Detection of Anti-Thrombopoietin Antibodies in Patients with Immune Thrombocytopenia

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 4187-4187
Author(s):  
Takashi Satoh ◽  
Koji Miyazaki ◽  
Naoki Shimada ◽  
Koki Nagane ◽  
Tomoya Inukai ◽  
...  
Keyword(s):  
Plasma Levels ◽  
Research Funding ◽  
Immune Thrombocytopenia ◽  
Healthy Controls ◽  
Plasma Samples ◽  
Elevated Plasma ◽  
Competition Assay ◽  
Platelet Production ◽  
Healthy Control ◽  
Control Plasma

Abstract Background: Immune thrombocytopenia (ITP) is an autoimmune disease characterized by the presence of autoantibodies against platelet membrane glycoproteins, which cause the autoantibody-mediated destruction of platelets and impaired platelet production. Thrombopoietin (TPO) binds to its receptor on the surface of hematopoietic stem cells and megakaryocytes and induces their maturation and proliferation. Patients with thrombocytopenia due to aplastic anemia have drastically elevated plasma levels of TPO, whereas patients with ITP have normal or slightly elevated plasma levels of TPO despite their low platelet count. Furthermore, based on the existence of a multitude of autoantibody reactivities in ITP, including antibodies against platelets and TPO receptors, the presence of anti-TPO antibodies in patients with ITP may be suspected. Objective: We developed assay systems to detect plasma anti-TPO antibodies and screen patients with ITP. We examined the clinical characteristics associated with anti-TPO antibodies and their pathogenic roles in patients with ITP. Methods: Plasma anti-TPO antibodies from 101 patients with ITP and 72 healthy controls were measured by enzyme-linked immunosorbent assay (ELISA) using recombinant human TPO (rhTPO) as an antigen. The specificity of anti-TPO antibody reactivity was confirmed by ELISA competition assay. The presence of anti-TPO antibodies was further examined using immunoprecipitation and immunoblotting using rhTPO. To investigate whether anti-TPO antibodies inhibited functional interactions between TPO and TPO receptors, we examined extracellular signal-regulated kinases (ERKs), downstream signals induced by TPO. The binding of TPO to TPO receptors induced the phosphorylation of ERK in TPO receptor-expressing UT-7/TPO cells. Results: The level of anti-TPO antibodies measured by ELISA was significantly greater in the samples from patients with ITP than in those from healthy controls (2.91 ± 3.64 units versus 1.45 ± 0.67 units, P < 0.001). Samples were classified as positive or negative for anti-TPO antibody, as determined by immunoprecipitation and immunoblotting. Thus, the ELISA positive-cutoff value was considered to be the mean plus 3.5 standard deviation (SD) of 72 healthy control plasma samples. Plasma anti-TPO antibodies were detected in twenty-four ITP patients (23.8%), but in none of the healthy controls. By ELISA competition assay, anti-TPO antibody reactivity was inhibited dose-dependently by preincubation of patient plasma with rhTPO. In addition, anti-TPO antibody-positive plasma samples inhibited the phosphorylation of ERK in UT-7/TPO cells. In contrast, healthy control plasma had no inhibitory effect. Furthermore, the number of megakaryocytes was decreased relatively in the anti-TPO antibody-positive ITP patients. There was no difference in the TPO levels in plasma between ITP patients with anti-TPO antibodies and patients without anti-TPO antibodies (63.6 ± 79.7 pg/ml versus 45.2 ± 49.3 pg/ml). Conclusion: Our results have thus demonstrated the presence of anti-TPO autoantibodies in patients with ITP. The ELISA using rhTPO was specific for the detection of anti-TPO antibodies and thus allows their easy and rapid measurement in clinical settings. These findings suggest that functional anti-TPO antibodies cause impaired megakaryocyte proliferation and platelet production in patients with ITP. Disclosures Higashihara: Bristol-Myers Squibb: Research Funding; Baxter: Research Funding; Teijin: Research Funding; Pfizer: Research Funding; Astellas: Research Funding; Yakurt: Honoraria; KyowaHakkoKirin: Honoraria, Research Funding; Chugai: Honoraria, Research Funding; Eisai: Honoraria; GlaxoSmithKline: Honoraria, Research Funding; Nippon Shinyaku: Research Funding; Shionogi: Honoraria, Research Funding; Novartis: Honoraria, Research Funding; Celgene: Honoraria; Takeda: Honoraria; Janssen　pharma: Honoraria, Research Funding; Alexion: Honoraria; Dainippon Sumitomo: Research Funding; Taisho Tomiyama: Research Funding.

