TPO Levels Research Articles

Tumor necrosis factor blocking therapy alters joint inflammation and hypoxia

To examine the effect of tumor necrosis factor (TNF) blocking therapy on hypoxia in vivo, macroscopic and microscopic inflammation, and magnetic resonance imaging (MRI) results in patients with inflammatory arthritis. Patients with inflammatory arthritis (n = 20) underwent full clinical assessment, arthroscopy, synovial biopsy, and MRI before and after initiation of biologic therapy. Macroscopic synovitis/vascularity was assessed with a visual analog scale, and tissue PO(2) (tPO(2) ) was measured at arthroscopy using a Licox probe. Cell-specific markers (CD4, CD8, CD68, CD20, and CD19) and blood vessel maturity were quantified by immunohistologic analysis and dual-immunofluorescence factor VIII/α-smooth muscle actin staining, respectively. Contiguous gadoteric acid-enhanced MRI of the target knee was used to assess synovial enhancement. Biologic therapy responders showed a significant increase of tPO(2) in vivo (P < 0.05). This response was associated with significant reductions in 28-joint Disease Activity Score using the C-reactive protein level (DAS28-CRP) (P = 0.012), macroscopic synovitis (P = 0.017), macroscopic vascularity (P = 0.05), CD4+ T cells (P < 0.041), and CD68+ macrophages (P < 0.011). Blood vessel numbers were also reduced in responders; however, this did not reach statistical significance. Strong inverse correlations were demonstrated between changes in tPo(2) levels and changes in DAS28-CRP (r = -0.53, P < 0.001), CD4 (r = -0.44, P < 0.026), CD68 (r = -0.46, P < 0.003), and macroscopic vascularity (r = -0.314, P = 0.049) after therapy. Furthermore, changes in inflammation as measured by MRI showed a strong inverse correlation with tPO(2) levels (r = -0.688, P < 0.002) and positive correlations with CRP levels (r = 0.707, P = 0.001), macroscopic synovitis (r = 0.457, P = 0.056), macroscopic vascularity (r = 0.528, P= 0.017), CD4 (r = 0.553, P < 0.032), and CD68 (r = 0.670, P < 0.002) after therapy. This is the first study to show that successful biologic therapy significantly improves in vivo synovial hypoxia. Changes are strongly associated with changes in macroscopic and microscopic measures of joint inflammation and MRI improvement. These data further strengthen the concept that hypoxia is an important event driving synovial inflammation.

Successful tumour necrosis factor (TNF) blocking therapy suppresses oxidative stress and hypoxia-induced mitochondrial mutagenesis in inflammatory arthritis

IntroductionTo examine the effects of tumour necrosis factor (TNF) blocking therapy on the levels of early mitochondrial genome alterations and oxidative stress.MethodsEighteen inflammatory arthritis patients underwent synovial tissue oxygen (tpO2) measurements and clinical assessment of disease activity (DAS28-CRP) at baseline (T0) and three months (T3) after starting biologic therapy. Synovial tissue lipid peroxidation (4-HNE), T and B cell specific markers and synovial vascular endothelial growth factor (VEGF) were quantified by immunohistochemistry. Synovial levels of random mitochondrial DNA (mtDNA) mutations were assessed using Random Mutation Capture (RMC) assay.Results4-HNE levels pre/post anti TNF-α therapy were inversely correlated with in vivo tpO2 (P < 0.008; r = -0.60). Biologic therapy responders showed a significantly reduced 4-HNE expression (P < 0.05). High 4-HNE expression correlated with high DAS28-CRP (P = 0.02; r = 0.53), tender joint count for 28 joints (TJC-28) (P = 0.03; r = 0.49), swollen joint count for 28 joints (SJC-28) (P = 0.03; r = 0.50) and visual analogue scale (VAS) (P = 0.04; r = 0.48). Strong positive association was found between the number of 4-HNE positive cells and CD4+ cells (P = 0.04; r = 0.60), CD8+ cells (P = 0.001; r = 0.70), CD20+ cells (P = 0.04; r = 0.68), CD68+ cells (P = 0.04; r = 0.47) and synovial VEGF expression (P = 0.01; r = 063). In patients whose in vivo tpO2 levels improved post treatment, significant reduction in mtDNA mutations and DAS28-CRP was observed (P < 0.05). In contrast in those patients whose tpO2 levels remained the same or reduced at T3, no significant changes for mtDNA mutations and DAS28-CRP were found.ConclusionsHigh levels of synovial oxidative stress and mitochondrial mutation burden are strongly associated with low in vivo oxygen tension and synovial inflammation. Furthermore these significant mitochondrial genome alterations are rescued following successful anti TNF-α treatment.

Thyroid Disorders in Patients with Inflammatory Bowel Diseases

Background: The rate of thyroid disorders is reported to be increased in patients with Ulcerative Colitis (UC) and Crohn’s disease (CD) than the normal population. The purpose of this study is to evaluate the prevalance of Thyroid disorders in a group of patients with inflammatory bowel diseases (IBD). Material and Methods: 146 IBD patients (113 UC and 33 CD patients) and 66 healthy control subjects were enrolled into the study. Serum free T4 (FT4), free T3 (FT3), TSH and anti TPO levels of the patients were analyzed retrospectively and and compared with a control group. These cases were also investigated with thyroid ultrasound and nuclear (scintigraphy) imaging. Results: The mean age of IBD patients (76 women) was 42.9 ± 12.4 years. Among the control group 42 were female and their mean age was 40.9 ± 12.1 years. Thyroid gland disease was found in 14 (9.5%, 8 female, 6 male) of 146 IBD patients. The frequency of thyroid disorders has been found to be higher than the control group in patients with control group (14/146 vs. 1/66, p = 0.042). No statistically significant difference has been observed at the thyroid disorder rates between the UC and CD patients (11/113 vs. 3/33, p = 0.912). Hashimoto thyroiditis has been found at 4 (2.7%) of the IBD patients. Conclusion: In accordance with several works published in literature, we have found a higher rate of thyroid disease apperance at IBD patients.

