bluuebunny/crossref_metadata_2025_split huggingface dataset:
The Role of the cAMP-Response Element in the Bcl-2 Promoter in the Regulation of Endogenous Bcl-2 Expression and Apoptosis in Murine B Cells.
Authors: Xiang Hong , Boxer Linda M. 
Published: April 2020

Abstract
We have previously shown in B cell lines that the cAMP-response element (CRE) is a major positive regulatory site in the bcl-2 promoter. This element is not only essential for bcl-2 deregulation in t(14;18) cells, but it is also responsible for the positive regulation of bcl-2 expression during the activation of mature B cells and the rescue of immature B cells from calcium-dependent apoptosis in vitro. However, the role of the CRE in the regulation of endogenous bcl-2 expression in vivo has not been characterized. We used gene targeting to generate knock-in mice in which a mutant CRE site was introduced into the bcl-2 promoter region. The mutant CRE reduced the expression of bcl-2 mRNA in several tissues, including thymus, kidney, lung, liver, brain and heart. The levels of bcl-2 mRNA and protein were also significantly lower in splenic B cells from the knock-in mice. Consistent with these results, the activation of B cells from the knock-in mice by anti-CD 40, lipopolysaccharide (LPS) or anti-IgM was reduced as compared to B cells from wild-type littermates. B cells with the mutant CRE were more susceptible to the induction of apoptosis with several different agents consistent with the decreased expression of bcl-2. Preliminary flow cytometric studies suggest that the number of B cells is decreased in the knock-in mice at 8 weeks of age. Quantitative chromatin immunoprecipitation assays revealed essentially no binding of CREB or ATF-2 and decreased binding of CBP and c-Rel to the mutant CRE site in the bcl-2 promoter. Our previous studies have shown that the CRE site in the bcl-2 promoter is linked to the mediation of signal transduction pathways in B cells, so we investigated the effect of forskolin, a cAMP-elevating agent. We found that treatment of the B cells from the knock-in mice with forskolin led to significantly more cell death than observed with wild-type B cells. Taken together, these findings indicate that the CRE site in the bcl-2 promoter has a functional role in the regulation of endogenous bcl-2 expression and plays an important role in the regulation of apoptosis in B cells.
Gene Expression Signature of Acute Myeloid Leukemia (AML) with T(8;16)(P11;P13) and MYST3-CREBBP Rearrangement: A Microarray Study Validated by Multiple Real-Time PCR.
Authors: Camos Mireia , Esteve Jordi , Colomer Dolors , Jares Pedro , Rozman Maria , Villamor Neus , Costa Dolors , Carrio Ana , Nomdedeu Josep F. , Montserrat Emili , Campo Elias 
Published: April 2020

Abstract
AML with t(8;16)(p11;p13) is an infrequent leukemia subtype with characteristic clinical-biological features. The t(8;16)(p11;p13) translocation leads to the fusion of MYST3 and CREBBP genes, probably resulting in a disturbed transcriptional program of a myelo-monocytic precursor. In this study, the genetic signature of MYST3-CREBBP AML was compared with other well-defined AML subtypes. Genotypic analyses using oligonucleotide U133A arrays (Affymetrix) were performed on RNA of 23 AML patients, including three MYST3-CREBBP cases, PML-RARa (n=3), RUNX1-CBF2T1 (n=3), CBFβ-MYH11 (n=3), t(9;11)/AF9-MLL (n=1), monocytic AML (FAB M4/M5), n=8, and two cases of AML with multilineage dysplasia. Forty-six genes differentially expressed in MYST3-CREBBP cases were analyzed by multiple real-time RT-PCR using low-density arrays in an additional series of 40 patients, which included 7 MYST3-CREBBP cases, 18 AML samples with well characterized rearrangements (PML-RARa, n=3; RUNX1-CBF2T1, n=3; CBFβ-MYH11, n=3, MLL-rearranged AML, n=9), and 15 patients with normal karyotype AML. After unsupervised analysis, MYST3-CREBBP cases clustered together, displaying a distinctive expression signature. The analysis by RT-PCR confirmed the gene expression pattern found in the high-density array study. Thus, overexpressed genes included oncogene RET, several homeobox (HOXA9, HOXA10), genes involved in apoptosis (DAP) and prolactin gene. In contrast, cyclinD2, STAT5A, STAT5B and WT1 were underexpressed. Interestingly, MYST3-CREBBP cases showed up-regulation of a subgroup of genes (HOXA9, MEIS1, AKR7A2, CHD3, FLT3 and APBA2) that were also found overexpressed in MLL-rearranged leukemias. In summary, this study showed the distinctive genetic signature of MYST3-CREBBP AML, which harbours some similarities with MLL-rearranged AML. In addition, the low-density array methodology validated the results of the microarray analysis and allowed to study a larger series of patients, including samples not suitable for microarray analysis.
Biological Differences in Peripheral T-Cell Lymphomas Identified by DNA Microarrays.
Authors: Martinez-Delgado Beatriz , Bahl Manisha , Cuadros Marta , Fernandez Victoria , Benitez Javier , Jaffe Elaine S. , Staudt Louis 
Published: April 2020

