bluuebunny/crossref_metadata_2025_split huggingface dataset:
AHSP Knockdown in Human Erythroleukemia Cell Line and Human Hematopoietic Stem Cells Results in Alpha Hemoglobin Chain Precipitation, Decreased Hemoglobin Levels and Increased Cell Death.
Authors: Pinho Flavia O. , Albuquerque Dulcineia M. , Saad Sara T.O , Costa Fernando F. 
Published: April 2020

Abstract
Alpha Hemoglobin Stabilizing Protein (AHSP) binds alpha hemoglobin chain (αHb), avoiding its precipitation and its pro-oxidant activity. In the presence of beta hemoglobin chain (βHb), the αHb-AHSP complex is dismembered and βHb displaces AHSP to generate the quaternary structure of hemoglobin. These data have been obtained in vitro and in mouse cells, but strongly suggest the importance of AHSP for normal hemoglobin synthesis in humans. To the best of our knowledge, the relationship between hemoglobin formation and alterations in AHSP expression has not yet been described in human red cells. Hence, to investigate the consequences of a reduced AHSP synthesis in human red cells, we established the RNA interference-mediated knockdown of AHSP expression in human erythroleukemia cell line (K562 cells) and human hematopoietic stem cells (CD34+ cells) induced to erythroid differentiation, and analyzed the consequent cellular and molecular aspects of AHSP knockdown in these cells. shRNA expression vectors, aimed at the AHSP mRNA target sequence, were cloned and transfected into K562 and CD34+ cells using a non-liposomal lipid reagent. Following transfection, K562 cells that stably expressed AHSP-shRNA and CD34+ cells that transiently expressed AHSP-shRNA were selected. K562 and CD34+ cells were stimulated to erythroid differentiation by hemin and erythropoietin (EPO) respectively. The cells were examined in terms of gene expression using quantitative real-time PCR; production of reactive oxygen species (ROS), apoptosis and hemoglobin production through flow cytometry assays; and immunofluorescence assays for globin chains. AHSP-shRNA hemin-induced K562 cells and AHSP-shRNA EPO-induced CD34+ cells presented 71% and 75% decreases in AHSP expression levels, respectively. The RNAi-mediated knockdown of AHSP expression resulted in a considerable αHb precipitation, as well as in a significant decrease in fetal hemoglobin formation. In addition, AHSP-knockdown cells demonstrated an increased ROS production and increased rate of apoptosis. These findings strengthen the hypothesis that AHSP stabilizes the alpha hemoglobin chain, avoiding its precipitation and its ability to generate ROS which implicate in cell death. Moreover, data indicate that AHSP may be highly significant for human hemoglobin formation and suggest that AHSP is a key chaperone protein during human erythropoiesis.
GVHD Abrogates T Cell Responses to Dendritic Cell Vaccines but Not Vaccine-Induced Proliferation.
Authors: Capitini Christian M. , Guimond Martin , Mackall Crystal L. , Fry Terry J. 
Published: April 2020

Abstract
BACKGROUND: Allogeneic bone marrow transplant (BMT) is a potent form of immunotherapy against hematopoietic and potentially solid tumors, but relapse of malignancy and Graft versus Host Disease (GVHD) remain a major cause of morbidity and mortality. Dendritic cell (DC) vaccines represent a potential strategy to enhance the potency and efficacy of the Graft versus Tumor (GVT) effect without exacerbating GVHD, but little is known regarding the impact of the allogeneic BMT environment on vaccine responses. Previous work in our lab has shown that DC can effectively expand tumor-antigen specific T cells following minor-mismatched allogeneic transplantation, resulting in delay of tumor growth. However, GVHD completely abrogates these responses. We hypothesized that the mechanism behind the loss of DC vaccine responses was through diminished T cell proliferation in the setting of GVHD.
