scholarly journals Bone Marrow-Derived MSCs Stimulated by IFN-γ Inhibited the Growth of ToxoplasmaGondii Via up-Regulation of GBP1

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 5143-5143
Author(s):  
Aiping Qin ◽  
De-Hua Lai ◽  
Weijun Huang ◽  
Mingshui Wu ◽  
Xiaoyong Chen ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Natural Science ◽  
Research Funding ◽  
Guangdong Province ◽  
Human Mscs ◽  
Dependent Manner ◽  
Rna Seq ◽  
Guangzhou City ◽  
Ifn Γ ◽  
Dose Dependent

Abstract Background Mesenchymal stromal cells (MSCs) are a heterogeneous cell population endowed with multi-lineage differentiation potential and extensive immunomodulatory properties. MSCs have been successfully used for prevention and treatment of immune disorders such as graft-versus-host disease. Emerging preclinical studies suggest that MSCs might also protect against infectious challenge. Aims This study aimed to rule out the potential mechanism of human MSCs against Toxoplasma gondii (T. gondii). Methods Human bone marrow-derived MSCs (hMSCs) were pretreated for 24h with a series of concentrations of IFN-γ and then infected with T. gondii strains of variant virulences (virulent RH and avirulent ME49). RNA-seq and westernblots were used to analyze gene and protein expression patterns of hMSCs in IFN-γ-stimulated and unstimulated conditions. The intracellular parasites (with fluorescence labeled) were counted microscopically at multiple time points postinfection. The short hairpin RNA (shRNA) expression was used to generate RNAi of GBP-1, GBP-2 and GBP-5. Results Human MSCs stimulated with IFN-γ were capable to inhibit the growth of T. gondii (eg: at IFN-γ 10ng/ml, the inhibition rates are 26.5% (RH) and 37.5% (ME49) 12hr postinfection) in a dose-dependent manner. Compared with the unstimulated MSCs (controls), IFN-γ treatment at 5, 10, 20ng/ml inhibited T. gondii (ME49) growth by percent of 27.1±7.9, 37.5±6.2, 47.0±7.6 (mean±SD, n=4) 12 hr postinfection and the inhibition rates are 54.5±2.1%, 62.5±4.9% and 78.5±2.1 at 24 hr postinfection, respectively. After 48 hr postinfection, the ratio between parasites per parasitophorous vacuole (PV) containing rosettes and single paraites in IFN-γ-stimulated MSCs was significantly reduced compared with that in the unstimulated MSCs (p<0.01, p<0.01, p<0.001 for ME49 at IFN-γ 5, 10, 20ng/ml, respectively). Furthermore, There was no significant effect of conditioned medium (CM) from IFN-γ-stimulated MSCs on T. gondii growth in comparison with CM from unstimulated MSCs (p=0.74 for RH and p=0.69 for ME49). We observed that the resistance in hMSCs does not depend on IDO (p=0.85 for RH and p=0.79 for ME49). RNA-seq data showed that IFN-γ-inducible p65 guanylate-binding proteins (GBPs) might play pivotal roles in the inhibition of T. gondii growth. Reads per kilobase-pairs per million (RPKM) mean values of GBP1, 2, 5 in IFN-γ-stimulated MSCs are 1093.3, 443.3, 348.2, respectively. By RNAi knockdown, the results showed that silencing of GBP1 (but not GBP2, GBP5) in hMSCs resulted in recovery of T. gondii growth inhibition at 12 hr and 24 hr postinfection (p<0.05 and p<0.001 for ME49). Conclusion: Human MSCs pre-stimulated with IFN-γ inhibited the growth of T. gondii in a dose-dependent manner via up-regulation of GBP-1 expression. Disclosures Liu: the project of the Zhujiang Science & Technology Star of Guangzhou city (2013027): Research Funding; the Technology Plan of Guangdong Province of China (2012B031800403): Research Funding; the project of health collaborative innovation of Guangzhou city (201400000003-4, 201400000003-1): Research Funding; Natural Science Foundation of Guangdong Province (S2012010009299): Research Funding; National Public Health Grand Research Foundation (201202017): Research Funding; National High Technology Research and Development Program of China (863 Program) (2011AA020105): Research Funding; National Natural Science Foundation of China (81270647, 81300445, 81200388): Research Funding.

Neutropenia during Hematopoietic Stem Cell Mobilization with G-CSF

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 5835-5835
Author(s):  
Lezhong Yuan ◽  
Hui Liu ◽  
Qiang Wang ◽  
Jing Sun ◽  
Qifa Liu ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Blood Sample ◽  
Natural Science ◽  
Research Funding ◽  
Guangdong Province ◽  
Routine Test ◽  
Guangzhou City ◽  
The Third ◽  
Blood Routine Test ◽  
Blood Routine

