Objective To explore the bone marrow stromal cells,anti-late antigen-4 (VLA-4) antibody (aVLA-4),cytarabine (Ara-C) on the proliferation and apoptosis of leukemia HL-60 cells.Methods The experiment was divided into five groups:HL-60 cells were cultured alone (control group),HL-60 cells and stromal cells group (stromal cells group),HL-60 cells + stromal cells + aVLA-4 (antibody group),HL-60 cells + stromal cells + Ara-C group (drug group),HL-60 cells + stromal cells + aVLA-4 + Ara-C group (antibody +drug group).Cell proliferation or inhibition rate was detected by CCK-8 method,the HL-60 cells apoptosis was detected by flow cytometry.The expression of anti-apoptotic gene bcl-2 in HL-60 cells was determined by Western blot.Results After 24 h and 48 h,treatment,the number of the stromal cells group HL-60 cells were higher than that of the control group with significant difference cultured [(7.2±0.3)×1O5/ml vs (5.3±0.4)×105/ml,(8.4±0.2)×105/ml vs (6.8±0.3)×105/m1,P ＜ 0.001],while the HL-60 cell proliferation inhibition rate [(24.3±2.1) ％,(37.0±2.6) ％,(65.6±3.8) ％] and apoptosis rate [(5.7±0.6) ％,(8.0±0.5) ％,(10.4±0.9) ％,(16.5±0.7) ％] of antibody group,drug group,antibody + drug group were higher than the control group with a difference of statistically significant (P ＜ 0.05),and the increase of antibody + drug group was most obvious.With the decreasing of the bcl-2 protein expression,which was most the decrease of antibody + drug group was most obvious.Conclusion Bone marrow stromal cells can stimulate the proliferation of leukemia cells,aVLA-4 interference the interaction between stromal cells and leukemia cells can enhance the chemosensitivity of leukemia cells to Ara-C.

Objective To observe the clinical efficacy and adverse reaction of the combination of fiudarabine,cytarabine and granulocytecolony-stimulating factor (G-CSF) (FLAG regime) therapy for refractory and relapsed acute leukemia in children. Methods From 2004 to date, a total of 9 patients with relapsed and refractory acute leukemia patients in our hospital accepted the treatment, in 9 cases 8 cases were AML,1cases were ALL; in 9 cases 5 cases were refractory acute leukemia, 4 cases were recurrent acute leukemia.Results Among the 9 cases,6 cases with 1 cycles of chemotherapy achieved complete remission (CR),CR rate was 66.7 ％ (6/9); partial remission (PR) rate was 22.2 ％ (2/9),total efficiency (CR+PR) was 88.9 ％.In 6 CR patients 2 underwent hematopoietic stem cell transplantation, are disease-free survival; this regimen' s main adverse reactions were infection,bone marrow depression and gastrointestinal reaction.Conclusion The remission rate of FLAG regimen in the treatment of children with refractory and relapsed acute leukemia is relatively high, adverse reactions were tolerable; the FLAG program is a choice for the treatment of children with refractory and relapsed acute leukemia,which provides the opportunity for subsequent hematopoietic stem cell transplantation.

AIM:To investigate the changes of CD27 and CD28 expression on cytokine-induced killer(CIK) cells and natural killer(NK) cells during cultivation.METHODS:The mononuclear cells were treated with interleukin-12(IL-2),IL-7,IL-15,stem cell factor and FLT3 ligand for four weeks. The CD27 and CD28 expression on CD3+CD56+ CIK cells and CD3-CD56+ NK cells were examined by flow cytometric analysis every week during four-week cultivation.RESULTS:The expression of CD27 on CIK and NK cells two weeks after cultivation reached the peak value at(44.57?4.07)% and(51.02?5.20)%,respectively. Then,their expression declined gradually to about 30% in the fourth week. The expression of CD28 on CIK and NK cells reached the peak at the third and the second week,the values were (4.40?0.66)% and (45.14?3.58)%,respectively,decreased rapidly and then were almost completely lost at the fourth week. CONCLUSION:According to the changes of expressions of CD27 and CD28 on CIK/NK cells,optimal harvest time of cultured CIK/NK cells should be at the third week of cultivation.

