bluuebunny/crossref_metadata_2025 huggingface dataset:
Mutant Ras-Expressing Fibroblasts Induce Expansion of Circulating Angio- and Lymphangiogenic Precursor Cells: Potential Role in Tumour Vascularisation.
Authors: Knight Robert J. , O’Brien Tracey A. , Lindeman Robert , Dolnikov Alla
Published: April 2020

Fibroblasts have a prominent role in the initiation, enlargement and metastasis of cancers. Their production of growth factors, chemokines and extracellular matrix facilitate the angiogenic recruitment of endothelial cells and pericytes. The oncogene Ras, known to be frequently mutated in many cancers, has also been shown to promote the expression of pro-angiogenic growth factors, chemokines and extracellular matrix components similar to those of fibroblasts. Here we show that mutant N-ras (N-rasm)/GFP - transduced NIH3T3 fibroblasts act to promote the ex vivo expansion of umbilical cord blood (UCB)-derived primitive endothelial progenitor cells through paracrine mechanisms. Biphasic expansion of both angiogenic and lymphangiogenic precursors was observed although they exhibited different dynamics in their expansion pattern. The first peak of expansion for both types of precursor cells was seen on day 10 with a 69-fold expansion of the CD34+VEGFR2+(endothelial) and a 23-fold expansion of CD34+VEGFR3+(lymphatic) cells co-cultured with media conditioned by N-rasm-transduced 3T3 cells. This compared to a 20-fold and 14-fold expansion respectively, in cells co-cultured with media conditioned by GFP (only)-transduced fibroblasts. Endothelial cell differentiation accounts for the dramatic reduction in the numbers of CD34+VEGFR2+ and CD34+VEGFR3+ precursor cells on day 14 with concomitant expansion of CD34−VEGFR2+ and CD34−VEGFR3+cells. The second peak for the expansion of endothelial precursor cells was seen on day 19 with a 214-fold expansion compared to 28-fold in the control cells. In addition, the expansion of CD14+ VEGFR2+ and CD14+VEGFR3+ cells was observed on days 14 and 19, the later correlated with the expansion of monocytic CD34−CD14+ cells promoted by N-rasm-transduced fibroblasts. The expanded monocytes appear to contribute to the expansion of endothelial and lymphatic precursor cells induced by N-rasm-transduced fibroblasts. We propose that the angiogenic factors secreted by mutant Ras-expressing fibroblasts in a tumour microenvironment promote tumour angiogenesis through the expansion of the circulated endothelial and lymphatic precursor cells. The recruitment of the expanded endothelial and lymphatic cells into the tumour’s vascular system is currently being investigated. We propose that targeting aberrant Ras signaling in tumour fibroblasts may represent an important target for cancer therapies.
Human CD34+ Myeloid Leukemic Progenitor Cells Are Susceptible to Lysis by Minor Histocompatibility Antigen LRH-1-Specific Cytotoxic T Lymphocytes.
Authors: Dolstra Harry , Overes Ingrid , Jedema Inge , Vos Annemieke , van Horssen-Zoetbrood Agnes , Norde Wieger , van der Voort Robbert , Falkenburg J.H. Frederik , Schattenberg Anton V. , de Witte T.M.
Published: April 2020

Allogeneic stem cell transplantation (SCT) is a specialized form of immunotherapy for treating patients with hematological malignancies. The curative potential is attributed to the graft-versus-tumor (GVT) response during which donor-derived cytotoxic T lymphocytes (CTL) eliminate malignant cells of the recipient. Minor histocompatibility antigens (MiHA) are the major targets of the GVT response, and expansion of MiHA-specific CTL has been shown to coincide with tumor remission following SCT. Recently, we identified a novel hematopoietic cell-restricted MiHA, designated LRH-1, which is presented by HLA-B7 and encoded by the P2X5 purinergic receptor gene (J. Clin. Invest.2005:115:3506–3516). Interestingly, tetramer analysis showed a direct association between in vivo expansion of LRH-1-specific CD8+ T cells and the disappearance of Bcr-Abl positive tumor cells in the CML patient from whom LRH-1-specific CTL was originally isolated. In addition, we detected in vivo expansion of LRH-1-specific CTL in an AML patient who was in clinical remission without GVHD. Furthermore, we demonstrated that P2X5 mRNA is significantly expressed in leukemic CD34+ progenitor cells from most CML as well as AML patients. These findings indicate a role for LRH-1 in inducing GVT immunity against myeloid leukemic progenitor cells. Here, we investigated the ex vivo responsiveness of myeloid leukemic CD34+ progenitor cells to LRH-1-specific CTL. First, we addressed this question using the CD34+ KG1 cell line which is positive for LRH-1. By using a CFSE-based survival assay we demonstrated that KG1 cells stably transfected with HLA-B7 could be efficiently lysed by LRH-1-specific CTL. Next, we determined responsiveness of purified CD34+ progenitor cells from HLA-B7+ CML patients to LRH-1 CTL-mediated killing. In the CFSE-based survival assay as well as a hematopoietic progenitor cell inhibition assay we showed that LRH-1-specific CTL efficiently recognize and kill CD34+ progenitor cells from LRH-1+ CML patients. In contrast, LRH-1-specific CTL did not inhibit the proliferation of CD34+ progenitor cells from LRH-1- CML patients. These findings illustrate that the P2X5-encoded LRH-1 antigen is an attractive target for adequate eradication of myeloid leukemic progenitor cells after allogeneic SCT.
MicroRNA hsa-mir-16 Contributes to Regulation of Myeloid Differentiation of Human CD34+ Cells.
Authors: Hildreth Richard , Georgantas Robert W. , Patel Roshan , Morisot Sebastien , Alder Jonathan , Civin Curt I.
Published: April 2020

