Interleukin 9 prevents immune thrombocytopenia in mice via JAK/STAT5 signaling.

Immune thrombocytopenia (ITP) is an autoimmune disorder characterized by autoimmune-mediated platelet destruction and impaired platelet production, which can lead to an increased risk of bleeding. The clinical management of ITP currently remains a challenge for hematologists. We explored the role of interleukin-9 (IL-9) in the treatment of CD41-induced ITP, and investigated its underlying mechanisms in a CD41-induced ITP mouse model. IL-9 treatment increased the numbers of mature megakaryocytes (CD41+CD42d+) and CD41+Sca-1+ cells in the bone marrow in these model mice, while IL-9 receptor (IL-9R) small interfering RNA (siRNA) inhibited the process. Moreover, phosphorylated signal transducer and activator of transcription 5 (STAT5), as a downstream molecule of IL-9R, was increased after IL-9 treatment. We next investigated the source of IL-9 in bone marrow, osteoblasts produced the highest level of IL-9. These results confirmed that IL-9 could prevent CD41-induced ITP in BALB/c mice by regulating osteoblasts and activating IL-9R/STAT5 signaling in megakaryocytes, thus providing further evidence for IL-9 as a promising therapeutic agent for the treatment of ITP.

Thrombopoietin Receptor Agonist Use for Immune Thrombocytopaenia.

Management of patients with corticosteroid-refractory immune thrombocytopaenia (ITP) possesses a significant challenge to practitioners. Until recently, options included splenectomy and immunosuppression. With improved knowledge of both thrombopoiesis and the pathophysiology of ITP, novel drug development with thrombopoietin-receptor agonists (TPO-RAs) was undertaken. Two agents, romiplostim and eltrombopag, are currently approved for use in patients with chronic ITP. Both agents have been shown to increase the platelet count, improve health-related quality of life and reduce bleeding symptoms and concomitant medication use. This review will highlight the discovery of TPO-RA agents, appraise key clinical trials and explore future directions.

Recurrent Immune Thrombocytopenia After Influenza Vaccination: A Case Report.

Immune thrombocytopenia (ITP) is an isolated autoimmune condition, often preceded by a viral infection. Vaccines, mainly the measles-mumps-rubella vaccine, have also been associated with an increased risk of developing the disease. Although some case reports of ITP after influenza immunization in adults have been published, epidemiologic studies examining the role of the influenza vaccine as a trigger of ITP have not conclusively proven causality. We report a child with 3 occurrences of ITP, each within 1 week of receiving the influenza trivalent inactivated vaccine. He recovered fully in-between the episodes, and no further episodes have occurred since discontinuation of seasonal influenza vaccination. To the best of our knowledge, this report is the first showing, with high probability, the influenza vaccine as a cause for ITP in a pediatric patient.

Indirubin Increases CD4+CD25+Foxp3+ Regulatory T Cells to Prevent Immune Thrombocytopenia in Mice.

Indirubin, a traditional Chinese medicine, is used to treat autoimmune diseases in clinics. However, the effects of indirubin on the immunosuppressive CD4+CD25+Foxp3+ regulatory T cells (Treg) have not been addressed. Thus, we aimed to investigate the effects of indirubin on CD4+CD25+Treg cells in immune thrombocytopenia (ITP) CBA mice, which were established by immunization with Wistar rat platelets. 50 mg/kg indirubin treatment daily for 4 weeks significantly decreased anti-platelet antibody production and prevented the decrease of platelets caused by immunization in ITP mice. Consistently, indirubin significantly enhanced the percentage and cell number of CD4+CD25+Foxp3+Treg cells in the peripheral blood, spleen and lymph nodes. We also observed a significant increase of the frequency and cell number of CD4+CD25+Foxp3+Treg cells in the thymus upon indirubin treatment. Furthermore, CD4+CD25+Treg cells from indirubin-treated mice showed similar immunosuppression on T effector cells as compared to those from control mice. Altogether, indirubin ameliorates ITP by enhancing CD4+CD25+Foxp3+Treg cell level with preserving immunosuppressive function.

The presence of KIR2DS5 confers protection against adult immune thrombocytopenia.

