bluuebunny/crossref_metadata_2025_split huggingface dataset:
High Molecular Weight Kininogen Fragments Stimulate the Secretion of Interleukin 1β through the Urokinase-Type Plasminogen Activator Receptor (uPAR) and CD11b/CD18 (Mac-1) in Human Blood Mononuclear Cells.
Authors: Khan Mohammad M. , Bradford Harlan N. , Isordia-Salas Irma , Espinola Ricardo , Colman Robert W. 
Published: October 2019

Abstract
High molecular weight kininogen (HK) is known to bind specifically and saturably to Mac-1 with a Kd = 9–18 nM for neutrophils and to uPAR with a Kd =30 nM for endothelial cells. However, the functional results of HK interaction with Mac-1 or uPAR on leukocytes is not fully understood. Kallikrein cleavage of single chain HK to a two chain form (HKa) with release of bradykinin (BK) occurs in sepsis, arthritis, and inflammatory bowel disease. We hypothesized that HKa stimulates secretion of inflammatory cytokines. Mononuclear cells were isolated from normal subjects by a Histopaque density gradient. We have expressed kininogen domain 3 (D3) and a fragment of domain 3, coded for by exon 7, E7P (aaG235-Q292), in E. Coli as glutathione S-transferase (GST) fusion proteins. HK and HKa were purified proteins. GST was recombinant. All proteins contained <0.01 EU/ml endotoxin. For all experiments, 2 X 106/ml mononuclear cells/ml were preincubated with monoclonal antibodies, murine IgG (both at 1.8 mM) or HANKS buffer containing 0.15 M NaCl, pH 7.4 for 30 minutes at 37°C. HK, HKa, GST-D3, GST-E7P, GST-D5 or GST all at 600 nM were added. Centrifugation allowed separation of the mononuclear cell suspension into cells and supernatant. The latter was used for assay of interleukin-1β (IL-1β) by ELISA. HK and all fragments tested stimulated secretion of IL-1β of 84.8 to 306.3 pg/ml when incubated with mononuclear cells for 30 minutes at 37°C. Anti-Mac-1 antibody inhibited IL-1β secretion by HK 100%, by HKa 89%, by GST-D3 78%, by GST-E7P 94% and by GST-D5 98%. Anti-uPAR antibody inhibited IL-1β release by HK 88%, by HKa 77%, by GST-D3 95%, by GST-E7P 85%, and by GST-D5 76%. Inhibition by both receptor antibodies is consistent with their known complex formation. A monoclonal antibody (mAb) to HK D5 (C11C1) and a mAb to HK D3 (2B5) both inhibited IL-1β release by HK, HKa, GST-D5 and GST-D3 indicate that both D3 and D5 are important in cytokine release. Murine IgG gave 0% inhibition in all studies. These results indicate that kininogen may contribute to the pathogenesis of inflammatory diseases by releasing IL-1β from human blood mononuclear cells.
Src Homology 2 Domain-Containing Inositol-5-Phosphatase 1 (SHIP1) Negatively Regulates TLR4 Mediated LPS Signaling through Phosphatase Activity and PI3K Independent Mechanism.
