Megakaryocytes and thrombocytopoiesis in idiopathic thrombocytopenic purpura

In idiopathic thrombocytopenic purpura (ITP) patients with histological signs of stronger auto-antibody synthesis (spleen follicle hyperplasia) more frequently revealed megakaryocyte changes due to antibodies and less frequently thrombocyte production compensatory increased than ITP patients with signs of weaker auto-antibody synthesis (spleen follicle normoplasia). The observation suggest that insufficient and frequently lacking compensatory increase of thrombocyte production in ITP is caused by an autoallergic disturbance of megakaryocyte maturation.     

Prognostic significance of splenic follicle size in splenectomized idiopathic thrombocytopenic purpura patients

The prognostic significance of splenic follicle (B-lymphocyte compartment) size was studied in 62 patients splenectomized for idiopathic thrombocytopenic purpura (ITP). Patients with hyperplasia of splenic follicles (mean follicle diameter greater than 500 micron) were more likely to relapse or to develop additional autoimmune disorders than patients without hyperplastic splenic follicles (mean follicle diameter less than 500 micron) (p less than 0.01). The enlargement of splenic follicles had a positive predictive value of 27% and a negative predictive value of 100% for a poor outcome of splenectomy. Thus, the histological examination of spleens surgically removed for ITP seems to be an appropriate method to obtain the first indication of the possible long-term effect of splenectomy almost immediately after the operation.     

Kinetics of megakaryocyte progenitor cells in idiopathic thrombocytopenic purpura

The number and proliferative state of megakaryocyte progenitor cells (CFU-Meg) were compared between 13 patients with idiopathic thrombocytopenic purpura (ITP) and hematologically normal controls. The mean frequency of CFU-Meg assayed by the plasma clot method was 27.8 +/- 12.2 (+/- SD)/2 x 10(5) bone marrow light-density cells for the ITP patients, which did not differ significantly from the control value of 31.9 +/- 16.1. The percentage of CFU-Meg in DNA synthesis estimated by the 3H-thymidine suicide technique was 41.3% +/- 9.2% in ITP, which was significantly greater than the control value of 27.1% +/- 7.4% (P less than 0.01). The megakaryocyte counts for histological sections prepared from bone marrow aspirates from the ITP patients and controls were 34.5 +/- 8.5/mm2 and 11.2 +/- 5.8/mm2, respectively, with the difference being highly significant (P less than 0.001). These results suggest that increased cycling activity in a quantitatively unchanged CFU-Meg pool may lead to increased megakaryocytes in the bone marrow of ITP patients.     

Long-term prognosis of the results of splenectomy in idiopathic thrombocytopenic purpura

The value of selected clinical and histological parameters for the prognosis of long-term results of splenectomies was investigated retrospectively in 50 patients with idiopathic thrombocytopenic purpura 4-14 years after surgery. Two parameters showed an association to the results of splenectomy, viz. spleen follicle size as well as site of thrombocytopenia degradation. Non-responders to splenectomy (n = 7) only turned up in spleen follicle hyperplasia, never in follicle normoplasia, however (p less than 0.01), four times more frequently in mainly extralienal thrombocyte degradation, also in predominantly lienal ones (p greater than 0.05).     

Association of interleukin-(IL)10 haplotypes and serum IL-10 levels in the progression of childhood immune thrombocytopenic purpura

Derangement of genetic and immunological factors seems to have a pivotal role in the pathophysiology of immune thrombocytopenic purpura (ITP). We investigated interleukin(IL)-10 genetically determined expression in children with an acute progression of ITP (n=41) compared to young patients with chronic ITP (n=44) and healthy controls (n=60), and attempted to correlate IL-10 production with the course of the disease. We genotyped our study population for three single nucleotide polymorphisms at positions -1082 (A/G), -819 (C/T) and -592 (C/A) in the promoter region of the IL-10 gene. IL-10 levels were measured by enzyme-linked immunoassay. The IL-10 production in our study population was significantly higher in patients carrying the GCC haplotype than those bearing ACC and ATA haplotypes (6.9 ± 1.5 vs 3.6 ± 0.8 vs 3.3 ± 0.3, p=0.03). The serum concentration of IL-10 was significantly higher in patients with an acute course of their disease, who mainly carried the GCC haplotype (92%), compared to chronic subjects, bearing the non-GCC haplotypes, and controls [17 pg/mL (1.7-18) vs 3.5 pg/mL (0.6-11) vs 3 pg/mL (1-7), p<0.01)]. Our findings show that patients carrying the GCC-high producer IL-10 haplotype have an acute development of ITP and that IL-10 levels might represent a useful predictive biomarker of the disease course.     

