1.Changes of DC Subsets and CD80 and CD86 Expression in Peri-pheral Blood of Patients with ITP and Their Correlation with Efficacy of Dexamethasone Treatment.
Xiao-Juan LI ; Xiao-Shuang SONG
Journal of Experimental Hematology 2018;26(6):1752-1756
OBJECTIVE:
To analyze the changes of DC subsets and the expression of CD80 and CD86 in peripheral blood of ITP patients and their correlation with dexamethasone efficacy.
METHODS:
Peripheral blood sample of 80 cases of ITP and 20 normal controls from June 2015 to June 2017 in our hospital were retrospectively analyzed. The specific distribution of DC subsets in the peripheral blood of all the subjects was detected by flow cytometry, and the expressions of CD80 and CD86 were detected by ELISA.
RESULTS:
The proportion of DC2 in DC subsets of ITP patients before treatment was significantly higher than that in normal control group (P<0.05). The proportion of DC2 in DC subset of ITP patients was still significantly higher than that of the control group (P<0.05). The level of CD80 expression on DC1 and DC2 in ITP patients before treatment was significantly higher than that in the normal control group (P<0.05), and the expression level of CD86 on DC2 was significantly higher than that of the normal control group (P<0.05). Both IL-2 and IFN- γ levels in the patients before the treatment were significantly higher than those in the normal control group (P<0.05), and the expression levels after treatment with dexamethasone decreased significantly. Before treatment, both IL-4 and IL-10 levels in ITP patients were significantly lower than those in the normal control group (P<0.05), and their expression levels after treatment with dexamethasone significantly increased (P<0.05).
CONCLUSION
The incidence of ITP patients closely relates with the level and dysfunction of DC subsets in peripheral blood and the expression levels of IL-2, IL-4, IL-10, IFN- γ, which significantly correlates with the efficacy of dexamethasone.
B7-1 Antigen
;
Dendritic Cells
;
Dexamethasone
;
Humans
;
Inosine Triphosphate
;
Retrospective Studies
2.Curative Efficacy of Rituximab for ITP Patients with Different Sensitivity to Hormone.
Jun-Xiu LIU ; Huan-Xin ZHANG ; De-Peng LI ; Wei-Wei XING ; Hu-Jun LI ; Wei CHEN ; Hai CHENG ; Jiang CAO ; Zhen-Yu LI ; Zhi-Ling YAN ; Kai-Lin XU
Journal of Experimental Hematology 2019;27(5):1602-1606
OBJECTIVE:
To investigatc the curative efficacy of low dose rituximab for glucocorticoid ineffective on dependent ITP patients and its relation with sensitivity to glucocorticoid so as to provide reference basis for rational use of drugs in clinical treatmant.
METHODS:
Seventy-ninth ITP patients enrolled in this study included the glucocorticoid-ineffective patients (19 cases) and glucocorticoid-dependent patients (60 cases). All ITP patients were treated with regimen consisted of high dose dexamethasone plus low dose rituximab (dexal-methasone 40 mg/d for 4 days per os, ritaximab 100 mg by intravenous infusion at D7, 14, 21 and 28 respectively). The patients after treatment were followed-up for 12 month, and the relation of patients sensitivity to glucocorticoid with therapentic response of rituximab was analyzed. The changes of Treg cell ratio and BAFF, IL-2 and sCD40L levels before and after treatment were detected by flow cytometry and ELISA respectively.