In a large microRNA-array and bioinformatics study, we determined all of the microRNAs (miRs) expressed by human CD34+ hematopoietic stem-progenitor cells (HSPCs) from bone marrow and G-CSF mobilized blood. When we combined miR expression data, mRNA expression data fro a previous study (Georgantas et al, Cancer Research 64:4434), and data from various published mir-target prediction algorithms, we were able to bioinformaticly predict the actions of miRs within the hematopoietic system. MicroRNA hsa-mir-16 was highly expressed in CD34+ HSPCs, and was predicted to target several HSPC-expressed mRNAs (CXCR4, HoxB7, Runx-1, ETS-1, and Myb) that encode proteins known to be critically involved specifically in myelopoiesis within the hematopoietic system. We first confirmed that protein expression from each of these putative target mRNAs was in fact regulated by mir-16. The 3′UTR sequence from each of these mRNAs was cloned behind a luciferase reporter. Each reporter construct was transfected into K562 cells, which strongly express mir-16. In all cases, protein expression from the predicted target mRNA was greatly reduced in K562 cells, as compared to controls. As a first determination of mir-16’s function in hematopoietic cells, HL60 and K562 cells were transduced with hsa-mir-16 lentivirus, then treated with various chemical differentiation inducers. As was predicted by bioinformatics, hsa-mir-16 halted myeloid differentiation of HL60 cells, but did not affect megakaryocytic differentiation or erythroid differentiation of K562 cells. These initial findings suggest that mir-16 is a specific negative regulator of myelopoiesis. We are currently evaluating the effects of mir-16 on normal human CD34+ cells by in vitro CFC and suspension culture assays, as well as in vivo by transplantation of hsa-mir-16 lentivirus transduced cells in immunodeficient mice.
Lenalidomide (CC-5013; Revlimid®) Promotes Erythropoiesis in Myelodysplastic Syndromes (MDS) by CD45 Protein Tyrosine Phosphatase (PTP) Inhibition.
Authors: List Alan F. , Estes Myka , Williams Ann , Sekharam Madhavi , Ozawa Ukiyasu , Gao Gui , Wu Jerry , Gao Gui , Sokol Lubomir
Published: April 2020

Lenalidomide displays erythroid and cytogenetic remitting activity in MDS that is karyotype-dependent, yielding a high response rate in patients with chromosome 5q deletion (del5q; 76%) whereas cytogenetic and pathologic improvement in non-del5q patients is less common despite transfusion response in a significant proportion of patients [NEJM2005;352:549–57]. We postulated that lenalidomide targets regulatory proteins that repress the erythropoietin (EPO) signal in non-del5q MDS while inhibiting a critical del5q-associated target to account for karyotype-specific clonal suppression. To evaluate the mechanism by which lenalidomide enhances erythropoiesis in non-del5q MDS, we investigated its effects in MDS [N=9] and normal [N=7] marrow specimens, and in an EPO-responsive UT7 cell line model. Pre-incubation exposure to lenalidomide augmented EPO-induced BFU-E recovery up to 2.5-fold in non-del5q MDS specimens [N=7] over a concentration range of 0.001–1 mM, whereas change in BFU-E recovery from normals was nominal. In UT7 cells, lenalidomide [1mM] enhanced clonogenic response to EPO in a supra-additive fashion. To characterize effects on receptor signal response, we investigated the interaction between lenalidomide and EPO on STAT5 activation, and corresponding EPO-receptor (R) associated kinases. Lenalidomide enhanced both EPO-induced STAT5 phosphorylation and DNA binding in UT7 and MDS CD71Hi MDS erythroid precursors with corresponding Bcl-XL up-regulation. The magnitude of lenalidomide enhancement of EPO/STAT5 phosphorylation was significantly higher in MDS CD71Hi erythroid precursors compared to normal donors (median log10 geometric MFI pSTAT5 EPO+CC5013:EPO, 0.989 [s.d.,0.25] MDS vs. 0.0195 [0.07] Normal; P=0.0005). Lenalidomide enhancement of the EPO-R/STAT5 signal was associated with increased Janus kinase-2 (Jak-2) and Lyn kinase auto-phosphorylation and was abolished only by dual Jak and Src kinase inhibition, implicating targeted inhibition of a common Jak/Lyn kinase regulatory molecule. Indeed, lenalidomide potentiation of the EPO/STAT5 axis was replicated by inhibition of the dual Jak-2/Lyn PTP, CD45, with the selective antagonist RWJ-60475. In a cell free assay employing rhu-CD45 and a pp60 c-Src reporter substrate, lenalidomide inhibited CD45 PTP activity in a time-dependent fashion with a 50% inhibitory concentration [IC50] of 10nM, whereas thalidomide was largely ineffective. PTP inhibition was CD45 selective, demonstrating no significant inhibition of rhu-SHP-2 PTP activity. Our data suggest that lenalidomide is a PTP inhibitor that enhances EPO receptor signaling in non-del5q erythroid precursors by relieving CD45 repression of ligand-dependent Jak2 and Lyn kinase mediated STAT5 activation. Moreover, these findings suggest that CD45 over-activity may contribute to impaired EPO-R signaling in MDS erythroid precursors.
Combination of Gene Expression Data from Myeloid Cell Lines with In Vitro Response to Molecularly Targeted Therapies.
Authors: Mills Ken I. , Gilkes Amanda F. , Walsby Beth J. , Woodward Eleanor L. , Austin Steve J. , Jenkins Chris , Knapper Steven , Burnett Alan K.
Published: April 2020

AML is currently classified using a combination of morphology and major cytogenetic abnormalities. There is considerable heterogeneity with respect to secondary cytogenetics, molecular mutations, response to therapy, remission rate, and overall survival. New molecular therapies are being developed constantly and are tested in vitro using patient samples in order to balance the drug-patient relationship. Use of the same drug produces patient specific responses, whereas different molecular drugs give drug specific responses.

