Differential Expression of Markers of Apoptosis in Platelets From Children with Acute Versus Chronic Immune Thrombocytopenia

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 1092-1092
Author(s):  
Markus Schmugge ◽  
Jeanine Winkler ◽  
Sabine Kroiss ◽  
Margaret L. Rand ◽  
Oliver Speer
Keyword(s):  
Platelet Count ◽  
Immune Thrombocytopenia ◽  
Caspase 3 ◽  
Pediatric Patients ◽  
Caspase 8 ◽  
Caspase 9 ◽  
Platelet Apoptosis ◽  
Chronic Itp ◽  
Acute Itp

Abstract Abstract 1092 Immune thrombocytopenia (ITP) is a common hematologic disorder in children that can lead to severe bleeding symptoms. In most children with ITP, platelet counts return to normal after weeks to months (acute ITP), however, in about 10–20% of patients, the low platelet counts persist for 12 months or longer (chronic ITP). No biological markers have been identified to predict the duration and/or severity of ITP. We have previously reported enhanced platelet apoptosis at the time of diagnosis of ITP in pediatric patients that was ameliorated after intravenous immunoglobulin (IVIg) (Winkler et al, Br J Haematol 2012;156:508–15). We have now investigated differences in the expression of markers of apoptosis in platelets from children with acute vs. chronic ITP. 23 pediatric patients with acute ITP were investigated and compared to 10 children with chronic ITP. In addition, from the initial group of acute ITP, 6 children developed chronic ITP and initial- and follow up results were compared. Markers of apoptosis, including activated caspase-3, caspase-8 and caspase-9, phosphatidylserine (PS) exposure, dissipation of the mitochondrial inner membrane potential (ΔYm), as well as microparticle formation, were analyzed by flow cytometry. At ITP diagnosis, the mean platelet count was 4×109/L (range: 1–14×109/L) and the proportions of platelets with activated caspase-3 (median, range) (20.4%, 1.4–64%, n=23), caspase-8 (16.7%, 1.0 – 42.7%, n=12) and caspase-9 (13.1%, 5 – 59.6%, n=12) were increased. While a higher mean platelet count was found in 10 children with chronic ITP (25×109/L, 4–60G/l), the proportions of platelets with activated caspase-3 (2.6%, 0.3–11.6%), caspase-8 (5.6%, 0.3–12.6%) and caspase-9 (4.3%, 0.3–15.6%) were significantly lower compared to children at diagnosis of acute ITP, but still higher compared to healthy controls (0.95%, 0 – 5.9%; 0.7%, 0.04 – 2.3% and 0.4%, 0.03 – 2.16%, respectively; n = 11) and children with thrombocytopenia due to chemotherapy (1.3%, 0.1 – 4.6%; 1.8%, 0.9 – 3.8%; and 1.8%, 0.6 – 2.9%, respectively; n = 11). Among the 6 children (26%) who developed chronic ITP from the initial cohort of 23 children, a mean platelet count of 29 (3–67×109/L) at >12 months after initial presentation was found. Except for one, none of the children with chronic ITP presented with bleeding symptoms; the median bleeding score was 2.5 (range: 1–3) at diagnosis and 1 (range: 0–2.5) at follow up during chronic ITP. In 5 of the children who developed chronic ITP, caspase activation was studied at diagnosis and at follow up >12 months after. In all of them, the proportions of platelets with activated caspase-3 (1.6%, 0.3–3.3%), caspase-8 (4.8%, 0.3–6.3%) and caspase-9 (4.1%, 0.3–7%) were found to be significantly lower at follow up compared to the time at diagnosis. In conclusion, although platelet apoptosis is enhanced at the time of diagnosis of pediatric ITP, this is not observed in platelets from patients with chronic ITP to the same degree. Further studies are needed to investigate other markers of apoptosis in platelets in the course of acute and chronic ITP. Disclosures: No relevant conflicts of interest to declare.

The North American Chronic Immune Thrombocytopenia Registry (NACIR): Demographics and Treatment Responses

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 2509-2509
Author(s):  
Rachael F. Grace ◽  
Ellis J. Neufeld ◽  
A. Kim Ritchey ◽  
Manjusha Kumar ◽  
Michael R. Jeng ◽  
...  
Keyword(s):  
Platelet Count ◽  
North American ◽  
Research Funding ◽  
Immune Thrombocytopenia ◽  
Evans Syndrome ◽  
Chronic Itp ◽  
The North ◽  
Acute Itp ◽  
Positive Direct

