Passive Immune Thrombocytopenia Research Articles

Progranulin facilitates the increase of platelet count in immune thrombocytopenia

IntroductionProgranulin (PGRN) is emerging as a critical immune mediator involved in a variety of autoimmune disorders. However, its role in immune thrombocytopenia (ITP) remains unclear. Materials and methodsIn this study, the enzyme-linked immunosorbent assay was used for determining the plasma levels of PGRN in ITP patients vs. healthy controls. In addition, the role of PGRN in ITP was investigated in two kinds of ITP murine models. Further, we explored whether PGRN functioned by affecting the number of T regulatory cells (Tregs) using flow cytometry. ResultsWe first observed that plasma levels of PGRN were significantly elevated in ITP patients (n = 52) compared to healthy controls (n = 40), and the levels of PGRN declined in patients after receiving treatment. Additionally, we found a negative correlation between plasma PGRN levels and platelet count of ITP patients, suggesting that PGRN is involved in the pathogenesis of ITP. PGRN deficiency further decreased platelet count in a passive-transfer ITP murine model. By contrast, administration of recombinant PGRN increased platelet count in SCID mice with chronic ITP. Meanwhile, PGRN deficiency impaired proliferation of Tregs in the passive transfer ITP murine model. These data suggest that PGRN may exert a protective role in ITP by promoting Treg proliferation. ConclusionOur study revealed a new regulator involved in the pathogenesis of ITP and provided a potential strategy for management of ITP.

Assessment of Th17/Treg cells and Th cytokines in an improved immune thrombocytopenia mouse model

ABSTRACTObjectives: The improved passive immune thrombocytopenia (ITP) mouse model has been extensively utilized for the study of ITP. However, how closely this model matches the human inflammation state and immune background is unclear. Our study aimed to explore the profile of Th cytokines and Th17/Treg cells in the model.Methods: We induced the ITP mouse model by dose-escalation injection of MWReg30. The serum levels of cytokines (IFN-γ, IL-2, IL-4, IL-10, IL-17A, and TGF-β1) were measured by enzyme-linked immunosorbent assay and the frequency of Th17 and Treg cells was measured by flow cytometry. The mRNA expression of Foxp3 and RORrt was measured by real-time PCR.Results: The serum levels of cytokines IFN-γ, TGF-β1, IL-4, and IL-10 were significantly lower in ITP mice. The secretion of serum proinflammatory cytokines IL-2 and IL-17A and the percentage of Th17 cells showed no statistically significant increase. In ITP mice the frequency of Treg cells and mRNA expression of Foxp3 was significantly lower in splenocytes.Conclusion: Our data suggest that the improved passive ITP mouse model does not mimic the autoimmune inflammatory process of human ITP. Compared with human ITP, this model has a similar change in frequency of Treg cells, which may directly or indirectly result from antibody-mediated platelet destruction due to attenuated release of TGF-β.

Monovalent Fc receptor blockade by an anti–Fcγ receptor/albumin fusion protein ameliorates murine ITP with abrogated toxicity

AbstractPatients with immune thrombocytopenia (ITP) commonly have antiplatelet antibodies that cause thrombocytopenia through Fcγ receptors (FcγRs). Antibodies specific for FcγRs, designed to inhibit antibody-FcγR interaction, had been shown to improve ITP in refractory human patients. However, the development of such FcγR-specific antibodies has stalled because of adverse events, a phenomenon recapitulated in mouse models. One hypothesis behind these adverse events involved the function of the Fc region of the antibody, which engages FcγRs, leading to inflammatory responses. Unfortunately, inhibition of Fc function by deglycosylation failed to prevent this inflammatory response. In this work, we hypothesize that the bivalent antigen-binding fragment regions of immunoglobulin G are sufficient to trigger adverse events and have reasoned that designing a monovalent targeting strategy could circumvent the inflammatory response. To this end, we generated a fusion protein comprising a monovalent human FcγRIIIA-specific antibody linked in tandem to human serum albumin, which retained FcγR-binding activity in vitro. To evaluate clinically relevant in vivo FcγR-blocking function and inflammatory effects, we generated a murine version targeting the murine FcγRIII linked to murine albumin in a passive murine ITP model. Monovalent blocking of FcγR function dramatically inhibited antibody-dependent murine ITP and successfully circumvented the inflammatory response as assessed by changes in body temperature, basophil activation, and basophil depletion. Consistent with our hypothesis, in vivo cross-linking of the fusion protein induced these inflammatory effects, recapitulating the adverse events of the parent antibody. Thus, monovalent blocking of FcγR function demonstrates a proof of concept to successfully treat FcγR-mediated autoimmune diseases.