AIM: To evaluate the biological characteristics and differentiating potentials of bone marrow-derived mesenchymal stem cells(MSCs) from sensitized mice by allogeneic splenocyte transfusion in vitro.METHODS: Adherent culture method was applied for culturing the bone marrow-derived MSCs from sensitized mice.The cell morphology was examined and the surface marker profiles were analyzed by flow cytometry.The differentiating potentials of the MSCs into osteogenic,adipogenic and myogenic lineages were explored.The bone marrow-derived MSCs from the normal mice were collected and served as controls.RESULTS: Both the bone marrow-derived MSCs from sensitized and normal mice were exhibited a homogeneous distinctive morphology and were positive for the surface markers CD29,CD105,CD44 and Sca-1,negative in CD 34 and CD11b.The abilities of both MSCs to differentiate into osteogenic,adipogenic and myogenic pathways in the same condition were also observed.CONCLUSION: There is no difference in the biological characteristics and induced differentiating potentials between the sensitized mouse bone marrow-derived MSCs by allogeneic splenocytes transfusion and the MSCs from normal mice.

AIM: To investigate the sequence expression o f CD158 molecule after tacrolimus (FK506), mycophenolate mofetil (MMF) combined with methylprednisone (MP) treatment for refractory chronic graft-versus-host di seases (cGVHD). METHODS: The efficacy and the side effect were observed in 6 chi ld patients with extensive cGVHD after allogeneic hematopoietic stem cell transp lantation treated with the combination of FK506, MMF and MP, meanwhile the chang es of the CD158 expressions on T lymphocytes and NK cells in peripheral blood be fore and after treatment were observed. RESULTS: The expression of CD4+CD158a+ and CD4+CD158b+ were very low before and after transplantation and treatment, there was no stati stical significance. The expression of CD3+CD158b+ and CD3+CD8+CD158b + were 4.97%?2.36% and 4.58%?2.90% respectively in five patients with acut e GVHD, and there was statistical significance compared with that of before-tran splantation (P

OBJECTIVE To investigate the characteristic and prognosis of nosocomial infection in children patients with malignances during deficiency stage of neutropenia. METHODS The clinical characteristic of infections and efficiency of antibiotics were analyzed retrospectively in 174 malignances children with nosocomial infection from Jan 2000 to Jun 2005. RESULTS The incidence rate of nosocomial infection in children patients with malignances during deficiency stage of neutropenia was 67.4%.Nosocomial infection was occurred mainly in the respiratory tract,oral cavity,blood,skin,and intestinal tract of patients.The main pathogens were bacteria(86.8%),and fungal infection was 13.2%.The Gram-negative bacilli were relatively sensitive to imipenem,amikacin,ceftazidine,piperacillin/tazobactam,and ticarcillin/clavulanic acid.The Gram positive cocci were sensitive to vancomycin,imipenem,meropenem,ampicillin/sulbactam,ciprofloxcin and clindamycin.The death rate due to nosocomial infection was 44.4%. CONCLUSIONS There is higher incidence of nosocomial infection in children patients with malignances during the neutropenia stage.Good nursing,intestines disinfection,the usage of granulocyte colony-stimulating factor,a reasonable usage of antibiotics and preventing the fungal infection are good to control nosocomial infection in children malignances.

Objective To investigate the susceptibilities of HCMV clinical strains isolated to ganciclovir from the patients after hematopoietic stem cell transplantation.Methods Eight HCMV clinical isolates were isolated from the blood or the urine of hematopoietic stem cell transplantation recipients who had been treated with GCV.Tissue cell infection median dose(TCID50) were calculated by Reed-Muench method.Drug susceptibility was determined by MTT assay.Results TCID50 values of eight HCMV clinical strains were 10-4.12/0.1ml,10-4.29/0.1ml,10-4.3/0.1ml,10-4.4/0.1ml 10-4.42/0.1ml,10-4.5/0.1ml,10-4.52/0.1ml and 10-4.62/0.1ml respectively.50％ inhibitory concentration(IC50) to GCV of eight HCMV clinical strains were 0.638,1.438,0.965,0.698,0.482,1.167,1.519,1.511 mg/L respectively.Conclusion Our results suggest that resistant HCMV strains are not prevalent in Guangzhou.Continuous monitoring of HCMV is needed to understand the antiviral resistance status of the virus in patients after hematopoietic stem cell transplantation and guide its clinical management.

