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 Extracellular purine metabolism and signaling of CD73-derived adenosine in murine Treg and Teff cells

2011 ◽  
Vol 301 (2) ◽  
pp. C530-C539 ◽  
Author(s):  
Michael Romio ◽  
Benjamin Reinbeck ◽  
Sabine Bongardt ◽  
Sandra Hüls ◽  
Sandra Burghoff ◽  
...  
Keyword(s):  
T Cells ◽  
Proinflammatory Cytokines ◽  
Wild Type ◽  
Murine Splenocytes ◽  
Gm Csf ◽  
A2a Receptors ◽  
Cytokines And Chemokines ◽  
Tnf Α ◽  
Ifn Γ ◽  
Macrophage Colony

CD73-derived adenosine acts as potent inhibitor of inflammation, and regulatory T cells (Treg) have been shown to express CD73 as a novel marker. This study explored the role of endogenously formed adenosine in modulating NF-κB activity and cytokine/chemokine release from murine Treg and effector T cells (Teff) including key enzymes/purinergic receptors of extracellular ATP catabolism. Stimulating murine splenocytes and CD4+ T cells with anti-CD3/anti-CD28 significantly upregulated activated NF-κB in CD73−/− T cells (wild type: 4.36 ± 0.21; CD73−/−: 6.58 ± 0.75; n = 4; P = 0.029). This was associated with an augmented release of proinflammatory cytokines IL-2, TNF-α, and IFN-γ. Similar changes were observed with the CD73 inhibitor APCP (50 μM) on NF-κB and IFN-γ in wild-type CD4+ T-cells. Treatment of stimulated CD4+ T-cells with adenosine (25 μM) potently reduced IFN-γ release which is mediated by adenosine A2a receptors (A2aR). AMP (50 μM) also reduced cytokine release which was not inhibited by APCP. In Teff, A2aR activation (CGS21680) potently inhibited the release of IL-1, IL-2, IL-3, IL-4, IL-12, IL-13, IFN-γ, TNF-α, granulocyte-macrophage colony-stimulating factor (GM-CSF), CCL3, and CCL4. However, in Treg, CGS21680 did not alter cytokine/chemokine release. In summary, CD73-derived adenosine tonically inhibits active NF-κB in CD4+ T-cells, thereby modulating the release of a broad spectrum of proinflammatory cytokines and chemokines. Downregulation of P2X7 and upregulation of CD73 in Treg after antigenic stimulation may be an important mechanism to maintain the ability of Treg to generate immunosuppressive adenosine.

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.

scholarly journals Natalizumab treatment in multiple sclerosis: marked decline of chemokines and cytokines in cerebrospinal fluid

2009 ◽  
Vol 16 (2) ◽  
pp. 208-217 ◽  
Author(s):  
J. Mellergård ◽  
M. Edström ◽  
M. Vrethem ◽  
J. Ernerudh ◽  
C. Dahle
Keyword(s):  
Multiple Sclerosis ◽  
Cerebrospinal Fluid ◽  
Plasma Levels ◽  
Therapeutic Effects ◽  
Gm Csf ◽  
Cytokines And Chemokines ◽  
Tnf Α ◽  
Natalizumab Treatment ◽  
Marked Decline ◽  
One Year

Natalizumab exerts impressive therapeutic effects in patients with multiple sclerosis (MS). The proposed main mode of action is reducing transmigration of leukocytes into the CNS, but other immunological effects may also be operative. Cytokines and chemokines are involved in the regulation of inflammatory responses and may reflect the disease process in MS. The objective of this study was to evaluate the effects of natalizumab treatment on cytokine and chemokine profiles systemically and intrathecally in multiple sclerosis. We used luminex to analyse a panel of cytokines (IL-1β, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, TNF-α, IFN-γ, GM-CSF) and chemokines (CXCL9, CXCL10, CXCL11, CCL17, CCL22) in blood and cerebrospinal fluid (CSF) from 31 patients with relapsing MS before and after one year of natalizumab treatment. There was a marked decline in CSF levels of cytokines and chemokines, thus including pro-inflammatory cytokines (IL-1β, IL-6 and IL-8) as well as chemokines associated with both Th1 (CXCL9, CXCL10, CXCL11) and Th2 (CCL22). Circulating plasma levels of some cytokines (GM-CSF, TNF-α, IL-6 and IL-10) also decreased after one year of treatment. This is the first study to show that natalizumab treatment is associated with a global decline in cytokine and chemokine levels at a protein level. This finding was most pronounced in CSF, in line with the reduced transmigration of cells into CNS, whereas reduction in plasma levels indicates other possible mechanisms of natalizumab treatment.

