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1.
Pigment Cell Melanoma Res ; 36(5): 365-377, 2023 09.
Article in English | MEDLINE | ID: mdl-37341054

ABSTRACT

An increasing number of cancer subtypes are treated with front-line immunotherapy. However, approaches to overcome primary and acquired resistance remain limited. Preclinical mouse models are often used to investigate resistance mechanisms, novel drug combinations, and delivery methods; yet most of these models lack the genetic diversity and mutational patterns observed in human tumors. Here we describe a series of 13 C57BL/6J melanoma cell lines to address this gap in the field. The Ohio State University-Moffitt Melanoma Exposed to Radiation (OSUMMER) cell lines are derived from mice expressing endogenous, melanocyte-specific, and clinically relevant Nras driver mutations (Q61R, Q61K, or Q61L). Exposure of these animals to a single, non-burning dose of ultraviolet B accelerates the onset of spontaneous melanomas with mutational patterns akin to human disease. Furthermore, in vivo irradiation selects against potent tumor antigens, which could prevent the outgrowth of syngeneic cell transfers. Each OSUMMER cell line possesses distinct in vitro growth properties, trametinib sensitivity, mutational signatures, and predicted antigenicity. Analysis of OSUMMER allografts shows a correlation between strong, predicted antigenicity and poor tumor outgrowth. These data suggest that the OSUMMER lines will be a valuable tool for modeling the heterogeneous responses of human melanomas to targeted and immune-based therapies.


Subject(s)
Cell Line, Tumor , Melanoma , Animals , Mice , Cell Line, Tumor/radiation effects , GTP Phosphohydrolases/genetics , Melanoma/drug therapy , Melanoma/genetics , Melanoma/pathology , Membrane Proteins/genetics , Mice, Inbred C57BL , Mutation/genetics , Proto-Oncogene Proteins B-raf/genetics
2.
Breast Cancer Res ; 22(1): 41, 2020 05 05.
Article in English | MEDLINE | ID: mdl-32370801

ABSTRACT

BACKGROUND: In utero endocrine disruption is linked to increased risk of breast cancer later in life. Despite numerous studies establishing this linkage, the long-term molecular changes that predispose mammary cells to carcinogenic transformation are unknown. Herein, we investigated how endocrine disrupting compounds (EDCs) drive changes within the stroma that can contribute to breast cancer susceptibility. METHODS: We utilized bisphenol A (BPA) as a model of estrogenic endocrine disruption to analyze the long-term consequences in the stroma. Deregulated genes were identified by RNA-seq transcriptional profiling of adult primary fibroblasts, isolated from female mice exposed to in utero BPA. Collagen staining, collagen imaging techniques, and permeability assays were used to characterize changes to the extracellular matrix. Finally, gland stiffness tests were performed on exposed and control mammary glands. RESULTS: We identified significant transcriptional deregulation of adult fibroblasts exposed to in utero BPA. Deregulated genes were associated with cancer pathways and specifically extracellular matrix composition. Multiple collagen genes were more highly expressed in the BPA-exposed fibroblasts resulting in increased collagen deposition in the adult mammary gland. This transcriptional reprogramming of BPA-exposed fibroblasts generates a less permeable extracellular matrix and a stiffer mammary gland. These phenotypes were only observed in adult 12-week-old, but not 4-week-old, mice. Additionally, diethylstilbestrol, known to increase breast cancer risk in humans, also increases gland stiffness similar to BPA, while bisphenol S does not. CONCLUSIONS: As breast stiffness, extracellular matrix density, and collagen deposition have been directly linked to breast cancer risk, these data mechanistically connect EDC exposures to molecular alterations associated with increased disease susceptibility. These alterations develop over time and thus contribute to cancer risk in adulthood.


