Serum from half of patients with immune thrombocytopenia trigger macrophage phagocytosis of platelets.

Humoral antiplatelet factors, such as autoantibodies, are thought to primarily clear platelets by triggering macrophage phagocytosis in immune thrombocytopenia (ITP). However, there are few studies characterizing the capacity and mechanisms of humoral factor-triggered macrophage phagocytosis of platelets using specimens from patients with ITP. Here, we assessed sera from a cohort of 24 patients with ITP for the capacity to trigger macrophage phagocytosis of normal donor platelets and characterized the contribution of humoral factors to phagocytosis. Sera that produced a phagocytosis magnitude greater than a normal human serum mean + 2 standard deviations were considered phagocytosis-positive. Overall, 42% (8/19) of MHC I alloantibody-negative ITP sera were phagocytosis-positive. The indirect monoclonal antibody immobilization of platelet antigens assay was used to detect immunoglobulin G (IgG) autoantibodies to glycoproteins (GP)IIb/IIIa, GPIb/IX, and GPIa/IIa. Autoantibody-positive sera triggered a higher mean magnitude of phagocytosis than autoantibody-negative sera. Phagocytosis correlated inversely with platelet counts among autoantibody-positive patients but not among autoantibody-negative patients. Select phagocytosis-positive sera were separated into IgG-purified and -depleted fractions via protein G and reassessed for phagocytosis. Phagocytosis was largely retained in the purified IgG fractions. In addition, we assessed serum concentrations of C-reactive protein, serum amyloid P, and pentraxin 3 as potential phagocytosis modulators. Pentraxin 3 concentrations correlated inversely with platelet counts among patients positive for autoantibodies. Taken together, sera from approximately half of the patients with ITP studied triggered macrophage phagocytosis of platelets beyond a normal level. An important role for antiplatelet autoantibodies in phagocytosis is supported; a role for pentraxins such as pentraxin 3 may be suggested.

THP-1 cells transduced with CD16A utilize Fcγ receptor I and III in the phagocytosis of IgG-sensitized human erythrocytes and platelets.

Fc gamma receptors (FcγRs) are critical effector receptors for immunoglobulin G (IgG) antibodies. On macrophages, FcγRs mediate multiple effector functions, including phagocytosis, but the individual contribution of specific FcγRs to phagocytosis has not been fully characterized. Primary human macrophage populations, such as splenic macrophages, can express FcγRI, FcγRIIA, and FcγRIIIA. However, there is currently no widely available monocyte or macrophage cell line expressing all these receptors. Common sources of monocytes for differentiation into macrophages, such as human peripheral blood monocytes and the monocytic leukemia cell line THP-1, generally lack the expression of FcγRIIIA (CD16A). Here, we utilized a lentiviral system to generate THP-1 cells stably expressing human FcγRIIIA (CD16F158). THP-1-CD16A cells treated with phorbol 12-myristate 13-acetate for 24 hours phagocytosed anti-D-opsonized human red blood cells primarily utilizing FcγRI with a lesser but significant contribution of IIIA while phagocytosis of antibody-opsonized human platelets equally utilized FcγRI and Fcγ IIIA. Despite the well-known ability of FcγRIIA to bind IgG in cell free systems, this receptor did not appear to be involved in either RBC or platelet phagocytosis. These transgenic cells may constitute a valuable tool for studying macrophage FcγR utilization and function.

Clinically approved IVIg delivered to the hippocampus with focused ultrasound promotes neurogenesis in a model of Alzheimer's disease.

