bluuebunny/crossref_metadata_2025 huggingface dataset:
Recapitulating the Expression Pattern of Jak2 in Transgenic Zebrafish Using Jak2 Promoters and a GFP Reporter Gene.
Authors: Zhang Jing , Lin Hui-Feng , Handin Robert I.
Published: April 2020

The non-receptor tyrosine kinase Jak2 plays an important role in regulating erythro and thrombopoiesis. There has been intense interest in Jak2 since the observation that an activating mutation V617F is present in almost all patients with Polycythemia vera and many patients with Essential Thrombocytosis and Myelofibrosis. The analysis of Jak2 function in vivo has been limited as the murine jak2 knockout is lethal at day 10.5 of embryogenesis. Our laboratory has taken advantage of an ancestral partial duplication of the zebrafish genome, which has yielded two jak2 alleles --- jak2a and jak2b to study jak2 expression and function. Whole mount in situ hybridization studies confirm that the jak2a gene is only expressed in hematopoietic tissues, while jak2b is expressed in the developing lens and nephritic ducts. We have cloned and characterized the full-length jak2a and jak2b cDNAs and characterized the jak2a and 2b genomic loci. The jak2b gene has 24 exons spanning 79kb of genomic DNA. We amplified a 4kb zebrafish genomic fragment upstream of the first exon of the jak2b gene and linked it to the enhanced green fluorescent protein (EGFP) cDNA reporter and then microinjected the construct into single-cell zebrafish embryos. At 24 hours post fertilization (hpf), we observed fluorescence in the lens and nephritic ducts of developing embryos, with some expression in skin, muscle and notochord. The jak2a gene locus is complex as the jak2a gene is linked to a gene of unknown function, STARD4, in a head-to-tail manner with a small intergenic region of 1kb. As observed with jak2b, the first exon contains the jak2b 5′-UTR and the second exon contains the translation initiation site. We cloned a 1.9kb DNA fragment that included exons 1 and 2 the intervening first intron and an additional 800bp upstream of exon 1. This 1.9 kb promoter fragment was sufficient to drive expression of enhanced green fluorescent protein (EGFP) in injected embryos in a manner that recapitulated the native expression pattern of jak2a. In injected embryos 24hpf, GFP+ cells were present in the anterior intermediate cell mass (ICM) and the lens. Fluorescent circulating blood cells, largely erythrocytes, were detected in 12 of 124 microinjected embryos 48 hours after fertilization. The jak2-EGFP transgenic zebrafish strains should be useful in the study of normal and pathologic hematopoiesis and in future studies of the pathogenesis of the MPDs.
STAT1 Promotes Megakaryocytic Differentiation in a GATA-1-Independent Manner.
Authors: Huang Zan , Crispino John D.
Published: April 2020

GATA-1 is a critical transcription factor that governs megakaryocyte development and differentiation. GATA-1-deficient megakaryocytes proliferate excessively and fail to undergo terminal differentiation, likely as a consequence of an aberrant gene expression program. In our studies to identify key signal transduction pathways that are dysregulated in the absence of GATA-1, we observed that STAT1, a gene of unknown function in megakaryocytes, was significantly downregulated in GATA-1-deficient megakaryocytes. Surprisingly, ectopic expression of STAT1, or IRF-1, a downstream effector of STAT1 signaling, promoted megakaryocytic differentiation of G1ME cells, a GATA-1-null and TPO-dependent erythromegakaryocytic cell line. Both STAT1- and IRF-1-transduced G1ME cells, but not GFP-expressing control cells, displayed multiple markers of terminal differentiation, including expression of CD42, as well as a significant fraction of polyploid cells. BrdU labeling assays confirmed that these transduced cells continued to effectively synthesize DNA, suggesting that STAT1 signaling switches these cells from mitosis to endomitosis. STAT1 and IRF-1 also modulated TPO signaling evidenced by enhanced STAT3 and STAT5 phosphorylation. Since STAT1 signaling is activated by IFN-g, we further investigated whether IFN-g-treatment also led to polyploidization of G1ME cells. IFN-g effectively promoted polyploidization of both G1ME cells and primary bone marrow-derived CD41+ cells in the absence of TPO. Finally, overexpression of STAT1 in primary bone marrow cells isolated from GATA-1 knockdown mice also promoted polyploidization in the absence of TPO. Taken together, these data reveal a GATA-1-independent mechanism by which STAT1 signaling promotes megakaryocyte differentiation.
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

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

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

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 (> 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

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 (>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

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 <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

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

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

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

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.
Clinical Disparities between Primary Versus Secondary MGUS in Veteran Patients.
Authors: Malani Ashok , Singh Jaswinder , Pabla Maninder , Patel Vijay , Kambhampati Suman
Published: April 2020

Background: Monoclonal gammopathy of unknown significance (MGUS) is characterized by low levels of a monoclonal protein (M protein) in the serum or urine that is produced by a single clone of plasma cells. Though published evidence indicates that the incidence of MGUS increases with age, the clinical differences between young versus older MGUS patients are not completely understood. We investigated the age-dependent clinical characteristics of veteran patients with MGUS.

