Objective:To observe the effect of combining transcranial direct current stimulation (tDCS) with functional electrical stimulation-assisted cycling (FES-cycling) on lower limb motor function early after a stroke.Methods:Thirty-seven survivors of a recent stroke were divided into a tDCS treatment group ( n=18) and a pseudo-stimulation group ( n=19). While receiving routine rehabilitation training and clinical drug treatment, the tDCS treatment group also cycled in response to functional electrical stimulation while simultaneously receiving tDCS anode stimulation of the motor cortex M1 area. The pseudo-stimulation group followed the same protocol but with the tDCS stimulation inactivated. Both groups were treated for 20min daily, 5 days weekly for 4 weeks. Before and after the 4 weeks of treatment, the lower limb motor function, walking ability and ability in the activities of daily living of both groups were evaluated using the Fugl-Meyer assessment scale for the lower extremities (FMA-LE), the timed up and go test (TUGT) and the modified Barthel index (MBI) respectively. Transcranial magnetic stimulation was used to detect each subject′s cerebral cortex motor threshold (CMT) , cortical latency (CL) and central motor conduction time (CMCT) as well as the amplitude (Amp) of the motor evoked potential of the lower limb primary motor cortex (M1 area). Results:After 4 weeks of treatment, the average FMA-LE and MBI scores and TUGT times of the two groups had improved significantly compared with those before treatment. The average FMA-LE score and TUGT time of the tDCS group were significantly better than those of the pseudo-stimulation group. The average CMT, CL and CMCT in both groups were significantly lower than those before the intervention, while the average Amp had increased significantly, but there were significant differences in the average CMT, Amp, CL and CMCT between the two groups after the 4 weeks of treatment.Conclusions:Transcranial direct current stimulation combined with cycling assisted by functional electrical stimulation can effectively stimulate excitability in the motor cortex soon after a stroke. That should promote the recovery of nerve activity and lower limb function.

The von Willebrand factor-cleaving protease (vWF-cp) is a newly identified plasma metalloproteinase and plays an important role in the pathogenesis of thrombotic microangiopathy. In the present study, the metalloproteinase domain of vWF-cp was expressed by IPTG-induced the recombinant engineered E.coli strain harbouring pET28a (+)-vWF-cp/MD and purified using chromatography on Ni-NTA column. Then the BALB/c mice were immunized with the recombinant protein to prepare the monoclonal antibodies (McAb) against vWF-cp and the obtained McAbs were characterized. Furthermore, the expression panels of vWF-cp in human normal tissues were investigated using immunohistochemistry. The results showed that high-level expression of the recombinant protein was achieved, which existed as inclusion body and amounted to 28% of total bacteria protein. Three monoclonal antibodies against the metalloproteinase domain of vWF-cp were obtained and two of them, SZ-112 and SZ-113, were further evaluated. Both of them belong to IgG(1). The concentration of them in ascites was 4 mg/ml, and their titers were as high as 1 x 10(-5). The data of ELISA showed that SZ-112 and SZ-113 recognized different epitopes of recombinant protein. The Western blot and immunoprecipitation data showed that the two monoclonal antibodies reacted not only with the recombinant protein, but also with a 200 kD protein in platelet lysate. Moreover, the vWF-cp was found to be present in the cytoplasm of many human tissues such as liver, prostate, ovary, etc. However, the protease could not be detected in brain tissue. In conclusion, the above-mentioned research work contributed not only to the further study of the structure and function of this protease, but also to the establishment of the method for quantifying the vWF-cp antigen in plasma.
ADAM Proteins ; biosynthesis ; genetics ; immunology ; ADAMTS13 Protein ; Animals ; Antibodies, Monoclonal ; biosynthesis ; immunology ; Binding Sites ; genetics ; Blotting, Western ; Epitopes ; immunology ; Escherichia coli ; genetics ; Humans ; Immunohistochemistry ; Immunoprecipitation ; Mice ; Mice, Inbred BALB C ; Recombinant Proteins ; biosynthesis ; immunology ; von Willebrand Factor ; biosynthesis ; genetics ; immunology