Unveiling the Regulatory Role of CD8+ T-Cells in the Pathogenesis and Effective Steroid Treatment in ITP

Background:Immune thrombocytopenia (ITP) is a common bleeding disorder. Autoantibodies against platelet GPIIbIIIa (integrin αIIbβ3, 70-80%) and GPIb-complex (20-40%) are considered to be the major mechanism leading to autologous platelet destruction. Recent studies demonstrated that in addition to autoantibodies, CD8+ cytotoxic T cells (CTLs) also contribute to thrombocytopenia, either through direct cytotoxicity against platelets or megakaryocytes. However, the roles of CD8+ regulatory T cells (Tregs) in ITP have not been adequately explored.Methods and Results: We developed the first animal models of steroid treatment in ITP, encompassing both the passive and active forms. In the passive model, we injected anti-β3 antibodies to induce transient antibody mediated thrombocytopenia. We found that a single intraperitoneal (IP) injection of steroids post-antibody injection was effective at rescuing platelet counts. We also adapted an active model of ITP whereby wild-type (WT) BALB/c mice were transfused with splenocytes from WT platelet immunized β3-/-mice. This model encompasses both antibody and cell-mediated ITP resulting in sustained thrombocytopenia. In this model, we found steroid treatment (prednisone and dexamethasone) administered daily either orally or through IP-injection were equally efficacious at ameliorating thrombocytopenia. Furthermore, immunophenotyping and cytokine analysis reveal a similar profile as reported of human ITP patients responsive to steroid treatments. Thus, successful steroid treatments in these animal models are representative of the therapeutic effects of steroid treatments seen in human ITP patients.To study the role of CD8+ T cells in the pathogenesis and response to steroid treatments in ITP, we depleted CD8+ T cells from splenocytes prior to its transfusion into WT mice. Unexpectedly,we found CD8+ T cell depleted splenocyte (lacking in CTL cells) engrafted mice had lower, but not higher, platelet counts and were less responsive to dexamethasone (DEX) treatment compared to non-depleted engrafted mice. Furthermore, in the passive ITP model, depletion of CD8+ T cells from mice prior to injection of anti-β3 antibodies resulted in more severe thrombocytopenia, compared with non-depleted mice. Conversely, transfusion of either antigen-primed CD8+ (isolated from immunized β3-/- splenocytes) or WT/β3-/- naïve CD8+ T cells alone was sufficient to rescue platelet counts and improve response to DEX in the passive ITP model. These results indicate for the first time that CD8+ T cells from both antigen-primed and naïve populations play a protective role in attenuating platelet clearance. In further support of these observations, we detected significant increased populations of both CTLs and CD8+ Tregs including, CD8+CD25+Foxp3+, CD8+CD103+, CD8+CD122+ and CD8+CD28- in the blood, and spleen of immunized β3-/- mice. Interestingly, the CD8+ Tregs populations were further increased while CTL population decreased following DEX treatment in the active ITP model. In vitro splenocyte cultures were used to explore putative regulatory mechanisms of CD8+ Tregs. It was found that antigen-primed CD8+ Tregs exerted significantly stronger inhibition CD4+ T- and CD19+ B cell proliferation, platelet apoptosis, and platelet associated IgG production in the presence of platelet antigens, while both antigen-primed and naïve CD8+ Tregs could effectively inhibit macrophage mediated phagocytosis of anti-β3 opsonized platelets.Conclusion: To the best of our knowledge, these are the first reported animal models of effective steroid treatment of ITP. Utilizing these models we uncovered a previously unidentified regulatory role of CD8+ T cells in both ITP and steroid treatment. The increased populations of various CD8+ Tregs following β3-/- immunization exerted a significant inhibitory function against other immune-cell mediated anti-platelet responses. In addition, therapeutic administration of both antigen-primed and naïve CD8+ T cells were able to rescue platelet counts in the passive ITP model. This suggests that CD8+ Treg may play a predominantly protective role in ITP. These data provides significant insights into the understanding of immunopathogenesis of ITP, which may be important in designing effective therapy including the potential usage of CD8+ Tregs as a cellular target in the treatment of ITP. DisclosuresNo relevant conflicts of interest to declare.

Megakaryocyte Abnormalities Occur In The Absence Of Cell-Mediated Immunity In a Murine Model Of Passive Immune Thrombocytopenia (ITP)

