Dimethyl fumarate inhibits antibody-induced platelet destruction in immune thrombocytopenia mouse

Background Immune thrombocytopenia (ITP) is an autoimmune disease characterized as a low platelet count resulting from immune-mediated platelet destruction. Dimethyl fumarate (DMF) is widely applied for the treatment of several autoimmune diseases with immunosuppressive effect. However, whether it ameliorates ITP is unclear. This study aims to evaluate whether DMF has a preventive effect on ITP in mice. Methods DMF (30, 60 or 90 mg/kg body weight) was intraperitoneally injected into mice followed by injection of rat anti-mouse integrin GPIIb/CD41antibody to induce ITP. Peripheral blood was isolated to measure platelet count and spleen mononuclear cells were extracted to measure Th1 and Treg cells along with detecting the levels of IFN-γ, and TGFβ-1 in plasma and CD68 expression in spleen by immuohistochemical staining. Additionally, macrophage cell line RAW264.7 was cultured and treated with DMF followed by analysis of cell apoptosis and cycle, and the expression of FcγRI, FcγRIIb and FcγRIV mRNA. Results DMF significantly inhibited antiplatelet antibody-induced platelet destruction, decreased Th1 cells and the expression of T-bet and IFN-γ, upregulated Treg cells and the expression of Foxp3 and TGF-β1 as well as reduced CD68 expression in the spleen of ITP mouse. DMF-treated RAW264.7 cells showed S-phase arrest, increased apoptosis and downregulated expression of FcγRI and FcγRIV. Meanwhile, in vitro treatment of DMF also decreased the expression of cyclin D1 and E2, reduced Bcl-2 level and increased Bax expression and caspase-3 activation. Conclusions In conclusion, DMF prevents antibody-mediated platelet destruction in ITP mice possibly through promoting apoptosis, indicating that it might be used as a new approach for the treatment of ITP.

Thioacetamide-induced liver damage and thrombocytopenia is associated with induction of antiplatelet autoantibody in mice

Thrombocytopenia is usually associated with liver injury, elevated plasma aspartate aminotransferase and alanine aminotransferase levels, and high antiplatelet immunoglobulin (Ig) titers, although the mechanism behind these effects remains elusive. Deciphering the mechanism behind acute liver disease–associated thrombocytopenia may help solve difficulties in routine patient care, such as liver biopsy, antiviral therapy, and surgery. To determine whether liver damage is sufficient per se to elicit thrombocytopenia, thioacetamide (TAA)-induced hepatitis rodent models were employed. The analysis results indicated that TAA treatment transiently induced an elevation of antiplatelet antibody titer in both rats and mice. B-cell-deficient (BCD) mice, which have loss of antibody expression, exhibited markedly less thrombocytopenia and liver damage than wild-type controls. Because TAA still induces liver damage in BCD mice, this suggests that antiplatelet Ig is one of the pathogenic factors, which play exacerbating role in the acute phase of TAA-induced hepatitis. TNF-α was differentially regulated in wild-type versus BCD mice during TAA treatment, and anti-TNF treatment drastically ameliorated antiplatelet Ig induction, thrombocytopenia, and liver injury, suggesting that the TNF pathway plays a critical role in the disease progression.

Antiplatelet Antibody Testing in Immune Thrombocytopenia and Evans Syndrome: Longitudinal Serologic Evolution and Relation to Clinical Features

