Efficacy and safety of low-dose rituximab combined with different dosage of glucocorticoids for immune thrombocytopenia / 中华血液学杂志

<p><b>OBJECTIVE</b>To compare the efficacy and safety of low-dose rituximab combined with different dosage of glucocorticoids for immune thrombocytopenia (ITP).</p><p><b>METHODS</b>Seventy-four patients (35 male, median age 34 years, range 18-70 years) including 60 newly-diagnosed, 6 persistent, 5 chronic and 3 refractory patients were enrolled in this study, and separated into control (36 cases) and experimental (38 cases) groups according to the dosage of glucocorticoids. Patients in both groups received dexamethasone 40 mg/day on days 1-4, followed by rituximab 100 mg on days 7, 14, 21, 28. The patients in experimental group also received decrements of prednisone 60, 30, 20, 10 mg/day on days 5-7, 8-14, 15-21, 22-28. The initial, long-term efficacy and safety were evaluated.</p><p><b>RESULTS</b>Platelet counts of all patients at day 4 remarkably increased, with the median platelet count from 11(1-26) × 10⁹/L to 84(23-132) × 10⁹/L in control group, and 10(2-20) × 10⁹/L to 80(22-115) × 10⁹/L in experimental group; the platelet counts of patients at day 14 in experimental group [163(19-262) × 10⁹/L] was higher than that of control group [98(18-238) × 10⁹/L] (P<0.05). The overall response (OR) rates at day 28 in experimental group (84.21%) was significantly higher than that of control group (66.67%, P = 0.03). There was no significant difference of sustained response (SR) rates in two groups (63.89%vs 65.79%, 58.33%vs 60.53%, P > 0.05) at six and twelve months follow-up points. Both groups showed similar incidence of adverse events, and no patients discontinued the treatment due to side effects.</p><p><b>CONCLUSION</b>Low-dose rituximab and glucocorticoids was an effective method for ITP patients, and maintenance treatment with decrements of prednisone contributed to improving earlier CR rate.</p>

Effect of different therapeutic regimens on regulatory T cells in patients of primary immune thrombocytopenia / 中华血液学杂志

<p><b>OBJECTIVE</b>To investigate the change of CD4⁺CD25(high)CD127(low) regulatory T cells (Tregs) percentage in patients with primary immune thrombocytopenia (ITP) treated by different methods.</p><p><b>METHODS</b>One hundred and thirty-eight newly diagnosed adult ITP patients (57 male, median age 40 years, range 18-70 years) were enrolled in this study, who were randomly separated into three regiment groups, namely prednisolone (PSL, 1.5 mg/kg for 2-4 weeks and subsequently stepwise reduction) group enrolled 49 patients, dexamethasone [(one course of high-dose dexamethasone (HDD) 40 mg/day, d1-4] 45 patients, and rituximab plus HDD (rituximab 100 mg on days 7, 14, 21, 28 and HDD) group 44 patients. Peripheral blood was taken in ITP patients of each group before treatment, 14 d and 28 d after treatment. The percentages of CD4⁺CD25(high)CD127(low) Tregs in 30 healthy controls and 138 patients were analyzed by flow cytometry.</p><p><b>RESULTS</b>Overall response (OR) rates of PSL, HDD and R+HDD groups at day 28 were 69.4%, 66.7% and 79.5% respectively with no difference. After the following 12 months, sustained response (SR) was more pronounced in R+HDD group compared to the other two groups (R+HDD vs PSL: 66.7% vs 37.8%, P<0.05; R+HDD vs HDD: 66.7% vs 22.7%, P<0.05). The percentage of CD4⁺CD25(high)CD127(low) Tregs in peripheral blood of ITP patients [(1.67±0.70)%] was significantly lower than in healthy control group; After treatment, the percentages of Tregs in peripheral blood of patients both at day 14 and 28 in R+HDD group remarkably decreased compared with before treatment [(4.28±1.09)% vs (1.68±0.68)%, P<0.05; (4.44±0.63)% vs (1.68±0.68)%]. The percentages of Tregs at day 14 in both other two groups decreased notably compared with before treatment. But the Tregs levels measured at day 28 in PSL and HDD groups were similar with before treatment.</p><p><b>CONCLUSION</b>The percentage of CD4⁺CD25(high)CD127(low) Tregs in peripheral blood of ITP patients was lower than healthy individual. The higher SR of patients treated by R+HDD was related to its ability to up-regulate the percentage of CD4⁺CD25(high)CD127(low) Tregs.</p>

