bluuebunny/crossref_metadata_2025_split huggingface dataset:
Activation of NK Cells Induces CD4 Expression and Allows HIV−1 Infection.
Authors: Harada Hideki , Goto Yumi , Dessouki Omar F. , Suzu Shinya , Okada Seiji 
Published: April 2020

Abstract
Natural killer (NK) cells play critical roles in immune surveillance without deliberate prior sensitization and restriction by major histocompatibility complex (MHC). Although function and cell number of NK cells are influenced in AIDS patients, direct interaction between HIV and NK cells is still controversial. Because steady-state NK cells are negative for CD4 which is a key molecule for HIV infection. In this study, we established a condition inducing CD4 expression and HIV-1 infection of NK cells in vitro.
CD4 was expressed on NK cells co-cultured with HFWT cells (NK cell-selective stimulating feeder cells) and IL-2. There were no differences in expression level of NK receptors, adhesion molecules and cytotoxicity between CD4+ and CD4- NK cell subpopulations. However, expression of activation markers, CD25 and HLA-DR, and size/granularity of the CD4+ NK cells were higher than CD4- NK cells. CD4+ NK cells expressed co-receptors for HIV-1, CCR5 and CXCR4. Thus, CD4 is induced on NK cells by activation, and the CD4+ NK cells are the possible target for HIV.
Next, we exposed HIV-1 clone (JR-FL) to the HFWT-activated NK cells, however, direct HIV infection to the NK cells was not observed. While, co-culture of activated NK cells with HIV infected T cells allowed HIV infection of NK cells. Because NK cell-specific marker, NKp46, was detected on p24+CD3-CD56+ cells but not on CD3+ cells, p24+CD3-CD56+ cells were certainly NK cells. These results demonstrate that NK cells are the targets of HIV.
Altogether, our data suggest that CD4+ T cells transfer HIV to NK cells during this cell-to-cell contact, which cause the NK cells to be the long-living viral reservoirs or modify the function of NK cells in HIV-infected patients. Our novel co-culture system using activated NK cells and HIV-infected T cells is the powerful tool to study the function of NK cells on HIV infection.
Novel Function of the TAL1/SCL Transcription Factor in Differentiation of Murine Bone Marrow Monocytes.
Authors: Dey Soumyadeep , Shi Yubin , Brandt Stephen J. 
Published: April 2020

Abstract
The TAL1 (or SCL) gene encodes a basic helix-loop-helix transcription factor that can activate or repress gene expression, depending on its interaction partners. TAL1 proteins are expressed in the vascular and hematopoietic systems and in erythroid, megakaryocytic, and mast cell precursors. TAL1 is essential for hematopoietic commitment and vascular remodeling during embryogenesis and is also important to the differentiation of erythroid and megakaryocytic progenitors postnatally. Although a mouse Tal1 cDNA was cloned from a bone marrow (BM) macrophage cDNA library and we reported expression of Tal1 protein in mouse bone marrow mononuclear cells (Blood83:1200, 1994), no studies have examined the function of this transcription factor in the monocyte-macrophage lineage. To address this issue, we characterized Tal1 expression in primary mouse BM monocytes and M1 monocytic leukemia cells induced to differentiate in vitro. We isolated BM cells from wild-type C57BL6/J mice and purified a mononuclear cell population by pronase treatment and density gradient centrifugation on horse serum. Monocytes were then induced to differentiate in culture, initially in the presence of both interleukin-3 (IL-3) and macrophage colony-stimulating factor (M-CSF) and subsequently in the presence of L-cell conditioned medium. Tal1 expression was maintained in these cultures, declining by only 14% over the full culture period, while calcitonin receptor expression, a marker of osteoclastic differentiation, was extinguished. In parallel, interleukin-6 receptor (IL-6R) and macrophage colony-stimulating factor receptor (M-CSFR) mRNA increased 3.5- and 114-fold, respectively. In M1 cells induced to differentiate with recombinant murine IL-6, Tal1 protein abundance decreased similarly with differentiation. To assess the function of Tal1 in this cell lineage, a full-length Tal1 cDNA was introduced into both primary BM monocytes and M1 cells via retroviral gene transfer. Puurified monocytes were transduced 24 hr after isolation with wild-type Tal1 or a DNA-binding-defective Tal1 mutant, T192P, in the MSCV-GFP vector. GFP-expressing cells were then sorted and induced to differentiate. Gene expression analysis using semi-quantitative PCR carried out after 8 days of culture showed increased expression of Tal1 mRNA in cells transduced with the wild-type Tal1 cDNA compared to the vector control. This was accompanied by upregulation of IL-6R and M-CSFR expression relative to both parental vector- and mutant-transduced cells. Northern blot analysis of M1 cells transduced with the Tal1 cDNA showed similar upregulation of M-CSFR mRNA relative to vector control cells. In summary, these studies show that the TAL1 transcription factor is expressed by murine monocytes during differentiation to macrophages and suggest a role for TAL1 in both gene expression and differentiation of this lineage. Further, they suggest a requirement for direct TAL1 DNA binding.
Adiponectin Produced by Lymphocytes Inhibits Granulopoiesis.
Authors: Crawford Lisa J. , Peake Roy W. , Price Susan , Morris Treen C. , Irvine Alexandra E. 
Published: April 2020