Introduction : Antiplatelet antibody (APA) testing is considered an adjunct laboratory test in the diagnosis of immune thrombocytopenia (ITP). While it is not routinely obtained owing to inconsistent sensitivity and specificity in prior studies (Neunert et al, 2011), the definition of ITP used in these studies was not standardized. Additionally, potential clinical utility of this testing beyond diagnosis has been suggested, such as correlations between certain serologic patterns and response to IVIG (Peng et al, 2014). In consideration of these prior findings, we undertook a retrospective analysis of APA testing [glycoprotein-specific testing done by the commercial PakAuto assay (Immucor, Brookfield, WI), a monoclonal antibody immobilization of platelet antigens (MAIPA) assay, with all testing performed by the same laboratory] utilizing standardized ITP and Evans syndrome (ES) diagnostic criteria for patient inclusion. We examined serologic evolution over time and relation of antibody positivity to disease severity and response to therapies. Methods : Data collected for analysis included dates and results of APA testing (including disease status and platelet count at time of testing), patient demographics, and disease characteristics. Satisfaction of the 2011 American Society of Hematology (ASH) ITP diagnostic criteria were required for ITP patient inclusion and standard definitions of disease severity and response to treatment from the ASH guidelines were used in analysis. Logistic regression was used to model the probability of disease severity (non-severe, severe, or refractory) and treatment response (to corticosteroids or IVIG) based on serologic findings. Longitudinal serologic evolution in patients with multiple APA assays were analyzed. Results : A total of 214 APA assays from 115 ITP patients and 12 ES patients were collected; results from eluate testing (direct assay) only were used in analysis. Baseline patient characteristics are listed in Table 1. Of 7 possible positive test serologic patterns, only 4 were seen (Figure 1); antibodies against both GPIIb/IIIa and GPIb/IX were required for the presence of antibodies against GPIa/IIa. A multinomial logistic regression model including disease severity, age, sex, duration of disease, and platelet count at time of APA assay found a statistically significant predictive relationship between an increasing number of positive antibodies and disease severity [relative to non-severe ITP, relative risk ratio for severe ITP and refractory ITP was 1.89 (P=0.001) and 2.38 (P=0.004), respectively, per one additional positive antibody, Figure 2]. Multiple logistic regression models including antibody positivity to each platelet glycoprotein (GPIIb/IIIa, GPIb/IX, or GPIa/IIa), age, sex, disease duration and splenectomy status found a significant predictive relation between presence of anti-GPIa/IIa and non-response to corticosteroid treatment (odds ratio, 0.082, P=0.008). No significant relation was found between an antibody and non-response to IVIG. Fifty patients had multiple (2 to 5) APA assays performed over months to years. In evaluation of serologic evolution over time, all 7 patients who entered clinical remission also converted from a positive to a negative serology; 22 patients had stable serologic findings over time; 5 patients had a reduction in number of positive antibodies; and 18 patients demonstrated evidence for epitope spreading, with an increase in the number of positive antibodies (Figure 3). The sensitivity of a positive APA assay for the presence of active ITP was 91%. The sensitivity and specificity of a negative APA assay for remission in a patient with previously confirmed ITP (N=40 assays in patients with clinical remission in the study) were 88% and 91%, respectively. Conclusions : The MAIPA-based direct APA assay is sensitive for the presence of active ITP in patients satisfying 2011 ASH diagnostic criteria. To our knowledge, this is the first study demonstrating that a higher number of positive glycoprotein-specific antibodies may predict for more severe disease as defined by 2011 ASH disease severity criteria. Anti-GPIa/IIa antibodies only occur in the setting of pre-existing positivity for both anti-GPIIb/IIIa and anti-GPIb/IX antibodies, possibly due to a distinctive sequence of epitope spreading. Serologic testing typically turns negative when a patient enters clinical remission. Disclosures Al-Samkari: Agios: Consultancy. Kuter:Novartis: Consultancy; Pfizer: Consultancy; Syntimmune: Consultancy; Argenx: Consultancy; Dova Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees; ONO: Consultancy; Amgen Inc.: Consultancy; Bioverativ: Consultancy, Research Funding; Principia: Research Funding; Rigel: Consultancy, Research Funding; BMS: Research Funding; Protalex: Research Funding.

Humanised effector-null FcγRIIA antibody inhibits immune complex-mediated proinflammatory responses

ObjectiveImmune complexes (ICs) play a critical role in the pathology of autoimmune diseases. The aim of this study was to generate and characterise a first-in-class anti-FcγRIIA antibody (Ab) VIB9600 (previously known as MEDI9600) that blocks IgG immune complex-mediated cellular activation for clinical development.MethodsVIB9600 was humanised and optimised from the IV.3 Ab. Binding affinity and specificity were determined by Biacore and ELISA. Confocal microscopy, Flow Cytometry-based assays and binding competition assays were used to assess the mode of action of the antibody. In vitro cell-based assays were used to demonstrate suppression of IC-mediated inflammatory responses. In vivo target suppression and efficacy was demonstrated in FcγRIIA-transgenic mice. Single-dose pharmacokinetic (PK)/pharmacodynamic study multiple dose Good Laboratory Practice (GLP) toxicity studies were conducted in non-human primates.ResultsWe generated a humanised effector-deficient anti-FcγRIIA antibody (VIB9600) that potently blocks autoantibody and IC-mediated proinflammatory responses. VIB9600 suppresses FcγRIIA activation by blocking ligand engagement and by internalising FcγRIIA from the cell surface. VIB9600 inhibits IC-induced type I interferons from plasmacytoid dendritic cells (involved in SLE), antineutrophil cytoplasmic antibody (ANCA)-induced production of reactive oxygen species by neutrophils (involved in ANCA-associated vasculitis) and IC-induced tumour necrosis factor α and interleukin-6 production (involved in rheumatoid arthritis). In FcγRIIA transgenic mice, VIB9600 suppressed antiplatelet antibody-induced thrombocytopaenia, acute anti-GBM Ab-induced nephritis and anticollagen Ab-induced arthritis. VIB9600 also exhibited favourable PK and safety profiles in cynomolgus monkey studies.ConclusionsVIB9600 is a specific humanised antibody antagonist of FcγRIIA with null effector function that warrants further clinical development for the treatment of IC-mediated diseases.

