ALBERT

Description: In 1998, Gasbarrini et al reported that in ITP cases with Helicobacter pylori (H.pylori) infection, elevation of platelet counts was observed by eradication of this bacterium. Since then, several reports from Italy and Japan confirmed the elevation of platelet counts after eradication. However, the characteristic background in the H.pylori positive ITP and eradication effects on platelet counts is unclear. On the other hand, reports from Spain, North Europe and USA could not show the evidence that eradication is effective on elevating platelet counts in H.pylori positive ITP. Therefore, we designed a nationwide retrospective study in Japan to evaluate the incidence of H.pylori positive ITP cases and the effects of eradication on platelet counts and to clear above problems. Four hundred and thirty-five ITP cases were enrolled over a period of one and half years (2002. 7~2003.12) from 12 hospitals. H. pylori infection was found in 300 cases(65%), who were significantly (P

Description: Helicobacter pylori (H. pylori), a gram-negative bacterium, is suspected to be involved in the pathogenesis of idiopathic thrombocytopenic purpura (ITP). Recent studies from Italy and Japan showed that more than a half of H. pylori-positive patients with ITP achieved a partial or complete platelet recovery after eradication of H. pylori. To examine therapeutic action of H. pylori eradication therapy, we performed a prospective study in which ITP patients were treated with a standard eradication regimen (a combination of lansoprazole, amoxicillin and clarithromycin for one week) irrespective of the presence or absence of H. pylori infection. Thirty-seven adult patients (mean 56.4 years of age) with chronic ITP and a platelet count below 50 x 109/L were enrolled. Platelet counts and circulating anti-GPIIb/IIIa antibody-producing B cell frequencies were monitored at 0, 1, 12, and 24 weeks after initiation of the eradication therapy. H. pylori infection was found in 26 (70%) patients by means of a urea breath test, while the remaining 11 patients were negative for all of a urea breath test, a stool antigen test, and a serum antibody test. Although H. pylori-positive patients tended to be older than H. pylori-negative patients (P = 0.06), other characteristics, such as disease duration, previous treatment regimens, platelet count and anti-GPIIb/IIIa antibody-producing B cells, were not different between these 2 groups. Twenty-five (96%) H. pylori-positive patients were successfully eradicated. At 24 weeks, a significant response (platelet count 〉 100 x 109/L) was observed in 16 (64%) of 25 eradicated patients, but not in a H. pylori-positive patient who failed in the eradication. In addition, a platelet count did not change at all in 11 H. pylori-negative patients, indicating that platelet recovery results from eradication of H. pylori itself, but not from immunomodulatory effects of the drugs used or eradication of microorganisms other than H. pylori. Anti-GPIIb/IIIa antibody-producing B cells were significantly reduced at 12 and 24 weeks in H. pylori-positive responders (P 〈 0.0001) as well as, to a lesser extent, in H. pylori-positive non-responders (P = 0.02), but not in H. pylori-negative patients (P = 1.0). In the majority of responders, a platelet count was already increased at one week when anti-GPIIb/IIIa antibody-producing B cells were not decreased. To further evaluate therapeutic action of H. pylori eradication, changes of anti-GPIIb/IIIa antibody-producing B cell frequency at one week after initiation of varuous therapies were additionally examined in ITP patients who responded to intravenous immunoglobulin (n = 6), corticosteroids (n = 7), or splenectomy (n = 7). The B cell frequency was significantly decreased after treatment with corticosteroids and splenectomy (both for P 〈 0.0001), whereas a stable B cell frequency observed at one week after H. pylori eradication was compatible with intravenous immunoglobulin that primarily inhibits Fc receptor-mediated platelet phagocytosis. In summary, this prospective study confirms effectiveness of H. pylori eradication for chronic ITP and a direct role of H. pylori infection in the pathogenesis of ITP. The platelet recovery after H. pylori eradication is likely to be mediated through complex processes; inhibition of Fc receptor-mediated phagocytosis followed by suppression of anti-platelet autoantibody production.

Description: Prolonged thrombocytopenia is one of late complications in patients underwent allogeneic hematopoietic stem cell transplantation (allo-HSCT), but its pathogenic process is unclear in the majority of cases. In this study, mechanisms for thrombocytopenia in allo-HSCT recipients were examined using a series of parameters useful for discriminating immune thrombocytopenia from bone marrow failure. Forty-one patients who underwent allo-HSCT and survived for 〉100 days without recurrence were enrolled. Of these, 20 (49%) had prolonged thrombocytopenia (

