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To investigate total blood loss (TBL), intraoperative blood loss (IBL), post-operative drainage (POD), and other related variables in total hip arthroplasty (THA) performed through direct lateral approach.This study was completed in Shanghai Sixth People's Hospital, Shanghai, China, between January and December 2010. We analyzed 113 patients treated by THA through direct lateral approach. Recordable blood loss (RBL) was measured and TBL was calculated according to the Gross formula. Ordinal logistic regression analysis was used for TBL, IBL, and POD, with gender, age, body mass index (BMI), disease duration, and operative time as independent variables.The average operative time was 51.5 +/- 10.4 (range: 35-70) minutes, with an average RBL of 454 +/- 144 (range: 150-180) ml. Average TBL was significantly higher (975 +/- 355, range: 430-2020 ml; p0.001). In the analysis of variables, BMI closely correlated with TBL, IBL, and POD, with odds ratios of 4.80 (95% CI: 2.63-8.78 [TBL]), 5.39 (95% CI: 2.84-10.25 [IBL]), and 4.37 (95% CI: 2.43-7.89 [POD]). Moreover, TBL (54.172), IBL (55.198), and POD (39.139) correlated with trend test BMI.The TBL, IBL, and POD closely correlate with BMI in patients undergoing THA through direct lateral approach.

With retrieval of articles on extraordinary acupoint that were published in domesticin recent five years, one hundred and eight articles of clinical application are screened out and one hundred and twenty-three extraordinary acupoints that are extensively recognized are collected. Of those acupoints, 23 acupoints are included in the latest national standard. Of the rest 100 extraordinary acupoints, 48 acupoints are located on the running courses of fourteen meridians, 4 acupoints are shared with the meridian points and the other 52 acupoints have not been clarified to be located on the running courses of meridians based on the literature data. It is found in the collection of these acupoints that there are many extraordinary acupoints that are extensively used in clinical practice. But the nomenclatures and locations of acupoints have not been unified. Hence, a further standardization on these aspects is anticipated.

This study was aimed to investigate the expression difference of serum cytokines in 20 patients receiving HLA-identical nonmyeloablative allogeneic hematopoietic stem cell transplantation (iNAHSCT) and HLA-haploidentical nonmyeloablative allogeneic hematopoietic stem cell transplantation (hiNAHSCT). IL-2, IL-4, IL-6, IL-10, TNF-α, γ-IFN and IL-17 were detected by flow cytometric bead array before and on week 1, 2, 4 after transplantation respectively. The results showed that the IL-2 level was found to be up-regulated at week 1 and 2 after transplantation in iNAHSCT group and in hiNAHSCT group respectively, but there was no difference between these two groups (P0.05). The γ-IFN levels was up-regulated at week 4 after transplantation in above-mentioned two groups, but no difference was found between these two groups. The IL-4 level increased at week 2 and 1 after transplantation in iNAHSCT and hiNAHCT groups respectively, but the IL-4 level in iNAHSCT group was higher than that in hiNAHSCT group. The IL-6 level rose at week 1 and 2 after transplant in above mentioned groups respectively, and reached to peak level at week 4 after transplantation, but IL-6 level in hiNAHSCT was higher than that in iNAHSCT group (P0.02). The IL-10 level was up-regulated at week 1 and 2 in iNAHSCT and hiNAHSCT groups respectively, but the IL-10 level in iNAHSC was higher than that in hiNAHSCT group. The TNF-α level was up-regulated at week 1 in hiNAHSCT group, but at week 2 in iNAHSCT group after transplantation. The TNF-α level in hiNHASCT group was higher than that in iNAHSCT group (P0.01). The IL-17 level was up-regulated at week 1 and week 4 after transplantation in hiNAHSCT and iNAHSCT groups respectively, the IL-17 level in hiNAHSCT group was high as compared with that in iNAHSCT group. It is concluded that the serum cytokine levels are obviously up-regulated in iNAHSCT and hiNHASCT groups, and reach to peak level at week 4 after transplantation. The IL-6, TNF-α and IL-17 level up-regulated significantly in hiNAHSCT group, but the IL-4 and IL-10 level up-regulated significantly in iNAHSCT.

To observe the therapeutic effect and major complications of haploidentical nonmyeloablative allogeneic peripheral blood stem cell transplantation (NST) for refractory or relapsed leukemia.The results of 30 patients, including 14 cases of acute myeloid leukemia (AML), 11 cases of acute lymphoblastic leukemia (ALL), 5 case of chronic myelogenous leukemia (CML) (accelerated and blastic phase) with refractory or relapsed leukemia (RF/RL) who underwent haploidentical NST from August 2000 to April 2009 were analyzed. The conditioning regimen consisted of fludarabine (flu), antithymocyte globulin (ATG), cyclophosphamide (CTX), total body irradiation (TBI) and cytarabine (Ara-C) or myleran (Bu). Graft-versus-host disease (GVHD) prevention programmes consisted of Cyclosporine (CsA), mycophenolate mofetil (MMF), CD25 monoclonal antibody combined with mesenchymal stem cells (MSC).Twenty six cases of patients were full donor engraftment and 4 cases mixed chimerism into full donor chimerism. The average duration of neutrophil0.5×10⁸/L after NST was 11 (9-16) days, and platelet20×10⁸/L 17 (12-60) days. Upon follow-up of 16 to 120 months, 12-month transplant-related mortality (TRM) was 46.7%, acute Ⅱ-Ⅳgraft-versus-host disease (aGVHD) incidence was 40.0%. The probability of 3-year disease relapse, EFS and overall survival (OS) rates were 16.7%, 46.2% and 50.0% respectively.Haploidentical NST could improve OS and EFS of refractory or relapsed leukemia and reducce TRM to some extent.目的 观察单倍体非清髓造血干细胞移植（NST）治疗难治复发性白血病的疗效及主要并发症。方法 对我中心2000年8月至2009年4月进行单倍体NST治疗的30例难治复发性白血病结果进行分析。其中急性髓系白血病14例,急性淋巴细胞白血病11例,慢性髓性白血病（加速、急变期）5例。预处理方案为氟达拉滨、抗胸腺细胞球蛋白、环磷酰胺、全身照射联合阿糖胞苷或白消安。移植物抗宿主病（GVHD）预防方案为环孢素、霉酚酸酯、CD25单抗联合间充质干细胞。结果 植入情况：30例患者中26例为完全供者嵌合,4例为混合嵌合转变为完全供者嵌合。移植后中性粒细胞＞0.5×109/L恢复平均时间为11（9~16）d,PLT＞20×109/L恢复平均时间为17（12~60）d。平均随访24（16~120）个月,12个月以内移植相关死亡率为46.7%,Ⅱ~Ⅳ度急性GVHD发生率为40.0%,累计3年总的疾病复发率为16.7%,无事件生存率为43.3%,总生存率为50.0%。结论 单倍体NST可以改善难治复发性白血病总生存率和无事件生存率,一定程度上减少移植相关死亡率。

To explore hemorrhage risk and the clinical significance of abnormal change of prothrombin time (PT), activated partial thromboplastin time (APTT), plasma fibrinogen (FIB), plasma thrombin time (TT) and d-dimer (D-D) in de novo acute leukemia (except for APL), the different bleeding manifestations of 114 cases of de novo acute leukemia with different coagulation indexes were analyzed retrospectively. The correlation between these blood coagulation indexes and the possible correlative clinical characteristics were analysed, including age, sex, type of acute leukemia, initial white blood cell(WBC) and platelet(Plt) count, the proportion of blast cells in bone marrow and cytogenetic abnormality of patients at diagnosis. The results indicated that the incidence of abnormal blood coagulation was as high as 78.1% for de novo AL patients. These patients with 5 normal blood coagulation indexes may have mild bleeding manifestation, but the more abnormal indexes, the more severe bleeding. Both PT and D-D were sensitive indexes for diagnosis of level II bleeding. Incidence of abnormal blood coagulation significantly correlates with the proportion of blast cells in bone marrow (χ(2) = 4.184, OR = 1.021, P0.05) and more with D-D (P0.01), while age, sex, type of AL, WBC count, Plt count and abnormality of cytogenetics did not correlate with abnormal blood coagulation. It is concluded that the coagulation and fibrinolysis are abnormal in most patients with de novo acute leukemia. More abnormal indexes indicate more severe bleeding, and both PT and D-D are sensitive indexes for diagnosis of level II bleeding. Higher proportion of blast cells in bone marrow predicts higher incidence of abnormal blood clotting. Acute leukemia with elderly age, high white blood cell count and adverse cytogenetics do not predict severer abnormal blood clotting. Detection of PT, APTT, TT, FIB, and D-D may help to judge whether the patients are in a state of hypercoagulability or disseminated intravenous coagulation, which will provide experiment evidences for early intervention and medication.