Objective: To identify age-dependent disparities in the clinical characteristics of veteran patients with primary versus secondary MGUS.

Methods: A retrospective chart review of 327 MGUS patients seen at the Kansas City Veteran Affairs Medical Center over the last 20 years was performed. “Primary” MGUS was defined as presence of monoclonal immunoglobulin protein (M-spike) in the absence of multiple myeloma (MM), primary amyloidosis, or leukemia/lymphoma. In this report, we use the term “Secondary” MGUS when an M-spike is associated with HIV, or non-HIV-infections, and/or autoimmune diseases. In the final analysis, we broadly subdivided patient with MGUS into age-group ≤ 55 years and ≥ 55 years.

Results: Of the total 327 patients, 14% (44/327) belonged to the age group ≤ 55 years. In our analysis, “Secondary” MGUS was more prevalent in patients ≤ 55 years (30/44; 68%) as compared to that in the ≥ 55 years age group (25/283; 8.5%; p-value <0.0001). In the veteran population, the majority of patients ≤ 55 years age had hepatitis C-associated “Secondary” MGUS (45%). HIV-associated MGUS was also more prevalent in the younger age group (4/44; 9%) as compared to age ≥ 55 years (0/283). Secondary MGUS co-existing with autoimmune disorders was comparatively more common in the younger age group than the older patients (13.6% versus 4.5%; p-value <0.05). IgA subtype was surprisingly not seen in MGUS patients with age ≤ 55 years. Most importantly, MGUS transformation into MM was not seen even after a prolonged follow-up of 20 years in the younger age group of patients.

Conclusion: MGUS in young patients (age ≤ 55 years) is often found in conditions associated with chronic antigenic stimulation or immunosuppression, such as chronic hepatitis B or C, HIV infections, and autoimmune diseases. We thus recommend that young patients (age ≤ 55 years) with MGUS undergo testing for hepatitis B/C, HIV infection if there is a possible history of exposure to these viruses. Our analysis also suggests that these so-called “secondary” MGUS are benign disorders.
Recombinant Human Mannose-Binding Lectin (rhMBL) Binds to Human and Mouse Cancer Cells and Elicits Antitumor Activity in a MBL Knockout Mouse Model of Murine Cancer.
Authors: Sapra Puja , Kraft Patricia , Mehlig Mary , Zhang Zhihua , Greenberger Lee M. , Horak Ivan D.
Published: April 2020

Mannose-binding lectin (MBL), a human plasma protein, plays a role in innate immune defense. MBL recognises microorganisms through surface carbohydrate structures leading to either direct opsonisation or activation of complement system. Since MBL deficiency is associated with increased susceptibility to infections in immunosuppressed individuals, e.g., during chemotherapy induced neutropenia, replacement therapy with recombinant human MBL (rhMBL) may be beneficial in this patient group. MBL has also been shown to bind to cancer cell lines. Therefore, we hypothesized that MBL binding to cancer cells may lead to direct opsonisation or activation of the innate immune system leading to cell kill. In this study, we aim to determine the binding and therapeutic efficacy of rhMBL in cancer cells and animals models respectively. The in vitro binding of rhMBL was evaluated on human cancer cell lines by flow cytometry using a specific anti-MBL antibody. rhMBL demonstrated binding to 6 of 7 cancer cells lines tested. These included human ovarian (OVCAR-3, SK-OV-3), pancreatic (MiaPaCa-2), colorectal (COLO-205), lymphoma (Daudi) and murine melanoma (B16-F0) cancer cells. The binding of rhMBL appeared to be specific since it was partially inhibited by 100mM mannose, 100mM N-acetyl glucosamine, 20mM mannan, use of Ca2+-free buffer or with 10mM EDTA. The antitumor effects of rhMBL were evaluated in MBL knockout (MBL/KO) transgenic mice, which lack both MBL-1 and MBL-2 genes, and corresponding MBL wildtype mice injected subcutaneously with B16-F0 mouse melanoma cells. MBL wildtype mice injected with B16-F0 mouse melanoma cells did not develop subcutaneous tumors. However, MBL/KO mice had robust tumor growth suggesting that MBL deficient individuals may be more susceptible to development of cancers.