Prospective study of combination therapy with low-dose thalidomide plus prednisolone ameliorating cytopenia in primary myelofibrosis

Recent studies reported that thalidomide attenuates anemia, thrombocytopenia and splenomegaly in patients with primary myelofibrosis (PMF) probably via anti-angiogenic effect [1–6]. However, many patients, who had been treated with 100–800 mg per day of thalidomide, discontinued therapy because of its toxicity. A combination therapy of low-dose thalidomide (50 mg/day) with prednisolone (PSL) conducted by Mesa et al. at Mayo Clinic [7] was well tolerated, and revealed an excellent clinical effect on anemia with a response rate of 62%. Based on the data, we have conducted the same prospective study for the treatment of Japanese patients with advanced PMF. According to the regimen conducted by Mesa et al. [7], patients received 50 mg daily dose of thalidomide together with a tapering regimen of PSL for the first 3 months (0.5 mg/kg/day for 1 month; 0.25 mg/kg/day for the second month; 0.125 mg/kg/day for the third month). This study was approved by the ethical committee of the Keio University School of Medicine, and written informed consent was obtained from all patients. Four patients were enrolled in this study from July 2003 to June 2004. At the time of study entry, genetic mutations of Jak2 could not be examined. One patient discontinued therapy within the first month because of grade 3 vertigo. The rest of three patients received thalidomide and PSL for longer than 1 month, and thus they were considered to be evaluable for response to therapy. The response and the changes in laboratory data during the treatment were summarized in Fig. 1a. Hemoglobin level was slightly increased in patient 1 while platelet counts increased in all three patients. However, the sizes of spleen measured by CT-based volumetry remained unchanged. Regarding side effects of this treatment, grade 3 vertigo was observed in a 76-year-old female as described above. Therefore, this combination therapy should be carefully performed especially in the elderly patients. Grade 3 lymphopenia also occurred in two patients, which was likely caused by PSL. Other toxicities were generally mild, including somnolence, constipation, fatigue, dyspnea, dry mouth, chest pain, common cold, headache, leukocytosis and thrombocytosis. In order to elucidate the mechanism of the hematological response, megakaryocyte count, microvessel density and degree of fibrosis in the bone marrow were examined (Fig. 1b). However, pathological examinations did not show consistent improvement of either one of them. Vertebral MRI scan also failed to demonstrate obvious improvement of bone marrow fibrosis or recovery of fatty component (data not shown). We also examined the plasma concentrations of hematopoietic growth factors including EPO and TPO before and after the therapy. Pre-treatment EPO and TPO levels were elevated compared to healthy controls in all three patients (Fig. 1a). Plasma EPO level was slightly increased in patient 1, but it was decreased in the other two patients after initiating thalidomide plus PSL Y. Hattori (&) Division of Clinical Physiology and Therapeutics, Faculty of Pharmacy, Keio University, 1-5-30, Shibakoen, Minato-ku, Tokyo 105-8512, Japan e-mail: hattori-yt@pha.keio.ac.jp

Megakaryocytes Possess a Functional Intrinsic Apoptosis Pathway That Must Be Restrained In Order to Survive and Produce Platelets

AbstractAbstract 550It is widely held that megakaryocytes undergo a specialized form of apoptosis in order to shed platelets. Conversely, it is also believed that a range of insults including chemotherapeutic agents, autoantibodies and viruses, cause thrombocytopenia by inducing the apoptotic death of megakaryocytes and their progenitors. However, the apoptotic pathways that megakaryocytes possess, and the role they play in survival and platelet production are ill-defined. We recently demonstrated that platelets contain a classical intrinsic mitochondrial apoptosis pathway that regulates their life span in vivo. The key components of this pathway are the Bcl-2 family pro-survival protein Bcl-xL, and pro-death Bak and Bax. Deletion of Bak and Bax—the gatekeepers of the intrinsic pathway—blocks platelet apoptosis in response to genetic mutation or pharmacological insult, and significantly extends circulating platelet life span.To elucidate the role of the intrinsic apoptosis pathway in megakaryocytes, we generated both hematopoietic- and megakaryocyte lineage-specific deletions of Bak and Bax in mice. Surprisingly, we found that the ability of Bak−/−Bax−/− animals to produce platelets, both at steady state and under conditions of thrombopoietic stress, was unperturbed. Megakaryocyte numbers, morphology and ploidy were normal. Bak−/−Bax−/− megakaryocytes cultured in vitro showed no impairment of pro-platelet formation. Thus, classical intrinsic apoptosis is not required by megakaryocytes for the process of platelet shedding. Given that in platelets, Bak and Bax must be kept in check to maintain survival, we reasoned that the same might be true of megakaryocytes. If so, then it would be expected that one or more members of the Bcl-2 family of pro-survival proteins restrain Bak and Bax. Since Bcl-xL fulfills this role in platelets, we generated mice lacking Bcl-xL in the megakaryocyte lineage. Platelet counts in Bcl-xPf4CΔ animals were approximately 2% of those observed in Bcl-xfl/fl littermates. Platelet life span was reduced to 5 hours, versus 5 days in controls, underscoring the requirement for Bcl-xL in mediating platelet survival. In addition, reticulated platelet analyses combined with mathematical modeling suggested that Bcl-xPf4CΔ mice had an underlying platelet production defect. Further examination revealed that megakaryocyte numbers were significantly increased in both the bone marrow and spleen of Bcl-xPf4CΔ animals relative to Bcl-xfl/fl controls. Megakaryocyte progenitor numbers were doubled, and serum TPO levels were dramatically reduced, indicating a megakaryocyte compartment under considerable thrombopoietic stress.In vitro cultures confirmed that Bcl-xPf4CΔ/Pf4CΔ megakaryocytes were able to develop and mature. Strikingly, however, at the point of pro-platelet formation, they underwent an abortive attempt to generate extensions and died. Death was accompanied by a dramatic increase in apoptotic effector caspase activity. This suggested that, like platelets, megakaryocytes possess a functional intrinsic apoptosis pathway that must be restrained in order to survive, and that Bcl-xL is the factor that does so during pro-platelet formation and platelet shedding. To establish whether Bak and Bax can mediate megakaryocyte death, we examined the effect on mature wild type megakaryocytes of three pharmacological agents that activate the intrinsic apoptosis pathway in other cell types: etoposide, staurosporine, and a BH3 mimetic that inhibits Bcl-xL, Bcl-2 and Bcl-w. All three triggered mitochondrial damage, caspase activation and cell death. Remarkably, genetic deletion of Bak and Bax rendered megakaryocytes resistant to etoposide and the BH3 mimetic, but not staurosporine. Our results demonstrate that megakaryocytes can undergo classical Bak- and Bax-mediated apoptotic death. They do not activate the intrinsic pathway to facilitate platelet shedding, rather, the opposite is true: they must restrain it in order to survive and generate platelets. These findings offer a potential mechanism for the death of megakaryocytes in response to insults such as cancer chemotherapy. They also suggest that additional megakaryocyte cell death pathways remain to be elucidated. Disclosures:Roberts:Abbott: Research Funding.