scholarly journals Fuzheng Qingjie Granules Inhibit Growth of Hepatoma Cells via Inducing Mitochondria-Mediated Apoptosis and Enhancing Immune Function

2016 ◽  
Vol 16 (3) ◽  
pp. 329-338 ◽  
Author(s):  
Xuzheng Chen ◽  
Zhiyun Cao ◽  
Youquan Zhang ◽  
Jinnong Li ◽  
Suqing Wang ◽  
...  
Keyword(s):  
T Cells ◽  
Cytochrome C ◽  
Immune Function ◽  
Nk Cells ◽  
Hepg2 Cells ◽  
Caspase 3 ◽  
Tumor Tissue ◽  
Hepatoma Cells ◽  
Tumor Bearing ◽  
Tnf Α

Fuzheng Qingjie (FZQJ) granules, a compound Chinese medicine, have been used as an adjuvant therapy for alimentary tract cancers. However, the underlying anticancer mechanisms are still not well understood. In the present study, HepG2 cells were treated with FZQJ-containing serum. Cell proliferation was evaluated using MTT assay. Apoptosis was analyzed using a flow cytometer. Cell ultrastructure was observed under a transmission electron microscope. The mitochondrial membrane potential (Δψ) was examined with JC-1 dye. In H22 tumor–bearing mice, CD4+ T cells, CD8+ T cells, CD3+ T cells, and natural killer (NK) cells in peripheral blood were evaluated cytometrically. Interleukin (IL)-2 and tumor necrosis factor (TNF)-α levels were measured using radioimmunoassay.The mRNA levels of Bax and Bcl-2 were examined by reverse transcription–polymerase chain reaction. The protein levels of Bax, Bcl-2, cytochrome C, caspase 3 and 9, PARP, and CD69 were examined by Western blotting. The apoptotic cells in tissues were observed using TUNEL method. Alanine transaminase (ALT), aspartate transaminase (AST), blood urea nitrogen (BUN), and creatinine (CRE) were detected by an automatic biochemical analyzer. The results showed that FZQJ-containing serum remarkably inhibited proliferation of HepG2 cells in dose- and time-dependent manners, induced HepG2 cell apoptosis and caused a decrease of Δψ. Analysis of tumor tissue showed that FZQJ-induced apoptosis was accompanied by downregulation of Bcl-2 and upregulation of Bax, release of cytochrome c, activation of caspase 3 and 9, and cleavage of PARP. In addition, FZQJ increased the percentages of CD4+ T and NK cells, the ratio of CD4+/CD8+ T cells as well as the levels of serum TNF-α. FZQJ also increased CD69 expression in tumor tissue. No hepatorenal toxicity was observed in H22 tumor–bearing mice. These results indicated that FZQJ could inhibit the growth of hepatoma cells via regulating immune function and inducing mitochondria mediated apoptosis.

Association Of Platelet Function Markers, Independent Of Platelet Count, With Bleeding Score In Patients With Immune Thrombocytopenia

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 3534-3534
Author(s):  
Andrew L. Frelinger ◽  
Anja J Gerrits ◽  
Michelle A. Berny-Lang ◽  
Travis Brown ◽  
Sabrina L. Carmichael ◽  
...  
Keyword(s):  
Platelet Count ◽  
Platelet Activation ◽  
Platelet Function ◽  
Research Funding ◽  
Immune Thrombocytopenia ◽  
Univariate Analysis ◽  
Blood Draw ◽  
Platelet Surface ◽  
Platelet Counts ◽  
Bleeding Score

