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1
Muller, A. M., and E. A. Dzierzak. "ES cells have only a limited lymphopoietic potential after adoptive transfer into mouse recipients." Development 118, no. 4 (August 1, 1993): 1343–51. http://dx.doi.org/10.1242/dev.118.4.1343.
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While hematopoietic stem cells from adult and fetal stages of murine development are capable of long term reconstitution of all mature blood lineages in vivo, embryonic hematopoietic stem cell repopulation in vivo has proved difficult. It is thought that there are many fewer hematopoietic stem cells in the embryo than in the fetal/adult stages of mouse development and that these cells possess a different developmental potential. One source of such cells are embryonic stem (ES) cells which can differentiate into most mature blood lineages in vitro. We have therefore used transplantation of differentiated ES cells to assess the hematopoietic potential of embryonic hematopoietic cells in vivo. We demonstrate here that precursors obtained from in vitro cultures of normal ES cells can contribute only to restricted and limited hematopoiesis in a mouse without leading to tumour formation. Repopulation occurs for greater than 6.5 months at levels ranging from 0.1% to 6% in B and T cell lineages in peripheral blood. In contrast to in vitro colony data demonstrating the myeloid lineage developmental potential of ES cells, no donor-derived myeloid repopulation was observed in CFU-S assays and no macrophage and mast cells were found in long term repopulated recipients. Thus, the hematopoietic potential of ES cells in vivo is limited to low levels of repopulation and is restricted to the lymphoid lineage.
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Denniss, Frances A. K., Nicola R. Hardwick, Ghulam J. Mufti, and Barbara A. Guinn. "An Unusual Expression: The Tumour Antigen SSX2IP Is Preferentially Expressed on the Surface of Acute Myeloid Leukaemia Cells during Early Mitosis." Blood 108, no. 11 (November 16, 2006): 4305. http://dx.doi.org/10.1182/blood.v108.11.4305.4305.
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Abstract The SSX family includes five functionally active and highly homologous members which are only expressed in the thyroid and testis in normal tissues. However these genes are expressed in several cancers including melanoma. Of note SSX1, SSX2, or SSX4 may be fused to the SYT gene as a result of the t(X;18) translocation in synovial sarcoma. Previous investigators used yeast two-hybrid systems to elucidate the SSX2 interacting protein (SSX2IP) through its association with SSX2. SSX2IP has been shown to be ubiquitously expressed in many normal tissues. We found SSX2IP through the immunoscreening of a testis cDNA library with pooled presentation M4 and M5 acute myeloid leukaemia (AML) sera. SSX2IP was found to be preferentially recognized by 62% of acute myeloid leukaemia (AML) sera (n=22) compared to 21% of normal donor sera (n=20). By RT-PCR SSX2IP was found to be expressed by four of 12 (30%) AML patient samples at presentation but none of eight normal donor haematopoietic samples (bone marrow and peripheral blood). By immunocytochemistry (ICC) we found SSX2IP protein expression in three of six myeloid leukaemia cell lines (K562, P39 and HL60), three of nine AML samples at presentation but none of three normal donor haematopoietic samples (peripheral blood). By confocal microscopy we noticed SSX2IP was predominantly expressed on the surface of K562 cells in early mitosis. We then synchronized K562 cells (as demonstrated by flow cytometry) using either serum starvation (0.1% fetal calf serum for 5 days) in the G0 phase or using 0.3mM hydroxyurea for 3 days at the G1/S interface of the cell cycle. On release back into the cell cycle (as shown by cell counts and flow cytometry) we observed a 3hr period, post-synchronisation, at 24–26 hours for serum starvation or 14–17 hours for hydroxyurea treatment during which time SSX2IP expression peaked, as detected by ICC and confocal microscopy. We transfected K562 cells with the cell surface expressed costimulatory molecule CD80 and demonstrated that SSX2IP was almost entirely restricted in its expression to the cell surface, mimicing the expression pattern of CD80. SSX2IP has a murine counterpart with 87% amino acid sequence similarity called ADIP. ADIP is a novel Afadin- and alpha-actinin-binding protein which localises at cell-cell adherens junctions. ADIP is at least partly involved in the physical association of nectins and cadherins and has more recently been implied to play a role in vesicle trafficking from the Golgi to the endoplasmic reticulum and through the Golgi complex. The mouse protein ADIP has been shown to interact with AF6, the human equivalent of which is involved in the human MLL-AF6 translocation in AML and acute lymphocytic leukaemia. However AF6 was not found to colocalise with SSX2IP in K562 cells as determined by confocal microscopy in our studies although both were expressed. P39 cells only expressed detectable levels of SSX2IP and not AF6. We have demonstrated a novel expression of the tumour antigen SSX2IP on the surface of AML cells in early mitosis. As such SSX2IP may provide a novel antibody target for the depletion of the AML cells from harvests prior to autologous transplant, when no suitable allogeneic donor can be found.
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Kaedei, Y., A. Fujiwara, F. Tanihara, Z. Namula, V. L. Vien, and T. Otoi. "39 IN VITRO DEVELOPMENT OF CANINE EMBRYOS PRODUCED BY INTERSPECIES SOMATIC CELL NUCLEAR TRANSFER USING ENUCLEATED BOVINE OOCYTES." Reproduction, Fertility and Development 23, no. 1 (2011): 126. http://dx.doi.org/10.1071/rdv23n1ab39.
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Interspecies somatic cell nuclear transfer (iSCNT) is an invaluable tool for studying nucleous-cytoplasm interactions, and may provide an alternative for cloning endangered animals, whose oocytes are difficult to obtain. Using readily available oocytes from domestic/farm animals as recipients for iSCNT would greatly benefit ongoing research on somatic cell reprogramming. However, little information is available concerning the development of canine iSCNT embryos reconstructed with bovine oocyte cytoplasm. In the first experiment, we investigated the influence of donor cell type on the development of canine iSCNT embryos reconstructed with enucleated bovine oocytes. Canine mammary gland tumour (MGT) cells and cumulus cells were used as donor cell. The bovine oocytes matured for 22 h were enucleated by the micromanipulator, and the donor cells were transferred into the perivitelline space adjacent to the plasma membrane of the oocyte. The couples were fused and activated simultaneously with a single DC pulse of 2.3 kV cm–1 for 30 μs, using an electro cell fusion generator. The reconstructed embryos were cultured for 72 h in the mSOF medium supplemented with 0.4% BSA. After 72 h of culture, only cleaved embryos were further co-cultured with bovine cumulus cells in mSOF supplemented with 5% fetal bovine serum (FBS) for an additional 5 days. In the second experiment, we examined the effects of serum type on the development of canine iSCNT embryos. The embryos reconstructed with canine cumulus cells were co-cultured with canine cumulus cells in mSOF supplemented with 5% FBS, and canine oestrous and diestrous serum for 5 days after 72 h of culture with 0.4% BSA. Data were analysed by chi-square analysis with a Yates’ correction. More than 75% of the canine somatic cells successfully were fused with bovine enucleated oocytes following electrofusion, irrespective of the types of the donor cells. There were no significant differences in the cleavage rates of iSCNT embryos between the cumulus cell and MGT cell (66.2% v. 62.6%). Although none of the embryos reconstructed with MGT cells (n = 123) developed to the 16-cell stage, 6% of embryos with cumulus cells (n = 133) reached at least the 16-cell stage. There were no significant differences in the cleavage rates of iSCNT embryos among the types of serum. The iSCNT embryos could not develop to the blastocyst stage, irrespective of the type of donor cell and serum. In conclusion, our results indicate that the bovine oocytes partly supported the remodelling and reprogramming of the canine somatic cell nuclei, but they were unable to support the development to the blastocyst stage of canine iSCNT embryos. Moreover, the development to the late embryonic stage of iSCNT embryos may be influenced by the type of donor cell but not serum.
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Youngster, Ilan, Erez Baruch, Lior Katz, Adi Lahat, Tal Brosh-Nissimov, Jacob Schachter, Omry Koren, Gal Markel, and Ben Boursi. "90. Fecal Microbiota Transplantation in Metastatic Melanoma Patients Resistant to Anti-PD-1 Treatment." Open Forum Infectious Diseases 6, Supplement_2 (October 2019): S7. http://dx.doi.org/10.1093/ofid/ofz359.014.
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Abstract Background Most metastatic melanoma patients treated with Programed cell Death (PD)-1 blockers fail to achieve a durable response. The gut microbiota profoundly affects host immunity, and fecal microbiota transplantations (FMT) have been shown to enhance anti-PD-1 effectiveness in murine models. We report initial safety and efficacy results from the first patients treated in a Phase I study of FMT and re-induction anti-PD-1 therapy in anti-PD-1 refractory metastatic melanoma. Methods FMT donors were two metastatic melanoma patients who achieved a durable complete response to treatment. FMT recipients were metastatic melanoma patients who failed at least one anti-PD-1 line of treatment. FMT was conducted by both colonoscopic and oral administration, followed by anti-PD-1 re-treatment. Each recipient underwent pre- and post-treatment stool sampling, tissue biopsy of both gut and tumor, and total body imaging. Results Five patients with treatment-resistant metastatic melanoma were recruited. No FMT-related or immunotherapy-related adverse events were observed. To assess engraftment of the new microbiota, recipients were paired with their respective donors and stool 16S rDNA gene sequence analysis was performed. Sequencing results demonstrated post-FMT compositional dissimilarity (Unweighted UniFrac, P = 0.04, FDR q = 0.22) between the two recipient–donor groups. Specific taxonomic dynamics included post-FMT increased abundance of Paraprevotellaceae, previously associated in descriptive studies with responsiveness to treatment, and significant reductions in abundance of β-proteobacteria, previously associated with reduced response to treatment. Immunohistochemical stains of biopsies demonstrated an increased post-FMT infiltration of antigen presenting cells (CD68+) in the gut (paired T-test, P = 0.008) and in the tumor (P = 0.0076). Post-treatment intra-tumoral CD8+ T-cell infiltration was also increased. Three patients had a partial or complete response to treatment post-FMT. Conclusion FMT in metastatic melanoma patients seems to be safe and may alter recipient gut microbiota to resemble that of a responder donor. This alteration may result in intra-tumoral T-cell activity, and conferred clinical and radiological benefit in several recipients previously unresponsive to treatment. Disclosures All Authors: No reported Disclosures.
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Zhang, Yuming, Xiaoqing Feng, Cuiling Wu, Wenling Guo, Huiping Li, and Shanshan Yuan. "Various Ages Thymus Transplantation Has Differential Ability of Anti Tumor Effects." Blood 124, no. 21 (December 6, 2014): 5799. http://dx.doi.org/10.1182/blood.v124.21.5799.5799.
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Abstract [Objective] Our previously work has demonstrated that allogeneic bone marrow transplantation (allo-BMT) combined with thymus transplantation (TT) was effective in restoring donor-derived T cell function and was beneficial for enhancing graft versus tumor (GVT) effects. However, since the thymic cell functions differ with age, the most effective age of thymus should be explored. In the present study, we examined the effects of allo-BMT plus thymus transplantation (TT) from various ages (fetal, newborn, adult) to determine it’s anti tumor effects. [Methods] BALB/c mice (H-2d ) bearing Meth-A sarcoma (H-2d )were treated with allo-BMT combined with or without TT from various age B6 mice(H-2b), the tumor size and survival period of the recipient BALB/c mice were examined, histological studies were performed in the liver, intestine, and the engrafted thymus from the recipients 4 weeks after the BMT. Surface markers on lymphocytes from the spleen were analyzed by 3-color fluorescence staining using a FACScan system to determine chimerism. Cytokine production was examined for monitoring lymphocyte function. [Results]. All mice treated with BMT with or without TT showed fully donor-derived chimerism. The tumor size were significantly smaller in the mice treated with BMT plus TT than those treated with BMT alone. Interestingly, the mice treated with BMT plus newborn or fetal thymus showed the greatest degree of tumor regression. The survival rate in mice treated with BMT plus newborn thymus was significantly prolonged compared with those treated with BMT plus adult thymus or BMT plus fetal thymus. Histologically, both the cortex and medullar areas were clearly shown in each group. Normal T-cell differentiation was also observed in the engrafted thymus. The number of CD4+ T cells significantly increased in the mice treated with BMT plus TT compared with those treated BMT alone. The numbers were highest in the mice treated with BMT plus newborn thymus or BMT plus fetal thymus. Microscopic founding of small intestine and liver indicated no evidence of GVHD in all mice treated with BMT combined with or without TT. The production of IL-2 and IFN-γ was significantly elevated in the mice treated with BMT plus TT compared with those treated with BMT alone. However, the production of IL-2 has no significantly difference in all various age thymus transplantation groups. In contrast, the production of IFN-γ was the highest in the mice treated with BMT plus newborn thymus transplantation. [Conclusion]. The present study indicated that allo-BMT combined with TT induces high thymopoiesis, elicit strong GVT effects, and is effective for the host with cancer. And the combination of allo-BMT with newborn thymus is the most effect. We thus found that donor-derived T cells play an important role in the treatment of leukemia. As human thymus tissue can be obtained from patient with congenital heart disease or from aborted fetuses, so the results of the present study suggest this strategy will become a new way for the treatment of malignant tumors in human. Disclosures No relevant conflicts of interest to declare.
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Garg, Tarun K., Susann Szmania, Paul Malbrough, Charles Ekworomadu, Katie Stone, Amberly Moreno-Bost, Emily Woods, et al. "Adoptively Transferred Expanded Natural Killer Cells Inhibit Myeloma Tumor Growth In Vivo." Blood 114, no. 22 (November 20, 2009): 953. http://dx.doi.org/10.1182/blood.v114.22.953.953.
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Abstract Abstract 953 Recently developed culture conditions for expanding and activating natural killer (NK) cells may improve immunotherapeutic options for patients with multiple myeloma. We have previously shown that co-incubation of NK cells from multiple myeloma patients and healthy donors with K562 cells genetically modified to express membrane-bound interleukin 15 and the co-stimulatory molecule 41BBL (K562-mb15-41BBL) leads to a dramatic increase in both NK cell number and anti-myeloma activity in vitro. In this study, we tested the anti-myeloma activity of these expanded NK cells in vivo using a murine model, which supports the growth of myeloma cells in a microenvironment that reproduces that of the human bone marrow. We first implanted human fetal bones subcutaneously into NOD/SCID/IL2Rγ null mice and allowed them to engraft. Luciferase transfected OPM2 myeloma cells were then injected into the human bone fragment. Tumor burden was followed by bio-imaging and ELISA for human Ig. NK cells were expanded from healthy donor PBMC by co-culture with irradiated K562-mb15-41BBL cells in the presence of 300U/ml IL2 for 10-12 days. Myeloma tumor bearing mice were dosed 6-7 days after OPM2 injection (bio-imaging confirmed myeloma engraftment) with expanded NK cells via tail vein injection followed by subcutaneous IL2 to prolong NK cell survival. Flow cytometry was used to track the human NK cells in blood. Histology was performed by H&E staining of formalin-fixed, paraffin embedded tissues harvested at the end of the study. Sufficient NK cells (3×109) for dosing mice were obtained from one unit of healthy donor blood and their ability to kill OPM2 myeloma was confirmed in vitro by chromium release assays. In experiment 1, OPM2-bearing mice received two tail vein injections, 48h apart, comprising PBS, 4 ×107, or 16 ×107 (total dose) expanded NK cells with 100U IL2 given twice per week. We observed significant myeloma growth inhibition in the cohort given 16 ×107 NK cells (p<0.04) and circulating human NK cells could be detected up to day 21 post-administration. Measurement of tumor burden by bio-imaging and ELISA were highly concordant (correlation coefficient = 0.995). Histologic analyses confirmed a dramatic reduction in tumor burden in the mice treated with expanded NK cells and revealed that bone loss was more pronounced in the implanted fetal bones of the control cohort not receiving NK cells. We observed in a subsequent experiment that increasing the IL2 dose to 1000U daily led to in vivo expansion of CSFE-labeled NK cells of up to 10 generations by day 6 post-infusion, which was associated with an enhanced anti-tumor effect in the 16 ×107 dose cohort. Furthermore, we found that multiple injections of NK cells (four injections of 4 ×107 NK cells versus two injections of 8 ×107) were better tolerated in the highest dose cohort (3/3 surviving versus 6/12 surviving). In conclusion, we observed a significant anti-myeloma effect against the aggressive plasma cell leukemia cell line OPM2 with expanded NK cells and low dose IL2. Adjusting the IL2 dose to 1000U daily led to in vivo expansion of NK cells, longer term NK cell persistence, and an increased anti-myeloma effect. Importantly, this IL2 dose is comparable to the 3×106 U daily dose well tolerated in a previous trial with non-expanded NK cells in humans. Single doses of NK cells >8×107 led to high morbidity in this model while multiple doses of 4×107 were well tolerated. Experiments are in progress testing the in vivo effect of expanded NK cells on primary myeloma cell tumors in this model. Our findings support the planned clinical application of ex vivo expanded NK cells. Disclosures: No relevant conflicts of interest to declare.
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Rettig, W. J., H. P. Erickson, A. P. Albino, and P. Garin-Chesa. "Induction of human tenascin (neuronectin) by growth factors and cytokines: cell type-specific signals and signalling pathways." Journal of Cell Science 107, no. 2 (February 1, 1994): 487–97. http://dx.doi.org/10.1242/jcs.107.2.487.
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The extracellular matrix protein tenascin (TN) is expressed with precise temporo-spatial patterns during embryonic and fetal development and is induced in healing wounds, inflammatory lesions and solid tumors. These tissue patterns suggest that TN synthesis may be modulated by soluble factors present in developing tissues or released from injured, inflammatory or neoplastic cells. To characterize the extrinsic control of human TN we examined the effects of several signalling molecules on cultured neural, melanocytic and fibroblastic cells. Results obtained with alpha TN antibodies in enzyme-linked immunosorbent and immunoprecipitation assays indicate that TN expression is tightly regulated in a cell type-specific manner: (1) Primitive neuroectodermal tumor (PNET) cells grown in chemically defined, serum-free media show up to > 100-fold TN induction in response to fibroblast growth factors (aFGF, bFGF, K-FGF) and phorbol ester, independent of changes in cell proliferation or total protein synthesis; no induction is seen in PNET cultures stimulated with serum or other growth and differentiation factors. (2) Normal melanocytes, which require FGF and phorbol ester for survival in vitro, fail to express TN; however, they produce TN following oncogenic transformation. (3) Fibroblasts derived from disparate tissues differ up to 100-fold in basal TN production; for example, fetal lung fibroblasts are TNhigh, but conjunctival fibroblasts derived from the same donors and fetal leptomeningeal cells are TNlow. (4) TNlow fibroblasts treated with interleukin-1, tumor necrosis factor-alpha, and interleukin-4 show up to > 100-fold increased TN secretion and TN incorporation into their extracellular matrix. Transforming growth factor-beta, which acts as an inducer of fibronectin, collagen, and integrin-type matrix receptors, has variable effects on fibroblast TN, ranging from increased deposition in the extracellular matrix of fetal conjunctival fibroblasts to reduced secretion in newborn foreskin fibroblasts. In contrast, FGFs (which are potent fibroblast mitogens), phorbol ester, bone morphogenetic proteins, and several other factors tested produced no discernible effects on fibroblast TN expression. These findings suggest that discrete sets of extrinsic signals modify TN expression in specific cell types, with the effects of a given ligand/receptor system determined by cell type-specific signalling pathways that may be linked to unique cis-regulatory elements of the TN gene. As a result, a limited set of regulatory peptides may produce highly diversified TN distribution patterns in developing and lesional tissues.
