[Prostaglandin E2 Receptor 4 Agonist Promotes Human CD34+ Cell Proliferation in vitro by Activating Wnt/ß-Catenin Signaling Pathway].

OBJECTIVE: To investigate the potential signaling pathway that regulates the proliferation of human CD34+ cells stimulated by prostaglandin E2 receptor 4 agonist (EP4A) in vitro. METHODS: Twenty samples of peripheral blood containing stem cells were collected from the G-CSF mobilized healthy donors in our department of hematology. Human CD34+ cells were isolated by magnetic activated cell sorting (MACS) microbeads kit. The Cell Counting Kit-8 (CCK8) assay was used to determine the optimal concentration and time of EP4A to promote human CD34+ cell proliferation in vitro. Under the optimal condition, quantitative real-time polymerase chain reaction (qRT-PCR) was used to detect mRNA level of ß-catenin, and Western blot was used to assay protein expression of ß-catenin and P-GSK-3ß in human CD34+ cells treated with EP4A. RESULTS: Culturing with 10 µmol/L EP4A for 72 h, it was found that EP4A promoted human CD34+ cell proliferation significantly, and the proliferation rate of human CD34+ cells was 1.36 times higher than that of the control(P=0.002). Under the optimal condition, it was also found that EP4A enhanced the ß-catenin expression at both mRNA and protein levels, and up-regulated phosphorylation of GSK-3ß in human CD34+ cells, but these effects could be inhibited by the EP4A antagonist EP4AA. CONCLUSION: EP4A can enhance human CD34+ cell proliferation in vitro by activating Wnt/ß-catenin signaling pathway.

[Clinical significance of abnormal protein bands in multiple myeloma treated with bortezomib-based induction regimen and autologous stem cell transplantation].

OBJECTIVE: To study the clinical significance of abnormal protein bands (APB) in multiple myeloma (MM) patients treated with bortezomib-based induction regimen and autologous stem cell transplantation (ASCT). METHODS: Sixty-eight MM patients submitted to bortezomib-based induction therapy and ASCT from January 2007 to July 2012 were retrospectively studied. Monoclonal protein was detected by immunofixation electrophoresis (IFE). RESULTS: Of all 68 patients, 33 (48.5%) patients had APB. At the first emergence of an APB, two patients with light chain type achieved CR and before transplantation, and thirty-one patients were after transplantation with median time of 104 (ranged 33-404) days. The median duration of APB appearance was 105 (ranged 35-801) days. Patients who developed APB compared with those without APB, had a significantly higher CR plus very good partial response (VGPR) rates (100.0% vs 85.7%%, P=0.017) and CR rates (87.9% vs 62.9%) (P=0.03). There were no significant differences in gender, age, HGB, ALB, ß2-microglobulin, M protein type, Durie-Salmon and ISS stages, the case number of first line or second line treatment, induction courses of bortezomib-based regimen, and the mode of ASCT. With a median follow-up of 33.4 (ranged 7.0-71.7) months, patients with APB tended to have a longer overall survival (OS) versus non-APB patients, although no significant difference obtained (P＞0.05). Among APB patients, OS was longer in patients whose appearance of APB occurred ＜6 months after transplantation than those ≥ 6 months, but the significant difference was not obtained yet (P＞0.05). CONCLUSIONS: Patients who developed APB had a significantly better response to bortezomib-based induction regimen followed ASCT. APB emergence has a good prognostic significance.

[Effects of killer immunoglobulin-like receptor and human leukocyte antigen class I ligand on the prognosis of related donor hematopoietic stem cell transplantation].

OBJECTIVE: To study the genotype distribution and the effects of killer immunoglobulin-like receptors (KIR) and human leukocyte antigen (HLA) class I ligand on related donor hematopoietic stem cell transplantation (HSCT). METHODS: The genotypes of donor/recipient HLA-Cw and donor KIR were determined by polymerase chain reaction-sequence specific primer (PCR-SSP) in 87 cases of related donor HSCT (40 cases were haploidentical HSCT, and the remaining 47 cases were HLA-identical sibling HSCT). RESULTS: All the donors possessed KIR2DL1, 2DL2/L3, 2DL4, 3DL2, and 3DL3, and 96.6% of donors possessed 3DL1. The rate of activating KIRs varied. 97.7% of the recipients expressed C1, while the rates of C2, Bw4, and HLA-A3/A11 were different. In haploidentical HSCT, KIR-HLA-mismatched group included 34 cases and the matched group included 6 cases. HLA-HLA-mismatched group included 31 cases and the matched group included 9 cases. In matched sibling donor HSCT, KIR-HLA-mismatched group included 42 cases and the matched group included 5 cases. KIR-HLA-mismatched group had higher 2-year disease-free survival (DFS) rate compared with KIR-HLA-matched group [ (71.5 +/- 6.5 ) % vs. (50.0 +/- 10.7)%, P < 0.05]. CONCLUSIONS: The rate of activating KIR is lower than inhibitory KIRs. Inhibitory KIR2DL1, 3DL1, and 3DL2 may play key roles in the natural killer cell alloreactivity. The DFS rate is higher in KIR-HLA-mismatched group than in KIR-HLA-matched group in related donor HSCT.

