bluuebunny/crossref_metadata_2025_split huggingface dataset:
G-CSF Receptor Activates Lyn through an Essential Temporal-Spatial Recruitment of Gab2 and Shp2.
Authors: Zhu Quan-sheng , Xia Ling , Corey Seth 
Published: April 2020

Abstract
The Hematopoietin/Cytokine Receptors, such as the Granulocyte Colony-Stimulating Factor (G-CSF) Receptor, signal through the Src and Janus kinases. Src activation involves the sequential regulation of positive and negative tyrosine phosphorylation sites. The C-terminal phosphotyrosine serves as a negative phosphorylation site via its interaction with an endogenous SH2 domain. How Lyn, the predominant Src kinase expressed in granulocytes, becomes activated following G-CSF Receptor engagement is not known. Because Lyn contains a negative phosphotyrosine site (pTyr507), we hypothesize that Shp2, an activating tyrosine phosphatase, might be involved. We report that G-CSF stimulation induces the dissociation of Lyn from the scaffolding protein Gab2, which leads to the dephosphorylation of pTyr507 and autophosphorylation of Tyr396. The dephosphorylation of Lyn pTyr507 was impaired in Shp2-deficient murine embryonic fibroblasts transfected with the G-CSF Receptor in response to G-CSF stimulation. Phosphorylation of Lyn Tyr 396 was impaired in cells treated with Gab2 siRNA. Gab2 constitutively associated with Shp2, and mutations of Tyr614 and Tyr643 in Gab2 abolished the recruitment of Shp2. The constitutively activated Shp2 E76A directed the dephosphorylation of Lyn pTyr507 in vitro. Based on these data, we propose that Gab2 forms a complex with Lyn and following G-CSF stimulation, Gab2 recruits Shp2, which dephosphorylates Lyn pTyr507, leading to Lyn activation. Because gain-of-function mutation in Shp2 or recruitment of Lyn in imatinib-resistant Bcr-Abl+ disease occurs in myeloid and lymphoid leukemias, aberrant pathways involving Src can now be targeted through small molecule inhibitors, such as dasatinib.
SCL/TAL1 Regulates Erythropoietin Receptor Expression.
Authors: Rogers Heather M. , Yu Xiaobing , Noguchi Constance Tom 
Published: April 2020

Abstract
The basic-helix-loop-helix transcription factor SCL/TAL1, is required for erythropoiesis during development, and conditional deletion in adult hematopoiesis results in hematopoietic stem cells with a competitive repopulation disadvantage and defective erythropoiesis in vitro. However, adult mice with a conditional SCL/TAL1 deletion survive with mild anemia, suggesting defective erythroid proliferation and indicating that SCL/TAL1 is important, but not essential in mature red blood cell production. We find that during erythroid differentiation of primary human hematopoietic CD34+ cells, SCL/TAL1 expression peaks at day 8–10 following erythropoietin (EPO) stimulation, concomitant with peak expression of GATA-1 and EKLF. Treatment with SCL/TAL1 antisense oligonucleotides during erythroid differentiation markedly decreases erythroid differentiation as indicated by decreased expression of GATA-1 and both b- and g-globin expression, along with the absence of the characteristic decrease in GATA-2. Microarray analysis of erythroid cells overexpressing SCL/TAL1 indicate increased gene expression for b- and g-globin, and other genes related to erythropoiesis including EPO receptor (EPO-R), and these results are confirmed in stable cell lines with increasing SCL/TAL1 expression. Examination of EPO-R transcription regulation indicates that E-boxes in the 5′ UTR can bind SCL/TAL1 in vitro and, in addition to the GATA-1 binding motif, provide transcription activity in reporter gene assays. These data indicate that in addition to the importance of SCL/TAL1 DNA binding for proliferation of BFU-E and expression of glycophorin A and protein 4.2, SCL/TAL1 is also necessary for high level expression of EPO-R. Reduction in EPO-R expression likely contributes to the anemia associated with the conditional adult deletion of SCL/TAL1 and to the proliferative defect of erythroid cells observed in vitro. Early expression of SCL/TAL1 in hematopoietic cells may activate expression of EPO-R prior to EPO stimulation of erythropoiesis and induction of GATA-1.
Oral Arginine Increases Erythrocyte Glutathione Levels in Sickle Cell Disease: Implications for Pulmonary Hypertension.
Authors: Morris Claudia R. , Suh Jung , Vichinsky Elliott P. , Klings Elizabeth S. , Steinberg Martin H. , Shigenaga Mark , Ames Bruce N. , Gladwin Mark T. , Kato Gregory J. 
Published: April 2020

