IL-4–producing CD4+ T cells in reactive lymph nodes during helminth infection are T follicular helper cells

Interleukin (IL)-4 is the quintessential T helper type 2 (Th2) cytokine produced by CD4⁺ T cells in response to helminth infection. IL-4 not only promotes the differentiation of Th2 cells but is also critical for immunoglobulin (Ig) G1 and IgE isotype-switched antibody responses. Despite the IL-4–mediated link between Th2 cells and B lymphocytes, the location of IL-4–producing T cells in the lymph nodes is currently unclear. Using IL-4 dual reporter mice, we examined the Th2 response and IL-4 production in the draining mesenteric lymph nodes during infection with the enteric nematode Heligmosomoides polygyrus. We show that although IL-4–competent Th2 cells are found throughout the B and T cell areas, IL-4–producing Th2 cells are restricted to the B cell follicles and associate with germinal centers. Consistent with their localization, IL-4 producers express high levels of CXCR5, ICOS, PD-1, IL-21, and BCL-6, a phenotype characteristic of T follicular helper (Tfh) cells. Although IL-4 was dispensable for the generation of Th2 and Tfh cells, its deletion resulted in defective B cell expansion and maturation. Our report reveals the compartmentalization of Th2 priming and IL-4 production in the lymph nodes during infection, and identifies Tfh cells as the dominant source of IL-4 in vivo.IL-4 is widely recognized as the canonical marker for Th2-polarized CD4⁺ T cells (1). IL-4 was originally identified by its function as a B cell–stimulating factor (2, 3), and numerous in vivo models of infection concur that IL-4 is critical for the isotype switch of B cells to IgE and IgG1 (4–10). IL-4 also promotes the Th2 polarization of naive CD4⁺ T cells in vitro (11, 12). However, in vivo studies have found that Th2 cells can develop in mice deficient for IL-4, the IL-4Rα chain, or the IL-4R–associated Stat 6 (4–8). Collectively, these observations suggest that IL-4 production in sentinel lymph nodes draining a site of infection may be more important to support type 2 B cell responses rather than to establish the underlying Th2 response. Therefore, it stands to reason that the production of IL-4 by Th2 cells should preferentially occur in B cell follicles to optimize B cell help. However, the localization and characterization of IL-4–producing T cells in the lymph nodes has not been assessed.T follicular helper (Tfh) cells are a subset of CD4⁺ T cells that migrate to B cell follicles after activation and promote germinal center formation and B cell Ig isotype switching in mice and humans (13–16). Phenotypically, Tfh cells are characterized by expression of CXCR5 (an obligatory receptor for the follicle-homing chemokine CXCL13) (17), the inducible co-stimulatory receptor ICOS (18, 19), and the inhibitory receptors PD-1 (also known as CD279) (14) and B and T lymphocyte attenuator (BTLA) (13). A previous study (13) using model antigens in adjuvant have suggested that Tfh cells are a distinct lineage of T helper cells that arises independently of Th1, Th2, and Th17 effector subsets. To date, however, no studies have examined the Tfh response during infection with a Th2-polarizing pathogen. To unambiguously identify and localize Th2 cells and IL-4 production in the reactive lymph nodes in vivo, we infected IL-4 dual reporter mice (20), in which cells that express IL-4 are marked by GFP and IL-4–producing subsets additionally display surface huCD2, with the mouse gastrointestinal helminth parasite Heligmosomoides polygyrus. We find that IL-4–producing Th2 cells localize to the B cell follicles and exhibit a Tfh cell phenotype in the draining lymph nodes of infected animals. In addition, we find that although IL-4 is dispensable for the generation of Tfh cells, it is critical for a mature B cell response. To our knowledge, this is the first report to show the location of IL-4–producing T cells in the reactive lymph nodes after helminth infection. Furthermore, our results challenge the prevailing concept that Tfh cells are a distinct T helper lineage. Rather, we suggest that Tfh cells act to support the demands of the impending immune response.     