Preclinical and clinical data support the use of focused ultrasound (FUS), in the presence of intravenously injected microbubbles, to safely and transiently increase the permeability of the blood-brain barrier (BBB). FUS-induced BBB permeability has been shown to enhance the bioavailability of administered intravenous therapeutics to the brain. Ideal therapeutics candidates for this mode of delivery are those capable of inducing benefits peripherally following intravenous injection and in the brain at FUS-targeted areas. In Alzheimer's disease, intravenous immunoglobulin (IVIg), a fractionated human blood product containing polyclonal antibodies, act as immunomodulator peripherally and centrally, and it can reduce amyloid pathology in the brain. Using the TgCRND8 mouse model of amyloidosis, we tested whether FUS can improve the delivery of IVIg, administered intravenously (0.4 g/kg), to the hippocampus and reach an effective dose to reduce amyloid plaque pathology and promote neurogenesis. Our results show that FUS-induced BBB permeability is required to deliver a significant amount of IVIg (489 ng/mg) to the targeted hippocampus of TgCRN8 mice. Two IVIg-FUS treatments, administered at days 1 and 8, significantly increased hippocampal neurogenesis by 4-, 3-, and 1.5-fold in comparison to saline, IVIg alone, and FUS alone, respectively. Amyloid plaque pathology was significantly reduced in all treatment groups: IVIg alone, FUS alone, and IVIg-FUS. Putative factors promoting neurogenesis in response to IVIg-FUS include the down-regulation of the proinflammatory cytokine TNF-α in the hippocampus. In summary, FUS was required to deliver an effective dose of IVIg to promote hippocampal neurogenesis and modulate the inflammatory milieu.

Daudi cell stroma: An alternative to dithiothreitol to resolve daratumumab interference in pretransfusion testing.

Treatment of red blood cells with dithiothreitol (DTT) or trypsin effectively denatures CD38; however, this treatment damages other antigens, some of which are of clinical importance. Thus, other avenues to deplete daratumumab (DARA) from plasma samples should be explored. STUDY DESIGN AND METHODS: The Daudi B-cell line was found to express high levels of CD38 and was sonicated in a sonication buffer to achieve complete cell lysis. The resulting stroma preparation was centrifuged at 20 000g for 20 minutes and then mixed with 250 µL of DARA-plasma and incubated for 10 minutes at 37°C. The stroma-DARA-plasma mixture was centrifuged again, and the supernatant was collected and subjected to four additional rounds of adsorption with fresh stroma. DARA-depleted plasma was tested by gel indirect antiglobulin test (IAT). RESULTS: CD38 expression on Daudi cells was confirmed by flow cytometry. Gel IAT analysis showed that the incubation of plasma from DARA-treated patients with Daudi cells stroma resulted in a significant depletion of DARA but allowing detection of other alloantibodies of interest such as anti-K, anti-Yta , and anti-Gya . CONCLUSIONS: Daudi cell stroma is inexpensive, easy to prepare in large batches, and can be used as an off-the-shelf reagent. Incubation of plasma from DARA-treated patients with Daudi cell stroma can efficiently overcome DARA interference in serologic testing without affecting DTT- or trypsin-sensitive antigens.

Targeting ABL1 or ARG Tyrosine Kinases to Restrict HIV-1 Infection in Primary CD4+ T-Cells or in Humanized NSG Mice.

BACKGROUND: Previous studies support dasatinib as a potent inhibitor of HIV-1 replication. However, a functional distinction between 2 kinase targets of the drug, ABL1 and ARG, has not been assessed. SETTING: We used primary CD4 T-cells, CD8-depleted peripheral blood mononuclear cells (PBMCs) from a treatment naïve HIV-1 patient, and a humanized mouse model of HIV-1 infection. We assessed the roles of ABL1 and ARG during HIV-1 infection and use of dasatinib as a potential antiviral against HIV-1 in humanized mice. METHODS: Primary CD4 T-cells were administered siRNA targeting ABL1 or ARG, then infected with HIV-1 containing luciferase reporter viruses. Quantitative polymerase chain reaction of viral integration of 4 HIV-1 strains was also assessed. CD8-depleted PBMCs were treated for 3 weeks with dasatinib. NSG mice were engrafted with CD34 pluripotent stem cells from human fetal cord blood, and infected with Ba-L virus after 19 weeks. Mice were treated daily with dasatinib starting 5 weeks after infection. RESULTS: siRNA knockdown of ABL1 or ARG had no effect on viral reverse transcripts, but increased 2-LTR circles 2- to 4-fold and reduced viral integration 2- to 12-fold. siRNA knockdown of ARG increased SAMHD1 activation, whereas knockdown of either kinase reduced RNA polymerase II activation. Treating CD8-depleted PBMCs from a treatment-naïve patient with 50 nM of dasatinib for 3 weeks reduced p24 levels by 99.8%. Ba-L (R5)-infected mice injected daily with dasatinib showed a 95.1% reduction in plasma viral load after 2 weeks of treatment. CONCLUSIONS: We demonstrate a novel nuclear role for ABL1 and ARG in ex vivo infection experiments, and proof-of-principle use of dasatinib in a humanized mouse model of HIV-1 infection.