Abstract
Previous studies by our group have shown that normal unstimulated lymphocytes produce a protein which inhibits colony formation of granulopoietic progenitors, but has no effect on erythroid progenitors. Therefore, this inhibitor was initially designated GIA (granulopoietic inhibitory activity). GIA was identified as a glycoprotein of approximately 30 kDa, with a pI of 7.9 - 8.4. Futhermore, we demonstrated that this inhibitor may have physiological significance in that its production is altered in patients with neutropenia. GIA has proved difficult to characterise to date since it is produced in relatively low amounts although it has a high specific biological activity. Adiponectin is an adipokine reported to share many of the inhibitory characteristics of GIA and has been demonstrated to act as a negative regulator of hematopoiesis and immune response. This study aimed to determine whether GIA is adiponectin or if it represents an adiponectin-like molecule. Lymphocyte conditioned medium (LCM) from lymhocytes cultured at 1 X 106 cells/ml in HL-1 minimal medium was used as a source of GIA. Inclusion of LCM as 10% of the top layer of agar in a myeloid colony assay inhibited growth of CFU-GM by 52 ± 11 % (n=3), confirming the presence of the inhibitory activity. RNA and protein from lymphocytes and LCM harvested over a 7 day culture period were subsequently investigated for adiponectin expression. Western blot analysis demonstrated a distinct banding pattern in days 3-7 LCM corresponding to monomers, dimers, trimers and greater. This is consistent with adiponectin which circulates as a multimer of trimers. Characterisation of GIA at the transcript level confirmed that GIA is in fact adiponectin. The N-terminal collagenous domain, C-terminal globular domain and full length adiponectin were amplified by RT-PCR analysis. Adiponectin is thought to be secreted exclusively from adipocytes and much of our current knowledge of this molecule relates to its metabolic functions. Our study provides evidence that adiponectin is also produced by lymphocytes and may play a role in the pathogenesis of neutropenia.
Abnormal Localization of Neutrophil Elastase in Patients with Severe Congenital Neutropenia.
Authors: Sato Takashi , Habara Masakazu , Kihara Hiroki , Kawaguchi Hiroshi , Miki Mizuka , Nakamura Kazuhiro , Okada Satoshi , Ishikawa Nobutsune , Ohno Norioki , Kobayashi Masao 
Published: April 2020

