bluuebunny/crossref_metadata_2025 huggingface dataset:
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Authors: Emanuel Peter Dean , Wang Zhuo , Cai Danying , Stofega Mary R. , Keck James G. , Estrov Zeev , Brown Gail L. , Ochoa-Bayona Jose L. , Callander Natalie
Published: October 2019

TLK199 is a novel glutathione analog that is a selective inhibitor of the enzyme glutathione S-transferase (GST) P1-1. TLK199 treatment induces hematopoietic cell proliferation and differentiation through activation of the MAP kinase signaling pathway leading to activation of JNK and ERK2. In rodent models of chemotherapy induced neutropenia, treatment with TLK199 accelerated recovery of white cell counts at rates comparable to treatment with G-CSF. We now report TLK199 treatment of myeloid progenitor cells isolated from normal human blood resulted in the increased formation of CFU-GM (46%) CFU-MK (47%) and BFU-E (142%) lineages over baseline. A corresponding increase in the percentage of cells expressing CD11b, a granulocyte and monocyte marker, was observed in the CFU-GM cells. Since TLK199 is currently being evaluated in a Phase 2a trial in patients with refractory MDS, we examined the effect of treatment on formation of BFU-E, CFU-GM and CFU-GEMM before and after TLK199 treatment at doses of 50 to 400 mg/m2. A significant increase in the number of hematopoietic progenitor cell colonies measured from patient peripheral blood and bone marrow was observed as early as Day 4 of Cycle 1 as compared to pretreatment baseline. Ten of 12 patients showed an increase in at least one colony forming type (BFU-E, CFU-GM and CFU-GEMM) and 7 of 12 had an increase in all three colony forming types following TLK199 administration. These results correlate with clinical improvement in hematological parameters in peripheral blood and bone marrow observed in MDS patients treated with TLK199. Studies are underway to define the mechanism of bone marrow and peripheral blood count recovery observed following treatment of MDS patients with TLK199 and the role of GST P1-1 as a regulatory element in myeloid proliferation and differentiation.
Evaluation of Cytokines Mobilizing Hematopoietic Progenitor Cells in Patients with Myelofibrosis with Myeloid Metaplasia (MMM).
Authors: Campanelli Rita , Massa Margherita , Meli Valentina , Rosti Vittorio , Ramajoli Isabella , Pecci Alessandro , Viarengo Gian Luca , Marchetti Monia
Published: October 2019

We have previously shown that in patients with MMM the number of circulating hematopoietic and endothelial progenitor cells is increased compared to other Ph-negative myeloproliferative disorders (Ph-CMPD). The aim of this study was to investigate the relevance of cytokines involved in the mobilization of progenitor cells from bone marrow to peripheral blood. We studied 120 patients with MMM, 22 patients with other Ph-CMPDs, and 30 healthy subjects. We evaluated by ELISA the plasma levels of stem cell factor (SCF), vascular endothelial growth factor (VEGF), and stromal derived factor-1alpha (SDF-1a). Moreover, the percentage of circulating CD34+ cells co-expressing the cytokine receptors CD117, VEGFR-2, or CXCR4 (for SCF, VEGF and SDF-1a, respectively) were assayed by flow cytometry. In all patients and controls, the percentage of circulating hematopoietic CD45+CD34+ cells and CD34+CD133+VEGFR-2+ endothelial progenitor cells were evaluated. SCF (median 872 pg/ml, range 281–2000), VEGF (median 38.9 pg/ml, range <15.6–2000), and SDF-1a (median, 3879 pg/ml, range <1560–18118) plasma levels were significantly higher (p<0.05, p<0.002, p<0.001, respectively) in patients with MMM than in normal controls (SCF, median 742 pg/ml, range 461–1167; VEGF, median <15.6 pg/ml, range <15.6–605; SDF-1a, median 1880 pg/ml, range <1560–8919). SDF-1a plasma levels of patients with MMM were also significantly higher (p<0.001) than those in other Ph-CMPDs (median 2896 pg/ml; range <1560–14144). In patients with MMM, cytokine plasma levels correlate with neither the percentage of CD34+ cells expressing their receptors, nor the percentage of circulating hemopoietic or endothelial progenitor cells. When clinical parameters were tested, we found a significant correlation between SDF-1a plasma levels and both the spleen size (n=113, R=0.20, p<0.05), and the number of immature myeloid cells (n=87, R=0.23, p<0.03). These data show increased plasma levels of SCF, VEGF, and SDF-1a in patients with MMM compared to normal controls; however, only SDF-1a characterizes patients with MMM with respect to those with other Ph-CMPDs. Neither the plasma levels of SDF-1a nor those of the other measured cytokines correlated with the extent of progenitor cells mobilization.
A Clinically Relevant SCID-hu in Vivo Model of Human Multiple Myeloma.
Authors: Tassone Pierfrancesco , Neri Paola , Carrasco Daniel R. , Burger Renate , Catley Laurence , Venuta Salvatore , Anderson Kenneth C. , Munshi Nikhil C.
Published: October 2019