This study was purposed to investigate the changes of Th1/Th2/Th17 in patients received non-myeloblastic haploidentical hematopoietic stem cell transplantation (NAHSCT). The levels of IL-2, IL-4, IL-6, IL-10, TNF-α and IFN-γ, as well as IL-17 level were determined by flow cytometric bead array (CBA) in samples from 18 patients underwent allo-peripheral NAHSCT at different time points before and after transplantation. The results showed that all cytokines changed obviously after transplantation, and their serum levels were higher than that before transplantation. The expression levels of IL-2, IL-4 and IL-17 changed early, and their obviously up-regulation was found after transplantation. The expression levels of IL-6, IL-10 and TNF-α changed significantly, and were high as compared with that before transplantation. The change of INF-γ serum level was observed late, its rising occurred at week 4 after transplantation. The expression of all cytokines kept increasing during 4 weeks after transplantation and peaked at week 4. It is concluded that the serum levels of all cytokines from the patients after NAHSCT increased significantly, in which the levels of IL-2, IL-4 and IL-17 increased early, but the level of INF-γ changed late. The detection of cytokines is helpful for deep understanding the pathophysiologic mechanism of transplant-related complications.

Gene therapy and nerve stem cells isolated from the developing human enteric nervous system (ENS) are significant. They may open the route for the cell therapy of Hirschsprung's disease (HD). We have constructed the recombinant adenovirus-carrying glial cell line-derived neurotrophic factor (GDNF) and endothelin receptor B (EDNRB) gene, and investigated the exosomatic coexpression in neural stem cells. The recombinant adenovirus Ad-GE coexpressing GDNF and EDNRB gene was constructed by the AdEasy system and confirmed by the reverse transcription polymerase chain reaction (RT-PCR) method. Expression of exogenous genes in neural stem cells after transfection was confirmed by the flow cytometry and real-time fluorescence quantitative PCR. Fragments of pAd Track-CMV-GE were consistent with GDNF and EDNRB. The green fluorescence of the positive cells was followed by fluorescence microscopy at 24 h after NSCs had been transfected, reaching a peak at 72 h after transfection. Flow cytometry showed that the efficiency of transfection was 15.0, 23.6, and 25.4% at 24, 48 and 72 h respectively. Real-time fluorescence quantitative PCR showed the expression levels of mRNA of GDNF and EDNRB in 48 and 72 h groups were obviously higher than that in 24 and 96 h groups. Recombinant adenovirus carrying GDNF and EDNRB genes are coexpressed in neural stem cells, which may offer the possibility of a novel approach to local combination gene therapy for Hirschsprung's disease.

Purpose Despite best current therapies, approximately half of patients with acute myeloid leukemia in first complete remission (AML-CR1) with no HLA-identical donors experience relapse. Whether HLA-mismatched stem-cell microtransplantation as a novel postremission therapy in these patients will improve survival and avoid graft-versus-host disease (GVHD) is still unknown. Patients and Methods One hundred one patients with AML-CR1 (9 to 65 years old) from four treatment centers received programmed infusions of G-CSF–mobilized HLA-mismatched donor peripheral-blood stem cells after each of three cycles of high-dose cytarabine conditioning without GVHD prophylaxis. Donor chimerism and microchimerism and WT1+CD8+ T cells were analyzed. Results The 6-year leukemia-free survival (LFS) and overall survival (OS) rates were 84.4% and 89.5%, respectively, in the low-risk group, which were similar to the rates in the intermediate-risk group (59.2% and 65.2%, respectively; P = .272 and P = .308). The 6-year LFS and OS were 76.4% and 82.1%, respectively, in patients who received a high dose of donor CD3+ T cells (≥ 1.1 × 108/kg) in each infusion, which were significantly higher than the LFS and OS in patients who received a lower dose (< 1.1 × 108/kg) of donor CD3+ T cells (49.5% and 55.3%, respectively; P = .091 and P = .041). No GVHD was observed in any of the patients. Donor microchimerism (2 to 1,020 days) was detected in 20 of the 23 female patients who were available for Y chromosome analysis. A significant increase in WT1+CD8+ T cells (from 0.2% to 4.56%) was observed in 33 of 39 patients with positive HLA-A*02:01 antigen by a pentamer analysis. Conclusion Microtransplantation as a postremission therapy may improve outcomes and avoid GVHD in patients with AML-CR1.

Natural killer (NK) cells are important immune cells in human body, which occupy an important place in adoptive immunotherapy for patients with malignancies due to their capacity of killing tumor cells without MHC limitation, as well as separating graft versus leukemia (GVL) and graft versus host disease (GVHD). Recent studies showed that different kinds of NK cell-surface receptors have been found, which transmit inhibiting signals or activating signals, the balance between them determines the functional status of NK cells. Researchers have focused on the study of NK cell-surface receptors recently in order to improve application of NK cells for adoptive immunotherapy. This review summaries the current advancement about NK cell-surface receptors and their clinical significance.

The optimal therapy for intermediate-risk patients with acute myeloid leukemia (AML) in first complete remission (CR1) is uncertain. Recent studies shown that microtransplantation (MST) can improve survival in AML-CR1 patients. However, a comparison study between the MST and nonmyeloablative stem cell transplantation (NST) is lacking. 156 intermediate-risk AML-CR1 patients aged 9 to 59 years were enrolled in this study. 57 patients who had a HLA-identical donors were assigned to receive NST therapy with graft-versus-host disease (GVHD) prophylaxis. The other 99 who had no HLA-identical donors including 86 family-related, 9 distantly related and 4 unrelated donor were assigned to receive MST therapy but without GVHD prophylaxis. The probabilities of 10-year overall survival and leukemia-free survival was comparable in the MST-group and NST-group (70.7% vs. 61.4% and 59.6% vs. 57.9%). The NST-group exhibited a higher full donor chimerism (96.5%) and higher GVHD (33.3%), whereas the MST-group produced a higher donor microchimerism (75%), slightly higher relapse (32.3% vs. 22.8%) and significantly lower non-relapse mortality (6.9% vs. 19.3%, P=0.021) but without GVHD. In the MST-group, the patients with increase of WT1+ CD8+ T cells exhibited significantly higher leukemia-free survival and lower relapse than those without (92.0% vs. 40.0%, P=0.003; 8.0% vs. 50%, P=0.009). These results indicate that, compared to NST, MST produced a comparable survival, less transplantation-related mortality, avoidance of clinical GVHD and overcome limitations of HLA-barrier, suggesting a much safe and effective therapy for intermediate-risk AML-CR1, particularly for those without a HLA-identical donor. Disclosures No relevant conflicts of interest to declare.

