Differential Hox expression in murine embryonic stem cell models of normal and malignant hematopoiesis

The Hox family are master transcriptional regulators of developmental processes, including hematopoiesis. The Hox regulators, caudal homeobox factors (Cdx1-4), and Meis1, along with several individual Hox proteins, are implicated in stem cell expansion during embryonic development, with gene dosage playing a significant role in the overall function of the integrated Hox network. To investigate the role of this network in normal and aberrant, early hematopoiesis, we employed an in vitro embryonic stem cell differentiation system, which recapitulates mouse developmental hematopoiesis. Expression profiles of Hox, Pbx1, and Meis1 genes were quantified at distinct stages during the hematopoietic differentiation process and compared with the effects of expressing the leukemic oncogene Tel/PDGFR;2. During normal differentiation the Hoxa cluster, Pbx1 and Meis1 predominated, with a marked reduction in the majority of Hox genes (27/39) and Meis1 occurring during hematopoietic commitment. Only the posterior Hoxa cluster genes (a9, a10, a11, and a13) maintained or increased expression at the hematopoietic colony stage. Cdx4, Meis1, and a subset of Hox genes, including a7 and a9, were differentially expressed after short-term oncogenic (Tel/PDGFR;2) induction. Whereas Hoxa4-10, b1, b2, b4, and b9 were upregulated during oncogenic driven myelomonocytic differentiation. Heterodimers between Hoxa7/Hoxa9, Meis1, and Pbx have previously been implicated in regulating target genes involved in hematopoietic stem cell (HSC) expansion and leukemic progression. These results provide direct evidence that transcriptional flux through the Hox network occurs at very early stages during hematopoietic differentiation and validates embryonic stem cell models for gaining insights into the genetic regulation of normal and malignant hematopoiesis

Gene expression during THP-1 differentiation is influenced by vitamin D3 and not vibrational mechanostimulation

Background: In injury or infection, monocytes migrate into the affected tissues from circulation and differentiate into macrophages which are subsequently involved in the inflammatory responses. Macrophage differentiation and activation have been studied in response to multiple chemokines and cytokines. However, mechanical, and physical stimuli can also influence macrophage differentiation, activation, cytokine production, and phagocytic activity. Methods: In this study the macrophage differentiation from THP-1 monocytes was assessed upon the stimulation with 1,25-dihydroxyvitamin D3 and 1,000 Hz vibrations, using qPCR for quantification of transcript expression. Vitamin D binds the vitamin D receptor (VDR) and subsequently modulates the expression of a variety of genes in monocytes. The effects of the 1,000 Hz vibrational stimulation, and the combined treatment of vitamin D3 and 1000 Hz vibrations were unknown. The differentiation of macrophages was assessed by looking at transcription of macrophage markers (e.g., CD14, CD36), antigen presenting molecules (e.g., HLA-DRA), transcription factors (e.g., LEF-1, TCF7L2), and mechanosensors (e.g., PIEZO1 and PKD2). Results: The results showed that vitamin D3 induced THP-1 macrophage differentiation, which was characterized by upregulation of CD14 and CD36, downregulation of HLA-DRA, upregulation of the PKD2 (TRPP2), and an inverse relationship between TCF7L2 and LEF-1, which were upregulated and downregulated respectively. The 1,000 Hz vibrations were sensed from the cells which upregulated PIEZO1 and TCF3, but they did not induce expression of genes that would indicate macrophage differentiation. The mRNA transcription profile in the cells stimulated with the combined treatment was comparable to that of the cells stimulated by the vitamin only. The 1,000 Hz vibrations slightly weakened the effect of the vitamin for the regulation of CD36 and HLA-DMB in the suspension cells, but without causing changes in the regulation patterns. The only exception was the upregulation of TCF3 in the suspension cells, which was influenced by the vibrations. In the adherent cells, the vitamin D3 cancelled the upregulating effect of the 1,000 Hz vibrations and downregulated TCF3. The vitamin also cancelled the upregulation of PIEZO1 gene by the 1,000 Hz vibrations in the combined treatment. Conclusion: The mechanical stimulation with 1,000 Hz vibrations resulted in upregulation of PIEZO1 in THP-1 cells, but it did not affect the differentiation process which was investigated in this study. Vitamin D3 induced THP-1 macrophage differentiation and could potentially influence M2 polarization as observed by upregulation of CD36 and downregulation of HLA-DRA. In addition, in THP-1 cells undergoing the combined stimulation, the gene expression patterns were influenced by vitamin D3, which also ablated the effect of the mechanical stimulus on PIEZO1 upregulation