Human multiple myeloma (MM) xenografts in immunodeficient mice have limitations as a model for the human disease since they lack the human bone marrow (huBM) microenvironment. In contrast, murine models harboring a huBM microenvironment with implantation of patient MM cells in the huBM recapitulate the in vivo pathophysiology of MM and have significant advantages over conventional murine models for pre-clinical evaluation of investigational drugs. However, there are significant limitations in using patient MM cells in such models since i) not all patient MM samples engraft in the huBM; ii) only a fraction of engrafted specimens produce measurable paraprotein and/or osteolytic lesions; and iii) a limited number of MM cells can be harvested from an individual patient, thus limiting the number of mice that can be injected with cells from the same patient. To overcome these limitations, we have developed a novel murine model of MM by engrafting INA-6, a cytokine-dependent human MM cell line into SCID mice previously implanted with a human fetal bone chip (SCID-hu mice). INA-6 cells require either exogenous IL-6 or interaction with the bone marrow stromal cells (BMSCs) to proliferate in vitro. In this model, we monitored the in vivo growth of INA-6 cells stably transfected with a green fluorescent protein (GFP) expression vector (INA-6GFP+). Serum soluble human IL-6 receptor (shuIL-6R) and fluorescence imaging of host animals were sensitive indicators of tumor burden with time dependent increase. Fluorescence imaging was able to detect the myeloma cell growth earlier than measurement of sIL-6R levels. INA-6 MM cells grew in SCID-hu mice, but not in SCID mice injected subcutaneously or intravenously without the human fetal bone. We have further confirmed the feasibility of this model in monitoring the response to therapeutic agents such as dexamethasone by detecting reduction in the intensity of the fluorescent lesions as well as shuIL-6R in SCID-hu mice following anti-MM treatment. This highly reproducible model therefore allows for evaluation of investigational drugs targeting MM cells in the huBM milieu.
AHOD00P1, a Pilot Study of Re-Induction Chemotherapy with Ifosfamide, and Vinorelbine (IV) in Children with Refractory/Relapsed Hodgkin Disease.
Authors: Trippett Tanya , De Alarcon Pedro , London Wendy , Sposto Richard , Chen Allen , Gardner Sharon , Schwartz Cindy
Published: October 2019

A COG Phase II pilot study was conducted to assess the efficacy of the novel re-induction regimen of ifosfamide/vinorelbine (IV) in pediatric patients with heavily pre-treated relapsed/refractory Hodgkin disease. The treatment regimen consisted of : Ifosfamide 3 gm/m2/d continuous 24-hr IV infusion on Days 1–4; MESNA 3 gm/m2/d continuous 24-hr IV infusion on Days 1–5; and vinorelbine 25 mg/m2/dose IV bolus on Days 1, 5. Filgrastim was administered on Day 6 of Cycle 1 at a dose of 5 μg/kg until ANC ≥ 1000/μl for 3 consecutive days or ≥ 10,000/μl for 1 day and on Day 6 of Cycle 2 at a dose of 10 μg/kg to continue daily until completion of peripheral stem cell mobilization. We report the results of the first phase of the study, that evaluated the re-induction regimen IV which was substantiated as an effective re-induction regimen based on acceptable toxicity profile, capacity to mobilize hematopoietic stem cells (CD34+), and response sufficient to allow subsequent stem-cell transplantation in a large fraction of patients. Twenty-nine patients were accrued on study; 12 male (41%), 17 female (59%); median age at time of enrollment was 16 (range 5–20 yrs). The protocol mandated two cycles of therapy, although some patients received an optional third cycle. This report is based on data from the first 2 cycles only. Twenty-five patients had complete toxicity data. The predominant toxicity was hematologic, specifically leukopenia/neutropenia. The frequencies of grade 3–4 hematologic toxicities included: anemia (62%), leukopenia (100%), neutropenia (95%), and thrombocytopenia (50%). Hypophosphatemia (3%), febrile neutropenia (34.9%) and infection with grade 3 or 4 neutropenia (21.6%), and neurotoxicity (3%), each occurred in a minority of patients. There were no toxic deaths. Harvest was attempted in 25 patients. At least 2.5 x 106 CD34+ cells/kg were collected in 19 (76%), and at least 5.0 x 106 were collected in 16 (64%). Complete response data were available for twenty-five patients. Seven patients attained a complete response (CR) and eleven a partial response (PR) (CR/PR rate 72%). IV was substantiated as an acceptable re-induction regimen for children with Hodgkin disease. It was effective, well tolerated, and allowed for collection of sufficient CD34+ cells to support peripheral stem cell transplantation. Based upon these findings, 35 additional patients will be accrued to more precisely establish the definitive response rate in pediatric patients. In addition, minimally pretreated patients will now be included, allowing assessment of response, toxicity and hematopoietic stem cells (CD34+) mobilization in minimally pre-treated patients as well as in the heavily pre-treated population.
p53 Silencing by Lentiviral Vector Mediated RNA Interference in Murine Hematopoietic Cells and the Development of a Myelogenous Proliferative Disease.
Authors: Woods Niels-Bjarne R. , Tergaonkar Vinay , Singer Oded , Bottero Virginie , Verma Inder M.
Published: October 2019