Immune thrombocytopenia (ITP) is an autoimmune disorder of unknown aetiology, characterised by an isolated low platelet count in the absence of other identifiable causes. Genes influencing activation of the immune system have been identified as influencing predisposition. Killer cell immunoglobulin-like receptors (KIR) control T-cell and natural killer (NK) cell function via inhibitory and activating signalling pathways. The inhibitory KIR2DL3, KIR3DL2 and KIR3DL1 are up-regulated in the T-cells of patients with ITP in remission relative to those with active disease, and an association of KIR2DS2 and KIR2DL2 with ITP has also been reported. No comprehensive KIR analysis in ITP has been reported. We performed genotyping of all currently known KIR genes using sequence specific primer polymerase chain reaction (SSP-PCR) on a cohort of 83 adult patients with ITP (chronic/persistent or relapsed primary ITP identified by defined criteria) and 106 age matched healthy white volunteers. Non-white patients were not included in the analysis. There was an over-representation of KIR2DS3 (known to be in linkage disequilibrium with KIR2DS2 and 2DL2) and under-representation of KIR2DS5 (also protective against other immune mediated disorders) in adult ITP [odds ratio (OR) = 0.16, confidence interval (CI) 0.08-0.32, P < 0.001]. By multivariable binary logistic regression to adjust for age, sex and the effects of other KIR genes, the presence of KIR2DS2/2DL2 with KIR2DS5 abrogated the risk of KIR2DS2/2DL2 and the protective benefit of KIR2DS5. Further studies are required to establish the mechanistic basis for these observations and their potential impact on ITP therapy.

Tpl2 kinase regulates FcγR signaling and immune thrombocytopenia in mice.

The MAPK3 Tpl2 controls innate and adaptive immunity by regulating TLR, TNF-α, and GPCR signaling in a variety of cell types. Its ablation gives rise to an anti-inflammatory phenotype characterized by resistance to LPS-induced endotoxin shock, DSS-induced colitis, and TNF-α-induced IBD. Here, we address the role of Tpl2 in autoimmunity. Our data show that the ablation and the pharmacological inhibition of Tpl2 protect mice from antiplatelet antibody-induced thrombocytopenia, a model of ITP. Thrombocytopenia in this model and in ITP is caused by phagocytosis of platelets opsonized with antiplatelet antibodies and depends on FcγR activation in splenic and hepatic myeloid cells. Further studies explained how Tpl2 inhibition protects from antibody-induced thrombocytopenia, by showing that Tpl2 is activated by FcγR signals in macrophages and that its activation by these signals is required for ERK activation, cytoplasmic Ca(2+) influx, the induction of cytokine and coreceptor gene expression, and phagocytosis.

Rozrolimupab, a first-in-class recombinant monoclonal antibody product for primary immune thrombocytopaenia.

INTRODUCTION: Over the last decade the field of medicine has dramatically changed with the introduction of biological therapies, among which monoclonal antibodies play a pivotal role in the management of many diseases. Rozrolimupab is the first of a new class of recombinant human monoclonal antibody mixtures, consisting of 25 genetically unique IgG1 antibodies, targeted against the RhD erythrocyte antigen. It is currently being investigated for its use in the treatment of primary immune thrombocytopenia. AREAS COVERED: This article outlines the impetus for the development of rozrolimupab in the treatment of ITP. In addition, the literature regarding the development of rozrolimupab and the recent clinical trials involving this agent are also reviewed. EXPERT OPINION: Although rozrolimupab is only in the very early phases of clinical development we believe that this agent represents a very interesting agent not only for its potential benefits in the management of ITP but potentially for the prevention of haemolytic disease of the foetus and newborn.

Immunoglobulin G dimers and immune complexes are dispensable for the therapeutic efficacy of intravenous immune globulin in murine immune thrombocytopenia.

