bluuebunny/crossref_metadata_2025 huggingface dataset:
Regulation of the PU.1 Gene by Sense and Functional Antisense RNAs Generated through the Same Chromatin Architecture.
Authors: Ebralidze Alex , Zhang Pu , Rosenbauer Frank , Huang Gang , Steidl Ulrich , Dayaram Tajhal , Guibal Florence , Klupp Johanna , Neel Dana , Bonifer Constanze , Tenen Daniel G.
Published: April 2020

The transcription factor PU.1 is an important regulator of hematopoiesis and correct expression levels in specific lineages are critical for normal hematopoietic development. Specifically, PU.1 is maintained or upregulated in specific lineages, and failure to downregulate PU.1 in other lineages can lead to a block in development of that lineage and/or leukemia. In vivo expression of PU.1 is dependent on an upstream regulatory element called the URE. Disruption of the URE leads to downregulation of PU.1 and development of leukemia and lymphoma, but the other distal elements regulating PU.1 have not been defined. Here we show that other phylogenetically conserved elements participate in the initiation of antisense transcription, and that these antisense RNAs function as important modulators of proper dosages of PU.1. Specifically, antisense transcripts originate from specific conserved sites in introns 1 and 3, and that the intron 3 site contains binding sites for transcription factors such as AML1 and Ets factors. The conserved intron 3 element also possesses anti-sense promoter activity. These antisense transcripts are present at about 15% of PU.1 sense transcripts in PU.1 expressing cells. They negatively regulate PU.1 sense RNA, as introduction of siRNA molecules which specifically target antisense transcripts lead to 3–5 fold increases in PU.1 sense RNA and protein. Both sense and antisense PU.1 gene RNAs are dependent on the URE and are transcribed from the same chromatin architecture, in which the conserved elements, including URE and sense and antisense promoters are located in the same nuclear fraction and can be shown to exist in the same nucleoprotein complex by chromosome conformation capture (3C). We are currently testing the mechanisms involved in the formation of such complexes, and specifically whether the complexes are mediated by binding of AML1 to the URE. Since we do not observe significant differences in antisense transcript levels between PU.1 high-expressing and PU.1 low-expressing cells, we hypothesize that the function of these antisense transcripts is to modulate rather than absolutely control PU.1 levels: in PU.1 high-expressing cells, such as myeloid cells, the antisense transcripts trim PU.1 levels to prevent overexpression, while in cell types in which PU.1 is not expressed, such as T cells, the antisense transcripts prevent any expression of PU.1. We propose that such a mechanism will likely be important in fine-tuning the regulation of many genes and may be the reason for the large number of overlapping complementary transcripts with so far unknown function.
Ferritin Heavy Chain Stimulates HbS-to-HbF Switching in Erythroid Precursor Cells from Sickle Cell Patients.
Authors: Broyles Robert H. , Belegu Visar , Roth Austin C. , Clarkson Emily J. , Williamson Kelly S. , Stewart Charles A. , Pye Quentin N. , Floyd Robert A. , Jani Klodiana , Trudel Marie , Santambrogio Paolo , Levi Sonia , Arosio Paolo , Cain Joan Parkhurst
Published: April 2020

We have found that ferritin heavy chain (FtH), an antioxidant/stress response/iron-storage protein, localizes to the nucleus in K562 cells and represses the human adult beta-globin promoter in transient assays in primate cells (Broyles et al., PNAS98: 9145, 2001). Since other work indicates FtH is also a gene activator of fetal-globin genes, we hypothesize that FtH is a long-sought developmental hemoglobin (Hb) switching factor and that delivery of FtH to human adult erythroid cell precursors will reverse the phenotype to HbF, offering a phenotypic cure for sickle cell disease (SCD). Chromatin immunoprecipitation (ChIP) assays, antisense treatments, and an FtH transgenic mouse have confirmed that FtH is a globin gene regulatory protein in vivo. With erythroid precursor cells from pediatric SCD patients, under an IRB-approved protocol, we have used a two-phase culture system for in vitro maturation of erythroid cells in the presence of FtH, delivered to the cells as pure protein, as an expression plasmid, or as a priority inducer compound that activates the endogenous FtH gene. HPLC with a PolyCAT A column was used to separate and quantify human Hbs. With each mode of delivery, FtH stimulated a complete switch from HbS to HbF. This result was repeatable in multiple experiments using erythroid precursor cells from three different SCD donors. Fluorescently-labeled recombinant human FtH protein was taken into red cell precursors in culture, suggesting that the purified protein can be directly delivered without gene therapy. This method of producing a phenotypic cure in SCD patients should be easy and inexpensive to deliver in vivo.
Survivin Is Required for Terminal Differentiation of Erythroid Cells as Well as for the Proliferation and/or Survival of Hematopoietic Progenitors.
Authors: Crispino John D. , Leung Cindy , Xu Yanfei , Mularski Bretton , Liu Hui , Gurbuxani Sandeep
Published: April 2020

