Assessment of the use of immunosuppressants combined with cord blood for severe aplastic anemia.

BACKGROUND: The objective of this study was two-fold: 1) to investigate the changes of cytokines concentration in relation to severe aplastic anemia (SAA) when treated with immunosuppressants combined with cord blood (IS + CBI). and 2) to assess the curative effect of umbilical cord blood chimerism engraftment. METHODS: We selected 43 patients with SAA all treated with IS + CBI (newly diagnosed group). Among them, a total of 33 patients were treated effectively (effective group) while 10 cases were treated invalidly (invalid group). An additional 20 healthy individuals were selected as control (control group). The expression levels of IL-17, IL-22 and other cytokines in each group were detected by ELISA. The engraftment of cord blood stem cells was detected by using short tandem repeat-polymerase chain reaction (STR-PCR). RESULTS: 1. IL-17, IL-22 and other cytokines expressions in the newly diagnosed group were significantly higher than in the control group. 2. After six months, the levels in the effective group were significantly lower than pre-therapy levels (P < 0.05). The levels in the invalid group did not differ to those observed prior treatment. 3. After one and three months of treatment, a small amount of engraftment was found in the effective group. However, after six months, transplant rejection was observed in all patients. No effective engraftment was observed in the invalid group. CONCLUSION: 1) Th17 and Th22 producing cells in SAA patients significantly increased indicating a positive correlation between these biomarkers and the progression of SAA. 2) During the IS + CBI treatment the maintenance of a normal hematopoietic function depended on immunesup-pressants. Early umbilical cord blood chimerism engraftment may promote hematopoietic recovery.

Mechanism of immunosuppressants combined with cord blood for severe aplastic anemia.

This study aims to explore the mechanism of immunosuppressants combined with cord blood (IS + CBI) for severe aplastic anemia. Selecting 30 patients with SAA and all treated with IS + CBI (newly diagnosed group). 23 patients who were treated effectively (effective group) while 7 cases were treated invalidly (invalid group). Another 20 healthy individuals were selected as control group. To detect the expression levels of IL-17, IL-22 and other cytokines by ELISA method in each group. To detect the engraftment of cord blood stem cells by using short tandem repeat-polymerase chain reaction (STR-PCR) method. 1. IL-17, IL-22 and other cytokines expressions in newly diagnosed group were significantly higher than in the control group. 2. After 6 months, the level in effective group was significantly lower than pretherapy (P < 0.05).The level in invalid group had no obvious difference than pretherapy. 3. After 1 month and 3 months of treatment, a small amount of engraftment was found in effective group. After 6 months, implant rejection was showed. No effective engraftment was observed in invalid group. 1. IL-17, IL-22 cells in SAA patients increased which might positively correlated with the progression of SAA. 2. During the treatment of IS + CBI, there is a bridging mechanism between the early stage of engraftment and the advanced stage of immunosuppressant adjustment. The first 3 months after treatment, it relies on the engraftment of cord blood stem cells to promote hematopoietic recovery and 3 months later, it relies on immunosuppressants to maintain normal hematopoietic function.

Increased immunosuppressive treatment combined with unrelated umbilical cord blood infusion in children with severe aplastic anemia.

A combination treatment of unrelated umbilical cord blood (UCB) and increased immunosuppressive treatment (IST) were investigated to reveal the potentially curative therapy for the severe aplastic anemia (SAA). A total of 36 children (2-17 ages) with SAA who received UCB infusion after an IST were analyzed. The treatment consisted of 100mg/kg cyclophosphamide, 12.5-15 mg/kg antithymocyte globulin and 3mg/kg cyclosporine. After 3 months, the hematologic complete response (CR) rate was 22.2% and partial response (PR) rate was 38.9%. After 6 months, the CR rate and PR rate was 50.4% and 26.3%, respectively. The probability of 3-year survival was 83.3%. There was no difference in the survival rate either between the horse-ATG and rabbit-ATG or between the SAA and VSAA. The results indicated that the increased IST combined with unrelated UCB infusion has an effective therapeutic potential for children with SAA who lack of compatible donor for transplantation.

Serum microRNA155 is increased in patients with acute graft-versus-host disease.

To explore the clinical relevance of three lymphocyte-related serum microRNAs (miR-155, miR-214, and miR-326) to the pathogenesis of graft-versus-host disease (GVHD), 64 subjects who received allogeneic peripheral blood stem cell transplantation (allo-PBSCT) were recruited in this study, of whom 19 subjects did not develop GVHD, 25 subjects were diagnosed with acute GVHD (aGVHD), and 20 subjects were diagnosed with chronic GVHD (cGVHD). Serum miRNAs were determined by real-time RT-PCR. Expression level of miRNAs and the expression signatures of miRNAs as a panel were analyzed among the three groups. The expression level of miR-214 and miR-326 showed no significant difference between GVHD and non-GVHD groups. However, miR-155 was significantly up-regulated in GVHD patients. There was a correlation between the level of miR-155 and the severity of aGVHD. Moreover, serum IFN-gamma, IL-17, and IL-9 levels were higher in aGVHD patients with high miR-155. In conclusion, the expression level of lymphocyte-related miR-155 in serum was significantly increased in aGVHD patients. The miR-155 may be considered as a potential targeted therapy for aGVHD patients.