Icaritin Provokes Serum Thrombopoietin and Downregulates Thrombopoietin/MPL of the Bone Marrow in a Mouse Model of Immune Thrombocytopenia

Immune thrombocytopenia (ITP) is a common acquired autoimmune disease, and thrombopoietin (TPO) is an important cytokine that regulates the production of megakaryocytes and platelets. We have identified a biologically active component, icaritin, from a Chinese herba epimedii extract. Icaritin promotes platelet production and regulates T cell polarization, but its mechanism is not clear. In this study, the BALB/c mouse model of ITP was established by injection of an antiplatelet antibody every other day for seven total times. The antiplatelet sera were derived from guinea pigs immunized with the platelets of BALB/c mice. Mice with ITP were treated with icaritin at low, moderate, or high doses of 4.73, 9.45, and 18.90 mg/kg, respectively, for fourteen consecutive days. The present study shows that icaritin can significantly increase peripheral blood platelet counts and thrombocytocrit, increase the TPO level in serum, attenuate splenomegaly, and reduce the abnormal proliferation of megakaryocytes in the spleen and bone marrow. Icaritin can also downregulate the expression of bone marrow TPO, myeloproliferative leukemia virus oncogene (MPL), and p-Stat3. Our results suggest that icaritin can significantly improve the health of mice with ITP via possible downregulation of p-Stat3 expression in the JAK2/Stat3 phosphorylation signaling pathway and regulation of bone marrow TPO/MPL metabolism.

Platelet interaction with lymphatics aggravates intestinal inflammation by suppressing lymphangiogenesis

Lymphatic failure is a histopathological feature of inflammatory bowel disease (IBD). Recent studies show that interaction between platelets and podoplanin on lymphatic endothelial cells (LECs) suppresses lymphangiogenesis. We aimed to investigate the role of platelets in the inflammatory process of colitis, which is likely to be through modulation of lymphangiogenesis. Lymphangiogenesis in colonic mucosal specimens from patients with IBD was investigated by studying mRNA expression of lymphangiogenic factors and histologically by examining lymphatic vessel (LV) densities. Involvement of lymphangiogenesis in intestinal inflammation was studied by administering VEGF-receptor 3 (VEGF-R3) inhibitors to the mouse model of colitis using dextran sulfate sodium and evaluating platelet migration to LVs. The inhibitory effect of platelets on lymphangiogenesis was investigated in vivo by administering antiplatelet antibody to the colitis mouse model and in vitro by coculturing platelets with lymphatic endothelial cells. Although mRNA expressions of lymphangiogenic factors such as VEGF-R3 and podoplanin were significantly increased in the inflamed mucosa of patients with IBD compared with those with quiescent mucosa, there was no difference in LV density between them. In the colitis model, VEGF-R3 inhibition resulted in aggravated colitis, decreased lymphatic density, and increased platelet migration to LVs. Administration of an antiplatelet antibody increased LV densities and significantly ameliorated colitis. Coculture with platelets inhibited proliferation of LECs in vitro. Our data suggest that despite elevated lymphangiogenic factors during colonic inflammation, platelet migration to LVs resulted in suppressed lymphangiogenesis, leading to aggravation of colitis by blocking the clearance of inflammatory cells. Modulating the interaction between platelets and LVs could be a new therapeutic means for treating IBD.

French Observatory of Adult' Chronic Immune Thrombocytopenia (ITP) Treated By Thrombopoietin Receptor Agonists (TPO-RAs)