Relation of B7-H3 molecule expression in multiple myeloma with poor prognosis and bone destruction / 中国实验血液学杂志

This study was purpose to investigate the B7-H3 expression in multiple myeloma cell lines and CD138 cells of patients with multiple myeloma, and explore its clinical significance. Three myeloma cell lines (RPMI8226, U266 and H929) were used. Forty-five patients with multiple myeloma were enrolled in the study. The expression of B7-H3 was detected by flow cytometry and RT-PCR. The relationship between B7-H3 and clinical prognostic factor was analyzed. The results showed that (1)In myeloma cell lines, high expression of B7-H3 was seen in RPMI8226 (92.30 ± 1.1)% and U266 (79.03 ± 1.2)% but not in H929 cell line (4.26 ± 0.2)%. (2) Exogenous IL-6 had no effect on upregulation of B7-H3 in myeloma cell lines. (3) In multiple myeloma patients, the proportions of B7-H3 positive cells in newly diagnosed, remission and relapsed patients were (48.58 ± 33.593)%, (22.16 ± 18.853)%, and (57.65 ± 28.296)%, respectively. The difference between the newly diagnosed and remission patients, and remission and relapsed patients was significant (P = 0.023, P = 0.004). (4)High B7-H3 expression was correlated with high numbers of bone destruction and high levels of serum calcium (P = 0.027, P = 0.046, respectively). It is concluded that the relation of B7-H3 molecule expression with prognosis of multiple myeloma may be negative, but with degree of bone destruction is positive, thus the high expression of B7-H3 may correlated with disease progression and bone destruction of patients with multiple myeloma.

In vitro effects of Wnt3a gene modification on mitigating damage of mouse bone marrow mesenchymal stem cells induced by Ara-C / 中国实验血液学杂志

This study was aimed to investigate the protective effect of Wit3a gene modification on mouse bone marrow mesenchymal stem cells against the injury induced by Ara-C. The gene-modified MSC steadily expressing Wnt3a were established by adenovirus system. The acute direct damage effects of different concentrations of Ara-C on the unmodified MSC and the gene-modified MSC were assessed by using an in vitro culture system, and the corresponding controls were set. The proliferation and apoptosis of MSC exposed to Ara-C were detected by cell count kit-8 (CCK-8) and flow cytometry. The expression of BCL-2 protein related with cell apoptosis was assayed by Western blot. The results indicated that as compared with unmodified MSC, Ara-C exhibited a less inhibitory effect on the proliferation of gene-modified MSC. There was obvious difference between unmodified MSC and gene-modified MSC (p < 0.05). The proliferation of gene-modified MSC began to recover at 72 hours after removal of Ara-C. However, unmodified MSC showed sustained suppression of proliferation after withdrawal of Ara-C. In apoptosis, the apoptosis rate of gene-modified MSC induced by Ara-C was significantly lower than those of unmodified MSC (p < 0.05). In addition, the expression levels of BCL-2 protein in gene-modified MSC were up-regulated compared with unmodified MSC (p < 0.05). It is concluded that Wnt3a gene modification can significantly mitigate the damage of mouse bone marrow MSC induced by Ara-C.