Authors: An Huazhang , Xu Hongmei , Zhou Jun , Zhang Minghui , Cao Xuetao 
Published: October 2019

Abstract
Lipopolysaccharide (LPS) is a cell wall component of Gram negative bacteria. LPS activates immune cells and triggers production of proinflammatory cytokines and other mediators through Toll-like receptor (TLR) 4. Src homology (SH) 2 domain-containing inositol-5-phosphatase 1 (SHIP1) plays important roles in negatively regulating the activation of immune cells primarily via phosphoinositide 3-kinase (PI3K) pathway by catalyzing PI3K product PtdIns-3,4,5P3 into PtdIns-3,4P2. However, little is known about the role of SHIP1 in TLR4-mediated LPS responses. We investigated the role of SHIP1 in LPS-induced MAPKs activation and IkappaB-alpha degradation by overexpression and RNA interfering experiments. SHIP1 becomes tyrosine phosphorylated and membrane translocated upon LPS stimulation in RAW264.7 macrophages. Stable SHIP1 transfection inhibits LPS-induced TNF-alpha and IL-6 production by 60% and 70% respectively. Transient SHIP1 transfection inhibits LPS-induced phospharylation of extracellular signal-regulated kinase (ERK1/2), p38, c-Jun NH2-terminal kinase (JNK) and degradation of IkappaB-alpha by about 20%, 50%, 40% and 40% respectively at the time point of 20 min after LPS stimulation. Disruption of SHIP1 phosphatase activity enhances LPS-induced activation of Akt, a downstream molecule of PI3K pathway, but fails to reverse SHIP1 mediated inhibition of LPS-induced activation of MAPKs and degradation of IkappaB-alpha. Consistently, LY 294002 and Wortmannin sufficiently inhibit LPS-induced Akt activation but can not diminish SHIP1 mediated inhibition of LPS-induced activation of MAPKs and degradation of IkappaB-alpha. On the contrary, SHIP1-specific RNA interfering inhibits SHIP1 expression and enhances LPS-induced phospharylation of ERK1/2, p38, JNK and degradation of IkappaB-alpha by about 2.6, 2.6, 3.0 and 8.0-fold respectively at the time point of 20 min after LPS stimulation. Even in the presence of LY 294002, SHIP1 RNA interfering could increase LPS-induced IL-6 production. Furthermore, SHIP1 overexpression nearly completely abolished TLR4 reconstitution conferred LPS responsiveness in COS7 cells. These results demonstrated that SHIP1 negatively regulates LPS responses in macrophage via phosphatase activity and PI3K-independent mechanism.
Cell Classification in Human Peripheral Blood Using the Amnis ImageStream® System.
Authors: Morrissey Philip , George Thaddeus , Basiji David , Frost Keith , Ortyn William , Williams Douglas 
Published: October 2019

Abstract
Amnis Corporation’s ImageStream® system combines the sample handling and quantitative power of flow cytometry with high-resolution brightfield, darkfield, and fluorescence cellular imagery. The system simultaneously generates up to six images of each cell in flow and can acquire data sets consisting of tens of thousands of cells in just a few minutes, while offering fluorescence sensitivity equal to or better than flow cytometry. The image data are analyzed using Amnis’ IDEAS® software, which automatically calculates over 200 morphometric and photometric features and associated statistics for each cell, identifying unique cell groups based not only on their fluorescence intensity signature but also on their morphological characteristics. The software offers the ability to view the imagery associated with any cell in a scatter plot, perform “virtual cell sorts” of user-specified sub-populations, and generate custom features of biological significance (e.g. N/C ratio). The ImageStream platform’s ability to quantitate morphologic and immunofluorescent differences between very large numbers of cells in suspension make it particularly well suited for hematology.
In the present study, human peripheral blood mononuclear cells were stained with a fluorescent DNA binding dye to reveal nuclear morphology, as well as fluorescently labeled mAb to various CD markers. Five images of each cell were acquired: brightfield (transmitted light), darkfield (laser side scatter), and three fluorescent colors for nuclear imagery and quantitation of the CD marker abundance. The object of the study was to identify morphometric parameters in the brightfield, darkfield, and nuclear imagery that would prove useful in hematologic cell type classification. The mAb to CD antigens provided a positive control for use in the evaluation of the of the various morphometric parameters. Parameters with discriminating power included cellular size and texture, darkfield intensity and granularity, and nuclear fluorescence intensity, texture, and shape. Cell types that could be automatically discriminated using these parameters in lieu of immunofluorescent markers included neutrophils, eosinophils, monocytes, and lymphocytes (including putative activated lymphocytes).