Description: Idiopathic thrombocytopenic purpura (ITP) is one of the major causes of thrombocytopenia. Currently, the diagnosis of ITP is principally based on the exclusion of other possible concurrent causes of thrombocytopenia. In the guidelines proposed by the American Society of Hematology, the panel recommended that no specific laboratory tests are considered necessary for the diagnosis. However, availability of reliable laboratory assays should be helpful in supporting the diagnosis of ITP. Recently, 2 of us (MK and YI) reported that erythrocyte count, leukocyte count, anti-GPIIb/IIIa antibody-producing B cells, platelet-associated anti-GPIIb/IIIa antibodies, reticulated platelets and thrombopoietin measured at first visit were useful to predict a future diagnosis of chronic ITP (manuscript submitted). To confirm this, we conducted a multicenter prospective study involving 113 patients with thrombocytopenia and a normal peripheral blood film at first visit. Patients with clinically apparent associated conditions that can cause thrombocytopenia were excluded. Each patient underwent physical examination and routine laboratory tests, and was prospectively followed for 〉6 months. Anti-GPIIb/IIIa antibody-producing B cells, platelet-associated anti-GPIIb/IIIa antibodies, reticulated platelets and plasma thrombopoietin were also examined at first visit. Clinical diagnosis was made in a blinded fashion based on bone marrow findings and the clinical course. Eighty-nine patients were diagnosed as having chronic ITP, and 24 had a non-ITP disorder, including 11 with aplastic anemia (AA), 10 with myelodysplastic syndrome (MDS), and one each with Fanconi anemia, May-Hegglin anomaly and myelofibrosis. Six laboratory findings were identified as initial parameters that discriminated future diagnosis of chronic ITP from non-ITP. These included the absence of anemia, absence of leukopenia, increased anti-GPIIb/IIIa antibody-producing B cell frequency, increased platelet-associated anti-GPIIb/IIIa antibodies, elevated reticulated platelet percentage and normal or slight increase of thrombopoietin (all for P 〈 0.001). Stepwise multiple regression analysis revealed that anemia, anti-GPIIb/IIIa antibody-producing B cell frequency, reticulated platelet percentage and thrombopoietin were factors that independently contributed to the later diagnosis of chronic ITP. Three or more of 6 ITP-associated laboratory findings were present at presentation in 78 (93%) patients later diagnosed as chronic ITP, compared with 6 (25%) patients whose disorder was non-ITP (P 〈 10−5). All 6 “false-positive” patients were diagnosed as AA or MDS, but 4 of them probably had overlapping immune thrombocytopenia. In summary, this multicenter prospective study confirms usefulness of 6 laboratory tests for the diagnosis of chronic ITP. The identification of ITP-associated laboratory findings encourages the future development of reliable diagnostic criteria for chronic ITP.

