Cocrystal structure of meningococcal factor H binding protein variant 3 reveals a new crossprotective epitope recognized by human mAb 1E6.

The 4 component meningococcus B vaccine (4CMenB) vaccine is the first vaccine containing recombinant proteins licensed for the prevention of invasive meningococcal disease caused by meningococcal serogroup B strains. 4CMenB contains 3 main recombinant proteins, including the Neisseria meningitidis factor H binding protein (fHbp), a lipoprotein able to bind the human factor H. To date, over 1000 aa sequences of fHbp have been identified, and they can be divided into variant groups 1, 2, and 3, which are usually not crossprotective. Nevertheless, previous characterizations of a small set (n = 10) of mAbs generated in humans after 4CMenB immunization revealed 2 human Fabs (huFabs) (1A12, 1G3) with some crossreactivity for variants 1, 2, and 3. This unexpected result prompted us to examine a much larger set of human mAbs (n = 110), with the aim of better understanding the extent and nature of crossreactive anti-fHbp antibodies. In this study, we report an analysis of the human antibody response to fHbp, by the characterization of 110 huFabs collected from 3 adult vaccinees during a 6-mo study. Although the 4CMenB vaccine contains fHbp variant 1, 13 huFabs were also found to be crossreactive with variants 2 and 3. The crystal structure of the crossreactive huFab 1E6 in complex with fHbp variant 3 was determined, revealing a novel, highly conserved epitope distinct from the epitopes recognized by 1A12 or 1G3. Further, functional characterization shows that human mAb 1E6 is able to elicit rabbit, but not human, complement-mediated bactericidal activity against meningococci displaying fHbp from any of the 3 different variant groups. This functional and structural information about the human antibody response upon 4CMenB immunization contributes to further unraveling the immunogenic properties of fHbp. Knowledge gained about the epitope profile recognized by the human antibody repertoire could guide future vaccine design.-Bianchi, F., Veggi, D., Santini, L., Buricchi, F., Bartolini, E., Lo Surdo, P., Martinelli, M., Finco, O., Masignani, V., Bottomley, M. J., Maione, D., Cozzi, R. Cocrystal structure of meningococcal factor H binding protein variant 3 reveals a new crossprotective epitope recognized by human mAb 1E6.

Expression of factor H binding protein in meningococcal strains can vary at least 15-fold and is genetically determined.

Factor H binding protein (fHbp) is a lipoprotein of Neisseria meningitidis important for the survival of the bacterium in human blood and a component of two recently licensed vaccines against serogroup B meningococcus (MenB). Based on 866 different amino acid sequences this protein is divided into three variants or two families. Quantification of the protein is done by immunoassays such as ELISA or FACS that are susceptible to the sequence variation and expression level of the protein. Here, selected reaction monitoring mass spectrometry was used for the absolute quantification of fHbp in a large panel of strains representative of the population diversity of MenB. The analysis revealed that the level of fHbp expression can vary at least 15-fold and that variant 1 strains express significantly more protein than variant 2 or variant 3 strains. The susceptibility to complement-mediated killing correlated with the amount of protein expressed by the different meningococcal strains and this could be predicted from the nucleotide sequence of the promoter region. Finally, the absolute quantification allowed the calculation of the number of fHbp molecules per cell and to propose a mechanistic model of the engagement of C1q, the recognition component of the complement cascade.

Human circulating influenza-CD4+ ICOS1+IL-21+ T cells expand after vaccination, exert helper function, and predict antibody responses.

Protection against influenza is mediated by neutralizing antibodies, and their induction at high and sustained titers is key for successful vaccination. Optimal B cells activation requires delivery of help from CD4(+) T lymphocytes. In lymph nodes and tonsils, T-follicular helper cells have been identified as the T cells subset specialized in helping B lymphocytes, with interleukin-21 (IL-21) and inducible costimulatory molecule (ICOS1) playing a central role for this function. We followed the expansion of antigen-specific IL-21(+) CD4(+) T cells upon influenza vaccination in adults. We show that, after an overnight in vitro stimulation, influenza-specific IL-21(+) CD4(+) T cells can be measured in human blood, accumulate in the CXCR5(-)ICOS1(+) population, and increase in frequency after vaccination. The expansion of influenza-specific ICOS1(+)IL-21(+) CD4(+) T cells associates with and predicts the rise of functionally active antibodies to avian H5N1. We also show that blood-derived CXCR5(-)ICOS1(+) CD4(+) T cells exert helper function in vitro and support the differentiation of influenza specific B cells in an ICOS1- and IL-21-dependent manner. We propose that the expansion of antigen-specific ICOS1(+)IL-21(+) CD4(+) T cells in blood is an early marker of vaccine immunogenicity and an important immune parameter for the evaluation of novel vaccination strategies.

