bluuebunny/crossref_metadata_2025 huggingface dataset:
Establishment of Cell Lines Stably Expressing HNA-1A, -1B, -2A, -4A, -4B, -5A and -5B Antigen with Low Background Reactivity in Flow Cytometric Analysis.
Authors: Yasui Kazuta , Miyazaki Toru , Watanabe Yoshihisa , Odagiri Miki , Matsuyama Nobuki , Furuta Rika A. , Kimura Takafumi , Ikeda Hisami , Hirayama Fumiya , Tani Yoshihiko , Shibata Hirotoshi
Published: October 2019

Antibodies to neutrophil antigens have been implicated in neonatal alloimmune neutropenia, autoimmune neutropenia, and transfusion-related acute lung injury. Although FCM analysis with a typed panel of neutrophils has been used to detect such antibodies, this procedure has two major limitations. First, only freshly isolated neutrophils can be used for the assay. Second, the assay cannot identify an HNA antibody in the presence of anti-HLA class I antibodies that react to panel neutrophils. To overcome these limitations, we established a panel of cell lines in which the HNA-1a (−1b; polymorphisms of HNA-1), HNA-2a, HNA-4a (−4b), or HNA-5a (−5b) gene was transduced using a retrovirus vector to confer stable transgene expression in K562 cells that dose not express HLA or HNA and not exhibit background reactivity to normal human serum samples. We confirmed the specificity of the one of each HNA expression on the K562 panel cells by using mAbs. We further showed that several well-characterized human sera containing antibodies against several HNAs were unambiguously identified by our panel cell lines. We also observed a lower background reactivity of the K562 panel cell lines compared with isolated neutrophils, which have been used for the panel to identify antibodies against HNA in human sera. These results indicate that the K562 panel cell lines provide a good panel for detecting HNA-reactive neutrophil antibodies in human sera.
Fatal Complication of Transfusion of No Irradiated Blood Product in an Immunocompetent Recipient.
Authors: Campos-Cabrera Gregorio , Campos-Cabrera Virginia , Campos-Cabrera Salvador , Campos-Villagomez Jose Luis
Published: October 2019

Transfusion-associated graft versus host disease (TA-GVHD) is a rare, but almost always a fatal complication. It has a mortality rate above 90%. For the development of TA-GVHD is need

immunocompetent cells in the blood product, incompatibility in HLA alloantigen, immune failure of the recipient against the donor cells.

The exact incidence is unknown, but more than 200 cases have been reported in the world literature and the molecular test for diagnostic where performed only in a very few of them. Thirty-five years old woman with multiple fractures in right leg from a car accident was treated in her rural town, she received a fresh whole blood transfusion from her sister and then went for surgery, antibiotics and analgesia where given; six days after she developed erytroderma in the upper chest, two days later generalized bone pain and weakness were aggregated, the next day erithroderma generalize and started with diarrhea and jaundice; her evolution was torpid with fever, more weakness and jaundice, pallor, purpura and oropharyngeal pain. She was sent for hematological evaluation to our tertiary care institution, and a TA-GVHD was considerate; a work up on that was performed and the CBC showed pancytopenia, LFT with elevation of bilirubins, transaminases and alkaline phosphatase; the bone marrow aspiration and biopsy with aplasia, the skin biopsy with lymphoid infiltration, junction of epidermis with dermis was intact and no leukocytoclastic vasculitis. She received methylprednisolone, cyclosporine, filgrastim, wide range antibiotics and amphotericin B, leukoreduced and irradiated blood products. Her evolution was torpid with deterioration of her conditions and 48 hour later she died of septic shock. There is no effective treatment for TA-GVHD and no difference between early o delayed diagnostic, the evolution is almost always fatal, that is why the prevention is needed with the leukoreduction and irradiation of blood products, specially in recipients with clear risk for development of TA-GVHD: congenital immunodeficiencies, fetuses y newborns, hematological cancers, solid tumors in chemotherapy, hemopoiectic and solid organ transplanted, and first and second degree relatives. Better policies, technology, education for the primary care physician and access to blood products with high quality is needed to prevent this type of complications, specially in rural areas where transfusions with whole fresh blood from relatives are performed commonly.

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A Pilot Trial To Assess College Student’s Willingness To Donate Blood.
Authors: Demmons Derrick G. , Crittenden Colleen P. , Lee Mark , Shaz Beth H. , Burnett Miriam , Hillyer Christopher D.
Published: October 2019

Background: Nationally, African Americans are significantly underrepresented in community blood donation programs. The need to understand the willingness to donate blood among African Americans is essential. In this study, we explore factors that serve as motivators and barriers to blood donation identified by college students at two Historical Black Colleges/Universities (HBCUs).

Methods: HBCU students at least 18 years of age were given the opportunity to complete a paper-based or on-line 41-item questionnaire to assess the participant’s demographic background, donation frequency, motivators and barriers toward donation, knowledge and beliefs regarding blood donation, and specific behavioral practices. No personal identifiers were used. Univariate and bivariate analyses were conducted to describe the sample.

Results: 409 individuals completed the survey. 95% self-identified as African-American and 45% of college students reported previously donating blood. Student’s felt that incentives, personal requests to donate from blood centers, blood drives sponsored by campus organizations, and the university involvement in promoting blood drives were significant motivating factors for donating blood (p<.05). Significant barriers against blood donation included believing donation was too painful, fear of feeling faint, dizzy, or nauseated, losing valuable time from school or work, too time consuming, and fear of deferral (p<.05).

Conclusions: College students are willing to donate blood based on convenience, accessibility, and support from their university. Campus-based recruitment methods geared towards students, specifically those of African American descent, may prove beneficial for increasing the general pool of African American blood donors.

