406 research outputs found

    Identification of stromal cells in spleen which support myelopoiesis

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    Stromal cells in spleen organize tissue into red pulp, white pulp and marginal zone, and also interact with hematopoietic cells to regulate immune responses. This study has used phenotypic information of a previously described spleen stromal cell line called 5G3, which supports restricted hematopoiesis in vitro, to identify an equivalent stromal cell subset in vivo and to test its capacity to support hematopoiesis. Using stromal cell fractionation, phenotypic analysis, as well as cell growth and hematopoietic support assays, the Sca-1+gp38+Thy1.2+CD29+CD51+ fraction of spleen stroma has been identified as an equivalent stromal subset resembling the 5G3 cell counterpart. While heterogeneity may still exist within that subset, it has been shown to have superior hematopoietic support capacity compared with the 5G3 cell line, and all other spleen stromal cell fractions tested.This work was supported by project grants to HO from the Australian Research Council (#DP130101703) and the National Health and Medical Research Council of Australia (#585443). HL was supported by an Australian National University Postgraduate Scholarship

    Niches for extramedullary hematopoiesis in the spleen

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    The spleen is a major site for extramedullary hematopoiesis and adult mouse and human spleens retain low numbers of hematopoietic stem cells (HSC). Hematopoiesis is however dependent on stromal microenvironments that provide appropriate molecular signals. Recent evidence now suggests that a distinct stromal cell type exists in the spleen which can function as a niche for hematopoiesis. This review considers the role of the spleen in hematopoiesis, and the possibility that the spleen can be utilized or amplified as a site for hematopoiesis during HSC transplantation. If stromal cells can be isolated and used to expand HSC 'in vitro', or provided as an ectopic niche 'in vivo' for the same purpose, then the potential exists to enhance hematopoiesis in patients undergoing myeloablative treatment, HSC transplantation or involution of lymphoid tissue with ageingNHMRC (National Health and Medical Research Council of Australia

    Estimating disease risk with genetic screening

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    It is more than a year since the complete sequence of the human genome was publicly announced (International Human Genome Sequencing Consortium, 2001). The impact of this milestone on medical research is already apparent. Scientists are geared up to adopt new experimental approaches in the race to combat human disease

    MCSF drives regulatory DC development in stromal co-cultures supporting hematopoiesis

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    Background: Splenic stroma overlaid with hematopoietic progenitors supports in vitro hematopoiesis with production of dendritic-like cells. Co-cultures of murine lineage-depleted bone marrow over the 5G3 stromal line produce two populations of cells, characterised as CD11b+CD11c+MHC-II− dendritic-like ‘L-DC’, and CD11b+CD11c+ MHC-II+ cells, resembling conventional dendritic cells (cDC). To date, the functional capacity of these two subsets has not been clearly distinguished. Results: Here we show both the L-DC and cDC-like subsets can be activated and induce proliferation of OT-I CD8+ T cells, being strong inducers of IL-2 and IFN-γ production. Both subsets lack ability to induce proliferation of OT-II CD4+ T cells. The cDC-like population is shown here to resemble regulatory DC in that they induce FoxP3 expression and IL10 production in OT-II CD4+ T cells, in line with their function as regulatory DC. L-DC did not activate or induce the proliferation of CD4+ T cells and did not induce FoxP3 expression in CD4+ T cells. L-DC can be distinguished from cDClike cells through their superior endocytic capacity and expression of 4-1BBL, F4/80 and Sirp-α. A comparison of gene expression by the two subsets was consistent with L-DC having an activated or immunostimulatory DC phenotype, while cDC-like cells reflect myeloid dendritic cells with inflammatory and suppressive properties, also consistent with functional characteristics as regulatory DC. When a Transwell membrane was used to prevent hematopoietic cell contact with stroma, only cDC-like cells and not L-DC were produced, and cell production was dependent on M-CSF production by stroma. Conclusion: Co-cultures of hematopoietic progenitors over splenic stroma produce two distinct subsets of dendritic-like cells. These are here distinguished phenotypically and through gene expression differences. While both resemble DC, there are functionally distinct. L-DC activate CD8+ but not CD4+ T cells, while the cDC-like population induce regulatory T cells, so reflecting regulatory DC. The latter can be enriched through Transwell co-cultures with cell production dependent on M-CSF. Keywords: Hematopoiesis, Dendritic cell, Regulatory dendritic cells, Regulatory T cellsThis work was supported by project grant #585443 to HO from the National Health and Medical Research Council of Australia. SP was supported by a graduate scholarship from the Royal Thai Government. PP was supported by an Australian National University Graduate Scholarship