METHODS: We established a minor histocompatibility antigen mismatched BMT model by transplanting B6 x C3H.SW (F1) thymectomized mice with either CD45.1+ B6 (allogeneic) or CD45.1+/45.2+ F1 (syngeneic) T cell-depleted bone marrow on day 0, followed by a donor lymphocyte infusion at day 14 to induce GVHD. On day 28, we adoptively transferred transgenic CD8+ (CD45.2, V beta 8.3+) or CD4+ (CD45.2, V beta 6+) T cells that only recognize components of the HY antigen to avoid cross-reactivity with allogeneic antigens. The T cells were labeled with CFSE prior to transfer to explore the impact of vaccine-induced proliferation in the presence or absence of GVHD.
RESULTS: In the allogeneic BMT setting, both CD8+ and CD4+ HY-transgenic T cells given with a DC vaccine begin proliferating 5 days (CD8+ 40%, CD4+ 65% divided) after adoptive transfer. The syngeneic BMT vaccinated control mice show similar kinetics (CD8+ 38%, CD4+ 65% divided), suggesting GVHD does not decrease T cell proliferation. Interestingly, although HY transgenic T cells do not normally undergo homeostatic peripheral expansion (HPE) during lymphopenia, the CD4+ T cells proliferate in lymph nodes even in the absence of a vaccine (50% divided), but this was not observed in the respective syngeneic BMT unvaccinated control mice (0% divided).
CONCLUSIONS: DC vaccines represent a strategy to improve the efficacy of the GVT response following allogeneic BMT, however, GVHD can abrogate DC vaccine responses. The negative impact of GVHD on vaccine responses does not appear to result from impaired DC vaccine-driven proliferation. Indeed, even in the absence of a vaccine, non-alloreactive CD4+ T cells proliferate via a mechanism that appears to be distinct from HPE. This data provides important insights towards optimizing antigen-specific vaccines administered in the allogeneic BMT setting.
Immunological and Clinical Responses in Patients with Acute Myeloid Leukemia (AML), Myelodysplastic Syndrome (MDS), Multiple Myeloma (MM) and Chronic Lymphocytic Leukemia (CLL) after RHAMM-R3 Peptide Vaccination.
Authors: Greiner Jochen , Schmitt Anita , Giannopoulos Krzysztof , Chen Jinfei , Goetz Marlies , Rojewski Markus , Ritter Gerd , Gnjatic Sascha , Guillaume Philippe , Ringhoffer Mark , Bommer Martin , Schlenk Richard F. , Liebisch Peter , Bunjes Donald , Shiku Hiroshi , Dohner Hartmut , Schmitt Michael 
Published: April 2020

Abstract
We initiated a phase I/II R3 peptide vaccination for patients with acute myeloid leukemia (AML), myelodysplastic syndrome (MDS), multiple myeloma (MM) and chronic lymphocytic leukemia (CLL) overexpressing the receptor for hyaluronic acid mediated motility (RHAMM). RHAMM is a leukemia associated antigen (LAA) that is strongly expressed in several hematological malignancies and induces humoral and cellular immune responses. In this study, patients with AML, MDS, MM or CLL were included with RHAMM expression but with a limited tumor load or a minimal residual disease. To date, 25 patients were enrolled. The first 12 patients were vaccinated with 300 mcg and further patients with 1000 mcg R3 peptide emulsified with the incomplete Freund’s adjuvant. The vaccine was given four times at a biweekly interval and GM-CSF was added for five days each vaccination. Only mild drug-related adverse events were observed such as erythema and induration of the skin at the site of injection. Immunological analysis were performed using enzyme linked immunospot (ELISpot) assays for Interferon gamma and Granzyme B, tetramer staining and chromium release assays. We detected specific immune responses in 70% of patients. In most patients, we found an increase of CD8+/HLA-A2/RHAMM R3 tetramer+/CD45RA+/CCR7−/CD27−/CD28− effector T cells in flow cytometry in accordance with an increase of R3-specific CD8+ T cells in ELISpot assays. In chromium release assays, a specific lysis of RHAMM-positive leukemic blasts was shown. Moreover, we measured IL-2 and IL-10 levels in sera before and after vaccination. While IL-10 levels remained at a rather low level, we detected an increase of IL-2 in four of ten patients who showed also clinical responses. RHAMM transcripts in bone marrow and peripheral blood were quantified by real-time RT-PCR. Responding patients showed a decrease of RHAMM after vaccination. We detected positive clinical effects in several patients with myeloid disorders showing a reduction of blasts in the bone marrow. One MDS patient did not need any longer erythrocyte transfusions. Two patients with MM showed a reduction of free light chain serum levels. Taken together, RHAMM-R3 peptide vaccination induced both immunological and clinical responses. Therefore, RHAMM constitutes a promising structure for further targeted immunotherapies in patients with different hematological malignancies.