Negative feedback control of the autoimmune response through antigen-induced differentiation of IL-10–secreting Th1 cells

Regulation of the immune response to self- and foreign antigens is vitally important for limiting immune pathology associated with both infections and hypersensitivity conditions. Control of autoimmune conditions can be reinforced by tolerance induction with peptide epitopes, but the mechanism is not currently understood. Repetitive intranasal administration of soluble peptide induces peripheral tolerance in myelin basic protein (MBP)–specific TCR transgenic mice. This is characterized by the presence of anergic, interleukin (IL)-10–secreting CD4⁺ T cells with regulatory function (IL-10 T reg cells). The differentiation pathway of peptide-induced IL-10 T reg cells was investigated. CD4⁺ T cells became anergic after their second encounter with a high-affinity MBP peptide analogue. Loss of proliferative capacity correlated with a switch from the Th1-associated cytokines IL-2 and interferon (IFN)-γ to the regulatory cytokine IL-10. Nevertheless, IL-10 T reg cells retained the capacity to produce IFN-γ and concomitantly expressed T-bet, demonstrating their Th1 origin. IL-10 T reg cells suppressed dendritic cell maturation, prevented Th1 cell differentiation, and thereby created a negative feedback loop for Th1-driven immune pathology. These findings demonstrate that Th1 responses can be self-limiting in the context of peripheral tolerance to a self-antigen.Antigens administered in a tolerogenic form have long been known to result in down-regulation of immune responses. In recent years, the potential of antigen-driven immunotherapy for the treatment of allergic and autoimmune diseases has been investigated in several experimental models. Administration of antigenic peptides via the intranasal (i.n.) route induces tolerance, and thus inhibits the development of both autoimmunity (Metzler and Wraith, 1993; Staines et al., 1996; Tian et al., 1996; Karachunski et al., 1997) and allergy (Hoyne et al., 1993). Possible mechanisms of tolerance induction include elimination of peptide-specific T cells by activation-induced cell death/apoptosis (Critchfield et al., 1994; Chen et al., 1995; Liblau et al., 1996) or modification of their function via induction of anergy (Kearney et al., 1994), TCR/coreceptor down-regulation (Schonrich et al., 1991), immune deviation (Guery et al., 1996), or secretion of immunoregulatory cytokines such as IL-10 and TGF-β (Miller et al., 1992; Sundstedt et al., 1997). Most immune cells, including monocytes, macrophages, DCs, NK cells, B cells, and T cells, are capable of secreting IL-10 under specific circumstances (Moore et al., 2001). Among these, IL-10–secreting CD4⁺ T cells are the best characterized because of their recently recognized role in immune regulation (O''Garra et al., 2004). Two phenotypically distinct CD4⁺ T regulatory (T reg) cell types have been described—naturally occurring FoxP3⁺ T reg cells that form an inherent part of the naive T cell repertoire (Sakaguchi et al., 1995) and induced, FoxP3⁻ IL-10-secreting T reg cells (for review see Roncarolo et al., 2006). Numerous subtypes of induced IL-10–secreting T reg cells with variable cytokine profiles have been generated in both murine and human systems. However, in contrast to T helper cells, the differentiation of induced T reg cells remains poorly defined.i.n. administration of a soluble peptide induces peripheral tolerance in TCR transgenic (Tg4) mice specific for the acetylated N-terminal peptide Ac1-9 of murine myelin basic protein (MBP). Increasing the affinity of the peptide for I-A^u greatly enhances the tolerogenicity of the peptide in the Tg4 mouse (Liu et al., 1995). After a single i.n. dose of a high-affinity analogue of the MBP epitope, Ac1-9[4Y], with a tyrosine substituting the lysine at position four, T cell deletion is only transient and incomplete (Burkhart et al., 1999). Instead, Tg4 CD4⁺ T cells become anergic and exhibit a shift in cytokine secretion profile toward IL-10 after repeated i.n. treatment with peptide (Burkhart et al., 1999). Evidence for the generation of CD4⁺ T cells with a regulatory phenotype in this model stems from both in vitro and in vivo suppression assays (Sundstedt et al., 2003). Thus, i.n. treatment with MBP Ac1-9[4Y] induces active tolerance in the form of IL-10–secreting T reg cells (IL-10 T reg cells) rather than deletion. A role for IL-10 in suppression in vivo and in experimental autoimmune encephalomyelitis protection was demonstrated by anti–IL-10 (Burkhart et al., 1999) and anti–IL-10R (Sundstedt et al., 2003) antibody administration. IL-10 has important immunosuppressive and antiinflammatory effects on immune responses to both foreign and self-antigens (Moore et al., 2001) that are primarily mediated by its inhibitory activities on the function of APCs (de Waal Malefyt et al., 1991). Although the role of IL-10 in suppression of experimental autoimmune encephalomyelitis in the Tg4 model is not known, the effect of IL-10 on antigen presentation and inflammation is a likely mechanism. Naturally occurring FoxP3⁺ T reg cells form a part of the Tg4 CD4⁺ T cell repertoire and may rely on IL-10 to mediate suppression, as previously shown in other inflammatory settings (Asseman et al., 1999). Even so, peptide-induced IL-10 T reg cells were found to be distinct in origin from naturally occurring T reg cells in that they do not express Foxp3 (Vieira et al., 2004). Genetic depletion of FoxP3⁺ T reg cells from the CD4⁺ T cell repertoire in the RAG-deficient Tg4 mouse gives rise to spontaneous EAE. However, the onset of disease can be prevented by repetitive treatment with i.n. peptide, correlating with the generation of IL-10 T reg cells (Nicolson et al., 2006).It has been proposed that induced IL-10 T reg cells arise from fully differentiated T effector cells that have lost the ability to secrete their hallmark cytokines as a result of chronic antigenic stimulation (O''Garra et al., 2004). Alternatively, induced IL-10 T reg cells could arise directly from naive precursors without a T effector phase. In this study, we investigate the ontogeny of induced IL-10 T reg cells generated by repeated i.n. peptide treatment. By following the differentiation pathway taken by CD4⁺ T cells over the course of tolerance induction, we demonstrate that peptide-induced IL-10 T reg cells are of Th1 origin and that IL-10 T reg cells complete the negative feedback loop of pathogenic Th1 responses in autoimmunity.     

