High-dose Chemotherapy Combined with Autologous Hematopoietic Stem Cell Transplantation as Frontline Therapy for Intermediate/High-risk Diffuse Large B Cell Lymphoma.

The role of autologous hematopoietic stem cell transplantation (auto-HSCT) following high-dose chemotherapy has been validated and accepted as a standard treatment for patients with relapsed diffuse large B-cell lymphoma (DLBCL). However, its clinical efficacy as frontline therapy remains to be elucidated. This study aimed to examine the feasibility of frontline auto-HSCT for newly diagnosed intermediate/high-risk DLBCL patients. We retrospectively reviewed the data of 223 patients treated with frontline auto-HSCT or chemotherapy alone (year 2008-2014) from four hospitals. The median follow-up time was 29.4 months. Between the two treatment arms among the intermediate/high-risk DLBCL patients, the 3-year overall survival (OS) and progression-free survival (PFS) rates of patients given frontline auto-HSCT were 87.6% and 81.9%, respectively, and the chemotherapy-alone group showed 3-year OS and PFS rates of 64.9% and 59.59%, respectively. Compared with the chemotherapy-alone group, the frontline auto-HSCT could eliminate the adverse impact of non-germinal center B-cell (GCB) type. In addition, in the frontline auto-HSCT group, patients who achieved complete response (CR) at auto-HSCT had a longer survival time than those who did not achieve CR. Our results suggested that frontline auto-HSCT could improve the prognosis of intermediate/high-risk DLBCL patients.

Study on the Prognostic Value of Aberrant Antigen in Patients With Acute B Lymphocytic Leukemia.

BACKGROUND: Approximately 30% to 60% of patients with acute B-lymphocytic leukemia (B-ALL) show as refractory or relapsed, which is one of the major causes of death in patients with B-ALL, but the methods of the treatment for relapsed/refractory B-ALL (R/R B-ALL) are limited. The chimeric antigen receptors redirected T cells (CAR-T cells) have showed a strong anti-leukemia role for B-ALL. About 90% of patients with R/R B-ALL treated with CD19-CAR-T cells achieved complete remission. However, 60% to 70% of patients relapsed after CAR-T cells treatment, which may be related to target antigen reduction or escape. New products are urgently needed to prevent and treat antigenic escapes causing recurrence. PATIENTS AND METHODS: In this article, we retrospectively analyzed the immunophenotype of patients with B-ALL initially diagnosed in our center from January 2010 to December 2015 to determine whether aberrant antigen expression was associated with the prognosis of patients in order to find new targets for immunotherapy. RESULTS: The results show that disease-free and overall survival in patients without aberrant antigen expression were better than patients with aberrant antigen expression. The most common abnormal antigens were CD123, CD13, and CD56. Correlation analysis showed a negative correlation between aberrant CD123 expression and both disease-free and overall survival. CONCLUSION: Therefore, in the construction of CAR-T cells in patients with R/R B-ALL, conventional CD19 can be combined with aberrant antigens such as CD123 to form CARs with bi-specific antigens or multi-specific antigens may achieve the purpose of improving efficacy. However, more clinical trials are needed.

Diagnostic challenges in T-lymphoblastic lymphoma, early T-cell precursor acute lymphoblastic leukemia or mixed phenotype acute leukemia: A case report.

RATIONALE: The diagnosis of hematological malignancies depends on laboratory analysis and often requires multiple experimental methods to judge, otherwise misdiagnosis is apt to happen. Lymph node biopsy immunohistochemistry (IHC) for T-lymphoblastic lymphoma (T-LBL) requires the establishment of antibody set screening. For identifying T-LBL and early T-cell precursor acute lymphoblastic leukemia (ETP-ALL) by lymph node biopsy and IHC, WHO has not yet proposed a better IHC antibody combination. PATIENT CONCERNS: Here we reported 1 case with tortuous diagnosis experience. Initially, a 51-year-old man was diagnosed as T-LBL by lymph node biopsy, but in another hospital acute myeloid leukemia (AML) was confirmed by bone marrow puncture. Finally, it was diagnosed as mixed phenotype acute leukemia (MPAL) through our comprehensive evaluation including bone marrow cell morphology, cytochemical staining and flow cytometry analysis. Importantly, the experience about differential diagnosis and our appreciation among the T-LBL, ETP-ALL and MPAL was discussed to enlighten readers. DIAGNOSES: The patient was diagnosed with mixed phenotype acute leukemia (T+My)-NOS. INTERVENTIONS: The patient received 1 cycle of VDCLP scheme treatment firstly. The effect of chemotherapy is satisfactory, and then he received continuous treatment and was currently in good condition. OUTCOMES: This patient is alive at present. The follow-up period has been 1 year. LESSONS: For the diagnosis of T-LBL, the molecular markers of the myeloid and lymphoid tissues need to be included, such as CD117, CD33, Lys and MPO. The bone marrow puncture also needs to be conducted to distinguish T-LBL and T-ALL. Secondly, to identify ETP-ALL and MPAL, bone marrow cell morphology, cytochemical staining as well as flow cytometric analysis were needed to make a clear diagnosis. It is recommended that at least CD8, CD1a, Lys and MPO should be included in the panel to identify ETP-ALL.