Survivin, the smallest member of the Inhibitor of Apoptosis (IAP) family of proteins, is expressed in proliferating cells, including fetal tissues, T-lymphocytes and hematopoietic progenitors. Survivin is also highly overexpressed in a wide spectrum of human cancers. Numerous studies have implicated survivin as a key mediator of the spindle assembly checkpoint and cytokinesis, while others have suggested an additional role as an inhibitor of apoptosis. In order to precisely examine the role of survivin in hematopoiesis in vivo, we induced acute deletion of the survivin gene in mice harboring either heterozygous or homozygous floxed alleles of survivin and the MX1-Cre transgene with polyinosinic-polycytidylic acid (pI-pC). Complete loss of survivin in homozygous floxed, MX1-Cre animals resulted in rapid death, which was associated with near total ablation of the bone marrow and marked hypocellularity of the spleen. The peripheral blood of these mutants displayed pancytopenia, including reticulocytopenia, and the presence of abnormal erythrocytes. Hematopoietic progenitors were markedly decreased, including c-kit+ cells, as well as CD71hiTer119hi erythroid precursors. Analysis of the cell cycle parameters revealed that a significant proportion of erythroid progenitors accumulated in G1, while others became polyploid. These defects could not be rescued by overexpression of the anti-apoptotic protein Bcl-2, consistent with a primary function of survivin in cell cycle progression. Bone marrow transplantation studies further confirmed that there exists a cell autonomous requirement for survivin in hematopoietic progenitors. Surprisingly, acute heterozygous deletion of survivin led to defects in erythropoiesis in approximately 20% of the animals. Upon pI-pC treatment, affected survivin heterozygous floxed mice with the MX1-Cre transgene exhibited a striking decrease in CD71−Ter119hi cells at the terminal stages of maturation. Morphological analysis of erythroid cells from neonatal spleens of these affected mice revealed a dramatic reduction in enucleated erythrocytes in the CD71−Ter119hi population compared to control animals. In addition, the majority of these erythroid cells were megaloblastic, with less condensed nuclear chromatin and nuclear-cytoplasmic dysynchrony. These observations suggest that there is a dose dependence for survivin. We predict that haploinsufficiency of survivin specifically affects “differentiation divisions” that occur concomitant with terminal maturation of red blood cells. Taken together, we show that homozygous deletion of survivin results in a dramatic loss of hematopoietic progenitors and rapid death, while heterozygous deletion leads to a selective defect in red blood cell development.
Transcriptome Classification of B-Cell Non-Hodgkins Lymphoma.
Authors: Aggarwal Mohit , Villuendas Raquel , Al-Shahrour Fatima , Aguilera Abel S. , Martinez Nerea , Ballesteros Elena R. , Camacho Francisca I. , Perez Alberto , Artiga Maria J. , Garcia-Marco Jose A. , Dopazo Joaquin , Piris Miguel A.
Published: April 2020

B-cell lymphomas are presently diagnosed according to the WHO criteria based on morphologic, immunophenotype and cytogenetic findings. However, the precise distinction among common lymphoma types is frequently a difficult task, and there are areas of overlapping and heterogeneity between them. Here we have analyzed whether gene expression profiling (GEP) data, solely considered, could be used to validate the currently used B-cell lymphoma classification, or proposing new lymphoma types, and for identifying functional signatures or genes defining these GEP-based lymphoma classification.

To this aim, we collected Gene Expression Profiling (GEP) for 173 cases of B-cell NHL, including BL (9), DLBCL (36), MALT (3), MCL (20), CLL (38), FL (33), MZL (6) and SMZL (29). The gene expression data for lymphoma cases was normalized against an average gene expression of reactive lymph nodes, except the SMZL which was normalized against normal spleen (3 cases). The analysis of the cases was done using Cluster Accuracy Analysis Tool (CAAT) (Cunningham P., 2005), that enabled us not only to compare gene expression between each node starting from root but also to identify new classes within existing lymphoma diagnosis defined by an internal validation method called Silhouette Width index (Julia Handl. et al, 2005). Using this approach each cluster could be represented by so called silhouette, which is based on the comparison of its tightness and separation. The average silhouette width could be applied for evaluation of clustering validity and can also be used to decide how good the number of selected clusters is.

Using this approach, we obtained the following categorization of lymphoma cases:

Figure Figure

Using T-Rex (Herrero J & Dopazo J., 2005) to compare differential expression between the categories obtained by CAAT, and FatiScan analysis (Al-Shahrour, F., 2006), we identified genes that were differentially expressed between molecular categories of lymphoma types, assigning them to Gene Ontology (GO) and KEGG (Kyoto Encyclopedia of Genes and Genomes)-defined pathways. The functional signatures that were identified as distinguishing between these lymphoma types were defining cell cycle, cytokine-cytokine receptor interaction, T-cell receptor, B-cell receptor, cell adhesion, NF-kB activation, and other significant interactions. Comparison between these lymphoma clusters following this definition yielded large number of genes distinguishing them, this list including already known genes and a large number of new potential markers.
Expression Levels of Glutathione Peroxidase 1 (GPx1) and Multi-Drug Resistance 1 (MDR1; ABCB1) Identify Patients with Diffuse Large B-Cell Lymphoma (DLBCL) That Have Superior Clinical Outcomes.
Authors: Andreadis Charalambos , Gimotty Phyllis , Hammond Rachel , Wahl Peter , Houldsworth Jane , Schuster Stephen , Rebbeck Timothy
Published: April 2020

Background: Standard chemotherapy fails in 40–50% of patients with DLBCL. Some patients can be salvaged with high-dose regimens, suggesting a role for drug resistance in this disease. While genes in the glutathione (GSH) pathway and ATP-dependent transporter (ABC) family have been implicated in drug resistance in other malignancies, their role in lymphoma, and DLBCL in particular, remains unclear.

Methods: We examined expression of 21 key members of these families in two tissue-based expression-microarray datasets of DLBCL patients, prior to CHOP chemotherapy. Expression signals derived from Affymetrix MAS 5.0 software (SF/NF=1) were publicly available for the primary dataset (Monti et al, Blood2005; 105: 1851; http://www.broad.mit.edu/cgi-bin/cancer/datasets.cgi) and were independently generated for the validation dataset (Houldsworth et al, Blood, 2004; 103: 1862). Univariable analysis was performed for freedom from progression (FFP) using a linear-effects Cox proportional-hazards model. Recursive partitioning with CART (Salford Systems, San Diego, CA) was used to develop a classification tree for the 2-year freedom-from-progression rate (FFP2) based on median-adjusted gene expression.