Plasma levels of sirtuin and adiponectin in patients with primary open-angle glaucoma, exfoliative glaucoma, and healthy controls

2021 ◽  
pp. 112067212110652
Author(s):  
Aysun Yucel Gencoglu ◽  
Murat Irkec ◽  
Sibel Kocabeyoglu ◽  
Z. Gunnur Dikmen ◽  
Jale Karakaya ◽  
...  
Keyword(s):  
Plasma Levels ◽  
Primary Open Angle Glaucoma ◽  
Open Angle Glaucoma ◽  
Healthy Controls ◽  
Plasma Samples ◽  
Open Angle ◽  
Control Subjects ◽  
Healthy Control ◽  
Exfoliative Glaucoma ◽  
Chi Square Analysis

Purpose To compare plasma levels of sirtuin 1 (SIRT1) and adiponectin (APN) in patients with primary open-angle glaucoma (POAG), exfoliative glaucoma (XFG), and healthy control subjects. Methods This prospective case-control study collected plasma samples from 118 participants. All subjects underwent a comprehensive ophthalmologic examination before the acquisition of a plasma sample. Plasma samples were obtained from 40 POAG, 38 XFG, and 40 healthy control subjects without any evidence of systemic or ocular disease. Serum SIRT1 and APN levels were estimated by an enzyme-linked immunosorbent assay, ELISA (Elabscience, Houston, USA) method. Statistical analysis of results relied on Kolmogorov-Smirnov, Kruskal-Wallis, Chi-square, analysis of variance (ANOVA) tests, and linear regression analysis, where appropriate. Results A significant decrease in SIRT1 levels was observed in POAG patients compared to healthy controls (p = 0.004, Dunn's test). In contrast, no difference was detected between XFG and POAG patients or healthy controls (p = 0.32 and p = 0.34, respectively, Dunn's test). There was no significant difference in plasma APN levels between the three groups under investigation (p = 0.59, ANOVA). Conclusion Alterations in serum level of SIRT1 may suggest a possible role in POAG via potential effects in neuroprotection and oxidative stress.

scholarly journals Evaluation of Platelet Function Defects in Patients with Immune Thrombocytopenia

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 1021-1021
Author(s):  
Elena Monzón Manzano ◽  
María Teresa Alvarez Román ◽  
Andres Ramirez Lopez ◽  
Elena G Arias-Salgado ◽  
Paula Acuña ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Sialic Acid ◽  
Platelet Function ◽  
Research Funding ◽  
Immune Thrombocytopenia ◽  
Healthy Controls ◽  
Activation Markers ◽  
Platelet Production ◽  
Neuraminidase Activity ◽  
Fibrinogen Receptor

Abstract Background: Primary immune thrombocytopenia (ITP) is a megakaryocytic (MK)/platelet-specific autoimmune disorder characterized by platelet count &lt;100×10 9/L with or without bleeding manifestations, and diagnosed by exclusion of other causes of thrombocytopenia. It is widely accepted the involvement of platelet autoantibodies on deterioration of platelets from patients with ITP. Moreover, an enhanced activity of neuraminidase may also reduce sialic acid from glycoside residues on platelet surface, especially from the highly glycosylated von Willebrand factor (vWF) receptor. Because controversial results regarding the functionality of platelets from ITP patients can be found in literature, we aimed to determine platelet ability to be stimulated by agonists. Moreover, we aimed to determine the way anti-platelet auto- antibodies (abs) and neuraminidase activity may affect the function of platelets derived from MKs of healthy controls. Methods: This observational, prospective and transversal study included 42 patients with chronic primary ITP and 55 healthy controls. Platelet fibrinogen and vWF receptors and activation markers (PAC1 binding to activated fibrinogen receptor and exposure of P-selectin after agonists treatment), were evaluated by flow cytometry. Presence of Antibodies (abs) against platelet's glycoproteins in ITP serum was analysed with a Luminex based assay (LifecodesPak Lx). Neuraminidase (NEU) activity in serum was determined with the substrate 20-(4-methylumbelliferyl)-a-D-N-(MUNANA). Human CD34 + cell-enriched population was obtained with CliniMACS (MiltenyiBiotec) from G-CSF mobilized peripheral blood of a healthy donor. For MK differentiation, CD34 + cells were cultured 12 days in StemSpan™ Serum-Free Expansion Medium II (SFEM II) with 50ng/ml of recombinant human thrompoietin. Then, 10% of serum from healthy controls (4) or ITP patients (4) were added to the culture of mature MKs and incubated for 3 days. Phenotypic analysis of MKs and culture derived-platelets was carried out using abs against CD34, CD41, CD42a and CD42b.Platelet-like particles were considered as CD41-positive events with a size (FSC) and granularity (SSC) scatter properties similar to blood platelets. Culture-derived platelets were stimulated with 100 µM TRAP and 10 µM ADP and activation markers were analyzed by flow cytometry. Results: Expression of fibrinogen receptor on platelets from ITP patients were similar to those from healthy controls but showed a reduced capacity to be activated. Impairment in platelet degranulation measured as exposition of P-selectin after agonist's stimulation was also observed in platelets from these patients (Figure 1). Of note, surface content of CD42b subunit of vWF receptor was reduced (Figure 1). To determine whether diminished platelet function might be due to a plasma component, we induced platelet production from MK of healthy controls as referred in Methods. Abs against platelets and neuraminidase activity were determined in serum samples. Serum from 4 healthy controls or from 4 ITP patients (1 with anti-CD42b, 1 with anti-GPIa-IIa and 2 with undetectable abs) were added to MKs culture. No differences existed in MK differentiation and platelet production between MKs incubated with serum from healthy controls or from ITP patients, but similarly as observed in platelets from ITP patients, MK-derived platelets had an impaired ability to be activated (Table 1). Platelets derived from MKs incubated with ITP serum with anti-platelet abs had also a diminished exposure of CD42b (73±8% of controls). Moreover, neuraminidase content of these samples was slightly higher than that from ITP samples without abs (130 vs 100 % of controls). Conclusion: Platelets from ITP patients had a diminished ability to be stimulated. In vitro study showed that megakaryopoiesis was normal in presence of ITP serum, but released platelets had a lower ability to be activated. Involvement of abs in this effect cannot be ruled out despite we detected abs only in 2 of the tested sera because efficiency of method to detect these abs is ~ 50%. On the other hand, reduced levels of CD42b might be due to the increased activity of neuraminidase. Reduction of sialic acid from CD42b might initiate its metalloproteinase-mediated cleavage or change affinity of the ab used for its detection. Research funded by ISCIII-Fondos FEDER PI19/00772 and Platelet Disorder Support Association Figure 1 Figure 1. Disclosures Alvarez Román: Pfizer: Consultancy, Honoraria, Research Funding; Octapharma: Consultancy, Honoraria, Research Funding; Sobi: Consultancy, Honoraria, Research Funding; Grifols: Consultancy, Honoraria, Research Funding; Biomarin: Consultancy, Honoraria, Research Funding; Amgen: Consultancy, Honoraria, Research Funding; Novartis: Consultancy, Honoraria, Research Funding; Bayer: Consultancy, Honoraria, Research Funding; CSL-Behring: Consultancy, Honoraria, Research Funding; Takeda: Consultancy, Honoraria, Research Funding; Novo-Nordisk: Consultancy, Honoraria, Research Funding. García Barcenilla: Roche: Speakers Bureau; Takeda: Speakers Bureau; Bayer: Speakers Bureau; SOBI: Speakers Bureau. Canales: Janssen: Consultancy, Honoraria, Speakers Bureau; Celgene/Bristol-Myers Squibb: Consultancy, Honoraria; Gilead/Kite: Consultancy, Honoraria; Eusa Pharma: Consultancy, Honoraria; Incyte: Consultancy; Karyopharm: Consultancy, Honoraria; Novartis: Consultancy, Honoraria; Sanofi: Consultancy; iQone: Honoraria; Sandoz: Honoraria, Speakers Bureau; F. Hoffmann-La Roche Ltd: Consultancy, Honoraria, Speakers Bureau; Takeda: Consultancy, Honoraria, Speakers Bureau. Jiménez-Yuste: Grifols: Consultancy, Honoraria, Research Funding; NovoNordisk: Consultancy, Honoraria, Research Funding; F. Hoffmann-La Roche Ltd: Consultancy, Honoraria, Research Funding; Takeda: Consultancy, Honoraria, Research Funding; Bayer: Consultancy, Honoraria, Research Funding; CSL Behring: Consultancy, Honoraria, Research Funding; Pfizer: Consultancy, Honoraria, Research Funding; BioMarin: Consultancy; Sobi: Consultancy, Honoraria, Research Funding; Octapharma: Consultancy, Honoraria, Research Funding; Sanofi: Consultancy, Honoraria, Research Funding. Butta: Novo-Nordisk: Speakers Bureau; Takeda: Research Funding, Speakers Bureau; Roche: Speakers Bureau; CSL-Behring: Research Funding.