GPIbα is required for platelet-mediated hepatic thrombopoietin generation.

Thrombopoietin (TPO), a hematopoietic growth factor produced predominantly by the liver, is essential for thrombopoiesis. Prevailing theory posits that circulating TPO levels are maintained through its clearance by platelets and megakaryocytes via surface c-Mpl receptor internalization. Interestingly, we found a two- to threefold decrease in circulating TPO in GPIbα-/- mice compared with wild-type (WT) controls, which was consistent in GPIbα-deficient human Bernard-Soulier syndrome (BSS) patients. We showed that lower TPO levels in GPIbα-deficient conditions were not due to increased TPO clearance by GPIbα-/- platelets but rather to decreased hepatic TPO mRNA transcription and production. We found that WT, but not GPIbα-/-, platelet transfusions rescued hepatic TPO mRNA and circulating TPO levels in GPIbα-/- mice. In vitro hepatocyte cocultures with platelets or GPIbα-coupled beads further confirm the disruption of platelet-mediated hepatic TPO generation in the absence of GPIbα. Treatment of GPIbα-/- platelets with neuraminidase caused significant desialylation; however, strikingly, desialylated GPIbα-/- platelets could not rescue impaired hepatic TPO production in vivo or in vitro, suggesting that GPIbα, independent of platelet desialylation, is a prerequisite for hepatic TPO generation. Additionally, impaired hepatic TPO production was recapitulated in interleukin-4/GPIbα-transgenic mice, as well as with antibodies targeting the extracellular portion of GPIbα, demonstrating that the N terminus of GPIbα is required for platelet-mediated hepatic TPO generation. These findings reveal a novel nonredundant regulatory role for platelets in hepatic TPO homeostasis, which improves our understanding of constitutive TPO regulation and has important implications in diseases related to GPIbα, such as BSS and auto- and alloimmune-mediated thrombocytopenias.

IVIG regulates the survival of human but not mouse neutrophils.

Intravenous immunoglobulin (IVIG) are purified IgG preparations made from the pooled plasma from thousands of healthy donors and are being tested in preclinical mouse models. Inherent challenges, however, are the pluripotency of IVIG and its xenogeneicity in animals. IVIG can alter the viability of human neutrophils via agonistic antibodies to Fas and Siglec-9. In this study, we compared the effects of IVIG on human and mouse neutrophils using different death assays. Different commercial IVIG preparations similarly induced cytokine-dependent death in human neutrophils, whereas they had no effects on the survival of either peripheral blood or bone marrow neutrophils from C57BL/6 or BALB/c mice. F(ab')2 but not Fc fragments of IVIG induced death of human neutrophils, whereas neither of these IVIG fragments, nor agonistic monoclonal antibodies to human Fas or Siglec-9 affected the viability of mouse neutrophils. Pooled mouse IgG, which exhibited a different immunoprofile compared to IVIG, also had no effect on mouse cells. Together, these observations demonstrate that effects of IVIG on neutrophil survival are not adequately reflected in current mouse models, despite the key role of these cells in human inflammatory and autoimmune diseases.