[Osteoclast differentiation regulated by receptor activator of nuclear factor kappaB probably through a novel signaling pathway].

This study was aimed to investigate the signaling pathways regulating osteoclast (OC) differentiation by receptor activator of nuclear factor kappa (RANK) under physiological condition so as to provide some theoretical basis for clarifying mechanism of bone destruction in multiple myeloma. A mutant TNFR(1)/RANK(2) (named RANK-Mu) chimera consisting of tumor necrosis factor receptor 1 (TNFR(1)) and RANK intramembrane domain was constructed by using deletion mutation for deleting IVVY amino acids in RANK intramembrane domain in accordance with (535-)IVVY(-538) as specific domain regulating OC differentiation by RANK. The RANK-Mu and TNFR(1)/RANK chimera without mutation (RANK-WT) were packaged by using plat E cell line to produce the retrovirus, which were transfected into bone marrow macrophages (BMMs) of TNFR(1)/TNFR(2) double knockout mice. After stimulation of these transfected BMMs with TNF-alpha, the differentiation of BMMs into OCs were observed, meanwhile the phosphorylation of NF-kappab, JNK, p38 and ERK was detected by Western blot after stimulation of these BMMs with TNF-alpha. The results showed that BMMs transfected with RANK-WT could be differentiated into OCs and phosphorylation of NF-kappaB, JNK, P38 and ERK were activated at 5 - 10 minutes after being stimulated by TNFalpha. BMMs transfected with RANK-Mu could not be differentiated into OCs, but phosphorylation of NF-kappaB, JNK, P38 and ERK were activated also. It is concluded that RANK regulates osteoclast differentiation probably not through 4 typical signaling pathways, named as NF-kappaB, JNK, P38 and ERK, in this process other new signaling pathways maybe participate.

[The effects and safety of bortezomib combined with dexamethasone in the treatment of primary systemic amyloidosis].

OBJECTIVE: To evaluate the effects and safety of the regimen of bortezomib combined with dexamethasone (VD) in the treatment of primary systemic (AL) amyloidosis. METHODS: Five newly diagnosed AL amyloidosis patients confirmed by renal biopsy with a median of 3 organs involved (3 to 5 organs) were treated by VD regimen for 3 (1-4) cycles. RESULTS: Among 3 evaluable patients, 1 was in stable condition and 2 had hematologic response (partial remission and complete remission) and organ function improvement. Hematologic responses were rapid (median 1.5 cycles) and median time to organ response was 2 cycles. Three cases were survived and the periods of follow up were 5, 4 and 4 months respectively. The other 2 died 2 and 14 months after diagnosis. The side effects were asthenia, diarrhea, constipation, edema aggravation and fever, all of which were in I grade. No treatment associating death was found. CONCLUSION: VD regimen might be an efficient, rapid effective and safe regimen in the treatment of AL amyloidosis.

[Efficacy of bortezomib combined dexamethasone in 24 patients with multiple myeloma].

