Clinical study of unrelated umbilical cord blood transplantation for 25 patients with hematological diseases / 中国实验血液学杂志

This study was purposed to investigate the engraftment, graft-versus-host disease (GVHD), transplantation related mortality (TRM), relapse and survival in hematologic patients received unrelated umbilical cord blood transplantation (UCBT). A total of 25 patients with hematological disease underwent UCBT, including 8 pediatric and 17 young adult patients. Among them 3 cases received single unit of UCBT and 22 cases received double units of UCBT. For donor/recipients human leukocyte antigen (HLA) matching: HLA 6/6 loci matched in 9 cases, HLA 4-5/6 loci matched in 16 cases. There were 19 patients with hematologic malignancies, including 3 cases in the period of disease progression and 6 cases of non-hematologic malignancies. Conditioning regimens were TBI/Cy ± Flu ± ATG or BuCy ± Flu ± ATG for 21 patients and Cy+Flu+ATG for 4 patients. For prophylaxis of acute graft-versus-host disease (aGVHD) the regimen of cyclosporine (CsA) as dominant drug was used. The results showed that among 16 patients (80.0%) achieved engraftment, 20 patients survived for more than 42 d after transplantation. The cumulative neutrophil recovery rate on day 42 after transplant was 64.0%, with a median time of 17.0 d;the cumulative platelet recovery rate on day 100 after transplant was 60.0 %, with a median time of 35.0 d. The cumulative rate of grade II-IV and III-IV aGVHD after transplantation 100 d was 44.0% and 30.7%, respectively. Until the end of the follow-up, the cumulative rate of TRM was 54.3%. For all the patients, overall survival rate was 42.7%. Out of 17 evaluable patients with hematologic malignancies 7 cases (41.2%) survived to date, and only 1 case relapsed, so event-free survival rate was 35.3%. Out of 5 evaluable patients with non-hematologic malignancies, 4 patients survived and 2 patients were in stable engraftment state, 2 cases with autologous hematopoietic recovery. Among 3 cases of hematologic malignancies at advanced stage, only 1 case survived to date. It is concluded that HLA-4-6/6 loci matched UCBT is an effective option to treat hematological diseases. Double cord blood transplantation (dUCBT) can overcome the disadvantage of insufficient cells of single cord blood UCBT to treat overweight children and adult.

Correlation of the level of Reg3α protein in plasma with gastrointestinal acute graft-versus-host disease / 中国实验血液学杂志

This study was purposed to explore the correlation of regenerating Islet-derived 3-alpha(Reg3α) protein level in plasma with the diagnosis and prognosis of the gastrointestinal acute graft-versus-host disease (GI-aGVHD) after all-HSCT, 103 patients who underwent allogeneic hematopoietic stem cell transplantation (allo-HSCT) were observed in our hospital from December 2011 to December 2012. Peripheral blood samples were routinely collected at 9 d before allo-HSCT, 0 d, 14 d, 28 d after allo-HSCT as well as in aGVHD and at the 1 and 4 weeks after aGVHD therapy. The plasma concentrations of Reg3α were measured by using ELISA kit. The results indicated that among the 103 patients, 17 cases never developed aGVHD symptoms (no-aGVHD), 27 cases presented with non-aGVHD associated diarrhea, 10 cases presented with isolated skin aGVHD, 17 cases developed grades I-II GI-aGVHD, 32 cases with grades III-IV GI-aGVHD. The plasma concentrations of Reg3α in group of patients with GI-aGVHD and group of non-aGVHD diarrhea were 111.5 (54.7-180.2) and 23.9 (14.5-89.5) ng/ml respectively with significant difference (P < 0.001). The plasma concentrations of Reg3α in 17 patients of grades III-IV GI-aGVHD who experienced a complete or partial response and 7 patients who had no response to therapy at 4 weeks were 137.2(51.7-205.4) and 679.4(122.3-896.8) ng/ml respectively with the significant difference (P = 0.028). All of the patients who had no response to therapy died of aGVHD associated multiple organ failure. The area under the ROC curve was 0.902 when plasma concentration of Reg3α was set at 87.73 ng/ml. The sensitivity was 81.48% and the specificity was 82.86% when the critical value was used in diagnosis of grades III-IV GI-aGVHD. The probability of grades III-IV GI-aGVHD had statistical difference above and below 87.73 ng/ml after allo-HSCT (P < 0.001). It is concluded that the increase of plasma Reg3α level after transplantation suggests the incidence of grades III-IV GI-aGVHD. The high level of plasma Reg3α protein in patients with grades III-IV GI-aGVHD after the immunosuppressive treatment for four weeks indicates a poor prognosis. The plasma concentrations of Reg3α can be used as a specific biomarker of GI-aGVHD.