Two cross-reactive monoclonal antibodies recognize overlapping epitopes on Neisseria meningitidis factor H binding protein but have different functional properties.

Factor H binding protein (fHbp) is one of the main antigens of the 4-component meningococcus B (4CMenB) multicomponent vaccine against disease caused by serogroup B Neisseria meningitidis (MenB). fHbp binds the complement down-regulating protein human factor H (hfH), thus resulting in immune evasion. fHbp exists in 3 variant groups with limited cross-protective responses. Previous studies have described the generation of monoclonal antibodies (mAbs) targeting variant-specific regions of fHbp. Here we report for the first time the functional characterization of two mAbs that recognize a wide panel of fHbp variants and subvariants on the MenB surface and that are able to inhibit fHbp binding to hfH. The antigenic regions targeted by the two mAbs were accurately mapped by hydrogen-deuterium exchange mass spectrometry (HDX-MS), revealing partially overlapping epitopes on the N terminus of fHbp. Furthermore, while none of the mAbs had bactericidal activity on its own, a synergistic effect was observed for each of them when tested by the human complement serum bactericidal activity (hSBA) assay in combination with a second nonbactericidal mAb. The bases underlying fHbp variant cross-reactivity, as well as inhibition of hfH binding and cooperativity effect observed for the two mAbs, are discussed in light of the mapped epitopes.

Impact of preexisting memory to seasonal A/H1N1 influenza virus on the immune response following vaccination against avian A/H5N1 virus.

Cross-protection against divergent strains of influenza virus is an objective of various vaccination approaches. B cells cross-neutralizing several influenza A heterosubtypes have been isolated from cultured human memory B cells (MBCs) and plasmablasts early after influenza vaccination or infection. However, a systematic assessment of the frequency of MBCs and plasmablasts in the blood of healthy individuals is lacking. Here, we show that under resting conditions about 45% of human adults never vaccinated nor exposed to avian A/H5N1 influenza have detectable circulating MBCs cross-reacting with H5N1. This proportion rises to 63.3% among subjects with a large pool of MBCs specific for seasonal H1N1 (i.e. frequency ≥1% of total IgG MBCs). Moreover, subjects with high baseline frequencies of H1N1-specific MBCs had an expansion of H5N1-specific MBCs producing H5-neutralizing antibodies already after the first dose of an MF59-adjuvanted H5N1 vaccine. These results suggest that H1N1-specific MBCs contain a subset of cells cross-reacting to H5. We propose that a proportion of human adults have a pool of H5/H1 cross-reactive MBCs that contribute to the rapid rise of the antibody response to divergent influenza strains. This may have implications on vaccination strategies aimed at counteracting future influenza pandemics.

Approach to discover T- and B-cell antigens of intracellular pathogens applied to the design of Chlamydia trachomatis vaccines.

Natural immunity against obligate and/or facultative intracellular pathogens is usually mediated by both humoral and cellular immunity. The identification of those antigens stimulating both arms of the immune system is instrumental for vaccine discovery. Although high-throughput technologies have been applied for the discovery of antibody-inducing antigens, few examples of their application for T-cell antigens have been reported. We describe how the compilation of the immunome, here defined as the pool of immunogenic antigens inducing T- and B-cell responses in vivo, can lead to vaccine candidates against Chlamydia trachomatis. We selected 120 C. trachomatis proteins and assessed their immunogenicity using two parallel high-throughput approaches. Protein arrays were generated and screened with sera from C. trachomatis-infected patients to identify antibody-inducing antigens. Splenocytes from C. trachomatis-infected mice were stimulated with 79 proteins, and the frequency of antigen-specific CD4(+)/IFN-γ(+) T cells was analyzed by flow cytometry. We identified 21 antibody-inducing antigens, 16 CD4(+)/IFN-γ(+)-inducing antigens, and five antigens eliciting both types of responses. Assessment of their protective activity in a mouse model of Chlamydia muridarum lung infection led to the identification of seven antigens conferring partial protection when administered with LTK63/CpG adjuvant. Protection was largely the result of cellular immunity as assessed by CD4(+) T-cell depletion. The seven antigens provided robust additive protection when combined in four-antigen combinations. This study paves the way for the development of an effective anti-Chlamydia vaccine and provides a general approach for the discovery of vaccines against other intracellular pathogens.

