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1.
Am J Pathol ; 193(12): 2031-2046, 2023 12.
Article in English | MEDLINE | ID: mdl-37689386

ABSTRACT

The pathophysiology of long-recognized hematologic abnormalities in Ebolavirus (EBOV) disease (EVD) is unknown. From limited human sampling (of peripheral blood), it has been postulated that emergency hematopoiesis plays a role in severe EVD, but the systematic characterization of the bone marrow (BM) has not occurred in human disease or in nonhuman primate models. In a lethal rhesus macaque model of EVD, 18 sternal BM samples exposed to the Kikwit strain of EBOV were compared to those from uninfected controls (n = 3). Immunohistochemistry, RNAscope in situ hybridization, transmission electron microscopy, and confocal microscopy showed that EBOV infects BM monocytes/macrophages and megakaryocytes. EBOV exposure was associated with severe BM hypocellularity, including depletion of myeloid, erythroid, and megakaryocyte hematopoietic cells. These depletions were negatively correlated with cell proliferation (Ki67 expression) and were not associated with BM apoptosis during disease progression. In EBOV-infected rhesus macaques with terminal disease, BM showed marked hemophagocytosis, megakaryocyte emperipolesis, and the release of immature hematopoietic cells into the sinusoids. Collectively, these data demonstrate not only direct EBOV infection of BM monocytes/macrophages and megakaryocytes but also that disease progression is associated with hematopoietic failure, notably in peripheral cytopenia. These findings inform current pathophysiologic unknowns and suggest a crucial role for BM dysfunction and/or failure, including emergency hematopoiesis, as part of the natural history of severe human disease.


Subject(s)
Ebolavirus , Hemorrhagic Fever, Ebola , Animals , Humans , Ebolavirus/physiology , Macaca mulatta , Bone Marrow , Disease Progression
2.
J Infect Dis ; 228(4): 371-382, 2023 08 16.
Article in English | MEDLINE | ID: mdl-37279544

ABSTRACT

BACKGROUND: Ebola virus (EBOV) disease (EVD) is one of the most severe and fatal viral hemorrhagic fevers and appears to mimic many clinical and laboratory manifestations of hemophagocytic lymphohistiocytosis syndrome (HLS), also known as macrophage activation syndrome. However, a clear association is yet to be firmly established for effective host-targeted, immunomodulatory therapeutic approaches to improve outcomes in patients with severe EVD. METHODS: Twenty-four rhesus monkeys were exposed intramuscularly to the EBOV Kikwit isolate and euthanized at prescheduled time points or when they reached the end-stage disease criteria. Three additional monkeys were mock-exposed and used as uninfected controls. RESULTS: EBOV-exposed monkeys presented with clinicopathologic features of HLS, including fever, multiple organomegaly, pancytopenia, hemophagocytosis, hyperfibrinogenemia with disseminated intravascular coagulation, hypertriglyceridemia, hypercytokinemia, increased concentrations of soluble CD163 and CD25 in serum, and the loss of activated natural killer cells. CONCLUSIONS: Our data suggest that EVD in the rhesus macaque model mimics pathophysiologic features of HLS/macrophage activation syndrome. Hence, regulating inflammation and immune function might provide an effective treatment for controlling the pathogenesis of acute EVD.


Subject(s)
Ebolavirus , Hemorrhagic Fever, Ebola , Lymphohistiocytosis, Hemophagocytic , Macrophage Activation Syndrome , Animals , Macrophage Activation Syndrome/therapy , Macaca mulatta
3.
Am J Pathol ; 192(1): 121-129, 2022 01.
Article in English | MEDLINE | ID: mdl-34626576

ABSTRACT

The pathogenesis of Ebola virus disease (EVD) is still incomplete, in spite of the availability of a nonhuman primate modelfor more than 4 decades. To further investigate EVD pathogenesis, a natural history study was conducted using 27 Chinese-origin rhesus macaques. Of these, 24 macaques were exposed intramuscularly to Kikwit Ebola virus and euthanized at predetermined time points or when end-stage clinical disease criteria were met, and 3 sham-exposed macaques were euthanized on study day 0. This study showed for the first time that Ebola virus causes uterine cervicitis, vaginitis, posthitis, and medullary adrenalitis. Not only was Ebola virus detected in the interstitial stromal cells of the genital tract, but it was also present in the epididymal and seminal vesicular tubular epithelial cells, ectocervical and vaginal squamous epithelial cells, and seminal fluid. Furthermore, as early as day 3 after exposure, Ebola virus replicative intermediate RNA was detected in Kupffer cells and hepatocytes. These findings in the nonhuman model provide additional insight into potential sexual transmission, possible disruption of sympathetic hormone production, and early virus replication sites in human EVD patients.