Significant relationships between barriers, motivators, and donor status Χ2 Sig. *p<.05 Barriers against blood donation Too painful 60.20 .000 Would feel faint, dizzy, or nauseated 42.15 .000 Will lose valuable time from school/work 16.82 .002 Previously been told not to donate 8.49 .004 Takes too long 37.41 .000 Motivators toward blood donation Incentive for donating 14.56 .006 Personal request to donate from blood center 10.15 .038 Blood drive sponsored by campus organization 26.29 .000 University involvement in promoting blood drives 5.36 .021
Muc1 Expression Identifies Human Self-Renewing Stem/Progenitor Populations and Is Elevated in AML CD34+ Cells.
Authors: Fatrai Szabolcs , Daenen Simon M.G.J. , Vellenga Edo , Schuringa Jan J.
Published: October 2019

Mucin1 (Muc1) is a membrane glycoprotein which is expressed on most of the normal secretory epithelial cells as well as on hematopoietic cells. It is involved in migration, adhesion and intracellular signalling. Muc1 can be cleaved close to the membrane-proximal region, resulting in an intracellular Muc1 that can associate with or activate various signalling pathway components such as b-catenin, p53 and HIF1a. Based on these properties, Muc1 expression was analysed in human hematopoietic stem/progenitor cells. Muc1 mRNA expression was highest in the immature CD34+/CD38− cells and was reduced upon maturation towards the progenitor stage. Cord blood (CB) CD34+ cells were sorted into Muc1+ and Muc1− populations followed by CFC and LTC-IC assays and these experiments revealed that the stem and progenitor cells reside predominantly in the CD34+/Muc1+ fraction. Importantly, we observed strongly increased Muc1 expression in the CD34+ subfraction of AML mononuclear cells. These results tempted us to further study the role of Muc1 overexpression in human CD34+ stem/progenitor cells. Full-length Muc1 (Muc1F) and a Muc1 isoform with a deleted extracellular domain (DTR) were stably expressed in CB CD34+ cells using a retroviral approach. Upon coculture with MS5 bone marrow stromal cells, a two-fold increase in expansion of suspension cells was observed in both Muc1F and DTR cultures. In line with these results, we observed an increase in progenitor counts in the Muc1F and DTR group as determined by CFC assays in methylcellulose. Upon replating of CFC cultures, Muc1F and DTR were giving rise to secondary colonies in contrast to empty vector control groups, indicating that self-renewal was imposed on progenitors by expression of Muc1. A 3-fold and 2-fold increase in stem cell frequencies was observed in the DTR and Muc1F groups, respectively, as determined by LTC-IC assays. To determine whether the above mentioned phenotypes in MS5 co-cultures were stroma-dependent, we expanded Muc1F and DTR-transduced cells in cytokine-driven liquid cultures. However, no proliferative advantage or increase in CFC frequencies was observed suggesting that Muc1 requires bone marrow stromal cells. In conclusion, our data indicate that HSCs as well as AML cells are enriched for Muc1 expression, and that overexpression of Muc1 in CB cells is sufficient to increase both progenitor and stem cell frequencies.
Cleaved High Molecular Weight Kininogen (HKa) and Kininostatin (D5) Induce Apoptosis and Inhibit Migration and Invasion of Human Prostate Cancer Cells by Preventing AKT Phosphorylation and Cyclin D1 Synthesis and Disrupting the Interaction of UPAR and EGFR.
Authors: Liu Yuchuan , Pixley Robin , Fusaro Mario , Colman Robert W.
Published: October 2019

Tumor metastasis is a major factor in the mortality rate in human prostate cancer. Upregulation and activation of EGFR and/or uPAR in a variety of cancers have been shown to be associated with poor prognosis. HK, a component of the plasma kallikrein-kinin system, can be hydrolyzed by plasma kallikrein to bradykinin and HKa. HKa and D5 both have been demonstrated to have potent anti-angiogenic activity in vitro and in vivo. We previously published that D5 directly inhibits human colon carcinoma cell (HCT-116) proliferation in vitro by blocking the G1/S transition in the cell cycle. We now show that HKa [100 nM] inhibits the migration of human prostate tumor cell (DU145) about 50%. Cyclin D1 can activate p21 and p27 with concomitant cell migration. DU145 cells rapidly increase cyclin D1 synthesis in response to bFGF [1.2 nM]. HKa suppresses cyclin D1 expression as shown by Western blotting as well as cell immunoflourescence. Stimulation by bFGF or VEGF results in clustering of uPAR and EGFR on the surface of DU145 cells. Immunoflourescence shows that the addition of HKa disrupts the co-localization of uPAR and EGFR. HKa or a monoclonal antibody against uPAR decreases the phosphorylation of EGFR at Tyr 1173. The phosphorylation of ERK and AKT, which are downstream effectors of EGFR, is also inhibited by HKa. Kininostatin [300nM] induced apoptosis of human prostate cancer cells challenged with uPA [50 nM] or EGF [6.7 nM]. Matrigel invasion assay reveals that HKa [100 nM] decreases the invading cell number by 90%. These novel data indicate that HKa and kininostatin induce apoptosis and inhibit migration and invasion of human prostate cancer cells, indicating the therapeutic potential of kininostatin in metastasis human prostate cancer.
Hematopoietic Stem Cell Transplantation in Acute Lymphocytic Leukemia Patients: Umbilical Cord Blood Versus Bone Marrow Transplant.
Authors: Sun Zimin , Liu Huilan , Geng Liangquan , Liu Xin , Yang Huizhi , Zhu Weibo , Ding Kaiyang , Yao Wen , Yuan Xiao
Published: October 2019