Immune thrombocytopenia (ITP) is an autoimmune bleeding disorder characterized by increased peripheral immune platelet destruction and megakaryocyte defects in the bone marrow. Although ITP was originally thought to be primarily due to humoral mediated autoimmunity it is now evident that T cells can also play a contributing role to the thrombocytopenia. In fact, the exact interplay between platelet destruction, megakaryocyte dysfunction, and the elements of both the humoral and cell mediated immune systems still remain incompletely defined. In murine passive models of ITP, the direct administration of anti-platelet antibodies can result in severe thrombocytopenia which is evident within 24 hours of injection. While most studies have focused on immune platelet destruction in the spleen, an additional possibility is that the anti-platelet antibody also has an effect on megakaryocytes. To unequivocally determine if antiplatelet antibodies have an effect on megakaryocytes in an in vivo model, BALB/c mice were intravenously administered 2 ug of an anti-GPIIbIIIa antibody (MReg30) or 50 uL of a high tittered anti-GPIIIa (anti-β3) serum from BALB/c GPIIIa (CD61) knockout mice immunized with wild type platelets. Platelet counts were assessed over time and the bone marrow and spleens were harvested for histological examination of megakaryocytes. Both preparations of antiplatelet antibodies significantly reduced platelet numbers within 1 day of antibody or serum administration. This thrombocytopenia could be rescued by administration of 2 g/kg of IVIg ip. Compared with naïve control mice, histological (H&E staining) examination of the bone marrow and spleens revealed that megakaryocytes were significantly increased in number and all exhibited abnormalities consistent with apoptosis e.g. pyknotic nuclei. IVIg administration completely prevented these megakaryocyte abnormalities. These results show that passively administered anti-platelet antibodies not only affect platelet counts but also significantly affect megakaryocyte physiology in the absence of cell mediated immunity. Disclosures:No relevant conflicts of interest to declare.

Amelioration of Murine Passive Immune Thrombocytopenia by IVIg and a Therapeutic Monoclonal CD44 Antibody Does Not Require the Myd88 Signaling Pathway

Immune thrombocytopenia (ITP) is an autoimmune bleeding disorder characterized by a low platelet count and the production of anti-platelet antibodies. The majority of ITP patients have antibodies to platelet integrin αIIbβ3 (GPIIbIIIa) which can direct platelet phagocytosis by macrophages. One effective treatment for patients with ITP is intravenous immunoglobulin (IVIg) which rapidly reverses thrombocytopenia. The exact mechanism of IVIg action in human patients is unclear, although in mouse models of passive ITP, IVIg can rapidly increase platelet counts in the absence of adaptive immunity. Another antibody therapeutic that can similarly increase platelet counts independent of adaptive immunity are CD44 antibodies. Toll-like receptors (TLRs) are pattern recognition receptors which play a central role in helping direct the innate immune system. Dendritic cells, which are notable for their expression of TLRs, have been directly implicated in IVIg function as an initiator cell, while CD44 can associate with TLR2 and TLR4. We therefore questioned whether IVIg, or the therapeutic CD44 antibody KM114, mediate their ameliorative effects in a manner dependent upon normal TLR function. Here, we demonstrate that the TLR4 agonist LPS does not inhibit IVIg or KM114 amelioration of antibody-induced thrombocytopenia, and that these therapeutics do not ameliorate LPS-induced thrombocytopenia. IVIg was able to significantly ameliorate murine ITP in C3H/HeJ mice which have defective TLR4. All known murine TLRs except TLR3 utilize the Myd88 adapter protein to drive TLR signaling. Employing Myd88 deficient mice, we found that both IVIg and KM114 ameliorate murine ITP in Myd88 deficient mice to the same extent as normal mice. Thus both IVIg and anti-CD44 antibody can mediate their ameliorative effects in murine passive ITP independent of the Myd88 signaling pathway. These data help shed light on the mechanism of action of IVIg and KM114 in the amelioration of murine ITP.

In Vivo Platelet Production Occurs from an Expanding Pool of Non-Apoptotic Megakaryocytes in a Mouse Model of Passive Immune Thrombocytopenia