Von Willebrand factor (vWF) is the unique substrate for the metalloprotease, ADAMTS-13, and plays a pivotal role in the pathology of von Willebrand disease (vWD) and thrombotic thrombocytopenic purpure (TTP). To study the pathogenesis of TTP and to establish a method to diagnose TTP, the DNA fragment of vWF-A2 domain was amplified and inserted into expression vector with 6 x His tag (pQE-30), the recombinant expression vector was transformed into E. coli (strain M15) and induced by IPTG. The recombinant fragment comprising residues 718-905 of mature vWF was designated as rvWF-A2. It was purified by Ni-NTA resin column chromatography and refolded in Tris buffer containing GSH and GSSG. The results demonstrated that rvWF-A2 was expressed successfully in E. coli M15, amounting to 42% of total bacterial protein with the purity over 98%. It was identified that rvWF-A2 can be efficiently cleaved by the citrated normal plasma while no cleavage can be detected by the TTP plasma or plasma with EDTA. It is concluded that rvWF-A2 expressed efficiently in E. coli demonstrated excellent biological activity, which lays a solid foundation for establishment of method to measure quantatively the activity of ADAMTS-13.
ADAM Proteins ; metabolism ; ADAMTS13 Protein ; Escherichia coli ; genetics ; Humans ; Peptide Fragments ; biosynthesis ; genetics ; Purpura, Thrombotic Thrombocytopenic ; diagnosis ; metabolism ; Recombinant Proteins ; biosynthesis ; isolation & purification ; von Willebrand Factor ; biosynthesis ; chemistry ; genetics

BACKGROUNDThrombotic thrombocytopenic purpura (TTP) is a rare thrombotic microangiopathy. In this study we investigated the von Willebrand factor-cleaving protease (vWF-cp) activity deficiency in patients with TTP.

METHODSThe plasma or serum vWF-cp activity was measured using a sensitive enzyme-linked immunosorbent assay (ELISA) by detecting the residual collagen binding activity (R-CBA) of von Willebrand factor (vWF) before and after digestion by vWF-cp. Multimers of vWF in plasma of patients with TTP were also analyzed by SDS-agarose electrophoresis. Moreover, the serum vWF-cp activities were compared between the patients with TTP and those with tumors.

RESULTSThe coefficient of variation for intra-batch and inter-batch of the assay were 3.60% and 8.35%. The plasma and serum vWF-cp activity in healthy individuals were (78.79 +/- 9.17)% (n = 30) and (79.47 +/- 10.78)% (n = 53), respectively, while the plasma vWF-cp activity in 5 patients with TTP was markedly decreased [(21.83 +/- 19.98)%, P < 0.001]. The unusually large vWF multimers were observed in two plasma samples of the patients with TTP. Although the vWF-cp activities in patients with benign and malignant tumors were also decreased (P < 0.03 and P < 0.001, respectively), they were relatively high in comparison with that of TTP patients (P < 0.001).

CONCLUSIONMeasurement of the vWF-cp activity using R-CBA is a simple and rapid method for diagnosing TTP. The vWF-cp activity in patients with TTP was markedly lower than those of patients with tumors.

ADAM Proteins ; ADAMTS13 Protein ; Adolescent ; Adult ; Aged ; Female ; Humans ; Male ; Metalloendopeptidases ; blood ; deficiency ; Middle Aged ; Neoplasms ; enzymology ; Purpura, Thrombotic Thrombocytopenic ; enzymology

ADAM Proteins ; biosynthesis ; genetics ; ADAMTS13 Protein ; Adult ; Aged ; Carcinoma, Hepatocellular ; metabolism ; Cell Line ; Female ; Humans ; Liver Neoplasms ; metabolism ; Middle Aged ; von Willebrand Factor ; biosynthesis ; genetics

