Clinical evaluation of posaconazole for preventing invasive fungal disease in the aplastic anemia patients receiving transplantation / 中国感染与化疗杂志

Objective To evaluate the efficacy and safety of posaconazole for preventing invasive fungal disease (IFD) in the aplastic anemia patients receiving allogeneic hematopoietic stem cell transplantation (HSCT). Methods A total of 46 aplastic anemia patients received allogeneic HSCT. They were treated with oral posaconazole 200 mg, three times a day from HSCT pretreatment to granulocyte recovery after transplantation. G test and GM test were done 1, 2 and 4 weeks after the end of posaconazole treatment, and chest CT scan repeated 4 weeks after the end of posaconazole treatment. Posaconazole prophylaxis was defined as successful if there were no clinical manifestations indicative of fungal infection. Results All the 46 patients experienced neutropenia. The median of absolute neutrophil count (ANC) nadir was 0.02 (0-0.05)×109/L for a median time of 10 (8-19) days. The median duration of posaconazole prophylaxis was 26 (15-41) days. Neutropenic fever was reported in 45 patients, which lasted a median time of 5 (1-13) days. Six patients (13.3％ of the patients with neutropenic fever) failed to respond to the empirical treatment of broad spectrum antibiotics with persistent fever over 7 days. Their treatment was switched to short-term empirical treatment with broad spectrum antifungal agents. However, subsequent G test, GM test and chest CT showed negative results. None of the six patient was consistent with IFD diagnosis. Breakthrough fungal infection was not considered. Oral posaconazole solution was resumed for preventing IFD. G test, GM test and chest CT scan did not show any sign of fungal infection 1, 2 and 4 weeks after the end of posaconazole prophylactic treatment in all the 46 patients, proving the success of oral posaconazole in preventing IFD. Posaconazole was not discontinued due to adverse drug reaction in any patient. Conclusions Posaconazole is effective for preventing IFD in the aplastic anemia patients receiving HSCT with good safety profile and few adverse reactions.

Effect of LSD1 knock-down by small hairpin RNA on the apoptosis and cell cycle of human acute myelogenous leukemia cells / 实用医学杂志

Objective To investigate the effect of down-regulation of lysine specific demethylase 1 (LSD1) by shRNA on the apoptosis and cell cycle of human acute myelogenous leukemia cells.Methods The lentiviral vector-mediated LSD1-shRNA was transfected into human acute promyelocytic leukemia HL-60 cells and acute monocytic leukemia SHI-1 cells.The expressions of LSD1 mRNA and protein were examined by real time quantitative PCR and Western blot,respectively.The flow cytometry was applied to detect the apoptosis and cell cycle distribution after AnnexinV-PE/7-AAD and PI dying,respectively.Results The expressions of LSD1 mRNA and protein in HL-60 and SHI-1 LSD1-shRNA group were significantly decreased compared with the blank control group and the negative shRNA group (P ＜ 0.01,respectively).The apoptosis levels of HL-60 and SHI-1 cells were significantly increased after knockdown of LSD1 (P ＜ 0.01).Moreover,the cell cycle distribution in the G0/G1 phases was also significantly increased(P ＜ 0.01).Conclusion LSDI-shRNA promotes cell apoptosis and increases the percentage of cells in the G0/G1 phases of human acute myelogenous leukemia cells.

Prediction of the target genes mediated by RYBP in the leukemic cell apoptosis / 实用医学杂志

Objective To investigate the target genes in RYBP-mediated leukemia cell apoptosis by high-flux sequencing. Methods The HL-60 cell line with knockdown of RYBP was set up. MRNA and microRNA sequencing was conducted by the next-generation sequencing technology. Result MRNAs and miRNAs were dys-regulated in HL-60 cell line with knockdown of RYBP. The results of KEGG analysis indicated that the down-regu-lated genes were associated with cancer transcription regulation and amino acid metabolism. SPI1 and miR-214-3p were shown downregulated after knockdown of RYBP. Conclusion The target genes in RYBP-mediated leukemia cell apoptosis include BBC3,BAI1,SESN2 ,CCNG2,JAK3,STAT4,SPI1,BCL6,CD11b and hsa-miR-214-3p.