Treatment of MBL/KO mice with 75 μg MBL/mouse (q2dx5) resulted in 63% tumor growth inhibition compared with the control mice (P<0.05). Moreover, MBL/KO mice treated with rhMBL had a significant prolongation of life span compared with the control group (P<0.05). In conclusion, rhMBL binds to mouse and human tumor cells and inhibits tumor cell growth in a MBL/KO murine melanoma model. This phenomenon may not take place in MBL-deficient individuals, and these individuals may thus be more susceptible to development of tumors. Replacement therapy with rhMBL in these individuals may have anticancer effects. rhMBL should thus be further evaluated for prevention and treatment of infection complications in MBL deficient immunocompromised patients as well as potential anticancer therapy.
Induction of Potentially Protective HIV-Specific T-Cell Responses In Vitro by Gag mRNA-Electroporated Dendritic Cells.
Authors: Berneman Zwi N. , Van Gulck Ellen R. , Heyndrickx Leo , Ponsaerts Peter , Van Tendeloo Viggo F.I. , Vanham Guido
Published: April 2020

Human immunodeficiency virus type 1 (HIV-1) infection is characterized by dysfunction of HIV-1-specific T-lymphocytes. In order to suppress the virus and delay evolution to AIDS, antigen-loaded antigen-presenting cells, including dendritic cells (DC) might be useful to boost and broaden HIV-1-specific T-cell responses. Monocyte-derived DC from 15 untreated (“naive”) and 15 highly active anti-retroviral therapy (HAART)-treated HIV-1-infected patients were electroporated with codon-optimized (“humanized”) mRNA encoding consensus HxB-2 (hHxB-2) Gag protein. These DC were co-cultured for 1 week with autologous peripheral blood leucocytes (PBL). Potential expansion of specific T-cells was measured by comparing ELISPOT responses of PBL before and after co-culture, using a pool of overlapping peptides, spanning the HxB-2 Gag. Expansion of specific PBL after co-culture was noted for T cells producing interferon (IFN)-gamma, interleukin (IL)-2 and perforin (Wilcoxon signed rank test p<0.05, except for IL-2 in naive patients). From all HIV-1-seropositive persons tested, 12 HAART-treated and 12 naive patients match in absolute number of CD4+ T-cells. A comparison of the increase of the response between day 0 and after 1 week of stimulation between those two groups showed that the response was higher in HAART-treated subjects for IFN-gamma and IL-2 but not for perforin in comparison to untreated subjects. Examining purified CD4+ and CD8+ T-cells after co-culture revealed that HxB-2 Gag peptides induced IFN-gamma in both subsets, that IL-2 was only secreted by CD4+ T-cells and that perforin was dominantly secreted by CD8+ T-cells. Remarkably, the perforin response in the treatment-naive persons was negatively correlated with the peripheral blood absolute CD4+ and CD8+ T-cell count (respectively R=0.618, p=0.014; and R=0.529, p=0.043). Furthermore, the nadir absolute CD4+ T-cell count in HAART-treated subjects was positively correlated with the IL-2 response (R=0.521, p=0.046) and negatively correlated with the perforin response (R=0.588, p=0.021). In conclusion, DC from HAART-treated and therapy-naive subjects, electroporated with hHxB-2 gag mRNA have the capacity to induce secondary T-cell responses. In an earlier study (Van Gulck ER et al. Blood2006;107:1818–1827), we already demonstrated ex vivo that CD4+ and CD8+ T-cells from non-treated HIV-1-infected subjects can be directly triggered by DC electroporated with autologous proviral-derived gag mRNA. Taken together, our results open the perspective for a DC immunotherapy for HIV disease.
C-Reactive Protein Binds to FcγRII and Impairs the Differentiation and Function of Dendritic Cells.
Authors: Qian Jianfei , Yang Jing , Wang Siqing , Zhang Liang , Hong Sungyoul , Han Xiaohong , Yi Qing
Published: April 2020