Enhanced Systemic Humoral Immune Response Induced in Mice by Generalized Modules for Membrane Antigens (GMMA) Is Associated with Affinity Maturation and Isotype Switching.

Generalized Modules for Membrane Antigens (GMMA) are outer membrane vesicles derived from Gram-negative bacteria that can be used to design affordable subunit vaccines. GMMA have been observed to induce a potent humoral immune response in preclinical and clinical studies. In addition, in preclinical studies, it has been found that GMMA can be exploited as optimal antigen carriers for both protein and saccharide antigens, as they are able to promote the enhancement of the antigen-specific humoral immune response when the antigen is overexpressed or chemically conjugated to GMMA. Here we investigated the mechanism of this GMMA carrier effect by immunizing mice and using factor H binding protein and GMMA of Neisseria meningitidis B as an antigen-GMMA model. We confirmed that the antigen displayed on the GMMA surface increased the antigen-specific IgG production and, above all, the antibody functionality measured by the serum bactericidal activity. We found that the enhancement of the bactericidal capacity induced by GMMA carrying the antigen on the surface was associated with the increase in antibody affinity to the antigen, and with the switching toward IgG subclasses with more bactericidal potential. Thus, we conclude that the potent carrier effect of GMMA is due to their ability to promote a better quality of humoral immunity.

Systems analysis of human responses to an aluminium hydroxide-adsorbed TLR7 agonist (AS37) adjuvanted vaccine reveals a dose-dependent and specific activation of the interferon-mediated antiviral response.

The candidate Adjuvant System AS37 contains a synthetic toll-like receptor agonist (TLR7a) adsorbed to alum. In a phase I study (NCT02639351), healthy adults were randomised to receive one dose of licensed alum-adjuvanted meningococcal serogroup C (MenC-CRM197) conjugate vaccine (control) or MenC-CRM197 conjugate vaccine adjuvanted with AS37 (TLR7a dose 12.5, 25, 50 or 100 µg). A subset of 66 participants consented to characterisation of peripheral whole blood transcriptomic responses, systemic cytokine/chemokine responses and multiple myeloid and lymphoid cell responses as exploratory study endpoints. Blood samples were collected pre-vaccination, 6 and 24 h post-vaccination, and 3, 7, 28 and 180 days post-vaccination. The gene expression profile in whole blood showed an early, AS37-specific transcriptome response that peaked at 24 h, increased with TLR7a dose up to 50 µg and generally resolved within one week. Five clusters of differentially expressed genes were identified, including those involved in the interferon-mediated antiviral response. Evaluation of 30 cytokines/chemokines by multiplex assay showed an increased level of interferon-induced chemokine CXCL10 (IP-10) at 24 h and 3 days post-vaccination in the AS37-adjuvanted vaccine groups. Increases in activated plasmacytoid dendritic cells (pDC) and intermediate monocytes were detected 3 days post-vaccination in the AS37-adjuvanted vaccine groups. T follicular helper (Tfh) cells increased 7 days post-vaccination and were maintained at 28 days post-vaccination, particularly in the AS37-adjuvanted vaccine groups. Moreover, most of the subjects that received vaccine containing 25, 50 and 100 µg TLR7a showed an increased MenC-specific memory B cell responses versus baseline. These data show that the adsorption of TLR7a to alum promotes an immune signature consistent with TLR7 engagement, with up-regulation of interferon-inducible genes, cytokines and frequency of activated pDC, intermediate monocytes, MenC-specific memory B cells and Tfh cells. TLR7a 25-50 µg can be considered the optimal dose for AS37, particularly for the adjuvanted MenC-CRM197 conjugate vaccine.

The respiratory syncytial virus (RSV) prefusion F-protein functional antibody repertoire in adult healthy donors.