Mesenchymal stem cells improve platelet counts in mice with immune thrombocytopenia

Immune thrombocytopenia (ITP) is a common autoimmune bleeding disorder. The breakdown of immune tolerance (regulatory T [Treg] cells and suppressor cytokines) plays an important role in ITP pathophysiology, especially in refractory ITP. Bone marrow–derived mesenchymal stem cells (BM-MSCs) show immunomodulatory properties and have been extensively utilized for autoimmune diseases. However, it has not been fully elucidated how BM-MSCs affect ITP. In this study, we explore the therapeutic mechanism of BM-MSCs on ITP in mice. Dose-escalation passive ITP mice were inducted by injection of MWReg30. BALB/c mice were randomly divided into two groups: ITP with BM-MSC transplantation and ITP controls. The serum levels of cytokines (interleukin 10 [IL-10] and transforming growth factor-β1 [TGF-β1]) were examined by enzyme-linked immunosorbent assays. The frequency of Treg cells in both peripheral blood and spleen mononuclear cells was analyzed by flow cytometry, and the forkhead box P3 (Foxp3) messenger RNA (mRNA) level was measured by real-time polymerase chain reaction. After BM-MSC treatment, the platelet (PLT) counts were significantly elevated. Meanwhile, cytokines (TGF-β1 and IL-10), the ratios of Treg cells, and the Foxp3 mRNA expression level were significantly higher in the BM-MSC group. Our results show that BM-MSCs can improve PLT counts mainly by secreting suppressive cytokines and upregulating Tregs, which may provide new therapeutic potential for human ITP.     

Changes in Follicular Helper T Cells in Idiopathic Thrombocytopenic Purpura Patients

Background: Idiopathic thrombocytopenic purpura (ITP) is a primary autoimmune disease with a decreased platelet count caused by platelet destruction mediated mainly by platelet antibodies. T follicular helper (TFH) cells have demonstrated important roles in autoimmune diseases. The aim of this study is to explore the might role of TFH cells in the patients of ITP.Methods: Twenty-three ITP patients and 12 healthy controls (HC) were enrolled in this study. The frequency of circulating TFH cells in both the patients and HC was analyzed by flow cytometry. Serum interleukin (IL)-21 and IL-6 levels were measured using ELISA, and platelet antibodies were tested using a solid phase technique. Additionally, IL-21, IL-6, Bcl-6 and c-Maf mRNA expressions in peripheral blood mononuclear cells (PBMCs) were detected using real-time PCR.Results: The percentages of circulating CXCR5⁺ CD4⁺TFH cells with ICOS^high or PD-1^high expression were significantly higher in the ITP patients than in the HC. Moreover, the frequencies of circulating CXCR5⁺ CD4⁺TFH cells with inducible costimulator (ICOS)^high or programmed death-1 (PD-1)^high expression were notably higher in ITP with platelet-antibody-positive ( ITP (+) ) patients than in ITP with platelet-antibody-negative ( ITP (-) ) patients and HC, as were the serum IL-21 and IL-6 levels (significant). Moreover, a positive correlation was found between the CXCR5⁺CD4⁺TFH cells with ICOS^high or PD-1^high expression and the serum IL-21 levels of ITP (+) patients. Additionally, the mRNA expression levels of IL-21, IL-6, Bcl-6 and c-Maf were significantly increased in ITP patients, especially in ITP (+) patients.Conclusions: This study demonstrated TFH cells and effector molecules might play an important role in the pathogenesis of ITP, which are possible therapeutic targets in ITP patients.     