Abstract Background Immune thrombocytopenia (ITP) patients with similarly low platelet counts differ in their tendency to bleed. Aim To determine if differences in platelet function in ITP patients with similarly low platelet counts partly account for the variation in bleeding tendency. Methods The relationship between bleeding scores and platelet function markers was investigated in a single center cross-sectional study of pediatric patients with ITP. Following informed consent, blood was collected from ITP patients and bleeding was graded using the Buchanan and Adix Score (J Pediatr 2002) at routine clinic visits or while admitted to the hospital. Bleeding scores were obtained by one of three hematologists blinded to platelet function results, and investigators performing platelet function tests were blinded to clinical results. Platelet function was assessed by whole blood flow cytometric measurement of unstimulated, ADP- or TRAP-stimulated platelet surface activated GPIIb-IIIa (as measured by PAC1 binding), P-selectin, and GPIb and by unstimulated, convulxin-, or ADP plus TRAP-stimulated platelet surface phosphatidylserine expression (as determined by annexin V binding). Platelet count, immature platelet fraction (IPF) and mean platelet volume (MPV) were determined by a Sysmex XE-2100, and platelet forward angle light scatter (FSC) was measured by flow cytometry. Results Platelet function and bleeding scores were evaluated in 34 consecutive consenting pediatric ITP patients (16 female, 18 male, age 9.7 ± 5.7 years [mean ± SD]). ITP was newly diagnosed (< 3 months) in 10 patients, persistent (3 -- 12 months) in 7 patients, and chronic (>12 months) in 17 patients. Platelet count at the time of the blood draw was 47 ± 55 x 109/L. The median bleeding score on day of blood draw was 1 (range 0 to 4). By univariate analysis, higher IPF, and lower platelet count were significantly associated with a higher bleeding score (odds ratio [OR] >1, p<0.05) but MPV was not. Multiple measures of platelet function were associated with bleeding scores by univariate analysis: higher levels of platelet FSC (a measure affected by multiple variables including size) surface GPIb on unstimulated, ADP- or TRAP-stimulated platelets, surface P-selectin on unstimulated platelets, and platelet FSC were associated with increased odds for higher bleeding scores (ORs each >1, p<0.05), while higher ADP- and TRAP-stimulated platelet surface activated GPIIb-IIIa and P-selectin were associated with reduced odds of higher bleeding scores (ORs each <1, p<0.05). After adjustment for platelet count, higher levels of platelet surface P-selectin on unstimulated platelets, GPIb on TRAP-stimulated platelets, and FSC remained significantly associated with increased odds for higher bleeding scores (Figure), but IPF did not. Similarly, after adjustment for platelet count, higher TRAP-stimulated percentage of P-selectin and activated GPIIb-IIIa positive platelets remained significantly associated with reduced odds of higher bleeding scores (Figure). These findings were independent of recent ITP-related treatment. Conclusions In this study of pediatric ITP patients, we identified selected platelet function markers which, independent of platelet count, are associated with increased (platelet FSC, platelet surface P-selectin on unstimulated platelets, and GPIb on TRAP-stimulated platelets) or decreased (TRAP-stimulated percent P-selectin and GPIIb-IIIa positive platelets) odds of high bleeding scores. Possible hypotheses to explain these associations are as follows: 1) Increased P-selectin on unstimulated platelets demonstrates in vivo platelet activation, possibly as a consequence of the recent bleeding. 2) Because platelet activation results in a reduction in platelet surface GPIb and increases in platelet surface activated GPIIb-IIIa and P-selectin, the ORs associated with all of these markers could be explained by reduced ability of platelets in patients with higher bleeding scores to respond to agonists. 3) While platelet FSC is partly related to size, the finding that MPV and IPF, adjusted for platelet count, were not associated with bleeding score suggests that factors other than size account for the association of platelet FSC with higher bleeding scores. Further study is required to validate these findings and determine if differences in platelet function are associated with future risk for bleeding. Disclosures: Off Label Use: Eltrombopag was given to WAS/XLT patients for treatment of thrombocytopenia. Neufeld:Shire: Consultancy, Research Funding; Novartis: Consultancy, Research Funding; Apopharma: Consultancy. Michelson:Sysmex: Honoraria.