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Richter, Guenther, Uwe E. Hattenhorst, Sabine Roessler, Martin S. Staege, Gesine Hansen, and Stefan Burdach. "Transcriptome Analysis of Pediatric cALL Versus Normal Fetal B Cells Reveals a Novel Signature of the Malignant Phenotype." Blood 106, no. 11 (November 16, 2005): 4352. http://dx.doi.org/10.1182/blood.v106.11.4352.4352.
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Abstract Differential gene expression profiling of pediatric common acute lymphoblastic leukemia (cALL) versus non-malignant tissues enables identification of aberrantly expressed genes in malignant cells, facilitating discrimination of leukemic from normal cells and possibly revealing specific disease mechanisms. Expression patterns of 29 pediatric cALL samples were analyzed by use of high-density DNA microarrays HG-U133A. Leukemic patients’ bone marrow samples were compared to sorted B cells from cord blood of healthy donors expressing CD19 and CD10 surface antigens. Principal component analysis clearly distinguished leukemia samples from normal controls. Significance analysis of microarrays revealed 723 genes significantly up-regulated, and 617 down-regulated genes in leukemic cells. Independent validation of deregulated genes by RT-PCR was chosen to address enrichment limitations of controls. A comparison to previous publications investigating genetically defined subsets of cALL revealed only 5 – 22% match with our differentially expressed genes. Furthermore, class prediction with only 14 differentially expressed genes correctly classified tumors and controls not included in the training set and hitherto not investigated samples including 3 leukemic tumor lines (NALM-6, CALL-2, 697) as cALL. Interestingly, terminal deoxynucleotidyl-transferase (DNTT) as well as in the context of cALL unknown genes, were found to be the strongest predictive genes for the malignant phenotype signifying the diagnostic value of our approach.
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King, W. A., B. G. Jeon, and D. H. Betts. "47 BLASTOCYST DEVELOPMENT RATE OF CLONED BOVINE EMBRYOS USING SERIAL NUCLEAR TRANSFER OF CELLS CONTAINING AN X-AUTOSOME-TRANSLOCATED CHROMOSOME t(Xp+;23q-)." Reproduction, Fertility and Development 18, no. 2 (2006): 132. http://dx.doi.org/10.1071/rdv18n2ab47.
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Somatic cell nuclear transfer (SCNT) has been utilized to study various genetic and epigenetic contributions of specific biomedical diseases and developmental events by using various donor cell types such as mature lymphocytes, brain tumor cells, and other unique genotypes. Previously, we produced cloned fetuses and offspring derived from SCNT of adult ear skin fibroblasts obtained from a sub-fertile cow harboring an X-autosome translocation as a model to study X-inactivation and chromosome dynamics during female meiosis. The aim of this study was to assess the cloning efficiency of the fibroblasts derived from a cloned calf with the X-autosome translocation t(Xp+;23q-) compared to the original adult fibroblast donor containing the same chromosome translocation. Primary cultures of cells were established in DMEM +15% fetal calf serum (FCS). To serve as nuclear donors, cells at 5-7 passages were cultured for 5 days until confluent. Oocytes matured for 18 h in TCM-199 with hormones were removed of their chromatin, and reconstructed by transfer of donor cells and fusion with two DC pulses (1.2 kV/cm, 15 �s), delivered by a BTX 2000 Electro Cell Minupulator (BTX, Inc., San Diego, CA, USA), in 0.28 M mannitol containing 0.01 mM MgCl2. After 1 h of fusion, the eggs were activated with 5.5 �M ionomycin for 5 min, followed by 11 �g/mL cyclohexamide for 5 h. The eggs were cultured for 9 days in L-SOF at 39�C in a humidified atmosphere of 5% CO2, 5% O2, 90% N2. Chi-square analysis revealed no significant (P > 0.05) differences in the rates of cleavage, blastocyst frequencies, and cell numbers between the 1st and 2nd generation cloned embryos. Cleavage rates were 87.4% and 85.4% for 1st and 2nd generation cloned embryos, respectively. The frequencies of blastocyst development and hatched blastocyst formation on Day 9 were 41.4% (91/220) and 38.7% (92/238), and 26.4% (58/220) and 22.7% (54/238) for the 1st and 2nd generation cloned embryos, respectively. The numbers of total cells and inner cell mass (ICM) cells of Day 9 blastocysts were 183 and 52, respectively, in the 1st generation embryos and 167 and 51 cells in the 2nd-generation cloned embryos. In summary, 2nd generation cloned embryos derived from fibroblasts of a cloned calf with an X-autosome translocated chromosome showed embryo development and cell numbers similar to those of the 1st generation clones. These results demonstrate that serial nuclear transfer does not improve the blastocyst development rate of cloned embryos containing the X-autosome translocation t(Xp+;23q-). This work was funded by OCAG, OMAF, and CRC.
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Pereira, Marta Isabel, and Artur Paiva. "Dendritic Cells in Cord Blood Transplantation: A Review." Stem Cells International 2011 (2011): 1–7. http://dx.doi.org/10.4061/2011/539896.
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Dendritic cells (DCs) are a heterogeneous population of antigen-presenting cells derived from hematopoietic progenitors that bridge the transition between the innate and adaptive immune responses, while maintaining self-tolerance and Th1/Th2 homeostasis, by priming other cells in either an immunogenic or tolerogenic direction. Through their role in both innate and adaptive immunity, DCs play a major part in transplant engraftment and rejection and in graft-versus-host disease (GvHD). Preferentially tolerogenic or immunogenic DC subtypes offer targets for immunotherapy, to optimize transplant success rates and prolong disease-free and overall survival. Cord blood DCs are immature and preferentially tolerogenic, due to maternal-fetal tolerance, leading to better graft acceptance and immune reconstitution and explaining the lower incidence and severity of GvHD in CB transplantation, despite donor-host mismatching. Manipulation of DC maturation and cell loading with tumor-antigens can direct antitumor immunity and target minimal residual disease, as demonstrated for acute myeloid leukemia, optimizing the graft-versus-leukemia effect.
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Warren, Edus H., Jeffrey K. Mito, Scott S. Tykodi, John A. Thompson, Brenda M. Sandmaier, Rainer Storb, Stanley R. Riddell, and Benoît Van den Eynde. "A Polymorphic Oncofetal Antigen Recognized by CD8+ CTL from Two Patients Experiencing Regression of Metastatic Renal Cell Carcinoma after Allogeneic HCT." Blood 106, no. 11 (November 16, 2005): 3097. http://dx.doi.org/10.1182/blood.v106.11.3097.3097.
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Abstract Regression of metatstatic renal cell carcinoma (RCC) has been observed in 8–57% of patients undergoing nonmyeloablative allogeneic hematopoietic cell transplant (HCT) from HLA-matched donors. Tumor regression in this setting typically occurs after complete donor T cell engraftment is established, and is correlated with the development of GVHD. We previously isolated from two patients experiencing regression of metastatic RCC after nonmyeloablative allogeneic HCT CD8+ CTL clones that recognized a RCC-associated minor histocompatibility (H) antigen presented by HLA-A*0201, and localized the gene encoding this antigen to chromosome 19q. To identify the gene that encodes this antigen, a cDNA expression library made from a HLA-A*0201+ minor H antigen-positive tumor cell line was screened using a transfection assay in COS-7 cells. After two rounds of screening, a 1.4 kb cDNA was identified that stimulated HLA-A*0201-dependent cytokine release from minor H antigen-specific CTL. Sequencing showed that this cDNA was derived from the MGC13170 gene on chromosome 19q, within the region of significant linkage. Sequencing of MGC13170 alleles in minor H antigen-positive and minor H antigen-negative cells revealed six single nucleotide polymorphisms (SNPs), inherited as a haplotype and all previously identified, that determined susceptibility or resistance to lysis by minor H antigen-specific CTL. Analysis of 5′ and 3′ truncation constructs derived from the MGC13170 cDNA identified an interval encoding a 48-residue open reading frame that contained the epitope and spanned one of the six SNPs (dbSNP rs3745526) that had been shown to correlate with CTL recognition. Further analysis of minigenes spanning this nonsynonymous T↔A SNP identified an interval containing T at the polymorphic position and encoding a predicted 11-residue peptide that stimulated maximal HLA-A2-dependent cytokine release from minor H antigen-specific CTL. The T↔A SNP is predicted to create a Ser↔Thr polymorphism in the encoded peptide. A synthetic 11-mer peptide containing Ser at the polymorphic residue, but not the homologous peptide containing Thr at this position, was recognized by minor H antigen-specific CTL when pulsed onto minor H antigen-negative HLA-A*0201+ cells, thus demonstrating that the T allele of MGC13170 encodes the minor H antigenic peptide. The function of MGC13170 is unknown, but a previous study (Zhou et al., Ai Zheng21:341–345, 2002) suggested a role in resistance of tumor cells to chemotherapeutic agents. The range of tissues in which the epitope-encoding MGC13170 sequence is expressed was evaluated in silico by probing the translated human EST database with the predicted sequence of the 48-residue polypeptide encoded by the epitope-containing ORF. Of the several hundred ESTs identified that encoded the Ser- or Thr-containing peptide sequence, 72% were derived from tumor cells or cell lines (including RCC), 5% from embryonic or fetal tissues, 9% from embryonic stem cells, and 11% from normal tissues. Thus, MGC13170 encodes a novel HLA-A*0201-restricted minor H antigen that is expressed in a wide variety of neoplastic, embryonic, and fetal cells. Further studies of MGC13170 as a possible target for antitumor immune responses after allogeneic HCT are underway.
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Hirano, Naoto, Marcus O. Butler, Zhinan Xia, Seiji Kojima, and Lee M. Nadler. "γ-Globin, a Tumor-Associated Antigen for Juvenile Myelomonocytic Leukemia (JMML): A Cell-Based Approach To Identify Tumor Antigenic Epitopes That Are Naturally Processed and Presented." Blood 104, no. 11 (November 16, 2004): 3418. http://dx.doi.org/10.1182/blood.v104.11.3418.3418.
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Abstract Juvenile myelomonocytic leukemia (JMML) is a rare clonal myeloproliferative disorder of early childhood. Although allogeneic stem cell transplantation can induce long-term remissions, relapse rates remain high, and innovative approaches are needed. Since donor lymphocyte infusion in JMML is efficacious, T cell mediated immunotherapy may be effective, and appropriate antigenic targets must be identified. One candidate tumor-associated antigen for the immunotherapy of JMML is γ-globin, which is expressed at high levels in most JMML patients. Most clonogenic JMML cells constitutively express this onco-fetal protein, which is not necessary for the normal erythropoesis of children and adults. To determine whether γ-globin can serve as a target for immunotherapy in JMML, we sought to determine whether γ-globin is naturally processed and presented by the HLA complex. Using conventional bioinformatic techniques and the T2 binding assay to predict candidate epitopes, we identified 4 γ-globin derived peptides (g031, g071, g105, and g106) that were predicted to bind to the HLA-A2 molecule in vitro. Since this strategy provides no evidence for which predicted epitopes are processed and presented by tumor cells in vivo, we employed a biochemical strategy to determine which peptides are naturally processed and presented. This step is critical in certifying that a candidate peptide epitope is an appropriate target for immunotherapy treatments. Using our K562-derived artificial APC (aAPC), an APC that expresses A2 and no other HLA allele, we introduced the EGFP-γ-globin fusion gene. We then acid stripped peptides directly from the surface of one billion aAPC/EGFP-γ-globin cells without subjecting the cells to detergent mediated lysis. Peptides less than 5 kDa in size were fractionated by reverse phased HPLC analysis and analyzed by mass spectrometry. We identified two mass spectrometry peaks which corresponded to γ-globin derived peptides, g031 and g105. Of these, the identity of one peak, g105, was successfully confirmed by peptide sequencing, providing strong evidence that g105 is naturally processed and presented by aAPC/EGFP-γ-globin cells. Next, to confirm that g105 is processed and presented by primary JMML cells, we generated γ-globin specific CD8+ cytotoxic T cells (CTL) from A2 positive healthy donors using synthetic g105 peptide. γ-Globin specific CTL were able to specifically cytolyze A2+ γ-globin+ JMML cells but not A2+ γ-globin- JMML cells. Specific cytotoxicity was blocked by anti-A2 mAb but not isotype control. These results show for the first time that the γ-globin derived peptide, g105, can serve as a target epitope for the CTL directed immunotherapy of JMML. Furthermore, these results illustrate an innovative aAPC based strategy that can identify the antigenic peptide epitopes of putative tumor associated antigens that are naturally processed by tumor cells, presented via HLA class I, and can serve as targets for effective anti-cancer immunotherapy.
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Mosby, Danielle, Patty Mcgraw, Chad Duffalo, Marci Drees, Fedele Depalma, Christine Herdman, Scott Myerson, and Alfred E. Bacon. "Factors Affecting Effectiveness of Fecal Microbiota Transplant." Open Forum Infectious Diseases 4, suppl_1 (2017): S386. http://dx.doi.org/10.1093/ofid/ofx163.959.
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Abstract Background Fecal microbiota transplant (FMT) is an effective treatment for relapsing Clostridium difficile infection (CDI). With more widespread use of this intervention, variable cure rates (70–95%) have been observed. We conducted this study to identify specific patient- and procedure-level factors affecting FMT effectiveness, hypothesizing that those patients with higher comorbidity, inadequate bowel preparation, and shorter retention of transplant would fail more frequently. Methods At our 2-hospital, >1100-bed community-based academic center, we prospectively followed patients pre/post-FMT between June 2014-April 2017. To undergo FMT, patients must have ≥2 CDI relapses and failed vancomycin taper. We entered all FMT patients into a registry and followed them regularly for up to 1 year, collecting age, Charlson Comorbidity Index, number of CDI relapses, Boston bowel prep score, and stool retention time. FMT donor stool was obtained from OpenBiome (Boston, MA). We defined failure as recurrent CDI requiring treatment ≤8 weeks after FMT. We used 1-sided t-tests to test our hypotheses. Results During the study period, 41 patients (mean age 65 years, SD 17.6) underwent FMT. Most (37, 90%) were performed via colonoscopy, 1 via upper endoscopy, and 3 via oral preparation (capsules). FMT failure occurred in 10 patients (24.4%). Nearly half (n = 20) reported adverse events, including constipation, gas, abdominal pain, blood in stool, and fatigue. Three patients expired from comorbid disease, and 3 were lost to follow-up. Patients with higher Charlson scores failed more frequently (P = 0.04), and history of tumor (P = 0.03) and pulmonary disease (P = 0.04) were both associated with failure. No other factors, including age, retention time, and Boston bowel prep score, were associated with failure. Conclusion This study found that patients with multiple comorbid conditions, as defined by the Charlson index, are at risk for FMT failure. However, quality of bowel prep and retention time did not predict FMT failure. Future studies should include larger samples of FMT patients to determine whether specific comorbidities such as history of tumor and pulmonary disease are clinically significant predictors of FMT failure. Disclosures All authors: No reported disclosures.
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Tan, Tira Jing Ying, Marcus O. Butler, Aaron Richard Hansen, David Hogg, Adrian G. Sacher, Philippe L. Bedard, Kendra Ross, et al. "Feasibility study of microbial ecosystem therapeutics (MET-4) to evaluate effects of fecal microbiome in patients on immunotherapy (MET4-IO)." Journal of Clinical Oncology 37, no. 15_suppl (May 20, 2019): TPS2664. http://dx.doi.org/10.1200/jco.2019.37.15_suppl.tps2664.
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TPS2664 Background: Differences in microbiome diversity and composition in immune checkpoint inhibitor (ICI)-responders vs non-responders have been demonstrated. Transplantation of responder feces in mouse models recapitulated the ICI-responsive phenotype. MET-4 is an oral alternative to fecal transplant consisting a well-defined mixture of intestinal bacteria isolated from healthy donor stool sample. We hypothesize that co-administration of MET-4 with ICI is safe and results in alterations of the gut microbiota. Methods: Three cohorts (n = 65) of subjects with any advanced solid tumor type treated with monotherapy anti- PD1/PD-L1 antibody outside of a therapeutic clinical trial will be enrolled. Group A: safety cohort of 5 subjects already on ICI will receive MET-4 in addition to standard of care (SOC) ICI. If < 2 subjects report adverse events of CTCAE grade ≥3 within 4 weeks at least possibly related to MET-4, this dose will be declared safe and groups B/C may start enrolling. Group B (n = 40): subjects with advanced solid tumors starting on ICI, randomized 3:1 to MET-4 plus SOC vs. SOC. Group C (n = 20): subjects with advanced solid tumors already on ICI with first unconfirmed disease progression randomized 1:1 to MET-4 plus ICI continuation vs. continuing ICI. Serial stool samples will be collected for taxonomic composition, diversity, metagenomics content and MET-4 species abundance. We anticipate the following analyses: 16S rRNA sequencing, shotgun metagenomics sequencing, qPCR, Nanostring nucleic acid detection and metabolomics profiling. Serial blood sampling for flow cytometry/CyTOF. Immune microenvironment of tumor specimen will be examined using immunohistochemistry. Other major inclusion criteria: willingness to provide correlative samples, RECIST v1.1 measurable disease and ECOG 0-2. Subjects unable to swallow oral medications are excluded. For the primary objective of cumulative relative abundance and changes of ICI-responsiveness associated species between baseline and day 12 MET-4, assuming a change of 0.5 standard deviation (SD) of microbial alpha diversity, our study will have ≥84% power to identify a significant difference given a significance level at 0.05 in group B. Assuming a change of 0.9 SD of microbial alpha diversity, we will have ≥83% power to identify a significant difference in group C. Response rates and progression free survival will be assessed per RECIST v1.1 and compared with historical data. Clinical trial information: NCT03686202.
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Murase, T., T. Hotta, H. Saito, and R. Ohno. "Effect of recombinant human tumor necrosis factor on the colony growth of human leukemia progenitor cells and normal hematopoietic progenitor cells." Blood 69, no. 2 (February 1, 1987): 467–72. http://dx.doi.org/10.1182/blood.v69.2.467.bloodjournal692467.