BACKGROUND: Hemorrhagic cystitis (HC) is one of common complications in patients undergoing hematopoietic stem cell transplantation (HSCT). It is of great value for improvement in the HSCT outcome to describe the clinical characteristics of HC and risk factors. OBJECTIVE: To investigate the incidence of HC in children after HSCT, and to analyze its clinical characteristics and risk factors.DESIGN: Case analysis SETTING: Center of Hematopoietic Stem Cell Transplantation, Department of Pediatrics, Second Affiliated Hospital of Sun Yat-sen University.PARTICIPANTS: Experiments were performed at the Center of Hematopoietic Stem Cell Transplantation, Department of Pediatrics of Second Affiliated Hospital of Sun Yat-sen University from October 1998 to June 2004. Eighty-eight patients receiving umbilical cord blood transplantation (UCBT) and peripheral blood stem cell transplantation (PBSCT) were enrolled; 49 were males and 39 were females. The age ranged from 2 to 18 years with an average of 8.0 years. Guardians of child patients signed informed consents. The experimental procedures were approved by Medical Ethics Committee.METHODS: ①Conditioning regimens included combination of cyclophosphamide (CY, 120-200 mg/kg) with busulphan (BU, 14-20 mg/kg)-based chemotherapy and combination of CY with total body irradiation (TBI, 2-8 Gy) or total lymphoid irradiation (TLI, 2-8 Gy)-based radiotherapy. ②HC was defined according to the criteria proposed by references 7 and 8. The incidence, clinical characteristics, laboratory examination, treatment and outcome for HC were described. The association of various clinical factors including age, gender, human leucocyte antigen (HLA) typing, diseases for transplant, the type of stem cell, the type of transplantation, the occurrence of acute graft-versus-host disease (aGVHD) and cytomegalovirus (CMV) infection with the development of HC were examined.MAIN OUTCOME MEASURES: ①Incidence of HC, ②HC patient characteristics and laboratory examination, ③HC treatment and outcome, and ④risk factors analysis. RESULTS: All 88 patients were included in the final analysis. ①The incidence of HC: 16 patients (18.2%, 16/88) developed HC post-transplant with the severity graded as mild in 11 cases (68.7%) and severe in 5 cases (31.3%). ②HC patient characteristics and laboratory examination: All had hematuria and 8 cases (50.0%) had typical pollakisuria, urinary urgency, odynuria and gross hematuria; 10 cases (62.5%) had gross hematuria and 11 had proteinuria (+ to +++); Leucocytes were detected in 7 cases. ③Treatment and outcome: All patients recovered at a median of 13.5 days (range 2-53 days). ④Risk factors analysis: The incidence of HC was significantly higher in the group of ≥ 6 years old, presence of aGVHD and development of cytomegalo-virus (CMV) infection (P < 0.05-0.01). CONCLUSION: ①HC has its own clinical characteristics following HSCT in children but with good prognosis. ②The risk factors for HC are ≥ 6 years old, presence of aGVHD and CMV infection.

