccinated within organized vaccination programmes, HPV vaccines drive the generation not only of NAbs to HPV vaccine types, but also of cross-NAbs. The bivalent vaccine induced significantly higher HPV16 and HPV18 NAb titres and more frequently and at higher titre HPV31 and HPV45 cross-NAbs than the quadrivalent vaccine.

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.

Improvement of CXCR3 ligand CXCL11/I-TAC measurement in human plasma and serum.

The chemokine receptor CXCR3 is involved in cell trafficking dysregulation associated with several inflammatory conditions, including autoimmune and viral diseases. Downregulation of CXCR3, through binding with its ligand CXCL11 (I-TAC), represents a key mechanism in lymphocyte recruitment. Determination of circulating I-TAC can provide useful information in the investigation of inflammatory/infectious conditions. The existing commercial kit does not measure CXCL11/I-TAC in complex matrices, such as human plasma and serum, as reliably as in in vitro-generated cell culture supernatants. We here describe means which lead to an improvement of CXCL11/I-TAC measurement in human plasma and serum.

Myeloid-derived suppressor cell role in tumor-related inflammation.

Chronic inflammatory state can create a proper environment for neoplastic onset and sustain cancer growth. The inflammatory state that arises at the tumor edge could contribute to immune escape phenomena in many ways. Myeloid-derived suppressor cells (MDSCs), a cell population that contributes to tumor escape, immune tolerance, and suppression, respond to a variety of pro-inflammatory and anti-inflammatory stimuli, which drive their recruitment and activation. Understanding how the inflammatory milieu favours tumor escape through the accumulation of MDSCs could be very useful to improve the efficacy of cancer immunotherapy.

Role of arginine metabolism in immunity and immunopathology.

A heterogeneous set of cells that are commonly grouped as "myeloid cells", interacts in a complex landscape of physiological and pathological situations. In this review we attempt to trace a profile of the "myeloid connection" through different normal and pathological states, by analyzing common metabolic pathways of the amino acid l-arginine. Myeloid cells exert various, often divergent, actions on the immune response through mechanisms that exploit mediators of this peculiar metabolic pathway, ranging from l-arginine itself to its downstream metabolites, like nitric oxide and polyamines. Various pathological situations, including neoplastic and autoimmune diseases, as well as injury repair and infections are discussed here, showing how l-arginine metabolism is able to play a dual role, both as an active protector and a possible threat to the organism.

Immunomodulatory effects of bovine colostrum in human peripheral blood mononuclear cells.

Human and bovine colostrum (BC) contain a remarkable amount of bioactive substances, including antibodies towards many common pathogens of the intestinal and respiratory tract as well as growth factors, vitamins, cytokines and other proteic, lipidic and glucidic factors. In this study we investigated whether BC had any immunomodulatory effect on human peripheral blood mononuclear cells (PBMC) from healthy donors. To this aim we focused on the production of IL-12 and IFN-gamma, cytokines involved in the Th1 polarization required for a successful immune response towards intracellular pathogens, such as bacteria and viruses. BC induced a dose-dependent production of IL-12 by CD14+ monocytes, but was unable to induce IFN-gamma production. However, BC differentially affected stimuli-induced IFN-gamma production: it enhanced IFN-gamma in response to weak antigenic stimulation and it inhibited IFN-gamma in response to strong antigenic stimulation. These effects were not dose-dependent. We also measured PBMC proliferation, which was substantially unaffected by BC. Our data suggest that the Th1-promoting activity of BC could contribute, together with the antibodies, to the protective effect of BC on the offspring. BC could also represent an inexpensive therapeutic tool in prevention and treatment of several human microbial infections, including influenza.

Favorable outcome of ex-vivo purging of monocytes after the reintroduction of treatment after interruption in patients infected with multidrug resistant HIV-1.

In multidrug resistant patients treatment interruptions allow the selection of archived wild-type drug-susceptible viruses that compete for the less fit drug-resistant strains. However, the selection of viruses with increased replicative capacity is often followed by a loss of CD4+ T cells. In addition, drug resistant variants later re-emerge limiting the overall clinical benefit of treatment interruption. Blood monocytes are a key component of the HIV reservoir and can be partially removed by a system for purging of myeloid cells (MYP). This study tested the safety and efficacy of MYP on multidrug resistant patients who underwent treatment interruption. Twelve patients were randomized to receive or not six cycles of MYP during treatment interruption. An optimized antiretroviral regimen was reintroduced after the reappearance of a drug susceptible genotype. Following therapy reintroduction, a long lasting increase in CD4+ T cell counts was observed only in the treatment interruption + MYP patients but not in the control patients. Five/six treatment interruption + MYP patients never experienced virological rebound during a median follow up period of 98 weeks. In contrast, 4/6 patients who did not receive MYP never reached complete viral suppression and had a virological rebound after a median of 16.5 weeks after treatment reintroduction. The difference between the two groups in the time to virological rebound was statistically significant (P = 0.021). A consistent decrease of HIV DNA load in CD14+ purified cells was observed only in treatment interruption + MYP patients. These data suggest that MYP can improve the immunological and virological response to treatment interruption.

A simplified flow cytometry method of CD4 and CD8 cell counting based on thermoresistant reagents: implications for large scale monitoring of HIV-infected patients in resource-limited settings.