Current advance in the treatment of viral infections by cytotoxic T-lymphocytes after allogeneic hematopoiefic stem cell transplantation / 白血病·淋巴瘤

Viral infections remain the common cause of morbidity and mortality in immunocompromised patients receiving allogeneic hematopoietic stem cell transplantation (allo-HSCT).Virus antigens can be recognized by cytotoxic T-lymphocytes (CTL) in the context of HLA class Ⅰ molecules.T-cell-based immunotherapies are now being included in the clinical practice of transplant recipients to prevent and treat viral infections and complications associated with CMV,AdV and EBV.Here,the current status and future feasibility of CTL immunotherapy for virus infections after allo-HSCT are discussed.

Current research advance on cellular immunotherapy for leukemia-review / 中国实验血液学杂志

Despite the chemotherapy is successful in inducing remission of hematologic malignancy, this disease also has a high probability of relapse; besides, the toxicity of chemotherapy for these patients can not be avoided. Researchers have been attempting to eliminate tumor cells by immunotherapy. Recently, various leukemia-associated antigens (LAA) that are recognized by cytotoxic T cell (CTL) in the context of HLA class I molecules have been identified. These LAA include WT1, PR-3, RHAMM, BCR-ABL and Aur-A. On the basis of these findings, various clinical trials of immunotherapy for hematologic malignancy including tumor peptide vaccination, adoptive T cell therapy, NK cell therapy and dendritic cells-cytokine induced killer (DC-CIK) cell therapy are on going. In this review, the current status and future feasibility of cellular immunotherapy for leukemia are discussed.

Ex vivo inducing cultured Epstein-Barr virus specific cytotoxic T lymphocytes and evaluation of their killing effect / 中国实验血液学杂志

This study was aimed to explore the method for induction and expansion of EB virus specific cytotoxic T lymphocytes (EBV-CTL) in vitro, and to detect their killing effect. Peripheral blood mononuclear cells (PBMNC) were collected from 6 EBV seropositive healthy donors, and EBV-transformed B lymphoblastoid cells (BLCL)were used as the antigen-presenting cells and antigen stimulant which was irradiated by 40 Gy (60)Co irradiator. The autologous PBMNC and irradiated BLCL were cultured to induce and expand the EBV-CTL, and the immunophenotype was identified by the flow cytometry. The killing effect of the EBV-CTL against the autologous BLCL (autoBLCL), the autologous PHA cultured B lymphoblastoid cells( PHA-BLCL), the allogeneic BLCL (alloBLCL) and the K562 cells were measured with LDH release assay under different effector-to-target ratio. The results showed that the 6 cell lines of EBV-CTL were induced and expanded from the EBV seropositive healthy donors, the overall increase in cell numbers varied from 18.6 to 55.0 times. After 10 stimulations, the specific killing efficiency of the EBV-CTL for the autoBLCL were 59.4%, 43.2% and 29.0% under the effector-to-target ratio of 20: 1, 10: 1 and 5: 1. The nonspecific killing efficiency for the PHA-blast, alloBLCL and K562 cells were 7.1%, 9.4% and 10.3% (P < 0.05) under the 20: 1 ratio; 6.6%, 8.3% and 8.1% (P < 0.05) under 10: 1; 5.4%, 7.3% and 6.3% (P < 0.05) under 5: 1, respectively. It is concluded that the EBV-CTL can be successfully induced and expanded ex vivo for specific killing of HLA matched BLCL and may become a potential treatment for EBV related post-transplant lymphoproliferative disorders.