Human C-reactive protein (CRP) is an acute-phase protein, and elevated levels of CRP are present in patients with infections, inflammatory diseases, necrosis such as myocardial infarction, or malignancies including multiple myeloma (MM), lymphoma, and carcinoma. CRP has many biological functions and is involved in host defense, regulation of inflammation, and modulation of autoimmune diseases. Although our current understanding of CRP interaction with complement and Fcγ receptors (FcγR) has elucidated a regulatory role of CRP in these disease situations, it is not clear whether CRP affects the function of immune cells such as dendritic cells (DCs). In this study, we investigated the effect of CRP on DC differentiation, maturation, and function. CD14+ monocytes isolated from human peripheral blood mononuclear cells were cultured in RPMI-1640 medium supplemented with GM-CSF and IL-4 for 5 days to generate immature DCs (imDCs), and were further treated with IL-1β and TNF-α for 2 additional days to produce mature DCs (mDCs). CRP (5–100 μg/mL) was added to the cultures during DC differentiation (on days 0 and 3) or maturation (on day 5). The presence of CRP in cultures reduced imDC cell yields in a dose-dependent manner. Significantly lower cell yields were detected in cultures with 5 to 10 μg/mL CRP. Compared with untreated controls, CRP treatment (10 μg/mL) led to inhibited surface expression of DC-related molecules HLA-ABC, CD1a, CD40, and CD54; increased secretion of IL-6, IL-8, and IL-10; reduced production of TGF-β by imDCs; and decreased secretion of IL-12 by mDCs. Furthermore, the function of CRP-treated DCs was also impaired, evident by the markedly decreased ability of imDCs to phagocytose apoptotic cells and to uptake and present soluble antigen to antigen-specific T cells. Compared with untreated controls, CRP-treated mDCs had reduced capacity at activating allospecific T cells, which consequently secreted significantly lower amounts of IFN-γ, IL-2, and TNF-α compared with T cells activated by normal mDCs. Western blot analysis showed that CRP treatment led to inhibited phosphorylation of ERK and p38 MAPKs, and inhibited NFκB activity in the differentiating cells. Monocytes and DCs all express FcγRI (CD64), FcγRII (CD32), and FcγRIII (CD16), and the expression of FcγRII, but not FcγRI and FcγRIII, were upregulated on CRP-treated DCs. The detrimental effects of CRP on DCs were abrogated by blocking antibody against CRP and by antibody against FcγRII, but not against FcγRI or FcγRIII. These results indicate that CRP affected DC differentiation via binding to cell surface FcγRII. Taken together, this study demonstrates for the first time that CRP at high concentrations has detrimental effects on in vitro differentiation and function of DCs. Further studies will be needed to examine the clinical and biological relevance of this observation.
A 17q25.3 Duplication Defines a New Dosage-Sensitive Congenital Neutropenia Locus and Implicates SOCS3 as a Candidate Gene for Cases Unexplained by ELA2 Mutation.
Authors: Mealiffe Matthew E. , Le Doan , Duan Zhijun , Papadaki Helen , Eliopoulos George , Badolato Raffaele , Horwitz Marshall S.
Published: April 2020

We have investigated the etiology of congenital neutropenia in a girl with de novo duplication of chromosome 17q25.3. She presented during the first year of life with neutropenia, episodic hypothermia, failure to thrive, and other congenital abnormalities. Peripheral blood karyotype demonstrated 46,XX,add(17)(q25.3), and molecular cytogenetic studies confirmed interstitial duplication of chr17-derived material. We excluded ELA2 mutation (the most common cause of hereditary neutropenia) and reasoned that the neutropenia and other medical problems most likely were the result of the chromosomal abnormality. In the duplicated 17q25.3 region, SOCS3 emerged as a promising candidate gene responsible for neutropenia, because SOCS3 is a well-characterized negative regulator of G-CSF-receptor signaling and, in murine conditional knockout models, acts as a physiologic negative regulator of granulopoiesis. As we had previously demonstrated that mutations of the Gfi1 transcriptional repressor are a rare cause of human neutropenia, we searched for potential Gfi1 binding sites in the SOCS3 promoter and noted a total of five, and chromatin immunoprecipitation analysis validated occupancy of the SOCS3 promoter by Gfi1 in Jurkat, HL-60, and U937 cells. Thus, several lines of evidence suggested SOCS3 as a plausible neutropenia gene. We confirmed that SOCS3 is indeed duplicated in this patient by both FISH and quantitative genomic PCR. In an effort to identify other patients with SOCS3-related neutropenia, we sequenced both exons of SOCS3 in a total of 66 patients with severe congenital neutropenia (SCN) or cyclic neutropenia (CN) and 94 patients with chronic idiopathic neutropenia of adults (CINA) in whom ELA2 mutations were absent. No coding mutations were detected, but we did identify several variants in the SOCS3 promoter and 5′- and 3′- UTRs, including a single base pair substitution (+1779AtoG) in the 3′-UTR in a SCN patient and deletion of a single G (-1318delG) occurring in a six-G tract immediately adjacent to a predicted STAT binding site in a highly conserved region of the SOCS3 promoter (found in heterozygous and homozygous form in several patients with CN and SCN). The 3′-UTR alteration was absent in 270 control chromosomes and is located within a predicted binding site for miR-449 that is well-conserved across mammalian species. In sum, we have identified a patient with congenital neutropenia and a de novo duplication of 17q25.3 defining a new candidate locus for congenital neutropenia, and the evaluation to date suggests that SOCS3 is a promising dosage sensitive candidate gene in this interval that additionally could be a key target in neutropenia associated with Gfi1 mutations.
Transgenic GATA1ie3.9int - DAPK2 Mice Exhibit Low-Grade Anemia, Deficient Stress Erythropoiesis, and Compromised Erythroblast Survival.
Authors: Fang Jing , Menon Madhu P. , Zhang Diya , Tschan Mario , Torbett Bruce , Pramanik Nayani , Houde Estelle , Wojchowski Don M.
Published: April 2020