Small molecule phagocytosis inhibitors for immune cytopenias.

Immune cytopenias are conditions characterized by low blood cell counts, such as platelets in immune thrombocytopenia (ITP) and red blood cells in autoimmune hemolytic anemia (AIHA). Chronic ITP affects approximately 4 in 100,000 adults annually while AIHA is much less common. Extravascular phagocytosis and massive destruction of autoantibody-opsonized blood cells by macrophages in the spleen and liver are the hallmark of these conditions. Current treatment modalities for ITP and AIHA include the first-line use of corticosteroids; whereas, IVIg shows efficacy in ITP but not AIHA. One main mechanism of action by which IVIg treatment leads to the reduction in platelet destruction rates in ITP is thought to involve Fcγ receptor (FcγR) blockade, ultimately leading to the inhibition of extravascular platelet phagocytosis. IVIg, which is manufactured from the human plasma of thousands of donors, is a limited resource, and alternative treatments, particularly those based on bioavailable small molecules, are needed. In this review, we overview the pathophysiology of ITP, the role of Fcγ receptors, and the mechanisms of action of IVIg in treating ITP, and outline the efforts and progress towards developing novel, first-in-class inhibitors of phagocytosis as synthetic, small molecule substitutes for IVIg in ITP and other conditions where the pathobiology of the disease involves phagocytosis.

A Rapid Screening Assay Identifies Monotherapy with Interferon-ß and Combination Therapies with Nucleoside Analogs as Effective Inhibitors of Ebola Virus.

To date there are no approved antiviral drugs for the treatment of Ebola virus disease (EVD). While a number of candidate drugs have shown limited efficacy in vitro and/or in non-human primate studies, differences in experimental methodologies make it difficult to compare their therapeutic effectiveness. Using an in vitro model of Ebola Zaire replication with transcription-competent virus like particles (trVLPs), requiring only level 2 biosafety containment, we compared the activities of the type I interferons (IFNs) IFN-α and IFN-ß, a panel of viral polymerase inhibitors (lamivudine (3TC), zidovudine (AZT) tenofovir (TFV), favipiravir (FPV), the active metabolite of brincidofovir, cidofovir (CDF)), and the estrogen receptor modulator, toremifene (TOR), in inhibiting viral replication in dose-response and time course studies. We also tested 28 two- and 56 three-drug combinations against Ebola replication. IFN-α and IFN-ß inhibited viral replication 24 hours post-infection (IC50 0.038µM and 0.016µM, respectively). 3TC, AZT and TFV inhibited Ebola replication when used alone (50-62%) or in combination (87%). They exhibited lower IC50 (0.98-6.2µM) compared with FPV (36.8µM), when administered 24 hours post-infection. Unexpectedly, CDF had a narrow therapeutic window (6.25-25µM). When dosed >50µM, CDF treatment enhanced viral infection. IFN-ß exhibited strong synergy with 3TC (97.3% inhibition) or in triple combination with 3TC and AZT (95.8% inhibition). This study demonstrates that IFNs and viral polymerase inhibitors may have utility in EVD. We identified several 2 and 3 drug combinations with strong anti-Ebola activity, confirmed in studies using fully infectious ZEBOV, providing a rationale for testing combination therapies in animal models of lethal Ebola challenge. These studies open up new possibilities for novel therapeutic options, in particular combination therapies, which could prevent and treat Ebola infection and potentially reduce drug resistance.

c-SRC protein tyrosine kinase regulates early HIV-1 infection post-entry.

