[The Study of Recombinant Interfering Lentiviruses and Overexpressed Adenovirus Vectors Targeting Human c-Cbl Gene： Construction, Identification and Efficacy].

OBJECTIVE: To construct recombinant lentivirus and adenovirus which regulate the expression of c-Cbl gene and evaluate their efficacy. METHODS: The interference lentivirus and overexpressed adenovirus targeting human c-Cbl gene were constructed by gene recombination technology. Quantitative PCR and western blotting were used to detect the expression changes in c-Cbl gene and its transcription after leukemia cells (HL60,THP1) were infected by virus. RESULTS: Three recombinant interfering lentiviral vectors targeting human c-Cbl genes to successfully constructed and were identified by DNA sequencing, and the titers of the packaged viruses were all greater than 1×108 TU/ml. Among them, shRNA-2 lentivirus had the highest interference efficiency, and the expression of c-Cbl gene and CBL protein were decreased about 95% and 60% respectively after leukemia cells were infected with shRNA-2; In addition, the recombinant overexpression adenovirus targeting human c-Cbl gene was packaged successfully with the virus titer greater than 1×109 TU/ml. When leukemia cells were infected with adenovirus, the expression of c-Cbl gene and CBL protein were up-regulated about 10 times and 1.5 times respectively. CONCLUSION: Both recombinant interfering lentivirus and overexpression adenovirus can efficiently infect leukemia cells and affect the expressions of c-Cbl gene and CBL protein. It will lay a preliminary foundation for the subsequent study on the function of c-Cbl gene in tumor cells.

miR-19a/b-3p promotes inflammation during cerebral ischemia/reperfusion injury via SIRT1/FoxO3/SPHK1 pathway.

BACKGROUND: Stroke affects 3-4% of adults and kills numerous people each year. Recovering blood flow with minimal reperfusion-induced injury is crucial. However, the mechanisms underlying reperfusion-induced injury, particularly inflammation, are not well understood. Here, we investigated the function of miR-19a/b-3p/SIRT1/FoxO3/SPHK1 axis in ischemia/reperfusion (I/R). METHODS: MCAO (middle cerebral artery occlusion) reperfusion rat model was used as the in vivo model of I/R. Cultured neuronal cells subjected to OGD/R (oxygen glucose deprivation/reperfusion) were used as the in vitro model of I/R. MTT assay was used to assess cell viability and TUNEL staining was used to measure cell apoptosis. H&E staining was employed to examine cell morphology. qRT-PCR and western blot were performed to determine levels of miR-19a/b-3p, SIRT1, FoxO3, SPHK1, NF-κB p65, and cytokines like TNF-α, IL-6, and IL-1ß. EMSA and ChIP were performed to validate the interaction of FoxO3 with SPHK1 promoter. Dual luciferase assay and RIP were used to verify the binding of miR-19a/b-3p with SIRT1 mRNA. RESULTS: miR-19a/b-3p, FoxO3, SPHK1, NF-κB p65, and cytokines were elevated while SIRT1 was reduced in brain tissues following MCAO/reperfusion or in cells upon OGD/R. Knockdown of SPHK1 or FoxO3 suppressed I/R-induced inflammation and cell death. Furthermore, knockdown of FoxO3 reversed the effects of SIRT1 knockdown. Inhibition of the miR-19a/b-3p suppressed inflammation and this suppression was blocked by SIRT1 knockdown. FoxO3 bound SPHK1 promoter and activated its transcription. miR-19a/b-3p directly targeted SIRT1 mRNA. CONCLUSION: miR-19a/b-3p promotes inflammatory responses during I/R via targeting SIRT1/FoxO3/SPHK1 axis.

An Immune Risk Score Predicts Survival of Patients with Acute Myeloid Leukemia Receiving Chemotherapy.