The effects of shRNA-mediated interference on histone lysine specific demethylase 1 on cell proliferation and apoptosis in acute leukemia cell lines HL-60 and SHI-1 / 白血病·淋巴瘤

Objective To investigate the effects of shRNA interference (RNAi) targeting the histone lysine specific demethylase 1 (LSD1) on the proliferation and apoptosis in acute leukemia (AL) cells. Methods LSD1 shRNA vectors were constructed and transduced into HL-60 and SHI-1 AL cell lines. Inhibitory efficiency of LSD1 was detected by real-time quantitative PCR (RT-qPCR) and Western blot respectively. Cell proliferation was assessed by methyl thiazolyl tetrazolium (MTT), and cell apoptosis was measured by flow cytometry. Results After interference of LSD1, the expression levels of LSD1 mRNA and protein in HL-60 and SHI-1 cells (mRNA: 0.242 ±0.023, 0.207 ±0.006; Protein: 0.256 ±0.015, 0.486 ±0.042) were decreased compared with blank control group without transfection process (mRNA: 1.021 ±0.178, 1.039 ±0.395; Protein:0.552 ±0.026, 0.754 ±0.060) and empty vector negative control group (shNC group) (mRNA: 0.935 ±0.136, 1.016±0.203;Protein: 0.500±0.026, 0.750±0.049) (P<0.05). The levels of their cell proliferation (absorbance value: 0.712±0.010, 0.549±0.007) were inhibited compared with blank control group (absorbance value:0.823±0.010, 0.625±0.005) and shNC group(absorbance value: 0.818±0.019, 0.621±0.003) (P< 0.05). While cell apoptosis rates were increased [(32.80 ±1.35) %, (23.49 ±1.40) %] compared with blank control group [(8.08 ±0.62) %, (7.28 ±1.17) %] and shNC group [(8.08 ±0.62) %, (7.28 ±1.17) %] (P< 0.05). Conclusions Lentivirus-mediated shRNA interferencing LSD1 can inhibit cells ' proliferation and promote apoptosis of HL-60 and SHI-1 AL cell lines, indicating that LSD1 may be a potential biological molecular marker and a new treatment target for AL.

Calibration of false positive result in detection of BCR/ABL using fluorescence in situ hybridization / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To explore the effect of false positive signals during detection of BCR/ABL fusion gene by fluorescence in situ hybridization (FISH), and develop a method for calibration.</p><p><b>METHODS</b>Normal specimens were mixed with BCR/ABL positive specimens in which presented signal pattern of 1-red-2-green-1-fusion (1R2G1F) using dual color dual fusion (DCDF) probes and 1-red-1-green-1-fusion (1R1G1F) using extra signal (ES) probes in different proportions. Mixed samples were detected using DCDF and ES probes. Results of DCDF probes, ES probe before calibration, ES probes after calibration and theoretical results were compared by binomial distribution in different proportions.</p><p><b>RESULTS</b>The rate of false positive signals has risen with increase of negative rate. A significant difference was found between theoretical proportion and results without calibration in negative level, 5%, 10% and 25% positive level (P<0.05). There was no significant difference between theoretical proportion and results without calibration in 50% and 90% positive level (P>0.05). Also there was no significant difference between theoretical proportion and calibrated results (P>0.05).</p><p><b>CONCLUSION</b>Calibration of FISH result can delimitate the effect of false positives, and can provide more reliable results in cases with low level positive rates.</p>

Immunophenotypic analysis and identification of hematogones and acute B lymphoblastic cells by flow cytometry / 白血病·淋巴瘤