Low-dose decitabine promotes megakaryocyte maturation and platelet production in healthy controls and immune thrombocytopenia

2015 ◽  
Vol 113 (05) ◽  
pp. 1021-1034 ◽  
Author(s):  
Hai Zhou ◽  
Yu Hou ◽  
Xuena Liu ◽  
Jihua Qiu ◽  
Qi Feng ◽  
...  
Keyword(s):  
Immune Thrombocytopenia ◽  
Low Dose ◽  
Methylation Status ◽  
Healthy Controls ◽  
Trail Expression ◽  
Platelet Production ◽  
Potential Therapeutic Role ◽  
Platelet Release

SummaryImpaired megakaryocyte maturation and insufficient platelet production have been shown to participate in the pathogenesis of immune thrombocytopenia (ITP). Our previous study demonstrated that low expression of tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) in megakaryocytes contributed to impaired platelet production in ITP. Decitabine (DAC), a demethylating agent, is known to promote cell differentiation and maturation at low doses. However, whether decitabine is potential in promoting megakaryocyte maturation and platelet release in ITP is unclear. In this study, we evaluated the effect of DAC on megakaryocyte maturation and platelet release in the presence of ITP plasma that has been shown to cause impaired megakaryocyte maturation and platelet production. We observed that low-dose DAC (10 nM) could significantly increase the number of mature polyploid (≥ 4N) megakaryocytes in cultures with plasma from healthy controls and more than one-half of ITP patients in vitro. Furthermore, the number of platelets released from these megakaryocytes significantly increased compared with those untreated with DAC. In these megakaryocytes, DAC significantly enhanced TRAIL expression via decreasing its promoter methylation status. These findings demonstrate that low-dose DAC can promote megakaryocyte maturation and platelet production and enhance TRAIL expression in megakaryocytes in healthy controls and ITP. The potential therapeutic role of low-dose DAC may be beneficial for thrombocytopenic disorders.H. Z. and Y. H. contributed equally to this work.

scholarly journals Low-dose decitabine modulates myeloid-derived suppressor cell function and restores immune tolerance in immune thrombocytopenia