The outcome of acute myeloid leukemia in elderly acute myeloid leukemia (EAML) patients is still unsatisfactory with a low complete remission (CR) rate and a poor overall survival due to prolonged pancytopenia and intrinsic resistance of leukemic blasts to therapeutic. The high/standard induction chemotherapy improved CR rate but remission is usually transient. Non-myeloablative stem cells transplantation or reduced-intensity conditioning transplantation shows curative effects but severe graft-versus-host disease (GVHD) still exists. Our clinical studies revealed that the microtransplantation (MST) could improve outcome and without GVHD in EAML. Recently, other centers worldwide used microtransplantation to young AML patients, MDS, non-Hodgkin's lymphoma and others. Here we introduce some important developments and in future directions about MST. 1. Microtransplantation is effective for EAML A seminal randomized study with 58 cases of EAML by Beijing group reported that MST result in a significantly higher CR rate and higher 2-year OS compared to chemotherapy. Another randomized muti-centres study with MST for EAML in China and USA is ongoing. Total of the fifteen EAML patients including 8 in chemotherapy-group and 7 in MST-group were registered with primary much better outcomes. Otherwise, another enlargement study about MST for EAML in China MST Cooperative Group showed overall CR rate was 77.1% which was no significantly difference among the patients with 60-65 years, 66-75 years or more. Fores reported three EAML patients get CR after MST. Gottlieb reported two EAML achieved CR. Otherwise, high CR rates (70-86%) was also found in other as-yet unpublished series of data included 5 cases of EAML from the University of Duke and 9 patients from the University of South California. 2. Microtransplantation is safe and hardly no GVHD From Ai, thirty EAML and 101 young AML patients did not develop GVHD. However, Ai reported one patient who developed a severe acute GVHD. No cytomegalovirus pneumocystis was reported. 3. Microtransplantation is no limit to donors Most patients received HLA partial/full-mismatched related/unrelated donor, which suggested that MST could overcome MHC barrier and without limitation of donors. 4. Microtransplantation mechanism study is ongoing Donor micro-chimerism has been detected by using Sry gene, HLA loci and G-6PD isoenzyme detected. Micro-chimerism generally becomes undetectable after weeks and months by a single infusion, and rarely persists beyond two or three years after several infusions. Other important experiment studies including: 1) Distribution, evolution and persistence of donor micro-chimerism was observed by molecular imaging in mouse. 2) Donor micro vesicle was found in host lymphocyte, who received mv from G-CSF mobilization donor mouse spleen cells infusion. 5. Anti-leukemia effects In addition to induce Graft-versus-leukemia effects, MST mediated recipient-versus-leukemia effects which may involve in host B-cell antibody synthesis and T/NK cytotoxicity. IL-2, Il-6, TNF and IL-10 are significantly increase in mouse leukemia model. In the future, Microtransplantation offers the benefit of allo-reactivity and hardly any GVHD. Further studies will focus on the mechanism of microtransplantation and multicentre cooperation study. It may become a standard weapon for EAML infuture. Disclosures No relevant conflicts of interest to declare.

Acute myeloid leukemia (AML) of older patients is an aggressive malignancy. Despite decitabine is performed some clinic trials in these patients, however there is little data on efficacy and is more controversial on it. Recent years, our novel microtransplantation (MST) clinical studies that regular chemotherapy combined with granulocyte colony-stimulating factor mobilized peripheral blood stem cells infusion of HLA mismatched related donor(GPBSC), without using any immunosuppressive agents, which support hematopoietic recovery, perform GVL effects and improve survival without GVHD appearance in elder patients with AML. Thus, we designed an open-label control clinical trial to compare the efficacy of decitabine plus MST to only MST for elder AML. The 61 patients in the registry were randomly assigned into two groups, 30 patients were in decitabine plus cytarabine/anthracycline (DCA) group, 31patients were in cytarabine/anthracycline (CA) group. In DCA group induction therapy included decitabine 30mg/m2/days1-4, cytarabine 100 mg/m2/days 1-7 and daunorubicin 45 mg/m2 days 1-3, or idarubicin 12 mg/m2 days 1-3, or mitoxantrone 10 mg/m2 days 1-3, or aclarubicin 10mg /m2 days 1-7 followed by intravenous infusion of GPBSC 24h after cytarabine therapy. Patients achieving CR received 2 further courses of post-remission therapy with decitabine (30 mg/m2 daily for 4 days) and medium-dose cytarabine therapy (cytarabine, 1-2 g/m2 , per 12 h intravenously, 3 hours infusion on days 1, 2, and 3) followed by infusion of GPBSC 24h after each cycle of cytarabine chemotherapy. Except for decitabine other treatments is same in CA group. The complete remission rate (CR) of patients in DCA group was lower than that of patients in CA group (70.0% vs.80.6 %, p=0.38). There was no statistical difference in CR rate in two groups. The median overall survival (OS) and diseases-free survival (DFS) in DCA group were not differences than those in the CA group (DFS, 26.5vs. 22.8months, p = 0.90; OS, 28.4months vs. 28.1months, p = 0.19,figure 1). The median duration of neutropenia was longer in the DCA group than in the CA group (14 vs. 10 days, p = 0.02). The median duration of thrombocytopenia was longer in the DCA group than in the CA group (16 vs. 14 days, p = 0.32).The rate of severe infection and TRM were not differences in two groups. This study suggests MST therapy results in a 71.4% CR in patients ≥60 years old a 21months DFS with acceptable toxicity. The decitabine did not produce superior outcomes than cytarabine and anthracyclines treatment, and resulted in prolonged suppression of bone marrow. Further studies involving a larger cohort of patients and longer follow-up are warranted. Figure 1. The probabilities of OS and PFS in the DCA group compared with CA groups. 1 thin line, DCA group; 2 thick line, CA group. DFS, 26.5vs. 22.8months, p = 0.90; OS, 28.4months vs. 28.1months, p = 0.19. The 12-month progression-free survival (PFS) for the MDS group and tAML group was 62.3% and 43.7%, respectively. The 24-month DFS for the DCA group and CA group was49.6% and 61.8%, respectively. Figure 1. The probabilities of OS and PFS in the DCA group compared with CA groups. 1 thin line, DCA group; 2 thick line, CA group. DFS, 26.5vs. 22.8months, p = 0.90; OS, 28.4months vs. 28.1months, p = 0.19. The 12-month progression-free survival (PFS) for the MDS group and tAML group was 62.3% and 43.7%, respectively. The 24-month DFS for the DCA group and CA group was49.6% and 61.8%, respectively. Disclosures No relevant conflicts of interest to declare.

The acute myeloid leukemia in elderly patients (EAML) is always associated with low complete remission (CR) rate and a poor survival due to prolonged pancytopenia and resistance of leukemic blasts to chemotherapy. The standard or high dose induction chemotherapy improved CR rate but remission is usually transient. Nonmyeloablative allergenic stem cell transplantation shows some curative effects but associated with severe graft-versus-host disease (GVHD). Recently, our clinical studies showed that HLA-mismatched microtransplantation (MST) could increase CR rate and improve the survival but without GVHD. However, it is still lack of data and multicentre studies about MST in such patients. Here, we designed a multicentre study about microtransplantation in combination with chemotherapy in such patients and elevate effects and toxicity. Method. Total of the 148 patients with AML at the age of 60 years or over from May 2005 to May 2014, were enrolled in this study from the 11 hematology disease centers in China. The diagnosed was defined by the French-American-British (FAB) and WHO criteria. Chromosomal and immunephenotyping analyses were performed by pre-treatment of bone marrow obtained. Patients with acute promyelocytic leukemia or with a blast crisis of chronic myeloid leukemia were excluded. High risk was defined by the presence of complex cytogenetic abnormalities (with 3 or more cytogenetic abnormalities), secondary AML, or a WBC count of 50,000/µL or above. Other patients were considered at a standard risk. In accordance with the Helsinki Declaration, written informed consent for enrollment in this study was obtained from the patients or their legal guardians and the donors. Induction therapy includes cytarabine and anthracycline followed by HLA-mismatched G-CSF Mobilization HLA-mismatched donor peripheral stem cells (G-PBSC) infusion. The patients who achieved complete remission (CR) further received two courses of cytarabine as post-remission chemotherapy. None of the patients received any GVHD prophylactic treatment and further maintenance therapy. The donor and recipient HLA-A, HLA-B, HLA-C, HLA-DRB1 and HLA-DQB1 alleles were genotyped using a PCR-SSP method. All of 148 patients had HLA-mismatched related or unrelated donors including 126 with a family-related donor, 16 with a distantly related donor and 6 with an unrelated donor. G-PBSC infusion: the median number of mononuclear cells, CD34+ and CD3+ T cells infused per course was 2.9 (1.2-5.2)´108/kg, 1.7(1.1-4.6)´106/kg and 0.9(0.5-2.6)´108/kg, respectively. Results. Total of 148 patients were divided into three groups with 60-65 years (n=54), 66-70 years (n=41) and over 71 years (n=53). The overall CR rate was 77% and CR rate of the first cycle of induction chemotherapy was 55.3%. The CR rate was not significantly different among the patients with 60-64 years (74.1%), 65-70 years (80.4%), and over 71 years (77.4%). The CR rate was much higher in the standard group than in the high risk group (88.2% vs. 63.3%, P=0.04). The early death rate and severe infection was no significantly different among three groups. The median recovery time of neutrophils and platelets was 12d and 14d, which were not significantly different among three groups. The probabilities of 12 months OS, 24 months and 36 months were not significantly different among the patients with 60-65 years (80.9%, 60.9% and 55.8%), 66-70 years (65.4%, 46.5% and 34.9%) and over 71 years (71.3%, 30.6% and 26.8%). No definite clinical acute or chronic GVHD was observed in all of the patients but two patients who developed a severe acute GVHD and dead on day 36. A frequencies of HLA-A0201/2402WT1+CD8+ T-cells was 0.23% which is no significantly difference between the patients with low 70 years or more. The donor micro-chimerism was detected in 11 of the 18 female patients with a range of 0.0000151- 0.0386 copies of gene expression. In conclusion, the microtransplantation combined conventional chemotherapy can result in a higher CR rate and OS with a shorter duration of pancytopenia, lower severe infections and hardly no GVHD, suggested that it is a much safer and effective new therapy for elderly AML patients including those who over 70 years old. Disclosures No relevant conflicts of interest to declare.

