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Two major subsets of rat NK cells can be distinguished based on their expression of the Ly49s3 or the NKR-P1B lectin-like receptor. Ly49s3(+) NK cells, but not NKR-P1B(+) NK cells, express a wide range of Ly49 receptors. Here, we have examined differences between these two subsets in their expression of certain NK cell-associated molecules as well as their responses to cytokines. A microarray analysis suggested several differentially expressed genes, including preferential expression of NKG2A/C receptors by NKR-P1B(+) NK cells. This was confirmed by staining with tetramers of RT.BM1, the putative ligand of CD94/NKG2, indicating that Ly49 and CD94/NKG2 receptors separate into distinct NK cell compartments. Further, expression of CD25 by Ly49s3(+) NK cells was associated with more rapid proliferation in response to IL-2 as compared with NKR-P1B(+) NK cells. Thus, certain inflammatory situations may preferentially expand the Ly49s3(+) NK cells. Moreover, freshly isolated Ly49s3(+) and NKR-P1B(+) NK cells produce similar amounts of cytokines, and a minor Ly49s3(-)NKR-P1B(-) double-negative NK subset appears to be hyporesponsive based on its significantly lower IFN-gamma production. Collectively, our data demonstrate divergent profiles of NKR-P1B(+) and Ly49s3(+) NK cells, indicating distinct tasks in vivo.

Oxytocin (OT) is becoming increasingly established as a prosocial neuropeptide in humans with therapeutic potential in treatment of social, cognitive, and mood disorders. However, the potential of OT as a general facilitator of human learning and empathy is unclear. The current double-blind experiments on healthy adult male volunteers investigated first whether treatment with intranasal OT enhanced learning performance on a feedback-guided item-category association task where either social (smiling and angry faces) or nonsocial (green and red lights) reinforcers were used, and second whether it increased either cognitive or emotional empathy measured by the Multifaceted Empathy Test. Further experiments investigated whether OT-sensitive behavioral components required a normal functional amygdala. Results in control groups showed that learning performance was improved when social rather than nonsocial reinforcement was used. Intranasal OT potentiated this social reinforcement advantage and greatly increased emotional, but not cognitive, empathy in response to both positive and negative valence stimuli. Interestingly, after OT treatment, emotional empathy responses in men were raised to levels similar to those found in untreated women. Two patients with selective bilateral damage to the amygdala (monozygotic twins with congenital Urbach-Wiethe disease) were impaired on both OT-sensitive aspects of these learning and empathy tasks, but performed normally on nonsocially reinforced learning and cognitive empathy. Overall these findings provide the first demonstration that OT can facilitate amygdala-dependent, socially reinforced learning and emotional empathy in men.

Nitric oxide (NO) mediates intercellular signaling through activation of its receptor, soluble guanylyl cyclase (sGC), leading to elevation of intracellular guanosine 3',5'-cyclic monophosphate (cGMP) levels. Through this signal transduction pathway, NO regulates a diverse range of physiological effects, from vasodilatation and platelet disaggregation to synaptic plasticity. Measurement of sGC activity has traditionally been carried out using end-point assays of cGMP accumulation or by transfection of cells with "detector" proteins such as fluorescent proteins coupled to cGMP binding domains or cyclic nucleotide gated channels. Here we report a simpler approach: the use of a fluorescently labeled substrate analog, mant-GTP (2'-O-(N-methylanthraniloyl) guanosine 5'-triphosphate), which gives an increase in emission intensity after enzymatic cyclization to mant-cGMP. Activation of purified recombinant sGC by NO led to a rapid rise in fluorescence intensity within seconds, reaching a maximal 1.6- to 1.8-fold increase above basal levels. The sGC inhibitor, ODQ (1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one), eliminated the fluorescence increase due to NO, and the synergistic activator of sGC, BAY 41-2272 (3-(4-amino-5-cyclopropylpyrimidin-2-yl)-1-(2-fluorobenzyl)-1H-pyrazolo[3,4-b]pyridine), increased the rate at which the maximal fluorescence increase was attained. High-performance liquid chromatography (HPLC) confirmed the formation of mant-cGMP product. This real-time assay allows the progress of purified sGC activation to be quantified precisely and, with refinement, could be optimized for use in a cellular environment.