The ability for siRNA containing lentiviral vectors to specifically downregulate gene expression in the hematopoietic system following hematopoietic stem cell transplantation provides a new tool for developing disease models of leukemia and other hematological disorders. The tumor suppressor gene, p53, has been implicated in the genesis or progression of hematological malignancies, such as AML, MDS, and CML, and disorders such as myeloproliferative disease (MPD). Following transduction of Ly5.1 hematopoietic stem cells using a lentiviral vector expressing small interfering RNAs specific for mouse p53, cells were transplanted into lethally irradiated syngeneic recipient mice. Peripheral blood analyses by FACS at 7 weeks posttransplant showed significantly increased numbers of Gr1 Mac1 double positive progenitor like cells in 4 of 4 mice (73±14% compared to control siGFP transplanted mice 5±0.5%). Interestingly, 3 of 4 transplanted mice showed signs of morbidity by 16 weeks posttransplant due to a myelogenous proliferative disease (MPD) phenotype characterized by high peripheral blood neutrophil counts (>70% of white blood cells) and anemia (<106 RBC/μL), and extensive neutrophil infiltration into spleen and liver. Furthermore, peripheral blood smears show an expansion of megakaryocytes. Bone marrow mononuclear cells from transplanted mice were analyzed for p53 protein expression following p53 stabilization by irradiation and significantly lower levels of p53 were detected in sip53-transduced mice compared to siGFP lentiviral vector control mice. No altering of normal hematopoietic cell differentiation was observed in control mice from nonspecific RNAi interactions, as determined by FACS or blood cell count analyses. These results demonstrate that downregulated expression of p53 in mice hematopoietic cells yields a myelogenous proliferative disease and creates a model for the study of p53 involvement in the genesis and progression of hematological disorders. Furthermore, these results demonstrate the ability of lentiviral vector mediated siRNA to efficiently downregulate endogenous gene expression in vivo and model human disease.
Atherosclerosis as a Risk Factor for Venous Thromboembolism (VTE): A Population-Based Cohort Study.
Authors: Petterson Tanya M. , Agmon Yoram , Meissner Irene , Khandheria Bijoy K. , Heit John A.
Published: October 2019

While atherosclerosis has been suggested as a risk factor for VTE, previously identified mechanisms for atherosclerosis and VTE differ dramatically.

Objectives: To compare (a) VTE-free survival and (b) observed vs. expected number of VTE events, among adult patients with and without aortic atherosclerosis.

Design/Setting: Population-based prospective cohort study in Olmsted County, MN.

Patients: Age- and sex-stratified random sample of the adult (≥45 years old) Olmsted County population identified over the three-year period, 1993–1995 (n=573), performed as part of the Stroke Prevention: Assessment of Risk in the Community (SPARC) study.

Measurements: The SPARC study included a baseline clinical evaluation (interviews, review of medical records), blood pressure measurements, blood sampling, and transesophageal echocardiography (TEE). The Risk Factors for VTE in the Community study identified all incident VTE among Olmsted County residents over the 32-year period, 1966–1997, from which expected VTE events by age and sex were calculated.

Results: Over 2830 person-years of follow-up, 12 SPARC patients developed VTE (7 idiopathic). The 4-year VTE-free survival was 99% and 98% for those with and those without any TEE-detected aortic atherosclerosis. Adjusting for age in a Cox proportional hazards model, lack of aortic atherosclerosis was significantly associated with subsequent VTE (p=0.01). Among the 276 patients without atherosclerosis observed for 1423 person-years, 7 VTE were observed compared to 2.6 expected (RR=2.7, 95%CI: 1.1, 5.6). Among the 291 patients with atherosclerosis (plaque ≥2mm), 4 VTE were observed compared to 5.8 expected (RR=0.7, 95%CI: 0.2, 1.8).

Conclusions: Atherosclerosis is not a risk factor for incident VTE.
Risk Factors Associated with Venous Thromboembolic Events in Patients with Malignancy.
Authors: Lin Jules , Li Hang , Wakefield Thomas W. , Henke Peter K.
Published: October 2019

Malignancy is a major risk factor for venous thromboembolic events (VTE), but not all patients with malignancy develop this complication. Who best to aggressively prophylax is thus not well defined. In the current study, 960 consecutive patients, 523 men and 437 women, admitted to the University of Michigan with malignancy between 1992–2000 were identified using ICD-9CM codes. Factors including cancer stage, type, therapy, and patient vital status were obtained from a database maintained prospectively by the University of Michigan Cancer Registry as well as a review of the medical record. Acute VTE, confirmed on radiological or ultrasound studies, occurred in 408 patients and were compared to 552 patients who did not experience any VTE using logistic regression analysis. Factors associated with VTE include solid tumors (Odds Ratio 5.0; 95% confidence interval 1.65–14.9, P =.004), infection (4.9; 1.2–19.8, P =.025), and advanced age (1.05; 1.03–1.08, P <.001). Interestingly, while leukopenia (4.2; 1.23–14.6, P =.023) was associated with an increased incidence of VTE, neutropenia was not, suggesting that a deficiency of a different class of leukocytes is more important in predisposing to primary VTE. Neutropenia was associated with recurrent, but not primary VTE (P =.034). Type of therapy, gender, and stage were not associated with VTE. Kaplan Meier estimated survival was decreased in patients with VTE as compared to those without (3.3 vs. 3.7 years, P=.18). Factors associated with a decreased survival include solid tumors (3.9; 1.8–8.4, P <.01), infection (3.3; 1.1–9.9, P =.03), advanced stage (1.6; 1.2–2.1, P <.01), and increased age (1.02; 1.0–1.04, P =.01). Patients with solid tumors, advanced age, and leukopenia have a significantly increased risk of VTE and might benefit from aggressive VTE prophylaxis, whereas other patients with malignancy can probably be carefully observed.
Oligosaccharide Expression and Modification Defines Novel Targets within the Vascular Tree: Selective Chondroitin Sulfate C Expression in the Tumor Microenvironment Generates a Novel Target of Otherwise Constitutive Receptors.
Authors: El-Sheikh Amr , Bhattacharjee Gourab , Borgstrom Per , Belting Mattias , Edgington Thomas
Published: October 2019

