bluuebunny/crossref_metadata_2025_split huggingface dataset:
Inhibition of HIV-1 by DpT-Based Iron Chelators
Authors: Debebe Zufan , Kumar Krishna , Ammosova Tatiana , Niu Xiaomei , Richardson Des R , Gordeuk Victor R. , Nekhai Sergei 
Published: October 2019

Abstract
HIV-1 transcription is activated by HIV-1 Tat protein, which recruits transcriptional co-activators to the HIV-1 promoter. Elongation of HIV-1 transcription is mediated by the interaction of Tat with host cell cycle-dependent kinase 9 (CDK9)/cyclin T1, which phosphorylates the C-terminal domain of RNA polymerase II. Tat itself is phosphorylated by host cell cycle-dependent kinase 2 (CDK2) [1] and inhibition of CDK2 by tridentate iron chelators such as 2-hydroxy-1-naphthylaldehyde isonicotinoyl hydrazone, (311) or ICL670 (deferasirox) inhibits HIV-1 transcription [2]. In addition to the inhibition of CDK2, 311 and ICL670 also prevent association of CDK9 with cyclin T1 [2], which could lead to inhibition of CDK9 activity and also to inhibition of HIV-1 transcription. Recently, a group of novel di-2-pyridylketone thiosemicarbazone (DpT) based tridentate iron chelators were shown to exhibit marked antiproliferative activity in vivo [3]. Here we screened DpT-based and also 2-benzoylpyridine thiosemicarbazone (BpT)-based tridentate iron chelators and identified three chelators, Dp44mT, Bp4eT and Bp4aT, that inhibited HIV-1 transcription but were not cytotoxic as determined by propidium iodide uptake, LDH release and calcein-AM uptake. The inhibition of HIV-1 transcription was observed in CEM HIV-1 LTR-GFP cells infected with Adeno-Tat and in 293T cells transiently transfected with HIV-1-LTR LacZ and Tat-expressing vectors with IC50s in the mid-nanomolar range. These new iron chelators also inhibited HIV-1 replication in CEM and THP-1 cells at 10 mM concentration. Analysis of the molecular mechanism of HIV-1 inhibition revealed that the DpT- and BpT-based iron chelators inhibited the activities of both CDK9/cyclin T1 and CDK2. The CDK9/cyclin T1 complex was disrupted in the cells treated with iron chelators, suggesting a possible mechanism for the inhibition of CDK9. In conclusion, our findings provide further evidence that iron chelators may inhibit HIV-1 transcription by deregulating CDK2 and CDK9. The projected therapeutic index of the selected DpT-based iron chelators was over 103 suggesting their potential usefulness as future anti-retroviral therapeutics.
Impact of Autologous Dendritic Cell Immunotherapy (Arcelis™) on B Cell Subsets in HIV-1-Infected Subjects Receiving Antiretroviral Therapy
Authors: Boulassel Mohamed-Rachid , Yassine-Diab Bader , Healey Don , Nicolette Charles , Sékaly Rafick-Pierre , Routy Jean Pierre 
Published: October 2019

Abstract
We demonstrated the enhancement of CD8-specific responses following the administration of an immune-based therapy consisting of dendritic cells (DC) electroporated with autologous amplified HIV-1 RNA and CD40 ligand (CD40 L) RNA manufactured by the Arcelis™ process in HIV patients receiving antiretroviral therapy (ART). We conducted a sub study on circulating B cell populations to further assess changes induced by this autologous DC therapy as CD40L is a major B cell co-stimulatory factor. To this end, we assessed B cell subset changes in relation to the proliferative capacity of CD4+ and CD8+ T cells response to DC targets containing the 4 HIV-1 antigens (Gag, Vpr, Rev, Nef). The co-expression of CD19, CD38, IgD, CD10, CD23, CD27, CD5, and CD138 were analyzed by multi-parametric flow cytometry to assess circulating B cell subsets such as naïve resting B-cells (Bm1), activated naïve B cells (Bm2), GC founder cells (Bm2’), centroblasts and centrocytes (Bm3 and Bm4), early memory B cells (eBm5), memory B cells (Bm5), IgD memory cells, plasma cells, and B-1 cells. Changes in B cells subsets were analyzed before and after the four intradermal injections of this immunotherapeutic product containing 1.2 × 107 DC. Ten ART treated subjects with undetectable viral load (< 50 copies/ml), median CD4+ count of 440 cells/μl (range: 316–1102), and with a CD4+ nadir > 200 cells/μl were studied. Throughout the study, no significant changes in CD4+ cell count, CD4/CD8 ratio, and no viral blips were noticed. The percentage of total B cells, Bm1, Bm2, Bm2′, eBm5, IgD memory, plasma cells, and B-1 cell subsets did not significantly change. However, a decrease in the percentage of Bm3 and Bm4 cells was found (0.36 [0.06–0.86] versus 0.11 [0.04–0.36]; P=0.05). Conversely, an important increase in the Bm5 cell subset was evidenced (10.4 [1.6–24.2] versus 18.1 [5.1–27.5]; P=0.005) suggesting a proliferation of B memory cells induced by DC immunization. In addition, the multifunctional and polyvalent CD8+ T cell proliferative responses to the 4 HIV genes used in this immunotherapy were noticed in 8 out of 9 subjects available for analysis and characterized by an effector memory phenotype. No CD4+ T cell immune responses were detected, consistent with the endogenous HLA class I loading of the antigens. Collectively, these results indicate that this immunotherapy induces an increase in the B memory cell population in the absence of inducing any clinically apparent autoimmunity along with strong HIV specific multifunctional CD8+ T cell specific immune responses.
Immunomodulatory Derivatives of Thalidomide (IMiD)-Induced Neutropenia Is Associated with PU.1 Downregulation and Myeloid Maturation Arrest
Authors: Pal Rekha , Roodman G. David , Mapara Markus , Mocsinski Lynn , List Alan F , Lentzsch Suzanne 
Published: October 2019