Respiratory syncytial virus (RSV) is the leading cause of death from lower respiratory tract infection in infants and children, and is responsible for considerable morbidity and mortality in older adults. Vaccines for pregnant women and elderly which are in phase III clinical studies target people with pre-existing natural immunity against RSV. To investigate the background immunity which will be impacted by vaccination, we single cell-sorted human memory B cells and dissected functional and genetic features of neutralizing antibodies (nAbs) induced by natural infection. Most nAbs recognized both the prefusion and postfusion conformations of the RSV F-protein (cross-binders) while a smaller fraction bound exclusively to the prefusion conformation. Cross-binder nAbs used a wide array of gene rearrangements, while preF-binder nAbs derived mostly from the expansion of B-cell clonotypes from the IGHV1 germline. This latter class of nAbs recognizes an epitope located between Site Ø, Site II, and Site V on the F-protein, identifying an important site of pathogen vulnerability.

Computational modeling of microfluidic data provides high-throughput affinity estimates for monoclonal antibodies.

Affinity measurement is a fundamental step in the discovery of monoclonal antibodies (mAbs) and of antigens suitable for vaccine development. Innovative affinity assays are needed due to the low throughput and/or limited dynamic range of available technologies. We combined microfluidic technology with quantum-mechanical scattering theory, in order to develop a high-throughput, broad-range methodology to measure affinity. Fluorescence intensity profiles were generated for out-of-equilibrium solutions of labelled mAbs and their antigen-binding fragments migrating along micro-columns with immobilized cognate antigen. Affinity quantification was performed by computational data analysis based on the Landau probability distribution. Experiments using a wide array of human or murine antibodies against bacterial or viral, protein or polysaccharide antigens, showed that all the antibody-antigen capture profiles (n = 841) generated at different concentrations were accurately described by the Landau distribution. A scale parameter W, proportional to the full-width-at-half-maximum of the capture profile, was shown to be independent of the antibody concentration. The W parameter correlated significantly (Pearson's r [p-value]: 0.89 [3 × 10-8]) with the equilibrium dissociation constant KD, a gold-standard affinity measure. Our method showed good intermediate precision (median coefficient of variation: 5%) and a dynamic range corresponding to KD values spanning from ~10-7 to ~10-11 Molar. Relative to assays relying on antibody-antigen equilibrium in solution, even when they are microfluidic-based, the method's turnaround times were decreased from 2 days to 2 h. The described computational modelling of antibody capture profiles represents a fast, reproducible, high-throughput methodology to accurately measure a broad range of antibody affinities in very low volumes of solution.

Antibody avidity, persistence, and response to antigen recall: comparison of vaccine adjuvants.

Differences in innate immune 'imprinting' between vaccine adjuvants may mediate dissimilar effects on the quantity/quality of persisting adaptive responses. We compared antibody avidity maturation, antibody/memory B cell/CD4+ T cell response durability, and recall responses to non-adjuvanted fractional-dose antigen administered 1-year post-immunization (Day [D]360), between hepatitis B vaccines containing Adjuvant System (AS)01B, AS01E, AS03, AS04, or Alum (NCT00805389). Both the antibody and B cell levels ranked similarly (AS01B/E/AS03 > AS04 > Alum) at peak response, at D360, and following their increases post-antigen recall (D390). Proportions of high-avidity antibodies increased post-dose 2 across all groups and persisted at D360, but avidity maturation appeared to be more strongly promoted by AS vs. Alum. Post-antigen recall, frequencies of subjects with high-avidity antibodies increased only markedly in the AS groups. Among the AS, total antibody responses were lowest for AS04. However, proportions of high-avidity antibodies were similar between groups, suggesting that MPL in AS04 contributes to avidity maturation. Specific combinations of immunoenhancers in the AS, regardless of their individual nature, increase antibody persistence and avidity maturation.

The respiratory syncytial virus fusion protein-specific B cell receptor repertoire reshaped by post-fusion subunit vaccination.

