Cells migrating through complex three-dimensional environments experience considerable physical challenges, including tensile stress and compression. To move, cells need to resist these forces while also squeezing the large nucleus through confined spaces. This requires highly coordinated cortical contractility.
View Article and Find Full Text PDFObjectives: Immunotherapies targeting natural killer (NK) cell receptors have shown promise against leukaemia. Unfortunately, cancer immunosuppressive mechanisms that alter NK cell phenotype prevent such approaches from being successful. The study utilises advanced cytometry to examine how cancer immunosuppressive pathways affect NK cell phenotypic changes in clinical samples.
View Article and Find Full Text PDFSuitable methods to assess immunogenicity and therapeutic efficacy of cancer vaccines in preclinical cancer models are critical to overcome current limitations of cancer vaccines and enhance the clinical applicability of this promising immunotherapeutic strategy. In particular, availability of methods allowing the characterization of T cell responses to endogenous tumor antigens is required to assess vaccine potency and improve the antigen formulation. Moreover, multiparametric assays to deeply characterize tumor-induced and therapy-induced immune modulation are relevant to design mechanism-based combination immunotherapies.
View Article and Find Full Text PDFGrowing interest surrounds adoptive cellular therapies utilizing Natural Killer (NK) cells, which can be obtained from various sources, including umbilical cord blood (UCB) and adult peripheral blood (APB). Understanding NK cell receptor expression and diversity in such cellular sources will guide future therapeutic designs. We used a 20-color flow cytometry panel to compare unstimulated and cytokine-activated UCB and APB NK cells.
View Article and Find Full Text PDFAutologous stem cell transplantation (ASCT) with subsequent lenalidomide maintenance is standard consolidation therapy for multiple myeloma, and a subset of patients achieve durable progression-free survival that is suggestive of long-term immune control. Nonetheless, most patients ultimately relapse, suggesting immune escape. TIGIT appears to be a potent inhibitor of myeloma-specific immunity and represents a promising new checkpoint target.
View Article and Find Full Text PDFMouse hematopoietic tissues contain abundant tissue-resident macrophages that support immunity, hematopoiesis, and bone homeostasis. A systematic strategy to characterize macrophage subsets in mouse bone marrow (BM), spleen, and lymph node unexpectedly reveals that macrophage surface marker staining emanates from membrane-bound subcellular remnants associated with unrelated cells. Intact macrophages are not present within these cell preparations.
View Article and Find Full Text PDFUrinary tract infections (UTI) frequently progress to chronicity in infected individuals but the mechanisms of pathogenesis underlying chronic UTI are not well understood. We examined the role of interleukin (IL)-17A in UTI because this cytokine promotes innate defense against uropathogenic Escherichia coli (UPEC). Analysis of UPEC persistence and pyelonephritis in mice deficient in IL-17A revealed that UPEC CFT073 caused infection at a rate higher than the multidrug resistant strain EC958.
View Article and Find Full Text PDFThese guidelines are a consensus work of a considerable number of members of the immunology and flow cytometry community. They provide the theory and key practical aspects of flow cytometry enabling immunologists to avoid the common errors that often undermine immunological data. Notably, there are comprehensive sections of all major immune cell types with helpful Tables detailing phenotypes in murine and human cells.
View Article and Find Full Text PDFInflammasomes are protein complexes activated by infection and cellular stress that promote caspase-1 activation and subsequent inflammatory cytokine processing and cell death. It has been anticipated that inflammasome activity contributes to autoimmunity. However, we previously showed that macrophages from autoimmune New Zealand Black (NZB) mice lack NLRP3 inflammasome function, and their absent in melanoma 2 (AIM2) inflammasome responses are compromised by high expression of the AIM2 antagonist protein p202.
View Article and Find Full Text PDFInflammasomes are large protein complexes formed in response to cellular stresses that are platforms for recruitment and activation of caspase 1. Central to most inflammasome functions is the adapter molecule ASC (apoptosis-associated speck-like protein containing a caspase-recruitment domain) that links the inflammasome initiator protein to the recruited caspases. ASC is normally diffuse within the cell but within minutes of inflammasome activation relocates to a dense speck in the cytosol.
View Article and Find Full Text PDFAsymmetric cationic amino acid-based dendrimers are highly branched chemically derived gene vectors developed to transport cargo such as plasmid DNA across the plasma membrane. We have previously demonstrated their propensity to enter cells that form caveolae, driven by positive charge density and promoted by arginine head groups. Caveolae are plasma membrane subdomains serving a number of cellular functions including endocytosis.
View Article and Find Full Text PDFInflammasomes are molecular complexes activated by infection and cellular stress, leading to caspase-1 activation and subsequent interleukin-1β (IL-1β) processing and cell death. The autoimmune NZB mouse strain does not express NLRP3, a key inflammasome initiator mediating responses to a wide variety of stimuli including endogenous danger signals, environmental irritants and a range of bacterial, fungal and viral pathogens. We have previously identified an intronic point mutation in the Nlrp3 gene from NZB mice that generates a splice acceptor site.