Acute lymphocytic leukemia (ALL) is a life threatening disease with high rate of relapse. To date, there is no ideal treatment for ALL. Recently, stem cell transplantation has been used as a potential therapy. However, it is unclear what the best source of donor stem cells is and whether it is safe or effective to use these regimens in human patients. In this study, we evaluated the safety and outcomes of 22 patients with ALL after umbilical cord blood transplantation (UCBT) or bone-marrow from related donors (RD). Eleven patients received stem cells from HLA 0–2 (A,B,DRB1) mismatched UCBT (median infused dose, 3.77×107 nucleated cell NC/kg; range, 3.40– 7.49 ×107 NC/kg). The other 11 patients received stem cells from bone marrow from RD (median infused dose, 4.08×108 NC/kg; range, 1.86–9.82×107 NC/kg). All patients in both groups were comparable, as shown in Table 1. All patients received myeloablative conditioning (busulfanum/cyclophosphamide was used for the RD transplantation and anti-thymus globulin 7.5 mg/kg was used for the UCB transplantation) followed by infusion of allogenetic hematopoietc stem cells from either bone marrow or cord blood. Cyclosporine A (CsA) and mycophenolate mofetil (MMF) were administered for the prophylaxis of graft-versus-host disease (GVHD). Overall survival (OS), leukemia-free survival (LFS), transplantation-related mortality (TRM) and relapse rate between RD and UCB were shown in Table 2. The results demonstrated that despite the higher HLA mismatch rate and lower number of infused hematopietic stem cells, OS, LFS, TRM and relapse rate in UCB group were 81.8%, 81.8%, 0% and 18.2% which were significantly better than in the RD group. These data strongly suggest that UCBT could be a safe and effective therapy for patients with ALL.

Table 1 The Profiles of Patients RD UCBT High Risk ALL 7/11 7/11 HLA 0/6 Mismatched (%) 72.7 9.1 HLA 1/6 Mismatched (%) 27.3 81.8 HLA 2/6 Mismatched (%) 0 9.1 Median Age (years, range) 30 (8–63) 25 (3–38) Median Body Weight (kg, range) 51 (26–70) 40 (13–84) Table 2 Outcomes of ALL patients after UCB transplantation RD UCB P value Number of patients 11 11 Median follow up (years) 2.0 2.0 OS 2 yr % (95% CI) 36.4 81.8 <0.05 LFS 2 yr % (95% CI) 27.3 81.8 <0.05 Relapse 1yr % (95% CI) 27.3 0 <0.05 TRM 1 yr % (95% CI) 63.6 18.2 <0.05
Cyclic ADP-Ribose Increases Human Mesenchymal Stem Cells Proliferation through the Modulation of Spontaneous Ca2+ Oscillation.
Authors: Tao Rong , Lau Chu-Pak , Lee Hon-Cheung , Li Gui-Rong
Published: October 2019

Human mesenchymal stem cells (hMSCs) play an important role in the regulation of hematopoietic microenvironment. Cyclic ADP-ribose (cADPR), a universal Ca2+ mobilizer produced by hMSCs in the bone marrow, has been demonstrated to be an important factor in the regulation of hematopoietic stem cells proliferation. However, its biological effects on hMSCs are not understood. The present study was designed to investigate the effects of cADPR on Ca2+ signaling, cell proliferation and differentiation in hMSCs. Ca2+ activity in hMSCs was measured with a confocal microscope. It was found that cADPR increased the frequency of spontaneous Ca2+ oscillatory transient, and the effect was abolished by pretreatment with the specific cADPR antagonist 8-Br-cADPR, but not by ryanodine, though the ryanodine receptors are the classic targets of cADPR in other types of cells. RT-PCR showed no gene expression for ryanodine receptors in hMSCs. Interestingly, the cADPR-induced increase of spontaneous Ca2+ oscillation was abolished by the TRPM2 channel inhibitors econazole and clotrimazole. The downregulation of TRPM2 channels with siRNA technique had no significant effects on spontaneous Ca2+ oscillation; however, it abrogated the cADPR-induced increase of spontaneous Ca2+ oscillation. The effects of cADPR on cell proliferation and differentiation were then investigated in hMSCs. We found that cADPR increased hMSCs proliferation, but had no significant effects on osteogeneic and adipogeneic differentiation. Importantly, the effect of cADPR on hMSCs proliferation was antagonized by 8-Br-cADPR or by the selectively knockdown of TRPM2 channels. In conclusion, our results demonstrate the novel information that cADPR regulate cell proliferation by increasing spontaneous Ca2+ oscillation mediated by TRPM2 channels, but has no effects on osteogeneic and adipogeneic differentiation in hMSCs.
The Biological Characteristics of Osteoblasts Derived from Myelodysplastic Syndrome Patients.
Authors: Chen Wen-ming , Chen Zi-xing , Cen Jian-nong , He Jun , Jiao Xiao-li , Wu Ya-fang , Zhang Jun , Qiu Qiao-cheng , Dai Lan
Published: October 2019

It was hypothesized that osteoblasts play a central role in hematopoiesis, and it has been shown that osteoblasts produce many factors essential for the survival, renewal, and maturation of hematopoietic stem cells (HSCs). By using human fetal osteoblastic cell line hFOB1.19 as a model of control, we investigated the biological characteristics of osteoblasts derived from patients with myelodysplastic syndrome (MDS) and their hematopoietic supportive function in vitro. MSCs isolated from bone marrow of MDS patients and normal donors were cultured and checked for their morphology, immunophenotype, CFU-F forming capacity and the expression of hematopoietic cytokines. A feeder layer was prepared by osteoblasts induced from 3rd generation of cultured MSCs and treated with mitomycin C. Ficoll-isolated bone marrow mononuclear cell from normal donors were then seeded on the feeder layer to co-culture in vitro without exogenous cytokines. FCM revealed that both MSCs and hFOB cells were positive for CD44, CD73(SH3), CD105(SH2) and CD90 (Thy1), but negative for CD34, CD45, HLA-DR. RT-PCR found that hFOB cells expressed mRNA of SCF, IL-6, IL-11, SDF-1, GM-CSF and G-CSF. MSCs obtained from MDS patients and normal donors were displaying fibroblastoid morphology. Their growth pattern, immunophenotype and CFU-F forming capacity were similar (P >0.05). Without exogenous cytokines, the osteoblasts derived from MDS could sustain GM-CFC survival for at least 3 weeks. The CFU-GM yield from cells in upper layer of co-culture was not different from those of control in hematopoiesis supportive experiments in vitro (P>0.05). RT-PCR clearly demonstrated that the cultured BM-MSCs from normal donor expressed mRNA of SCF, SDF-1, IL-6, and IL-11. As the MSCs differentiated toward osteoblasts, the expression of G-CSF could be detected, whereas GM-CSF remained undetectable. The same expression profile of above cytokines were also seen in osteoblasts induced from BM-MSCs of MDS patients. In conclusion, osteoblasts may play a pivotal role in hematopoiesis. The biological characteristics of osteoblasts from bone marrow of MDS patients were generally not different from those of osteoblasts in bone marrow of normal controls. Both of them could support survival of GM-CFC hematopoietic progenitor cells in vitro, according to their expression of multiple cytokines. These findings suggested that the osteoblasts derived from MDS patients may not be involved in the malignant process.
Nitric Oxide from Mesenchymal Stem Cells Regulated T-Cells through Inhibiting Expression of the T-bet.
Authors: Liu Gexiu , He Dongmei , Tan Guangxiao
Published: October 2019