BACKGROUND: Several mechanisms have been proposed to explain the therapeutic effects of intravenous immunoglobulin (IVIG) in immune thrombocytopenia (ITP). Noteworthy, a major role has been attributed to immunoglobulin (Ig)G dimers present in IVIG. It has also been suggested that immune complexes formed between IVIG and the patient's proteins after infusion could contribute to the therapeutic effect of IVIG in several autoimmune disorders. We recently observed that in-house preparations of polyclonal human IgG derived from small pools of plasma and devoid of IgG dimers were as efficient as IVIG in a mouse model of thrombocytopenia. In this work, we revisited the role of IgG dimers in the therapeutic effects of IVIG in ITP. STUDY DESIGN AND METHODS: We used the passive mouse model of ITP to determine the therapeutic efficacy of human IgG preparations devoid of IgG dimers and of dimer-enriched and -depleted commercial IVIG. Immune complex formation between IVIG and mouse plasma proteins was evaluated using a combination of chromatography and immunoprecipitation procedures. RESULTS: All preparations tested showed the same efficacy to alleviate ITP, regardless of their dimer contents. Significant amounts of immune complexes formed between IVIG and mouse plasma proteins were detected. However, the amount of immune complexes detected using the in-house preparation of human polyclonal IgG and mouse plasma was significantly lower, although the in-house preparation exhibited the same therapeutic efficacy as commercial IVIG. CONCLUSION: IgG dimers and immune complexes are dispensable to prevent thrombocytopenia in a mouse model of the disease.

Fully recombinant IgG2a Fc multimers (stradomers) effectively treat collagen-induced arthritis and prevent idiopathic thrombocytopenic purpura in mice.

INTRODUCTION: Soluble immune aggregates bearing intact Fc fragments are effective treatment for a variety of autoimmune disorders in mice. The better to understand the mechanisms by which Fc-bearing immune complexes suppress autoimmunity, and to develop a platform for clinical translation, we created a series of fully recombinant forms of polyvalent IgG2a Fc, termed stradomers, and tested their efficacy in a therapeutic model of collagen-induced arthritis (CIA) and preventive models of both idiopathic thrombocytopenic purpura (ITP) and graft-versus-host disease (GVHD). METHODS: Stradomers were created by engineering either the human IgG2 hinge sequence (IgG2H) or the isoleucine zipper (ILZ) onto either the carboxy or amino termini of murine IgG2a Fc. Multimerization and binding to the canonical Fc receptors and the C-type lectin SIGN-RI were evaluated by using sodium dodecylsulfate-polymerase chain reaction (SDS-PAGE) and Biacore/Octet assays. The efficacy of stradomers in alleviating CIA and preventing ITP and GVHD was compared with "gold standard" therapies, including prednisolone and intravenous immune globulin (IVIG). RESULTS: Stradomers exist as both homodimeric and highly ordered sequential multimers. Higher-order multimers demonstrate increasingly stable associations with the canonic Fcγ receptors (FcγRs), and SIGN-R1, and are more effective than Fc homodimers in treating CIA. Furthermore, stradomers confer partial protection against platelet loss in a murine model ITP, but do not prevent GVHD. CONCLUSION: These data suggest that fully human stradomers might serve as valuable tools for the treatment of selected autoimmune disorders and as reagents to study the function of Fc:FcR interactions in vivo.

[Clinical efficacy of lower dose rituximab for chronic refractory immune thrombocytopenic purpura].

OBJECTIVE: To investigate the efficacy and safety as well as the effects of lower dose of rituximab on B-lymphocytes, serum immunoglobulin, and platelet glycoprotein-specific antibodies in patients with chronic refractory immune thrombocytopenic purpura (ITP). METHODS: Twenty chronic refractory ITP patients, median age 47 (20 to 60) years old, received intravenous rituximab at the dose of 100mg once weekly for 4 consecutive weeks. Laboratory studies included complete blood cell count, regular monitoring of liver and kidney functions, blood coagulation and serum concentrations of IgG, IgM and IgA. CD3(+), CD4(+), CD8(+), CD19(+), CD20(+) cell numbers were assayed by flow cytometry prior to and following rituximab. Platelet glycoprotein antibodies were detected by ELISA. The detection of indicators were compared by paired T test, with P < 0.05 as statistically significant. RESULTS: There was significant difference of the average platelet count between prior- \[(13 ± 5) × 10(9)/L\] and post-treatment \[(124 ± 106) × 10(9)/L\] with lower dose rituximab (P < 0.01). Reaching PLT peak period was of (24 ± 7) d with median time of 18 d. The responses were of 11(55%) CR, 4 (20%) R and 5 (25%) NR, respectively, with median response duration of 8 months (5 - 23 months). There were no significant changes of peripheral blood white blood cell count, hemoglobin, serum immunoglobulin, as well as CD3(+), CD4(+), CD8(+) lymphocyte counts during prior- and post-treatment. CD19(+)/CD20(+) cells were almost depleted in all patients \[(125.65 ± 14.12) × 10(6)/L vs (50.53 ± 29.11) × 10(6)/L, P < 0.01)\]. Expectedly, three cases of positive detection of platelet antibodies were negative after 4 weeks of lower dose of rituximab; one patient experienced infusion-related reaction. CONCLUSION: Treatment with lower dose rituximab may be an effective and safe approach in patient with chronic refractory ITP. However, the optimal therapeutic schedule, long-term efficacy and adverse events need further investigation.