View Article and Find Full Text PDFResponses to cytosolic DNA can protect against both infectious organisms and the mutagenic effect of DNA integration. Recognition of invading DNA is likely to be fundamental to eukaryotic cellular life, but has been described only in mammals. Introduction of DNA into chicken macrophages induced type I interferon mRNA via a pathway conserved with mammals, requiring the receptor cGAS and the signalling protein STING.
View Article and Find Full Text PDFCytosolic DNA can indicate infection and induces type I interferon (IFN) and AIM2 inflammasome responses. Characterization of these responses has required introduction of DNA into the cytosol of macrophages by either chemical transfection or electroporation, each of which has advantages in different applications. We describe here optimized procedures for both electroporation and chemical transfection, including the centrifugation of chemical transfection reagent onto cells, which greatly increases the speed and strength of responses.
View Article and Find Full Text PDFInflammasomes mediate inflammatory and cell death responses to pathogens and cellular stress signals via activation of procaspases-1 and -8. During inflammasome assembly, activated receptors of the NLR or PYHIN family recruit the adaptor protein ASC and initiate polymerization of its pyrin domain (PYD) into filaments. We show that ASC filaments in turn nucleate procaspase-8 death effector domain (DED) filaments in vitro and in vivo.
View Article and Find Full Text PDFComplications arising from dengue virus infection include potentially fatal vascular leak, and severe disease has been linked with excessive immune cell activation. An understanding of the triggers of this activation is critical for the development of appropriately targeted disease control strategies. We show here that the secreted form of the dengue virus nonstructural protein 1 (NS1) is a pathogen-associated molecular pattern (PAMP).
View Article and Find Full Text PDFInflammasomes are protein complexes that promote caspase activation, resulting in processing of IL-1β and cell death, in response to infection and cellular stresses. Inflammasomes have been anticipated to contribute to autoimmunity. The New Zealand Black (NZB) mouse develops anti-erythrocyte Abs and is a model of autoimmune hemolytic anemia.
View Article and Find Full Text PDFDefence against invading DNA occurs in both mammals and bacteria. Recognition of stray DNA can initiate responses to infection, but may also protect against potentially mutagenic integration of transposons or retrotransposons into the genome. Double-stranded DNA detected in the cytosol of mammalian macrophages can elicit inflammatory cytokines and cell death following assembly of the AIM2 inflammasome.
View Article and Find Full Text PDFInflammasomes are large protein complexes induced by a wide range of microbial, stress, and environmental stimuli that function to induce cell death and inflammatory cytokine processing. Formation of an inflammasome involves dramatic relocalization of the inflammasome adapter protein apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) into a single speck. We have developed a flow cytometric assay for inflammasome formation, time of flight inflammasome evaluation, which detects the change in ASC distribution within the cell.
View Article and Find Full Text PDFUnlabelled: The mosquito-borne West Nile virus (WNV) is responsible for outbreaks of viral encephalitis in humans, horses, and birds, with particularly virulent strains causing recent outbreaks of disease in eastern Europe, the Middle East, North America, and Australia. Previous studies have phylogenetically separated WNV strains into two main genetic lineages (I and II) containing virulent strains associated with neurological disease. Several WNV-like strains clustering outside these lineages have been identified and form an additional five proposed lineages.
View Article and Find Full Text PDFBackground: The draft genome of the domestic pig (Sus scrofa) has recently been published permitting refined analysis of the transcriptome. Pig breeds have been reported to differ in their resistance to infectious disease. In this study we examine whether there are corresponding differences in gene expression in innate immune cells
Results: We demonstrate that macrophages can be harvested from three different compartments of the pig (lungs, blood and bone-marrow), cryopreserved and subsequently recovered and differentiated in CSF-1.
Mouse p202 containing two hemopoietic expression, interferon inducibility, nuclear localization (HIN) domains antagonizes AIM2 inflammasome signaling and potentially modifies lupus susceptibility. We found that only HIN1 of p202 binds double-stranded DNA (dsDNA), while HIN2 forms a homotetramer. Crystal structures of HIN1 revealed that dsDNA is bound on face opposite the site used in AIM2 and IFI16.
View Article and Find Full Text PDFHuman and mouse monocyte can be divided into two different subpopulations based on surface marker expression: CD14/16 and Ly6C/CX3CR1, respectively. Monocyte subpopulations in the pig were identified based on reciprocal expression of CD14 and the scavenger receptor CD163. The two populations, CD14(hi)-CD163(low) and CD14(low)-CD163(hi), show approximately equal abundance in the steady-state.
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