The first definitive cell fate decision in development occurs at the blastocyst stage with establishment of the trophoblast and embryonic cell lineages. In the mouse, lineage commitment is achieved by epigenetic regulation of a critical gatekeeper gene, the transcription factor Elf5, that reinforces placental cell fate and is necessary for trophoblast stem (TS) cell self-renewal. In humans, however, the epigenetic lineage boundary seems to be less stringent since human embryonic stem (ES) cells, unlike their murine counterparts, harbour some potential to differentiate into trophoblast derivatives. Here, we show that ELF5 is expressed in the human placenta in villous cytotrophoblast cells but not in post-mitotic syncytiotrophoblast and invasive extravillous cytotrophoblast cells. ELF5 establishes a circuit of mutually interacting transcription factors with CDX2 and EOMES, and the highly proliferative ELF5(+)/CDX2(+) double-positive subset of cytotrophoblast cells demarcates a putative TS cell compartment in the early human placenta. In contrast to placental trophoblast, however, ELF5 is hypermethylated and largely repressed in human ES cells and derived trophoblast cell lines, as well as in induced pluripotent stem cells and murine epiblast stem cells. Thus, these cells exhibit an embryonic lineage-specific epigenetic signature and do not undergo an epigenetic reprogramming to reflect the trophoblast lineage at key loci such as ELF5. Our identification of the trophoblast-specific transcriptional circuit established by ELF5 will be instrumental to derive human TS cell lines that truly reflect early placental trophoblast and that will be most beneficial to gain insights into the aetiology of common pregnancy complications, including intra-uterine growth restriction and pre-eclampsia.

Spatial organization of the genome is non-random. Preferential chromatin interactions, both in cis and in trans and between transcriptionally active and silent regions, influence organization.

Traditionally, researchers have believed that axons are highly dependent on their cell bodies for long-term survival. However, recent studies point to the existence of axon-autonomous mechanism(s) that regulate rapid axon degeneration after axotomy. Here, we review the cellular and molecular events that underlie this process, termed Wallerian degeneration. We describe the biphasic nature of axon degeneration after axotomy and our current understanding of how Wld(S)--an extraordinary protein formed by fusing a Ube4b sequence to Nmnat1--acts to protect severed axons. Interestingly, the neuroprotective effects of Wld(S) span all species tested, which suggests that there is an ancient, Wld(S)-sensitive axon destruction program. Recent studies with Wld(S) also reveal that Wallerian degeneration is genetically related to several dying back axonopathies, thus arguing that Wallerian degeneration can serve as a useful model to understand, and potentially treat, axon degeneration in diverse traumatic or disease contexts.

Rac1 and Rac2 GTPases transduce signals from multiple receptors leading to cell migration, adhesion, proliferation, and survival. In the absence of Rac1 and Rac2, B cell development is arrested at an IgD- transitional B cell stage that we term transitional type 0 (T0). We show that T0 cells cannot enter the white pulp of the spleen until they mature into the T1 and T2 stages, and that this entry into the white pulp requires integrin and chemokine receptor signaling and is required for cell survival. In the absence of Rac1 and Rac2, transitional B cells are unable to migrate in response to chemokines and cannot enter the splenic white pulp. We propose that loss of Rac1 and Rac2 causes arrest at the T0 stage at least in part because transitional B cells need to migrate into the white pulp to receive survival signals. Finally, we show that in the absence of Syk, a kinase that transduces B cell antigen receptor signals required for positive selection, development is arrested at the same T0 stage, with transitional B cells excluded from the white pulp. Thus, these studies identify a novel developmental checkpoint that coincides with B cell positive selection.