To understand the gastroscopic and pathological characteristics of patients with miasis in Poyang Lake area, and to explore the relationship between schistosomiasis and pathological changes of gastric mucosa.Volunteers with or without schistosomiasis were recruited and divided into a case group and a control group. All the objects were examined by electronic gastroscopy and pathological examinations.Two hundred and fifty-three volunteers diagnosed with chronic or advanced schistosomiasis in the case group showed different degrees of gastric mucosal changes, including 7 cases with schistosomal eggs deposited beneath the gastric mucosa (with an incidence of 2.77%) , 33 cases with dysplastic hyperplasia and intestinal metaplasia (with an incidence of precancerous lesion of 13.04%), and 1 case with gastric cancer. While in the 200 volunteers without schistosomiasis in the control group, the results showed milder gastric mucosal changes, 33 cases were detected with dysplastic hyperplasia and intestinal metaplasia (with an incidence of 7.50%), and 1 case was diagnosed gastric cancer. The difference between the incidences of precancerous lesion in the two groups had no statistic significance (P0.05).The incidence and extent of gastric mucosal changes in schistosomiasis patients are higher and more serious than those in non-schistosomiasis patients, and gastrointestinal schistosomiasis is not related to gastric cancer.

This study was aimed to investigate the effect of recombinant human granulocyte colony-stimulating factor(G-CSF) on murine thymocyte emigration and cell cycle alteration after a sublethal dose of gamma-irradiation. Female BALB/c mice were given 6.0 Gy γ-ray total body irradiation and then randomly divided into G-CSF and control groups. Mice in the G-CSF group were injected recombinant human G-CSF 100 µg/(kg·d) subcutaneously once daily for 14 consecutive days and mice in the control group were given the same volume of phosphate buffered solution. Thymocyte cycle alteration and the proportion of apoptosis cells were detected by flow cytometry within 72 hours after irradiation. Real-time PCR was used for detection and quantitation of murine T cell receptor rearrangement excision circles (sjTREC) of the thymic cells at 30 and 60 day after the irradiation. The results showed that at 6 hour after irradiation G-CSF could significantly increase the thymic cells in G(0)/G(1) phase, G-CSF vs control: (82.0 ± 5.0)% vs (75.9 ± 2.8)% (p0.05), and decrease the thymic cells in S phase, G-CSF vs control: (10.2 ± 4.8)% vs (15.7 ± 2.3)% (p0.05), but G-CSF seemed have no evident effects on the percentage of thymic cells in G(2)/M phase. G-CSF could also protect thymocytes from apoptosis at 6 hour and 12 hour after irradiation the percentages of apoptosis cells in G-CSF group were (11.5 ± 2.4)% and (15.5 ± 3.3)%, respectively, which were significantly lower than that of the control group (16.5 ± 2.2)% and (22.6 ± 0.7)%, respectively (p0.05). The sjTREC copy amount was conspicuously higher in G-CSF group than that in the control at 30 day after irradiation (p0.01), but the preponderance disappeared 60 days later. It is concluded that G-CSF has a positive effect on the thymic cell cycle alteration to protect thymocytes from apoptosis and enhance the recent thymocyte emigration, which may contribute to the central immune reconstitution after irradiation.

This study was purposed to investigate the effect of G-CSF on the proliferation, differentiation, and cell cycle distribution of thymocytes in sublethally irradiated mice. Female BALB/c mice were exposed to 6.0 Gy γ-ray irradiation and then randomly divided into control and G-CSF treatment group. In the treatment group rhG-CSF 100 µg/(kg·d) was given subcutaneously for 14 continuous days and to make sure the first injection was given within 1 hour after irradiation. Cell cycle distribution and apoptosis of thymocytes were detected within 72 hours after irradiation. Subpopulations of CD4(-)CD8(-) cells and sequential changes in the distribution of CD4(+)CD8(+), CD8(+)CD4(-), CD8(-)CD4(+) cells were detected by a three-color flow cytometry during a four-weeks period after irradiation. The results showed that in G-CSF treatment group marked increase of cells in G(0)/G(1) phase (G-CSF vs control: 82.0 ± 5.0% vs 75.9 ± 2.8%) (p0.05) and a decrease of cells in S phase (G-CSF vs control: 10.2 ± 4.8% vs 15.7 ± 2.3%) (p0.05)could be observed as early as 6 hours after irradiation, but G-CSF seems have no evident effects on the cells in G(2)/M phase. G-CSF could also protect thymocytes against apoptosis. 6 and 12 hours after irradiation the apoptosis rates of thymic cells in G-CSF treatment group were 11.5 ± 2.4% and 15.5 ± 3.3% respectively, while in the control group the apoptosis rates were 16.5 ± 2.2% and 22.6 ± 0.7% respectively. Comparison between the two group demonstrated significant difference (p0.05). CD4(-)CD8(-) double negative thymocytes (DN)can be defined as DN1-4 according to their maturation. G-CSF treatment resulted in a significant increase in DN1 thymocytes and promoted their proliferation and differentiation to a more mature DN3 and DN4 stage. G-CSF could enhance the recovery of CD4(+)CD8(+) thymocytes and mitigate their relapse during reconstitution. The percentage of CD4(+)CD8(+) thymocytes in the G-CSF treatment group 28 days after irradiation was significantly higher than that of the control group (71.0 ± 6.3% vs 25.5 ± 6.3%) (p0.05). It is concluded that G-CSF has a positive effects on the thymic cell cycle distribution, proliferation and differentiation, which may contribute to the reconstitution of central immune system after acute irradiation.

This study was aimed to investigate the expression level of NOV and BNIP3 mRNA in mice myelomonocytic leukemia (AML-M(4)) and its significance. The mice were inoculated intravenously with myelomonocytic leukemia cells of WEHI-3, and divided randomly into chemotherapy group and control (untreated) group. Bone marrow samples were then collected from both groups at different times. The NOV and BNIP3 mRNA expression were detected by TaqMan quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR), and the relationship between these expression levels and clinical significance in leukemia incidence and progression were analyzed with β-actin as the housekeeping gene. The results showed that the mean values of NOV and BNIP3 increased gradually from 2 weeks after inoculation and achieved highest level at death in control group. Expression level of NOV increased from 1.85E-05 before inoculation to 3.57E-02 at death (p0.05), and BNIP3 from 3.44E-03 to 3.48E-02. While 2 gene expression in the chemotherapy group decreased quickly to 2.51E-05 and 1.58E-03 (p0.05) respectively after chemotherapy, which were close to the level before inoculation (p0.05). The 2 gene expressions again rose at relapse, and difference of expression level between 2 group at death were no statistically significant (p0.05). It is concluded that the expression of NOV and BNIP3 in leukemia AML-M(4) is significantly higher than that in normal controls, of which high level expression is an important factor in the development of leukemia. Close relation between the therapeutic effect and expression level of these two genes suggests the great value in prognostic evaluation and MRD detection.