OBJECTIVE: We investigated whether HIV-1 inhibition by SRC-family kinase inhibitors is through the non-receptor tyrosine kinase pp60 (c-SRC) and its binding partner, protein tyrosine kinase 2 beta (PTK2B). DESIGN: CD4 T-lymphocytes were infected with R5 (JR-FL) or X4 (HXB2) HIV-1. We used SRC-family kinase inhibitors or targeted siRNA knockdown of c-SRC and PTK2B, then monitored effects on the early HIV-1 lifecycle. METHODS: Four SRC-family kinase inhibitors or targeted siRNA knockdown were used to reduce c-SRC or PTK2B protein expression. Activated CD4 T-lymphocytes were infected with recombinant, nef-deficient, or replication-competent infectious viruses. Knockdown experiments examined early infection by monitoring: luciferase activity, expression of host surface receptors, reverse transcriptase activity, p24 levels and qPCR of reverse transcripts, integrated HIV-1, and two-long terminal repeat (2-LTR) circles. RESULTS: All SRC-family kinase inhibitors inhibited R5 and X4 HIV-1 infection. Neither c-SRC nor PTK2B siRNA knockdown had an effect on cell surface receptors (CD4, CXCR4, and CCR5) nor on reverse transcriptase activity. However, using JR-FL both decreased luciferase activity while increasing late reverse transcripts (16-fold) and 2-LTR circles (eight-fold) while also decreasing viral integration (four-fold). With HXB2, c-SRC but not PTK2B siRNA knockdown produced similar results. CONCLUSIONS: Our results suggest c-SRC tyrosine kinase is a major regulator of HIV-1 infection, participating in multiple stages of infection post-entry: Reduced proviral integration with increased 2-LTR circles is reminiscent of integrase inhibitors used in combination antiretroviral therapy. Decreasing c-SRC expression and/or activity provides a new target for antiviral intervention and the potential for repurposing existing FDA-approved kinase inhibitors.

Relaxin receptor antagonist AT-001 synergizes with docetaxel in androgen-independent prostate xenografts.

Androgen hormones and the androgen receptor (AR) pathway are the main targets of anti-hormonal therapies for prostate cancer. However, resistance inevitably develops to treatments aimed at the AR pathway resulting in androgen-independent or hormone-refractory prostate cancer (HRPC). Therefore, there is a significant unmet need for new, non-androgen anti-hormonal strategies for the management of prostate cancer. We demonstrate that a relaxin hormone receptor antagonist, AT-001, an analog of human H2 relaxin, represents a first-in-class anti-hormonal candidate treatment designed to significantly curtail the growth of androgen-independent human prostate tumor xenografts. Chemically synthesized AT-001, administered subcutaneously, suppressed PC3 xenograft growth by up to 60%. AT-001 also synergized with docetaxel, standard first-line chemotherapy for HRPC, to suppress tumor growth by more than 98% in PC3 xenografts via a mechanism involving the downregulation of hypoxia-inducible factor 1 alpha and the hypoxia-induced response. Our data support developing AT-001 for clinical use as an anti-relaxin hormonal therapy for advanced prostate cancer.

Cytokine profiles in mouse models of experimental immune thrombocytopenia reveal a lack of inflammation and differences in response to intravenous immunoglobulin depending on the mouse strain.