CD4+ T helper cells use CD154–CD40 interactions to counteract T reg cell–mediated suppression of CD8+ T cell responses to influenza

CD4⁺ T cells promote CD8⁺ T cell priming by licensing dendritic cells (DCs) via CD40–CD154 interactions. However, the initial requirement for CD40 signaling may be replaced by the direct activation of DCs by pathogen-derived signals. Nevertheless, CD40–CD154 interactions are often required for optimal CD8⁺ T cell responses to pathogens for unknown reasons. Here we show that CD40 signaling is required to prevent the premature contraction of the influenza-specific CD8⁺ T cell response. CD40 is required on DCs but not on B cells or T cells, whereas CD154 is required on CD4⁺ T cells but not CD8⁺ T cells, NKT cells, or DCs. Paradoxically, even though CD154-expressing CD4⁺ T cells are required for robust CD8⁺ T cell responses, primary CD8⁺ T cell responses are apparently normal in the absence of CD4⁺ T cells. We resolved this paradox by showing that the interaction of CD40-bearing DCs with CD154-expressing CD4⁺ T cells precludes regulatory T cell (T reg cell)–mediated suppression and prevents premature contraction of the influenza-specific CD8⁺ T cell response. Thus, CD4⁺ T helper cells are not required for robust CD8⁺ T cell responses to influenza when T reg cells are absent.Primary CD8⁺ T cell responses often require help from CD4⁺ T cells, which produce cytokines and provide co-stimulation, including the engagement of CD40 by its ligand CD154 (Bennett et al., 1998; Ridge et al., 1998; Schoenberger et al., 1998). In one model, CD4⁺ T cells engage CD40 on DCs and license them to become efficient antigen-presenting cells for naive CD8⁺ T cells (Bennett et al., 1998; Ridge et al., 1998; Schoenberger et al., 1998). However, other models suggest that CD4⁺ T cells provide help to CD8⁺ T cells by activating B cells and promoting CD40-dependent antibody responses (Bachmann et al., 2004) or that they engage CD40 on CD8⁺ T cells (Bourgeois et al., 2002) and directly promote CD8⁺ T cell activation or survival.Interestingly, CD4⁺ T cell help is not required to prime all CD8⁺ T cells responses. Whereas CD8⁺ T cell responses to noninflammatory antigens are impaired in the absence of CD4⁺ T cells or CD40 signaling (Bennett et al., 1998; Ridge et al., 1998; Schoenberger et al., 1998; Feau et al., 2011), primary responses to some pathogens occur independently of CD4⁺ T cells or CD40 signaling (Whitmire et al., 1996, 1999; Shedlock and Shen, 2003; Shedlock et al., 2003; Sun and Bevan, 2003), possibly because of the direct activation of DCs through pathogen recognition receptors (Hamilton et al., 2001). Curiously, primary CD8⁺ T cell responses to influenza virus require CD40 signaling (Lee et al., 2003a) but not CD4⁺ T cells (Belz et al., 2002), suggesting that other cell types may express CD154 and license CD40-expressing targets in the absence of CD4⁺ T cells. Consistent with this view, activated CD8⁺ T cells (Hernandez et al., 2007; Wong et al., 2008) and natural killer T cells (NKT) express CD154 (Tomura et al., 1999) and may license DCs (Hernandez et al., 2007, 2008; Wong et al., 2008) and help B cells (Chang et al., 2012) in the absence of CD4⁺ T cells. In addition, CD154 is expressed on activated DCs (Johnson et al., 2009) and may directly activate CD40-expressing CD8⁺ T cells. However, the actual role of CD40 signaling and the cellular basis of CD40-mediated help to CD8⁺ T cells help are not fully understood.Whereas helper CD4⁺ T cells promote T and B cell responses, FoxP3-expressing CD4⁺ regulatory T cells (T reg cells) suppress them (Kim et al., 2007; Campbell and Koch, 2011; Chung et al., 2011; Dietze et al., 2011; Linterman et al., 2011). Although the potent suppressive activity of T reg cells is neutralized during infection to allow robust immune responses to pathogens, T reg cells are also involved in the late stages of immune responses to resolve inflammation and curtail immunopathology (Suvas et al., 2003; Fulton et al., 2010; McNally et al., 2011). However, the relationship between CD40-mediated CD4⁺ T cell help and the immunosuppressive activity of T reg cells in CD8⁺ T cell responses to pathogens remains unexplored.Here we determined what cells use CD40–CD154 interactions and how CD40 signaling promotes CD8⁺ T cell responses to influenza. We found that CD4⁺ T cells were the only cells to functionally express CD154 and that DCs were the only cells that required CD40 for optimal CD8⁺ T cell responses to influenza. However, rather than licensing DCs to prime naive CD8⁺ T cells, CD40 signaling was required to prevent the early contraction of the CD8⁺ T cell response. Despite the necessity for CD154 on CD4⁺ T cells, we also observed apparently normal CD8⁺ T cell responses in the absence of CD4⁺ T cells. Finally, we showed that CD8⁺ T cell responses were normal or even enhanced when T reg cells were depleted and that additional CD40 blockade did not change the CD8⁺ T cell response. Thus, our data demonstrate that CD154-expressing CD4⁺ T cells stimulate DCs through CD40 to counteract T reg cell–mediated suppression of the CD8⁺ T cell response during the contraction phase of the immune response.     

