The first suspected disseminated Hormographiella aspergillata infection in China, diagnosed using metagenomic next-generation sequencing: a case report and literature review.

Hormographiella aspergillata is a rare and emerging cause of invasive mould infections in patients with haematological malignancies, with a mortality rate of approximately 70%. Here, we present the first reported case of suspected disseminated H. aspergillata infection in China. The patient experienced a second relapse of acute myeloid leukaemia and developed neutropenia, fever, discrepant blood pressure between limbs, and cutaneous lesions limited to the left upper extremity. Since lung tissue biopsy was not feasible, metagenomic next-generation sequencing (mNGS) and panfungal polymerase chain reaction (PCR) analysis of bronchoalveolar lavage fluid and blood samples were performed, which indicated probable H. aspergillata pulmonary infection. Histopathology of cutaneous lesions revealed numerous fungal hyphae within dermal blood vessels. mNGS of a skin biopsy sample identified H. aspergillata sequences, and the fungi was subsequently recovered from fungal culture, proving cutaneous H. aspergillata infection. Despite combined antifungal therapy, the patient died owing to disease progression. Additionally, 22 previously reported cases of invasive H. aspergillata infection were reviewed in patients with haematological malignancies. Thus, mNGS is a powerful diagnostic tool for the early and effective detection of invasive H. aspergillata infections, with the advantage of sequencing all potential pathogens, and providing results within 24 h.

[Clinical Analysis of Post-Transplant Lymphoproliferative Disorder after Hematopoietic Stem Cell Transplantation in Severe Aplastic Anemia Patients].

OBJECTIVE: To analyze the clinical characteristics of SAA patients with post-transplantation lymphoproliferative disease (PTLD) after allogeneic hematopoietic stem cell transplantation, and to improve diagnosis and treatment of PTLD. METHODS: The clinical data of 192 patients with SAA patients who underwent HSCT in a single center from September 2010 to September 2017 were analyzed retrospectively. All patients were received antithymocyte globulin（ATG） conditioning regimen and mesenchymal stem cell(MSC) infusion. RESULTS: Among 192 cases, PTLD occurred in 14 cases, the incidence was 7.29％， 9 of them were diagnosed by pathology， and 5 were diagnosed clinically. EBV infection occurred with a median time of 72（35-168） days, Viral load higher than 1×104 copies/ml occured in all PTLD patients. The incidence of probable PTLD in patients ≤12 years old and ＞12 years old was 11.11%, 2.38%, respectively (P<0.01）. Univariate and multivariate analysis that the EBV infection, patients age≤12 years old, HLA-mismatch in URD-HSCT, grade II to IV aGVHD were the independent risk factors for PTLD. All PTLD patients were treated with rituximab(RTX) when EBV-DNA load higher than 1×104 copies/ml, or reducted the use of immunosuppression(RIS), patients with poor therapeutic effect were treated combined with EBV-specific CTLs(EBV-CTL) and chemotherapy. All patients were treated effectively, and the total effective rate was 100％. The median follow-up time was 65(62-115) months, and the overall survival rate was 92.85%. One patients died of cerebral hemorrhage, 7 months after PTLD curred. CONCLUSION: The incidence of PTLD after HSCT with SAA who used ATG and MSC in conditioning regimen closely relates to EBV infection, age of patients≤12 years, HLA-mismatch in URD-HSCT, grade II to IV GVHD. Rituximab combined with RIS may reduce the incidence of PTLD, combined EBV-CTL and chemotherapy may be the useful and most important treatment for PTLD.

[Haploidentical Hematopoietic Stem Cell Transplantation with Co-Infusion of Mesenchymal Stromal Cells for Acquired Severe Aplastic Anemia: A Report of 127 Cases].