    Antigen presenting capacity of murine splenic myeloid cells

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    BACKGROUND: The spleen is an important site for hematopoiesis. It supports development of myeloid cells from bone marrow-derived precursors entering from blood. Myeloid subsets in spleen are not well characterised although dendritic cell (DC) subsets are clearly defined in terms of phenotype, development and functional role. Recently a novel dendritic-like cell type in spleen named ‘L-DC’ was distinguished from other known dendritic and myeloid cells by its distinct phenotype and developmental origin. That study also redefined splenic eosinophils as well as resident and inflammatory monocytes in spleen. RESULTS: L-DC are shown to be distinct from known splenic macrophages and monocyte subsets. Using a new flow cytometric procedure, it has been possible to identify and isolate L-DC in order to assess their functional competence and ability to activate T cells both in vivo and in vitro. L-DC are readily accessible to antigen given intravenously through receptor-mediated endocytosis. They are also capable of CD8(+) T cell activation through antigen cross presentation, with subsequent induction of cytotoxic effector T cells. L-DC are MHCII(−) cells and unable to activate CD4(+) T cells, a property which clearly distinguishes them from conventional DC. The myeloid subsets of resident monocytes, inflammatory monocytes, neutrophils and eosinophils, were found to have varying capacities to take up antigen, but were uniformly unable to activate either CD4(+) T cells or CD8(+) T cells. CONCLUSION: The results presented here demonstrate that L-DC in spleen are distinct from other myeloid cells in that they can process antigen for CD8(+) T cell activation and induction of cytotoxic effector function, while both L-DC and myeloid subsets remain unable to activate CD4(+) T cells. The L-DC subset in spleen is therefore distinct as an antigen presenting cell

    Hematopoiesis leading to a diversity of dendritic antigen-presenting cell types

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    Hematopoietic stem cells (HSCs) undergo expansion and differentiation, giving rise to all terminally differentiated blood cells throughout life. HSCs are found in distinct anatomical sites during development, and in adults, hematopoiesis occurs predominantly on the luminal side of the bone cavity in bone marrow. Millions of newly formed blood cells are generated per second to accommodate the short half-life of hematopoietic cells. For this to happen, HSCs must sustain their self-renewal capacity as well as their capability to commit and differentiate toward multiple cell lineages. Development of the hematopoietic system is finely regulated as the animal ages, so that it does not become exhausted or misdirected. This review covers aspects of hematopoietic development from the embryonic period through adult life in relation to development of dendritic cells. It also considers a role for HSCs in extramedullary sites and their possible role in myelopoiesis, with formation of tissue-specific antigen-presenting cells.NHMRC (National Health and Medical Research Council of Australia

    Comparing the diagnostic and clinical utility of WGS and WES with standard genetic testing (SGT) in children with suspected genetic diseases: A systematic review and meta-analysis

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    Rare genetic diseases remain a significant cause of infant mortality worldwide. Despite the established use of classic newborn screening (NBS) methodologies, many genetic diseases escape detection, necessitating the exploration of alternative diagnostic approaches. Whole-genome sequencing (WGS) and whole-exome sequencing (WES) have emerged as promising techniques for diagnosing genetic diseases in children. This study aimed to systematically evaluate the diagnostic and clinical utility of WGS and WES compared to standard genetic testing (SGT) in children suspected of having genetic diseases and discuss their potential impact on the expansion of NBS. A thorough search of EMBASE, MEDLINE, PubMed, Scopus, Web of Science, Cochrane Central Register of Controlled Trials, and references of included full-text articles was conducted until October 21, 2021. Studies reporting the diagnostic yield or rate of change in management using WGS and/or WES were included for analysis. The Preferred Reporting Items for Systematic Reviews and Meta-analyses (PRISMA) reporting guideline was followed for data extraction, and the Quality Assessment of Diagnostic Accuracy Studies (QUADAS-2) tool was used to assess study quality. Meta-analysis was performed using a random-effects model to calculate pooled proportions and odds ratios. The main outcomes of interest were the diagnostic utility and clinical utility of WGS, WES, and SGT. Diagnostic utility was defined as the identification of pathogenic or likely pathogenic variants strongly or moderately associated with the patient's clinical phenotype, which were subsequently reported to the clinician. Clinical utility encompassed any changes observed in medical or surgical management, as assessed through clinician questionnaires or Electronic Health Record reviews. The analysis included 43 studies involving 6,168 children. The pooled diagnostic utility of WES (0.40, 95% CI 0.34-0.45, I2=90%) exhibited a qualitative superiority over WGS (0.34, 95% CI 0.29-0.39, I2=79%) and SGT (0.19, 95% CI 0.13-0.25, I2=64%). Regarding clinical utility, WGS (0.74, 95% CI 0.56-0.89, I2=93%) demonstrated a qualitative advantage over WES (0.72, 95% CI 0.61-0.81, I2=86%), while both outperformed SGT (0.69, 95% CI 0.38-0.94). In conclusion, our findings suggest that WGS and WES should be considered as primary diagnostic tools for identifying genetic diseases in children. Furthermore, the integration of WGS and WES into NBS holds promise; however, additional investigations are warranted to assess the cost-effectiveness of this approach. This study sheds light on the potential of advanced sequencing methods to revolutionize the diagnosis and management of genetic diseases, thus impacting the field of human genetics significantly

    Murine spleen contains a diversity of myeloid and dendritic cells distinct in antigen presenting function

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    The spleen contains multiple subsets of myeloid and dendritic cells (DC). DC are important antigen presenting cells (APC) which induce and control the adaptive immune response. They are cells specialized for antigen capture, processing and presentation to naıve T cells. However, DC are a heterogeneous population and each subset differs subtly in phenotype, function and location. Similarly, myeloid cell subsets can be distinguished which can also play an important role in the regulation of immunity. This review aims to characterize splenic subsets of DC and myeloid cells to better understand their individual roles in the immune response.NHMRC (National Health and Medical Research Council of Australia
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