Results: In univariable analysis, expression of GPx1 had the most significant adverse association with FFP in the primary dataset (n=130), hazard ratio (HR) 1.68 (95% CI: 1.26 – 2.22, p<0.001). This effect remained highly statistically significant after controlling for the Monti biological signature, the LLMPP cell of origin signature, and the IPI score, and was confirmed in the validation dataset (n=39) (HR 1.5, 95% CI: 1.01 – 2.3, p=0.048). None of the ABC transporters examined were associated with FFP in the univariable analysis. Recursive partitioning identified a group of patients (n=34) with low-level expression of GPx1 and ABCB1 that had no early failures (FFP2: 100%, 95% CI: 90% – 100%). Notably, this group was comprised of patients in all IPI and LLMPP signature subgroups and achieved superior long-term FFP compared to the entire cohort (p<0.001).

Conclusions: Overall, our findings suggest an important role for redox-stress defense and drug elimination in the treatment failure of DLBCL and identify GPx1 and ABCB1 as potentially powerful biomarkers of early response and response duration. Coordinate expression of these genes delineates a subgroup of patients with superior clinical outcomes regardless of other clinical and biological risk stratification in current use. Prospective, treatment-based studies with uniform inclusion and response criteria are needed to validate these markers in clinical practice.
T Cell Lymphopenia in Rhoh−/− Mice Results from Combined Defects of T Cell Migration and IL-7-Regulated Naïve T Cell Homeostasis.
Authors: Li ShuShun , Hildeman David A. , Grimes H. Leighton , Williams David A. , Gu Yi
Published: April 2020

The Rho family GTPases are increasingly implicated for their important roles in T cell development and function. We have found that RhoH, a hematopoietic-specific member of this family is essential for thymocyte development (Gu et al, Nat Immunol, in press). Further, Rhoh−/− mice showed T cell lymphopenia. In particular, naïve T cells were reduced in secondary lymphoid organs and blood both in unchallenged and LCMV-challenged Rhoh−/− mice compared to WT controls. These findings suggest a possible defect in T cell emigration from thymus and peripheral T cell homeostasis in Rhoh−/− mice. To study the role(s) of RhoH in T cell migration and homeostasis, purified splenic T cells were adoptively transferred into common γ−/−; Rag2−/− mice. T cells were then recovered from spleens 3d and 8d post-transplantation. The recovery of Rhoh−/− T cells was decreased by 60% compared with WT cells. In vitro migration of Rhoh−/− T cells towards SDF-1α, a homeostatic chemokine for T cells, in a transwell migration assay was significantly reduced compared to WT cells. Flow analysis showed decreased number of CXCR4 expressing and reduced expression levels of CXCR4 on Rhoh−/− T cells. Furthermore, apoptotic T cells were increased twofold in Rhoh−/− mice compared to controls. CFSE staining of adoptively transferred T cells demonstrated a comparable proliferation rate between Rhoh−/− and WT cells in the recipient mice. Our data suggest that RhoH plays an important role in T cell homeostasis via regulating cell survival and SDF-1α-mediated migration. To further investigate how RhoH regulates T cell survival, we focused on IL-7, an essential factor for prolonged survival of naïve T cells. One way IL-7 exerts its effect is through upregulating the Bcl-2 family of antiapoptotic proteins. Our data showed that in vitro survival of Rhoh−/− T cells in response to IL-7 was impaired, and expression of IL-7Rα and Bcl-2 were both decreased in Rhoh−/− T cells. To further study a potential role of RhoH in regulation of IL-7R expression, FACsvantage sorted naïve T cells (CD4+CD44low, or CD8+CD44low) were cytokine starved overnight, then cultured with or without the addition of IL-7 for 6 hrs, and analyzed for IL-7Rα expression by flow cytometry. In WT cells, during cytokine starvation CD4 and a subset of CD8 naïve cells upregulated IL-7Rα expression, but this upregulation was reduced followed IL-7 stimulation. In contrast, Rhoh−/− T cells failed to show either up- or subsequent down-regulation of IL-7Rα in response to cytokine starvation and IL-7 exposure. These data may indicate a role for RhoH in regulating IL-7R expression in naïve CD4 T cells. Taken together, these findings suggest that RhoH is required for IL-7-mediated T cell survival and SDF-1α-mediated homing and/or emigration from thymus. Thus, deficiency of naïve T cells in Rhoh−/− mice likely results from combined defects in T cell migration and homeostasis.
Monitoring of Clopidogrel Using Multiple Electrode Aggregometry.
Authors: Calatzis Andreas , Theisen Franz , Reininger Armin J. , Spannagl Michael
Published: April 2020

A control of clopidogrel response is one proposed strategy for an improvement of anti-platelet therapy. Different methods have been evaluated for this indication. We assessed ADP induced aggregation in whole blood in healthy blood donors and patients treated with clopidogrel 75 mg qd using a new monitoring method.

Methods: Platelet function was determined using multiple electrode aggregometry (MEA) on the Multiplate analyzer (Dynabyte, Munich, Germany). This device uses a single use test cell with two separate impedance sensors, consisting of a total of 4 electrodes and has 5 channels for parallel tests. Aggregation was triggered using ADP (6.4 μM, ADPtest, Dynabyte) and using a combination of ADP (6.4 μM) and prostaglandin E1 (20 nM) (ADPtest high sensitivity = ADPtest HS, Dynabyte). The combination of ADP + prostaglandin E1 (PGE1) is used to enhance sensitivity for the effects of clopidogrel onto ADP induced platelet activation. PGE1 reduces intracellular calcium mobilisation and therefore platelet activation and acts thus synergistical to the effect of clopidogrel. For the analysis 300 μl of blood are analyzed with the addition of 300 μl of saline. Aggregation was quantified by the area under the curve in arbitrary U (1 U corresponds to 10 AU*min). Following IRB approval venous blood was collected from 120 blood donors without anamnestic intake of platelet inhibitors in the 2 weeks before the analysis and 160 patients taking clopidogrel 75 mg qd using the direct thrombin inhibitor Melagatran in a final concentration of 15 μg/ml as the anticoagulant.