Immune Thrombocytopenia (ITP) is an autoimmune disorder characterized by isolated low platelet count (<100x109/L) with a variable risk of bleeding. The prevalence of ITP in France is 25/100000. While drug therapy is indicated in patients with platelet count less than 30x109/L and or with bleeding symptoms, discussions remain about therapeutic strategy to be implemented. The SATURNE study aimed to describe the current therapeutic management of adult ITP patients, to focus on TPO-RAs treated patients, and to assess changes in real life treatment strategy since TPO-RAs' approval. This study was carried out in referral and non-referral centersby hematologists and internists from hospitals or clinics in France. Enrolled patients were adults suffering from persistent (3 to 12 months after diagnosis) or chronic (>12 months) ITP. Patients with a newly diagnosed ITP (<3 months) and patients with secondary ITP (viral infection, lupus, etc.) were excluded. Data were collected online by investigators through an eCRF at inclusion (M0). A subgroup of patients initiating a TPO-RAs treatment during the study period was followed up at M3, M6, M12, M18 and M24. Only M0 data are presented in this abstract as interim analysis. Overall, 48 investigators included 333 patients (278 with chronic ITP and 55 with persistent ITP) over a 19 months period (2012 to 2013). Figure 1 displays the main characteristics including comorbidities and laboratory tests performed at diagnosis. Half ofthe patients (53%) had bleeding manifestations at ITP diagnosis; 10% at time of inclusion. ITP was mainly diagnosed by a hematologist (53%) or an internist (32%) and less frequently by a general practitioner (11%). Patients completed a median of 2 treatment lines before entering the study. Figure 2 shows treatment-lines distribution according to the ITP phase. Most patients had been treated with corticosteroids ± intravenous immunoglobulin (IVIG) (83%) as 1st line treatment. Rituximab was the preferred 2nd line option, far prior to splenectomy (44% vs 14%). A total of 144 patients (123 chronic/ 21 persistent ITP) received TPO-RAs (39% romiplostim/ 33% eltrombopag / 15% both / 13% non specified): 6%, 20%, 34%, 28% and 12% respectively as a 1st line treatment, 2nd, 3rd, 4th, 5th and beyond. At inclusion 75% were still on TPO-RAs. Recently diagnosed patients received 2nd line TPO-RAs in higher proportions: 40% (of 46 patients diagnosed <2 years) vs 15% (of 65 patients diagnosed 2-5 yrs ago) and 7% (of 72 patients diagnosed >5 yrs). TPO-RAs became the 3rd line most used treatment (over 76% for diag. <2 yrs). In parallel, the use of splenectomy decreased from 31% (diag. >5 yrs) to 9% (<2 yrs) in 2nd line, and from 16% to 5% in 3rd line. TPO-RAs treated patients had a more severe ITP, in particular at diagnosis. More patients in this group showed platelet counts less than 30.109/L (71% vs 51%, p<0.0001 at diagnosis / 23% vs 10%, p<0.001 at inclusion), and bleeding manifestations (64% vs 44%, p<0.001 at diagnosis/12% vs 8%, p=0.24 at inclusion). They received an average of 3.2 lines of treatment (against 1.7 in TPO-RAs' non-treated patients, p<0.0001). The SATURNE study supports epidemiological trends observed in current practice in terms of patients and ITP characteristics, and provides current data on comorbidities. The results highlight the increasing use of TPO-RAs as 2nd and 3rd lines for ITP treatment and the decrease of splenectomy use over time. Initiated in 2012, respectively 1 and 2 years after eltrombopag and romiplostim approval, the SATURNE study points out the changes of the management of adult ITP in France. Table 1. N=333 Age 57 ± 20yr Women 190 (57%) Main ITP characteristics Chronic/Persistent ITP 278 (84%)/55 (16%) Mean ITP duration* 6 ±8yr Platelet count* - diagnosis - inclusion 33±31.109/L 100±83.109/L Hemorrhagic manifestations- diagnosis- baseline 176 (53%) 32 (10%) White blood cell* 8±3.109/L Hemoglobin* 14±5g/dl Globular volume* 90±6fl Comorbidities since ITP diagnosis At least once 36% Hypertension 17% Diabetes 8% Benign/malignant tumors 8% Cardiovascular disease 6% Diagnostic tests performed at ITP onset Viral serology tests 96% Blood smear 93% Blood coagulation 93% Marrow aspirate 78% Antiplatelet antibody 52% ITP treatment (at least once since diagnosis all lines combined) Corticosteroids and/or IVIG 275 (83%) Rituximab 146 (44%) TPO-RAs- eltrombopag- and/or romiplostim 144 (43%) 69 (48%) 78 (54%) Splenectomy 59 (18%) *mean ± SD Figure 1. Figure 1. Disclosures Michel: Roche: Research Funding; AMGEN: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; GSK: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees. Adoue:GSK: Other: Symposium presentations; AMGEN: Other: Symposium presentations; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees; OCTAPHARMA: Other: Symposium presentations; LFB: Other: Symposium presentations; PFIZER: Other: Symposium presentations; ACTELION: Other: Symposium presentations. Cheze:Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees. Coppo:Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees. Leclerc-Teffahi:Novartis: Employment. Fernandes:Novartis: Other: CRO. Texier:Novartis: Other: CRO.

Splenic TFH expansion participates in B-cell differentiation and antiplatelet-antibody production during immune thrombocytopenia

Key Points Human splenic TFH expansion during ITP participates in B-cell differentiation and antiplatelet-antibody production. IL-21 and CD40 are key TFH molecules that could be promising targets in the treatment of ITP.