Human Neutrophil Elastase Stimulating CD4+ and CD8+ T Cells Is a Potential Protein Vaccine for Leukemia Patients with Diverse HLA Types.
Authors: Le Quan , Melenhorst J. Joseph , Hensel Nancy , Eniafe Rhoda , Barrett A. John 
Published: April 2020

Abstract
The detection of naturally occurring T cells responding to proteins overexpressed in myeloid leukemia suggests that vaccines could boost the immune response to leukemia and achieve disease control. Human neutrophil elastase (HNE) is a myeloid-restricted protein highly expressed in myeloid leukemia cells. HNE protein can induce both CD4+ and CD8+ T cell responses in healthy individuals including HLA-A*0201 negative individuals. To develop HNE protein vaccine, and to identify major histocompatibility complex (MHC) class II restricted or HLA-A*0201 negative MHC class I restricted epitopes in HNE, we stimulated peripherial blood mononuclear cells (PBMC) from 6 myeloid leukemia pre-transplant patients with purified full length HNE protein. HNE protein induced a response in CD4+ and CD8+ T cells from 4 patients with acute myeloid leukemia (AML), as measured by IFN-γ protein expression in flow cytometric assay. However, no response was detected in CD4+ and CD8+ T cells from 2 pre-transplant patients with chronic myeloid leukemia (CML). Responses of CD4+ and CD8+ T cells to HNE protein stimulation were enhanced by HNE expressing antigen-presenting T cells (T-APC), as measured by flow cytometric assay. HNE protein induced significant IFN-γ expression in CD4+ and CD8+ T cells at 540X and 10X above background in an (HLA-A11, 31) AML patient (patient #1 in the figure), and at 6X and 3.5X above background in an (HLA-A*0205, 03*) AML patient (patient #2 in the figure), as measured by quantitative Real-Time RT-PCR assay (QRT-PCR). HNE protein also induced significant IL-2 production in CD4+ and CD8+ T cells at 7.7X and 6.1X above background in patient #1. Finally, we showed by QRT-PCR that the T cell responses to HNE protein stimulation were associated with the levels of HNE gene transcripts in the PBMC. Thus we found that full-length HNE protein stimulated both CD4+ and CD8+ T cell responses in HLA-A*0201 negative patients with AML. These results provide the rationale for using the full-length HNE protein to vaccinate patients with myeloid leukemias pre- or post-stem cell transplantation to improve graft-versus-leukemia effects.
Figure Figure
Determining the Mechanism of Transformation of Follicular Lymphoma into Diffuse Large B Cell Lymphoma.
