Peripheral Blood Plasma Of Patients Research Articles

Study on the TGF-β Ligand Trap Enhances Erythroid Differentiation in HEL Cell By Inhibiting Smad3 Phosphorylation

Objective: Anemia is the most common clinical manifestation of MF, and its pathogenesis may be closely related to the over activation of TGF-β signaling pathway. This study aims to establish an erythroid differentiation model by inducing HEL cells with hemin and investigate the mechanism of the TGF-β ligand trap luspatercept enhances erythroid differentiation in HEL cell. Methods: 1.ELISA was used to detect the levels of GDF11 in peripheral blood plasma in patients with MF and healthy people. 2.The erythroid differentiation model was established by inducing HEL cells with hemin. The differentiation and proliferation levels of cells were detected by benzidine staining and CCK8. The expression levels of mRNA and protein in the cells were detected by RNA-seq and TMT proteomic analysis, and verified by flow cytometry, RT-PCR and WB. 3.HEL cells were induced with 40 μM hemin as a model of erythroid differentiation. 100ng/mL GDF11 interfered with the over activation of TGF-β signal pathway in differentiated HEL cells. 5μg/mL luspatercept blocked the activation of TGF-β signaling pathway by GDF11. Divided them into three groups, named: hemin group , GDF11 group, and luspatercept group. WB was used to detect the protein levels of pSmad3 and Smad4 in nuclear. Benzidine staining and flow cytometry were used to detect the levels of cell differentiation. RT-PCR and WB were used to detect the expression levels of GATA-1 and KLF1. The changes of RNA expression were detected by RNA-seq and verified by WB. 4.All experimental data were analyzed by SPSS 22.0 statistical software. Results: 1.The GDF11 levels of plasma of MF patients were higher than those of normal people ( P=0.0411). 2.The erythroid differentiation model of HEL cells induced with 40 μM hemin was successfully constructed. The results of RNA-seq and TMT proteome analysis showed that compared with the control group, the expression of hematopoietic related genes such as GATA-1, KLF1 and EPOR in hemin group were up-regulated, and the intracellular proteins related to erythroid differentiation such as HBγ- 2, HB-α and CD235a were up-regulated. Flow cytometry indicated that the expression of CD235a in hemin group was significantly higher than that of the control group (33.83% vs81%, P<0.01). RT-PCR and WB experiment showed that the expression of GATA-1 and KLF1 in hemin group were higher than those in control group. 3.WB showed that compared with hemin group, the hyperphosphorylation of smad3 and Smad4 in nuclear levels were increased in GDF11 group. And luspatercept could inhibit the hyperphosphorylation of Smad3 and reduce Smad4 in nuclear levels of GDF11 group. Luspatercept could inhibit GDF11 mediated activation of TGF-β signaling pathway in HEL cells. 4.Compared with hemin group, the benzidine positivity rate of cells and the expression of CD235a on the cell surface in GDF11 group were significantly decreased (benzidine: 13.0% vs 9%, P<0.01; CD235a: 16.8% vs 33.83%, P<0.01), and the expression of GATA-1 and KLF1 were also decreased. GDF11 could inhibit the erythroid differentiation of HEL cells. 5.Compared with GDF11 group, the benzidine positivity rate of cells and the expression of CD235a on the cell surface in luspatercept group were significantly increased (benzidine: 29.0% vs0%, P<0.01; CD235a: 31.1% vs 16.8%, P<0.01), and the expression of GATA-1 and KLF1 were increased. Luspatercept could reverse the inhibition of GDF11 on the erythroid differentiation of HEL cells. 6.Compared with the hemin group, the expression of GATA-1, KLF1, EPOR and other genes related to hematopoiesis in GDF11 group were down-regulated, and the differentially expressed genes were related to the biological functions of ribosomal metabolism process, chromosome regulation, erythropoiesis, oxygen binding, oxidoreductase activity and so on, mainly involving erythroid differentiation, oxidative phosphorylation, iron metabolism, TGF- β, JAK-STAT and other channels. Luspatercept could restore the expression of GATA-1, KLF1, EPOR and other genes by inhibiting the pathway over-activated by GDF11. Conclusion: Hemin could induce the erythroid differentiation of HEL cells. The TGF-β ligand trap luspatercept could inhibit GDF11-mediated hyperphosphorylation of Smad3 in HEL cells and promote the erythroid differentiation of HEL cells. Luspatercept could be a therapeutic tool for patients with myelofibrosis associated anemia.