SENIEUR status of the originating cell donor negates certain ‘anti-immunosenescence’ effects of ebselen and N-acetyl cysteine in human T cell clone cultures

BACKGROUND: Damage to T cells of the immune system by reactive oxygen species may result in altered cell function or cell death and thereby potentially impact upon the efficacy of a subsequent immune response. Here, we assess the impact of the antioxidants Ebselen and N-acetyl cysteine on a range of biological markers in human T cells derived from a SENIEUR status donor. In addition, the impact of these antioxidants on different MAP kinase pathways in T cells from donors of different ages was also examined. METHODS: T cell clones were derived from healthy 26, 45 and SENIEUR status 80 year old people and the impact of titrated concentrations of Ebselen or N-acetyl cysteine on their proliferation and in vitro lifespan, GSH:GSSG ratio as well as levels of oxidative DNA damage and on MAP kinase signaling pathways was examined. RESULTS: In this investigation neither Ebselen nor N-acetyl cysteine supplementation had any impact on the biological endpoints examined in the T cells derived from the SENIEUR status 80 year old donor. This is in contrast to the anti-immunosenescent effects of these antioxidants on T cells from donors of 26 or 45 years of age. The analysis of MAP kinases showed that pro-apoptotic pathways become activated in T cells with increasing in vitro age and that Ebselen or N-acetyl cysteine could decrease activation (phosphorylation) in T cells from 26 or 45 year old donors, but not from the SENIEUR status 80 year old donor. CONCLUSIONS: The results of this investigation demonstrate that the biological phenotype of SENIEUR status derived human T cells negates the anti-immunosenescence effects of Ebselen and also N-acetyl cysteine. The results highlight the importance of pre-antioxidant intervention evaluation to determine risk-benefit

Acceptability and feasibility of a computer-based application to help Aboriginal and Torres Strait Islander Australians describe their alcohol consumption

We examined acceptability and feasibility of a tablet application (“App”) to record self-reported alcohol consumption among Aboriginal and Torres Strait Islander Australians. Four communities (1 urban; 3 regional/remote) tested the App, with 246 adult participants (132 males, 114 females). The App collected (a) completion time; (b) participant feedback; (c) staff observations. Three research assistants were interviewed. Only six (1.4%) participants reported that the App was “hard” to use. Participants appeared to be engaged and to require minimal assistance; nearly half verbally reflected on their drinking or drinking of others. The App has potential for surveys, screening, or health promotion

A study of the granulocyte-macrophage colony stimulating factor receptor (GM-CSFR) in leukaemia

Mutation of signaling molecules such as Ras have been shown to confer a growth advantage in blast cells from patients with acute myeloid leukaemia (AML) and may contribute to the pathogenesis of the disease. Alterations in other signaling molecules could also play a role in leukaemogenesis. GM-CSF is a key growth factor in haemopoiesis which exerts its influence on target cells via membrane bound receptors. The GM-CSF receptor (GM-CSFR) is composed of oligomers of ligand-specific a chains and p chains which are common to the GM-CSF/IL-3 and IL-5 receptors (βC). The intracytoplasmic tail of the βC chain is essential for the activation of several downstream signaling pathways and alterations in this region could deregulate normal signaling processes. RT-PCR-SSCP analysis was used to look for mutations in the βC chain tail (nts 1281-2816) in RNA from 35 patients with acute myeloid leukaemia (AML) and 10 haematologically normal controls. Six nucleotide substitutions were detected, three of which were silent (Ser426, Pro648 and Pro800) and three which altered the amino acid residue at that position in the receptor sequence (Gly647>Val, Val652>Met and Pro603Thr). However all substitutions were detected in normal controls and were thought to be polymorphisms, with allele frequencies of 0.23 and 0.13 found for two of the most common silent substitutions. RNA from patients with juvenile chronic myeloid leukaemia (JCML) was also screened for mutations in the entire GM-CSFR α and β chain coding sequences using SSCP analysis as studies have shown progenitors from JCML patients have a hypersensitive growth response to GM-CSF in culture. Two nucleotide substitutions accounted for all a chain abnormalities (Ala17Gly and silent Val333) with both previously detected in normal controls. Four base substitutions were detected in the β chain. Three were polymorphisms previously described in AML patients and normal controls. A further nucleotide mutation which resulted in a Glu249Gln substitution was also found but was not thought to be of pathological significance as a Gin residue is present at this amino acid in both mouse β chains. During the course of these studies a novel isoform of the βC chain with a truncated intracytoplasmic tail (βIT) was isolated by our group. Transcripts of this alternatively spliced isoform were shown to be present in relatively high levels in blast cells from AML patients. Polyclonal antibodies were raised against a novel 23 amino acid sequence in the tail of the βIT chain and screened in COS-7 cells transfected with plasmids coding for βC or βIT chain before being used to demonstrate expression of the truncated receptor in a range of haemopoietic cells. The βIT-specific antibodies were then used to investigate GM-CSFR α and β chain stoichiometry and demonstrated that α chain homodimerization occurred without ligand stimulation in primary haemopoietic cells or GM-CSFR α chain in transfected COS-7 cells