Abstract Abstract 2509 Background: Chronic pediatric immune thrombocytopenia (ITP) has an incidence of 1–2/100,000. Due to its low incidence, large studies in pediatric chronic ITP are difficult. This registry includes patients from 16 sites in the US and Canada and represents one of the largest longitudinal datasets of children/adolescents with chronic ITP. Objective: To describe the North American Chronic ITP Registry (NACIR) study population and evaluate univariate predictors of platelet count response to therapies, including IVIG, anti-D globulin (anti-D), steroids (5–14 day course), and splenectomy. Registry methods and patient characteristics: After local IRB approval, 550 patients with chronic ITP enrolled in the NACIR between January 2004 and June 2010. Eligibility included: ages 6 months-18 years at ITP diagnosis, clinical diagnosis of ITP, and ITP duration >6 months. Primary ITP was defined as isolated thrombocytopenia without associated conditions. Secondary ITP included those patients with immune thrombocytopenia associated with other immune-mediated medical conditions, including Evans Syndrome. Treatment response was defined as a post-treatment platelet count ≥50,000/uL within 7 days of IVIG, 10 days of anti-D, 14 days of steroids, and 30 days of splenectomy. 365 subjects had at least one 6 month follow-up report after enrollment; median duration of follow-up was 2.1 yrs. Demographics of participants include: 46% male; 84% Caucasian, 7% Black, and 7% Asian; and 20% Hispanic. Mean age at diagnosis of acute ITP was 8.7 ± SD 5.2 years. At the time of enrollment in NACIR, subjects had received a median of 2 prior treatments (range 0–7). Results: The median platelet count at diagnosis of acute ITP was 11,000/uL (IQR 6,000–31,000/uL) and at chronic ITP was 35,000/uL (IQR 18,000–66,000/uL). 69 (12.5%) patients had secondary ITP or Evans syndrome. Of those tested, 25.6% (98/359) of patients had a positive ANA (titer > 1:40), and 75/337 (22.3%) had a positive direct anti-globulin test (DAT). 27.5% of patients had an antecedent viral illness. Of the 550 subjects, 2 (0.4%) experienced life-threatening bleeding. Patients were treated as follows: 259 (47.1%) with steroids, 253 (46%) with IVIG, 189 (34.4%) with anti-D, and 64 (11.6%) with splenectomy. Overall responses to therapy included: 69.1% response to steroids, 74.3% response to IVIG, 66.7% response to anti-D, and 85.9% response to splenectomy. Univariate predictors of response to treatments are shown in Table I. Higher platelet count at chronic ITP diagnosis and DAT positive predicted a platelet response to a short course of steroids in univariate analysis. This was confirmed in multivariable analysis of potential confounders, using logistic regression with a backwards elimination procedure. Response to one type of therapy was often strongly associated with a response to a second therapy. Gender, ethnicity, race, older age, and platelets ≥20,000/uL at acute ITP diagnosis were not associated with response to any single therapy. Conclusion: The demographics and laboratory findings of the large, well characterized NACIR population are consistent with other reports of young people with chronic ITP. The novel finding that DAT positivity predicts steroid response, even with multivariable adjustment for confounders, provides evidence that the NACIR is a robust and useful tool for trying to predict response to ITP treatment strategies. Disclosures: Klaassen: Novartis: Research Funding; Cangene: Research Funding. Lambert: Cangene: Membership on an entity's Board of Directors or advisory committees.

The Role of Oxidative Stress in Pediatric Immune Thrombocytopenia

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 3319-3319
Author(s):  
Clara Lo ◽  
Bing Zhang ◽  
Kristina Cusmano-Ozog ◽  
Wendy Wong ◽  
Michael Jeng ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Immune Thrombocytopenia ◽  
Pediatric Patients ◽  
Protein Carbonyl ◽  
Carbonyl Content ◽  
Protein Carbonyl Content ◽  
Chronic Itp ◽  
Redox Capacity ◽  
Acute Itp

Abstract Abstract 3319 Background: An unpredictable subset of patients (∼20–30%) with pediatric immune thrombocytopenia (ITP) progress to chronic ITP; this increases the risk of morbidity and mortality from bleeding, long-term immunomodulation, and/or splenectomy. Furthermore, treatments such as chronic steroid therapy often result in intolerable side effects, raising the need for targeted therapies. We previously tested a novel list of genes that might predict progression to chronic ITP (Zhang et al Blood 2011). Oxidative stress (OS)-related pathways were among those most significantly perturbed in chronic ITP. For further evaluation of the role of OS in ITP, we measured glutathione as a marker of redox capacity and protein carbonyl content as a marker of oxidative cell damage. Methods: Pediatric patients with primary ITP were included, with exclusion of subjects with secondary thrombocytopenia, other autoimmune disorders (ie, lupus), or other chronic illnesses. Healthy pediatric volunteers were recruited as controls. Patients had blood draws within 1 month from ITP diagnosis. Reduced (GSH) to oxidized (GSSG) glutathione ratios were measured from whole blood by tandem mass-spectrometry. Protein carbonyl content (PCC) levels were measured from platelet-rich plasma by enzyme-linked immunosorbent assay (ELISA). Subjects were followed up to 15 months from diagnosis and monitored for disease resolution or progression. Chronic ITP was defined as thrombocytopenia (platelets <100,000/μL) lasting at least 12 months from diagnosis (Rodegheiro et al Blood 2009). Acute ITP was defined as thrombocytopenia resolving within 12 months from diagnosis. Statistical significance was defined as p<0.05. Results: Between July 2009 and December 2011, 67 pediatric patients with ITP were recruited. Thirty-four patients had acute ITP, and 33 patients progressed to chronic ITP. The median age of patients was 7 years (range 18 months – 17 years). Sixty-three percent were female, 37% were male. Twenty-four pediatric controls were also recruited (46% female, 54% male). The median age of controls was 8 years (range 5 years – 17 years). Patients with ITP had significantly lower GSH:GSSG ratios compared to controls, and patients with chronic ITP had lower GSH:GSSG ratios compared to those with acute ITP (Figure 1). Furthermore, patients with ITP had significantly higher PCC levels compared to controls (Figure 2). Conclusions: This data provides further evidence for a role of oxidative stress (OS) in the pathophysiology of ITP. Furthermore, decreased redox capacity, as evidenced by the decreased glutathione ratios, may be associated with progression to chronic ITP. Reactive oxidative species (ROS) may be important in the pathogenesis of autoimmunity in ITP; oxidatively altered cellular by-products induce pathogenic antibodies and become immunogenic. This also raises a potential anti-oxidant mechanism of therapy, which may play a greater role in chronic ITP treatment. Increased understanding of OS in pediatric ITP may reveal markers of disease progression, highlighting those at greatest risk for chronic ITP and creating a role for targeted therapy. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Plant Food Anthocyanins Induced Platelet Apoptosis Via BCL-2/BCL-XL Pathway