Subject(s)
Ebolavirus/physiology , Hormones/metabolism , Liver/virology , Tropism/physiology , Virus Replication/physiology , Animals , Chromaffin Cells/pathology , Chromaffin Cells/virology , Disease Models, Animal , Epididymis/pathology , Epididymis/virology , Epithelial Cells/pathology , Epithelial Cells/virology , Female , Hepatocytes/pathology , Hepatocytes/virology , Kupffer Cells/pathology , Kupffer Cells/virology , Macaca mulatta , Male , Uterine Cervicitis/pathology , Uterine Cervicitis/virology , Vaginitis/pathology , Vaginitis/virology
4.
J Immunol ; 204(4): 796-809, 2020 02 15.
Article in English | MEDLINE | ID: mdl-31900342

ABSTRACT

TLR7 is associated with development of systemic lupus erythematosus (SLE), but the underlying mechanisms are incompletely understood. Although TLRs are known to activate type I IFN (T1IFN) signaling, the role of T1IFN and IFN-γ signaling in differential regulation of TLR7-mediated Ab-forming cell (AFC) and germinal center (GC) responses, and SLE development has never been directly investigated. Using TLR7-induced and TLR7 overexpression models of SLE, we report in this study a previously unrecognized indispensable role of TLR7-induced IFN-γ signaling in promoting AFC and GC responses, leading to autoreactive B cell and SLE development. T1IFN signaling in contrast, only modestly contributed to autoimmune responses and the disease process in these mice. TLR7 ligand imiquimod treated IFN-γ reporter mice show that CD4+ effector T cells including follicular helper T (Tfh) cells are the major producers of TLR7-induced IFN-γ. Transcriptomic analysis of splenic tissues from imiquimod-treated autoimmune-prone B6.Sle1b mice sufficient and deficient for IFN-γR indicates that TLR7-induced IFN-γ activates multiple signaling pathways to regulate TLR7-promoted SLE. Conditional deletion of Ifngr1 gene in peripheral B cells further demonstrates that TLR7-driven autoimmune AFC, GC and Tfh responses and SLE development are dependent on IFN-γ signaling in B cells. Finally, we show crucial B cell-intrinsic roles of STAT1 and T-bet in TLR7-driven GC, Tfh and plasma cell differentiation. Altogether, we uncover a nonredundant role for IFN-γ and its downstream signaling molecules STAT1 and T-bet in B cells in promoting TLR7-driven AFC, GC, and SLE development whereas T1IFN signaling moderately contributes to these processes.


Subject(s)
Autoimmunity/immunology , B-Lymphocytes/immunology , Interferon-gamma/immunology , Lupus Erythematosus, Systemic/immunology , Lymphocyte Activation/immunology , Signal Transduction/immunology , Animals , Germinal Center/immunology , Interferon Type I , Membrane Glycoproteins/immunology , Mice , Toll-Like Receptor 7/immunology
5.
Am J Pathol ; 190(9): 1867-1880, 2020 09.
Article in English | MEDLINE | ID: mdl-32479821

ABSTRACT

The most commonly reported symptom of post-Ebola virus disease syndrome in survivors is arthralgia, yet involvement of the joints in acute or convalescent Ebola virus infection is not well characterized in human patients or animal models. Through immunohistochemistry, we found that the lining synovial intima of the stifle (knee) is a target for acute infection by Ebola virus/Kikwit, Ebola virus/Makona-C05, and Marburg virus/Angola in the rhesus macaque model. Furthermore, histologic analysis, immunohistochemistry, RNAscope in situ hybridization, and transmission electron microscopy showed that synoviocytes of the stifle, shoulder, and hip are a target for mouse-adapted Ebola virus/Yambuku-Mayinga infection during acute disease in rhesus macaques. A time course of infection study with Ebola virus/Kikwit found that the large joint synovium became immunopositive beginning on postinfection day 6. In total, the synovium of 28 of 30 rhesus macaques with terminal filovirus disease had evidence of infection (64 of 96 joints examined). On the basis of immunofluorescence, infected cell types included CD68+ type A (macrophage-like) synoviocytes and CD44+ type B (fibroblast-like) synoviocytes. Cultured primary human fibroblast-like synoviocytes were permissive to infection with Ebola and Marburg viruses in vitro. Because synovial joints include immune privileged sites, these findings are significant for future investigations of filovirus pathogenesis and persistence as well as arthralgias in acute and convalescent filovirus disease.