BACKGROUND: Mouse models of human immune thrombocytopenia (ITP) have been used for years to investigate the mechanism of intravenous immunoglobulin (IVIG) to ameliorate ITP; however, how closely these experimental mouse models mirror the human autoimmune inflammatory disease is unclear. The aim of this study was to assess the cytokine profiles in experimental ITP with and without IVIG treatment. STUDY DESIGN AND METHODS: We examined the production of 23 cytokines that included pro- and anti-inflammatory cytokines, in two different mouse strain models of ITP, BALB/c and C57BL/6J, with and without IVIG treatment. RESULTS: In contrast to human ITP, in mouse models of ITP generated by passive transfer of an alloantibody we find no evidence of inflammatory disease even when ITP is robust, suggesting that while these models capture the innate response phase of the disease, they may not be capturing the adaptive mechanisms of autoimmune initiation of the disorder. Regardless of the mouse strain examined, interleukin (IL)-1α, -2, -6, -17, and -23; granulocyte-macrophage-colony-stimulating factor (GM-CSF); monocyte chemoattractant protein (MCP)-1; macrophage inflammatory protein (MIP)-1ß; RANTES; tumor necrosis factor (TNF)-α; and interferon-γ remain at negligible levels after ITP induction. IVIG treatment in the absence of ITP induced significant levels of IL-4, -10, -11, -17, and -23; GM-CSF; MCP-1; and TNF-α in BALB/c mice, but only IL-11 was elevated in C57BL/6J mice. In response to IVIG treatment of ITP, both strains produced IL-4, -10, -11, and -23; GM-CSF; MCP-1; MIP-1ß; and TNF-α; however, only BALB/c exhibited increased MCP-3 and RANTES. IL-11 levels were the highest of any cytokine after IVIG administration, given either alone or as treatment of ITP. CONCLUSIONS: We conclude that mouse models for human ITP do not capture the full range of autoimmune inflammatory mechanisms of this disease. Furthermore, cytokine profiles differ in response to IVIG depending on the mouse strain used.

c-Src and Pyk2 protein tyrosine kinases play protective roles in early HIV-1 infection of CD4+ T-cell lines.

BACKGROUND: During early HIV-1 infection of CD4 T-lymphocytes, many host protein tyrosine kinases become activated within minutes, including phosphoprotein pp60 (c-Src) and the focal adhesion kinase family member, proline-rich tyrosine kinase 2 (Pyk2). Whether their activation facilitates or impedes infection remains to be determined. METHODS: c-Src kinase inhibitors (SU6656, PP1, and PP2), adenovectors [wild-type and dominant-negative (DN) c-src] or siRNA (targeting c-src or pyk2) were used to inhibit, compete with or knockdown c-Src in Jurkat C, Jurkat E6-1, Hut 78 or Kit225 T-cell lines. Cells were then infected with HIV-1 luciferase reporter virus expressing VSV-G or HXB2(X4) envelope, and luciferase activity was measured after 2 days. Reverse transcriptase activity and viral cDNA were measured 1 hour after infection, whereas integrated virus was measured 12 hours after infection. RESULTS: Pretreating Jurkat T-cells with SU6656 led to increased VSV-G luciferase activity. In the adenovector experiments, T-cells overexpressing dominant-negative c-Src, but not wild-type c-Src, showed increased luciferase activity after VSV-G infection. siRNA knockdown of c-Src or Pyk2, followed by HXB2 infection in Jurkat T-cells, lead to increased reverse transcriptase activity, viral cDNA, integrated virus, and increased luciferase activity. CONCLUSIONS: Pyk2 is known to interact with c-Src. Thus, Pyk2 activation that coincides with increased c-Src activity during HIV-1 infection could be responsible for c-Src activation. Reduced c-Src activation increases HIV-1 reverse transcription, integration, and/or transcription, suggesting the high c-Src activity seen early in HIV-1 infection may be a cellular response to slow or prevent early infection in CD4 T-cells.

Acute hemolysis after intravenous immunoglobulin amid host factors of ABO-mismatched bone marrow transplantation, inflammation, and activated mononuclear phagocytes.