The expression of certain proteoglycans or modification of proteins within the vascular tree, dependent on the microenvironment, is critical for elucidating the biology of endothelium specificity and development. It also facilitates the targeting of physiologic and therapeutic agents to different addresses within the vascular map. Using in vivo panning, we have identified a truncated heparin-binding domain (HBDt) that recognizes its target selectively in tumor vasculature. Here we show that it localizes selectively to the endothelial cells of intra-tumoral blood vessels of various murine tumor models, such as CT26, LLC, N202, and Tramp-L1. The HBDt, as a part of the VEGF heparin-binding domain, is conserved throughout evolution and is known to bind the VEGFR-2/Npn-1 complex. Although the VEGFR-2/Npn-1 complex is expressed elsewhere in the vascular tree, this domain only localizes to a target in tumor vasculature. We have analyzed the basis of this selectivity in vitro and in vivo. In vitro analysis has shown that chondroitin sulfates are the most potent inhibitors of HBDt binding to heparin. We also show, using Western blot and confocal microscopy analyses, that VEGFR-2 and Npn-1, although expressed in different organs, are only recognized by HBDt when coexpressed with chondroitin sulfate C (C6S) in the tumor vasculature. The HBDt colocalized with VEGFR-2, Npn-1, and C6S but with not bFGFR or heparan sulfates in the intravasculature of different tumor models. Furthermore, the selective expression of C6S oligosaccaharide, in conjunction with VEGFR-2 and Npn-1, during the angiogenesis of tumor endothelium defines the target for the HBDt but not during aortic angiogensis. Therefore, our data demonstrate that the expression of C6S, as part of the HBDt receptor, is an example of the tumor microenvironment conditioning, which imparts association of a novel target on endothelium surfaces of tumors.
APRIL Promotes Survival and Proliferation of T Cells: Implications for T-Cell Lymphoma.
Authors: Yang Zhi-Zhang , Ansell Stephen M. , Witzig Thomas E. , Novak Anne J.
Published: October 2019

Background: Cutaneous T-cell lymphoma (CTCL) is an incurable lymphoproliferative disorder characterized by the clonal expansion of malignant CD4+ T cells. Currently there is a poor understanding of the underlying mechanisms in the development and progression of this disease. Members of the TNF superfamily have been shown to be important mediators in the formation and regulation of normal T cell responses. A proliferation-inducing ligand (APRIL), a newly identified member of the TNF superfamily, has been shown to stimulate tumor cell growth as well as proliferation of primary B and T lymphocytes. APRIL transcript levels are reported to be low in normal tissues with the highest levels found in peripheral blood leucocytes. In contrast, higher mRNA levels have been detected in tumor cell lines and primary tumor tissues and various tumor cell lines have been shown to respond to APRIL with increased tumor cell proliferation and decreased apoptosis. The striking effects of APRIL on tumor cell growth and survival raise the possibility that the APRIL and its receptors-TACI and BCMA may be involved in the pathogenesis and maintenance of T cell malignancies.

Goal: To explore the involvement of APRIL on normal and malignant T cell growth and survival.

Results: Under resting conditions, there was minimal APRIL binding to normal T-cells and no TACI or BCMA expression. However, stimulation of resting T cells with OKT3 (anti-CD3) or a combination of PMA and ionomycin resulted in an increase in APRIL binding and TACI expression on a subset of activated T-cells. When CD3+ resting human T cells were cultured in the presence of APRIL, IL-2, or OKT3 alone or a combination, normal T cells had greater viability compared to the nil control and APRIL mediated-T cell survival was similar level to that seen with IL-2 or OKT3 alone. CSFE labeled T cells were activated with suboptimal dose of OKT3 and incubated in the presence of APRIL and /or IL-2. Both APRIL and IL-2 alone induced cell proliferation, while treatment with the combination of APRIL and IL-2 had a synergistic effect on T cell proliferation. Malignant T cells isolated from the blood of patients with Sezary Syndrome exhibited a similar pattern of APRIL binding and APRIL also enhanced their survival. Gene array experiments were performed to identify genes involved in APRIL-mediated survival. In normal and primary malignant T cells the anti-apoptotic genes BCL2A1, BIRC6, CHK1, MCL-1, and MDM2 were upregulated and the pro-apoptotic genes CASP5, DR5, and TNFR2 were downregulated in response to APRIL.

Conclusion: APRIL promotes the survival of normal and malignant T cells and co-stimulates CD3-mediated T cell proliferation. The gene array experiments suggest that APRIL regulates the expression of anti-apoptotic and pro-apoptotic genes in both normal and malignant T cells. Taken together, these data indicate an important role for APRIL in the biology of normal T cells and in the pathophysiology of T cell lymphoma.
A Population Pharmacokinetic Model Describing the Activity-Time-Course of PEG-Asparaginase in Children.
Authors: Hempel Georg , Lanvers-Kaminsky Claudia , Mueller Hans-Joachim , Wuerthwein Gudrun , Boos Joachim
Published: October 2019