Subject(s)
Filoviridae Infections/virology , Synoviocytes/virology , Animals , Cells, Cultured , Filoviridae , Humans , Macaca mulatta
6.
J Infect Dis ; 222(10): 1745-1755, 2020 10 13.
Article in English | MEDLINE | ID: mdl-32498080

ABSTRACT

Neurological signs and symptoms are the most common complications of Ebola virus disease. However, the mechanisms underlying the neurologic manifestations in Ebola patients are not known. In this study, peripheral ganglia were collected from 12 rhesus macaques that succumbed to Ebola virus (EBOV) disease from 5 to 8 days post exposure. Ganglionitis, characterized by neuronal degeneration, necrosis, and mononuclear leukocyte infiltrates, was observed in the dorsal root, autonomic, and enteric ganglia. By immunohistochemistry, RNAscope in situ hybridization, transmission electron microscopy, and confocal microscopy, we confirmed that CD68+ macrophages are the target cells for EBOV in affected ganglia. Further, we demonstrated that EBOV can induce satellite cell and neuronal apoptosis and microglial activation in infected ganglia. Our results demonstrate that EBOV can infect peripheral ganglia and results in ganglionopathy in rhesus macaques, which may contribute to the neurological signs and symptoms observed in acute and convalescent Ebola virus disease in human patients.


Subject(s)
Hemorrhagic Fever, Ebola/complications , Hemorrhagic Fever, Ebola/pathology , Nerve Degeneration/complications , Nerve Degeneration/pathology , Peripheral Nervous System Diseases/complications , Peripheral Nervous System Diseases/pathology , Animals , Antigens, CD , Antigens, Differentiation, Myelomonocytic , Disease Models, Animal , Ebolavirus , Female , Ganglia , Ganglia, Spinal/pathology , Ganglia, Spinal/virology , Ganglion Cysts/pathology , Hemorrhagic Fever, Ebola/virology , Humans , Immunohistochemistry , Leukocytes, Mononuclear , Macaca mulatta , Macrophages/pathology , Male , Microglia/pathology , Microglia/virology , Necrosis , Parasympathetic Nervous System/pathology , Peripheral Nervous System Diseases/virology , Sensory Receptor Cells/pathology , Sensory Receptor Cells/virology , Sympathetic Nervous System/pathology
7.
Am J Physiol Renal Physiol ; 318(2): F509-F517, 2020 02 01.
Article in English | MEDLINE | ID: mdl-31904280

ABSTRACT

Endothelial dysfunction, characterized by reduced bioavailability of nitric oxide and increased oxidative stress, is a hallmark characteristic in diabetes and diabetic nephropathy (DN). High levels of asymmetric dimethylarginine (ADMA) are observed in several diseases including DN and are a strong prognostic marker for cardiovascular events in patients with diabetes and end-stage renal disease. ADMA, an endogenous endothelial nitric oxide synthase (NOS3) inhibitor, is selectively metabolized by dimethylarginine dimethylaminohydrolase (DDAH). Low DDAH levels have been associated with cardiac and renal dysfunction, but its effects on DN are unknown. We hypothesized that enhanced renal DDAH-1 expression would improve DN by reducing ADMA and restoring NOS3 levels. DBA/2J mice injected with multiple low doses of vehicle or streptozotocin were subsequently injected intrarenally with adenovirus expressing DDAH-1 (Ad-h-DDAH-1) or vector control [Ad-green fluorescent protein (GFP)], and mice were followed for 6 wk. Diabetes was associated with increased kidney ADMA and reduced kidney DDAH activity and DDAH-1 expression but had no effect on kidney DDAH-2 expression. Ad-GFP-treated diabetic mice showed significant increases in albuminuria, histological changes, glomerular macrophage recruitment, inflammatory cytokine and fibrotic markers, kidney ADMA levels, and urinary thiobarbituric acid reactive substances excretion as an indicator of oxidative stress, along with a significant reduction in kidney DDAH activity and kidney NOS3 mRNA compared with normal mice. In contrast, Ad-h-DDAH-1 treatment of diabetic mice reversed these effects. These data indicate, for the first time, that DDAH-1 mediates renal tissue protection in DN via the ADMA-NOS3-interaction. Enhanced renal DDAH-1 activity could be a novel therapeutic tool for treating patients with diabetes.