Establishment of a high expressing system of human coagulant factor VIII in vitro / 中华血液学杂志

<p><b>OBJECTIVE</b>To construct a recombinant lentiviral vector (pXZ208-BDDhFVIII) mediating B-domain-deleted human coagulation factor VIII (BDDhFVIII) gene and investigate its expression in HLF, Chang-Liver and MSC cells.</p><p><b>METHODS</b>BDDhFVIII gene fragment was separated by endonuclease digestion and was cloned into the multiple cloning sites of pXZ208 to construct a recombinant lentiviral vector pXZ208-BDDhFVIII. Viral particles were prepared by means of three-plasmid cotransfection of 293T package cells by calcium phosphate precipitation. After infection, the coagulant activity of human FVIII in the culture medium of 293T, HLF, Chang-Liver and MSC cells was assayed by one-stage method. The gene transduction efficiency was assayed by flow cytometry (FCM). Furthermore, PCR was performed to test the integration of BDDhFVIII.</p><p><b>RESULTS</b>The infection rates of HLF, Chang-Liver and MSC were (74.52 +/- 7.57)%, (27.24 +/- 6.53)% and (42.34 +/- 5.84)% respectively. The activities of FVIII in supernatants of HLF, Chang-Liver and MSC were (54.1 +/- 5.6)%, (22.5 +/- 2.9)% and (12.5 +/- 2.7)% respectively. BDDhFVIII gene integration was detected in all the infected cells.</p><p><b>CONCLUSION</b>The recombinant lentiviral vector pXZ208-BDDhFVIII was successfully constructed and efficiently integrated into target cells to express human FVIII activity in vitro.</p>

Expression of green fluorescent protein gene in mouse T lymphocytes mediated by lentiviral vector / 中国实验血液学杂志

This study was purposed to constructe the three-plasmid system of the lentiviral vector carrying the green fluorescent protein (GFP) gene and to investigate the expression of GFP in T lymphocytes of the mouse. The polypurine tract (PPT) element, ubiquinone promoter (PUB) and GFP were ligated to plasmid pLO134 using subcloning technology to construct plasmid pTK153. Human kidney 293T cells were co-transfected with the three-plasmid system containing packaging plasmid DeltaNRF, plasmid pTK153 and envelope plasmid VSV-G by using calcium phosphate DNA precipation and the expression of GFP was observed under fluorescence microscope after 12 hours. The viral particles were collected after transfection 72 hours, were frozen at -80 degrees C and were used to infect mouse T lymphocytes at multiplicity of infection (m.o.i.) of 3. The expression of GFP in mouse T lymphocytes was observed by fluorescence microscopy and fluorescence-activated cell sorting (FACS). The results showed that the transfection efficacy was 63.04 +/- 7.24% in 293T cells analysed by FACS and the viral titer was (3.09 +/- 0.61) x 10(6) U/ml. The expression of GFP was also evident in mouse T lymphocytes and the transduction efficacy was (37.98 +/- 6.26)%. It is concluded that the three-plasmid system of lentiviral vector containing GFP gene is successfully constructed and the transduction efficacy is high in mouse T lymphocytes.

Expression of B domain-deleted human coagulant factor VIII gene in 293T cells mediated by lentiviral vector in vitro / 中国实验血液学杂志

This study was aimed to construct a lentiviral vector carrying human coagulant factor VIII (FVIII) and to investigate its expression in 293T cells. B-domain-deleted factor VIII gene fragment (BDDhFVIIIcDNA) was obtained by enzyme digestion and cloned into lentiviral vector pXZ208 to establish the expression vector pXZ208-BDDhFVIII. Recombinant viral particles were prepared by cotransfection with packaging plasmid delta NRF and envelope plasmid VSV-G using calcium phosphate precipitation method. 293T cells were transfected by viral supernatant. Coagulant activity of FVIII, BDDhFVIIImRNA and genome integration were assayed by one-step method, RT-PCR and PCR after transfection. The results showed that 293T cells could be transfected by recombinant virus. The transfection rate of 293T was 59.57%. After transfection, the cells expressed FVIII efficiently. Detection confirmed that the activity of FVIII was 12%, 43% and 87% respectively at 24, 48 and 72 hours after infection. BDDhFVIII transcription was detected by RT-PCR from the infected cells. The gene integration in the targeted cells was also observed. It is concluded that the successfully constructed lentiviral vector is able to generate high level expression of human FVIII in 293T cells, which may provide a potential application of gene therapy to haemophilia A.