RESULTS:
The overall response rate (ORR) of patients treated with above- mentioned regemen at 1, 3, 6 and 12 months after treatment was 79.7% (63/79), 69.6% (55/79), 63.3% (50/79) and 60.8% (48/79) respectivcly, out of which the ORR of glucocorticoid ineffective and glucocorticoid-dependent ITP patients treated with above-mentioned regimen at 1, 3, 6 and 12 months after treatment was 47.4% (9/19) vs 90.0% (54/60), 36.8% (7/19) vs 80.0% (48/60), 21.1% (4/19) vs 76.7% (46/60), 21.1% (4/19) vs 73.3% (44/60), and the difference between 2 groups was statistically significant. The detection of T reg cell showed that the T reg cell ratio in glucocorticoid- ineffective and dependent patients at 1, 3, 6 and 12 months after treatment was (1.70±0.43)% vs (3.47±0.72)%, (1.66±0.33)% vs (4.29±0.91)%, (1.71±0.37)% vs (4.44±0.97)%, (3.36±0.54)% vs (4.29±1.04)%, respectively. The detection of cytokines showed that the levels of BAFF, IL-2 and sCD40L in plasma of glucocorticoid-dependent patients at 1 month after treatment significanlly decreased (P<0.05), the levels of BAFF, IL-2 and sCD40L in plasma of glucocorticoid-ineffective patients although decreased at 1 mouth after treatment, but there was no statistical difference as compared with glucocosticoid-depenment patients.
CONCLUSION
The treatment of glucocorticoid-dependent ITP patients with rituximab is more effective. The regulatory effect of rituximab on the T-reg cells, BAFF, IL-2 and sCD40L may be one of its mechanisms.
Dexamethasone
;
Glucocorticoids
;
Humans
;
Inosine Triphosphate
;
Purpura, Thrombocytopenic, Idiopathic
;
drug therapy
;
Rituximab
;
therapeutic use
3.Analysis of nucleotides and their derivatives in renal tissue of rat during ischemia by HPLC.
Yeungnam University Journal of Medicine 1992;9(1):90-101
In rat kidney, the changes in concentrations of nucleotides and their derivatives during ischemia induced by renal artery ligation was measured quantitatively with high performance liquid chromatography (HPLC). After the ligation of renal artery for 60minutes, the concentrations of the nucleotides and derivatives to 80.7±18.39 µg (p<0.01); ATP, 307.2±56.68 µg to 47.6±5.95µg (p<0.01); ADP+AMP, 227.1±7.98 µg to 61.4±3.92 µg (P<0.01); NAD+, 217.9±4.49 µg to 126.6±10.44 µg (P<0.01); GTP, 202.5±23.76 µg to 117.7±14.24 µg (P<0.05); GMP, 54.5±9.03µg to 23.7±0.46 µg (p<0.05), and inosine, 16.6±3.45 µg to 7.8±0.87 µg (P<0.05). But hypoxanthine and xanthine were significantly increased from 113.0±15.58µg to 159.7±12.97µg (P<0.05) and from 87.7±6.77µg to 173.1±12.52µg (P<0.01). In ischemic kidney, concentration of ATP was decreased to 39.9% of control at 10 minutes, 19.8% at 30 minutes, and 15.5% at 60 minutes, and ADP+AMP were decreased to 70.3% of control at 10 minutes, 67.3% at 30 minutes, and to 27.0% at 60 minutes, but hypoxanthine and xanthine were increased to 121.5% and 127.1% at 10 minutes, 126.0% and 174.4% at 30 minutes, and 141.4% and 197.3% at 60 minutes. Total adenosine nucleotides were decreased to 20.3% of control during 60 minutes of ischemia, but hypoxanthine and xanthine were increased to 157.5% of control. These results suggest that the changes in the concentration of nucleotides and their metabolic derivatives are useful indices of the extents of tissue ischemia in rat kidney.
Adenosine
;
Adenosine Triphosphate
;
Animals
;
Chromatography, High Pressure Liquid*
;
Chromatography, Liquid
;
Guanosine Triphosphate
;
Hypoxanthine
;
Inosine
;
Ischemia*
;
Kidney
;
Ligation
;
Nucleotides*
;
Rats*
;
Renal Artery
;
Xanthine
4.Effect of Decitabine on Megakaryocyte Culture of Steroid-resistant ITP Patients.
You-Ping LIAO ; Yu-Xia LIU ; Qing-Zhao LI ; Guo-Yu HU
Journal of Experimental Hematology 2015;23(4):1092-1096
OBJECTIVETo investigate the effect of decitabine and plasma of ITP patients on in vitro cultrue of megakaryocytes in bone marrow of steroid-resistant ITP patients.