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The effects of recombinant human tumor necrosis factor (rH-TNF) on the colony growth of human leukemia progenitor cells (L-CFU), granulocyte- macrophage progenitor cells (CFU-GM), and erythroid progenitor cells (BFU-E) were studied. L-CFU was assayed with leukemia cells obtained from patients with acute myelogenous leukemia. CFU-GM and BFU-E were assayed with bone marrow cells obtained from hematologically normal donors and patients with acute leukemia or non-Hodgkin's lymphoma in complete remission. A dose-dependent growth inhibition of L-CFU as well as CFU-GM and BFU-E was observed by rH-TNF at concentrations of 1 to 100 U/mL. The inhibitory effect on L-CFU was significantly greater than that on CFU-GM. No correlation was observed between the inhibitory effect on L-CFU and the number of colonies formed in the cultures without rH-TNF. Preincubation of the progenitor cells in culture medium containing 20% fetal calf serum with up to 1,000 U/mL of rH-TNF for 24 hours did not result in the inhibition of colony growth of L-CFU or CFU- GM. The inhibitory effect of rH-TNF was neutralized by an anti-rH-TNF murine monoclonal antibody.
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Murase, T., T. Hotta, H. Saito, and R. Ohno. "Effect of recombinant human tumor necrosis factor on the colony growth of human leukemia progenitor cells and normal hematopoietic progenitor cells." Blood 69, no. 2 (February 1, 1987): 467–72. http://dx.doi.org/10.1182/blood.v69.2.467.467.
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Abstract The effects of recombinant human tumor necrosis factor (rH-TNF) on the colony growth of human leukemia progenitor cells (L-CFU), granulocyte- macrophage progenitor cells (CFU-GM), and erythroid progenitor cells (BFU-E) were studied. L-CFU was assayed with leukemia cells obtained from patients with acute myelogenous leukemia. CFU-GM and BFU-E were assayed with bone marrow cells obtained from hematologically normal donors and patients with acute leukemia or non-Hodgkin's lymphoma in complete remission. A dose-dependent growth inhibition of L-CFU as well as CFU-GM and BFU-E was observed by rH-TNF at concentrations of 1 to 100 U/mL. The inhibitory effect on L-CFU was significantly greater than that on CFU-GM. No correlation was observed between the inhibitory effect on L-CFU and the number of colonies formed in the cultures without rH-TNF. Preincubation of the progenitor cells in culture medium containing 20% fetal calf serum with up to 1,000 U/mL of rH-TNF for 24 hours did not result in the inhibition of colony growth of L-CFU or CFU- GM. The inhibitory effect of rH-TNF was neutralized by an anti-rH-TNF murine monoclonal antibody.
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Hiwarkar, Prashant, Stuart Adams, Kimberly Gilmour, Ramya Nataraj, Denise Bonney, Kay Poulton, and Robert Wynn. "Cord blood CD8+ T-cell expansion following granulocyte transfusions eradicates refractory leukemia." Blood Advances 4, no. 17 (September 4, 2020): 4165–74. http://dx.doi.org/10.1182/bloodadvances.2020001737.
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Abstract The action of hematopoietic cell transplantation in controlling leukemia is principally mediated by donor T cells directed against residual recipient malignant cells. However, its utility is limited by graft-versus-host disease (GVHD), where alloreactivity is extended beyond leukemic and marrow cells. In a human/murine chimeric model, we previously showed that the preferential infiltration of cord blood (CB) CD8+ T cells eradicates an Epstein-Barr virus–driven lymphoblastoid tumor without causing xenogeneic GVHD. In the clinic, however, cord blood CD8+ T-cell reconstitution is significantly delayed, and the observation of such a robust antileukemia effect mediated by cord blood CD8+ T cells has not been reported. We describe an observation of very early T-cell expansion in 4 high-risk pediatric leukemia patients receiving third-party, pooled granulocytes after T cell–replete CB transplantation (CBT). The T-cell expansion was transient but robust, including expansion of CD8+ T cells, in contrast to the delayed CD8+ T-cell expansion ordinarily observed after T cell–replete CBT. The CD8+ T cells were polyclonal, rapidly switched to memory phenotype, and had the ability to mediate cytotoxicity. This phenomenon is reproducible, and each patient remains in long-term remission without GVHD. The results suggest that fetal-derived CB CD8+ T cells can be exploited to generate robust antileukemia effects without GVHD.
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Macleod, Kay F., and Benjamin T. Spike. "Defects in Management of Labile Iron and Oxidative Stress Underpin Red Cell Enucleation Defects in Rb Null Mice." Blood 106, no. 11 (November 16, 2005): 1351. http://dx.doi.org/10.1182/blood.v106.11.1351.1351.
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Abstract The Rb tumor suppressor is critically required for end-stage red cell maturation under conditions of oxidative stress, including in the developing fetal liver, in the bone marrow of aging mice, in the spleen and bone marrow of young mice treated with phenylhydrazine to induce hemolytic anemia, and in lethally irradiated mice reconstituted with donor tissue [1]. Loss of Rb resulted in a failure of end-stage red cells to enucleate, accumulation of red cells with a 4N DNA content and aberrant chromatin structure [1]. The molecular basis of these defects is not defined nor do we understand the reasons why pRb should be required under stress conditions, but not during normal “steady-state” erythropoiesis. The work presented will address both of these questions. In determining why pRb is critically required for stress erythropoiesis but not for steady-state erythropoiesis, we have demonstrated increased levels of reactive oxygen species (ROS) and labile iron in Rb null erythroblasts relative to wild-type control erythroblasts derived from E12.5 fetal liver. Furthermore, we show that quenching of ROS in Rb null erythroblasts by treatment of mice with the anti-oxidant N-acetyl cysteine (NAC) rescued aspects of the erythroid defect, including red cell enucleation and also extended the lifespan of Rb null mice. Similarly, chelation of labile iron with desferroxiamine restored enucleation capacity to Rb null erythroblasts. Furthermore, we show that the transferrin receptor (CD71) is transcriptionally repressed by pRb/E2F and examine whether deregulated expression of CD71 contributes to increased labile iron and oxidative stress in Rb null erythroblasts. These results suggest that loss of pRb limits the ability of erythroblasts to manage labile iron and oxidative stress, in part through deregulated expression of CD71, and that this contributes to the enucleation defect observed in Rb null mice. Given that pRb is itself regulated by ROS, we present a model in which the timely induction and repression of the CD71 receptor in differentiating erythroblasts is required to manage labile iron, oxidative stress and to coordinate cell cycle exit with end-stage maturation.
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Gillissen, Marijn A., Martijn Kedde, Greta de Jong, Etsuko Yasuda, Sophie E. Levie, Arjen Q. Bakker, Marie Jose Kersten, et al. "Tumor Specific Glycosylated CD43 Is a Novel and Highly Specific Target for Acute Myeloid Leukemia and Myelodysplastic Syndrome." Blood 128, no. 22 (December 2, 2016): 1646. http://dx.doi.org/10.1182/blood.v128.22.1646.1646.
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Abstract Background: Acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS) are high-risk diseases with a poor prognosis. Even with intensive treatment regimens less than 50% of patients can be cured, and for the majority of patients - those over 65 years of age and/or patients with comorbidities - such intensive regimens are not feasible. Novel therapeutic approaches such as immunotherapy directed against a specific tumor target are highly needed. Aims: The aim of our study was to identify antibodies that are highly specific for AML and to discover novel tumor-specific antigens, widely expressed on AML and MDS but not on healthy hematopoietic and non-hematopoietic cells. Methods: Allogeneic bone marrow transplantation is an immunotherapy with proven therapeutic efficacy. We selected a patient with high-risk AML who remained disease free, now more than 5 years after receiving an allogeneic HSCT and therefore can be considered to have mounted a potent graft versus leukemia response. To study the antibody repertoire of this patient we isolated CD27+ IgG+ memory B lymphocytes, about 2 years after the transplant. These cells were transduced with Bcl-6 and Bcl-xL to generate plasmablast B cell clones that produce antibodies and express the B cell receptor on the cell surface. Supernatants of these B cell clones were used to screen for binding to surface antigens on the AML cell line THP-1. Results: We identified an donor derived IgG1 antibody, AT1413, that specifically bound to AML cell lines THP-1, MOLM-13, SH-2 and others, but not to normal bone marrow cells or non-hematopoietic cells. The antibody also interacted with AML blasts from the allogeneic HSCT patient from whom the antibody was derived, and with leukemic blasts isolated from newly diagnosed AML and MDS patients. Biochemical analysis revealed that AT1413 recognizes a sialylated epitope on CD43 which is specifically expressed on all types of AML and MDS cells, as illustrated by its reactivity with blasts of each of 60 randomly selected AML and MDS patients in our clinic. Since the target it is also expressed by CD34+ hematopoietic progenitor cells obtained from fetal liver and fetal bone marrow, but not by post-natal hematopoietic progenitor cells, it can be considered to be a oncofetal epitope. AT1413 induced antibody-dependent cell-mediated cytotoxicity and complement dependent cytotoxicity of AML cell lines and primary blasts. Summary and conclusion: We have identified oncofetal-sialylated CD43 (CD43os) as a novel tumor-specific target that is widely expressed on AML and MDS blasts. Antibodies against this target have therefore high potential as therapeutic antibodies, either as a naked antibody or manufactured into an antibody-drug conjugate, bispecific T cell engager or CAR (chimeric antigen receptor) T cell. Disclosures Kersten: Celgene: Research Funding; Amgen: Honoraria.
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Oropeza, M., B. Petersen, N. Hornen, D. Herrmann, and H. Niemann. "306 GENERATION OF HUMAN A20 GENE-TRANSGENIC PORCINE FETAL FIBROBLASTS FOR SOMATIC CELL NUCLEAR TRANSFER." Reproduction, Fertility and Development 20, no. 1 (2008): 233. http://dx.doi.org/10.1071/rdv20n1ab306.
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The aim of this project was to produce transgenic pigs with improved features in xenotransplantation, by expressing the human A20 gene to modulate the acute vascular rejection (AVR) reaction ocurring after porcine-to-human xenotransplantation. The A20 gene was originally characterized as a tumor necrosis factor-inducible gene in human umbilical vein endothelial cells (Opipari AW et al. 1990 J. Biol. Chem. 25, 14 705–14 708). The gene is both anti-apoptotic and anti-inflammatory in endothelial cells (Ferran C 2006 Transplantation 82(1 Suppl.), S36–S40) and could thus prevent endothelial cell activation leading to AVR. The hA20-expression vector driven by the CAGGS hybrid promoter (chicken β-actin–rabbit β-globin) containing an IRES-neomycin resistance cassette (9.1 kb) was transfected into porcine fetal fibroblasts (PFF) derived from German Landrace porcine fetal explant cultures. Transfection of 3 � 106 cells was accomplished at 450 V and 350 µF with 10 µg of plasmid DNA. Then, G418-resistant cell clones (800 µg mL–1) were screened by PCR with hA20-specific primers for hA20 integration. Eighty clones were A20-positive in PCR screening from 4 rounds of transfection. One cell clone was verified by DNA sequencing and subsequently used as donor cells in somatic cell nuclear transfer. One hundred sixty-nine embryos were transferred to 2 synchronized peripuberal German Landrace gilts, respectively. Ultrasound examination of recipient sows on Day 22 after embryo transfer confirmed established pregnancies in both recipients. One pregnancy was allowed to go to term and 7 healthy piglets were born, whereas the second pregnancy was terminated on Day 70 of pregnancy for detailed expression analysis of the 8 isolated fetuses. Results showed that the A20 vector can be integrated in PFF, and A20-transgenic PFF can be successfully used in somatic cell nuclear transfer to establish pregnancies. Further analysis will focus on the expression levels and patterns in A20-positive cell clones and the biological function in transgenic piglets. Functional assays will be conducted in vitro and in vivo. We thank Prof. Beyaert of Ghent University, Belgium for providing us with the expression vector pCAGGSEhA20.
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Schallmoser, Katharina, Eva Rohde, Andreas Reinisch, Anna C. Obenauf, Christina Bartmann, Gerhard Lanzer, Werner Linkesch, and Dirk Strunk. "Genomic Stability and Safety of MSCs after Animal Serum-Free Humanized Clinical Scale Propagation." Blood 112, no. 11 (November 16, 2008): 2307. http://dx.doi.org/10.1182/blood.v112.11.2307.2307.
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Abstract Human multipotent mesenchymal stromal cells (MSCs) are currently tested in a growing number of clinical trials to determine their safety and efficiency as an immune modulating and organ regenerative therapy. Repeated observations of genomic instability in the commonly used fetal bovine serum (FBS)-driven MSC cultures and consecutive tumor formation by transformed MSCs in experimental animals have raised serious safety concerns. We and others have recently established alternative clinical scale MSC expansion protocols with pooled human platelet lysate (pHPL) as a substitute for fetal bovine serum. This study was performed to determine the genomic stability of MSCs expanded under humanized conditions ex vivo. Small volume (14–17mL) bone marrow aspirates of four donors (3 male: 30, 36 and 47 years; 1 female: 13 years) were seeded without manipulation directly in heparinized minimum essential medium just supplemented with pHPL and L-glutamine. Clinical scale propagation was done in a newly developed humanized cell expansion system. MSC quality, identity, purity and function were assessed according to a defined panel of release criteria. Array-comparative genomic hybridization (array-CGH) was carried out using a whole genome oligonucleotide microarray platform with female reference DNA. Samples were labeled and scanned images were analyzed using CGH Analytics software. Results confirmed that pHPL is highly efficient in stimulating MSC expansion resulting in the recovery of 780 ± 150 million MSCs (mean ± SEM) after one culture phase. Starting from 15 ± 0.6 mL bone marrow we were able to produce four application doses of MSCs (defined as 2 mio. MSCs/kg x 100kg) in a unique standardized single culture phase within 13.5 ± 1.0 days with a minimum of manipulation and without antibiotics in three of four expansions. MSC viability was ≥ 95%. Flow cytometry revealed virtually pure MSC products with >95% CD73/90/105 reactivity, <2% hematopoietic contamination with CD14/19/34/45-or HLA-DR-reactive cells and intact adipo-, osteo-and chondrigenic differentiation potential. Microbiologic safety measures included negative bacterial/ fungal/mycoplasma testing despite antibiotic/antimycotic-free culture and endotoxin levels <0.025 EU/mL. Array-CGH analysis revealed balanced profiles for all propagated MSC products. Five copy number variations (CNVs; >60kb) that were detected were not documented in the database of genomic variants. Several small (7kb–1.8Mb; n=33) autosomal CNVs were also observed previously in normal individuals and were not associated with phenotype changes. These data extend earlier results showing that MSCs expanded under humanized conditions did not form tumors in experimental animals in vivo. Our data show that despite high proliferation rate MSCs propagated in a human platelet-derived growth factor-driven system are genomically stable in array-CGH and do not form tumors in vivo. This indicates superior safety of the rapidly available humanized MSC transplants compared to currently used FBS-expanded MSCs.
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Wang, Qian, and Ping Zhu. "High Doses of Moternal Lymphocyte Infusions to Treat EBV-Associated Lymphoma in Pediatric Patients." Blood 114, no. 22 (November 20, 2009): 785. http://dx.doi.org/10.1182/blood.v114.22.785.785.
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Abstract Abstract 785 Introduction: The ability of allogeneic T cells to effect anti tumor responses had greatly improved the outcome of malignant EBV-associated lymphoproliferations. But high doses of DLI (donor lymphocyte infusion) could arise severe GVHD (graft versus host disease), and affect clinical outcomes. Recently, we successfully treated four EBV-associated lymphoma children with high doses of DLI from their IPA (inherited paternal antigens)-positive mothers, with no obvious GVHD occurred, which indicated that high doses of maternal leukocytes infusions could be an effective and safe therapy for EBV-associated lymphoma. Epstein-Barr virus (EBV) associated lymphoproliferative disorders(LPD) or hemophagocyticlymphohistiocytosis(EBV-HLH) in pediatric patients is classified into malignant lymphoma due to 2008 WHO International Working Formulation for lymphoma classification. In pediatric patients, cure is rarely get through conventional therapy such as chemotherapy. Approaches using adoptive immunotherapies offer very attractive alternative options for this subgroup of patients. The rationale for this strategy was that most EBV seropositive individuals have a high frequency of EBV specific precursors so that transfer of unmanipulated donor lymphocyte populations should be able to restore the immune response to EBV. However, these products may also contain a high frequency of alloreactive T cells so there is a significant risk of GVHD. Reciprocal cell traffic between mother and fetus during pregnancy gives rise to long-term postpartum fetal–maternal lympho-hematopoietic microchimerism (MC). Recent experimental evidence have suggested the association of MC with acquired immunologic hyporesponsiveness, which would benefit solid organ and hematopoietic stem cell transplantation. Some research have showed that mother-to-child transplantations involved significantly less GVHD and better outcome than father-to-child transplantations. Based on this fetal-maternal tolerance theory, we treated four EBV-associated lymphoma children with high doses of DLI from their mothers. Material and methods: Our four patients were all developed with fever over than 6 months, accompanied with lymphadenopathy, hepatosplenomegaly, two of them with skin lesion. The diagnosis were confirmed by skin or lymphnode biopsies. EBV-DNA is positive in all of the patients' plasma at the quantitation of >1×104 copies /ml. One of the patient had accepted chemotherapy in the past but got no remission. Using sex determining region Y chromosome (SRY) gene and insertion-deletion (InDel) polymorphism as fetal markers, child's DNA (IPAs) were detected through nested PCR and QT-PCR technology in all of the four mothers' blood at the level of 1: 104-105. With the approval of Ethics Committee, we treated these patients with infusions of unmanipulated leukocytes from their HLA-haploidentical, EBV-seropositive mothers. First at the doses below 107 PBNC cells/kg, while no side effects occurred, we began infusing high doses of mother's PBMC at above 108 PBNC cells /kg per time. Results: Clinical remissions were achieved in all of the patients within 3 to 10 days after the infusions, with remarkable reduction of the lymphnode and spleen, the EBV-DNA were undetectable in the patients' plasma. The remissions were sustained without further therapy for 5-10 months except one patient who got recurrance of lymphadenopathy and fever about two months after each infusion. We had monitored the quantitation of his mother's lymphocyte in the child's blood and found no detectable maternal MC a week after the infusion. During the process, none of our patients developed obvious GVHD. Conclusions: High doses of maternal lymphocyte infusions could be an effective and safe treatment for EBV-associated lymphoma. Further research is needed to improve the sustained effect. Disclosures: No relevant conflicts of interest to declare.
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Cozzolino, F., M. Torcia, D. Aldinucci, A. Rubartelli, A. Miliani, AR Shaw, PM Lansdorp, and R. Di Guglielmo. "Production of interleukin-1 by bone marrow myeloma cells." Blood 74, no. 1 (July 1, 1989): 380–87. http://dx.doi.org/10.1182/blood.v74.1.380.380.