Abstract
Peripheral T-cell lymphomas (PTCL) constitute a heterogeneous and aggressive group of tumors whose pathogenic alterations remain largely unknown. They show great morphologic, immunophenotypic and clinical differences. Expression profiling has been demonstrated to be a useful tool for the molecular classification of tumours. We used DNA microarrays (Affymetrics, Lymph_DX) to look for molecular differences in peripheral T-cell lymphomas.
We analysed the expression of 19 pathologically confirmed PTCL (11 PTCL, unspecified (u) and 8 angioimmunoblastic, AILT). A time-course of stimulation with anti-CD3 and anti-CD28 was performed in CD4+ and CD8+ isolated peripheral blood lymphocytes, and the gene expression of tumors was compared with resting and stimulated normal T-cells. Cases of classical Hodgkin’s lymphoma, with which PTCL may be confused, were included.
Gene expression of PTCL revealed several clusters of genes with biological significance showing differential expression within the tumors. We defined a “Cytotoxic Response” cluster characterized by the expression of cytotoxic molecules PRF1 and GZMB and GZMH, a “B-cell-related genes” cluster which included B-cell specific genes PAX5 or CD19, and an interesting “Resting-cells genes” cluster. These three clusters were negatively correlated with a “Proliferation” signature. On the other hand, the “Proliferation” signature significantly correlated with the expression of TNFRSF8 (CD30) (r=0.63, p=0.003) and the expression of CD3G (r=0.51, p=0.022). Other interesting genes were also found associated specifically to the proliferation of PTCL.
Comparison between PTCLu and AILT showed differences in the expression of CD21 and other genes reflecting the higher presence of dendritic cells in AILT, and also revealed differences in expression of relevant chemokines, such us CXCL5, CXCL13 and CCL19.
Intriguingly, compared with normal CD4 and CD8 T-cells, PTCL showed lower expression of most of the typical genes associated with T-cell biology, suggesting that PTCL often lose the common genetic program of T-cells. PTCL expression profiles are difficult to interpret due to the significant proportion of other infiltrating cells accompanying the tumor. Our results showed that microarrays are a helpful tool to dissect the PTCL expression profile, identifying those genes expressed by infiltrating cells and those expressed by tumor cells.
Antigen-Specific T Lymphocyte Function Following Unrelated Cord Blood Transplantation (UCBT).
Authors: Parkman Robertson , Cohen Geoff , Carter Shelley L. , Weinberg Kenneth I. , Masinsin Bernadette , Guinan Eva C. , Kurtzberg Joanne , Wagner John E. , Kernan Nancy A. 
Published: April 2020

Abstract
The T lymphocytes contained in cord blood are naïve and do not have antigen-specific function. Since the antigen-specific T lymphocytes contained in other hematopoietic stem cell (HSC) source may contribute to protective cellular immunity following transplantation, it has been hypothesized that the recipients of cord blood transplantation (CBT) might be at increased risk of opportunistic infections. The development of antigen-specific T lymphocyte function was measured in 153 recipients of unrelated cord blood transplants (UCBT) by determining antigen-specific T lymphocyte proliferation to the herpes viruses (CMV, HSC, VZV) for 3 years following UCBT. The first antigen-specific response was detected 29 days following transplantation to HSV. Positive T lymphocyte proliferative responses were detected in 66 recipients: 40 to VZV, 36 to HSV and 22 to CMV. Recipients with and without documented herpes infection/reactivation were equally likely to develop positive proliferative responses. The development of antigen-specific T lymphocyte function following UCBT documents successful immune reconstitution since the antigen-specific T lymphocytes are derived from naïve T lymphocytes or HSC, but not donor-derived antigen-specific T lymphocytes. The assessment of antigen-specific function following CBT is a model for the evaluation of post-transplant immune reconstitution without the compounding influence of donor derived antigen-specific function.
Synergy between Nonmyeloablative Doses of Intravenous Busulfan and Post-Transplantation Cyclophosphamide for Induction of Tolerance to MHC-Compatible Stem Cell Allografts.
Authors: Zhou Xiaotao , Wang Jie , Levy Moshe Y. , Symons Heather , Fuchs Ephraim J. 
Published: April 2020