scholarly journals MicroRNA regulate immunological pathways in T-cells in immune thrombocytopenia (ITP)

Blood ◽  
2013 ◽  
Vol 121 (11) ◽  
pp. 2095-2098 ◽  
Author(s):  
Margareta Jernås ◽  
Intawat Nookaew ◽  
Hans Wadenvik ◽  
Bob Olsson
Keyword(s):  
T Cells ◽  
Plasma Levels ◽  
Immune Thrombocytopenia ◽  
Target Gene ◽  
Key Points

Key Points MicroRNA and plasma levels of the target gene CXCL13 differ between ITP and controls indicating that microRNA may be important in ITP.

Immunoprofile of Patients with Chronic Myeloid Leukemia Treated with Imatinib, Nilotinib or Dasatinib,

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 3764-3764
Author(s):  
Yoshiki Hayashi ◽  
Hirohisa Nakamae ◽  
Takako Katayama ◽  
Takahiko Nakane ◽  
Hideo Koh ◽  
...  
Keyword(s):  
T Cells ◽  
Chronic Myeloid Leukemia ◽  
Plasma Levels ◽  
Myeloid Leukemia ◽  
Research Funding ◽  
Nk Cell ◽  
Chronic Phase ◽  
Interquartile Range ◽  
Cell Reactivity ◽  
Gm Csf

Abstract Abstract 3764 Recent reports showed that dasatinib induces significant immunostimulation with clonal expansion of large granular lymphocytes (LGL) which, in chronic myeloid leukemia (CML), is related to both better prognosis and to autoimmune-like side effects. It is speculated that lower levels of circulating T-regulatory cells play a partial role in LGL proliferation in patients receiving dasatinib. The immunoprofile was studied using flow cytometry to evaluate lymphocyte subsets and NK-cell reactivity in the peripheral blood of 61 patients in the chronic phase of CML during treatment with a tyrosine kinase inhibitor (TKI) (Median age: 58 years; imatinib 36, nilotinib 9, dasatinib 16). Furthermore, we measured plasma levels of 27 types of cytokines or chemokines in 58 patients in the chronic phase of CML so that a comprehensive comparison could be made of the differences in immunoprofile among the patients receiving these three TKIs. There were no significant differences between the three TKI-treated groups in terms of CD4/8 ratios or the number of T-cells (CD3+CD8+ or CD4+) and NK cells (CD3-CD56+). However, the number of NK-LGL (CD56+CD57+) and T-LGL (CD3+CD57+) increased significantly in the group that received dasatinib. Furthermore, dasatinib significantly enhanced NK-cell reactivity as compared to imatinib and nilotinib. In contrast nilotinib significantly suppressed NK-cell reactivity (E/T ratio =10:1: Median (interquartile range), 8.7% (5.0–16.2), 5.2% (4.8–11.4), 20.8% (13.4–33.3), for imatinib, nilotinib and dasatinib, respectively). In addition, the number of regulatory T-cells (CD4+CD25int-hiCD127low) was similar among the three groups (Median (interquartile range), 36/mm3 (27–53), 48/mm3 (34–60), 39/mm3 (26–53), for imatinib, nilotinib and dasatinib, respectively). Furthermore, in the analysis of cytokines and chemokines, plasma levels of IL-8, IP-10, and MCP-1 were significantly elevated while the level of PDGF-bb was significantly decreased in all three groups compared to those of healthy control. Plasma levels of IL-1 beta, IFN-gamma, and FGF-basic were significantly decreased in only the dasatinib group compared to those of control (P=.02, P=.04, P=.03, respectively). In addition, plasma levels of GM-CSF were significantly elevated in the imatinib and dasatinib groups (Median (interquartile range), 6.1 pg/ml (2.7–11.7) and 7.9 pg/ml (4.5–8.2), P=.02, and P=.03, respectively) but not in the nilotinib group (P=.34) when compared to those of control. In the multiple regression analysis that evaluated the relationship between NK-reactivity and cytokines or chemokines in the patients receiving dasatinib, only plasma levels of GM-CSF were significantly associated with NK-reactivity (P=.03). Notably, in our data, dasatinib and nilotinib exerted opposite effects on NK-cell reactivity, expansion of LGL, and showed different cytokine and chemokine profiles. Based on our results, the activation of NK-cell reactivity induced by dasatinib might be caused by a mechanism other than a decrease in the number of regulatory T-cells. Additionally, in an unphysiological immunological status mediated by dasatinib, GM-CSF might make some contribution to NK-cell reactivity. Disclosures: Nakamae: BMS: Research Funding, Speakers Bureau; Novartis: Research Funding, Speakers Bureau. Hino:BMS: Research Funding, Speakers Bureau; Novartis: Research Funding, Speakers Bureau.