In order to rationalise and enhance the drug-patient relationship we have, using MTS assays, tested a range of molecular inhibitors against FLT3, PKC, HSP90, aminopeptidases and NFκB as well as modified nucleoside analogues on a panel of myeloid cell lines. These cell lines have also been profiled for gene expression. The combination of drug response and gene expression levels, were analysed by statistical algorithms including SAM, ANOVA and T-test to identify genes that differentiated between molecular responses or the overall therapeutic index (TI). Forty one genes distinguished between the molecular responses of FLT3, PKC, HSP90, and NFkB inhibitors. Around 100 genes were needed for the Ara-C response. The gene expression profiles of 187 diagnostic AML samples were also available with full clinical and molecular analysis. Some of these samples have been tested for in vitro responses to the molecular therapies, such as those targeted to FLT3 or HSP90. The TI gene list was used to analyse AML sub-groups including elderly patients (>60 yrs old); younger patients (<60 yrs old); cytogenetic risk groups, and those patients with FLT3 mutations (either ITD or TKD mutations) in an attempt to identify patients who may respond to specific molecularly targeted therapy. The Ara-C genes were used to predict whether a CR response was achieved. In patients less than 60 yrs old this correlation was not significant due to the great majority of patients achieving a complete remission. However, this was significant (p<0.031) in patients >60 yrs. Around 12% of patients were predicted to be responsive to HSP90 inhibitors whilst 18% might respond to FLT3 inhibitors. Several patients were in both groups, so 17% of patients were sensitive to a least one of the drugs. Whilst the potential added value of identifying patients sensitive to one of these drugs was only an additional 1–3%, this study shows the potential of using expression data to identify patients for specific molecular therapies and increasing the possibility of achieving a remission.
Dasatinib (BMS354825) Inhibits IgE-Dependent Activation and Histamine Release in Human Blood Basophils.
Authors: Kneidinger Michael , Valent Peter , Gleixner Karoline V. , Florian Stefan , Boehm Alexandra , Sonneck Karoline , Weghofer Margit , Lupinek Christian , Vrtala Susanne , Vales Anja , Valenta Rudolf , Superti-Furga Giulio , Lee Francis Y.
Published: April 2020

Dasatinib (BMS354825) is a small molecule-type inhibitory drug that blocks several tyrosine kinases including PDGFR, KIT, BCR/ABL, and several src kinases. Dasatinib has recently been introduced as a new effective antileukemic drug in patients with imatinib-resistant chronic myeloid leukemia. However, because of the many targets of dasatinib and the broad range of potential target-cells in physiologic and pathologic tissues, dasatinib has recently been examined for its immunosuppressive and anti-inflammatory effects. We here report that dasatinib at 1 μM completely blocks the anti-IgE-induced release of histamine in human blood basophils in healthy subjects. The effects of dasatinib on histamine secretion in basophils were dose-dependent (IC50: 50–100 nM). Dasatinib was also found to block recombinant allergen-induced release of histamine from human basophils in sensitized individuals. In addition, as assessed by flow cytometry, dasatinib (1 μM) was found to inhibit the IgE-dependent upregulation of several activation-linked antigens, including aminopeptidase N (CD13), LAMP-3 (CD63), endolyn (CD164), and the basophil-specific ectoenzyme E-NPP3 (CD203c) on the cell surface of human basophils. The effects of dasatinib on upregulation of basophil differentiation antigens were found to be dose-dependent and were seen in normal subjects as well as in allergic individuals. Together, these data show that dasatinib blocks IgE-mediated activation and histamine release in human blood basophils. These anti-allergic effects of dasatinib may have clinical implications and may point to additional indications for this multifunctional drug.
Analysis of Aurora Kinase Expressions and Cell Cycle Regulation by Aurora-C in Leukemia Cells.
Authors: Kobayashi Miki , Nakamura Satoki , Ono Takaaki , Sugimoto Yuya , Sahara Naohi , Shinjo Kaori , Shigeno Kazuyuki , Ohnishi Kazunori
Published: April 2020

Background: The conserved Aurora family kinases, a family of mitotic serine/threonine kinases, have three members (Aurora-A, -B and -C) in mammalian cells. The Aurora kinases are involved in the regulation of cell cycle progression, and alterations in their expression have been shown to associate with cell malignant transformation. Aurora A localizes to the centrosomes during anaphase, and it is required for mitotic entry. Aurora B regulates the formation of a stable bipolar spindle-kinetochore attachment in mitosis. The function of Aurora-C in mammalian cells has not been studied extensively. In this study, we investigated that human leukemia cells expressed all 3 Aurora kinases at both protein and mRNA level, and the mechanisms of cell cycle regulation by knock down of Aurora C in leukemia cells.

Methods: In this study, we used the 7 human leukemia cell lines, K562, NB4, HL60, U937, CEM, MOLT4, SUP-B15 cells. The expression levels of mRNA and proteins of Aurora kinases were evaluated by RT-PCR and western blot. The analysis of proliferation and cell cycle were performed by MTT assay and FCM, respectively.

Results: The mRNA of Aurora-A and Aurora-B are highly expressed in human leukemia cell lines (K562, NB4, HL60, U937, CEM, MOLT4, SUP-B15 cells), while the mRNA of Aurora C is not only expressed highly in all cells. In contrast, an increase in the protein level of the 3 kinases was found in all cell lines. These observations suggested posttranscriptional mechanisms, which modulate the expression of Aurora C. In cell cycle analysis by flow cytometory, the knock down of Aurora C by siRNA induced G0/G1 arrest and apoptosis in leukemia cells, and increased the protein levels of p27Kip1 and decreased Skp2 by western blot. In MTT assay, it was revealed that the growth inhibition of leukemia cells transfected with siRNA Aurora C compared with leukemia cells untransfected with siRNA Aurora C. Moreover, We showed that Aurora C was associated with Survivin and directly bound to Survivin by immunoprecipitation and western blot.