AbstractAbstract 1214Immune thrombocytopenia (ITP) is the most common acquired thrombocytopenia in children. Typically, external triggers as infections or vaccinations cause the rise of antibodies that crossreact with antigens expressed on the platelet surface. These anti-platelet antibodies are mostly directed against glycoprotein complexes GPIIb/IIIa or GPIb/IX/V, resulting in an increased turnover of antibody-decorated platelets which are then sequestered by the reticuloendothelial system. Recently, it has been suggested that thrombocytopenia might also be due to an insufficient platelet production as serum of some patients with ITP can impair the maturation of CD34+ hematopoietic stem cells to bone marrow megakaryocytes (MKs) in vitro or abrogate the formation of proplatelets in an in vitro culture system. The accelerated platelet turnover demands the generation of platelets de novo. Bone marrow smears often reveal normal or slightly increased MKs, although they seem to be smaller and of altered morphology. However, very little is known about the consequences of anti-platelet antibodies on bone marrow MKs in vivo and in situ. Here, we took advantage of a simple animal model of passive ITP by single or multiple intraperitoneal injections of an anti-GPIb antibody into mice. MKs were evaluated by multi-color immunofluorescence histology on whole femur sections in a modified staining procedure that bypasses decalcification. MK numbers on day 3 were doubled in response to a single injection and tripled on day 8 when mice were injected additionally on day 3 and 7. In these mice platelet counts were up to 2000/nL on day 10, indicating the power to produce platelets. MK area per section was transiently upregulated on day 3 in single injected mice and quadrupled after multiple injections on day 8 before shrinking below norm on day 14. Staining with an anti-rat IgG antibody showed that the antibody was present on MKs within the bone marrow several hours to days after injection. The signal was present for 5 days and no antibody was detected on day 7. MKs had an overall normal morphology and showed no signs of apoptosis or DNA blebbing. All MKs analyzed were negative for TdT in a classical TUNEL assay, indicating that there were no single strand breaks. As platelet counts rose markedly while the antibody was still present on the MK surface, we sought to identify whether the pool of MKs is expanded or formed de novo. To address this, mice where fed with nucleotide analogue EdU for up to 12 days and femur sections stained with Click-It-647 reagent to stain for newly incorporated DNA while mice were treated with anti-platelet antibody or isotype control. We found EdU-positive MKs after 12 days in control isotype-injected mice indicating the de novo formation from hematopoietic stem cells. In antibody-injected mice, newly formed MKs were negative or stained weakly for EdU on day 12, suggesting that they arise partially from an existing pool of progenitors. Finally, we analyzed platelet formation in vivo by imaging of the cranial bone marrow of GPIIb-eYFP-heterozygous mice. The depletion antibody was labeled with Atto-590-fluorophore and injected hours before imaging. Vasculature was counterstained by Quantum dots. We found that MKs residing at the bone marrow were decorated with the antibody and released pre- and proplatelets into the vasculature, indicating that platelet biogenesis can occur in the presence of anti-platelet antibodies on MKs. Our data thus provide novel insight into the pathomechanism of platelet production in patients with ITP. Disclosures:No relevant conflicts of interest to declare.

Transplantation of adipose‐derived mesenchymal stem cells into a murine model of passive chronic immune thrombocytopenia

Immune thrombocytopenia (ITP) is a bleeding disorder characterized by antibody-opsonized platelets (PLTs) being prematurely destroyed by macrophages in the reticuloendothelial system. T helper (Th) cells and different Th cytokines play an important role in the pathophysiology of ITP. As immunomodulators, adipose-derived mesenchymal stem cells (ADSCs) regulate Th cells and show therapeutic effects in autoimmune diseases. However, it is not clear how ADSCs affect ITP. In this study, we explored the specific effects of ADSCs on ITP in mice. BALB/c mice were randomly divided into three groups: normal controls, ITP controls, and ITP with ADSC transplantation. PLT levels were monitored by an automatic blood cell counter, and the cytokines interferon-γ (IFN-γ); interleukin (IL)-2, -4, -10, and -17; and transforming growth factor-β1 (TGF-β1) were analyzed by enzyme-linked immunosorbent assays. Compared to the untreated ITP mice, the PLT level of the ITP mice significantly increased after ADSC treatment. In the ADSC group, IFN-γ, IL-2, and IL-17 significantly decreased, while IL-4, IL-10, and TGF-β1 increased. These findings constitute the first experimental evidence that ADSCs are efficacious in improving PLT levels and reducing the related Th cytokines mediating proinflammatory response in ITP mice, which may provide a scientific basis for using ADSCs as a new therapy for ITP.

Enrichment of Sialylated IgG by Lectin Fractionation Does Not Enhance the Efficacy of Immunoglobulin G in a Murine Model of Immune Thrombocytopenia

Intravenous immunoglobulin G (IVIg) is widely used against a range of clinical symptoms. For its use in immune modulating therapies such as treatment of immune thrombocytopenic purpura high doses of IVIg are required. It has been suggested that only a fraction of IVIg causes this anti immune modulating effect. Recent studies indicated that this fraction is the Fc-sialylated IgG fraction. The aim of our study was to determine the efficacy of IVIg enriched for sialylated IgG (IVIg-SA (+)) in a murine model of passive immune thrombocytopenia (PIT). We enriched IVIg for sialylated IgG by Sambucus nigra agglutinin (SNA) lectin fractionation and determined the degree of sialylation. Analysis of IVIg-SA (+) using a lectin-based ELISA revealed that we enriched predominantly for Fab-sialylated IgG, whereas we did not find an increase in Fc-sialylated IgG. Mass spectrometric analysis confirmed that Fc sialylation did not change after SNA lectin fractionation. The efficacy of sialylated IgG was measured by administering IVIg or IVIg-SA (+) 24 hours prior to an injection of a rat anti-mouse platelet mAb. We found an 85% decrease in platelet count after injection of an anti-platelet mAb, which was reduced to a 70% decrease by injecting IVIg (p<0.01). In contrast, IVIg-SA (+) had no effect on the platelet count. Serum levels of IVIg and IVIg-SA (+) were similar, ruling out enhanced IgG clearance as a possible explanation. Our results indicate that SNA lectin fractionation is not a suitable method to enrich IVIg for Fc-sialylated IgG. The use of IVIg enriched for Fab-sialylated IgG abolishes the efficacy of IVIg in the murine PIT model.