Authors: Carlotti Emanuela , Wrench David , Iqbal Sameena , O’Shea Derville , Davies Andrew , Virappane Priya , Hart Jason , Lai Raymond , Gribben John G. , Lister Andrew T. , Fitzgibbon Jude 
Published: April 2020

Abstract
The molecular mechanisms involved in histological transformation of follicular lymphoma (FL) to diffuse large B-cell lymphoma (DLBCL) are largely unknown. Here we investigated the clonal relationship in FL and DLBCL in 17 cases of paired FL/DLBCL samples and also in a bone marrow transplant (BMT) model. The aim of this study was to determine the cell of origin of the transformed DLBCL (t-DLBCL) and whether this cell always arises as a clonal evolution from the major FL clone. The immunoglobulin variable heavy chain (IgVH) was amplified by PCR using family specific primers, the major clone identified by homo/heteroduplex and genealogical trees generated. As expected 70% of FL/DLBCL cases used VH3, 24% VH4 and 6% VH5. The median degree of somatic hypermutation (SHM) was 15% (range 4 to 43%). In 3/17 cases the pattern of SHM was identical between FL and DLBCL. In 6/17 cases a clear pattern of direct evolution was evident. Surprisingly for 8/17 cases the pattern of SHM was compatible with the FL and DLBCL arising from a precursor cell rather than direct evolution. Clone specific primers and probes were designed to investigate whether there was evidence of the t-DLBCL in the earlier biopsy. Quantification by RQ-PCR demonstrated that in most cases the transformed clonotype was already present at levels between 10-4 and 10-2. We further examined an interesting case in which both the father (donor) and the son (recipient) developed FL and t-DLBCL 3 and 10 years after transplantation respectively. Sequencing confirmed the presence of an identical t(14;18) translocation in donor and recipient and that both cases used IGHV3-48*03. Analysis of the genealogical trees of SHM in these cases was also consistent with the evolution of t-DLBCL from a common precursor cell rather than direct evolution. These data are consistent with two distinct patterns of transformation of FL to t-DLBCL. About 50% of our cases had evidence of direct evolution while in the remaining 50% the pattern is more suggestive of the FL and t-DLBCL arising from a common precursor cell. The long latency period after BMT is in agreement with the requirement for additional genetic events within the t(14;18) bearing post germinal centre precursor lymphoma cell for the development of these diseases. The finding of the t-DLBCL clonotype in the FL sample before transformation suggests that the transforming event may occur in a minor subclone of the disease or earlier in a lymphoma precursor cell. Ongoing experiments seek to investigate the genetic events arising in these cells that result in transformation.
Apoptosis Inducing Factor Acts as a Survival Factor in Acute Myelogenous Leukemia through Mitochondrial Complex 1.
Authors: Wang Rui-yu , Samudio Ismael J. , McQueen Teresa , Schober Wendy D. , Ruvolo Vivian R. , Kornblau Steven , Konopleva Marina , Andreeff Michael 
Published: April 2020

Abstract
Apoptosis inducing factor (AIF) is a mitochondrial flavoprotein that is released into the cytoplasm to form an active DNase and then translocates into the nucleus to induce DNA fragmentation when cells undergo apoptosis. AIF also acts as an essential NADPH oxidase, a function which is distinct from its proapoptotic role. We have shown previously that AIF expression level increased with increasing blast counts in primary acute myelogenous leukemia (AML) patient samples. Here we report that AIF expression levels are increased in primary CD34+ AML cells compared with CD34− cells. To investigate the role of AIF in primary AML, bone marrow or peripheral blood samples from AML patients (n=8) with CD34+ or CD34− cell populations were examined for changes in reactive oxygen species (ROS), mitochondrial membrane potential (ΔψM), apoptosis and chemo-sensitivity in the CD34+ and CD34− compartments after treatment with the mitochondrial complex 1 inhibitor rotenone. Similar experiments were performed following knockdown of AIF with lentiviral shRNA directed against AIF in K562 cells.
Results demonstrate:
significantly increased apoptosis rate (39.2% Annexin V+ cells in CD34+ vs. 14.1% in CD34− cells following 2 uM rotenone, p=0.05), and decreased ΔψM in CD34+ cells (44.5% ΔψM cells in CD34+ vs. 78.2% in CD34− cells, p=0.01). MCL-1 expression levels were significantly decreased in CD34+ but not in CD34− cells (p=0.007). The apoptotic effect of rotenone in CD34+ cells was potentiated by AraC (1uM). Profound inhibition of O2 consumption in AIF knockdown K562 cells (−5.2±0.3) compared with K562-neo (neo transfected K562, −13.1±0.3) cells (p=0.001).