Birth weight corrected for gestational age is related to the incidence of Down's syndrome pregnancies.

Three recent studies reported that early depletion of the primordial follicle pool is likely to be an independent risk factor for Down's syndrome pregnancies. The size of the primordial follicle pool at birth is determined by oogenesis and by the rate of follicle atresia during the intra uterine period. Since intra uterine growth retardation was reported to be associated with a significantly reduced primordial follicle pool at birth, we investigated the possibility of a relation between low birth weight for gestational age and the risk of a Down's syndrome pregnancy. In a case control study, 95 women with a history of a Down's syndrome pregnancy and 85 controls provided information on their own birth weight and length of gestation. Birth weight standard deviation scores, indicating the difference in birth weight from a reference group, were significantly lower in Down's syndrome mothers than in controls. These findings illustrate that the risk of a Down's syndrome pregnancy is related to a low birth weight corrected for gestational age, possibly by a causal relation between intra uterine growth retardation and the size of the primordial follicle pool.     

A study of beta-thromboglobulin and platelet factor-4 plasma levels in steady state sickle cell patients

To evaluate the platelet function in sickle cell syndromes we measured the beta-thromboglobulin (beta-TG) and platelet factor 4 (PF-4) plasma values of 45 patients suffering from homozygous sickle cell anaemia (10) and sickle cell beta-thalassaemia (35) in steady state. The results were compared to those of 32 normal controls. Both the beta-TG and PF-4 levels were found to be significantly higher in patients than in controls but the beta-TG:PF-4 ratio was significantly lower in the patients group. This finding and the absence of any statistical correlation between platelet number and beta-TG or PF-4 indicate that platelets seem to be somehow activated in sickle cell syndromes, both in homozygotes and sickle cell/beta-thalassaemia heterozygotes. This platelet activation seems to exist even in steady state sickle cell disease patients, regardless of the functional status of the spleen.     

Detection of Circulating B Cells Producing Anti-GPIb Autoantibodies in Patients with Immune Thrombocytopenia

Background

We previously reported that an enzyme-linked immunospot (ELISPOT) assay for detecting anti-GPIIb/IIIa antibody-secreting B cells is a sensitive method for identifying patients with immune thrombocytopenia (ITP). Here we assessed the clinical significance of measuring circulating B cells producing antibodies to GPIb, another major platelet autoantigen.

Methods

Anti-GPIb and anti-GPIIb/IIIa antibody-producing B cells were simultaneously measured using ELISPOT assays in 32 healthy controls and 226 consecutive thrombocytopenic patients, including 114 with primary ITP, 25 with systemic lupus erythematosus (SLE), 30 with liver cirrhosis, 39 with post-hematopoietic stem cell transplantation (post-HSCT), and 18 non-ITP controls (aplastic anemia and myelodysplastic syndrome).

Results

There were significantly more circulating anti-GPIb and anti-GPIIb/IIIa antibody-producing B cells in primary ITP, SLE, liver cirrhosis, and post-HSCT patients than in healthy controls (P<0.05 for all comparisons). For diagnosing primary ITP, the anti-GPIb ELISPOT assay had 43% sensitivity and 89% specificity, whereas the anti-GPIIb/IIIa ELISPOT assay had 86% sensitivity and 83% specificity. When two tests were combined, the sensitivity was slightly improved to 90% without a reduction in specificity. In primary ITP patients, the anti-GPIb antibody response was associated with a low platelet count, lack of Helicobacter pylori infection, positive anti-nuclear antibody, and poor therapeutic response to intravenous immunoglobulin.

Conclusion

The ELISPOT assay for detecting anti-GPIb antibody-secreting B cells is useful for identifying patients with ITP, but its utility for diagnosing ITP is inferior to the anti-GPIIb/IIIa ELISPOT assay. Nevertheless, detection of the anti-GPIb antibody response is useful for subtyping patients with primary ITP.     

Increased number of Tc17 and correlation with Th17 cells in patients with immune thrombocytopenia

Background

IL-17-secreting CD8+ T cells (Tc17 subset) have recently been defined as a subpopulation of effector T cells implicated in the pathogenesis of autoimmune diseases. The role of Tc17 and correlation with Th17 cells in the pathophysiology of immune thrombocytopenia (ITP) remain unsettled.