OBJECTIVE: To analyze the prognostic factors of haplo-HSCT combined with MSC in the treatment of SAA. METHODS: 127 SAA patients who had undergone haplo-HSCT with co-infusion of MSC in our center from January 2014 to August 2019 were analyzed retrospectively. Median age was 11 (1-37) years, and median follow-up time was 39.8 (1-74) months. RESULTS: The median time for neutrophil and platelet engraftment was 14 d and 18 d respectively. The cumulative incidences of grade III-IV aGVHD was 4.4%±1.9% at day +100. The 2-year cumulative incidence of extensive cGVHD was 8.3%± 2.7%. The estimated 3-year OS was 86.1%±3.1%. Univariate analysis showed that high-dose CD34+ cells (>6.69×106/kg) could promote the engraftment of neutrophil (97.9%±0.05% vs 88.6%±0.13% at day +21, P=0.0006) and platelet (81.2%±0.33% vs 70.8%±0.26% at day +28, P=0.002) and did not increase the incidence of aGVHD (10.4%±0.1% vs 18.9%±0.1% at day +100, P=0.18). More nucleated cells (>12.78×108/kg) caused a lower incidence of grade II-IV aGVHD (8.6%±0.13% vs 21.7%±0.25% at day+100, P=0.04) and a higher incidence of 3-year OS (91.3%±3.2% vs 78.1%±6.5%, P=0.03) than less nucleated cells (≤12.78×108/kg). Younger patients (age≤12 y) had faster neutrophil engraftment (94.9%±0.06% vs 87.5%±0.24% at day+21, P=0.02), higher 3-year OS (93.6%±2.8% vs 75.9%±6.4%, P=0.006) and higher 3-year FFS (93.6%±2.8% vs 68.3%±7.1%, P=0.000) than older patients (age>12 y). The shorter the time from diagnosis to HSCT (≤29.5 months), the higher the 3-year FFS of patients (88.8%±3.5% vs 74.2%±7.2%, P=0.028). Male patients with female donors had higher cumulative incidence of extensive cGVHD than others (20.0%±0.8% vs 4.6%±0.1%, P=0.01). CONCLUSION: In the haplo-HSCT of SAA, the prognosis of children patients is better than that of adults patients. More CD34+ cells and nucleated cells can promote engraftment, reduce the incidence of aGVHD and improve OS. HSCT should be performed as early as possible, and the occurrence of cGVHD should be reduced in male patients by avoiding female donors.

[Clinical Safety of NK Cell in the Prevention of Leukemia Relapse Post-transplantation and in Treatment of the Elderly Leukemia Patients].

OBJECTIVE: To observe the safety of donor NK cell infusions in the settings of hematopoietic stem cell transplantation and after consolidation chemotherapy in elderly patients with acute myeloid leukemia (AML). METHODS: Forty patients with AML were included, in which 21 patients aged over 60 years were at the stage of complete remission (CR) and 19 patients that received allogeneic hematopoietic stem cell transplantation (allo-HSCT). Mononucleated cells were isolated from peripheral blood from the donors (for allo-HSCT) or healthy immediate family members (elderly AML). The cells were seeded into the flasks pre-coated with NK cell specific activators, and expanded in media containing recombinant human IL-15 and IL-2 for 14 days. The cells were transfused intravenously after the identification of quality control. Trypan blue exclusion test was used for the determination of cell viability and counting. Flow cytometry analysis was performed to assess the surface antigenic profile. Seventy-eight infusions of the cell products were received by the elderly patients with AML after consolidation chemotherapy, 11 infusions were received by the patients during allo-HSCT and 32 infusions 3 moths after transplantation. The safety of cell therapy, body temperature, blood pressure and other indexes were observe during and 48 hours after cell transfusion. Meanwhile, the occurrence and severity of acute graft-versus-host disease (GVHD) were documented. RESULTS: Flow cytometry analysis showed that the proportion of NK cells (CD3-CD56+) in the mononucleated cells before culture was (14.10±4.22)% (n=121), and the proportion increased dramatically up to (87.29±8.75)% (n=121) after culture for 14 days, the number of NK cells increased to 753.47±140.13 times (n=121). The doses of the infused NK cells was (7.58±2.50)×107/kg per infusion. Moderate fever occurred in three cases after multiple infusions, and the temperature restored to normal on the same day after treatment. Fever was observed in one patient after every infusion of four times in total. The temperature reached to 38.5-39.0 ℃ and returned to normal within 1-2 hours after adequate antipyretic treatment, and then there was no discomfort. No GVHD was observed in the elderly AML patients, while 6 cases that received allo-HSCT developed moderate acute GVHD, among them grade I in 5 cases and grade II in 1 case. No other severe toxicities were observed. CONCLUSION: NK cell products with a high-purity could be obtained by ex vivo expansion with this protocol. The transfusion of these expanded cells is generally safe in the elderly patients with AML that have received chemotherapy or patients that received hematopoietic stem cell transplantation.

Successful desensitization of high level donor-specific anti-HLA antibody in patients with hematological diseases receiving haploidentical allografts.