Effects of plasma lipopolysaccharide changes on platelet release of vascular endothelial growth factor and thromobospondin-1 in patients with cirrhotic portal hypertension after TIPS procedure

Objective: To investigate the effects of plasma lipopolysaccharide (LPS) concentration changes on platelet release of vascular endothelial growth factor (VEGF) and thrombospondin (TSP)-1 in patients with decompensated cirrhotic portal hypertension after transjugular intrahepatic portosystemic shunt (TIPS) procedure. Methods: 169 cases with cirrhotic portal hypertension were enrolled, of which 81 cases received TIPS treatment. LPS, VEGF, and TSP-1 concentrations with different Child-Pugh class in peripheral blood plasma of patients were measured. After pre-incubation of normal human platelets with different concentrations of LPS and stimulated by collagen in vitro, platelet PAC-1 expression rate, VEGF, and TSP-1 concentrations were detected. PAC-1 expression rate and the concentrations of LPS, VEGF and TSP-1 in peripheral blood plasma of patients before and after TIPS procedure were detected. The relationship between plasma LPS, VEGF and TSP-1 concentrations and Child-Pugh score changes in patients after TIPS procedure was analyzed. Statistical analysis was performed by t-test, one-way ANOVA or Pearson's rho according to different data. Results: Plasma LPS and TSP-1 concentrations were significantly higher in Child-Pugh class C patients than class A and B, but the concentration of plasma VEGF was significantly lower than class A and B (P < 0.01). In vitro experiments showed that concentration of LPS, TSP-1, and platelet PAC-1 expression rate was higher in the supernatant, but the difference in the concentration of VEGF in the supernatant was not statistically significant. Portal vein pressure and platelet activation were significantly decreased (P < 0.01) in patients after TIPS procedure. Portal venous pressure, platelet activation, plasma LPS, and TSP-1 levels were significantly decreased continuously, while VEGF levels were significantly increased continuously after TIPS procedure. Plasma LPS concentration was positively correlated with TSP-1 concentration (r = 0.506, P < 0.001), and negatively correlated with VEGF concentration (r = -0.167, P = 0.010). Child-Pugh score change range was negatively correlated with change range of plasma VEGF concentration (r = -0.297, P = 0.016), and positively correlated with change range of plasma TSP-1 concentration (r = 0.145, P = 0.031) after TIPS. Conclusion: Portal venous pressure gradient, plasma LPS concentration and corresponding platelet activation was decreased in cirrhotic portal hypertension after TIPS procedure, and with TSP-1 reduction and VEGF elevation it is possible to reduce the liver function injury caused by portal venous shunt.

Concentration of Circulating Cell-Free DNA in the Peripheral Blood Plasma of Patients with Acute Endogenous and Exogenous Etiology Psychoses

Introduction: cell-free plasma DNA (cfDNA) is used as a marker refl ecting the level of apoptosis in the human body under stress. Acute psychosis caused by endogenous (schizophrenia) and exogenous (alcohol intoxication) factors in the patient’s body is associated with oxidative stress. Presumably, cfDNA concentration in the blood plasma of patients with acute psychoses of endogenous and exogenous etiology is increased. The purpose of the study: comparative analysis of the cfDNA concentration in the blood plasma of treated and untreated patients with paranoid schizophrenia during the disease exacerbation, patients with alcoholic psychosis and healthy volunteers. Patients and methods: the concentration of cfDNA was determined in the blood plasma samples of 476 people: control group (n = 95); patients with schizophrenia in the acute stage of the disease (n = 334); patients with alcoholic psychosis (n = 47). Results: the concentrations of cfDNA in the plasma of patients with schizophrenia (median 931 ng/ml) is 2.2 times higher than in the control group (median 428 ng/ml) and 1.8 times higher than in the patients with alcoholic psychosis (504 ng/ml). For the patients with schizophrenia with high PANSS, we found the highest values of the cfDNA concentration in the blood plasma during psychosis, which indicates a more pronounced systemic process, which is accompanied by the cell death level increase. Conclusions: the concentration of cfDNA in the blood plasma could be used as a biochemical marker that refl ects the severity of the schizophrenia patient’ state upon admission to the hospital.

Circulating cell-free DNA in the peripheral blood plasma of patients is an informative biomarker for multiple myeloma relapse.