Abstract A 36-year-old female was referred to our hematology department for HSC donation by leukapheresis of peripheral blood after mobilization. On the first day she was informed of adverse events of HSC harvesting and then signed an agreement. She denied of having any severe or chronic disorders before. No abnormal signs are found in physical and laboratory examination. Laboratory data showed normal blood routine test (WBC 3.77X10E9/L, LYM 0.61X10E9/L NEU 1.68X10E9/L, HGB 128 g/L and PLT 174X10E9/L), negative test for HBV, HCV, HIV, Toxoplasmosis and Treponemapallidum. Quantitation of both cytomegalovirus and Epstein-Barr virus DNA were <500 copies/mL. She was administrated with 300u G-CSF (FILGRASTIM, Kirin-Amgen) by subcutaneous injection at 16:00, then once a day in the morning. Norethisterone tablets were given 5000ug TID daily to delay the coming menstruation, which had been used routinely for many years safely for this purpose in our hospital, altogether total dose of Norethisterone tablets 30000ug in 48 hours. On the third day of G-CSF administration, blood routine test amazingly showed a sharply declined Neutrophilic Granulocyte count (NEU) of 0.54X10E9/L, reduced lymphocyte count (LYM) of 0.77X10E9/L and normal Monocyte count (MON) of 0.56X10E9/L, normal Hematoglobin (HGB) of 127 g/L and normal platelet count (PLT) of 150X10E9/L. She was diagnosed with Neutropenia. During re-inquiring for her medical history, she admitted that she was diagnosed with Adult Onset Still’s Disease (AOSD) when she was 18 y.o. which relapsed at 24. She was given aspirin and achieved remission of the symptoms. She denied any history of allergy. The donor did not complain about any symptoms and her body temperature was 36°C. Norethisterone tablets administration was stopped. At 16:00 of the third day, additional 300u G-CSF was administrated for Neutropenia. At 17:21, blood sample showed NEU 0.50X10E9/L, Agranulocytosis indicated, and an evaluated MON of 0.77X10E9/L; normal coagulation function, evaluated ESR of 31mm/h, slightly reduced complement 3 of 0.87 g/L (normal: 0.9 to 1.8 g/L) and evaluated total complement of 50.1 U/mL (normal: 23.0 to 46.0 U/mL) was showed; on the next day afternoon, strong positive (3+) test for anti-RO52 and negative test for ANA, anti-DS-DNA, anti-Jo-1, ANCA, anti-SS-A or anti-SS-B in autoimmune antibody was reported. Abdominal ultrasonography reported normal size of her liver and spleen. On the fourth day, 300u G-CSF was administrated the fifth time at 10:00. After that, blood sample was collected and showed WBC of 2.22X10E9/L, LYM of 0.86X10E9/L, NEU of 0.52X10E9/L, MON of 0.83X10E9/L, HGB of 133 g/L and PLT of 153X10E9/L. Bone marrow aspiration showed myeloid hyperplasia (-), hypoplasia and abnormal maturation in granule cell, left shift with toxic granulation. CD34+ cell ratio reported 1.4% (marrow) and 0.2% (peripheral blood), which revealed HSC mobilization was failed. On the fifth day, G-CSF was not administrated. Blood sample collected at 12:00, 26 hours after the last G-CSF administration, revealed WBC of 3.94X10E9/L, LYMX1.31 10E9/L, NEU 1.08 X10E9/L, MON 1.48X10E9/L, HGB of 125 g/L and PLT of 151X10E9/L. The donor seemed recovering from Neutropenia. At 12:00 on the sixth day, bone marrow was collected for transplantation. Blood routine test showed WBC of 4.03X10E9/L, LYM 0.84X10E9/L, NEU 2.38X10E9/L, MON 0.78X10E9/L, HGB of 94 g/L and PLT of 129X10E9/L. We described a HSC donor with AOSD history who developed Neutropenia subsequent to subcutaneous injection of G-SCF and recovered in 2 days after the last administration. Neutropenia caused by Norethisterone tablets was not common and we didn’t find any report that combined medication of norethisterone tablets and G-CSF would cause neutropenia. The donor didn’t complain any symptoms and we couldn’t find any typical signs in the process, which indicated Neutropenia might be unrelated with infection or allergy. It may be the first case of this situation, we have not yet confirmed the cause of her Neutropenia. We preserved samples of blood and marrow aspiration under the donor’s consent and we will have further research and follow-up with this case. Disclosures Liu: National Natural Science Foundation of China (81270647, 81300445, 81200388): Research Funding; National High Technology Research and Development Program of China (863 Program) (2011AA020105): Research Funding; National Public Health Grand Research Foundation (201202017): Research Funding; Natural Science Foundation of Guangdong Province (S2012010009299): Research Funding; the project of health collaborative innovation of Guangzhou city (201400000003-4, 201400000003-1): Research Funding; the Technology Plan of Guangdong Province of China (2012B031800403): Research Funding; the project of the Zhujiang Science & Technology Star of Guangzhou city (2013027): Research Funding.