BACKGROUND & OBJECTIVE: Bortezomib, a potent and reversible proteasome inhibitor, induces apoptosis of myeloma cells, resulting in durable responses in patients with multiple myeloma (MM). This study was to explore the medical effects and side effects of bortezomib combined dexamethasone in treating newly diagnosed and relapsed or refractory MM, and evaluate the safety of this regimen in the patients with renal impairment. METHODS: Twenty-four MM patients were treated with bortezomib and dexamethasone in a 21-day cycle. The patients received a median of 3 cycles (range, 1-8 cycles) of the treatment. Response to bortezomib was evaluated according to the criteria of the European Group for Blood and Marrow Transplantation (EBMT); adverse events were graded according to the National Cancer Institute Common Toxicity Criteria. RESULTS: During the follow-up with a median of 4 months, 19 (79.2%) patients responded to the treatment. The complete remission (CR) rate was significantly higher in the patients of light-chain type than in those of non-light-chain type (57.1% vs. 5.9%, P=0.014). The response rates of the patients with and without renal impairment were similar (100% vs. 70.6%, P=0.272), and the renal functions were ameliorated in the patients with renal impairment during chemotherapy. Grade III-IV adverse events, including leucocytopenia (8.3%), thrombocytopenia (33.3%), diarrhea (8.3%) and debility (4.2%), could be relieved by symptomatic treatment or delayed chemotherapy. CONCLUSIONS: The combination of bortezomib and dexamethasone shows obvious effects on MM, especially in the patients with light-chain type. The adverse events can be tolerant in most patients, and this regimen is also safe in the patients with renal impairment.

[Impact of donor KIR2DS5 genotype on outcome following haploidentical hematopoietic stem cell transplantation].

The aim of this study was to evaluate the impact of donor killer cell immunoglobulin-like receptor (KIR) and recipient HLA genotypes on outcome following haploidentical hematopoietic stem cell transplantation (HSCT). 26 patients with hematologic diseases received non T-cell-depleted (TCD) in vitro transplant from haploidentical donor. Donor/recipient HLA and donor KIR genotypes were determined by polymerase chain reaction-sequence-specific primer (PCR-SSP). Donor/recipient KIR/HLA subgroup was assessed by donors KIR and recipients HLA-Bw4, Cw1 group and Cw2 group alleles. Hematopoietic reconstitution, incidence of graft versus host disease (GVHD), disease-free survival (DFS), infection and transplant-related mortality (TRM) were analyzed between every two groups. The influence of donor activating KIR on outcome following haploidentical HSCT also has been studied. The results showed that hematopoietic reconstitution, incidence of GVHD, DFS, infection and TRM were not significantly different between every two groups (p>0.05). There were 4 cases of severe GVHD in C2 mismatched group. The donor activating KIR2DS5 positive group had higher 2-year DFS compared with the negative group [(85.7+/-13.2)% vs (31.2+/-12.8)%, p<0.05]. It is concluded that KIR/HLA genotypes between donor and recipient influence the outcome following haploidentical HSCT. Donor activating KIR2DS5 may improve DFS in non TCD haploidentical HSCT.

[Role of alloreactive natural killer cell in mouse MHC haploidentical bone marrow transplantation].

OBJECTIVE: To study the effect of alloreactive natural killer (NK) cells used in conditioning regimen on elimination of recipient-type T cell and granulocyte, reconstitution of hematopoiesis, engraftment and graft-versus-host disease (GVHD) in murine major histocompatibility complex (MHC) haploidentical bone marrow transplantation (BMT). METHODS: The murine model of MHC haploidentical BMT was established by using (C57BL/6 x BALB/c) BCF(1) (H-2(d/b)) mouse as the donor, and BALB/c (H-2(d)) mouse as the recipient. Recipient mice were divided into 8.5 Gy control group and 7, 6 and 5 Gy experimental groups according to different irradiation dose and different kinds of NK cell treatment. The control group was further subdivided into untreated and BMT groups, while each experimental group was subdivided respectively into untreated group, BMT group, non-allo-reactive NK cells (non-allo NK) group and alloreactive NK cells (allo NK) group. The effect of adding alloreactive NK cell to conditioning regimen was assessed by peripheral white blood cell and platelet counts, recipient type H-2(d+) T cells and granulocytes counts, expression of H-2(d/b+) cells and pathohistological examination. RESULTS: Survival time was (6.00 +/- 0.82) days for 8.5 Gy untreated group, and beyond 60 days for all the other groups. No clinical and histopathological evidence of GVHD was observed in all the groups. The reconstitution of hematopoiesis was faster in allo NK groups than in other groups (P < 0.05). On day 1 after BMT, in allo NK groups with different irradiation dose, bone marrow and spleen recipient type H-2(d+) granulocytes and T cells were significantly decreased compared with identical BMT groups and non-allo NK groups (P < 0.05). The engraftment rates of H-2(d/b+) cells were significantly higher in 7, 6 and 5 Gy allo NK groups than in identical BMT groups and non-allo NK groups (P < 0.05, respectively). CONCLUSIONS: In mouse MHC haploidentical BMT, alloreactive NK cell can eliminate recipient-type T cell and granulocyte, promote reconstitution of hematopoiesis, enhance engraftment while not induce GVHD.

