Browsing by Subject "Flow Cytometry"

Now showing 1 - 20 of 42
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    A cell-based multiplex immunoassay platform using fluorescent protein-barcoded reporter cell lines.
    (Communications biology, 2021-11) Song, Shengli; Manook, Miriam; Kwun, Jean; Jackson, Annette M; Knechtle, Stuart J; Kelsoe, Garnett
    Multiplex immunoassays with acellular antigens are well-established based on solid-phase platforms such as the Luminex® technology. Cell barcoding by amine-reactive fluorescent dyes enables analogous cell-based multiplex assays, but requires multiple labeling reactions and quality checks prior to every assay. Here we describe generation of stable, fluorescent protein-barcoded reporter cell lines suitable for multiplex screening of antibody to membrane proteins. The utility of this cell-based system, with the potential of a 256-plex cell panel, is demonstrated by flow cytometry deconvolution of barcoded cell panels expressing influenza A hemagglutinin trimers, or native human CCR2 or CCR5 multi-span proteins and their epitope-defining mutants. This platform will prove useful for characterizing immunity and discovering antibodies to membrane-associated proteins.
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    A discrete chromatin loop in the mouse Tcra-Tcrd locus shapes the TCRδ and TCRα repertoires.
    (Nat Immunol, 2015-10) Chen, Liang; Carico, Zachary; Shih, Han-Yu; Krangel, Michael S
    The locus encoding the T cell antigen receptor (TCR) α-chain and δ-chain (Tcra-Tcrd) undergoes recombination of its variable-diversity-joining (V(D)J) segments in CD4(-)CD8(-) double-negative thymocytes and CD4(+)CD8(+) double-positive thymocytes to generate diverse TCRδ repertoires and TCRα repertoires, respectively. Here we identified a chromatin-interaction network in the Tcra-Tcrd locus in double-negative thymocytes that was formed by interactions between binding elements for the transcription factor CTCF. Disruption of a discrete chromatin loop encompassing the D, J and constant (C) segments of Tcrd allowed a single V segment to frequently contact and rearrange to D and J segments and dominate the adult TCRδ repertoire. Disruption of this loop also narrowed the TCRα repertoire, which, we believe, followed as a consequence of the restricted TCRδ repertoire. Hence, a single CTCF-mediated chromatin loop directly regulated TCRδ diversity and indirectly regulated TCRα diversity.
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    A model for harmonizing flow cytometry in clinical trials.
    (Nat Immunol, 2010-11) Maecker, Holden T; McCoy, J Philip; FOCIS Human Immunophenotyping Consortium; Amos, Michael; Elliott, John; Gaigalas, Adolfas; Wang, Lili; Aranda, Richard; Banchereau, Jacques; Boshoff, Chris; Braun, Jonathan; Korin, Yael; Reed, Elaine; Cho, Judy; Hafler, David; Davis, Mark; Fathman, C Garrison; Robinson, William; Denny, Thomas; Weinhold, Kent; Desai, Bela; Diamond, Betty; Gregersen, Peter; Di Meglio, Paola; Nestle, Frank O; Peakman, Mark; Villanova, Federica; Ferbas, John; Field, Elizabeth; Kantor, Aaron; Kawabata, Thomas; Komocsar, Wendy; Lotze, Michael; Nepom, Jerry; Ochs, Hans; O'Lone, Raegan; Phippard, Deborah; Plevy, Scott; Rich, Stephen; Roederer, Mario; Rotrosen, Dan; Yeh, Jung-Hua
    Complexities in sample handling, instrument setup and data analysis are barriers to the effective use of flow cytometry to monitor immunological parameters in clinical trials. The novel use of a central laboratory may help mitigate these issues.
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    A Protocol for the Comprehensive Flow Cytometric Analysis of Immune Cells in Normal and Inflamed Murine Non-Lymphoid Tissues.