Steroids Are Less Effective In Treating Thrombocytopenia Caused by Immune Responses Against Platelet GPIbα: A Comparative Study Using Passive and Active ITP Models.

AbstractAbstract 1433 Introduction:Immune thrombocytopenia (ITP) is a common autoimmune disorder in which autoantibodies are generated against a patient's platelets, leading to decreased platelet counts and bleeding diatheses. Autoantibodies in ITP mainly target platelet receptors GPIIbIIIa (αIIbβ3 integrin) and GPIbα. Current first-line therapy for ITP patients is steroids, while intravenous immunoglobulin (IVIG) is a common second-line therapy. However, not all patients are responsive and we lack useful exclusion criteria for steroid therapy. Appropriate identification of non-responsive patients would facilitate proper treatment and limit side effects. We previously showed anti-GPIbα (versus anti-GPIIbIIIa) mediated ITP is less responsive to IVIG (Blood. 2006;108(3):943-6). Our preliminary data from human patients also suggests that patients with anti-GPIbα antibodies are less responsive to steroids. However, these data need to be confirmed and mechanisms remain to be elucidated. Methods:To examine whether antibody specificity (GPIIbIIIa or GPIbα) correlates with responsiveness to steroids and determine the target immune cells for steroid action, we used two murine ITP models (passive antibody transfer model and active splenocyte transfer model (Blood. 2010;115:1247-53)). In both models, mice are treated with the corticosteroid Dexamethasone (DEX) and platelet counts are monitored. In the passive antibody transfer model: BALB/c or C57 mice were pretreated for 3 days with 0.1mg/kg, 1mg/kg, or 10 mg/kg of DEX prior to injection with our newly developed mouse-anti-mouse monoclonal antibodies (mAbs) targeting GPIIbIIIa or GPIbα. A second group of mice were treated with DEX 1 day following mAb injection (i.e. after becoming thrombocytopenic) and daily thereafter. In both groups, platelet counts were monitored for 4 consecutive days post mAb injection. In the active splenocyte transfer model: β3-/- and GPIbα-/- mice were immunized with wild-type (WT) platelets resulting in an antibody titer of >1:12,800. Purified splenocytes from immunized mice were transferred into syngeneic WT or SCID recipients, such that they developed ITP. Recipient mice were treated with 1mg/kg or 10 mg/kg of DEX starting 1 day after splenocyte transfer, and daily thereafter. Platelet counts were monitored. Results and Discussion:In the passive ITP model, irrespective of DEX dose, route of administration (i.p. or s.c.) or mouse strain, pretreatment with DEX was unable to ameliorate ITP induced by either anti-GPIIbIIIa or anti-GPIbα mAbs. In addition to pretreating with DEX, administration of DEX following ITP induction also appeared to be unable to ameliorate ITP induced by either anti-GPIIbIIIa or anti-GPIbα mAbs. Similar results were obtained in the passive model when using the corticosteroid Prednisolone in a different experimental setting. In the active splenocyte transfer model, we found that varying doses of DEX ameliorated ITP in mice engrafted with anti-GPIIbIIIa reactive splenocytes while untreated mice remained thrombocytopenic. Importantly, DEX failed to show a significant effect on ITP amelioration in mice engrafted with anti-GPIbα reactive splenocytes. Since the passive model involves exogenous administration of mAb, the effect of steroid treatment is largely limited to effects on the reticuloendothelial system (RES) and destruction of opsonized platelets. Conversely, the splenocyte transfer model allows for steroid effects on active immune responses (i.e. it may affect dendritic cells, T cells, B cells, etc.) in addition to the RES. As DEX was efficacious in only the splenocyte transfer model, this suggests steroids primarily affect upstream of the immune responses in ITP. Ongoing experiments involving depletion of CD4+, CD8+, or CD19+ splenocytes will help elucidate specific immune cells as targets of DEX. Conclusions:Our data clearly demonstrated that DEX ameliorated anti-GPIIbIIIa mediated ITP but was not significantly effective for amelioration of anti-GPIbα mediated ITP in the active splenocyte transfer model. We also demonstrated that steroid therapy failed in our passive ITP model. This finding is consistent with our preliminary data in human patients (patients with anti-GPIbα antibodies may be refractory to both IVIG and steroids). This may explain the clinical variability seen in response to steroids and lead to new diagnostic/therapeutic approaches. Disclosures:No relevant conflicts of interest to declare.