Subject(s)
Adenoviridae/genetics , Amidohydrolases/biosynthesis , Arginine/analogs & derivatives , Diabetes Mellitus, Experimental/therapy , Diabetic Nephropathies/prevention & control , Genetic Therapy , Genetic Vectors , Kidney/enzymology , Albuminuria/enzymology , Albuminuria/genetics , Albuminuria/prevention & control , Amidohydrolases/genetics , Animals , Arginine/metabolism , Cytokines/genetics , Cytokines/metabolism , Diabetes Mellitus, Experimental/complications , Diabetes Mellitus, Experimental/enzymology , Diabetes Mellitus, Experimental/genetics , Diabetic Nephropathies/enzymology , Diabetic Nephropathies/etiology , Diabetic Nephropathies/genetics , Fibrosis , Inflammation Mediators/metabolism , Kidney/pathology , Male , Mice, Inbred DBA , Nitric Oxide/metabolism , Nitric Oxide Synthase Type III/genetics , Nitric Oxide Synthase Type III/metabolism , Oxidative Stress , Signal Transduction , Thiobarbituric Acid Reactive Substances/metabolism
8.
J Infect Dis ; 219(11): 1818-1822, 2019 05 05.
Article in English | MEDLINE | ID: mdl-30517671

ABSTRACT

Lassa fever (LF) survivors develop various clinical manifestations including polyserositis, myalgia, epididymitis, and hearing loss weeks to months after recovery from acute infection. We demonstrate a systemic lymphoplasmacytic and histiocytic arteritis and periarteritis in guinea pigs more than 2 months after recovery from acute Lassa virus (LASV) infection. LASV was detected in the arterial tunica media smooth muscle cells by immunohistochemistry, in situ hybridization, and transmission electron microscopy. Our results suggest that the sequelae of LASV infection may be due to virus persistence resulting in systemic vascular damage. These findings shed light on the pathogenesis of LASV sequelae in convalescent human survivors.


Subject(s)
Lassa Fever/virology , Lassa virus/immunology , Animals , Convalescence , Disease Models, Animal , Disease Progression , Female , Guinea Pigs , Humans , Immunohistochemistry , Inflammation , Lassa Fever/pathology , Male
9.
J Immunol ; 199(12): 4001-4015, 2017 12 15.
Article in English | MEDLINE | ID: mdl-29118245

ABSTRACT

Mer tyrosine kinase (Mer) signaling maintains immune tolerance by clearing apoptotic cells (ACs) and inducing immunoregulatory signals. We previously showed that Mer-deficient mice (Mer-/-) have increased germinal center (GC) responses, T cell activation, and AC accumulation within GCs. Accumulated ACs in GCs can undergo necrosis and release self-ligands, which may influence the outcome of a GC response and selection. In this study, we generated Mer-/- mice with a global MyD88, TLR7, or TLR9 deficiency and cell type-specific MyD88 deficiency to study the functional correlation between Mer and TLRs in the development of GC responses and autoimmunity. We found that GC B cell-intrinsic sensing of self-RNA, but not self-DNA, released from dead cells accumulated in GCs drives enhanced GC responses in Mer-/- mice. Although self-ligands directly affect GC B cell responses, the loss of Mer in dendritic cells promotes enhanced T cell activation and proinflammatory cytokine production. To study the impact of Mer deficiency on the development of autoimmunity, we generated autoimmune-prone B6.Sle1b mice deficient in Mer (Sle1bMer-/-). We observed accelerated autoimmunity development even under conditions where Sle1bMer-/- mice did not exhibit increased AC accumulation in GCs compared with B6.Sle1b mice, indicating that Mer immunoregulatory signaling in APCs regulates B cell selection and autoimmunity. We further found significant expansion, retention, and class-switching of autoreactive B cells in GCs under conditions where ACs accumulated in GCs of Sle1bMer-/- mice. Altogether, both the phagocytic and immunomodulatory functions of Mer regulate GC responses to prevent the development of autoimmunity.


Subject(s)
Autoimmunity/immunology , Germinal Center/immunology , Self Tolerance/physiology , c-Mer Tyrosine Kinase/physiology , Animals , Antigen Presentation , Apoptosis , B-Lymphocyte Subsets/immunology , Female , Immunization , Immunoglobulin Class Switching , Kidney/pathology , Male , Membrane Glycoproteins/deficiency , Mice , Mice, Knockout , Myeloid Differentiation Factor 88/immunology , RNA/immunology , Specific Pathogen-Free Organisms , Toll-Like Receptor 7/deficiency , Toll-Like Receptor 9/deficiency , c-Mer Tyrosine Kinase/deficiency , c-Mer Tyrosine Kinase/genetics
10.
Vet Pathol ; 56(5): 799-806, 2019 09.
Article in English | MEDLINE | ID: mdl-31060453