BACKGROUND: Hemolysis may follow intravenous immunoglobulin (IVIG), with product, dosing, and host factors contributing. The importance of recipient features remains unclear. CASE REPORT: A 52-year-old obese woman, 10 years after ABO-mismatched (recipient O, donor A) marrow transplantation, presented with immune thrombocytopenia (ITP). IVIG at 100 g/day × 2 days was followed by hemoglobinuria and angina and dyspnea, with frank hemoglobinemia and anemia (hemoglobin 12.9 to 8.4 over 24 hr, to a nadir of 6.9 g/dL). STUDY DESIGN AND METHODS: Serologic methods established ABO, A1, Lewis, and Secretor type, while monocyte monolayer assay (MMA) examined erythrophagocytosis with control or patient monocytes, and the implicated IVIG lot to opsonize control (group A1, A2, B, O) or patient red blood cells (RBCs). Baseline, hemolytic, and convalescent markers (including cytokines) were assessed. RESULTS: Passive anti-A was identified on reverse type and eluted from sensitized RBCs (immunoglobulin G 1+, C3d-). Le(a-b+) typing and saliva confirmed H Secretor status. MMA revealed significant activity between patient RBCs, monocytes, and IVIG. However, normal A1 cells opsonized with IVIG were not significantly phagocytosed by either normal or patient monocytes. Proinflammatory markers were significantly elevated before and after IVIG. CONCLUSIONS: Synergizing host factors (including obesity-unadjusted dosing and existing inflammation) marked this severe post-IVIG hemolytic crisis. Group A antigen restriction to myeloid tissues, with H Secretor phenotype, may have contributed, rendering this bone marrow transplant chimera vulnerable to anti-A in a manner analogous to the idiosyncratic effect of therapeutic anti-D in certain D+ ITP recipients. However, MMA suggested a macrophage activation state as contributory, perhaps precipitated by existing inflammation.

Characterization of SHP-1 protein tyrosine phosphatase transcripts, protein isoforms and phosphatase activity in epithelial cancer cells.

We identified 7 SHP-1 (PTPN6) transcripts using epithelial cancer-derived cell lines. Four were shown to utilize the epithelial promoter 1 to transcribe a full-length, a partial (exon 3) or complete (exons 3 & 4) deletion of the N-SH2 domain, and also a non-coding transcript having a stop codon caused by a frame shift due to intron 2 retention. Three additional transcripts were shown to utilize the hematopoietic promoter 2 to transcribe a full-length, a partial (exon 3) deletion of the N-SH2 domain and a non-coding transcript with intron 2 retention. We show that endogenous proteins corresponding to the open-reading-frame (ORF) transcripts are produced. Using GST-fusion proteins we show that each product of the ORF SHP-1 transcripts has phosphatase activity and isoforms with an N-SH2 deletion have increased phosphatase activity and novel protein-protein interactions. This study is the first to document utilization of promoter 2 by SHP-1 transcripts and a noncoding transcript in human epithelial cells.

Disulfide linked pyrazole derivatives inhibit phagocytosis of opsonized blood cells.

Immune thrombocytopenia (ITP) is caused by production of an autoantibody to autologous platelets. ITP can be treated either by reducing platelet destruction or by increasing platelet production. Fcγ receptor mediated phagocytosis of the opsonized blood cells is a well-accepted mechanism for the underlying pathogenesis of ITP and inhibition of this phagocytosis process with small molecules is a potential strategy for the development of drugs against ITP. A broad screen indicated that 4-methyl-1-phenyl-pyrazole derivative (1) could inhibit the phagocytosis of opsonized blood cells with weak potency. We reveal here the discovery of the polysulfide products, synthesis of various 1-phenyl-pyrazole derivatives, and the biological evaluation of pyrazole derivatives as inhibitors of phagocytosis for potential use as therapeutics for ITP. Substitution at C4 of the pyrazole moiety in the disulfide-bridged dimers influenced the potency in the increasing order of 10 ~/= 11~/= 16 < 19 < 20. A novel scaffold, 20 with an IC(50) of 100 nM inhibiting opsonized blood cell phagocytosis was identified as a potential candidate for further studies. Confirmation of the disulfide bridge additionally provides clues for the non-thiol or non-disulfide bridge carrying ligands targeting ITP and other similar disorders.

ABO blood groups influence macrophage-mediated phagocytosis of Plasmodium falciparum-infected erythrocytes.