PEG-Asparaginase is an important part of many treatment protocols for ALL. In many centres Asparaginase activity is measured after administration of PEG-asparaginase. However, a predictive pharmacokinetic model is lacking. Such a model would be helpful for dose adjustment and decision making when to switch to another preparation due to the development of inactivating antibodies. Previously described models like linear one-compartment [V.I. Avramis et al., Blood 2002, 99: 1986–1994] or a one-compartment Michaelis-Menten model [H.J. Mueller et al., Cancer Chemother. Pharmacol. 2002, 49, 149–154] describe the data sufficiently for one dose alone, but cannot account for the phenomenom that the time to reach a lower activity limit after administration is not increasing with increasing the dose. Therefore, we analysed 1189 serum activity measurements in 185 children from the ALL-BFM 95 study. Patients received 500, 1000 or 2500 U/m2 PEG-Asp on up to 9 occasions. Serum asparaginase activity was measured using a semi-automatic enzymatic assay with a limit of quantification of 2 U/l [C. Lanvers et al. Anal. Biochem. 2002, 309, 117–126]. Data analysis was done using nonlinear mixed effects modelling (NONMEM Vers. V). Different models like Michaelis-Menten, linear first-order, Weibull and gamma models were tested. The best model applicable to all dosing groups was a modified first-order one-compartmental model with clearance increasing with time according to the formula: Cl = Cli*exp(0.0853*t) with Cli=initial clearance, and t=time. Addition of a second compartment did not improve the model. A typical activity-time course of a patient receiving 1000 U/m2 is shown below displaying the typical shape observable in all patients and in all doing groups. The population parameters found were: Volume of distribution (V) 1.05 ± 27.3% l/m2, Cli 60.3 ± 70.8% ml/day/m2 (mean ± interindividual variability). Interoccasion variability was substantial with 0.223 l/m2 for V and 37.7 ml/day/m2 for Cl, respectively. A subgroup of one third of the patients is identifiable showing a high clearance probably due to the development of inactivating antibodies. Drug monitoring of serum PEG-Asparaginase activity is required to identify these patients who do not benefit from PEG-Asp therapy. The pharmacokinetic model presented here should help to reduce the number of required serum samples per patient.

Figure Figure
Differential Effects of Delta1 and Jagged1 in Early Human Myelopoiesis: Correlation with Distinct Gene-Expression Profiles in CD34+ Cells.
Authors: Neves Hélia , Weerkamp Floor , Naber Brigitta A.E. , Gameiro Paula , Becker Jörg D. , Lucio Paulo , Clode Nuno , van Dongen Jacques J.M. , Staal Frank J.T. , Parreira Leonor
Published: October 2019

The Notch ligands Delta1 and Jagged1 are known to differentially affect the development of B and T-cell lineages but their function in the myelopoiesis is still unclear. Here we show that Delta1 and Jagged1, expressed by bone-marrow-derived stromal cells, differentially affect de myeloid development of human CD34+ cells: Jagged1 increases their clonogenic potential both at the level of bi- (CFU-GM) and uni-potent progenitors (CFU-G and CFU-M), without quantitatively affecting terminal cell differentiation; Delta1 strikingly reduces the number of CFU-GM and differentiated monocytic cells.

Gene expression profiling of CD34+ cells cultured in contact with Jagged1 or Delta1, reveals that the observed phenotypic/functional differences correlate with the emergence of distinct transcription profiles for genes related to hematopoietic-differentiation programs, receptors for growth-factors and Notch-signalling.

The comparative analysis of different CD34+ sub-populations revealed that: cells with similar phenotypes (CD34+CD7−), but emerging in distinct environments (Delta1 or Jagged1), have different “lineage-specific” transcription profiles, Delta1-CD34+ cells exhibiting an almost “pure” myeloid program, whereas Jagged1-CD34+ cells are enriched in transcripts for B- and myeloid lineages; the same environment (Delta1) originates CD34+ cells with different phenotypes, as to CD7 expression, which differ in their lineage-specific transcription profiles: Delta1-CD34+CD7+ cells show a high expression of T/NK-cell related genes in contrast with their CD7− counterparts which express a myeloid-lineage program. Furthermore, Jagged1 and Delta1-stromas have differential effects in the expression of genes coding for receptors for several hematopoietic growth factors. Overall, these transcription profiles agree with the specific cell-lineage-affiliation of the corresponding CD34+ cell sub-population. Quantitative analysis of transcripts from Notch-signalling-related genes in different CD34+ sub-populations showed that cells with similar phenotypes (CD34+CD7−), but emerging in distinct environments (Delta1 or Jagged1), have different expression profiles of receptors and targets of Notch. In the case of Delta1 environment, we observe a predominance of Notch3 and Hes1 transcripts, whereas Jagged1-CD34+ cells are enriched in Notch1 and Deltex transcripts. In the same stromal environment (Delta1) but different in CD34+ cell sub-populations Notch3 receptor was up-regulated. Delta1, contrary to Jagged1, promotes up-regulation of modulators of Notch signaling.

Together, the data show that Delta1 and Jagged1 modulate gene-expression programs in CD34+ cells in ways that contribute not only to cell-lineage-affiliation but also to the functional interactions between developing cells and their micro-environment.
A Novel NF-kB:NFAT Enhanceosome Synergistically Regulate CD154 Gene in Aggressive B Cell Lymphomas.
Authors: Pham Lan , Tamayo Archito , Yoshimura Linda , Lin-Lee Yen-Chiu , Ford Richard J.
Published: October 2019