Gene expression during THP-1 differentiation is influenced by vitamin D3 and not vibrational mechanostimulation

BackgroundIn injury or infection, monocytes migrate into the affected tissues from circulation and differentiate into macrophages which are subsequently involved in the inflammatory responses. Macrophage differentiation and activation have been studied in response to multiple chemokines and cytokines. However, mechanical, and physical stimuli can also influence macrophage differentiation, activation, cytokine production, and phagocytic activity.MethodsIn this study the macrophage differentiation from THP-1 monocytes was assessed upon the stimulation with 1,25-dihydroxyvitamin D3 and 1,000 Hz vibrations, using qPCR for quantification of transcript expression. Vitamin D binds the vitamin D receptor (VDR) and subsequently modulates the expression of a variety of genes in monocytes. The effects of the 1,000 Hz vibrational stimulation, and the combined treatment of vitamin D3 and 1000 Hz vibrations were unknown. The differentiation of macrophages was assessed by looking at transcription of macrophage markers (e.g.,CD14,CD36), antigen presenting molecules (e.g.,HLA-DRA), transcription factors (e.g.,LEF-1,TCF7L2), and mechanosensors (e.g.,PIEZO1andPKD2).ResultsThe results showed that vitamin D3 induced THP-1 macrophage differentiation, which was characterized by upregulation ofCD14andCD36, downregulation ofHLA-DRA, upregulation of thePKD2(TRPP2), and an inverse relationship betweenTCF7L2andLEF-1, which were upregulated and downregulated respectively. The 1,000 Hz vibrations were sensed from the cells which upregulatedPIEZO1andTCF3, but they did not induce expression of genes that would indicate macrophage differentiation. The mRNA transcription profile in the cells stimulated with the combined treatment was comparable to that of the cells stimulated by the vitamin only. The 1,000 Hz vibrations slightly weakened the effect of the vitamin for the regulation ofCD36andHLA-DMBin the suspension cells, but without causing changes in the regulation patterns. The only exception was the upregulation ofTCF3in the suspension cells, which was influenced by the vibrations. In the adherent cells, the vitamin D3 cancelled the upregulating effect of the 1,000 Hz vibrations and downregulatedTCF3. The vitamin also cancelled the upregulation ofPIEZO1gene by the 1,000 Hz vibrations in the combined treatment.ConclusionThe mechanical stimulation with 1,000 Hz vibrations resulted in upregulation ofPIEZO1in THP-1 cells, but it did not affect the differentiation process which was investigated in this study. Vitamin D3 induced THP-1 macrophage differentiation and could potentially influence M2 polarization as observed by upregulation ofCD36and downregulation ofHLA-DRA. In addition, in THP-1 cells undergoing the combined stimulation, the gene expression patterns were influenced by vitamin D3, which also ablated the effect of the mechanical stimulus onPIEZO1upregulation.</jats:sec

A Truncated Isoform of the Human β Chain Common to the Receptors for Granulocyte-Macrophage Colony-Stimulating Factor, Interleukin-3 (IL-3), and IL-5 With Increased mRNA Expression in Some Patients With Acute Leukemia