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Abstract Plasma cells isolated from bone marrow (BM) aspirates of 12 patients with multiple myeloma (MM) and nine patients with monoclonal gammopathy of undetermined significance (MGUS) were analyzed for production of cytokines with bone-resorbing activity, such as interleukin-1 (IL-1), tumor necrosis factor (TNF), and lymphotoxin (LT). Culture supernatants of plasma cells from MM, but not from MGUS or normal donor, invariably contained high amounts of IL-1-beta and lower amounts of IL-1-alpha. With a single exception, TNF/LT biologic activity was not detected in the same supernatants. IL-6 was present in two of five supernatants tested. Normal B lymphocytes released both IL-1 and TNF/LT activities for four days after activation in vitro; however, production of these cytokines ceased at the final stage of plasma cell. Unexpectedly, the mRNA extracted from MM plasma cell hybridized with TNF- and LT- specific, as well as IL-1-specific probes, although the culture supernatants did not contain detectable TNF/LT biologic activity. When tested in the fetal rat long bone assay, MM plasma cell supernatants displayed a strong osteoclast-activating factor (OAF) activity, which was greatly reduced but not completely abolished by neutralizing anti- IL-1 antibodies. Anti-TNF or anti-LT antibodies were ineffective in the same test. We conclude that the IL-1 released in vivo by malignant plasma cells has a major role in pathogenesis of lytic bone lesions of human MM.
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Cozzolino, F., M. Torcia, D. Aldinucci, A. Rubartelli, A. Miliani, AR Shaw, PM Lansdorp, and R. Di Guglielmo. "Production of interleukin-1 by bone marrow myeloma cells." Blood 74, no. 1 (July 1, 1989): 380–87. http://dx.doi.org/10.1182/blood.v74.1.380.bloodjournal741380.
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Plasma cells isolated from bone marrow (BM) aspirates of 12 patients with multiple myeloma (MM) and nine patients with monoclonal gammopathy of undetermined significance (MGUS) were analyzed for production of cytokines with bone-resorbing activity, such as interleukin-1 (IL-1), tumor necrosis factor (TNF), and lymphotoxin (LT). Culture supernatants of plasma cells from MM, but not from MGUS or normal donor, invariably contained high amounts of IL-1-beta and lower amounts of IL-1-alpha. With a single exception, TNF/LT biologic activity was not detected in the same supernatants. IL-6 was present in two of five supernatants tested. Normal B lymphocytes released both IL-1 and TNF/LT activities for four days after activation in vitro; however, production of these cytokines ceased at the final stage of plasma cell. Unexpectedly, the mRNA extracted from MM plasma cell hybridized with TNF- and LT- specific, as well as IL-1-specific probes, although the culture supernatants did not contain detectable TNF/LT biologic activity. When tested in the fetal rat long bone assay, MM plasma cell supernatants displayed a strong osteoclast-activating factor (OAF) activity, which was greatly reduced but not completely abolished by neutralizing anti- IL-1 antibodies. Anti-TNF or anti-LT antibodies were ineffective in the same test. We conclude that the IL-1 released in vivo by malignant plasma cells has a major role in pathogenesis of lytic bone lesions of human MM.
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Shah, Mrinal, Aparna Vasanthakumar, Natalie Barnes, and Lucy A. Godley. "Expression of a Truncated DNMT3B Protein, DNMT3B7, Highlights the Sensitivity of Hematopoietic Progenitor Cell Function to Hormonal Milieu." Blood 114, no. 22 (November 20, 2009): 83. http://dx.doi.org/10.1182/blood.v114.22.83.83.
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Abstract Abstract 83 Epigenetic changes, including DNA methylation and histone modifications, alter chromatin structure and regulate gene transcription in numerous cellular processes, including stem cell differentiation, mammalian embryogenesis, genomic imprinting, X-chromosome inactivation, and in cancer cells. Our laboratory studies the molecular basis for the abnormal distribution of DNA methylation in tumors. We found that cancer cells exhibit aberrant splicing of the DNMT3B gene, which encodes one of the de novo DNA methyltransferase enzymes. Aberrant DNMT3B transcripts encode truncated proteins, some of which lack the C-terminal catalytic domain. We hypothesize that aberrant DNA methylation in cancer cells is due in part to the presence of these truncated, catalytically inactive DNMT3B proteins. To test the in vivo effects of expression of a truncated DNMT3B protein, we engineered transgenic mice to express DNMT3B7, a truncated isoform expressed in cancer cells, and tested its influence on murine hematopoiesis. Since homozygous DNMT3B7 transgenic mice die in mid-gestation or within hours of birth, we propagated transgenic fetal liver cells (FLCs) in lethally irradiated recipients to bypass the animals' developmental abnormalities. DNMT3B7 was expressed in E14.5 FLCs, and we achieved approximately 80% donor chimerism in all recipients. Cells from wild-type (WT) embryos engrafted normally regardless of recipient gender. However, pancytopenia occurred at 2 weeks, with anemia and leucopenia persisting until 8 weeks post-transplant when females received DNMT3B7 homozygous cells. Male recipients displayed normal peripheral blood counts regardless of donor cell genotype. For example, females receiving WT or hemizygous cells had hemoglobin levels of 10 g/dL, whereas those receiving homozygous cells had levels of about 6 g/dL. Anemia was not seen in male recipients, where hemoglobin levels were 11-12 g/dL across all donor genotypes. When DNMT3B7 homozygous cells were transplanted into female recipients, neutropenia and lymphopenia were observed. Normal white blood cell recovery was seen in male recipients. Additionally, thrombocytopenia was observed at 2 weeks in female recipients of homozygous DNMT3B7 transgenic cells, but the platelet count normalized in these animals by 4 weeks. In preliminary experiments to further examine the role of hormonal milieu, oophorectomized female recipients demonstrated loss of the previously observed effect. At 2 weeks, oophorectomized females transplanted with DNMT3B7 homozygous cells showed recovery of hemoglobin levels to levels around 11 g/dL, the same level seen in normal female recipients transplanted with WT or hemizygous cells. Oophorectomized females receiving homozygous cells also showed improvement in white blood cell count. This suggests that the female hormonal milieu is suppressive to DNMT3B7-expressing hematopoietic progenitor cell function, rather than male-specific hormones augmenting hematopoiesis. We hypothesize that DNMT3B7 alters the DNA methylation patterns, and consequently, the gene expression profiles of hematopoietic progenitor cells, revealing a dependence of these cells on a particular hormonal milieu in recipient animals. Preliminary gene expression profiling of DNMT3B7-expressing versus WT E14.5 fetal liver cells reveals 29 genes are differentially expressed. These genes fall into interesting gene ontogenies, including chromatin modification genes (GSG2, SUZ12), cell cycle, programmed cell death, cell differentiation and proliferation. The defective hematopoiesis seen up to 8 weeks after transplantation in female recipients of DNMT3B7-expressing progenitor cells, suggests that there is an important relationship between progenitor cell function and hormonal milieu. Our hope is that we will be able to use our understanding of the molecular basis for the influence of hormonal milieu on hematopoiesis to augment stem/progenitor cell function in patients undergoing stem cell transplantation and chemotherapy. Disclosures: No relevant conflicts of interest to declare.
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Woo, J. S., Y. G. Ko, H. N. Kim, J. G. Yoo, B. S. Yang, H. C. Lee, J. Y. Lee, et al. "83 THE ENDOMETRIUM AND EXTRAEMBRYONIC TISSUES RESPOND DIFFERENTLY TO CLONED v. NATURAL MATING EMBRYOS ON EARLY PREGNANCY." Reproduction, Fertility and Development 22, no. 1 (2010): 200. http://dx.doi.org/10.1071/rdv22n1ab83.
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Porcine cloning by somatic cell nuclear transfer (SCNT) has limitations because of the high incidence of fetal failure after embryo transfer to recipient. The fetal failure may originate from disturbed embryo-uterine interactions in the early implantation period. In this study, we compared apoptotic-related gene expression and cytokines using real-time RT-PCR and protein chip array. Ovaries were obtained from prepubertal crossbred gilts at a local slaughterhouse. Oocytes were matured for 40 to 44 h at 38.5°C under 5% CO2 in air. As donor cells, fibroblast cells were cultured from ear skin tissue of 8-month-old MHC inbred miniature pig. After the enucleation and injection process, eggs were held in TCM-199. For fusion, two DC pulses of 1.2 kV cm-1 were applied for 30 μs. SCNT embryos were cultured under PZM-3 medium to the 1- to 4-cell stage. For embryo transfer, approximately 200 SCNT eggs were transferred into the oviduct of each synchronized recipients. We obtained the extraembryonic (embryo origin) and endometrium (maternal origin) tissues from 3 cloned fetus with that from normal fetus by natural mating at Day 30. Each experiment was repeated at least 3 times and the data were presented as mean ± SE. Values of P < 0.05 were considered statistically significant. Data from real-time RT-PCR are expressed as log (fold change) ± SE. Expression of Bax-α, p53, and caspase-3 mRNA was significantly higher in the SCNT group than in the control group (P < 0.05). Five cytokines were analyzed: Interleukin (IL)4, IL8, tumor necrosis factor-α, and epidermal growth factor were lower in the control group than in the cloned group (P < 0.05). However, the level of MCP1 was higher in the SCNT group (P < 0.05). Numbers of trophoblast cells in the cloned group were also lower than in the control group (P < 0.05). These findings indicate that failure of endometrium and extraembryonic tissues in cloned pregnancies may originate from abnormal embryo-maternal communication that develops during the implantation period. This work received grant support from the Agenda Program (no. 200901FHT010305535), Rural Development Administration, Republic of Korea.
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Sun, Kun, Peiyong Jiang, K. C. Allen Chan, John Wong, Yvonne K. Y. Cheng, Raymond H. S. Liang, Wai-kong Chan, et al. "Plasma DNA tissue mapping by genome-wide methylation sequencing for noninvasive prenatal, cancer, and transplantation assessments." Proceedings of the National Academy of Sciences 112, no. 40 (September 21, 2015): E5503—E5512. http://dx.doi.org/10.1073/pnas.1508736112.
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Plasma consists of DNA released from multiple tissues within the body. Using genome-wide bisulfite sequencing of plasma DNA and deconvolution of the sequencing data with reference to methylation profiles of different tissues, we developed a general approach for studying the major tissue contributors to the circulating DNA pool. We tested this method in pregnant women, patients with hepatocellular carcinoma, and subjects following bone marrow and liver transplantation. In most subjects, white blood cells were the predominant contributors to the circulating DNA pool. The placental contributions in the plasma of pregnant women correlated with the proportional contributions as revealed by fetal-specific genetic markers. The graft-derived contributions to the plasma in the transplant recipients correlated with those determined using donor-specific genetic markers. Patients with hepatocellular carcinoma showed elevated plasma DNA contributions from the liver, which correlated with measurements made using tumor-associated copy number aberrations. In hepatocellular carcinoma patients and in pregnant women exhibiting copy number aberrations in plasma, comparison of methylation deconvolution results using genomic regions with different copy number status pinpointed the tissue type responsible for the aberrations. In a pregnant woman diagnosed as having follicular lymphoma during pregnancy, methylation deconvolution indicated a grossly elevated contribution from B cells into the plasma DNA pool and localized B cells as the origin of the copy number aberrations observed in plasma. This method may serve as a powerful tool for assessing a wide range of physiological and pathological conditions based on the identification of perturbed proportional contributions of different tissues into plasma.
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Tricot, Guido J., Ming Zeng, Ye Yang, Maurizio Zangari, Hongwei Xu, and Fenghuang Zhan. "The Effect of ICAM-1 Antibody Therapy in the SCID-Hu Mouse Model Using Primary Myeloma Cells." Blood 118, no. 21 (November 18, 2011): 2914. http://dx.doi.org/10.1182/blood.v118.21.2914.2914.
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Abstract Abstract 2914 Background: Myeloma (MM) - stromal cell-cell interactions play a crucial role in MM cell growth, survival, and drug resistance. Intercellular adhesion molecule-1 (ICAM-1) is up-regulated in different cancers, including MM, and represents one of the major adhesion molecules mediating tumor-stromal cell-cell contacts. Previous investigations have shown that ICAM antibodies induce antitumor activity in SCID mice xenografted with MM cell lines, likely involving Fc :Fc gamma receptor dependent anti-tumor mechanisms. We have examined the anti-MM activity in the SCID-hu mouse model of a specific humanized ICAM1 antibody (BI-505, Bio-Invent, Sweden) using primary MM cells. Materials and Methods: The expression of ICAM1 was examined in plasma cells from 22 donors, 351 newly diagnosed MMs, and 9 MM cell lines using Affymetrix U133 Plus2 chips. Human fetal femurs and tibias, obtained at 17 to 22 gestational weeks, were cut into fragments and implanted subcutaneously in 16 SCID mice (SCID-hu) at age 6 to 8 weeks. Four weeks after bone implantation, approximately 5 ×106 CD138+ plasma cells from MM patients were injected directly into the human bone of the SCID-hu mice in final volume of 30 to 40μl of phosphate-buffered saline(PBS). Human immunoglobin (hIg) levels by ELISA were used as an indicator of myeloma cell growth. When hIg level reached 10μg/mL or higher in 2 consecutive samples after 4 weeks of injection of the tumor cells, the mice were divided into four groups (n=4), Control group (not injected MM cells and no drug treatment); the other 3 groups were injected with MM cells: the isotype control group (isotype IgG 2mg/kg, i.p., twice weekly), BI-505 group (2mg/kg, i.p., twice weekly) and bortezomib group (1mg/kg, i.p., twice weekly). Bone remolding was detected by X-radiography and by measuring bone mineral density (BMD). Tumor cells were detected by immunohistochemistry using the CD138 antibody. Results: High expression of ICAM1 was observed in normal plasma cells, primary MM cells and MM cell lines. With a follow- up of 10 weeks, the tumor burden in the mice treated with BI-505 or bortezomib was significantly lower compared with the isotype control group (p: BI505 < 0.01; p: bortezomib < 0.01). Also, the number of MM tumor cells stained with the CD138 antibody was significantly less in samples treated with either BI-505 or bortezomib than in the isotype control group (p < 0.01). In addition, 6 weeks after injection of tumor cells, X-rays showed severe bone resorption in the isotype-control group, while there was no obvious bone resorption in the fetal bones after treatment with BI-505 or bortezomib. The BMD (0.0715±0.006 g/cm2) of isotype control was significantly lower than that in other 3 groups including control: 0.1278±g/cm2, BI-505 group: 0.102±0.0064 g/cm2, and bortezomib group: 0.106±0.0059g/cm2. Importantly, the number of TRAP-positive cells was significantly higher in the isotype control group than in the other 3 groups (p < 0.01). Conclusion: ICAM1 expression presents in all subtypes of myeloma. The ICAM1 antibody BI-505 significantly inhibits primary MM cell growth and bone destruction in the SCID-hu mice to the same degree as bortezomib, indicating its clinical applicability in therapy of MM. Disclosures: No relevant conflicts of interest to declare.
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Liu, Qifa, Xiaodan Liu, Meiqing Wu, Yanwen Peng, Xiaoyong Chen, Jing Sun, Fen Huang, et al. "Improvement in Poor Graft Function After Allogeneic Hematopoietic Stem Cell Transplantation Upon Administration of Mesenchymal Stem Cells From Third-Party Donors: A Pilot Prospective Study." Blood 120, no. 21 (November 16, 2012): 1904. http://dx.doi.org/10.1182/blood.v120.21.1904.1904.
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Abstract Abstract 1904 Background Poor graft function (PGF) is a refractory complication that occurs in 5–27% patients, and is associated with considerable morbidity and mortality related to infections or hemorrhagic complications after allogeneic hematopoietic stem cell transplantation (allo-HSCT). Mesenchymal stem cells (MSCs) possess the capacity to differentiate into several types of mesenchymal tissues, to suppress immunological responses, and to support hematopoiesis. Clinical applications of MSCs are evolving rapidly with goals of improving hematopoietic engraftment, preventing and treating graft-versus-host disease (GVHD) after allo-HSCT and so on. In the present study, we prospectively evaluated the efficacy and safety of MSCs expanded from the bone marrow of a third-party donor to patients with PGF after allo-HSCT. Methods Twenty patients, including primary PGF in 7 and secondary in 13, were enrolled in this prospective multicenter trial. Bone marrow-derived MSCs were obtained from HLA-unrelated third-party donors. MSCs were cultured and expanded with human MSCs growth medium (low-glucose Dulbecco's modified Eagle's medium [L-DMEM], Hyclone, Logan and UT) with 10% (v/v) fetal bovine serum. MSCs were harvested after 4–5 passages for clinical administration. MSCs were given at 4 weeks intervals at the dose of 1×106 cells/kg. If ANC counts did not achieve >1.5×109/L and PLT counts not >50×109/L within 28 d after MSCs treatment, a second course of MSCs treatment was given. Flow cytometry was used to examine the lymphocyte subsets in peripheral blood pre- and post-MSCs treatment. Results Seventeen patients were responsive and 3 (primary PGF in 2 and secondary in 1) were not to 1–3 cycles of MSCs treatment. Within the first 100 d after MSCs treatment, 13 patients developed 20 episodes of infections. Moreover, 5 patients experienced cytomegalovirus-DNA viremia and 7 experienced Epstein-Barr virus (EBV)-DNA viremia within the first 100 d after MSCs treatment. Within a median follow-up of 125 d (range 11–579 d) after MSCs treatment, 6 patients (primary PGF in 3 and secondary in 3) experienced CMV-DNA viremia, and 8 (primary PGF in 2 and secondary in 6) developed EBV-DNA viremia, including 3 with EBV-associated post-transplant lymphoproliferative disorders (PTLD). One patient developed gradeII acute GVHD and 2 developed local chronic GVHD after MSCs treatment; 2 developed acute GVHD and chronic GVHD, respectively, after donor lymphocyte infusion because of PTLD. The proportions of CD3+ T cells and CD3+CD4+ T cells at 56 d after MSCs treatment were higher than those prior to MSCs treatment (88.05 ± 1.26% vs 79.05 ± 3.23%, P=0.015 and 24.09 ± 1.95% vs 17.40 ± 1.57%, P=0.012, respectively). The proportion of CD3+CD8+ T cells at 56 d after MSCs treatment was lower than that prior to MSCs treatment (51.91 ± 2.86% vs 61.39 ± 3.43%, P=0.043), and the ratio of CD3+CD4+ to CD3+CD8+ T cells was higher after MSCs treatment (0.31 ± 0.04 vs 0.51 ± 0.06, P=0.01). The proportions of CD4+CD25+ FoxP3 regulatory T cells, CD19+ B cells, and NK cells did not change significantly after MSCs treatment. With a median follow-up time of 508 d (range, 166–904 d) post-transplantation, 9 patients were alive and 11 died. No short-term toxic side effects were observed after MSCs treatment. Two patients experienced leukemic relapse. With the exception of 3 patients with PTLD, no secondary tumors occurred. Conclusions MSCs derived from the bone marrow of a third-party donor are effective to both primary and secondary PGF after allo-HSCT. However, whether such treatment might increase the risks of EBV infection and reactivation or the development of EBV-associated PTLD should be studied further. Disclosures: No relevant conflicts of interest to declare.