Donor-derived CAR-T Cells Serve as a Reduced-intensity Conditioning Regimen for Haploidentical Stem Cell Transplantation in Treatment of Relapsed/Refractory Acute Lymphoblastic Leukemia: Case Report and Review of the Literature.

BACKGROUND: Reduced-intensity conditioning (RIC) regimens with low tolerable toxicities have been used for allogeneic hematopoietic stem cell transplantation (allo-HSCT). However, the relapse rate by this treatment is high. Treatment of CD19 B-cell relapsed/refractory acute lymphoblastic leukemia (r/r ALL) with allogeneic chimeric antigen receptor-modified T (CAR-T) cells is safe and effective. Use of allogeneic CD19-CAR-T cells as a part of RIC regimens for treatment of r/r ALL patients with haploidentical HSCT has not been investigated yet. CASE PRESENTATION: A 12-year-old girl with CD19 r/r ALL underwent haploidentical HSCT. The patient received fludarabine, busulfan, and cyclophosphamide combined with haploidentical donor-derived CD19-CAR-T cells as the conditioning regimen. Granulocyte colony-stimulating factor-mobilized peripheral blood stem cells and granulocyte colony-stimulating factor-mobilized bone marrow were infused on days 1 and 2, respectively. Mycophenolate mofetil and tacrolimus were administered on day 1, antithymocyte globulin was administered on days +14 and +15, and a short course of methotrexate was administered to prevent graft-versus-host disease. The time of peak CAR-T cell proliferation was detected after the first infusion of CAR-T cells on day 7. The patient's engraftment and full-donor cell engraftment were established. The disease was in complete remission with minimal residual disease, which was undetectable by flow cytometry. No graft-versus-host disease or serious cytokine-release syndrome was found. CONCLUSIONS: Treatment of r/r ALL with RIC including CD19-CAR-T cells followed by allo-HSCT was safe and effective, which suggest that CAR-T cells can be used as a part of RIC regimens in the treatment of r/r ALL in haploidentical HSCT.

High-dose methotrexate in the mobilization of hematopoietic stem cells for patients with non-Hodgkin's lymphoma: a twelve-year study in a single center.

BACKGROUND: High-dose chemotherapy followed by autologous hematopoietic stem cell transplantation (auto-HSCT) is a promising approach for non-Hodgkin's lymphoma (NHL). Higher cell doses have been associated with a faster blood count recovery and a reduction in transfusion requirements, infection rates, and hospitalization times. Mobilization failure constitutes one of the main reasons for avoiding auto-HSCT. The role of high-dose methotrexate (MTX) as mobilization regimen is still unclear. STUDY DESIGN AND METHODS: The effect of high-dose MTX as a mobilization regimen for 67 adult patients with NHL who received auto-HSCT was studied between January 2001 and October 2012. The stem cells were mobilized using combination chemotherapy including MTX plus granulocyte-colony-stimulating factor (G-CSF) in 33 patients (Group A), and the stem cells of the other 34 patients were mobilized using the same combination chemotherapy plus G-CSF without MTX (Group B). RESULTS: All of the patients were successfully mobilized in Group A; however, two patients failed in Group B. The median numbers of CD34+ cells collected were 14.36 × 10(6) and 5.3 × 10(6) cells/kg for Groups A and B, respectively (p < 0.05). All of the patients experienced a stable neutrophil and platelet (PLT) engraftment. The times to white blood cell engraftment were 8.0 days in Group A and 11.0 days in Group B, and the times to PLT engraftment were 12.0 days in Group A and 13.0 days in Group B (p < 0.05 for both variables). CONCLUSION: High-dose MTX is a powerful regimen component for stem cell mobilization in adult patients with NHL.

