[Investigation of Laboratory and Clinical Feature in the Patients with Myeloproliferative Neoplasm Co-expression of BCR-ABL1 and JAK2 V617F].

OBJECTIVE: To analyze the comprehensive laboratory test data of BCR-ABL1 fusion gene and JAK2 V617F mutation co-expressed in myeloproliferative neoplasm (MPN) patients, and investigate its relative clinical significance. METHODS: Data of 1 332 MPN patients were comprehensively analyzed, BCR-ABL1 (P190/P210/P230) fusion gene and JAK2 V617F mutation were detected by real time-polymerase chain reaction (RT-PCR) technique, the CALR, MPL, JAK2 12 and 13 exon mutations were detected by the First Generation Sequencing, the bone marrow cell morphology and pathological characteristics were evaluated by bone marrow smear and biopsy technique, the immune phenotypes of bone marrow cells were evaluated by flow cytometry, the chromosome karyotypes of bone marrow cells were analyzed by chromosome G banding technique. RESULTS: Four of the 1 332 patients were found to have the co-existence of BCR-ABL1 fusion gene and the JAK2 V617F mutation, with a 0.3% incidence and a median age of 70 years old, including 2 cases of polycythemia vera, 1 case of primary myelofibrosis, and 1 case of chronic myeloid leukemia-accelerated phase. The clues of double positive genes of such patients at the time of initial diagnose could not be cued only by age, physical signs and cell morphology, they should be analyzed by comprehensive test data. CONCLUSION: The co-existence of BCR-ABL1 fusion gene and JAK2 V617F mutation in the same case is a kind of disease with special clinical significance. The application of multiple detection methods can improve the detection of this disease, which is conducive to early detection, reasonable diagnosis and treatment by clinicians.

[Time-course effect and region-specificity of endoplasmic reticulum stress in rat brains acutely exposed by methylmercury].

OBJECTIVE: To explore time-course effect and region-specificity of endoplasmic reticulum stress in rat brain acutely exposed by methylmercury (MeHg). METHODS: Forty-two SD rats were randomly divided into seven groups, and the rats intraperitoneally injected at the dose of 4 mg/kg bw. MeHg were decapitated at the times of 0.5, 1, 3, 6, 12 and 24 h, then rats in control group intraperitoneally injected by the corresponding volume of normal saline were decapitated. The cerebellum, cerebral cortex, brain stem, hippocampus and striatum were dissected out and weighted on ice at once. The expressions of Grp78 protein which is a marker of endoplasmic reticulum stress, were determined by Western blotting analysis. The contents of reduced glutathione (GSH) of cerebral cortex were also determined. RESULTS: After exposure to MeHg, the tendencies of expression of Grp78 were consistent in various brain regions. It began to increase at the time of 0.5 h and the peak levels reached at the times of 6 h or 12 h, then it began to decline. All expression levels returned nearly to the control levels at the time of 24h. The alternations of Grp78 protein in cerebral cortex and brain stem were statistically significant in comparison with those of control groups at the time of 0h among brain regions. Increases of Grp78 protein in cerebral cortex reached peak levels at the time of 6h after MeHg exposure, and the expressions of Grp78 protein corresponded to 150% of control levels. Increases of Grp78 protein in brain stem reached peak levels at the time of 12 h after MeHg exposure, and the expressions of Grp78 protein corresponded to 140% of control levels. Further, the contents of GSH in cerebral cortex showed the tendencies of first decreases and then gradually showed increases. These changes were inversely correlated to the change of Grp78 protein in cerebral cortex (r = -0.77). CONCLUSION: Rats acutely exposed with MeHg could show endoplasmic reticulum stress in a time dependent and region-specific pattern, and this alteration could be associated with oxidative stress in cerebral cortex.

ABCA1 expression in macrophages of allogeneic hematopoietic stem cell transplantation patients with severe infection undergoing continuous blood purification.