Design and Methods

We studied 47 ITP patients (20 newly-diagnosed and 27 with complete response) and 34 healthy controls. IL-17-producing CD3+CD8+ cells (Tc17) and IL-17-producing CD3+CD8− cells (Th17) were evaluated by flow cytometry and expressed as a percentage of the total number of CD3+ cells. Specific anti-platelet glycoprotein (GP) GPIIb/IIIa and/or GPIb/IX autoantibodies were measured by modified monoclonal antibody specific immobilization of platelet antigens. Peripheral blood mononuclear cells of ITP patients were isolated, incubated in the presence of 0, 0.25, 0.5, or 1 µmol/L of dexamethasone for 72 h, and collected to detect Tc17 and Th17 cells by flow cytometric analysis.

Results

IL-17 was expressed on CD3+CD8− and CD3+CD8+ T cells. The percentages of Tc17 and Th17 cells in newly-diagnosed patients were significantly elevated compared to controls, and Tc17 was decreased after clinical treatment. The Th17∶Tc17 ratio was significantly lower in newly-diagnosed patients compared with controls, and was increased in patients who had complete response. There was a significantly positive correlation between Tc17 and Th17 cells in the control group, but not in the ITP patients. A positive correlation existed between Tc17 and the CD8∶CD4 ratio, as well as CD8+ cells in patients with ITP. The frequencies of Tc17 were marginally higher in autoantibody-negative patients than autoantibody-positive patients. Moreover, both Tc17 and Th17 cell percentages decreased as the concentration of dexamethasone in the culture media increased in ITP patients.

Conclusions

Tc17 and the Th17 subset are involved in the immunopathology of ITP. Blocking the abnormally increased number of Tc17 may be a reasonable therapeutic strategy for ITP.     

The humoral regulation of megakaryocytopoiesis and platelet production in vivo

We examined the effects of the urinary extracts from aplastic anemia (AA) patients, idiopathic thrombocytopenic purpura (ITP) patients, and normal subjects on murine megakaryocyte/platelet production in vivo and in vitro. In the first study, single doses of AA urinary protein (65%-90% ethanol precipitate) were individually injected intraperitoneally into rats and mice. Blood platelet counts in rats increased significantly 24 hours after the injection. Total megakaryocyte colony-forming units (CFU-Meg) in mouse spleens increased by 24 hours postinjection, peaked at 48 hours and returned to normal levels at 96 hours. Changes in the number of megakaryocyte colonies showed similar patterns of increasing, peaking and returning to normal levels postinjection. In the second study, we compared the effects of some urinary extracts on murine megakaryocyte/platelet production. These observations provided the evidence that AA urinary extracts contain a factor that directly stimulates megakaryocyte progenitor cell proliferation in mouse spleen in vivo as well as the release of platelets from megakaryocytes, and ITP urinary extracts do not contain increased levels of Meg-CSF and/or some other factor that directly stimulates CFU-Meg in vivo, and the decreased blood platelet mass that is clinically characteristic of ITP is not a primary in vivo determinant of the elaboration of these factors.     

Effect of a neutrophilia-inducing tumor on hemopoietic stem cells in mice

The influence of neutrophilic stimulation on hemopoietic stem cells was studied in mice with tumor-induced neutrophilia. Transfusions of marrow cells from normal and neutrophilic tumor-bearing mice into lethally irradiated normal and tumor-bearing mice were performed. The number and the erythroid:granuloid (E:G) ratio of day 7 colonies in the recipient spleens and bones as well as the size of spleen colonies of recipient animals were determined. The E:G ratio of spleen and bone marrow colonies between normal and tumor-bearing mouse recipients and the number of spleen colonies did not differ significantly in either experiment. However, spleen colonies which developed in tumor-bearing irradiated mice were significantly larger than those which developed in normal recipients in both experiments. These studies indicated that while the line of differentiation taken by hemopoietic stem cells was not affected by the neutrophilic influence of the tumor, the tumor-bearing host environment appeared to enhance proliferation of transfused stem cells and/or their descendants. The stimulators of granulocytopoiesis in this model of neutrophilia appear to act on a population of progenitor cells more mature than the stem cells capable of forming 7-day colonies in the spleen and bone marrow of irradiated recipient mice.     

