Search

To the editor: Several studies have indicated that platelet recovery occurs in a subgroup of immune thrombocytopenia (ITP) patients after successful Helicobacter pylori ( H pylori) eradication.[1][1],[2][2] Interestingly, a higher response rate to H pylori eradication therapy has been reported in

Antiphospholipid syndrome (APS) is an autoimmune prothrombotic disorder in association with autoantibodies to phospholipid (PL)-binding plasma proteins, such as beta(2)-glycoprotein I (beta(2)GPI). We have recently found that CD4(+) T cells autoreactive to beta(2)GPI in patients with APS preferentially recognize a cryptic peptide encompassing amino acid residues 276-290 (p276-290), which contains the major PL-binding site, in the context of DR53. However, it is not clear how previously cryptic p276-290 becomes visible to the immune system and elicits a pathogenics autoimmune response to beta(2)GPI. Here we show that presentation of a disease-relevant cryptic T-cell determinant in beta(2)GPI is induced as a direct consequence of antigen processing from beta(2)GPI bound to anionic PL. Dendritic cells or macrophages pulsed with PL-bound beta(2)GPI induced a response of p276-290-specific CD4(+) T-cell lines generated from the patients in an HLA-DR-restricted and antigen-processing-dependent manner but those with beta(2)GPI or PL alone did not. In addition, the p276-290-reactive T-cell response was primed by stimulating peripheral blood T cells from DR53-carrying healthy individuals with dendritic cells bearing PL-bound beta(2)GPI in vitro. Our finding is the first demonstration of an in vitro mechanism eliciting pathogenic autoreactive T-cell responses to beta(2)GPI and should be useful in clarifying the pathogenesis of APS.

Recently, we have identified a primitive cell population termed monocyte-derived multipotential cells (MOMCs), which have a fibroblast-like morphology and a unique phenotype positive for CD14, CD45, CD34, and type I collagen. MOMCs are derived from in vitro culture of circulating CD14+ monocytes, and contain progenitors capable of differentiating into a variety of mesenchymal cells, neuron, and endothelium. Thus, human MOMCs are attractive candidates for an autologous transplantable cell source for therapeutic tissue regeneration. In vitro induction of MOMCs from circulating CD14+ monocytes requires their binding to matrix proteins such as fibronectin, and exposure to soluble factors derived from peripheral blood CD14− cells, but detailed processes remain unknown. Here, we investigated molecular factors involved in MOMC induction using human peripheral blood monocyte cultures. Induction of MOMCs was defined as having all of features, including fibroblastic morphology, CD34+ phenotype, and potentials of differentiating into multiple cell lineages. First, we set up MOMC cultures on fibronectin, type I collagen, laminin, or poly-L-lysine, and found that MOMCs were efficiently obtained exclusively in the presence of fibronectin. Since fibronectin is a ligand for β1 integrins, we examined effects of blockade of these interactions on MOMC generation. Induction of MOMCs was completely inhibited by anti-α5-integrin monoclonal antibody, but not by anti-α4-integrin antibody. In addition, a synthetic peptide that competed with the RGD domain of fibronectin suppressed generation of MOMC, but a CS-1 domain peptide had no effect, indicating that binding of α5β1 integrin to the RGD domain of fibronectin is required for monocytes to acquire a multipotential property. Next, highly enriched monocytes were cultured on fibronectin with various peripheral blood cell subsets to identify cells producing soluble factors required for MOMC generation. As a result, MOMCs were obtained exclusively in the presence of platelets. MOMC induction was also observed when conditioned medium prepared by stimulating platelets with various agonists, such as thrombin and ADP, was used instead of whole platelets. When platelet-derived condition medium was fractionated based on the molecular weight (MW), MOMC induction activity was retained in the fraction of MW < 30,000. Then, we selected a list of 16 candidate factors that are released from activated platelets and < 30,000 in MW, and screened them using two different strategies: evaluation of MOMC induction efficiency in monocyte cultures by adding individual candidate factors; and FACS sorting of circulating monocytes into cells with high and low expressions of receptor for individual candidate factors, followed by evaluation of MOMC induction. As a result, SDF-1was selected as a sole factor with capacity to promote generation of MOMCs. Moreover, MOMC generation was significantly more prominent in cultures with monocytes with high expression of CXCR4, a receptor for SDF-1, than in cultures of monocytes with low CXCR4 expression. In summary, circulating monocytes are able to transdifferentiate into multipotential cells through the fibronectin-α5β1 integrin and SDF-1-CXCR4 interactions. This information is helpful in establishing an optimal MOMC culture condition used in cell transplantation for tissue regeneration.

Recent studies from Italy and Japan demonstrated that more than a half of patients with idiopathic thrombocytopenic purpura (ITP) infected with Helicobacter pylori (H. pylori) achieved a partial or complete platelet recovery after eradication of H. pylori, suggesting a causal link between H. pylori and ITP. To examine the role of H. pylori infection in the pathogenic process of ITP, autoimmune response to platelet GPIIb/IIIa as well as phenotypic and functional properties of circulating monocytes were serially evaluated in ITP patients who 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-five adult patients with chronic ITP and a platelet count below 100 × 109/L were enrolled. Platelet counts, proportion of reticulated platelets, plasma thrombopoietin, expression levels of FcγRI, FcγRII, CD86 and HLA-DR on circulating monocytes assessed by flow cytometry, capacity of circulating monocytes to uptake FITC-dextran, circulating anti-GPIIb/IIIa antibody-producing B cells, and T-cell proliferative responses to GPIIb/IIIa and tetanus toxoid were evaluated at 0, 1, 12, and 24 weeks after initiation of the eradication therapy. H. pylori infection was found in 24 (69%) patients by means of a urea breath test. At baseline, circulating monocytes from H. pylori-positive patients exhibited up-regulated expression of FcγRI and HLA-DR, down-regulated expression of FcγRII, and enhanced phagocytic capacity, compared with those from H. pylori-negative patients (all for p < 0.05). All H. pylori-positive patients were successfully eradicated, and a significant platelet response was observed in 13 (54%) eradicated patients, but in none of 11 H. pylori-negative patients. Anti-GPIIb/IIIa antibody-producing B cells and proportion of reticulated platelets were significantly reduced at 12 and 24 weeks in 13 H. pylori-positive responders as well as, to a lesser extent, in 11 H. pylori-positive non-responders (p < 0.05), but not in 11 H. pylori-negative patients. In H. pylori-positive responders, up-regulated FcγRI expression and enhanced phagocytic capacity of monocytes were decreased at 12 and 24 weeks (p < 0.05), but HLA-DR expression remained high. On the other hand, down-regulated FcγRII expression on monocytes was significantly increased at week 24 (p = 0.02). These changes were not observed in H. pylori-negative patients, although similar trends were found in H. pylori-positive non-responders without statistical significance. When gene expression of a stimulatory receptor FcγRIIA and an inhibitory receptor FcγRIIB was independently assessed on the sorted monocytes by quantitative real-time PCR, the FcγRIIA/IIB expression ratio was significantly reduced in H. pylori-positive responders at one week, but not in H. pylori-negative patients. In summary, H. pylori infection plays a role in the ITP pathogenesis by altering FcγR balance of monocytes/macrophages in favor of activating receptors. A platelet recovery observed in a subset of ITP patients after the H. pylori eradication is likely to be mediated by a change in the FcγR balance toward the inhibitory FcγRIIb.

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.

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.

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 (