INTRODUCTION: More recently, experimental evidence and preliminary clinical studies have demonstrated that mesenchymal stem cells (MSCs) have an important immune modulatory function. Many reports have shown that MSCs regulate functions of T-cells and suppress its proliferation, but the molecular mechanisms are poorly understood. Here We found that T-bet expression in T cells is suppressed in the presence of MSCs, and nitric oxide (NO) is involved in the regulation of T-cell status and T-bet expression.

METHODS: Adult C57BL/6 mouse bone marrow MSCs were characterized by surface molecules, the typical spindle-shaped morphology, and the ability of differentiation. T-lymphocytes were isolated from the spleen of rats by using mouse CD4, CD8, and CD19 MACS beads, of which the purity determined by flow cytometry was more than 85%. Responding recipient cells (1×10 5) and an equal number of irradiated (3000 cGy) donor stimulating cells or MSCs were cocultured. Cell proliferation was assessed using enzyme-linked immunosorbent assay (ELISA), in accordance with the BrdU user’s manual.

RESULTS: Flow cytometry showed that expression of the activation markers CD25 and CD69 on CD4 or CD8 T cells increased significantly in the presence of stimulating cells, and the production of IFN-and expression of its T-bet in T cells in response to allogeneic stimulation increased to 18 times and 15times, respectively. However, after 24 hours in the presence of MSCs, IFN-γ in T cells was diminished, expression of the T-bet was reduced more than 10 times, T cell proliferation was suppressed by 73–92% in response to allogeneic stimulation or in response to ConA activation. Reverse transcriptase-polymerase chain reaction and Western blot analysis showed that the expression of iNOS in MSCs cocultured with activated splenocytes increased significantly, but not in MSCs alone, or splenocytes alone. The immunofluorescence studies showed that iNOS was exclusively expressed by large adherent CD45− cells, which correspond to MSCs. To further determine a functional immunosuppressive role for NO expressed by MSCs, N-nitro-L-arginine methyl ester, the NO-specific inhibitor was used. As a result, it reversed the suppression of T-cell proliferation and the T-bet expression.

CONCLUSION: Taken into account of T-bet’s functions in T cells, these results suggest that NO produced by MSCs is one of the major mediators of T-cell suppression by MSCs.
Long-Term Reversal of Renal Anemia in Rat by Erythropoietin Gene Modified Mesenchymal Stem Cell Transplantation Beneath the Renal Capsule, Which Is Effectively Regulated by Hypoxia.
Authors: Liu Gexiu , He Dongmei , Tan Guangxiao
Published: October 2019

INTRODUCTION: Patients with chronic renal failure usually require exogenous erythropoietin (EPO) to alleviate anaemia resulting from inadequate epo production by the kidneys. Although this treatment is effective, recombinant EPO could cause hypertension, its antibodies, and other side-effects. EPO gene therapy is more attractive since it could be more economical and more convenient for the long-term management of the disease. As targets for gene therapy, mesenchymal stem cells (MSCs) are more easily expanded, lower immunogenic, and more stable for expressing exogenous genes than other cells. Here we describe an approach of transplantation of the erythropoietin gene modified mesenchymal stem cells beneath the renal capsule to treate Wistar rats with relative erythropoietin deficiency.

METHODS: we constructed an EPO-expressed vector pGenesil-HRE-EPO directed by hypoxia response element (HRE)/SV40 promoter, and transduced it into MSCs prepared by adherently culturing rat bone marrow. The EPO-gene modified MSCs (Epo+) in vitro growed normally and stably secreted high level of functional EPO in hypoxia condition, which produced from 3.23 × 10(5) to 9.98 × 10(5) mIU of EPO per 10(6) cells per 24 hr. To determine the role of these MSCs to treate renal anemia in vivo, EPO+, EPO (vector pGenesil-1 or normal) MSCs were injected into rats with renal anemia beneath the renal capsule, the skin, or in muscles (4 × 10(7) cells/rat). Renal anemia rats had been induced by feeding with adenine, whose hematocrit levels were at 33.5 +/− 4.1%, the plasma Epo concentration was 6.5 ± 1.7 mU/ml. EPO concentration was determined by ELISA. During whole experiment, we measured rat body weight and blood pressure, and obtained blood samples regularly.

RESULTS: 2wk after transplantation, the hematocrit levels in anemia rats injected Epo+ MSCs beneath the renal capsule, the skin, or in muscles markedly increased to 67.2 ± 5.9%, 61.5 ± 5.7%, and 53.8 ± 5.2%, and 10wk after transplantation to 68.0 ± 5.6%, 52.7 ± 5.8%, and 40.6 ± 5.5%, respectively. Moreover, the plasma Epo concentration of anemic rats injected Epo+MSCs beneath the renal capsule, the skin, or in muscles was considerably increased to 158 ± 35 mU/ml, 136 ± 36 mU/ml, and 114 ± 42 mU/ml at 2 wk, whereas that of anemic rats injected EPO MSCs was not increased significantly. All rats injected Epo+MSCs did not have polycythemia or severe hypertension. Survival time of anemic rats injected Epo+MSCs beneath the renal capsule is longest among anemic rats. Histological examination by fluorescent microscopy showed that implanted MSCs beneath the renal capsule survived best.

