Seasonal H1N1 2007 influenza virus infection is associated with elevated pre-exposure antibody titers to the 2009 pandemic influenza A (H1N1) virus.

The new influenza strain detected in humans in April 2009 has caused the first influenza pandemic of the 21st century. A cross-reactive antibody response, in which antibodies against seasonal H1N1 viruses neutralized the 2009 pandemic influenza A (H1N1) virus (2009 pH1N1), was detected among individuals aged >60 years. However, factors other than age associated with such a cross-reactive antibody response are poorly documented. Our objective was to examine factors potentially associated with elevated pre-exposure viro-neutralization and hemagglutination-inhibition antibody titers against the 2009 pH1N1. We also studied factors associated with antibody titers against the 2007 seasonal H1N1 virus. One hundred subjects participating in an influenza cohort were selected. Sera collected in 2008 were analysed using hemagglutination inhibition and viro-neutralization assays for the 2009 pH1N1 virus and the 2007 seasonal H1N1 virus. Viro-neutralization results were explored using a linear mixed-effect model and hemagglutination-inhibition results using linear-regression models for interval-censored data. Elevated antibody titers against 2009 pH1N1 were associated with seasonal 2007 H1N1 infection (viro-neutralization, p 0.006; hemagglutination-inhibition, p 0.018). Elevated antibody titers were also associated with age in the viro-neutralization assay (p <0.0001). Seasonal 2007 H1N1 infection is an independent predictor of elevated pre-exposure antibody titers against 2009 pH1N1 and may have contributed to lowering the burden of the 2009 pH1N1 pandemic.

Temporal trends in baseline characteristics and peri-procedural complications of percutaneous drug-eluting stent implantation for unprotected left main disease: a single high-volume center experience.

AIM: Percutaneous drug-eluting stent (DES) implantation is commonly used in patients with unprotected left main (ULM) disease. As this procedure has been performed routinely in Turin Center since 2002, this article aimed to summarize a five year-experience in DES implantation in the ULM. METHODS: Baseline, procedural and in-hospital outcome data of all patients with ULM undergoing percutaneous coronary intervention (PCI) with DES between July 2002 and October 2006 at Turin Center have been collected. Patients were randomized into four groups: A (patients treated between July 2002 and December 2003), B (treated in 2004), C (treated in 2005) and D (treated in 2006). The baseline surgical risk features was to be compared with the European System for Cardiac Operative Risk Evaluation, disease location in the ULM, and in-hospital major adverse cerebro-cardiovascular events (MACCE), defined as death, myocardial infarction, repeat percutaneous revascularization, coronary artery bypass grafting, stroke, or stent thrombosis. RESULTS: Out of a total of 4 432 coronary interventional procedures 198 patients treated with DES in the ULM were identified. There was a significant increase in the number of patients treated (P=0.00095), but no difference in EuroSCORE across groups (P=0.14). Conversely, there was a significant temporal trend in the incidence of bifurcational ULM being treated with DES (P=0.03). Intriguingly, despite this increase in adverse lesion characteristics, no significant increase was found in the rate of in-hospital MACCE (P=0.93). CONCLUSION: In this single-center study, the number of patients being treated with DES for ULM disease has risen across the years, although keeping a similar surgical risk profile. Distal ULM involvement is no longer considered an absolute contraindication to PCI, as testified by the increasing frequency of such lesion among patients undergoing DES implantation at this Institution, with remarkably low rates of adverse events.

FDF03, a novel inhibitory receptor of the immunoglobulin superfamily, is expressed by human dendritic and myeloid cells.

