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: 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.

Description: We recently identified CD4+ T cells that are autoreactive to β2-glycoprotein I (β2GPI) and that promote antiphospholipid antibody production in patients with antiphospholipid syndrome (APS). In this study, T-cell receptor (TCR) β chains of β2GPI-reactive T cells were examined in 8 β2GPI-responders, including 5 patients with APS and 3 healthy subjects, using polymerase chain reaction and single-strand conformation polymorphism (PCR-SSCP) analysis combined with in vitro stimulation of peripheral blood T cells with recombinant β2GPI. The TCR Vβ segments that expanded oligoclonally after stimulation with β2GPI varied among responders, but the Vβ7 and Vβ8 segments were commonly detected in 6 and 4 β2GPI-responders, respectively. Analysis of the complementarity-determining region 3 sequence of β2GPI-reactive T cells revealed limited diversity, and all Vβ7+ TCRs had an amino acid motif of TGxxN/Q or minor variations. The Vβ8+ TCRs had another motif, PxAxxD/E. Surprisingly, an identical Vβ7+ TCRβ chain was used by β2GPI-reactive T cells in 3 patients with APS. There was no apparent difference in the TCRβ usage between APS patients and healthy responders. Some of the Vβ7+ TCRs with the TGxxN/Q motif detected by PCR-SSCP analysis were also used by β2GPI-specific CD4+ T-cell clones responsive to an immunodominant epitope containing the major phospholipid-binding site. Depletion of Vβ7+ or Vβ8+ T cells from the peripheral blood mononuclear cell cultures significantly inhibited in vitro anti-β2GPI antibody production in response to β2GPI. Our results indicate preferential usage of TCRβ chains by β2GPI-reactive T cells. These TCRβ chains can be reasonable targets for TCR-based immunotherapy for patients with APS.

Description: It was recently reported that autoreactive CD4+ T cells to glycoprotein IIb-IIIa (GPIIb-IIIa) mediate antiplatelet autoantibody production in patients with immune thrombocytopenic purpura (ITP). To further examine the antigenic specificity of the GPIIb-IIIa–reactive T cells, 6 recombinant fragments encoding different portions of GPIIbα or GPIIIa were generated and tested for their ability to stimulate antigen-specific T-cell proliferation and anti–GPIIb-IIIa antibody production in vitro. T cells from the peripheral blood of 25 patients with ITP and 10 healthy donors proliferated in response to recombinant GPIIb-IIIa fragments in various combinations. The amino-terminal portions of both GPIIbα and GPIIIa (IIbα18-259 and IIIa22-262) were frequently recognized (60% and 64%, respectively) compared with other fragments (4%-28%) in patients with ITP, but this tendency was not detected in healthy donors. In subsequent analyses in patients with ITP, T-cell reactivities to IIbα18-259 and IIIa22-262 were consistently detected, whereas those to other fragments were sometimes lost. In vitro antigenic stimulation of peripheral blood mononuclear cells with IIbα18-259 or IIIa22-262 promoted the synthesis of anti–GPIIb-IIIa antibodies in patients with ITP, but not in healthy donors. Of 15 CD4+ T-cell lines specific for platelet-derived GPIIb-IIIa generated from 5 patients with ITP, 13 lines recognized IIbα18-259, IIIa22-262, or both. T-cell lines reactive to IIbα18-259 or IIIa22-262 promoted the production of anti–GPIIb-IIIa antibodies that were capable of binding to normal platelet surfaces. These results indicate that the immunodominant epitopes recognized by pathogenic CD4+ T cells in patients with ITP are located within the amino-terminal portions of both GPIIbα and GPIIIa.

Description: Autoreactive CD4+ T cells to β2-glycoprotein I (β2GPI) that promote antiphospholipid antibody production were recently identified in patients with antiphospholipid syndrome (APS). To further examine antigen recognition profiles and T-cell helper activity in β2GPI-reactive T cells, 14 CD4+ T-cell clones specific to β2GPI were generated from 3 patients with APS by repeated stimulation of peripheral blood T cells with recombinant β2GPI. At least 4 distinct T-cell epitopes were identified, but the majority of the β2GPI-specific T-cell clones responded to a peptide encompassing amino acid residues 276 to 290 of β2GPI (KVSFFCKNKEKKCSY; single-letter amino acid codes) that contains the major phospholipid-binding site in the context of the DRB4*0103 allele. Ten of 12 β2GPI-specific T-cell clones were able to stimulate autologous peripheral blood B cells to promote anti-β2GPI antibody production in the presence of recombinant β2GPI. T-cell helper activity was exclusively found in T-cell clones capable of producing interleukin 6 (IL-6). In vitro anti-β2GPI antibody production induced by T-cell clones was inhibited by anti-IL-6 or anti-CD40 ligand monoclonal antibody. In addition, exogenous IL-6 augmented anti-β2GPI antibody production in cultures of the T-cell clone lacking IL-6 expression. These results indicate that β2GPI-specific CD4+ T cells in patients with APS preferentially recognize the antigenic peptide containing the major phospholipid-binding site and have the capacity to stimulate B cells to produce anti-β2GPI antibodies through IL-6 expression and CD40-CD40 ligand engagement. These findings are potentially useful for clarifying the pathogenesis of APS and for developing therapeutic strategies that suppress pathogenic antiphospholipid antibody production in these patients.