Clinical study of umbilical cord-derived mesenchymal stem cells for treatment of nineteen patients with steroid-resistant severe acute graft-versus-host disease / 中华血液学杂志

<p><b>OBJECTIVE</b>To evaluate the safety and efficacy of umbilical cord-derived mesenchymal stem cells (MSCs) infusion in patients with steroid-resistant severe acute graft-versus-host disease (aGVHD).</p><p><b>METHODS</b>A total of 19 patients with steroid-resistant severe aGVHD received MSCs infusion treatment. The treatment response, transplantation-related mortality, events associated with infusion and relapse rate were analyzed.</p><p><b>RESULTS</b>Two patients with grade II, 5 patients with grade III and 12 patients with grade IV aGVHD received a total of 58 infusions of MSCs. The mean total dose of MSCs was 2.13 (range 0.60 - 7.20)×10(6) cells per kg bodyweight. Seven patients received one infusion, 2 patients received two infusions, and 10 patients received three or more infusions. Eleven patients had a complete response and 4 had a partial response and 4 had no response. No patients had side-effects during or immediately after infusions, and no MSCs related tumorigenesis was detected to date. Eleven patients survived and 8 died, 4 for aGVHD, 1 for infection and 2 for aGVHD with concomitant infection and 1 for underlying leukemia relapse. The cell viability of freshly prepared MSCs is 93% (92% - 95%) by trypan blue staining. The cell viability of programmatically frozen and thawed MSCs is 72% (70% - 74%).</p><p><b>CONCLUSION</b>Infusion of umbilical cord-derived MSCs expanded in vitro is an effective therapy for patients with steroid-resistant severe aGVHD without negative impact on relapse. Freshly prepared MSCs are superior to frozen and thawed cells in terms of cell viability.</p>

The chimerism status of bone marrow mesenchymal stem cells following double-unit cord blood stem cell transplantation / 中华器官移植杂志

ObjcetiveTo study the chimerism status ot bone marrow mesenchymal stem cells (BMSC) following double-unit cord blood stem cell transplantation (DCBT).MethodsBone marrow mononuclear cells were isolated by ficoll density gradient centrifugation from the patient following DCBT and healthy donors.After cultured for about 20 days,BMSC surface makers,the ability to differentiate into adipocytes and osteoblast and the expression of hematopoietic and immune molecules were detected,moreover,chimerism status of BMSC were also evaluated by STR-PCR.ResultsMSC derived from the patient were similar to normal adult BMSC in morphology,surface and differentiation ability.Besides,MSC from the patient can express a variety of hematopoietic and immune molecules normally.The results of STR-PCR showed that the patient demonstrated complete donor chimerism,hematopoiesis in BM and PB were from a single unit cord blood (donor chimerism was 96.4％ and 95.7％,respectively).But the predominant cord blood chimerism of BMSC was 5.4％,the remaining part was from the patient himself.ConclusionSustained hematopoiesis was derived from a single unit cord blood following DCBT.The majority of BMSC were origin from host tissue,mixed chimerism donor part of MSC stemed from the engrafted unit of cord blood.

Diagnosis and treatment procedures of congenital neutropenia / 中国实验血液学杂志

To explore the reasonable procedures and strategies of diagnosis and treatment of congenital neutropenia (CN), clinical data and laboratory examination results of a boy suspected of CN were collected; gene ELA2, GFI1, HAX1, and WASp of whom were sequenced, granulocyte colony-stimulating factor receptor (G-CSFR) expression on neutrophil was analyzed, and cytoplasmic domain of G-CSFR was sequenced. The results showed that the diagnosis of non-syndromic variants of CN (NSVCN) was made on this patient according to the criteria; sequencing results revealed no mutation occurred in ELA2, GFI1, HAX1 and WASp; a normal expression level of G-CSFR on neutrophil from this patient was detected and no truncated mutation was found in the intracellular domain of G-CSFR. It is concluded that reasonable procedure of diagnosis and treatment of CN is established, and a sporadic NSVCN with no recognized pathogenic mutation is confirmed in this patient.