ABSTRACT

During a screen for vascular phenotypes in aged laboratory mice, a unique discrete phenotype of hyaline arteriolosclerosis of the intertubular arteries and arterioles of the testes was identified in several inbred strains. Lesions were limited to the testes and did not occur as part of any renal, systemic, or pulmonary arteriopathy or vasculitis phenotype. There was no evidence of systemic or pulmonary hypertension, and lesions did not occur in ovaries of females. Frequency was highest in males of the SM/J (27/30, 90%) and WSB/EiJ (19/26, 73%) strains, aged 383 to 847 days. Lesions were sporadically present in males from several other inbred strains at a much lower (<20%) frequency. The risk of testicular hyaline arteriolosclerosis is at least partially underpinned by a genetic predisposition that is not associated with other vascular lesions (including vasculitis), separating out the etiology of this form and site of arteriolosclerosis from other related conditions that often co-occur in other strains of mice and in humans. Because of their genetic uniformity and controlled dietary and environmental conditions, mice are an excellent model to dissect the pathogenesis of human disease conditions. In this study, a discrete genetically driven phenotype of testicular hyaline arteriolosclerosis in aging mice was identified. These observations open the possibility of identifying the underlying genetic variant(s) associated with the predisposition and therefore allowing future interrogation of the pathogenesis of this condition.


Subject(s)
Aging , Arteriosclerosis/veterinary , Hyalin/metabolism , Rodent Diseases/pathology , Testicular Diseases/veterinary , Animals , Arteriosclerosis/genetics , Arteriosclerosis/pathology , Female , Genetic Predisposition to Disease , Male , Mice , Mice, Inbred Strains , Rodent Diseases/genetics , Testicular Diseases/genetics , Testicular Diseases/pathology , Testis/pathology
11.
J Infect Dis ; 218(suppl_5): S423-S433, 2018 11 22.
Article in English | MEDLINE | ID: mdl-30053050

ABSTRACT

Previously, several studies have been performed to delineate the development and progression of Marburg virus infection in nonhuman primates (NHPs), primarily to clarify the mechanisms of severe (fatal) disease. After the 2013-2016 Ebola virus disease (EVD) epidemic in Western Africa, there has been a reassessment of the available filovirus animal models and the utility of these to faithfully recapitulate human disease. The high lethality of the NHP models has raised doubts as to their ability to provide meaningful data for the full spectrum of disease observed in humans. Of particular interest are the etiologic and pathophysiologic mechanisms underlying postconvalescent sequelae observed in human survivors of EVD and Marburg virus disease (MVD). In the current study, we evaluated the lesions of MVD in NHPs; however, in contrast to previous studies, we focused on the potential for development of sequelae similar to those reported in human survivors of MVD and EVD. We found that during acute MVD in the macaque model, there is frequent inflammation of peripheral nerves, autonomic ganglia, and the iris of the eye. Furthermore, we demonstrate viral infection of the ocular ciliary body and retina, testis, epididymis, ovary, oviduct, uterine endometrium, prostate, and mammary gland. These findings are relevant for both development of postconvalescent sequelae and the natural transmission of virus.


Subject(s)
Marburg Virus Disease/pathology , Animals , Disease Models, Animal , Eye/pathology , Female , Ganglia/pathology , Humans , Macaca mulatta , Male , Mammary Glands, Human/pathology , Peripheral Nerves/pathology , Urogenital System/pathology
12.
Cancer Immunol Immunother ; 67(4): 639-652, 2018 Apr.
Article in English | MEDLINE | ID: mdl-29332158

ABSTRACT

Tumor-reactive T lymphocytes can promote the regression of established tumors. However, their efficacy is often limited by immunosuppressive mechanisms that block T cell accumulation or function. ACT provides the opportunity to ameliorate immune suppression prior to transfer of tumor-reactive T cells to improve the therapeutic benefit. We evaluated the combination of lymphodepleting whole body irradiation (WBI) and agonist anti-CD40 (αCD40) antibody on control of established autochthonous murine neuroendocrine pancreatic tumors following the transfer of naïve tumor-specific CD8 T cells. Sublethal WBI had little impact on disease outcome but did promote T cell persistence in the lymphoid organs. Host conditioning with αCD40, an approach known to enhance APC function and T cell expansion, transiently increased donor T cell accumulation in the lymphoid organs and pancreas, but failed to control tumor progression. In contrast, combined WBI and αCD40 prolonged T cell proliferation and dramatically enhanced accumulation of donor T cells in both the lymphoid organs and pancreas. This dual conditioning approach also promoted high levels of inflammation in the pancreas and tumor, induced histological regression of established tumors, and extended the lifespan of treated mice. Prolonged survival was entirely dependent upon adoptive transfer, but only partially dependent upon IFNγ production by donor T cells. Our results identify the novel combination of two clinically relevant host conditioning approaches that synergize to overcome immune suppression and drive strong tumor-specific T cell accumulation within well-established tumors.