In this study, we describe human FDF03, a novel member of the Ig superfamily expressed as a monomeric 44-kDa transmembrane glycoprotein and containing a single extracellular V-set Ig-like domain. Two potential secreted isoforms were also identified. The gene encoding FDF03 mapped to chromosome 7q22. FDF03 was mostly detected in hemopoietic tissues and was expressed by monocytes, macrophages, and granulocytes, but not by lymphocytes (B, T, and NK cells), indicating an expression restricted to cells of the myelomonocytic lineage. FDF03 was also strongly expressed by monocyte-derived dendritic cells (DC) and preferentially by CD14+/CD1a- DC derived from CD34+ progenitors. Moreover, flow cytometric analysis showed FDF03 expression by CD11c+ blood and tonsil DC, but not by CD11c- DC precursors. The FDF03 cytoplasmic tail contained two immunoreceptor tyrosine-based inhibitory motif (ITIM)-like sequences. When overexpressed in pervanadate-treated U937 cells, FDF03 was tyrosine-phosphorylated and recruited Src homology-2 (SH2) domain-containing protein tyrosine phosphatase (SHP)-2 and to a lesser extent SHP-1. Like engagement of the ITIM-bearing receptor LAIR-1/p40, cross-linking of FDF03 inhibited calcium mobilization in response to CD32/FcgammaRII aggregation in transfected U937 cells, thus demonstrating that FDF03 can function as an inhibitory receptor. However, in contrast to LAIR-1/p40, cross-linking of FDF03 did not inhibit GM-CSF-induced monocyte differentiation into DC. Thus, FDF03 is a novel ITIM-bearing receptor selectively expressed by cells of myeloid origin, including DC, that may regulate functions other than that of the broadly distributed LAIR-1/p40 molecule.

APCs express DCIR, a novel C-type lectin surface receptor containing an immunoreceptor tyrosine-based inhibitory motif.

We have identified a novel member of the calcium-dependent (C-type) lectin family. This molecule, designated DCIR (for dendritic cell (DC) immunoreceptor), is a type II membrane glycoprotein of 237 aa with a single carbohydrate recognition domain (CRD), closest in homology to those of the macrophage lectin and hepatic asialoglycoprotein receptors. The intracellular domain of DCIR contains a consensus immunoreceptor tyrosine-based inhibitory motif. A mouse cDNA, encoding a homologous protein has been identified. Northern blot analysis showed DCIR mRNA to be predominantly transcribed in hematopoietic tissues. The gene encoding human DCIR was localized to chromosome 12p13, in a region close to the NK gene complex. Unlike members of this complex, DCIR displays a typical lectin CRD rather than an NK cell type extracellular domain, and was expressed on DC, monocytes, macrophages, B lymphocytes, and granulocytes, but not detected on NK and T cells. DCIR was strongly expressed by DC derived from blood monocytes cultured with GM-CSF and IL-4. DCIR was mostly expressed by monocyte-related rather than Langerhans cell related DC obtained from CD34+ progenitor cells. Finally, DCIR expression was down-regulated by signals inducing DC maturation such as CD40 ligand, LPS, or TNF-alpha. Thus, DCIR is differentially expressed on DC depending on their origin and stage of maturation/activation. DCIR represents a novel surface molecule expressed by Ag presenting cells, and of potential importance in regulation of DC function.

Interleukin-17.

The particular interest of IL-17, a homodimeric cytokine of about 32 kDa, is the strict requirement for an activation signal to induce its expression from a rather restricted set of cells, human memory T cells or mouse alpha beta TCR+CD4-CD8- thymocytes. In contrast with the tightly controlled expression pattern of this gene, the IL-17 receptor, a novel cytokine receptor, is ubiquitously distributed but apparently more abundant in spleen and kidney. In addition to its capture by the T lymphotropic Herpesvirus Saimiri (HVS), this cytokine is inducing the secretion of IL-6, IL-8, PGE2, MCP-1 and G-CSF by adherent cells like fibroblasts, keratinocytes, epithelial and endothelial cells. IL-17 is also able to induce ICAM-1 surface expression, proliferation of T cells, and growth and differentiation of CD34+ human progenitors into neutrophils when cocultured in presence of irradiated fibroblasts. In vitro, IL-17 synergizes with other proinflammatory signals like TNF alpha for GM-CSF induction, and with CD40-ligand for IL-6, IL-8, RANTES and MCP-1 secretion from kidney epithelial cells. In vivo, injection of IL-17 induces a neutrophilia, except in IL-6-KO mice. The involvement of IL-17 in rejection of kidney graft has also been demonstrated. The role of this T cell secreted factor in various inflammatory processes is presently investigated.

Cross-linking of antigen receptor via Ig-beta (B29, CD79b) can induce both positive and negative signals in CD40-activated human B cells.