Apoptosis of neuronal cells induced by serum of patients with acute brain injury: a new in vitro prognostic model

Objective To investigate whether serum draining from the jugular bulb of patients with traumatic or haemorrhagic brain injury induced apoptosis of neuronal PC12 cells in vitro and whether the apoptotic rate correlated with patients' outcome at 6 months. Design and setting Prospective clinical investigation in a 21-bed intensive care unit (ICU) in a university hospital. Patients Seventy patients who had suffered from acute brain injury requiring intensive care. Interventions Jugular bulb vein and systemic samples were obtained on admission to the ICU and after 48 h. PC12 cells were incubated in the presence of 10% of heat-inactivated patient's sera and apoptotic rate was determined by flow cytometry using annexin V and 7-aminoactinomycin D. Results Regional serum draining from the lesions induced higher early apoptosis of PC12 cells than systemic serum. Early apoptotic rate, Glasgow coma score, APACHE II score and the presence of pupil abnormalities were associated with mortality at 6 months in univariate statistical analyses. In logistic regression analysis only early apoptotic rate was an independent factor associated with mortality at 6 months (odds ratio: 1.502, 95% CI 1.2–1.9; p < 0.001). The final model has a sensitivity of 82.4% and a specificity of 84.8% for predicting death within 6 months. Conclusions We developed a simple and reproducible in vitro model for predicting outcome in patients with traumatic or haemorrhagic brain injury that survived in the early phase. Our in vitro model combined with clinical and radiological measurements might improve the value of prognostic models to predict acute brain injury patients' outcome.     

IL-17–induced CXCL12 recruits B cells and induces follicle formation in BALT in the absence of differentiated FDCs

