Eosinophils, but not neutrophils, exibit an efficient DNA repair machinary and high nucleolar activity

BACKGROUND AND OBJECTIVES: Traditionally eosinophils have been considered terminally differentiated cells that play a role in host protection against parasites. However, there is some evidence showing that eosinophils are, in fact, multifunctional leukocytes involved in inflammatory responses, as well as in tissue homeostasis. We characterized the transcriptome profile of human eosinophils, and, for the purpose of comparison, the transcriptome profile of neutrophils, monocytes and hematopoietic progenitor cells. Moreover, we studied the activation of selected cellular processes for which a significant differential expression was demonstrated. DESIGN AND METHODS: We profiled gene expression using Affymetrix GeneChips. DNA repair capacity was tested using the comet assay. Nucleoli and their activity were characterized by transmission electron microscopy analysis, silver staining of nucleolus regions (AgNOR) and RNA staining. RESULTS: Gene expression profiling showed that eosinophils appear hierarchically closer to monocytes than to neutrophils. Gene ontology mapping of differentially expressed genes revealed that eosinophils express categories very similar to those expressed by monocytes, related to DNA repair and nucleolar functions. Moreover, our data show that eosinophils and monocytes maintain the ability to repair both double and single strand DNA breaks, whereas neutrophils lack this capacity. Furthermore, eosinophils exhibit nucleolar activity, which is lacking in neutrophils, but resembles that in monocytes. INTERPRETATION AND CONCLUSIONS: The presence of large, active nucleoli in eosinophils, coupled to marked activity of DNA repair systems, suggests that eosinophils are not terminally differentiated cells. Indeed, their transcriptome profile and functional properties are more similar to those of non-terminally differentiated cells such as monocytes, rather than to neutrophils

miR-382-5p Controls Hematopoietic Stem Cell Differentiation Through the Downregulation of MXD1

microRNAs are key regulators of gene expression that control stem cell fate by posttranscriptional downregulation of hundreds of target genes through seed pairing in their 3' untranslated region. In fact, miRNAs tightly regulate fundamental stem cell processes, like self-renewal, proliferation, and differentiation; therefore, miRNA deregulation may contribute to the development of solid tumors and hematological malignancies. miR-382-5p has been found to be upregulated in patients with myeloid neoplasms, but its role in normal hematopoiesis is still unknown. In this study, we demonstrated that miR-382-5p overexpression in CD34(+) hematopoietic stem/progenitor cells (HSPCs) leads to a significant decrease of megakaryocyte precursors coupled to increase of granulocyte ones. Furthermore, by means of a computational analysis using different prediction algorithms, we identified several putative mRNA targets of miR-382-5p that are downregulated upon miRNA overexpression (ie, FLI1, GATA2, MAF, MXD1, RUNX1, and SGK1). Among these, we validated MXD1 as real target of miR-382-5p by luciferase reporter assay. Finally, we showed that MXD1 knockdown mimics the effects of miR-382-5p overexpression on granulocyte and megakaryocyte differentiation of CD34(+) cells. Overall, our results demonstrated that miR-382-5p expression favors the expansion of granulocyte lineage and impairs megakaryocyte commitment through MXD1 downregulation. Therefore, our data showed for the first time that the miR-382-5p/MXD1 axis plays a critical role in myelopoiesis by affecting the lineage choice of CD34(+) HSPCs

Genomic expression during human myelopoiesis

<p>Abstract</p> <p>Background</p> <p>Human myelopoiesis is an exciting biological model for cellular differentiation since it represents a plastic process where multipotent stem cells gradually limit their differentiation potential, generating different precursor cells which finally evolve into distinct terminally differentiated cells. This study aimed at investigating the genomic expression during myeloid differentiation through a computational approach that integrates gene expression profiles with functional information and genome organization.</p> <p>Results</p> <p>Gene expression data from 24 experiments for 8 different cell types of the human myelopoietic lineage were used to generate an integrated myelopoiesis dataset of 9,425 genes, each reliably associated to a unique genomic position and chromosomal coordinate. Lists of genes constitutively expressed or silent during myelopoiesis and of genes differentially expressed in commitment phase of myelopoiesis were first identified using a classical data analysis procedure. Then, the genomic distribution of myelopoiesis genes was investigated integrating transcriptional and functional characteristics of genes. This approach allowed identifying specific chromosomal regions significantly highly or weakly expressed, and clusters of differentially expressed genes and of transcripts related to specific functional modules.</p> <p>Conclusion</p> <p>The analysis of genomic expression during human myelopoiesis using an integrative computational approach allowed discovering important relationships between genomic position, biological function and expression patterns and highlighting chromatin domains, including genes with coordinated expression and lineage-specific functions.</p