    (PloS one, 2016-01) Yu, Yen-Rei A; O'Koren, Emily G; Hotten, Danielle F; Kan, Matthew J; Kopin, David; Nelson, Erik R; Que, Loretta; Gunn, Michael D
    Flow cytometry is used extensively to examine immune cells in non-lymphoid tissues. However, a method of flow cytometric analysis that is both comprehensive and widely applicable has not been described. We developed a protocol for the flow cytometric analysis of non-lymphoid tissues, including methods of tissue preparation, a 10-fluorochrome panel for cell staining, and a standardized gating strategy, that allows the simultaneous identification and quantification of all major immune cell types in a variety of normal and inflamed non-lymphoid tissues. We demonstrate that our basic protocol minimizes cell loss, reliably distinguishes macrophages from dendritic cells (DC), and identifies all major granulocytic and mononuclear phagocytic cell types. This protocol is able to accurately quantify 11 distinct immune cell types, including T cells, B cells, NK cells, neutrophils, eosinophils, inflammatory monocytes, resident monocytes, alveolar macrophages, resident/interstitial macrophages, CD11b- DC, and CD11b+ DC, in normal lung, heart, liver, kidney, intestine, skin, eyes, and mammary gland. We also characterized the expression patterns of several commonly used myeloid and macrophage markers. This basic protocol can be expanded to identify additional cell types such as mast cells, basophils, and plasmacytoid DC, or perform detailed phenotyping of specific cell types. In examining models of primary and metastatic mammary tumors, this protocol allowed the identification of several distinct tumor associated macrophage phenotypes, the appearance of which was highly specific to individual tumor cell lines. This protocol provides a valuable tool to examine immune cell repertoires and follow immune responses in a wide variety of tissues and experimental conditions.
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    Airway fibroblasts in asthma manifest an invasive phenotype.
    (American journal of respiratory and critical care medicine, 2011-06) Ingram, Jennifer L; Huggins, Molly J; Church, Tony D; Li, Yuejuan; Francisco, Dave C; Degan, Simone; Firszt, Rafael; Beaver, Denise M; Lugogo, Njira L; Wang, Ying; Sunday, Mary E; Noble, Paul W; Kraft, Monica

    Rationale

    Invasive cell phenotypes have been demonstrated in malignant transformation, but not in other diseases, such as asthma. Cellular invasiveness is thought to be mediated by transforming growth factor (TGF)-β1 and matrix metalloproteinases (MMPs). IL-13 is a key T(H)2 cytokine that directs many features of airway remodeling through TGF-β1 and MMPs.

    Objectives

    We hypothesized that, in human asthma, IL-13 stimulates increased airway fibroblast invasiveness via TGF-β1 and MMPs in asthma compared with normal controls.

    Methods

    Fibroblasts were cultured from endobronchial biopsies in 20 subjects with mild asthma (FEV(1): 90 ± 3.6% pred) and 17 normal control subjects (FEV(1): 102 ± 2.9% pred) who underwent bronchoscopy. Airway fibroblast invasiveness was investigated using Matrigel chambers. IL-13 or IL-13 with TGF-β1 neutralizing antibody or pan-MMP inhibitor (GM6001) was added to the lower chamber as a chemoattractant. Flow cytometry and immunohistochemistry were performed in a subset of subjects to evaluate IL-13 receptor levels.

    Measurements and main results

    IL-13 significantly stimulated invasion in asthmatic airway fibroblasts, compared with normal control subjects. Inhibitors of both TGF-β1 and MMPs blocked IL-13-induced invasion in asthma, but had no effect in normal control subjects. At baseline, in airway tissue, IL-13 receptors were expressed in significantly higher levels in asthma, compared with normal control subjects. In airway fibroblasts, baseline IL-13Rα2 was reduced in asthma compared with normal control subjects.

    Conclusions

    IL-13 potentiates airway fibroblast invasion through a mechanism involving TGF-β1 and MMPs. IL-13 receptor subunits are differentially expressed in asthma. These effects may result in IL-13-directed airway remodeling in asthma.
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    Boosting high-intensity focused ultrasound-induced anti-tumor immunity using a sparse-scan strategy that can more effectively promote dendritic cell maturation.