Results: The distribution of the aggregation values is shown in Fig. 1. The median (min-max) was 83 (36–143) for the blood donors in the ADPtest and 68 (1–130) in the ADPtest HS. In the patient group the median (mix–max) was 30 (2–147) in the ADPtest and 10 (0–108) in the ADPtest HS. If a non-response rate of 25% is assumed, the cut-off between clopidogrel responders and non-responders would be 51 for the ADPtest and 21 for ADPtest HS.

Discussion: The comparison of the results of clopidogrel-treated patients and healthy controls (blood donors) reveals a higher sensitivity of ADPtest HS vs. ADPtest for the effects of clopidogrel onto ADP induced aggregation in whole blood. Due to the use of a direct thrombin inhibitor as the anticoagulant in our study platelet function was determined under physiological levels of ionized calcium.

For both test methods a significant proportion of patients does not show adequate reductions in their vitro platelet aggregation. Prospective trials are required to show whether a clopidogrel-non-response in this particular method is associated with an increased occurrence of arterial thromboembolism.

Fig. 1 Fig. 1.
Use of Therapeutic Dose Low Molecular Weight Heparin in Patients with Renal Insufficiency: Analysis of Anti-Xa Levels and Creatinine Clearance.
Authors: Lim Wendy , Sridhara Manasa , Wang Luqi , Kinnon Krystyna , Douketis James , Crowther Mark
Published: April 2020

Low molecular weight heparin (LMWH) is predominantly eliminated by the kidneys. In patients with severe renal impairment, use of therapeutic dose LMWH may be associated with accumulation and a resultant bleeding risk. Tinzaparin may be less dependent on renal clearance due to its higher molecular mass and greater negative charge compared to other LMWHs. The objective of this prospective cohort study was to serially measure the anti-Xa anticoagulant effect of therapeutic dose tinzaparin over 5–7 days, used for the initial treatment of venous thromboembolism (VTE) in patients with varying degrees of renal insufficiency. We present the anti-Xa results from the first 78 patients enrolled in the study, correlated with renal function. In this study, consecutive in- and outpatients with objectively confirmed VTE requiring anticoagulation were enrolled and stratified into 4 groups based on the calculated Cockcroft-Gault creatinine clearance (CrCl): > 60 mL/min, 30–60 mL/min, ≤ 30 mL/min and hemodialysis-dependent. Tinzaparin 175 IU/kg was administered subcutaneously once daily for 5–7 days or until the INR ≥ 2.0 with warfarin therapy. Trough anti-Xa levels were measured prior to the 3rd, 5th and/or 7th tinzaparin doses. Patients with anti-Xa level > 0.5 IU/mL received tinzaparin dose adjustment using a standardized nomogram. Bleeding and recurrent VTE events were recorded. The relationship between anti-Xa levels and CrCl is shown in Figure 1. Based on our predefined anti-Xa threshold of 0.5 IU/mL, 5 of 78 patients (6.4%) required dose adjustment; 1 hemodialysis dependent patient, 2 patients with CrCl < 30 mL/min, 1 patient each with CrCl 30–60 and > 60 mL/min, respectively. None of these patients developed bleeding or recurrent VTE. Among all 78 patients, 1 hemodialysis-dependent patient developed a hematoma following a traumatic line insertion, and no patients developed recurrent VTE. In conclusion, in a cohort of 78 patients with differing degrees of renal function including patients requiring hemodialysis, use of therapeutic-dose tinzaparin for the initial treatment of VTE resulted in accumulation (defined by trough anti-Xa > 0.5 IU/mL) in 6% of patients. There appears to be a weak inverse relationship between renal function and trough anti-Xa levels, but does not result in clinically significant accumulation when tinzaparin is used for up to 7 days. Further evaluation of tinzaparin in patients with severe renal insufficiency is required.

Figure Figure
Active Site-Inhibited rFVIIa (ASIS) Blocks Platelet Adhesion/Aggregation on Intact Endothelium Activated with TNFα and Shiga Toxin-1.
Authors: Grabowski Eric F. , Nestoridi Eirini , Bussel James B.
Published: April 2020

Human blood flowing over monolayers of endothelial cells (ECs) in vitro under controlled flow conditions constitutes a promising model of blood-EC interactions, especially in a system permitting real-time imaging of single platelets (1 μm resolution or better) and platelet aggregates. We have developed such a model and have used it to study platelet adhesion/aggregation on monolayers of human renal microvascular ECs grown on optically clear vinyl slides and activated with TNFα (20 ng/ml; hr 0 to 22) and Shiga toxin-1 (Stx; 10 pM; hr 18 to 22), in simulation of probable events in the childhood hemolytic uremic syndrome (HUS). Normal donor blood, collected into 4U/ml low MW heparin (dalteparin) and 10 μM mepacrine (platelet, EC, and granulocyte label), was drawn at shear rates of 270–650 sec−1 through a parallel-plate flow chamber for which one wall was one of the above slides. Such shear rates give rise to shear stresses which approximate the 20–25 dynes/cm2 estimated to exist in glomerular arterioles. Platelets, ECs, and granulocytes were imaged in real time using epifluorescence digital videomicroscopy. With activated, but not with control ECs, clusters of platelets deposit on the monolayers in strings (Fig.1, upper right, with flow from top to bottom) superposed upon ECs, for which the cytoplasm is fluorescent. The strings are tethered at the upstream end, as has been observed by others using platelet-rich plasma. Granulocytes, identifiable by size (Fig. 1, upper middle), also adhere to activated ECs. In five paired experiments, monolayer preincubation × 30 min with 50 nM active-site inactivated recombinant factor VIIa (ASIS; courtesy of Dr. Ulla Hedner, Novo Nordisk Pharmaceuticals) largely eliminated the platelet strings, while reducing the number of adherent granulocytes to zero. In control experiments, preincubation of monolayers with 100 nM of a monoclonal antibody directed against human tissue factor similarly largely eliminated both platelet strings and adherent granulocytes. In other control experiments, the use of the above concentration of low MW heparin did not by itself block fibrin formation (by immunostaining) in the boundary region near the slide-blood interface. These findings may be explained on the basis that ASIS both interrupts the tissue factor pathway of coagulation and blocks tissue factor-associated upregulation of endothelial cell E-selectin. ASIS therefore may have promise as a therapy for the prothrombotic and proinflammatory effects of childhood HUS. Studies with the TAB monoclonal antibody and ALEXA 555 are in progress to permit platelet-specific labeling and quantitation of platelet adhesion/aggregation with and without ASIS.