Features and clinical outcomes in 40 patients with mixed-lineage acute leukemia in a single center.

Mixed-lineage acute leukemia (MAL) is characterized as acute leukemia involving acute myeloid cells and lymphoid cells at the same time. It is easily misdiagnosed because of the dual characteristics involving both lymphoid and myeloid cells and has a poor prognosis. We retrospectively analyzed the features and treatment effectiveness in a single center in 40 patients with MAL. The morphology was consistent with acute lymphoblastic leukemia (ALL) (47.5%) or acute myeloid leukemia (AML) (20%) or was inconclusive (32.5%). Twenty-two patients were characterized as B/myeloid, and 18 patients as T/myeloid. Cytogenetics showed t(9;22)/(Ph(+)) (12.5%) and 11q23/MLL rearrangements (6.25%). The rate of first complete remission for patients undergoing chemotherapy based on the features of both ALL and AML and of either ALL or AML was 71.4 and 42.9%, respectively. The 1-year overall survival rates were 37.5 and 60.0% for chemotherapy and chemotherapy followed by haploidentical hematopoietic stem cell transplantation (HSCT), respectively. The 1-year disease-free survival rates were 25.0 and 50.0% for chemotherapy and chemotherapy followed by HSCT, respectively. These results showed that MAL is confirmed to be a poor-risk disease. The chemotherapy for remission induction should be based on both myeloid cells and lymphoid cells. Transplantation should be performed after the first remission.

Human umbilical cord blood-derived stromal cells are superior to human umbilical cord blood-derived mesenchymal stem cells in inducing myeloid lineage differentiation in vitro.

Stromal cells and mesenchymal stem cells (MSCs), 2 important cell populations within the hematopoietic microenvironment, may play an important role in the development of hematopoietic stem/progenitor cells. We have successfully cultured human umbilical cord blood-derived stromal cells (hUCBDSCs). It has been demonstrated that MSCs also exist in hUCB. However, we have not found any reports on the distinct characteristics of hUCBDSCs and human umbilical cord blood-derived mesenchymal stem cells (hUCBDMSCs). In this study, hUCBDSCs and hUCBDMSCs were isolated from the cord blood of full-term infants using the same density gradient centrifugation and cultured in the appropriate medium. Some biological characteristics and hematopoietic supportive functions were compared in vitro. hUCBDSCs were distinct from hUCBDMSCs in morphology, proliferation, cell cycle, passage, immunophenotype, and the capacity for classical tri-lineage differentiation. Finally, quantitative real-time polymerase chain reaction analysis revealed that granulocyte colony-stimulating factor (G-CSF) gene expression was higher in hUCBDSCs than that in hUCBDMSCs. Enzyme-linked immunosorbent assay revealed that the secretion of G-CSF, thrombopoietin (TPO), and granulocyte macrophage colony-stimulating factor (GM-CSF) by hUCBDSCs was higher than that by hUCBDMSCs. After coculture, the granulocyte/macrophage colony-forming units (CFU-GM) of hematopoietic cells from the hUCBDSC feeder layer was more than that from the hUCBDMSC feeder layer. Flow cytometry was used to detect CD34(+) hematopoietic stem/progenitor cell committed differentiation during 14 days of coculture; the results demonstrated that CD14 and CD33 expression in hUCBDSCs was significantly higher than their expression in hUCBDMSCs. This observation was also true for the granulocyte lineage marker, CD15. This marker was expressed beginning at day 7 in hUCBDSCs. It was expressed earlier and at a higher level in hUCBDSCs compared with hUCBDMSCs. In conclusion, hUCBDSCs are different from hUCBDMSCs. hUCBDSCs are superior to hUCBDMSCs in supporting hematopoiesis stem/progenitor cells differentiation into myeloid lineage cells at an early stage in vitro.

Factors influencing engraftment in HLA-haploidentical/mismatch related transplantation with combined granulocyte-colony stimulating factor-mobilized peripheral blood and bone marrow for patients with leukemia.

