Role of L-Arginine on the Expression of Coagulation Factor VIII Gene.

Objective: To explore the key sites in which L-arginine affects the expression of human coagulation factor VIII gene, and to create new drug targets for the treatment of hemophilia. Methods: A total of 5 human FVIII genes (A1, A2, A3, C1 and C2) with B domain deletion were transfected into human umbilical vein endothelial cells (HUVECs) as promoters. Run-on assay and ELISA analysis were performed to observe the driving effect of each domain gene on chloramphenicol acetyl transferase (CAT) gene transcription and expression, and the effect of L-arginine on each promoter. Results: In co-culture with L-arginine, transcriptional expression of the CAT gene was not detected in the PCAT3-Basic group (negative control without promoters), PA3-CAT3-Enhancer group or PC1-CAT3-Enhancer group. The transcriptional expression of CAT gene in the PCAT3-Control group (positive control with promoters) and PA1-CAT3-Enhancer group was unchanged compared with the non-L-arginine intervention, while the transcriptional expression of CAT gene in the PA2-CAT3-Enhancer group was significantly enhanced. Conclusions: A1 and A2 domain genes had promoter function and could initiate the transcription and expression of CAT gene, but A3, C1 and C2 domain genes could not. Moreover, L-arginine can significantly enhance transcription and expression of human coagulation factor VIII via A2 domain.

Preliminary Study on Apoptotic Proteins in Platelet from Adult Patients with Chronic Immune Thrombocytopenic Purpura.

BACKGROUND: Adult chronic idiopathic thrombocytopenic purpura (ITP) is a chronic and usually lifelong hemorrhagic disorder in which enhanced platelet destruction and -weakened platelet production lead to thrombocytopenia. In this study, the p38 mitogen-activated protein kinase (p38-MAPK), early growth response 1 (EGR-1), p53, Bcl-xL, Bak, Bax, and reactive oxygen species (ROS) in platelets from adult patients with chronic ITP were investigated. METHODS: Platelets were isolated from blood samples collected from 20 adult patients with chronic ITP and 20 healthy volunteers. p38-MAPK, EGR-1, p53, Bcl-xL, Bak, Bax, and ROS were determined by flow cytometry, and the results were analyzed by EXPO32 ADC. RESULTS: Flow cytometry showed the expression levels of p38-MAPK (61.66 ± 19.38% vs. 27.52 ± 14.34%), EGR-1 (62.22 ± 20.48% vs. 9.05 ± 5.79%), p53 (56.82 ± 20.07% vs. 4.35 ± 2.04%), Bak (39.86 ± 11.45% vs. 20.82 ± 11.85%), Bax (36.85 ± 15.99% vs. 6.69 ± 5.01%), and ROS (19.98 ± 1.47% vs. 1.29 ± 0.10%) were all elevated (p < 0.05 compared with healthy volunteers). In addition, pro-survival Bcl-xL (5.38 ± 1.52% vs. 21.20 ± 6.04%) was decreased markedly in platelets from adult patients with chronic ITP (p < 0.05 compared with healthy volunteers). CONCLUSIONS: Our findings reveal that platelets in adults with chronic ITP display a proapoptotic gene expression phenotype, based on the enhanced expression of p38-MAPK, EGR-1, p53, Bak, Bax, and ROS, and attenuated expression of Bcl-xL, suggesting increased sensitivity toward apoptosis.

Role of Ca2+, Calnexin and Calreticulin in Platelet from Adult Patients with Chronic Immune Thrombocytopenic Purpura.

BACKGROUND: Adult chronic immune thrombocytopenia (chronic ITP) is a common autoimmune hemorrhagic disease characterized by decreased platelet production and increased platelet destruction, leading to thrombocytopenia. In this study, Ca2+, calnexin (CNX) and calreticulin (CRT) within platelets from adult patients with chronic ITP were investigated. METHODS: Platelets were isolated from blood specimen collected from 20 adult patients with chronic ITP and 20 healthy volunteers. Ca2+, CNX and CRT were determined by flow cytometry, and the results were analyzed with EXPO32 ADC software. RESULTS: Flow cytometry showed the expressions of Ca2+ (74.19±19.40% vs 22.79±10.47%) was elevated (P<0.05). However, CNX (15.10±7.32% vs 41.79±14.45%) and CRT (25.11±12.66% vs 38.58±12.02%) were decreased markedly in platelets from adult patients with chronic ITP (P<0.05 compared with healthy volunteers). CONCLUSION: Based on enhanced expression of Ca2+ and attenuated expression of CNX and CRT in patients with chronic ITP, Ca2+ concentration and its associated down-regulated proteins may be important regulatory signals in the pathogenesis of chronic ITP.

Investigation of Lymphocyte Subsets in Peripheral Blood of Patients with Dyslipidemia.

OBJECTIVE: In order to evaluate the effect of dyslipidemia on cellular or humoral immunity in patients, changes in the absolute number of lymphocyte subsets were detected. METHODS: Flow cytometry was applied to determine the absolute value of lymphocyte subsets: B cell, NK cell, CD4+ T cell including the functional subset (CD4+CD28+), native subset (CD4+CD45RA+CD62L+), memory T cell subset (CD4+CD45RA-), CD8+ T cell including the functional subset (CD8+CD28+) and activated subsets (CD8+CD38+ and CD8+DR+). The relationship between lymphocyte subsets and hypercholesterolemia and hypertriglyceridemia was analyzed. RESULTS: The absolute values of CD19+ B cell, CD3+ T cell, CD4+ Th cell, CD4+CD28+ cell, naive CD4+ T cell and memory CD4+ T cell in patients with dyslipidemia were markedly higher than those in healthy controls (P<0.05). There was no significant difference between healthy controls and dyslipidemia patients in other lymphocyte subsets (P>0.05). The absolute values of CD3+ T cell and naive CD4+ T cell were significantly positively correlated with hypercholesterolemia in peripheral blood (r=0.291 and 0.306, respectively, all P<0.05). There was no significant correlation between hypertriglyceridemia and lymphocyte subsets (P>0.05). CONCLUSION: Dyslipidemia has potential effects on immune profiles in lymphocytes subsets, and changes in lymphocyte subsets in dyslipidemia patients may lead to immune dysfunction.