The Role of Recipient Platelets In the Prevention of Antibody-Mediated Transfusion Related Acute Lung Injury (TRALI)

AbstractAbstract 3351Transfusion related acute lung injury (TRALI) is a serious complication of transfusion. The pathogenesis of TRALI is not fully understood but previous findings have suggested that platelet depletion can protect mice in a two-hit model of TRALI (Looney et al J Clin Invest 119:3450, 2009). To further understand the role of platelets in preventing antibody-mediated TRALI, two mouse models of immune thrombocytopenia (ITP) were utilized. In the passive ITP model, SCID mice were injected with a monoclonal anti-platelet antibody (MWReg30) intraperitoneally (ip, 18 h before TRALI induction) or intravenously (iv, 2 h before TRALI induction). In the active ITP model, SCID mice were transferred with splenocytes from anti-CD61 immune GPIIIa-knockout mice and thrombocytopenia occurred within 2 weeks post transfer (Chow et al Blood 115;1247, 2010). TRALI induction was performed by injecting the various thrombocytopenic SCID mice with a murine monoclonal MHC class I antibody (mAb, 34-1 -2s) iv and several parameters were observed for up to 2 h post antibody injection. In control, non-thrombocytopenic SCID mice, 34-1 -2s injection caused severe systemic shock as noted by reduced rectal temperatures which was associated with significant lung damage and mortality (45%) within 1 hour of 34-1 -2s infusion as previously shown (Fung et al. Blood DOI 10.1182/blood-2010-05-284570). In contrast, while SCID mice depleted of platelets by the passive ip route had systemic shock, lung damage and a 60% mortality rate, those mice made thrombocytopenic by the iv route were completely protected from mortality. On the other hand, in the active ITP model, where the induced thrombocytopenia is associated with a proinflammatory anti-platelet immune response, no mortality was observed in those mice made thrombocytopenic by antibody-mediated immune mechanisms whereas 80% of mice rendered thrombocytopenic by CD8+ T cell-mediated immunity were dead within 1 hr post 34-1 -2s infusion. These results suggest that thrombocytopenia in itself does not protect against antibody-mediated TRALI severity but the nature of the thrombocytopenia induction (e.g. acute passive iv infusion or active ITP immune transfer) is important. In fact, depending on the inflammatory milieu associated with the thrombocytopenia, platelets may actually increase the severity of TRALI. Disclosures:No relevant conflicts of interest to declare.

Thrombopoietic effect of VPAC1 inhibition during megakaryopoiesis

Megakaryocytes and platelets express the stimulatory G protein (Gs)-coupled VPAC1 receptor, for which the pituitary adenylyl cyclase-activating peptide (PACAP) and vasoactive intestinal peptide (VIP) are agonists. The neuropeptide PACAP and VPAC1 were previously found to negatively regulate megakaryopoiesis, and inhibition of their physiological pathway was found to have a thrombopoietic effect in conditions where megakaryopoiesis and thrombopoiesis were impaired, such as chemotherapy-induced thrombocytopenia and congenital thrombocytopenia. The present study explored the thrombopoietic effect of VPAC1 inhibition in a murine model of syngeneic bone marrow transplantation (BMT) and in passive immune thrombocytopenia. Treatment of donor mice with a neutralizing anti-VPAC1 antibody stimulated the initial, most critical recovery of the platelets in irradiated mice. In the passive immune thrombocytopenia model, we observed a thrombopoietic effect, resulting in a less severe platelet drop after induction of their removal in the spleen by an anti-platelet antibody. We concluded that inhibition of the physiological PACAP/VPAC1 pathway could stimulate in vivo megakaryopoiesis. This inhibition can be applied to attenuate thrombocytopenia in conditions where platelets are destroyed as the major pathogenetic mechanism, e.g. immune thrombocytopenia purpura, or need to be produced de novo, e.g. after irradiation and BMT.

Comparison of the effects of antibody-coated liposomes, IVIG, and anti-RBC immunotherapy in a murine model of passive chronic immune thrombocytopenia

AbstractThe present work evaluated antibody-coated liposomes as a new treatment strategy for immune thrombocytopenic purpura (ITP) through the use of a mouse model of the disease. Effects of antimethotrexate antibody (AMI)–coated liposomes and intravenous immunoglobulin (IVIG)–coated liposomes (15, 30, 60 µmol lipid/kg) were compared with the effects of IVIG (0.4, 1, 2 g/kg) and anti–red blood cell (anti-RBC) monoclonal antibody immunotherapy (TER119, 5, 15, 25, and 50 µg/mouse) on MWReg30-induced thrombocytopenia. Each treatment was found to attenuate thrombocytopenia in a dose-dependent manner and, consistent with previous work, IVIG was found to increase antiplatelet antibody clearance in a dose-dependent manner. TER119 demonstrated greater effects on thrombocytopenia relative to other therapies (peak platelet counts: 224% ± 34% of initial platelet counts for 50 µg TER119/mouse versus 160% ± 34% for 2 g/kg IVIG, 88% ± 36% for 60 µmol lipid/kg AMI-coated liposomes, and 80% ± 25% for 60 µmol lipid/kg IVIG-coated liposomes). However, the effects of TER119 were associated with severe hemolysis, as TER119 decreased RBC counts by approximately 50%. The present work demonstrated that antibody-coated liposomes attenuated thrombocytopenia in this model at a much lower immunoglobulin dose than that required for IVIG effects and, in contrast with TER119, antibody-coated liposomes increased platelet counts without altering RBC counts.