Conclusion: We found that human leukemia cells expressed all 3 members of the Aurora kinase family. These results suggest that the Aurora kinases may play a relevant role in leukemia cells. Among these Aurora kinases, Aurora C interacted with Survivin and prevented apoptosis of leukemia cells, and induced cell cycle progression. Our results showed that Aurora-C may serve as a key regulator in cell division and survival. These results suggest that the Aurora C kinase may play an important role in leukemia cells, and may represent a target for leukemia therapy.
Inhibition of T315I Bcr-Abl and Other Imatinib-Resistant Bcr-Abl Mutants by the Selective Abl Kinase Inhibitor SGX70393.
Authors: O’Hare Thomas , Eide Christopher A. , Tyner Jeffrey W. , Wong Matthew J. , Smith Caitlyn A. , Corbin Amie S. , Buchanan Sean , Jessen Katayoun A. , Tang Crystal , Holme Kevin , Burley Stephen K. , Deininger Michael W.N.
Published: April 2020

Imatinib effectively inhibits the tyrosine kinase activity of Bcr-Abl, the molecular driver of CML. Emergence of imatinib resistance due to mutations within the Bcr-Abl kinase domain (KD) has prompted the development of new Abl kinase inhibitors. A particularly important target is Bcr-Abl(T315I), which accounts for 15–20% of patients with resistance. To address this unresolved need, we profiled the novel Abl kinase inhibitor SGX70393 against native and mutant Bcr-Abl. Methods: We assessed the efficacy of SGX70393 in cellular and biochemical assays against a panel of KD mutants. Cell proliferation assays and Bcr-Abl tyrosine phosphorylation immunoblot analyses were performed for parental Ba/F3 cells, Ba/F3 cells expressing unmutated Bcr-Abl, or Ba/F3 cells expressing a single Bcr-Abl KD mutation (M244V, G250E, Q252H, Y253F, Y253H, E255K, E255V, F311L, T315I, F317L, M351T, F359V, V379I, L387M, H396P, or H396R). The resistance profile of SGX70393 was also evaluated using a recently developed accelerated, cell-based mutagenesis assay (Bradeen, et al. Blood, June 2006; doi:10.1182). Results: SGX70393 inhibited growth of cells expressing Bcr-Abl(T315I) (IC50: 7.3 nM) or unmutated Bcr-Abl (IC50: 12 nM). Sensitivity of Bcr-Abl mutants to SGX70393 partitioned into three categories: high (IC50<25 nM: M244V, T315I, F359V, V379I, L387M, H396P, and H396R), medium (IC50<300 nM: Q252H, Y253H, E255K, and F311L), and low (IC50>500 nM: G250E, Y253F, E255V, and F317L). A cell-based mutagenesis screen for Bcr-Abl kinase domain escape mutants emerging in the presence of SGX70393 revealed a concentration-dependent reduction in surviving clones, with five previously reported Bcr-Abl mutations (L248M; G250E; Y253F; E255V; F317V) accounting for almost all resistance. Conclusions: (a) SGX70393 is a potent inhibitor of native and T315I mutant Bcr-Abl. (b) SGX70393 coverage extends to most clinically relevant mutants except mutations of the p-loop and F317.
Use of RNA Electroporated DC for Activation of LRH-1 Specific Cytotoxic T Lymphocytes in the Treatment of Lymphoid Malignancies.
Authors: Overes Ingrid , Fredrix Hanny , de Rijke Bjorn , van Horssen-Zoetbrood Agnes , van Krieken Han , Raymakers Reinier , van der Voort Robbert , de Witte T.M. , Dolstra Harry
Published: April 2020

Allogeneic hematopoietic stem cell transplantation (HSCT) is a potent treatment for patients with hematological malignancies. The therapeutic efficacy is attributed to the graft-versus-tumor (GVT) response during which donor-derived cytotoxic T lymphocytes (CTL) eliminate malignant cells of the recipient. Minor histocompatibility antigens (MiHA) are the major targets of the GVT response, and expansion of MiHA-specific CTL has been shown to coincide with tumor remission following SCT. Unfortunately, GVT response is often accompanied by graft-versus-host disease (GVHD) causing severe damage to skin, liver and gut. Therefore, it would be highly beneficial to direct GVT immunity to MiHA that are selectively expressed by “malignant” hematopoietic cells. Recently, we identified a novel lymphoid lineage-restricted MiHA, designated LRH-1, which is derived from a frame shift polymorphism in the P2X5 purinergic receptor protein (J. Clin. Invest.2005:115:3506–3516). Here, we examined by real-time quantitative RT-PCR mRNA expression of P2X5 in patient samples and cell lines from numerous lymphoid malignancies. We observed that P2X5 mRNA is highly expressed in tumor cells from all stages of lymphoid development. Furthermore, we demonstrated that LRH-1−specific CTL efficiently lyse LRH-1+ tumor cells and cell lines of lymphoid origin. These findings illustrate that LRH-1 is an attractive target for specific immunotherapy after allogeneic HSCT. A potentially efficient strategy is the application of MiHA-loaded dendritic cells (DC) to boost MiHA-specific T cell responses already primed in vivo after HSCT. To facilitate efficient presentation of LRH-1 by DC, we have optimized RNA electroporation of DC using in vitro-transcribed mRNA. We observed that RNA electroporation of mature DC resulted in a high yield of viable cells, high expression of co-stimulatory molecules, no loss of migratory capacity towards lymph node-specific chemokines, and high protein expression of the introduced antigens. Furthermore, we demonstrated that RNA-electroporated DC display long-lasting peptide presentation to LRH-1− specific CTL and induce proliferation of LRH-1− specific effector-memory CTL ex vivo. These data will be of importance in designing LRH-1− based immunotherapy in transplanted patients with lymphoid malignancies.
Bortezomib Inhibits Human Osteoclastogenesis.
Authors: Zavrski Ivana , Hecht Monica , Krebbel Holger , Fleissner Claudia , Mieth Maren , Kaiser Martin , Heider Ulrike , Jakob Christian , Sterz Jan , Possinger Kurt , Sezer Orhan
Published: April 2020