scholarly journals The profile of leucocytes, CD3+, CD4+, and CD8+ T cells, and cytokine concentrations in peripheral blood of children with acute asthma exacerbation

2017 ◽  
Vol 45 (6) ◽  
pp. 1658-1669 ◽  
Author(s):  
Thuy Nguyen-Thi-Dieu ◽  
Huong Le-Thi-Thu ◽  
Sy Duong-Quy
Keyword(s):  
Flow Cytometry ◽  
T Cells ◽  
Cd8 T Cells ◽  
Peripheral Blood ◽  
Asthma Exacerbation ◽  
Acute Asthma ◽  
Gm Csf ◽  
Paediatric Patients ◽  
Tnf Α ◽  
Acute Asthma Exacerbation

Objective To determine the leucocyte profile and cytokine concentrations in the peripheral blood of children with an acute asthma exacerbation (AAE). Methods This descriptive, cross-sectional study enrolled paediatric patients admitted to hospital for AAE. The severity of AAE was assessed using the paediatric asthma score (PAS). Peripheral blood samples were collected for automatic quantification of white blood cell counts, CD3+, CD4+, and CD8+ T cells populations by flow cytometry and cytokine concentrations by flow cytometry-assisted immunoassay. Results A total of 127 children with AAE and 30 healthy control subjects were included in the study. The proportion of paediatric patients with decreased CD3+, CD4+ and CD8+ T cells was significantly higher in those with severe AAE compared with those with mild-to-moderate AAE. The concentrations of interleukin (IL)-2, IL-8, IL-12, and IL-4 in paediatric patients with rhinovirus infection were significantly higher than in those without rhinovirus infection. IL-2, IL-4, IL-6, TNF-α and GM-CSF concentrations during AAE were significantly lower than control. IL-5 and IL-13 concentrations during AAE were significantly higher than control. Conclusions The decrease of CD3+, CD4+, CD8+ T cells and IL-2, IL-4, IL-6, TNF-α, and GM-CSF combined with the increase of IL-5 and IL-13, were associated with AAE in children with asthma.

scholarly journals Active Immune Thrombocytopenia (ITP) Disease Is Characterised By a Reduced Treg:CD8 Effector T Cell Ratio Which Is Modulated By Thrombopoietin-Receptor Agonists (TPO-RA)

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 2430-2430
Author(s):  
Anwar A. Sayed ◽  
Amna Malik ◽  
Ahmad Khoder ◽  
George Adams ◽  
Nichola Cooper
Keyword(s):  
T Cells ◽  
Platelet Count ◽  
T Cell ◽  
Cd8 T Cells ◽  
Immune Thrombocytopenia ◽  
Cd8 T Cell ◽  
T Cell Subsets ◽  
P Value ◽  
Cell Ratio ◽  
Thrombopoietin Receptor