Unmanipulated HLA-haploidentical/mismatch related transplantation with combined granulocyte-colony stimulating factor-mobilized peripheral blood stem cells (G-PBSCs) and granulocyte-colony stimulating factor-mobilized bone marrow (G-BM) has been used as an alternative transplantation strategy for patients without an HLA-matched donor. In this transplantation setting, factors associated with hematopoietic recovery have not been defined completely. The aim of this study was to investigate the factors influencing the engraftment in this transplantation setting for patients with leukemia. The study group comprised 104 patients with leukemia who underwent transplantation at a single institution between 2005 and 2008. Factors correlating with neutrophil and platelet engraftment post-transplantation were analyzed retrospectively. All patients achieved an absolute neutrophil count of 500/µL with a mean time of 13.6days (range 8-20days) and a platelet count over 20×10(9)/L with a mean time of 20.2days (range 16-26days). In univariate analysis, donor and age were associated with increased risk of neutrophil engraftment, but their significance was lost upon multivariate analysis. The sex, age, donor, CD34(+) cell dose, conditioning regimen, mismatched locus, ABO mismatched and diagnosis have no effect on platelet engraftment. Our results suggest that it is an ideal approach to treat patients with leukemia with HLA-haploidentical/mismatched related transplantation with combined G-PBSCs and G-BM for a high level of stem cells without delayed engraftment.

Regulation of acute graft-versus-host disease by human umbilical cord blood derived stromal cells in haploidentical stem cell transplantation in mice through very late activation antigen-4.

Human umbilical cord blood derived stromal cells (hUCBDSCs), a novel resource isolated by our laboratory, have been shown to exert an immunologic regulation. Very late activation antigen-4 (VLA-4) has been associated with graft-versus-host disease (GVHD). This study aimed to investigate the possible mechanism by in vitro co-cultured splenocytes of donor mice with hUCBDSCs and in haploidentical stem cell transplantation in mice with acute GVHD. Both hUCBDSCs and human bone marrow stromal cells (hBMSCs) elicited decreased lymphocyte expression of VLA-4, but this decrease was stronger with hUCBDSCs than with hBMSCs (p<0.05). Cotransplantation of bone marrow with hUCBDSCs significantly decreased the expression of VLA-4 compared with control mice (p<0.05). A significant reduction of VLA-4 labeling in the target organs of GVHD was evident in haploidentical mice cotransplanted with hUCBDSCs. Our study shows that hUCBDSCs may protect mouse recipients of haploidentical stem cell transplantation from aGVHD via downregulating the expression of VLA-4.

Human umbilical cord blood-derived stromal cells, a new resource in the suppression of acute graft-versus-host disease in haploidentical stem cell transplantation in sublethally irradiated mice.

Human umbilical cord blood-derived stromal cells (hUCBDSCs), a novel population isolated from CD34(+) cells by our laboratory, exerted an immunosuppressive effect on xenogenic T cells. This study aimed to investigate whether hUCBDSCs play a critical role in the suppression of acute graft-versus-host disease (aGVHD). The hUCBDSCs were co-cultured with splenocytes (SPCs) of donor C57BL/6 mice. The aGVHD in the recipient (B6×BALB/c) F1 mice was induced by the infusion of bone marrow cells and SPCs from donor mice following sublethal irradiation. The shift in vivo for hUCBDSCs was detected. The proliferation and cell cycle of SPCs were tested by cell counting kit-8 and flow cytometry, respectively. The expression of CD49b natural killer (NK) cells and CD3 T cells was detected by flow cytometry in co-culture and post-transplantation. IL-4, and IFN-γ were detected by ELISA in the serum of co-culture and post-transplantation. The survival time, body weight, clinical score, and histopathological score were recorded for mice post-transplantation. The hUCBDSCs promoted the proliferation of SPCs and significantly increased the ratio of the S and G(2)/M phase (p < 0.05). The hUCBDSCs significantly increased the expression of CD49b NK cells and IL-4 protein and decreased the expression of CD3 T cells and IFN-γ protein both in vitro and in vivo. The survival time of mice with co-transplantation of hUCBDSCs was significantly prolonged, and decreased clinical and histopathological scores were also observed. The hUCBDSCs were continually detected in the target organs of GVHD. These results suggest that hUCBDSCs possess the capability of suppressing aGVHD, possibly via their influence on CD3 T cells, NK cells, and cytokines.