[Effects of mesenchymal stem cells on expansion potential and adhesion molecules expression of cord blood CD34+ cells].

OBJECTIVE: To explore the effects of bone marrow mesenchymal stem cells (MSCs) on in vitro expansion potential, the adherent molecules expression of cord blood (CB) CD34(+) cells. METHODS: MSCs were obtained from human bone marrow and their differentiation function and phenotype were identified. CB CD34(+) cells were expanded in culture systems with or without MSC layer. Hematopoietic progenitor cells and adhesion molecules expression were assessed by semisolid culture assay and flow cytometry. RESULTS: Thy-1, SH2, SB10, CD44, CD13, CD49e and CD29 were highly expressed on MSCs with no expressions of CD34, CD45, HLA-DR, CD14 and CD31. The MSCs could differentiate into adipocytes and osteoblasts under specific induction conditions. After culturing on MSCs layer with supplement of cytokines for 8 days, the absolute numbers of nuclear cells, CD34(+), CD34(+)CD38(-), CD34(+)CD62L(+) cells and CFU-Cs were increased by 145.57 +/- 17.89, 37.47 +/- 13.78, 69.78 +/- 50.07, 10.74 +/- 5.89 and 20.73 +/- 5.54-folds, respectively, being significantly higher than that cultured with cytokines alone. The expression of ALCAM, VLA-alpha4, VLA-alpha5, VLA-beta1, HCAM, PECAM and LFA-1 on CD34(+) cells remained unaffected. The expressions of ICAM-1 and L-selectin were downregulated during expansion, while the absolute numbers of CD34(+)CD62L(+) and CD34(+)CD54(+) cells were increased. CONCLUSIONS: MSCs layer improves expansion of CB CD34(+) cells while inhibiting their differentiation and retaining their homing ability.

[Comparative study on various subpopulations in mesenchymal stem cells of adult bone marrow].

To explore the difference of biological characteristics between two subpopulations of adult bone marrow mesenchymal stem cells (MSC), this study was designed to observe the morphological feature and immunophenotype of the adult MSC in the ex vivo culture, the mononuclear cells isolated from normal adult bone marrow were cultured in DMEM with 10% fetal bovine serum. Cell morphology, immunophenotype and cell cycle of two different subgroups were investigated. Cells from 80% confluence were passed through a 10 microm filter, then the fillered cells were cultured in the semisolid methylcellulose medium. The results showed that (1) two different subpopulations were observed in the ex vivo culture. The fibro-like cell was called mature MSC (mMSC) and the smaller round cell was defined rapidly as MSC self-renewing cells (RS cells); (2) the average proportion of cells in G(0)/G(1) of RS cells was approximately 99%, but that of mMSCs was 90%; (3) both of the two populations were negative on the lineage-committed antigen (such as CD34, CD45, CD3, CD19, CD33, HLA-DR, CD38), while positive on the expression of CD90, CD105, C166, CD29, CD44, CD49e, CD54, CD13. However, the expression of these antigens on RS cells was weaker than that on mMSC, but CD117 and KDR were higher expressed when compared with the mMSC; (4) after 4 to 5 week semisolid culture, no hematopoietic progenitor cell colonies were observed. It is concluded that adult MSCs are heterogeneous in that distinct morphological populations exist. The RS cells appear to be the more primitive with greater potential for self-renewal and multilineage differentiation.

[T-bet gene expression in acute graft versus host disease].

OBJECTIVE: To study the role of T-bet [a T helper 1 (Th1) lymphocyte transcription factor] gene expression in predicting acute graft-versus-host disease (aGVHD) and evaluate the correlation between T-bet gene and aGVHD. METHODS: Twenty patients who underwent allogeneic stem cell transplantation (allo. HSCT) entered this study. The expression of T-bet gene was examined with reverse transcription (RT)-PCR in bone marrow samples collected from patients on the day before conditioning, and day 0, day 14, day 28, and day 42 after HSCT. RESULTS: The expression level of T-bet in patients developed aGVHD was increased compared with that before conditioning (P = 0.043). The incidence of aGVHD was 91.7% in patients whose T-bet expression level was increased on day 14 after transplant while was 12.5% in those whose T-bet gene expression level was not increased on that day (P < 0.001). CONCLUSION: Patients with increased expression levels of T-bet after allo-HSCT may have an increased possibility to develop aGVHD. T-bet expression level may serve as an advisable guide to the clinician in predicting aGVHD and monitoring treatment.