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 2089-2089
Author(s):  
Xiaofei Ni ◽  
Lingjun Wang ◽  
Tianshu Yu ◽  
Haoyi Wang ◽  
Yu Hou ◽  
...  
Keyword(s):  
Immune Tolerance ◽  
Immune Thrombocytopenia ◽  
Low Dose ◽  
Cell Function ◽  
Suppressor Cell ◽  
Healthy Controls ◽  
Suppressive Activity ◽  
Wild Type ◽  
Myeloid Derived Suppressor Cell ◽  
Healthy Control

Abstract Low-dose decitabine modulates myeloid-derived suppressor cell function and restores immune tolerance in immune thrombocytopenia Primary immune thrombocytopenia (ITP) is an autoimmune disorder characterized with increased risk of bleeding. Myeloid-derived suppressor cells (MDSCs) are heterogeneous immature cells and natural inhibitors of adaptive immunity. Metabolic changes within MDSCs elucidate a direct influence on immunologic consequences of their suppressive activity. Liver kinase B1 (LKB1) is a tumor suppressor gene of STK11/LKB1 coding serine/Sue, and LKB1 signaling pathway plays an important role as a "bridge" between metabolic balance and functional homeostasis of immune cells. Our previous studies demonstrated that low dose decitabine, a hypomethylating agent, significantly increased the number of mature polyploidy megakaryocytes and exhibited long-term clinical efficacy. Besides, it also increased the production of Treg and enhanced their immunosuppressive function in ITP. However, whether decitabine could regulate the metabolic and suppressive activity of MDSCs in ITP is unknown. The percentage of MDSCs in peripheral blood mononuclear cells (PBMCs) was determined by flow cytometry, which was shown to be significantly lower in ITP compared with that in healthy controls. We then investigated the effect of low-dose decitabine in patients with active ITP, where decitabine induced a significant expansion of MDSCs in line with an impressive platelet response. In the in vitro experiments, MDSCs were isolated from PBMCs of ITP patients or healthy controls and cultured with different concentrations of decitabine (0/10nM/50nM/100 nM/1uM/10μM) for 7 days. A concentration gradient from 50nM to 1uM stimulated MDSCs amplification in a dose-dependent manner, and we chose an optimal concentration of 100 nM. Moreover, we found the mRNA expression level of LKB1, AMPKα1, AMPKα2, AMPKβ1, AMPKβ2, AMPKγ1, and AMPKγ2 was significantly lower in ITP patients than that in healthy control subjects. After incubation with decitabine (100nM), the relative expression of the above molecules were significantly increased compared to untreated levels. We also analyzed oxygen consumption rate (OCR) and key parameters of mitochondrial function within MDSCs. Overall, the OCR curve of ITP patients was lower than that of the healthy control subjects, and the OCR curve of ITP patients significantly improved after treatment with decitabine. We sorted the cultured MDSCs and co-cultured them with CFSE-labeled CD4 +CD25 - T cells to evaluate the suppressive activity of MDSCs. Results indicated that the inhibitory function of decitabine-modulated MDSCs was corrected in line with metabolic rewriting. We further established the ITP murine model by transferring splenocytes of C57BL/6 CD61 knockout mice, immunized against platelets from wild-type syngeneic C57BL/6 mice, into severe combined immune deficient (SCID) mice. MDSCs were sorted from the bone marrow of wild-type mice and incubated with PBS or decitabine, respectively. SCID mice were divided into three groups and received the same numbers of splenocyte transfer, two groups were given additional transfer of PBS-treated or decitabine-treated MDSCs. Our data showed that the decitaine-treated MDSCs group had significantly higher platelet counts compared with control group and PBS-treated MDSCs group. In summary, our findings suggest that the immune function and metabolic characteristics of MDSCs in ITP patients are impaired. These data shed new light on the molecular mechanism of decitabine action by regulating immune function and aerobic metabolism via LKB1, which supervises the immunosuppressive functions of MDSCs. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

scholarly journals Effects of Rituximab and Dexamethasone on Regulatory and Pro-Inflammatory B-Cell Subsets in Patients with Primary Immune Thrombocytopenia

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 1378-1378
Author(s):  
Sif Gudbrandsdottir ◽  
Marie Brimnes ◽  
Tania Kollgaard ◽  
Hans Carl Hasselbalch ◽  
Claus Henrik Nielsen
Keyword(s):  
B Cells ◽  
Autoimmune Diseases ◽  
B Cell ◽  
Research Funding ◽  
Immune Thrombocytopenia ◽  
Lower Proportion ◽  
Surface Markers ◽  
Healthy Controls ◽  
Cell Functions ◽  
Primary Immune Thrombocytopenia