The N-methyl-D-aspartate receptor (NMDAR) is critical for learning-related synaptic plasticity in amygdala and hippocampus. As a consequence, there is considerable interest in drugs targeting this receptor to help enhance amygdala- and hippocampus-dependent learning. A promising candidate in this respect is the NMDAR glycine-binding site partial agonist D-cycloserine (DCS). Accumulating clinical evidence indicates the efficacy of DCS in the facilitation of amygdala-dependent fear extinction learning in patients with phobic, social anxiety, panic, and obsessive-compulsive disorder. An important unresolved question though is whether the use of DCS can also facilitate hippocampus-dependent declarative learning in healthy people as opposed to being restricted to the fear memory domain.

Long-Term Potentiation (LTP) of synapses is thought to be due in part to a change in AMPA Receptor trafficking leading to an increase in the number of AMPA Receptors at the synapse. LTP onset occurs within seconds after the induction signal. A particle-based stochastic simulation software is used to investigate the effect of Brownian diffusion of glutamate receptors on receptor incorporation into the synaptic specialisation and the time-course of LTP expression. The model of the dendritic spine includes receptors diffusing within the membrane, scaffold molecules within the synaptic specialisation capable of binding receptors and a molecular picket-fence surrounding the synaptic membrane area, all features found within the biological system.

Most cellular signal transduction mechanisms depend on a few molecular partners whose roles depend on their position and movement in relation to the input signal. This movement can follow various rules and take place in different compartments. Additionally, the molecules can form transient complexes. Complexation and signal transduction depend on the specific states partners and complexes adopt. Several spatial simulator have been developed to date, but none are able to model reaction-diffusion of realistic multi-state transient complexes.

MicroRNAs (miRNAs) play an important role in diverse physiological processes and are potential therapeutic agents. Synthetic oligonucleotides (ONs) of different chemistries have proven successful for blocking miRNA expression. However, their specificity and efficiency have not been fully evaluated. Here, we show that peptide nucleic acids (PNAs) efficiently block a key inducible miRNA expressed in the haematopoietic system, miR-155, in cultured B cells as well as in mice. Remarkably, miR-155 inhibition by PNA in primary B cells was achieved in the absence of any transfection agent. In mice, the high efficiency of the treatment was demonstrated by a strong overlap in global gene expression between B cells isolated from anti-miR-155 PNA-treated and miR-155-deficient mice. Interestingly, PNA also induced additional changes in gene expression. Our analysis provides a useful platform to aid the design of efficient and specific anti-miRNA ONs for in vivo use.

Transcription in the eukaryotic nucleus has long been thought of as conforming to a model in which RNA polymerase complexes are recruited to and track along isolated templates. However, a more dynamic role for chromatin in transcriptional regulation is materializing: enhancer elements interact with promoters forming loops that often bridge considerable distances and genomic loci, even located on different chromosomes, undergo chromosomal associations. These associations amass to form an extensive 'transcriptional interactome', enacted at functional subnuclear compartments, to which genes dynamically relocate. The emerging view is that long-range chromosomal associations between genomic regions, and their repositioning in the three-dimensional space of the nucleus, are key contributors to the regulation of gene expression.

The guanosine triphosphatases (GTPases) of the immunity-associated protein (GIMAP) family of putative GTPases has been implicated in the regulation of T-lymphocyte development and survival. A mouse conditional knockout allele was generated for the immune GTPase gene GIMAP1. Homozygous loss of this allele under the influence of the lymphoid-expressed hCD2-iCre recombinase transgene led to severe (> 85%) deficiency of mature T lymphocytes and, unexpectedly, of mature B lymphocytes. By contrast there was little effect of GIMAP1 deletion on immature lymphocytes in either B or T lineages, although in vitro studies showed a shortening of the survival time of both immature and mature CD4(+) single-positive thymocytes. These findings show a vital requirement for GIMAP1 in mature lymphocyte development/survival and draw attention to the nonredundant roles of members of the GIMAP GTPase family in these processes.

MicroRNA are emerging as key regulators of the development and function of adaptive immunity. These 19-24 nucleotide regulatory RNA molecules have essential roles in multiple faucets of adaptive immunity, from regulating the development of the key cellular players to the activation and function in immune responses.