Subject(s)
Endocrine Disruptors/toxicity , Extracellular Matrix/pathology , Mammary Glands, Animal/pathology , Prenatal Exposure Delayed Effects/pathology , Stromal Cells/pathology , Animals , Benzhydryl Compounds/toxicity , Estrogens, Non-Steroidal/pharmacology , Extracellular Matrix/drug effects , Extracellular Matrix/immunology , Female , Fibroblasts/immunology , Fibroblasts/pathology , Mammary Glands, Animal/drug effects , Mammary Glands, Animal/immunology , Mammary Glands, Animal/metabolism , Mice , Phenols/toxicity , Pregnancy , Prenatal Exposure Delayed Effects/chemically induced , Prenatal Exposure Delayed Effects/metabolism , Stromal Cells/drug effects , Stromal Cells/immunology , Transcriptome
3.
J Virol ; 92(10)2018 05 15.
Article in English | MEDLINE | ID: mdl-29514902

ABSTRACT

The mouse model of West Nile virus (WNV), which is a leading cause of mosquito-borne encephalitis worldwide, has provided fundamental insights into the host and viral factors that regulate viral pathogenesis and infection outcome. In particular, CD8+ T cells are critical for controlling WNV replication and promoting protection against infection. Here, we present the characterization of a T cell receptor (TCR)-transgenic mouse with specificity for the immunodominant epitope in the WNV NS4B protein (here referred to as transgenic WNV-I mice). Using an adoptive-transfer model, we found that WNV-I CD8+ T cells behave similarly to endogenous CD8+ T cell responses, with an expansion phase in the periphery beginning around day 7 postinfection (p.i.) followed by a contraction phase through day 15 p.i. Through the use of in vivo intravascular immune cell staining, we determined the kinetics, expansion, and differentiation into effector and memory subsets of WNV-I CD8+ T cells within the spleen and brain. We found that red-pulp WNV-I CD8+ T cells were more effector-like than white-pulp WNV-I CD8+ T cells, which displayed increased differentiation into memory precursor cells. Within the central nervous system (CNS), we found that WNV-I CD8+ T cells were polyfunctional (gamma interferon [IFN-γ] and tumor necrosis factor alpha [TNF-α]), displayed tissue-resident characteristics (CD69+ and CD103+), persisted in the brain through day 15 p.i., and reduced the viral burden within the brain. The use of these TCR-transgenic WNV-I mice provides a new resource to dissect the immunological mechanisms of CD8+ T cell-mediated protection during WNV infection.IMPORTANCE West Nile Virus (WNV) is the leading cause of mosquito-borne encephalitis worldwide. There are currently no approved therapeutics or vaccines for use in humans to treat or prevent WNV infection. CD8+ T cells are critical for controlling WNV replication and protecting against infection. Here, we present a comprehensive characterization of a novel TCR-transgenic mouse with specificity for the immunodominant epitope in the WNV NS4B protein. In this study, we determine the kinetics, proliferation, differentiation into effector and memory subsets, homing, and clearance of WNV in the CNS. Our findings provide a new resource to dissect the immunological mechanisms of CD8+ T cell-mediated protection during WNV infection.


Subject(s)
Brain/immunology , CD8-Positive T-Lymphocytes/immunology , Viral Nonstructural Proteins/immunology , West Nile Fever/immunology , West Nile virus/immunology , Adoptive Transfer , Animals , Brain/virology , CD8-Positive T-Lymphocytes/cytology , Cell Differentiation/immunology , Cells, Cultured , Disease Models, Animal , Immunodominant Epitopes/immunology , Immunologic Memory/immunology , Interferon-gamma/immunology , Mice , Mice, Inbred C57BL , Mice, Transgenic , Receptors, Antigen, T-Cell/genetics , Receptors, Antigen, T-Cell/immunology , Tumor Necrosis Factor-alpha/immunology , Viral Load/immunology , West Nile Fever/virology
4.
Cell Physiol Biochem ; 39(4): 1271-80, 2016.
Article in English | MEDLINE | ID: mdl-27606466