Donor-specific anti-human leukocyte antigen (HLA) antibody (DSA) is associated with a higher incidence of graft failure and mortality in HLA-mismatched allograft settings. However, the optimal protocol of desensitization for patients with positive DSA remains uncertain. We investigated the effectiveness of a desensitization protocol, including rituximab, high-dose intravenous immunoglobulin (IVIG), and a single session of plasma exchange (PE), for haploidentical allograft recipients with a high mean fluorescence intensity (MFI) level of DSA (≥ 5,000). Eleven patients with hematological disease who had positive DSA (median, 11,676, range 5387-20,435) were desensitized by the protocol. All of the patients achieved hematopoietic recovery. The median times for neutrophil and platelet engraftment were 13 (range, 11-26) days and 19 (range, 11-90) days, respectively. Grade II-IV acute graft-versus-host disease (GVHD) was seen in one patient and was controlled completely. Chronic cutaneous GVHD was seen in eight patients. Nine patients are alive with good performance so far. One patient suffered extramedullary relapse, and one patient died of transplantation-associated thrombotic microangiopathy. The 1-year probability of overall survival was 81.8%. These results suggest that successful desensitization could be obtained by a combination of rituximab, high-dose IVIG, and PE for haploidentical allograft recipients with high MFI levels of DSA.

Infusion of haploidentical HSCs combined with allogenic MSCs for the treatment of ALL patients.

Although haploidentical stem cell transplantation (haplo-HSCT) offers almost all acute lymphoblastic leukaemia (ALL) patients an opportunity for immediate transplantation, it exhibits a higher incidence of graft failure and graft versus host disease (GVHD). Mesenchymal stem cells (MSCs) are characterised by their haematopoiesis-promoting and immunomodulatory capacity. Thus, we designed a combination of haplo-HSCT and MSCs for ALL patients. ALL patients (n = 110) were given haploidentical HSCs combined with allogenic MSCs, and ALL patients without MSC infusion (n = 56) were included as controls. The 100-day cumulative incidences of grade ≥2 acute GVHD (aGVHD) and grade ≥3 aGVHD were 40.00% and 9.09% compared to 42.32% (P = 0.79) and 22.79% (P = 0.03) in patients without MSC infusion, respectively. The 3-year cumulative incidences of chronic GVHD (cGVHD) and extensive cGVHD were 22.27% and 10.27% compared to 32.14% (P = 0.19) and 22.21% (P = 0.04) in patients without MSC infusion, respectively. No significant differences in the 3-year relapse incidence, nonrelapse mortality, leukaemia-free survival or overall survival in groups with and without MSC cotransplantation were observed. Multivariate analysis showed that MSC infusion contributed to a lower risk of developing extensive cGVHD. Our data suggested that haplo-HSCT combined with MSCs may provide an effective and safe treatment for ALL patients.

Infusion of haploidentical hematopoietic stem cells combined with mesenchymal stem cells for treatment of severe aplastic anemia in adult patients yields curative effects.

BACKGROUND AIMS: Despite the great advances in immunosuppressive therapy for severe aplastic anemia (SAA), most patients are not completely cured. Haploidentical hematopoietic stem cell transplantation (haplo-HSCT) has been recommended as an alternative treatment in adult SAA patients. However, haplo-HSCT presents a higher incidence of graft failure and graft-versus-host disease (GVHD). The authors designed a combination of haplo-HSCT and umbilical cord-derived mesenchymal stem cells (UC-MSCs) for treatment of SAA in adult patients and evaluated its effects. METHODS: Adult patients (≥18 years) with SAA (N = 25) were given HLA-haploidentical hematopoietic stem cells (HSCs) combined with UC-MSCs after a conditioning regimen consisting of busulfan, cyclophosphamide, fludarabine and anti-thymocyte globulin and intensive GVHD prophylaxis, including cyclosporine, basiliximab, mycophenolate mofetil and short-term methotrexate. Additionally, the effects of the protocol in adult SSA patients were compared with those observed in juvenile SAA patients (N = 75). RESULTS: All patients achieved myeloid engraftment after haplo-HSCT at a median of 16.12 days (range, 11-26). The median time of platelet engraftment was 28.30 days (range, 13-143). The cumulative incidence of grade II acute GVHD (aGVHD) at day +100 was 32.00 ± 0.91%. No one had grade III-IV aGVHD at day +100. The cumulative incidence of total chronic GVHD was 28.00 ± 0.85%. The overall survival was 71.78 ± 9.05% at a median follow-up of 42.08 months (range, 2.67-104). Promisingly, the protocol yielded a similar curative effect in both young and adult SAA patients. CONCLUSIONS: The authors' data suggest that co-transplantation of HLA-haploidentical HSCs and UC-MSCs may provide an effective and safe treatment for adult SAA.