The New CXCR4 Inhibitor MDX-1338 Exerts Anti-Tumor Activity in Multiple Myeloma

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 1844-1844 ◽  
Author(s):  
Aldo M Roccaro ◽  
Antonio Sacco ◽  
Michelle Kuhne ◽  
AbdelKareem Azab ◽  
Patricia Maiso ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
Tumor Cells ◽  
Mouse Models ◽  
Research Funding ◽  
Therapeutic Agents ◽  
Dependent Manner ◽  
Apoptotic Pathways ◽  
Dose Dependent

Abstract Abstract 1844 Background. We have previously shown the SDF1/CXCR4 axis plays a major role in homing and trafficking of multiple myeloma (MM) to the bone marrow (BM), and disruption of the interaction of tumor cells with the BM leads to enhanced sensitivity to therapeutic agents. We hypothesize that the novel anti-CXCR4 antibody, BMS936564/MDX-1338, may prevent the homing and adhesion of MM cells to the BM and will sensitize them to therapeutic agents. Methods. Primary MM cells (CD138+); MM cell lines (MM.1S, RPMI.8226); and primary MM bone marrow stromal cells (BMSCs) were used. Migration towards SDF-1 and BMSCs has been evaluated. Cytotoxicity and DNA synthesis were measured by MTT and thymidine uptake, respectively. Cell signaling and apoptotic pathways were studied by Western Blot. Synergism was calculated using the Chou-Talalay method. In vivo MM tumor growth was evaluated with xenograft mouse models. Results. MDX-1338 inhibited migration of MM cells toward SDF-1a and primary MM BMSCs, in a dose-dependent manner. Adhesion of primary MM cells to BMSCs was also inhibited by BMS936564/MDX-1338 in a dose-dependent manner, while also inducing cytotoxicity on primary BM-derived CD138+ cells. BMS936564/MDX-1338 targeted MM cells in the context of BM milieu by overcoming BMSC-induced proliferation of tumor cells. In addition, BMS936564/MDX-1338 synergistically enhanced bortezomib-induced cytotoxicity in MM cells. BMS936564/MDX-1338-dependent activation of apoptotic pathways in MM cells was documented, as shown by cleavage of caspase-9 and PARP. SDF-1a-induced ERK-, Akt-, and Src-phosphorilation was inhibited by BMS936564/MDX-1338 in a dose-dependent manner. Importantly, BMS936564/MDX-1338 inhibited MM cell proliferation in vivo in xenograft mouse models. Conclusion. These studies therefore show that targeting CXCR-4 in MM by using BMS936564/MDX-1338 represents a valid therapeutic strategy in this disease. Disclosures: Roccaro: Roche:. Kuhne:BMS: Employment. Pan:Bristol-Myers Squibb: Employment. Cardarelli:Bristol-Myers Squibb: Employment. Ghobrial:Noxxon: Research Funding; Bristol-Myers Squibb: Research Funding; Millennium: Research Funding; Noxxon:; Millennium:; Celegene:; Novartis:.

Autologous HSCT Followed By Immunotherapy and Maintenance Chemotherapy Compared with Allogeneic HSCT for Intermediate-Risk Molecules/Cytogenetics Acute Myeloblastic Leukemia in First Complete Remission

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 3949-3949 ◽  
Author(s):  
Xiao Zhai ◽  
Li Xuan ◽  
Jing Sun ◽  
Zhiping Fan ◽  
Yu Zhang ◽  
...  
Keyword(s):  
Natural Science ◽  
Research Funding ◽  
Development Program ◽  
Guangdong Province ◽  
Technology Research ◽  
Maintenance Chemotherapy ◽  
Post Transplantation ◽  
Guangzhou City ◽  
Leukemia Relapse