To explore the relationship between WT1-induced T-cell subsets and graft-versus-host disease (GVHD) after nonmyeloablative allogeneic hematopoietic stem cell transplantation (NST).Peripheral blood mononucleated cells (PBMCs) from 19 patients who expressed WT1 and developed GVHD after NST were simulated by WT1126-134 peptide in vitro, and proportions of WT1-induced-T-cell subsets (Tc1, Tc2, Th1, Th2 cells) before and after transplant were detected by intracellular cytokine staining (ICCS) assay. WT1-specific CD8(+) CTLs of 14 patients with HLA-A*0201 were detected by HLA-A*0201/WT1 pentamer.(1) 17 of 19 patients developed GVHD, among whom proportions of Tc1 and Th1 cells, achieved peak value in 16 patients at occurrence of GVHD (P = 0.039); (2) The peak proportions of Tc1 and Th1 cells in patients with aGVHD above grade II were higher than those with grade I, but being no statistical difference (P = 0.900 and P = 0.140, respectively); (3) The peak proportion of Th1 cells (P = 0.004), but not Tc1 cells (P = 0.060) in patients with extensive cGVHD was significantly higher than that in patients with limited one; (4) Proportions of Tc1, Th1 and WT1(+)CD8(+)CTL in patients without GVHD were similar to those in patients with Grade I aGVHD, but lower than those in aGVHD above grade II.GVHD promotes the generation of WT1-induced GVL effect, and the intensity of the latter maybe correlated with the intensity of GVHD, especially cGVHD. Th1 cells play a more important role in the enhancement of WT1-induced GVL effect in extensive cGVHD patient than in limited cGVHD patients.

To investigate the effects of exogenous NO donors sodium nitroprusside (SNP) and 3-morpholinosydnonimine (SIN-1) on automaticity of the rabbit sino-atrial node in vitro and the action mechanism.The intracellular microelectrode technique is used to record the action potentials of rabbit sino-atrial node and APA (amplitude of AP), V(max) (maximal rate of depolarization), VDD (velocity of diastolic depolarization), RPF (rate of pacemaker firing) are analyzed.SNP(10(-5) - 10(-2) mol/L) increased its RPF and VDD dose-dependently. 10(-3) mol/L SNP increased RPF (beats/min) from 163 +/- 10.8 to 195.0 +/- 13.1 increased VDD (mV/s) from 50.3 +/- 9.6 to 70.2 +/- 12.1 (P0.01). SIN-1(10(-3) - 10(-2) mol/L) also increased RPF and VDD (P0.01).10(-4) mo/L Methylene blue (MB), a blocker of GMP cyclase, prevented the positive chronotropic effect and increasement of VDD induced by 10(-3) mol/L SNP totally (P0.01). 2. CsCl (2 mmol/L), a blocker of I(f) prevented the increasement of RPF and VDD in part (P0.05). 3. NIF (0.46 micromol/L), a blocker of I(Ca-L, had no significant effects on chronotropic effect and increasement of VDD (P0.01).Exogenous NO can increase the automaticity of rabbit sino-atrial node in vitro. The chronotropic effect is involved in NO-cGMP pathway and results from increasement of I(f) in the sino-atrial node at least in part; I(ca-L) is unlikely to play a major role in this effect.

Using double immunohistochemical method for Fos and tyrosine hydroxylase(TH) to examine the effects of intracerebroventricular (icv) administration of adrenomedullin (AM) on catecholaminergic neurons and the expression of c-fos gene in rat brain nuclei involved in cardiovascular regulation in order to define whether the effects of central administration of adrenomedullin (AM) were induced by activating the catecholaminergic neurons.(1) Following icy administration of AM (3 nmol/kg), Fos-like immunoreactivity neurons were markedly increased in several brain areas of the rat, including the brainstem, the hypothalamus and the forebrain. (2) Following icy administration of AM (3 nmol/kg), double-labeled neurons for Fos and TH increased significantly in the area postrema (AP), the nucleus of the solitary tract (NTS), the nucleus paragigantocellularis lateralis (PGL) and the locus coeruleus (LC). (3) Pretreatment with calcitonin gene-related peptide receptor antagonism CGRP (8-37) (30 nmol/kg) significantly reduced the action of AM (3 nmol/kg) in the brain.AM activates the nuclei involved in cardiovascular regulation in the forebrain, the hypothalamus and the brainstem, and that the central actions of AM are induced by activating the catecholaminergic neurons of brainstem nuclei involved in cardiovascular regulation. CGRP receptor can mediate the effects of AM in brain.

The anterior cruciate ligament (ACL) is one of the most commonly injured knee ligaments. Even following ACL reconstruction, significant articular cartilage degeneration can be observed and most patients suffer from premature osteoarthritis. Articular cartilage degeneration and osteoarthritis development after ACL injury are regarded as progressive process that are affected by cyclic loading during frequently performed low-intensity daily activities. The purpose of this study was to perform a meta analysis on studies assessing the effects of ACL reconstruction on kinematics, kinetics and proprioception of knee during level walking.This meta analysis was conducted according to the methodological guidelines outlined by the Cochrane Collaboration. An electronic search of the literature was performed and all trials published between January 1966 and July 2010 comparing gait and proprioception of a reconstructed-ACL group with an intact-ACL group were pooled for this review. Thirteen studies were included in the final meta analysis.There was no significant difference in step length, walking speed, maximum knee flexion angle during loading response, joint position sense and threshold to detect passive motion between the reconstructed-ACL group and the intact-ACL group (P0.05). However, there was a significant difference in peak knee flexion angle, maximum angular knee flexion excursion during stance, peak knee flexion moment during walking and maximum external tibial rotation angle throughout the gait cycle between the reconstructed-ACL group and the intact-ACL group (P0.05).Step length, walking speed, maximum knee flexion angle during loading response, joint position sense and threshold to detect passive motion usually observed with ACL deficiency were restored after the ACL reconstruction and rehabilitation, but no significant improvements were observed for peak knee flexion angle, maximum angular knee flexion excursion during stance, peak knee flexion moment during walking and maximum external tibial rotation angle throughout the gait cycle.

The chemical compositions of the rainwater collected in Shanghai in Summer of 2008-2009 were investigated. The chemical character and pollutant source of rainwater were evaluated depended on HYSPLIT model, ions tracer techniques, correlation and principal component analysis. The results showed that: (1) the mean pH in rain was 4.72 and 4.68; (2) the frequency of acid rain was 53.30% and 63.30%, respectively, in 2008 and 2009; (3) ionic concentration was SO4(2-)NH4+NO3-Cl-Ca2+Na+Mg2+K+, in which the secondary components like SO4(2-), NO3- and NH4+ contributed significantly to total ions of rainwater and they accounted for 55.01% and 65.97% of total ions in 2008 and 2009, respectively, which indicate the severe secondary pollution in Shanghai; (4) the ratio of SO4(2-) to NO3- in Summer precipitation in 2008 and 2009 was 3.19 and 2.13, respectively, which implies sulfuric-nitrous mixed type of precipitation; (5) the content of DOC varied from 1.36 mg/L to 10.69 mg/L and average value was 2.44 mg/L in rainwater; (6) SO4(2-) and NO3- were mainly in the form of (NH4) 2SO4 and NH4NO3, which showed the dominant neutralization effect of NH4+ over Ca2+ in Summer. Source identification indicated that SO4(2-), NH4+, NO3-, K+ and most Ca2+ derived from anthropogenic sources, while Mg2+ and Cl- derived from both marine and non-marine but non-marine was over marine. The chemistry of precipitation in Shanghai was impacted by local pollutants and the long-and moderate-range transport by Southwest monsoon according to backward trajectory analysis.

To investigate the molecular biological mechanism of chronoexercise regulating circadian.Expressions of mPer1 and mPer2 in the diencephalon of golden hamster were determined 2 hours after acute exhaustive exercise (circadian time 6) by quantitative RT-PCR.Chronoexercise at CT6 significantly decreased expressions of mPer1 and mPer2 in the diencephalon of golden hamster.Inhibitory effect of chronoexercise on Per1 and Per2 mRNA levels in the diencephalon of golden hamster at CT6 may be achieved transcription-translation-based autoregulatory negative feedback loop.

The results of haploidentical hematopoietic stem cell transplantation (HSCT) have been disappointing due to the high incidence of severe graft-versus-host disease (GVHD) and infectious complications. It is well known that mesenchymal stem cells (MSCs) can prevent severe acute GVHD in HSCT. However, there is a controversy concerning whether MSC-mediated suppression of T cell functions is accompanied by inducing T cells maturation effects after HSCT. The CB6F1((H-2bd)) female mice irradiated with 8 Gy (60)Co γ-rays were divided into two groups: mice in the MSCs group were infused with MSCs labeled with cm-DiI and mononuclear cells from the bone marrow and spleen of BALB/c((H-2d)) mice; the control group was infused with only the mononuclear cells of BALB/c((H-2d)) mice. After transplantation, chimerisms of donor MSCs were observed in the recipients. The recovery of the T-lymphocyte subpopulation, the proliferative activity of T-cells after stimulation with ConA, the mixed lymphocytes' reaction between donor and recipient and three parts, and the number of apoptosis thymus cells were compared in two groups. The results showed that MSCs preferentially homed to the thymus and grew there, a more rapid recovery of T-cells in the peripheral blood, and decreased the apoptosis of the thymocytes. Thus MSCs may affect the thymus in order to improve T-cells maturation and immune system recovery.