Human umbilical cord blood-derived stromal cells prevent graft-versus-host disease in mice following haplo-identical stem cell transplantation.

BACKGROUND AIMS: Human umbilical cord blood-derived stromal cells (hUCBDSC) comprise a novel population of CD34(+) cells that has been isolated in our laboratory. They have been shown previously not only to be non-immunogenic but also to exert immunosuppressive effects on xenogenic T cells in vitro. This study investigated the role of hUCBDSC in immunomodulation in an acute graft-versus-host disease (GvHD) mouse model after haplo-identical stem cell transplantation. METHODS: Acute GvHD was induced in recipient (B6 × BALB/c)F(1) mice by irradiation (750 cGy) followed by infusion of bone marrow cells and splenocytes from donor C57BL/6 mice. hUCBDSC were co-transplanted in the experimental group. The survival time, body weight and clinical and histopathologic scores were recorded after transplantation. The expression of surface markers [major histocompatibility complex (MHC) I, MHC II, CD80 and CD86] on CD11c(+) dendritic cells (DC), and the percentage of CD4(+) regulatory T cells (Treg), in the spleens of recipient mice were examined by flow cytometry. RESULTS: The survival time was significantly prolonged, and the clinical and histopathologic scores were reduced in mice co-transplanted with hUCBDSC. The expression levels of the surface markers on DC were significantly lower in mice transplanted with hUCBDSC compared with those without. The proportion of CD4(+) Treg in the spleen was also increased in mice transplanted with hUCBDSC. CONCLUSIONS: These results from a GvHD mouse model are in agreement with previous in vitro findings, suggesting that hUCBDSC possess immunosuppressive properties and may act via influencing DC and CD4(+) Treg.

Cost and outcome in stem cell collections in HLA-haplo identical/mismatched related transplantation with combined granulocyte-colony stimulating factor-mobilized blood and bone marrow for patients with hematologic malignancies.

Unmanipulated HLA-haplo identical/mismatched related transplantation with combined granulocyte-colony stimulating factor-mobilized peripheral blood stem cells (G-PBSCs) and granulocyte-colony stimulating factor-mobilized bone marrow (G-BM) has been developed as an alternative transplantation strategy for patients without an HLA-matched related or unrelated donor. In this transplantation setting, the cost and outcome of stem cell collections have not been defined completely. The aim of this study was to compare the cost and outcome of stem cell collection in HLA-haplo identical/mismatched related transplantation with combined G-PBSCs and G-BM to the HLA-identical/matched transplantation with G-PBSCs alone for patients with hematologic malignancies. Hundred and fifty-two healthy donors received twice-daily granulocyte-colony stimulating factor (G-CSF) subcutaneously for 5 days. The PBSCs were collected on day 4 and 5 of G-CSF treatment for HLA-identical/matched transplantation from unrelated/related donors. The PBSC collections and BM harvests was performed on day 4 and 5 of G-CSF treatment for HLA-haplo identical/mismatched related transplantation from related donors, respectively. There was no difference in the major characteristics between groups. More stem cells were harvested in HLA-haplo identical/mismatched related donors than that of HLA-identical/matched donors and a lower cost was seen in the former. The HLA-haplo identical/mismatched related transplantation with combined G-PBSCs and G-BM was a feasible approach with high cell harvest and low cost of stem cell collection for patients with hematologic malignancies.

Human bone marrow mesenchymal stem cells expressing SDF-1 promote hematopoietic stem cell function of human mobilised peripheral blood CD34+ cells in vivo and in vitro.

PURPOSE: There is mounting evidence demonstrating that stromal cell derived factor-1 (SDF-1) plays an important role in homing of hematopoietic progenitor cells to bone marrow. This study was aimed to assess whether bone marrow mesenchymal stem cells overexpressing exogenous SDF-1 could synergistically promote the homing of CD34(+) (Cluster of Differentiation [CD]) cells to bone marrow of lethally irradiated severe combined immunodeficiency (SCID) mice. METHODS: Human SDF-1 complementary Deoxyribonucleic acid (cDNA) was transfected into bone marrow-derived mesenchymal stem cells with recombinant lentiviral vector. The expression of SDF-1 was detected by real-time Polymerase Chain Reaction (PCR) and Enzyme-Linked Immunosorbent Assay (ELISA), and the ex vivo chemotaxis function on CD34(+) cells was measured by coculture system and Transwell system. SDF-1 gene-modified mesenchymal stem cell (MSC) and CD34(+) cells were infused into lethally irradiated SCID mice and the hematopoietic reconstitution in the recipient mice was examined. RESULTS: Messenger ribonucleic acid (mRNA) and protein of SDF-1 in infected MSC were significantly higher than that of the non-infected control MSC (p < 0.05). The infected MSC have significant chemotaxis effect on CD34(+) cells in vitro and promote hematopoietic reconstitution after CD34(+) cell transplantation in vivo. CONCLUSION: MSC with high-level expression of SDF-1 can synergistically promote hematopoietic reconstitution after CD34(+) cell transplantation in lethally irradiated SCID mice.