Subject(s)
Antibodies, Monoclonal/therapeutic use , CD40 Antigens/immunology , CD8-Positive T-Lymphocytes/immunology , Chemoradiotherapy , Lymphocyte Activation/immunology , Pancreatic Neoplasms/therapy , Whole-Body Irradiation , Adoptive Transfer , Animals , Female , Male , Mice , Mice, Inbred C57BL , Pancreatic Neoplasms/immunology , Pancreatic Neoplasms/pathology , Tissue Donors
13.
Chem Res Toxicol ; 31(11): 1111-1118, 2018 11 19.
Article in English | MEDLINE | ID: mdl-30260214

ABSTRACT

Ovarian cancer ranked second in incidence among gynecologic cancers, but it causes more deaths than any other gynecologic cancer; at present there is no curative treatment beyond surgery. Animal models that employ carcinogens found in the human environment can provide a realistic platform to understand the mechanistic basis for disease development and to design rational chemopreventive/therapeutic strategies. We and others have shown that the administration of the environmental pollutant and tobacco smoke constituent dibenzo[ def,p]chrysene (DBP) to mice by several routes of exposure can induce tumors in multiple sites including the ovary. In the present study we compared, for the first time, the tumorigenicity and DNA damage induced by DBP and its metabolites DBP-dihydrodiol (DBPDHD) and DBP-dihydrodiol epoxide (DBPDE) in the mouse ovary. Compounds were dissolved in dimethyl sulfoxide (DMSO) as the vehicle and administered by topical application into the mouse oral cavity three times per week for 38 weeks. No tumors were observed in mice treated with DMSO. At equal dose (24 nmol/30 µL DMSO), the incidence of ovarian tumors induced by DBPDHD was higher (60.7%), although not significantly, than that induced by DBP (44.8%). Similarly the levels of DNA damage induced by DBPDHD in the ovary were higher than those observed with DBP. We did not observe any histological abnormality in the ovary of mice treated with DBPDE, which is consistent with lack of DNA damage. Our results suggested that both DBP and DBPDHD can be metabolized in the mouse ovary leading to the formation of DBPDE that can damage DNA, which is a prerequisite step in the initiation stage of carcinogenesis.


Subject(s)
Benzopyrenes/toxicity , DNA Damage/drug effects , Ovarian Neoplasms/etiology , Ovary/drug effects , Administration, Topical , Animals , Benzopyrenes/metabolism , Carcinogens/metabolism , Carcinogens/toxicity , Chromatography, High Pressure Liquid , DNA Adducts/analysis , Female , Mice , Ovarian Neoplasms/mortality , Ovarian Neoplasms/veterinary , Ovary/pathology , Survival Rate , Tandem Mass Spectrometry
14.
Am J Physiol Renal Physiol ; 313(2): F522-F534, 2017 Aug 01.
Article in English | MEDLINE | ID: mdl-28515179

ABSTRACT

Novel therapeutic interventions for preventing or attenuating kidney injury following ischemia-reperfusion injury (IRI) remain a focus of significant interest. Currently, there are no definitive therapeutic or preventive approaches available for ischemic acute kidney injury (AKI). Our objective is to determine 1) whether renal arginase activity or expression is increased in renal IRI, and 2) whether arginase plays a role in development of renal IRI. The impact of arginase activity and expression on renal damage was evaluated in male C57BL/6J (wild type) and arginase-2 (ARG2)-deficient (Arg2-/- ) mice subjected to bilateral renal ischemia for 28 min, followed by reperfusion for 24 h. ARG2 expression and arginase activity significantly increased following renal IRI, paralleling the increase in kidney injury. Pharmacological blockade or genetic deficiency of Arg2 conferred kidney protection in renal IRI. Arg2-/- mice had significantly attenuated kidney injury and lower plasma creatinine and blood urea nitrogen levels after renal IRI. Blocking arginases using S-(2-boronoethyl)-l-cysteine (BEC) 18 h before ischemia mimicked arginase deficiency by reducing kidney injury, histopathological changes and kidney injury marker-1 expression, renal apoptosis, kidney inflammatory cell recruitment and inflammatory cytokines, and kidney oxidative stress; increasing kidney nitric oxide (NO) production and endothelial NO synthase (eNOS) phosphorylation, kidney peroxisome proliferator-activated receptor-γ coactivator-1α expression, and mitochondrial ATP; and preserving kidney mitochondrial ultrastructure compared with vehicle-treated IRI mice. Importantly, BEC-treated eNOS-knockout mice failed to reduce blood urea nitrogen and creatinine following renal IRI. These findings indicate that ARG2 plays a major role in renal IRI, via an eNOS-dependent mechanism, and that blocking ARG2 activity or expression could be a novel therapeutic approach for prevention of AKI.