Critical role of CD4(+)CD25(+) regulatory T cells in preventing murine autoantibody-mediated thrombocytopenia.

Autoimmune response suppression by regulatory T cells (Tregs) helps to maintain peripheral immune tolerance, and defects in this mechanism are thought to play a role in the pathogenesis of various autoimmune diseases. In patients with immune thrombocytopenia, naturally occurring CD4(+)CD25(+) Tregs are both functionally impaired and reduced in number. This study was undertaken to investigate Tregs' role in preventing immune thrombocytopenia in mice. Treg-deficient mice were prepared by inoculation of Treg-depleted CD4(+)CD25(-) T cells isolated from BALB/c mice into syngeneic nude mice intravenously. Platelet count, proportion of reticulated platelets, platelet-associated IgG, platelet-associated anti-platelet antibodies, and IgG anti-platelet antibody production in splenocyte cultures were examined by flow cytometry. Of 69 Treg-deficient mice, 25 (36%) spontaneously developed thrombocytopenia that lasted at least 5 weeks. The platelet-associated IgG level and proportion of reticulated platelets were elevated in the thrombocytopenic mice. Platelet eluates and splenocyte culture supernatants prepared from thrombocytopenic mice, but not from nonthrombocytopenic mice, contained IgG antibodies capable of binding to intact platelets. Simultaneous transfer of Tregs completely prevented the onset of thrombocytopenia, but Treg transfer after the onset of thrombocytopenia had no apparent effect. Treatment with IgG anti-cytotoxic T lymphocyte-associated antigen 4 antibody canceled this Treg-governed suppressive effect. In summary, these results indicate that Tregs play a critical role in preventing murine autoantibody-mediated thrombocytopenia by engaging cytotoxic T lymphocyte-associated antigen 4.

The neonatal Fc receptor (FcRn) is not required for IVIg or anti-CD44 monoclonal antibody-mediated amelioration of murine immune thrombocytopenia.

To definitively determine whether the neonatal Fc receptor (FcRn) is required for the acute amelioration of immune thrombocytopenia (ITP) by IVIg, we used FcRn-deficient mice in a murine ITP model. Mice injected with antiplatelet antibody in the presence or absence of IVIg displayed no difference in platelet-associated IgG between FcRn deficient versus C57BL/6 mice. FcRn-deficient mice treated with high-dose (2 g/kg) IVIg or a low-dose (2 mg/kg) of an IVIg-mimetic CD44 antibody were, however, protected from thrombocytopenia to an equivalent extent as wild-type mice. To verify and substantiate the results found with FcRn-deficient mice, we used ß(2)-microglobulin-deficient mice (which do not express functional FcRn) and found that IVIg or CD44 antibody also protected them from thrombocytopenia. These data suggest that for both high-dose IVIg as well as low-dose CD44 antibody treatment in an acute ITP model, FcRn expression is neither necessary nor required.

Rozrolimupab, symphobodies against rhesus D, for the potential prevention of hemolytic disease of the newborn and the treatment of idiopathic thrombocytopenic purpura.