METHODSBone marrow mononuclear cells were isolated from 20 steroid-resistant ITP patients, both methyl cellulose semisolid culture system (to observe and count the number of megakaryocytes colony-forming unit) and liquid culture system (to analysis the expression rate of CD41a(+) cells) were used for megakaryocyte cultrue. The experiments were divided into 4 groups according to the different components of the culture system, group A was control, group B was added with decitabine, group C with ITP plasma, group D with both decitabine and ITP plasma, and the rest of the culture components were the same in the 4 groups except the above-mentioned materials. Morphology of megakaryocytes was observed by inverted and light microscopy. The expression rate of CD41a⁺ cells in culture was analysed by flow cytometric.
RESULTSDifferent concentration of decitabine showed different effect on megakaryocyte growth of steroid-resistant ITP patients and the optimal concentration to differentiate into megakaryocyte for bone marrow mononuclear cells is 3.0 µmol/L. Compared with group A, both megakaryocyte colony forming units (CFU) and expression rate of CD41a⁺ cells in group B were statistically significantly higher (P < 0.05). As compared with group A, the megakaryocyte colony-forming units in group C decreased with statistically significant difference, while compared with group C, the megakaryocyte colony-forming units in group D obviously increased with statistically significant difference.
CONCLUSIONSDecitabine is able to induce bone marrow mononuclear cells of steroid-resistant ITP patients to differentiate into megakaryocyte and the optimal concentration is 3.0 µmol/L; ITP plasma is able to inhibit the megakaryocyte growth of steroid-resistant ITP patients.
Azacitidine ; analogs & derivatives ; Bone Marrow ; Bone Marrow Cells ; Drug Resistance ; Flow Cytometry ; Hematopoietic Stem Cells ; Humans ; Inosine Triphosphate ; Megakaryocytes ; Stem Cells ; Steroids
5.In Vitro Perfusion Studies on Coronary Function of Cardiac Ischemia-Reperfusion in Spontaneously Hypertensive Rat Heart.
Korean Circulation Journal 1995;25(1):59-67
BACKGROUND: Myocardial ischemia in human hypertension and in various animal models of hypertension may be due to abnormal maximal coronary vasodilator reserve and disturbaces of coronary vasomotion. The vascular reactivity defects in hypertension have been associated with the defective endothelium and sympathetic neural activation. However, such abnormalities in hypertension need to be elucidated. In the present study the effectsof cardiac ischemia reperfusion on coronary circulation, intramyocytic adenylates and purine nucleosides were examined in Langendorff-perfused Sprague Dawley rat (SD) and spontaneously hypertensive rat (SHR) hearts. Coronary venous and cardiac lactate and cardiac pyruvate were also measured. It should be noted that in the regulation of coronary flow the intrinsic flow autoregulation is highly variable due to coexisting metabolic flow control, and that natural coronary flow and cardiomyocytic energy state are normally reciprocally related in perfused heart. METHODS: For the Langendorff heart perfusion, bicarbonate perfusion buffer (pH 7.40+/-0.02,37degrees C) was equilibrated with 95% O2 : 5% CO2 and contained 5mM glucose (+5U/1 insulin) and 2mM pyruvate as energy-yielding substrates. Global hypoperfusion ischemia was induced by lowering coronary perfusion pressure of 100 to 40 cmH2O, followed by 20 min reperfusion. RESULTS: During the ischemia and reperfusion, metabolic acidosis and enhanced venous lactate output in SHR were observed with increases in coronary vascular resistance and myocardial oxygen consumption.In addition, coronary reactive hyperemia during reperfusion was depressed. Although ischemia-induced increase in combined adenosine plus inosine were abolished during prolonged reperfusion, SD still exhibited coronary vasodilation. The depressed reactive hyperemia in SHR was associated with decreases in cardiac adenosine triphosphate (ATP) pool and creatine phosphate/inorganic phosphate (CrP/Pi) ratio and an increase in cardiac lactate/pyruvate ratio. CONCLUSION: This abnormal vascular reactivity during ischemia and reperfusion in SHR may be in part due to an alteration in the cardiac energy state and hence to a mismatch between myocardial metabolic demand and supply.