Abstract
Previous clinical trials have shown that immunomodulatory derivatives of thalidomde (IMiDs) including lenalidomide or pomalidomide induce high response rates in patients with relapsed or refractory multiple myeloma. However, the most dose limiting adverse effect of IMiDs is severe neutropenia. This is in contrast to our findings that IMiDs promote a shift in hematopoietic progenitor lineage commitment toward myeloid precursors with a subsequent maturation stop.
In colony forming assays using human CD34+ hematopoietic precursors treated with IMiDs, we observed a highly significant increase in myeloid progenitor formation at the expense of erythroid colonies. Treatment of hematopoietic progenitors with IMiDs showed an upregulation of CD33, a marker for immature myeloid cells. Further, in vitro treatment with IMiDs resulted in downregulation of the transcription factor PU.1. PU.1 gene knock-down models show increased granulopoiesis with impaired neutrophil maturation, resulting in an accumulation of promyelocytes. In accordance with that, Cathepsin G as a promyelocytic marker was highly upregulated under treatment with IMiDs measured by high-density oligonucleotide microarray, quantitative RT-PCR, western blot and ELISA, confirming the maturation arrest of neutrophil granulocytes. To evaluate medullary changes in granulopoiesis in human subjects treated with lenalidomide, we evaluated sequential bone marrows from 6 patients comparing bone marrow features prior to treatment, during treatment, and at the time of grade 4 neutropenia. Marrows at the time of neutropenia showed an accumulation of myeloid precursors (myelocytes, metamyelocytes) supporting impaired myeloid maturation. In 5 out of 6 patients the myeloid:erythroid (M:E) ratio increased with an M:E median of 1.95 at pretreatment and 9.2 at the time of the neutrophil nadir. At the same time bone marrow cellularity was unchanged.
In summary we show that IMiDs downregulate PU.1, a key transcription factor involved in granulocyte differentiation. The loss of PU.1 results in medullary accumulation of immature myeloid precursors, with corresponding neutropenia in the peripheral blood. Further high amounts of azurophilic granules of promyelocytes subsequently increase Cathepsin G and neutrophil elastase 2 resulting in enhanced platelet aggregation and a possible higher risk for venous thromboembolism. Our findings show that IMiDs do not induce neutropenia by marrow suppression but rather by transient block of maturation, which might be overcome by application of G-CSF.
Recombinant Human HPA-1A Antibodies for Treatment of Fetomaternal Alloimmune Thrombocytopenia (FMAIT): Proof of Principle in An in Vivo Murine Model and Human Volunteer Studies
Authors: Ghevaert Cedric , Wilcox David A. , Fang Juan , Armour Kathryn , Clark Mike R , Ouwehand Willem H. , Williamson Lorna M 
Published: October 2019