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 4988-4988
Author(s):  
Yang Yan ◽  
Ma Jing ◽  
Tian Jinju ◽  
Chen Liyi ◽  
Songmei Yin ◽  
...  
Keyword(s):  
Platelet Activation ◽  
Caspase 3 ◽  
Caspase 8 ◽  
Plant Food ◽  
Intrinsic Apoptotic Pathway ◽  
Caspase 9 ◽  
Cytochrome C Release ◽  
Apoptotic Pathway ◽  
Platelet Apoptosis ◽  
Activated Platelets

Abstract Background: Platelets are versatile cells and play important roles in hemostasis/thrombosis, inflammation, and atherosclerosis. The pathogenesis of cardiovascular diseases (CVDs) is linked to platelet hyperactivity which is considered an independent risk factor for CVDs. Platelets are critical for promoting the progression of CVDs, and platelet apoptosis have been reported to be involved in platelet activation. Anthocyanins are major phytochemicals abundant in plant food and have been shown to play a protective role against CVDs. Our previous studies demonstrated that anthocyanins from plant food significantly inhibited platelet activation, adhesion, aggregation and granule secretion, as well as attenuated thrombus growth at both arterial and venous shear stresses in vitro and in vivo, however, the effects of anthocyanin on platelet apoptosis and its mechanisms have not been explored. In the present study, we examined whether anthocyanin Cyanidin-3-glucoside (Cy-3-g) affect platelet apoptosis and the BCL-2/BCL-XL intrinsic apoptotic pathway. Methods: Cy-3-g, the predominant bioactive compound of anthocyanin preparations, was obtained from Polyphenol AS Company in Norway.Purified gel-filtered platelets from healthy volunteers were incubated at 37oC for 40 minutes with different concentrations of Cy-3-g (0.5、5、50μM) or PBS buffer as a control. the activated platelets were triggered with 0.5U thrombin for 15min to induce apoptosis. Mitochondria membrane potential (Δψm) and membrane phospholipid phosphatidylserine (PS) exposure in both activated and resting platelets were assessed by flow cytometry. Cytochrome C release, activation of caspase-3, caspase-8, caspase-9, cleavage of gelsolin, the levels of anti-apoptotic BCL-2 family proteins such as BCL-2, BCL-XL and proapoptotic BCL-2 family proteins Bax, Bak, Bad, Bid and tBid in both activated and resting platelets were measured by western blotting. Results: Cy-3-g at 5μM and 50μM directly induced significant ΔΨm dissipation in activated platelets dose dependently. Correspondingly, 50μM Cy-3-g increased cytochrome C release compared to control. The expression of pro-caspase-8 and pro-caspase-9 decreased, activation of caspase-3, caspase-8 and caspase-9 was induced in activated platelets in both 5μM and 50μM Cy-3-g groups. Both PS exposure and the cleavage of gelsolin increased in activated platelets, however these effects were only observed at Cy-3-g doses as high as 50μM. Cy-3-g did not induce the above changes in resting platelets. The intrinsic apoptotic pathway was initiated by Cy-3-g treatment in activated platelets; Cy-3-g significantly inhibited the expression of BCL-2, BCL-XL and increased the levels of Bax, Bak, Bad and Bid in activated platelets dose dependently. No significant difference was observed in resting platelets. Conclusions: Our data demonstrate for the first time that purified anthocyanin Cy-3-g directly accelerated apoptosis in activated platelets via the BCL-2/BCL-XL pathway. Anthocyanins may possess therapeutic potential for patients suffering from thrombotic conditions. Disclosures No relevant conflicts of interest to declare.

Can Anti-Thyroid Antibodies Influence the Outcome of Primary Chronic Immune Thrombocytopenia in Children?