Human umbilical cord blood-derived stromal cells: multifaceted regulators of megakaryocytopoiesis.

It has been demonstrated that stromal cell precursors exist in human umbilical cord blood. After being cultured in vitro, these cells are called human umbilical cord blood-derived stromal cells (hUCBDSCs). However, the role of hUCBDSCs in hematopoiesis is still unclear. We have previously shown that hUCBDSCs are superior to human bone marrow stromal cells (hBMSCs) at enhancing the expansion of megakaryocyte colony forming units (CFU-Meg). Based on this observation, we postulated that hUCBDSCs might promote megakaryocytopoiesis. to test this hypothesis, we developed a megakaryocyte/hUCBDSC co-culture model and a hematopoietic microenvironment injury model in nude mice. We explored the ability and mechanisms by which hUCBDSCs promoted the proliferation of megakaryocytes in vitro, and we also explored their capacity to restore the hematopoietic microenvironment in vivo. As expected, hUCBDSCs were more efective than hBMSCs at enhancing the proliferation of megakaryocyte lines from HeL cells and restoring megakaryocytopoiesis in a hematopoietic microenvironment injury model in nude mice. thrombopoietin (tpo) and stromal cell derived factor-1 (SDF-1) are two of the key factors underlying this capacity. We also found that gap junction intercellular communication (GJIC) between HeL cells and hUCBDSCs might be partially absent. our data provide the first evidence that hUCBDSCs play a regulatory role during megakaryocytopoiesis, which might be important for designing treatments for patients with megakaryocytic injury.

[Intracerebral infiltration by monoclonal plasmacytoid cells in Waldenstrom's macroglobulinemia: a case report and review of the literature].

OBJECTIVE: To sum up the clinical experience of the diagnosis and treatment of intracerebral infiltration by monoclonal plasmacytoid cells in Waldenstrom's macroglobulinemia(Bing-Neel syndrome). METHODS: The clinical data of the diagnosis and treatment of a case of Bing-Neel syndrome was analyzed. RESULTS: A 56-year-old male was diagnosed as Waldenstrom's macroglobulinemia one year ago, and presented with persistent headache during the treatment period. Magnetic resonance imaging showed a high intensity area on T2-weighed images in the right frontal lobe which was well enhanced by gadolinium-diethylenetriaminepenta-acetic acid. Infiltration of neoplastic cells was confirmed by biopsy. Immunohistochemical examination showed that mature plasmacytoid cells in the cerebral parenchyma were immunoglobulin M positive. CONCLUSION: Infiltration in CNS (Bing-Neel syndrome) is uncommon in Waldenstrom's macroglobulinemia. As there is no effective therapy for this Bing-Neel syndrome, combination of radiation and chemotherapy should be considered for this situation.

Human umbilical cord blood-derived stromal cells suppress xenogeneic immune cell response in vitro.