Abstract
Mutations in the ELA2 gene encoding neutrophil elastase (NE) in patients with severe congenital neutropenia (SCN) are involved in the pathogenesis of this disorder, possibly due to the abnormal protein trafficking and accelerated apoptosis of myeloid cells. In this study we precisely examined the localization of NE in neutrophils and myeloid precursor cells in bone marrow in patients with SCN using immunofluorescence microscopy equipped with three-dimensional analysis program. Three patients with SCN were enrolled in this study. All patients with SCN showed heterozygous mutation in the ELA2 gene. In normal subjects the pattern of localization of NE in mature neutrophils was almost similar to those of myeloperoxidase (MPO), proteinase 3, lysosomal associated membrane protein 2 (LAMP2). Administration of G-CSF to normal subjects did not affect the pattern of the localization of these proteins in neutrophils. In contrast, mature neutrophils elicited by the administration of G-CSF in patients with SCN NE predominantly localized to the plasma membranes. A small part of NE was detected in the cytoplasmic compartment. The pattern of localization of NE was significantly different from those of MPO, proteinase 3, and LAMP2 in SCN patients, suggesting the abnormal traffic of NE to granules. Adaptor proteins 3 (AP3) specifically shuttles transmembrane cargo proteins from the trans-Golgi to lysosomes. AP3 of myeloid progenitor cells enriched for CD33-positive cells in normal bone marrow was localized in both cytoplasm and plasma membranes. The localization pattern of AP3 was completely consistent with those of NE, MPO, and LAMP2. The localization of AP3 of promyelocytes in patients with SCN was observed in both plasma membranes and cytoplasm. This finding was completely similar to that in normal myeloid precursor cells. However, the localization of NE of promyelocytes in SCN patients was predominantly in plasma membrane. The figures merged apparently presented the different localization of NE and AP3. This result was confirmed by the 3-dimensional analysis with histogram. The localizations of other constituents of primary granules, MPO, poteinase 3, and LAMP2, were consistent with those of AP3. These observations suggest that the mislocalization of NE in myeloid precursor cells in SCN patients does not result from a generalized impairment of protein trafficking but is specific to the mutant NE. The abnormal localization of NE may be involved in the pathogenesis of SCN associated with ELA2 mutation.
Prospective Isolation of Human Eosinophil-Committed Progenitors.
Authors: Mori Yasuo , Iwasaki Hiromi , Yoshimoto Goichi , Okeda Aki , Miyamoto Toshihiro , Mizuno Shin-ichi , Harada Mine , Takatsu Kiyoshi , Akashi Koichi 
Published: April 2020

Abstract
Eosinophils play an important role in the pathogenesis of allergic reactions or chronic inflammatory diseases by releasing various types of cytokines and chemical mediators. Recently, we have identified murine eosinophil-committed progenitors (mEoPs) in mouse bone marrow. The expression of receptor for IL-5, a critical cytokine for proliferation and differentiation of eosinophils, was a key marker to isolate mEoPs: mEoP was IL-5Ra+Lineage(lin)-CD34+c-Kitlow population in murine bone marrow (J Exp Med.201, 1891ndash;7, 2005). Here we report that EoPs are prospectively isolatable also in human bone marrow. We analyzed the expression of human IL-5Ra in human stem and progenitor populations, and found that a fraction of common myeloid progenitor (CMP; lin-CD34+CD38+CD45RA-IL-3Ra+) population expressed hIL-5Ra on their surface by using anti-human IL-5Ra monoclonal antibodies. IL-5Ra protein and mRNA were undetectable in hematopoietic stem cells (HSCs; lin-CD34+CD38-), common lymphoid progenitors (CLPs; lin-CD34+CD38+CD10+), megakaryocyte/erythrocyte progenitors (MEPs; lin-CD34+CD38+CD45RA-IL-3Ra-), or granulocyte/monocyte progenitors(GMPs; lin-CD34+CD38+CD45RA+IL-3Ra+) by FACS and RT-PCR, respectively. The IL-5Ra+ cells within the CMP fraction constituted only ~0.04% of steady-state bone marrow mononuclear cells, and gave rise only to pure eosinophil colonies. Thus we termed this population as human EoP (hEoP). Both HSCs and the IL-5Ra- fraction of CMPs gave rise to IL-5Ra+ hEoPs in vitro in the presence of IL-3 and GM-CSF, while MEPs or GMPs never generated hEoPs, indicating that human eosinophil pathway diverges at the CMP stage, and that the eosinophil potential was lost at the GMP or MEP stage. Accordingly, the human eosinophil pathway is different from that in murine hematopoiesis where mEoPs develop from the GMP stage. Strikingly, the number of hEoPs in the bone marrow of patients with hypereosinophilic syndrome was significantly (~4-fold) increased as compared to that in normal bone marrow, suggesting that hEoP represents a critical stage for eosinophilia in vivo. Thus, the hEoP is an attractive candidate for therapeutic target in eosinophil-related allergic and inflammatory disorders. This population might also be very useful to study the molecular mechanism of human eosinophil development.
Kruppel-Like Factor 4 Upregulates p21 and Downregulates Proliferation of Human and Mouse HSPCs, but Is Not Essential for Mouse HSPC Repopulation.
Authors: Alder Jonathan K. , Georgantas Robert W. , Hildreth Richard L. , Yu Xiaobing , Civin Curt I. 
Published: April 2020