ABSTRACT

BACKGROUND/AIMS: Viral infections represent a global health problem with the need for new viral therapies and better understanding of the immune response during infection. The most immediate and potent anti-viral defense mechanism is the production of type I interferon (IFN-I) which are activated rapidly following recognition of viral infection by host pathogen recognition receptors (PRR). The mechanisms of innate cellular signaling downstream of PRR activation remain to be fully understood. In the present study, we demonstrate that CASP2 and RIPK1 domain-containing adaptor with death domain (CRADD/RAIDD) is a critical component in type I IFN production. METHODS: The role of RAIDD during IFN-I production was investigated using western blot, shRNA mediated lentiviral knockdown, immunoprecipitation and IFN-I driven dual luciferase assay. RESULTS: Immunoprecipitation analysis revealed the molecular interaction of RAIDD with interferon regulatory factor 7 (IRF7) and its phosphorylating kinase IKKε. Using an IFN-4α driven dual luciferase analysis in RAIDD deficient cells, type I IFN activation by IKKε and IRF7 was dramatically reduced. Furthermore, deletion of either the caspase recruitment domain (CARD) or death domain (DD) of RAIDD inhibited IKKε and IRF7 mediated interferon-4α activation. CONCLUSION: We have identified that the adaptor molecule RAIDD coordinates IKKε and IRF7 interaction to ensure efficient expression of type I interferon.


Subject(s)
CRADD Signaling Adaptor Protein/genetics , I-kappa B Kinase/genetics , Interferon Regulatory Factor-7/genetics , Toll-Like Receptor 3/genetics , Animals , CRADD Signaling Adaptor Protein/immunology , Caspase Activation and Recruitment Domain , Gene Expression Regulation , Genes, Reporter , Genetic Vectors/chemistry , Genetic Vectors/metabolism , HEK293 Cells , Humans , I-kappa B Kinase/immunology , Interferon Regulatory Factor-7/immunology , Interferon-alpha/genetics , Interferon-alpha/immunology , Interferon-beta/genetics , Interferon-beta/immunology , Lentivirus/genetics , Lentivirus/metabolism , Luciferases/genetics , Luciferases/metabolism , Mice , Plasmids/chemistry , Plasmids/metabolism , Poly I-C/pharmacology , Recombinant Proteins/genetics , Recombinant Proteins/immunology , Signal Transduction , Toll-Like Receptor 3/immunology
5.
J Gastroenterol Hepatol ; 29(4): 878-86, 2014 Apr.
Article in English | MEDLINE | ID: mdl-24224701

ABSTRACT

BACKGROUND AND AIM: Hepatocyte growth factor (HGF) is a pleiotropic cytokine related with cell proliferation and survival; however, its role in viral hepatitis is not elucidated. In this study, we studied HGF immune role in viral hepatitis. METHODS: Mice received hydrodynamically delivered HGF plasmid or control plasmid and then infected with adenovirus, and parameters of immune-mediated liver damage were evaluated. We studied dendritic cell (DC) activation in the presence of HGF. T cells collected from infected mice were restimulated with virally infected DC to measure cytokine production in vitro. RESULTS: HGF ameliorated the liver inflammation during viral hepatitis as alanine transferase, intrahepatic lymphocytes, and splenocyte counts were diminished by HGF. Lower histological scores of liver pathology were observed in the HGF group. DC from the HGF group expressed reduced CD40. The hepatic expression and serum concentration of IL-12p40 were diminished in HGF-transfected mice. In vitro experiments with DC confirmed that HGF diminished CD40 expression and IL-12p40 production. The expression and serum levels of IFN-γ, IL-6 and CXCL9 were significantly decreased in the HGF group. HGF overexpression diminished the expression and concentration of IL-10 and TGF-ß. The frequency of PD-1(+) Tim-3(+) in CD8 T cells was decreased by HGF overexpression. Moreover, T cells in the HGF group at day 14 secreted more IFN-γ and TNF-α than those in the control group when restimulated with virally infected DC. CONCLUSION: HGF modulated DC activation and T cell priming, thereby limiting the immune-mediated damage in the liver. However, viral clearance was not compromised by HGF.