AIM: To explore immunological properties of human umbilical cord blood-derived stromal cells (hUCBDSC) and their effect on xenogeneic immune cells in vitro. METHODS: Immunological phenotype of freshly isolated and cryopreserved hUCBDSCs was evaluated by flow cytometry. Xenogeneic splenic T-cells were stimulated by phytohemaglutinin A (PHA) or dendritic cells in the absence or presence of hUCBDSCs. T-cell proliferation was measured by cell counting kit-8 after 7-day incubation. The proportion of apoptotic cells and CD4+CD25+Foxp3+ regulatory T-cells (Tregs) was determined in T-cells activated by PHA in the absence or presence of hUCBDSCs by flow cytometry. Phenotype of dendritic cells, cultured alone or with hUCBDSCs, was analyzed by flow cytometry. RESULTS: Levels of immune molecule expression on freshly isolated hUCBDSCs were as follows: human leukocyte antigen-I (HLA-I) (84.1+/-2.9%), HLA-II (1.6+/-0.3%), CD80 (0.8+/-0.1%), CD86 (0.8+/-0.1%), CD40 (0.6+/-0.1%), and CD40L (0.5+/-0.1%), which was not influenced by cryopreservation. T-cell proliferation in the presence of hUCBDSCs was significantly lower than that of positive control. The coculture led to a 10-fold increase (from 1.2+/-0.3% to 12.1+/-1.4%, P<0.001) in the proportion of CD4+CD25+Foxp3+ regulatory T-cells (Tregs) and a reversion of mature dendritic cells, as indicated by the down-regulation of major histocompatibility complex (MHC)-II molecule (49.3% vs 25.9%, P=0.001), CD80 (47.2% vs 23.3%, P=0.001), and CD86 (40.6% vs 25.1%, P=0.002). When subjected to annexin V binding and propidium iodide uptake assay, the hUCBDSCs did not show the ability to induce apoptosis of xenogeneic T-cells. CONCLUSION: These results demonstrate low immunogenicity and immunomodulation effect of the hUCBDSCs. Reversion of mature dendritic cells and increase in Treg proportion, but not cell apoptosis, can possibly contribute to the suppression of xenogeneic T-cell proliferation by the hUCBDSCs.

[Contribution of SDF-1/CXCR4 axis on proliferation of megakaryocyte co-cultured with human umbilical cord blood-derived stromal cells].

In order to investigate the effect of stromal cell derived factor-1 (SDF-1)/CXCR4 on the proliferation of megakaryocytic line-HEL cells co-cultured with human umbilical cord blood-derived stromal cells (hUCBSCs) and to further elucidate the mechanism of SDF-1/CXCR4-mediated functions, the HEL cells were co-cultured with hUCBSCs or human bone marrow stromal cells (hBMSCs), the suspended HEL was used as control. The concentrations of SDF-1 in supernatant of hUCBSCs and hBMSCs were detected by ELISA assay. The expression of CXCR4 membrane-bound protein of HEL cells was detected by laser confocal scanning microscopy and flow cytometry, and the expression of CXCR4 mRNA was detected by RT-PCR. The result showed that the concentrations of SDF-1 in different groups were the same at the early stage of culturing. But at 6 days after seeding, the concentrations of SDF-1 increased significantly in the hUCBSCs group, even though the passage was done. By means of laser confocal microscopy, the expression of CXCR4 protein and also red dots of fluorescence could be detected in cytoplasm of HEL cells co-cultured with hUCBSCs. However, there was no significant differences of the CXCR4 mRNA level between different groups (p > 0.05). It is concluded that hUCBSCs may play important roles in secreting high level of SDF-1 and regulating megakaryocyte expression of CXCR4.

HLA-haploidentical blood and bone marrow transplantation with anti-thymocyte globulin: long-term comparison with HLA-identical sibling transplantation.

We present an update of our results regarding related HLA-haploidentical and HLA-identical sibling hematopoietic stem cell transplantation (HSCT) in patients with leukemia. We compared the outcomes of 107 patients with leukemia undergoing HLA-identical sibling (n=51) or related HLA-haploidentical (n=56) HSCT performed during the same time period. Patients received BU-CY/CY-TBI in HLA-identical sibling HSCT or TBI+Ara-C+CY+ATG/CCNU+Ara-C+Bu+CY+ATG in haploidentical HSCT as conditioning regimens, followed by unmanipulated marrow and/or peripheral blood (PB) transplantation. All patients achieved full engraftment. The cumulative incidence of grades II through IV acute graft-versus-host disease (aGvHD) in the matched and haploidentical cohorts was 13.7% and 26.8% (P<0.05), respectively. The risk of developing cGvHD was not different between HLA-matched and haploidentical patients (P>0.05). The two-year incidence of treatment-related mortality for matched and haploidentical patients was 7.8% and 12.5%, respectively, with P>0.05, and the incidence of relapse was 17% and 22%, respectively, with P>0.05. The two-year adjusted leukemia-free survival for matched versus haploidentical patients was 76% and 68%, respectively, with P>0.05, and the overall survival for matched versus haploidentical patients was 80% and 70%, respectively, with P>0.05. Multivariate analyses showed that only advanced disease stage and a diagnosis of acute leukemia were related to increased risk of relapse, treatment failure, and overall mortality. In summary, HSCT performed with related HLA-haploidentical donors is a feasible approach with acceptable outcomes.