Abstract
The goal of conditioning for nonmyeloablative allogeneic stem cell transplantation, or NST, is to permit the stable engraftment of donor cells with a minimum of toxicity to the patient. We have previously developed a minimal conditioning regimen, consisting of low dose total body irradiation (TBI; 100–200 cGy) and post-transplantation cyclophosphamide (Cy), that permits the induction of stable mixed hematopoietic chimerism following the transplantation of modest doses of MHC-identical allogeneic stem cells in mice. The goal of the current study was to determine whether intravenous (IV) busulfan (Busulfex; ESP Pharma, Edison, NJ), an agent with myelotoxic but minimal immunosuppressive activity, could be used in place of TBI in this regimen and still promote allograft tolerance. TBI was more immunosuppressive than Busulfex, because engraftment of allogeneic stem cells could be achieved in mice conditioned with 700 cGy TBI alone but not in mice conditioned with an equivalently myelotoxic dose of 60 mg/kg Busulfex IV. However, when post-transplantation Cy was added, stable mixed hematopoietic chimerism was achieved in mice conditioned with doses as low as 100 cGy TBI or 20 mg/kg Busulfex. Busulfex and post-transplantation Cy were both required for the induction of allograft tolerance, because graft rejection occurred in transplanted mice treated with either agent alone. The capacity of Busulfex to promote allograft tolerance in this model could not be explained by effects of the drug on the host immune system, because the graft rejection response was not restored in Busulfex-conditioned mice reconstituted with mature host lymphocytes at the time of transplantation. These results demonstrate that Busulfex effectively substitutes for TBI in promoting allograft tolerance, and suggests that the creation of marrow “space” promotes alloengraftment in conditions of suboptimal host immunosuppression.
A Non-Human Primate Model To Study Anti-HIV Gene Therapy Strategies.
Authors: Trobridge Grant D. , Beagles Karen , Beard Brian , Li Ming-Jie , Yee Jiing-Kuan , Rossi John , Hu Shiu-lok , Kiem Hans-Peter 
Published: April 2020

Abstract
AIDS remains a significant health problem worldwide despite the advent of highly active antiretroviral therapy (HAART). Although substantial efforts have been made to develop a vaccine there is still no cure and alternative strategies are needed to treat HIV infection and to control its spread. Our goal is to evaluate lenti and foamy retroviral vectors that inhibit HIV replication by RNA interference (RNAi) in a non-human primate SHIV model to develop a hematopoietic stem cell (HSC) gene therapy for AIDS. SHIV is a chimeric virus comprised of an SIV genome that contains the tat, rev and env genes of HIV and infects both T lymphocytes and macrophages. Infection of non-human primates with SHIV results in significant decreases in CD4+ T cells as early as 4 weeks post infection, and is currently the best large animal model available to test gene therapy strategies for AIDS. We are developing methylguanine-DNA-methyltransferase (MGMT) selection strategies for HSCs in the primate model to allow for high level marking with vectors containing anti-SHIV/HIV transgenes. We have obtained marking levels over 90% in granulocytes and over 30% in lymphocytes. To determine the effectiveness of an anti tat/rev shRNA to inhibit SHIV in vitro, a human T cell/B cell hybrid cell line (CEMx174) was transduced with a lentiviral vector expressing a short-hairpin RNA (shRNA) targeted to both HIV tat and rev sequences that also contained either a GFP reporter gene or a MGMT(G156A) resistance gene. Polyclonal populations of CEMx174 cells transduced with the GFP and MGMT(G156A) vectors were challenged with a 2.15x103 TCID50 dose of SHIV. Expression of both tat and rev transcripts was reduced 88% and 97% respectively in these cultures as measured by real-time PCR and replication of SHIV was inhibited as evidenced by inhibition of p27 production. Although others reported a block to transduction of M. mulatta CD34+ cells with an HIV-based lentiviral vector, we observed efficient transduction rates (~45%) of M. nemestrina CD34+ cells, comparable to transduction rates observed in human CD34+ cells (~55%). Thus M. nemestrina monkeys provide a powerful model to test lenti and foamy virus mediated anti-HIV gene therapy strategies.
Risk of Relapse (REL) after Umbilical Cord Blood Transplantation (UCBT) in Patients with Acute Leukemia: Marked Reduction in Recipients of Two Units.
Authors: Verneris Michael R. , Brunstein Claudio , DeFor Todd E. , Barker Juliet , Weisdorf Daniel J. , Blazar Bruce R. , Miller Jeffrey S. , Wagner John E. 
Published: April 2020