Biological characteristics of bone marrow mesenchymal stem cells and JAK2 mutation in myeloproliferative neoplasms / 中华血液学杂志

<p><b>OBJECTIVE</b>To study the biological characteristics of bone marrow mesenchymal stem cells (BMSCs) and detect JAK2 mutation in BMSCs from myeloproliferative neoplasms (MPN) patients.</p><p><b>METHODS</b>JAK2 V617F mutation and exon 12 mutation in 70 MPN patients' blood or bone marrow samples were detected. Isolated BMSCs were then characterized their phenotype, mesenchymal differentiation capacity and existence of JAK2 mutation.</p><p><b>RESULTS</b>BMSCs derived from the patients were similar with healthy donors in terms of morphology, surface antigen and differentiation ability. Of them, 38 patients' blood or bone marrow samples harbored JAK2 V617F, and identified that 3 V617F-negative-patients' samples existed JAK2 exon 12. No patients' BMSC harbored JAK2 mutation though their blood or bone marrow samples carried JAK2 mutation.</p><p><b>CONCLUSION</b>BMSCs from MPN patients had similar biological characteristics with healthy donors. BMSCs from MPN patients known to bear JAK2 mutation in blood or bone marrow cells didn't carry the mutation.</p>

Comparison of the biological characteristics of serum-free and fetal bovine serum-contained medium cultured umbilical cord-derived mesenchymal stem cells / 中华血液学杂志

<p><b>OBJECTIVE</b>To compare the differences of biological characteristics between human umbilical cord-derived mesenchymal stem cells (UC-MSCs) cultured by serum-free medium or fetal bovine serum-contained complete medium to establish a xenogeneic protein-free UC-MSCs culture system.</p><p><b>METHODS</b>Healthy human umbilical cord segments were digested with collagenase. UC-MSCs were cultured by serum-free MesenCult-XF medium and FBS-based αMEM complete medium, then analyzed the morphology, immunophenotype, expansion potential, lineage differentiation potential, karyotype and immunosuppression of early passages.</p><p><b>RESULTS</b>The average cell diameters of UC-MSCs in suspension cultured by serum-free medium and FBS-based medium were 26 (18 - 39) µm and 35 (20 - 61) µm, respectively. Cell expansion folds with serum free medium and FBS-based medium were (5.2 ± 0.2) and (3.5 ± 0.1) respectively, in the first five passages. The expansion potential of serum-free medium cultured UC-MSCs was significantly higher than FBS-based medium cultured ones (P < 0.05). A panel of markers CD29, CD44, CD90, CD73, CD105 and HLA-ABC expressed on human UC-MSCs. Hematopoietic lineage markers CD34, CD45 and HLA-DR were not detectable on UC-MSCs. The cpm were (4.57 ± 0.14)×10(4), (2.04 ± 0.16)×10(4) and (0.42 ± 0.04)×10(4), respectively when serum-free medium cultured MSCs were added to the cultures at MSCs/T cell ratios of 1:100, 1:10 and 1:5. While the cpm was (4.57 ± 0.14)×10(4), (2.04 ± 0.16)×10(4) and (0.42 ± 0.04)×10(4), respectively when serum-free medium cultured UC-MSCs were added to the cultures. The immunosuppressive potential of serum-free medium-cultured UC-MSCs was higher than serum-contained medium cultured ones at three different MSC/T cell ratios (P < 0.05).</p><p><b>CONCLUSION</b>Compare with serum-contained medium cultured early passages of UC-MSCs, the cell diameter of serum-free medium cultured UC-MSCs was smaller with higher expansion potential. No xenogeneic proteins were presented in UC-MSCs preparations when cultured with serum-free medium. Human UC-MSCs suppressed T-cell proliferation in a dose-dependent manner. The immunosuppressive potential of serum-free medium cultured UC-MSCs was higher than FBS-based medium cultured ones.</p>

Culture and pluripotentiality of murine compact bone-derived mesenchymal stem cells / 中国实验血液学杂志