The study was purposed to analyze the relationship between the content of T-cell receptor excision DNA circles (TREC) and bcr-abl mRNA levels in CML patients and to evaluate the prognostic significance of recent thymic output function detection in patients with chronic myelogenous leukemia (CML). Quantitative detection of TREC and bcr-abl fusion gene transcripts in peripheral blood from 15 CML patients were preformed by real-time PCR. The change of bcr-abl levels in 6 patients was followed-up for two years. The results showed that there was no significant correlation between TREC and bcr-abl mRNA levels in peripheral blood from CML patients for the first attack. Patients who had higher TREC at diagnosis had a larger reduction of bcr-abl after 2 years of follow-up. While out of 2 patients who underwent haemopoietic stem cell transplantation (HSCT), one patient with higher level of TREC before transplantation was confirmed to express undetectable level of TREC by three consecutive detections after transplantation, other one patient was identified to express low level of bcr-abl. It is concluded that high thymic output function in CML patients can be beneficial for killing the residual CML cells.
Adolescent ; Adult ; Aged ; Female ; Fusion Proteins, bcr-abl ; biosynthesis ; genetics ; Gene Rearrangement ; Humans ; Leukemia, Myelogenous, Chronic, BCR-ABL Positive ; immunology ; Male ; Middle Aged ; Proto-Oncogene Proteins c-abl ; biosynthesis ; genetics ; Proto-Oncogene Proteins c-bcr ; biosynthesis ; genetics ; RNA, Messenger ; biosynthesis ; genetics ; Receptors, Antigen, T-Cell ; analysis ; immunology ; T-Lymphocytes ; chemistry ; immunology ; Thymus Gland ; immunology

The objective of this study was to analyze the level of bcr-abl mRNA in peripheral blood (PB) after allogeneic stem cell transplantation (allo-SCT) in chronic myeloid leukemia patients, providing a experimental basis for diagnosing early relapse. bcr-abl mRNA levels in 78 PB and bone marrow (BM) samples from 15 CML patients after allo-SCT were detected by using real-time quantitative PCR. The results indicated that levels of bcr-abl mRNA before transplantation were high (median 29.303%) and decreased greatly (median 0) at the first month after allo-SCT. During the first year after allo-SCT, the patterns of serial bcr-abl transcripts varied in number, but the overall bcr-abl transcript levels significantly decreased at 6 months after allo-SCT. Majority of patients with undetectable or very low levels of bcr-abl mRNA were monitored after 1 year following transplantation. The hematological features of BM and PB in all detected patients remained normal. PB and BM bcr-abl values were not different significantly and had the similar trend of changes. It is concluded that the bcr-abl mRNA levels in CML patients change greatly early after allograft. Serial monitoring measurements for bcr-abl mRNA contribute to understanding the trend of change and effect of transplantation, also can be a guidance for starting therapy. But detectable levels of bcr-abl mRNA during the first 6 months do not indicate relapse. Measurements of bcr-abl mRNA of PB may be more suitable for routine monitoring long-term disease status in CML after allo-HSCT.
Adolescent ; Adult ; Bone Marrow ; metabolism ; Fusion Proteins, bcr-abl ; blood ; metabolism ; Hematopoietic Stem Cell Transplantation ; Humans ; Leukemia, Myelogenous, Chronic, BCR-ABL Positive ; metabolism ; therapy ; Neoplasm, Residual ; diagnosis ; RNA, Messenger ; blood ; metabolism ; Reverse Transcriptase Polymerase Chain Reaction ; Young Adult

BACKGROUNDvon Willebrand factor (vWF) mediates the initial capture of platelets to vascular subendothelium and is essential for platelet aggregation under high fluid shear stress as in arterial stenosis. On release from endothelial cells, vWF is rapidly cleaved by ADAMTS13/vWF-cleaving protease (vWF-CP). We investigated the clinical significance of changes in plasma vWF and vWF-CP activities in chronic renal disease.

METHODSPlasma vWF and vWF-CP activities were measured using enzyme-linked immunosorbent assay (ELISA) and residual collagen binding assay respectively in patients with lupus nephritis (n = 31), primary nephritic syndrome (n = 25), diabetic nephropathy (n = 45), chronic glomerulonephritis (n = 38) and 40 normal controls. The relation of their levels with pathological and renal status was analyzed.

RESULTSIn all diseased patients the levels of vWF were significantly higher and vWF-CP activity significantly lower than the controls (both P < 0.01). vWF in the four subgroups did not correlate with the stage of disease but correlated negatively with vWF-CP activity. vWF-CP activity was not changed two weeks after renal transplantation. Renal biopsy demonstrated that the vWF level in stage IV was higher than in stages II and III while vWF-CP activity was lower in patients with lupus nephritis. After eight-week treatment, the vWF level significantly decreased and the vWF-CP activity significantly increased in systemic lupus erythema, disease activity index < 9, but not with index = 9. Even though the vWF-CP activity was significantly lower in membranous nephropathy than in minimal change disease, mesangial proliferative glomerulonephritis or IgA glomerulonephritis, the vWF level was not significantly different.