Ectopic lymphoid tissue, such as bronchus-associated lymphoid tissue (BALT) in the lung, develops spontaneously at sites of chronic inflammation or during infection. The molecular mechanisms underlying the neogenesis of such tertiary lymphoid tissue are still poorly understood. We show that the type of inflammation-inducing pathogen determines which key factors are required for the formation and maturation of BALT. Thus, a single intranasal administration of the poxvirus modified vaccinia virus Ankara (MVA) is sufficient to induce highly organized BALT with densely packed B cell follicles containing a network of CXCL13-expressing follicular DCs (FDCs), as well as CXCL12-producing follicular stromal cells. In contrast, mice treated with P. aeruginosa (P.a.) develop BALT but B cell follicles lack FDCs while still harboring CXCL12-positive follicular stromal cells. Furthermore, in IL-17–deficient mice, P.a.-induced BALT largely lacks B cells as well as CXCL12-expressing stromal cells, and only loose infiltrates of T cells are present. We show that Toll-like receptor pathways are required for BALT induction by P.a., but not MVA, and provide evidence that IL-17 drives the differentiation of lung stroma toward podoplanin-positive CXCL12-expressing cells that allow follicle formation even in the absence of FDCs. Taken together, our results identify distinct pathogen-dependent induction and maturation pathways for BALT formation.Bronchus-associated lymphoid tissue (BALT) represents a major component of the larger network of mucosa-associated lymphoid tissues, which essentially contribute to the initiation and maintenance of protective immune responses against invading pathogens. BALT is defined as aggregations of immune cells in the lung, containing a follicle-like structure composed of B cells which is surrounded by parafollicular T cell areas (Randall, 2010). To be classified as BALT, such lymphoid aggregates have to be situated adjacent to a bronchus and next to a vein and an artery. Furthermore, BALT contains some myeloid cells, in particular DCs, a network of stromal cells, and, depending on its state of maturation, follicular DCs (FDCs) within the B cell follicles (Kocks et al., 2007; Halle et al., 2009; Randall, 2010). The formation of high endothelial venules and lymphatic vessels facilitates the recirculation of lymphocytes (Xu et al., 2003).Although the lungs of naive mice (as well as adult humans) are typically devoid of BALT, pulmonary infection in mice with pathogens such as influenza virus (Moyron-Quiroz et al., 2004), murine herpesvirus 68 (Kocks et al., 2009), Mycobacterium tuberculosis (Kahnert et al., 2007), or modified vaccinia virus Ankara (MVA) (Halle et al., 2009) have been described to induce these tertiary lymphoid structures. Despite morphological and functional similarities between lymph nodes and BALT, developmental pathways that control the formation and maintenance of the latter remain largely unknown. The development of lymph nodes requires the presence of lymphotoxin (LT) α₁β₂-expressing lymphoid tissue inducer (LTi) cells (van de Pavert and Mebius, 2010) to stimulate the differentiation of stromal organizer cells, which in turn express lymphocyte-recruiting chemokines such as CXCL13 and CCL21. In contrast, induction of BALT and the presence of CXCL13- and CCL21-expressing cells within these structures seem to occur independently of LTi cells (Moyron-Quiroz et al., 2004), instead relying on DCs (Halle et al., 2009) expressing LTα₁β₂ (GeurtsvanKessel et al., 2009).The chemokine receptor CXCR4 is expressed on B cells and facilitates their efficacious migratory response toward its ligand CXCL12 (Okada et al., 2002). In combination with two other homeostatic chemokine receptors, CXCR5 and CCR7, CXCR4 is regulating the homing of B cells to Peyer’s patches (Okada et al., 2002), but so far the functional relevance of the CXCL12–CXCR4 axis for B cell homing toward or follicle formation within BALT has not been addressed.Recently, a model for BALT induction based on the repeated intranasal sensitization of newborn mice with LPS has been reported to be dependent on the production of IL-17 by T cells (Rangel-Moreno et al., 2011). At the same time, we demonstrated that the formation of MVA-induced BALT occurs completely independent of this cytokine (Fleige et al., 2012). Collectively, these data strongly suggested that at least two different signaling pathways can independently control BALT induction.In the present study, we used MVA and P. aeruginosa (P.a.) to dissect the different pathways for BALT induction and maturation. While MVA-induced BALT harbored CXCL13⁺ FDCs in WT, Il17a/f^−/−, and Myd88^−/−Trif^−/− mice, BALT induced by P.a. generally failed to develop FDCs and, within Il17a/f^−/− and Myd88^−/−Trif^−/− mice, showed an impaired morphological development and maturation.Mechanistically, P.a. was found to induce, in an IL-17–dependent process, follicular stromal cells to express CXCL12 which enabled B cell recruitment and follicle formation in BALT even in the absence of FDCs. Furthermore, our study identified γδ T cells within BALT as a major source for IL-17, triggering the differentiation of stromal cells into podoplanin⁺ (gp38) follicular cells that express CXCL12.     