Outcomes of haploidentical bone marrow transplantation in patients with severe aplastic anemia-II that progressed from non-severe acquired aplastic anemia.

Severe aplastic anemia II (SAA-II) progresses from non-severe aplastic anemia (NSAA). The unavailability of efficacious treatment has prompted the need for haploidentical bone marrow transplantation (haplo-BMT) in patients lacking a human leukocyte antigen (HLA)-matched donor. This study aimed to investigate the efficacy of haplo-BMT for patients with SAA-II. Twenty-two patients were included and followed up, and FLU/BU/CY/ATG was used as conditioning regimen. Among these patients, 21 were successfully engrafted, 19 of whom survived after haplo-BMT. Four patients experienced grade II-IV aGvHD, including two with grade III-IV aGvHD. Six patients experienced chronic GvHD, among whom four were mild and two were moderate. Twelve patients experienced infections during BMT. One was diagnosed with post-transplant lymphoproliferative disorder and one with probable EBV disease, and both recovered after rituximab infusion. Haplo-BMT achieved 3-year overall survival and disease-free survival rate of 86.4% ± 0.73% after a median follow-up of 42 months, indicating its effectiveness as a salvage therapy. These promising outcomes may support haplo-BMT as an alternative treatment strategy for patients with SAA-II lacking HLA-matched donors.

A study of human leukocyte antigen-haploidentical hematopoietic stem cells transplantation combined with allogenic mesenchymal stem cell infusion for treatment of severe aplastic anemia in pediatric and adolescent patients.

The clinical applications of human leukocyte antigen (HLA) haploidentical hematopoietic stem cells transplantation (haplo-HSCT) have offered most of the young severe aplastic anemia (SAA) patients an opportunity to accept curative therapy at the early stage of bone marrow lesions. However, the outcome of juvenile SAA patients received haplo-HSCT remain to be improved due to high incidence of graft failure and graft vs host disease (GVHD). Mesenchymal stem cells (MSCs) have been characterized by their hematopoiesis-supporting and immunomodulatory properties. In the current study, we designed a combination of haplo-HSCT with allogenic MSC for treatment of SAA in pediatric and adolescent patients and evaluated its effects. Juvenile patients (<18 years) with SAA (n = 103) were given HLA-haploidentical HSC combined with allogenic MSC after a conditioning regimen consisting of busulfan, cyclophosphamide, fludarabine, and antithymocyte globulin and an intensive GVHD prophylaxis, including cyclosporine, short-term methotrexate, mycophenolate mofetil, and basiliximab. Neutrophil engraftment was achieved in 102 of 103 patients in a median time of 14.3 days (range 9-25 days). The median time of platelet engraftment was 25.42 days (range 8-93 days). The cumulative incidence of II-IV acute GVHD at day +100 was 26.32% ± 0.19% and III-IV acute GVHD was 6.79% ± 0.06% at day +100, respectively. The cumulative incidence of chronic GVHD was 25.56% ± 0.26%. The overall survival was 87.15% ± 3.3% at a median follow-up of 40 (1.3-98) months. Our data suggest that cotransplantation of HLA-haploidentical HSC and allogenic mesenchymal stem cell may provide an effective and safe treatment for children and adolescents with SAA who lack matched donors.

Thrombopoietin enhances hematopoietic stem and progenitor cell homing by impeding matrix metalloproteinase 9 expression.

We reported a novel function of recombinant human thrombopoietin (TPO) in increasing hematopoietic stem and progenitor cell (HSPC) homing to the bone marrow (BM). Single doses of TPO treatment to the recipients immediately after BM transplantation showed significantly improved homing of HSPCs to the BM, which subsequently resulted in enhanced short- and long-term engraftment of HSPCs in mice. We found that TPO could downregulate the expression and secretion of matrix metalloproteinase 9 in BM cells. As a result, SDF-1α level was increased in the BM niche. Blocking the interaction of SDF-1α and CXCR4 on HSPCs by using AMD3100 could significantly reverse the TPO-enhanced HSPC homing effect. More importantly, a single dose of TPO remarkably promoted human HSPC homing and subsequent engraftment to the BM of nonobese diabetic/severe combined immunodeficiency mice. We then performed a clinical trial to evaluate the effect of TPO treatment in patients receiving haploidentical BM and mobilized peripheral blood transplantation. Surprisingly, single doses of TPO treatment to patients followed by hematopoietic stem cell transplantation significantly improved platelet engraftment in the cohort of patients with severe aplastic anemia (SAA). The mean volume of platelet and red blood cell transfusion was remarkably reduced in the cohort of patients with SAA or hematological malignancies receiving TPO treatment. Thus, our data provide a simple, feasible, and efficient approach to improve clinical outcomes in patients with allogenic hematopoietic stem cell transplantation. The clinical trial was registered in the Chinese Clinical Trial Registry website (www.chictr.org.cn) as ChiCTR-OIN-1701083.