Death-associated protein kinases (DAPKs) include DAPK-1, DAPK-2, ZIPK and death receptor associated kinase 1 & 2 (DRAK-1 and -2). DAPK-1 and -2 are highly homologous in their shared serine-threonine kinase domains, and calmodulin and phospho-serine regulatory regions. DAPK1 is cytoskeletally associated via ankyrin repeats, and modulates a p19ARF/p53 pathway. DAPK2 is cytoplasmic, less widely distributed, and is expressed predominantly in erythroid cells. Hypothesized effects of DAPK2 on erythroblast formation presently were tested by preparing and studying several independent lines of GATA1ie3.9int-DAPK2 transgenic mice. At steady-state, hematocrits in GATA1ie3.9int-DAPK2 mice were mildly yet significantly decreased (while other lineages were essentially unaffected). Reticulocyte production also was modestly increased, and spleen morphologies were altered. During hemolytic anemia, however, DAPK2 mice exhibited comparably sharp deficiencies in stress erythropoiesis including prolonged decreases in hematocrits (as observed in repeated experiments at varied phenylhydrazine dosing). The basis for this stress-associated erythropoietic defect due to DAPK2 was investigated further in primary bone marrow erythroid progenitor cells ex vivo. A stage-specific defect in erythroblast formation was discovered specifically at KitnegCD71highTer119neg stage. Later stage CD71highTer119pos DAPK2-erythroblasts also were somewhat under-represented (but perhaps as a consequence of decreased KitnegCD71high pools.) In addition, DAPK2-progenitors gave rise to increased frequencies of Annexin V positive apoptotic erythroblasts. These first-time analyses of DAPK2 function in a relevant primary cell model reveal negative regulation of stage-specific erythroblast pool selectively during stress erythropoiesis, and in part via pro-apoptotic mechanisms.
Phenotypic Changes of Hematopoietic Stem Cells and Stromal Cells by Aberrant Expression of GATA−2; Possible Role for the Development of Aplastic Anemia.
Authors: Okitsu Yoko , Harigae Hideo , Takahashi Shinichiro , Minegishi Naoko , Suzuki Norio , Kameoka Junichi , Yamamoto Masayuki , Sasaki Takeshi
Published: April 2020

(Introduction) Aplastic anemia (AA) is characterized by a fatty bone marrow and the reduced number of hematopoietic stem cell (HSC)s, resulting in peripheral pancytopenia. Several clinical studies have reported that the expression of transcription factor GATA−2 is significantly decreased in CD34 positive cells in AA, suggesting that downregulation of GATA−2 in HSCs may cause the reduction of number of HSCs in AA. Of note, GATA−2 has been shown to be essential for the maintenance of immaturity of preadipocytes as well as HSCs. If GATA−2 functions in the differentiation of stromal preadipocytes in bone marrow, GATA−2 may be involved in the formation of a fatty bone marrow in AA. In order to explore the role of GATA−2 for development of AA, phenotypic changes of HSCs and stromal preadipocytes by aberrant expression of GATA−2 were examined in this study.