Platelet homeostasis is regulated by platelet expression of CD47 under normal conditions and in passive immune thrombocytopenia

AbstractInteraction between target cell CD47 and the inhibitory macrophage receptor signal regulatory protein α (SIRPα) counteracts macrophage phagocytosis of CD47-expressing host cells. As platelets also express CD47, we asked whether inhibitory CD47/SIRPα signaling regulates normal platelet turnover and clearance of platelets in immune thrombocytopenic purpura (ITP). CD47-/- mice had a mild spontaneous thrombocytopenia, which was not due to a decreased platelet half-life as a result of increased expression of P-selectin, CD61, or phosphatidylserine. In contrast, CD47-/- platelets were rapidly cleared when transfused into CD47+/+ recipients, whereas CD47+/- platelets had a nearly normal half-life in CD47+/+ mice under nonautoimmune conditions. CD47-/- mice were more sensitive to ITP, as compared with CD47+/+ mice. In vitro, macrophage phagocytosis of immunoglobulin G (IgG)–opsonized CD47-/- platelets was significantly higher than that for equally opsonized CD47+/+ platelets. However, when SIRPα was blocked, phagocytosis of CD47+/+ platelets increased to the level of CD47-/- platelets. Phagocytosis of opsonized CD47+/- platelets was higher than that for CD47+/+ platelets, but lower than that for CD47-/- platelets, suggesting a gene-dose effect of CD47 in this system. In conclusion, we suggest that inhibitory CD47/SIRPα signaling is involved in regulating platelet phagocytosis in ITP, and that targeting SIRPα may be a new means of reducing platelet clearance in ITP.

Passive immune thrombocytopenia in neonates of mothers with idiopathic thrombocytopenic purpura: incidence and risk factors.

The aim of this study was to evaluate risk factors for occurrence of neonatal passive immune thrombocytopenia (PIT) in pregnancy complicated by idiopathic thrombocytopenic purpura (ITP). We studied 63 pregnant women with ITP and the 66 neonates retrospectively. Neonatal platelet counts were compared with maternal platelet counts, platelet-associated gamma G immunoglobulin (PAIgG) values, and the presence of antiplatelet antibody in the maternal circulation, history of previous PIT, maternal treatments for ITP, and other maternal or neonatal factors. PIT (platelet counts <100 x 10(3)/microL) was observed in 9 (14.3%) of 63 pregnancies. Presence of circulating antiplatelet antibody in maternal blood, splenectomy prior to pregnancy, and history of previous PIT were observed more frequently with statistical significance in patients giving birth to neonates who developed PIT. No effect on occurrence of PIT was found by the administration of corticosteroids or immunoglobulin. Splenectomy prior to pregnancy was found by logistic regression analysis to be a single significant variable (p = 0.021, odds ratio 7.20, confidence intervals: 1.35 to 38.3) among the risk factors for PIT.

Risk factors for neonatal thrombocytopenia in pregnancy complicated by idiopathic thrombocytopenic purpura.

The aim of this study was to evaluate risk factors for the occurrence of fetal/neonatal passive immune thrombocytopenia (PIT) in pregnancy complicated with ITP. We studied 52 pregnancies with ITP and the 54 neonates retrospectively. Neonatal platelet counts were compared with maternal platelet counts, platelet-associated IgG (PAIgG) values and the presence of antiplatelet antibody in maternal circulation, history of previous PIT, maternal treatments for ITP, and other maternal/neonatal factors including gestational age and birth weight. Logistic regression analysis for multivariables was performed. PIT (platelet counts < 100 x 10(3)/microl) without neonatal mortality or any morbidity was observed in eight (15.4%) of 52 pregnancies. The presence of circulating antiplatelet antibodies in maternal blood, splenectomy prior to pregnancy, and a history of previous PIT were observed more frequently with statistical significance in women giving birth to neonates who developed PIT. By logistic regression analysis, splenectomy prior to pregnancy was found to be the single significant variable (p=0.017, odds ratio 9.33) among the risk factors for PIT. Thus, splenectomy prior to pregnancy is related to increased risk for PIT in ITP-complicated pregnancy.

Perinatal management of idiopathic thrombocytopenic purpura in pregnancy: risk factors for passive immune thrombocytopenia.

