Antigen presenting cells recognize pathogens via pattern recognition receptors (PRR), which upon ligation transduce intracellular signals that can induce innate immune responses. Because some C-type lectin-like receptors (e.g. dectin-1 and DC-SIGN) were shown to act as PRR for particular microbes, we considered a similar role for dectin-2. Binding assays using soluble dectin-2 receptors showed the extracellular domain to bind preferentially to hyphal (rather than yeast/conidial) components of Candida albicans, Microsporum audouinii, and Trichophyton rubrum. Selective binding for hyphae was also observed using RAW macrophages expressing dectin-2, the ligation of which by hyphae or cross-linking with dectin-2-specific antibody led to protein tyrosine phosphorylation. Because dectin-2 lacks an intracellular signaling motif, we searched for a signal adaptor that permits it to transduce intracellular signals. First, we found that the Fc receptor ␥ (FcR␥) chain can bind to dectin-2. Second, ligation of dectin-2 on RAW cells induced tyrosine phosphorylation of FcR␥, activation of NF-B, internalization of a surrogate ligand, and up-regulated secretion of tumor necrosis factor ␣ and interleukin-1 receptor antagonist. Finally, these dectin-2-induced events were blocked by PP2, an inhibitor of Src kinases that are mediators for FcR␥ chain-dependent signaling. We conclude that dectin-2 is a PRR for fungi that employs signaling through FcR␥ to induce innate immune responses.

IntroductionT-cell activation is dependent on signals delivered by antigenpresenting cells (APCs) to the antigen (Ag)-specific T-cell receptor (TCR) and accessory receptors on T cells. 1 The principal stimulatory accessory signal is transmitted by B7-1 (CD80) or B7-2 (CD86) on APCs to the CD28 receptor on T cells. 2 Interestingly, engagement of the same B7-1 or B7-2 ligand to CTLA-4 (CD152) on T cells markedly attenuates T-cell responses. 3,4 The importance of CTLA-4 as an inhibitory regulator of T-cell activation is illustrated by death of CTLA-4-deficient mice within 4 weeks of birth because of massive lymphocytic infiltration destroying critical organs. 5 More recently, other inhibitory regulators of T-cell activation were identified, including PD-L1 (B7-H1) and PD-L2 (B7-DC) on APCs and PD-1 on T cells, 6 BTLA on B cells and T helper 1 (Th1) effector cells and its ligand (herpes virus entry mediator) on T cells, 7,8 and Tim-3 on APCs and Th1 effector cells and Tim-3 ligand on CD4 ϩ T cells. [9][10][11] The T-cell ligands possess a single immunoglobulin (Ig)-like variable (IgV) domain, and the APC receptors contain both IgV and Ig constant (IgC) domains. 12 Interactions between ligand-receptor pairs are mediated predominantly by residues of Ig-like domains. 12 Because of their structural and functional similarities to B7 molecules, these ligands/receptors are considered members of the B7 receptor superfamily. 12 Ligation of PD-1 on T cells leads to inhibited T-cell responses that can be rescued by exogenous IL-2 or CD28 costimulation, [13][14][15] although one report showed that binding of PD-L1 (B7-H1) to PD-1 stimulated T-cell proliferation and IL-10 secretion. [16][17][18] PD-1 deficiency leads to exaggerated autoimmunity since PD-1 knockout mice develop splenomegaly, increased numbers of B and myeloid cells, increased serum IgG and IgA, and a lupus erythematosuslike disease with age. 19,20 These mice are also markedly susceptible to Ag-induced experimental autoimmune encephalomyelitis (EAE). 19,20 BTLA knockout mice do not exhibit developmental Tor B-cell defects, but their lymphocytes have heightened responses to anti-CD3 antibody (Ab) and to anti-IgM Ab; 8 these mice are also prone to developing EAE. 8 In the case of the Tim-3 pathway, its blockade by monoclonal Ab (mAb), Fc-fused soluble receptor, or gene disruption leads to exacerbated Th1-mediated autoimmune diabetes mellitus in nonobese diabetic (NOD) mice. 10,11 T-cell expression of PD-1, BTLA, or Tim-3 resembles CTLA-4 in that it is not constitutive, but is induced by activation. 21 Moreover, the costimulation delivered by each appears to be mediated through the TCR. 12 By contrast, expression of PD-1, BTLA, Tim-3, or their ligands differ from CTLA-4 in that it is not restricted to T cells, but is expressed more widely to include B cells and APCs. Indeed, some of these ligands (PD-L1 and PD-L2) are also expressed in nonlymphoid tissues. 12 Such broad expression profiles suggest that these molecules can modulate immune responses in secondary lympho...