AbstractWe report here a naturally occurring isoform of the human β chain common to the receptors for granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin-3 (IL-3), and IL-5 (GMRβC) with a truncated intracytoplasmic tail caused by deletion of a 104-bp exon in the membrane-proximal region of the chain. This β intracytoplasmic truncated chain (βIT) has a predicted tail of 46 amino acids, instead of 432 for βC, with 23 amino acids in common with βC and then a new sequence of 23 amino acids. In primary myeloid cells, βIT comprised approximately 20% of the total β chain message, but was increased up to 90% of total in blast cells from a significant proportion of patients with acute leukemia. Specific anti-βITantibodies demonstrated its presence in primary myeloid cells and cell lines. Coexpression of βIT converted low-affinity GMRα chains (KD 2.5 nmol/L) to higher-affinity αβ complexes (KD 200 pmol/L). These could bind JAK2 that was tyrosine-phosphorylated by stimulation with GM-CSF. βITdid not support GM-CSF–induced proliferation when cotransfected with GMRα into CTLL-2 cells. Therefore, it may interfere with the signal-transducing properties of the βC chain and play a role in the pathogenesis of leukemia.</jats:p

A Truncated Isoform of the Human β Chain Common to the Receptors for Granulocyte-Macrophage Colony-Stimulating Factor, Interleukin-3 (IL-3), and IL-5 With Increased mRNA Expression in Some Patients With Acute Leukemia

We report here a naturally occurring isoform of the human β chain common to the receptors for granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin-3 (IL-3), and IL-5 (GMRβC) with a truncated intracytoplasmic tail caused by deletion of a 104-bp exon in the membrane-proximal region of the chain. This β intracytoplasmic truncated chain (βIT) has a predicted tail of 46 amino acids, instead of 432 for βC, with 23 amino acids in common with βC and then a new sequence of 23 amino acids. In primary myeloid cells, βIT comprised approximately 20% of the total β chain message, but was increased up to 90% of total in blast cells from a significant proportion of patients with acute leukemia. Specific anti-βITantibodies demonstrated its presence in primary myeloid cells and cell lines. Coexpression of βIT converted low-affinity GMRα chains (KD 2.5 nmol/L) to higher-affinity αβ complexes (KD 200 pmol/L). These could bind JAK2 that was tyrosine-phosphorylated by stimulation with GM-CSF. βITdid not support GM-CSF–induced proliferation when cotransfected with GMRα into CTLL-2 cells. Therefore, it may interfere with the signal-transducing properties of the βC chain and play a role in the pathogenesis of leukemia.</jats:p

Canonical Wnt Signaling Promotes Early Hematopoietic Progenitor Formation and Erythroid Specification during Embryonic Stem Cell Differentiation

<div><p>The generation of hematopoietic stem cells (HSCs) during development is a complex process linked to morphogenic signals. Understanding this process is important for regenerative medicine applications that require <i>in vitro</i> production of HSC. In this study we investigated the effects of canonical Wnt/β-catenin signaling during early embryonic differentiation and hematopoietic specification using an embryonic stem cell system. Our data clearly demonstrates that following early differentiation induction, canonical Wnt signaling induces a strong mesodermal program whilst maintaining a degree of stemness potential. This involved a complex interplay between β-catenin/TCF/LEF/Brachyury/Nanog. β-catenin mediated up-regulation of TCF/LEF resulted in enhanced brachyury levels, which in-turn lead to Nanog up-regulation. During differentiation, active canonical Wnt signaling also up-regulated key transcription factors and cell specific markers essential for hematopoietic specification, in particular genes involved in establishing primitive erythropoiesis. This led to a significant increase in primitive erythroid colony formation. β-catenin signaling also augmented early hematopoietic and multipotent progenitor (MPP) formation. Following culture in a MPP specific cytokine cocktail, activation of β-catenin suppressed differentiation of the early hematopoietic progenitor population, with cells displaying a higher replating capacity and a propensity to form megakaryocytic erythroid progenitors. This bias towards erythroid lineage commitment was also observed when hematopoietic progenitors were directed to undergo myeloid colony formation. Overall this study underscores the importance of canonical Wnt/β-catenin signaling in mesodermal specification, primitive erythropoiesis and early hematopietic progenitor formation during hematopoietic induction.</p> </div