2020 ◽  
Vol 20 (3) ◽  
pp. 351-355 ◽  
Author(s):  
Paola Giordano ◽  
Maurizio Delvecchio ◽  
Giuseppe Lassandro ◽  
Federica Valente ◽  
Valentina Palladino ◽  
...  
Keyword(s):  
Autoimmune Thyroiditis ◽  
Immune Thrombocytopenia ◽  
Pediatric Patients ◽  
Pediatric Population ◽  
Clinical Signs ◽  
Thyroid Antibodies ◽  
Chronic Itp ◽  
Immune Mediated

Background: Immune thrombocytopenia (ITP) is an acquired immune mediated disorder characterized by isolated thrombocytopenia. Pediatric ITP patients can develop autoantibodies such as anti-thyroglobulin (TG) and anti-thyroperoxidase (TPO), even in the absence of clinical signs of autoimmune disease. Objective: The purpose of this article is to provide a review about: 1) the prevalence of positivity of anti-thyroid antibodies (TPO and TG) in pediatric patients with chronic ITP; 2) the role of autoimmune thyroiditis on the outcome of chronic ITP. Method: The authors individually completed a review of the literature for this article. Retrospective and prospective clinical studies with pediatric cohorts were considered. Results: From the analysis of data, we found 4 papers which included studies only on pediatric population, and which corresponded to selected criteria. Pediatric ITP patients have been shown to have a statistically significant prevalence of anti-thyroid antibodies over healthy controls (11.6-36% versus 1.2-1.3%). No correlation has been found between the platelet count and the prevalence of positive anti-thyroid antibodies at any time of the follow up. Conclusion: The results of our bibliographic research demonstrated that: a) pediatric patients with chronic ITP tend to have a statistically significant prevalence of anti-thyroid antibodies positivity respect to general pediatric population; b) there are no clear data about the role of autoimmune thyroiditis as prognostic factor for chronic course of ITP in pediatric age.

scholarly journals Immunoglobulin Levels As Predictors of the Development of Chronic Disease in Pediatric Immune Thrombocytopenia

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 388-388
Author(s):  
Amanda Bell Grimes ◽  
Taylor Olmsted Kim ◽  
Jenny M. Despotovic ◽  
Susan Kirk
Keyword(s):  
Chronic Disease ◽  
Autoimmune Disease ◽  
Immune Thrombocytopenia ◽  
Spontaneous Resolution ◽  
Systemic Autoimmune Disease ◽  
Chronic Itp ◽  
Age Of Diagnosis ◽  
Acute Itp ◽  
Immunoglobulin Levels

Background: Immune thrombocytopenia (ITP) is the most common acquired bleeding disorder in children. Although ~75% of these patients will go on to have spontaneous resolution of disease, up to 25% will develop chronic ITP, with significant and prolonged bleeding symptomatology and impaired quality of life. At this time, there is no definitive method to predict the development of chronic disease at the time of ITP diagnosis. Aims: We aim to identify clinical biomarkers predictive of the development of chronic ITP at the time of diagnosis among pediatric ITP patients. Methods: The clinical records of 280 pediatric ITP patients enrolled in a biological banking study at a large pediatric tertiary care referral center from July 1, 2015 to July 1, 2019 were reviewed in accordance with IRB-approved protocols. ITP diagnosis and chronicity of disease (vs. spontaneous resolution within 1 year of diagnosis) was confirmed by a pediatric hematologist, as defined by current international expert committee standards (Neunert et al, Blood, 2011;117(16):4190-207; Provan et al, Blood, 2010;115(2):168-186). Patients with non-classical ITP were excluded (1 child with drug-induced ITP in the setting of acyclovir therapy, and 1 neonate with passive ITP in the setting of maternal lupus); and patients who were lost to follow-up or had ongoing disease of &lt;1 year duration were subsequently excluded. Patient characteristics including age, gender, ethnicity, presence of concurrent autoimmune disease, and time to resolution were collected. Pertinent biomarkers including immunoglobulin levels (IgG, IgA, and IgM), anti-nuclear antibody (ANA), C-reactive protein (CRP), and immature platelet fraction (IPF) which were obtained at the time of diagnosis were documented, if if obtained prior to the administration of any ITP-directed therapy, and within 2 weeks of diagnosis. Chi-squared test or Fisher's exact test was utilized to compare nonparametric categorical data. Mann-Whitney U test was used to compare nonparametric continuous data. A multivariate backwards logistic regression model was conducted to determine independent associations with the development of chronic ITP. Statistical analyses were performed using SPSS Statistics 26 (IBM, Armonk, New York). A Bonferroni correction was applied to correct for multiple comparisons. A p value &lt; 0.05 was defined as statistically significant. Results: We identified 251 pediatric ITP patients with sufficient length of follow-up to define acute (&lt;1 year) vs. chronic (&gt;1 year) disease within our 4-year study period. This included 132 patients with acute ITP (53%) and 119 patients who went on to develop chronic ITP (47%). Mean age of diagnosis among those with acute ITP was 5.5 years, while mean age of diagnosis among those with chronic ITP was 7.7 years. Within the entire cohort, 126 (50%) were male, and 124 (49%) were of Hispanic ethnicity. Fifty-eight had ITP attributable to systemic autoimmune disease - 17% within the Acute ITP group, and 30% within the Chronic ITP group. Among all patients (n=251), 188 with evaluable biomarkers (immunoglobulin levels, ANA, CRP, or IPF) at the time of diagnosis were identified. These included 174 patients with Ig levels at diagnosis, 43 patients with ANA titers at diagnosis, 25 patients with CRP levels at diagnosis, and 78 patients with IPF at diagnosis. By univariate analysis, we found that abnormalities in immunoglobulin levels at diagnosis were significantly associated with the development of chronic ITP. Low IgG levels (p = 0.015) were more prevalent in chronic (10.3%) vs. acute ITP (1.9%). High IgG levels (p = 0.015) were more prevalent in chronic (6.7%) vs. acute ITP (1.9%). High IgM levels (p = 0.03) were more prevalent in chronic (26.5%) vs. acute ITP (13.3%). Of note, the presence of Evans syndrome (p = 0.0005) and other systemic autoimmune disease (p = 0.011) were also significantly associated with the development of chronic ITP. Subsequent multivariate analysis identified low IgG level at diagnosis to be independently predictive of chronic ITP (p = 0.043, with an odds ratio of 5.7). Conclusion: Although further study is needed within a larger international pediatric ITP cohort, the findings from this institutional study indicate that biomarkers obtained in routine clinical care at the time of ITP diagnosis, specifically immunoglobulin levels, can be utilized to help predict development of chronic disease among pediatric ITP patients. Disclosures Despotovic: Amgen: Research Funding; Dova: Honoraria; Novartis: Research Funding.