    (J Transl Med, 2010-01-27) Liu, Fang; Hu, Zhenlin; Qiu, Lei; Hui, Chun; Li, Chao; Zhong, Pei; Zhang, Junping
    BACKGROUND: The conventional treatment protocol in high-intensity focused ultrasound (HIFU) therapy utilizes a dense-scan strategy to produce closely packed thermal lesions aiming at eradicating as much tumor mass as possible. However, this strategy is not most effective in terms of inducing a systemic anti-tumor immunity so that it cannot provide efficient micro-metastatic control and long-term tumor resistance. We have previously provided evidence that HIFU may enhance systemic anti-tumor immunity by in situ activation of dendritic cells (DCs) inside HIFU-treated tumor tissue. The present study was conducted to test the feasibility of a sparse-scan strategy to boost HIFU-induced anti-tumor immune response by more effectively promoting DC maturation. METHODS: An experimental HIFU system was set up to perform tumor ablation experiments in subcutaneous implanted MC-38 and B16 tumor with dense- or sparse-scan strategy to produce closely-packed or separated thermal lesions. DCs infiltration into HIFU-treated tumor tissues was detected by immunohistochemistry and flow cytometry. DCs maturation was evaluated by IL-12/IL-10 production and CD80/CD86 expression after co-culture with tumor cells treated with different HIFU. HIFU-induced anti-tumor immune response was evaluated by detecting growth-retarding effects on distant re-challenged tumor and tumor-specific IFN-gamma-secreting cells in HIFU-treated mice. RESULTS: HIFU exposure raised temperature up to 80 degrees centigrade at beam focus within 4 s in experimental tumors and led to formation of a well-defined thermal lesion. The infiltrated DCs were recruited to the periphery of lesion, where the peak temperature was only 55 degrees centigrade during HIFU exposure. Tumor cells heated to 55 degrees centigrade in 4-s HIFU exposure were more effective to stimulate co-cultured DCs to mature. Sparse-scan HIFU, which can reserve 55 degrees-heated tumor cells surrounding the separated lesions, elicited an enhanced anti-tumor immune response than dense-scan HIFU, while their suppressive effects on the treated primary tumor were maintained at the same level. Flow cytometry analysis showed that sparse-scan HIFU was more effective than dense-scan HIFU in enhancing DC infiltration into tumor tissues and promoting their maturation in situ. CONCLUSION: Optimizing scan strategy is a feasible way to boost HIFU-induced anti-tumor immunity by more effectively promoting DC maturation.
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    Characterization of CD4 and CD8 T cell responses in MuSK myasthenia gravis.
    (J Autoimmun, 2014-08) Yi, JS; Guidon, A; Sparks, S; Osborne, R; Juel, VC; Massey, JM; Sanders, DB; Weinhold, KJ; Guptill, JT
    Muscle specific tyrosine kinase myasthenia gravis (MuSK MG) is a form of autoimmune MG that predominantly affects women and has unique clinical features, including prominent bulbar weakness, muscle atrophy, and excellent response to therapeutic plasma exchange. Patients with MuSK MG have predominantly IgG4 autoantibodies directed against MuSK on the postsynaptic muscle membrane. Lymphocyte functionality has not been reported in this condition. The goal of this study was to characterize T cell responses in patients with MuSK MG. Intracellular production of IFN-gamma, TNF-alpha, IL-2, IL-17, and IL-21 by CD4+ and CD8+ T cells was measured by polychromatic flow cytometry in peripheral blood samples from 11 Musk MG patients and 10 healthy controls. Only one MuSK MG patient was not receiving immunosuppressive therapy. Regulatory T cells (Treg) were also included in our analysis to determine if changes in T cell function were due to altered Treg frequencies. CD8+ T cells from MuSK MG patients had higher frequencies of polyfunctional responses than controls, and CD4+ T cells had higher IL-2, TNF-alpha, and IL-17. MuSK MG patients had a higher percentage of CD4+ T cells producing combinations of IFN-gamma/IL-2/TNF-gamma, TNF-alpha/IL-2, and IFN-gamma/TNF-alpha. Interestingly, Treg numbers and CD39 expression were not different from control values. MuSK MG patients had increased frequencies of Th1 and Th17 cytokines and were primed for polyfunctional proinflammatory responses that cannot be explained by a defect in CD39 expression or Treg number.
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    Chlamydia trachomatis immune evasion via downregulation of MHC class I surface expression involves direct and indirect mechanisms.