Objective To discriminate morphology and immunophenotype differences between hematogones and lymphoblast to provide some references for the correct identification of hematogones and minimal residual leukemia cells.Methods Immunophenotypes were detected by flow cytometry in a total of 132 bone marrow from 58 patients with acute B lymphoblastic leukemia during diagnosis,remission and relapse.Hematogones were identified based on combination of CD34/CD10/CD19/CD45 or CD34/CD10/CD45/CD19/CD20/CD38.Results Among 132 specimens,45 (34 ％) were identified hematogones,the detection range was 0-36 ％.Three specimens appeared in diagnosis patients,one in relapse,and the remaining 41 cases in remission.The detection rate of hematogones was 62 ％ (41/66) in the remission cases.More than 5 ％ leukemia cells of nucleated cells were detected in diagnosis and relapse,and less than 5 ％ residual leukemia cells was in 24 specimens from remission patients.In 28 specimens,the co-existence of hematogones and leukemia cells was found,including three in diagnosis,one in relapse and the remaining 24 in remission.Hematogones were characterized in term of variable expression of CD45 and very low side scatter.The early hematogones expressed CD34.With maturation increasing,hematogones gradually lacked CD34.CD19 and CD10 were presented in whole hematogones stage.Early hematogones had expression of CD10.Lymphoblasts showed maturation arrest and more homogeneous populations.SSC values of hematogones were higher than that of normal B cell progenitors.Antigen overexpression or underexpression was not found in normal hematopoietic progenitor B cells,and hematopoietic progenitor B cells did not appear cross-lineage markers,CD20+ cells exhibited continuous distribution from negative to weak positive for normal hematogones.Conclusions Hematogones were present in diagnosis,remission and relapse cases with acute B lymphoblastic leukemia,especially abundant in bone marrow after chemotherapy.It should be careful to diagnose and discriminate the malignant cells from benign cells.By comprehending continuous and complete maturation spectrum of antigen expression for normal hematogones,knowing phenotype of leukemia cells drift change patterns and using multiparameter flow cytometry and optimal antibody combination,it is significant in identifying residual lymphoblasts from hematogones and improving the detection accuracy in minimal residual disease.

Efficacy and safety of 0.05％ cyclosporine A ophthalmic emulsion in treatment of dry eye / 中华实验眼科杂志

Background Dry eye is a common disease worldwide.Cyclosporine A(CsA) is provided to be a immunosuppressive agent and is effective on dry eye.But in China,0.05％ CsA is not yet applied in dry eye treatment.Objective This study was to evaluate the efficacy and safety of 0.05％ CsA eye drops in the treatment of dry eye.Methods This was a randomized,double-blind,vehicle-controlled parallel group study.Forty eyes of 40 patients with moderate to severe dry eye were randomly divided into two groups,with the corresponding treatment of 0.05％ CsA eye drops or the vehicle emulsion.The patients in both the groups received non-preserved artificial tear.Symptoms and signs were observed before administration,(7±1),(28±2),(56±3),and (84±3) days and also 14 days after withdrawal.The clinical effective rate was considered as the primary outcome.The subjective assessment of the patients including total symptom scores and ocular surface disease index (OSDI) scores,Schirmer Ⅰ test (S Ⅰ t) with topical anaesthesia,tear film breakup time (BUT),rose Bengal and fluorescein staining scores were evaluated.The safety profile was evaluated by adverse events,visual acuity and ocular tolerance.Results At the end of this trial,the ocular symptoms scores,conjunctival hyperemia,BUT,S Ⅰ t and keratoconjunctiva staining scores of the two groups had statistically significant difference.The total effective rate of 0.05％ CsA treatment group was 75％ (15/20) and vehicle group was 25％ (5/20).There was a statistically significant difference between groups (P =0.000),and the 95％ confidence interval (C1) of the difference value of total effectiveness between the two groups was 30.80％-53.75％.At the end of this trial,there was no statistically significant difference in visual acuity distribution (P =0.890).No obvious discomfort was found in the patients received 0.05％ CsA eye drops.There were no adverse events during the follow-up duration.Conclusions 0.05％ CsA ophthalmic emulsion is an effective and safe treatment for dry eyes.

Influence of different types of corneal laser refractive surgeries on corneal hysteresis and corneal resistance factor in myopic eyes / 中华实验眼科杂志