Prolonged mechanical ventilation–induced neuroinflammation affects postoperative memory dysfunction in surgical mice

IntroductionPatients undergoing surgery frequently develop neuropsychological disturbances, including cognitive decline or memory impairment, and routine clinical procedures such as mechanical ventilation (MV) may affect acute-phase brain outcome. We aimed to investigate the effect of the prolonged MV on postoperative memory dysfunction in surgical mice.MethodsMale C57BL/6 mice were randomly divided into the following three groups: (1) The control group (group C) comprised anesthetized, unventilated animals; (2) the surgery group (subgroups S1h, S3h and S6h) was unventilated animals that underwent surgery under general anesthesia; and (3) the MV group (subgroups MV1h, MV3h and MV6h) was made up of animals under MV for 1 hour, 3 hours or 6 hours after surgery. Separate cohorts of animals were tested for memory function with fear conditioning tests or were killed at 6 hours, 1 day or 3 days postsurgery or post-MV to examine levels systemic and hippocampal interleukin (IL)-1β, IL-6 and tumor necrosis factor α (TNFα), and assessed synaptic structure and microglial activation. Nuclear factor κB (NF-κB) p65, cytochrome c, cleaved caspase-3 and cleaved poly(ADP-ribose) polymerase (PARP) activation were analyzed by Western blotting.ResultsThe MV6h group showed increased CD11b-immunopositive cells, synapse degeneration, cytochrome c release, cleaved caspase-3 and cleaved PARP-1 activation after surgery, as well as a decrease in freezing time after surgery. At 6 hours and 1 day post-MV, MV6h increased NF-κB activation and levels of systemic and hippocampal IL-1β, IL-6 and TNFα after surgery.ConclusionsProlonged MV after surgery further aggravates cognitive decline that may stem from upregulation of hippocampal IL-1β, IL-6 and TNFα, partially via activation of gliocytes in the surgical mouse hippocampus.     

HLA-E–restricted regulatory CD8+ T cells are involved in development and control of human autoimmune type 1 diabetes

A key feature of the immune system is its ability to discriminate self from nonself. Breakdown in any of the mechanisms that maintain unresponsiveness to self (a state known as self-tolerance) contributes to the development of autoimmune conditions. Recent studies in mice show that CD8⁺ T cells specific for the unconventional MHC class I molecule Qa-1 bound to peptides derived from the signal sequence of Hsp60 (Hsp60sp) contribute to self/nonself discrimination. However, it is unclear whether they exist in humans and play a role in human autoimmune diseases. Here we have shown that CD8⁺ T cells specific for Hsp60sp bound to HLA-E (the human homolog of Qa-1) exist and play an important role in maintaining peripheral self-tolerance by discriminating self from nonself in humans. Furthermore, in the majority of type 1 diabetes (T1D) patients tested, there was a specific defect in CD8⁺ T cell recognition of HLA-E/Hsp60sp, which was associated with failure of self/nonself discrimination. However, the defect in the CD8⁺ T cells from most of the T1D patients tested could be corrected in vitro by exposure to autologous immature DCs loaded with the Hsp60sp peptide. These data suggest that HLA-E–restricted CD8⁺ T cells may play an important role in keeping self-reactive T cells in check. Thus, correction of this defect could be a potentially effective and safe approach in the therapy of T1D.     

IL-12–induced IFN-γ is dependent on caspase-1 processing of the IL-18 precursor

IL-12 and IL-18 are IFN-gamma-inducing cytokines. In the present study, the role of endogenous IL-18 in the induction of IFN-gamma by IL-12 was investigated in mice. In the presence of a specific inhibitor of caspase-1 (also known as IL-1beta-converting enzyme, or ICE) IL-12-induced IFN-gamma from splenocytes was reduced by 85%. Using splenocytes from ICE-deficient mice, IL-12-induced IFN-gamma was reduced by 80%. However, the role of ICE was not through processing and release of IL-1beta. Neutralizing anti-IL-18 IgG reduced IL-12-induced IFN-gamma in splenocytes by 85%. Splenocytes cultured in vitro spontaneously released IL-18 into the extracellular compartment over time. Extracellular levels of IL-18 significantly correlated with IL-12-induced IFN-gamma and were reduced in cells obtained from ICE-deficient mice. In vivo, IL-12 administration increased circulating levels of IL-18 in wild-type mice but not in ICE-deficient mice. Both neutralization of IL-18 and ICE deficiency significantly reduced induction of circulating IFN-gamma in mice receiving IL-12. The IL-18 precursor was constitutively expressed in the livers and spleens of untreated mice. Furthermore, administration of IL-12 significantly increased liver-associated IL-18 levels. These data demonstrate that endogenous, ICE-cleaved IL-18 significantly contributes to induction of IFN-gamma by IL-12.     