Objective To research the antiviral activity of artesunate (ART) in vitro fighting against both standard laboratory strains and ganciclovir(GCV)-resistance strains of human cytomegalovims(HCMV) and to explore whether fractionation dosage method can obviously enhance the antiviral effect of ART.Methods 1.Cytotoxicity assay to ART was performed by the use of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) colorimetry.The 0％ toxic concentration (TC0) were determined,and median cytotoxic concentration (TC50) was calculated with Probit regression method.2.Antiviral activity assays of ART against HCMV:human embryonic lung fibroblast cells (HELs) were infected with standard laboratory strains and GCV-resistance strains of HCMV,respectively,after which virus was removed and overlays of dulbecco's modified eagle medium(MEM) containing different antiviral drugs were added to the wells.All cells were cultured continuously at 37 ℃ in a 50 mL/L CO2 humidified atmosphere for 7-10 days and the cytopathic effect (CPE) was observed under a microscope.When the degree of CPE was clear (+ + +-+ + + +),the values of absorbency at 490 nm of all cell wells were measured by MTT colorimetry.The cell survival rate (CSR)and drug inhibitory rate (IR) for HCMV were calculated.By Probit regression method,the median inhibitory concentration (IC50) of 2 drugs was calculated respectively.3.To explore whether fractionation dosage method could obviously enhance the antiviral effect of ART against HCMV,the experiment was divided into 3 groups and compared with GCV group,respectively:Group 1:ART antiviral compounds were added to cell layers by one dosage.Group 2:Total drug dosage was divided into 3 parts,and each part was added to cell layers once a day for 3 days.Group 3:Total antiviral compounds were divided into 6 and delivery 2 times a day.The values of absorbency at 490 nm of all cell wells were measured by MTT colorimetry.The CSR and viral inhibitory rates were calculated.All data were statistically analyzed by One-Way ANOVA analyzing using SPSS 18.0 statistical software.P value of ＜0.05 was considered to indicate statistical significance.Results 1.Cytotoxicity assay showed that cytotoxicity was not found in the relevant range of ART concentrations under 62.5 μmol/L.TC0 and TC50 value of ART were 62.5 μmol/L and 171.7 μmol/L.2.In concentration of 5 μmol/L,15 μmol/L and 30 μmol/L,ART and GCV could obviously inhibit growth of HCMV AD169 strains.There was no significant difference between them.The value of GCV IC50 was 3.49μmol/L,and the value of ART IC50 was 2.17 μmol/L.Treatment index (TI) of ART was 28.8,and GCV was 716.3.ART could still obviously inhibit growth of HCMV resistant strains,but GCV couldn't.Differences between them were statistically significant.The value of GCV IC50 to HCMV resistant strains was 44.4 μmol/L,and the value of ART IC50 was 2.5 μmol/L.3.Fractionation dosage method (2 times a day) of ART could improve the inhibition rate of virus significantly compared to that used once a day and single dose method.Difference was statistically significant(P ＜ 0.01).GCV delivered as the same method had little different changes in virus suppression ratio(P ＞ 0.05).Conclusions 1.Cytotoxicity was not found in the relevant range of ART concentrations under 62.5 μmol/L.2.ART could obviously inhibit growth of HCMV resistant strains and standard laboratory strains.3.Fractionation dosage method (2 times a day) of ART could improve the inhibition rate of virus significantly compared to that used once a day and single dose method.4.Because the action mode of ART is different from other anti-HCMV drugs,and ART has a high biological activity and fewer side effects,it is expected to become a kind of new antiviral drugs for HCMV infections.

Objective To investigate the effect of fungus Aspergillus fumigatus and Candida albicans on the expression of endocellular interferon-γ (IFN-γ) and interleukin-4 (IL-4) in cytokineinduced natural killer (NK) cells.Methods NK cells were cultured with Aspergillusfumigatus or Candida albicans by non-contact or direct-contact methods with a ratio of NK cells to fungus of 10 ∶ 1.The expressions of IFN-γ and IL-4 in NK cells were evaluated by flow cytometry after co-cultured for 6 h.Analysis of variance or SNK-q test was used to compare the expressions of IFN-γ and IL-4 among different groups.Results The IFN-γexpression rates in NK cells with direct contacting to Aspergillus fumigatus hyphae,or to different morphotypes of Candida albicans were (20.12 ± 0.53) ％,(20.69 ± 0.34) ％ and (20.8 ±0.37)％ respectively,while IFN-γexpression in NK cells with indirect contacting to fumigatus hyphae,or to different morphotypes of Candida albicans were (21.40 ± 0.53) ％,(20.57 ± 1.09) ％ and (20.20 ±0.51) ％ respectively,and all were significantly higher than that in the blank group [(15.11 ± 2.60) ％,all P ＞ 0.05].The IFN-γ expression rates in the Aspergillus fumigatus spores direct and indirect contacting groups were (14.33 ± 0.98) ％ and (14.97 ± 1.53) ％,which were not of significant difference compared with the blank group (P ＞ 0.05).The IL-4 expression rates in NK cells with direct contacting to different morphotypes of Aspergillus fumigatus and Candida albicans were (1.25 ± 0.06) ％,(1.21 ± 0.03) ％,(1.22 ± 0.46) ％ and (1.26 ± 0.11) ％,while those in indirect contacting groups were (1.21 ± 0.06) ％,(1.25 ±0.04)％,(1.27 ±0.03)％ and (1.26 ±0.1)％,which were not of significant difference compared with the blank group [(1.23 ± 0.05) ％,all P ＞ 0.05].Conclusion Fungus stimuli can reduce the secretion of IFN-γ in NK cells,but have not significant influence on the secretion of IL-4.