Multiple myeloma (MM) is an incurable hematological malignancy. Despite the introduction of several novel drugs, most patients relapse. Biomarkers to identify the early signs of relapse will make it possible to adjust the therapeutic strategy before the disease worsens. Although understanding genetic changes is important for the treatment of MM, currently known biomarkers of relapse, including serum free-light chains and monoclonal paraproteins, are not associated with genetic changes. We therefore performed a multicenter study to examine the usefulness of circulating cell-free DNA (cfDNA) present in the peripheral blood (PB) plasma of patients as a biomarker for MM relapse. We identified several driver mutations by combined analysis of next-generation sequencing and existing databases of candidate oncogenes. Furthermore, relapse was detected more sensitively by monitoring the circulating cfDNA with these driver mutations than by conventional serum free-light chain examination. These results suggest the potential utility of cfDNA in the PB plasma of patients as a relevant early biomarker for MM relapse.

The Occurrence of Valvular Atrial Fibrillation: Involvement of NGF/TrKA Signaling Pathway

Objective Nerve growth factor (NGF) and tropomyosin kinase receptors A (TrKA) exert a crucial effect on the regulation of autonomic nervous system which contributes to the progress of atrial fibrillation (AF). Valvular heart disease (VHD) patients are more easily to induce the AF. We investigated whether NGF/TrKA could impact the occurrence of AF in VHD patients. Materials and methods Atrial tissues were resected from 30 VHD patients with chronic AF (n = 15, AF >6 months) or sinus rhythm (SR, n = 15). The expression of NGF, TrKA, protein kinase B (PKB/Akt), beta-isoforms of glycogen synthase kinase-3 (GSK3β), Serine473 phosphorylation of Akt (p-Ser473 Akt), Serine9 phosphorylation of GSK-3β (p-Ser9 GSK3β) in right atrial tissues and peripheral blood lymphocyte were quantified by Western blot. The localization of those genes expression was measured by immunohistochemistry. Double sandwich enzyme-linked immunosorbent assay was used to observe the trace changes of NGF-β in peripheral plasma. Results Our results revealed that the NGF expression was markedly elevated in the tissue of right atrial appendage and peripheral blood lymphocytes from AF patients compared with the SR patients. But, the expression of TrKA, GSK3β, p-Akt and p-GSK3β were decreased. There was no difference about the expression of Akt from the AF patients and the SR patients. The NGF-β level in peripheral blood plasma of patients with AF and SR was not statistical difference. Conclusion Thus, we thought that NGF/TrKA signaling pathway may be involved in the AF in the patients with VHD, inactivation of GSK3β could increase the incidence of AF, but not relevant to phosphorylation.

Expression and significance of HSP90 in plasma of patients with multiple myeloma

This study was purposed to investigate the expression of heat shock protein 90 (HSP90) in peripheral blood plasma of patients with multipl myeloma (MM), and to explore its possible role in the pathogenesis of MM, and its relationship with treatment, prognosis and the outcome of patients. The peripheral blood samples from 58 patients with MM and 20 healthy volunteers were collected. The plasma concentration of HSP90 in patients and healthy volunteers was measured by ELISA. The results showed that the concentration of HSP90 in peripheral blood of patients with MM was significantly higher than that in the healthy volunteers [(32.398 ± 3.674) vs (25.762 ± 2.916) ng/ml] (P < 0.001). The concentration of HSP90 showed positively correlation with International Staging System(ISS) stage, therapeutic response, frequency of plasmocyte, globulin, immune globulin, M-protein, β2 micro-globulin, and light chain of MM patients (P < 0.05) ; while it showed little correlation with sex, age and type of MM patients (P > 0.05) . It is concluded that the HSP90 may be involved in the occurrence and development of MM. Detection of HSP90 in plasma would contribute to judge the clinical course, therapeutic efficacy and prognosis of MM patients.

Elevated expression of IL-12 and IL-23 in patients with primary immune thrombocytopenia

Immune thrombocytopenia (ITP) is an acquired autoimmune disorder. Both impaired platelet production and T-cell-mediated effects play a role in ITP. A T-helper1 (Th1) polarization of the immune response and up-regulation of Th17 cells have been demonstrated in ITP patients. Interleukin (IL)-12 and IL-23 produced by antigen presenting cells are essential for inducing and sustaining Th1 and Th17 effector cells via different pathways. However, less is known with regard to the levels of expression and synthesis of these two cytokines in patients with ITP. This was determined in this study in 46 patients with ITP as well as in 22 healthy controls. Our results showed that an increased expression of IL-12 p40, IL-12 p35, and IL-23 p19 mRNA was observed in bone marrow mononuclear cells and peripheral blood mononuclear cells of patients with ITP compared with controls. Consequently, higher levels of IL-12 and IL-23 were also found in bone marrow plasma and peripheral blood plasma in patients with ITP than in controls. Afterwards, a markedly higher level of IL-12 and IL-23 in bone marrow plasma or peripheral blood plasma was found in patients with chronic ITP than in patients with acute ITP. Furthermore, the peripheral blood plasma levels of IL-12 and IL-23 were negative correlated with platelet counts in ITP patients. Therefore, the augmented expression of IL-12 and IL-23 in patients with ITP may play an important role in the pathogenesis of this disease.