Tumor necrosis factor α in aGVHD patients contributed to the impairment of recipient bone marrow MSC stemness and deficiency of their hematopoiesis-promotion capacity.

BACKGROUND: Though accumulated evidence has demonstrated visceral organ involvement in acute graft-versus-host disease (aGVHD), how aGVHD influences the bone marrow (BM) niche and the reconstitution of hematopoiesis post-hematopoietic stem cell transplantation remains largely unknown. METHODS: In the current study, the cell morphology, immunophenotype, multi-differentiation capacity, self-renewal capacity, and hematopoiesis promotion of the MSCs from aGVHD and non-aGVHD patients were investigated. Additionally, the stemness and hematopoiesis-promoting property of healthy donor-derived MSCs were evaluated in the presence of BM supernatant from aGVHD patients. Mechanistically, antibodies targeting inflammatory cytokines involved in aGVHD were added into the MSC culture. Furthermore, a recombinant human tumor necrosis factor (TNF-α) receptor-Ig fusion protein (rhTNFR:Fc) was used to protect healthy donor-derived MSCs. Moreover, mRNA sequencing was performed to explore the underlying mechanisms. RESULTS: The aGVHD MSCs exhibited morphological and immunophenotypic characteristics that were similar to those of the non-aGVHD MSCs. However, the osteogenic and adipogenic activities of the aGVHD MSCs significantly decreased. Additionally, the colony formation capacity and the expression of self-renewal-related genes remarkably decreased in aGVHD MSCs. Further, the hematopoiesis-supporting capacity of aGVHD MSCs significantly reduced. The antibody neutralization results showed that TNF-α contributed to the impairment of MSC properties. Moreover, rhTNFR:Fc exhibited notable protective effects on MSCs in the aGVHD BM supernatants. The mRNA sequencing results indicated that the TNF-α pathway and the Toll-like receptor pathway may be activated by TNF-α. CONCLUSIONS: Thus, our data demonstrate MSCs as cellular targets of aGVHD and suggest a potential role of TNF-α blockage in maintaining the BM niche of aGVHD patients.

Impact of prophylactic/preemptive donor lymphocyte infusion and intensified conditioning for relapsed/refractory leukemia: a real-world study.

Prophylactic/preemptive donor lymphocyte infusion (p/pDLI) and intensified conditioning have shown promising results in experimental studies of refractory/relapsed acute leukemia (RRAL), but real-world data remain scarce. We conducted a multicenter, population-based analysis of 932 consecutive patients. The three-year leukemia-free survival (LFS) rates were 56% for patients receiving both p/pDLI and intensified myeloablative conditioning (MAC) (intenseMAC) and 30% for those who received neither therapy per landmark analysis. Multivariable analyses were run separately for acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL), and p/pDLI treatment was linked to significantly higher LFS than non-DLI for both AML and ALL patients without increasing the nonrelapse mortality. IntenseMAC was associated with significantly lower relapse and higher LFS than nonintensified MAC despite higher nonrelapse mortality rates in ALL, while there was no impact of intenseMAC observed in AML. p/pDLI achieved superior outcomes in both matched-sibling donor (MSD) and haploidentical donor transplantation, while intenseMAC only influenced MSD outcomes. Data suggest that RRAL patients receiving "total therapy" by way of p/pDLI and intensified conditioning treatment have an improved chance for LFS, with p/pDLI being safer with a more extensive impact relative to intenseMAC. Patients with RRAL can tolerate both interventions and achieve a reasonable outcome.

[Effect of TGF-ß1 and IL-10 on the Immunoregulatory Function of Extracellular Vesicles Derived from Mesenchymal Stem Cells].