PURPOSE: Prediction models for acute myeloid leukemia (AML) are useful, but have considerable inaccuracy and imprecision. No current model includes covariates related to immune cells in the AML microenvironment. Here, an immune risk score was explored to predict the survival of patients with AML. EXPERIMENTAL DESIGN: We evaluated the predictive accuracy of several in silico algorithms for immune composition in AML based on a reference of multi-parameter flow cytometry. CIBERSORTx was chosen to enumerate immune cells from public datasets and develop an immune risk score for survival in a training cohort using least absolute shrinkage and selection operator Cox regression model. RESULTS: Six flow cytometry-validated immune cell features were informative. The model had high predictive accuracy in the training and four external validation cohorts. Subjects in the training cohort with low scores had prolonged survival compared with subjects with high scores, with 5-year survival rates of 46% versus 19% (P < 0.001). Parallel survival rates in validation cohorts-1, -2, -3, and -4 were 46% versus 6% (P < 0.001), 44% versus 18% (P = 0.041), 44% versus 24% (P = 0.004), and 62% versus 32% (P < 0.001). Gene set enrichment analysis indicated significant enrichment of immune relation pathways in the low-score cohort. In multivariable analyses, high-risk score independently predicted shorter survival with HRs of 1.45 (P = 0.005), 2.12 (P = 0.004), 2.02 (P = 0.034), 1.66 (P = 0.019), and 1.59 (P = 0.001) in the training and validation cohorts, respectively. CONCLUSIONS: Our immune risk score complements current AML prediction models.

[Role of allograft inflammatory factor-1 in regulating the proliferation, migration and apoptosis of colorectal cancer cells].

OBJECTIVE: To investigate the role of allograft inflammatory factor-1 (AIF-1) in colorectal cancer (CRC) progression and explore the possible mechanism. METHODS: The expression levels of AIF-1 in 70 CRC tissues and paired adjacent tissues were detected using immunohistochemistry and Western blotting, and the correlation of AIF-1 expression with the clinicopathological features of the patients was analyzed. In the CRC cell line SW480, the functional role of AIF-1 in regulating tumor progression was investigated by transfecting the cells with an AIF-1-overexpressing plasmid (AIF-1) and a negative control plasmid (NC). EdU proliferation assay and flow cytometry were used to assess the cell proliferation and cell cycle changes; Transwell migration assay and Annexin V-APC/7-AAD apoptosis assay kit were used to analyze the cell migration and apoptosis. The changes in the biological behaviors of the cells were observed after application of SB203580 to block the p38 MAPK pathway. The expression levels of CDK4, cyclin D1, P21, P27, MMP2, MMP9, Bax, Bcl2, Bcl-xl, p38 and p-p38 were detected using Western blotting. RESULTS: AIF-1 was down-regulated in CRC tissues compared with the adjacent normal tissues, and its expression level was positively correlated with lymph node metastasis (P=0.008), TNM stage (P=0.003) and tumor size (P=0.023). Overexpression of AIF-1 in SW480 cells significantly reduced EdU-positive cells and caused obvious cell cycle arrest in G1 phase (P<0.05). AIF-1 overexpression resulted in significantly lowered protein expressions of CDK4 and cyclin D1, enhanced expressions of P21 and P27, attenuated cell migration ability (P<0.001), and decreased protein levels of MMP2 and MMP9. AIF-1 overexpression also induced obvious apoptosis of SW480 cells (P<0.01), significantly increased the protein levels of Bax and p-p38, and decreased the protein levels of Bcl-2 and Bcl-xl; SB203580 significantly attenuated the apoptosis-inducing effect of AIF-1 overexpression. CONCLUSION: AIF-1 plays the role of a tumor suppressor in CRC by inhibiting cell proliferation, suppressing cell migration and inducing cell apoptosis. AIF-1 overexpression promotes the apoptosis of CRC cells by activating the p38 MAPK pathway.

Sunitinib Induces NK-κB-dependent NKG2D Ligand Expression in Nasopharyngeal Carcinoma and Hepatoma Cells.