Lipid Peroxidation Products In Bone Marrow Plasma (BMp) and Peripheral Blood Plasma (PBp) From Patients with Low Risk Myelodysplastic Syndrome (MDS)

AbstractAbstract 2924Patients with Low-risk MDS have ineffective hematopoiesis, which results in refractory cytopenias requiring frequent blood transfusions for anemia treatment, nevertheless iron overload can occur even without transfusions. Recent research showed that reactive oxygen species were higher while reduced glutathione GSH was lower in RBC and platelets of MDS patients,when compared to normal individuals. The presence of highly reactive free radicals may play a role in the peroxidation of membrane lipids. Indirect evidence supports the role of oxidative DNA damage in the pathogenesis of MDS. Studies of the expression of antioxidant enzymes suggest that many CD34+ progenitors have inadequate oxidative stress defence. In this study we evaluated lipid peroxidation products measuring the thiobarbituric acid reactive substances (TBARS) in bone marrow plasma (BMp) and peripheral blood plasma (PBp) in low risk MDS patients. Materials and Methods:BMp and PBp for TBARS determination were obtained from 15 Low and Int-1 Risk MDS patients: 4 males, 11 females, mean age 65.5 years (range 50–83) at least 30 days from the last transfusion. Ferritin (FE), Transferrin (Tf), Tf Saturation (Tfs) were performed on the same day. As a control for TBARS determination we used (BMp) and (PBp) from 15 healthy bone marrow adult donors for transplant, age range 20– 55 years. Plasma levels of lipid peroxidation products were estimated according to the method of Ohkawa et al. as modified by Yagi. Lipid peroxides were expressed as nmol of malondialdehyde (MDA) per ml of plasma. Statistical analysis were performed using unpaired and paired Student's t-test and Pearson correlation was used to establish the relationship between variables (SAS V.9.2). Results:In MDS patients BMp and PBp TBARS levels were significantly increased: 10.59±0.84 and 5.25±0.55 respectively compared with controls: 4.49±0.47 and 1.89±0.26 (p<0.0001). Eighty-six percent of the patients (13/15) showed high serum FE concentration, mean 1341.3 ng/ml (range 631–3000ng/ml) We detected a positive and significant correlation between TBARS in PBp and BMp with FE levels (r=0.68, p=0.0049) and (r=0.54, p=0.0363) respectively. Additionally, both TBARS levels were highly correlated with the Tfs: r=0.81 (p=0.0025) and r=0.87 (p=0.0005) for BMp and PBp respectively. Also the patients showed high correlation between Tfs and FE (r=0.73, p=0.011).When patients were grouped according to the amount of blood transfusions in greater or less than 20, FE levels were 1793.5±224 and 824±188 (p=0.0063), and Tfs were 72.3%±3 and 45%±8 (p=0.0036) respectively. However, in both groups TBARS in BMp and PBp did not show significant differences. TBARS levels showed no significant correlations with haemoglobin concentration nor platelet number. Conclusions:To date, the role of oxidative stress in MDS has not been fully elucidated. The current results show elevated levels of TBARS in BMp and PBp in patients with low risk MDS, as an evidence of increased oxidative stress, probably caused by the cellular alterations observed in MDS. We can speculate that iron overload may be a contributing factor, as we found a positive correlation between FE and Tfs with TBARS in both BMp and PBp in our patient population. Disclosures:No relevant conflicts of interest to declare.

Influence of Intravenous Chemotherapy on Cytokine in Peripheral Blood Plasma in Patients with Advanced Digestive Tract Cancer

Objective To investigate the change of cytokines in plasma after intravenous chemotherapy in patients with digestive tract cancer and to discuss its clinical significance.Methods Using ELISA method,we assessed the change of cytokines in plasma in patients with digestive tract cancer after chemotherapy.Results Cytokines in plasma in patients with digestive tract cancer showed that the concentration of IL-2 was decreased with statistical significance in patients with digestive tract cancer compared with normal person,while the concentration of IL-10 and TNF-α could be higher than those of normal person and without statistical significance.All the variations were related with clinical stage of tumor.One week after intravenous chemotherapy in effective patients with advanced digestive tract cancer,the concentration of IL-2 was significantly decreased;two weeks later,the concentration of IL-2 was recovered and closed to normal level;three weeks later,it was even higher than the concentration before chemotherapy.IL-2 of patients in non-effective group was slightly increased after chemotherapy but still lower than before.All the variations of IL-10 and TNF-α after chemotherapy were showed the trend declined after rising first.Conclusion Patients with advanced-stage cancer have a poor immune response which was related with clinical stages of tumor.After intravenous chemotherapy,in early stage(about 1-2 weeks)patients were both in an immunosuppressant state.While three weeks later,patients with different results showed different immune states.It may be followed by "rebound-overshoot" recovery of immune function in patients with favorable clinical course.