Abstract
Background: Pulmonary hypertension (PHT) is a strong predictor of mortality in sickle cell disease (SCD), and is associated with dysregulation of the arginine-nitric oxide (NO) pathway. This is in part the consequence of hemolysis, as erythrocyte release of plasma hemoglobin consumes NO, while simultaneous release of arginase consumes arginine, the substrate for NO production. The arginase-induced shift towards ornithine metabolism may then contribute to the proliferative changes in the lungs and vasculature associated with PHT through excess production of proline and polyamines. Glutathione (GSH) depletion may contribute to oxidative stress and pre-dispose sickle erythrocytes to hemolysis. We have found that erythrocyte GSH depletion is associated with severity of anemia and PHT measured by Doppler echocardiography (Morris C, Klings E, unpublished data). Kaul et al recently demonstrated that arginine supplementation in BERK mice markedly reduced hemolysis (&gt; 60% reduction in plasma hemoglobin), increased NO generation, and decreased COX-2 expression and PGE2 levels. Since short-term arginine therapy improves PHT in SCD, we hypothesize that oral arginine therapy may impact GSH, a key erythrocyte anti-oxidant.
Methods: Total erythrocyte GSH was analyzed using a sensitive liquid chromatography coupled to tandem mass spectrometric technique before and after arginine therapy at 0.1 mg/kg three times daily for 1 month, followed by 0.2 mg/kg three times daily for two more months, in 8 SCD patients already on stable hydroxyurea therapy.
Results: Erythrocyte GSH levels increased significantly in SCD patients after arginine therapy (Mean±SEM: 1222±150 vs. 1593± 144mM, p=0.03; Figure 1).
Conclusion: Arginine therapy increases erythrocyte GSH levels, which may decrease oxidative stress and hemolysis. Therapies that attenuate hemolysis and oxidative stress will likely benefit PHT in SCD.
Figure 1: Arginine Therapy Figure 1:. Arginine Therapy
Reduced Regulatory T Cell to Activated T Cell Ratio in a Mouse Model of Immune-Mediated Bone Marrow Failure and Prevention of Aplastic Anemia by Infusion of T Regulatory Cells.
Authors: Chen Jichun , Young Neal S. 
Published: April 2020

Abstract
We have produced a murine model of immune-mediated bone marrow (BM) failure based on minor histocompatability antigen mismatch. Infusion of C57BL/6 (B6) lymph node (LN) cells into congenic C.B10 mice results 2–5 weeks later in severe marrow hypoplasia and fatal pancytopenia. Expansion of pathogenic T cells, especially cytotoxic T cells specific for the dominant minor antigen H60, is critical in disease progression: infusion of LN cells from H60-matched congenic B6 donors failed to induce BM failure in C.B10 recipients. We observed that the proportion of CD4+CD25+ T cells in the BM increased significantly in LN-cell-infused animals, coordinate with BM invasion by CD4 and CD8 T cells. However, a very large fraction (60–80%) of CD4+CD25+ T cells from BM failure mice did not express intracellular FoxP3, in contrast to CD4+CD25+ cells from normal BM which were 80–90% FoxP3+. In the mouse model, there were significant declines in the ratios between regulatory T cells and total T cells in the BM: CD4+CD25+FoxP3+ (Treg) to CD4+CD25+FoxP3− (activated CD4 cells) ratio decreased from 1 : 0.29 to 1 : 2.10; CD4+CD25+FoxP3+ to CD4+ ratio decreased from 1 : 7.01 to 1 : 19.87 whereas CD4+CD25+FoxP3+ to CD8+ ratio decreased from 1 : 7.94 to 1 : 46.71 when unaffected animals and BM failure mice were compared. Infusion of as few as 5000 CD4+CD25+ T cells obtained from normal B6 mouse spleen (&gt;80% FoxP3+) with allogeneic B6 LN cells prevented H60-specific T cell expansion in C.B10 recipients and suppressed marrow destruction. A reduced regulatory T cell to activated T cell ratio accompanies pathogenic T cell expansion in this model of immune-mediated marrow destruction. Added regulatory T cells can suppress T cell expansion and prevent murine aplastic anemia. Our model concurs with recent observations in human aplastic anemia, in which numbers and activity of circulating regulatory T cells are much reduced in comparison to healthy controls (Solomou EE et al. ASH abstract submitted). Regulatory T cells might be useful in cellular therapy of autoimmune diseases.
Figure Figure
Modifications of the Coagulation System Alter Inflammation in Murine Sickle Cell Disease.
Authors: Uy Teresa , Wandersee Nancy J. , Hillery Cheryl A. 
Published: April 2020