Abstract
Relapse remains a major obstacle that limits the success of hematopoietic stem cell transplantation. Therefore, we analyzed the outcome of patients with acute leukemia transplanted with myeloablative conditioning at a single institution who had a minimum follow-up of 1 year to determine if there were any unique risk factors associated with relapse after UCBT. 96 consecutive patients were evaluated, 39 were &gt;18 years of age, 50 were male; 46 were CMV seropositive; 50 had acute lymphoblastic leukemia (11 CR1, 27 CR2, 9 CR3+, 4 relapse), and 46 had acute myeloid leukemia (11 CR1, 20 CR2, 3 CR3+, 11 relapse). Patients received two different conditioning regimens. Regimen A consisted of cyclophosphamide 120 mg/kg, TBI 1320–1375 cGy, and ATG, followed by cyclosporine A (CSA)/methylprednisolone immunosuppression (n=53). Regimen B consisted of cyclophosphamide 120 mg/kg, fludarabine 75 mg/m2, TBI 1320–1375 cGy pre-transplant and CSA/mycophenolate mofetil immunosuppression (n=43). Patients received either one (n=67) or two (n=29) 4–6/6 HLA-matched UCB units, so that the total MNC count was &gt;2.5 x 107/kg. Accordingly, the demographics for recipients of one or two UCB units were similar except for older age (median age 24 yrs. [range 13–42] vs. 8 yrs. [range 1–52 yrs], p&lt;0.01) and greater weight (median wt. 70 kg [range 48–120 kg] vs. 32 kg [range 10–108 kg], p&lt;0.01) for recipients of two UCB units. Potential risk factors for relapse that were evaluated included: age, gender, recipient CMV sero-status, diagnosis, disease risk, HLA disparity, regimen (A vs. B), and TNC (or CD34+) dose of the unit responsible for sustained engraftment. Notably, in Cox regression analysis two factors were associated with lower relapse risk: disease risk (CR1/CR2 vs CR3+/REL [RR 0.32, p=0.02]) and transplantation of two UCB units (RR 0.3, p=0.03). Importantly, the diagnosis, UCB graft cell dose, and presence of acute GVHD had no demonstrable impact. All recipients of regimen A received a single UCB unit (relapse was 28% (95% CI, 15–41%)), thus interactions between regimen A and number of UCB units could not be assessed. For patients who received regimen B and were in CR1/2, there was a significantly lower risk of relapse for recipients of two UCB units compared to one unit (11% (95% CI, 0–25%) vs. 54% (95% CI, 23–85%), p&lt;0.01). No significant difference in relapse risk could be discerned for patients with advanced disease (CR3-REL) when comparing single vs double UCBT (p=0.48). This report is the first to suggest that the use of double unit UCB grafts may be associated with a reduced risk of relapse in patients with acute leukemia. Larger studies are needed to confirm this clinical experience and to investigate the potential mechanisms by which double unit grafts could mediate protection against relapse.
Gene Therapy for Tolerance to Factor VIII Induced with B Cells and Bone Marrow Transduced with FVIII Domains.
Authors: Scott David W. , Lei Tie Chi 
Published: April 2020