Background Refractive surgery has propelled itself forward to become widely performed surgical procedure nowadays.After the surgery,corneal biomechanics decreases lead to keratoconus and corneal ectasia.Doctors pay more attention to biomechanics changes after refractive surgeries.Objective This clinical study was to investigate the influence of different laser refractive surgeries on corneal biomechanics.Methods A prospective nonrandomized and controlled clinical study was designed.One hundred and sixty-four eyes of 82 patients with moderate myopia were enrolled.The patients were divided into sub-Bowman keratomileusis (SBK) group (60 eyes of 30 patients),laser in situ keratomileusis (LASIK) group (54 eyes of 27 patients) and laser subepithelial keratomileusis (LASEK) group (50 eyes of 25 patients),with the matched demography among the three groups.Corneal hysteresis (CH) and corneal resistance factor (CRF) were detected by ocular response analyzer (ORA) before and 1 week,5 months after refractive surgery.The correlations between stromal ablation depth and postoperative changing values of CH or CRF were analyzed.Results Significant differences were found in CH and CRF at different time points in the three groups (Ftime =41.90,P =0.00;Ftime =49.65,P =0.00),and the CH and CRF values were significantly lower 1 week and 5 months after surgery than those before surgery (all at P =0.00).However,no significant difference of CH or CRF was seen at all time points among the three groups (Fgroup =2.17,P =0.08;Fsroup =2.67,P =0.07).No correlation was found between corneal ablation depth and CH in 1 week and 5 months after surgery (both at R2 =0.000),however,weaker correlations were seen between corneal ablation depth and CRF 1 week and5 months after surgery (Y=3.253+ 0.010X,R2=0.007;Y=1.073+0.021 X,R2=0.004).Conclusions SBK,LASIK and LASEK lead to the change of corneal biomechanics by altering CH and CRF,they play the same influence on cornea.CRF appears to be an useful indicator in evaluating corneal biomechanical changes after laser refractive surgery.

Immunophenotypic analysis of multiparametric flow cytometric in multiple myeloma and minimal residual disease / 国际检验医学杂志

Objective To investigate the detectable significance of multiparameter flow cytometry (MFC) for the first visiting and minimal residual disease (MRD) in the patients with multiple myeloma .Methods MFC was used to identify the plasma cells by the expression of CD138 or CD38 antigen in 74 patients with multiple myeloma .By combining surface antigens like CD45 ,CD56 , CD19 ,CD20 ,CD117 and the cytoplasm Kappa and Lambda light chain ,the aberrant myeloma cells were differentiated from normal plasma cells .Results In the 44 first visiting cases ,the positive expression of CD138 can be detected in all cases ,while the expres‐sion of CD19 was negative and 42 cases (95% ) were negative or weak positive expression for CD45 .The detection rates of CD38 , CD56 ,CD20 and CD117 were 98% ,93% ,45% and 41% ,respectively .The cytoplasm Kappa and Lambda light chains were showed the limited expression .Of the patients with MM ,14 cases were used for evaluating the change of immunophenotype at first visiting and during the treatment process ,among them ,11 cases(79% ) appeared the changes in at least one of aberrant phenotypes .4 cases (29% ) had the significant enhancement of antigen marker fluorescence intensities after chemotherapy and 7 cases (50% ) had sig‐nificant decrease of antigen marker fluorescence intensities after chemotherapy .CD45 ,CD19 and cytoplasm immunoglobulin light chains were the most stable marker ,no obvious antigen marker changes were found during the treatment ,while there was a signifi‐cant antigen density change in 2 cases of CD38 (14% ) ,7 cases of CD56 (50% ) ,4 cases of CD20 (29% ) and 2 cases of CD117 (14% ) .Of the 30 cases for evaluating MRD immunophenotype ,the abnormal myeloma cells were detected in 25 cases .In 5 cases ,no expression of limited Kappa and Lambda light chains was found and the ratio of Kappa and Lambda was 0 .5 - 2 ,which were identi‐fied as negative for MRD .Conclusion The multiparameter flow cytometry has important significance in evaluating the diagnosis , therapeutical effect and prognosis .The detection by adopting cytoplasm immunoglobulin light chains can improve the accuracy in MRD detection .

Establishment of a HL-60 cell line with stable RYBP silencing / 白血病·淋巴瘤

Objective To establish stable HL-60 cell line with stable RYBP gene silencing using lentivirus-mediated RNA interference. Methods Five special shRNAs for RYBP gene were cloned to lentivirus vector.Recombinate lentivirus vectors were packed into lentivirus,which were used to infect HL-60 cells, and took empty vector and non-specific shRNA as control groups. Stable infected cells were selected with puromycin in 8 μg/ml concentration.The expression levels of RYBP were analyzed by Western blot,and confirmed the most effective RYBP shRNA.Then the level of mRNA was analyzed by real-time PCR.Results Stable infected cells were selected by puromycin successfully.Comparing to control groups,the expression of RYBP were reduced at different degrees (P ＜ 0.01). And RYBP shRNA2 took the most silencing effect, the RYBP mRNA was decreased by more than 95 ％ (P ＜ 0.05).Conclusion The shRNA2 targeting RYBP gene can effectively inhibit the expression of RYBP. HL-60 cell line with stable RYBP gene silencing were constructed successfully,which had provided experiment fundament for further studying the function of RYBP.