Figure Figure
Hematopoietic Stem Cells Regulate Niche Development.
Authors: Taichman Russell S. , Jung Younghun , Williams Benjamin , Song Junhui , Krebsbach Paul H.
Published: April 2020

Events localized to endosteal surfaces are critical for the maintenance of hematopoetic stem cells (HSCs). Here we explored whether HSCs themselves regulate their microenvironment directly by regulating cytokine expression by osteoblasts (OBs) in response to physiologic demands, or influence the developmental pattern of mesenchymal lineages and thereby indirectly modulate cytokine expression. To test the these possibilities, marrow was isolated from mice 48h after stressing the animals with a single acute bleed (removing 30% of the calculated blood volume by jugular vein venipucture) and in a second control group of non-stressed (puncture only) animals. The Sca-1(+) hematopoietic cells were co-cultured with confluent murine bone marrow stromal (BMSC) and calvarial digested OBs. The presence of HSCs stimulated the basal production of IL-6, SDF-1 and osteoclacin by OBs and BMSCs as determined by ELISA. Co-cultures of HSCs derived from the stressed group produced more IL-6, SDF-1 and osteocalcin relative to the non-stressed group. Newborn dermal fibroblasts did not respond in a similar fashion. To determine if HSCs influence the developmental pattern of the marrow, HSCs derived from stressed and non-stressed animals were separated from either murine OBs or BMSCs using TranswellR membranes and the ability of target cells to differentiate along the osteoblastic lineage was evaluated. A significant proportion of the colonies established from calvarial-derived OB cultures were able to mineralize their extracellular matrix relative whole BMSC population (CFU-OB). In the presence of HSCs, the proportion of non-mineralized (CFU-F) and mineralized colonies from the OB populations significantly increased. Further enhancement of both colony types were induced by the HSCs derived from stressed and non-stressed animals. Adherent cells derived from mixed BMSCs also responded to the presence of HSCs by increasing the generation of CFU-F and OBs relative to the No HSC groups. HSCs derived from stressed vs. the non-stressed groups of animals were better able to induce CFU-OB differentiation. Microarray of HSCs derived from stressed vs. the non-stressed groups, and QRT-PCR of highly purified HSCs (CD150(+), CD48(−), CD41(−), Sca-1(+), cKit(+)) suggested that BMP-2 and BMP-6 were responsible for the activities. Antibody neutralization studies confirmed these observations that BMP-2 and BMP-6 derived from the HSCs themselves alters the developmental pattern of the marrow microenvironment. In conclusion, cross-talk between HSCs-OBs is essential for the development of both cellular populations. These studies demonstrate that at least 2 mechanisms whereby HSCs might set up a paracrine loop with OBs to establish the HSC niche; (i) HSCs directly regulate cytokine expression by OBs in response to physiologic demands or, (ii) HSCs may influence the developmental pattern of mesenchymal lineages and thereby indirectly modulate cytokine expression in the marrow.
Impaired Maturation and Survival of T Lymphocytes, B Lymphocytes, and NK Cells in Mice Lacking Gimap5/Ian5.
Authors: Schulteis Ryan D. , Dai Xuezhi , Chu Haiyan , Edwards Brandon , Haribhai Dipica , Williams Calvin B. , Malarkannan Subramanium , Martin Hessner J. , Anne Kwitek E. , Lernmark Åke , Gorski Jack , Wang Demin , Weiler Hartmut
Published: April 2020

In biobreeding rats (BB), the lyp mutation in Gimap5/Ian5 is associated with T-cell lymphopenia and autoimmune insulinitis. To delineate the role of Gimap5 in peripheral T cell survival, we generated Gimap5-deficient mice by targeted disruption. Gimap5−/− mice are viable to adulthood, but develop fulminant autoimmune hepatitis, become moribund, and die at a median age of 12 weeks. Gimap5−/− mice are lymphopenic, displaying an overall 7-fold reduction in peripheral T cells and a 43-fold reduction in peripheral CD8+ T cells. Additionally, Gimap5−/− mice exhibit an early, complete block in both NK and NKT lineage development and a partial block in B cell commitment to T1, T2, and mature subsets. Data from lethally irradiated congenic strains reconstituted with Gimap5−/− bone marrow will also be presented. Taken together, these data indicate that Gimap5 is necessary for peripheral survival of T lymphocytes and development of B lymphocytes and NK cells. Furthermore, these data suggest that Gimap5 deficiency plays a role in the development of autoimmune pathology in the mouse.
In Vitro and In Vivo Evidence That the PP2A Inhibitor SET Regulates IFN-γ Production in Monokine-Stimulated Natural Killer Cells.
Authors: Trotta Rossana , Col Jessica Dal , Allard Jeffrey , Neviani Paolo , Santhanam Ramasamy , Mao Hsiaoyin , Becknell Brian , Yu Jianhua , Modi Aalok , Blaser Bradley W. , Perrotti Danilo , Caligiuri Michael A.
Published: April 2020