Description: Abstract 381 Immune thrombocytopenic purpura (ITP) is a T cell-mediated autoimmune disorder, in which IgG autoantibodies to platelet surface glycoproteins promote platelet clearance in the reticuloendthelial system. Since CD4+CD25+ regulatory T cells (Tregs) are known to play a crucial role in the maintenance of immune homeostasis to self-antigens, it has been believed that Treg dysfunction contributes to the development of a various forms of human autoimmune disorders. Several lines of recent evidence have shown that Tregs are decreased in number and are functionally impaired in patients with ITP. However, it remains unclear how Treg alteration is involved in the pathophysiology of ITP. Recently, we have found that a group of Treg-deficient mice develop autoantibody-mediated thrombocytopenia. For preparation of Treg-deficient mice, Treg-depleted T cells were prepared from BALB/c splenocytes by serial purification steps consisting of a positive selection of CD4+ T cells and a negative selection of CD25+ cells using magnetic bead-based cell sorting, and were transferred into syngeneic T cell-deficient nude mice via tail vein. Treg-depleted T cell fraction transferred contained 〉99% CD4+CD25− cells, and was confirmed to lack expression of Foxp3, a typical Treg marker. Three weeks after transfer, approximately one third of the recipient mice spontaneously developed thrombocytopenia, which sustained for 〉 20 weeks. Thrombocytopenic mice represented elevated platelet-associated IgG and increased proportion of reticulated platelets, but non-thrombocytopenic mice did not. In addition, platelets eluates and culture supernatants of splenocytes prepared from thrombocytopenic mice contained IgG antibodies capable of binding to intact platelets, which were not detected in non-thrombocytopenic mice. The presence of anti-platelet antibodies and increased platelet turnover observed in thrombocytopenic Treg-deficient mice are analogous to ITP patients. Treg-deficient mice prepared by transfer of a less number of Treg-depleted T cells resulted in reduced prevalence of thrombocytopenia, suggesting that onset of thrombocytopenia depends on the number of conventional T cells transferred. Treg-deficient mice are known to frequently develop autoimmune gastritis, another autoimmune disease mediated by IgG anti-parietal cell antibodies, but anti-parietal cell antibodies were almost equally detected in plasma from thrombocytopenic and non-thrombocytopenic mice (70% versus 60%). Transplantation of Tregs together with Treg-depleted T cells completely prevented the onset of thrombocytopenia, but Treg transplantation was not effective as a treatment once thrombocytopenia occurred. To further investigate how Tregs exert the regulatory function, Treg-depleted T cells and Tregs were simultaneously transferred in the presence of antibodies that blocked engagement of cytotoxic T lymphocyte-associated antigen 4 (CTLA4). This treatment cancelled Treg function and resulted in development of thrombocytopenia in recipient nude mice, while mock treatment with control antibodies had no effect. In summary, these results together indicate that CD4+CD25+Foxp3+ Tregs play a critical role in preventing the development of murine autoantibody-mediated thrombocytopenia, in part, through CTLA4 engagement. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 524 Background: Immune thrombocytopenia (ITP) is a T cell-mediated autoimmune disorder, in which IgG autoantibodies to platelet surface glycoproteins promote platelet clearance in the reticuloendthelial system. CD4+CD25+Foxp3+ regulatory T cells (Tregs) are known to play a crucial role in the maintenance of immune homeostasis to self-antigens. Several lines of recent evidence have shown that Tregs are decreased in number and are functionally impaired in patients with ITP. Recently, we have found that approximately one third of Treg-deficient mice spontaneously develop thrombocytopenia with increased platelet-associated IgG and proportion of reticulated platelets. Platelets eluates and culture supernatants of splenocytes prepared from thrombocytopenic mice contain IgG antibodies capable of binding to intact platelets, which are not detected in non-thrombocytopenic mice. The main target of anti-platelet autoantibodies is GPIb, and some mice also produce anti-GPIIIa antibodies. However, detailed mechanisms that elicit ITP during immune reconstitution through homeostatic proliferation in the absence of Tregs remain uncertain. Purpose: To evaluate T-helper (Th) cell balance that promotes anti-platelet autoantibody response in a Treg-deficient mouse model for ITP. Methods: Treg-deficient mice were prepared by inoculation of Treg-depleted CD4+ T cells obtained from BALB/c mice into syngeneic T cell-deficient nude mice. Platelet count was determined using flow cytometry 4 weeks after inoculation, and Treg-deficient mice with platelet count 〈 0.33 × 106/ul were regarded as ITP mice. Treg-deficient mice without thrombocytopenia were also used as a control. To evaluate cytokine profiles of Th cells, proportions of Th subsets in the freshly prepared splenic CD4+ T cells were evaluated by intracellular staining for IFN-γ, IL-4, and IL-17 followed by flow cytometry. Th1, Th2, and Th0 cells were defined as IFN-γ+IL-4−, IFN-γ−IL-4+, IFN-γ+IL-4+ cells, respectively, and Th17 and Th1/17 cells were defined as IFN-γ−IL-17+ and IFN-γ+IL-17+, respectively. In addition, CD4+ T cells were isolated from splenocytes using magnetic activated cell sorting, and were stimulated with phorbol 1,2-myristate 1,3-acetate and ionomycin for 4 days. The culture supernatants were subjected to a cytokine bead array to measure levels of interleukin (IL)-2, IL-4, IL-6, IL-10, IL-17, interferon (IFN)-γ, and tumor necrosis factor (TNF). Finally, to determine IgG subclasses of anti-platelet autoantibodies, splenocyte culture supernatants were incubated with platelets derived from BALB/c mice, followed by incubation with fluorescence-conjugated antibodies to IgG1, IgG2a, IgG2b, or IgG3. Then, the antibodies bound to platelets was detected by flow cytometry. Results: Fourteen ITP mice and 8 control mice were used at 6–8 weeks after inoculation. The proportions of Th1, Th2, and Th0 cells did not differ significantly between ITP and control mice, while the Th1/Th2 ratio was significantly increased in ITP mice than in control mice (8.3 versus 3.2, p 〈 0.01). The proportions of Th17 and Th1/17 cells were comparable between ITP and control mice. There was no difference in the in vitro production levels of cytokines except IL-4, which was lower in ITP mice compared to control mice (140 versus 600 pg/ml, p = 0.02). Increase in the IFN-γ/IL-4 ratio was noted in the culture supernatants from ITP mice, compared to those from control mice (15.6 versus 9.2, p = 0.04). The Th1/Th2 ratio detected by flow cytometric measurement and the IFN-γ/IL-4 ratio in in vitro cultures were correlated with each other (r = 0.85, p 〈 0.01). IgG subclasses of anti-platelet autoantibodies were heterogeneous among individual ITP mice, but IgG2a was the predominant subclass in the majority of ITP mice. Interestingly, a high Th1/Th2 ratio was associated with production of IgG2b anti-platelet antibodies, while the mice with a low Th1/Th2 ratio produced IgG1 anti-platelet antibodies. Conclusions: These findings suggest that induction of IgG anti-platelet autoantibody response in Treg-deficient mice is associated with Th1 bias, which is analogous to the Th balance in patients with primary ITP. The Th1/Th2 balance may modulate the autoimmune responses during expansion of CD4+ T cells in the absence of Tregs. Disclosures: No relevant conflicts of interest to declare.