Excessive production of IFN-gamma in patients with systemic lupus erythematosus and its contribution to induction of B lymphocyte stimulator/B cell-activating factor/TNF ligand superfamily-13B

Expression and immunological significance of IFN-gamma, a pivotal cytokine in murine lupus, have not been clearly demonstrated in human systemic lupus erythematosus (SLE). In the present study we investigated the expression of IFN-gamma in peripheral blood T cells from patients with SLE and its role in the production of the soluble B lymphocyte stimulator (sBLyS). Peripheral blood T cells from patients with SLE expressed significantly larger amounts of IFN-gamma in response to stimulation with anti-CD3 mAb plus anti-CD28 mAb than those from normal controls as shown by three analytical methods, including ELISA, flow cytometry, and quantitative RT-PCR. The ratio of IFN-gamma-producing T cells to effector memory T cells in CD3(+)CD4(+) and CD3(+)CD8(+) populations in patients with SLE was significantly higher than that of normal controls. The T-box expressed in T cells (T-bet) mRNA/GATA-binding protein-3 (GATA-3) mRNA ratio was significantly higher in patients with SLE than in normal controls. T cell culture supernatants from patients with SLE contained significantly higher sBLyS-inducing activity than normal controls; this was almost completely inhibited by the addition of anti-human IFN-gamma mAb. Percentages of BLyS-expressing peripheral blood monocytes in patients with SLE were significantly higher than those of normal controls. Monocytes from patients with SLE produced significantly larger amounts of sBLyS in response to IFN-gamma than those from normal controls. Taken together, these data strongly indicate that the overexpression of IFN-gamma in peripheral blood T cells contributes to the immunopathogenesis of SLE via the induction of sBLyS by monocytes/macrophages, which would promote B cell activation and maturation.     

Detection of platelet-associated IgG in chronic immune thrombocytopenic purpura using antibody-coated polyacrylamide beads

Platelet-associated IgG (PAIgG) was detected by means of anti-human IgG coated polyacrylamide beads ("Immunobeads") technique in 32 patients with chronic ITP. Both a direct test (with in vivo sensitized platelets) and an indirect test (with in vitro loaded platelets) were carried out. The percent of rosette forming beads was both in the direct test (41.2%) and in the indirect test (32.6%) significantly higher in the cases of chronic ITP patients than in the controls (2.5% and 3.2%, respectively). These results confirm the diagnostic value of this new, relatively simple and rapid method in routine clinical practice.     

脾动脉灌注长春新碱加栓塞治疗ITP

目的探讨慢性特发性血小板减少性紫癜（ＩＴＰ）新的有效治疗方法,方法３例慢性ＩＴＰ患者行脾动脉灌注长春新碱（ＶＣＲ）加脾动脉栓塞术,观察术后临床症状及血小板计数,结果３例术后临床症状消失,血小板计数上升,随诊６－８个月以上仍稳定于正常水平,无感染发生,结论脾动脉灌注ＶＣＲ加栓塞治疗慢性ＩＴＰ疗效,无明显副作用。     

Contributions of T Lymphocyte Abnormalities to Therapeutic Outcomes in Newly Diagnosed Patients with Immune Thrombocytopenia