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Dees, Kory, Hyunmin Koo, J. Fraser Humphreys, Joseph Hakim, David Crossman, Michael Crowley, Louis Nabors, Etty Benveniste, Casey Morrow, and Braden McFarland. "TMOD-19. INDIVIDUAL SPECIFIC HUMAN GUT MICROBE COMMUNITIES INFLUENCE RESPONSE TO IMMUNOTHERAPY IN A HUMANIZED MICROBIOME MOUSE MODEL OF GLIOMA." Neuro-Oncology 22, Supplement_2 (November 2020): ii232. http://dx.doi.org/10.1093/neuonc/noaa215.970.
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Abstract Although immunotherapy works well in glioblastoma (GBM) pre-clinical mouse models, the therapy has not demonstrated efficacy in GBM patients. Since recent studies have linked the gut microbial composition to the success with immunotherapy for other cancers, we utilized a novel humanized microbiome (HuM) model in order to study the response to immunotherapy in a pre-clinical mouse model of GBM. We used five healthy human donors for fecal transplantation of gnotobiotic mice since it is now recognized that microbe strain level differences render individual humans with a unique microbial community composition. After the transplanted microbiomes stabilized, the mice were bred to generate 5 independent humanized mouse lines (humanized microbiome HuM1-HuM5). Analysis of shotgun metagenomic sequencing data from fecal samples revealed a unique microbiome composition with significant differences in diversity and microbial composition among HuM1-HuM5 lines. We next analyzed the growth of intracranial glioma cells in the HuM lines. All HuM mouse lines were susceptible to GBM transplantation, and exhibited similar median survival ranging from 19-26 days. Interestingly, we found that HuM lines responded differently to the immune checkpoint inhibitor anti-PD-1. Specifically, we demonstrate that HuM1, HuM4, and HuM5 mice are non-responders to anti-PD-1 resulting in the death of the mice from the intracranial tumors, while HuM2 and HuM3 mice are responsive to anti-PD-1 and displayed significantly increased survival compared to isotype controls. Bray-Curtis cluster analysis of the 5 HuM gut microbial communities revealed that HuM2 and HuM3 were closely related. Detailed taxonomic comparison analysis at the top 5 across all HuM mouse lines revealed that Bacteroides cellulosilyticus was commonly found between HuM2 and HuM3 with high abundances. The results of our study establish the utility of humanized microbiome mice as avatars to delineate features of the host interaction with gut microbe communities needed for effective immunotherapy against GBM.
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Dege, Carissa, Kathleen E. McGrath, Melissa M. Berrien-Elliott, Katherine H. Fegan, Julia Alexandra Wagner, Todd A. Fehniger, James Palis, and Christopher Sturgeon. "Ontogeny As a Critical Determinant of Natural Killer Cell Potential and Function." Blood 132, Supplement 1 (November 29, 2018): 1271. http://dx.doi.org/10.1182/blood-2018-99-119812.
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Abstract Hematopoietic development during mammalian embryogenesis is comprised of a restricted primitive program of primitive erythroid, megakaryocytic, and macrophage lineages, and a definitive program of definitive erythroid, myeloid and lymphoid potential emerging from hematopoietic stem cell (HSC)-independent and dependent processes. Interestingly, progenitors of natural killer (NK) cells, but not B- or T-cells, have been found in the early human yolk sac, suggesting that NK cells may arise from HSC-independent sources. NK cells recognize and kill virally infected cells and tumor cells, making them a highly desirable cell-type for adoptive immunotherapy. To bypass donor-related issues, human pluripotent stem cell (hPSC)-derived NK cells offer the possibility of uniform activity in a renewable "off-the-shelf" cell product. As the differentiation of hPSCs recapitulates early developmental processes, we sought to characterize the developmental origin of hPSC-derived NK cells. Studies in mice indicate that NK cells in the adult are derived from hematopoietic stem cells (HSCs) that commit to a lymphoid differentiation pathway. However, while NK cells, like HSCs, have been found in the fetal liver, the developmental origin of the fetal NK cell lineage remains poorly understood. We have developed a stage-specific hPSC differentiation method that separates WNT-independent (WNTi) hematopoietic progenitors that harbor "primitive" hematopoietic potential from WNT-dependent (WNTd) erythro-myeloid-(T-)lymphoid "definitive" hematopoietic progenitors. Using this system, we find that CD34+ cells from both populations harbor NK cell potential. NK cells from hPSC WNTi progenitors (WNTi-NK cells) mature rapidly, are significantly more granular, and express very high levels of CD16 in comparison to their hPSC WNTd counterparts (WNTd-NK cells) and cord blood-derived NK (cbNK) cells. Further, WNTi CD34+ progenitors always gave rise to a granulocyte population alongside NK cells, suggesting they may be derived from a myeloid progenitor. Both WNTi-NK and WNTd-NK cells robustly respond to tumor targets, antibody-dependent cell-mediated cytotoxicity (ADCC), and PMA/ionomycin stimulation in comparison to cbNK cells. In all cases, WNTi-NK cells exhibited a strong bias for cytolytic degranulation over cytokine production, while WNTd-NK cells were biased for IFNg secretion. Similarly, WNTi-NK cells exhibit superior ADCC-mediated cell killing of Raji cells. We then turned to the well-characterized murine embryo to determine whether HSC-independent NK cell progenitors are developmentally conserved. Assessing NK cell potential via explant culture, we found that as early as E7.5, yolk sac explants give rise to NK cells, as well as primitive and definitive erythroid progenitors. Further, we find that murine E9.5 yolk sac kit+CD41+CD16/32+ erythro-myeloid progenitors (EMP) give rise to NK cells ex vivo. Similar to hPSC WNTi-NK cells, EMP-derived NK cells were larger and more granular, and emerged alongside a granulocyte population in explant culture. Thus, the murine yolk sac harbors unique NK cell potential, from a committed myeloid progenitor, prior to HSC emergence. Collectively, these studies suggest that ontological origin is an unexpectedly important consideration in the design of hPSC-derived NK cell-based therapeutics, and raise new questions regarding the potential of early hematopoietic progenitors in the mammalian embryo. Disclosures Fehniger: Celgene: Research Funding; Cyto-Sen Therapeutics: Consultancy; Altor BioScience: Research Funding; Affimed: Research Funding; NIH/NCI: Other: R01 CA205239, P50CA171963. Palis:Rubies Therapeutics: Consultancy.
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Vicari, Perla, Maria Aparecida E. Noguti, Vania M. Morelli, Rodolfo D. Cançado, and Maria Stella Figueiredo. "Inflammatory Cytokines: TNFα, IL-1β, IL-6 and IL-8 in Pulmonary Hypertension of Sickle Cell Disease." Blood 110, no. 11 (November 16, 2007): 3787. http://dx.doi.org/10.1182/blood.v110.11.3787.3787.
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Abstract Background: Although pulmonary hypertension (PHT) is a common complication in patients with sickle cell disease (SCD), the rate of development of PHT and the factors that affect disease progression are unknown. Objectives: To evaluate the plasma levels of cytokines in SCD comparing with healthy subjects and to find out if there is a relationship among cytokines and PHT. Patients and methods: Dosage of interleukin-6 (IL-6), tumor necrosis factor-alpha (TNFα), interleukin-8 (IL-8) and interleukin-1 beta (IL-1β) had been evaluated in 107 steady-state sickle cell patients, from the UNIFESP/EPM and College of Medical Sciences of the Santa Casa de Misericórdia, and in 108 blood donors. The presence of PHT was evaluated in sickle cell patients by transthoracic Doppler echocardiogram and the results were associated with age, hemoglobin and fetal hemoglobin, lactic dehydrogenase and cytokines levels. Results: The presence of PHT was significantly related with age, low hemoglobin and high lactic dehydrogenase levels. There were no difference of plasma levels of IL-6, IL-1β and TNFα between patients and controls. The IL-8 levels were predominantly higher in patients than in controls (p=0.0001). Among patients with SCD, IL-8 levels were significantly higher in those with PHT (p=0.02). In addition, increased levels of IL-8 (>95th percentile of the control group) were detected in 33.3% in patients with PHT in comparison with 10.8% in those without (OR: 4.1; 95%CI 1.3–12.7). Conclusion: These results indicate that age, hemoglobin, lactic dehydrogenase levels, and elevated levels of IL-8 could be risk factors of PHT in SCD. However a larger study with SCD patients with PHT is required to clarify this question. (Funded by Fapesp 04/04498–4).
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Sarfaty, Michal, Christopher A. Desjardins, Paul Giardina, Kankana Bardhan, Swarna Pandian, Keith Halley, Elizabeth M. Halvorsen, et al. "Assessment of cancer-specific microbiome signature of response to immune checkpoint inhibitors." Journal of Clinical Oncology 39, no. 15_suppl (May 20, 2021): 2574. http://dx.doi.org/10.1200/jco.2021.39.15_suppl.2574.
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2574 Background: Clinical and preclinical experiments suggest that the gut microbiome can affect outcome in cancer patients treated with immune checkpoint inhibitors (ICI). Most data to date has been in melanoma, so the relationship of the gut microbiome with treatment outcome in other cancers is poorly understood. Here, we evaluated the microbiome composition in correlation to ICI response in patients with metastatic lung, urothelial, or renal cancer, as well as metastatic melanoma. Methods: Fecal microbiome samples were obtained from patients with metastatic melanoma, lung, urothelial, or renal cancer immediately before ICI therapy was initiated. Bacterial genomic DNA was isolated and profiled by whole metagenome sequencing. Sequence data were analyzed using a custom implementation of MetaPhlAn2. Response to ICI was defined as partial or complete response or remaining on therapy for more than 6 months. Results: Samples were prospectively collected from 94 patients, including metastatic melanoma (n = 17), lung (n = 44), urothelial (n = 23), or renal cancer (n = 10). Treatment included anti-PD(L)1 monotherapy (n = 51), anti-PD1 + anti-CTLA4 combination therapy (n = 17), or a combination of anti-PD1 and chemotherapy (n = 26). Clinical response was observed in 58% of patients, including partial or complete response (45%) and on treatment for more than 6 months (55%, with 31% on treatment for more than 1 year). Although the variance in the composition of pretreatment microbiome samples did not explain response alone (R vs NR, PERMANOVA, p = 0.273), a significant portion of the variance in microbiome composition was explained by the interaction of cancer type and outcome (PERMANOVA, p = 0.014), suggesting a cancer-specific microbiome relationship. Notably, there was some similarity in the signature of NR across three cancer types (lung, urothelial and melanoma). One sample in this NR cluster was from a patient whose metastatic NSCLC was nonresponsive to pembrolizumab and carboplatin/pemetrexed. This microbiome sample was evaluated in vivo using subcutaneous MC38 and CT26 tumor models in germ-free mice. In contrast to mice colonized with stool from a healthy donor, mice colonized with stool from this patient yielded a nonresponsive result upon treatment with anti-PD1 or anti-PD-L1 in combination with anti-CTLA4. Conclusions: Analysis of the fecal microbiome composition from patients with metastatic lung, urothelial, renal cancer, and melanoma identified a cancer-specific signature of R and NR to ICI. Across three cancer types, a consistent signature of NR was identified and corroborated experimentally in preclinical models.
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de Carvalho, Fabricio, Veruska Lia, Fook Alves, Walter Moisés, Tobias Braga, Celso Vieira Xavier, and Gisele Wally Braga Colleoni. "Cancer/Testis Antigens MAGE-C1/CT7 and MAGE-C2/CT10 Are Expressed in 90% of Multiple Myeloma Samples and Can Be Explored in Combined Immunotherapy for This Malignancy." Blood 120, no. 21 (November 16, 2012): 4974. http://dx.doi.org/10.1182/blood.v120.21.4974.4974.
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Abstract Abstract 4974 Background: Multiple Myeloma (MM) is a malignant proliferation of B-cells with plasma cell differentiation. Vaccines formulated with antigens associated with myeloma cells can instruct the immune system to eliminate malignant cells. Can/Testis Antigens (CTAs) tumor-associated antigens are identified in several human malignant neoplasms and in normal testis, fetal ovary and trophoblast cells. MAGE CTA genes are recognized by CTL (cytotoxic T cell) and are strictly tumor-specific. MAGE-C1/CT7 and MAGE-C2/CT10 are highly immunogenic and both CTAs have been previously shown in various human tumors, suggesting its potential role to evoke both humoral and cellular immune responses. Objective: We evaluated MAGE-C1/CT7 and MAGE-C2/CT10 expression in bone marrow (BM) aspirates collected from patients with MM, solitary plasmacytomas, MGUS (monoclonal gammopathy of undetermined significance) and normal controls to evaluate potential combination of CTA genes for immunotherapy in this disease by RT-PCR (reverse transcription-polymerase chain reaction). Material and Methods: BM aspirates from 20 MM patients [30% ISS (International Staging System) stage 1–2 and 70% stage 3], five solitary plasmacytomas, four MGUS and five normal BM aspirates. RNA was prepared from total BM aspirates using TRIzol, followed by synthesis of cDNA with SuperScript III, according to the manufacturer's instructions. MAGE-C1/CT7 and MAGE-C2/CT10 were analyzed by RT-PCR and 2% agarose gel electrophoresis and visualized by Sybr Safe. Results: MAGE-C1/CT7 was positive in 75% (15/20) and MAGE-C2/CT-10 in 70% (14/20) of MM cases. In 11 out of 20 MM cases (55%), both CTAs were positive and in 18 out of 20 samples (90%) the expression of at least one of the genes could be detected by RT-PCR. In the other monoclonal gammopathies (solitary plasmacytomas and MGUS), MAGE-C1/CT7 and MAGE-C2/CT10 were expressed in at least one of the analyzed cases. All BM aspirates collected from healthy donors were negative for both CTAs evaluated, showing restricted expression of these genes in monoclonal gammopathies. Considering the International Staging System (ISS), the data show that expression of both CTA genes were widely expressed in MM samples studied, independently on the stage of disease. Conclusions: These findings suggest that both MAGE-C1/CT7 and MAGE-C2/C10 CTA genes can have a biological role in MM distinct clinical phases and maybe contribute to malignant plasma cell development. The high frequency found in MM patients suggests that the subfamily of MAGE-C has a role on the development of myeloma. Although the function of these genes is still poorly understood, several studies have shown that MAGE-C1/CT7 and MAGE-C2/CT10 genes are able to elicit spontaneous immune responses; therefore they can be considered potential targets for cell therapy in this incurable disease. Disclosures: No relevant conflicts of interest to declare.
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Oshima, Motohiko, Satoru Miyagi, Shuhei Koide, George Russel Wendt, Nagisa Hasegawa, Shogo Yabata, Changshan Wang, Yutaka Suzuki, and Atsushi Iwama. "Ezh2 Is Required for Developmental Stage-Specific Silencing of Oncofetal Genes in Hematopoietic Stem/Progenitor Cells." Blood 124, no. 21 (December 6, 2014): 2900. http://dx.doi.org/10.1182/blood.v124.21.2900.2900.
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Abstract In contrast to adult bone marrow (BM) hematopoiesis, fetal liver (FL) hematopoiesis involves mainly erythropoiesis and myelopoiesis, with limited lymphopoiesis. Fetal hematopoietic stem cells (HSCs) are known to have greater repopulating capacity as compared with adult HSCs. Polycomb group (PcG) protein Ezh2 participates in gene silencing by catalyzing the trimethylation on H3K27 (H3K27me3). We previously reported that in transplantation assays, Ezh2-deficient FL hematopoietic cells have greater reconstitution capacity and establish significantly higher ratios of myeloid to lymphoid reconstitution relative to wild-type (WT). From these results, we hypothesized that adult BM hematopoietic cells repopulated with Ezh2-deficient FL cells partially retain the features of fetal hematopoietic cells as compared with WT. To address this hypothesis, we transplanted Cre-ERT;Ezh2fl/fl or WT FL hematopoietic cells and deleted Ezh2 (Ezh2 KO) via intraperitoneal administration of tamoxifen at 4 weeks after transplantation. At 3 months post-deletion of Ezh2, donor-derived lin-Sca-1+c-Kit+ (LSK) HSC/MPP fraction were recovered and subjected to microarray analysis, together with LSK cells from WT E15.5 FL and adult BM. Within genes which showed higher expression in Ezh2 KO BM LSK cells compared with WT (453 genes: Ezh2 KO-gene), and higher in FL compared with adult BM LSK cells (1139 genes: FL-gene), 102 genes (23% of Ezh2 KO-gene and 9% of FL-specific gene) were overlapped (p-value < 1.0x10-16). Recently, Copley et al. (Nat Cell Biol, 2013) revealed that Lin28b is exclusively expressed in FL HSCs and the Lin28b/let7/Hmga2 axis plays a critical role in controlling developmental changes in HSC properties. As reported, we found that Lin28b was detected only in FL LSK cells, but of note, remarkably higher expression was observed in Ezh2 KO relative to WT LSK cells in adult BM. Lin28b is known to block the maturation of let-7 microRNAs, thereby up-regulates expression of let-7 target genes. Within 1648 putative let-7 miRNA target genes, 136 genes were highly expressed in FL LSK cells (FL-LSK let-7 target). Gene set enrichment analysis (GSEA) showed that FL-LSK let-7 target gene set was ectopically enriched in Ezh2 KO LSK and also GMP cells but not in WT cells in adult BM. We next performed ChIP-seq analysis to evaluate the direct target of Ezh2 in adult BM, Lin28b promoter region appeared to be marked with PcG histone marks H3K27me3 and H2AK119Ub1 but not in FL. The levels of PcG histone marks were significantly low in Ezh2-deficient BM LSK cells and GMPs relative to WT. These findings indicate that Lin28 is directly regulated by PcG histone modifications in adult BM. Interestingly, Within FL-LSK let-7 target, 16 genes underwent H3K27me3 histone modifications in adult BM and several of them escaped gene silencing in the absence of Ezh2, including Igf2bp3, Hmga2, Jam3, Dcbld1 and Zfp354a., in the same way as Lin28b. These results demonstrated that a part of let-7 target genes are negatively regulated in BM not only by let-7 but also by PcG histone modifications in adult BM. Recent analyses have revealed that inactivating somatic mutations in epigenetic regulator genes such as EZH2 or TET2 occur frequently in patients with myelodysplastic disorders. We previously reported that Ezh2 is largely dispensable for adult HSCs in contrast to fetal hematopoiesis but after a long latency, deletion of Ezh2 in adult hematopoietic cells induces myelodysplastic/myeloproliferative neoplasm (MDS/MPN)-like disorders characterized by myelodysplasia with higher repopulating capacity of HSCs. Furthermore, concurrent depletion of Ezh2 and Tet2 markedly accelerated the development of myelodysplastic syndrome (MDS) and MDS/MPN. Lin28b and its targets Igf2bp1 and Igf2bp3 are characterized as ‘oncofetal’ genes, which is highly expressed not only in early embryogenesis but also in various tumors. Corresponding to these findings, FL-LSK let-7 target genes were highly and more significantly enriched in Tet2KD/KDEzh2Δ/Δ(DKO) LSK and GMPs from MDS or MDS/MPN mice relative to Ezh2 KO in adult BM. Taken together, Ezh2 is indispensable for the developmental stage-specific regulation of the Lin28b-let7 pathway, and Ezh2 deletion results in insufficient suppression of a subset of the oncofetal genes, which may contribute to the FL-like characteristics and the development of hematopoietic disorders. Disclosures No relevant conflicts of interest to declare.