Acidosis
;
Adenosine
;
Adenosine Triphosphate
;
Animals
;
Coronary Circulation
;
Creatine
;
Endothelium
;
Glucose
;
Heart*
;
Homeostasis
;
Humans
;
Hyperemia
;
Hypertension
;
Inosine
;
Ischemia
;
Lactic Acid
;
Models, Animal
;
Myocardial Ischemia
;
Oxygen
;
Perfusion*
;
Purine Nucleosides
;
Pyruvic Acid
;
Rats
;
Rats, Inbred SHR*
;
Reperfusion
;
Vascular Resistance
;
Vasodilation
6.Mycophenolic Acid Induced Apoptotic Signal Transduction in Molt-4 T-cells.
Soo JinNa CHOI ; Sang Young CHUNG ; Shin Kon KIM
Journal of the Korean Surgical Society 2002;62(1):8-17
PURPOSE: Mycophenolic acid (MPA), a selective inhibitor of inosine monophosphate dehydrogenase (IMPDH), is the active metabolite of the immunosuppressive drug, mycophenolate mofetil (MMF). MMF is used to prevent an immune- mediate rejection response following organ transplantation via the inhibition of the IMPDH and GTP biosynthesis pathway. This study was designed to elucidate the mechanism by which MPA exerts its cytotoxic effect on human T lymphocytic and monocytic cell lines. METHODS: MOLT-4 and U937 cell lines were treated with MPA. Cell viability, expression of Bcl2 family proteins and Fas/Fas-L, effects of antioxidants and intracellular Ca2+ regulating agents and apoptosis were measured using a variety of microscopic and biochemical techniques. RESULTS: MPA induced the death of U937 and MOLT-4 cells in dose and time dependent manners, which was revealed an apoptosis with a characteristic ladder pattern of DNA fragmentation. In addition, BAPTA/AM, an intracellular Ca2+ chelator protected MOLT-4 cells from MPA treated apoptosis, although it did not have an additive with thapsigargin, and increases cytosolic Ca2+ stores. However, antioxidants including reduced glutathione (GSH) and N-acetyl-L-cysteine (NAC) did not inhibit the apoptosis of cells by MPA. Furthermore, guanosine suppressed MPA induced apoptosis of MOLT-4 lymphocytes, although adenosine did not. MPA also increased the catalytic activity of caspase family cysteine proteases including caspase-8, 9 and 3 proteases in MOLT-4 cells. Sequential activation indicated that the cleavage of caspase-8 and 9 precedes those of caspase-3. CONCLUSION: The results suggest that MPA induces the apoptotic death of MOLT-4 lymphocytes via the activations of caspase family proteases and the depletion of GTP.
Acetylcysteine
;
Adenosine
;
Antioxidants
;
Apoptosis
;
Caspase 3
;
Caspase 8
;
Cell Line
;
Cell Survival
;
Cysteine Proteases
;
Cytosol
;
DNA Fragmentation
;
Glutathione
;
Guanosine
;
Guanosine Triphosphate
;
Humans
;
Inosine Monophosphate
;
Lymphocytes
;
Mycophenolic Acid*
;
Organ Transplantation
;
Oxidoreductases
;
Peptide Hydrolases
;
Signal Transduction*
;
T-Lymphocytes*
;
Thapsigargin
;
Transplants
;
U937 Cells
7.Vasodilating effect and its mechanism of ethanol on isolated rat thoracic aorta at different resting tension.
Xiao-Chen RU ; Ling-Bo QIAN ; Jie CUI ; Yun QIAN ; Qin GAO ; Qiang XIA
Chinese Journal of Applied Physiology 2008;24(3):269-273
AIMTo investigate the vasodilating effect and its mechanism of ethanol on isolated rat thoracic aorta at different resting tension.