This study was aimed to explore the effectiveness of sequential and quantitative detection method for analysing donor chimerism (DC). In order to simulate a mouse model of haploidentical stem cell transplantation, C57BL/6 male mice were used as donors, while CB6F1 female mice were used as recipients and were divided into 2 groups. The two groups of recipients were irradiated with 2 Gy and 6 Gy from (⁶⁰CO gamma-ray source respectively, and then were inoculated intravenously with bone marrow cells (BMCs) and spleen mononuclear cells (SPMNCs). Quantitative analyses of DC were performed with real-time PCR or flow cytometry (FCM) on different days after transplantation. The results showed that real-time PCR and FCM both have advantages and disadvantages in the detection of DC. When DC amount in group of 6 Gy was90% with stable macrochimerism for more than 3 months, the efficacy of detection by FCM was well; while the DC amount in group of 2 Gy was below 10% and gradually transformed to different forms of microchimerism until disappeared, in which condition the use of real time-PCR was more appropriate. It is concluded that FCM can detect macrochimerism with high accuracy but would fail when DC amount is less than 1% due to sensitivity limitation, while the real time-PCR is more sensitive for detecting microchimerism but lack of accuracy for detecting macrochimerism. Combination of the two methods can afford sensitive and accurate tool for quantitative analysis of chimerism in mouse model of transplantation and adoptive immunotherapy.

This study was purposed to investigate the value of combination of pentamer and intracellular IFNgamma staining in the qualitative and quantitative detection of circulating antigen-specific T cells. WT1 expressions in 14 HLA-A*0201+ patients and their matched donors were detected by RT-PCR, and circulating WT1 specific T cells were assayed by HLA-A*0201/WT1 pentamer combined with intracellular IFNgamma+ staining. The results showed that the low level of WT1 expression was found only in 2 cases out of 14 donors, but different levels of WT1 expression could be observed in all leukemic patients. The WT1+CD8+ CTL and WT1+IFNgamma+ cells did not detected in all 14 donors, but WT1+CD8+ CTL cells in 2 patients and WT1+IFNgamma+ cells in 3 patients could be detected before transplantation respectively, there was no significant difference between them, while the WT1+CD8+ CTL cells and WT1+IFNgamma+ cells both could be detected in all 14 patients after transplantation, the positive detection rate after transplantation was obviously higher than that before transplantation. The WT1+CD8+ and WT1+ IFNgamma+ cells could be detected within 30 days after transplantation, but the positive detection rate of WT1+IFNgamma+ cells was higher than that of WT1+CD8+ CTL cells (p=0.014). The median peak value of WT1+CD8+ CTL cells was 0.18% in 14 patients, and the median peak value of WT1+IFNgamma+ cells was 0.83% in 14 patients, the later was significantly higher than former. The median peak time of WT1+CD8+ CTL cells was 75 days after transplantation, while the WT1+IFNgamma+ cells was 105 days after transplantation, there was no significant difference between them. It is concluded that pentamer and intracellular IFNgamma staining may effectively detect circulating WT1 specific T cells in leukemic patients, and the combination of these two methods profit to the exact qualitation and quantitation of circulating antigen-specific T cells.

The aim of this study was to investigate the effects and mechanisms of mesenchymal stem cells (MSCs) on haematopoietic reconstitution in reducing bone marrow cell apoptosis effects in irradiated mice, and to research the safe and effective dosage of MSCs in mice with total body irradiation (TBI). After BALB/c mice were irradiated with 5.5 Gy cobalt-60 gamma-rays, the following were observed: peripheral blood cell count, apoptosis rate, cell cycle, colony-forming unit-granulocyte macrophage (CFU-GM) and colony-forming unit-fibroblast (CFU-F) counts of bone marrow cells and pathological changes in the medulla. The survival of mice infused with three doses of MSCs after 8.0 Gy or 10 Gy TBI was examined. The blood cells recovered rapidly in the MSC groups. The apoptotic ratio of bone marrow cells in the control group was higher at 24 h after radiation. A lower ratio of G0/G1 cell cycle phases and a higher ratio of G2/M and S phases, as well as a greater number of haematopoietic islands and megalokaryocytes in the bone marrow, were observed in the MSC-treated groups. MSCs induced recovery of CFU-GM and CFU-GM and improved the survival of mice after 8 Gy TBI, but 1.5 x 10(8) kg(-1) of MSCs increased mortality. These results indicate that MSCs protected and treated irradiated mice by inducing haematopoiesis and reducing apoptosis. MSCs may be a succedaneous or intensive method of haematopoietic stem cell transplantation under certain radiation dosages, and could provide a valuable strategy for acute radiation syndrome.

This study was purposed to investigate the reconstitution of immune system in patients with acute lymphocyte leukemia (ALL) or acute myeloid leukemia (AML) after HLA-mismatched nonmyeloablative hematopoietic stem cell transplantation (NHSCT) and its relation with infection and GVHD. 6 ALL and 4 AML patients having HLA-mismatched related donors received the nonmyeloablative precondition regimen composed of fludarabine (Fln), ATG, Ara-C, CTX and total body irradiation (TBI) in dose 2 Gy. The GVHD was prevented and treated by CsA, anti-CD25 antibody and mycophenolic mofetil (MMF) before and after transplantation. The flow cytometry was used to detect the changes of total T cells, help/inducer T cells, suppressor/killer T cells, gamma/delta T cells, B cells, NK cells, NKT cells, regulatory T cells, activated T cells, naive T cells, memory T cells and ratio of CD4/CD8 in patients with remission resulting from chemotherapy before transplantation, and analyse the relation of immunofunctional cells to infection and GVHD after transplantation, compare the difference in recovery of immune system of ALL and AML patients. The results showed that the recovery of total lymphocytes and lymphocyte subsets displayed one's own regular pattern. As compared with patients without GVHD, the counts of lymphocyte subsets in patients with GVHD was higher, while the counts of gamma/delta T cells, regulatory T cells, NK cells, the counts of B cells, NK cells, naive cells and CD4/CD8 ratio as well as the counts of B cells, naive T cells and NK cells were lower at 1 month, 2 - 3 months and 6 - 8 months after transplantation respectively. The total T cells and subsets recovered slowly, but NK cells and NKT cells recovered rapidly in patients with infection at early period after transplantation, the B cells and naive B cells recovered rapidly at 3 months after transplantation. There was no difference in lymphocyte recovery between ALL and AML patients. It is concluded that the analysis of each lymphocyte subsets may indirectly show the recovery of thymus function in patients, the changes of NK cells, B cells and naive T cells have an important significance for identifying and forecasting the GVHD and infection.

To observe the treatment effect and toxicity of nonmyeloablative allogeneic stem cell transplantation (NST) for hematologic diseases.A total of 243 hematologic diseases patients received HLA-identical NST were enrolled in this study from 9 transplant centers of NST Cooperative Group in China. Nonmyeloablative conditioning regimen was based on fludarabine (Flud), rabbit anti-human thymocyte globulin (ATG), cyclophosphamide (CTX) (FAC), and plus cytarabine or busulfan (BU) etc. Graft-versus-host disease (GVHD) prophylaxis included cyclosporin A (CsA) and mycophenolate mofetil (MMF).Among the 243 patients, 219 (90.1%) achieved full donor chimerism (FDC), 2(0.8%) engraftment failure. 78 (32.1%) had mixture chimerism (MC) at 4 weeks after NST, out of which 56 switched to FDC, 16 remained MC and 6 (2.5%) developed graft rejection. The incidence of acute GVHD was 34.2%, including 6.6% of grade III-IV acute GVHD. Chronic GVHD developed in 78 (32.1%) patients. The follow-up durations were 3 - 99 months, 162 (66.7%) were still alive and the overall survival rates were 76.5%, 73.9%, 70.7%, and 27.8% for MDS/SAA, chronic myeloid leukemia, acute leukemia at first remission, and refractory or relapsed leukemia, respectively.The nonmyeloablative allogeneic stem cell transplantation based on FAC conditioning results in sustained engraftment and mild aGVHD, providing a new feasible curative therapy for hematology diseases.