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Munson, Paul, Juraj Adamik, and Lisa Butterfield. "829 Tumor alpha-fetoprotein inhibits cholesterol and steroid metabolism in monocyte-derived dendritic cells." Journal for ImmunoTherapy of Cancer 8, Suppl 3 (November 2020): A880—A881. http://dx.doi.org/10.1136/jitc-2020-sitc2020.0829.
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BackgroundHepatocellular carcinoma (HCC) is a particularly lethal malignancy in part due to the potently immune-suppressive tumor microenvironment. The weak immune response is due in part to the presence of tumor alpha-fetoprotein (tAFP), a fetal glycoprotein that is produced by a majority of HCC tumors.1 Previously, we showed that tAFP potently inhibited the differentiation of monocytes to dendritic cells when compared to cord blood-derived normal AFP (nAFP) and ovalbumin (OVA).2 Additionally, we demonstrated that tAFP inhibits lipid metabolism by limiting the expression of fatty acid metabolic enzymes.3 To identify the mechanism whereby tAFP alters dendritic cell metabolism, we analyzed microarray data by a functional enrichment pathway analysis with g: Profiler.4MethodsMonocytes from healthy donors (n=4) were isolated with CD14 magnetic beads and differentiated for five days in the presence of IL-4 and GM-CSF with OVA, nAFP, or tAFP. After five days, we isolated RNA for microarray analysis using an Affymetrix HG-U133A array. R studio generated principal component analysis. Differentially expressed (DE) genes were identified as a 1 log fold change and had adjusted p values ofResultsPrincipal component analysis of the gene expression data revealed that tAFP clustered separately from OVA and nAFP based on PC1 (p = 0.016) and PC2 (p = 0.009) (figure 1). In total, 688 DE genes were identified with 495 upregulated and 193 downregulated (figure 2). Downregulated DE genes between tAFP versus nAFP yielded significantly down regulated pathways including cholesterol (p = 10e-7.5), steroid (p = 10e-7.5), and lipid biosynthesis (p = 10e-6) (figure 3). Interestingly, upregulated DE genes between tAFP versus nAFP included many pathways specific to stress response to metal ions including zinc (p = 10e-10.5) and copper (p = 10e-10) (figure 4).Abstract 829 Figure 1tAFP induces a distinct gene expression profile in monocyte-derived DC’sAbstract 829 Figure 2Identifying differentially expressed genes in OVA, nAFP, and tAFP treated DC’sAbstract 829 Figure 3tAFP downregulates cholesterol and steroid metabolism in DC’sAbstract 829 Figure 4tAFP upregulates stress response to metal ions in DC’sConclusionsIn addition to validating previous data demonstrating tAFP inhibited lipid biosynthesis generally, this is the first report to our knowledge of tAFP inhibiting gene signatures associated with cholesterol and sterol synthesis specifically. Furthermore, we identified significant upregulation of gene pathways corresponding to the stress response genes to metal ions. Notably, functional assays are underway to confirm these gene pathway data. These findings shed new insight into how tAFP perturbs monocyte and DC metabolism and thereby limits differentiation of monocytes to immature dendritic cells. Future insights into how tAFP limits innate immunity could lead to improved immunotherapies for HCC.Ethics ApprovalSamples were collected with informed consent at the University of Pittsburgh (Pitt IRB #UPCI 04-001 and UPCI 04-111).ReferencesChan SL, Mo FKF, Johnson PJ, Hui EP, Ma BBY, Ho WM, et al. New utility of an old marker: serial alpha-fetoprotein measurement in predicting radiologic response and survival of patients with hepatocellular carcinoma undergoing systemic chemotherapy. J Clin Oncol Off J Am Soc Clin Oncol 2009;27:446–52. https://doi.org/10.1200/JCO.2008.18.8151Pardee AD, Shi J, Butterfield LH. Tumor-derived α-fetoprotein impairs the differentiation and T cell stimulatory activity of human dendritic cells. J Immunol 2014;193:5723–32. https://doi.org/10.4049/jimmunol.1400725Santos PM, Menk AV, Shi J, Tsung A, Delgoffe GM, Butterfield LH. Tumor-derived α-fetoprotein suppresses fatty acid metabolism and oxidative phosphorylation in dendritic cells. Cancer Immunol Res 2019;7:1001–12. https://doi.org/10.1158/2326-6066.cir-18-0513Raudvere U, Kolberg L, Kuzmin I, Arak T, Adler P, Peterson H, et al. g:Profiler: a web server for functional enrichment analysis and conversions of gene lists (2019 update). Nucleic Acids Res 2019;47:W191–8. https://doi.org/10.1093/nar/gkz369
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Lichtman, Eben, Hongwei Du, Barbara Savoldo, Soldano Ferrone, Guangming Li, Lishan Su, and Gianpietro Dotti. "Pre-Clinical Evaluation of B7-H3-Specific Chimeric Antigen Receptor T-Cells for the Treatment of Acute Myeloid Leukemia." Blood 132, Supplement 1 (November 29, 2018): 701. http://dx.doi.org/10.1182/blood-2018-99-113468.
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Abstract Background: The development of safe and effective chimeric antigen receptor T-cell (CAR-T) therapy for acute myeloid leukemia (AML) remains an elusive goal. Whereas CD19-directed CAR-T therapies have shown great promise for the treatment of B-cell malignancies, the identification of AML-associated surface antigens that can be safely and effectively targeted by CAR T-cells has remained a challenge. Because most AML-associated surface antigens are also expressed on normal myeloid progenitors, the potential for unacceptable hematopoietic toxicity has been a major limitation. The identification of cell surface antigens that are absent on all normal myeloid progenitors and yet expressed on all subtypes of AML is not likely. On the other hand, it seems plausible that some antigens not detected on early myeloid lineage cells may be preferentially overexpressed in certain AML subtypes. We have identified B7-H3 as one such candidate. B7-H3 (B7-homolog 3, or CD276) is a coreceptor belonging to the B7 family of immune checkpoint molecules. B7-H3 protein expression in normal tissues is largely restricted to certain antigen-presenting cells. In multiple human cancers, however, B7-H3 protein is significantly overexpressed. This includes a substantial subset of AML, and B7-H3 expression appears to be higher in AML with a monocytic immunophenotype. Furthermore, B7-H3 on tumor cells, and on myeloid-derived suppressor cells in the tumor microenvironment, is likely to play an immunosuppressive role, and may drive immune escape in multiple cancer types. This suggests that targeting B7-H3 could also enhance anti-tumor adaptive immune responses. We therefore hypothesized that B7-H3-specific CAR-Ts (B7-H3.CARs) could be effective in eliminating B7-H3-expressing AML cells and would not cause unacceptable hematopoietic toxicity. Methods and Results: We obtained bone marrow aspirates from patients with monocytic/myelomonocytic AML (n=10) and demonstrated surface expression of B7-H3 on a median of 62.5% (range 27.8 to 98.5) of primary AML blasts. We also showed that B7-H3 is highly expressed on monocytic/myelomonocytic AML cell lines (THP1, U937, OCI-AML2, OCI-AML3), and that B7-H3 expression compares favorably to that of other previously identified candidate antigens for AML-directed CAR-T therapy. Next, we generated B7-H3.CARs via retroviral transduction of CD3/CD28-activated T-cells, followed by expansion in vitro with interleukin- (IL) 7 and IL-15. When B7-H3.CARs (n=3-5 donors) were cocultured with B7-H3-positive AML cell lines (listed above) and with primary AML blasts (n=10 patients), B7-H3.CARs proliferated, released high amounts of IL-2 and interferon-γ, and demonstrated efficient B7-H3-specific cytotoxicity. Autologous B7-H3.CARs also demonstrated significant cytotoxicity against primary AML blasts (n=4). Additionally, B7-H3.CARs controlled tumor cell proliferation and prolonged survival in xenograft mouse models of disseminated AML using OCI-AML2 (p=0.0025, n=5 mice per group) and THP1 (p<0.0001, n=10 mice per group). Next, we showed that B7-H3 is not significantly expressed on hematopoietic stem cells or myeloid progenitor cell populations in normal human bone marrow samples (n=2). We also evaluated the effects of B7-H3.CARs (n=4 donors) on normal hematopoietic stem cells via in vitro colony formation assays using umbilical cord-blood (n=4 donors) derived CD34+ cells, and showed that B7-H3.CARs did not significantly inhibit the formation of myeloid progenitor colonies. We then showed in a humanized mouse model (using fetal liver-derived hematopoietic stem cells) that B7-H3.CARs did not lead to significant reductions in the populations of circulating CD45/CD14-positive or CD45/CD33-positive cells. Conclusions: B7-H3 is expressed on a significant proportion of AML blasts from patients with monocytic AML. Adoptive transfer of B7-H3.CARs could be an effective treatment option for patients with B7-H3-positive AML, since i) we have previously demonstrated limited expression of B7-H3 in normal tissues, and ii) the present results show that B7-H3.CARs are unlikely to cause significant hematopoietic toxicity. Given variable expression of B7-H3 in AML, however, it may be necessary to develop a dual-targeting approach, combining B7-H3 with a second target AML-associated surface antigen. Disclosures Du: N/A: Patents & Royalties: Patent filed for B7-H3 chimeric antigen receptor. Ferrone:N/A: Patents & Royalties: Patent filed for B7-H3 chimeric antigen receptor. Dotti:University of North Carolina: Patents & Royalties: Patent filed for B7-H3 chimeric antigen receptor.
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Ali, Hiba, Weijie Ma, Shruti Khurana, Zhi-Dong Jiang, Herbert L. DuPont, Pablo Okhuysen, Anusha Thomas, and Yinghong Wang. "Safety and efficacy of fecal microbiota transplantation (FMT) in the management of recurrent clostridioides difficile infection (rCDI) in cancer patients." Journal of Clinical Oncology 38, no. 15_suppl (May 20, 2020): e24048-e24048. http://dx.doi.org/10.1200/jco.2020.38.15_suppl.e24048.
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e24048 Background: Cancer patients are at a significantly increased risk of rCDI by virtue of a compromised immune function from underlying malignancy, anti-cancer therapy and frequent antibiotic use for opportunistic infections. Furthermore, cancer patients have shown to have a lower response rate to standard oral antibiotics for CDI. Data is limited in regards to the safety and efficacy of FMT in managing rCDI in cancer patients. We aim to describe our experience of the same at a tertiary care cancer center. Methods: We conducted a retrospective study of cancer patients who underwent FMT for rCDI at the MD Anderson Cancer Center 06/2017-01/2020. FMT was performed through colonoscopy with universal donors’ stool. Baseline clinical data were collected and analyzed. Results: Our sample comprises 19 patients of whom 12 had solid tumors and 7 hematological malignancies, most of which were stage 4 at the time of FMT. The mean age was 66.5 years. Most patients received proton pump inhibitor, antibiotics and cancer therapy within 3-6 months of FMT. On average, each patient had 3 episodes of CDI, received 4 courses of antibiotic treatments, and required 4 CDI related hospitalizations within 1 year prior to FMT. Majority of the CDI episodes were managed with a combination of antibiotics. Bezlotoxumab was used in 4 cases. 18 patients received FMT once, while one patient was treated with FMT thrice. Clinical response was seen in 74% patients with a median time of 1 day. 5 patients had refractory CDI including 3 recurrent rCDI, and 2 persistent symptoms. Compared to patients with good response, these refractory cases received more frequent antibiotics following FMT (100% vs 43%, p=0.033). Side effects were mostly mild GI complaints in 15.8% patients with no serious adverse events or mortality related to FMT. Conclusions: Our study shows that FMT is a safe and effective treatment for rCDI in cancer patients and provides rapid resolution of symptoms; Subsequent antibiotic use for the management of cancer related complications can negatively affect the efficacy of FMT. [Table: see text]
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Huang, Stephen, Minjiang Xu, Edward Bruno, Giovanni Barosi, Josef Prchal, and Ronald Hoffman. "HMGA2, a Member of the High Mobility Group Gene Family, Is Expressed at the Protein Level in Peripheral Blood CD34+ Cells of Patients with Idiopathic Myelofibrosis and Polycythemia Vera." Blood 104, no. 11 (November 16, 2004): 798. http://dx.doi.org/10.1182/blood.v104.11.798.798.
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Idiopathic myelofibrosis (IM) is a chronic myeloproliferative disorder associated with increased numbers of CD34+ cells circulating in the peripheral blood (PB). To characterize these cells, we transplanted CD34+ or CD34− cells from either G-CSF mobilized PB or IM PB into NOD/SCID mice to test their hematopoietic stem cell function. IM CD34+ cells, but not CD34−, cells engrafted in NOD/SCID mice, demonstrating that IM PB CD34+ cells contain true bone marrow repopulating cells. Furthermore, the differentiation program of IM CD34+ cells was quite different than that of CD34+ cells isolated from normal donors receiving G-CSF. G-CSF mobilized PB CD34+ cells generated predominantly CD19+ cells (B-lymphocytes), while IM PB CD34+ cells generated predominantly myeloid cells as well as larger numbers of CD41+ cells (megakaryocytes), but few CD19+ cells. The molecular basis for this stem cell defect in IM remains poorly defined. We hypothesized that the High Mobility Group Gene, HMGA2, might play a role in the biogenesis of IM. HMGA2 is a nuclear protein, normally expressed only during embryonic and fetal development, which acts as an architectural transcription factor important in the growth and differentiation of cells of mesenchymal origin. It has been reported that this gene has a direct effect on chromatin configuration by promoting DNA relaxation and that it may control the transcriptional activities of several genes. Rearrangement of the HMGA2 gene has frequently been detected in human benign tumors of mesenchymal origin including lipomas and sarcomas. In addition, the gene has been shown to be overexpressed in squamous cell carcinomas of the oral cavity. Previous work has suggested that HMGA2 is overexpressed in the PB mononuclear cells of patients with IM at the mRNA level (Andrieux, J. et al, Genes, Chromosomes & Cancer, 39: 82, 2004). Since IM and polycythemia vera (PV) are hematological malignancies that originate at the level of the hematopoietic stem cell, we examined the expression of HMGA2 in CD34+ and CD34− cell fractions isolated from the PB of patients with IM and PV as well as G-CSF mobilized normal donors. Using Western Blotting, we were unable to detect HMGA2 protein in either G-CSF mobilized CD34+ or CD34− cells. By contrast, HMGA2 was clearly expressed in the CD34+ cell fraction, but not the CD34− fraction isolated from IM and PV patients. These data indicate that increased HMGA2 protein levels are unique to IM and PV CD34+ cells and that the aberrant expression of this gene in the CD34+ progenitor cells may contribute to the stem cell defect in these myeloproliferative disorders.
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Bernardo, Maria Ester, Maria Antonietta Avanzini, Cesare Perotti, Angela M. Cometa, Antonia Moretta, Elisa Lenta, Claudia Del Fante, et al. "Platelet-Lysate for In Vitro Expansion of Human Multipotent Mesenchymal Stromal Cells in Approaches of Cell-Therapy." Blood 108, no. 11 (November 16, 2006): 2577. http://dx.doi.org/10.1182/blood.v108.11.2577.2577.
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Abstract There is large interest in the use of mesenchymal stromal cells (MSCs) in approaches of cell-therapy and tissue engineering. MSCs are currently expanded in vitro in the presence of fetal calf serum (FCS); however, FCS raises concerns in the case of clinical grade cellular preparations because of the theoretical risk of transmission of zoonoses and triggering immune reactions in the host. Therefore, the identification of a serum-free medium appropriate for both the extensive expansion necessary to reach the large numbers of MSCs required for clinical application, and the exclusion of risks connected with the use of animal products, is warranted. Aim of this study was to evaluate whether MSCs expanded in medium supplemented with platelet-lysate (PL) are endowed with biological properties appropriate for cell-therapy approaches. MSCs were generated from bone-marrow of 8 healthy hematopoietic stem cell donor; 4 different culture conditions were tested: 10 % FCS; 5% PL; 2,5% PL; 1% PL. MSCs were harvested when reaching ≥ 80% confluence and replated for expansion at 4.000 cells/cm² until passage 5. CFU-F frequency, proliferative capacity, morphology, surface phenotype and differentiation capacity were evaluated. In particular, the immune regulatory effect on alloantigen-specific immune response, the kinetics of cytokine production and the resistance to spontaneous transformation into tumor cells of MSC expanded in the presence of either PL or FCS were investigated. Our results demonstrate that MSCs expanded in either FCS or PL display comparable morphology, phenotype and differentiation capacity, while PL-MSCs were superior in terms of clonogenic efficiency and proliferative capacity. Immune-regulatory effect of MSCs was investigated on alloantigen-specific immune response in mixed lymphocyte culture (MLC). We found that MSCs-PL are comparable to MSCs-FCS in their capacity to: decrease alloantigen-induced cytotoxic activity; favor differentiation of CD4+ T-cell subsets expressing Treg phenotype; increase early secretion of IL-10 in MLC supernatant, as well as to induce a striking augmentation of IL-6 production. As compared with MSCs-PL, MSCs-FCS were more efficient in suppressing alloantigen-induced lymphocyte subset proliferation and in reducing early IFNg-secretion. Resistance to spontaneous transformation into tumor cells of expanded MSCs was demonstrated by both molecular karyotyping (array-comparative genomic hybridization) and maintenance of normal morphology/phenotype after prolonged in vitro culture. Our data support the hypothesis of a remarkable immune functional plasticity of human MSCs and suggest that the use of MSCs-PL, which seem to be endowed with a relatively low immune suppressive activity, could be more appropriate in approaches of reparative/regenerative cell-therapy or in strategies aimed at improving hematopoietic/immune recovery after hematopoietic stem cell transplantation (HSCT). On the contrary, as MSCs-FCS seem to display a more pronounced immune suppressive function, they might be more suitable for preventing or treating alloreactive-related immune complications, such as severe Graft-versus-host disease (GvHD) in HSCT and graft rejection in HSCT and solid organ transplantation.
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Garg, Tarun K., Junaid Khan, Susann Szmania, Amy D. Greenway, Joshuah D. Lingo, Amberly Moreno-Bost, Katie Stone, et al. "Autologous Expanded Natural Killer Cells As a New Therapeutic Option for High-Risk Myeloma." Blood 118, no. 21 (November 18, 2011): 2918. http://dx.doi.org/10.1182/blood.v118.21.2918.2918.