Role of miR-34a-5p in Hematopoietic Progenitor Cells Proliferation and Fate Decision: Novel Insights into the Pathogenesis of Primary Myelofibrosis

Primary Myelofibrosis (PMF) is a chronic Philadelphia-negative myeloproliferative neoplasm characterized by a skewed megakaryopoiesis and an overproduction of proinflammatory and profibrotic mediators that lead to the development of bone marrow (BM) fibrosis. Since we recently uncovered the upregulation of miR-34a-5p in PMF CD34+ hematopoietic progenitor cells (HPCs), in order to elucidate its role in PMF pathogenesis here we unravelled the effects of miR-34a-5p overexpression in HPCs. We showed that enforced expression of miR-34a-5p partially constrains proliferation and favours the megakaryocyte and monocyte/macrophage commitment of HPCs. Interestingly, we identified lymphoid enhancer-binding factor 1 (LEF1) and nuclear receptor subfamily 4, group A, member 2 (NR4A2) transcripts as miR-34a-5p-targets downregulated after miR-34a-5p overexpression in HPCs as well as in PMF CD34+ cells. Remarkably, the knockdown of NR4A2 in HPCs mimicked the antiproliferative effects of miR-34a-5p overexpression, while the silencing of LEF1 phenocopied the effects of miR-34a-5p overexpression on HPCs lineage choice, by favouring the megakaryocyte and monocyte/macrophage commitment. Collectively our data unravel the role of miR-34a-5p in HPCs fate decision and suggest that the increased expression of miR-34a-5p in PMF HPCs could be important for the skewing of megakaryopoiesis and the production of monocytes, that are key players in BM fibrosis in PMF patients

Co-culture of hematopoietic stem/progenitor cells with human osteblasts favours mono/macrophage differentiation at the expense of the erythroid lineage

Hematopoietic stem cells (HSCs) are located in the bone marrow in a specific microenvironment referred as the hematopoietic stem cell niche, where HSCs interact with a variety of stromal cells. Though several components of the stem cell niche have been identified, the regulatory mechanisms through which such components regulate the stem cell fate are still unknown. In order to address this issue, we investigated how osteoblasts (OBs) can affect the molecular and functional phenotype of Hematopoietic Stem/Progenitor Cells (HSPCs) and vice versa. For this purpose, human CD34+ cells were cultured in direct contact with primary human OBs. Our data showed that CD34+ cells cultured with OBs give rise to higher total cell numbers, produce more CFUs and maintain a higher percentage of CD34+CD38- cells compared to control culture. Moreover, clonogenic assay and long-term culture results showed that co-culture with OBs induces a strong increase in mono/macrophage precursors coupled to a decrease in the erythroid ones. Finally, gene expression profiling (GEP) allowed us to study which signalling pathways were activated in the hematopoietic cell fraction and in the stromal cell compartment after coculture. Such analysis allowed us to identify several cytokine-receptor networks, such as WNT pathway, and transcription factors, as TWIST1 and FOXC1, that could be activated by co-culture with OBs and could be responsible for the biological effects reported above. Altogether our results indicate that OBs are able to affect HPSCs on 2 different levels: on one side, they increase the immature progenitor pool in vitro, on the other side, they favor the expansion of the mono/macrophage precursors at the expense of the erythroid lineage

Valproic acid triggers erythro/megakaryocyte lineage decision through induction of GFI1B and MLLT3 expression

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Platelet Function Testing in Patients with Acute Ischemic Stroke: An Observational Study