Abstract Background B-cell depletion with rituximab (RTX) is widely accepted as first- or second-line therapy in primary immune thrombocytopenia (ITP), but it is still unclear how RTX mediates its positive effect in ITP patients. RTX has been reported to induce a reduced titer of platelet antibodies. However, this finding is inconsistent and other B-cell functions, such as the ability to secrete cytokines or to function as antigen-presenting cells for T cells, may be involved in the pathogenesis of ITP. Evidence suggests that B cells participate in the regulation of autoimmune diseases by virtue of their ability to produce the regulatory cytokines interleukin (IL)-10, IL-35, or transforming growth factor β. The various functions of B cells involved in the pathogenesis of autoimmune diseases can in part be deducted by their phenotype as recognized by measurement of specific surface markers and cytokine secretion. Materials and Methods We previously conducted a trial involving 137 newly diagnosed adult ITP patients randomized to treatment with RTX (375 mg/m2/week for 4 weeks) + dexamethasone (DXM) (40 mg/day for 4 days repeated up to 6 cycles) or DXM monotherapy. From this cohort, we identified 16 patients with available samples of peripheral blood mononuclear cells (PBMCs) at baseline and 12 months after treatment; 9 patients from the RTX+DXM group, 7 patients from the DXM group. Seven anonymous blood donors served as healthy controls. PBMCs were incubated for 18 h at 37°C under 5% CO2 in RPMI-1640 containing 10% (v/v) serum from healthy blood group AB donors, either alone or stimulated with 10 µg/ml CpG oligodeoxynucleotides. Expression of the cell-surface markers CD5, CD27, CD25 and CD19, and intracellular content of IL-6 and IL-10 were measured by flow cytometry. Results All patients responded to therapy and were in complete or partial remission at 12 months. Patient characteristics are listed in table I. We observed a significant increase in the proportion of CD5+ B cells 12 months after treatment with RTX+DXM compared to baseline (p < 0.01, Fig. 1A). The percentage of CD27+ memory B cells was significantly decreased at 12 months compared to baseline in patients receiving RTX+DXM (p < 0.05, Fig. 1B), and there was an inverse correlation between platelet numbers and the proportion of CD27+ B cells (R = -0.71; p < 0.05). The proportion of CD25+ B cells tended to decrease in patients treated with RTX+DXM, and was lower at 12 months than in patients treated with DMX only (p < 0.05, Fig 1C). PBMCs from ITP patients contained a lower proportion of IL-10+ B cells (p < 0.01) as well as a lower proportion of B cells producing IL-6 (p < 0.01) at baseline than PBMCs from healthy controls. At 12 months the low proportions had normalized in both treatment groups (Fig. 2). Conclusion B cells from ITP patients treated with RTX+DXM contained a high proportion of CD5+ B cells and low proportions of CD25+ and CD27+ B cells. Before treatment, B cells from ITP patients contained low frequencies of IL-10+ and IL-6+ B cells. Treatment with RTX + DXM or DXM alone reverted these aberrancies to normal. The increase in IL-10+ B cells as well as CD5+ B cells, which may represent overlapping subsets, is compatible with induction of Bregs and may support Treg development. Given the role of CD5+ B cells in maintenance of tolerance, the high frequency of these cells, which has also been observed after RTX therapy in rheumatoid arthritis, is compatible with amelioration of disease. Table 1 Table 1. Disclosures Gudbrandsdottir: GSK: Research Funding; Amgen: Research Funding.

scholarly journals Deep Immune Profiling of Patients with STAT1 Gain-of-Function: Revealing New Mechanisms of Pathology

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 2067-2067
Author(s):  
Saara Kaviany ◽  
Todd Bartowiak ◽  
Daniel E. Dulek ◽  
Yasmin Khan ◽  
Jonathan Michael Irish ◽  
...  
Keyword(s):  
T Cell ◽  
Research Funding ◽  
Coiled Coil ◽  
Therapeutic Interventions ◽  
Healthy Controls ◽  
Gain Of Function ◽  
Inborn Errors ◽  
Clinical Presentations ◽  
Healthy Control ◽  
Immune Profiling