Bortezomib Induced Thrombocytopenia Might Be Due to the Inhibition of Proplatelet Formation of Megakaryocyte.

AbstractAbstract 3696 Background:Bortezomib is potent and reversible proteasome inhibitor that has been extensively used for multiple myeloma. Several clinical studies demonstrated that overall response rates using bortezomib alone to relapsed or refractory patients with multiple myeloma were 33 to 50%. The most common grade 3 adverse event was a cyclic thrombocytopenia, which was reported in 20–30% of patients in several clinical studies. The mechanism by which bortezomib causes thrombocytopenia remains unknown. In this study, we evaluated the effect of bortezomib on megakaryocytic progenitor cells, megakaryocytopoiesis, megakaryocyte and platelet production in mice. Method:All animal procedures were approved by the Institutional Animal Care and Use Committee in Iwate Medical University. Male ddY at 8 weeks of age mice were used in all experiments. In vivo experiments: (a) The mice received 2.5 mg/kg bortezomib via tail-vein injection. Blood was obtained and the following experiments were carried out at day 2, 4, 6, 8, 10 after intravenous injection (n=9, each group). Complete blood counts were measured. Reticulated platelet (RP) was analyzed by flow cytometry using thiazole orange (TO) to evaluate platelet kinetics. Plasma TPO level were measured by ELISA. Bone marrow megakaryocyte's number and morphology from femur in bortezomib- and control-treated mice were observed by microscopy. Femur was fixed in 10% buffered formalin, decalcificated, embedded in paraffin and stained for Hematoxylin-Eosin (H-E). (b) Bortezomib (2.5 mg/kg) was administrated via tail-vein to mice. After 24hr, bone marrow cells were cultured in MegaCult®-C at 5% CO2 and 20% O2for 7 days. The megakaryocytic colonies (CFU-Megs-in vivo) were counted. In vitro experiments: (c) Bone marrow cells, obtained from non-treated mice, were cultured at 37°C in 5% CO2and 20% O2 for 7 days with bortezomib (0.01, 0.1, 1, 10, 100 ng/ml). CFU-Megs were counted (CFU-Megs-in vitro). (d) Proplatelet formation: Murine megakarocytes were partially purified from bone marrow using BSA gradient. They were plated in 96 micro-well culture plates (300 megakaryocyte)well) and cultured in IMDM in duplicates, supplemented with 1 × ITS-G (Life technologies) and each concentration of bortezomib (0.01, 0.1, 1, 10, 100 ng/ml), at 37°C in 5% CO2and 20% O2. After 24 hr incubation, the megakaryocytes with proplatelets in each well were counted. Results:(a) Control mice did not have any significant change in platelet counts, % reticulated platelets and plasma TPO levels at days 0, 2, 4, 6, 8, 10. While, bortezomib treated mice (2.5mg/kg) had a significant reduction in platelet counts at day 2 (470 ± 210 × 109/L. P<0.001), at day 4 (667 ± 118 × 109/L, P<0.001). The platelet counts returned to normal value at day 6 (903 ± 548 × 109/L) and day 10 (1122 ± 187 × 109/L). RP (%) began to increase at day 6 (8.8 ± 4.0 %). Plasma TPO levels tend to increase at day 4. Means megakaryocytes's number in one field of femur was similar in between bortezomib non-treated and –treated mice. The megakaryocytes were similar in morphology at each day, too. (b) CFU-Megs-in vivo were similar in number between bortezomib non-treated and –treated mice (38.0 ± 6.1 vs 34.5 ± 5.6 per 1 × 105 bone marrow cells respectively). (c) CFU-Megs-in vitro were not decreased significantly at 0.001 to 1 ng/ml and decreased significantly (p<0.01) at 10 and 100 ng/ml of bortezomib. (d) Proplatelet formation (PPF) were decreased significantly at 0.01, 0.1, 1, 10, 100 ng/ml bortezomib (0 mg)ml: 25.2 ± 4.8%, 0.01ng/ml: 23.8 ± 4.9%, 0.1 ng/ml: 18.4 ± 3.1% p<0.01, 1 ng/ml: 13.2 ± 3.8% p<0.001, 10 ng/ml: 13.3 ± 2.1% p<0.001, 100ng/ml: 5.9 ± 1.4 % p<0.001). Discussion & Conclusion:Bortezomib did not adversely affect on megakaryocytic prognitors nor megakaryocytes. It did inhibit PPF, that is, the step of platelet production, even when bortezomib plasma concentration levels have gone down. Plasma TPO level showed an inverse relationship against circulating platelet counts. Based on the evidence in which Cmax of plasma bortezomib concentration was under 100 ng/ml in bortezomib-injected mice (2.5mg/kg), bortezomib induced thrombocytopenia might be due to the inhibition of proplatelet formation of megakaryocyte. Disclosures:No relevant conflicts of interest to declare.