Monokines (i.e. IL-12, IL-18 and IL-15) induce natural killer (NK) cells to produce interferon-γ (IFN-γ), which is critical for monocyte clearance of infectious pathogens and tumor surveillance. To identify new regulators of IFN-γ production we performed oligonucleotide array analysis of unstimulated and IL-12- and IL-18-stimulated NK92 cells. Among the subset of mRNAs differentially regulated in monokine-stimulated cells, we found SET, a potent inhibitor of the protein phosphatase type 2A (PP2A). SET mRNA and/or protein levels were upregulated in IL-12/IL-18- and IL-12/IL-15-stimulated primary human NK cells. Interestingly, the SET protein is also selectively increased in the resting CD56bright NK subset, which is a potent producer of IFN-γ relative to the CD56dim NK subset. To determine whether SET positively regulates IFN-γ production by inhibiting PP2A activity, we employed RNAi and interfered with SET expression in NK92 cells. SET downregulation inhibited IFN-γ secretion by IL-12/IL-18, IL-12/IL-15- or IL-15/IL-18-stimulated NK92 cells. By contrast, ectopic SET expression increased IFN-γ production in monokine-stimulated NK92 and primary human NK cells. Because downregulation of SET augmented PP2A activity in NK92 cells, we sought to investigate whether pharmacologic activation of PP2A inhibits the ability of NK cells to produce IFN-γ. Indeed, suppression of IFN-γ expression and secretion was also observed upon treatment of NK92 and primary NK cells with 1,9-dideoxy-forskolin, a known inducer of PP2A activity. Accordingly, NK cells from mice treated with 1.9-dideoxy-forskolin produced less IFN-γ in response to in vivo monokine stimulation than did NK cells from vehicle-treated mice. Mechanistically, activation of PP2A by SET knock-down or 1,9-dideoxy-forskolin treatment leads to inhibition of ERK1/2, p65RelA and STAT5 activity in monokine-stimulated NK cells. Because these signaling molecules are important for IFN-γ production by monokine-stimulated NK cells, our results strongly suggest that monokine induction of SET expression in NK cells is essential for limiting PP2A activity that, otherwise, would negatively impact the ability of NK cells to produce and release optimal levels of IFN-γ.
Protein Tyrosine Phosphatase Receptor-Type O Truncated (PTPROT) Regulates SYK Phosphorylation, Proximal B-Cell Receptor Signaling and Cellular Proliferation.
Authors: Chen Linfeng , Juszczynski Przemyslaw , Takeyama Kunihiko , Aguiar Ricardo C.T. , Shipp Margaret A.
Published: April 2020

The strength and duration of B-cell receptor (BCR) signaling depends upon the balance between protein tyrosine kinase (PTK) activation and protein tyrosine phosphatase (PTP) inhibition. BCR-dependent activation of the SYK PTK initiates downstream signaling events and amplifies the original BCR signal. Although BCR-associated SYK phosphorylation is clearly regulated by PTPs, SYK has not been identified as a direct PTP substrate. In an earlier screen for genes that might contribute to the pathogenesis of diffuse large B-cell lymphoma (DLBCL), we identified and preliminarily characterized a lymphoid PTP termed PTPROt (truncated). PTPROt is a member of the PTPRO family (also designated GLEPP, PTP-ϕ, PTP-oc and PTPu2), a group of highly conserved receptor-type PTPs that are thought to function as tumor suppressor genes. In earlier functional assays, lymphoma cells overexpressing PTPROt exhibited markedly increased G0/G1 arrest, providing the first functional evidence that this PTP regulated tumor growth. We have now used PTPROt substrate trapping mutants to identify SYK as a major substrate of this tissue-specific and developmentally regulated PTP. Co-immunoprecipitation studies confirmed that SYK is also an in vivo PTPROt substrate. To examine the effects of PTPROt on SYK-mediated BCR signaling, we generated B-lymphoma cell lines that could be induced to express either wild type PTPROt (PTPROt-wt) or mutant forms of the phosphatase (D/A, C/S) following doxycycline treatment. Dox-induced overexpression of PTPROt-wt inhibited baseline and BCR-triggered SYK phosphorylation whereas the inactive PTPROt mutants had no effect. More specifically, PTPROt-wt overexpression inhibited the phosphorylation of SYK TYR352, the initial TYR residue phosphorylated upon BCR signaling. Thereafter, we showed that overexpression of PTPROt-wt inhibited BCR-induced activation of two direct SYK targets, the SHC and BLNK adaptor proteins, and downstream signaling events including calcium mobilization and MAPK/ERK activation. BCRs also transmit low-level survival signals in the absence of receptor engagement and BCR-proximal PTPs and SYK likely modulate this “tonic” BCR signaling. These observations and the basal phosphorylation of SYK TYR352 in certain B-lymphoma cell lines prompted us to examine the effects of PTPROt-wt and the PTPROt D/A and C/S mutants on B-cell lymphoma proliferation and apoptosis. PTPROt-wt overexpression, but not that of PTPROt D/A or C/S, dramatically inhibited lymphoma cell proliferation and induced apoptosis in the absence of BCR crosslinking. Taken together, these data indicate that PTPROt modulates SYK phosphorylation and tonic BCR signaling and highlights the importance of this pathway for tumor cell survival.
Hierarchy of Human B Cell Regeneration after Rituximab Therapy.
Authors: Wirths Stefan , Ruprecht Claudia , Bertoni Francesco , Kanz Lothar , Lanzavecchia Antonio
Published: April 2020

Constant immunoglobulin levels throughout life-time may be maintained by either long lived plasma cells or continuous formation of immunoglobulin secreting cells (ISC). Rituximab therapy targets CD20+ B cells, thus selectively spares B progenitor cells and plasma cells, providing an in vivo model to investigate human B cell homeostasis.