Validating a model of row–column scanning

Purpose: For individuals with severe motor and communicative disabilities, single switch scanning provides a way to access a computer and communicate. A model was developed that utilizes scanning interface settings, error tendencies, error correction strategies, and the matrix configuration to predict a user’s communication rate. Method: Five individuals who use single switch scanning transcribed sentences using an on-screen keyboard configured with the settings from their communication devices. Data from these trials were used as input to a model that predicted TER for the baseline configuration and at least three other system configurations. Participants transcribed text with each of these new configurations and the predicted TER was compared to the actual TER. Results: Results showed that predicted TER was accurate to within 90% on average. The scan rate was also entered into a previously published model which assumes error-free performance. For our model, the average error for each participant was 10.49%, compared to 79.7% for the model assuming error-free performance. Conclusions: Our model of row–column scanning was much more accurate than a model that did not consider the likelihood of an error occurring. There is still room for improvement, however, and the results of the study will lead to additional modifications of the model.

Implications for Rehabilitation

• Although row-column scanning is a very slow method of selection, changes in the configuration of the interface can produce noticeable changes in performance.

• When configuring a row-column scanning interface, clinicians should consider the type of errors their client is likely to commit to target interface features to their client's specific needs.

• Some clients who use row-column scanning may not benefit from advanced interface features, even if they are available.

     

Caspase-3–mediated splenic lymphocyte apoptosis in a porcine model of cardiac arrest

Background

Postresuscitation immunologic dysfunction contributes to the low survival rate after successful resuscitation, but its mechanism remains poorly understood. The mitochondrial apoptosis pathway is initiated by the Bcl-2/Bax-controlled and caspase-3–mediated pathway, this study investigated whether mitochondrial pathway-mediated splenic lymphocyte apoptosis is involved in the postresuscitation immunosuppression in a porcine model of cardiac arrest.

Methods

Twenty-eight Wuzhishan miniature pigs were randomly divided into 2 groups: return of spontaneous circulation (ROSC; n = 22) and sham-operated (n = 6). Return of spontaneous circulation was initiated after 8 minutes of untreated ventricular fibrillation. After successful ROSC, CD4⁺ and CD8⁺ lymphocyte subsets were determined by flow cytometry. Surviving pigs were randomly assigned to be humanely killed at 24 and 72 hours after ROSC (n = 8 per group). Spleens were removed for histopathologic analysis, Western blotting, quantitative real-time polymerase chain reaction, and terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling assay.

Results

A high degree of splenic lymphocyte apoptosis was observed in the ROSC group. Expression of Bax and activated caspase-3 was markedly increased in splenic tissue, whereas Bcl-2 was significantly decreased in the post-ROSC group compared with the sham-operated group (P < .05) at 24 and 72 hours after ROSC. The messenger RNA levels of activated caspase-3 of splenic tissue were significantly elevated at 24 and 72 hours after ROSC.

Conclusion

These results demonstrates that Bcl-2/Bax and caspase-3–mediated mitochondrial apoptosis signaling pathway may contribute to abnormal splenic lymphocyte apoptosis, which may be one of the main pathologic mechanisms of postresuscitation disturbance of immunologic function in a porcine model of cardiac arrest.     

Tension–type headache in 40–year-olds: a Danish population–based sample of 4000

The aim of this study was to evaluate the one–year prevalence of tension–type headache in the general population. Three thousand men and one thousand women aged 40 years from the Danish population were included. They received a mailed questionnaire and the response rate was 87%. The selfreported one–year prevalence of tension– type headache was 84.7%. The one–year prevalence of infrequent episodic, frequent episodic and chronic tension–type headache was 48.2%, 33.8% and 2.3%, respectively. No tension–type headache and infrequent episodic tension–type headache was significantly more frequent in men than women (p<0.0005 and p=0.004), while frequent and chronic tension–type headache was significantly more frequent in women than men (p<0.0005 and p<0.0005). No tension– type headache and infrequent tension–type headache was significantly more frequent among those without than with self–reported migraine (no headache, men, p<0.0005 and women, p=0.002 and infrequent, men, p<0.0005 and women, p<0.0005), while episodic frequent and chronic tension–type headache was significantly more frequent among those with than those without self–reported migraine, with the exception of chronic tension–type in women (frequent episodic, men, p<0.0005 and women, p<0.0005 and chronic, men, p<0.0005 and women, p=0.08). Women are more prone to tensiontype headache than men and they have it more frequently than men. Self–reported migraine increases the risk for frequent episodic and chronic tension–type headache.     