METHODSThe tension of the isolated Sprague-Dawley rat thoracic aorta rings perfused with different concentrations of ethanol was measured using organ bath technique.
RESULTSAt different resting tension (1.0, 1.5, 2.0, 2.5, 3.0, 3.5 and 4.0 g), ethanol (0.1-7.0 per thousand) caused a concentration-dependent relaxation on endothelium-denuded aortic rings precontracted with KCl (6 x 10(-2)mol/L) or phenylephrine (PE, 10(-6) mol/L), and the vasodilating effect was the most potent when the aortic rings were at the resting tension of 3 g. Ethanol had much less vasodilating effect on endothelium-intact aortic rings. Ethanol at 3 per thousand (the maximum-effect concentration) inhibited the CaCl2 induced contraction and downward shifted concentration-response curve of endothelium-denuded aortic rings pre-contracted with KCI or PE at the resting tension of 3 g. Incubation of aorta with ruthenium red (10(-5) mol/L) or heparin (50 mg/L) decreased the vasodilating effect of ethanol (3.0 per thousand) on endothelium-denuded aorta precontracted with PE at the resting tension of 3 g.
CONCLUSIONEthanol induces endothelium-independent relaxation on rat thoracic aorta, which is concerned with the resting tension. This effect of ethanol may be mediated by the inhibition of voltage-dependent and receptor-operated Ca2+ channels in the vascular smooth muscle cells. The inhibition of the ryanodine receptor and trisphosphate inositol (IP3) pathway may also contribute to this effect.
Animals ; Aorta, Thoracic ; drug effects ; Calcium Channel Blockers ; pharmacology ; Ethanol ; pharmacology ; In Vitro Techniques ; Inosine Triphosphate ; metabolism ; Male ; Muscle, Smooth, Vascular ; drug effects ; Rats ; Rats, Sprague-Dawley ; Ryanodine Receptor Calcium Release Channel ; drug effects ; Vasodilation ; drug effects
8.Mizoribine-mediated Apoptosis Signaling in Jurkat T Cells.
Kyoung Won SEO ; So Hwan CHUNG ; Sang Young CHUNG ; Shin Kon KIM ; SooJin Na CHOI
Journal of the Korean Surgical Society 2004;66(4):259-270
PURPOSE: Mizoribine (MZR), an inhibitor of Inosine monophosphate (IMP) dehydrogenase which depletes cellular GTP, is clinically used as an immunosuppressive drug. This study was designed to evaluate the mechanism by which MZR exerts the cytotoxic effect on Jurkat T cells. METHODS: Jurkat T cell is a human T lymphocytic cell line. It was obtained from the Korean Type Culture Collection. Cell viability was measured by the MTT assay and flow cytometry. Caspase activity assay, Western blotting, 2-D PAGE, and mitochondrial membrane potential were detected using biochemical analysis. Morphologic finding was observed by Hoechst staining. RESULTS: The data demonstrated that the treatment of MZR decreased cell viability in a dose- and time-dependent manner. MZR-induced cell death was confirmed as apoptosis, which was characterized by chromatin condensation and H2AX phosphorylation. MZR increased the catalytic activity of caspase-3 protease, -8 protease and -9 proteases. The activation of caspase-3 protease was further confirmed by the degradation of polymerase (PARP), a substrate of caspase-3 protease by MZR in Jurkat T cells. Furthermore, MZR induced the changes of the mitochondrial transmembrane potential (MTP) and the cytosolic release of cytochrome c from the mitochondria. In addition, MZR induced the decrease of Bcl-X(L) expression whereas the increase of Bcl-X(S), Bak and Bim expression. Guanosine markedly inhibited cell viability and apoptosis through consistent suppression of the activity of caspase-8 protease, an upstream caspase among the caspase family, H2AX phosphorylation and PARP cleavage in MZR-treated cells. Also, I have screened the expression profile of proteins in the Jurkat T cells by using two-dimensional (2-D) gel electrophoresis. Among 300 spots resolved in the 2-D gels, the comparison of the control versus apoptotic cells revealed that the signal intensity of 10 spots was decreased and 5 spots was increased. CONCLUSION: The results suggest that MZR functions as an inhibitor of IMP dehydrogenase in apoptosis of Jurkat T cells via activation of intrinsic caspase cascades as well as mitochondrial dysfunction.