Abstract
There is evidence for increased activity of both the coagulation and inflammatory pathways in patients with sickle cell disease (SCD). The interrelationships between inflammation and coagulation likely set up a vicious cycle that further propagates the end organ pathology observed in SCD. The aim of this study was to determine whether alterations of the coagulation system change the inflammatory system in mice with SCD. For this study, we generated SCD mice that were deficient in either tissue factor (SCD-TFLow mice) or fibrinogen (SCD-Fib−/− mice) by transplantation of hematopoietic stem cells from Berkeley SCD fetal livers into tissue factor-deficient (TFLow) mice or fibrinogen-deficient (Fib−/−) mice. Because tissue factor and thrombin have potent pro-inflammatory effects, we hypothesized that SCD-TFLow mice (low in both tissue factor and subsequent thrombin generation) have decreased inflammation while SCD-Fib−/− mice (with increased thrombin levels due to afibrinogenemia) have increased inflammation. We measured levels of the following markers of vascular and systemic inflammation: (1) pro-inflammatory or Th1 cytokines (IL-17, TNF-α, IFN-γ, and IL-2); (2) a Th2 cytokine that may limit inflammation (IL-4); and (3) soluble vascular cell adhesion molecule-1 (sVCAM), a measure of endothelial injury. Plasma levels of these markers were measured with commercially available ELISA kits (R&D systems) or BioPlex cytokine assay kits. We compared plasma from SCD-TFLow mice, SCD-Fib−/− mice, transgenic SCD mice (SCD mice), and mice that express only normal human hemoglobin (HbA mice). In comparison to control HbA mice, SCD mice had elevated levels of IL-17, TNF-α, IL-2, and sVCAM, consistent with a pro-inflammatory state. SCD-Fib −/− mice had further elevations of these inflammatory markers. In contrast, SCD-TFLow mice had decreased levels of IL-17, TNF-α, IL-2, and sVCAM in comparison to SCD mice, suggesting that tissue factor deficiency decreases inflammation. IL-4 remained low for all groups of mice, suggesting that these manipulations of the hemostatic pathway did not affect this potentially anti-inflammatory cytokine.
Mean plasma concentration* IL-17 (pg/mL) TNF-α (pg/mL) IFN-γ (pg/mL) IL-2 (pg/mL) IL-4 (pg/mL) sVCAM (ng/mL) *n=2–6 HbA 1.2 22 1.0 0.8 0.2 403 SCD 31 35 0.7 1.5 0.1 696 SCD-Fib-/- 102 188 6.0 1.9 0.7 807 SCD-TFLow 1.3 9.3 1.6 0.3 &lt;0.1 593
This pattern of change in the plasma levels of these selected inflammatory markers provides evidence that
at baseline, SCD is a pro-inflammatory state; afibrinogenemia has the potential to worsen inflammation in SCD, likely through increased thrombin generation previously described in human and murine afibrinogenemia; and tissue factor deficiency decreases systemic and vascular inflammation in SCD. These data indicate that modifications of the coagulation system may affect the inflammatory state in SCD, and suggest the potential for therapeutic manipulation of coagulation in order to reduce inflammation in SCD.
A Novel Mutation 774del4 in IFNGR1, Which Produces Truncated Form of Interferon γ Receptor 1, Causes a Dominant-Negative Effect on Interferon γ Signal Transduction.
Authors: Okada Satoshi , Ishikawa Nobutsune , Shirao Kenichirou , Kawaguchi Hiroshi , Tsumura Miyuki , Ohno Yoshinori , Yasunaga Shin’ichiro , Ohtsubo Motoaki , Takihara Yoshihiro , Kobayashi Masao 
Published: April 2020