[Comparison of autologous and allogeneic hematopoietic stem cell transplantation for 140 patients with de novo acute leukemia in first complete remission].

OBJECTIVE: To evaluate the outcome of patients with de novo acute leukemia (AL, no AML-M(3)) in CR(1) undergone autologous hematopoietic stem cell transplantation (auto-HSCT) or HLA-identical sibling allogeneic HSCT (allo-HSCT). METHODS: Forty-six AL patients received allo-HSCT and 94 received auto-HSCT in CR(1). The conditioning regimens mainly consisted of TBICy, BuCy and MAC. Cyclosporine plus methotrexate, or cyclosporine alone, or FK506 alone was used for graft-versus-host disease (GVHD) prophylaxis. Among auto-HSCT group, 39 patients received purged autologous bone marrow and 38 received immunotherapy and/or maintenance chemotherapy after transplant. RESULTS: Myeloid reconstitution was achieved in all patients. After a median of 700 (range, 18 approximately 5563) days follow-up, the probabilities of leukemia-free survival (LFS) at 5 year were not significantly different in these two groups: (51.5 +/- 5.4)% for auto-HSCT group and (52.8 +/- 7.6)% for allo-HSCT group (P > 0.05). There was a lower cumulative relapse incidence (RI) [(26.3 +/- 6.9)% vs. (52.0 +/- 5.5)%, P > 0.05] but a significantly higher cumulative transplant-related mortality (TRM) [(37.6 +/- 7.8% vs. (14.4 +/- 4.1)%, P < 0.05] in the allo-HSCT group than in auto-HSCT group. Among auto-HSCT group, the patients received purged autografts and/or post-transplant therapy had significantly better LFS and lower RI (P < 0.05) than those received unpurged autografts or no post-transplant treatments [5-y LFS: (62.8 +/- 6.8)% and (38.4 +/- 8.4)%; RI: (37.7 +/- 6.8)% and (74.2 +/- 8.7)%, respectively]. CONCLUSION: The long-term LFS of auto-HSCT was comparable to that of allo-HSCT in the management of patients with AL in CR(1), because autograft purging and post-transplant treatment can significantly decrease relapse of auto-HSCT patients and auto-HSCT has lower therapy-related toxicities.

[Second allogeneic transplant for leukemia relapsed after first allogeneic transplantation].

OBJECTIVE: To evaluate the efficacy of second allogeneic hematopoietic stem cell transplantation (allo-HSCT) for treatment of leukemia relapsed after first allo-HSCT. METHODS: Nine patients with relapsed acute leukemia (5 AML, 4 ALL) and one with chronic myelogenous leukemia (CML) who showed cytogenetic relapse after first allo-HSCT received second allo-HSCT. The median relapse time from the first allo-HSCT was 141 days. Conditioning regimens for second allo-HSCT were combination chemotherapy based on moderate-dose Ara-C (n = 5), Bu (n = 3), conventional-dose Ara-C (n = 1) and Flud/Mel (n = 1). Prophylaxis for acute graft-versus-host disease (aGVHD) were CsA alone (n = 2), CsA/MTX (n = 1), FK506 (n = 1), and no prophylaxis in 6. The median number of peripheral blood mononuclear cells transfused was 6.1 x 10(8)/kg. RESULTS: Eight cases were evaluable. All of them were engrafted and 7 developed aGVHD (grade I 4, grade II 3). The median time for absolute neutrophil count (ANC) > 0.5 x 10(9)/L and platelets > 20 x 10(9)/L were 11 and 12 days, respectively. Five cases developed localized chronic GVHD. Of all the 10 cases received second allo-HSCT, 8 died from interstitial pneumonia (n = 2), multiple-organ failure (n = 1), sepsis (n = 1), fungous pneumonia (n = 1), and leukemia relapse (n = 3), and 2 survived without leukemia for +986 and +1913 days, respectively. The leukemia free survival, transplantation related mortality and relapse rate at 2 year were 20%, 50% and 30%, respectively. CONCLUSION: Second allo-HSCT is a therapeutic alternative for selected patients with relapsed leukemia after first allo-HSCT.