CONCLUSIONS: EPO gene-modified mesenchymal stem cells can be transplanted in renal anemia rats, and can produce sufficiently active EPO to correct anemia. Transplantation of the cells beneath the renal capsule is best.
Maintenance and Expansion of Hematopoietic Stem/Progenitor Cells in a Biomimetic Hematopoietic Cells Niche.
Authors: Liu Ting , Tan Jing , Hou Li , Meng Wentong , Wang Yuchun
Published: October 2019

As main hematopoietic organ, bone marrow have three dimensional microenvironment for hematopoietic stem/progenitor cells grow in, so call “hematopoietic cells niche”, which are composed by stromal cells and extracelluar matrix. The interactions of cell to cell and cell to matrix between stem/progenitor cells with hematopoietic niche are facilitated by its three dimensional conformation. The biology behaviors of hematopoietic stem cells are mediate by many signal transductions between stem/progenitor cells with their corresponding microenvironment. Now there are strong evidence from animal model study suggests that osteoblasts might play an essential role in creation of a hematopoietic stem cell niche and thereby regulation of stem cell maintenance, proliferation, and maturation. In light of the structure-function relationship of bone marrow topography, we conceived a biomimetic culture system (3D culture system) with bio-derived bone as framework, composited with human marrow mesenchymal stem cells, and induced the cells into osteoblasts to simulate the effects of hematopoietic osteobalst niche. CD 34+ cells or mononuclear cells separated from umbilical cord blood were cultured for 2∼5 weeks in the 3D culture system and also in a conventional 2D culture system as control without additional cytokine supplement. Based on our results, higher expression of extracelluar matrix and N-cadherin were observed in 3D culture system compared to 2D system. At 2 weeks culture, 3D culture system showed higher number of CD34+ cells and CD34+/CD38- cells when compared with the input (P<0.05), the increased cells were predominant CD34+/CD38-cells. Although CD34+ cells were decreased at 5 weeks culture; nevertheless, CD34+/CD38- cells were still maintained at high level. We also observed that imbedding MNCs with a higher percentage of CD34+/CD38-cells cultured in 3D system (P<0.05), which may represent a down regulation of CD38 phenotype during culture. The function of cultured cells was evaluated in colony forming unit (CFU) assay and long term culture initial cell (LTC-IC) assay. 3D system produced higher expansion of CFU progenitors than 2D system (7.13–8.89 times vs. 1.22–1.31times) after 2 weeks culture. Of note, the colony distribution of 3D system manifested higher percentage of BFU-E and CFU-GEMM, while 2D system showed mainly CFU-GM. LTC-IC represents the primitive progenitor, 3D system showed a 6.2 times increment over input after 2 weeks culture. Furthermore, it was competent to maintain the immaturation of hematopoietic progenitor cells (HPCs) over 5 weeks. This study demonstrated that our 3D culture system constructed with the bio-derived bone composited with induced osteoblast is capable to allow maintenance and expansion of primitive hematopoietic progenitor cells in vitro. It may open a new avenue to study HSCs/HPCs behaviors and to achieve sustained primitive progenitor cell expansion.
Transvection Mediated by the Translocated Cyclin D1 Gene in Mantle Cell Lymphoma.
Authors: Epner Elliot M. , Liu Hui , Wang Jing , Thayer Mathew
Published: October 2019

Cyclin D1 expression in B cells is deregulated by chromosome translocations involving the immunoglobulin heavy chain (IgH) locus in mantle cell lymphoma (MCL). Gene targeting experiments produced MCL cell lines that had lost the translocated t(11;14) and no longer expressed cyclin D1. In these cyclin D1 (−) cells, the nonrearranged cyclin D1 (CCND1) locus reverts from CpG hypomethylated to hypermethylated. Reintroduction of the translocated chromosome by somatic cell hybrid fusion induces loss of methylation at the unrearranged CCND1 locus. Thus, the translocated chromosome exerts a transallelic effect on the unrearranged CCND1 locus in B cells that resembles transvection in Drosophilia. Control somatic cell fusion experiments with a nontranslocated cyclin D1 locus do not demonstrate transvection effects. We also have evidence for pairing of the translocated and nontranslocated cyclin D1 loci in MCL cell lines and MCL patient samples. This pairing is not related to DNA replication, as it is observed in flow sorted G1 cells and not in cyclin D1 expressing breast cancer cells or in B lymphocytes. In addition, to pairing of the cyclin D1 loci, we also have demonstrated translocation specific small RNAs in MCL cells upstream of the cyclin D1 gene.
Involvement of STAT3 Transcription Factor in Disseminated Nasal-Type Natural Killer Cell Lymphomas.
Authors: Coppo Paul , Gouilleux-Gruart Valérie , Bouchet Sandrine , Huang Yenlin , Dartigues Peggy , Gaulard Philippe , Agbalika Felix , Douay Luc , Lassoued Kaiss , Gorin Norbert C.
Published: October 2019