Enhanced osteoclastogenesis in cancer-induced bone disease may be caused by intercellular interactions between tumor cells and cells of the bone marrow microenvironment. In multiple myeloma, overexpression of RANKL in the bone marrow microenvironment may lead to the activation of TRAF-signaling and in consequence to increased NF-κB and AP-1 transcriptional activities in osteoclastic lineage cells. This results in enhanced osteoclast differentiation, activation and increased bone resorption. In this study, we have examined the effects of two NF-κB inhibitors towards their inhibitory potency on human osteoclastogenesis: proteasome inhibitor bortezomib and selective IKK inhibitor PS-1145. CD14+ osteoclast precursors from peripheral blood were stimulated with RANKL and M-CSF up to four weeks. Using MTT- and TUNEL-assays, cytotoxicity levels of each drug were determined on the differentiation stage day +1 (early osteoclast precursors), day +8 (preosteoclasts) and day +21 (osteoclasts). To evaluate the effects of both drugs considering osteoclast differentiation and -function, 2 sub-apoptotic doses of bortezomib, one sub-apoptotic and one low-apoptotic dose of PS-1145 were used. As revealed by the microscopic quantification of mature osteoclasts (TRAP-positive and multi-nucleated cells), the osteoclast differentiation was diminished by both drugs, whereas the effects were dose- and time-dependent. The microscopic quantification of resorption lacunae on dentine pits revealed that the resorptional activity was reduced by 65% for 0.1 nM bortezomib (p=0.007), by 79% for 1 nM bortezomib (p<0.0005), by 60% for 1 μM PS-1145 (p=0.023) and by 91% for 10 μM PS-1145 (p<0.0005). As shown by immunoblotting and by ELISA-based methods, the subcellular mechanisms of action involved in inhibition of early osteoclast differentiation were found to be related to the inhibition of p38 mitogen-activated protein kinase (MAPK) pathways, whereas the advanced differentiation and activation occurred in course of inhibition of AP-1 and NF-κB activation. The AP-1 blockade contributed to significant reduction of osteoclastic vascular endothelial growth factor (VEGF) production. In conclusion, our data demonstrate that proteasomal inhibition should be considered as a novel therapeutical principle of cancer-induced lytic bone disease.
Characterization of Intercellular Junctional Complexes between Human Hematopoietic and Mesenchymal Stem Cells.
Authors: Wuchter Patrick , Saffrich Rainer , Straub Beate , Boda-Heggemann Judit , Miesala Katrin , Diehlmann Anke , Wagner Wolfgang , Seckinger Anja , Krause Ulf , Franke Werner W. , Ho Anthony D.
Published: April 2020

The interaction between hematopoietic stem cells and their niche is essential for the balance between self-renewal and differentiation. We previously demonstrated that intercellular connections in mesenchymal stem cells (MSC) are realized by occasional gap junctions and frequent adherens junctions, comprising specific cadherin-catenin-complexes. Using MSC-feeder-layer as a surrogate model for the hematopoietic stem cell (HSC) niche, we have analyzed the intercellular junctional complexes between HSC and MSC.

MSC were obtained from bone marrow aspirates from healthy voluntary donors. HSC were isolated from umbilical cord blood. Using advanced confocal laser scanning in combination with deconvolution and volume rendering software, we were able to produce 3D-images of intercellular junctions between HSC and MSC. We used a panel of antibodies specific for various components of tight, gap and adherens junctions. Additionally, we compared the data to human and bovine bone marrow tissue in situ.

We could show that intercellular connections between HSC and MSC are mainly realized by podia formation of the HSC linking to the adjacent MSC. These podia vary greatly in length and shape (uropodia, filopodia). Along these podia and especially at the contact zone to the MSC, we have identified the cytoplasmic plaque proteins alpha- and beta-catenin and protein p120ctn, as well as the transmembrane glycoprotein N-cadherin.

This study provided solid evidence for the direct and intimate cellular interaction of HSCs with their niche. Direct cell contact represents a key factor for the regulation of self-renewal versus differentiation. The examination of the specific function of catenins, p120ctn and N-cadherin in this process is concurrently underway.
Metalloproteinase Regulation Determines Integrin αIIbβ3 Function and Enhances the Capacity of ES Cell-Derived Platelets.
Authors: Nishikii Hidekazu , Eto Koji , Kanaji Taisuke , Ware Jerry , Nakauchi Hiromitsu
Published: April 2020

To resolve the lack of sources for products used in blood transfusion therapy, the utilization of embryonic stem cells (ESCs) has been proposed. However, the use of ESC-derived cells has potential drawbacks including, immunological rejection and teratoma formation. Being fully differentiated cells lacking a nucleus, platelets can be irradiated to eliminate passenger ESCs and other lineages before transfusion. We previously established in vitro differentiation system whereby proplatelet-producing megakaryocytes can be derived from murine ESCs on OP9 stroma, but the platelets were generated with a low efficiency and exhibited weak agonist-induced integrin αIIbβ3 activation and spreading on immobilized fibrinogen. Here, we describe an improved method to obtain ESC-derived platelets of potential use for hemostasis and thrombosis in vivo. We generated a novel murine ESC line wherein the GPIbα promoter regulates EGFP expression as a marker (GP-G ESCs). The combination of flow cytometry with GP-G-derived embryoid bodies and OP9 co-culture enabled us to enrich integrin αIIb + c-kit + CD9 + Sca-1 − progenitors on day 5–6 of culture as a major fraction capable of differentiating into GFP+ mature megakaryocytes on day 8–10 in the presence of TPO. This strategy increased platelet production by 10-fold (P<0.01) compared to our previous report (PNAS, 2002, 99:12819). Interestingly, αIIb+platelets included 35–40% GPIbα-negative population besides GPIbα-positive on day 13–15, which was caused by an increased activity of matrix metalloproteinase (MMP) and TACE (ADAM17) to shed GPIbα in culture. Inhibition of MMP and TACE with GM6001 or TAP1 delayed the clearance rate on post transfusion recovery in vivo as transfused into a platelet-depleted mouse model. Maintaining GPIbα expression in the presence of MMP/TACE inhibitors restored the specific fibrinogen-bound to integrin αIIbβ3 and platelet spreading on fibrinogen as evidenced by the generation of filopodia and lamellipodia. Collectively, these results suggest that 1) ESC-derived αIIb+ c-kit + CD9+ Sca-1 − progenitors may contribute to enrich megakaryocytopoiesis at high efficiency; 2) inhibition of metalloproteinase activity to retain GPIbα expression is required for αIIbβ 3 integrin inside-out and outside-in signaling in ESC-derived platelets; and 3) these platelets may be useful alternatives for future clinical application.
The Expression of AML1-ETO9a Isoform in Acute Myeloid Leukemia M2 Subtype and Its Clinical Significance.
Authors: Miao Yu Q. , Chen Zi X. , Cen Jian N. , Yan Ming , He Jun , Qiu Qiao Ch. , Zhang Dong E.
Published: April 2020