CONCLUSIONSThe alterations of plasma vWF and vWF-CP activities were associated with different renal pathologies. Injury to endothelial cells and autoantibodies against vWF-CP activity may result in higher vWF level and lower vWF-CP activity in chronic renal disease and thus a mechanism for worsening of chronic renal disease and thrombosis.

ADAM Proteins ; blood ; ADAMTS13 Protein ; Adolescent ; Adult ; Aged ; Chronic Disease ; Female ; Humans ; Kidney Diseases ; blood ; Kidney Transplantation ; Lupus Nephritis ; blood ; Male ; Middle Aged ; von Willebrand Factor ; analysis

von Willebrand factor-cleaving protease (vWF-cp) is a newly identified metalloproteinase. The activity of vWF-cp would vary in different physiological or pathological states. To explore activity detectron of von Willebrand factor-cleaving protease and its clinical application, the vWF-cp activity was measured by a sensitive enzyme-linked immunoadsorbent assay to detect the residual collagen binding activity (R-CBA) of von Willebrand factor before and after digestion with vWF-cp. Moreover, its activity deficiency in patients with thrombotic thrombocytopenic purpura (TTP) and solid tumors was also investigated. The results showed that the residual collagen binding assay was sensitive enough to measure the serum or plasma vWF-cp activity in 87 health individuals, 79 patients suffering from TTP and solid tumors. The coefficient of variation within and between the batches was were 3.60% and 8.35%, respectively. The serum and plasma vWF-cp activity in health individuals was (79.47 +/- 10.78)% (n = 53) and (78.79 +/- 9.17)% (n = 30), respectively, whereas the vWF-cp activity in patients with TTP, benign and malignant tumors was significantly decreased (P values were less than 0.001, 0.03 and 0.001, respectively). It is concluded that the vWF-cp activity in plasma or serum of patients with TTP and solid tumors markedly decrease, especially in patients with TTP. Assay of the vWF-cp activity using R-CBA is a simple method.
ADAM Proteins ; blood ; metabolism ; ADAMTS13 Protein ; Adolescent ; Adult ; Aged ; Enzyme-Linked Immunosorbent Assay ; Female ; Humans ; Male ; Middle Aged ; Purpura, Thrombotic Thrombocytopenic ; diagnosis ; enzymology ; Stomach Neoplasms ; diagnosis ; enzymology ; von Willebrand Factor ; metabolism

The purpose of this study was to investigate the influence of nitro oxide (NO) from mesenchymal stem cells (MSC) on the proliferative responses of allogeneic lymphocytes and its mechanism. MSCs were isolated and cultured from human bone marrow. Selected surface antigens of MSCs were detected by flow cytometry and their morphologic characteristics were determined by microscopy. Mitomycin C-treated MSCs were plated in dishes and then mixed lymphocyte cultures (MLC) were set up. After 4 days, lymphocyte proliferation was determined by CCK-8 assays; NO secretion in coculture supernatant was determined by Griess reagent kit; the level of FOXP3 mRNA expression was detected by real-time quantitative PCR. The results indicated that in MSC/MLC coculture experiment, the lymphocyte proliferation decreased significantly with of IOD value 0.49+/-0.03, NO production increased obviously (21.05+/-1.14 micromol/L) and FOXP3 mRNA expression was increased [(1.56+/-0.34)%] as compared with MLC coculture without MSC. There were significant difference between these two groups. It is concluded that NO production in human MSC culture up-regulates FOXP3 mRNA expression and thus inhibits lymphocyte proliferation response.
Adult ; Bone Marrow Cells ; cytology ; Cell Proliferation ; Cells, Cultured ; Forkhead Transcription Factors ; metabolism ; Humans ; Lymphocyte Count ; Lymphocyte Culture Test, Mixed ; Lymphocytes ; cytology ; immunology ; metabolism ; Mesenchymal Stromal Cells ; cytology ; Nitric Oxide ; metabolism