In treating of leukemia and controlling of minimal-residual disease (MRD), graft versus leukemia effect (GVL) plays a critical role, and complicated mechanisms are involved in this immunology process. When graft cells are infused into recipients, the evoked GVL effect must be inevitably influenced by many factors derived from allogeneic effect between donor and receptor. To utilize GVL more efficiently in future clinical practice and to improve the curative effect of allo-HSCT, it is necessary to recognize these factors. Some potential factors influencing GVL such as chimerism patterns, autocytotoxic cells, dynamics of immune cells in patients, the cytokines and so on are reviewed in this article.

To investigate the expressions of vascular endothelial growth factors (VEGF)-A, -C and -D and their prognostic significance and relation to angio- and lymphangiogenesis in gastric cancer.The expression of VEGF-A, -C and -D in 123 primary gastric cancers was detected by immunohistochemical staining. The lymphatic vessel density (LVD) and microvessel density (MVD) were assessed after immunohistochemical double-staining with D2-40 and CD34, respectively. The correlation between the expression of those VEGF factors and clinicopathological parameters were analyzed by univariate method. The overall survival was evaluated by Kaplan-Meier method and log-rank test. Multivariate analysis was carried out using Cox proportion hazard model.The positive expression rate of VEGF-A, -C and -D in primary gastric cancer samples were 64.2%, 65.9% and 41.5%, respectively. High expression of VEGF-A, or -C or -D, or any two of them was correlated with high LVD (P0.05). High expression of both VEGF-A and -C was associated with high MVD, lymph node metastasis, LVI and MVI (P0.05). Both VEGF-C and -D high expression was correlated with LVI and lymph node metastasis (P0.05). The patients with high expression of these factors had a statistically shorter overall survival (P0.05). The patients with both VEGF-A and -C expression had the shortest survival (56 months). Multivariate analysis showed that VEGF-A high expression, MVD, lymph node metastasis and depth of tumor invasion were independent survival predictors (P = 0.033, 0.002, 0.019 and P0.001, respectively).High expression of both VEGF-A and -C imply high potential of lymphangiogenesis, metastasis and poorer survival in gastric cancer patients. High expression of VEGF-C and -D may induce lymphangiogenesis and promote lymph node metastasis, but only VEGF-A is an independent predictor of survival.

This study was purposed to investigate the proliferation and antitumor activity of rhG-CSF-mobilized peripheral blood mononuclear cells (G-PBMNCs) activated by interleukin 21 (IL-21) alone or in combination with interleukin 15 (IL-15)/interleukin 2 (IL-2) and to evaluate the feasibility and value of tumor immunotherapy with cytokine combinations. G-PBMNCs were activated by IL-21 alone or in combination with IL-15/IL-2 in vitro, and the proliferation of the activated G-PBMNCs was analyzed by CCK-8 assay. The cytotoxicity of the activated G-PBMNC to the K562 cells was studied by the test principle which is based on target cell labeling with 5-(6)-carboxy-fluorescein succinimidyl ester (CFSE) and subsequent DNA-labeling with propidium iodide (PI) for identification of target cells with compromised cell membranes. The phenotypes of the activated G-PBMNCs were assayed by flow cytometry. The results showed that the cytotoxicity of IL-21 group had no difference from which of IL-2 group. When G-PBMNCs were exposed to the combinations of IL21+IL15/IL21+IL15+IL2, the cytotoxicity was significantly enhanced at E:T ratio of 25:1, as compared with combination of IL21+IL2 (p0.05). The cytotoxicity of the cytokines combinations was significantly higher than that in cytokine used alone at E:T ratio of 50:1 (p0.05). The cryopreservative and resuscitative G-PBMNCs showed the same result with the fresh G-PBMNCs in cytotoxicity test. The proportions of CD3+ and CD8+ T cells were increased when G-PBMNCs were incubated with cytokines for 72 hours. CD4, CD3-56+ and CD3+56+ counts were significantly elevated when G-PBMNCs were exposed to IL21 + IL15 (p0.05). It is concluded that IL-21 alone enhance the antitumor activity of G-PBMNCs, which further strengthens when IL-21 combinated with IL-15.

The aim of this study was to investigate the effect of mesenchymal stem cells (MSCs) on cell cycle and apoptosis of thymus, spleen and bone marrow cells in mice totally irradiated with sublethal dose, and to explore its mechanisms. BALB/c mice irradiated with 5.5 Gy 60Co gamma-ray were randomly divided into control group and MSC group. Mice in MSC group were infused with 0.4 ml containing 2.5x10(7)/kg of MSCs through tail vein at 1 hour after irradiation. Mice in control group were infused with 0.4 ml normal saline. The cell apoptosis and cell cycle of thymus, spleen and bone marrow cells were detected by flow cytometry at 6, 12, 24 and 72 hours after irradiation and the P53 protein expressions in thymus and bone marrow cells were assayed by immunohistochemistry at 12 hours after irradiation. The results showed that the arrest of cells in G0/G1 and G2/M phase, and decrease of cells in S phase appeared at 6 hours after irradiation, those reached peak respectively at 12 hours in thymus cells, 6 hours in spleen and 24 hours in bone marrow, then the cell counts in G0/G1 phase decreased and the cell counts in S and G2/M phases increased. At 72 hours the cell counts in G0/G1 phase were less than the normal level and the cell counts in S phase were more than the normal level. The above changes of cell cycle in thymus and spleen were more rapid in spleen and more obvious in amplitude than that in bone marrow, the change of cell cycle in MSC group was more rapid and obvious than those in control group. After irradiation the apoptosis cells increased from 6 hours, reached the highest level at 12 hours and decreased to the normal level gradually after 24 hours in two groups; the apoptosis rates in spleen and thymus cells were higher than that in bone marrow cells. In comparison with the control group, the apoptosis rate in thymus cells at 12 hours, in spleen cells at 12 and 24 hours, and in bone marrow cells at 24 hours were fewer in MSC group. The cells expressing P53 protein in control group were more than that in MSC group. It is concluded that the MSCs accelerate the running of cell cycle in these hematopoietic tissue cells of irradiated mice, reduce the cell apoptosis and promote the recovery from injuries in hematopietic and immunological organs, thus protect the irradiated mice at early stage.

To explore immunoregulatory mechanism of mesenchymal stem cells (MSCs) in H-2 haploidentical bone marrow cells transplantation mice.BALB/c female mice irradiated with 8Gy 60Co gamma-rays were divided into two groups: MSCs group, infused cm-DiI labeled MSCs from female CB6F1 mice and monocytes from the bone marrow and spleen of male CB6F1; Control group, only infused monocytes from the bone marrow and spleen of male CB6F1. T-lymphocyte subpopulation of peripheral blood cells, T and B cells proliferation stimulated by ConA and LPS, mixed lymphocyte reaction between donor and recipient and third part, the sry-gene chimerism of bone marrow, spleen and thymus of the recipient, the distribution of MSCs in the recipient, the incidence rate of GVHD and survival were observed.The CD3 at +90 d the percent of CD3+ CD4+ cells, and CD4/CD8 at +30 d in the MSCs group were higher than that in control post-transplantation, respectively (P0.05). The proliferation activity of B cells recovered more rapidly and that of T cells recovered comparably in MSCs group as compared with that in control group. The result of MLR between donor and recipient was lower in MSCs group than that in the control; and that between recipient and the third part had no difference. The sry-gene chimerism of bone marrow and spleen of the recipient was higher in MSCs group than in control at +30 d. The MSCs mainly distributed in intestine, thymus, bone marrow, liver, heart of the recipient after transplantation. The incidence of acute GVHD was higher and the survival rate was lower in MSCs group than that in control group (P0.05). Chronic GVHD occurred in the control group at +90 d, while in the MSCs group at +120 d.MSCs might improve stem cell engraftment, promote lymphocyte and humoral immunity recovery, decrease incidence of GVHD and increase survival by inducing specific immunologic tolerance and repairing organs injuries.