Homeostatic control of the immune system involves mechanisms that ensure the self-tolerance, survival and quiescence of hematopoietic-derived cells. In this study, we demonstrate that the GTPase of immunity associated protein (Gimap)5 regulates these processes in lymphocytes and hematopoietic progenitor cells. As a consequence of a recessive N-ethyl-N-nitrosourea-induced germline mutation in the P-loop of Gimap5, lymphopenia, hepatic extramedullary hematopoiesis, weight loss, and intestinal inflammation occur in homozygous mutant mice. Irradiated fetal liver chimeric mice reconstituted with Gimap5-deficient cells lose weight and become lymphopenic, demonstrating a hematopoietic cell-intrinsic function for Gimap5. Although Gimap5-deficient CD4(+) T cells and B cells appear to undergo normal development, they fail to proliferate upon Ag-receptor stimulation although NF-kappaB, MAP kinase and Akt activation occur normally. In addition, in Gimap5-deficient mice, CD4(+) T cells adopt a CD44(high)CD62L(low)CD69(low) phenotype and show reduced IL-7ralpha expression, and T-dependent and T-independent B cell responses are abrogated. Thus, Gimap5-deficiency affects a noncanonical signaling pathway required for Ag-receptor-induced proliferation and lymphocyte quiescence. Antibiotic-treatment or the adoptive transfer of Rag-sufficient splenocytes ameliorates intestinal inflammation and weight loss, suggesting that immune responses triggered by microbial flora causes the morbidity in Gimap5-deficient mice. These data establish Gimap5 as a key regulator of hematopoietic integrity and lymphocyte homeostasis.

During T cell-dependent responses, B cells can either differentiate extrafollicularly into short-lived plasma cells or enter follicles to form germinal centers (GCs). Interactions with T follicular helper (Tfh) cells are required for GC formation and for selection of somatically mutated GC B cells. Interleukin (IL)-21 has been reported to play a role in Tfh cell formation and in B cell growth, survival, and isotype switching. To date, it is unclear whether the effect of IL-21 on GC formation is predominantly a consequence of this cytokine acting directly on the Tfh cells or if IL-21 directly influences GC B cells. We show that IL-21 acts in a B cell-intrinsic fashion to control GC B cell formation. Mixed bone marrow chimeras identified a significant B cell-autonomous effect of IL-21 receptor (R) signaling throughout all stages of the GC response. IL-21 deficiency profoundly impaired affinity maturation and reduced the proportion of IgG1(+) GC B cells but did not affect formation of early memory B cells. IL-21R was required on GC B cells for maximal expression of Bcl-6. In contrast to the requirement for IL-21 in the follicular response to sheep red blood cells, a purely extrafollicular antibody response to Salmonella dominated by IgG2a was intact in the absence of IL-21.

Voltage-gated proton currents regulate generation of reactive oxygen species (ROS) in phagocytic cells. In B cells, stimulation of the B cell antigen receptor (BCR) results in the production of ROS that participate in B cell activation, but the involvement of proton channels is unknown. We report here that the voltage-gated proton channel HVCN1 associated with the BCR complex and was internalized together with the BCR after activation. BCR-induced generation of ROS was lower in HVCN1-deficient B cells, which resulted in attenuated BCR signaling via impaired BCR-dependent oxidation of the tyrosine phosphatase SHP-1. This resulted in less activation of the kinases Syk and Akt, impaired mitochondrial respiration and glycolysis and diminished antibody responses in vivo. Our findings identify unanticipated functions for proton channels in B cells and demonstrate the importance of ROS in BCR signaling and downstream metabolism.