scholarly journals Cytokine and Chemokine Levels Correlate with Apoptosis Markers in Platelets of Pediatric Patients with Immune Thrombocytopenia

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 1371-1371
Author(s):  
Nadine Goelz ◽  
Jeannine Winkler ◽  
Julia J.M. Eekels ◽  
Margaret L. Rand ◽  
Oliver Speer ◽  
...  
Keyword(s):  
T Cells ◽  
Platelet Count ◽  
Platelet Activation ◽  
Plasma Levels ◽  
Immune Thrombocytopenia ◽  
Caspase 3 ◽  
Gm Csf ◽  
Cytokines And Chemokines ◽  
Tnf Α ◽  
Acute Itp

Abstract Immune thrombocytopenia (ITP) is defined as an autoimmune disease that leads to platelet clearance by macrophages. It is well known that auto-reactive B cells and CD4+ T-helper (Th) cells, and in particular their cytokines, have been associated with ITP. Cytokines and chemokines are key players in our immune response to recruit different cells, e.g. macrophages and monocytes, to respond to certain inflammatory signals. Specific cytokines (i.e. TNF-a, IFN-g) are known to induce apoptosis in nucleated blood cells. To understand the role of cytokines and chemokines in acute ITP, we studied their plasma levels in 10 pediatric ITP patients at diagnosis with a median platelet count of 3 x 109/L (range < 1 to 22 x 109/L) and compared them with controls: healthy children (n=9; platelet count > 150 x 109/L); and chemotherapy-induced thrombocytopenia patients (cTP; n=9; median platelet count of 12 x 109/L; range: 3-53 x 109/L). ITP patients fulfilled the criteria for acute ITP and presented with mild to moderate bleeding symptoms. The median age at diagnosis was 3.4 yrs (range: 1.6 - 6.5 yrs); blood samples were taken prior to treatment. Luminex technology was used to measure plasma levels of 42 cytokines and chemokines. Markers of platelet apoptosis - activated caspase-3, -8 and -9 - and of platelet activation - CD62P and CD63 expression and PAC-1 binding - were measured by flow cytometry. Distinct plasma cytokine/chemokine patterns were observed in ITP patients compared with controls. Significantly increased levels of the Th1 cell commitment cytokines TNF-α (p < 0.01) and IFN-g (p < 0.05), as well as of the Th2 cytokines IL-6 (p < 0.01), IL-10 (p < 0.01) and IL-13 (p < 0.05), were identified in ITP patients. We have previously shown that there is activation of platelet caspase-3, -8 and -9 at diagnosis in acute paediatric ITP patients compared with controls (Winkler et al, Br J Haematol 2012;156:508). In ITP patients, but not in controls, a negative correlation between eotaxin and caspase- 3 (r2 = 0.72), -8 (r2 = 0.76) and -9 (r2 = 0.53) activity were observed, as well as a negative correlations between GM-CSF and caspase-8 (r2 = 0.52) and -9 (r2 = 0.33). Furthermore, we found a correlation between IL-13 and platelet activation as measured by CD62P (r2 = 0.87) and CD63 (r2 = 0.67) expression in ITP patients but not in controls. In summary, increased plasma levels of the cytokines TNF-α, IFN-g, IL-6, IL-10, and IL-13 were observed in ITP patients at initial presentation, suggesting that these cytokines contribute to the pathogenesis of the disease. Since IL-6 and IFN-g are known to activate macrophages, while higher levels TNF-α, IL-10 and IL-13 in the plasma are signs for activated T cells, our findings are consistent with the current model of ITP, in which activated macrophages induce B and T cells to produce anti-platelet autoantibodies. Our goal is to study the interplay between the immune system and the reduction of platelet count in ITP and to further define apoptosis/activation-related pathways in ITP platelets. Furthermore, we showed a correlation in ITP patients between the chemokine eotaxin and GM-CSF with caspase-3, -8 and -9 activity, and a correlation between IL-13 and platelet activation. Our results imply, that apoptosis and platelet activation at diagnosis in ITP play a role in the development of ITP, but the underlying mechanisms are still unknown and needs to be further evaluated by increasing the patient cohort in our study. Disclosures No relevant conflicts of interest to declare.

scholarly journals Response to Steroids Predicts Response to Rituximab In Pediatric Chronic Immune Thrombocytopenia.