Abstract Background: Immune thrombocytopenia (ITP) is an autoimmune disorder characterized by isolated low platelet count and a skewed proinflammatory Th1/Th17 profile. However, little is known about the involvement of CD8+ cytotoxic T cells in ITP pathophysiology and whether they are regulated by regulatory T cells (Treg). Immunosuppressive therapy has been the mainstay treatment in ITP. More recently, Thrombopoietin receptor agonists (TPO-RA); Romiplostim (Romi) and Eltrombopag (EPAG), have been increasingly used to stimulate megakaryocytopoiesis to produce more platelets. TPO-RAs are reported to induce complete remission in up to 30% of cases, with limited understanding of their impact on the immune system. Here we describe changes in T cell subsets in patients with ITP: how these changes are affected by disease activity and how TPO-RA may induce remission through modulating the immune system. Methods: Multi-color flow cytometric panels were designed to characterize peripheral blood T cell subsets, including CD8+ T cell and Treg subsets, phenotypically as well as functionally through intracellular cytokine expression. To determine whether CD8+ cells were platelet specific, an IFNγ ELISpot assay was performed using platelets from a healthy donor and PBMC from both HC and patients. Forty patients with ITP were included: 13 were on Romi, 11 on EPAG and 16 on no treatment at the time of analysis. Of these 40 patients, 15 patients had active disease (AD) (platelet-count less than 30 x 109/L) and 25 had stable disease (SD) (> 30 x 109/L). These patients were compared with 26 age and gender-matched healthy controls (HC). Data were presented as median values; Mann Whitney U and Kruskal Wallis tests were used with Dunn's multiple comparisons correction; a P value of < 0.05 was considered significant. Results: CD4/CD8 T cell ratio was significantly lower in patients compared to HC [1.77 vs. 3.97; P value < 0.001]. CD45RA+CD62L- Terminally-differentiated CD8+ T cells were significantly higher in patients compared to HC [66.3% vs. 8.56%; P value < 0.001]. This finding was more prominent in AD patients than those with SD [66% vs. 44.4%; P value < 0.05]. This effector population is polyfunctional, expressing high levels of proinflammatory cytokines including TNFα, IFNγ and Granzyme B when compared to HC [P value < 0.05]. Additionally, this population lacks the exhaustion markers PD-1 and Tim-3. Furthermore, these cells were reactive to platelets showing higher IFNγ-producing cells when co-cultured with platelets. CD3+CD4+CD25hiCD127lo Treg frequency did not differ between patients and HC [P value>0. 05]. Treg functionality was preserved in these patients; no important changes were observed in their capacity to express interlukin2 intracellularly, nor in the cell surface receptor (CD25) [P value > 0. 05]. However, Tregs were significantly lower in AD patients with compared to SD patients [2.32% vs. 4.46%; P value < 0.05]. Treg function is interactive with other T cell subsets and depends on its relative abundance in relation to other subsets; The Treg/effector CD8+ T cell ratio was significantly lower in patients compared to HC [0.06 vs. 0.16; P value < 0.01] and was also significantly lower in AD compared to SD patients [0.03 vs. 0.09 ; P value < 0.05]. EPAG-treated patients had a significantly lower effector CD8+ T cells compared to Romi-treated patients [42.4% vs 76.8%; P value <0.01]. Although EPAG did not have a direct effect on the frequency of Treg, the Treg/effector CD8+ T cell ratio was significantly lower than that in patients on Romi [0.04 vs. 0.11; P value < 0.05] because of the CD8+.T cell difference. The Treg/effector CD8+ T cell ratio in EPAG-treated patients was comparable to the ratio of HC [0.11 vs. 0.16; P value > 0.05]. Conclusion: While Th1/Th2 cell ratio is often considered as driving ITP, these results demonstrate the involvement of cytokine secreting effector CD8+ T cells in the disease pathogenesis. The imbalance in immune tolerance is also highlighted in the form of a significant reduction in the Treg/effector CD8 T cell ratio. The differences seen in T cell subsets between EPAG- and Romi-treated patients suggest the potential for an additional, differential immunomodulatory effect between the two agents which is currently being explored. Acknowledgment: The authors wish to thank Prof James B Bussel for his insightful comments and support of this work. Disclosures Cooper: Amgen, Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees.

scholarly journals Platelet Apoptosis in Adult Immune Thrombocytopenia. Relationship with Auto-Antibodies, Platelet Function and Treatment