Description: Circulating CD14+ monocytes are known to be precursors of phagocytes, such as macrophages and dendritic cells. We have recently identified a novel CD14+CD45+CD34+type I collagen+ cell fraction derived from human circulating CD14+ monocytes, monocyte-derived mesenchymal progenitor (MOMP), which contains progenitors capable of differentiating into a variety of mesenchymal cells, including bone, cartilage, fat and skeletal muscle (J Leukoc Biol2003;74:833). Here, we investigated a differentiation potential of human MOMPs along endothelial, cardiomyocytic, and neuronal lineages. MOMPs treated with angiogenic factors for 7 days underwent a change in their morphology from spindle-shaped to caudated. Transmission electron microscopic analysis revealed that these cells displayed rod-shaped microtubulated structures corresponding to Weibel-Palade bodies. Almost every cell expressed CD31, VE-vadherin, VEGFR2, Tie-2, von Willeband factor (vWF), eNOS and CD146, but CD14/CD45 expression was markedly down-regulated. Functional characteristics, including vWF release upon histamine stimulation, acetylated LDL uptake, and up-regulated expression of VEGFR1 in response to hypoxia, were indistinguishable between MOMP-derived endothelial-like cells and human umbilical vein endothelial cells. We further performed xenogenic transplantation studies using a SCID mouse model, in which syngeneic colon carcinoma cells were injected subcutaneously with or without human MOMPs. Tumors generated from carcinoma cells alone showed central necrosis and less blood vessel formation, but co-transplantation with MOMPs resulted in promotion of blood vessel formation and no areas of necrosis. Immunohistochemical analysis using human specific antibodies to CD31 and vWF demonstrated that 〉50% of blood vessels incorporated MOMP-derived endothelial cells. To investigate whether MOMPs were able to differentiate along cardiomyocytic and neuronal lineages, pre-labeled human MOMPs were co-cultivated with primary cultures of rat cardiomyocytes or neurons. Shortly after co-cultivation with rat cardiomyocytes, the majority of MOMPs expressed cardiomyocyte-specific transcription factors, Nkx2.5, GATA-4, eHAND and MEF2, together with CD14/CD45. Subsequently, a subpopulation of MOMPs expressed troponin I and atrial natriuretic peptide and lost CD14/CD45 expression. Spontaneously beating cells formed gap junctions with adjacent rat cardiomyocytes and exhibited electrophysiological properties of ventricular myocytes. MOMPs co-cultured with rat neurons for 3 days expressed neuron-specific transcription factors, Ngn-2, NeuroD, Mash1 and nestin. At day 7, these cells expressed neuron-specific markers, NeuN and Hu. At day 18, a subpopulation of the cells exhibited a neuron-like morphology, including characteristic axons and a refractile round cell body, and expressed MAP2 and β3-tubulin. Co-cultivation of MOMPs with rat cells induced to express GFP by adenoviral gene transfer resulted in appearance of human cardiomyoocytes and neurons without GFP staining, suggesting that our observations are not solely explained by cell fusion. In summary, human MOMPs are capable of differentiating along endothelial and cardiomyocytic lineages as well as a neuronal lineage of an ectoderm-origin. Circulating CD14+ monocytes can be an abundant and easily accessible source for autologous cell transplantation for tissue regeneration.

Description: The potential immunosuppressive effect of an anti-CD154 monoclonal antibody (mAb) on the pathogenic autoreactive T-cell response was evaluated using an in vitro culture system with glycoprotein IIb/IIIa (GPIIb/IIIa)–reactive T cells from patients with immune thrombocytopenic purpura (ITP). The anti-CD154 mAb did not inhibit T-cell proliferation, but suppressed anti-GPIIb/IIIa antibody production, in bulk peripheral blood mononuclear cell cultures stimulated with GPIIb/IIIa. Repeated antigenic stimulation of GPIIb/IIIa-reactive CD4+ T-cell lines in the presence of anti-CD154 mAb resulted in the loss of proliferative capacity and helper function for promoting anti-GPIIb/IIIa antibody production. These anergic T-cell lines showed a cytokine profile of low interferon γ and high interleukin 10 and suppressed anti-GPIIb/IIIa antibody production. Our results indicate that blockade of the CD40/CD154 interaction induces generation of autoantigen-specific anergic CD4+ T cells with regulatory function and could be a therapeutic option for suppressing pathogenic autoimmune responses in patients with ITP.