Rotenone treated AIF knockdown K562 cells and K562-neo cells revealed that decreased AIF expression led to higher ΔψM and higher superoxide generation (p=0.0002). In contrast, total mitochondrial mass (NAO) decreased slightly in K562KD cells. Data suggested that
AIF contributes to leukemia cell survival by modulating mitochondrial metabolism, independent of its proapoptotic activity, which is consistent with recent reports demonstrating its importance in mitochondrial complex 1 activity. Decreased AIF levels are associated with increased generation of superoxide anion, suggesting that AIF critically regulates mitochondrial function in AML cells. Rotenone selectively decreases AIF and induces apoptosis in CD34+ cells in AML. AIF functions as a survival factor in AML strategies targeting AIF expression or function may be beneficial in the treatment of myeloid leukemia.
The RARa-PLZF Oncogenic Protein Inhibits C/EBPa Function in Myeloid Cells.
Authors: Tremblay Mathieu , Girard Nathalie , Haman Andre , Grondin Benoit , Bouchard Nathalie , Labrecque Jean , Chen Bing , Chen Zhu , Chen Sai-Juan , Hoang Trang 
Published: April 2020

Abstract
In acute promyelocytic leukemia (APL), the variant t(15;17) translocation is responsive to differentiation therapy with retinoic acid (RA) while the t(11;17) APL is a more aggressive disease with poor prognosis. The latter produces two fusion proteins, PLZF-RARa and RARa-PLZF, and both proteins are required for leukemogenesis. To define the role of RARa-PLZF, we ectopically expressed the fusion gene in 32D cells and in primary bone marrow cells. First, our results show that RARa-PLZF inhibits myeloid gene expression, specifically CEBPa targets, which fulfill important function in cell survival and differentiation along the granulocytic lineage. Second, we found that repression by RARa-PLZF is dependent on the binding of C/EBPa to its cognate sequence in the promoter of CEBPa target gene, GCSFR. Third, we confirmed by chromatin immuprecipitation that RARa-PLZF associate with C/EBPa on DNA. Fourth, we showed that as PLZF, RARa-PLZF interact directly with HDAC1 and that this interaction causes a deacetylation of histone H3 at the promoter. This inhibition is reversed by treatment with histone deacetylase inhibitor (TSA) both in vitro and in vivo. Thus, this repression is dependent on direct interaction of RP with C/EBPa and recruitment of HDAC1, causing histone deacetylation at C/EBPa target loci. Finally, our data indicate that C/EBPa activity is severely impaired in leukemic cells from patients with t(11;17) APL, as compared to the t(15;17) APL, which is more amenable to therapy. In summary, our study indicates that the oncogene RARa-PLZF inhibits C/EBPa function through direct protein-protein interaction, and thus contributes to leukemogenesis in t(11;17) APL.
The Presence of Central Nervous System Disease at Diagnosis in Pediatric Acute Myeloid Leukemia Does Not Affect Outcome.
Authors: Johnston Donna L. , Alonzo Todd A. , Gerbing Robert B. , Lange Beverly J. , Woods William G. 
Published: April 2020

Abstract
Introduction: The presence of disease in the central nervous system (CNS) in pediatric acute myeloid leukemia (AML) is often thought to confer a worse prognosis. This study examined the outcome of children with AML who had CNS disease at diagnosis.
Methods: Patients enrolled on Children’s Oncology Group protocols 2891 (N=836)and 2961 (N=901) being treated for de novo AML were classified for the presence of CNS disease at diagnosis as: CNS1 (less than 5 WBC in the CSF with no blasts), CNS2 (less than 5 WBC in the CSF with blasts) or CNS3 (5 or more WBC in the CSF with blasts). CNS disease at diagnosis was then analysed for survival, patient characteristics and outcome using univariate analysis. Patients on these protocols were treated with intrathecal chemotherapy and not radiation therapy for their CNS disease.
Results: For both AML protocols, the overall survival and event free survival were highest in patients with CNS2 disease but this was not significantly different from the survival of the CNS1 and CNS3 patients.