Currently under codevelopment by Symphogen and Swedish Orphan Biovitrum, rozrolimupab is the first in a new class of recombinant polyclonal antibodies, known as symphobodies, produced using a proprietary technology from Symphogen. Rozrolimupab is being investigated for the prevention of hemolytic disease of the fetus and newborn (HDFN) and for the treatment of idiopathic thrombocytopenic purpura (ITP). Rozrolimupab comprises 25 genetically unique IgG1 antibodies, all of which are specific for the rhesus D (RhD) erythrocyte protein. In preclinical studies, rozrolimupab demonstrated binding to erythrocytes that was comparable with that of two plasma-derived anti-D Ig preparations. In a phase I clinical trial in healthy male volunteers, treatment with rozrolimupab was not associated with serious adverse events. In a phase II clinical trial of rozrolimupab in healthy, male, RhD-negative volunteers, rozrolimupab dose-dependently cleared RhD-positive erythrocytes from the circulation. Phase II clinical trials in ITP and HDFN are currently ongoing. Phase III clinical trials are necessary to establish the efficacy and safety profile of rozrolimupab compared with standard plasma-derived anti-D Ig preparations.

Long-term follow-up of idiopathic thrombotic thrombocytopenic purpura treated with rituximab.

Rituximab may be used to treat patients with thrombotic thrombocytopenic purpura (TTP) refractory to plasma exchange or recurrent disease. While initial response rates are reported to be high, long-term follow-up data of patients treated with rituximab are not available to date, however important to estimate the safety and benefit of this treatment. Twelve patients with non-familial idiopathic TTP refractory to plasma exchange or with recurrent disease treated with rituximab between 2000 and 2008 were reexamined. The median follow-up was 49.6 months, ranging from 11 to 97 months. All patients achieved initial complete remission after application of rituximab. During follow-up, nine patients remained disease-free and three patients suffered from recurrent disease. All patients with recurrent disease responded to subsequent rituximab therapy. No long-term side effects were noted during the follow-up period. In conclusion, rituximab represents an effective second-line treatment option in relapsing or refractory TTP. Still, patients need to be closely monitored for relapses with extended follow-up.

Combination immunosuppressant therapy for patients with chronic refractory immune thrombocytopenic purpura.

Treatment options for patients with chronic refractory immune thrombocytopenic purpura (ITP) are limited. Because combination immunosuppressant therapy appeared to be effective in ITP and other disorders, we used this approach in patients with particularly severe and refractory ITP. In this retrospective, observational study, we determined the response (platelet count above 30 x 10(9)/L and doubling of baseline) among 19 refractory ITP patients. Treatment consisted of azathioprine, mycophenolate mofetil, and cyclosporine. The patients had failed a median of 6 prior treatments, including splenectomy (in all except 1). Of 19 patients, 14 (73.7%) achieved a response lasting a median of 24 months, after which time 8 (57.1%) relapsed. Of the 8 relapsing patients, 6 responded to additional treatments. Of the 14 patients who achieved an initial response, 2 (14.3%) remained in remission after eventually stopping all medications. Severe adverse events did not occur. Combination immunosuppressant therapy can produce a rise in the platelet count that is sometimes sustained in refractory ITP patients.

Direct inhibitors of coagulation proteins - the end of the heparin and low-molecular-weight heparin era for anticoagulant therapy?

Heparins, either unfractionated or low-molecular-weight (UFH and LMWHs), and vitamin K antagonists (VKAs) are currently the anticoagulants of choice for the prevention of post-operative venous thromboembolism (VTE) and for the treatment of acute venous and arterial thromboembolism. While VKAs are widely used in the US, LMWHs are the standard of care in the EU. Although efficacious, these agents are associated with a number of drawbacks, such as the risk of heparin-induced thrombocytopenia, the need for frequent coagulation monitoring in the case of UFH and VKAs, and the parenteral mode of administration in the case of heparins, which can lead to problems associated with patient compliance. There is a need for new anticoagulants that overcome these limitations. Direct, small-molecule inhibitors of coagulation proteins targeting a single enzyme in the coagulation cascade - particularly thrombin or Factor Xa - have been developed in recent years. Two agents, the direct thrombin inhibitor dabigatran and the direct Factor Xa inhibitor rivaroxaban, have recently been approved in the EU and several other countries for the prevention of VTE after total hip or knee replacement surgery. Here we will review data that suggest that the antithrombin-independent mechanism of action of these agents, particularly that of direct Factor Xa inhibitors, leads to increased efficacy with similar safety profiles compared with the antithrombin-dependent heparins. Although the end of the heparins era is not to be expected, the new anticoagulants presented in this review potentially represent the future of anticoagulation.