Disseminated nasal-type natural killer (NK) cell lymphoma is an aggressive disease with very poor prognosis. The usual chemoresistance of this disease led us to explore the possible role of the transcription factor STAT3 in oncogenesis. For this, we established and characterized a continuous interleukin (IL)2-dependent NK cell line (MEC04) from a patient with a fatal nasal-type NK cell lymphoma. MEC04 cells phenotype was CD3−, CD7+, TCR−, CD16−, CD56+bright, p58−, p70−, NKP−, and NKG2A+, they harbored poor cytotoxic activity against K562 cells, and spontaneously secreted interferon-γ, IL-10 and vascular-endothelium growth factor in vitro, suggesting that malignant cells arise from the CD56+bright regulatory NK cell population. Injection of MEC04 cells to NOD/SCID mice led to a fatal multiorgan infiltration by malignant cells. We show by immunohistochemistry that STAT3 is phosphorylated in Y705 dimerization residue in MEC04 and YT cell lines, and on biopsies from 6/6 patients with nasal-type NK cell lymphoma, suggesting that STAT3 may be oncogenic in this disease. By contrast, Y705 residue was not phosphorylated on 2 patients with hepatosplenic γδT-cell lymphoma. By confocal microscopy, we showed that STAT3 was located in nucleus in MEC04 cells. Y705 STAT3 phosphorylation involved JAK2, since exposure of cells to AG490 inhibitor inhibited Y705 STAT3 phosphorylation, and correlated in a dose-dependent growth inhibition at 24 hours and 48 hours. By using transducible TAT-STAT3b or TAT-STAT3Y705F recombinant proteins (a dominant-negative form of STAT3 [STAT3b isoform], and a STAT3 protein mutated on Y705 residue which prevents STAT3 dimerization [STAT3Y705F]), we were able to demonstrate that inhibition of STAT3 activation increased mortality and decreased proliferation by more than 50 p. cent at 24 hours and 48 hours, which correlated with decreased expression of 2 STAT3 target genes (Bcl-XL and c-Myc). These results suggest that STAT3 protein has a major role in the oncogenic process of nasal-type NK cell lymphomas, and may thus represent a promising therapeutical target.
Synergistic Effect of Magnetic Nanopartical Fe304, Au and Daunomycin on K562/A02.
Authors: Chen Bao-An , Dai Yong-Yuan , Wang Xue Mei , Zhang Ren-Yun , Gao Feng , Chen Jian , Gao Chong , Sun Yun-Yu , Wang Jun , Zhao Gang , Ding Jia-Hua , Chen Ning-Na , Pei Xiao-Ping
Published: October 2019

Objective: This paper was to study the reversal effect of magnetic Nano-Fe3O4 or Nano-Au with DNR, on multidrug resistance cell line K562/A02 and to investigate the reversal mechanism of this combination, and to provide theoretic evidence for the clinical application of them as resistance modifying agents.

Method: The IC50 (the concentration causing 50% inhibition of cell growth) of DNR, Nano-Fe3O4 and Nano-Au respectively were assayed by MTT method.The drug-loaded nanoparticles were prepared by solvent diffusion method.Some nanoparticales, volume ratio from 1.5% to 50%, were combined with some DNR to find the best combination to prepare best drug-loaded nanopartilces.At last, the K562/A02 cells was treated with the composite of 25% nanoparticales and 10mg/L DNR, which MDR1 mRNA was assayed by RT-PCR;intracellular drug concentration and the apoptosis was determined by fluorometry and confocal fluorescence microscope.

Results: The IC50 of DNR for K562/A02 and K562 cells were 23.23mg/L and0.307mg/L respectively.Two nanoparticles themselves have not evident cytotoxic effect to K562/A02 and K562 cells.Pretreating K562/A02 cells with the composite of 25% nanoparticales and 10mg/L DNR for 48 hours partially restored the sensitivity of K562/A02 cells to DNR;K562/A02 showed apoptotic characteristics after treated with this composite;drug-loaded nanopartilces elevated the intracellular DNR accumulation in K562/A02 and its MDR1 mRNA were down regulated.Data was analyzed by SPSS 11.5 software and expressed as mean ± SD.

Conclusions:

Nano- Fe3O4 or Nano-Au can increases the intracellular free DNR concentration of the K562/A02 cells, which lead to more K562/A02 cells apoptosis. Two nanoparticles themselves could not lower the MDR1 gene expression of the K562/A02 cells, but they degraded the MDR1 gene level with combine DNR.

These results suggested that Nano- Fe3O4 or Nano-Au with DNR can reverse the resistance of K562/A02 cells significantly.
Research of Synergetic Reversal in Adriamycin - Resistance by the Application of Magnetic Nanoparticle of Fe3O4 and Tetrandrine in K562/A02 Leukemic Cells.
Authors: Chen Bao-An , Shun Qian , Wang Xue-Mei , Zhang Ren-Yu , Gao Feng , Chen Jian , Gao Chong , Wang Jun , Ding Jia-Hua , Zhao Gang , Chen Ning-Na , Pei Xiao-Ping , Dai Yong-Yuan , Shun Ning
Published: October 2019

Objective: To prepare functionalized Fe3O4-magnetic nanoparticles(Fe3O4-MNPs) loaded with adriamycin(ADM) or Fe3O4-MNPs co-polymerized with ADM and tetrandrine(Tet) to investigate whether the temperature, time or ratio of drug to nanoparticles influences the polymerization. To study the reversal role that the drug-loaded nano-composites play in K562 and one of its resistant cell line K562/A02 and to learn the reversal mechanism of this kind of treatments so as to provide theoretic evidence for the clinical application of them as resistance modifying agents.

Method: The drug-loaded nanoparticles were prepared using mechanical absorption polymerization process in different condition of 4° or 37° for 24h or 48h. To investigate whether Fe3O4-MNPs loaded with ADM and Tet would play a synergetic reverse role in multidrug resistant cell, the drug-loaded nanoparticles by mechanical absorption polymerization were prepared to act with K562 and one of its resistant cell line K562/A02. The survival of cells which were cultured with drug-loaded nano-composites for 48h was observed through MTT assay, the growth inhibition efficacy of cells was calculated then. Using cells under the same condition described before, we took use of fluorescence microscope to measure fluorescence intensity of intracellular ADM at a wavelength of 488nm. P-glycoprotein (P-gp) was analyzed with flow cytometer. The expression of mdr1 mRNA was measured by RT-PCR.