Antigen-dependent activation of B lymphocytes is mediated through surface immunoglobulins and their associated molecules Ig-alpha (CD79a, Mb1) and Ig-beta (CD79b, B29). Here we show that an antibody directed against the extracellular part of human Ig-beta can, when cross-linked by CD32-transfected L cells, induce an IL-2-dependent proliferation of tonsil B cells. With the use of L cells stably transfected with both CD32 and CD40L, anti-Ig-beta activation of B cells was combined with CD40 triggering, an important component of the T cell-dependent B cell activation. This dual cellular activation resulted in two different phases, with initially synergistic proliferative effects, both without and with IL-2 or IL-10. Then, after 5-6 days of culture, cells stimulated with both anti-Ig-beta and CD40L underwent massive cell death, in contrast to B cells activated with CD40L alone. Cell death was not prevented by the addition of IL-2 or IL-10, but was prevented by the addition of IL-4. These results are discussed in the context of positive and negative selection of mature B cells.

Antibodies to HLA class I alpha1 domain trigger apoptosis of CD40-activated human B lymphocytes.

We analyzed herein whether antibodies to HLA class I alpha1 domain, which trigger apoptosis of activated T cells, may also control the growth/survival of human B lymphocytes. Addition of monoclonal antibody (MoAb) 90 (mouse IgG1) or YTH862 (rat IgG2b) was found to strongly inhibit the proliferation of CD40-activated total tonsil B cells as well as that of purified naive, germinal center, and memory B-cell subsets. This inhibitory effect was not prevented by addition of B-cell tropic factors, such as interleukin-2 (IL-2), IL-4, and IL-10, and was a result of induced B-cell apoptosis as shown by using a TUNEL assay and DNA electrophoresis. In contrast, engagement of another epitope of the alpha1 domain, as well as that of the alpha2 and alpha3 domains by specific anti-HLA class I MoAbs, failed to inhibit DNA synthesis and to induce apoptosis of CD40-activated B cells. As recently reported for acquisition of sensitivity to Fas (APO-1/CD95) -dependent apoptosis, susceptibility to MoAb90-and YTH862-induced death was restricted to CD40-activated B cells, because resting and anti-IgM-activated B cells did not undergo apoptosis after HLA class I engagement. Moreover, ligation of the B-cell receptor protected CD40-activated B cells from both HLA class I- and Fas-mediated growth inhibition and apoptosis. Taken together, these results show that engagement of the alpha1 domain of HLA class I induces apoptotic cell death of CD40-activated, but not of antigen-activated B cells, and would, therefore, suggest a possible role for HLA class I molecules in the control of B-cell homeostasis.

Human interleukin 4 is a glycosaminoglycan-binding protein.

Using different binding assays we examined the interaction of the cytokine interleukin 4 (IL-4) with basement membrane. Equilibrium binding analysis revealed a high-affinity site characterized by a dissociation constant (Kd) of 0.3 nM. This interaction was confirmed by native polyacrylamide gel electrophoresis, which also indicated that the binding sites are composed of glycosaminoglycans (GAGs). In competition studies, N-sulfated GAGs (heparin and heparan sulfate) displayed a higher affinity than other GAGs for IL-4, and therefore may constitute the physiological ligand. Furthermore, the enzymatic and chemical cleavage of heparan sulfate demonstrated that only few peculiar domains (i.e. N-sulfated rich sequences) within heparan sulfate chains, displayed a significant affinity for IL-4. These data indicate a possible role of GAGs in storing IL-4 and modulating the cellular response to this cytokine.

Increased incidence of neutralizing autoantibodies against interleukin-1 alpha (IL-1 alpha) in nondestructive chronic polyarthritis.

Cytokines such as IL-1 and tumor necrosis factor alpha (TNF alpha) play a critical role in chronic joint inflammation and destruction. To study their regulation, we looked for circulating antiproinflammatory cytokine autoantibodies in 318 patients with chronic arthritis by immunoprecipitation with protein G. Anti-IL-1 alpha but not anti-IL-1 beta or anti-TNF alpha IgG antibodies were detected in 9% of blood donors and 18.9% of chronic arthritis patients. These antibodies were found more commonly and at a higher level in patients with nondestructive arthritis. Negative correlations were observed between the antibody levels and indices of disease activity and joint destruction. There was a negative association between the presence of anti-IL-1 alpha antibodies and that of HLA-DR4. These circulating anti-IL-1 alpha antibodies were not complexed with IL-1 alpha and could block specifically the biological activity of IL-1 alpha and its binding to membrane IL-1 receptors. These results indicate that these antibodies are beneficial, suggesting their contribution in the clinical presentation.