[Comparison of protein expression profiles between bortezomib-resistant JurkatB cells with PSMB5 mutation and their parent cells].

This study was purposed to investigate the differences of cyto biological characteristics and protein expression levels between bortezomib-resistant T-lymphoblastic lymphoma/leukemia cell lines JurkatB containing PSMB5 G322A mutation and their parent cell line Jurkat, The cytotoxicities of bortezomib and chemotherapeutic drugs to JurkatB5 cells (end selection concentration of bortezomib was 500 nmol/L), JurkatB8 (end selection concentration 800 nmol/L) and Jurkat cells were analyzed. The cell growth curves were drawn with viable cell counts by trypan blue assay, the colony formation rate were assayed by soft-agar colony culture, and the cell distributions in cell cycle were analyzed by flow cytometry, mRNA expression levels of multidrug resistance (MDR) genes MDR1, LRP and MRP were measured by real-time fluorescence quantitative RT-PCR, the differences of protein expression levels were detected by SpringBio antibody microarray containing 720 proteins. The results showed that the drug resistance multiples for 48 hours of JurkatB5 and JurkatB8 cells (relative to Jurkat) to bortezomib were increased by 33.52 and 39.04 times, respectively. JurkatB5 and JurkatB8 cells did not display significant cross-resistance to daunorubicin, adriamycin, vindesine, and etoposide after exposure for 48 hours. There were no significant differences in the cell growth curve, colony formation rate and cell distributions in cell cycle between JurkatB5, JurkatB8 and Jurkat cells (p > 0.05). There were no significant differences of mRNA expression levels of MDR1, LRP, MRP between JurkatB5 and Jurkat cells (p > 0.05). There were 264 analyzable expression points detected by antibody microarray. Among them, 252 protein expression levels were not significantly different between JurkatB5, JurkatB8 and Jurkat cells (< 2-fold), including 15 drug resistance-related proteins. 12 proteins were detected at higher or lower expression levels in JurkatB5 or JurkatB8 cells then that in Jurkat cells (cell division cycle protein 34, cell division cycle protein 37, CD34 Type II, matrix metalloproteinase-2, tenascin, Golgi complex, involucrin, histone deacetylase 1, perforin, prolactin, retinoic acid receptor ß, integrin ß-1), but no proteins were detected in JurkatB5 and JurkatB8 cells with higher or lower expression levels than that in Jurkat cells. It is concluded that there are no significant differences in the characteristics of cellular biology between Jurkat and JurkatB with bortezomib-resistant and PSMB5 G322A mutation. There are no significant phenotype change of MDR and overexpression of genes related to MDR in PSMB5 mutated cells. There are no significantly differential expressions of a majority of known proteins related to drug resistance, tumor cells growth, proliferation, apoptosis, malignancy degree, aggressiveness.

[Blocking ICOS-B7h signal pathway by ICOS-Ig fusion protein inhibits function of allogeneic T lymphocytes].

Inducible costimulatory molecule (ICOS), a CD28 family member expressed on activated T cells, plays an important roles in T cell activation and effector function. This study was purposed to investigate the effect of blocking ICOS-B7h signal pathway by ICOS-Ig fusion protein on allogeneic reactive T cells and its mechanism. CHO cells stably and highly expressing ICOS-Ig were established, while the human ICOS-Ig fusion protein was harvested and purified from supernatant of CHO cells transfected with pSecTag2/Hygro A-ICOS-Ig. The CD4(+) cells from spleen of C57BL/6 mice were used as reactive cells, the bone marrow derived dendritic cells (DCs) from BALB/C mice were used as stimulatory cells, these cells were treated with different concentrations of ICOS-Ig or human Ig (h-Ig) as control. The results showed that the target protein with molecular weigh 54 kD and endotoxin level < 10 EU/ml was gained. The ICOS-Ig (> or = 10 microg/ml) could significantly inhibited the proliferative effect of allogeneic reactive T cells resulting from stimulation of DCs (p < 0.01). ICOS-Ig did not influence the activation of CD4(+) T cells. ICOS-Ig concentration positively related to the apoptosis of CD4(+) T cells. The percentages of CD4(+) Annexin V(+)PI(-) cells in simple stimulated group, ICOS-Ig 10 microg/ml group and ICOS-Ig 20 microg/ml group were 15.1%, 26.4% and 33.6% respectively. ICOS-Ig decreased secretion of TNFalpha and increased secretion of IL-4. It is concluded that the ICOS-Ig fusion protein has bioactivity of inhibiting T cell proliferation and altering the polarization of T helper cells to Th2 cells which promotes the apoptosis of allogeneic reactive T cells but had no effect on the activation of allo-reactive CD4(+) T cells.