Abstract
Tolerance induction for hemophilia inhibitor formation has recently been achieved by gene therapy using B cells transduced with a fusion protein of C2 and A2 domains of factor VIII with an IgG heavy chain. We have shown that inhibitor titers can be reduced over 90% by this approach in both naïve and highly immunized FVIII knockout mice (FVIII−/−). This process requires the presence of CD25+ regulatory cells for induction, and is stable for at least three months (Lei, T-C. and Scott, D.W. 2005. Induction of tolerance to fVIII inhibitors by gene therapy with immunodominant A2 and C2 domains presented by B-cells as Ig fusion proteins. Blood, 105: 4865–4870). Herein, we wished to establish whether retroviral transduction and integration of the fusion Ig was necessary for tolerance. In addition, we also tested whether transduction of bone marrow could lead to long-term tolerance. B cells (resting and LPS blasts) from FVIII sufficient C57Bl/6 and FVIII−/− mice were pulsed with FVIII and injected into naïve FVIII−/− recipients that were subsequently challenged with therapeutic doses of FVIII in an immunogenic protocol. We reasoned that C57Bl/6 mice were FVIII sufficient and could display FVIII processed epitopes within their MHC class II. Moreover, pulsing with FVIII should lead to presentation of processed epitopes transiently in class II MHC. We found that specific tolerance to FVIII was only induced with B cells transduced with a combination of A2-IgG and C2-IgG, indicating that transient expression of FVIII epitopes and presentation by B cells is not sufficient for tolerance induction.
Resting B-cells pulsed with fVIII in vitro are not tolerogenic Resting B-cells pulsed with fVIII in vitro are not tolerogenic
In further studies, we examined whether tolerance could be induced by bone marrow transplantation of C2-Ig and A2-Ig transduced hematopoietic stem cells under a non-myeloablative regimen with busulfan in immunized FVIII−/− mice. This protocol could lead to long-term tolerance if pluripotential stem cells were transduced and engrafted. Our results indicate that tolerance to FVIII domains could be achieved even though the level of expression of GFP positive cells in peripheral blood of busulfan-treated mice was less than 1% (compared to &gt;50% positive cells in lethally irradiated recipients of similarly transduced bone marrow cells). These results have implications for long-term therapy of inhibitor formation in hemophilia A. (Supported by NIH grants HL061883, AI035622 and a Lab Grant from the National Hemophilia Foundation.)
E2f-2 Regulates Caspase-3 Expression and Mitotic Checkpoint Control during End-Stage Erythroid Maturation.
Authors: Dirlam Alexandra , Spike Benjamin T. , Macleod Kay F. 
Published: April 2020

Abstract
E2f-2 is a member of the E2F superfamily of transcription factors that plays multiple roles in regulating cell cycle progression, cell cycle exit and checkpoint control [1]. While over-expression studies have ascribed a role for E2f-2 as a transcriptional activator of cell cycle genes, our loss of function studies performed in vivo have revealed a novel role for E2f-2 in repressing cellular proliferation and mediating differentiation. Our expression analyses demonstrate that E2f-2 is up-regulated in hematopoietic tissues, and is most strongly expressed in differentiating erythroblasts. Furthermore, E2f-2 is the major pRb-associated E2F in hematopoietic tissues, suggesting E2f-2 can function as a co-repressor of cell cycle genes during end-stage differentiation. Compound Rb−/−;E2f2−/− mice were generated to understand how deregulated E2f-2 activity contributes to the erythroid defects in Rb null mice. Loss of E2f-2 restored end-stage erythroid differentiation and enucleation to Rb null erythroblasts and extended survival of Rb null mice. Loss of E2f-2 was sufficient to bypass the mitotic arrest observed in Rb null erythroblasts [2] and was associated with reduced expression of known E2f target genes involved in M phase checkpoint control, such as Aurora B and Survivin. In addition, we observed a significant increase in caspase-3 expression in E2f-2 null erythroblasts and demonstrate, for the first time, direct regulation of caspase-3 expression by E2f-2. Given known roles for caspases in promoting erythroid differentiation and enucleation, we have over-expressed caspase-3 in Rb null erythroblasts and are currently analyzing its effects on cell cycle and maturation. Thus, our work identifies caspase-3 as a key downstream target of E2f-2 during end-stage erythroid differentiation whose expression may alleviate enucleation and mitotic defects in Rb null erythroblasts. Future work aims to understand which E2f-2 target genes ensure mitotic checkpoint control, and the molecular mechanism by which caspase-3 coordinates cell cycle control with erythroid differentiation.