In addition to forming the basis for an advanced ImageStream hematology platform, it is envisioned that the automated morphometric classification of blood cells will act as the foundation for a wide range of image-based cellular assays performed in peripheral blood (e.g. NF-kB translocation, apoptosis, mAb compartmentalization), allowing the differential quantitation of assay results in various cell types for the purposes of basic research, drug discovery, and clinical diagnostics.
Deletion of Bcl11a Gene Reveals Roles in Lymphoid, Myeloid, and Leukemic Cell Development.
Authors: Ortiz Mariaestela , Liu P. , Tessarollo L. , Rachel R.A. , Nakamura T. , Stull Steven W. , Jenkins N.A. , Copeland N.G. , Keller J. R. 
Published: October 2019

Abstract
B-cell lymphoma/leukemia 11a (Bcl11a) gene, encodes a zinc finger protein that is expressed in normal B- and T-lymphocytes, monocytes, megakaryocytes, and physically interacts with the human B-cell proto-oncogene, BCL6. Bcl11a is activated by fusion of IG the locus in human B-cell lymphomas carrying the (2;14)(p13:q32.3) translocation. To determine the function of Bcl11a in normal hematopoiesis and proliferation of leukemic cells, we generated a Bcl11a knockout mouse model. This showed that Bcl11a is essential for postnatal development and normal lymphopoiesis. While Bcl11a+/− mice are fertile, Bcl11a−/− die a few hours after birth from unknown causes. To evaluate the role of Bcl11a in hematopoiesis, fetal liver (FL) cells were analyzed by FACS for the expression of lineage specific cell surface markers. B-cell maturation was blocked prior to the development of pro-B cells (B220+/CD43+), while myeloid and erythroid lineages were normal. The Bcl11a−/− FL also showed defects in T-cell maturation (reduced CD4+ CD8− and increased CD4− CD8+ fetal thymocytes). Lethally irradiated mice transplanted with Bcl11a−/− FL cells showed normal myeloid and erythroid development, while B- and T-cell development was blocked. Thus, defects in lymphoid development were intrinsic to the hematopoietic stem cells (HSC). We established that this defect in lymphopoiesis was not due to homing to the thymus, as shown by failure of T-cell repopulation when Bcl11a−/− FL cells were injected into the thymus. Greater than 80% of the mice transplanted with Bcl11a−/− FL cells died between 15 and 27 weeks, while 100% mice transplanted with Bcl11a+/− survived. Histopathology of moribund mice showed enlarged thymus with lymphoblasts infiltrating the bone marrow, liver, lung, heart, and kidney. Furthermore, analysis by real time-PCR of thymocytes from the sick mice showed increased expression of Notch1, a negative regulator of lymphoid development. Leukemic cells were tumorogenic when injected into SCID-NOD mice, and were immortalized by co-culturing on OP9 stromal cells. Immortalized leukemic cell lines showed high expression of T-cell markers CD3e, CD4, CD8, and B-cell marker CD43. Finally, myeloid progenitor cell growth (CFU-c, 3H-Thymidine incorporation, and viable cell growth) was inhibited by over-expression of Bcl11a in bone marrow cells from 5-FU treated mice. Thus, these data show that Bcl11a is required for B and T cell differentiation and may also function as a T-cell tumor suppressor gene. Furthermore, these results are the first indication of a possible suppressive role for Bcl11a during normal myeloid progenitor development and differentiation. This project has been funded in part from NCI, NIH Contract # NO1-CO-56000, with SAIC-Fredrick, Inc.
Retrovirus-Transduced T Cell Blasts Have Not Only Antigen-Presenting Capabilities but Also Suppressor Regulatory T Cell-Inducing Capability.
Authors: Okamura Takayuki , Leen Ann M. , Silli Uluhan , Gottschalk Stephen M. , Vigouroux Stephane , Heslop Helen E. , Brenner Malcolm K. , Rooney Cliona M. 