Respiratory syncytial virus (RSV) is the major cause of acute lower respiratory illness in children of less than 5 years of age which usually results in hospitalization or even in death. Vaccine development is hampered in consequence of a failed vaccine trial with fatalities in the 1960s. Even though research has been more focused on the RSV fusion protein in its pre-fusion conformation, maternal vaccination with post-fusion protein (post F) was considered as a promising vaccine strategy for passive immunization of babies, because post F preserves very potent neutralizing epitopes. We extensively analyzed post F-binding B cell receptor (BCR) repertoires of three vaccinees who received a post F-subunit vaccine in the context of a first-in-human, Phase 1, randomized, observer-blind, placebo-controlled clinical trial (ClinicalTrials.gov Identifier: NCT02298179). In order to compare the vaccine-induced BCR repertoires with BCR repertoires induced by natural infection, we also analyzed pre F- and post F-binding BCRs isolated from a healthy blood donor with relatively high F-binding memory B cell (MBC) frequencies. Analysis of the vaccine-induced repertoires revealed that preferentially VH4-encoded BCRs were expanded in response to vaccination. Estimation of antigen-driven selection further demonstrated that expanded BCRs accumulated positively selected replacement mutations which substantiated the hypothesis that post F-vaccination induces diversification of VH4-encoded BCRs in germinal centers. Comparison of the vaccine-induced BCR repertoires with clonally related pre and post F-binding BCRs of the healthy blood donor suggested that the vaccine expanded pre/post F cross-reactive MBCs. Interestingly, several vaccine-induced BCRs shared stereotypic VDJ gene junctions with known neutralizing Abs. Once expressed for functional characterization, the selected monoclonal Abs demonstrated the predicted neutralization activities in plaque reduction neutralization assays indicating that the post F-vaccine induced expansion of neutralizing BCRs.

CT043, a protective antigen that induces a CD4+ Th1 response during Chlamydia trachomatis infection in mice and humans.

Despite several decades of intensive studies, no vaccines against Chlamydia trachomatis, an intracellular pathogen causing serious ocular and urogenital diseases, are available yet. Infection-induced immunity in both animal models and humans strongly supports the notion that for a vaccine to be effective a strong CD4(+) Th1 immune response should be induced. In the course of our vaccine screening program based on the selection of chlamydial proteins eliciting cell-mediated immunity, we have found that CT043, a protein annotated as hypothetical, induces CD4(+) Th1 cells both in chlamydia-infected mice and in human patients with diagnosed C. trachomatis genital infection. DNA priming/protein boost immunization with CT043 results in a 2.6-log inclusion-forming unit reduction in the murine lung infection model. Sequence analysis of CT043 from C. trachomatis human isolates belonging to the most representative genital serovars revealed a high degree of conservation, suggesting that this antigen could provide cross-serotype protection. Therefore, CT043 is a promising vaccine candidate against C. trachomatis infection.

Dual role of mitochondrial reactive oxygen species in hypoxia signaling: activation of nuclear factor-{kappa}B via c-SRC and oxidant-dependent cell death.

Hypoxia is a prominent feature of solid tumor development and is known to stimulate mitochondrial ROS (mROS), which, in turn, can activate hypoxia-inducible transcription factor-1alpha and nuclear factor-kappaB (NF-kappaB). Because NF-kappaB plays a central role in carcinogenesis, we examined the mechanism of mROS-mediated NF-kappaB activation and the fate of cancer cells during hypoxia after mitochondrial reduced glutathione (mGSH) depletion. Hypoxia generated mROS in hepatoma (HepG2, H35), neuroblastoma (SH-SY5Y), and colon carcinoma (DLD-1) cells, leading to hypoxia-inducible transcription factor-1alpha-dependent gene expression and c-Src activation that was prevented in cells expressing a redox-insensitive c-Src mutant (C487A). c-Src stimulation activated NF-kappaB without IkappaB-alpha degradation due to IkappaB-alpha tyrosine phosphorylation that was inhibited by rotenone/TTFA or c-Src antagonism. The c-Src-NF-kappaB signaling contributed to the survival of cells during hypoxia as c-Src inhibition or p65 down-regulation by small interfering RNA-sensitized HepG2 cells to hypoxia-induced cell death. Moreover, selective mGSH depletion resulted in an accelerated and enhanced mROS generation by hypoxia that killed SH-SY5Y and DLD-1 cells without disabling the c-Src-NF-kappaB pathway. Thus, although mROS promote cell survival by NF-kappaB activation via c-Src, mROS overgeneration may be exploited to sensitize cancer cells to hypoxia.

Intracellular reactive oxygen species activate Src tyrosine kinase during cell adhesion and anchorage-dependent cell growth.