T cell abnormalities have been reported to play an important role in pathogenesis of immune thrombocytopenia (ITP) besides specific autoantibodies towards platelet. The aim of this study was to explore the clinical importance of T lymphocyte subsets in adult patients with newly diagnosed ITP before and after first-line treatment. Elderly ITP patients were also studied and we tried to analyze the relationships between these items and therapeutic outcomes. The patients were treated with intravenous immunoglobulin (IVIG) plus corticosteroids and therapeutic responses were evaluated. As a result, compared with the controls, absolute lymphocyte counts in ITP patients decreased significantly before treatment. After treatment, lymphocyte counts restored to control level regardless of their treatment outcomes. In addition, we observed increased IgG and CD19⁺ cell expression and decreased CD4⁺/CD8⁺ cell ratio in both whole ITP group and elderly group before treatment. After treatment, the increased IgG and CD19⁺ cell expression could be reduced in both respond and non-respond group regardless of patient age, while CD4⁺/CD8⁺ cell ratio could not be corrected in non-respond ITP patients. In non-respond ITP patients, increased CD8⁺ cell expression was noticed and could not be corrected by first-line treatment. Furthermore, even lower NK cell expression was found in non-respond elderly patients after treatment when compared with that in controls. Our findings suggest that ITP patients usually had less numbers of peripheral lymphocytes and patients with higher levels of CD8⁺ cells or lower levels of CD4⁺/CD8⁺ cell ratio were less likely to respond to first-line treatment. Lower levels of NK cells made therapies in elderly ITP patients even more difficult.     

Alteration of Immune Responses of Rabbits Infected with Bovine Immunodeficiency-Like Virus

Nine 3-month-old rabbits were inoculated with bovine immunodeficiency-like virus (BIV) to study the pathogenesis of BIV and alteration of the immune responses in experimentally infected rabbits. BIV proviral DNA and anti-BIV antibodies were detected from all rabbits inoculated with BIV-infected bovine embryo spleen (BESP) cells. Rabbits inoculated with spleen cells of the BIV-infected rabbit also converted to proviral DNA-positive and BIV-antibody-positive. The blastogenic responses to concanavalin A of peripheral blood mononuclear cells prepared from BIV-infected rabbits were not significantly different from those from uninfected controls at 2 and 4 months post-inoculation (PI). The humoral immune responses against bovine serum albumin (BSA) were depressed in two of four BIV-infected rabbits at 1 to 3 months PI. The antibody responses against sheep red blood cells (SRBCs) were significantly depressed in all BIV-infected rabbits at 2 to 4 months PI. BIV was rescued by cocultivation of spleen cells of infected rabbits with BESP cells. Distinct development of lymphoid follicle was observed in lymph nodes and spleens of uninfected rabbits which received BSA and SRBCs. In contrast, moderate lymphoid cell depletion was observed in BIV-infected rabbits which received the same immunogens.     

Mathematical analysis of the relative contributions of decreased production and increased peripheral destruction in idiopathic thrombocytopenic purpura and implications in splenectomy

We utilize a model of platelet concentration kinetics and bone marrow production based on three terms (a constant loss term, a random loss term and a higher order loss term) to compare a hypoplastic bone marrow patient and a patient with Idiopathic Thrombocytopenic Purpura (ITP) for the same platelet concentration. We compare this model to published data and show that in many ITP patients there is an overall decrease in platelet production. However, for almost all cases of ITP there is an increase in peripheral platelet destruction, even in those cases where total bone marrow production is less than that in a normal individual or is severely depressed. We are able to graphically depict the variable contributions of decreased production and increased peripheral destruction in patients with ITP and hence give insight into their relative contributions in a given patient. We apply a unique feature of our model, the newly postulated destruction term proportional to the platelet concentration squared (the higher order loss term), to explain cases of antibody negative ITP. Application of our model to data on patients splenectomized as treatment for ITP shows promise in predicting which patients are likely to respond.     

Abnormal perception of food size in anorexia nervosa

The hypothesis that patients with anorexia nervosa exaggerate the perceived size of food was tested. Video recordings of five items of food and four neutral objects of a similar size were made such that the size of each object increased steadily from half to twice its normal size. Each of the nine objects was placed on a ledge inside a dummy television screen next to the video screen, the food items alternating with the neutral objects, and 20 female patients with anorexia nervosa and 20 female controls matched for age were asked to adjust the size of the video recording to that of the real object. Although there was no overall difference in perceptual accuracy between patients and controls, both groups perceived the food items as being bigger than the neutral objects, the patients exaggerating the size of the food significantly more than the controls.These results imply that patients with anorexia might start eating more easily when admitted if their food were presented as small portions on large plates.