    (Infectious diseases in obstetrics and gynecology, 2011-01) Ibana, Joyce A; Schust, Danny J; Sugimoto, Jun; Nagamatsu, Takeshi; Greene, Sheila J; Quayle, Alison J
    Genital C. trachomatis infections typically last for many months in women. This has been attributed to several strategies by which C. trachomatis evades immune detection, including well-described methods by which C. trachomatis decreases the cell surface expression of the antigen presenting molecules major histocompatibility complex (MHC) class I, MHC class II, and CD1d in infected genital epithelial cells. We have harnessed new methods that allow for separate evaluation of infected and uninfected cells within a mixed population of chlamydia-infected endocervical epithelial cells to demonstrate that MHC class I downregulation in the presence of C. trachomatis is mediated by direct and indirect (soluble) factors. Such indirect mechanisms may aid in priming surrounding cells for more rapid immune evasion upon pathogen entry and help promote unfettered spread of C. trachomatis genital infections.
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    Comparison of interlaboratory variation in absolute T-cell counts by single-platform and optimized dual-platform methods.
    (Cytometry B Clin Cytom, 2010-05) Hultin, Lance E; Chow, Marianne; Jamieson, Beth D; O'Gorman, Maurice RG; Menendez, Frederick A; Borowski, Luann; Denny, Thomas N; Margolick, Joseph B
    BACKGROUND: Previous studies have reported that the adoption of a single-platform flow cytometry cell counting method resulted in lower interlaboratory variation in absolute T cell counts as compared to predicate dual-platform flow cytometry methods which incorporate independent automated lymphocyte counts (Schnizlein-Bick et al., Clin Diagn Lab Immunol 2000;7:336-343; Reimann et al., Clin Diagn Lab Immunol 2000;7:344-351). In the present study, we asked whether use of a single-platform method could reduce variation in absolute cell counts across the laboratories in the Multicenter AIDS Cohort Study (MACS) (n = 4), as suggested by the studies cited. METHODS: Identical study samples were shipped overnight to the MACS laboratories either by the National Institute of Allergy and Infectious Diseases, Division of AIDS Immunology Quality Assessment (NIAID- IQA) proficiency-testing program (n = 14), or by the Los Angeles site of the MACS (n = 10). For each sample, two tubes of blood were received; one was used for an automated complete blood count and differential, and the other for flow cytometry. The latter was performed using both our current dual-platform method (three-color CD45 gating and automated hematology) and the single-platform method (with TruCOUNT beads to generate the absolute counts). RESULTS: The median percent coefficients of variation (%CVs) for the dual-platform and single-platform methods were 6.6 and 9.9, respectively, for CD4 T cell counts, and 5.9 and 8.5, respectively, for CD8 T cell counts (n = 24). These differences were not statistically significant. The differences in absolute T-cell counts between the MACS sites and the median of all laboratories participating in the NIAID-IQA were smaller for the dual-platform than for single-platform absolute count method. CONCLUSION: In contrast to previous reports, we did not observe lower interlaboratory variation across the MACS sites for single-platform absolute lymphocyte subset counting relative to dual-platform methods. This result may be at least partly explained by the lower interlaboratory variation with the optimized dual-platform method in this study relative to the previous reports.
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    Cryptococcal cell morphology affects host cell interactions and pathogenicity.
    (PLoS Pathog, 2010-06-17) Okagaki, Laura H; Strain, Anna K; Nielsen, Judith N; Charlier, Caroline; Baltes, Nicholas J; Chrétien, Fabrice; Heitman, Joseph; Dromer, Françoise; Nielsen, Kirsten
    Cryptococcus neoformans is a common life-threatening human fungal pathogen. The size of cryptococcal cells is typically 5 to 10 microm. Cell enlargement was observed in vivo, producing cells up to 100 microm. These morphological changes in cell size affected pathogenicity via reducing phagocytosis by host mononuclear cells, increasing resistance to oxidative and nitrosative stress, and correlated with reduced penetration of the central nervous system. Cell enlargement was stimulated by coinfection with strains of opposite mating type, and ste3aDelta pheromone receptor mutant strains had reduced cell enlargement. Finally, analysis of DNA content in this novel cell type revealed that these enlarged cells were polyploid, uninucleate, and produced daughter cells in vivo. These results describe a novel mechanism by which C. neoformans evades host phagocytosis to allow survival of a subset of the population at early stages of infection. Thus, morphological changes play unique and specialized roles during infection.
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    Dectin 1 activation on macrophages by galectin 9 promotes pancreatic carcinoma and peritumoral immune tolerance.