Estimation of thyroid hormone status and thyroid antibodies in type-1 diabetes mellitus

Autoimmune thyroid diseases are frequent in patients with type-1 diabetes mellitus. The aim of the study is to determine the incidence of autoimmune thyroid diseases (AIT) in type 1 diabetes mellitus patients which helps to provide early care and treatment. Thirty cases of type-1 diabetes mellitus were included. Serum TSH, serum FT3, serum FT4, anti Tg Ab, anti TPO levels were measured. A significant increase in the levels of anti Tg Ab, anti TPO and serum FT3 was observed. There was no significant difference in the levels of serum FT4 and serum TSH. These findings suggest that there is a strong association between type 1 DM and autoimmune thyroid disorders. The detection of these abnormalities in early stage will help the patients to improve quality of life and reduce the morbidity rate. Keywords: Serum TSH, serum FT3, serum FT4, anti TPO antibodies, anti Tg antibodies.

Plasma cytokines associated with low platelet counts in aplastic anemia and immune thrombocytopenia

Abstract Abstract 1317 Poster Board I-333 Thrombocytopenia occurs in aplastic anemia (AA) as a result of hematopoietic failure, in which decreased or absent megakaryocytes are observed in a hypocellular bone marrow. In immune thrombocytopenia (ITP), thrombocytopenia primarily occurs secondary to peripheral destruction and marrow cellularity is normal with megakaryocytes numerically normal or increased. In both AA and ITP, platelet function is normal and the disturbance in hemostasis is the result of the diminution in the platelet count. We recently reported that high plasma concentrations of Tpo and G-CSF, in combination with low levels of CD40L, CXCL5, CCL5, CXCL11, EGF, VEGF, and eotaxin, served as a cytokine signature profile for AA when compared to MDS or healthy controls (Blood 2008;112:380-381). In 8 patients with severe AA who responded to immunosuppressive therapy, the levels of CD40L, CXCL5, EGF, and CCL5 in plasma correlated with platelet recovery, as compared with absolute neutrophil count and hemoglobin recovery. We hypothesized that the plasma levels of CD40L, CXCL5, EGF, and CCL5 reflected platelets or their precursor megakaryocytes. We measured these cytokines in the plasma of 41 ITP patients using the Luminex assay, for comparison with 33 untreated AA patients and 52 healthy controls ( Figure 1 ). Results were assessed by the Kruskal-Willis one-way analysis of variance statistic. ITP patients showed similar low levels of circulating CCL5, CD40L, CXCL5, and EGF as did AA patients, when compared to healthy controls. These low plasma levels of cytokines/chemokines in AA and ITP may not reflect inflammation but rather the common feature of deficiency of platelets. Soluble CD40L appears to be a platelet agonist and may promote coagulation; CCL5-deficient mice model demonstrate that CCL5 blockade compromises systemic immune responses, delays macrophage-mediated viral clearance and impairs normal T cell function; CXCL5 attracts leukocytes and activates platelets; and EGF upregulates tissue factor and acts as a procoagulant. Levels of Tpo, G-CSF, VEGF, and eotaxin were not statistically different between ITP and healthy controls, but the levels of these cytokines were different between AA patients and healthy controls, indicating that Tpo levels are high when thrombocytopenia is due to megakaryocyte deficiency and low when due to increased platelet destruction. High Tpo, and G-CSF levels in AA represent a compensatory physiologic mechanism operating to counter impaired blood hematopoiesis. In addition, ITP patients showed higher levels of IP-10 than both AA patients and healthy controls, suggesting the involvement of interferon-gamma related responses in ITP. We compared cytokine gene expression in platelets, differentiated CD61+ cells (megakaryocytes), and CD61- cells (non-megakaryocytes) from bone marrow CD34+ cells of healthy controls by real-time PCR. Platelets and megakaryocytes showed higher mRNA expression of CXCL5 (440 and 16.3-fold), CD40L (9.6 and 51.9-fold), EGF (171 and 112-fold) and CCL5 (526 and 27.7-fold) than did CD61- cells (non-megakaryocytes), respectively. Together, our study provides evidence that platelets as well as megakaryocytes appear to be sources of CXCL5, CCL5, EGF, and CD40L; these cell types may contribute to the physiological levels of these cytokines. Similarities in cytokine levels between AA and ITP may reflect the autoimmune destruction of targets at varying stages of differentiation. Measurement of platelet-associated cytokines allows inferences concerning pathophysiology in quantitative platelet disorders. Download : Download high-res image (167KB) Download : Download full-size image Figure 1 . Comparison of plasma cytokine levels among patients with immune thrombocytopenia (ITP), aplastic anemia (AA), and healthy controls (HC). Cytokine levels in the plasma samples from 41 ITP, 33 untreated AA patients and 52 HC were measured using Luminex assay. The bars represent median values. ***, p Disclosures No relevant conflicts of interest to declare.

