Despite a more than 20-year experience of therapeutic benefit, the relevant molecular and cellular targets of intravenous immunoglobulin (IVIg) in autoimmune disease remain unclear. Contrary to the prevailing theories of IVIg action in autoimmunity, we show that IVIg drives signaling through activating Fc gamma receptors (Fc gammaR) in the amelioration of mouse immune thrombocytopenic purpura (ITP). The actual administration of IVIg was unnecessary because as few as 10(5) IVIg-treated cells could, upon adoptive transfer, ameliorate ITP. IVIg did not interact with the inhibitory Fc gammaRIIB on the initiator cell, although Fc gammaRIIB does have a role in the late phase of IVIg action. Notably, only IVIg-treated CD11c+ dendritic cells could mediate these effects. We hypothesize that IVIg forms soluble immune complexes in vivo that prime dendritic-cell regulatory activity. In conclusion, the clinical effects of IVIg in ameliorating ITP seem to involve the acute interaction of IVIg with activating Fc gammaR on dendritic cells.

MicroRNAs (miRNAs) are single-stranded regulatory RNAs, frequently expressed as clusters. Previous studies have demonstrated that the six-miRNA cluster miR-17~92 has important roles in tissue development and cancers. However, the precise role of each miRNA in the cluster is unknown. Here we show that overexpression of miR-17 results in decreased cell adhesion, migration and proliferation. Transgenic mice overexpressing miR-17 showed overall growth retardation, smaller organs and greatly reduced haematopoietic cell lineages. We found that fibronectin and the fibronectin type-III domain containing 3A (FNDC3A) are two targets that have their expression repressed by miR-17, both in vitro and in transgenic mice. Several lines of evidence support the notion that miR-17 causes cellular defects through its repression of fibronectin expression. Our single miRNA expression assay may be evolved to allow the manipulation of individual miRNA functions in vitro and in vivo. We anticipate that this could serve as a model for studying gene regulation by miRNAs in the development of gene therapy.

Intravenous Ig (IVIg) mediates protection from the effects of immune thrombocytopenic purpura (ITP) as well as numerous other autoimmune states; however, the active antibodies within IVIg are unknown. There is some evidence that antibodies specific for a cell-associated antigen on erythrocytes are responsible, at least in part, for the therapeutic effect of IVIg in ITP. Yet whether an IVIg directed to a soluble antigen can likewise be beneficial in ITP or other autoimmune diseases is also unknown. A murine model of ITP was used to determine the effectiveness of IgG specific to soluble antigens in treating immune thrombocytopenic purpura. Mice experimentally treated with soluble OVA + anti-OVA versus mice treated with OVA conjugated to rbcs (OVA-rbcs) + anti-OVA were compared. In both situations, mice were protected from ITP. Both these experimental therapeutic regimes acted in a complement-independent fashion and both also blocked reticuloendothelial function. In contrast to OVA-rbcs + anti-OVA, soluble OVA + anti-OVA (as well as IVIg) did not have any effect on thrombocytopenia in mice lacking the inhibitory receptor FcgammaRIIB (FcgammaRIIB(-/-) mice). Similarly, antibodies reactive with the endogenous soluble antigens albumin and transferrin also ameliorated ITP in an FcgammaRIIB-dependent manner. Finally, broadening the significance of these experiments was the finding that anti-albumin was protective in a K/BxN serum-induced arthritis model. We conclude that IgG antibodies directed to soluble antigens ameliorated 2 disparate IVIg-treatable autoimmune diseases.

Q3G is a natural derivative of quercetin and is already widely used in various foods and drinks. Our results clearly demonstrated that Q3G exerts antiviral activity against ZIKV in both tissue culture and knockout mice, and that post-exposure in vivo treatment with Q3G could have a beneficial effect. In the future, Q3G should be tested in human cell lines (such as Huh-7, HeLa, or K048, a fetal brain neural stem cell line) to provide further data supporting its potential efficacy in humans; in addition, live viral loads or viremia should be tested in treated animals to supplement the survival results observed in this study. Although the treatment regimens will need to be further optimized (i.e., dosage, frequency of treatment, and administration routes), our results support the results of Q3G efficacy studies in nonhuman primates against ZIKV infection. Further studies will also be needed to investigate the mechanism of Q3G antiviral action, in order to obtain valuable insights into the design of novel targets for antiviral therapeutics in the future.