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 3681-3681
Author(s):  
Rachael F. Grace ◽  
Carolyn M. Bennett ◽  
A. Kim Ritchey ◽  
Michael R. Jeng ◽  
Courtney Thornburg ◽  
...  
Keyword(s):  
Platelet Count ◽  
Research Funding ◽  
Immune Thrombocytopenia ◽  
Univariate Analysis ◽  
Multivariable Analysis ◽  
Evans Syndrome ◽  
Chronic Itp ◽  
Predictors Of Response ◽  
Count Response ◽  
Acute Itp

Abstract Abstract 3681 Background: Pediatric Immune Thrombocytopenia (ITP) has an incidence of 4–6/100,000 with 1/3 of cases becoming chronic. Treatment choice is arbitrary, because few studies are powered to identify predictors of therapy response. Increasingly, rituximab is becoming a treatment of choice in those refractory to other therapies (Neunert CE, et al. Pediatr Blood Cancer 2008; 51(4):513). Previous studies in ITP have not examined predictors of response to rituximab or whether response to prior treatments predicts response. Objective: To evaluate univariate and multivariable predictors of platelet count response to rituximab. Methods: After local IRB approval, 550 patients with chronic ITP enrolled in the longitudinal, North American Chronic ITP Registry (NACIR) between January 2004 and June 2010. Eligibility included: ages 6 months-18 years at ITP diagnosis, clinical diagnosis of ITP, and ITP duration >6 months. Primary ITP was defined as isolated thrombocytopenia without associated conditions. Secondary ITP included those patients with immune thrombocytopenia associated with other immune-mediated medical conditions, including Evans Syndrome. Treatment response was defined as a post-treatment platelet count ≥50,000/uL within 16 weeks of rituximab and within 14 days of steroids. Steroids were prescribed as 1–4 mg/kg prednisone or adult equivalent over 4–14 days with or without taper. The NACIR captured treatment responses both retrospectively prior to enrollment and then prospectively, and both periods were included in this analysis. The multivariable logistic regression modeling process utilized SAS 9.1 using binary variables which were either significant in the univariate analysis or clinically important. A backwards elimination procedure was used to select the final model. Results: Seventy-six (13.8%) patients were treated with rituximab. Demographics of the patients treated with rituximab include: 42% male; 81% Caucasian, 17% Black, and 2% Asian. The mean age at diagnosis of ITP was 8.4 ± SD 5.1 years. The median platelet count at diagnosis of acute ITP was 10,000/uL (IQR 5,000-20,000/uL). 19 (25%) patients had secondary ITP or Evans syndrome. Treatment with rituximab had an overall response rate of 63.2% (48/76). Univariate predictors of response to rituximab are shown in Table I. The strongest univariate predictor of response to rituximab was response to steroids. Gender, ethnicity, and race were not predictive of response to rituximab. Furthermore, other variables which did not predict rituximab response include: history of a bleeding score ≥3 (Buchanan and Adix, J Pediatr 2002; 141: 683), symptoms ≥1 month prior to ITP diagnosis, older age (age >5 years), platelets ≥20,000/uL at acute ITP diagnosis, and a positive ANA. In multivariable analysis, response to steroids remained a strong predictor of response to rituximab with an OR 6.2 (95% CI 1.8–21.3, p=0.004). Secondary ITP also remained a strong a predictor of a positive response to rituximab with an OR 5.9 (95% CI 1.2–33.3, p=0.03). Conclusion: In the NACIR, response to steroids and secondary ITP were strong predictors of response to rituximab, a finding not previously reported in children or adults. Although this finding requires further validation, this result may provide evidence that rituximab should be most considered in patients previously responsive to steroids. Disclosures: Off Label Use: Rituximab for chronic ITP. Lambert:Cangene: Membership on an entity's Board of Directors or advisory committees. Klaassen:Novartis: Research Funding; Cangene: Research Funding. Neufeld:Novartis, Inc: Research Funding.

scholarly journals A Comparative Prospective Observational Study of Children and Adults with Immune Thrombocytopenia: 2-Year Follow-up

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 3741-3741 ◽  
Author(s):  
Thomas Kuehne ◽  
Alexandra Schifferli
Keyword(s):  
Platelet Count ◽  
Research Funding ◽  
Immune Thrombocytopenia ◽  
Initial Diagnosis ◽  
Diagnostic Methods ◽  
Newly Diagnosed ◽  
Time Points ◽  
Platelet Counts ◽  
Chronic Itp