Abstract
Several Kruppel-like factor family members, including KLF1, KLF2, KLF3, and KLF6 have pivotal roles in hematopoiesis. Experiments in zebrafish have suggested that KLF4 may play a similar role. Here we found that enforced expression of KLF4 in hematopoietic cells induced cell cycle arrest without triggering apoptosis. Based on the high levels of expression of KLF4 in mouse and human hematopoietic stem-progenitor cells (HSPCs), we hypothesized and demonstrated that KLF4 regulates proliferation of these cells through regulation of p21cip1/waf1 (p21). Nevertheless, KLF4−/− mouse fetal liver cells had normal numbers of all mature lineages and provided radioprotection, similar to wild type (wt) controls. Furthermore, in long-term competitive repopulation assays, KLF4−/− mouse HSPCs demonstrated hematopoietic potency equivalent to wt. We found that KLF2 is expressed at higher levels than KLF4 in mouse HSPCs and is a more potent activator of p21, suggesting that KLF2 (and/or other KLF family members) may play a compensatory role in KLF4−/− HSPCs. Thus, although is not essential for their normal development and function, KLF4 expression is sufficient to induce p21-mediated cell cycle arrest in hematopoietic cells.
Identification of Key Cellular Regulators That Interact with Id1 To Control Hematopoietic Stem Cell Behavior.
Authors: Jankovic Vladimir , Ciarrocchi Alessia , DeBlasio Tony , Benezra Robert , Nimer Stephen D. 
Published: April 2020

Abstract
The ability of hematopoietic stem cells to tightly regulate the transition from relative quiescence and self-renewal to the transiently amplifying, differentiating progenitor fate is critical for HSC homeostasis as well as their regenerative capacity. We have recently described the diminished frequency and rapid exhaustion of HSC self-renewal capacity in the absence of the dominant negative helix-loop-helix molecule Id1. Furthermore, Id1 null HSCs have an increased rate of cycling, coupled with accelerated myeloid commitment both in vivo and in vitro. This is reflected in the elevated expression of myelo-erythroid transcription factors (c/EBPalpha and GATA1) within the Lin−c-kit+Sca-1+ population - “myeloid priming”. The major targets of Id1 mediated transcriptional repression are the ubiquitous E protein E2A as well as Ets transcription factors (Ets1 and Ets2). We hypothesized that the unrestrained activity of these and/or other targets of Id1 transcriptional repression leads to premature HSC commitment in Id1 null animals. Indeed, we show that HSC differentiation in culture can be delayed by transduction of E2A directed shRNA specifically in Id1 null, but not in wild-type Id1 expressing cells. This indicates an abnormal E2A activity in Id1 null HSCs that could be responsible for their increased differentiation status. To further define the transcriptional deregulation in Id1 null HSCs, we have used the Affymetrix microarray technology. We observed ~3 fold increased expression of the CDK inhibitor p21 in freshly isolated Id1 null HSCs and have confirmed this result by multiple independent qPCR measurements. The transcriptional induction of p21 by E2A as well as its repression by Id1 have been well established. Therefore, the observed p21 induction could be explained by the elevated level of E2A activity in HSCs in the absence of Id1 expression. To explore the functional significance of Id1 mediated p21 regulation in HSCs, we have generated p21/Id1 double knockout animals. Surprisingly, despite its reported function in restricting the cell cycle entry of normal HSCs, we show that in the context of Id1 loss, p21 expression is required for the accelerated HSC cycling, and unlike Id1 single null HSCs, p21/Id1 double knockout HSCs do not show accelerated myeloid differentiation in culture. Therefore, we propose that Id1 actively represses E2A activity in HSCs, as well as the induction of p21, which could be an important component of the HSC commitment program. Further studies will be presented defining the in vivo relevance of the Id1/p21 genetic interaction for HSC growth and differentiation.
Dose Dependent Effects of STAT5 on Proliferation, Differentiation and Self Renewal of Hematopoietic Stem/Progenitor Cells.
Authors: Wierenga Albertus T.J. , Rozenveld-Geugien Marjan , Vellenga Edo , Schuringa Jan Jacob 
Published: April 2020