Abstract Background Autologous hematopoietic stem cell transplantation (auto-HSCT) is an alternative choice for acute myeloblastic leukemia (AML) with intermediate-risk molecules / cytogenetics (IR). Its disadvantage is high relapse rate, compared with allogeneic HSCT (allo-HSCT). To reduce leukemia relapse, we introduced a strategy of auto-HSCT followed by immunotherapy and maintenance chemotherapy for IR AML. Methods One hundred and seventy-six IR AML in first complete remission (CR1) undergoing HSCT between January 2001 and December 2010 at our single institute were enrolled in this study. The choice of auto-HSCT or HLA-matched sibling transplantation was based on the donor source and patients’ desire. The conditioning regimen included BuCY (busulfan 4 mg/kg/day P.O. or 3.2 mg/kg/day I.V. on days -7 to -4, and cyclophosphamide 60 mg/kg/day on days -3 and -2) and BuF (busulfan 4 mg/kg/day P.O. or 3.2 mg/kg/day I.V. on days -7 to -4, and fludarabine 30mg/m2/day on days −6 to −2). Cyclosporine A and methotrexate were administered for GVHD prophylaxis. For patients undergoing auto-HSCT, interleukin-2 (IL-2) was administered from day 0 at a dose of 3×106 U/day (three times a day) subcutaneously for 3 weeks. One or more subsequent cycles were given after a 30-day interval for up to 6 cycles unless the patient developed ≥ grade 3 toxicity. Standard chemotherapy was administered from three months post-transplantation. One or more subsequent cycles were given after a 90-day interval for up to 3 cycles. Survival, leukemia relapse and quality of life were compared between auto-HSCT and allo-HSCT. Results Of the 176 IR AML-CR1, 102 patients received auto-HSCT, and 74 received allo-HSCT. The 5-year overall survival (OS) and disease-free survival (DFS) post-transplantation were 73.8%±4.3% and 67.2%±4.7%, 69.1%±6.3% and 69.1%±6.3%, respectively, in auto-HSCT and allo-HSCT.There were no difference in OS and DFS between auto-HSCT and allo-HSCT (P=0.533, P=0.948). The 5-year cumulative incidence of leukemia relapse was 25.8%±4.6% and 13.5%±4.8%, respectively (P=0.171). The 5-year non-relapse mortality was 10.4%±3.1% and 19.9%±5.7%, respectively, in auto-HSCT and allo-HSCT (P=0.157). The performance status, measured using the Karnofsky performance score, was observed in 85 and 55 patients surviving 3 years in auto-HSCT and allo-HSCT. Of the auto-HSCT and allo-HSCT recipients, 98.8% and 89.1% had normal or near-normal activity scores (Karnofsky assessment 90–100%) in 3 years post-transplantation, and there was significant difference between the two groups (P=0.015). Conclusion Auto-HSCT followed by immunotherapy and maintenance chemotherapy might reduce relapse for IR AML-CR1 post-transplantation, and have similar survival compared with allo-HSCT. It is superior to allo-HSCT in quality of life. Disclosures Xuan: It was supported by National Natural Science Foundation of China (81270647, 81300445, 81200388); National High Technology Research and Development Program of China (863 Program) (2011AA020105): Research Funding. Liu:It was supported by National Natural Science Foundation of China (81270647, 81300445, 81200388); National High Technology Research and Development Program of China (863 Program) (2011AA020105); National Public Health Grand Research Foundation (201202017);: Research Funding; It was supported by Natural Science Foundation of Guangdong Province (S2012010009299); the project of health collaborative innovation of Guangzhou city (201400000003-4, 201400000003-1);: Research Funding; It was supported by the Technology Plan of Guangdong Province of China (2012B031800403); the project of the Zhujiang Science & Technology Star of Guangzhou city (2013027).: Research Funding.

Role of Allogeneic Hematopoietic Stem Cell Transplantation for None Remission / Advanced Acute Myeloid Leukemia with Flt3-Itd Mutations: Outcomes of 15 Newly Diagnosed Patients from a Single Institution

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 5936-5936
Author(s):  
Zhiping Fan ◽  
Qifa Liu ◽  
Jing Sun ◽  
Yu Zhang ◽  
Fen Huang ◽  
...  
Keyword(s):  
Stem Cell ◽  
Hematopoietic Stem Cell Transplantation ◽  
Stem Cell Transplantation ◽  
Cell Transplantation ◽  
Natural Science ◽  
Research Funding ◽  
Guangdong Province ◽  
Hematopoietic Stem ◽  
Guangzhou City ◽  
Disease Free

Abstract Backgrouds : Acute myelogenous leukemia (AML) patients with FLT3-ITD mutations have an inferior survival compared to AML patients with wild-type (WT) FLT3, primarily because of an increased relapse rate. Allogeneic hematopoietic stem cell transplantation (allo-HSCT) represents a postremission therapy that is effective at reducing the risk of relapse for many cases of poor-risk AML. Whether or not allo-HSCT can improve outcomes for patients with none remission/advanced FLT3-ITD mutation AML is not known. Objective : To compare the effect of allo-HSCT between none remission/advanced FLT3-ITD positive AML and other none remission/advanced AML excluding FLT3-ITD mutations. Patients and methods : We analyzed 49 patients who underwent allo-HSCT with a diagnosis of none remission/advanced AML on FLT3-ITD mutations between February 2012 and Apiril 2014. Fifteen patients were FLT3-ITD positive and 24 were FLT3-ITD negative. Transplantations were performed in none remission/advanced status after myeloablative conditioning or intensified conditoning. Results : Patient’s characteristics were similar in the two groups, including leukocyte count at diagnosis, interval from CR to transplant, disease status, donor type, stem cell resource, prepare regimens, and graft versus host disease (GVHD) prophylactic protocols. All patients achieved hematopoietic engraftment. The time to neutrophil and platelet engraftment was similar between the two groups. The incidences of acute GVHD and chronic GVHD were comparable between the two groups. At 2 year after transplantation, cumulative relapse incidence (33.3% ± 14.9% v 18.5% ± 7.7%; P =0.335) and disease-free survival (DFS) were not different (51.9% ± 15% v 61.4% ± 8.9%; P =0.749) in FLT3/ITD-positive compared with FLT3/ITD-negative patients. The overall survival between the two groups was also similar (57.0% ± 14.8% v 62.1% ± 9.3%; P =0.834). Conclusions : FLT3-ITD didn’t affect the outcome of HSCT for none remission/advanced patients in the same direction it does after chemotherapy; and more than half of the patients harboring this mutation who received transplants were alive and disease free at 2 years. Disclosures Liu: National Natural Science Foundation of China (81270647, 81300445, 81200388): Research Funding; National High Technology Research and Development Program of China (863 Program) (2011AA020105): Research Funding; National Public Health Grand Research Foundation (201202017): Research Funding; Natural Science Foundation of Guangdong Province (S2012010009299): Research Funding; the project of health collaborative innovation of Guangzhou city (201400000003-4, 201400000003-1): Research Funding; the Technology Plan of Guangdong Province of China (2012B031800403): Research Funding; the project of the Zhujiang Science & Technology Star of Guangzhou city (2013027): Research Funding.