AML-ETO (AE) fusion protein caused by t(8;21) is believed to be involved in the development of AML. Most recent studies using a mouse models indicate that expression of a truncated version of AE by alternative splicing, namely AML1/ETO9a (AE9a), may be more closely related to development of leukemia. To validate the hypothesis, we have studied the expression of AE9a in AML-M2 patients and its prognostic value. The Expression of AE fusion gene and AE9a was detected by using RT-PCR in 40 cases of newly diagnosed patients and 30 cases in complete remission. The expression pattern of these fusion genes in M2 patients was compared with that in other subtypes of AML, MDS and healthy controls. In addition, we have also examined the expression level of AE and AE9a in 40 cases of M2 patients during follow-up and analyzed the correlation with clinical manifestations. Our Results demonstrated that 19 of 70 cases of AML-M2 patients simultaneously expressed AE and AE9a while 10 cases expressed only AE; 2 cases expressed AE9a alone; the rest expressed neither. 19 of 40 cases of newly diagnosed patients expressed AE9a (47.5%). Neither 30 AML-M2 cases in complete remission nor the other subtypes of AML, Myelodysplastic syndrome (MDS) and healthy controls expressed AE9a. After treatment by 1–3 course of conventional chemotherapy (MA, DA, DA or AAG regimen), 4 of 19 AE9a positive cases attained complete remission (21%), 7 cases relapsed after relieved (36.8%), 3 cases died after transplantation, 3 cases failed to attained remission and gave up(15.8%), 2 cases lost connection after transplant. While after same conventional chemotherapy, 17 of 21 AE9a negative cases (81%) attained remission, 3 cases(14.3%)relapsed after relieved, 1 case (4.8%) died of giving up treatment after attaining remission. These results confirmed that AE9a was closely related to AML-M2, and it may promote the development of leukemia in combination with AE fusion gene. The remission rate in AML1/ETO9a positive patients is remarkable lower than that in negative’s. Thus AE9a expression may indicate an unfavorable prognosis of AML-M2 patients.
Estradiol Is Concentrated in Human Platelets and Is Released upon Activation.
Authors: Sarabia Stephen F. , Raya Joe L. , Hoogeveen Ron C. , Bray Paul F.
Published: April 2020

Background and Aim: Estrogens are prothrombotic in both normal and atherosclerotic vessels. There is evidence that exogenous estrogen modifies platelet reactivity and signaling pathways, but very little is known about sex hormone metabolism in platelets or whether platelet estrogen stores contribute to atherothrombosis. To begin to characterize the role of sex hormones in platelet biology, we measured estradiol (E2), estrone (E1) and testosterone (T) concentrations within platelets and plasma of normal healthy men and women. Additionally, we tested the hypothesis that platelets have the capacity to release E2 during thrombin-stimulated platelet aggregation and activation.

Methods and Results: Platelets and plasma were prepared from platelet-rich plasma (PRP) samples of 16 male (age 24–63), 13 premenopausal female (age 25–34) and 8 menopausal female (age 39–65) donors; no females were using exogenous hormones. E2, E1 and T were determined by Gas Chromatography-Mass Spectrometry. Hormone content in platelets and plasma was expressed per mL of PRP. Plasma E2, E1 and T levels were within the range of values previously reported in the literature. E2 concentrations were significantly higher in platelets compared to plasma in men (p<0.01) but not in women (premenopausal p=0.33; menopausal p=0.16). The geometric mean and 95% CI for platelet and plasma E2 levels were 199 (114–349) and 55 (37–81) pM for men, 360 (154–831) and 235 (158–357) pM for premenopausal women, and 107 (52–224) and 40 (15–118) pM for menopausal women, respectively. Conversely, T concentrations were significantly lower in platelets than in plasma in both men and women (p<0.01). E1 levels in platelets and plasma were not significantly different within men and within women. In a subset of participants, PRP samples were incubated with 0.5 U/mL thrombin for 10 minutes at 37oC and estradiol levels were measured in the non-clotted plasma+platelet releasate. Compared to no thrombin, thrombin-treated PRP from male subjects (n=7) yielded a 1.6 to 6.7 fold increase in E2 levels (p<0.01). The incubation of PRP from premenopausal (n=5) and menopausal women (n=4) with thrombin did not produce a significant change in E2 release.