Results: The results showed that the growth inhibition efficacy of both the two cells increased with augmenting concentration of Fe3O4-MNPs which were loaded with drugs. The condition of 4° and 48h was significantly better than that of 37° and 24h respectively. Both Fe3O4-MNPs loaded with ADM or Fe3O4-MNPs co-polymerized with ADM and Tet elevated the intracellular ADM accumulation in K562/A02. However, no linear correlation was found between fluorescence intensity of intracellular adriamycin and augmenting concentration of Fe3O4-MNPs in K562/A02. Tet could downregulate the level of mdr-1 gene and decrease the expression of P-gp. Furthermore, Tet polymerized with Fe3O4-MNPs reinforced this downregulation, causing a 100-fold more decrease in mdr1 mRNA level, but did not reduce total P-gp content.

Conclusions: Fe3O4-MNPs can load ADM or both ADM and Tet by mechanical absorption polymerization, which depends on proper temperature and time. Furthermore, the drug-loaded nano-composites have the ability in multidrug resistance reversal. Fe3O4-MNPs loaded with ADM or Tet can enhance the effective accumulation of the drugs in K562/A02 and Fe3O4-MNPs loaded with Tet obviously reverse multidrug resistance by reinforcing mdr1 gene downregulation. Functionalized Fe3O4-MNPs loaded with Tet probably have synergetic effect on reversal in multidrug resistance.
Development as Anti-Leukemic Agents, a Novel Synthesis of Phospha Sugar Derivatives in Therapy for Leukemias, and Analysis of Their Characterizations.
Authors: Nakamura Satoki , Okinaka Keiji , Hirano Isao , Ono Takaaki , Shigeno Kazuyuki , Shinjo Kaori , Fujie Michio , Iguchi Yukiko , Niimi Taishi , Ito Satoru , Suyama Takuya , Asai Kazuhide , Yamashita Mitsuji , Ohnishi Kazunori
Published: October 2019

[Background] Sugar derivatives whose oxygen atom in the hemiacetal ring is replaced by a carbon, nitrogen, sulfur atom, and etc., are celled as pseudo sugars. The synthesis and bioassay of these pseudo sugars are well investigated and are known as potential bioactive materials. Phospha sugar, which is one of pseudo sugars, having a phosphorus atom instead of the oxygen atom in the hemiacetal ring, is little known. The synthesis and bioassay of phospha sugars are not well studied in detail. We synthesized the 2, 3, 4-tribromo-3-methyl-1-phenyl phospholane 1-oxide (TMPP) and 2, 3-dibromo-3-methyl-1-phenyl phospholane 1-oxide (DMPP) by the nucleophilic substitution reactions, and found their anti-leukemic activities.

[Purpose] The aims of the present study were to evaluate the inhibition of proliferation and induction of apoptosis in leukemia cell treated with TMPP or DMPP, and define the target molecules for TMPP in leukemia cells.

[Methods] The cells used in this study were human leukemia cell lines, K562, U937, and YRK2 cells. For proliferation analysis, MTT assays were performed in leukemia cells treated with TMPP. For cell cycle analysis, flow cytometory analysis was performed in leukemia cells treated with TMPP or DMPP by PI staining. For analysis of mitotic regulatory proteins (p27, p21, Skp2, Cdc25B, Cyclin D1, Survivin, and Aurora kinase B), Western blotting was performed in leukemia cells treated with TMPP.

[Results] In leukemia cell lines, DMPP and TMPP significantly inhibited the cell proliferation, and TMPP more strongly inhibited the cell proliferation than DMPP. 10 μM TMPP significantly induced G2/M phase arrest, and 25 μM TMPP induced apoptosis in leukemia cells. In leukemia cells, 10 μM TMPP reduced protein of Aurora kinase B, Survivin, Cyclin D1, Skp2 KIS, and FoxM1, while increased protein expression of p21and p27 by western blot analysis. Moreover, 25 μM TMPP activated caspase-3 and caspase-9, cleaved PARP, and reduced Bcl-2.

[Conclusions] In this study, we report in the first time the possibility of phospha sugar derivatives as anti-leukemic agents in therapy for leukemias, and analysis of their characterizations. DMPP significantly inhibited the proliferation, and induced apoptosis of leukemia cells. These results demonstrated that the FoxM1, which is an essential transcription factor for cell growth and regulates expression of the mitotic regulators, is one of the targets for DMPP. Therefore, DMPP or TMPP has possibility as new agents for FoxM1 in leukemia therapy.
Reversal of P-Glycoprotein-Dependent Resistance to Adriamycin by 5-bromotetrandrine in K562/A02 Cell Line.
Authors: Chen Bao-An , Wang Jue-Qiong , Ding Jia-Hua , Gao Feng , Chen Jian , Gao Chong , Sun Yun-Yu , Zhao Gang , Chen Ning-Na , Pei Xiao-Ping , Dai Yong-Yuan , Shun Qian
Published: October 2019

Objective: The present study aimed to evaluate the MDR reversal activity of bromotetrandrine (BrTet), a bromized derivative of tetrandrine (Tet), in vitro.

Methods: Drug sensitivity was determined using the MTT assay. Adriamycin (ADM) accumulation, the protein levels of P-glycoprotein (P-gp) and the apoptotic changes were analyzed by fluorospectrophotometry, respectively. The mRNA levels of P-gp was determined by RT-PCR.

Results: BrTet at 0.25, 0.5 and reversed ADM resistance in MDR K562/A02 cells dose-dependently and its potency was greater than that of Tet at the same concentrations. The IC50 of ADM for K562 and K562/A02 cells were 55.122 mg/l and 1.1373 mg/l, respectively. Treating K562/A02 cells with BrTet(1uM)and TTD(1uM)both for 48 hours partially restored the sensitivity of K562/A02 cells to ADM (IC50 were 4.7729 mg/l and 13.584 mg/l respectively) but had not effect on K562 cells. The fold reversal (FR) were 11.55 and 4.06 respectively. K562/A02 cells showed apoptotic characteristics after treated with Brtet and Tet both for 48 hours compared with control group(apoptosis rate was 61.1%, 11.1% and 9.9%,respectively); Fluorospectrophotometric assay showed that BrTet significantly increased the intracellular accumulation of ADM in K562/A02 cells in a dose-dependent manner. The fluorescence intensity of intracellelar ADM in K562/A02 cells treated with ADM(1mg/L)was 33% of that in K562 cells. BrTet and Tet elevated the intracellular ADM concentration in K562/A02 cells up to 52% and 69%,respectively. BrTet also inhibited the overexpression of P-gp in K562/A02 cells. The fluorescence intensity of P-gp in K562 and K562/A02 cells was 0.5 and 97.97.The P-gp expression was down after treated with BrTet and TTD (65.05 and 54.86). The mdr1 mRNA was also down regulated.