OBJECTIVE: To investigate the effect of immune regulatory molecules TGF-ß1 and IL-10 on the immunoregulatory activities of extracellular vesicles(EV) secreted from mesenchymal stem cells (MSCs). METHODS: MSC were isolated from human umbilical cord and expanded, then were treated with TGF-ß1 and IL-10 for 72h, and MSC-EVs in supernatants were isolated. The total protein content of all samples was determined by BCA methed. The morphological structure was observed by transmission electron microscopy. The surface markers of MSC-EV were analyzed by flow cytometry. The apoptosis of peripheral blood mononuclear cells(PBMNC) stimulated by ConA and the proportion of CD4+CD25+/CD127- (Treg) cells were detected by flow cytometry after incubation with MSC-EV for 72 h. The CBA and ELISA kit were used to detect the contents of IL2, IL4, IL6, IL10, IFN-γ, TNF-α, Th17A and TGF-ß1 in PBMC supernatants and MSC-EV. RESULTS: All the samples showed that the typical cup-shaped membrane-like structure was observed by transmission electron microscopy, and CD9, CD44, CD63 and CD81 expressed. After TGF-ß1 treatment, the MSC-EV displayed the strongest ability to promote PBMNC apoptosis（P<0.01）, and in all the samples the proportion of Treg cells increased. MSC-EV could increase the content of IL-10 in the supernatants of PBMNC culture, the content of TGF-ß1 in PBMNC supernatants after MSC treatment with TGF-ß1 was lower than that in untreated group(P<0.05). The content of IL-6 in MSC-EV increased significantly after treatment with TGF-ß1, and the content of TGF-ß1 decreased. CONCLUSION: TGF-ß1 alters the immnomodulatory function of MSC-EV and its underlying mechanisms need to be clarified in further investigations.

[Effect of Graft Composition on Acute Graft-Versus-Host Disease in Aplastic Anemia after Haploidentical Hematopoietic Stem Cell Transplantation].

OBJECTIVE: To investigate the relationship between acute graft-versus-host disease and graft composition in patients with aplastic anemia(AA) after haploidentical hematopoietic stem cell transplantation. METHODS: Fifty-seven cases of AA after haploidentical hematopoietic stem cell transplantation were retrospectively analyzed. All the patients were divided into 2 groups according to whether presence or absence grade Ⅱ-Ⅳ aGVHD, the relationship between aGVHD and graft composition was analyzed by comparing the differences of graft components between the 2 groups. RESULTS: Fourteen out of 57 patients had grade Ⅱ-Ⅳ aGVHD and the other 43 did not have grade Ⅱ-Ⅳ aGVHD. The mononuclear cells, CD3+, CD4+, CD8+, NK cells, NKT cells, B cells and Treg cells were not significantly different between the 2 groups (P>0.05), the CD34+ cell count in the patients with grade Ⅱ-Ⅳ aGVHD was 3.85（1.73-10.61）×106/kg, which was significantly lower than that without grade Ⅱ-ⅣaGVHD: 6.31（2.98-19.35）×106/kg (P<0.05). CONCLUSIONS: The incidence of grade Ⅱ-Ⅳ aGVHD may be related with CD34+ cell count in AA after haploidentical hematopoietic stem cell transplantation..

Exosomes originating from MSCs stimulated with TGF-ß and IFN-γ promote Treg differentiation.

Mesenchymal stem cells (MSCs) have been approved as a cellular drug for the treatment of a variety of immune-related diseases by the government of many countries'. Previous investigations, including ours, have shown that exosomes secreted by MSCs (MSC-ex) are one of the main factors responsible for the therapeutic effect of MSCs. However, the immune modulation activities and the contents of MSC-ex derived from cells under different incubation conditions differ dramatically. Therefore, the optimal way to ensure effectiveness is by identifying and preparing MSC-ex with confirmed potent immunosuppressive activity. The aim of this study was to investigate and analyze the composition and function of MSC-ex secreted by MSCs stimulated by different cytokines to obtain exosomes with more potent immunosuppressive activity. To achieve this aim, umbilical cord-derived MSCs were treated with PBS, TGF-ß, IFN-γ, or TGF-ß plus IFN-γ for 72 hr. Then, exosomes were isolated from the culture supernatants. Common exosome markers, such as CD9, CD63, and CD81, were detected and analyzed by FCM. At the same time, the TGF-ß, IFN-γ, IDO, and IL-10 content in exosomes was detected, and the influence of exosmes from defferent groups on the induction of mononuclear cell transformation into Tregs was analyzed via FCM. Our results show that the TGF-ß combined with IFN-γ exosome group more effectively promoted the transformation of mononuclear cells to Tregs, and the analysis showed that IDO may play an important role. This study might provide a novel strategy to treat GVHD as well as other immune-associated disorders.

[Detection of ATP Level in CD4+ T Lymphocytes and Its Clinical Significance in Allogeneic Hematopoietic Stem Cell Transplantation Recipients].