scholarly journals Bone Marrow Stromal Cells Protect Both FLT3-ITD and FLT3-WT Primary AML Cells from the Anti-Leukemic Activity of the FLT3 Inhibitor AC220

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 4377-4377
Author(s):  
Cedric Dos Santos ◽  
Georges Habineza Ndikuyeze ◽  
Michael Nisssan ◽  
Chenghui Zhou ◽  
Xiaochuan Shan ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Growth Inhibition ◽  
Stromal Cells ◽  
Research Funding ◽  
Critical Role ◽  
Annexin V ◽  
Dependent Manner ◽  
Induced Apoptosis ◽  
The Impact ◽  
Dose Dependent

Abstract FTL3 mutations are found in about 30% of AML patients, conferring a leukemic blast growth advantage, drug therapy resistance in the bone marrow (BM) and poor outcome. Mesenchymal stem/stromal cells (MSCs) are essential components of the bone marrow microenvironment, and growing evidence suggest that MSCs play a critical role in AML chemo-resistance, although the molecular mechanisms involved are poorly understood. The purpose of the study was to (1) establish an novel in vitro co-culture system between primary AML blasts and healthy donor BM-MSCs (HD-MSCs) or AML patient-derived MSCs (AML-MSCs), (2) evaluate the impact of culture with BM-MSCs on the sensitivity of AML cells to AC220 using patients samples with FLT3-ITD (n=4) or FLT3-WT (n=3). We first cultured HD-MSCs (n=5) and AML-MSC (n=3) and observed no phenotypical differences (CD14- CD34- CD45- CD73+ CD90+ CD105+), although HD-MSCs grew faster. We evaluated the effect of co-culturing AML samples (n=6) with HD-MSCs or AML-MSCs for 5 and 12 days on leukemic cell growth and found that both types of MSCs significantly and equally enhanced AML cell proliferation while maintaining blast phenotype. Using clonogenic assays on 4 AML specimens cultured alone or with either HD- or AML-MSCs for 5 and 12 days, we found that co-culture with either source of BM-MSCs drastically increased colony-forming cells number at day 5 and day 12 while CFC number decreased in the absence on BM-MSCs (no colonies at day 12 for the 4 samples), indicating that AML co-culture with HD/AML-MSCs supports the survival and/or proliferation of AML stem/progenitor cells. We next assessed the effect of increasing doses of AC220 (1, 10, 50, 100 and 500nM) on the apoptosis of FLT3-ITD (n=3) and FLT3-WT (n=4) AML cells cultured alone or with HD-MSCs. Exposure to AC220 for 72 hours significantly, and in a dose-dependent manner, increased the apoptosis of AML FLT3-ITD cells in monoculture (n=3, 21±1% of Annexin V positive cells for control, AC220 1nM 29±3.7%, 10nM 31±2.5%, 50nM 32±1.5%, 100nM 34±1.7% and 500nM 38±3.6%). In contrast, AML FLT3-ITD cells co-cultured with HD-MSCs were resistant to the drug (n=3, 21±2.6% of Annexin V positive cells for control, AC220 1nM 23±3%, 10nM 22±3%, 50nM 25±5.7%, 100nM 30±8.3% and 500nM 33±9.5%). Interestingly, we found that AML FLT3-WT are much less sensitive to increasing doses of AC220 compared to ITD samples (n=4, 27±3.9% of Annexin V positive cells for control, AC220 1nM 30±6.5%, 10nM 35±14%, 50nM 37±11%, 100nM 39±13% and 500nM 43±11%), and co-culture with BM-MSCs further decreased the sensitivity of AML FLT3-WT cells to AC220-induced apoptosis (n=4, 19±3.2% of Annexin V positive cells for control, AC220 1nM 17±3.9%, 10nM 20±3.4%, 50nM 19±3.7%, 100nM 21±4.5% and 500nM 26±1%). AC220 treatment for 3 days significantly, and in a dose-dependent manner, inhibited CFCs in AML FLT3-ITD (n=4, with 26±8%, 46±6%, 60±9%, 69±10% and 86±3% inhibition with 1, 10, 50, 100 and 500nM of AC220 respectively) while AML FLT3-ITD co-culture with HD-MSCs were less sensitive (n=4, with 9±10%, 30±6%, 42±9%, 57±11% and 72±7% inhibition with 1, 10, 50, 100 and 500nM of AC220, respectively). Similarly to the AC220-induced apoptosis, we observed that AML FLT3-WT CFCs are less sensitive to AC220-induced growth inhibition compared to ITD samples, although a 3 days exposure to AC220 significantly, and in a dose-dependent manner, inhibited AML FLT3-WT CFCs (n=3, with 38±16%, 44±14%, 58±12%, 70±21% and 81±19% inhibition with 1, 10, 50, 100 and 500nM of AC220, respectively). Interestingly, we observed that co-culture of AML FLT3-WT with stromal cells were significantly more resistant to increasing doses of AC220 (n=3, with 22±7%, 36±5%, 43±8%, 46±8% and 57±6% inhibition with 1, 10, 50, 100 and 500nM of AC220, respectively). Altogether, these results suggest that AML FLT3-ITD cells in monoculture are more sensitive to AC220 treatment compared to AML FLT3-WT primary cells, but more importantly, upon interaction with primary HD-MSCs, both WT and FLT3-ITD primary samples are protected from apoptosis and growth inhibition induced by AC220, indicating a critical role for the BM microenvironment in AC220 resistance. We are currently testing the impact of BM-MSCs co-culture on leukemic stem cell sensitivity to AC220 using transplantation in NSG mice. We will also evaluate if this co-culture model can be predictive of the response to in vivo treatment with AC220 in a patient-derived xenograft model. Disclosures Dos Santos: Janssen R&D: Research Funding. Danet-Desnoyers:Janssen R&D: Research Funding.