Abstract Background: Patients with heterozygous signal transducer and activator of transcription 1 (STAT1) gain-of-function (GOF) pathogenic variants exhibit an array of clinical phenotypes including susceptibility to multiple infections, autoimmunity, and cancer predisposition. Previous studies to characterize the pathways involved and explore therapeutic interventions have been constrained by the technology to perform in-depth immunophenotyping. Mass cytometry has allowed us to perform extensive immune profiling of patients with inborn errors of immunity (IEI) to help gain a better understanding of disease pathology. STAT1 gain-of-function (GOF) mutations have demonstrated higher levels of phosphorylated STAT1 in response to type I and II interferons, but the response to other cytokines is less understood. Using advanced cytometry, we demonstrate a unique pattern of STAT1 phosphorylation in response to IL-6 stimulation in T-cell subsets and this differential pattern may play a role in T-cell differentiation and memory in STAT1 GOF patients. Cases: We report two patients with heterozygous STAT1 GOF mutations in the coiled-coil domain. For both patients, the clinical phenotype was largely consistent with other STAT1 GOF patients, one (P1, c.800C&gt;T; p.ala267Val) presented with secondary HLH due to histoplasmosis, and the second (P2, c.866A&gt;G; p.Tyr289Cys) presented with presumed vaccine strain varicella zoster virus (VZV) meningitis and subsequent history of recurrent herpes simplex virus (HSV) skin lesions. Patient peripheral blood mononuclear cells (PBMCs) were evaluated by fluorescence flow cytometry and cytometry by time of flight (CyTOF) as previously described (Roussel et al. J Leukoc Biol. 2017) to evaluate the impact of STAT1 GOF mutations on T-cell immunophenotype and cytokine signaling. Results: Utilizing cytometric data, we were able to identify similar patterns of T cell distribution on t-distributed stochastic neighbor embedding (t-SNE) plots for both patients with STAT1 GOF that were distinct compared to healthy controls (Fig 1a). In the T-cell compartment, both patients had decreased Th17 and Treg populations and an increased Th1/Th2 ratio compared to healthy donor (Fig 1b). In response to stimulation with IFNg or IL-6, there were also clear patterns with the two patients compared to healthy controls. Levels of p-STAT1 and p-STAT3 were assessed in STAT1 GOF and health donor PBMCs at several times points between 15 and 120 minutes, after stimulation with either IFNg or IL-6. Using fluorescence flow, we found that IL-6 stimulation led to greater than anticipated p-STAT1 response at all timepoints compared to a much more muted response to IFNg. The cell subsets highlighted in the t-SNE after IL-6 stimulation differ from the cell subsets that respond to IFNg stimulation in patients and healthy control (Fig 2a), suggesting that distinct cell populations are driving the response to IL-6. By evaluating these IL-6 responsive subsets in comparison with healthy control by CyTOF, we identified an exaggerated p-STAT1 response to IL-6 in the memory T-cell populations in P2 (Fig 2b). Conclusions: These two unique clinical presentations demonstrate that with similar mutations in the coiled-coil domain of STAT1, yet largely differing clinical presentations, the immune profiling patterns of the patients compared to healthy controls can drive further work on disease characterization and therapeutic interventions. This is relevant for this patient cohort, as treatment recommendations for STAT1 GOF are not well established. Long-term outcomes with JAK inhibition are lacking and transplant survival rates to date have been very poor compared to other immune diseases including familial HLH. With identification of IL-6 signaling playing a potential role in T-cell maturation, further studies will need to be performed to determine if IL-6 modulation could be used as a treatment modality. We conclude that performing deep immunophenotyping of patients with inborn errors of immunity (IEIs) such as STAT1 GOF can point to new disease mechanisms of human immunity and inflammation and lead to improved understanding of the role of cytokine and cellular signaling in normal hematopoiesis and cellular maturation. Figure 1 Figure 1. Disclosures Rathmell: Sitryx: Consultancy, Current equity holder in publicly-traded company, Research Funding; Caribou: Consultancy, Current equity holder in publicly-traded company, Current holder of stock options in a privately-held company; Nirogy: Consultancy, Current holder of stock options in a privately-held company; Merck: Speakers Bureau; Pfizer: Speakers Bureau; Mitobridge: Consultancy; Incyte: Research Funding; Calithera: Research Funding; Tempest: Research Funding.

scholarly journals Immune Profiling of Immune Thrombocytopenia (ITP) Patients: Evidence for CD8 T Cell Involvement in the Disease

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 2259-2259
Author(s):  
Monica Escorcio-Correia ◽  
Andrew Provan ◽  
Daniel J Pennington
Keyword(s):  
T Cells ◽  
B Cells ◽  
Research Funding ◽  
Immune Thrombocytopenia ◽  
Cytotoxic T Cells ◽  
Granzyme B ◽  
Cd8 T Cell ◽  
Interferon Γ ◽  
Healthy Controls ◽  
Platelet Autoantibodies

Abstract Introduction: Immune thrombocytopenia (ITP) is a bleeding disorder caused by an autoimmune response against platelets. In the majority of cases, ITP is thought to be caused by the presence of autoreactive B cells that produce anti-platelet autoantibodies and target platelets for destruction by phagocytic cells. However, in about 40% of ITP patients platelet autoantibodies cannot be detected and there is some evidence that cytotoxic cells might also be responsible for platelet death. Indeed, many patients repeatedly fail to respond to current immunosuppressive therapies that target B cells and their autoantibodies. As a consequence, these patients retain very low platelet counts with increased bleeding diathesis. In this study we have immunophenotyped a group of adult chronic ITP patients that have not responded to traditional immunosuppressive therapies and we identified 2 subgroups of patients with either an increase or decrease in the frequency of CD8+ T effector memory CD45RA+ cells (CD8TEMRA) compared to healthy controls. Methods: PBMCs were isolated from blood samples of 14 ITP patients with platelet counts <100x109/L and 14 matched healthy controls. The cells were phenotyped using a variety of antibodies including: CD3, CD4, CD8, CD45RA, CCR7, CD127, CD25, CD14, CD16 and CD19. In addition, at least 5x106 PBMCs were stimulated with PMA (50ng/ml) and ionomycin (1µg/ml) for 5 hours at 37°C, 5% CO2 and stained with antibodies against CD3 and CD8, then fixed and permeabilised before staining with antibodies specific to Granzyme B and Interferon-γ. Results and discussion: In our cohort of ITP patients we were able to identify two subgroups of patients based on their frequency of CD8TEMRA cells, identified as CD45RA+ CCR7- cells, gated on CD3+ CD8+ cells. Compared to healthy controls (mean=16.33%), 6/14 patients had significantly lower frequencies of CD8TEMRA cells (mean=11.31%) and 8/14 patients showed a significant increase (mean=31.50%). Interestingly, these two groups of patients also show significant differences between them in the frequency of CD19+ B cells (gated on CD3- cells), as the group with the lowest CD8TEMRA frequency showed a significant increase in B cells compared to the high CD8TEMRA group. Considering that CD8TEMRA cells are described as highly differentiated cytotoxic T cells, these results suggest that in patients with active ITP in which the CD8TEMRA population is more prevalent and the frequency of B cells is reduced, cytotoxic T cells might play an important role in platelet destruction. Although an increase in the frequency of CD8TEMRA with age has been described we did not find a correlation between these two variables in our cohort of patients. In the low CD8TEMRA group we also observed a significant increase in the frequency of T regulatory cells (Tregs) and monocytes when compared to healthy controls, whereas the trend in the high CD8TEMRA group was for frequencies closer to controls. In addition, when analysing the production of Granzyme B and Interferon-γ after a short in vitro stimulation, we found that the trend was for the CD8+ T cells in the high CD8TEMRA group to produce higher levels of both Granzyme B and Interferon-γ when compared to the patients in the low CD8TEMRA group. This would support the hypothesis that in patients with increased frequency of CD8TEMRA there has been an expansion of cells with cytotoxic properties. Further work will be required to confirm that in this cohort of patients there is a CD8+ T cell population that can specifically target and lyse platelets, thus contributing to ITP pathogenesis. Disclosures Provan: UCB: Consultancy; GSK: Equity Ownership, Honoraria, Research Funding; Amgen: Honoraria, Research Funding; Medimmune: Consultancy.