Hematologic Manifestations and Changes of Cytokines or Hematopoietic Growth Factors in Mice with Iron Overlaod.

Abstract 5103 BackgroundIron overload by repeated transfusions induced organ toxicity including liver, heart. We investigated hematologic manifestations and cytokines or hematopoietic growth factors in murine secondary hemochromatosis. Materials and methodsWe established murine secondary hemochromatosis model using 6 week-old male C57/BL6 (H-2b) with iron dextran. Mice (n=10∼12) were intraperitoneally injected with 10 mg of iron dextran for 2 or 4 weeks. We divided five groups: control (PBS injection), iron 100mg, iron 200mg, iron 200mg with deferasirox (DFX) 300mg, and only DFX 300mg. We examined hematocrit, platelet counts and plasma iron concentration (PIC) in peripheral blood, and liver iron contents (LIC) by atomic absorption spectrophotometer. We evaluated colony forming capacity from bone marrow according to experimental group. For cytokines and hematopoietic growth factors, we performed real-time PCR for IL-1b, iNOS, IFN-g, TNF-a, TGF-b, SCF, TPO, GM-CSF, and IL-11 in bone marrow. We compared each values of relative ratio with b-actin. ResultsThere was no difference of hematocrit among experimental groups. The platelet counts were significantly decreased in iron 200mg among groups (P<0.05), and showed increased trends after administration of DFX. The levels of LIC and PIC were dependent on cumulative dose of iron loaded, and decreased by DFX (P<0.01). This findings showed positive correlation between PIC and LIC (P<0.01, R2=0.726). The CFU-GEMM and CFU-GM decreased in iron 200mg, iron 200mg+DFX300mg, and DFX300mg compared with control and iron 100mg (P<0.01). Most colonies in DFX300mg were not observed except CFU-GM. In cytokines, there was shown no difference for IL-1b, iNOS, IFN-g, TNF-a, TGF-b according to experiments (P>0.05). However, SCF was shown diminished expressions for treated mice compared with control (P=0.02). The levels of TPO were increased in hemochromatosis, and decreased after administration of DFX (P=0.05). The GM-CSF was observed significantly lower in iron 200mg, iron 200mg plus DFX, DFX than control and iron 100mg (P<0.01). ConclusionsOur results suggested that iron overload might affect hematopiesis and these findings were due to effects of hematopoietic growth factors including SCF, TPO, GM-CSF, not inhibitory cytokines. Also, we need further study for DFX in hematopoiesis. DisclosuresNo relevant conflicts of interest to declare.

Plasma Cytokines Associated with Low Platelet Counts in Aplastic Anemia and Immune Thrombocytopenia.

Abstract 1317Poster Board I-333Thrombocytopenia occurs in aplastic anemia (AA) as a result of hematopoietic failure, in which decreased or absent megakaryocytes are observed in a hypocellular bone marrow. In immune thrombocytopenia (ITP), thrombocytopenia primarily occurs secondary to peripheral destruction and marrow cellularity is normal with megakaryocytes numerically normal or increased. In both AA and ITP, platelet function is normal and the disturbance in hemostasis is the result of the diminution in the platelet count. We recently reported that high plasma concentrations of Tpo and G-CSF, in combination with low levels of CD40L, CXCL5, CCL5, CXCL11, EGF, VEGF, and eotaxin, served as a cytokine signature profile for AA when compared to MDS or healthy controls (Blood 2008;112:380-381). In 8 patients with severe AA who responded to immunosuppressive therapy, the levels of CD40L, CXCL5, EGF, and CCL5 in plasma correlated with platelet recovery, as compared with absolute neutrophil count and hemoglobin recovery. We hypothesized that the plasma levels of CD40L, CXCL5, EGF, and CCL5 reflected platelets or their precursor megakaryocytes. We measured these cytokines in the plasma of 41 ITP patients using the Luminex assay, for comparison with 33 untreated AA patients and 52 healthy controls (Figure 1). Results were assessed by the Kruskal-Willis one-way analysis of variance statistic. ITP patients showed similar low levels of circulating CCL5, CD40L, CXCL5, and EGF as did AA patients, when compared to healthy controls. These low plasma levels of cytokines/chemokines in AA and ITP may not reflect inflammation but rather the common feature of deficiency of platelets. Soluble CD40L appears to be a platelet agonist and may promote coagulation; CCL5-deficient mice model demonstrate that CCL5 blockade compromises systemic immune responses, delays macrophage-mediated viral clearance and impairs normal T cell function; CXCL5 attracts leukocytes and activates platelets; and EGF upregulates tissue factor and acts as a procoagulant. Levels of Tpo, G-CSF, VEGF, and eotaxin were not statistically different between ITP and healthy controls, but the levels of these cytokines were different between AA patients and healthy controls, indicating that Tpo levels are high when thrombocytopenia is due to megakaryocyte deficiency and low when due to increased platelet destruction. High Tpo, and G-CSF levels in AA represent a compensatory physiologic mechanism operating to counter impaired blood hematopoiesis. In addition, ITP patients showed higher levels of IP-10 than both AA patients and healthy controls, suggesting the involvement of interferon-gamma related responses in ITP. We compared cytokine gene expression in platelets, differentiated CD61+ cells (megakaryocytes), and CD61- cells (non-megakaryocytes) from bone marrow CD34+ cells of healthy controls by real-time PCR. Platelets and megakaryocytes showed higher mRNA expression of CXCL5 (440 and 16.3-fold), CD40L (9.6 and 51.9-fold), EGF (171 and 112-fold) and CCL5 (526 and 27.7-fold) than did CD61- cells (non-megakaryocytes), respectively. Together, our study provides evidence that platelets as well as megakaryocytes appear to be sources of CXCL5, CCL5, EGF, and CD40L; these cell types may contribute to the physiological levels of these cytokines. Similarities in cytokine levels between AA and ITP may reflect the autoimmune destruction of targets at varying stages of differentiation. Measurement of platelet-associated cytokines allows inferences concerning pathophysiology in quantitative platelet disorders. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Thrombocytopenia in Patients With Chronic Hepatitis C: A Possible Role of HCV on Platelet Progenitor Cell Maturation