Conclusion: Our results show that platelets have the capacity to concentrate E2 and may serve as a vascular vehicle for the delivery of this prothrombotic factor upon platelet activation.
Regulation of Homocysteine Transport in Vascular Cells.
Authors: Jiang Xiaohua , Yang Xiao-feng , Brailoiu Eugen , Jakubowski Hieronim , Schafer Andrew I. , Durante William , Wang Hong
Published: April 2020

Increased levels of plasma homocysteine is an independent risk factor for cardiovascular disease and has cell-type distinct proatherosclerotic effects on vascular cells. In this study, we characterized L- homocysteine transport in cultured human aortic endothelial and aortic smooth muscle cells. L-homocysteine was transported into vascular cells in a time-dependent fashion. L-homocysteine transport activity was about 2-fold higher in aortic smooth muscle cells. In addition, L-homocysteine transport in both cell types was mediated by sodium-dependent and independent carrier systems. Competition studies revealed that the neutral amino acids cysteine, glycine, serine, tyrosine, alanine, leucine, and methionine, and inhibitors of the cysteine transport systems inhibited L-homocysteine uptake in both cell types, but the inhibition was greater in endothelial cells. Eadie-Hofstee plots demonstrated that L-Hcy transport in endothelial cells had a Michaelis constant (Km) of 79mM and a maximum transport velocity (Vmax) of 873 pmol/mg protein/min. In contrast, homocysteine transport in aortic smooth muscle cells had a lower affinity (Km=212mM) but a higher transport capacity (Vmax=4192 pmol/mg protein/min). Interestingly, increases in pH (pH 6.5–8.2) only inhibited L-homocysteine uptake in endothelial cells. Moreover, L-homocysteine transport in endothelial cells was partially inhibited by lysosomal inhibitors. Our studies indicate that L-homocysteine shares transporter systems with cysteine and can be inhibited for transport by multiple neutral amino acids in vascular cells, and that L-homocysteine transport involves lysosomal transport in endothelial cells. The specific lysosomic feature of L-homocystein transport in endothelial cells may contribute to cell type specific growth inhibitory effects and therefore play a role in homocysteine atherogenic potential.
Role of Activated Platelets in the Homing, Maturation, and Differentiation of Endotheleal Progenitor Cells (EPCs) to Vascular Subendotheleal Matrix.
Authors: Lev Eli I. , Dong Jing-fei , Bujak Marcin , Aboulfatova Khatira , Kleiman Neal S. , Harris David , Estrov Zeev
Published: April 2020

We and others have found that platelets play an important role in the recruitment of endothelial progenitor cells to sights of vascular injury. However, it is not clear whether the EPCs mature and differentiate to endothelial cells following recruitment to the vascular injury sites. In addition, there is limited in vivo data to support the role of EPCs in re-endothlialization following vascular injury. We conducted in vitro experiments to investigate the maturation of EPCs on platelet based-media and in vivo experiments to evaluate the recruitment of EPCs following vascular injury. In in vitro experiments human EPCs were isolated from donated buffy coats by magnetic microbeads and flow cytometry cell sorting using CD133 and VEGFR-2, respectively, as cell markers. Isolated viable EPCs (CD133+, VEGFR-2+ cells) were plated on human fibronectin or a monolayer of washed human platelets. Cell colonies were counted 7 days after plating and stained for the endothelial cell markers CD31 (PECAM-1) and CD144 (VE-cadherin). The mean number of colony-forming cells was 35±2.6 colonies/106 cells on platelets, which was significantly higher than 18±4.2 colonies/106 cells on fibronectin (n = 4, P<0.01). Apart from the difference in colony numbers, the EPC colonies grew faster on the platelet substrate, were larger, and had more spindle-shaped cells (Figure 1 - staining of EPC colonies for CD31 and CD144). In the in vivo experiments a model of transluminal injury to mouse femoral arteries was used. Femoral artery denudation was performed by 0.25-mm-diameter angioplasty guidewire. Injured femoral arteries were compared to the contra-lateral controls (uninjured), and were harvested 1.5 hours following the injury and immunostaining performed with an anti-VEGFR-2 antibody. Four experiments showed a markedly higher number of VEGFR-2+ cells in the artery that has undergone denudation. These experiments indicate that a media composed of platelets promotes the maturation and differentiation of EPCs. Furthermore, in vivo, EPCs are recruited early following vascular injury. Thus, homing, maturation, and differentiation of EPCs are mediated by platelets.
Upshaw-Schulman Syndrome Revealed by Pregnancy.
Authors: Seike Masako , Kozuka Teruhiko , Kokame Koichi , Miyata Toshiyuki , Matsuyama Tomomi , Matsumoto Masanori , Fujimura Yoshihiro , Hara Masamichi
Published: April 2020

Background:

Thrombotic thrombocytopenic purpura (TTP) is a rare systemic disease. Its mechanism has been unclear for yeas. Recently, it has been reported that the deficiency of ADAMTS 13 (a disintegrin-like domain, and metalloprotease, with thrombospondin type1 motif 13) activity cause TTP. Congenital TTP (Upshaw-Schulman syndrome: USS) is caused by ADAMTS 13 gene mutation and secondary TTP is caused by anti-ADAMTS 13 antibody. Only five families of USS have been reported in Japan, previously. Here, we report a case of USS patient who has gene mutations.

Case report:

On 23 weeks of gestation, a Japanese pregnant woman was pointed out thrombocytopenia (8.2 x 10E7/mL), and was diagnosed as idiopathic thrombocytopenic purpura. On 31 weeks of gestation, she developed headache, vomiting, high grade fever, disorientation and additional vanishing fetal movement. Unfortunately, intrauterine fatal death was confirmed. The diagnosis of TTP was confirmed, plasma exchange, the administration of methylprednisolone, dipyridamole and high-dose intravenously immunoglobulin was started. She has recovered day by day and the stabilization of platelet count was obtained. Her ADAMTS 13 activity on admission day was lower than detection limit (< 3%), and her father, mother and sister’s low ADAMTS 13 activity (47.4%, 35.8% and 34.8%, respectively) was proved later. Because ADAMTS 13 inhibitor was not detected, she was diagnosed as USS and preventive FFP infusions were started. In accordance with the increase of ADAMTS 13 activity, the platelet count also has a tendency to increase. She has performed regular FFP infusions every two weeks at this time of reporting. This family’s ADAMTS 13 gene mutations were analyzed by DNA sequencing. Patient has compound heterozygote mutations; R193W from father and A606P from mother. Sister has Q606A too, and brother has normal gene.