Multitargeted tyrosine kinase inhibitors (MTKIs) have been shown to combine with natural killer (NK) cell adoptive transfer for the treatment in various cancers. MTKIs sensitize cancer cells to NK cell therapy through upregulation of nature killer group 2 member D ligands (NKG2DLs) on tumor cells. However, the molecular mechanism of MTKIs-mediated upregulation of NKG2DLs is still unknown. In this study, we confirmed sunitinib induced downregulation of its targets, such as vascular endothelial growth factor, platelet-derived growth factor, and c-kit in multiple-drug-resistant nasopharyngeal carcinoma cell line CNE2/DDP and hepatoma cell line HepG2. Then, we further showed sunitinib induced cell proliferation inhibition, apoptosis, and DNA damage in CNE2/DDP and HepG2 cells. Coculture experiments showed that sunitinib-treated CNE2/DDP and HepG2 cells were able to increase the activation and cytotoxicity of NK cells. Quantitative polymerase chain reaction results showed that sunitinib upregulated NKG2DLs, apoptotic genes, DNA damage repair genes, and nuclear factor (NF)-κß family genes. Silencing of NF-κß1, NF-κß2, or RelB (NF-κß pathway) inhibited sunitinib-induced upregulation of NKG2DLs. Taken together, we concluded that sunitinib upregulated NKG2DLs through NF-κß signaling noncanonical pathway which might mediate higher cytotoxic sensitivity of CNE2/DDP and HepG2 cells to NK cells.

[Clinical Efficacy of Sorafenib Combined with Low Dose Cytarabine for Treating Patients with FLT3+ Relapsed and Refractory Acute Myeloid Leukemia].

OBJECTIVE: To study the efficacy and safety of sorafenib combined with low dose cytarabine for treating patients with FLT3(+) relapsed and refractory acute myeloid leukemia (FLT3(+) RR-AML). METHODS: Seven patients with FLT3(+) RR-AML were treated with sorafenib and low dose cytarabine. The curative rate and adverse effects were observed in these patients. RESULTS: Out of 7 RR-AML patients after treatment, 5 patients achieved complete remission (CR), 2 patients achieved partial remission (PR), and the overall response rate (ORR) after one course of therapy was 100%. No severe bleeding, nausea, vomiting and other side effects were found in these patients. CONCLUSION: Sorafenib combined with low dose cytarabine can effectively induce the remission of FLT3(+) RR-AML patients, and is worth for further clinical trails to verify its safty and efficiency.

[Clinical Summarization of Allogeneic Hematopoietic Stem Cell Transplantation for Leukemia: A Report of 100 Cases].

OBJECTIVE: To analyze the treatment outcome of a consecutive series of 100 leukemia patients received allogeneic hematopoietic stem cell transplantation (allo-HSCT). METHODS: The clinical data of leukemia patients received allo-HSCT were analyzed retrospectively, the therapeutic efficacy was summarized. 100 evaluable cases of leukemia included 47 cases of AML, 33 cases of ALL, 2 cases of AL (biphenotypic), 16 CML and 2 CMML. Before transplantation, 76 cases were in first complete remission, 9 cases in second or greater complete remission and 15 cases in non-remission or relapse. All the patients received peripheral blood hematopoietic stem cell transplantation (PBHSCT). The conditioning regimen of human leukocyte antigen (HLA)-matched allo-HSCT group was modified BuCy, but in HLA-mismatched group Fludarabine and anti-human thymocyte globulin (ATG) was added. CsA+MTX regimen was used for prophylaxis of graft-versus-host disease (GVHD) in HLA-identical allo-HSCT, while additional MMF was added in HLA-mismatched group. The average time of follow-up was 13 months. RESULTS: At the last follow-up, 66.0% (66/100) patients survived, 53.0% (53/100) patients survived without leukemia, 28.0% (28/100) patients relapsed and 34.0% (34/100) patients died, 44.1% patients of them died from infectious pulmonary complications. During transplantation, 65.0% of the patients were suffered from lung infection. The overall survival (OS) and disease-free survival (DFS) of all cases was 60.9% and 48.8%, respectively. The recurrence rate was significantly higher in non-remission (66.7%) than in CR (21.2%) patients (P < 0.05). The cumulative incidence of GVHD in HLA-mismatched transplantation was 60.8%, which was significantly higher than that of HLA-matched transplantation (38.8%) (P < 0.05). CONCLUSION: Allo-HSCT can cure a significant proportion of leukemia patients, especially for those in CR status. Since the incidence of infectious pulmonary complications after allo-HSCT is still high, much more attention should be paid to it. The comprehensive prognosis of HLA-matched transplantation is better than the HLA-mis-matched transplantation.

