Improved Mouse Models for the Study of Treatment Modalities using Sulfur-containing Small-molecular-Weight Molecules for Passive Immune-mediated Thrombocytopenia

Immune thrombocytopenic purpura (ITP) is an autoimmune disease characterized by autoantibody-mediated platelet destruction. To test the efficacy of novel sulfur compounds as alternative treatments for ITP, we used a mouse model of passive immune thrombocytopenia (PIT). Using this model, the platelet nadir could not be maintained, with platelet counts rising after day 4, despite daily anti-platelet antibody administration. We examined reticulated platelet counts by flow cytometry, and found increased thrombopoiesis in the bone marrow to be at least partially responsible for this platelet rebound. Consequentially, two improved mouse models of PIT were developed, where the platelet rebound is circumvented. The first model employs sublethal total body gamma-irradiation in combination with daily antibody administration, while the second model employs gradual escalation of the daily antibody dose. Finally, we show that none of the tested candidate compounds show efficacy in elevating platelet counts in vivo, likely due to their limited solubility.MAS

The Inhibitory Fc[gamma] Receptor is Unnecessary for IVIG Efficacy

Intravenous gamma globulin (IVIG) is used as an effective therapy for many different autoimmune and inflammatory diseases. The current paradigm for the mechanism underpinning anti-inflammatory effects links IVIG administration to increased expression and activity of the inhibitory Fc[gamma] receptor (Fc[gamma]RIIB) on splenic macrophages. This hypothesis has been disputed in the literature. Here we show that IVIG administered in the context of passive antibody-mediated thrombocytopenia to be therapeutically efficacious in both splenectomized and unmanipulated Balb/c mice despite no upregulation of Fc[gamma]RIIB mRNA in the spleen. Moreover, IVIG effectively ameliorated immune thrombocytopenia (ITP) in Fc[gamma]RIIB-deficient Balb/c mice, but not in Fc[gamma]RIIB-/- or Fc[gamma]RIIB+/+ control mice with a C57BL6/129S background. Our results demonstrate that Fc[gamma]RIIB is not relevant to the beneficial effects of IVIG. We anticipate that investigators will now shift their research focus away from Fc[gamma]RIIB and seek out other possible mechanism(s) to explain the plethora of diseases and conditions treatable with IVIG

The Inhibitory Fcγ Receptor is Unnecessary for IVIG Efficacy

Intravenous gamma globulin (IVIG) is used as an wild-type mice In contrast to the system described above, the more-commonly studied mouse model of ITP involves administration of monoclonal anti-platelet antibody, 6A6 or MWReg30, with the consequent induction of a ~50% drop in platelet number. Importantly, in the original study 6 , this drop occurs primarily during the initial 2-8 hours, platelet counts recovering to normal or near normal numbers by 24 hours later. Also, in this and other mouse models of ITP, IVIG is usually administered prior to induction of thrombocytopenia. By contrast, in human ITP, platelet counts usually fall to very low levels and their levels remain low until therapeutic intervention. Thus our mouse model of passive antibody-induced ITP 17 more closely resembles the human condition, the monoclonal antibody evoking a significant (up to 80%-90%) reduction in platelet number by 24 hours that is sustained until IVIG is administered. Importantly, the major evidence supporting the importance of FcγRIIB to IVIG effects was derived using FcγRIIB knockout mice to show that the effect is lost in the context of FcγRIIB deficiency. However, using our mouse model of ITP, we have found the effects of IVIG on thrombocytopenia to be no different in FcγRIIB-deficient compared to wild-type mice on either the C57BL6/129S or Balb/c backgrounds A lack of FcγRIIB contribution to IVIG therapeutic effect is also supported by our finding that FcγRIIB expression is unaltered in the spleen of wild-type Balb/c mice following IVIG treatment and the lack of any detectable difference in IVIG therapeuti

Verotoxin A Subunit Protects Lymphocytes and T Cell Lines against X4 HIV Infection in Vitro

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Verotoxin A Subunit Protects Lymphocytes and T Cell Lines against X4 HIV Infection in Vitro

Our previous genetic, pharmacological and analogue protection studies identified the glycosphingolipid, Gb₃ (globotriaosylceramide, P^k blood group antigen) as a natural resistance factor for HIV infection. Gb₃ is a B cell marker (CD77), but a fraction of activated peripheral blood mononuclear cells (PBMCs) can also express Gb₃. Activated PBMCs predominantly comprise CD4⁺ T-cells, the primary HIV infection target. Gb₃ is the sole receptor for <i>Escherichia coli </i>verotoxins (VTs, Shiga toxins). VT1 contains a ribosome inactivating A subunit (VT1A) non-covalently associated with five smaller receptor-binding B subunits. The effect of VT on PHA/IL2-activated PBMC HIV susceptibility was determined. Following VT1 (or VT2) PBMC treatment during IL2/PHA activation, the small Gb₃⁺/CD4⁺ T-cell subset was eliminated but, surprisingly, remaining CD4⁺ T-cell HIV-1_IIIB (and HIV-1_Ba-L) susceptibility was significantly reduced. The Gb₃^-Jurkat T-cell line was similarly protected by brief VT exposure prior to HIV-1_IIIB infection. The efficacy of the VT1A subunit alone confirmed receptor independent protection. VT1 showed no binding or obvious Jurkat cell/PBMC effect. Protective VT1 concentrations reduced PBMC (but not Jurkat cell) proliferation by 50%. This may relate to the mechanism of action since HIV replication requires primary T-cell proliferation. Microarray analysis of VT1A-treated PBMCs indicated up regulation of 30 genes. Three of the top four were histone genes, suggesting HIV protection via reduced gene activation. VT blocked HDAC inhibitor enhancement of HIV infection, consistent with a histone-mediated mechanism. We speculate that VT1A may provide a benign approach to reduction of (X4 or R5) HIV cell susceptibility