Up-regulation of a death receptor renders antiviral T cells susceptible to NK cell–mediated deletion

Antiviral T cell responses in hepatotropic viral infections such as hepatitis B virus (HBV) are profoundly diminished and prone to apoptotic deletion. In this study, we investigate whether the large population of activated NK cells in the human liver contributes to this process. We show that in vitro removal of NK cells augments circulating CD8⁺ T cell responses directed against HBV, but not against well-controlled viruses, in patients with chronic hepatitis B (CHB). We find that NK cells can rapidly eliminate HBV-specific T cells in a contact-dependent manner. CD8⁺ T cells in the liver microcirculation are visualized making intimate contact with NK cells, which are the main intrahepatic lymphocytes expressing TNF-related apoptosis-inducing ligand (TRAIL) in CHB. High-level expression of the TRAIL death receptor TRAIL-R2 is found to be a hallmark of T cells exposed to the milieu of the HBV-infected liver in patients with active disease. Up-regulation of TRAIL-R2 renders T cells susceptible to caspase-8–mediated apoptosis, from which they can be partially rescued by blockade of this death receptor pathway. Our findings demonstrate that NK cells can negatively regulate antiviral immunity in chronic HBV infection and illustrate a novel mechanism of T cell tolerance in the human liver.T cell responses are tightly regulated to maintain immune homeostasis and limit damage to vital organs. T cells in the liver, in particular, are subjected to potent tolerizing mechanisms. Although these mechanisms prevent overzealous responses causing tissue injury, they may be exploited by hepatotropic pathogens to subvert antiviral immunity (Protzer et al., 2012). There have been major recent advances in our understanding of the multiple co-inhibitory pathways driving T cell exhaustion in the liver and perpetuating persistent viral infections (Protzer et al., 2012). However, the potential for NK cells to regulate T cell immunity has not been defined in human viral infections.NK cells can contribute to the containment of many infections by intracellular pathogens (Orange et al., 2002; Khakoo et al., 2004; Lodoen and Lanier, 2006; Alter et al., 2011), acting though cytolytic or noncytolytic effects on target cells or by promoting adaptive immunity (Vivier et al., 2008). Accumulating data highlight the capacity of NK cells to also exert a negative regulatory effect on T cells (Su et al., 2001) through inhibition of antigen presentation (Andrews et al., 2010), production of IL-10 (Lee et al., 2009), or direct killing of T cells. Several receptor–ligand interactions between NK cells and T cells have been found to be capable of leading to autologous lysis of activated T cells (Rabinovich et al., 2003; Cerboni et al., 2007; Lu et al., 2007; Soderquest et al., 2011). More recently, NK cells have been shown to limit T cell immunity in a mouse model of chronic viral infection (Waggoner et al., 2010; Lang et al., 2012; Waggoner et al., 2012).In this study, we sought to investigate the impact of NK cells on antiviral T cell responses in the setting of persistent infection with a human hepatotropic virus. Activated NK cells are markedly enriched in the liver microcirculation, where we hypothesized they would come into prolonged, close contact with infiltrating T cells. Although NK cells in patients with chronic hepatitis B (CHB) infection have impaired noncytolytic antiviral function, we have previously shown that they maintain their cytotoxic potential and up-regulate the death ligand TRAIL, particularly in the intrahepatic compartment (Dunn et al., 2007; Peppa et al., 2010). HBV-specific CD8⁺ T cells, which are essential for viral control, are profoundly depleted in these patients (Maini et al., 2000; Boni et al., 2007). Here, we demonstrate that hepatitis B virus–specific T cells up-regulate a death receptor for TRAIL and become susceptible to NK cell–mediated killing, thereby contributing to the failure of antiviral immunity in CHB.     

Efficacies of calcium–EDTA in combination with imipenem in a murine model of sepsis caused by Escherichia coli with NDM-1 β-lactamase

We evaluated the efficacy of ethylenediamine-N,N,N′,N′-tetraacetic acid, disodium calcium salt (Ca-EDTA), as an inhibitor for New Delhi metallo-β-lactamase-1 (NDM-1) in vitro antibiotic susceptibility and in a mouse model of sepsis caused by Escherichia coli. Ca-EDTA drastically reduced the MICs of carbapenems for all NDM-producing bacteria [imipenem (IPM) ≤1–2 μg/ml; meropenem (MEPM) ≤1–4 µg/ml]. In the neutropenic murine model of sepsis, the bacterial burden was further reduced by combination therapy using imipenem/cilastatin sodium (IPM/CS) and Ca-EDTA to 2.3 × 10³ CFU/liver, compared with 2.9 × 10⁴ CFU/liver for IPM/CS alone. These data demonstrated the possibility of Ca-EDTA for clinical applications. In our understanding, this is the first report examining the effect of Ca-EDTA on a mouse sepsis model caused by NDM-1-producing bacteria.