Subject(s)
Acute Kidney Injury/enzymology , Arginase/metabolism , Reperfusion Injury/enzymology , Acute Kidney Injury/pathology , Adenosine Triphosphate/metabolism , Animals , Arginase/antagonists & inhibitors , Blood Urea Nitrogen , Creatinine/blood , Kidney/pathology , Male , Mice, Inbred C57BL , Mitochondria/metabolism , Nitric Oxide Synthase Type III/metabolism , Nitrites/metabolism , Oxidative Stress , Peroxisome Proliferator-Activated Receptor Gamma Coactivator 1-alpha/metabolism , Reperfusion Injury/pathology
15.
Am J Physiol Renal Physiol ; 313(4): F899-F905, 2017 Oct 01.
Article in English | MEDLINE | ID: mdl-28446459

ABSTRACT

Diabetes is the leading cause of end-stage renal disease, resulting in a significant health care burden and loss of economic productivity by affected individuals. Because current therapies for progression of diabetic nephropathy (DN) are only moderately successful, identification of underlying mechanisms of disease is essential to develop more effective therapies. We showed previously that inhibition of arginase using S-(2-boronoethyl)-l-cysteine (BEC) or genetic deficiency of the arginase-2 isozyme was protective against key features of nephropathy in diabetic mouse models. However, those studies did not determine whether all markers of DN were dependent only on arginase-2 expression. The objective of this study was to identify features of DN that are associated specifically with expression of arginase-1 or -2. Elevated urinary albumin excretion rate and plasma urea levels, increases in renal fibronectin mRNA levels, and decreased renal medullary blood flow were associated almost completely and specifically with arginase-2 expression, indicating that arginase-2 selectively mediates major aspects of diabetic renal injury. However, increases in renal macrophage infiltration and renal TNF-α mRNA levels occurred independent of arginase-2 expression but were almost entirely abolished by treatment with BEC, indicating a distinct role for arginase-1. We therefore generated mice with a macrophage-specific deletion of arginase-1 (CD11bCre /Arg1fl/fl ). CD11bCre /Arg1fl/fl mice had significantly reduced macrophage infiltration but had no effect on albuminuria compared with Arg1fl/fl mice after 12 wk of streptozotocin-induced diabetes. These results indicate that selective inhibition of arginase-2 would be effective in preventing or ameliorating major features of diabetic renal injury.


Subject(s)
Arginase/metabolism , Diabetic Nephropathies/enzymology , Albuminuria/enzymology , Albuminuria/etiology , Animals , Diabetic Nephropathies/complications , Fibronectins/metabolism , Macrophages/enzymology , Male , Mice , Renal Circulation , Tumor Necrosis Factor-alpha/metabolism
16.
Kidney Int ; 91(3): 671-682, 2017 03.
Article in English | MEDLINE | ID: mdl-27914709

ABSTRACT

Inflammation is a central pathophysiologic mechanism that contributes to diabetes mellitus and diabetic nephropathy. Recently, we showed that macrophages directly contribute to diabetic renal injury and that pharmacological blockade or genetic deficiency of chemokine (C-C motif) receptor 2 (CCR2) confers kidney protection in diabetic nephropathy. However, the direct role of CCR2 in kidney-derived cells such as podocytes in diabetic nephropathy remains unclear. To study this, we developed a transgenic mouse model expressing CCR2 specifically in podocytes (Tg[NPHS2-Ccr2]) on a nephropathy-prone (DBA/2J) and CCR2-deficient (Ccr2-/-) background with heterozygous Ccr2+/- littermate controls. Diabetes was induced by streptozotocin. As expected, absence of CCR2 conferred kidney protection after nine weeks of diabetes. In contrast, transgenic CCR2 overexpression in the podocytes of Ccr2-/- mice resulted in significantly increased albuminuria, blood urea nitrogen, histopathologic changes, kidney fibronectin and type 1 collagen expression, podocyte loss, and glomerular apoptosis after nine weeks of streptozotocin-induced diabetes. Interestingly, there was no concurrent increase in kidney macrophage recruitment or inflammatory cytokine levels in the mice. These findings support a direct role for CCR2 expression in podocytes to mediate diabetic renal injury, independent of monocyte/macrophage recruitment. Thus, targeting the CCR2 signaling cascade in podocytes could be a novel therapeutic approach for treatment of diabetic nephropathy.