Background: The measurement of platelet reactivity in patients with stroke undergoing antiplatelet therapies is not commonly performed in clinical practice. We assessed the prevalence of therapy responsiveness in patients with stroke and further investigated differences between patients on prevention therapy at stroke onset and patients naive to antiplatelet medications. We also sought differences in responsiveness between etiological subtypes and correlations between Clopidogrel responsiveness and genetic polymorphisms. Methods: A total of 624 stroke patients on antiplatelet therapy were included. Two different groups were identified: "non-naive patients", and "naive patients". Platelet function was measured with multiple electrode aggregometry, and genotyping assays were used to determine CYP2C19 polymorphisms. Results: Aspirin (ASA) responsiveness was significantly more frequent in naive patients compared with non-naive patients (94.9% versus 82.6%, P < .0010). A better responsiveness to ASA compared with Clopidogrel or combination therapy was found in the entire population (P < .0010), in non-naive patients (P < .0253), and in naive patients (P < .0010). Multivariate analysis revealed a strong effect of Clopidogrel as a possible "risk factor" for unresponsiveness (odds ratio 3.652, P < .0001). No difference between etiological subgroups and no correlations between responsiveness and CYP2C19 polymorphisms were found. Conclusion: In our opinion, platelet function testing could be potentially useful in monitoring the biological effect of antiplatelet agents. A substantial proportion of patients with stroke on ASA were "resistant", and the treatment with Clopidogrel was accompanied by even higher rates of unresponsiveness. Longitudinal studies are needed to assess whether aggregometry might supply individualized prognostic information and whether it can be considered a valid tool for future prevention strategies

Ultrastructural analysis reveals differences in the secretory activity among four regions of amniotic membrane

Human Amniotic Epithelial Cells (hAEC) from term placenta are a promising source of stem cells for regenerative medicine. In a previous study we observed histological heterogeneity, together with different expression of pluripotency markers and content in lipid granules among four regions of amniotic membrane (AM). To better investigate cell heterogeneity among different cell populations, we performed an ultrastructural study with Transmission Electron Microscopy. Term placentae from healthy women were collected after caesarean section and AM samples were freshly isolated from four regions: R1 (close to the umbilical cord); R2 (intermediate); R3 (peripheral to the placental disc); R4 (reflected amnion). Ultrastructural analysis revealed an epithelium of variable thickness, cellular shape, amount and type of vesicles in the four regions. The epithelium showed columnar hAEC with increased height in R1 and R3 and a multi-layered organization in R3, whereas it was a monolayer in the other regions. The highest amount of granules and vesicles was observed in R3, although R4 showed granules with a different density. Furthermore, in R1, R3 and R4 we noticed several vesicles of 100-150 nm in diameter, probably exosome-like structures, suggesting a consistent secretory activity. All along its length the epithelium was rich in microvilli both on the side facing the amniotic fluid and in lateral contacts (narrow desmosomal junctions) between cells. This in situ investigation shows for the first time differences in secretory activity and granules appearance along the AM as a proof of its heterogeneity. This could be relevant in clinical applications as the choice of the area could improve the effectiveness of AM/hAEC transplantation

Analytic and Dynamic Secretory Profile of Patient-Derived Cytokine-Induced Killer Cells

Adoptive immunotherapy with cytokine induced killer (CIK) cells has shown antitumor activity against several kinds of cancer in preclinical models and clinical trials. CIK cells are a subset of ex vivo expanded T lymphocytes with T-NK phenotype and MHC-unrestricted antitumor activity. The literature provides scant information on cytokines, chemokines and growth factors secreted by CIK cells. Therefore, we investigated the secretory profile of CIK cells generated from tumor patients. The secretome analysis was performed at specific time points (d 1, d 14 and d 21) of CIK cell expansion. Mature CIK cells (d 21) produce a great variety of interleukins and secreted proteins that can be divided into three groups based on their secretion quantity: high (interleukin [IL]-13, regulated on activation normal T cell expressed and secreted [RANTES] chemokine, MIP-1 alpha and 1 beta), medium (IL-1Ra, IL-5, IL-8, IL-10, IL-17, IP-10, INF-gamma, vascular endothelial growth factor [VEGF] and granulocyte-macrophage colony-stimulating factor [GM-CSF]) and low (IL-1 beta, IL-4, IL-6, IL-7, IL-9, IL-12, IL-15, eotaxin, platelet-derived growth factor-bb, basic fibroblast growth factor, G-CSF and monocyte chemoattractant protein [MCP]-1). Moreover, comparing peripheral blood mononuclear cells (PBMCs) (d 1) and mature CIK cells (d 14 and 21) secretomes, we observed that IL-5, IL-10, IL-13, GM-CSF and VEGF were greatly upregulated, while IL-1 beta, IL-6, IL-8, IL-15, IL-17, eotaxin, MCP-1 and RANTES were downregulated. We also performed a gene expression profile analysis of patient-derived CIK cells, showing that mRNA for the different cytokines and secreted proteins was modulated during PBMC-to-CIK differentiation. We highlight previously unknown secretory properties and provide, for the first time, a comprehensive molecular characterization of CIK cells. Our findings provide a rationale to explore the functional implications and possible therapeutic modulation of CIK secretome