Role of antithymocyte globulin and granulocyte-colony stimulating factor-mobilized bone marrow in allogeneic transplantation for patients with hematologic malignancies.

The main obstacle for allogeneic transplantation is delayed hematologic reconstitution and serious graft-versus-host disease (GVHD). The results of 128 patients with hematologic malignancies undergoing HLA-identical (n=52) or HLA-haploidentical/mismatched (n=76) hematopoietic stem cell transplantation (HSCT) performed during the same time period were compared. Patients with HLA-identical HSCT received unmanipulated granulocyte-colony stimulating factor-mobilized peripheral blood stem cells (G-PBSCs). Forty-six patients with HLA-haploidentical related HSCT received antithymocyte globulin (ATG) in conditioning regimens followed by the transplantation of the combination of unmanipulated G-PBSCs and granulocyte-colony stimulating factor-mobilized bone marrow (G-BM) and 30 patients with HLA-mismatched unrelated HSCT received ATG in conditioning regimens followed by the transplantation of unmanipulated G-PBSCs. All patients got successful hematopoietic engraftment. The cumulative incidences of grades I to II acute GVHD (aGVHD) on day 100 in the identical, haploidentical related and mismatched unrelated cohorts were 21.2%, 43.5%, and 53.3%, respectively. The cumulative incidences of chronic GVHD (cGVHD) in the identical, mismatched unrelated, and haploidentical related cohorts were 34.6%, 33.3%, and 10.9%, respectively. The 2-year relapse and treatment-related mortality (TRM) rates were 19.2%, 23.9%, 23.3%, and 9.6%, 8.7%, 10% for patients who underwent identical, HLA-haploidentical related, and mismatched unrelated transplantation, respectively. The 2-year probabilities of leukemia-free survival and overall survival were 72.2%, 70.6%, 68.1%, and 76.5%, 77.8%, 70.0% after identical, haploidentical related and mismatched unrelated transplantations, respectively. Multivariate analyses showed that only advanced disease stage and a diagnosis of disease had increased risk of relapse, treatment failure, and overall mortality. In conclusion, it is a feasible approach with acceptable outcomes for patients undergoing HLA-haploidentical related HSCT by the combination of G-PBSCs and G-BM with conditioning regimens including ATG.

[Influence of different gelatin concentration and lymphocyte isolation liquid on primary culture of umbilical cord blood derived adhesive cells].

In order to study the influence of different gelatin concentrations, and lymphocyte isolation liquid on primary culture of umbilical cord blood-derived adhesive cells (hCBACs), the red blood cells of umbilical cord blood was separated by 3% and 6 % gelatin for detecting the effectiveness of sedimentation, then the adhesion rate at 48 hours, the day of initial expansion and the rate of culture success were detected for hCBACs cultured with CD34(+) cells after the mononuclear cells were separated by 6% gelatin followed by Ficoll and Percoll, and the morphological characteristics and growth status were observed by invert microscopy. Cytochemistry stain for nonspecific esterase stain (NSE), peroxidase (POX), periodic acid Schiff reaction (PAS) and alkali phosphatase (ALP) and immunocytochemistry labeling for CD31, CD45, CD68 and fibronectin (Fn) were detected. The results showed that 6 % gelatin was better than that 3% gelatin for red blood sedimentation. The Percoll was predominant over Ficoll in adhesion rate at 48 hours, the day of initial expansion, the time of initial formation of adhesive cell colony units, the time of maximal numbers of adhesive cell colony units, the the cell fusion time and ratio of culture success. 60% fibroblast-liked cells, 36% macrophage liked cells and 4% small-round cells were observed in cells isolated by both isolated methods. The cytochemistry stain for NSE, POX, PAS and ALP was similar in two groups, the difference was not statistically significant between these two groups. The immunocytochemistry labeling for CD31, CD45, CD68 and Fn was also similar in both groups and the difference was also not statistically significant between these two groups. It is concluded that the combination of 6% gelatin with Percoll is an ideal separation method for primary culture of hCBACs, which provides basic information for clinical application.