Abstract Introduction It is widely accepted that immune thrombocytopenia (ITP) of children differs from that of adults in the clinical course, such as the rate of spontaneous remission, the bleeding risk and the need of treatment. However, this assumption is limited by incongruity of study populations and divergences of collected information, definitions, study objectives and end-points. Surprisingly, data of the Pediatric and Adult Registry on Chronic ITP (PARC-ITP) at initial diagnosis demonstrated far less differences in clinical and laboratory findings between children and adults than expected (Kühne et al. Haematologica 2011). This suggests that newly diagnosed ITP may be driven by similar pathophysiological mechanisms. Differences may occur in the ability of restoring tolerance. We analyzed 6-, 12-, and 24-month follow-up data of children and adults recorded in the PARC-ITP Registry. Design and Methods PARC-ITP is an international multi-center registry designed to collect data prospectively of children and adults with newly diagnosed ITP, and was opened in May 2004. Demographic information, diagnostic methods, clinical data, and efficacy and safety of management are continuously registered at the time of diagnosis, 6 and 12months and then yearly. Patients younger than 3 months (n=167) and those with a platelet count of >100x109/l were excluded from the analysis. Patients with missing follow-up data at certain time-points were not excluded. Remission of ITP was defined as a platelet count of >100x109/l at any time point and regardless of therapy. Platelet counts of chronic ITP were defined as being <100x109/l at 12 or 24 months. The data were analyzed with descriptive statistics. Results A total of 3'780 evaluable patients with the initial diagnosis of primary ITP were recorded in the PARC-ITP database between 2004 and 2015. There were 3360 children (3 months - 16 years) and 420 adults (≥16 years). The pediatric female: male ratio was 1:1.09, and that of adults was 1:0.54. Follow-up information was available for 67% of children at the 6-month, 49% at the 12-month and 31 % at the 24-month evaluation and in adults in 77%, 64%, and 47%, respectively. In children remission was seen at 6, 12 and 24 months in 70%, 70%, and 71%, and in adults in 45%, 49%, and 56%, respectively. Of the patients with a platelet count of <100x109/l at 6 months, 212/590 children (36%) and 42/152 adults (28%) achieved again a remission at 12-months. The platelet counts of children and adults with chronic ITP at 12 months were 46±30x109/l and 51 ±26x109/l. Adults with a diagnosis of chronic ITP at 12 and 24 months reported having no bleeding in 69% and 65% for the last follow-up period, children in 37% at both time-points. Children with thrombocytopenia at 6, 12 and 24-months received platelet-enhancing drugs in 58%, 46% and 47% and adults in 58%, 52% and 40%, respectively. The diagnosis of secondary ITP and other causes of thrombocytopenia was reported for 123 children, i.e. 3.5%, 1.9% and 1.3% at 6, 12 and 24 months, respectively and 21 adults, i.e. 3.7%, 2.3% and 1.7% at 6, 12 and 24 months, respectively. The reported cause was an infectious disease in both children (49%) and adults (52%). Discussion The PARC-ITP Registry is the first cohort of ITP patients including a mixed pediatric and adult population. Limitations include the variety of participating centers (n=74), data registration on a voluntary basis, a high percentage of loss of follow-up and an unbalanced number of children and adults. Preliminary analyses of follow-up data demonstrate similarities between children and adults in much more areas, than previously assumed. Differences in remission rates where confirmed but in a smaller extent than expected. Treatment requirement in patients with active disease was very similar in both age groups. Surprisingly, adults with a diagnosis of chronic disease exhibited a greater number of a non-bleeding phenotype than children. Conclusion Understanding differences or similarities among children and adults with ITP may guide in finding immune modulatory strategies with the goal of achieving early sustained responses. Disclosures Kuehne: Amgen: Research Funding; UCB Biosciences GmbH: Consultancy. Schifferli:Amgen: Research Funding.

Efficacy and Safety Of Eltrombopag In Korean Adult Patients With Refractory Chronic Immune Thrombocytopenia

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 4758-4758
Author(s):  
Yeo-Kyeoung Kim ◽  
Sung-Hoon Chung ◽  
Jae-Sook Ahn ◽  
Deok-Hwan Yang ◽  
Il-Kwon Lee ◽  
...  
Keyword(s):  
Platelet Count ◽  
Treatment Outcome ◽  
Maintenance Dose ◽  
Immune Thrombocytopenia ◽  
Conflicts Of Interest ◽  
Platelet Counts ◽  
Chronic Itp ◽  
Insufficient Response ◽  
Korean Adult