Modification of histone acetylation and its regulation effect to the expression of mismatch repair genes in acute leukemia / 白血病·淋巴瘤

Objective To explore the status of histone acetylation modification and their regulatory effect to hMSH2 gene and hMLH1 gene expression in acute leukemia. Methods Reverse transcription-polymerase chain reaction (RT-PCR) was used to measure the expression of hMSH2 and hMLH1 mRNA, and Western blot was used to measure the expression of histone H3, H4, HDACi, hMSH2 and hMLH1 protein in mononuclear cells of 56 acute leukemia patients and 30 healthy volunteers. The mononuclear cells of 30 acute leukemia patients were treated with histone deacetylase inhibitors trichostatin A (TSA), and measured the expression difference of histone H3, H4, HDAC1, hMSH2 and hMLH1 in the mononuclear cells treated with TSA. Results The protein expression levels of hMSH2, hMLH1, histone H3 and histone H4 in those mononuclear cells of acute leukemia patients were 0.4610±0.1211, 0.4013±0.1143, 0.4103±0.1241 and 0.4251±0.1081, respectively, which were significantly decreased comparing with those of healthy volunteers (0.9461±0. 1841, 0.996±0.2021, 0.8971±0. 1194 and 0.9513±0.1953) (t = 3.341, 3.935, 2.843 and 3.575,respectinely, P ＜0.05). The protein expression levels of HDAC1 (0.8841±0.2018) of acute leukemia patients was significantly increased comparing with those of healthy volunteers (0.5142±0.1340) (t= 2.634, P ＜0.05).After treatment with TSA for 48 hours, the protein expression of hMSH2 was increased nearly 1.5-fold, hMLH1 about 1.6-fold, H3 about 2.9-fold and H4 about 3.4-fold comparing with the negative control groups (P ＜0.05),while the protein expression of HDAC1 were decreased comparing with the negative control groups by 40 %.Conclusion There was an low expression phenomenon of histone acetylation in acute leukemia, and histone acetylation played an important role in regulation of the mismatch repair gene expression in acute leukemia.

Sibling and unrelated allogeneic hematopoietic stem cell transplantation for multiple myeloma patients under 45-year-old at early stage / 白血病·淋巴瘤

Objective To evaluate the effectiveness and side effects in sibling and unrelated HLA identical allogeneic hematopoietic stem cell transplantation for multiple myeloma patients under 45-year-old at early stage. Methods Three patients with multiple myeloma ranged from 38 to 44-year-old received two courses of chemotherapies and achieved partial remission. Sibling HLA identical allogeneic hematopoietic stem cell transplantations were underwent in case 1 and 2, and unrelated were in case 3. The conditioning regimens for case 1 and 2 included fludarabine, busulfan plus cyclophosphamide, and of case 3 included modified busulfan, cyclophosphamide plus antithymocyte globulin. Cycloporine A combined with methotrexate were used to prevent GVHD in the case 1 and 2, and methotrexate, mycophenolate and cycloporine A were used in case 3. Results All patients achieved full donor chimerism without graft failure. Grade Ⅱ acute GVHD and extensive chronic GVHD were found in case 1, but not in case 2 and 3. The period of follow-up of case 1, 2 and 3 were 48, 27 and 6 months, respectively, and all of them were alive with no signs of relapse. Conclusion The multiple myeloma patients under 45-year-old underwent sibling and unrelated HLA identical allogeneic hematopoietic stem cell transplantation at early stage after chemotherapy remission have the low treatment-related mortality, high complete remission rate and may prolong long-term survival.