Anti-inflammatory Effect of Mesenchymal Stromal Cell Transplantation and Quercetin Treatment in a Rat Model of Experimental Cerebral Ischemia.

Here, we have investigated the synergistic effect of quercetin administration and transplantation of human umbilical cord mesenchymal stromal cells (HUMSCs) following middle cerebral artery occlusion in rat. Combining quercetin treatment with delayed transplantation of HUMSCs after local cerebral ischemia significantly (i) improved neurological functional recovery; (ii) reduced proinflammatory cytokines (interleukin(IL)-1ß and IL-6), increased anti-inflammatory cytokines (IL-4, IL-10, and transforming growth factor-ß1), and reduced ED-1 positive areas; (iii) inhibited cell apoptosis (caspase-3 expression); and (iv) improved the survival rate of HUMSCs in the injury site. Altogether, our results demonstrate that combined HUMSC transplantation and quercetin treatment is a potential strategy for reducing secondary damage and promoting functional recovery following cerebral ischemia.

Overexpression of EPS8 is associated with poor prognosis in patients with acute lymphoblastic leukemia.

Molecular markers have become an invaluable tool in monitoring disease status particularly of leukemias, as bone marrow samples can be easily collected for analysis during all stages of disease development including diagnosis, treatment, and follow-up. Two genes that have been used as prognostic markers in acute leukemia are Wilms' tumor (WT1) and multidrug resistance-1 (MDR1). A novel gene, epidermal growth factor receptor pathway substrate 8 (EPS8), is often over-expressed and associated with poor outcome in some solid tumor types. However, whether EPS8 is also associated with the development of acute lymphoblastic leukemia (ALL) is unclear. Here, quantitative real-time PCR was used to evaluate the expression of EPS8, MDR1, and WT1 in bone marrow samples of adult ALL patients (n=107) and non-leukemia controls (n=22). EPS8, MDR1, and WT1 were detected in ALL patients, and significant correlations were found between expression profiles for EPS8 and MDR1, EPS8 and WT1, and MDR1 and WT1. In general, high expression of EPS8, MDR1, or WT1 in patients was associated with a higher risk of relapse. Furthermore, when patients were stratified based on high or low expression of the genes, Kaplan-Meier survival analysis indicated that disease-free survival of patients with the high-EPS8/high-WT1/high-MDR1 profile was significantly shorter than in patients with the low-EPS8/low-WT1/low-MDR1 profile or those excluded from either of these groups (P<0.0001). Thus, EPS8, as MDR1 and WT1, may be a clinically valuable biomarker for assessing the outcome of ALL patients.

Association between the RAD51 135 G>C polymorphism and risk of cancer: a meta-analysis of 19,068 cases and 22,630 controls.

BACKGROUND: RAD51 135G>C can modify promoter activity and the penetrance of BRCA1/2 mutations, which plays vital roles in the etiology of various cancer. To date, previous published data on the association between RAD51 135G>C polymorphism and cancer risk remained controversial. Recent meta-analysis only analyzed RAD51 135G>C polymorphism with breast cancer risk, but the results were also inconsistent. METHODS: A meta-analysis based on 39 case-control studies was performed to investigate the association between cancer susceptibility and RAD51 135G>C. Odds ratios (OR) with 95% confidence intervals (CIs) were used to assess the association in different inheritance models. Heterogeneity among studies was tested and sensitivity analysis was applied. RESULTS: Overall, no significant association was found between RAD51 135G>C polymorphism and cancer susceptibility in any genetic model. In further stratified analysis, significantly elevated breast cancer risk was observed in BRCA2 mutation carriers (recessive model: OR = 4.88, 95% CI = 1.10-21.67; additive model: OR = 4.92, 95% CI = 1.11-21.83). CONCLUSIONS: This meta-analysis suggests that RAD51 variant 135C homozygote is associated with elevated breast cancer risk among BRCA2 mutation carriers. Moreover, our work also points out the importance of new studies for RAD51 135G>C association in acute myeloid leukemia, especially in Caucasians, where at least some of the covariates responsible for heterogeneity could be controlled, to obtain a more conclusive understanding about the function of the RAD51 135G>C polymorphism in cancer development.