T cell interleukin-17 induces stromal cells to produce proinflammatory and hematopoietic cytokines.

Analysis of the cDNA encoding murine interleukin (IL) 17 (cytotoxic T lymphocyte associated antigen 8) predicted a secreted protein sharing 57% amino acid identity with the protein predicted from ORF13, an open reading frame of Herpesvirus saimiri. Here we report on the cloning of human IL-17 (hIL-17), the human counterpart of murine IL-17. hIL-17 is a glycoprotein of 155 amino acids secreted as an homodimer by activated memory CD4+ T cells. Although devoid of direct effects on cells of hematopoietic origin, hIL-17 and the product of its viral counterpart, ORF13, stimulate epithelial, endothelial, and fibroblastic cells to secrete cytokines such as IL-6, IL-8, and granulocyte-colony-stimulating factor, as well as prostaglandin E2. Furthermore, when cultured in the presence of hIL-17, fibroblasts could sustain the proliferation of CD34+ hematopoietic progenitors and their preferential maturation into neutrophils. These observations suggest that hIL-17 may constitute (a) an early initiator of the T cell-dependent inflammmatory reaction; and (b) an element of the cytokine network that bridges the immune system to hematopoiesis.

Inability to produce IL-6 is a functional feature of human germinal center B lymphocytes.

In response to Ag encounter, B lymphocytes undergo a complex maturation process yielding phenotypically distinct subpopulations that are located in highly organized compartments of secondary lymphoid organs. This study describes the patterns of cytokine secretion of naive, memory, and germinal center (GC) human tonsillar B lymphocytes, activated either through CD40 or B cell receptor or with Staphylococcus aureus Cowan I particles. The three B cell subpopulations produced comparable levels of IL-10 and TNF-alpha, regardless of the stimulation pathway. Interestingly, activated GC B lymphocytes fail to express IL-6, as determined both at mRNA and at protein levels, whereas both naive and memory B cells can be induced to secrete IL-6. Likewise, naive B lymphocytes undergoing dual ligation of CD40 and B cell receptor fail to express IL-6, since they acquire a GC-like phenotype. IL-6 receptors are up-regulated on both ex vivo-purified GC B lymphocytes and in vitro generated GC-like B cells, following CD40 activation. Consistent with this, addition of exogenous IL-6 sustains growth of CD40-stimulated GC B lymphocytes. Taken together, these results demonstrate that loss of IL-6 secretion is a functional characteristic of human GC B lymphocytes. The swap from an autocrine to a paracrine IL-6 response may permit a better control of B cell growth and differentiation during the germinal center reaction.

Generation and characterization of a human monoclonal autoantibody that acts as a high affinity interleukin-1 alpha specific inhibitor.

Interleukin-1 (IL-1) defines two polypeptides, IL-1 alpha and IL-1 beta, that possess a wide spectrum of biological effects. Two natural antagonists of IL-1 action have been characterized: the IL-1 receptor antagonist (IL-1Ra) and a soluble form of the type II IL-1 receptor. Neutralizing autoantibodies to IL-1 alpha have also been detected in sera of healthy individuals and patients with autoimmune or inflammatory diseases. To characterize such antibodies molecularly, we attempted to generate B cell clones producing anti-IL-1 alpha human monoclonal antibody (HuMAb) by combining Epstein-Barr virus-immortalization and CD40-activation of B lymphocytes from individuals with circulating anti-IL-1 alpha. We describe herein the generation and properties of a natural IgG4/kappa anti-IL-1 alpha monoclonal autoantibody, HuMAb X3, that bound specifically to human IL-1 alpha, but not to IL-1 beta and IL-1Ra, with a high affinity (Kd = 1.2 x 10(-10)M). HuMAb X3 inhibited IL-1 alpha binding to IL-1 receptors and neutralized biological activities of both recombinant and natural forms of IL-1 alpha. A recombinant form of HuMAb X3 was found to display identical specific IL-1 alpha antagonism. The presence of somatic mutations within X3 variable regions suggests an antigen-driven affinity maturation. This study extends the demonstration of the presence of high affinity neutralizing anti-IL-1 alpha autoantibodies that can function as a third type of IL-1 antagonist.