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Abstract Abstract 2918 Natural killer cells (NK) have the unique ability to kill target cells without priming. While their therapeutic potential against various malignancies is becoming more apparent, it has been restricted to the allogeneic setting; NK cells are inhibited by autologous targets by engaging killer immunoglobulin-like receptors with their ligands. Another major challenge to the clinical utility of NK cells is obtaining a sufficient number of NK cells for infusion. Co-culture of blood mononuclear cells (PBMNC) with the leukemic cell line K562, genetically modified to express membrane-bound IL15 and the co-stimulatory molecule 41BBL (K562mbIL15-41BBL) in the presence of IL2 results in robust expansion and activation of NK cells. To determine if NK cells derived from myeloma (MM) patients can be used therapeutically in the autologous setting, we explored the expansion of NK cells from MM patients, their gene expression profiles (GEP), and their ability to kill autologous and allogeneic MM cells from high-risk patients in vitro and in vivo, and compared these to NK cells from healthy donors (HD). PBMNC from MM patients (N=30) co-cultured with irradiated K562mbIL15-41BBL cells expanded a median of 351 fold (range20–10, 430), comparable to the expansion of HD-derived NK cells (N=15, median 803, range 127–1, 727; p=0.5). GEP of MM non-exp-NK differed from HD non-exp-NK in the expression of only one gene (PRKCi), underexpessed in MM (false discovery rate (FDR) <0.05, p-value <3×10−10). GEP of exp-NK cells from both MM patients and HD was very different from non-exp-NK cells (8 pairs each, 10, 639 differentially overexpressed and 26, 057 underexpressed probe sets, FDR <0.05). Genes associated with proliferation, cytolytic activity, activation, adhesion, migration and cell cycle regulation were highly up-regulated in exp-NK cells. Standard chromium release assays demonstrated that MM exp-NK cells killed both allogeneic and autologous primary MM cells more efficiently compared to non-exp-NK cells, via a perforin mediated mechanism. Blocking studies revealed that the natural cytotoxicity receptors, activating receptors, and DNAX accessory molecule (DNAM-1) played a central role in target cell lysis. The killing ability of MM patient and HD derived exp-NK cells was very similar against allogeneic targets, while primary MM targets were more resistant to killing by autologous exp-NK. The anti-MM activity of allogeneic and autologous exp-NK cells was further examined in vivo. NOD/SCID/IL2R γ-null mice were implanted subcutaneously with a human fetal bone, and primary MM cells or luciferase-transfected OPM2 MM cell line were engrafted into the bone. The tumor burden was determined by ELISA for human Ig and/or bio-imaging. The mice were randomized to control and exp-NK treatment groups. A total of 160 ×106 exp-NK cells, in 4 doses 48 hrs apart, were injected in the exp-NK treatment group via tail vein injection. The mice were administered 1000U of IL2 subcu daily to support the NK cells. The mice were bled on days 7, 14, 21 & 28 for the assessment of human Ig by ELISA and enumerating circulating NK cells by flow cytometry. Exp-NK treated mice had a significantly reduced MM burden by ELISA (p<0.04) on day 21, and exp-NK could be detected in the murine blood up to day 28 post-administration in both primary MM and OPM2 tumor bearing mice. The mice were sacrificed and the tumors were harvested after 4 weeks. A noticeable reduction in tumor burden in the exp-NK cell treated mice was confirmed by histology. NK cells were detected by immunohistochemistry (CD57 or CD16) in the hu-bone implants harvested 28 days after infusion. In conclusion, MM patient-derived NK cells have a similar expansion potential, and MM exp-NK cells have cytolytic activity against allogeneic targets similar to those of HD exp-NK cells, and somewhat reduced activity against autologous targets. These exp-NK cells have significant activity against the aggressive cell line OPM2 and high-risk autologous primary MM cells in vivo. Exp-NK cells trafficked to MM tumors and persisted in the myelomatous hu bone microenvironment for 4 weeks. The anti-MM activity of autologous exp-NK cells is exciting and avails a new therapeutic avenue for patients with GEP-defined high-risk disease. A phase II clinical trial of allogeneic and autologous exp-NK cell therapy for relapsed/refractory high-risk MM is in progress at our institution. Disclosures: No relevant conflicts of interest to declare.
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Laje, Pablo, William H. Peranteau, Masayuki Endo, Philip W. Zoltick, and Alan W. Flake. "In Utero Transplantation of Transduced Hematopoietic Stem Cells Results in Deletion of Transgene-Specific T Cells." Blood 108, no. 11 (November 16, 2006): 3266. http://dx.doi.org/10.1182/blood.v108.11.3266.3266.
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Abstract The developing fetal immune system provides a unique opportunity to manipulate normal immunologic development for therapeutic prenatal and anticipated postnatal interventions. In previous studies we have shown that allogeneic in utero hematopoietic cell transplantation (IUHCT) results in donor specific tolerance that can subsequently facilitate non-myeloablative postnatal cellular or organ transplants. It follows that in utero injection of transduced hematopoietic stem cells (HSC) could potentially induce tolerance to a transgene encoded protein. We hypothesized that expression of a transduced antigenic protein by HSC and their progeny would alter thymic T cell development resulting in deletion of antigen specific T-cells. To test this hypothesis, we used the mammary tumor virus (MTV) superantigen system to evaluate the effect of IUHCT of transduced HSC on T cell development. In this system, expression of different MTV oncogenes by different I-E+ strains of mice results in deletion of T cells expressing the relevant Vβ T cell receptor. Specifically, mice which are Mtv7+ delete T cells expressing the Vβ6 T-cell receptor. In this study, CD150+CD48− enriched Balb/c (I-E+ Mtv7−) HSC were transduced with an HIV-based lentivirus expressing MTV7 under an MND promoter. 1.5E+05 transduced cells were injected intravascularly via the vitelline vein into E14 Balb/c fetuses. Non-injected age matched naive Balb/c mice served as the control group. The peripheral blood (PB) and thymuses of injected fetuses and control mice were harvested at day of life (DOL) 10, 20 and 60 and analyzed by flow cytometry for T lymphocyte Vβ6 expression. Additionally, the T cell composition of the thymus was assessed at DOL10 for CD4 and CD8 single positive (SP) and CD4/CD8 double positive (DP) cells. Thymic flow cytometric analysis at DOL10 revealed that greater than 98% of the T cells were CD4CD8 DP, a stage that has not yet undergone negative selection. No significant difference was noted in the percentage of thymic Vβ6+ DP T-cells at this time point or at DOL20 and DOL60. In contrast, there was a significant decrease in the percentage of Vβ6+ peripheral blood SP cells in those mice injected with MTV7 transduced HSC relative to control mice at DOL10, DOL20 and DOL60 (p<0.05) (Fig 1). The current study supports the ability of enriched transduced HSC to induce deletion of transgene specific T cells after IUHCT. In the future, this strategy may be useful to promote tolerance for pre or postnatal cellular or gene therapy. Figure Figure
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Couch, Tyler A., Zachary C. Murphy, Michael Getman, Ryo Kurita, Yukio Nakamura, and Laurie A. Steiner. "Human Erythroblasts with c-Kit Activating Mutations Have Reduced Cell Culture Costs and Remain Capable of Terminal Maturation and Enucleation." Blood 132, Supplement 1 (November 29, 2018): 2315. http://dx.doi.org/10.1182/blood-2018-99-117157.
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Abstract There is a constant need for red blood cells for transfusion therapy in the treatment of anemias and acute injury. As all blood products for transfusion come from donors, there are concerns over shortages and safety. Furthermore, many patients with transfusion-dependent anemias risk alloiumminization. The in vitro production of red blood cells would address these problems, especially as they can be genetically engineered to prevent alloimmunization. Numerous erythroid culture systems now exist for the in vitro production of red blood cells. Hematopoietic stem and progenitor cells (HSPCs) obtained from umbilical cord or peripheral blood can be differentiated into erythrocytes, however, they are limited in expansion. While umbilical cord HSPCs have greater expandability than peripheral blood, the resulting erythrocytes contain fetal globins. Pluripotent stem cells can also be used as a starting source, however only a small percentage of the cells can be differentiated into erythroblasts which also suffer from low enucleation rates. Presently, the cost of in vitro production of a unit of red cells is greater than an order of magnitude higher than obtaining it from a donor largely due to the medium and cytokine costs (Timmins & Nielsen, Trends Biotechnol, 2009). A relatively new approach of immortalizing early erythroblasts allowing unlimited expansion as well as terminal maturation and enucleation shows great therapeutic promise (Kurita et al., PLoS One, 2013; Huang et al., Mol Ther, 2014; Trakarnsanga et al., Nat Commun, 2017). However, these immortalized erythroblasts are still reliant on two costly cytokines: stem cell factor (SCF) and erythropoietin (Epo). Mutations in exon 17 of the receptor tyrosine kinase gene KIT are frequently seen in acute myeloid leukemias, gastrointestinal stromal tumors, and mast cells leading to mastocytosis. These mutations cause the c-Kit protein to spontaneously activate and transduce signal in the absence of SCF (Kit-ligand). To generate an SCF-independent HUDEP-2 cell line (Kurita et al., PLoS One, 2013), we used CRISPR/Cas9 to introduce missense and frameshifting mutations within the vicinity of Asp816 in exon 17 of the KIT gene. The resulting monoclonal cell lines were selected for by removing SCF from the expansion medium and were subsequently named KIT-CAT (KIT with Constitutively Activating Transformation). To better understand what KIT mutations allowed or impaired terminal maturation, monoclonal cell lines were genotyped by Sanger sequencing. Three cell lines with unique genotypes were chosen for further analysis. All three KIT-CAT lines had a shorter doubling time compared to HUDEP-2 cells (16.7 vs 18.9 hrs, p=0.020) and were no longer dependent on SCF or Epo. However, two of the three KIT-CAT lines showed more robust proliferation with Epo in the expansion medium. The addition of SCF to the medium caused no increase in c-Kit activation by Western blotting for phosphorylation at Tyr703. Furthermore, the low molecular weight and immature form of c-Kit is also phosphorylated in KIT-CAT cells, but not HUDEP-2 cells, indicating c-Kit activation occurs before trafficking to the cell membrane where SCF would bind (Tabone-Eglinger et al., Clin Cancer Res, 2008). Key features of erythroblast maturation are the decrease in cell and nuclear size which can be measured using imaging flow cytometry (McGrath et al., Methods, 2017). While in expansion phase, all 3 cell lines were larger in cell and nuclear area compared to the parental HUDEP-2 line. By day 6 of maturation, all three cell lines had statistically significant decreases in cell and nuclear size indicating maturation. By day 13 of culture, Wright-Giemsa staining showed that the majority of the cells were orthochromatic erythroblasts or enucleate reticulocytes. Reducing cell culture costs is needed for in vitro manufacturing of red blood cells to be economically feasible. These results show that a c-Kit activating mutations in human erythroblasts removes the cost of SCF and reduces the cost of Epo while still allowing for terminal maturation and enucleation. Disclosures No relevant conflicts of interest to declare.
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Chiu, Martina, Denise Toscani, Emanuela Vicario, Roberta Andreoli, Giuseppe Taurino, Valentina Marchica, Paola Storti, Massimiliano Bianchi, Ovidio Bussolati, and Nicola Giuliani. "Glutamine Depletion By Addicted Myeloma Cells Inhibits Osteoblastic Differentiation of Bone Marrow Mesenchymal Stromal Cells Limiting Asparagine Availability: A Possible New Mechanism for Myeloma Bone Disease." Blood 134, Supplement_1 (November 13, 2019): 4339. http://dx.doi.org/10.1182/blood-2019-128034.
Full textAbstract:
Metabolic alterations of cancer cells, aimed at sustaining their growth, may also influence the behavior of the tumor microenvironment. Our group has recently demonstrated that multiple myeloma (MM) is a highly glutamine(Gln)-addicted tumor that utilize huge amounts of Gln to fuel its metabolism through the enzyme glutaminase (GLS). For this reason, MM cells exhibits increased Gln uptake, mainly through the ASCT2 transporter. Interestingly, lower bone marrow (BM) plasma Gln concentration (down to a median value of 0.4 mM vs. a median value of 0.6 mM) was found in MM patients as compared with smoldering MM (SMM) and Monoclonal Gammopathy of Uncertain Significance (MGUS). The main feature of MM BM microenvironment is the suppression of osteoblastic (OB) differentiation leading to the development of osteolytic bone lesions, the hallmark of MM. Most recently, it has been demonstrated that Gln metabolism is needed to sustain bone mass formation in murine models and that GLS inhibition decreases OB differentiation of human mesenchymal stromal cells (hMSCs). However, no information is yet available on the role of Gln depletion imposed by MM cell metabolism on OB differentiation into the BM. This topic has been investigated in the present study. Firstly, human MM cells were co-cultured with BM hMSCs, and Gln medium concentration was evaluated with mass spectrometry (MS), demonstrating a MM-induced depletion of the amino acid. Upon Gln depletion, MSC exhibited a sustained induction of Glutamine Synthetase (GS). On the contrary, when differentiated in osteogenic medium (D-MEM + 5% Fetal Bovine Serum, supplemented with 2 mM Gln, ascorbic acid and dexamethasone), GS was suppressed. Conversely, GLS (both KGA and GAC isoforms) and SLC38A2, the gene for the concentrative Gln transporter SNAT2, were induced. These data suggest that hMSCs differentiation in OBs is associated with an increased dependence upon extracellular Gln. Consistent with this conclusion, the activity of SNAT2 was absent in undifferentiated hMSCs but well detectable after 14 days of OB differentiation, when total Gln uptake was also increased. Under the same conditions, OB differentiation markers (RUNX2, COL1A1, ALPL expression and ALPL activity or staining) were significantly induced but their expression was blunted by incubation in low-Gln (0.4 mM) medium or in the presence of the SNAT2 inhibitor MeAIB. The incubation in Gln-free D-MEM suppressed the induction of GLS and SLC38A2 along with OB differentiation, which was restored by the supplementation of Non-Essential Amino Acids (NEAA). Among NEAA, only asparagine (Asn) was able to rescue OB differentiation in the absence of Gln. The determination of intracellular amino acids with MS indicated that OB differentiation was associated with the increase of cell Asn, without significant changes of Gln, glutamate (Glu) or aspartate (Asp). Asparagine Synthetase (ASNS), the Gln-dependent enzyme that accounts for Asn synthesis, was also found induced during OB differentiation of hMSCs. Gene Expression Profiles of primary BM hMSCs and OBs from bone biopsies of both healthy donors (n=7) and MM patients (n=16) indicated that GLS, ASNS, and SLC38A2 are more expressed in OBs, while the expression of GLUL, the gene for GS, is higher in undifferentiated hMSCs from healthy donors. Overall, these results indicate that (1) OB differentiation of hMSCs is Gln-dependent; (2) the partial Gln depletion, imposed by Gln-addicted MM cells in the BM microenvironment, contributes to the impairment of osteoblastic differentiation of hMSCs; (3) hindrance of differentiation may depend on the limited availability of intracellular Asn derived from Gln-dependent ASNS. These results support the evidence that Gln addiction of MM cells affects bone microenvironment leading to the inhibition of OB differentiation and, consequently, to the development of MM bone disease. Disclosures Giuliani: Janssen: Research Funding.
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Zhu, Xiongpeng, Jianda Hu, Xu Lin, Ting Yang, Zhengshu Xu, Lulu Lv, Chuntuan Li, and Zhizhe Chen. "In Vitro Induction of Anti-Leukemia CTL Effect by Dendritic Cells Infected with Recombinant Adenovirus Vectors Containing Human Survivin Genes." Blood 108, no. 11 (November 16, 2006): 5483. http://dx.doi.org/10.1182/blood.v108.11.5483.5483.
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Abstract Relapse after chemotherapy could lead to poor prognosis in patients with leukemia. By using tumor-specific immunotherapy to eradicate minimal residual diseases could prevent disease relapse. Our study aims at exploring whether peripheral blood derived dendritic cells (DC) expressing human survivin can enhance their capability to induce human T lymphocytes into tumor-specific cytotoxic T lymphocytes (CTL) for immunotherapy. In our experiments the full length survivin cDNA was obtained from recombinant plasmid pcDNA3.0-survivin by PCR. The PCR products were double-digested with restriction endonucleases KpnIand HindIII, and inserted orientationally into pshuttle-CMV plasmid. The plasmid of pshuttle-CMV-survivin was lined with PmeI, and the fragments containing survivin genes were reconfirmed by DNA sequencing. The plasmid of pshuttle-CMV-survivin was transfected into E.coli BJ5183. After homologous recombination in bacteria, the extracted plasmids of positive clones were lined with PacI, transfected into HEK293 cells with liposome Lipofectaminei 2000 and then identified by PCR method. The high-titer adenovirus supernatants were harvested. Mononuclear cells were obtained from healthy donors by density centrifuge. The washed mononuclear cells were cultured in complete medium (CM) supplemented with 10% fetal calf serum (FCS) at a final concentration of 2×106 cells/ml. After incubated for 120minutes, the adherent cells were used for inducing CTL, and cultured in CM with 1000 U/ml GM-CSF, 1000 U/ml IL- 4 and 50 u/ml TNF-α. The morphology of DC was dynamically analyzed with an inverted microscope and an electron-microscope, and their surface immunophenotyping were determined by flow cytometry. The function of dendritic cells was assayed by mixed leukocyte reaction (MLR) tests. DCs were collected on day 7 and divided into two groups, ad-survivin group and the control group. Two days later, 5×105 cells of each group were placed to a 24-well plate as stimulation cells, and then 6×105 of T lymphocytes (responding cell) were added to each well. On day 14, cells were collected as effector cells for cytotoxicity test. LDH release test was used to evaluate cytotoxicity of CTL. The results showed that the recombined adenovirus-survivin was constructed successfully and its titer was about 2.65×109 pfu/ml. Under inverted microscope and electron-microscope the morphology of these cells is consistent with typical DCs. They are non-adherent and have dendrictic projections. The immunophenotypic analysis of these cells showed positive for CD80, CD 86, CD83 and HLA-DR. The levels of IL-12 and IFN-γ in the culture supernatants, assayed by ELISA were 50.5pg/ml and 82.5pg/ml, respectively. The expression of survivin in transfected dentritic cells was confirmed by Western blot analysis. A 16.5×103 KD band was detected by Western blot. In MLR assay, DCs infected with Ad-survivin can induce high allogeneic lymphocytes reaction at the ratios of 1 to 5, 1 to10, 1 to 50 and 1 to 100. DCs infected with Ad-survivin have much higher activity of CTL to HL-60 cells than control DC. Our conclusion is that DCs infected with Ad-survivin can induce ant-leukemic cells CTL response in vitro. Therefore, adenovirus vectors containing survivin might be the promising candidates for leukemic vaccine development.