    (Nature medicine, 2017-05) Daley, Donnele; Mani, Vishnu R; Mohan, Navyatha; Akkad, Neha; Ochi, Atsuo; Heindel, Daniel W; Lee, Ki Buom; Zambirinis, Constantinos P; Pandian, Gautam Sd Balasubramania; Savadkar, Shivraj; Torres-Hernandez, Alejandro; Nayak, Shruti; Wang, Ding; Hundeyin, Mautin; Diskin, Brian; Aykut, Berk; Werba, Gregor; Barilla, Rocky M; Rodriguez, Robert; Chang, Steven; Gardner, Lawrence; Mahal, Lara K; Ueberheide, Beatrix; Miller, George
    The progression of pancreatic oncogenesis requires immune-suppressive inflammation in cooperation with oncogenic mutations. However, the drivers of intratumoral immune tolerance are uncertain. Dectin 1 is an innate immune receptor crucial for anti-fungal immunity, but its role in sterile inflammation and oncogenesis has not been well defined. Furthermore, non-pathogen-derived ligands for dectin 1 have not been characterized. We found that dectin 1 is highly expressed on macrophages in pancreatic ductal adenocarcinoma (PDA). Dectin 1 ligation accelerated the progression of PDA in mice, whereas deletion of Clec7a-the gene encoding dectin 1-or blockade of dectin 1 downstream signaling was protective. We found that dectin 1 can ligate the lectin galectin 9 in mouse and human PDA, which results in tolerogenic macrophage programming and adaptive immune suppression. Upon disruption of the dectin 1-galectin 9 axis, CD4+ and CD8+ T cells, which are dispensable for PDA progression in hosts with an intact signaling axis, become reprogrammed into indispensable mediators of anti-tumor immunity. These data suggest that targeting dectin 1 signaling is an attractive strategy for developing an immunotherapy for PDA.
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    Detection of amino-terminal extracellular domain of somatostatin receptor 2 by specific monoclonal antibodies and quantification of receptor density in medulloblastoma.
    (Hybridoma (Larchmt), 2009-12) Kuan, Chien-Tsun; Wikstrand, Carol J; McLendon, Roger E; Zalutsky, Michael R; Kumar, Ujendra; Bigner, Darell D
    Somatostatin receptor 2 (SSTR2) is expressed by most medulloblastomas (MEDs). We isolated monoclonal antibodies (MAbs) to the 12-mer (33)QTEPYYDLTSNA(44), which resides in the extracellular domain of the SSTR2 amino terminus, screened the peptide-bound MAbs by fluorescence microassay on D341 and D283 MED cells, and demonstrated homogeneous cell-surface binding, indicating that all cells expressed cell surface-detectable epitopes. Five radiolabeled MAbs were tested for immunoreactive fraction (IRF), affinity (KA) (Scatchard analysis vs. D341 MED cells), and internalization by MED cells. One IgG(3) MAb exhibited a 50-100% IRF, but low KA. Four IgG(2a) MAbs had 46-94% IRFs and modest KAs versus intact cells (0.21-1.2 x 10(8) M(-1)). Following binding of radiolabeled MAbs to D341 MED at 4 degrees C, no significant internalization was observed, which is consistent with results obtained in the absence of ligand. However, all MAbs exhibited long-term association with the cells; binding at 37 degrees C after 2 h was 65-66%, and after 24 h, 52-64%. In tests with MAbs C10 and H5, the number of cell surface receptors per cell, estimated by Scatchard and quantitative FACS analyses, was 3.9 x 10(4) for the "glial" phenotype DAOY MED cell line and 0.6-8.8 x 10(5) for four neuronal phenotype MED cell lines. Our results indicate a potential immunotherapeutic application for these MAbs.
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    Establishment of normative ranges of the healthy human immune system with comprehensive polychromatic flow cytometry profiling.