scholarly journals Active and Ultra-Large Von Willebrand Factor Is Significantly Elevated in Patients with Immune Thrombotic Thrombocytopenic Purpura

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 523-523
Author(s):  
Wenjing Cao ◽  
Alicia Veninga ◽  
Elizabeth M. Staley ◽  
Adam Miszta ◽  
Nicole Kocher ◽  
...  
Keyword(s):  
Thrombotic Thrombocytopenic Purpura ◽  
Von Willebrand Factor ◽  
Plasma Levels ◽  
Acute Episode ◽  
Healthy Controls ◽  
Plasma Samples ◽  
Adamts13 Activity ◽  
Thrombocytopenic Purpura ◽  
Von Willebrand ◽  
Willebrand Factor

Abstract Background: Immune thrombotic thrombocytopenic purpura (iTTP), a potentially fatal hematological emergency, is primarily caused by acquired deficiency of ADAMTS13 activity due to autoantibodies. Immunoglobulin G (IgG)-type autoantibodies bind ADAMTS13 and inhibit its ability to cleave endothelium-derived ultra large von Willebrand factor (ULVWF). However, it remains poorly understood whether plasma VWF status can be used as a disease marker for diagnosis and monitoring therapy in patients with acute iTTP. Objective: To address this question, we determined plasma levels of VWF antigen (VWF:Ag), collagen-binding activity (VWF:CB), active forms of VWF (VWF:Ac), and VWF multimers in iTTP patients during acute episode and in early remission. Patients and Methods: From the Alabama registry, we identified 69 unique patients with a confirmed diagnosis of iTTP in whom plasma ADAMTS13 activity was <10 U/dL with positive inhibitors and elevated anti-ADAMTS13 IgGs. Of 69 patients, 21 had longitudinal plasma samples collected. Plasma samples from 56 healthy individuals, who did not have a hematological disease, cancer, and infection, were recruited as controls. Plasma levels of VWF:Ag, VWF:CB, and VWF:Ac were determined by an ELISA-based assay. Plasma VWF multimer distribution was assessed by an in-gel Western blotting assay following electrophoresis on a 1% SDS-agarose gel. Results: The mean age for our cohort iTTP patients was 43.9 ± 13.4 years. Twenty-six patients were male and 43 were female with male to female ratio of 1 to 1.7. Fifty-three patients were African American descents, 14 Caucasians, 1 Hispanic, and 1 unknown race. Plasma levels of VWF:Ag in acute iTTP patients were 289.4 ± 17.7%, significantly increased compared with those in the healthy controls (144.9 ± 7.6%) (p<0.0001); plasma levels of VWF:CB in these patients were 241 ± 17.9%, also significantly elevated compared with those in the healthy controls (149.9 ± 12.01%) (p=0.0001); additionally, plasma levels of VWF:Ac (304.6 ± 23.2%), assessed by its ability to bind anti-VWF-A1 nanobody, were more dramatically elevated compared with those in the controls (101.6 ± 5.9%) (p<0.0001). More interestingly, while the ratios of VWF:CB to VWF:Ag in patients with acute iTTP (0.8 ± 0.04) were lower than those in the healthy controls (1.0 ± 0.05) (p=0.0036), the ratios of VWF:Ac to VWF:Ag were significantly higher in patients with acute episode (1.2 ± 0.1) than those in the controls (0.8 ± 0.05) (p=0.0003). Furthermore, there was no statistically significant difference in the patient plasma levels of VWF:Ag (p=0.69) and VWF:CB (p=0.08) during acute episode and during early remission. However, the plasma levels of VWF:Ac in patients with acute disease were significantly higher than those in the early remission (p=0.002). Surprisingly, 90% (36/40) of out iTTP patients during acute episode showed the presence of ULVWF in their plasma using in-gel Western blotting, which allows the ULVWF to be detected without the transfer step to avoid any potential loss of larger VWF multimers during protein transfer. These ULVWF multimers disappeared in 3/4 iTTP patients in remission when ADAMTS13 activity recovered. In 28 healthy control samples, only one showed ULVWF. Conclusion: Our results demonstrate, for the first time in a large cohort, that active forms of VWF and ultra large VWF multimers are present in iTTP patient's plasma during the acute period, which is reduced or disappears during the early remission. Therefore, measuring active forms of VWF and ultra large VWF multimers may aid in diagnosis of iTTP and help monitoring of disease processes following therapy. Our ongoing study is to determine whether these biomarkers can be used to predict responses to treatment and long-term outcome. Disclosures Zheng: Alexion: Research Funding, Speakers Bureau.