scholarly journals Veronica austriaca L. Extract and Arbutin Expand Mature Double TNF-α/IFN-γ Neutrophils in Murine Bone Marrow Pool

Molecules ◽  
2020 ◽  
Vol 25 (15) ◽  
pp. 3410
Author(s):  
Petya Dimitrova ◽  
Kalina Alipieva ◽  
Tsvetinka Grozdanova ◽  
Milena Leseva ◽  
Dessislava Gerginova ◽  
...  
Keyword(s):  
Bone Marrow ◽  
High Performance ◽  
Colorimetric Assay ◽  
Major Constituent ◽  
Dependent Manner ◽  
Murine Bone Marrow ◽  
Traditional Remedies ◽  
Tnf Α ◽  
Ifn Γ ◽  
Dose Dependent

Plants from the Veronica genus are used across the world as traditional remedies. In the present study, extracts from the aerial part of the scarcely investigated Veronica austriaca L., collected from two habitats in Bulgaria—the Balkan Mountains (Vau-1) and the Rhodopi Mountains (Vau-2), were analyzed by nuclear magnetic resonance (NMR) spectroscopy. The secondary metabolite, arbutin, was identified as a major constituent in both extracts, and further quantified by high-performance liquid chromatography (HPLC), while catalpol, aucubin and verbascoside were detected at lower amounts. The effect of the extracts and of pure arbutin on the survival of neutrophils isolated from murine bone marrow (BM) were determined by colorimetric assay. The production of cytokines—tumor necrosis factor (TNF)-α and interferon (IFN)-γ was evaluated by flowcytometry. While Vau-1 inhibited neutrophil vitality in a dose-dependent manner, arbutin stimulated the survival of neutrophils at lower concentrations, and inhibited cell density at higher concentrations. The Vau-1 increased the level of intracellular TNF-α, while Vau-2 and arbutin failed to do so, and expanded the frequency of mature double TNF-α+/IFN-γhi neutrophils within the BM pool.

scholarly journals The Role of Nestin+ Mesenchymal Stem Cells (MSCs) in Bone Marrow Chronic Graft-Versus-Host Disease

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 2039-2039
Author(s):  
Haiyan Zhang ◽  
Kaibo Yang ◽  
Yanqiu Chen ◽  
Hua Jin ◽  
Qifa Liu
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Mesenchymal Stem Cells ◽  
Graft Versus Host Disease ◽  
Natural Science ◽  
Research Funding ◽  
Guangdong Province ◽  
Host Disease ◽  
Graft Versus Host ◽  
Tgf Β1