Abstract
Patients with interferon γ receptor 1 (IFNγR1) deficiency are characterized by disseminated Bacilli Calmette-Guérin (BCG) infections and severe non-tuberculosis mycobacterium infections, and show remarkable genetic heterogeneity. It is known as one of inherited immunodeficiency disorders with recessive or dominant form. So far known dominant forms of IFNγR1 deficiency are associated with heterozygous mutation in the intracellular domain of IFNγR1, among of which 818del4 is known as a major mutation in IFNGR1, leading to frameshift and premature stop codon. We describe here a novel heterozygous IFNGR1 mutation in a patient with recurrent mycobacterium infections, resulting in impairment of receptor degradation. The proband, a 12-year old Japanese girl, developed multiple osteomyelitis due to M. avium infection. She had suffered from BCG lymphadenitis, which occurred 2 months after BCG vaccination, for 3 years. DNA sequence analysis of IFNGR1 demonstrated that the patient had a heterozygous mutation 774del4, producing a truncated protein lacking intracellular component of IFNγR1. IFNγR1 was overexpressed on the surface of CD14-positive cells in the peripheral blood of the patient, and STAT1 phosphorylation was partially defected in response to high dose IFN-γ stimulation. Impaired TNF-α production in response to IFN-γ stimulation was also observed. T cells from the patient presented decreased IFN-γ production in response to IL-12 stimulation.
In order to define the characterization of this truncated form of IFNγR1, we cloned 774del4 and 818del4 mutations as well as the wild-type into mammalian expression vector. These constructs were transfected into HEK293 cells. Cycloheximide (CHX) treatment enabled detection of IFNγR1 expression by flow cytometry and Western blot analysis. In contrast to the wild-type IFNγR1 protein, which rapidly disappeared after CHX treatment, 774del4 mutant protein was stably retained on the cell surface as was observed in 818del4 mutant. These observations suggest that 774del4 mutant causes overexpression of dominant-negative form of IFNγR1 on the cell surface through impairment of receptor degradation.
Deoxyadenosine Inhibits T-Cell Activation in ADA-SCID Patients through a cAMP/PKA-Dependent Pathway.
Authors: Cassani Barbara , Mirolo Massimiliano , Cattaneo Federica , Benninghoff Ulrike , Carlucci Filippo , Tabucchi Antonella , Bordignon Claudio , Roncarolo Maria Grazia , Aiuti Alessandro 
Published: April 2020

Abstract
Adenosine deaminase (ADA) is a key enzyme of the purine salvage pathway, metabolizing adenosine (Ado) and deoxyadenosine (dAdo). ADA deficiency results in increased levels of Ado and dAdo in plasma and cells, leading to severe combined immunodeficiency (SCID). However, the role of intracellular accumulation of dAdo and/or an aberrant extracellular signaling, mediated by G-coupled adenosine receptors, in the pathogenesis of the disease remains to be elucidated. Retroviral-mediated gene transfer of ADA into hematopoietic stem/progenitor cells has been recently shown to be an effective treatment for ADA-SCID children, thus providing a unique model to investigate the influence of purine metabolism in the survival and functions of T lymphocytes. Using untransformed bulk or CD4+ T-cell lines established from ADA-SCID patients before and after gene therapy, we investigated the biochemical pathways responsible for the pathogenesis of the disease and the ability of gene therapy to restore normal metabolic and immunological functions. We found that the expression of functional ADA in gene corrected T cells resulted in the restoration of SAHH activity and in the normalization of apoptosis induced by exposure to Ado/dAdo in defective cells. Interestingly, while a nucleoside transporter inhibitor could prevent Ado-induced cell toxicity, dAdo exerted its cytotoxic effect via the accumulation of intracellular cAMP, mediated by the engagement of Gs-protein coupled adenosine receptors. Functional studies revealed that both proliferative responses and Th1/Th2 cytokine production were impaired in ADA-deficient cells, but were restored in T cells generated after gene therapy. Such impairment was consequent to a defective TCR signalling, as indicated by the intrinsically reduced activation of p44/p42 MAPK pathway after anti-CD3/anti-CD28 mAb stimulation. Furthermore, a reduced transcription of cytokine genes was observed in ADA−/− CD4+ T cells, which was associated to a defective activation of early transcription factor CREB and possibly to an altered nuclear recruitment of NF-kB, as predicted by the decreased phosphorylation of IkBa. Remarkably, in ADA-deficient T cells, but not in gene corrected or healthy donor cells, exposure to non-toxic concentrations of dAdo resulted in a strong inhibition of p44/p42 MAPK activation and complete abrogation of TCR/CD28-driven proliferation and cytokine production. The effects of dAdo were reverted in the presence of a cAMP/PKA inhibitor, highlighting a key role for PKA hyperactivation and involvement of A2 receptors in causing T-cell dysfunctions. Collectively, these findings provide a more clear understanding of the physiopathology of ADA-SCID and confirm the efficacy of ADA-gene transfer in restoring normal metabolic pathways and immunological functions.
HIV-1 Nef Inhibits M-CSF Signals by Down-Regulating Its Receptor Fms through Hck.
Authors: Suzu Shinya , Hiyoshi Masateru , Harada Hideki , Okada Seiji 
Published: April 2020