Published: October 2019

Abstract
EBV-associated T/NK-lymphoproliferative diseases (LPD) are relatively rare but their prognosis is very poor. In order to understand the pathogenesis and rationalize immunotherapy, it is important to know whether EBV-infected T/NK cells have antigen-presenting capacities and how they escape the EBV-specific immunity that controls virus-infected B cells and epithelial cells. As a model for T cell antigen presenting cells (T-APC), we used activated T cells expressing pp65, the immunodominant antigen of CMV, after retroviral transduction. Two to three days after activation with CD3/28, T cells were transduced with retrovirus supernatants for 24h to 48 hr on retronectin-coated plates, and then expanded for one to two weeks with IL-2 before use as APCs or target cells. As control APCs, we used LCLs transduced with a retrovirus vector expressing pp65 (pp65-LCL). Pp65-expressing T cells functioned as target cells, since they were readily killed by pp65-specific cytotoxic T cells (CTL), reactivated using Adenovirus-pp65-pulsed dendritic cells. Furthermore, pp65T-APCs could reactivate pp65-specific CD8+ CTL from autologous PBMC. However, the pp65T-APC could not sustain prolonged pp65-specific CTL expansion. The ratio of CD4+: CD8+ T cells increased in responders after the 2nd stimulation with pp65-T-APC, and after the 3rd stimulation, the CD8+ CTL had virtually disappeared so that most of live cells were CD4+ T cells. The majority of the CD4+ T cells co-expressed CD25, the glucocoticoid-induced TNF receptor (GITR) and intracellular antigen CD152, all markers of T regulatory cells. To determine if they had regulatory function, the CD4+CD25+ population was isolated and shown to inhibit the proliferation of autologous PBMCs stimulated by pp65-LCL. In contrast, the CD4+CD25− fraction did not inhibit proliferation. Transwell experiments revealed that the inhibition required cell-to-cell contact. CFSE analysis showed that the CD4+CD25+ cells expanded while inhibiting the proliferation of stimulated PBMC. Taken together, these findings indicated that T-APC could reactivate antigen specific CD8+ CTL, but at the same time they activated regulatory CD4+ T cells, which prevented the long-term expansion of the CD8+ CTL. Similar results were found when the LMP2 antigen of EBV, which is expressed in T/NK-LPD, was used as antigen. Thus the immune escape of EBV-associated T/NK-LPD might result in part from their induction of regulatory T cells which inhibit T cell-specific CTL generation and function. T cells in general may evade immune-mediated recognition of their “novel” T cell receptors in the same way.
Sensitization of Human T Cells with Overlapping Pentadecapeptides Spanning the Wt1 Protein Induces Expansion of Leukemocidal T Cells Specific for Both Previously Identified and Novel WT1 Epitopes.
Authors: Doubrovina Ekaterina , Dupont Jacob , Trivedi Deepa , Kanaeva Elena , O’Reilly Richard J. 
Published: October 2019

Abstract
The oncofetal protein, WT1, is differentially expressed in 60–80% of acute leukemias and CML. Previously, four peptide epitopes presented by HLA-A0201 or HLA-A2402 have been identified. We used a pool of 15-mers with 10 amino acid overlaps spanning the full sequence of WT1, loaded on autologous monocyte-derived dendritic cells or EBV-transformed BLCL to sensitize and raise T cell lines from a series of normal donors expressing or not expressing HLA-A0201 or HLA-A2402. Specific 15-mers eliciting responses were identified by quantitating IFNg+ CD8+ or CD4+ T cell responses to secondary stimulation with 15-mer subpools organized in a mapping grid with single overlaps. Responses against targets loaded with the identified stimulating 15-mer and sharing single HLA class I or II alleles permitted identification of restricting HLA alleles. Patients inheriting HLA-A0201 have generated CD8+ T cells in response to the complete pool which are selectively reactive against single 15-mers containing the known immunogen 252–260RMFPNAPYL. However, in certain donors, responses to 15-mer 28, containing a heretofore unreported epitope 136–144ALLPAVPSL, (which has a high predicted affinity for HLA-A0201) have been dominant. By mapping responses of T cells sensitized with the complete pool, defining the presenting HLA alleles and delineating immunogenic sequences within specific 15-mers, additional novel epitopes presented by HLA- B0801 and DRB10301 have also been defined. T cells generated by this approach also lyse both peptide-loaded targets as well as primary WT1+ leukemic cells expressing the restricting HLA allele. Thus, sensitization with pools of overlapping synthetic 15-mers spanning WT1 may selectively stimulate WT1-specific CD4+ and CD8+ T cell lines restricted by HLA alleles other than HLA- A0201 or A2402 that are leukemocidal and therefore of potential use for adoptive immunotherapy.