Abstract
To study the dose dependent effects of ectopically expressed STAT5A in cordblood (CB) CD34+ cells, we developed a tamoxifen-inducible STAT5A expression system. Constitutively active STAT5A (STAT5A1*6) was fused to the ER domain to facilitate its activation upon stimulation of the transduced cells by the addition of 4OHT. Transactivation and DNA binding studies revealed that STAT5A could be activated to different levels in a controlled way by addition of different concentrations of 4OHT. CB CD34+ cells were retrovirally transduced with this construct and the effects on proliferation, differentiation and self renewal was studied.
In an MS5 coculture system, we demonstrated that a high dose of 4OHT (500nM) resulted in a rapid burst of erythropoiesis and a block in myeloid differentiation (as previously demonstrated using STAT5A1*6 without an ER domain). Moderate levels of 4OHT (20nM) however, resulted in a much higher proliferation of not yet differentiated cells, ultimately resulting in a higher expansion. This higher expansion coincided with a higher frequency of early (day10) cobblestones (without 4OHT: 1,5 cobblestones per 104 plated cells, 20nM 4OHT: 1750/104, 500nM 4OHT:200/104). RT-PCR of STAT5A1*6-ER transduced cells revealed a rapid downregulation of C/EBPalpha mRNA upon stimulation with 4OHT, with a dose response curve inversely correlated to the number of early cobblestones, suggesting that these two processes are linked.
When STAT5A1*6-ER transduced cells were cultured in methylcellulose, stimulation with increasing concentrations of 4OHT resulted in decreasing numbers of colonies (myeloid as well as erythroid). Interestingly, whereas colonies from unstimulated (control) cultures and 500nM 4OHT stimulated cultures could not be secondary plated, cultures stimulated with only very low concentrations of 4OHT (1 nM) could be secondary plated, giving rise to only myeloid colony formation. These findings demonstrate that the level of activation of STAT5 affects the proliferation and differentiation in a separate manner; low levels of STAT5A activation results in self renewal of the progenitor/stem cell pool cells, whereas high levels of STAT5A induces a rapid differentiation at the expense of proliferation and self renewal.
These data correspond well to our findings in AML cells. Downregulation of the moderate constitutive STAT5 activation in CD34+ AML cells by STAT5 RNAi affects especially the proliferative capacity of the AML cells. In conclusion, we hypothesise that low constitutive levels of active STAT5 might contribute to leukemic transformation.
Mutant Ras-Expressing Fibroblasts Induce Expansion of Circulating Angio- and Lymphangiogenic Precursor Cells: Potential Role in Tumour Vascularisation.
Authors: Knight Robert J. , O’Brien Tracey A. , Lindeman Robert , Dolnikov Alla 
Published: April 2020

Abstract
Fibroblasts have a prominent role in the initiation, enlargement and metastasis of cancers. Their production of growth factors, chemokines and extracellular matrix facilitate the angiogenic recruitment of endothelial cells and pericytes. The oncogene Ras, known to be frequently mutated in many cancers, has also been shown to promote the expression of pro-angiogenic growth factors, chemokines and extracellular matrix components similar to those of fibroblasts. Here we show that mutant N-ras (N-rasm)/GFP - transduced NIH3T3 fibroblasts act to promote the ex vivo expansion of umbilical cord blood (UCB)-derived primitive endothelial progenitor cells through paracrine mechanisms. Biphasic expansion of both angiogenic and lymphangiogenic precursors was observed although they exhibited different dynamics in their expansion pattern. The first peak of expansion for both types of precursor cells was seen on day 10 with a 69-fold expansion of the CD34+VEGFR2+(endothelial) and a 23-fold expansion of CD34+VEGFR3+(lymphatic) cells co-cultured with media conditioned by N-rasm-transduced 3T3 cells. This compared to a 20-fold and 14-fold expansion respectively, in cells co-cultured with media conditioned by GFP (only)-transduced fibroblasts. Endothelial cell differentiation accounts for the dramatic reduction in the numbers of CD34+VEGFR2+ and CD34+VEGFR3+ precursor cells on day 14 with concomitant expansion of CD34−VEGFR2+ and CD34−VEGFR3+cells. The second peak for the expansion of endothelial precursor cells was seen on day 19 with a 214-fold expansion compared to 28-fold in the control cells. In addition, the expansion of CD14+ VEGFR2+ and CD14+VEGFR3+ cells was observed on days 14 and 19, the later correlated with the expansion of monocytic CD34−CD14+ cells promoted by N-rasm-transduced fibroblasts. The expanded monocytes appear to contribute to the expansion of endothelial and lymphatic precursor cells induced by N-rasm-transduced fibroblasts. We propose that the angiogenic factors secreted by mutant Ras-expressing fibroblasts in a tumour microenvironment promote tumour angiogenesis through the expansion of the circulated endothelial and lymphatic precursor cells. The recruitment of the expanded endothelial and lymphatic cells into the tumour’s vascular system is currently being investigated. We propose that targeting aberrant Ras signaling in tumour fibroblasts may represent an important target for cancer therapies.