This study was aimed to construct the soluble HLA-A*0201-PR1 complex for preparation of HLA-A*0201-PR1 tetramer. The recombinant HLA-A*0201-BSP (BirA substrate peptide) fusion protein as heavy chain and beta(2)-microglobulin (beta(2) m) as light chain were expressed highly as insoluble aggregates in Escherichia coli and then purified with gel filtration, and the final purity reached above 90%. The two subunits were refolded to form an HLA-A*0201-peptide complex by dilution method in the presence of an antigenic peptide PR1, a HLA-A2-restricted peptide from proteinase 3 (aa 169 - 177, VLQELNVTV). Refolded HLA-A*0201-PR1 complex was biotinylated using a BirA enzyme and purified by anion exchange chromatography on a Q-Sepharose (fast flow) column. The extent of reconstitution of the HLA-A*0201-PR1 complex was analyzed by HPLC gel filtration. The refolded and biotinylated products were detected by Western blot and ELISA with monoclonal antibody BB7.2 that recognized the natural conformations of HLA-A2 and streptavidin. The results showed that the refolded complex was composed of HLA-A*0201-BSP aggregate, HLA-A*0201-PR1 complex and beta(2) m, and reconstitution yields of 18% with PR1 was obtained. Refolded HLA-A*0201-PR1 complex could be confirmed by practical immunological method and biotinylated efficiently. It is concluded that the refolding and biotinylation of HLA-A*0201-PR1 complex is successfully obtained. This work provides the basis for the preparation of HLA-A*0201-PR1 tetramer.

Hepatocyte growth factor (HGF) is a pleiotropic cytokine, its roles in the physiology and pathology of immune system, have been investigated thoroughly, great deal of data have been documented on its immunoregulatory activity. In this review, according to advance of study on HGF in recent years, the role of HGF in the immune regulation, such as immunoregulatory effects of HGF on T lymphocytes, B lymphocytes and dendritic cell, modulation of HGF on specific humoral and cellular immune response, control of acute GVHD and acceleration of myeloid and immunologic reconstitution in allogenetic bone marrow transplantation models, promotion of tissue repair and regeneration, and alleviation of immune rejection in allogeneic organ transplantation including the heart, liver and kidney transplantation, prevention of grafts from injury as well as applicably useful of HGF in the therapy of autoimmune disorders were summarized.

The aim of study was to select the best scheme of G-CSF to mobilize peripheral stem/progenitor cells in healthy donors. The clinical data of 60 cases received nonmyeloablative allogenic hematopoietic stem cell transplantation was analyzed retrospectively. The results indicated that the counts of MNC and CD34(+) cells were significantly higher in the 10 microg/(kg.d) group than that in the 5 microg/(kg.d) group (P0.05). The counts of MNC and CD34(+) cells which were collected after day 4 or 5 in the 10 microg/(kg.d) groups were not significantly different. The percentage of CD3(+) cells, CD4(+) cells and CD8(+) cells were not different in different groups. It is concluded that the scheme using 10 microg/(kg.d) G-CSF is more efficient than that in the 5 microg/(kg.d) group in mobilizing stem cells. It may reduce days for mobilization and decrease expense for collection of cells after 4 days of mobilization. Scheme using 10 microg/(kg.d) G-CSF for cells collecting after 4 days is more efficient.

High-yield production of HLA-A*0201 heavy chain is a prerequisite to the preparation of HLA-A2 tetramer. The present study was aimed to construct the expression vector of recombinant HLA-A*0201-BSP fusion gene for preparing HLA-A2 tetramers. The extracellular region HLA*0201 was cloned by RT-PCR from HLA-A2(+) donor, and a 15-amino acid biotin-protein ligase (BirA) substrate peptide (BSP) for BirA-dependent biotinylation was added to the COOH-terminus of HLA-A*0201 heavy chain. Then the fusion gene was cloned into pBV220 vector at EcoRI and Bam HI sites and its sequence was confirmed by DNA sequence analysis. The recombinant plasmid pBV220-HLA-A*0201-BSP was transformed to the competent cells of E.coli DH5alpha. The results showed that the HLA-A*0201-BSP fusion protein was successfully expressed in the form of inclusion body and amounted to over 28% of total cell proteins via induction at 42 degrees C. After washed with triton X-100 and urea, the inclusion body was dissolved with 8 mol/L urea and then purified with Sepharcyl S-300 HR, and the final purity reached above 90%. It is concluded that the HLA-A*0201-BSP fusion gene was cloned successfully and expressed efficiently in E.coli DH5alpha. This work establishes a convenient approach for purification of large quantity of recombinant HLA-A*0201-BSP. This provides the basis for the preparation of HLA-A2 tetramers.

Flk-1(+)CD31(-)CD34(-) mesenchymal stem cells (MSCs) are multipotent cells with extensive proliferation ability and immunomodulatory function. On the basis of our previous work showing their potential application in tissue engineering and immune diseases, we designed a preclinical and Phase I trial to evaluate the feasibility and safety of intravenous infusion of these cells after ex vivo expansion. Rhesus monkey and human Flk-1(+)CD31(-)CD34(-) MSCs were isolated from bone marrow and passaged a maximum of six passages. Karyotype analysis showed Flk-1(+)CD31(-)CD34(-) MSCs remained normally diploid within six passages of expansion. Expanded cells were transplanted into rhesus monkeys and human volunteers, respectively. During the infusion of these cells, the life signs of the recipients were normal. Laboratory tests for blood, bone marrow, kidney, and liver function were conducted and no significant changes were observed before and after cell infusion. Identification of the sry sequence from the male donor in female recipients showed that allogeneic rhesus Flk- 1(+)CD31(-)CD34(-) MSCs were capable of homing to and establishing residence within the recipients' bone marrow. No significant changes were observed in lymphocyte cell subpopulation before and after infusion. These result showed that Flk-1(+)CD31(-)CD34(-) MSCs have no obvious side effects after intravenous administration, encouraging investigators to perform further studies in stem cell therapy.

Human beta(2)-microglobulin (beta(2)m) is the light chain of major histocompatibility complex (MHC) class I molecule. High-yield production of this protein is a prerequisite to the preparation of MHC class I tetramer. The present study was aimed to obtain recombinant human beta(2)m expressed in Escherichia coli (E. coli) for preparing MHC class I tetramers. For cloning of human beta(2)m gene, a pair of specific primers was designed based on the published sequence of this gene. A 300 bp specific DNA fragment corresponding to the encoding region of beta(2)m lack of the signal peptide sequence was obtained by RT-PCR from the total RNA of human leukocytes. The amplified cDNA was inserted into the IPTG-inducible expression plasmid pET-17b by Nde I and Bam H I sites and its sequence was confirmed by DNA sequence analysis. The recombinant plasmid pET-beta(2)m was transformed to the competent cells of E. coli BL21 (DE3). The results showed that beta(2)m was expressed in the form of inclusion body and amounted to over 32% of total cell proteins after IPTG induction. After washing with triton X-100 and urea, the inclusion body was dissolved with 4 mol/L urea and then purified with Sephacryl S-200 HR, and the final purity reached above 95%. The denatured protein was renatured by dilution method. Western blot assay indicated that the monoclonal antibody against human native beta(2)m could react specifically with the recombinant protein. In conclusion, the human beta(2)m gene was cloned successfully and expressed efficiently in E. coli BL21 (DE3). This work establishes a convenient approach for renaturation and purification of large quantity of recombinant beta(2)m. This provides the basis for the preparation of MHC tetramers.

This study was aimed to analyze the mRNA expression of cytokines (TGF-beta, IL-2, IL-6, IL-10, IFN-gamma, TNF-alpha, FAS-L) in five rhesus treated with haploidentical peripheral blood stem cell transplantation after nonmyeloablative preparative regimens and to explore the role of these cytokines in the development and pathology of acute graft-versus-host-disease (aGVHD). Five rhesus monkeys received nonmyeloablative haploidentical peripheral blood stem cells transplantation. Semi-quantitative reversed transcription polymerase chain reaction (RT-PCR) was used to analyze the kinetics of cytokine mRNA expression in the transplantation and aGVHD. The results showed that five rhesus monkeys acquired hematopoietic reconstitution successfully. The graft was rejected in one monkey which survived without disease, the other four achieved mixed chimerism and full donor chimerism. Chimerism of low centigrade in one monkey achieved high centigrade at 35 days after donor stem cell infusion. Intestinal aGVHD grade III developed in one monkey. Cytokines of Th1 and Th2 changed after transplantation. In period of aGVHD, expression of TGF-beta decreased but all others increased in various levels. When donor chimerism decreased, the cytokines decreased accordingly. It is concluded that the decrease of TGF-beta mRNA may be an indicator to predict aGVHD, and can be used as a differential diagnostic indicator for intestinal GVHD.