After weekly administration of rituximab in patients with follicular lymphoma, peripheral blood B cells are virtually absent (<0,01%), and no ISC are detected. In contrast, during rituximab maintenance therapy (administered in two months intervals) small numbers of B cells can be detected. These carry the phenotype of new formed plasma blasts (CD27++/CD38++/icIg+++ and Ki67+) and are identified as ISC in ex vivo ELISPOT assay.

Surprisingly, ISC detected in treated patients were the same as in healthy donors in absolute numbers, while B cells were still reduced by 100-fold. Only 4 to 6 months after therapy human naive and memory B cells were detected in peripheral blood again. Thus, regeneration of human peripheral blood B lymphocytes follows a hierarchy: Recirculating ISC recover rapidly to normal steady state levels, while B cells with naive or memory phenotype require longer time periods. This hierarchy may represent two elements within the B cells system: a primary system sustaining constant antibody levels and a secondary system providing a diverse repertoire for adaptive immune responses.
Stimulation of TNF-α Production from Monocytes by Anti-Moesin Antibodies in the Serum of Patients with Aplastic Anemia: A Novel Mechanism of Myelosuppression by Autoantibodies.
Authors: Takamatsu Hiroyuki , Feng Xingmin , Lu Xuzhang , Chuhjo Tatsuya , Okawa Katsuya , Nakao Shinji
Published: April 2020

Although aplastic anemia (AA) is a T-cell mediated disease, recent studies have revealed the presence of antibodies (Abs) specific to proteins derived from hematopoietic progenitor cells in the serum of AA patients. It is as yet unclear whether these auto-Abs play some roles in the pathophysiology of AA. We previously demonstrated that Abs specific to moesin, a membrane-cytoskeleton linker protein in the cytoplasm, were detectable in approximately 37% of AA patients. Some reports identified moesin-like molecules on the surface of blood cells such as T cells and macrophages. It is therefore conceivable that anti-moesin Ab in AA patients may bind these immune cells and modulate hematopoietic function of AA patients. To test these hypotheses, we first studied the expression of moesin on various types of blood cells using monoclonal Ab specific to moesin (clone 38/87). Flow cytometry detected the expression of the protein recognized by anti-moesin Ab on T cells and monocytes from healthy individuals, acute monocytic leukemia cells lines including U937 and THP-1, and an acute T-lymphoblastic leukemia cell line, Molt-4, but failed to detect the molecule on CD34+ cells from healthy individuals and myeloid leukemia cell lines as well as B-lymphoblastic leukemia cell lines. Treatment of THP-1 cells with phorbol 12-myristate 13-acetate (PMA)/lipopolysaccharide (LPS) augmented the expression level of moesin. To confirm the expression of the moesin-like protein on the surface of monocytic leukemia cell lines, Molt-4 and THP-1 were treated with sulfo-NHS-SS-biotin, and the cell surface proteins were isolated with avidin-fixed column, and were subjected to Western blotting and peptide mass fingerprinting. Western blotting with anti-moesin monoclonal Abs showed two clear bands of proteins (75 kD and 80 kD); an amino acid sequence compatible with moesin was confirmed in the protein eluted from the 80 kD band. Next, we purified anti-moesin Abs from AA patients’ sera using affinity chromatography with recombinant moesin protein. Western blotting showed binding of the serum-derived Abs to a fraction of surface proteins of Molt-4, U937 and THP-1. When THP-1 cells were incubated in the presence of PMA and LPS with 5 αg/ml of control IgG or anti-moesin Abs derived from an AA patient’s serum, TNF-α production from THP-1 cells stimulated by anti-moesin Abs was 1.9–2.3 times as much as that from the control culture depending on the concentration of LPS. Incubation of THP-1 cells in the presence of monoclonal anti-moesin Abs showed the similar augmentation of TNF-α production. These results indicate that anti-moesin Abs may be involved in the suppression of hematopoiesis of AA patients by stimulating TNF-α production from monocytes.
Regulation of Erythrocyte Volume by Cell Surface Associated Protein Disulfide Isomerase.
Authors: Romero Jose R. , Bicho Manuel D. , Pong Lin-Chien , Greenbowe Joel , Rivera Alicia
Published: April 2020

Acute and chronic clinical manifestations of sickle cell disease (SCD) are based on vaso-occlusion and impaired blood flow. Dense erythrocytes are believed to be important contributors to the vaso-occlusive manifestations of SCD. However, the physiological regulation of erythrocyte hydration status in SCD is not entirely clear. The Gardos channel and K/Cl cotransport are major contributors to sickle erythrocyte dehydration. Protein disulfide isomerase (PDI) on the cell surface catalyzes disulfide formation, causes redox modifications and has been observed to be up-regulated under hypoxic conditions. We now report the detection of PDI in red cells. Western blot analysis revealed a prominent band, migrating at 55 kDa, in ghost preparation from both sickle and normal erythrocytes. To evaluate the role of PDI in Gardos channel activation, we measured charybdotoxin-sensitive K+ influx in the presence of bacitracin, a well-known blocker of PDI activity. When sickle erythrocytes are exposed to bacitracin, Gardos channel activity is significantly reduced (1.4 ±0.2 to 0.8± 0.05 mmol/L cell x min, n=6, P<0.01). We also observed that Gardos channel activity was attenuated by 0.25 mM bacitracin and was maximally inhibited by 3 mM in sickle and normal erythrocytes. Similar results were observed with phenyl arsine oxide and acetyl-thyroxin, two other well-known inhibitors of cell surface PDI activity. We then studied the effects of PDI inhibition on red cell density profiles of sickle and normal human red cells. Analysis of the baseline density profile indicates that erythrocytes have a median density of 1.10 g/mL. This value significantly increased to 1.125 g/mL (n=2) after 3 h of oxygenation/de-oxygenation cycles. However, in the presence of 3 mM bacitracin, the cellular density profile markedly shifted to the left (1.098 g/mL) following deoxygenation/oxygenation. We also investigated the reductive capacity of freshly isolated human erythrocytes by the ferrocyanide-production method. The reductive capacity of normal Hb A red cells was significantly lower than in Hb S containing cells (2.8 ± 1.1 vs 5.1 ± 1.3 mmol/L cell x h, n=18, p<0.0001). Similar results were observed in Santillies and NYKO1, two transgenic mouse models of sickle cell disease, when compared to C57 mice. These results strongly implicate cell surface associated PDI in cellular dehydration and formation of dense sickle erythrocytes by activating K+ efflux via the Gardos channel and suggest that aberrant regulation of PDI activity and/or its expression may contribute to the pathophysiology of Sickle Cell Disease.
p19Arf Cooperation in Shp2-Induced Leukemogenesis.
Authors: Chen Xiaolan , Huang Yuhui , Yan Ling , Wang Zhizhang , Brault Marie , Thompson Maryann , Yang Elizabeth
Published: April 2020