[An enzyme-linked immunosorbent assay for determining serum anti-themocyte globulin concentration].

OBJECTIVE: To establish an enzyme-linked immunosorbent assay (ELISA) for determining anti-themocyte globulin (ATG) levels in serum samples. METHODS: The microplate was coated with mouse anti-rabbit IgG monoclonal antibody, and sheep anti-rabbit polyclonal antibody conjugated with HRP was used as the second antibody for detecting the serum ATG levels in patients undergoing allogeneic hematopoietic stem cell transplantation. RESULTS: The optimal concentration of the coating antibody and dilution ratios of the serum samples and IgG-HRP conjugate were 0.2 microg/ml, 1:40 and 1:2500, respectively. The lower sensitivity limit of the assay was 31.25 ng/ml for ATG detection. A linear relationship was established within the concentration range from 40 to 1000 ng/ml, with the coefficients of variation of 7.91 within assay and 5.22 between assays, respectively. Seven patients undergoing stem cell transplantation with ATG pretreatment showed gradually decreased concentration of ATG, and after 90 days ATG could still be detected. CONCLUSION: The sandwich ELISA we established provides a specific and sensitive method for quantitative measurement of ATG in the clinical setting. In patients undergoing stem cell transplantation with ATG pretreatment, the ATG concentration gradually decreases but remains detectable 90 days after the administration.

[Effect of small interfering RNA targeting multidrug resistance-related protein and bcl-2 on drug resistance and apoptosis of K562 and K562/ADM cells].

OBJECTIVE: To observe the effect of small interfering RNA (siRNA) targeting multidrug resistance-related protein (MRP) and bcl-2 genes in modulating drug resistance and apoptosis of K562 and K562/ADM cells. METHODS: Two siRNA constructs targeting respectively bcl-2 and MRP genes, were synthesized and transfected either alone or in combination into K562 and K562/ADM cells via lipofectamine2000. MTT assay was used to evaluate the viability of the transfected cells at 24, 48 and 72 h Post-fransfection, and RT-PCR was performed to determine the mRNA levels of bcl-2 and MRP. The effects of MRP siRNA and bcl2 siRNA on the apoptosis and the protein expression of Bcl-2 and MRP were evaluated with flow cytometry. RESULTS: In K562/ADM cells, the IC (50) decreased from 12.81 microg/ml (ADM group) to 3.74 microg/ml (ADM+MRP siRNA group), 6.82 microg/ml (ADM+bcl2 siRNA group) and 2.51 microg/ml (ADM+MRP siRNA+bcl2 siRNA). Similarly, in K562 cells, the IC50 decreased significantly from 6.75 microg/ml (ADM) to 3.22 microg/ml (ADM+MRP siRNA), 3.56 microg/ml (ADM+bcl2 siRNA) and 1.84 microg/ml (ADM+MRP siRNA+bcl2 siRNA) (P<0.05). Flow cytometry demonstrated significantly increased apoptosis of the cells following MRP siRNA and bcl2 siRNA transfection, which also resulted in significantly decreased expressions of MRP and bcl-2 proteins (P<0.05). CONCLUSION: Treatment with both MRP and bcl-2 siRNAs inhibits the target gene expression, and increases the drug sensitivity and apoptosis of K562 and K562/ADM cells.