Abstract Chronic graft-versus-host disease (cGVHD) is a major cause of morbidity and mortality after allogeneic hematopoietic stem cell transplantation (allo-HSCT). cGVHD is a syndrome of variable clinical features resembling autoimmune, involving not only the epithelial target tissues (skin, lung, gastrointestinal tract and liver) but also any other organ system (musculoskeletal, joint, ocular and genital tissues). Bone marrow (BM) suppression without infection is often observed in patients undergoing allo-HSCT as GVHD symptoms appear, suggesting that BM could be a target of GVHD (Yusuke S. et. al. Blood 2010). However, the BM cGVHD is rarely reported and the mechanism for BM cGVHD is still unclear. Nestin+ mesenchymal stem cells (MSCs) are capable of differentiating into cells of multiple cell lineages and involved in the formation of fibrotic lesions (Miaohua M. et. al. Cell Mol Life Sci 2016). To determine whether Nestin+ MSCs play an important role in the development of BM cGVHD. We used two murine models of cGVHD, including MHC-matched BALB/c (H-2d) recipients given B10D2 (H-2d) grafts, and MHC-mismatched BALB/c (H-2d) recipients given C57BL/6 (H-2b) grafts. We observed that BM displayed a significant increase in collagen and reticulin fibers deposition around blood vessels in cGVHD group compared to non-cGVHD group. The decreased absolute numbers of monocytes and neutrophils in the BM indicated the broad BM suppression in the cGVHD group. We further demonstrated that Nestin+MSCs located in perivascular BM niche underwent tremendous expansion in cGVHD group (p<0.001). Nestin+ MSCs in cGVHD group acquired expression of alpha smooth muscle actin (a-SMA), indicating myofibroblast differentiation. Moreover, transforming growth factor- beta1 (TGF-β1) concentrations also increased in cGVHD BM microenvironment (p<0.0001). High concentrations of TGF-β1 induced formation of Nestin+ MSC clusters and more transition from Nestin+ MSCs to a-SMA+ cells (p<0.05). Besides, expanded Nestin+ MSCs were surrounded by many macrophages and expressed high level of TGF-β1 receptor (p<0.001 and p<0.005). Taken together, these results indicate that Nestin+ MSCs participate BM cGVHD development through transition to a-SMA+ cells induced by high TGF-β1 in cGVHD microenvironment. Disclosures Zhang: National Natural Science Foundation of China (No. 81600141, No. 81770190) and Natural Science Foundation of Guangdong Province (No. 2016A030310390): Research Funding. Yang:National Natural Science Foundation of China (No. 81600141, No. 81770190) and Natural Science Foundation of Guangdong Province (No. 2016A030310390): Research Funding. Chen:National Natural Science Foundation of China (No. 81600141, No. 81770190) and Natural Science Foundation of Guangdong Province (No. 2016A030310390): Research Funding.

scholarly journals Mesenchymal Stem Cells Ameliorate Thymic Functions in aGVHD Patients after Allogenetic Haematopoietic Stem Cell Transplantation

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 42-42
Author(s):  
Ke Zhao ◽  
Xiuli Wu ◽  
Qian Fan ◽  
Zhiping Fan ◽  
Shaohua Chen ◽  
...  
Keyword(s):  
Natural Science ◽  
Research Funding ◽  
Development Program ◽  
Guangdong Province ◽  
Complete Response ◽  
Third Party ◽  
Control Group ◽  
Technology Research ◽  
Guangzhou City ◽  
Before And After

Abstract Background Mesenchymal stem cells (MSCs) have been considered as a promising strategy for the prevention and treatment of graft-versus-host disease (aGVHD), but the mechanism of MSCs ameliorating GVHD is still not fully understood. In mice aGVHD model, some studies proposed that MSCs ameliorated GVHD via enhancements in proliferation and function of thymic epithelial cells, but it is not performed in patients. Methods Twenty-five refractory aGVHD patients were enrolled in this study, including 13 patients receiving MSCs treatment and 12 without MSCs treatment as the control group. MSCs derived from bone marrow (BM) of a third-party donor and given at a median dose of 1×106 cells/kg once weekly until aGVHD got complete response (CR) or MSCs were infused for a total of 8 doses. CD4+CD25+Foxp3+ regulatory T cells (Tregs) in peripheral blood were analyzed by flow cytometry and signal joint T-cell receptor excision circle (sjTREC) was monitored by real-time polymerase chain reaction (PCR) before and 4, 8, 12, 16, 24 and 36 weeks after treatments. Results In MSCs group,the overall response (OR) rate for aGVHD was 76.9%, including complete response (CR) in 53.8% and partial response (PR) in 23.1%, while the OR rate in non-MSCs group was 33.3%, including CR in 25% and PR in 8.3%. The OR rate in MSC group is significantly higher than those in the control group (P=0.028). No evident difference of CD4+CD25+Foxp3+Tregs and sjTREC were shown between the patients in MSCs and the control groups before the study treatment (P=0.762, P=0.870). In the evaluated patients, the level of sjTREC in MSCs group is significantly higher at 8, 16, 24 weeks than the control group after treatments (P=0.043, P=0.0301 and P=0.039, respectively.). The level of sjTREC significantly increased at 8 weeks after MSCs treatment, compared with pre-MSCs treatment (P=0.034), and sustained to 36 weeks after MSCs treatment. In the control group, sjTREC increased, but did not change significantly before and after treatments (P>0.05). The level of sjTREC in CR patients is significantly higher than non-response patients at 8, 12, 16, and 24 weeks after MSCs treatment, and sjTREC levels correlated with the response of MSCs treatment (r=0.782). With respect to CD4+CD25+Foxp3+Tregs, they were significantly higher in MSC group at 8, 12 weeks after treatment than the control group (P=0.026 and P=0.029). In MSCs group, the proportion of CD4+CD25+Foxp3+Tregs showed a significant increase at 8 weeks compared with pre-MSCs treatment (P=0.012), and sustained to 36 weeks after MSCs treatment. In the control group, CD4+CD25+Foxp3+Tregs did not significantly change before and after treatment. Conclusions MSCs derived from BM of a third-party donor are effective to refractory aGVHD. MSCs ameliorated aGVHD via repairing thymus, including the enhancements of thymic output function and inducing the generation of Tregs. Disclosures Wu: It was supported by National Natural Science Foundation of China (81270647, 81300445, 81200388); National High Technology Research and Development Program of China (863 Program) (2011AA020105): Research Funding; It was supported by the Technology Plan of Guangdong Province of China (2012B031800403); the project of the Zhujiang Science & Technology Star of Guangzhou city (2013027).: Research Funding. Liu:It was supported by National Natural Science Foundation of China (81270647, 81300445, 81200388); National High Technology Research and Development Program of China (863 Program) (2011AA020105); National Public Health Grand Research Foundation (201202017);: Research Funding; It was supported by Natural Science Foundation of Guangdong Province (S2012010009299); the project of health collaborative innovation of Guangzhou city (201400000003-4, 201400000003-1);: Research Funding; It was supported by the Technology Plan of Guangdong Province of China (2012B031800403); the project of the Zhujiang Science & Technology Star of Guangzhou city (2013027).: Research Funding.