Thirty-nine pregnant women with idiopathic thrombocytopenic purpura (ITP) were studied in order to evaluate the influence of therapies for maternal ITP on fetal passive immune thrombocytopenia (PIT). Neonatal platelet counts were also compared with platelet counts, amount of PAIgG, and presence of circulating antiplatelet antibody in maternal blood. Eight of 41 neonates (19.5%) presented PIT without neonatal mortality. A higher incidence of PIT was observed in women with prior splenectomy than in women without splenectomy (66.7% vs 11.4%). Neither a therapeutic effect nor an increased risk of PIT was observed with steroids or gammaglobulin administration. No correlation was found between neonatal platelet counts and maternal platelet counts or maternal PAIgG, while positive cases for circulating antiplatelet antibody assay presented a higher incidence of PIT than negative cases. Additionally, a higher incidence of PIT was observed in women with a history of previous PIT than in women with a history of normal delivery. Prior splenectomy, presence of antiplatelet antibody in maternal blood, and a history of previous PIT seem to be risk factors for fetal PIT.

Failure of Intravenous Gammaglobulin for Passive Immune Thrombocytopenia in a Neonate

Sir .—Intravenous gammaglobulin (IVIG) therapy has been used with success in cases of neonatal thrombocytopenia caused by transplacentally transferred maternal autoantibodies 1,2 and in neonatal isoimmune thrombocytopenia. 3 Recently we reported a persistent increase in platelet count after 0.4 g/kg per day of intact polyethylene glycol–treated IVIG during the first 5 days of life in a neonate with immune thrombocytopenia, passively acquired from his mother. 4 In another case, IVIG treatment was without benefit in a neonate with passive immune thrombocytopenia. In this case, the infant's mother had mild thrombocytopenia during the pregnancy (platelet count, 70 × 10 9 /L). Her first child was noted to have thrombocytopenia at 2 months that spontaneously resolved after 2 months. A male infant was born at term (gestational age, 39 weeks) by vaginal delivery to a woman with a normal pregnancy. His birth weight was 3740 g; Apgar scores were 9 at

The use of intravenous γ-globulin in idiopathic thrombocytopenic purpura

In 1981, Imbach et al. ( Lancet, 1, 1228–1231) reported that infusion of intravenous immunoglobulin (IVIG) would substantially elevate platelet counts in children with acute or chronic idiopathic thrombocytopenic purpura (ITP). Subsequent studies confirmed these findings and extended the effect to adults and to newborns with passive immune thrombocytopenia. Studies in children with acute ITP demonstrated that administration of IVIG was the fastest way to increase a patient's platelet count, and that this agent could be given at doses as high as 1 g/kg/day so that the course of therapy and response to treatment would be more rapid. Reports of effective treatment of patients with autoimmune neutropenia and autoimmune hemolytic anemia by IVIG broadened the scope of its usefulness. In addition, several studies in children and adults with chronic ITP suggested that repeated infusions were a safe and effective way to maintain an adequate platelet count in such patients, and might also gradually lead to lasting improvement. These studies and Imbach's controlled trial in children with acute ITP suggested that IVIG therapy might provide a curative effect in addition to the acute effect. To combine all of these clinical effects with a multitude of in vitro observations to explain the mechanism of action of IVIG is complicated. Fehr et al. ( N. Engl. J. Med. 306, 1254–1258, 1982) showed that Fc receptor blockade occurs following administration of IVIG. This effect is clearly demonstrated in vivo by the delayed removal from the vascular space of antibody-coated red blood cells following infusion of IVIG. In addition, many less well-defined effects occur in relation to immunoglobulin production, induction of suppressor cells, antiplatelet antibody levels, and bone marrow platelet production. Studies continue to try to define which effects actually underlie the clinical effects seen, and which are merely test-tube phenomena.

Intravenous Gamma Globulin for Prolonged Passive Immune Thrombocytopenia

AbstractThe effectiveness of intravenous gamma globulin was evaluated in prolonged thrombocyto penia in two infants of mothers with autoimmune thrombocytopenic purpura. Case 1, de spite unresponsiveness to steroid therapy, showed a slight response to intravenous gamma globulin infused at 15–19 days of age. A second administration of intravenous gamma globu lin at 70–74 days of age completely reversed the thrombocytopenia. In case 2, passive immune thrombocytopenia persisting over one month was reversed by the administration of intravenous gamma globulin at 37–39 days of age. A comparison of the effects of intra venous gamma globulin on the increase in platelet counts in the newborn infant (case 1) and in his mother showed that passive immune thrombocytopenia in neonates can be more resistant to therapy than autoimmune thrombocytopenic purpura in the mother. Adverse effects were not observed in either case. The effectiveness of and indications for intravenous gamma globulin in passive immune neonatal thrombocytopenia are discussed.