Introduction Eltrombopag is a synthetic non-peptide thrombopoietin receptor agonist (TPO-RA), which shows an excellent treatment outcome in immune thrombocytopenia (ITP) patients. For East Asians, starting dose of eltrombopag is generally recommended as a 25 mg/day and maximal dose is restricted as a 50 mg/day because of higher plasma eltrombopag AUC values. Furthermore, sensitivity to TPO-RA may increase or decrease over time. Here, we attempted to analyze the effective eltrombopag doses to achieve and maintain safe platelet counts in Korean ITP patients. Methods A total of twelve adult chronic ITP patients were enrolled, which showed insufficient response to previous ITP treatments (platelet counts less than or equal to 30,000/uL). All patients were allowed to take concomitant low dose prednisolone or danazol. Ten patients started eltromobopag at a dose of 25 mg/day and two patients started 25 mg every other day (EOD) because of previously detected hepatic problem. The doses increased every two weeks to achieve a target platelet count (equal to or more than 50,000/uL). For patients achieving platelet count within the range of 50,000 to 200,000/uL, firstly, we reduced concomitant ITP medications and then reduced eltrombopag doses to find the lowest effective dose of eltrombopag to maintain platelet counts more than 50,000/uL (maintenance dose). Results Before starting eltrombopag treatment, patients received median 4 (ranges; 3-9) ITP treatments including 1 case of splenectomy (Table 1). Bone marrow (BM) examinations including reticulin / Masson-trichrome staining were performed and all patients confirmed not to have BM fibrosis before eltrombopag treatment. Follow-up BM examinations were performed at 1 and 2 year of eltrombopag medication. Of total 12 patients, 10 (83.3%) achieved platelet counts more than 100,000/uL, 1 (8.3%) achieved platelet between 50,000-100,000/uL and 1 (8.3%) failed to increase platelet counts. One failed patient was diagnosed as ITP 114 months ago and received eight ITP treatments before starting eltrombopag. Eleven (91.7%) patients who achieved the target platelet counts (equal to or more than 50,000/uL) could quit or reduce the dose of concomitant ITP medications and median time to achieve the target platelet counts was 21 days (6-151 days). Most common initially required dose to achieve the target platelet counts was 25 mg/day (63.6%). Others were 25 mg EOD (9.1%), 50 mg/day (18.2%) and 75 mg/day (9.1%). After achieving the target platelet counts, most common adjusted maintenance dose was 25 mg/day (63.6%). Others were 25 mg twice a week (27.3%) and 50 mg/day (9.1%). There was no more than gr. 2 bleeding episode during eltrombopag treatment. One patient who required 75 mg/day to achieve the target platelet was diagnosed as ITP 91 months ago and received nine ITP treatments before starting eltrombopag. In 11 responded patients, 9 discontinued eltrombopag medication. Among them, 6 (66.7%) were relapsed and median relapse-free survival (RFS) was 11 days (6-574 days). In an aspect of adverse events, seven (58.3%) showed hepatobiliary laboratory abnormalities (HBLA, two gr. 3), however, all HBLA resolved after reduction or short-term discontinuation of eltrombopag. Eight patients underwent follow-up BM assessment. In 2 year BM, one patient revealed gr. 1 BM fibrosis during eltrombopag medication, however, there was no significant hematologic abnormality or lactate dehydrogenase (LDH) elevation in peripheral blood and no definitive abnormal immature cell clusters in his BM specimen. Conclusions Eltrombopag was generally well tolerated and showed an excellent treatment outcome in refractory chronic ITP patients in Korea. Low doses eltrombopag (25 mg/day or 25 mg twice a week) were effective to maintain safe platelet counts in most Korean patients. However, some of the patients needed longer time and higher doses (50 or 75 mg/day) to initially achieve and to maintain the target platelet counts, especially in heavily pre-treated patients with longer time from diagnosis to starting eltrombopag treatment. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Inclusion Complex of Zerumbone with Hydroxypropyl-β-Cyclodextrin Induces Apoptosis in Liver Hepatocellular HepG2 Cells via Caspase 8/BID Cleavage Switch and Modulating Bcl2/Bax Ratio

2013 ◽  
Vol 2013 ◽  
pp. 1-16 ◽  
Author(s):  
Nabilah Muhammad Nadzri ◽  
Ahmad Bustamam Abdul ◽  
Mohd Aspollah Sukari ◽  
Siddig Ibrahim Abdelwahab ◽  
Eltayeb E. M. Eid ◽  
...  
Keyword(s):  
Inclusion Complex ◽  
Mitochondrial Membrane ◽  
Morphological Study ◽  
Caspase 3 ◽  
Anticancer Agent ◽  
Caspase 8 ◽  
Caspase 9 ◽  
Solubility In Water ◽  
Anticancer Effects ◽  
First Time

Zerumbone (ZER) isolated fromZingiber zerumbetwas previously encapsulated with hydroxypropyl-β-cyclodextrin (HPβCD) to enhance ZER’s solubility in water, thus making it highly tolerable in the human body. The anticancer effects of this new ZER-HPβCD inclusion complex via apoptosis cell death were assessed in this study for the first time in liver hepatocellular cells, HepG2. Apoptosis was ascertained by morphological study, nuclear stain, and sub-G1 cell population accumulation with G2/M arrest. Further investigations showed the release of cytochrome c and loss of mitochondrial membrane potential, proving mitochondrial dysfunction upon the ZER-HPβCD treatment as well as modulating proapoptotic and anti-apototic Bcl-2 family members. A significant increase in caspase 3/7, caspase 9, and caspase 8 was detected with the depletion of BID cleaved by caspase 8. Collectively, these results prove that a highly soluble inclusion complex of ZER-HPβCD could be a promising anticancer agent for the treatment of hepatocellular carcinoma in humans.

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