scholarly journals α-Lipoic acid suppresses osteoclastogenesis despite increasing the receptor activator of nuclear factor κB ligand/osteoprotegerin ratio in human bone marrow stromal cells

2005 ◽  
Vol 185 (3) ◽  
pp. 401-413 ◽  
Author(s):  
Jung-Min Koh ◽  
Young-Sun Lee ◽  
Chang-Hyun Byun ◽  
Eun-Ju Chang ◽  
Hyunsoo Kim ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Lipoic Acid ◽  
Nuclear Factor Κb ◽  
Human Bone ◽  
Human Bone Marrow ◽  
Mouse Bone Marrow ◽  
Dependent Manner ◽  
Nuclear Factor ◽  
Receptor Activator ◽  
Dose Dependent

Growing evidence has shown a biochemical link between increased oxidative stress and reduced bone density. Although α-lipoic acid (α-LA) has been shown to act as a thiol antioxidant, its effect on bone cells has not been determined. Using proteomic analysis, we identified six differentially expressed proteins in the conditioned media of α-LA-treated human bone marrow stromal cell line (HS-5). One of these proteins, receptor activator of nuclear factor κB ligand (RANKL), was significantly up-regulated, as confirmed by immunoblotting with anti-RANKL antibody. ELISA showed that α-LA stimulated RANKL production in cellular extracts (membranous RANKL) about 5-fold and in conditioned medium (soluble RANKL) about 23-fold, but had no effect on osteoprotegerin (OPG) secretion. Despite increasing the RANKL/OPG ratio, α-LA showed a dose-dependent suppression of osteoclastogenesis, both in a coculture system of mouse bone marrow cells and osteoblasts and in a mouse bone marrow cell culture system, and reduced bone resorption in a dose-dependent manner. In addition, α-LA-induced soluble RANKL was not inhibited by matrix metalloprotease inhibitors, indicating that soluble RANKL is produced by α-LA without any posttranslational processing. In contrast, α-LA had no significant effect on the proliferation and differentiation of HS-5 cells. These results suggest that α-LA suppresses osteoclastogenesis by directly inhibiting RANKL–RANK mediated signals, not by mediating cellular RANKL production. In addition, our findings indicate that α-LA-induced soluble RANKL is not produced by shedding of membranous RANKL.

scholarly journals MIR2111-5 locus and shoot-accumulated mature miR2111 systemically enhance nodulation depending on HAR1 in Lotus japonicus

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Nao Okuma ◽  
Takashi Soyano ◽  
Takuya Suzaki ◽  
Masayoshi Kawaguchi
Keyword(s):  
Root Nodules ◽  
Lotus Japonicus ◽  
Dependent Manner ◽  
Rna Seq ◽  
Signaling System ◽  
Nodule Number ◽  
Knockout Mutants ◽  
Mutualistic Relationship ◽  
Dose Dependent

Abstract Legumes utilize a shoot-mediated signaling system to maintain a mutualistic relationship with nitrogen-fixing bacteria in root nodules. In Lotus japonicus, shoot-to-root transfer of microRNA miR2111 that targets TOO MUCH LOVE, a nodulation suppressor in roots, has been proposed to explain the mechanism underlying nodulation control from shoots. However, the role of shoot-accumulating miR2111s for the systemic regulation of nodulation was not clearly shown. Here, we find L. japonicus has seven miR2111 loci, including those mapped through RNA-seq. MIR2111-5 expression in leaves is the highest among miR2111 loci and repressed after rhizobial infection depending on a shoot-acting HYPERNODULATION ABERRANT ROOT FORMATION1 (HAR1) receptor. MIR2111-5 knockout mutants show significantly decreased nodule numbers and miR2111 levels. Furthermore, grafting experiments using transformants demonstrate scions with altered miR2111 levels influence nodule numbers in rootstocks in a dose-dependent manner. Therefore, miR2111 accumulation in leaves through MIR2111-5 expression is required for HAR1-dependent systemic optimization of nodule number.

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