IntroductionIntravenous immunoglobulin (IVIg) is used widely in the treatment of various autoimmune conditions, including immune thrombocytopenic purpura (ITP). Many mechanisms have been suggested to explain the therapeutic effectiveness of IVIg in increasing platelet counts in ITP. The predominant hypotheses are Fc␥ receptor (Fc␥R) blockade, anti-idiotypic regulation, and immunomodulation. [1][2][3][4][5] It also has been suggested that the major histocompatibility complex (MHC) class I-like Fc receptor (FcRn) may play a role in the action of IVIg. 6 Recently, the ratio of activating Fc␥RIII and the inhibitory Fc␥RIIB expression on splenic macrophages has been proposed to be a key to the therapeutic effectiveness of IVIg, as IVIg up-regulates Fc␥RIIB expression in wild-type mice (Fc␥RIIB ϩ/ϩ ) and the absence of Fc␥RIIB (Fc␥RIIB Ϫ/Ϫ ) completely abrogates the therapeutic efficacy of IVIg in thrombocytopenic mice. 7 IVIg and anti-D may use different pathways to produce their therapeutic effects. [7][8][9][10][11][12][13][14][15] We have previously demonstrated that 2 monoclonal anti-erythrocyte antibodies, TER119 and M1/69, mimic the effect of anti-D in reversing thrombocytopenia in a murine model of ITP; amelioration of thrombocytopenia correlated with an impaired ability to clear sensitized erythrocytes. 16 The current study shows that, unlike IVIg, these therapeutic anti-erythrocyte monoclonal antibodies reverse thrombocytopenia in a Fc␥RIIB-independent fashion, possibly by blocking or decreasing the level of functional Fc␥RIIIA expression. This suggests that, in contrast to IVIg, anti-D-like monoclonal antibodies ameliorate ITP via inhibition of activating Fc␥ receptors. Study design MiceFc␥RIIB Ϫ/Ϫ mice (B6;129S4-Fcgr2b tm1Rav /J) and C57BL/6 mice were purchased from The Jackson Laboratory (Bar Harbor, ME) and housed at the St Michael's Hospital Research Vivarium. Induction and reversal of ITPThrombocytopenia was induced by daily intraperitoneal injection of 2 g rat anti-mouse integrin ␣ IIb antibody (PharMingen, Mississauga, ON, Canada) as previously described. [16][17][18] On day 2, mice were injected intravenously with 50 g monoclonal antibody TER119 or M1/69 (PharMingen) or intraperitoneally with 2 g/kg IVIg (Gamimune, 10%; Bayer, Elkhart, IN). Whole blood was collected daily via the saphenous vein into capillary tubes preloaded with 5 L 1% EDTA (ethylenediaminetetraacetic acid) in phosphate-buffered saline (PBS); pH 7.2; 50 L blood was diluted in 1200 L 1% EDTA in PBS (1:25), and centrifuged at room temperature at 170g for 2 minutes to isolate platelet-rich plasma (PRP). Fifty microliters of PRP was diluted into 9.95-mL Isoton II diluent (Coulter Corporation, Miami, FL) and platelet count determined using a Beckman Z2 Coulter Counter (Coulter Corporation). Induction and prevention of erythrocyte clearanceTo block erythrocyte clearance, mice were injected intravenously with 50 g 2.4G2 (an Fc␥RIIB/Fc␥RIIIA blocking antibody) 19,20 or rat IgG as negative control, or intraperitoneally with 2 g/kg IVIg. Aft...

Arrhythmogenic right ventricular cardiomyopathy (ARVC) is a myocardial disease characterized by fibro-fatty replacement of myocardium in the right ventricular free wall and frequently results in life-threatening ventricular arrhythmias and sudden cardiac death. A heterozygous missense mutation in the transmembrane protein 43 (TMEM43) gene, p.S358L, has been genetically identified to cause autosomal dominant ARVC type 5 in a founder population from the island of Newfoundland, Canada. Little is known about the function of the TMEM43 protein or how it leads to the pathogenesis of ARVC. We sought to determine the distribution of TMEM43 and the effect of the p.S358L mutation on the expression and distribution of various intercalated (IC) disc proteins as well as functional effects on IC disc gap junction dye transfer and conduction velocity in cell culture. Through Western blot analysis, transmission electron microscopy (TEM), immunofluorescence (IF), and electrophysiological analysis, our results showed that the stable expression of p.S358L mutation in the HL-1 cardiac cell line resulted in decreased Zonula Occludens (ZO-1) expression and the loss of ZO-1 localization to cell-cell junctions. Junctional Plakoglobin (JUP) and α-catenin proteins were redistributed to the cytoplasm with decreased localization to cell-cell junctions. Connexin-43 (Cx43) phosphorylation was altered, and there was reduced gap junction dye transfer and conduction velocity in mutant TMEM43-transfected cells. These observations suggest that expression of the p.S358L mutant of TMEM43 found in ARVC type 5 may affect localization of proteins involved in conduction, alter gap junction function and reduce conduction velocity in cardiac tissue.

Intravenous Ig (IVIg) mediates protection from the effects of immune thrombocytopenic purpura (ITP) as well as numerous other autoimmune states; however, the active antibodies within IVIg are unknown. There is some evidence that antibodies specific for a cell-associated antigen on erythrocytes are responsible, at least in part, for the therapeutic effect of IVIg in ITP. Yet whether an IVIg directed to a soluble antigen can likewise be beneficial in ITP or other autoimmune diseases is also unknown. A murine model of ITP was used to determine the effectiveness of IgG specific to soluble antigens in treating immune thrombocytopenic purpura. Mice experimentally treated with soluble OVA + anti-OVA versus mice treated with OVA conjugated to rbcs (OVA-rbcs) + anti-OVA were compared. In both situations, mice were protected from ITP. Both these experimental therapeutic regimes acted in a complement-independent fashion and both also blocked reticuloendothelial function. In contrast to OVA-rbcs + anti-OVA, soluble OVA + anti-OVA (as well as IVIg) did not have any effect on thrombocytopenia in mice lacking the inhibitory receptor FcγRIIB (FcγRIIB-/-mice). Similarly, antibodies reactive with the endogenous soluble antigens albumin and transferrin also ameliorated ITP in an FcγRIIB-dependent manner. Finally, broadening the significance of these experiments was the finding that anti-albu-min was protective in a K/BxN serum-induced arthritis model. We conclude that IgG antibodies directed to soluble antigens ameliorated 2 disparate IVIg-treatable autoimmune diseases.

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