Effectiveness of Splenectomy and Platelet Autoantibodies.
Authors: Sikorska Anna , Maslanka Krystyna , Misiak Andrzej , Konopka Lech , Zupanska Barbara 
Published: October 2019

Abstract
The role of the spleen in pathophysiology of Idiopatic Thrombocytopenic Purpura (ITP) remains incompletely defined. Splenectomy has a fairly favourable initial response rate of 60% to 80%, and the longer the follow-up, the lower the success rate. As yet, there is no consistently effective method of predicting an individual response to splenectomy. So far not many observations have been published and if so, they were based on very small groups of ITP.
The aim of this work was to evaluate the response to splenectomy in 151 patients with ITP in relation to the detection of platelet autoantibodies. 151 ITP patients before and after splenectomy were observed for a period of 16 years.
Autoantibodies were examined by the platelet immunofluoresce test (PIFT) and/or by the monoclonal antibody immunobilization of platelet antigen (MAIPA) assay, sometimes in addition the HPA genotyping was performed using the PCR.
The response to splenectomy was regarded to be very good if platelet count was >150 G/l during at least 6 months follow-up. However, some patients were followed-up even for 15 years. Overall, platelet antibodies were detected in 34.3% (52/151) of patients. In 76% out of 52 patients the antibodies were detected by PIFT, in 63% by MAIPA; both tests were positive in 27% of patients. Most antibodies reacted with GPIIbIIIa.
Splenectomy was effective in 57% (30/52) of patients with antibodies and in 75.8% (75/99) of patients without detectable antibodies (p<0.01). In the effective splenectomy and antibodies group, in 23 patients the antibodies were examined during a follow-up and in 10 of them (43.5%) were not detected. In the uneffective splenectomy group only in 4 out of 18 (22.2%) examined patients the antibodies were not found. The differences between these groups were not significant.
Our data show that autoantibody platelet examination is not very helpful for predicting the effectiveness of splenectomy.
Erythroid Inhibition by AML1-ETO: Repression of GATA-1 Function by a Novel Non-Transcriptional Mechanism Involving Targeted Protein Degradation.
Authors: Choi Youngjin , Elagib Kamaleldin E. , Goldfarb Adam N. 