OBJECTIVE: To explore the clinical value of detecting adenosine triphosphate (ATP) level in CD4+ T lymphocytes (Immuknow ATP) of patients on early stage after allogeneic hematopoietic stem cell transplantation (allo-HSCT). METHODS: The base-line ATP value in CD4+ T lymphocytes in cases of hematological malignancies and the ATP level in CD4+ T lymphocytes of acute leukemia patients before allo-HSCT were detected. Allo-HSCT recipients were devided into 3 groups with different level of immunereactivity according to ATP concentraiton in month 3 (day 90±5) after allo-HSCT. The clinical characteristics of patients in 3 groups were analyzed. RESULTS: The mass concentration of Immuknow ATP in 15 cases of hematological malignancies before allo-HSCT ranged from 56.21-435.71 ng/ml, with a mean of 203.98±112.72 ng/ml. The ATP level in 46 cases after allo-HSCT ranged from 1.69-333.09 ng/ml, with a median of 41.96 ng/ml. Both 91.26 ng/ml (mean-SD) and 316.70 ng/ml (mean+SD) were used as cutoff, and 36 allo-HSCT recipients (78.3%) were assigned to low immunereactivity group, 8 recipients (17.4%) to middle group and 2 recipients (4.3%) to high group. The incidence of infection in low immunereactivity group was significantly higher than that in middle immunereactivity group (86.1% vs 50.0%)(P=0.022), and also significantly higher than that in high immunereactivity group (86.1% vs 0%)(P=0.002). There were no statistical differences in the incidences of severe infection among 3 groups. The incidence of grade II or higher acute graft versus host disease (aGVHD) in high immunereactivity group was superior to that in low immunereactivity group statistically (100% vs 13.9%)(P=0.002). Immune-mediated organ injury occurred more frequently in high immunereactivity group as compared with low and middle immunereactivity groups (100% vs 0% and vs 0%)(P=0.000; P=0.002). There were no significant differences in relapse rates of leukemia among 3 groups. The percentage of patients with increased trough blood concentration of cyclosporine A(CsA) was not significantly different among 3 groups (P=0.720). CONCLUSION: Detection of ATP level in CD4+ T lymphocytes on early stage after allo-HSCT possesses clinical significance for predicting infection, severity at aGVHD and immune-mediated organ injury.

[Clinical Study on Treatment of Juvenile Myelomonocytic Leukemia with Haploidentical-Hematopoietic Stem Cell Transplantation].

OBJECTIVE: To analyze the therapeutic efficacy of haploidentical-hematopoietic stem cell transplantation (hi-HSCT) for patients with juvenile myelomonocytic leukemia (JMML). METHODS: The engraftment of hematopoietic stem cells, incidence of graft versus host disease (GVHD), infection, relapse, and survival of 6 JMML patients received hi-HSCT were retrospectively analyzed. RESULTS: Six (6 males) JMML patients received hi-HSCT from haplo-HLA-matched related donors. The results showed that the hematopoictic stem cells in all 6 patients were grafted successfully. Two cases of JMML died of pulmenary infections, other 4 cases survive without disease. Acute GVHD occurred in 3 patients and chronic GVHD occurred in 1 patients. The overall survival, disease free survival and relapse rates were 66.7%, 66.7%, 0%, respectively. CONCLUSION: The hi-HSCT is an effective method for treatment of patients with JMML, but there also is a serial problems to be resolved.

[Clinical Features and Prognosis of t(8;21) AML Patients in China: A Multicenter Retrospective Study].

OBJECTIVE: To summarize the clinical characteristics of peripheral blood, immune phenotypes, fusion genes and cytogenetics of patients with t(8;21) acute myeloid leukemia(AML) through the retrospective analysis of 586 patients with t(8;21) AML from 15 blood disease research centers in Northern area of China. METHODS: The factors affecting prognosis of patients with t(8;21) AML were investigated by using univariate and multivariate COX regression. RESULTS: The immune type of t(8;21) AML patients was mainly with HLA-DR+, CD117+, CD34+, MPO+, CD38+, CD13+ and CD33+ (>95%), part of them with CD19+ and CD56+; the most common accompanied mutation of t(8;21) AML patients was C-KIT mutation (37.8%); in addition to t(8;21) ectopic, the most common chromosomal abnormality was sex chromosome deletions (38.9%). The univariate analysis revealed a significant survival superiority of OS and PFS in t(8;21) AML patients of WBC≤3.5×109/L without C-KIT mutation, the newly diagnosed ones achieved HSCT(P<0.05), only survival superiority on OS in t(8;21) AML patients with extramedullary infiltration and CD19 positive; the results of multivariate analysis showed a significant survival superiority on OS and PFS in t(8;21) AML patients with WBC≤3.5×109/L(P<0.05). CONCLUSION: The clinical features of t(8;21) AML patients in China are similar to those in other countries, WBC≤3.5×109/L is a good prognostic factor while the C-KIT mutation is a poor one in t(8;21) AML patients.