BACKGROUND: Excessive activation of the inflammatory mediator cascade after allogeneic hematopoietic stem cell transplantation (allo-HSCT) in patients is associated with high mortality. Many studies have shown that continuous blood purification (CBP) could improve the prognosis of allo-HSCT patients with severe infection. However, the exact mechanism remains unclear. The aim of this study was to observe the effect of CBP on the expression of ATP-binding cassette transporter A1 (ABCA1) in macrophages, and to investigate the interventional effects of CBP on serum cytokine in allo-HSCT patients with severe infection. METHODS: A total of 26 allo-HSCT patients with severe infection were included in this study. Before CBP and after CBP, blood samples were collected to observe hepatic and renal function, and the serum levels of TNF-α, IL-1, IL-6, and IL-10 were detected via ELISA. The THP-1 macrophages were exposed to serum samples obtained from patients at specific time points during CBP to test the changes of ABCA1 in macrophages by real-timePCR and Western blotting. RESULTS: Serum creatinine, alanine aminotransferase, and C reaction protein (CRP) levels decreased significantly after CBP. Moreover, TNF-α, IL-1, and IL-6 serum levels decreased significantly, but IL-10 level increased significantly after CBP (P<.05). After CBP, ABCA1 expression levels were higher than those before CBP, and ABCA1 expression was significantly increased with the supplementation of CBP (P<.05). CONCLUSIONS: CBP improved the condition of allo-HSCT patients with severe infection. CBP may be a potent up-regulator of the ABCA1 levels in macrophages of allo-HSCT patients with severe infection.

[The variation of T-cell clonal repertoire in patients with systemic lupus erythematosus (SLE) following autologous peripheral blood hematopoietic stem cell transplantation].

OBJECTIVE: Analyze the variation of T cell receptor(beta) (TCRbeta) CDR3 gene expressing repertoire in systemic lupus erythematosus (SLE) patients before and after autologous peripheral blood hematopoietic stem cell transplantation (APBSCT), to search the pathogenesis of SLE and characteristics of T cell reconstitute immune after APBSCT. METHODS: Reverse transcriptase - polymerase chain reaction (RT-PCR) amplified 25 subfamily genes of TCRbeta prom peripheral blood lymphocytes (PBLs) of SLE patient with APBSCT and run denaturation polyacrylamide sequencing gel electrophoresis, set up a map of TCRbetaCDR3 gene expressing repertoire. The bands of TCRbeta gene repertoire in the electrophoresis gel can be used to analyze the variety of T cell clones which expanded and disappeared in cases of SLE. Cut the bands and sequencing. Compare sequences of TCRbetagenes in normal and abnormal T cell clones to understand the relationship between TCRbeta gene and autoimmune diseases. RESULTS: In the normal control group, TCRbeta gene repertoires show more than 10 bands in each of 25 TCRbetaV families, characterized as Gaussian distribution. TCRbeta gene repertoire in the 8 patients with SLE were abnormally. Four patients expressed TCRbetaV13.1 gene preferentially. Three patients had TCRbetaV8, TCRbetaV9 and TCRbetaV18 genes over expressed. These over expressive genes represented the oligoclonal expansion of T cell populations. After APBSCT the TCRbeta gene repertoires regained CDR3 polymorphism in 5 patients, some of them recovered to normal completely. The sequencing analyse show dense and strong band in map of TCRbeta gene repertoires were T cell monoclonal or oligoclonal expansion frequently. In a case of SLE an amino acid sequence of TCRbetaV8 CDR3 was same to a sequence of disease correlative T cell clone with ankylosing spondylitis that had been reported in a literature before. CONCLUSION: SLE patients have oligoclonal expansion of T cell that related with the pathogenesis of autoimmune disease. After APBSCT, T cell clones recover to normal distribution. T cell clones that related with disease have been suppressed during immunological reconstitution. It may be a main reason the SLE get remission after transplantation.

Hormetic effects of acute methylmercury exposure on grp78 expression in rat brain cortex.