We studied bovine subjects that exhibited a moderate uncompensated anemia with hereditary spherocytosis inherited in an autosomal incompletely dominant mode and retarded growth. Based on the results of SDS-PAGE, immunoblotting, and electron microscopic analysis by the freeze fracture method, we show here that the proband red cells lacked the band 3 protein completely. Sequence analysis of the proband band 3 cDNA and genomic DNA showed a C → T substitution resulting in a nonsense mutation (CGA → TGA; Arg → Stop) at the position corresponding to codon 646 in human red cell band 3 cDNA. The proband red cells were deficient in spectrin, ankyrin, actin, and protein 4.2, resulting in a distorted and disrupted membrane skeletal network with decreased density. Therefore, the proband red cell membranes were extremely unstable and showed the loss of surface area in several distinct ways such as invagination, vesiculation, and extrusion of microvesicles, leading to the formation of spherocytes. Total deficiency of band 3 also resulted in defective Cl Ϫ /HCO 3 Ϫ exchange, causing mild acidosis with decreases in the HCO 3

We previously produced mice with human hepatocyte (h-hep) chimeric livers by transplanting h-heps into albumin enhancer/promoter-driven urokinase-type plasminogen activator-transgenic severe combined immunodeficient (SCID) mice with liver disease. The chimeric livers were constructed with h-heps, mouse hepatocytes, and mouse hepatic sinusoidal cells (m-HSCs). Here, we investigated the morphological features of the chimeric livers and the h-hep gene expression profiles in the xenogeneic animal body. To do so, we performed immunohistochemistry, morphometric analyses, and electron microscopic observations on chimeric mouse livers, and used microarray analyses to compare gene expression patterns in hepatocytes derived from chimeric mouse hepatocytes (c-heps) and h-heps. Morphometric analysis revealed that the ratio of hepatocytes to m-HSCs in the chimeric mouse livers were twofold higher than those in the SCID mouse livers, corresponding to twin-cell plates in the chimeric mouse liver. The h-heps in the chimeric mouse did not show hypoxia even in the twin-cell plate structure, probably because of low oxygen consumption by the h-heps relative to the mouse hepatocytes (m-heps). Immunohistochemical and electron microscopic examinations revealed that the sinusoids in the chimeric mouse livers were normally constructed with h-heps and m-HSCs. However, a number of microvilli projected into the intercellular clefts on the lateral aspects of the hepatocytes, features typical of a growth phase. Microarray profiles indicated that ∼82% of 16 605 probes were within a twofold range difference between h-heps and c-heps. Cluster and principal component analyses showed that the gene expression patterns of c-heps were extremely similar to those of h-heps. In conclusion, the chimeric mouse livers were normally reconstructed with h-heps and m-HSCs, and expressed most human genes at levels similar to those in human livers, although the chimeric livers showed morphological characteristics typical of growth.

Interphotoreceptor retinoid-binding protein (IRBP) secreted by photoreceptors plays a pivotal role in photoreceptor survival with an unknown mechanism. A mutation in the human IRBP has been linked to retinitis pigmentosa, a progressive retinal degenerative disease. Mice lacking IRBP display severe early and progressive photoreceptor degeneration. However, the signaling pathway(s) leading to photoreceptor death in IRBP-deficient mice remains poorly understood. Here, we show that amounts of tumor necrosis factor-␣ (TNF-␣) in the interphotoreceptor matrix and retinas of Irbp Ϫ / Ϫ mice were increased more than 10-fold and fivefold, respectively, compared with those in wild-type mice. Moreover, TNF-␣ receptor 1, an important membrane death receptor that mediates both programmed apoptosis and necrosis, was also significantly increased in Irbp Ϫ / Ϫ retina, and was colocalized with peanut agglutinin to the Irbp Ϫ / Ϫ cone outer segments. Although these death signaling proteins were increased, the caspase-dependent and independent apoptotic pathways were mildly activated in the Irbp Ϫ / Ϫ retinas, suggesting that other cell death mechanism(s) also contributes to the extensive photoreceptor degeneration in Irbp Ϫ / Ϫ retina. We found that receptor interacting protein 1 and 3 (RIP1 and RIP3) kinases, the intracellular key mediators of TNF-induced cellular necrosis, were elevated at least threefold in the Irbp Ϫ / Ϫ retinas. Moreover, pharmacological inhibition of RIP1 kinase significantly prevented cone and rod photoreceptor degeneration in Irbp Ϫ / Ϫ mice. These results reveal that RIP kinase-mediated necrosis strongly contributes to cone and rod degeneration in Irbp Ϫ / Ϫ mice, implicating the TNF-RIP pathway as a potential therapeutic target to prevent or delay photoreceptor degeneration in patients with retinitis pigmentosa caused by IRBP mutation.

No abstract

We found that hesperidin, a plant-derived bioflavonoid, may be a candidate agent for neuroprotective treatment in the retina, after screening 41 materials for anti-oxidative properties in a primary retinal cell culture under oxidative stress. We found that the intravitreal injection of hesperidin in mice prevented reductions in markers of the retinal ganglion cells (RGCs) and RGC death after N-methyl-D-aspartate (NMDA)-induced excitotoxicity. Hesperidin treatment also reduced calpain activation, reactive oxygen species generation and TNF-α gene expression. Finally, hesperidin treatment improved electrophysiological function, measured with visual evoked potential, and visual function, measured with optomotry. Thus, we found that hesperidin suppressed a number of cytotoxic factors associated with NMDA-induced cell death signaling, such as oxidative stress, over-activation of calpain, and inflammation, thereby protecting the RGCs in mice. Therefore, hesperidin may have potential as a therapeutic supplement for protecting the retina against the damage associated with excitotoxic injury, such as occurs in glaucoma and diabetic retinopathy.

Greater cognitive and affective decline occurs in patients with AD-MetS than in those without. Further, insulin resistance and vascular endothelial dysfunction are strongly correlated with AD-MetS before pathological white matter changes can be observed.