A total of 30 patients with chronic hepatitis C (HCV) thrombocytopenia (TP) and 20 healthy controls were studied. Both groups were subjected to complete medical history, clinical examination in addition to assessment of hepatitis markers: level of thrombopoietin (Tpo), Geimsa-stained bone marrow smears, and in vitro short-term megakaryocytic progenitors culture (CFU-MK). Serum Tpo level was significantly elevated in patients with TP HCV. Short-term CFU-MK showed an evident depression in the colony-forming unit-megakaryocyte (CFU-meg). There is a positive correlation between the number of CFU-meg and the platelet count and between serum Tpo level and prothrombin time, transaminase, albumin, and the Child Pugh score of liver disease; a negative correlation between serum Tpo level and the number of CFU-meg and between serum Tpo level and the platelet count. Thus, the level of Tpo could be an indicator of intact functional response of the hepatocytes.

Serum anti — TPO levels in benign and malignant breast tumors

Breast cancer is a hormone dependent neoplasm. Conflicting results regarding the clinical correlation between breast cancer and thyroid diseases have been reported. The objective of this study was to determine the association of anti - TPO levels in patients having complaints of a lump in breast. Serum samples and Fine needle aspiration cytology (FNAC) samples were collected from 31 female patients with a lump in breast between the age group of 20-75 years. 31 age matched normal healthy controls were also examined for the same parameters. Serum samples were analyzed for its anti - TPO levels. FNAC reports confirmed patients as having duct cell carcinoma. They had raised serum anti - TPO levels compared to controls. FNAC results of others (n=26) were reported as fibroadenoma whose anti - TPO levels were less than the controls.

Reticulated platelets and thrombopoietin in schistosomiasis patients

Schistosomiasis mansoni is a non-cirrhotic liver disease. In cirrhosis patients with portal hypertension, a decreased number of reticulated platelets associated with increased thrombopoietin serum levels were reported. We previously reported a 120/nl platelet cutoff level as a marker of clinically significant portal hypertension in schistosomiasis patients. To evaluate reticulated platelet counts and thrombopoietin serum levels (TPO) in schistosomiasis patients and correlate them with portal hypertension markers. Thirty-three schistosomiasis patients without co-morbidities were endoscopically classified as those with (n = 19) or without (n = 14) clinically significant portal hypertension. Flow cytometric determination of reticulated platelets was performed using CD41 antibody and thiazol orange. Ultrasonographic examinations were performed according to the Niamey protocol. TPO and hyaluronic acid serum levels were determined in duplicate using ELISA methods. The platelet number of 120/nl discriminates the two groups with 100% accuracy and 100% positive and negative predictive values, and correlates with spleen length and portal and splenic vein diameters. Differences in reticulated platelets and hyaluronic acid serum levels between both groups were significant (P = 0.025 and 0.012, respectively), but thrombopoietin serum levels were not (P = 0.769). Schistosomiasis patients with portal hypertension have increased reticulated platelets associated with normal TPO serum levels.

Spontaneous Resolution of Extreme Essential Thrombocytosis in a Child

Essential Thrombocytosis (ET) is rare in children and its clinical course is considered to be different from the adult disease. Unlike adults, most children with ET do not need treatment; however, children with extreme thrombocytosis present the treating physician with a therapeutic dilemma. We report a girl with a spontaneous resolution of extreme essential thrombocytosis. A 13 year old previously healthy girl presented to our ER with a 3 months history of intermittent mild abdominal pain with short episodes of diarrhea, intermittent episodes of paraesthesia in lower limbs and 3000 × 109/L platelets on CBC with pseudohyperkalemia of 6.4 mEq/L. Physical examination was unremarkable except for some diffuse mild tenderness on abdominal palpation. Laboratory work-up revealed normal WBC and Hb, Platelets 4100 × 109/L, mild microcytosis, Iron - 21.9 mcg/dL, transferin saturation 5.6%, serum potassium − 6.5 mEq/L, LDH 1000 IU. Liver, renal and thyroid function tests were within normal limits. PTT was mildly elevated 49.3 seconds (normal – 24.3–35), LAC-R mildly positive – 1.205 (0.8–1.20), KCT 58.2 sec. Anticardiolipin IgG −4.82 U/ml (negative), IgM - 11.24 U/ml (negative < 7 U/ml). Anti dsDNA and Anti nuclear antibodies were negative. Studies for hypercoagulable state included Protein C, Antitrombin, Factor VIII, IX, XI, Factor V R506Q, and Apolipoprotein a; all were normal. A heterozygosity for PT G20210A was found, as well as homozygosity for MTHFR C667T with a normal level of Homocysteine (7.7mmole/L). Bone marrow aspiration showed increased cellularity of all cell lines, with many young megakaryocytes including some in clusters. No malignant cells were seen. Reticulin score on bone marrow biopsy was 0. Platelets function tests with adenosine di-phosphate and epinephrine were normal. Serum TPO level was 31.0 pg/ml (normal). A karyotype examination revealed a normal 46XX female. FISH for BCR/ABL was negative. DNA mutations for JAK2 on both the 12 and the14 exons and c-mpl mutations were not found. Spontaneous clone formation was normal. Helicobacter pylori IgG was 2.89 U/ml (normal) and stool was negative for parasites. Imaging studies included high resolution abdominal contrast CT scan, upper gastrointestinal tract imaging with gastrografin, Doppler sonography of splanchnic vessels and capsule endoscopy; all were interpreted as normal. Aspirin 75 mg/d was started without antimetabolite therapy. Abdominal pain and diarrhea gradually improved. After 3 month of antiagregant therapy, platelets count declined, gradually but steadily. One year after presentation the child is asymptomatic with platelet count of 600 × 109/L. The spontaneous remission of the extreme ET achieved by conservative treatment with no complications adds to the growing evidence, that childhood ET is a unique entity with different biology than adult ET, which might not need antimetabolite therapy.