Subject(s)
Hepatitis, Viral, Animal/drug therapy , Hepatitis, Viral, Animal/immunology , Hepatocyte Growth Factor/physiology , Liver/immunology , Acute Disease , Animals , CD40 Antigens/metabolism , CD8-Positive T-Lymphocytes , Cells, Cultured , Chemokine CXCL9/metabolism , Dendritic Cells/immunology , Gene Expression , Hepatitis, Viral, Animal/pathology , Hepatitis, Viral, Animal/virology , Hepatocyte Growth Factor/administration & dosage , Hepatocyte Growth Factor/genetics , Hepatocyte Growth Factor/pharmacology , Inflammation Mediators/metabolism , Interferon-gamma/metabolism , Interleukin-10/metabolism , Interleukin-12 Subunit p40/metabolism , Interleukin-6/metabolism , Liver/pathology , Liver/virology , Lymphocyte Count , Mice , Mice, Inbred C57BL , T-Lymphocytes/immunology , Transforming Growth Factor beta/metabolism , Tumor Necrosis Factor-alpha/metabolism
6.
J Immunol ; 190(11): 5666-75, 2013 Jun 01.
Article in English | MEDLINE | ID: mdl-23630360

ABSTRACT

Molecules containing damage-associated molecular patterns play an important role in many pathogenic processes. In this study, our aim was to investigate the role of IL-33, a damage-associated molecular pattern molecule, in adenovirus (Ad)-induced liver inflammation. Ad-infected mice exhibited a steadily increased IL-33 and its receptor IL-1R-like 1 expression in the liver during the first week of infection. Treatment of exogenous IL-33 resulted in a great decrease in the serum alanine aminotransferase levels and the number of Councilman bodies in the liver. Attenuated liver injury by IL-33 correlated with an increase in T regulatory cells but with a decrease in macrophages, dendritic cells, and NK cells in the liver. IL-33 enhanced both type 1 (IL-2 and IFN-γ) and type 2 (IL-5 and IL-13) immune responses in infected mice. However, IL-33 inhibited TNF-α expression in hepatic T cells and macrophages, and significantly reduced TNF-α levels in the liver. We found that in addition to its direct effects, IL-33 strongly induced novel nuocytes in the livers and spleens of infected mice. When cocultured with nuocytes, hepatic T cells and macrophages expressed lower levels of TNF-α. The IL-33-treated mice also demonstrated a slight delay, but no significant impairment, in eliminating an intrahepatic infection with Ad. In conclusion, this study reveals that IL-33 acts as a potent immune stimulator and a hepatoprotective cytokine in acute viral hepatitis. Its direct immunoregulatory functions and ability to induce novel nuocytes further suggest to us that it may be a potentially promising therapeutic candidate for the management of viral hepatitis.


Subject(s)
Hepatitis, Viral, Animal/immunology , Hepatitis, Viral, Animal/metabolism , Interleukins/metabolism , Adoptive Transfer , Animals , Disease Models, Animal , Female , Hepatitis, Viral, Animal/pathology , Immunity, Innate/drug effects , Interferon-gamma/metabolism , Interleukin-33 , Interleukins/pharmacology , Liver/immunology , Liver/metabolism , Liver/pathology , Mice , T-Lymphocytes/immunology , T-Lymphocytes/metabolism , Tumor Necrosis Factor-alpha/metabolism
7.
Blood ; 117(4): 1408-14, 2011 Jan 27.
Article in English | MEDLINE | ID: mdl-21119114