Different transplantation strategies for acute lymphoblastic leukemia in adult patients:Preliminary observations / 中国组织工程研究

AIM:Allogeneic hematopoietic stem cell transplantation for treatment of acute lymphoblastic leukemia characterized with high rate of relapse,especially in Ph+ patients.Presently,researchers focus on how to resolve relapse.Transplantation time,transplantation schedule and adoptive immunotherapy after transplantation are important.The study was performed to preliminarily observe treatment effectiveness after myeloablative stem cell transplantation,non-myeloablative transplantation after donor lymphocyte infusion and non-myeloablative transplantation followed by low-doses of Cyclosporin A.METHODS:Five patients were admitted at Department of Haematology of First People's Hospital between December 1998 and May 2007.Acute lymphoblastic leukemia patients were informed consent for allogeneic hematopoietic stem cell transplantation.The experiment was approved by hospital ethics committee.Among them,one patient was used the traditional preconditioning of busulfan and cyclophosphamide.Four cases were performed with non-myeloablative hematopoietic stem cell transplantation,and one of them was treated with reduced intensity regimen based on anti-thymocyte globulin donor lymphocyte infusion after transplantation;Three cases with fludarabine-based non-myeloablative transplantation were used low-dose Cyclosporin A after engraftment.Graft-versus-host disease prevention regimen was consisted of short-range methotrexate combined with Cyclosporin A.Haematopoiesis,chimerism,graft-versus-host disease and infection were observed after transplantation.RESULTS:All patients achieved successful engraftment.①One patient with mixed chimerism received eight donor lymphocyte infusion based on anti-thymocyte globulin-non-myeloablative transplantation and gradually converted into full donor chimerism with disease-free survival,and complicated acute graft-versus-host disease of skin and liver.②Three patients achieved full donor chimerism based on fuladarabine-non-myeloablative transplantation,one patient relapsed without graft-versus-host disease,and other two cases eliminated BCR/ABL fusion gene-positive cells with acute and chronic graft-versus-host disease.③One case after myeloablative transplantation relapsed and complicated with acute and chronic graft-versus-host disease.CONCLUSION:①The traditional,anti-thymocyte globulin or fludarabine-based non-myeloablative conditioning for transplantation in the treatment of adults with acute leukemia will be eligible for the successful implantation,and adoptive immunotherapy have graft-versus-leukemia effect.②The efficacy and complications of three transplantation strategies should be further studied.

Transplantation of non-myeloablative double umbilical cord blood units for severe aplastic anemia in eight adult patients / 中国组织工程研究

Eight patients with severe aplastic anemia received unrelated cord blood transplantation at the Guangzhou First People’s Hospital from June 1998 to December 2007. The patients were conditioned with decreased dosage of immunosuppressive agents of cyclophosphamide and antilymphocytic globulin. The median infused donor total nucleated cell were 5.69?10 7/kg of recipient weight, and the CD34+ cell was 4.10?105/kg of recipient weight. Methotrexate and corticoid methylprednisolone were used for prophylaxis of graft versus host disease (GVHD). The time to reach an absolute neutrophil count of 0.5?109/L ranged from 7 to 25 days (median: 17 days) and the time to reach a platelet count of 20?109/L ranged from 13 to 102 days (median: 35 days) after transplantation. DNA finger print map of 7 patients showed donor and recipient chimera. Two patients developed grade I to II acute GVHD, which was controlled. One patients developed grade II chronic GVHD, which was controlled by using methylprednisolone. Five patients had lived for 10-108 months, with no diseases. Taken together, unrelated umbilical cord blood transplantation is effective for adult patients. Partial conditioning regiment could ensure engraft of unrelated umbilical cord blood transplantation.