Increased Myelofibrosis in Thrombospondin-1-Null Mice Is Associated with Enhanced TGF-β1-Mediated Response by Bone Marrow Fibroblasts

In Primary Myelofibrosis, several lines of evidence suggest that pleiotropic cytokine TGF-β1, released by clonal proliferation of pathological megakaryocytes and/or monocytes, plays a prominent role in reticulin fibers deposition. This cytokine is synthesized as a biologically inactive molecule that needs to be activated in order to trigger biological responses. However, the mechanisms involved in local TGF-β1 activation within the hematopoietic environment remain unclear. Since TGF-β1 and thrombospondin-1 (TSP-1) are synthesized and stored within the same organelles in megakaryocytes, one can speculate that the abnormal release of both molecules leads to pathological local TGF-β1 activation that becomes ultimately responsible of fibrosis development in the vicinity of these cells. To investigate the role of TSP-1 in local TGF-β1 activation, we used the TPOhigh murine model of bone marrow (BM) fibrosis. BM cells from wild-type (WT) or Tsp-1-null male littermates were infected with a retrovirus encoding the murine TPO protein and engrafted into lethally irradiated WT or Tsp-1-null female hosts, respectively, leading to the following engraftment combinations, WT/WT (WT TPOhigh mice, n=21) and Tsp-1-null/Tsp-1-null (Tsp-1-null TPOhigh mice, n=17). Lethally-irradiated hosts were engrafted with 4 to 8 × 106 cells in 3 independent experiments. Peripheral blood was analyzed every 4 weeks during 3 months and mice were killed for histological analysis at week 8 and 12 post-engraftment. The magnitude of plasma TPO level increase was comparable regardless of the TPOhigh mice groups. Chimerism levels, analyzed in recipients by FISH on the presence of the donor Y chromosome in whole nucleated BM cells, were more than 90% in either WT or Tsp-1-null TPOhigh mice. We report here that all TPOhigh mice developed a similar myeloproliferative syndrome associated with TGF-β1 overproduction. Surprisingly, we were able to detect the active form of TGF-β1 in BM and spleen extracellular fluids in all mice, including Tsp-1-null TPOhigh mice, suggesting that alternative mechanisms are mainly responsible for local TGF-β1 activation in this murine model of myelofibrosis. We then confirmed that Tsp-1-null platelets are able to activate TGF-β1 in vitro in response to thrombin. As predicted by the detection of the active form of TGF-β1, Tsp-1-null TPOhigh mice developed BM and spleen fibrosis which appears, intriguingly, to be of a greater grade than the one displayed by WT TPOhigh mice. Since TSP-1 is a potent inhibitor of angiogenesis, we investigate whether this increased fibrosis could be correlated with an augmentation of neoangiogenesis. The microvascular density (MVD) in control Tsp-1-null BM were higher than in control WT one (10±4.7 vs 0.6±0.2; p<0.001), as expected. However, MVD displayed by Tsp-1-null TPOhigh mice (8.3±4.4) did not rise above the one displayed by control Tsp-1-null mice and was similar to MVD observed in WT TPOhigh mice (5.7±2.9). Thus, the increase of myelofibrosis in Tsp-1-null TPOhigh mice cannot be explained by an augmentation of neoangiogenesis. Since TGF-β1 levels were similar in both TPOhigh groups, we hypothesized that this increase could be related to an enhanced TGF-β1-mediated response by Tsp-1-null BM fibroblasts. Indeed, we could show that Tsp-1 deficiency is associated with sustained phospho-Smad3 levels and a 10-fold increase in collagen III transcription level by BM fibroblasts in response to TGF-β1. Together, our resultsshow that TSP-1 is not the major activator of TGF-β1 in this in vivo model of myelofibrosis;suggest that other mechanisms are involved in this activation;shed light on a possible new mechanism of TGF-β1 regulation by one of its own activator.

Effects of Sepsis on Neonatal Thrombopoiesis

We serially evaluated the effects of sepsis and/or necrotizing enterocolitis (NEC) on neonatal thrombopoiesis, using a panel of tests that included platelet counts, thrombopoietin concentrations (Tpo), circulating megakaryocyte progenitor concentrations (CMPs), and reticulated platelets (RPs). Variables analyzed included sepsis type, time after onset of sepsis, platelet counts, and gestational (GA) and postconceptional ages (PCA). Twenty neonates were enrolled. Ten had Gram-negative, six had Gram-positive, and four had presumed sepsis. Four neonates had NEC stage II or higher, and six developed thrombocytopenia. Overall, septic neonates had significantly elevated Tpo concentrations and circulating megakaryocyte progenitors. The highest Tpo levels were associated with Gram-negative or presumed sepsis. RP percentages were increased only in neonates with low platelet counts, while RP counts (RP% x platelet count) were elevated in neonates with high platelet counts. Our findings suggest that septic neonates up-regulate Tpo production, leading to increased megakaryocytopoiesis and platelet release, although the degree of upregulation is moderate. The changes in RP% and RP count most likely reflect increased thrombopoiesis with variable degrees of platelet consumption. In addition, our findings suggest that different factors, likely including level of illness and/or specific platelet or bacterial products, can down-regulate the magnitude of the thrombopoietic response.