Abstract
Fetomaternal alloimmune thrombocytopenia (FMAIT) is caused by maternal generation of antibodies specific for paternal platelet antigens and can lead to fetal intracranial hemorrhage. A single nucleotide polymorphism (SNP) in the gene encoding integrin β3 causes a clinically important maternal-paternal antigenic difference; Leu33 generates the human platelet antigen 1a (HPA-1a), whereas Pro33 generates HPA-1b. As a potential treatment to prevent fetal intracranial hemorrhage in HPA-1a alloimmunized pregnancies, we generated an antibody that blocks the binding of maternal HPA-1a–specific antibodies to fetal HPA-1a1b platelets by combining a high-affinity human HPA-1a–specific scFv (B2) with an IgG1 constant region modified to minimize Fcγ receptor–dependent platelet destruction (G1Δnab). B2G1Δnab saturated HPA-1a+ platelets at micromolar concentration and substantially inhibited binding of clinical HPA-1a–specific sera to HPA-1a+ platelets. The response of monocytes to B2G1Δnab-sensitized platelets was substantially less (<15%) than their response to unmodified B2G1, as measured by chemiluminescence. In addition, B2G1Δnab inhibited chemiluminescence induced by B2G1 and HPA-1a–specific sera. We set out to prove the principle of our strategy in vivo using a murine model. Beta3-deficient mice (WT) were transplanted with littermate bone marrow transduced with a lentiviral vector containing the human ITGB3 encoding either Leucine-33 or Proline-33. B2G1 and polyclonal Ig preparations from clinical HPA-1a–specific sera reduced circulating HPA-1a+ platelets, concomitant with transient thrombocytopenia whilst WT mice and HPA-1b+ mice remained unaffected. Given the differences between murine and human Fcγ receptors, F(ab)2 B2G1 was used as a non-destructive proof of principle blocking antibody and prevented the in vivo platelet destruction seen with B2G1 and polyclonal HPA-1a–specific antibodies. These results provide rationale for human clinical studies, which are now underway. Eight HPA-1a1b heterozygotes volunteers have been recruited and from each of them we generate 2 pools of autologous platelets. Each pool is sensitized with either B2G1 or B2G1Δnab and radiolabeled with either Cr-51 or In-111. After re-injection into the volunteer, platelet survival is followed by the means of the radiolabel in order to show the differential effect on platelet destruction of the G1Δnab constant region when compared to the reference IgG1 destructive antibody. Full data of the volunteer studies will be presented in support of further clinical trials.
Identification of Mir-145 and Mir-146a as Micrornas Involved in the Pathogenesis of 5q- Syndrome
Authors: Starczynowski Daniel T. , Kuchenbauer Florian , Argiropoulos Bob , Sung Sandy , Morin Ryan , Muranyi Andrew L , Hirst Martin , Hogge Donna E. , Marra Marco , Wells Richard A. , Lam Wan , Humphries R. Keith , Karsan Aly 
Published: October 2019

Abstract
Deletion of the long arm of chromosome 5 is the most common cytogenetic alteration in myelodysplastic syndromes and is classified as a distinct subtype of the disease (5q- syndrome). Patients with 5q- syndrome present with macrocytic anemia, variable neutropenia, normal or elevated platelet counts, and megakaryocyte dysplasia. Over time these patients progress to marrow failure or acute myeloid leukemia. The commonly deleted region in 5q- syndrome contains numerous genes that are implicated in hematopoiesis, however no single gene loss recapitulates all features of 5q- syndrome in vivo. We examined the expression of microRNAs (miR) within the deleted region of chromosome 5q and found reduced expression of miR-145 and miR-146a in patients with del (5q). Knockdown of miR-145 or miR-146a in normal mouse hematopoietic cells resulted in increased megakaryopoiesis. We identified TIRAP and TRAF6 as critical mRNA targets of miR-145 and miR-146a, respectively. Expression of TIRAP alone resulted in autoactivation of TRAF6, implicating activation of a common pathway with loss of either miR-145 or miR-146a. Transplantation of lethally-irradiated mice with marrow cells expressing retrovirally-transduced TRAF6 resulted in neutropenia, anemia, thrombocytosis, and hypolobulated megakaryocytes. Long-term follow up of chimeric mice over 12 months revealed progression in approximately half the mice to marrow failure or acute leukemia. Further experiments revealed that recapitulation of 5q- syndrome-like features were mediated by paracrine effects induced by IL-6, as well as by cell autonomous TRAF6 effects. These findings suggest that several of the features of 5q- syndrome may be secondary to loss of microRNAs within the deleted region of chromosome 5q.
A Stapled BIM BH3 Helix Overcomes the MCL-1-Mediated Apoptotic Blockade of Refractory Hematologic Cancers
Authors: Labelle James L. , Lawrence Chelsea E. , Bird Gregory H. , Walensky Loren D. 
Published: October 2019