Erythrocyte polymorphisms associated with a survival advantage to Plasmodium falciparum infection have undergone positive selection. There is a predominance of blood group O in malaria-endemic regions, and several lines of evidence suggest that ABO blood groups may influence the outcome of P. falciparum infection. Based on the hypothesis that enhanced innate clearance of infected polymorphic erythrocytes is associated with protection from severe malaria, we investigated whether P. falciparum-infected O erythrocytes are more efficiently cleared by macrophages than infected A and B erythrocytes. We show that human macrophages in vitro and mouse monocytes in vivo phagocytose P. falciparum-infected O erythrocytes more avidly than infected A and B erythrocytes and that uptake is associated with increased hemichrome deposition and high molecular weight band 3 aggregates in infected O erythrocytes. Using infected A(1), A(2), and O erythrocytes, we demonstrate an inverse association of phagocytic capacity with the amount of A antigen on the surface of infected erythrocytes. Finally, we report that enzymatic conversion of B erythrocytes to type as O before infection significantly enhances their uptake by macrophages to observed level comparable to that with infected O wild-type erythrocytes. These data provide the first evidence that ABO blood group antigens influence macrophage clearance of P. falciparum-infected erythrocytes and suggest an additional mechanism by which blood group O may confer resistance to severe malaria.

SHP-1 protein tyrosine phosphatase associates with the adaptor protein CrkL.

SHP-1, encoded by the PTPN6 gene, is a protein tyrosine phosphatase with two src-homology-2 (SH2) domains that is implicated as providing suppression of hematopoietic malignancies. A number of reports have shown protein-protein interactions between SHP-1 SH2 domains and tyrosine-phosphorylated proteins. However, despite its having three proline-rich, potential SH3-binding motifs, no reports of protein-protein interactions through src-homology-3 (SH3)-binding domains with SHP-1 have been described. Herein we show that the SH3 domain-containing CT10 regulator of kinase-like (CrkL) adaptor protein associates with SHP-1. We also provide results that suggest this association is due to CrkL binding to PxxP domains located at amino acid residues 158-161 within the SHP-1 C-terminal SH2 domain, and amino acid residues 363-366 within its phosphatase domain. This study is the first to identify and define an interaction between SHP-1 and an SH3 domain-containing protein. Our findings provide an alternative way that SHP-1 can be linked to potential substrates.

Evaluating the role of IL-11, a novel cytokine in the IL-6 family, in a mouse model of spinal cord injury.

BACKGROUND: Spinal cord injury (SCI) is a devastating condition with substantial functional and social morbidity. Previous research has established that the neuroinflammatory response plays a significant role in cord damage post-SCI. However, global immunosuppressive therapies have demonstrated mixed results. As a result, more specific therapies modulating inflammation after injury are needed. In this regard, research into cytokine signaling has demonstrated that cytokines of the gp130 family including IL-6 and leukemia inhibitory factor (LIF) play key roles in mediating damage to the spinal cord. Since members of the gp130 family all share a common signal transduction pathway via the JAK/STAT system, we performed the first study of a relatively new member of the gp130 family, IL-11, in SCI. METHODS: A validated clip-compression mouse model of SCI was used to assess for temporal changes in expression of IL-11 and its receptor, IL-11Rα, post-SCI. To elucidate the role of IL-II in the pathophysiology of SCI, we compared differences in locomotor recovery (Basso Mouse Score; CatWalk), electrophysiological spinal cord signaling, histopathology, and the acute inflammatory neutrophil response in IL-11Rα knockouts with littermate wild-type C57BL/6 mice. RESULTS: We found an increase in gene expression of IL-11 in the spinal cord to a peak at twenty-four hours post-SCI with increases in IL-11Rα gene expression, peaking at seven days post-SCI. In spite of clear changes in the temporal expression of both IL-11 and its receptor, we found that there were no significant differences in motor function, electrophysiological signaling, histopathology, or neutrophil infiltration into the spinal cord between wild-type and knockout mice. CONCLUSIONS: This is the first study to address IL-11 in SCI. This study provides evidence that IL-11 signaling may not play as significant a role in SCI as other gp130 cytokines, which will ideally guide future therapy design and the signaling pathways those therapies target.