This study aims to explore the expression of GRP78, a marker of endoplasmic reticulum (ER) stress, in the cortex of rat brains acutely exposed to methylmercury (MeHg). Thirty Sprague-Dawley (SD) rats were randomly divided into six groups, and decapitated 6 hours (h) after intraperitoneal (i.p.) injection of MeHg (2, 4, 6, 8 or 10 mg/kg body weight) or normal saline. Protein and mRNA expression of Grp78 were detected by western blotting and real-time PCR, respectively. The results showed that a gradual increase in GRP78 protein expression was observed in the cortex of rats acutely exposed to MeHg (2, 4 or 6 mg/kg). Protein levels peaked in the 6 mg/kg group (p < 0.05 vs. controls), decreased in the 8 mg/kg group, and bottomed below the control level in the 10 mg/kg group. Parallel changes were noted for Grp78 mRNA expression. It may be implied that acute exposure to MeHg induced hormetic dose-dependent changes in Grp78 mRNA and protein expression, suggesting that activation of ER stress is involved in MeHg-induced neurotoxicity. Low level MeHg exposure may induce GRP78 protein expression to stimulate endogenous cytoprotective mechanisms.

[Effects of autologous hematopoietic stem cell transplantation on immune function in patient with systemic lupus erythematosus].

To investigate the effects of autologous hematopoietic stem cell transplantation (AHSCT) on immune index in patient with systemic lupus erythematosus (SLE), and evaluate its treatment outcome, the flow cytometry (FCM) and enzyme linked immunosorbent assay (ELISA) were used to detect the leukocyte differentiation antigen, sIL-2R, IL-6, C3, C4, autoantibodies, immunoglobulin for 33 case of SLE before transplantation and at 1, 3, 6, 12 months after transplantation. The results showed that the ratio of CD4(+), CD19(+) cell, the level of sIL-2R, IL-6 and the positive rate of autoantibodies were significantly lower, CD8(+), CD16(+)CD56(+) cell and C3, C4 were higher than those before transplantation. Out of the 33 patients, 26 achieved CR, 3 reached PR and 4 relapsed at 4 - 6 months after transplantation. It is concluded that the immune indexes of patients with SLE changed significantly following AHSCT. These immune indexes may be indications to predict the status of remission in patient with SLE.

Autologous stem cell transplantation for systemic lupus erythematosus.

Systemic lupus erythematosus (SLE) is responsive to treatment with immunosuppressives and steroids, but often pursues a relapsing or refractory course resulting in increasing incapacity and reduced survival. Autologous stem cell transplantation (ASCT) following immunoablative chemotherapy is a newer therapy for autoimmune disease of potential use in severe SLE. A retrospective registry survey was carried out by the European Blood and Marrow Transplant and European League Against Rheumatism (EBMT/EULAR) registry. Data was collected from 53 patients with SLE treated by ASCT in 23 centres. Disease duration before ASCT was 59 (2-155) months (median, range), 44 (83%) were female, and median age was 29 (9-52) years. At the time of ASCT a median of seven American College of Rheumatology (ACR) diagnostic criteria for SLE were present (range 2-10) and 33 (62%) had nephritis. Peripheral blood stem cells were mobilized with cyclophosphamide and granulocyte colony stimulating factor in 93% of cases. Ex vivo CD34 stem cell selection was performed in 42% of patients. Conditioning regimens employed cyclophosphamide in 84%, anti-thymocyte globulin in 76% and lymphoid irradiation in 22%. The mean duration of follow-up after ASCT was 26 (0-78) months. Remission of disease activity (SLEDAI < 3) was seen in 33/50 (66%; 95%CI 52-80) evaluable patients by six months, of which 10/31 (32%; 95%CI 15-50) subsequently relapsed after six (3-40) months. Relapse was associated with negative anti-double stranded DNA (anti-dsDNA) antibodies before ASCT (P = 0.007). There were 12 deaths after 1.5 (0-48) months, of which seven (12%; 95%CI 3-21) were related to the procedure. Mortality was associated with a longer disease course before ASCT (P = 0.036). In conclusion, this registry study demonstrates the efficacy of ASCT for remission induction of refractory SLE, although mortality appeared high. The safety of this procedure is likely to be improved by patient selection and choice of conditioning regimen. The return of disease activity in one-third of patients might be reduced by long-term immunosuppressive therapy post-ASCT.