Platelet-activating factor and arachidonic acid metabolites in psoriatic inflammation

Platelet-activating factor (PAF), as well as PAF acetylhydrolase (PAF-AH) activity in the peripheral blood plasma of patients with psoriasis and palmoplantar pustolosis, was measured with a radioimmunoassay technique, and compared with leukotriene (LT) B4, LTC4, LTD4 and E4 (LTD4/E4), thromboxane (TX) B2 and prostaglandin (PG) E2 levels. In a normal healthy group (n = 12) PAF level was 25.9 +/- 6.5 pg/0.1 ml plasma (mean +/- standard error of the mean: SEM), and this was elevated in patients with psoriasis (68.1 +/- 11.8, n = 25, P < 0.01), without a change in the PAF-AH level. LTB4 showed a similar increase (115.0 +/- 21.6 pg/ml vs. 68.2 +/- 11.8 pg/ml, P < 0.05), while TXB2 and PGE2 showed insignificant (P > 0.05) changes. LTC4 and LTD4/E4 were around the level of the limit of detection. Patients with palmoplantar pustulosis (n = 33) demonstrated similar, but milder and statistically insignificant, increases in PAF, LTB4, TXB2 and PGE2 levels. Modulation of the mediator levels before and after treatment was compared in 16 patients with psoriasis and 11 with palmoplantar pustulosis. PAF in psoriasis significantly decreased after treatment (70.9 +/- 17.1 to 25.1 +/- 5.5, P < 0.05) and this was moderately correlated (r = 0.298) with clinical improvement as indicated by the psoriasis area and severity index (38.5 +/- 7.5 to 10.9 +/- 4.2, P < 0.01). TXB2 (180.2 +/- 100.4 to 34.1 +/- 13.5), PGE2 (3.7 +/- 0.7 to 2.9 +/- 0.5) and LTB4 (120.1 +/- 31.1 to 84.2 +/- 8.2), in psoriasis, mildly decreased without statistical significance. Patients with palmoplantar pustulosis demonstrated a similar decrease in all mediators without statistical significance. The results obtained suggest a role of PAF in psoriasis. As the priming effects of PAF have been shown, for leucocytes and endothelial cells, to enhance their inflammatory response, we assume that PAF has roles in the acute phase of psoriatic and leucotactic inflammation.

Arachidonic acid metabolites on peripheral blood plasma in patients with bronchial asthma

To assess the role of arachidonic acid metabolites in pathogenesis of bronchial asthma, we measured thromboxane B2 (TxB2), 6-keto PGF1 alpha, leukotriene (LT) C4, and LTB4 in venous blood plasma in patients with bronchial asthma and in controls. The level of TxB2 was significantly higher in 18 asthmatics with attacks than that in 11 controls (77.3 +/- 48.8 pg/ml vs 48.8 +/- 9.4 pgml). The levels of 6-keto PGF1 alpha in both asthmatics with attacks (17.8 +/- 6.7 pg/ml) and without an attack (16.4 +/- 9.7) were significantly higher than that in controls (11.6 +/- 3.9 pg/ml). The levels of LTC4 in asthmatics with attacks (0.84 +/- 0.11 ng/ml, n = 11) were significantly higher than that in controls, furthermore the level of LTC4 in asthmatics without an attack. The level of LTB4 was significantly higher in asthmatics with attacks (295.0 +/- 160.7 pg/ml, n = 26) than that in controls (161.7 +/- 25.3 pg/ml, n = 12) and asthmatics without an attack (182.4 +/- 97.9 pg/ml, n = 22). These findings suggest that the arachidonic acid metabolites are related to the asthma attack and are associated with the pathogenesis of bronchial asthma.

Androgen metabolites in blood of patients with prostatic carcinoma

Dehydroepiandrosterone, androsterone and etiocholanolone sulphate esters have been determined in peripheral blood plasma of patients with prostatic carcinoma. A significant decrease in the plasma level of steroid was observed. A diminution in the excretion rate of certain 17-oxosteroid conjugates was also found in the same patients. There was no correlation between the steroid levels and orchidectomy or heterosexual hormone treatment.