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 2792-2792 ◽  
Author(s):  
Geraldine Contrufo ◽  
Ana C Glembotsky ◽  
Nora P Goette ◽  
Paola R Lev ◽  
Matías Grodzielski ◽  
...  
Keyword(s):  
Platelet Count ◽  
Platelet Activation ◽  
Immune Thrombocytopenia ◽  
Calcium Ionophore ◽  
Activation Parameters ◽  
Calcium Ionophore A23187 ◽  
Ionophore A23187 ◽  
Platelet Surface ◽  
Platelet Apoptosis ◽  
Cell Penetrating

Abstract Similarly to nucleated cells, platelet life span is also controlled by an intrinsic apoptotic program that triggers collapse of the mitochondrial inner membrane potential, activation of caspases-3, -8 and -9, phosphatidylserine (PS) externalization and microparticle shedding. The aim of the present study was to investigate platelet apoptosis in adult patients with immune thrombocytopenia (ITP) under different treatment conditions and to search for its relationship with the type of auto-antibody and the platelet-activation status. Twenty-four patients with chronic ITP, age 42 (21-80) years (median and range) diagnosed according to current criteria (Rodeghiero et al, 2009) were included after written informed consent in accordance with the Declaration of Helsinki. The study was approved by the Ethics Committee from Instituto de Investigaciones Medicas Alfredo Lanari. Platelet count was 38x109/L (6-85). Platelet apoptosis was evaluated by phosphatidylserine (PS) exposure on the platelet surface using FITC-conjugated Anexin-V, mitochondrial electrochemical potential changes (ΔΨm) using the cell penetrating lipophilic cationic fluorochrome JC-1, and activated caspase-3 (a-casp3) measured by the cell-penetrating carboxyfluorescein-labelled fluoromethyl ketone tetrapeptide (FAM-DEVD-FMK). These parameters were studied in resting platelets and after stimulation with calcium ionophore (A23187). Platelet activation was evaluated by FITC-PAC-1 binding to activated GPIIb-IIIa and GPIb-IX internalization using PE-CD42b, in resting conditions and after stimulation either with ADP or TRAP. Apoptosis and activation parameters were evaluated by flow cytometry. In resting conditions, platelets from ITP patients showed increased PS expression and a- casp3 and abnormal ΔΨm (table 1). TablePSΔΨma-casp3Patients19.6 (1.9-82.0)31.2 (5.8-92.4)11.3 (1.8-40.9)Controls4.7 (1.9-10.8)10.3 (2.2-27.5)4.3 (1.9-8.2)p (Mann-Whitney)0.002<0.00010.012n212312 After stimulation with A23187, ITP platelets had similar levels of PS expression (p=0.305, n=20) and ΔmΨ (p=0.383, n=25) compared to normal controls. However, an increased sensitivity to the apoptotic stimulus was evidenced by elevated levels of a-casp3 at low and high A23187 concentrations (1-3 mM, p=0.097; 6-10 mM, p=0.002). Platelet apoptosis was not related to platelet activation, as PAC-1 binding was not increased in ITP platelets (basal p=0.847, ADP-induced p=0.059, TRAP-induced p=0.103, n=16). Besides, internalization of GPIb-IX after ADP and TRAP stimulation was also normal (p=NS, n=9 for both agonists). Platelets from ITP patients bearing two of the most frequently found auto-antibodies (5 with anti-GPIIb-IIIa and 1 with anti-GPIb-IX) had similar levels of PS expression and ΔmΨ at resting conditions than those who were negative for these auto-antibodies (n=14) (p=0.265 and 0.148, respectively). There were no differences in apoptosis markers either in resting platelets or after stimulation when comparing untreated patients (n=9) vs patients under any kind of treatment (n=15) (resting conditions, PS p=0.737; ΔmΨ p=0.270; stimulated, PS p=0.966; ΔmΨ p=0.987), although platelet count was similar in both groups. Normal platelets incubated during 1 hour with plasma from ITP had higher a-casp3 than those incubated with normal plasma (n=12 and 9, respectively, p=0.027), suggesting a plasmatic component could be responsible for the apoptotic stimulus. In conclusion, increased platelet apoptosis in ITP patients could be induced by a plasmatic factor, contributing to thrombocytopenia in this entity. Disclosures Riveros: Roche: Speakers Bureau.

Sign in / Sign up

Export Citation Format

Share Document