Shp2 is a protein-tyrosine phosphatase which has both proliferation and apoptosis functions in hematopoietic cells. Gain-of-function (GOF) Shp2 mutations are found in Juvenile Myelomonocytic Leukemia (JMML) and Noonan syndrome patients predisposed to JMML, as well as in common childhood leukemias. Genetic data suggest that Shp2 alone is insufficient for leukemogenesis, and other genetic alterations are required, such as disruption of tumor suppressor pathways. We hypothesized that Arf deletion will enhance GOF Shp2-induced leukemogenesis. In colony forming cell assays (CFC), E76K Shp2 expression in arf−/− bone marrow cells led to increased colony number and size compared to E76K Shp2 expression in arf+/− cells or arf−/− cells with vector alone. To assay leukemia induction, arf+/− or arf−/− bone marrow cells transduced with E76K Shp2 mutant were transplanted into lethally irradiated WT C57BL/6 mice. We found that mice transplanted with E76K Shp2/arf−/− bone marrow cells developed leukemia/lymphoma at an accelerated rate compared to mice transplanted with E76K Shp2/arf+/− or vector/arf−/− bone marrow. While the most common hematological disease in mice transplanted with either E76KShp2/arf+/− or vector/arf−/− bone marrow was myeloproliferation, mice with E76K Shp2/arf−/− bone marrow developed predominantly B220+ leukemia. Compared to leukemia in E76K Shp2/arf+/− reconstituted mice, leukemias arising from E76K Shp2/arf−/− bone marrow were much more aggressive, effacing the bone marrow and spleen, infiltrating the liver diffusely and into surrounding tissue. Diseases arising from vector/arf−/− bone marrow were either localized lymphoma or mild myeloproliferation. Both E76K Shp2 expression and arf deletion were associated with the presence of BrdU-positive immature hematopoietic progenitors in the spleens of pre-leukemic mice, consistent with extramedullary hematopoiesis. These findings suggest that arf deletion cooperated with E76K Shp2’s proliferative function to accelerate leukemogenesis.
The Various Procoagulant Effects of PolyP Require Different Minimal Polymer Lengths.
Authors: Smith Stephanie A. , Huyck Jillian , Choi Sharon H. , Morrissey James H.
Published: April 2020

Introduction: Polyphosphates (polyP) are negatively charged, linear phosphate polymers found throughout biology. PolyP in prokaryotes and unicellular eukaryotes is typically hundreds of phosphate units long, while polyP in dense granules of human platelets is ∼75 phosphates long. PolyP is secreted upon platelet activation, and we recently reported that it is a potent hemostatic regulator, acting at multiple points in the clotting cascade: PolyP triggers the contact pathway, accelerates factor V activation, and abrogates the anticoagulant function of tissue factor pathway inhibitor (TFPI). Our previous experiments utilized relatively heterodisperse polyP preparations. We have now isolated polyP fractions of defined polymer lengths and here report the size dependence of the various procoagulant effects of polyP.

Methods: PolyP preparations were carefully size-fractionated by gel electrophoresis (Clark and Wood, Anal. Biochem. 1987, 161:280–290). Clotting assays were performed using pooled normal plasma with or without added recombinant TFPI or 40–50 μM polyP. Clotting was initiated by tissue factor (TF), factor Xa (FXa), or CaCl2 alone (contact pathway).

Results: Of the polyP procoagulant activities tested, triggering the contact pathway required the longest polymers: We observed weak triggering of the contact pathway with ∼75mers and increasing procoagulant activity as polymer lengths increased, with maximal contact pathway activation requiring 450mers or longer. On the other hand, the downstream procoagulant effects of polyP required significantly shorter polyP polymers. (When clotting is triggered by either TF or FXa, further shortening of clotting times by polyP is attributed to accelerated factor V activation.) Minimum polymer lengths required to shorten TF or FXa clot times were ∼37mers, with maximal activity requiring 75mers or longer. Finally, the ability of polyP to abrogate TFPI anticoagulant function required the shortest minimum polymer lengths: Detectable anti-TFPI activity was observed with 11mers, with maximal activity requiring 49mers or longer.

Conclusions: PolyP exhibited different minimum length requirements for its various procoagulant effects, consistent with the idea that polyP interacts with different proteins at various points in the clotting cascade. Contact pathway activation required very long polymers that were similar in size to those in infectious microorganisms. Therefore, triggering of the contact pathway by polyP may contribute to procoagulant/inflammatory responses to infection. In contrast, acceleration of factor V activation by polyP was maximal with polymers of the size released from human platelets. Even shorter polyP polymers abrogated TFPI function, suggesting that at least some of polyP’s anti-TFPI effect may be mediated by mechanisms other than (or in addition to) its ability to accelerate factor V activation.