Non-Hodgkin’s Lymphoma (NHL) causes many deaths world wide, and is one of the few cancers that have a continual increase in incidence and mortality rates over the last few decades. Diffused large B cell lymphoma (DLBCL) is an aggressive B cell lymphoma that accounts approximately 40% of all NHL-B cells. We have previously shown that dysregulated CD40 ligand (CD40L/CD154) expression in DLBCL maintains lymphoma cell growth and cell survival. CD40 ligand is a member of the TNF superfamily of proteins that has a wide variety effects throughout the immune system and is critical for both cellular and humoral immunity. It was originally thought that CD154 expression was restricted to activated CD4+ T lymphocytes. However, expression of CD154 has also been demonstrated in non-lymphocytic leukocytes such as mast cells, basophils, eosinophils, dendritic cells and macrophages. More importantly, dysregulated CD154 expression has been noted in a number of diseases, including systemic lupus erythematosus (SLE), Alzheimer’s, and in B cell lymphomas. Although the functional analysis of CD154 has been extensive, little is known about the mechanisms controlling CD154 expression in activated T cells, let alone in normal and malignant B cells. In this study, we show that constitutive NF-kB and NFAT activation in aggressive lymphoma B cells directly interact and synergistically regulate CD154 gene expression. Immunoprecipitation and confocal analyses indicate that NFATc1 and NF-kB (p65 and c-rel) complex and colocalize in lymphoma B cells. Chromatin Immunoprecipitation (CHIP) assays analysis demonstrate that NFATc1, p65 and c-rel bind to the CD154 promoter, that is enhanced by HDAC inhibitor (TSA or SAHA) treatments. HDAC inhibitors also enhance CD154 promoter activity, providing evidence that CD154 transcriptional regulation involves histone acetylation. Over-expression studies show a synergistic effect by NFATc1 and NF-kB (c-rel) on the CD154 promoter. Promoter deletion and site direct mutagenesis studies on the CD154 promoter reveal that the transcriptional regulation of CD154 requires two sites, the distal kB site at −1180 and a proximal NFAT site at −250. These sites bind to both, NFAT and NF-kB proteins, as wells as the DNA architectural protein HMG-1, indicating that the CD154 promoter DNA may loop and form an enhanceosome-like complex. Studies using siRNA to p65, c-rel, or NFATc1 repress CD154 promoter activity, indicating that these transcription factors are necessary and sufficient for CD154 gene transcription. These findings strongly suggest a novel control mechanism for CD154 gene transcription, therefore, providing potential treatment modalities for lymphoma B cells as well as other disorders involving dysredulated CD154 expression.
Damaged Murine Brain Induces ES Cells into Migration and Proliferation.
Authors: Srivastava Anand S. , Shenouda Steve , Carrier Ewa
Published: October 2019

An understanding of how ES cells differentiate in vivo into neural cells is essential for developing and refining transplantation strategies for the nervous system, as well as for understanding the molecular mechanism underlying neurogenesis. We hypothesized that damaged neural tissues will create an environmental niche and stimulate ES cells to migration and differentiation into the neural cells. We induced damage in murine (C57BL/6) brain by injecting 2.5 μl of PBS into the left frontal and right caudal regions and confirmed neural damage by immunohistochemistry. Yellow fluorescent protein (EYFP) expressing embryonic stem cells (50,000 cells/1μl) (Nagy et al., Cell Biol., 2001; 115:49–58.) were injected into the non-damaged left caudal portion of the brain. Using histochemistry (anti-GFP peroxidase, cross-reacting with YFP) and fluorescent microscopy, we observed migration of ES cells from the injection site (left caudal) to the damaged site (right caudal and left frontal) at 5 days post injection. The survival of ES cells was demonstrated by the real-time PCR assay of stemness genes such OCT4, SOX2 and FGF4. The portions of the damaged neural tissues containing ES cells demonstrated 4-fold increase in expression of these genes one week of post injection in comparison to non-injected ES cell murine brain suggesting proliferation. Increased PDGF protein expression and histochemistry studies demonstrated that ES cells responded to damaged neural tissues, migrated to the opposite site of the brain and proliferated. Differentiation studies are in progress and will be presented. This work demonstrates the responsiveness of undifferentiated ES cells to the damaged neural tissues and provides a model of in vivo stem cellà stem cell niche interactions.
Ex-Vivo Generation and Expansion of Both Lymphoid and Myeloid Lineages from Human Cord Blood (CB) HSC Using a Serum-Free Human Mesenchymal Stem Cell Based Culture System.
Authors: Frias Ana , Porada Christopher D. , Crapnell Kirsten B. , Cabral Joaquim M.S. , Zanjani Esmail D. , Almeida-Porada Graca
Published: October 2019

The in vitro culture of a hematopoietic stem cell (HSC) graft with either media containing animal-derived components or a feeder layer with ill-defined pathogenic potential such as xenogeneic cell lines or cells modified by viral transformation poses risks that concern scientists and regulatory agencies. In the present studies, we avoided these risks by evaluating the ability of a human stromal-based serum free culture system (hu-ST) to support the ex-vivo expansion/maintenance of human CB HSC. CB CD34+ enriched cells were cultured in serum free medium in the presence of hu-ST with SCF, bFGF, LIF and Flt-3, and the cultures were analyzed for expansion, phenotype and clonogenic ability. We have previously reported the ability of this culture system to allow the successful expansion/maintenance of HSC along the myeloid pathway. In the present study, we investigated whether we could further develop this culture system to simultaneously expand myelopoiesis and lymphopoiesis in vitro. To this end, cord blood CD34+ cells were cultured for a total of 28 days and analyzed every 3 days for expansion and phenotype. There was a progressive increase in CD34 cell number with time in culture. The differentiative profile was primarily shifted towards the myeloid lineage with the presence of CD33, CD15, and CD14. However, a significant number of CD7+ cells were also generated. At week 2 of culture, we observed that 30% of the cells in the culture were CD7 positive. These CD7+CD2-CD3-CD5-CD56-CD16-CD34- cells were then sorted and either plated on top of new irradiated hu-ST layers in the presence of SCF, FLT-3, IL-7, IL-2, and IL-15, or cultured with IL-4, GM-CSF, and FLT-3 in the absence of stroma. Both of these cultures were maintained for an additional 2 weeks. In both sets of cultures, further expansion in the total cell number occurred with the time in culture, and by the end of the week 2, we observed that 25.3±4.18% of the cells had become CD56+ CD3-, a phenotype consistent with that of NK cells. Furthermore, cytotoxicity assays were performed and showed cytotoxic activity that increased in an E:T ratio-dependent fashion. 38.6% of the CD7+ cells grown in the presence of IL-4, GM-CSF, and FLT-3 became CD123+CD11c-, a phenotype characteristic of nonactivated dendritic cells, while 7.3–12.1% adopted an activitated dendritic cell phenotype CD83+CD1a+. In summary, we developed an in vitro culture system that reproducibly allows the effective ex vivo expansion of human cord blood HSCs while maintaining the capability of generating both myeloid and lymphoid hematopoiesis in vitro.
The CALM/AF10 Fusion: Molecular Analysis of the Fusion Transcripts in 13 Cases of AML and ALL; Gene Expression Profiling Reveals HOX Gene Deregulation.
Authors: Krause Alexandre , Kohlmann Alexander , Haferlach Torsten , Schoch Claudia , Schnittger Susanne , Mecucci Cristina , Ludwig Wolfgang D. , Bohlander Stefan K.
Published: October 2019