Conclusion:

Clinical signs of TTP are often masqueraded as a simple thrombocytopenia until 20-30 weeks of gestation. USS is a life threatening disease to both mother and fetus, an early diagnosis is critical. Therefore, pregnant women developed thrombocytopenia need to be determined ADAMTS 13 activity routinely.
Childhood ITP: A Single Institution Review of Treatment and Outcomes.
Authors: Houser Laura , Luchtman-Jones Lori
Published: April 2020

Immune thrombocytopenia purpura (ITP) is diagnosed in approximately 50 per 1 million children per year. Although thrombocytopenia can be associated with serious, life-threatening bleeding, the outcome for most patients with ITP is excellent. Approximately 80% of children with ITP will recover by 6 – 12 months from diagnosis. Large randomized, controlled trials have not been done to demonstrate the optimal management strategy, either for safety or efficacy. Some patients are managed with observation, given the benign ultimate outcome of most cases, but, because of existing or potential bleeding, the majority of patients with severe thrombocytopenia receive treatment with glucocorticoids, IVIG, or anti-D globulin. At our institution, we do not have a standardized strategy for management of patients with childhood ITP. We performed a 10-year retrospective review of our institutional experience with childhood ITP. The approximately 400 eligible patients were aged 0.5 to 18 years, and were managed between January 1996 and January 2006 at St. Louis Children’s Hospital or Washington University Pediatric Hematology/Oncology Clinic. Data from the medical records of these patients was collected and will be analyzed to determine the following primary endpoints:

Time to reach target platelet count, stratified by initial therapy type; Rate of re-treatment and/or addition of second therapy over the first 28 days, stratified by initial therapy type and dosage; Incidence of treatment-related toxicities, Incidence of hemorrhage, anemia and/or transfusion, stratified by therapy choice, Rates and duration of hospitalization, Time to resolution of normal platelet count, stratified by initial therapy.

The management and outcome of patients without platelet recovery by 6 and 12 months (i.e. chronic ITP) and will be detailed. Results from this study will be used as a framework for developing institutional guidelines for management of childhood ITP.
The Role of GBPIalpha in Phagocytosis of Platelets by Macrophages.
Authors: Badlou Bahram Alamdary , Spierenburg Gerit , Ulricht Hans , Deckmyn Hans , Smid W. Martin , Akkerman J.W.N. , Nieuwenhuis H. Karel
Published: April 2020

Platelet storage at 04 C suppresses bacterial multiplication, but induces clusters of glycoprotein (GP) Ibalpha that trigger their phagocytosis by macrophages and reduce their survival after transfusion. We searched for a method that detects cold-induced changes in GPIbalpha involved in phagocytosis.

STUDY DESIGN AND METHODS: Human platelets were isolated and stored for up to 48 hrs at 0C. Binding of a PE-labeled antibody directed against amino acids 1–35 on GPIbalpha (AN51-PE) was compared with phagocytosis of platelets by matured monocytic THP-1 cells, analyzed by FACS.

RESULTS: Freshly isolated platelets were detected as a single population of AN51-PE positive particles and showed < 5% phagocytosis. Cold storage led to a decrease in AN51-PE binding and an increase in phagocytosis. N-acetylglucosamine (GlcNAc), known to interfere with macrophage recognition of GPIbalpha clusters, restored normal AN51-PE binding to cold-stored platelets and suppressed phagocytosis.

CONCLUSIONS: We conclude that binding of an antibody against AA 1–35 on GPIbalpha reflects changes in GPIbalpha that make platelets targets for phagocytosis by macrophages.
Double Knockouts Reveal That Protein Tyrosine Phosphatase 1B Is a Physiological Substrate of Calpain-1 in Platelets.
Authors: Kuchay Shafi M. , Fay William P. , Chishti Athar H.
Published: April 2020

Calpains are ubiquitous calcium-regulated cysteine proteases that have been implicated in cytoskeletal organization, cell proliferation, apoptosis, cell motility, and hemostasis. Previously we used gene-targeting to evaluate the physiological function of mouse calpain-1, and established that its inactivation results in reduced platelet aggregation and clot retraction, potentially by causing dephosphorylation of platelet proteins. Here, we present data showing that calpain-1 null platelets accumulate protein tyrosine phosphatase 1B (PTP1B) that correlates with enhanced tyrosine phosphatase activity and dephosphorylation of multiple substrates in platelets. Using antibodies specific for phosphotyrosines 747 and 759 of the b3 subunit of αIibβ3 integrin, we show that the tyrosine phosphorylation of both tyrosine residues at positions 747 and 759 in the cytoplasmic domain of b3 subunit is reduced by approximately 60–70% in the calpain-1 null platelets. Treatment of calpain-1 null platelets with DMHV, an inhibitor of tyrosine phosphatases, corrected the aggregation defect and recovered impaired clot retraction. Importantly, platelet aggregation, clot retraction, and tyrosine dephosphorylation defects were rescued in the double knockout mice lacking both calpain-1 and PTP1B. Consistent with this paradigm, treatment of wild type mouse platelets as well as human platelets with the tyrosine phosphatase inhibitor DMHV enhanced their aggregation at low doses of thrombin. Conversely, MDL, a cell permeable inhibitor of calpains, potently inhibited aggregation of wild type mouse platelets in a dose-dependent manner upon thrombin activation. Further evaluation of mutant mice by ferric chloride induced arterial injury model suggests that the calpain-1 null mice are relatively resistant to thrombosis in vivo. Finally, the calpain-1 mediated regulation of PTP1B appears to be a systemic event as evident by the enhanced tyrosine dephosphorylation of B lymphocytes and their resistance to apoptosis in calpain-1 null mice. Together, our results demonstrate that PTP1B is a physiological substrate of calpain-1 and suggest that a similar mechanism may regulate calpain-1 mediated tyrosine dephosphorylation in other cells.