[Thermotherapy enhances the sensitivity of lymphoma cell line Raji to chemotherapy in vitro].

OBJECTIVE: To observe the effect of thermotherapy on the intracellular adriamycin concentration and apoptosis of Raji cells in vitro. METHODS: The working concentration of adriamycin against Raji cells was determined with MTT assay. Raji cells were subjected to thermotherapy (at 40 degrees Celsius;, 41 degrees Celsius; or 42 degrees Celsius;) and chemotherapy with adriamycin alone or in conjunction, and the cell survival rates were determined 48 h after the treatment. The cell growth inhibition effect of the treatment was evaluated with MTT assay, and the apoptotic rates of Raji cells and alteration of intracellular adriamycin concentration were determined by flow cytometry. RESULTS: The IC(50) of adriamycin was defined as the working concentration in the experiment. Thermotherapy at 40, 41 and 42 degrees Celsius; for 60 min in conjuction with chemotherapy significantly inhibited the growth of Raji cells (P<0.01). The results of flow cytometry showed that thermotherapy and adriamycin chemotherapy, used either alone or in combination, significantly increased the apoptotic rates of Raji cells (P<0.01), and thermotherapy remarkably increased the intracellular concentration of adriamycin. CONCLUSION: Adriamycin chemotherapy combined thermotherapy for 60 min can increase the intracellular concentration of adriamycin and the apoptosis rates of Raji cells.

[The outcomes of the thirty patients with refractory leukemia treated with related HLA haploidentical stem cells transplantation].

OBJECTIVE: To assess the outcomes of the therapy for patients with refractory leukemia with HLA haploidentical stem cells transplantation. METHODS: To analyze the outcomes of 30 patients with refractory leukemia who underwent HLA haploidentical peripheral blood stem cells transplantation from August 1998 to August 2004. RESULTS: Thirty refractory leukemia patients including 13 cases of acute non-lymphocytic leukemia, 10 cases of acute lymphocytic leukemia (ALL), 6 cases of chronic myeloid leukemia and 1 case of phase IV non-Hodgkin's lymphoma underwent HLA haploidentical peripheral blood stem cells transplantation. The median age was 25 years old (3-52 years old). Twelve patients received stem cells from parent donors, four from daughter or son donors and the remaining from sibling donors. Three HLA loci mismatched in twelve cases, two HLA loci mismatched in thirteen cases and one HLA locus mismatched in five cases. The conditioning regime consisted of fludara (25 mg/m(2) x 5 d), busulfan (4 mg/kg x 4 d) and cyclophosphamide (60 mg/kg x 2 d). Rabbit anti-human lymphocyte globulin (5 mg/kg x 5 d) was added in some patients in the conditioning regime. A mean of 5.0 (2.9-8.0) x 10(8)/kg mononucleated cells was grafted. The number of mean CD(34)(+) cells was 5.5 (3.0-6.5) x 10(6)/kg. Twenty-seven patients were successfully grafted, one failed to graft, one died from severe fungal infection at day 2 and one died from severe veno-occlusive disease at day 28. The mean time of white cell count more than 1.0 x 10(9)/L was 14 (11-18) days and platelet count more than 20 x 10(9)/L was 15 (11-18) days. ALL the 27 successfully grafted patients got complete remission. Severe acute graft versus host disease occurred in six patients and four of them died. Seven patients suffered from chronic graft versus host disease. Seven patients relapsed and died. The median relapse time was 10 (3-24) months. Fourteen patients are still surviving, and ten have disease free survival. CONCLUSION: It is concluded from our observation that HLA haploidentical peripheral blood stem cells transplantation may be an effective therapy for refractory and relapse leukemia. Some patients with refractory and relapse leukemia treated with HLA haploidentical stem cells transplantation may have disease free survival. Graft versus leukemia effect may be strong in patients receiving HLA haploidentical blood stem cells transplantation and leukemia will probably be relapsed when the patient without complete remission was treated with this therapy.

[Experimental study on NK cells promoting donor marrow engraftment and hematopoietic reconstitution after MHC haploidentical BMT in mice].