Recessive mutations at the mouse pirouette (pi) locus result in hearing loss and vestibular dysfunction due to neuroepithelial defects in the inner ear. Using a positional cloning strategy, we have identified mutations in the gene Grxcr1 (glutaredoxin cysteine-rich 1) in five independent allelic strains of pirouette mice. We also provide sequence data of GRXCR1 from humans with profound hearing loss suggesting that pirouette is a model for studying the mechanism of nonsyndromic deafness DFNB25. Grxcr1 encodes a 290 amino acid protein that contains a region of similarity to glutaredoxin proteins and a cysteine-rich region at its C terminus. Grxcr1 is expressed in sensory epithelia of the inner ear, and its encoded protein is localized along the length of stereocilia, the actin-filament-rich mechanosensory structures at the apical surface of auditory and vestibular hair cells. The precise architecture of hair cell stereocilia is essential for normal hearing. Loss of function of Grxcr1 in homozygous pirouette mice results in abnormally thin and slightly shortened stereocilia. When overexpressed in transfected cells, GRXCR1 localizes along the length of actin-filament-rich structures at the dorsal-apical surface and induces structures with greater actin filament content and/or increased lengths in a subset of cells. Our results suggest that deafness in pirouette mutants is associated with loss of GRXCR1 function in modulating actin cytoskeletal architecture in the developing stereocilia of sensory hair cells.

The molecular triggers for axon degeneration remain unknown. We identify endogenous Nmnat2 as a labile axon survival factor whose constant replenishment by anterograde axonal transport is a limiting factor for axon survival. Specific depletion of Nmnat2 is sufficient to induce Wallerian-like degeneration of uninjured axons which endogenous Nmnat1 and Nmnat3 cannot prevent. Nmnat2 is by far the most labile Nmnat isoform and is depleted in distal stumps of injured neurites before Wallerian degeneration begins. Nmnat2 turnover is equally rapid in injured Wld(S) neurites, despite delayed neurite degeneration, showing it is not a consequence of degeneration and also that Wld(S) does not stabilize Nmnat2. Depletion of Nmnat2 below a threshold level is necessary for axon degeneration since exogenous Nmnat2 can protect injured neurites when expressed at high enough levels to overcome its short half-life. Furthermore, proteasome inhibition slows both Nmnat2 turnover and neurite degeneration. We conclude that endogenous Nmnat2 prevents spontaneous degeneration of healthy axons and propose that, when present, the more long-lived, functionally related Wld(S) protein substitutes for Nmnat2 loss after axon injury. Endogenous Nmnat2 represents an exciting new therapeutic target for axonal disorders.

T helper (Th) 17 cells have been classified as a new lineage, distinct from Th1, Th2 and Treg. Their development requires a unique combination of cytokines and depends on distinct intracellular events, resulting in the production of the signature cytokines interleukin (IL)-17A, IL-17F and IL-22. The differential cytokine expression patterns in Th cells suggest a division of labour in the response against a variety of pathogens. Th17 have an important function in the host-defense-response against extracellular pathogens, but they also have become notorious for their role in the pathogenesis of many autoimmune and allergic disorders. Animal models of autoimmune disorders have shown that Th17 effector molecules and transcription factors play a crucial role in both development and maintenance of the disease. The discovery of Th17 not only enhanced our insight into these disorders but also placed a Th subset at the interface between the innate and adoptive immune systems with the potential to regulate subsequent immunity against pathogens.

Follicular helper T cells have recently emerged as a separate CD4(+) T helper lineage specialised in provision of help to B cells. They develop independently from Th1, Th2 and Th17 cells and are critical for humoral immunity, including the generation of long-lived and high affinity plasma cells and memory cells crucial for long-term protection against infections. A stepwise differentiation programme has emerged in which T cell receptor (TCR) signalling strength, CD28-mediated costimulation, B cell-derived inducible costimulator ligand signals, induction of c-maf and actions of cytokines, including interleukin (IL)-6 and IL-21, lead to upregulation of the transcriptional repressor B cell lymphoma 6 (Bcl-6) that drives T follicular helper (Tfh) cell differentiation. Bcl-6 turns on a repression programme that targets Blimp-1, transcriptional regulators of other helper lineages and microRNAs. Their concerted actions modulate expression of chemokine receptors, surface molecules and cytokines critical for follicular homing and B cell helper functions. Here, we review the nature of Tfh cells providing help to B cells during the two phases of B cell activation that occur in the outer T zone and, for some B cells, in germinal centres (GC). Recent insights into the signalling events that drive terminal differentiation of Tfh cells critical for selecting somatically mutated GC B cells and the consequences of Tfh dysregulation for immunodeficiency and autoimmune pathology are discussed.