Abstract
MCL-1 is an anti-apoptotic member of the BCL-2 family of proteins and has emerged as a major resistance factor in hematologic cancer. Pharmacologic targeting of anti-apoptotic proteins is an effective strategy for reactivating apoptosis in cancers that evade cell death by neutralizing pro-apoptotic BCL-2 family proteins. Anti-apoptotic proteins contain a hydrophobic groove of differing specificity that binds and sequesters the critical BH3 the BH3-only members such as BID and BIM that transmit signals of cellular stress to that oligomerize upon activation to permeabilize the outer mitochondrial membrane. Small molecules such as ABT-737 that selectively inhibit the anti-apoptotic proteins BCL-2 and BCL-XL induce apoptosis of tumors that are especially dependent upon this subset of survival proteins. However, the compounds are rendered ineffective by cellular expression of alternate anti-apoptotic proteins such as MCL-1, which lie outside their range of binding specificity. We previously developed Stabilized Alpha Helices of BCL-2 domains (SAHBs) that are structurally stable, protease-resistant, and cell-permeable compounds capable of targeting BCL-2 family proteins in situ with high affinity. By inserting a hydrocarbon staple into the BH3 domain of pro-apoptotic BIM, we have generated a broad spectrum BCL-2 family targeting agent that binds all anti-apoptotic proteins with nanomolar affinity and also directly triggers the activation of pro-apoptotic BAX. Here we report that BIM SAHB, but not a point mutant control, induces dosedependent apoptosis of ABT-737-resistant acute myeloid leukemia, acute lymphoblastic leukemia, chronic myelogenous leukemia, diffuse large B-cell lymphoma, and multiple myeloma cell lines. Importantly, we demonstrate by co-immunoprecipitation that BIM SAHB broadly targets BCL-2 family proteins in these MCL-1-expressing hematologic cancers. Thus, by exploiting the native binding spectrum of BIM BH3 for both anti- and pro-apoptotic BCL-2 family members, we demonstrate the therapeutic potential of BIM SAHB in overcoming MCL-1-mediated resistance in hematologic cancers.
p38MAPK Inhibits Neutrophil Development through Phosphorylation of C/EBPα on Serine 21
Authors: Buitenhuis Miranda , Geest Christian R. , Laarhoven Annemieke G. , Bruin Marrie C.A. , Bierings Marc B. , Vellenga Edo , Coffer Paul J. 
Published: October 2019

Abstract
Many extracellular stimuli regulate growth, survival and differentiation responses through activation of the dual specificity kinase MAPK kinase 3 (MKK3) and its downstream effector p38 Mitogen-Activated Protein Kinase (MAPK). Using CD34+ hematopoietic progenitor cells, here we describe a novel role for MKK3-p38MAPK in the regulation of myelopoiesis. Inhibition of p38MAPK utilising the pharmacological inhibitor SB203580, enhanced neutrophil development ex-vivo, but conversely reduced eosinophil differentiation. In contrast, constitutive activation of MKK3 dramatically inhibited neutrophil differentiation. Transplantation of β2-microglobulin−/− NOD/SCID mice with CD34+ cells ectopically expressing constitutively active MKK3 resulted in reduced neutrophil differentiation in vivo, whereas eosinophil development was enhanced. Inhibitory phosphorylation of C/EBPα on serine 21 was induced upon activation of p38MAPK. Moreover, ectopic expression of a non-phosphorylatable C/EBPα mutant was sufficient to abrogate MKK3 induced inhibition of neutrophil development. Furthermore, treatment of CD34+ progenitors from patients with severe congenital neutropenia with SB203580 restored neutrophil development. These results establish a novel role for MKK3-p38MAPK in the regulation of lineage choices during myelopoiesis through modulation of C/EBPα activity. This signaling module may thus provide an important therapeutic target in the treatment of bone marrow failure.
Autosomal Recessive Hereditary Thrombocytosis: Functional Analysis of a Novel C-Mpl Mutation Resulting in An Unsual Mode of Constitutive Receptor Activation
Authors: Stockklausner Clemens , Gehring Niels , Echner Nicole , Mulla Naima Ali Al , Kulozik Andreas E. 
Published: October 2019