Published: October 2019

Abstract
AML1-ETO (A/E) is the fusion product of a chromosomal translocation, t(8;21) frequently associated with FAB M2 acute myeloid leukemia (AML). The fusion combines the runt domain of the hematopoietic transcription factor RUNX1 with almost the entire transcriptional repressor ETO. Clinical cases of AML with t(8;21) are distinguished by blockade in erythroid differentiation. In addition, enforced expression of A/E in primary human erythroid progenitors impairs differentiation. Existing paradigms postulate that A/E exerts its leukemogenic effects through recruitment to RUNX binding sites of cofactors such as corepressors, histone deacetylases (HDACs), and DNA methyltransferases (DNMTs), causing repression of RUNX target genes. However, this paradigm fails to explain effects of A/E on erythropoiesis as erythroid genes generally lack functional RUNX sites. We have published a physical and functional interplay between RUNX1 and the erythroid master regulator GATA-1 (Blood 101:4333). Furthermore, A/E physically interacted and functionally interfered with GATA-1. In the current studies we have examined domain and cofactor requirements for A/E inhibition both of GATA-1 function and of erythroid differentiation. Deletional mutagenesis of A/E demonstrated that the zinc finger (NH4) and runt domains were absolutely required for GATA-1 inhibition. Treatment with HDAC and DNMT inhibitors failed to affect A/E repression of GATA-1. RNAi knockdown of all known NH4 interactors, HDACs 1-3, N-CoR, SMRT-A, and SMRT-B also failed to affect A/E inhibition of GATA-1. Inducible expression of A/E in MEL cells caused downregulation of endogenous GATA-1 protein and mRNA, an effect dependent on induction of erythroid differentiation. A coexpressed GATA-1-GFP fusion showed downregulation with identical kinetics to endogenous GATA-1. Interestingly, proteasome-specific inhibitors effectively prevented the downregulation of endogenous GATA-1 and GATA-1-GFP caused by induction of A/E coupled with erythroid differentiation. Fluorescence microscopy showed a striking relocation of GATA-1-GFP from the nucleus to discrete, paranuclear bodies upon joint induction of A/E expression and erythroid differentiation. Our findings indicate that A/E inhibition of GATA-1 occurs through a previously undescribed mechanism that involves GATA-1 redistribution to novel cellular structures followed by proteasome-mediated degradation. These findings expand the paradigm of A/E leukemogenicity to include a non-transcriptional mechanism in which a growth inhibitor/tumor suppressor, GATA-1, is targeted by A/E for proteolytic degradation in a manner reminiscent of human papilloma virus E6 targeting of p53 for degradation in cervical carcinogenesis.
Murine Platelets Retain High Amounts of Alpha Granule Proteins on Their Surface upon Stimulation with Multiple Agonists.
Authors: Jobe Shawn M. , Leo Lorie , Dicknite Gerhard , Lentz Steven R. , Di Paola Jorge 
Published: October 2019

Abstract
Human platelets exposed to two strong agonists have recently been noted to form a unique subpopulation characterized by increased amounts of bound alpha granule constituents, including fibrinogen and factor V. We have identified and characterized a similar population of platelets in the mouse utilizing thrombin and the GPVI agonist, convulxin, as dual stimulants. We demonstrate that these platelets are analogous to those previously characterized in humans and examine their formation in both Fc Receptor γ-chain (FcRγ) and Factor XIIIa null mice. C57/Bl6 mouse platelets were exposed to convulxin, thrombin, and ionomycin singly or in combination at concentrations previously demonstrated to give maximal aggregation. Platelets were then examined by flow cytometry. Upon stimulation by any of the agonists, all platelets exhibited moderate amounts of fibrinogen binding and equivalent P-selectin exposure. Approximately 70% of convulxin and thrombin stimulated platelets (CoaT platelets) and 10% of thrombin stimulated platelets retained high amounts of alpha granule proteins, including fibrinogen and vWF. Interestingly, despite their high amount of bound fibrinogen, CoaT platelets demonstrated decreased binding of the GPIIb/IIIa activated state-specific antibody, JON/A, relative to single agonist stimulated platelets. Further characterization identified this platelet population as having high phosphatidylserine exposure (increased annexin V binding) and increased ability to bind the actin specific marker, phalloidin. All of these characteristics were present within two minutes of agonist stimulation. CoaT platelets could not be generated in the FcRγ-chain knockout mouse demonstrating the importance of this signaling pathway in their formation. Since human CoaT platelet formation is inhibited both by transglutaminase inhibitors and a monoclonal antibody to Factor XIIIa, CoaT platelet formation was examined in the FXIIIa null mouse. The absence of FXIIIa did not affect CoaT platelet formation, aggregation, or phosphatidylserine exposure. These findings demonstrate the presence of a unique subpopulation of platelets in the mouse formed on exposure to dual agonists analogous to the previously described CoaT platelets. This population is characterized by high amounts of bound alpha granule proteins, decreased binding of a GPIIb/IIIa activation specific mAb, and increased phosphatidylserine exposure; and in the mouse is not dependent on FXIIIa.