Clinical study on graft-versus-host disease post non-myeloablative stem cell transplantation for haematologic diseases / 中华器官移植杂志

Objective To explore clinical features of graft-versus-host disease (GVHD) after allogeneic non-myeloablative stem cell transplantation (NST) for haematologic diseases.Methods Eighteen patients were divided into three groups. Group A: Six severe aplastic anemia (SAA) adult patients underwent unrelated umbilical cord blood transplantation (UCBT). Group B: Combined transplantation of G-CSF primed allogeneic bone marrow and peripheral blood stem cells (PBSCs) was performed for 5 SAA patients. Group C: Seven malignant haematologic patients underwent transplantation of bone marrow cells for 3 patients or PBSCs for 4 patients. The protocol consisted of nonmyeloablative conditioning regimens based on anti-themocyte globulin (ATG) or anti-T-lymphocyte globulin (ALG). GVHD prophylaxis consisted of cyclosprine (CSA) and methylprednisolone (MP) for groups A and B, and methotrexate and CSA for group C. Mixed chimerism (MC) patients in group C were subjected to donor lymphocyte infusion (DLI).Results Four patients in group A achieved and sustained MC status, among them, one patient died of fungal septemia and one patient left hospital voluntarily. Three patients in group B achieved short period MC with donor chimerism more than 94 % at early stage post transplantation and converted into full donor chimerism (FDC) with long-term disease-free survival (DFS) and one patient developed chronic GVHD (cGVHD) 8 month post- trasplantation . Another two patients receiving donor stem cell infusion (DSI), one died of secondary mediastina lymphoma after 6 months and one patient recovered haematopoiesis. All patients achieved MC with haematologic partial remission (PR), and did not complicated acute GVHD (aGVHD) prior to DLI. Two cases died of severe infection and lost follow-up respectively. Another 5 patients gradually converted into FDC and achieved haematologic complete remission after 4, 3, 7, 5 and 4 DLIs, but they developed cGVHD (n=4), aGVHD (n=2) and myelosurppression (n=2).Conclusion The treatment of NST for SAA patients achieved better clinical effect with lower incidence of GVHD, and characterized by lower incidence of aGVHD and early mortality and higher incidence of cGVHD and infection for malignant haematologic diseases.

Donor lymphocyte infusion after nonmyeloablative stem cell transplantation in patients with hematologic malignancy / 中华器官移植杂志

Objective To evaluate the application of donor lymphocyte infusion (DLI) after nonmyeloablative stem cell transplantation (NSCT) for hematologic malignancies. Methods Donor lymphocyte infusion was performed on 5 hematologic malignant patients with mixed chimera (MC) and hemato logic partial remission of case 1, 2, 3, 4 or progression of case 5 after NSCT. The patients received the first DLI on the 4th to 5 th week posttransplant. The first T cell dose of ( 0.5 ～ 1.0 )?10 5/kg was followed by the escalated doses to the range of ( 0.5 ～ 2.0 )?10 8/kg. The total of procedures were performed at an average of 4.6 procedures (range 3～8) at intervals of 3～4 weeks. Results The MC was converted gradually into complete chimera (CC) after DLI in case 1, 2, 3 and 4 who were subjected to 7, 2, 3, 3 procedures respectively, and eventually converted into hematologic complete remission (CR), while case 5 remained MC and progression. The micro residual disease (MRD) of case 2 and 3 disappeared after DLI. Grade Ⅰ/Ⅱ acute graft versus host disease (aGVHD) in case 1, 2 and extensive/limited chronic graft versus host disease (cGVHD) in case 3 and 4 were found, and myelosuppression in case 2 and 4 was found as well. Conclusion Transient mixed donor recipient hematopoietic cell MC may be successfully converted into complete CC by DLI post transplant, and DLI can eliminate MRD. GVHD and myelosuppression remains major complications of DLI.

Partial conditioning regimen for stable mixed chemerism after unrelated cord blood transplantation in adult patients with severe aplastic anemia / 中华器官移植杂志

Objective To study and observe the engraftment of donor cells from unrelated cord blood into adult patients with severe aplastic anemia (SAA) and the outcome of allo-CBSCT. Methods Six patients received cord blood provided by Guangzhou Cord Blood Bank, for three of which one unit of cord blood was given in a procedure, whereas for other 3 patients, 2 units of cord blood were infused at the same time in a transplant protocol. In all 9 units of umbilical cord blood (UCB) infused, UCB units contained 1.6 ～ 10.7 nucleated cells/kg body weight of the recipient after thawing. The patients were conditioned with decreased dosage of immunosuppressive agents of CTX (60?mg/kg) and ALG (120?mg/kg). The engraftment state of the donor cells into recipients was confirmed by microsatellite DNA fingerprinting and fluorescent quantitative PCR analysis. Results Engrafted evidence has been found in 5 patients by molecular biology analyses showing donor-recipient mixed chimerism post transplant which was stable and persistent. After a median follow-up of 21 months (range 7～50), 4 patients were alive and disease free. One patient died of severe infection in the 3rd month from transplant, though the evidence of engraftment was obvious. Another patient also died in the early stage posttransplant of serious aspergillus infection without the engrafted proof.Conclusion Durable donor-recipient stable mixed chimerism can be achieved by unrelated UCBT in patients with SAA. Umbilical cord blood could be employed as a source of hematopoietic stem cell for adult transplantation.