The t(10;11)(p13;q14) is a recurring translocation associated with the CALM/AF10 fusion gene which is found in undifferentiated leukemia, acute myeloid leukemia, acute lymphoblastic leukemia and malignant lymphoma with poor prognosis. The CALM/AF10 fusion protein was reported to be the most common fusion protein in T-ALL with TCR gamma delta rearrangement. We have analyzed samples from 9 patients with different types of leukemia: case 1 (AML M2), case 2 (AML M0), case 3 (Pre T-ALL), case 4 (Acute Undifferentiated Leukemia), case 5 (PreT-ALL), case 6 and 7 (ProT-ALL), case 8 (T-ALL), case 9 (AML), with a t(10;11) translocation suggesting a CALM/AF10-rearrangement. The samples were analyzed for the presence of the CALM/AF10 and AF10/CALM mRNA by RT-PCR and sequence analysis. All these patients were found positive for the CALM/AF10 fusion.

In addition, we analyzed a series of twenty-nine patients with T-ALL with gamma delta rearrangement. Among these patients, four were positive for CALM/AF10 transcripts, indicating a high incidence of CALM/AF10 fusions in this group of leukemia. We found three different breakpoints in CALM at nucleotide 1926, 2091 and a new exon, with 106 bases inserted after nt 2064 of CALM in patient 4. In AF10 four breakpoints were identified: at nucleotide position 424, 589, 883 and 979. In seven patients it was also possible to amplify the reciprocal AF10/CALM fusion transcript (case 1, 3, 4, 8, 9, 10 and 11). There was no correlation between disease phenotype and breakpoint location. The patients were 5 to 46 years old (median 25). Ten CALM/AF10 positive patients were further analyzed using oligonucleotide microarrays representing 33,000 different genes (U133 set, Affymetrix). Analysis of microarray gene expression signatures of these patients revealed high expression levels of the homeobox gene MEIS1 and the HOXA cluster genes HOXA1, HOXA4, HOXA5, HOXA7, HOXA9, and HOXA10. The overexpression of HOX genes seen in these CALM/AF10 positive leukemias is reminiscent of the pattern seen in leukemias with rearrangements of the MLL gene, and complex aberrant karyotypes suggesting a common effector pathway (i.e. HOX gene deregulation) for these diverse leukemias. It is known that alhambra, the Drosophila homologue of AF10 can act on polycomb group responsive elements, which play a critical role in the regulation of the HOX gene clusters. It is thus conceivable that the CALM/AF10 fusion proteins acts in a dominant negative fashion on wild type AF10 function relieving the repression that is presumably normally exerted by AF10 on the expression of HOX genes.
Towards the In Vivo Identificaton of Leukemogenic Fusion Proteins.
Authors: Chen Ying , Froehlich Nicole , Bohlander Stefan K.
Published: October 2019

Currently there are no methods available to identifiy leukemogenic fusion proteins in vivo. All available methods, like Southern blotting, PCR, FISH or Western blotting, require the destruction of the cells that are assayed. A method for the in vivo detection of leukemogenic fusion proteins would be highly desirable because it would open up new approaches to study leukemia and might lead to novel treatment strategies.

We have developed a strategy for the in vivo detection of the BCR/ABL fusion protein. BCR/ABL is found in virtually all cases chronic myeloid leukemia (CML) and a large proportion of acute lymphoblastic leukemia (ALL). Animal model have shown that the BCR/ABL fusion protein is required for the induction and maintenance of leukemia. The fact that BCR/ABL fusion protein is crucial for the development of leukemia makes this fusion protein an attractive target for therapy development. Our BCR/ABL detection strategy is based on protein-protein interactions and a proof of principle for the strategy was implemented in the yeast system. Two detection proteins are expressed in the cells: 1) protein A, a Gal4-DNA binding domain/BCR interacting protein fusion protein and 2) protein B, a Gal4-activation domain/ABL interacting protein fusion protein. Only when BCR/ABL is present in the cell, do protein A, protein B, and BCR/ABL form a trimeric complex which activates the transcription of reporter genes under the control of Gal4-upstream activating sequence (UAS).

Yeast cells (strain CG1945) transformed with a protein A expressing plasmid (pGBT9-BCR-interactor), a protein B expressing plasmid (pGAD424-ABL1-interactor), and a BCR/ABL expressing plasmid (pES1-BCR/ABL) showed expression of the reporter genes HIS3 and LACZ. The expression of the HIS3 reporter gene was assayed by growth of the yeast cells on medium lacking histidine. The expression of the LACZ gene was verified by a beta-galactosidase filter assay.