Src tyrosine kinases are central components of adhesive responses and are required for cell spreading onto the extracellular matrix. Among other intracellular messengers elicited by integrin ligation are reactive oxygen species, which act as synergistic mediators of cytoskeleton rearrangement and cell spreading. We report that after integrin ligation, the tyrosine kinase Src is oxidized and activated. Src displays an early activation phase, concurrent with focal adhesion formation and driven mainly by Tyr527 dephosphorylation, and a late phase, concomitant with reactive oxygen species production, cell spreading, and integrin-elicited kinase oxidation. In addition, our results suggest that reactive oxygen species are key mediators of in vitro and in vivo v-Src tumorigenic properties, as both antioxidant treatments and the oxidant-insensitive C245A and C487A Src mutants greatly decrease invasivity, serum-independent and anchorage-independent growth, and tumor onset. Therefore we propose that, in addition to the known phosphorylation/dephosphorylation circuitry, redox regulation of Src activity is required during both cell attachment to the extracellular matrix and tumorigenesis.

A phase I, randomized, controlled, dose-ranging study of investigational acellular pertussis (aP) and reduced tetanus-diphtheria-acellular pertussis (TdaP) booster vaccines in adults.

Despite high vaccination coverage worldwide, pertussis has re-emerged in many countries. This randomized, controlled, observer-blind phase I study and extension study in Belgium (March 2012-June 2015) assessed safety and immunogenicity of investigational acellular pertussis vaccines containing genetically detoxified pertussis toxin (PT) (NCT01529645; NCT02382913). 420 healthy adults (average age: 26.8 ± 5.5 years, 60% female) were randomized to 1 of 10 vaccine groups: 3 investigational aP vaccines (containing pertussis antigens PT, filamentous hemagglutinin [FHA] and pertactin [PRN] at different dosages), 6 investigational TdaP (additionally containing tetanus toxoid [TT] and diphtheria toxoid [DT]), and 1 TdaP comparator containing chemically inactivated PT. Antibody responses were evaluated on days 1, 8, 30, 180, 365, and approximately 3 years post-booster vaccination. Cell-mediated immune responses and PT neutralization were evaluated in a subset of participants in pre-selected groups. Local and systemic adverse events (AEs), and unsolicited AEs were collected through day 7 and 30, respectively; serious AEs and AEs leading to study withdrawal were collected through day 365 post-vaccination. Antibody responses against pertussis antigens peaked at day 30 post-vaccination and then declined but remained above baseline level at approximately 3 years post-vaccination. Responses to FHA and PRN were correlated to antigen dose. Antibody responses specific to PT, toxin neutralization activity and persistence induced by investigational formulations were similar or significantly higher than the licensed vaccine, despite lower PT doses. Of 15 serious AEs, none were considered vaccination-related; 1 led to study withdrawal (premature labor, day 364; aP4 group). This study confirmed the potential benefits of genetically detoxified PT antigen. All investigational study formulations were well tolerated.

Protein tyrosine phosphorylation and reversible oxidation: two cross-talking posttranslation modifications.

In addition to protein phosphorylation, redox-dependent posttranslational modification of proteins is emerging as a key signaling system, conserved throughout evolution, and influencing many aspects of cellular homeostasis. Recent data have provided new insight about the interplay between phosphorylation- and redox-dependent signaling, and reactive oxygen species have been included among intracellular signal transducers of growth factor and extracellular matrix receptors. Both tyrosine phosphorylation and thiol oxidation are reversible and dynamic, and this review will particularly focus on the cross-talk between these posttranslational protein regulatory means. Although these modifications share their reversibility, their effects on enzymatic activity of protein tyrosine phosphatases (PTPs) and protein tyrosine kinases (PTKs) may be even opposite. Indeed, while tyrosine phosphorylation is frequently correlated to enzyme activation, thiol oxidation leads to inactivation of PTPs and to superactivation of PTKs. Several papers describe that both these modifications occur during the same input, (i.e., cell proliferation and motility induced by numerous growth factors and cytokines). The review will discuss several aspects of phosphorylation\oxidation interplay, describing both convergent and divergent features of the integrated and coordinated function of PTPs and PTKs during signaling.

Beta-catenin interacts with low-molecular-weight protein tyrosine phosphatase leading to cadherin-mediated cell-cell adhesion increase.