Negative selection of human germinal center B cells by prolonged BCR cross-linking.

The antigen receptors on T and B lymphocytes can transduce both agonist and antagonist signals leading either to activation/survival or anergy/death. The outcome of B lymphocyte antigen receptor (BCR) triggering depends upon multiple parameters which include (a) antigen concentration and valency, (b) duration of BCR occupancy, (c) receptor affinity, and (d) B cell differentiation stages. Herein, using anti-immunoglobulin kappa and lambda light chain antibodies, we analyzed the response of human naive, germinal center (GC) or memory B cells to BCR cross-linking regardless of heavy chain Ig isotype or intrinsic BCR specificity. We show that after CD40-activation, anti-BCR (kappa + gamma) can elicit an intracellular calcium flux on both GC and non-GC cells. However, prolonged BCR cross-linking induces death of CD40-activated GC B cells but enhances proliferation of naive or memory cells. Anti-kappa antibody only kills kappa + GC B cells without affecting surrounding gamma + GC B cells, thus demonstrating that BCR-mediated killing of GC B lymphocytes is a direct effect that does not involve a paracrine mechanism. BCR-mediated killing of CD40-activated GC B cells could be partially antagonized by the addition of IL-4. Moreover, in the presence of IL-4, prestimulation through CD40 could prevent subsequent anti-Ig-mediated cell death, suggesting a specific role of this combination in selection of GC B cells. This report provides evidence that in human, susceptibility to BCR killing is regulated along peripheral B cell differentiation pathway.

Bcl-2+ tonsillar plasma cells are rescued from apoptosis by bone marrow fibroblasts.

Plasma cells represent the final stage of B lymphocyte differentiation. Most plasma cells in secondary lymphoid tissues live for a few days, whereas those in the lamina propria of mucosa and in bone marrow live for several weeks. To investigate the regulation of human plasma cell survival, plasma cells were isolated from tonsils according to high CD38 and low CD20 expression. Tonsillar plasma cells express CD9, CD19, CD24, CD37, CD40, CD74, and HLA-DR, but not CD10, HLA-DQ, CD28, CD56, and Fas/CD95. Although plasma cells express intracytoplasmic Bcl-2, they undergo swift apoptosis in vitro and do not respond to CD40 triggering. Bone marrow fibroblasts and rheumatoid synoviocytes, however, prevented plasma cells from undergoing apoptosis in a contact-dependent fashion. These data indicate that fibroblasts may form a microenvironment favorable for plasma cell survival under normal and pathological conditions.

CD40 and B cell antigen receptor dual triggering of resting B lymphocytes turns on a partial germinal center phenotype.

Phenotypic alterations occur when resting human B lymphocytes become germinal center (GC) cells. These include the induction of surface CD38, CD95 (FAS/APO-1), and carboxy-peptidase-M (CPM), a recently described GC marker. However, the factors that govern the in vivo induction of these surface molecules on B cells remain unknown. Here, we purified resting (CD38-) human B lymphocytes from tonsils in an attempt to establish culture conditions resulting in the induction of these three GC markers. We show that interferon (IFN) alpha or IFN-gamma, as well as antibodies against the B cell antigen receptor (BCR), could induce CD38 on resting B lymphocytes, a phenomenon further enhanced by CD40 stimulation. Concomitantly, CD95 was upregulated by CD40 ligation and, to a lesser extent, by IFN-gamma. By contrast, CPM expression could be upregulated only through BCR triggering. This CPM induction was specifically enhanced by CD19 or CD40 ligation. CD40 + BCR stimulation of resting B cells with CD40 ligand-transfected fibroblastic cells in the presence of cross-linked anti-BCR monoclonal antibodies resulted in the coexpression of CD38, CD95, and CPM. As GC cells, these cells also expressed CD71, CD80 (B7.1), and CD86 (B7.2), but not CD24. However, CD10+ or CD44- B cells could not be detected in these culture conditions, suggesting that yet other signals are required for the induction of these GC markers. Consistent with a GC phenotype, CD40 + BCR-stimulated cells exhibited reduced viability when cultured for 20 h in the absence of stimulus. These results first demonstrate that cotriggering of resting B cells through BCR and CD40 induces both phenotypic and functional GC features. They also show that IFN and CD19 triggering of resting B cells specifically modulate the expression of GC markers.