This study was purposed to culture murine compact bone-derived mesenchymal stem cell (MSC) and analyze the immunological and trilineage differentiation potential. Tibia and femur were extracted. Bone marrow cells were flushed out and compact bone fragments were digested with collagenase. The digested cells were cultured in 6-well plates. The immunophenotype, immunosuppressive function and trilineage differentiation potential were analysed by flow cytometry, mixed lympocyte reaction and Oil red O, von Kossa and alcian blue straining, respectively. The results indicated that the pure compact bone MSC could be isolated with in 3 weeks. The resulting MSC had trilineage differentiation potential and immunosuppressive effect on mixed lymphocyte reaction. The count per minute (CPM) value in control group of BALB/c T cells cocultured with irradiated C57BL/6 T cells was (2.56 ± 0.31) × 10(4), while CPM values of mixed lymphocyte cocultured with C57BL/6 compact bone MSC at ratios of 100:1 and 10:1 were (0.47 ± 0.12) × 10(4) and (0.28 ± 0.09) × 10(4). The CPM value of control group was higher than those of MSC cocultured group (P < 0.001). Compact bone-MSC had an immunosuppressive effect on mixed lymphocyte reaction in a dose dependent manner. It is concluded that murine compact bone has rich MSC and the primary MSC is contaminated with less hematopoietic cells. Murine compact bone-MSC have immunosuppressive effect on mixed lymphocyte reaction and trilineage differentiation potential. Compact bone-MSC have promising experimental study value.

Genetic diagnosis of a patient with non-syndromic variants of congenital neutropenia / 中华内科杂志

ObjectiveTo explore the procedures and methods for genetic diagnosis in one nonsyndromic variants of congenital neutropenia (NSVCN) patient and its pathogenic mutation.Methods Genomic DNA was prepared from one NSVCN patient who had progressed to chronic myelomonocytic leukemia and ELA2,HAX1,WASp and GFI1 genes were amplified and sequenced.Results A novel compound heterogeneous mutation consisting of two frame-shift mutations (c.430-1insG and c.655- 9del5bp) was found in HAX1 gene.ConclusionA practically genetic diagnosis procedure for NSVCN has been established,and the novel HAX1 gene mutation may contribute to the etiology of NSVCN.

Analysis of tyrosine kinases gene mutations in core binding factor related acute myeloid leukemia and its clinical significance / 中华血液学杂志

<p><b>OBJECTIVE</b>To assess the prevalence of several tyrosine kinases (TKs) gene mutations including c-Kit, FLT3 and JAK2 V617F in core binding factor related acute myeloid leukemia (CBF-AML), and analyze their impact on clinical characteristics and prognosis.</p><p><b>METHODS</b>Mutations of c-Kit, FLT3-ITD and FLT3-TKD were detected by genomic DNA PCR and sequencing, and JAK2 V617F mutation screening by allele-specific PCR in 58 newly diagnosed CBF-AML patients [28 AML with inv(16) and 30 with t(8;21)], and analyze the patients clinical characteristics and prognoses.</p><p><b>RESULTS</b>c-Kit aberrations were detected in 32.8% cases, including 6 cases mutated in exon 8 (mutKIT8) and 13 mutated in exon 17 (mutKIT17). MutKIT8 was more prominent in inv(16) than in t(8;21) patients (21.4% vs 0, P = 0.009). Only 2 cases had FLT3-ITD and 7 (12.1%) FLT3-TKD mutations. The result of JAK2 V617F mutation screenings in these CBF-AML patients was negative. The frequency of receptor tyrosine kinases(RTK) mutations was 46.6% and only one case had two kinds of missense mutations (mutKIT8 & TKD(+)). Median age of onset was higher for mutKIT17 than for wide-type c-Kit (wtKIT) patients (55 vs 31, P = 0.003). c-Kit mutations were significantly associated with decreased overall survival (OS) and continuous complete remission (CCR) rates (P = 0.053, and 0.048 respectively), and so did more for exon17 mutated patients reduced (P = 0.005, and 0.013 respectively). FLT3-TKD mutation showed no effects on prognosis of CBF-AML patients.</p><p><b>CONCLUSIONS</b>RTK mutations are common in patients with CBF-AML. c-Kit mutations frequently and JAK2V617F mutation rarely appear in CBF-AML. c-Kit mutations, especially mutKIT17 confers higher relapse risk and poorer prognosis.</p>