Beta-catenin plays a dual role as a major constituent of cadherin-based adherens junctions and also as a transcriptional coactivator. In normal ephitelial cells, at adherens junction level, beta-catenin links cadherins to the actin cytoskeleton. The structure of adherens junctions is dynamically regulated by tyrosine phosphorylation. In particular, cell-cell adhesion can be negatively regulated through the tyrosine phosphorylation of beta-catenin. Furthermore, the loss of beta-catenin-cadherin association has been correlated with the transition from a benign tumor to an invasive, metastatic cancer. Low-molecular-weight protein tyrosine phosphatase (LMW-PTP) is a ubiquitous PTP implicated in the regulation of mitosis and cytoskeleton rearrangement. Here we demonstrate that the amount of free cytoplasmic beta-catenin is decreased in NIH3T3, which overexpresses active LMW-PTP, and this results in a stronger association between cadherin complexes and the actin-based cytoskeleton with respect to control cells. Confocal microscopy analysis shows that beta-catenin colocalizes with LMW-PTP at the plasma membrane. Furthermore, we provide evidence that beta-catenin is able to associate with LMW-PTP both in vitro and in vivo. Moreover, overexpression of active LMW-PTP strongly potentiates cadherin-mediated cell-cell adhesion, whereas a dominant-negative form of LMW-PTP induces the opposite phenotype, both in NIH3T3 and in MCF-7 carcinoma cells. On the basis of these results, we propose that the stability of cell-cell contacts at the adherens junction level is positively influenced by LMW-PTP expression, mainly because of the beta-catenin and LMW-PTP interaction at the plasma membrane level with consequent dephosphorylation.

During muscle ageing the activation of the mitogenic signalling is not sufficient to guarantee cellular duplication.

Satellite cells are quiescent cells that can be induced to proliferate by a variety of stimuli such as injury and exercise, providing in this way a source of new myoblasts that repopulate the damaged muscle. It is well known that, as senescence progresses, the muscle regenerative potential progressively diminishes, but the molecular mechanisms underlying this process are not yet completely defined. Many growth factors, including Platelet Derived Growth Factor (PDGF-BB)*, have been associated to satellite cells activation, acting as potent mitogenic agents for these cells. The aim of this study is to explore if the diminished response of senescent myoblasts to growth stimuli could be due to the inability to receive and transduce hormonal signals. Herein, we demonstrate that that although PDGF-r expression is down-regulated during senescence, the receptor is fully able to be phosphorylated and to transmit the signal. Although senescent myoblasts display increased level of phosphotyrosine phosphatases (PTPs), neither the PDGF receptor (PDGF-r) phosphorylation level nor the citosolic signal transduction machinery is affected. Indeed, we demonstrated that senescent human myoblasts are able to initiate a proper mitogenic signalling cascade, since the activation of mitogen-activated protein kinases (MAPK) and phosphatydil inositole 3 kinase (PI-3K) pathways is similar in young and senescent cells. Our data underline that, despite a conserved capability to activate PDGF-r after agonist stimulation and a functional signal transduction machinery, the mitogenic signal initiated by growth factors in senescent cells does not lead to cell division, being unable to overcome the cell cycle block, likely caused by the accumulation of the inhibitor p21WAF1.

Ex vivo analysis of human memory B lymphocytes specific for A and B influenza hemagglutinin by polychromatic flow-cytometry.

Understanding the impact that human memory B-cells (MBC), primed by previous infections or vaccination, exert on neutralizing antibody responses against drifted influenza hemagglutinin (HA) is key to design best protective vaccines. A major obstacle to these studies is the lack of practical tools to analyze HA-specific MBCs in human PBMCs ex vivo. We report here an efficient method to identify MBCs carrying HA-specific BCR in frozen PBMC samples. By using fluorochrome-tagged recombinant HA baits, and vaccine antigens from mismatched influenza strains to block BCR-independent binding, we developed a protocol suitable for quantitative, functional and molecular analysis of single MBCs specific for HA from up to two different influenza strains in the same tube. This approach will permit to identify the naive and MBC precursors of plasmablasts and novel MBCs appearing in the blood following infection or vaccination, thus clarifying the actual contribution of pre-existing MBCs in antibody responses against novel influenza viruses. Finally, this protocol can allow applying high throughput deep sequencing to analyze changes in the repertoire of HA⁺ B-cells in longitudinal samples from large cohorts of vaccinees and infected subjects with the ultimate goal of understanding the in vivo B-cell dynamics driving the evolution of broadly cross-protective antibody responses.