Abstract
Nef, an accessory protein of HIV−1, has been shown to be critical for both high viral loads and progression to AIDS. We recently demonstrated that Nef inhibited the bioactivities of M-CSF, a monocytes/macrophages-specific cytokine (Blood105: 3230–3237, 2005). The inhibitory effect of Nef is a likely explanation for dysregulated functions in HIV−1−infected macrophages. Here, we examined the specificity and molecular mechanism of the newly-characterized function of Nef, to clarify its relevance to the pathogenetic activity of Nef. By analyzing cytokine-dependent cell lines expressing the conditionally active Nef, we found that Nef had a profound inhibitory effect on M-CSF signal, but not on IL-4- and GM-CSF signals. We also found that Nef down-regulated the surface expression of M-CSF receptor (M-CSFR) but not of GM-CSF receptors. The M-CSFR down-regulation by Nef was reproducible in 293 cells when co-transfected with Hck, a member of Src kinases. Interestingly, the down-regulation of cell surface M-CSFR in the presence of Nef and Hck correlated with the concomitant increase of the immature form of M-CSFR. These results suggested that Nef perturbed M-CSFR maturation through Hck and thereby selectively inhibited M-CSF signal.
Expression of Immune Memory Markers CD8 α α and Interleukin−7 Receptor on CD8+ T Cell Subsets among HIV−1 Infected Patients.
Authors: Routy Jean Pierre , Mercier Francois , Galal Ahmed , Boulassel Med-Rachid 
Published: April 2020

Abstract
Evidence from animal models suggests that the expression of CD8α α homodimer on CD8+ T-cells plays a key role in the generation of long-lived memory cells. However, very little information is available in the human clinical setting. Here, we examined immunophenotypic patterns of CD8+ T-cell subsets expressing CD8α α with other markers involved in generating and maintaining memory cells such as interleukin-7 receptor (IL-7Rα ) and circulating levels of IL-7 and IL-15, in three well-defined groups of human immunodeficiency virus-1 (HIV-1)-infected individuals including aviremic (n=15), viremic (n=31) and slow-progressor (n=15). In addition, immunophenotypic patterns were correlated with immune activation markers (CD38/HLA-DR), which are known to be an important factor in HIV-1 disease pathogenesis. Cell-surface expression of CD8α α , IL-7Rα and CD38/HLA-DR on CD8+ naïve, central memory, pre-terminal and terminal effector memory T-cells was measured by eight-color flow cytometry on freshly peripheral blood samples. IL-7 and IL-15 levels were measured by ELISA and viral loads were assessed by PCR. Group differences in the CD8+ T-cell subsets expressing each antigen tested were evaluated using the unpaired nonparametric Mann Whitney U test. Correlations were determined by Spearman’s correlation tests. Compared to slow-progressor subjects, expression of CD8α α was significantly reduced in aviremic and viremic patients and this reduction occurred mainly within naïve and central memory T-cell subsets and not in effector memory compartments. In contrast, persistent antigenemia in viremic patients appeared to lead to IL-7Rα loss mainly on central and effector memory subsets and not on naive T-cells. Compared to aviremic and viremic patients, slow-progressor subjects had lower levels of circulating IL-7, normal levels of IL-15, CD8α α and IL-7Rα , and reduced activated T-cells. Overall, expression of CD8α α was not significantly related to IL-7Rα although negative associations were evidenced within all CD8+ T-cell subsets. However, in viremic patients, naïve and central memory cell subsets expressing CD8α α were positively correlated with viral load but not with CD8+ T-cell subsets expressing immune activation markers. Together, these results provide new insights into the role of CD8α α /IL-7Rα along with immune activation markers in maintaining memory populations during HIV-1 infection. The inter-relationships between these immune memory markers require further investigations, which may help understanding the mechanisms of antiviral control.