Association of isocitrate dehydorgenase-1 (IDH-1) mutations with elevated oncometabolite 2-hydroxyglutarate (2HG) in advanced colorectal cancer.

2016 ◽  
Vol 34 (4_suppl) ◽  
pp. 627-627 ◽  
Author(s):  
Nazanin Fallah-Rad ◽  
Philippe L. Bedard ◽  
Lillian L. Siu ◽  
Suzanne Kamel-Reid ◽  
Helen Chow ◽  
...  
Keyword(s):  
Plasma Levels ◽  
Advanced Colorectal Cancer ◽  
Matched Control ◽  
Illumina Miseq ◽  
Tandem Mass ◽  
Plasma Samples ◽  
Elevated Plasma ◽  
Laboratory Plasma ◽  
Kras Mutations ◽  
Archived Samples

627 Background: IDH-1 mutations are common in AML, gliomas, and intrahepatic cholangiocarcinoma. The enzyme product of the IDH-1 R132 variant preferentially catalyzes the NADPH-dependent reduction of alpha-ketoglutarate to the oncometabolite R-2-Hydroxyglutarate (R-2HG), resulting in accumulation of R-2HG, relative to its enantiomer, S-2HG. Elevated plasma levels of 2HG and/or higher ratios of R/S have been observed in AML and intrahepatic cholangiocarinoma, but not in gliomas. Only a few cases of IDH-1/2 mutations have been reported in advanced colorectal cancers (CRC). We investigated plasma levels of 2HG and relative ratios of R/S in CRC patients harboring IDH-1/2mutations. Methods: Between 2012 and 2015, 428 patients with advanced CRC were molecularly profiled through the COMPACT and IMPACT programs at the Princess Margaret Cancer Centre. Tumor DNAs were isolated from FFPE archived samples and genotyped using a customized Sequenom panel or Illumina MiSeq TruSeq Amplicon Cancer Panel in a CLIA-certified laboratory. Plasma samples from patients harboring IDH-1/2mutations and 4 gender/age matched control patients were analyzed for 2HG, R-2HG and S-2HG using HPLC tandem mass spectrometry coupled with a CHIROBIOTIC R column. Results: Of 428 patients with advanced CRC, 4 (0.9%) patients were identified to harbor IDH-1 mutation. No IDH-2 mutations were detected. The variants identified were R132C (2 patients), R132S, and R132H. 3 of 4 patients also harbored KRAS mutations. Patients with IDH-1 mutations had higher levels of 2HG (319 ± 102 ng/ml vs 195 ± 43 ng/ml, p = 0.043). The ratio of R/S were 0.44, 0.75, 1.59, and 2.60 in 4 patients with IDH-1 mutations and 1.3, 0.90, 0.41 and 0.67 in 3 patients without IDH-1mutations. Conclusions: IDH-1 mutations are rare in advanced CRC. 3 of 4 patients in our study had concurrent KRAS mutations. Patients with IDH-1 mutations had higher levels of oncometabolite 2HG compared with controls with no difference in R/S ratio.

PLASMA LEVELS OF TESTOSTERONE IN BULLS

1978 ◽  
Vol 88 (4) ◽  
pp. 787-792 ◽  
Author(s):  
Anne Sundby ◽  
P. A. Torjesen
Keyword(s):  
Testosterone Level ◽  
Plasma Levels ◽  
Leydig Cells ◽  
Sharp Decrease ◽  
Plasma Testosterone ◽  
Plasma Samples ◽  
Elevated Plasma ◽  
Plasma Testosterone Level ◽  
Testosterone Levels ◽  
Testosterone Production

ABSTRACT Administration of 6000 IU HCG to 4 bulls was followed by an elevation of plasma testosterone lasting for 9–13 days. When HCG administration was repeated, the testosterone response was shortened to 4–6 days in 3 bulls due to the formation of antibodies against HCG. The appearance of HCG antibodies coincided with a sharp decrease in the plasma testosterone level, indicating that Leydig cells have to be under continuous HCG stimulation to maintain increased testosterone production. No antibody against bovine LH was detected in the plasma samples containing antibodies against HCG. In one bull the response following the second HCG injection was similar to the plasma testosterone pattern following the first. No antibodies against HCG were found in this bull. Five bulls received 750 IU HCG twice. Following the period with elevated plasma testosterone levels, subnormal levels were observed after both injections. One injection led to decreased levels without development of antibodies against HCG while the second HCG injection led to subnormal testosterone levels concomitant with measurable antibodies against HCG.

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