Overall survival from 2891 study entry for all de novo patients Overall survival from 2891 study entry for all de novo patients Overall survival from 2961 study entry for all de novo patients Overall survival from 2961 study entry for all de novo patients
Patients with CNS 2 and 3 disease were of significantly lower median age than CNS1 patients (p=0.001 in 2891 and p=0.005 in 2961). There was a significantly higher WBC count at diagnosis (p=0.001 in both studies), presence of hepatomegaly (p=0.001 in both studies), presence of splenomegaly (p=0.003 in 2891 and p=0.001 in 2961), FAB M2 morphology (p=0.001 in both studies) and FAB M4 morphology (p=0.001 in both studies) in patients with CNS2 and 3 disease compared to CNS 1 patients. Also, CNS2 and 3 patients had a significantly higher incidence of abnormal chromosome 16 (p=0.002 in 2891 and p=0.001 in 2961). In protocol 2891 significantly more CNS2 and 3 patients had hyperdiploid cytogenetics compared to CNS1 patients (p=0.016), and in protocol 2961 there were significantly more patients with t(8;21) in the CNS1 group (p=0.007). In terms of overall outcome, there was a significantly higher incidence of isolated CNS relapse in patients with CNS3 disease at diagnosis in the 2891 protocol (p=0.001), and in patients with both CNS2 and 3 disease at diagnosis in the 2961 protocol (p=0.001).
Conclusion: Patients with CNS disease at diagnosis have similar survival to those without CNS disease, although they have an increased incidence of isolated CNS relapse. More aggressive CNS directed therapy may be warranted in this patient population.
High Frequency of 1p36.32 Deletion or Loss of Heterozygosity in Follicular Lymphoma (FL).
Authors: Cheung K.-John , Telenius Adele , Lai Betty , Johnson Nathalie , Relander Thomas , Steidl Christian , Baross Agnes , Qian Hong , Schein Jacquie , Marra Marco , Connors Joseph M. , Gascoyne Randy D. , Horsman Douglas E. 
Published: April 2020

Abstract
Background: The initial genetic event in ∼85% of follicular lymphomas (FL), the most common B-cell lymphoma in North America, is the t(14;18)(q32;q21) resulting in over-expression of the anti-apoptotic protein Bcl-2. The secondary events associated with disease progression are not well understood. Alterations affecting the p arm of chromosome 1 are evident by standard karyotype analysis in ∼20% of FL. We have further examined the relationship between 1p deletion and FL using high resolution genomic analyses.
Methods: The prevalence of 1p alterations was investigated in 139 cases of indolent and transformed FL using whole genome tiling path BAC array Comparative Genomic Hybridization (array CGH). Array-based single nucleotide polymorphism analysis was performed on a subset of cases using Affymetrix 500K SNP arrays.
Results: Array CGH identified a minimum region of deletion spanning ∼0.5MB within 1p36.32 in 51 cases (37%). In 38 cases (27%) this loss was exhibited in the transformed sample but not the pre-transformation sample. The majority of cases displayed heterozygous deletion, while two cases showed homozygous deletion. The mechanisms of loss included simple deletions, unbalanced translocations with various partner chromosomes and eleven cases with an unbalanced t(1;1)(p36;q12). The Affymetrix 500 SNP array analyses showed copy neutral loss of heterozygosity or acquired uniparental disomy (aUPD) in three of ten cases that were negative for loss by aCGH. Contained within the 1p36.32 minimally deleted region are only a few candidate genes including tumor necrosis factor receptor superfamily 14 (TNFRS14), which has been implicated in growth inhibition of HT-29 human colon adenocarcinoma cells and induction of Fas-mediated apoptosis in non-Hodgkin’s lymphoma.
Conclusions: Our data indicate that loss of heterozygosity at 1p36.32 through deletion or aUPD constitutes the most common secondary cytogenetic event in FL. LOH at 1p36 may represent an important step in the progression of indolent to transformed FL. Further studies have been initiated to investigate other possible gene inactivation events such as methylation and mutation.