Conclusions: BrTet showed significant MDR reversal activity in vitro. Its activity may be related to the inhibition of P-gp overexpression and the increase in intracellular accumulation of anticancer drugs. BrTet may be a promising MDR modulator for eventual assessment in the clinic.
Difference of Drug-Resistance between Single-Factor Resistance Cell Line K562/MDR1 Transfected with mdr1 Gene and Multi-Factor Resistance Cell Line K562/A02 Induced by Doxorubicin.
Authors: Gong Yuping , Ye Xueshi , Liu Ting
Published: October 2019

Objective To study difference of drug-resistance between single-factor resistance cell line K562/MDR1 transfected with mdr1 gene and multi-factor resistance cell line K562/A02 induced by doxorubicin.

Methods Retroviral virions carrying the complete sequence of mdr1 gene cDNA were produced and infected drug-sensitive leukemia cell line K562 and mdr1 single-factor resistance cell line K562/MDR1 was established. The difference of drug-resistance between K562/MDR1 and K562/A02, a kind of multi-factor resistance cell line induced by doxorubicin, was studied by checking the expression of mdr1 gene and Pgp, daunorubicin efflux rate, MTT drug sensitivity to chemotherapeutic drug. Lentiviral vector encoding shRNA which targeted MDR1 gene was transfected into two kinds of cell lines and effect of RNAi on reversing drug resistance was detected.

Results The results of Q-PCR and flow cytometry demonstrated that there were high expression of mdr1 mRNA and Pgp in both kinds of drug-resistance cell lines and no difference between them. The function of Pgp detected by daunorubicin efflux rate is higher in K562/MDR1 (90.93%) than K562/A02 (78.67%). The results of MTT test showed that IC50 of K562/MDR1 and K562/A02 is 0.55 and 1.22μmol/L respectively and this confirmed that drug-resistance in K562/A02 is higher than that in K562/MDR1. After RNA interference, the expression of the mdr1 gene and Pgp in K562/MDR1 markedly was down-regulated and the drug resistance was restored and IC50 is 0.16μmol/L, similar to K562 sensitive cell line. The expression of the mdr1 gene and Pgp in K562/A02 markedly was downregulated too, and drug resistance to anticancer drug is reduced to some extent but IC50 is 0.56μmol/L, it is still higher than that in sensitive cell line.

Conclusion Drug-resistance in K562/A02 induced by anticancer-drug was made of many factors and it is more resistance to anticancer-drug than that in K562/MDR1 caused by mdr1 gene. Due to only mdr1 resistance, K562/MDR1 is better cell model to make mdr1/Pgp research.
Addition of PTK787/ZK 222584 Can Lower the Dosage of Amsacrine To Achieve Equal Amounts of AML Cell Death.
Authors: Weidenaar Alida C. , de Jonge Hendrik J.M. , Fidler Vaclav , Elst Arja ter , Meeuwsen Tiny , Douwes Jenny , Bouma Jessica C.A. , Hählen Karel , Kamps Willem A. , de Bont Evelina S.
Published: October 2019

Acute myeloid leukemia (AML) is a disease with a poor prognosis. At this moment conventional chemotherapy is the gold standard for the treatment of AML. It is demonstrated that AML cells express VEGFA and VEGFC as well as KDR (VEGFR2), the main receptor for downstream effects, resulting in an autocrine pathway for cell survival. The present study investigates the role of PTK787/ZK 222584, a potent inhibitor of VEGF receptor tyrosine kinases, upon leukemic cell death, and the possibility of an additional effect upon cell death achieved by a conventional chemotherapeutic drug Amsacrine. In 3 AML-cell lines and 33 pediatric AML patient samples we performed total cell kill assays to determine the percentage of cell death achieved by PTK787/ZK 222584 (5–100 μM) and/or Amsacrine (0.001–2 μg/ml). Both drugs induced AML cell death. The LC50 values (drug concentration needed to kill 50% of the leukemic cells) for HL-60, TF-1 and THP-1 were 27 μM, 49 μM and 24 μM respectively. An excellent survival was seen on a KDR-negative cell line demonstrating that the cell death induced by PTK787/ZK 222584 is not a toxic effect. The primary blasts were overall 5–10 times more sensitive to PTK787/ZK 222584 than the cell lines, with a median LC50 value of 5.1 μM. Patient samples with a LC50 value below the median did not differ from patients above the median regarding age, sex, FAB classification or WBC count. With a response surface analysis we investigated the additional effect of PTK787/ZK 222584 upon cell death achieved by Amsacrine; we estimated the concentration combinations resulting in the same cell survival. We could show that, in cell lines as well as in primary AML blasts, the concentration of Amsacrine can be lowered and replaced for a certain dose of the potentially less toxic VEGFR inhibitor to achieve the same percentage of leukemic cell death. This study shows that PTK787/ZK 222584 might have more clinical potential in AML when combined with chemotherapy, e.g. Amsacrine. In addition, it is known from literature that topoisomerase inhibitors, e.g. Amsacrine, can cause treatment related AMLs. Moreover, many survivors of childhood or adolescent cancer experience treatment related cardiovascular complications that can impair their quality of life years after treatment. Therefore, reduction of complications and long-term effects of chemotherapy is warranted and might be achieved by lowering dosages of Amsacrine and replace it by other drugs with a lower toxicity profile, such as PTK787/ZK 222584.