[Clinical Observation of Haploidentical Hematopoietic Stem Cell Transplantation Combined with Bone-marrow Derived Mesenchymal Stem Cells Transfusion for Treatment of Children with Severe Aplastic Anemia].

OBJECTIVE: To investigate the efficacy and safety of haploidentical hematopoietic stem cell transplantation(hi-HSCT) combined with bone-marrow derived mesenchymal stem cell (BM-MSC) tranfusion for treatment of children with severe apastic anemia(SAA). METHODS: The clinical data of 25 children with SAA undergoing hi-HSCT and BM-MSC tranfusion were retrospectively analyzed from August 2014 to July 2016. RESULTS: neutrophil engraftment was achieved in all 25(100%) children, with the median time 12(11-22) days. The median time of platelet engraftment was 21(11-130) days in 23(92%) children. Acute graft-versus-host disease(aGVHD) was observed in 16(64%) cases, including 11 case of grade I and 5 cases of aGVHD grade II-IV, and one of them died of grade IV of skin, gut and liver at day 115; 5 cases of chronic GVHD were found, all of them were limited cGVHD. Cytomegalovirus (CMV) viremia was observed in 23(92%) cases, but no CMV disease was developed after therapy. 3 cases of post-transplant lymphoroliferative disease with 23 EBV viremia positive occurred, all of them were cured after rituximab. Hemorrhagic cystitis appeared in 9 cases with only one case of grade III, 22 children suffered from infection, involving 10 cases in lung and 4 cases in liver, 1 patient was diagnosed as Guillain-Barre syndrome. Autoimmune hemolytic anemia was recorded in 1 patient, 22 children survived during a median following-up time of 14(3-27) months. CONCLUSION: The hi-HSCT combined with BM-MSC transfusion for treatment of children with SAA has been confirmed to be safe and feasible.

[Efficacy and Safety of Co-transplantation of Haploidentical-HSC with Umbilical Cord Mesenchymal Stem Cell in Children with Hematologic Malignancies].

OBJECTIVE: To analyse the efficacy and safety of co-transplantation of umbilical cord mesenchymal stem cell(UC-MSC) with haploidentical hematopoietic stem cell transplantation(hi-HSCT) in children with hematologic malignancy. METHODS: The clinical data of 47 children undergoing hi-HSCT were retrospectively analyzed from November 2003 to November 2014, among them 34 patients received UC-MSC from October 2011 to November 2014, and another 13 patients without UC-MSC from November 2003 to September 2011. The median follow-up time was 20(0.5-67) months. RESULTS: No adverse events were observed after the UC-MSC transplantation. The engraftment rate, the median neutrophils engraftment time and platelet engraftment time all were not significantly different between hi-HSCT and hi-HSCT+UC-MSCT(P>0.05). The three-years cumulative overall survival (70.6% vs 23.1%),(P=0.004), three-years cumulative disease-free survival(52.9% vs 0) (P=0), and early cytomegalovirus (CMV) viremia (91.2% vs 38.5%) (P=0) in UC-MSC+hi-HSCT group were statistically significantly higher than that in the conventional hi-HSCT group. The morbidity of aGVHD (44.1% vs 92.3%) (P=0.003), I-II aGVHD (26.5% vs 61.5%) (P=0.041) and transplantation-related mortality (11.8% vs 46.2%) (P=0.017) in UC-MSC+hi-HSCT group was statistically significantly lower than that in hi-HSCT group, however, the morbidity of III-IV aGVHD (17.6% vs 30.8%), cGVHD (26.5% vs 30.8%), HC (35.3% vs 7.7%), pulmonary infection (52.9% vs 46.2%) and relapse rate (32.4% vs 53.8%) were not statistically significantly different (P>0.05) between the 2 groups. CONCLUSION: The application of umbilical cord mesenchymal stem cell in children undergoing hi-HSCT is safe, the UC-MSC can improve the overall survival, disease-free survival and reduce transplantation-related mortality. UC-MSC can reduce the morbidity of aGVHD, but increase the early infection of CMV, however it is nothing for the pulmonary infection and relapse in the children after hi-HSCT.