OBJECTIVE: To validate a simplified flow cytometry assay for CD4 and CD8 T cell counting based on monoclonal antibodies which are made resistant to high temperatures (simplified thermoresistant assay (STRA)). METHOD: The STRA employs FITC-conjugated anti-CD4 and anti-CD8 monoclonal antibodies, predispensed into test tubes and chemically treated to be resistant to high temperatures. Five correlation studies were performed in three different laboratories on a total of 560 blood samples from HIV-1 infected patients. Each study correlated the STRA with either double or single platform assays currently available. Accelerated stability tests on the FITC-conjugated monoclonal antibodies were performed to assess the resistance of the STRA to high temperatures. RESULTS: Comparison of STRA with both single platform and double platform assays gave correlation coefficients ranging 0.957-0.987 for CD4+ T cells and 0.946-0.968 for CD8+ T cells. In all correlation studies there was a perfect data overlapping in the low-pathological interval of CD4+ T cells (0-400 cells/ml). The FITC-conjugated CD4 and CD8 monoclonal antibodies maintained intact binding activity and fluorescence brightness after storage for 4 weeks at 45 degrees C and can be stored for up to 8 years in regular conditions (+4 degrees C). CONCLUSIONS: The STRA correlates well with both single-platform and double-platform flow-cytometry assays currently used to assess CD4+ T cells. The test procedure is simple, rapid, and easy to perform. The reagents can be stored under unfavorable environmental conditions for long period of time. These features should facilitate access to flow cytometry testing in resource-poor settings.

In vivo modulation of leukocyte trafficking receptor following therapeutic purging of myeloid cells: implications for treatment of HIV infection and other immune disorders.

Therapeutic purging of myeloid cells (monocytes and granulocytes) (MYP) has been proposed as a treatment of severe inflammatory conditions like ulcerative colitis and rheumatoid arthritis. Although direct purging of inflammatory cells contributes to its efficacy, the precise mechanism of action is still unclear. We have tested MYP in a pilot study on 12 patients with chronic HIV infection, of whom 6 underwent MYP. Three/6 MYP patients and none of the controls displayed a strong and long-lasting decrease of cells expressing CXCR3, a major chemokine receptor responsible for trafficking of inflammatory cells. In these three patients, the number of circulating CD4 T cells increased during treatment. The data provide a rational for the use of MYP as a therapeutic tool acting via the modulation of immune cell trafficking.

CD30 ligation differentially affects CXCR4-dependent HIV-1 replication and soluble CD30 secretion in non-Hodgkin cell lines and in gamma delta T lymphocytes.

We studied whether signaling through CD30, a member of the TNF receptor family, affected acute infection with HIV-1, encompassing its entire replicative cycle. Several non-Hodgkin cell lines, targets of CXCR4-dependent (X4) HIV-1 infection, were positive for CD30 expression. CD30 ligation induced up-regulation of viral replication only in certain CD30+ cell lines. Enhancement of X4 virus replication by CD30 engagement inversely correlated with both CD30 surface density and constitutive NF-kappaB activation. Conversely, expression of CD30, but not of other members of the TNF receptor family, was proportional to constitutive NF-kappaB binding. Concomitantly, secretion of soluble (s) CD30 increased in all cell lines by CD30 ligation. sCD30 release was enhanced by engagement of CD30 alone and, to a greater extent, by co-engagement of CD3 also in primary gamma delta T lymphocytes, along with complementary modulations of their surface CD30 expression. sCD30-containing supernatant specifically inhibited HIV-1 expression induced by CD30 engagement in chronically infected ACH-2 T cells; thus sCD30 may act as a negative feed-back molecule. In conclusion, we have delineated novel features of CD30 biology and underline the peculiar link of CD30 expression to constitutive NF-kappaB activation which is pivotal to both HIV replication and cell survival.

Double-edged effect of Vgamma9/Vdelta2 T lymphocytes on viral expression in an in vitro model of HIV-1/mycobacteria co-infection.

A reciprocal influence exists between mycobacteria and HIV: HIV-infected individuals are more susceptible to mycobacterial infections and, on the other hand, mycobacterial infection results inacceleration of HIV disease progression. Vgamma9/Vdelta2 T lymphocytes are known to participate in the defense against intracellular pathogens, including Mycobacterium tuberculosis. Indeed, they kill mycobacteria-infected macrophages and, upon recognition of mycobacterial Ag, release TNF-alpha and IFN-gamma, which are also up-regulators of HIV expression. To assess whether mycobacteria-activated gamma delta T lymphocytes contribute to the enhancement of HIV replication, we established an in vitro model mimicking HIV and mycobacteria co-infection with the latently HIV-infected promonocytic U1 cell line and Vgamma9/Vdelta2 peripheral lymphocytes stimulated with mycobacterial Ag. gamma delta T cell activation determined two distinct, but connected effects, namely U1cell death and HIV expression. Both effects were mainly mediated by release of TNF-alpha and IFN-gamma from activated gamma delta lymphocytes, although Fas-FasL interaction also contributed to U1 apoptosis. The final outcome on U1 survival, and thus, on HIV expression, highly depended on mycobacterial Ag concentration coupled to the differential secretory potency of gamma delta cells. In particular, the induction of viral expression prevailed at low Ag concentration and with lower cytokine production by mycobacteria-activated gamma delta cells. Notably, during the course of HIV infection, Vgamma9/Vdelta2 lymphocytes are reported to be functionally impaired and may thus indirectly influence the progression of HIV disease. In addition, a predominant inhibition of viral replication was encountered when mycobacteria-activated gamma delta T cells were co-cultured with primary HIV-infected macrophages. Thus, we suggest that specific recognition of mycobacterial Ag by gamma delta T lymphocytes in co-infected individuals may modulate viral replication through the complex array of soluble factors released.

Expression of CD4 on human peripheral blood neutrophils.

CD4, 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 gammadelta 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.