OBJECTIVE: To explore the effect of donor-derived NK cells added to pretreatment conditioning regimen on hematopoietic reconstitution after MHC haplotype-mismatched BMT in mice. METHODS: Murine model of MHC haplotype-mismatched BMT was established by using BALB/c(H-2d) x C57BL/6(H-2b) (CB6F(1)(H-2d/b)) mouse as recipient, and C57BL/6(H-2b) mouse as donor. Fifty recipient mice were divided into 5 groups. The mice in the first three groups were each infused 1 x 10(6), 5 x 10(5), 2 x 10(5)/mouse donor-derived NK cells, respectively before TBI ((60)Co, 9.0 Gy) and then conditioned with TBI, followed by infusion of C57BL/6(H-2b) mice bone marrow cells four hours later. The mice in the fourth group received TBI only, and in the fifth group, TBI and BMT at the some doses as the first three groups. Hematopoietic reconstitution, survival time, body weight, histopathology of the recipients were followed up. RESULTS: (1) Survival time was (5.15 +/- 0.66) days for the fourth group, and > 30 days for the other 4 groups. (2) Leukocyte and platelet counts at day 10 after BMT were (0.99 +/- 0.22) x 10(9)/L and (61.0 +/- 7.27) x 10(9)/L respectively for the fifth group and (2.01 +/- 0.21) x 10(9)/L, (101.50 +/- 16.34) x 10(9)/L; (1.98 +/- 0.29) x 10(9)/L, (99.50 +/- 16.41) x 10(9)/L and (1.97 +/- 0.21) x 10(9)/L, (98.0 +/- 16.19) x 10(9)/L for the first three groups, respectively. Histopathology displayed no GVHD in all the groups. CONCLUSION: Donor-derived NK cells could promote hematopoietic reconstitution after MHC haplotype-mismatched BMT in mice.

[Decrease of chronic graft-versus-host disease by adding anti-human thymocyte globulin to the conditioning regimen].

OBJECTIVE: To observe the influence of adding anti-human thymocyte globulin (ATG) into conditioning regimen on graft-versus-host disease (GVHD) and life quality of the patients of allo-peripheral blood stem cell transplantation (PBSCT). METHODS: Patients were distributed into study (19 cases) and control (24 cases) groups at random. Median dose of rabbit ATG was added to the conditioning regimen based on the fludara, busufan and cyclophosphamide (FBC) in study group, and no ATG in the control group. Acute and chronic GVHD disease and Karnofsky scores were compared between two groups after allo-PBSCT. RESULTS: The patients in the study group received a mean of 6.0 (3 - 9) x 10(8)/kg mononucleated cells and 5.5 (4.5 - 7.5) x 10(6)/kg in the control group. The mean CD(34)(+) cells number was 5.5 (3.0 - 6.5) x 10(5)/kg in the study and 5.0 (3.0 - 7.0) x 10(6)/kg in the control group respectively. Eighteen patients in the study group and in the control group were successfully engrafted. The mean time of absolute neutrophil count recovered more than 500/ micro l was 13 days and 12 days respectively. Acute GVHD occurred in 6 patients of the study group, and 15 of the control group. Seven patients suffered from chronic GVHD and 14 got 90 Kanrofsky scores in a mean of 250 days follow-up in the study group, and 19 patients GVHD and 4 patients respectively in a mean of 440 days follow-up in the control group. There was a significant difference for acute and chronic GVHD and life quality between the two groups. CONCLUSIONS: Addition of anti-thymocyte globulin to the FBC conditioning regimen had no effect on stem cells engraftment but could decrease acute and chronic GVHD and improve patients life quality.

A model to induce low temperature trauma for in vitro astrogliosis study.