    (PloS one, 2019-01) Yi, John S; Rosa-Bray, Marilyn; Staats, Janet; Zakroysky, Pearl; Chan, Cliburn; Russo, Melissa A; Dumbauld, Chelsae; White, Scott; Gierman, Todd; Weinhold, Kent J; Guptill, Jeffrey T
    Existing normative flow cytometry data have several limitations including small sample sizes, incompletely described study populations, variable flow cytometry methodology, and limited depth for defining lymphocyte subpopulations. To overcome these issues, we defined high-dimensional flow cytometry reference ranges for the healthy human immune system using Human Immunology Project Consortium methodologies after carefully screening 127 subjects deemed healthy through clinical and laboratory testing. We enrolled subjects in the following age cohorts: 18-29 years, 30-39, 40-49, and 50-66 and enrolled cohorts to ensure an even gender distribution and at least 30% non-Caucasians. From peripheral blood mononuclear cells, flow cytometry reference ranges were defined for >50 immune subsets including T-cell (activation, maturation, T follicular helper and regulatory T cell), B-cell, and innate cells. We also developed a web tool for visualization of the dataset and download of raw data. This dataset provides the immunology community with a resource to compare and extract data from rigorously characterized healthy subjects across age groups, gender and race.
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    Flow cytometric methods for prenatal and neonatal diagnosis.
    (J Immunol Methods, 2011-01-05) Curtis, Michèle G; Walker, Brooke; Denny, Thomas N
    Flow cytometry offers a promising alternative to the current methods of amniocentesis or chorionic villus sampling (CVS) for fetal cell sorting for prenatal diagnosis. While flow cytometric methods have been greatly improved to be more sensitive at detecting fetal cells within the maternal circulation, there are still several challenges that need to be overcome before application in prenatal diagnosis. However, flow cytometry is a powerful tool that can be used to enhance molecular testing and other diagnostic testing modalities in prenatal and neonatal diagnosis. It remains the gold standard to identify cellular immunodeficiencies and, for some immunological disorders with established biomarkers, flow cytometric assays can be used to make a definitive diagnosis. In this review, the advantages and disadvantages of using MACS and FACS analysis for fetal cell sorting are discussed. This review also includes an overview of the current flow cytometric assays and biomarkers that may be used for prenatal and neonatal diagnosis of common immunological and hematological abnormalities and the role of flow cytometry in treatment monitoring after bone marrow and stem cell transplantation.
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    FlowKit: A Python Toolkit for Integrated Manual and Automated Cytometry Analysis Workflows.
    (Frontiers in immunology, 2021-01) White, Scott; Quinn, John; Enzor, Jennifer; Staats, Janet; Mosier, Sarah M; Almarode, James; Denny, Thomas N; Weinhold, Kent J; Ferrari, Guido; Chan, Cliburn
    An important challenge for primary or secondary analysis of cytometry data is how to facilitate productive collaboration between domain and quantitative experts. Domain experts in cytometry laboratories and core facilities increasingly recognize the need for automated workflows in the face of increasing data complexity, but by and large, still conduct all analysis using traditional applications, predominantly FlowJo. To a large extent, this cuts domain experts off from the rapidly growing library of Single Cell Data Science algorithms available, curtailing the potential contributions of these experts to the validation and interpretation of results. To address this challenge, we developed FlowKit, a Gating-ML 2.0-compliant Python package that can read and write FCS files and FlowJo workspaces. We present examples of the use of FlowKit for constructing reporting and analysis workflows, including round-tripping results to and from FlowJo for joint analysis by both domain and quantitative experts.
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    Fluorescent-Activated Cell Sorting (Flow Cytometry)
    (2022) Carrillo, Luis
    This volume provides a comprehensive reference guide for researchers to study the applications of labeled antibodies.
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    In vivo Modeling Implicates APOL1 in Nephropathy: Evidence for Dominant Negative Effects and Epistasis under Anemic Stress.