Expression of recombinated canine factor VIII in vitro mediated by lentiviral vector / 中国实验血液学杂志

The study was purposed to prepare the recombinant lentiviral vector pTK161 and pTK162 carrying B-domain-deleted canine factor (BDDcFVIII) gene, and to investigate whether the canine FVIII (cVIII) can be expressed in vitro. The BDDcFVIII gene was ligated behind PUB and 2OH1 promotors to create lentiviral vectors pTK161 and pTK162. Meantime lentiviral vectors pTK161' and pTK161' were produced by cloning a green fluorescent protein (GFP) into pTK151 and pTK152, which was driven by PUB and 2OH1 promotors respectively. Vector supernatant were prepared by using transfer calcium phosphate mediated-cotransfection of 293T cells. The virus vector, DeltaNRF packaging-plasmid, and VSV-G envelope-plasmid was assayed by titers and cFVIII activity in cell culture supernatant after infection into 293T cells. pTK161, pTK162, pTK161' and pTK161' were identified by restriction enzyme analyzing. The results showed that the lentiviral vectors pTK161, pTK162, pTK161' and pTK161' were successfully constructed, and the titers of pTK161' and pTK161' reached to 1.54 x 10(6) U/ml and 2.83 x 10(6) U/ml; the activity of cFVIII could be detected at 24 hours after infection of 293T cells by pTK161 and pTK162, and achieved the highest level at 72 hours later. The higher level of cFVIII activity was achieved by transfected with pTK162 than that of pTK161 (p < 0.05), which closed to the cFVIII activity in normal dog plasma. 1/4 of the highest level could be detected 6 weeks later. It is concluded that the prepared HIV1-based lentiviral vectors can infect 293T cells to express cFVIII effectively. The results provide the basis for further studying HIV-1-based lentiviral vector gene therapy for hemophilia A.

In vitro killing effect of mutant thymidine kinase mediated by lentiviral vector on T lymphocytes / 中华血液学杂志

<p><b>OBJECTIVE</b>To explore the killing effect of the mutant herpes simplex virus thymidine kinase (HSV-sr39tk) and its wild-type (HSV-tk) mediated by lentiviral vector on T lymphocytes in vitro and compare T cell survival rate after GCV or ACV treatment.</p><p><b>METHODS</b>The three-plasmid lentiviral vector system including packaging plasmid DeltaNRF, envelope plasmid VSV-G and vector plasmid (pTK151 + HSV-sr39tk or pTK151 + HSV-tk) were cotransfected into human embryonic kidney 293T cells using modified calcium phosphate precipitation methods. The packaged virus was harvested 72 h later. The survival of T cells expressing HSV-sr39tk or HSV-tk was measured by MTT assay after 4 day-culture against a gradient of GCV or ACV concentrations.</p><p><b>RESULTS</b>The three plasmids were effectively cotransfected and a high titre of lentivirus was obtained (2 x 10(6) IU/ml). 39tk(+) T cell survival rates declined promptly when the prodrug GCV/ACV concentrations increased from 0 micromol/L to 10 micromol/L. The T cell survival rates in GCV group declined from (96.04 +/- 3.23)% to (36.76 +/- 4.38)% while in ACV group from (97.31 +/- 4.61)% to (43.75 +/- 8.99)%. However, when GCV/ACV concentrations were more than 10 micromol/L, further decline of 39tk(+) T cell survival rates became unobvious. The growth rate of 39tk(+) T cell exposed to GCV or ACV was obviously lower than that in un-transfected T cells (P < 0.05). Tk(+) T cells were sensitive to GCV (P < 0.05), but not to ACV (P > 0.05). There was a significant difference in killing effects between 39tk(+) T cell + GCV group and tk(+) T cell + GCV group (P < 0.05).</p><p><b>CONCLUSION</b>The lentiviral vectors containing HSV-sr39tk gene could infect T lymphocytes effectively and stably without affecting the proliferation of the transduced cell. In contrast to HSV-tk gene, T cells infected HSV-sr39tk were more sensitive not only to GCV but also to ACV.</p>