Anti-T-lymphocyte globulin based non-myeloablative stem cell transplantation followed by HLA-identical donor lymphocyte infusion for hematologic malignancies / 中华医学杂志(英文版)

<p><b>OBJECTIVE</b>To evaluate the application of anti-T-lymphocyte globulin (ATG) based nonmyeloablative but profoundly immunosuppressive regimens followed by donor lymphocyte infusion (DLI) for the treatment of hematologic malignancies.</p><p><b>METHODS</b>The protocol was designed to minimize the intensity of the conditioning regimen to the range of nonmyeloablative therapies based on ATG with low-dose busulfan (Bu) and Cytoxan (CTX) (15 - 19.5 mg/kg, 8 mg/kg and 80 mg/kg, respectively). The patients received the first lymphocytic infusion from HLA-identical sibling donors on days 28 - 30 after transplant, and the first T cell dosage of 10(6)/kg followed by the escalated dosage in the range of (0.5 - 1.5) x 10(8)/kg. The total number of procedures were performed at a median of 4.2 procedures (range of 2 - 8 procedures).</p><p><b>RESULTS</b>Engraftment was documented in all six patients in the form of donor-recipient hematopoietic cells mixed chimera at early-stage posttransplant, which was converted gradually into complete chimera by DLI in four patients. Graft-versus-host disease (GVHD) developed in three of six cases, only one of which was severe. To date, four patients are disease free and alive.</p><p><b>CONCLUSIONS</b>Allogeneic donor stem cell engraftment into host can be achieved by nonmyeloablative conditioning regimen based on ATG. Transient mixed donor-recipient hematopoietic cell mixed with chimeras may be successfully converted into complete chimerism by DLI posttransplant. GVHD remains major clinical concern in our study.</p>

Clinical application of antithymocyte globulin and antilymphocyte globulin in nonmyeloablative stem cell transplantation for hematologic disease / 中国医师杂志

Objective To evaluate the effectiveness and side-effects of antithymocyte globulin(ATG) and antilymphocyte globulin(ALG) in nonmyeloablative stem cell on transplantation complication.Methods Fourteen cases of hematologic malignancies and 11 cases of sever aplastic anemia(SAA) were treated with allogenic bone marrow transplantation or cord blood haemopoietic stem cell transplantation based on ATG/ALG.Five patients with malignant hematonosis were received donor lymphocyte infusion(DLI) after transplantation.The protocols for graft-versus-host disease(GVHD) prophylaxis consisted of Cyclosporin A(CSA) and methotrexate(MTX) for malignant hematonosis patients or CSA and methylprednisolone(MP) for patients with SAA.Results Three patients had not evidence of engraftment and died from infection at early stage.Other patients recovered haematopoiesis.The mean time of ANC more than 0.5?10~9/L and Plt more than 20?10~9/L were 12.1(3～29) and 20.1(5～79) days posttransplant respectively.Three patients achieved donor complete chimera(CC).Five malignant patients with transient mixed chimerism(MC) grdually converted into complete chimerism by DLI post transplant.Nineteen patients achieved MC and four of them coverted into donor haematopoietic cell complete chimerism.There was no aGVHD in early stage post transplant.There were 1 patient with Ⅰgrade aGVHD,3 with Ⅱgrade aGVHD,2 with skin local cGVHD and 2 with extensive cGVHD after DLI,respectively.There were 2 patients with bacteria infection complications,4 with virous infection and 5 with fungal infection.All patients complicated with chills and fever in the use of ATG and ALG.Conclusion Treatment with ATG and ALG is safety and tolerant for the patients,and can enhance the engraftment of haemopoietic stem cell and decrease the aGVHD.