(Method) First, to analyze phenotypic changes of HSCs by decreased expression of GATA-2, lineage negative, sca-1 positive, c-kit positive (LSK) cells were sorted both from wild-type and GATA-2 haplodeficient mice (kindly provided Dr SH Orkin), and the gene expression profile was compared by cDNA array. The difference of expression level on cDNA array was confirmed by RQ-PCR. Next, in order to examine the role of GATA-2 in the differentiation of stromal preadipocytes, the GATA-2-GFP expression vector was transfected into TBR343, a mouse stromal preadipocyte cell line, and GFP positive cells were sorted. The differentiation capacity of these GATA-2 overexpressing TBR343 cells was compared to that of control cells. In addition, GATA-2 expression was suppressed by using siRNA in TBR343, and the phenotypic change was also examined.

(Results) By comparing the gene expression profile, the expression levels of cyclin D1, D3, p21, E2F, c-Myc were found to be decreased in GATA-2 haplodeficient LSKs, suggesting GATA-2 down-regulation in HSCs may inhibit the cell cycle progression. When GATA-2 was overexpressed in TBR343, the oil drop formation and adipocyte-specific gene expression was significantly suppressed. Conversely, When GATA-2 was suppressed in TBR343 by siRNA, the oil drop formation and adipocyte-specific gene expression was significantly accelerated, suggesting that decrease of GATA-2 expression accelerates the differentiation of stromal preadipocytes.

(Conclusion) These results suggest that suppression of GATA-2 in the bone marrow leads to an inactive cell cycle of HSCs and the accumulation of mature adipocytes, which may contribute to the expression of the representative features of AA, a fatty bone marrow and the reduced number of HSCs.
HematideTM, a Synthetic Peptide-Based Erythropoiesis Stimulating Agent (ESA), Assessed for Correction of Anemia in Oncology Patients Receiving Chemotherapy.
Authors: Pickering Lisa M. , Cwiertka Karel , Jassem Jacek , Petera Jiri , Pettengell Ruth , Ramlau Rodryg , Vorlicek Jiri , Vyzula Rostislav , Andresen Corina , Chang Stephen , Duliege Anne-Marie , Leong Robert , Stead Richard , Harper Peter G.
Published: April 2020

Introduction: Hematide™, a novel synthetic PEGylated peptidic compound, binds to and activates the erythropoietin receptor. It is being developed for the treatment of anemia associated with chronic kidney disease and cancer.

Objective: To assess the subcutaneous (SC) Hematide™ dose required to increase hemoglobin (Hgb) by ≥1 g/dL in anemic cancer patients on chemotherapy. Safety, PD and PK assessment of Hematide™ are also planned.

Method: In an ongoing phase 2, openlabel, multicenter, dosefinding study, Hematide™ is being given SC in 15 patient cohorts every 3 wks for 4 doses. The initial dose is 0.10 mg/kg. Entry criteria include confirmed solid tumor malignancy or lymphoma, ≥9 weeks of chemotherapy, baseline (BL) Hgb ≥8 and <11 g/dL, and adequate iron, folate, and B12 stores.

Results: Preliminary results are presented for the 1st cohort. Mean body weight for the 8 female and 7 male patients was 67.8Kg. Nine out of 15 patients completed 12 weeks follow-up. Six patients terminated early for non-study drug related SAEs (2), remission of cancer (1), or transfusions (3, Hgb values posttransfusion excluded from this report) . Week 7: 10 patients completed Week (Wk) 7 with data available. Their mean ± SD Hgb increased from BL by 1.6 ± 1.8 g/dL. Seven had a ≥1.0 g/dL Hgb increase (range 1.7–3.7 g/dL) at Wk 7. Of the remaining 3 patients, 2 had a Hgb increase of 1.65 and 1.1 g/dL, respectively at Wk 9; the 3rd had a Hgb increase of 1.7 g/dL at Wk 12. One patient with missing Wk 7 data had Hgb increases of 1.8 and 3.4 g/dL at Wk 6 and 9 respectively. Week 9: 11 patients had Wk 9 data that show a mean ±SD Hgb increase from baseline of 2.2 ±1.79 g/dL. Of these, 10 had Hgb increases of ≥1.0 g/dL (range 1.1–5.2 g/dL). The one patient who did not have ≥1.0 g/dL Hgb increase at Wk 9 had a Hgb increase of 1.7 g/dL at Wk 12. Four SAEs (none related to study drug) were reported in 3 patients.

Conclusion: Preliminary data from the 1st cohort indicate that Hematide™ given every 3 wks appears to be well tolerated and results in an increase in Hgb of ≥ 1g/dL by Wk 7 in the majority of this study population. Additional data will be presented as it becomes available.