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Reinisch, Andreas, Nicole A. Hofmann, Anna C. Obenauf, Karl Kashofer, Eva Rohde, Katharina Schallmoser, Daniela Thaler, et al. "Making Functional Endothelial Progenitors: Humanized Large-Scale Animal Serum-Free Propagated Adult Blood-Derived Endothelial Colony-Forming Cells Assemble Stable Perfused Vessels in Vivo." Blood 112, no. 11 (November 16, 2008): 1882. http://dx.doi.org/10.1182/blood.v112.11.1882.1882.
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Abstract Circulating angiogenic cells (CACs) and several related hematopoietic cell types can mimic an endothelial progenitor cell (EPC) phenotype and facilitate vascular regeneration mainly by humoral and cell mediated support functions but do not form vessels. Despite a documented risk of tumor support and pre-metastatic niche formation, various types of hematopoietic CACs are currently tested in ongoing clinical trials. In sharp contrast, endothelial colony forming cells (ECFCs) have recently been described as the prototype of blood- and vessel-derived EPCs with a robust proliferative potential. Their profound vessel-forming capacity makes ECFCs a promising tool to study human vascular homeostasis, regeneration and tumor angiogenesis. This study was initiated to develop the first animal protein-free large-scale expansion system for adult human blood-derived ECFCs and to test their functionality in vitro and in vivo. We isolated ECFCs directly from whole blood with a novel recovery strategy. ECFC propagation was done under animal protein-free culture conditions with pooled human platelet lysate (pHPL) replacing fetal bovine serum (FBS) for clinical-scale expansion. ECFC long-term proliferation potential was monitored and phenotype was analyzed in detail by flow cytometry as well as immune cytochemistry. Functionality was studied during vascular network assembly in vitro and in two models for human vessel formation in immune-deficient mice in vivo. Genomic stability was assayed with chromosome G-banding and array-comparative genomic hybridization (array-CGH). A mean of four ECFC colonies/mL peripheral blood could be recovered repeatedly in seven donors. The progeny of these oligoclonal cultures could be expanded to mean 1.5 ± 0.5 x 108 ECFCs within 11–25 days in the humanized animal protein-free large scale culture system. Consecutive analysis confirmed ECFC purity, immune phenotype and sustained proliferation potential for >30 population doublings. Preserved progenitor hierarchy after oligoclonal large-scale expansion was confirmed by a mean 74% of high proliferation potential (HPP) and a mean 26% of low proliferation potential (LPP) colonies comprising >500 and 51–500 cells per day 14 colony, respectively. Monoclonal ECFC progeny could also be generated but was less suited for ECFC mass production than oligoclonal ECFCs due to the lower cumulative cell number recovered. Genomic stability was confirmed by karyotyping and array-CGH. Large-scale expanded ECFCs functioned even after cryopreservation to form complex vascular networks in vitro and assembled stable CD31+/Vimentin+/von Willebrand factor+ human vessels in vivo. These human vessels where firmly connected to the mouse circulation for the entire seven week study period as indicated by a rich content of Ter119+ murine red blood cells. This demonstrates for the first time that proliferating, functional, storable and genomically stable human ECFCs can be expanded to a relevant clinical quantity under GMP-compliant conditions in an animal protein-free system. This novel humanized procedure for largescale ECFC propagation represents a promising tool to develop innovative, experimental, diagnostic and therapeutic strategies. It should help to set a new standard to study therapeutic applicability and risk profile of vessel-forming EPC-based investigational new drugs.
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Wheat, Justin C., Daniela S. Krause, Jianfeng Wang, Xi Chen, Thomas Shin, and David Sweetser. "The Transcriptional corepressorTle4 Is a Novel Regulator Of Murine Hematopoietic Stem and Progenitor Cells and Their Niche." Blood 122, no. 21 (November 15, 2013): 588. http://dx.doi.org/10.1182/blood.v122.21.588.588.
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Abstract We previously identified Transducin-like/Enhancer of Split 4 (TLE4) as a potential tumor suppressor gene in acute myeloid leukemia whose loss appears to complement AML1-ETO induced leukemia. In this study we examined the role of Tle4 in normal hematopoiesis. Using Tle4 knockout mice we demonstrated that Tle4 is critical for maintaining a supportive hematopoietic stem cell niche, and also has intrinsic effects on HSC survival and B-cell differentiation. Tle4 knockout mice exhibit profound defects in bone mineralization Tle4 knockout mice become runted by 2 weeks of age and generally die by the time of weaning. These mice exhibit a profound defect in mineralization of the bone and bone marrow failure by 3.5 weeks. At birth KO mice showed a significant reduction in the degree of calcification of the skull and long bones, despite no significant differences in overall body size or weight at this early time point. This was confirmed by Von Kossa staining for calcified bone of one-day old tibiae from KO and WT mice. At 21-28 days after birth trabecular bone was almost absent in tibiae and femurs with thin cortices of the long bones. These data indicate Tle4is critical for proper bone mineralization. In further support of this novel role of Tle4 in skeletal development, a mutation in TLE4 has recently been identified in a father and daughter with congenital vertebral malformations including delayed anterior fontanelle closure, shortened vertebral pedicles and kyphoscoliosis. Tle4 deficient bone marrow stroma is defective in supporting hematopoietic stem and progenitor cells To determine if the hematopoietic abnormalities in Tle4 KO mice might in part be due to the observed defects in the skeletal or stromal compartment of the BM, we cultured WT LKS cells on WT or KO stromal cells obtained at 2 weeks, prior to the observation of bone marrow hypoplasia. In WT cocultures, 6-15% of recovered cells were positive for both c-kit and Sca-1. In stark contrast, less than 1% of cells were C-kit+Sca-1+, when WT LKS cells were plated in KO stromal cocultures. Long-term coculture experiments in methylcellulose revealed an even more pronounced effect, with cells recovered from WT cocultures generating on average 10-fold more colonies than cells recovered from KO cocultures. Some KO cocultures failed to exhibit any colony forming ability. Therefore, these data suggest that Tle4-/- stromal cells cannot maintain and support HSPC growth as efficiently as WT stromal cells. In addition, Western blots of whole bone lysates from two week old mice showed that the majority of Tle4 KO mice had a reduction in Scf protein levels. Tle4 deficient hematopoietic stem cells exhibit cell intrinsic defects in B-cell differentiation and survival Due to the progressive leukopenia in primary Tle4 KO mice, and the extrinsic effects noted above, we sought to determine whether loss of Tle4 intrinsically affected self-renewal and repopulation efficiency of HSC in serial transplantation assays using BM and fetal liver cells. Whole BM from two-week-old KO and WT animals transplanted into lethally irradiated allogeneic CD45.1 mice showed similar levels in donor chimerism in blood, but significant reductions in the frequency of B cells in KO transplanted mice at 16 weeks. At 32 weeks after transplant, recipient mice of KO BM developed significant leucopenia, mostly due to a decreased frequency of B cells. To exclude any effects of the bone marrow stroma we also performed serial transplantations from fetal livers of embryonic day 13.5 WT and KO fetuses. In this transplant model, both peripheral leukopenia and specific B cell lymphopenia were observed in primary transplant recipients, recapitulating the phenotype observed in recipients of KO BM. Although in the primary transplants no difference in the frequency of HSC between recipients of KO or WT BM was observed, progressively with secondary and tertiary transplants we observed a striking hematopoietic stem cell exhaustion with reduction in LKS cells, CD34-LKS cells, and LKS CD34-CD48-CD150+ long term HSC. Taken together, these data indicates Tle4 is a critical regulator of hematopoiesis acting by maintaining supportive function of the bone marrow niche as well as intrinsically affecting B-cell differentiation and the survival of hematopoietic stem cells. Disclosures: No relevant conflicts of interest to declare.
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Mi, Yingchang, Wenbin Wu, Qing Zhang, Yan Li, Xiaoyan Li, Zheng Tian, and Min Wang. "Expression of Kindlins in Acute Myeloid Leukemia." Blood 118, no. 21 (November 18, 2011): 4910. http://dx.doi.org/10.1182/blood.v118.21.4910.4910.
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Abstract Abstract 4910 The Kindlin family of intracellular proteins has recently emerged as key regulators of cellular functions and cell-matrix interactions. They comprise of three evolutionarily conserved members, kindlin-1, kindlin-2 and kindlin-3, they share considerable sequence and structural similarities. A few of study revealed that Kindlin-2 influences solid tumor cell invasion and resistance. With regard to AML, the influence of Kindlins is still unknown. To evaluate the clinical significance of Kindlin-2 in acute myeloid leukemia (AML), we investigated the expression of Kindlin-2, kindling-3 in AML cells. 1. Materials and methods K562, KG-1a, HL60, U937, Jurkat cell lines were cultured in RPMI 1640 medium, supplemented with 10% fetal bovine serum (FBS, GIBCO) at 37°C in a humidified atmosphere of 5% CO2. Bone marrow (BM) samples were obtained from 88 patients with de novo AML from Blood Diseases Hospital, Chinese Academy of Medical Sciences & Peking Union Medical College (CAMS & PUMC). Samples of 9 normal donors and ITP were used as the control group. Bone marrow mononuclear cells (BMMCs) were prepared by Ficoll-Hypaque density gradient centrifugation. Expressions of Kindlin-2, Kindlin-3 were detected by RQ-PCR. The following primers for real-time PCR were used: (a) Kindlin-2 sense primer, 5'-CCGCTCGAGCTATGCGTATCCCCGTAG-3'; (b) Kindlin-2 antisense primer, 5'-CGACGCGTCTAGCGAGGGGTTGTC-3'; (c) Kindlin-3 sense primer, 5'-CCGCTCGAGCTATGCGTATCCCCGTAG-3'; (d) Kindlin-3 antisense primer, 5'-CGACGCGTCTAGCGAGGGGTTGTC-3'; (e) GAPDH sense primer, 5'-GAAGGTGAAGGTCGGAGTC-3'; (f) GAPDH antisense primer, 5'-GAAGATGGTGATGGGATTTC-3'. Analysis was performed using ABI 7500 Sequence Detection software (Applied Biosystems). The expression of Kindlin-2 and Kindlin-3 were showed as RQ value calculated through ΔΔCt method [ΔΔCt = (CtKindlin □ CtGAPDH)sample □ (CtKindlin □ CtGAPDH)calibrator]. The ΔCt (CtKindlin □ CtGAPDH) of K562 was defined as calibrator, and the RQ of calibrator was 1.000. Relationships between Kindlin-2, Kindlin-3 and the patients' clinical data were analyzed. 2. Results Expression of Kindlins in newly diagnosis AML The level of Kindlin-2 in AML (0.163±1.665) was significantly lower than that in non-AML (1.683±1.395) controls (p=0.010). No significant difference was found between the AML and controls in levels of Kindlin-3 (p=0.216). Out of the 79 patients who accepted treatment, 61 patients achieved complete remission (CR) and 18 patients were NR. Patients with higher expression of Kindlin-2 had a higher CR rate (86.8% vs 68.3%) (p=0.050). Expression of kindling 3 was unrelated to CR rate. Both of kindling-2 and kindling-3 increased after CR. This finding implicates Kindlin-2 as a potential prognostic factor of AML. Disclosures: No relevant conflicts of interest to declare.
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Yang, Jing, Jingda Xu, Zhiqiang Liu, Jin He, Huan Liu, Pei Lin, Robert Z. Orlowski, Larry W. Kwak, and Qing Yi. "Activation Of Autophagy By Bone Marrow Adipocytes Protects Myeloma Cells From Chemotherapy-Induced Apoptosis." Blood 122, no. 21 (November 15, 2013): 1915. http://dx.doi.org/10.1182/blood.v122.21.1915.1915.
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Abstract Currently, chemotherapy is the most effective treatment for multiple myeloma (MM). Although some new drugs have been shown to prolong survival in MM patients, these patients are prone to rapid relapse after high-dose treatment. Recent studies show that several bone marrow (BM) stromal cells are potentially involved in drug resistance. However, the role of other stromal cells is unclear. Adipocytes (ADs) are a major component of BM stromal cells. ADs have been shown to be involved in tumor rapid growth, metastasis, and apoptosis. Clinical studies suggest that BM ADs are associated with an increased risk of MM. Moreover, ADs isolated from patient BM biopsies were shown to support MM proliferation and migration. However, no published study has examined the importance of ADs in MM drug resistance. In addition, autophagy activation has been shown to induce drug resistance in cancer patients. We hypothesized that BM ADs protect MM cells from chemotherapy drug-induced apoptosis by autophagy activation. To examine the role of ADs in MM drug resistance, MM cells were cocultured with ADs at a ratio of 1:5 for 24 hours in medium with melphalan, dexamethasone, or bortezomib, the commonly used drugs for the treatment of MM. MM cells included primary MM cells isolated from BM aspirates of 5 MM patients and 6 MM cell lines. Human ADs were generated from mesenchymal stem cells derived from the BM mononuclear cells of healthy human fetal bones or BM aspirates of MM patients or healthy adult donors, cultured in AD medium for 2 weeks. ADs generated in vitro contained cytoplasmic Oil red O+ lipid droplets and produced triglycerol. Our results showed less drug-induced MM apoptosis in cocultures of MM cells and ADs compared with cultures of MM cells alone. Western blot analysis showed that treatment with melphalan upregulated the levels of cleaved caspase-9 and -3, but not -8, and PARP in MM cells. Compared with cultures alone, cocultures with ADs showed significantly lower levels of cleaved caspase-9, -3, and PARP in melphalan-treated MM cells. Mechanistic studies further showed that cocultures of ADs, compared with cultures alone, significantly upregulated the expression of autophagy proteins LC3B, Atg3, Atg5, and LAMP-1, but not Beclin-1. The addition of autophagy inhibitors 3-methyl adenine and chloroquine diphosphate to the cocultures remarkably enhanced apoptosis and caspase activation. Furthermore, we observed that cocultures of MM cells and ADs with either cell-cell contact or those separated by transwell inserts conferred similar protection from drug-induced apoptosis. We identified that AD-produced adipokines such as adiponection, leptin, adipsin, IL-6, MCP-1, TNF-a, and IGF-1, but not VEGF and CRP, were abundant in all examined ADs. Among these adipokines, adiponection, leptin, and adipsin were mainly produced from ADs and not from BM stromal cells, whereas other adipokines were produced from both cells. The addition of antibodies against these adipokines to the cocultures enhanced apoptosis and reduced autophagy, whereas addition of these adipokines to the cultures alone inhibited apoptosis and enhanced autophagy. In vivo studies validated these findings that injection of BM-derived ADs into the implanted human bones of SCID-hu mice bearing primary MM cells reduced response to treatment with melphalan and induced autophagy activation. Taken together, our findings elucidate a novel mechanism of MM drug resistance, through BM ADs. Our studies also provide evidence that targeting BM ADs may be a new approach to improve the efficacy of chemotherapy for the treatment of MM. Disclosures: No relevant conflicts of interest to declare.
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Mellstedt, Hakan, Mohammad Hojjat-Farsangi, Ladan Mansouri, Anders Osterborg, and Hodjattallah Rabbani. "ROR1 Isoforms Are Constitutively Phosphorylated in Chronic Lymphocytic Leukemia (CLL) - a Survival Factor for CLL Cells." Blood 118, no. 21 (November 18, 2011): 1778. http://dx.doi.org/10.1182/blood.v118.21.1778.1778.
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Abstract Abstract 1778 Background: Phosphorylation of receptor tyrosine kinases (RTK) plays an important role for many aspects of cell life, as cell-cycle progression, differentiation, apoptosis, intercellular communication and cell survival. RTKs as e.g. EGFR, HER2/neu, VEGFR, IGFR etc. are important structures contributing to the malignant phenotype of many tumors. Targeting RTKs by monoclonal antibodies (MAb) or small inhibitory molecules has been successful for the treatment of various cancers. ROR is one of the twenty RTK families and consists of two members, ROR1 and ROR2. ROR1 and ROR2 have important functions during embryogenesis. ROR1 is uniquely expressed in CLL cells compared to normal tissues. ROR1 siRNA transfection of CLL cells induced specific apoptosis of the leukemic cells. Aims: To study ROR1 protein variants in CLL, ROR1 phosphorylation and relation to clinical activity of the disease. Methods: Phosphorylation of primary human CLL cells from patients with non-progressive and progressive disease, and a series of cell lines were studied applying immunoprecipitation (IP) of the ROR1 molecule, using anti ROR1 and irrelevant MAbs by Western blotting as well as in intracytoplasmic staining of the cells by flowcytometry, using an anti-phospho ROR1 (pROR1) MAb (a MAb specifically recognizing a phospho-peptide of the cytoplasmic TK domain of ROR1) and semi quantification of the staining intensity as well as by p-tyrosine and p-serine MAbs. Results: IP of the ROR1 molecule of fresh leukemic cells using ROR1 specific antibodies and subsequent Western blot with anti ROR1 MAbs, showed various protein bands. Two bands had the size of 105 and 130 KDa probably representing unglycosylated or partially glycosylated ROR1 and the fully glycosylated glycoform respectively. One of the two bands dominated in individual patients. A band of 260 KDa could also be detected in a large number of patients probably representing dimerized monomers. Finally a band of 64 KDa could also be noted which may represent an intracellular part of ROR1, as has been described in fetal and adult human CNS, leukemia and lymphoma cell lines (Reddy UR et al, Oncogene. 1996; 13:1555–9). The 64, 105 and 130 KDa bands were constitutively phosphorylated in all patients both at tyrosine and serine residues. The intensity of phosphorylation of the 130 KDa band was significantly higher in patients with progressive disease vs. non-progressive disease (p=0.0001). There was no relation between the pattern of the different ROR1 protein bands and disease activity. Using 9 cell lines of different hematologic malignancies including CLL, similar protein bands and phosphorylation status as for fresh CLL cells were noted. We have also produced specific mouse monoclonal antibodies against defined epitopes of the extracellular parts of ROR1. One of those MAbs (anti-KNG, IgG1) induced a high degree of specific apoptosis of the CLL cells (ASH Annual Meeting Abstracts, Nov 2010; 116: 916) and could also be shown to induce a specific dephosphorylation of the cytoplasmic TK domain which was noted already after 20 min and gradually increased up to 4h. No effects were noted using irrelevant MAbs or healthy donor lymphocytes. Conclusion: Our results showed that the ROR1 molecule in CLL cells are expressed in various protein variants probably representing different glycosylation patterns (105–130 KDa), dimerized monomers (260 KDa) and a truncated protein variant (64 KDa). ROR1 was constitutively phosphorylated (64, 105 and 130 KDa) both at serine and tyrosine residues. Treatment of CLL cells in vitro with a ROR1 specific antibody induced specific apoptosis as well as rapid dephosphorylation of ROR1. Collectively the data suggest that phosphorylated ROR1 might be an important structure for the growth potential of CLL cells and an interesting structure to target in a therapeutic intervention. Disclosures: Mellstedt: Kancera AB: Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Hojjat-Farsangi:Kancera AB: Equity Ownership. Rabbani:Kancera AB: Equity Ownership.