Familial essential thrombocythemia with spontaneous megakaryocyte colony formation and acquired JAK2 mutations

Essential thrombocythemia (ET) is a chronic myeloproliferative disorder, characterized by increased proliferation of megakaryocytes and elevated platelet count that usually occurs sporadically. We report a family with seven affected individuals in three generations, including one individual with a phenotype resembling polycythemia vera, a related disorder. Megakaryocyte (CFU-MK) colony formation occurred in the absence of added cytokines in cultures of peripheral blood from affected family members. Some reports of familial ET have identified mutations in THPO and MPL, the genes for a cytokine (thrombopoietin, TPO) that regulates platelet production and its receptor (c-MPL), respectively. In this family, the MPL gene was excluded by linkage analysis. Although TPO levels were elevated in most affected family members and evidence for linkage was found between the disease and THPO (theta=0.0, Z(max)=3.0), a THPO mutation was not identified by DNA sequencing. The JAK2 V617F mutation that has been associated with 50% of sporadic cases of ET was identified as a somatic mutation, an acquired defect, in peripheral blood of the two most severely affected family members. These patients also had elevated TPO levels. Further study of familial myeloproliferative diseases will help elucidate the initiating genetic events underlying ET.

425: Parity in relation to antithyroid peroxidase antibodies

CATHERINE SPONG, DONALD MCINTIRE, KENNETH LEVENO, F. GARY CUNNINGHAM, University of Texas Southwestern Medical Center at Dallas, Dallas, Texas, National Institutes of Health (NIH), Bethesda, Maryland OBJECTIVE: Antithyroid peroxidase antibodies (TPO) are associated with chronic thyroiditis. Since hypothyroidism occurs more commonly in women, it has been hypothesized that fetal-to-maternal cell trafficking is responsible for this gender disparity. If so, this suggests a link between parity and TPO status and our objective was to evaluate the relationship between maternal parity and TPO. STUDY DESIGN: Between November 2000 and April 2003, all women who presented for prenatal care underwent thyroid screening. Excess serum was frozen and stored at 80°C. Serum samples from women without clinical hypothyroidism who had been screened in the first 20 weeks of gestation and who delivered a singleton infant weighing 500 g or more were analyzed for concentrations of thyroid peroxidase antibodies. Serum TPO levels were determined using a chemiluminescent assay (Immulite 2000 Analyzer, Diagnostic Products Corporation). Maternal parity was evaluated in relationship to positive TPO level ( 50 IU/mL). RESULTS: Serum samples from 17,298 women were tested. Of these, 1,012 (6%) women were identified with TPO. Women with TPO were older and more often parous. Figure A: After adjustment for maternal age, TPO status and parity were not related (P .92). Figure B: After adjustment for parity, TPO status and age remained significantly associated (P .001).

Thyroid dysfunction in patients with systemic lupus erythematosus (SLE): relation to disease activity

We examined the prevalence of thyroid dysfunction and the production of anti-thyroid antibodies (ATA) in patients with systemic lupus erythematosus (SLE) and assessed the association between ATA production and SLE disease activity status. Seventy-seven patients who met the American College of Rheumatology classification criteria for SLE participated in the study. Fifty-two individuals served as a control group. Demographic, clinical information and SLE disease activity (SLEDAI) status were collected from all patients. The sera of all participants were tested for free thyroxine (FT4), thyroid-stimulating hormone (TSH), anti-thyroglobulin (ATg) and anti-thyroid peroxidase (TPO). A SLEDAI score of > or =6 was considered clinically significant. The results of the thyroid function tests and ATA were compared between the study group and the control group. ATA levels were compared between the patients with a SLEDAI score of > or =6 to those with a SLEDAI score of <6. Hypothyroidism was detected in 11.6% of SLE patients compared to 1.9% in the control group. None of the patients or controls had evidence of hyperthyroidism. No statistically significant difference was observed in the levels of ATg or TPO between the study group and the control group. No correlation was found between ATA levels and the degree of the disease activity. Among the different variables tested in this study, hypothyroidism was the only significant abnormal finding in SLE patients. No association was found between the SLEDAI score and the prevalence of ATA production. Larger controlled, longitudinal studies are necessary to confirm these findings and elucidate the role played by ATA in the pathogenesis of thyroid dysfunction in SLE patients.

Thrombopoietin is not uniquely responsible for thrombocytosis in inflammatory disorders

A few studies in patients with reactive thrombocytosis identified levels of the hormone higher than expected, and suggested that TPO behaves as an acute-phase protein and was responsible for increased platelet count. At the opposite, other studies did not find any significant rise of the hormone in patients who similarly developed reactive thrombocytosis. To gain further information on this topic, we compared TPO levels and platelet counts in two series of patients hospitalized for acute illnesses: one with strong elevation of both erythrocyte sedimentation rate (ESR) and C-reactive protein (CRP), and the other with normal values. Within the group of subjects with high ESR and CRP, 38 had normal platelet counts, while 15 had thrombocytosis. No thrombocytosis was observed in control patients. Patients with high acute phase indexes had significantly higher TPO levels and platelet counts than control patients. We identified significant positive correlations between ESR and CRP, and between TPO and CRP. Interestingly, no significant relationship between platelet counts and TPO levels was find. When we grouped patients with acute-phase reaction according to absence or presence of thrombocytosis, we found similar TPO values. Conversely, positive correlations between platelet count and IL-6 and between TPO and IL-6 have been identified. All together our results confirm that TPO acts as an acute phase protein but exclude the possibility that it is uniquely responsible for thrombocytosis of inflammatory disorders, which might recognize in IL-6 a credible candidate as a cooperating factor.