Fas ligation induces apoptosis of CD40-activated human B lymphocytes.

Since CD40/CD40 ligand (CD40Lig) interactions are essential in vivo for the generation of germinal center B cells that express Fas (Apo-1/CD95), we explored whether CD40 engagement may modulate Fas expression and function on human B lymphocytes. Resting tonsil B cells, isolated by density gradient centrifugation, express either absent or low levels of Fas. They could be induced to promptly express Fas after ligation of their CD40, however, using either a recombinant human CD40Lig or a cross-linked anti-CD40 mAb. In contrast, engagement of the B cell antigen receptor by immobilized anti-kappa and -lambda antibodies did not turn on Fas expression. Addition of anti-Fas mAb CH11 inhibited the later phases of CD40-induced B cell growth as a result of apoptotic cell death. Furthermore, Fas ligation inhibited proliferation and Ig secretion of CD40-activated B cells in response to recombinant cytokines such as interleukin (IL)-2, IL-4, and IL-10, as well as a cytokine-rich supernatant of phytohemagglutinin-activated T cells, indicating that none of those B cell tropic factors were able to prevent the Fas-induced death. Taken together, the present results show that engagement of CD40 antigen on B cells induces Fas expression and sensitizes them to Fas-mediated apoptosis. The delayed functional response to Fas ligation after CD40 activation may represent a way to limit the size of a specific B cell clone that is generated during T-B cell interactions.

Regulatory effects of prostaglandin E2 on the growth and differentiation of human B lymphocytes activated through their CD40 antigen.

We have studied the effects of prostaglandin E2 (PGE2) on the growth and differentiation of human tonsillar B lymphocytes cultured in the CD40 system with or without IL-4 or IL-10. PGE2 (10(-9) to 10(-6) M) enhanced proliferation of B cells activated through their CD40 Ag, but not their Ig secretion. PGE2 further potentiated both IL-4- and IL-10-induced B cell growth as determined by [3H]TdR uptake and cellular enumeration. The IL-10-induced IgM, IgG, and IgA secretion was enhanced twofold to fourfold after addition of PGE2, whereas IL-4-induced IgG and IgE secretion was inhibited. The IgE production was particularly sensitive as an approximately 90% inhibition was obtained for 10(-7) M PGE2. In addition, PGE2 inhibited IgE production by naive surface IgD+ B cells cultured in the CD40 system, suggesting that PGE2 may interact with mechanisms involved in IgE switching. PGE2 displayed similar effects on cytokine-induced proliferation and Ig secretion of B cells activated by anti-CD40 Abs used in a soluble form. Finally, the PGE2 effects were mimicked by agents increasing cAMP, indicating that the PGE2 activities are likely to depend on the activation of the cAMP pathway. Altogether, the present data indicate that PGE2 stimulates human CD40-activated B cell growth, but differently modulates cytokine-induced differentiation. Thus, in microenvironments supporting the development of an immune response, the secretion of PGE2 by competent cells such as macrophages may participate in the regulation of the humoral response.

Interleukin 10 (IL-10) upregulates functional high affinity IL-2 receptors on normal and leukemic B lymphocytes.

Interleukin 10 (IL-10) has recently been shown to induce normal human B lymphocytes to proliferate and differentiate into immunoglobulin (Ig)-secreting cells. Herein, we show that IL-10 also promotes DNA synthesis and IgM production by anti-CD40 activated B cell chronic lymphocytic leukemia (B-CLL). Most strikingly, IL-2 and IL-10 were found to synergize to induce the proliferation and differentiation of B-CLL cells. This synergy between IL-2 and IL-10 was also observed with normal B cells which proliferated strongly and secreted large amounts of IgM, IgG, and IgA. The observed synergy is likely to be due to the IL-10-induced increase of high affinity IL-2 receptors on both normal and leukemic B cells. This increase of high affinity receptor is associated to an increase of Tac/CD25 expression that can be detected by flow cytometric analysis. Taken together, these results indicate that IL-10 permits anti-CD40 activated B cells to respond to IL-2 through an induction of high affinity IL-2 receptors. This effect of IL-10 may partly explain how T cells, which activate B cells in a CD40-dependent fashion, induce B cell proliferation and differentiation mostly through IL-2.