    (PLoS Genet, 2015-07) Anderson, Blair R; Howell, David N; Soldano, Karen; Garrett, Melanie E; Katsanis, Nicholas; Telen, Marilyn J; Davis, Erica E; Ashley-Koch, Allison E
    African Americans have a disproportionate risk for developing nephropathy. This disparity has been attributed to coding variants (G1 and G2) in apolipoprotein L1 (APOL1); however, there is little functional evidence supporting the role of this protein in renal function. Here, we combined genetics and in vivo modeling to examine the role of apol1 in glomerular development and pronephric filtration and to test the pathogenic potential of APOL1 G1 and G2. Translational suppression or CRISPR/Cas9 genome editing of apol1 in zebrafish embryos results in podocyte loss and glomerular filtration defects. Complementation of apol1 morphants with wild-type human APOL1 mRNA rescues these defects. However, the APOL1 G1 risk allele does not ameliorate defects caused by apol1 suppression and the pathogenicity is conferred by the cis effect of both individual variants of the G1 risk haplotype (I384M/S342G). In vivo complementation studies of the G2 risk allele also indicate that the variant is deleterious to protein function. Moreover, APOL1 G2, but not G1, expression alone promotes developmental kidney defects, suggesting a possible dominant-negative effect of the altered protein. In sickle cell disease (SCD) patients, we reported previously a genetic interaction between APOL1 and MYH9. Testing this interaction in vivo by co-suppressing both transcripts yielded no additive effects. However, upon genetic or chemical induction of anemia, we observed a significantly exacerbated nephropathy phenotype. Furthermore, concordant with the genetic interaction observed in SCD patients, APOL1 G2 reduces myh9 expression in vivo, suggesting a possible interaction between the altered APOL1 and myh9. Our data indicate a critical role for APOL1 in renal function that is compromised by nephropathy-risk encoding variants. Moreover, our interaction studies indicate that the MYH9 locus is also relevant to the phenotype in a stressed microenvironment and suggest that consideration of the context-dependent functions of both proteins will be required to develop therapeutic paradigms.
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    Inflammation triggers emergency granulopoiesis through a density-dependent feedback mechanism.
    (PLoS One, 2011) Cain, Derek W; Snowden, Pilar B; Sempowski, Gregory D; Kelsoe, Garnett
    Normally, neutrophil pools are maintained by homeostatic mechanisms that require the transcription factor C/EBPα. Inflammation, however, induces neutrophilia through a distinct pathway of "emergency" granulopoiesis that is dependent on C/EBPβ. Here, we show in mice that alum triggers emergency granulopoiesis through the IL-1RI-dependent induction of G-CSF. G-CSF/G-CSF-R neutralization impairs proliferative responses of hematopoietic stem and progenitor cells (HSPC) to alum, but also abrogates the acute mobilization of BM neutrophils, raising the possibility that HSPC responses to inflammation are an indirect result of the exhaustion of BM neutrophil stores. The induction of neutropenia, via depletion with Gr-1 mAb or myeloid-specific ablation of Mcl-1, elicits G-CSF via an IL-1RI-independent pathway, stimulating granulopoietic responses indistinguishable from those induced by adjuvant. Notably, C/EBPβ, thought to be necessary for enhanced generative capacity of BM, is dispensable for increased proliferation of HSPC to alum or neutropenia, but plays a role in terminal neutrophil differentiation during granulopoietic recovery. We conclude that alum elicits a transient increase in G-CSF production via IL-1RI for the mobilization of BM neutrophils, but density-dependent feedback sustains G-CSF for accelerated granulopoiesis.
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    Integrin-mediated interactions with extracellular matrix proteins for nucleus pulposus cells of the human intervertebral disc.
    (J Orthop Res, 2013-10) Bridgen, DT; Gilchrist, CL; Richardson, WJ; Isaacs, RE; Brown, CR; Yang, KL; Chen, J; Setton, LA
    The extracellular matrix (ECM) of the human intervertebral disc is rich in molecules that interact with cells through integrin-mediated attachments. Porcine nucleus pulposus (NP) cells have been shown to interact with laminin (LM) isoforms LM-111 and LM-511 through select integrins that regulate biosynthesis and cell attachment. Since human NP cells lose many phenotypic characteristics with age, attachment and interaction with the ECM may be altered. Expression of LM-binding integrins was quantified for human NP cells using flow cytometry. The cell-ECM attachment mechanism was determined by quantifying cell attachment to LM-111, LM-511, or type II collagen after functionally blocking specific integrin subunits. Human NP cells express integrins β1, α3, and α5, with over 70% of cells positive for each subunit. Blocking subunit β1 inhibited NP cell attachment to all substrates. Blocking subunits α1, α2, α3, and α5 simultaneously, but not individually, inhibits NP cell attachment to laminins. While integrin α6β1 mediated porcine NP cell attachment to LM-111, we found integrins α3, α5, and β1 instead contributed to human NP cell attachment. These findings identify integrin subunits that may mediate interactions with the ECM for human NP cells and could be used to promote cell attachment, survival, and biosynthesis in cell-based therapeutics.