ABSTRACT

Antiphospholipid (aPL)/anti-ß(2) glycoprotein I (anti-ß(2)GPI) antibodies stimulates tissue factor (TF) expression within vasculature and in blood cells, thereby leading to increased thrombosis. Several cellular receptors have been proposed to mediate these effects, but no convincing evidence for the involvement of a specific one has been provided. We investigated the role of Apolipoprotein E receptor 2 (ApoER2') on the pathogenic effects of a patient-derived polyclonal aPL IgG preparation (IgG-APS), a murine anti-ß(2)GPI monoclonal antibody (E7) and of a constructed dimeric ß(2)GPI I (dimer), which in vitro mimics ß(2)GPI-antibody immune complexes, using an animal model of thrombosis, and ApoER2-deficient (-/-) mice. In wild type mice, IgG-APS, E7 and the dimer increased thrombus formation, carotid artery TF activity as well as peritoneal macrophage TF activity/expression. Those pathogenic effects were significantly reduced in ApoER2 (-/-) mice. In addition, those effects induced by the IgG-APS, by E7 and by the dimer were inhibited by treatment of wild-type mice with soluble binding domain 1 of ApoER2 (sBD1). Altogether these data show that ApoER2 is involved in pathogenesis of antiphospholipids antibodies.


Subject(s)
Antiphospholipid Syndrome/genetics , LDL-Receptor Related Proteins/physiology , Thrombosis/genetics , Animals , Antibodies, Antiphospholipid/adverse effects , Antibodies, Antiphospholipid/metabolism , Antibodies, Antiphospholipid/pharmacology , Antibodies, Monoclonal/administration & dosage , Antibodies, Monoclonal/pharmacology , Antibodies, Phospho-Specific/administration & dosage , Antibodies, Phospho-Specific/adverse effects , Antibodies, Phospho-Specific/pharmacology , Antiphospholipid Syndrome/complications , Antiphospholipid Syndrome/pathology , Antiphospholipid Syndrome/prevention & control , Disease Models, Animal , Drug Evaluation, Preclinical , Humans , Immunoglobulin G/administration & dosage , Immunoglobulin G/adverse effects , Immunoglobulin G/pharmacology , LDL-Receptor Related Proteins/genetics , LDL-Receptor Related Proteins/metabolism , Mice , Mice, Inbred C57BL , Mice, Knockout , Middle Aged , Thrombosis/etiology , Thrombosis/pathology , beta 2-Glycoprotein I/immunology
8.
BMC Genomics ; 11: 667, 2010 Nov 25.
Article in English | MEDLINE | ID: mdl-21108814

ABSTRACT

BACKGROUND: Adherent and invasive Escherichia coli (AIEC) are commonly found in ileal lesions of Crohn's Disease (CD) patients, where they adhere to intestinal epithelial cells and invade into and survive in epithelial cells and macrophages, thereby gaining access to a typically restricted host niche. Colonization leads to strong inflammatory responses in the gut suggesting that AIEC could play a role in CD immunopathology. Despite extensive investigation, the genetic determinants accounting for the AIEC phenotype remain poorly defined. To address this, we present the complete genome sequence of an AIEC, revealing the genetic blueprint for this disease-associated E. coli pathotype. RESULTS: We sequenced the complete genome of E. coli NRG857c (O83:H1), a clinical isolate of AIEC from the ileum of a Crohn's Disease patient. Our sequence data confirmed a phylogenetic linkage between AIEC and extraintestinal pathogenic E. coli causing urinary tract infections and neonatal meningitis. The comparison of the NRG857c AIEC genome with other pathogenic and commensal E. coli allowed for the identification of unique genetic features of the AIEC pathotype, including 41 genomic islands, and unique genes that are found only in strains exhibiting the adherent and invasive phenotype. CONCLUSIONS: Up to now, the virulence-like features associated with AIEC are detectable only phenotypically. AIEC genome sequence data will facilitate the identification of genetic determinants implicated in invasion and intracellular growth, as well as enable functional genomic studies of AIEC gene expression during health and disease.


Subject(s)
Bacterial Adhesion/genetics , Escherichia coli/genetics , Escherichia coli/pathogenicity , Genome, Bacterial/genetics , Genomics/methods , Sequence Analysis, DNA/methods , Base Sequence , Biological Transport/genetics , Escherichia coli/classification , Genes, Bacterial , Genomic Islands/genetics , Iron/metabolism , Molecular Sequence Data , Mutation/genetics , Phylogeny , Plasmids/genetics , Virulence/genetics , Virulence Factors/genetics
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