Astrogliosis is an inevitable and rapid response of astrocytes to physical, chemical and pathological injuries. To study astrogliosis, we developed a reproducible in vitro model in which low temperature injury to cultured astrocytes could be induced by placing the culture dish onto a copper pipe pre-cooled by liquid nitrogen. Using this model, the relationship between the temperature decline and the severity of cellular damage was analyzed. An increase in the expression of some known injury-related proteins, such as glial fibrillary acidic protein (GFAP), immediate early response genes (IEGs), and heat shock proteins 70 (HSP70), was demonstrated in astrocytes after low temperature trauma. With the use of this low temperature trauma model, the flexibility in the temperature control and injury area may allow researchers to evaluate cryotherapy and cryosurgery, which could be applicable to future development of quality health care.

[Treatment of refractory leukemia by combined chemotherapy and halpotype lymphocytes infusion].

BACKGROUND & OBJECTIVE: The complete remission of refractory leukemia treated with conventional chemotherapy is below 50 percent. The high dose chemotherapy can cause more mortality of patients with refractory leukemia. Cytolysis of leukemia cells induced by halpotype lymphocytes was observed in vitro in our previous experiment. In order to improve the complete remission of refractory leukemia and decrease the complication of chemotherapy,the authors treated the patients with refractory leukemia by combined chemotherapy and halpotype lymphocytes infusion and to assess the therapeutic effects and the side effects of this modality. METHODS: Sixteen patients with refractory leukemia were treated by combined chemotherapy. Halpotype lymphocytes irradiated by 7.5 Gygamma radial were infused when patient's white cells count was at the lowest after the chemotherapy. A mean number of 1x10(8)/kg (range:0.8-1.2x10(8)/kg) of halpotype lymphocytes irradiated by 7.5 Gygamma radial was infused. The side effects of infusion of halpotype lymphocyte and completed remission rate were observed. RESULTS: Out of thirteen patients with refractory acute non-lymphocyte leukemia, eleven cases got complete remission and two partial remissions. Out of three patients with refractory acute lymphocyte leukemia, two got complete remission and one no reaction. The total remission rate was 81.2%. No severe side effects and no transfusion related graft versus host disease was observed. CONCLUSION: The results show that chemotherapy combined with 7.5 Gy irradiated halpotype lymphocyte infusion could improve the complete remission of refractory leukemia and decrease the complications caused by chemotherapy.

[Treatment of malignant hematologic diseases by peripheral blood stem cell transplantation combined with halotype lymphocyte infusion].

In order to observe the curative and side effects in malignant hematologic diseases treated with autologous peripheral blood stem cell transplantation (auto-PBSCT) combined with halotype lymphocyte infusion, auto-PBSCs were mobilized, harvested and stored at -196 degrees C from patients in first CR or PR with intensive chemotherapy (Ara-C 1.0 g/m(2) x 5 days or cyclophosphamide 60 mg/kg x 2 days) and G-CSF. Unpurged auto-PBSCs were infused when patients received the conditioning regimen with busulfan, total irradiation or cyclophosphamide. Halotype lymphocytes [mean 5.0 x 10(7)/kg, (4.5 - 6.5) x 10(7)/kg] irradiated with 7.5 Gy were infused to patients when WBCs were more than 1 x 10(9)/L. Hematopoietic recovery and survival of patients were observed. The results showed that in 12 cases accepted this protocol, five patients with acute non-lymphocytic leukemia got to durable remission, of which 2 had durable remission of more than 50 months. One of three patients with non-Hodgkin's lymphoma IVb reached durable remission, and two relapsed and died on 4 and 6 months after treatment, respectively. Two CML patients were also achieved durable remission. One patient with multiple myeloma relapsed on 36 months later, but he still survived disease-free with treatment of thalidomide. In a follow-up survey of 25 months, the disease-free survival was 83%. No severe side effects were observed except platelet delayed recovery after halotype lymphocyte infusion. STR-PCR analysis showed that infused donor lymphocytes disappeared in 3 recipients at 72 hours after infusion. It is concluded that auto-PBSCT combined with halotype lymphocyte infusion could decrease the relapse of malignant hematologic diseases and improve the effect of auto-PBSCT. Recovery of platelet, however, could be delayed by halotype lymphocyte infusion.