Yeast cells that were transformed with the pES1 plasmid without the BCR-ABL coding region did not show activation of the reporter genes. Several other negative controls were also negative. Thus the method was able to clearly distinguish between BCR/ABL expressing cells and cells did not express BCR/ABL.

We are presently adapting this system for use in mammalian cells. The flexibility of our strategy allows us to freely choose the reporter or effector genes. Therapeutically more useful effector genes are suicide genes, which encode pro-drug converting enzymes (e.g. HSV thymidine kinase), or markers that can easily be assayed (e.g. green fluorescent protein).
Cytogenetic Remission Status after Induction Chemotherapy for AML and High-Risk MDS Predicts Long-Term Outcome.
Authors: Balleisen Sebastian , Barbara Hildebrand , Brigitte Royer-Prokora , Andrea Kuendgen , Norbert Gattermann , Ulrich Germing , Rainer Haas
Published: October 2019

Cytogenetic findings at diagnosis have an important impact on prognosis in AML and MDS. Therefore, treatment is commonly stratified according to karyotype. Another important prognostic factor is remission status after induction chemotherapy. However, the diagnosis of remission, and the resulting treatment decisions, are usually not based on cytogenetic findings, but rely on peripheral blood counts and bone marrow blast cell counts. We analysed the prognostic impact of cytogenetic remission status in 115 patients with abnormal karyotype who received induction chemotherapy because of AML (n=102) or MDS (n=13). Initial karyotypes were as follows: 14x t(15;17), 9x t(8;21), 5x inv(16), 55 intermediate-risk, and 32 complex karyotypes. The following induction protocols were used: 38x TAD, 4x HAM, 25x Ida-Ara, 36x ICE, and 3x other protocols. Eleven patients received induction chemotherapy plus ATRA. 23 patients underwent allogeneic transplantation after induction therapy, either as late consolidation in cytologic remission (CR), or as standard treatment in high-risk patients. 78 patients achieved CR, 21 achieved partial remission (PR), and 16 patients were non-responders (NR).

There were 26 patients who reached cytological CR but still showed an abnormal karyotype after induction. 17 of these 26 patients had been classified as standard-risk and therefore did not receive intensified consolidation. In 59 patients, a normal karyotype was found after induction therapy. 24 of 28 patients belonging to low-risk cytogenetics (t15;17, t8/21, or inv16) achieved cytogenetic complete remission (CCR). The CCR rate was much lower in patients with intermediate-risk cytogenetics (24/55) and patients with complex karyotypes (11/32). Median survival in the group achieving CCR was 40 months, as compared to 11 months in patients with persistence of abnormal karyotype after induction (p<0,00005). The difference remained highly significant when calculated only for patients with complex or intermediate-risk karyotypes (median survival 29 vs 11 months).

Conclusions: Cytogenetic analysis after induction chemotherapy is useful to detect residual disease and therefore provides meaningful information in addition to cytology. Achieving cytogenetic remission after induction therapy is strongly correlated with a better long-term outcome. Patients with a persisting abnormal karyotype must be regarded as high-risk patients who should receive intensified treatment.
XPD Lys751Gln Polymorphism and Etiology and Outcome of Childhood Acute Myeloid Leukemia.
Authors: Mehta Parinda A. , Alonzo Todd A. , Gerbing Robert B. , Perentesis John P. , Lange Beverly J. , Davies Stella M.
Published: October 2019

Genetic polymorphism results in inter-individual variation in DNA repair capacity, and may in part account for susceptibility of a cell to genotoxic agents, and to malignancy. The DNA repair protein xeroderma pigmentosum complementation group D (XPD) is a major participant in the neucleotide excision repair pathway and is also involved in transcription initiation and in the control of cell cycle and apoptosis. Several studies have found associations between ineffective DNA repair and cancer risk. It is also thought that ineffective DNA repair may lead to additional genomic instability and tumors that are more aggressive. Several single nucleotide polymorphisms including an adenine to cytosine polymorphism leading to Lys751Gln in exon 23 of XPD have been shown to be associated with elevated frequency of chromosomal aberrations and variety of environmentally induced cancers in adults. We hypothesized that XPD polymorphism may play a role in causation of childhood AML. In addition, there are data that XPD polymorphism influences outcome of chemotherapy in adults with solid tumors. Therefore, we also evaluated its effect on outcome of AML therapy. Methods: 457 children treated for AML on CCG studies 2941 and 2961 (intensive induction with daunorubicin, idarubicin, Ara-C, 6-thioguanine, etoposide, dexamethasone, +/− fludarabine, Ara-C, idarubicin; consolidation with high-dose Ara-C and L-asparaginase or HLA-matched sibling BMT) were genotyped for XPD exon 23 polymorphism. Genotype frequencies were compared with published normal control frequencies. In addition, patient outcomes were analyzed according to genotype. Results: Comparison of gene frequencies in AML patients and reported normal controls showed similar frequencies (42.5% AA, 44.2% AC, 13.3% CC in patients vs. 44.2% AA, 45% AC, 10.8% CC in controls; p=0.54). There were no significant difference in OS (p=0.56), EFS (p=0.61), TRM (p=0.5) or relapse rate (p=0.88) between patients with 751AA genotype vs. 751AC/751CC genotypes, in contrast to reports in adult malignancies. Stratification of cases by age at diagnosis, white blood count at diagnosis, AML subtype, or cytogenetics revealed no difference in genotype frequencies. Conclusion: These data, representing the only data in pediatric AML, suggest that XPD genotype does not affect the etiology or outcome of childhood AML.