Apoptosis*
;
Blotting, Western
;
Caspase 3
;
Caspase 8
;
Cell Death
;
Cell Line
;
Cell Survival
;
Chromatin
;
Cytochromes c
;
Cytosol
;
Electrophoresis
;
Flow Cytometry
;
Gels
;
Guanosine
;
Guanosine Triphosphate
;
Humans
;
IMP Dehydrogenase
;
Inosine Monophosphate
;
Membrane Potential, Mitochondrial
;
Membrane Potentials
;
Mitochondria
;
Oxidoreductases
;
Peptide Hydrolases
;
Phosphorylation
;
T-Lymphocytes*
9.Mizoribine-mediated Apoptosis Signaling in Jurkat T Cells.
Kyoung Won SEO ; So Hwan CHUNG ; Sang Young CHUNG ; Shin Kon KIM ; SooJin Na CHOI
Journal of the Korean Surgical Society 2004;66(4):259-270
PURPOSE: Mizoribine (MZR), an inhibitor of Inosine monophosphate (IMP) dehydrogenase which depletes cellular GTP, is clinically used as an immunosuppressive drug. This study was designed to evaluate the mechanism by which MZR exerts the cytotoxic effect on Jurkat T cells. METHODS: Jurkat T cell is a human T lymphocytic cell line. It was obtained from the Korean Type Culture Collection. Cell viability was measured by the MTT assay and flow cytometry. Caspase activity assay, Western blotting, 2-D PAGE, and mitochondrial membrane potential were detected using biochemical analysis. Morphologic finding was observed by Hoechst staining. RESULTS: The data demonstrated that the treatment of MZR decreased cell viability in a dose- and time-dependent manner. MZR-induced cell death was confirmed as apoptosis, which was characterized by chromatin condensation and H2AX phosphorylation. MZR increased the catalytic activity of caspase-3 protease, -8 protease and -9 proteases. The activation of caspase-3 protease was further confirmed by the degradation of polymerase (PARP), a substrate of caspase-3 protease by MZR in Jurkat T cells. Furthermore, MZR induced the changes of the mitochondrial transmembrane potential (MTP) and the cytosolic release of cytochrome c from the mitochondria. In addition, MZR induced the decrease of Bcl-X(L) expression whereas the increase of Bcl-X(S), Bak and Bim expression. Guanosine markedly inhibited cell viability and apoptosis through consistent suppression of the activity of caspase-8 protease, an upstream caspase among the caspase family, H2AX phosphorylation and PARP cleavage in MZR-treated cells. Also, I have screened the expression profile of proteins in the Jurkat T cells by using two-dimensional (2-D) gel electrophoresis. Among 300 spots resolved in the 2-D gels, the comparison of the control versus apoptotic cells revealed that the signal intensity of 10 spots was decreased and 5 spots was increased. CONCLUSION: The results suggest that MZR functions as an inhibitor of IMP dehydrogenase in apoptosis of Jurkat T cells via activation of intrinsic caspase cascades as well as mitochondrial dysfunction.
Apoptosis*
;
Blotting, Western
;
Caspase 3
;
Caspase 8
;
Cell Death
;
Cell Line
;
Cell Survival
;
Chromatin
;
Cytochromes c
;
Cytosol
;
Electrophoresis
;
Flow Cytometry
;
Gels
;
Guanosine
;
Guanosine Triphosphate
;
Humans
;
IMP Dehydrogenase
;
Inosine Monophosphate
;
Membrane Potential, Mitochondrial
;
Membrane Potentials
;
Mitochondria
;
Oxidoreductases
;
Peptide Hydrolases
;
Phosphorylation
;
T-Lymphocytes*