Abstract
Thrombopoiesis is tightly regulated by the interaction between thrombopoietin (TPO) and its receptor c-Mpl. Ligand binding results in dimerization of the receptor, internalization of the complex, and activation of downstream signaling. This activation pathway also leads to the clearance of TPO from the plasma thus establishing a negative feedback loop. Gain of function mutations of TPO and c-Mpl are exceedingly rare but provide interesting models for the biological understanding of the molecular interactions that are required for a functional TPO pathway. Here, we describe a consanguineous Qatari family with hereditary thrombocytosis. Genotype analysis revealed a novel c-Mpl P106L mutation that was found homozygously in 3 affected patients with platelet counts between 700–800/nl. The TPO gene sequence was normal. Notably, platelet counts in heterozygous relatives were within the normal range. Surprisingly, TPO plasma levels were elevated to 10 to 15-fold in homozygotes but within the normal range in heterozygotes. A simple gain of function mechanism of the c-Mpl mutation could thus be excluded. Therefore, we next functionally analyzed and compared the normal and the P106L mutated c-Mpl receptor. An analysis of HeLa and BaF3 cells that were transiently or stably, respectively, transfected with c-Mpl expression vectors showed that the P106L mutation leads to abnormal subcellular receptor distribution. Whereas normal c-Mpl was detected in the Golgi-apparatus and in the plasma membrane, c-Mpl P106L was predominantly found within the ER. Furthermore, we showed that c-Mpl P106L cannot be processed to the normal, tunicamycin sensitive, strongly glycosylated form. Functional analysis of the TPO/c-Mpl signaling pathway in stably transfected BaF3 cells demonstrated an elevated phosphorylation of Stat5 with P106L when compared to normal c-Mpl. Moreover, cells transfected with the mutant but not with the normal receptor gene were proliferating independently of extracellular growth factors such as IL3. These data indicate that c-Mpl P106L activates downstream signaling and thus stimulates cell division and platelet production in a ligand-independent fashion. The recessive mode of inheritance suggests that the presence of normal receptor molecules prevents this abnormal function and promotes the maintenance of the negative feedback loop regulating TPO plasma levels by an unknown mechanism. The raised TPO plasma levels in homozygotes and the lack of detectable mutated c-Mpl in the plasma membrane indicate that the mutated receptor does not bind and internalize its normal ligand. In conclusion, we hypothesize that the novel c-Mpl P106L receptor mutation results in spontaneous dimerization and ligand independent activation even before the receptor reaches the Golgi apparatus and is fully glycosylated and transported to the plasma membrane.
Nation-Wide Survey of Infant Leukemia in Japan: A Report from the Japanese Pediatric Leukemia/Lymphoma Study Group (JPLSG).
Authors: Miyamura Takako , Koh Katsuyoshi , Tomizawa Daisuke , Sugita Kanji , Kato Koji , Sato Takashi , Takahashi Yoshihiro , Ogawa Atsushi , Hirayama Masahiro , Kikuchi Akira , Oda Megumi , Hasegawa Daiichiro , Koike Kazutoshi , Saikawa Yutaka , Hatanaka Michiki , Horibe Keizo , Ishii Eiichi 
Published: October 2019

Abstract
Infant leukemia consists of wide spectrum of disease phenotype which includes acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML) and mixed lineage leukemia, and has unique biological and clinical features. However, there are few studies which have focused on its diversity from the cross-sectional view of this specific age group. We here conducted a nation-wide survey to analyze the disease distribution, characteristics, clinical courses, and outcomes of these patients in Japan. Questionnaires were sent to 170 institutions participating in the JPLSG which covers more than 95% of the hospitals in Japan, and total of 225 cases with infant leukemia and related disorders aged less than 24 months at onset who were diagnosed between 2004 and 2007 were registered. Median age at onset was 12 months, ranged from 0 to 22 months. No gender differences were observed (male, 118 cases; female, 107 cases). Thirty-nine were diagnosed as ALL with positive MLL gene rearrangements (MLL-R ALL), 74 as ALL with germline MLL (MLL-G ALL), 83 as AML, 9 as mixed lineage leukemia, and 20 as other disorders. In MLL-R ALL group, the frequency of t(4;11) was high in patients of <12 months of age (18/29 cases), whereas hyperdiploidy was detected in 20% of cases of MLL-G ALL group (12/61 cases). In AML, positive MLL gene rearrangements were detected in only 15% (13/83 cases), while t(1;22)(p13q13) or OTT-MAL was detected in 4 cases. Regardless of disease groups, early infant group of <6 months of age showed poor prognosis compared with other age groups. Particularly, all cases of ALL diagnosed at <4 weeks after birth harbored MLL gene rearrangements and either died or relapsed, indicating that more appropriate strategy is needed to improve the outcome for this specific group. Patients with MLL-G ALL less than 12 months old showed a favorable prognosis compared to those with MLL-R ALL. In AML, majority of cases were myelomonocytic/monocytic leukemia (M4/M5) or megakaryoblastic leukemia (M7), and showed favorable outcome. In conclusion, infant leukemia is still a challenging disease harboring diverse features, therefore, it is necessary to keep on-going the prospective trials, to register and follow-up these cases systematically and to standardize its treatment including supportive cares, especially for those with younger age onset.