Subject(s)
Diabetes Mellitus, Experimental/metabolism , Diabetes Mellitus, Type 1/metabolism , Diabetic Nephropathies/metabolism , Podocytes/metabolism , Receptors, CCR2/metabolism , Albuminuria/genetics , Albuminuria/metabolism , Albuminuria/prevention & control , Animals , Apoptosis , Blood Urea Nitrogen , Collagen Type I/genetics , Collagen Type I/metabolism , Diabetes Mellitus, Experimental/complications , Diabetes Mellitus, Experimental/genetics , Diabetes Mellitus, Experimental/pathology , Diabetes Mellitus, Type 1/complications , Diabetes Mellitus, Type 1/genetics , Diabetes Mellitus, Type 1/pathology , Diabetic Nephropathies/genetics , Diabetic Nephropathies/pathology , Diabetic Nephropathies/prevention & control , Fibronectins/genetics , Fibronectins/metabolism , Fibrosis , Genetic Predisposition to Disease , Inflammation Mediators/metabolism , Macrophages/metabolism , Male , Mice, Inbred DBA , Mice, Knockout , Mice, Transgenic , Monocytes/metabolism , Phenotype , Podocytes/pathology , RNA, Messenger/genetics , RNA, Messenger/metabolism , Receptors, CCR2/deficiency , Receptors, CCR2/genetics , Signal Transduction , Streptozocin , Up-Regulation
17.
J Hepatol ; 66(1): 75-85, 2017 01.
Article in English | MEDLINE | ID: mdl-27520877

ABSTRACT

BACKGROUND & AIMS: We have established a clinically relevant animal model of hepatocellular cancer (HCC) in immune competent mice to elucidate the complex dialog between host immunity and tumors during HCC initiation and progression. Mechanistic findings have been leveraged to develop a clinically feasible anti-tumor chemoimmunotherapeutic strategy. METHODS: Intraperitoneal injection of carbon tetrachloride and intrasplenic inoculation of oncogenic hepatocytes were combined to induce progressive HCCs in fibrotic livers of immunocompetent mice. Immunization and adoptive cell transfer (ACT) were used to dissect the tumor antigen-specific immune response. The ability of the tyrosine kinase inhibitor sunitinib to enhance immunotherapy in the setting of HCC was evaluated. RESULTS: This new mouse model mimics human HCC and reflects its typical features. Tumor-antigen-specific CD8+ T cells maintained a naïve phenotype and remained responsive during early-stage tumor progression. Late tumor progression produced circulating tumor cells, tumor migration into draining lymph nodes, and profound exhaustion of tumor-antigen-specific CD8+ T cells associated with accumulation of programmed cell death protein 1 (PD-1)hi CD8+ T cells and regulatory T cells (Tregs). Sunitinib-mediated tumoricidal effect and Treg suppression synergized with antibody-mediated blockade of PD-1 to powerfully suppress tumor growth and activate anti-tumor immunity. CONCLUSION: Treg accumulation and upregulation of PD-1 provide two independent mechanisms to induce profound immune tolerance in HCC. Chemoimmunotherapy using Food and Drug Administration-approved sunitinib with anti-PD-1 antibodies achieved significant tumor control, supporting translation of this approach for the treatment of HCC patients. LAY SUMMARY: In the current study, we have established a clinically relevant mouse model which mimics human liver cancer. Using this unique model, we studied the response of the immune system to this aggressive cancer. Findings from this trial have led to the development of an innovative and clinically feasible chemoimmunotherapeutic strategy.


Subject(s)
Carcinoma, Hepatocellular , Immunotherapy/methods , Indoles/pharmacology , Liver Neoplasms , Pyrroles/pharmacology , Adoptive Transfer , Animals , CD8-Positive T-Lymphocytes/immunology , Carcinoma, Hepatocellular/drug therapy , Carcinoma, Hepatocellular/immunology , Carcinoma, Hepatocellular/pathology , Cytotoxicity, Immunologic/physiology , Disease Models, Animal , Enzyme Inhibitors/pharmacology , Histocompatibility Antigens Class II/immunology , Immune Tolerance , Liver Neoplasms/drug therapy , Liver Neoplasms/immunology , Liver Neoplasms/pathology , Mice , Neoplasm Staging , Programmed Cell Death 1 Receptor/metabolism , Sunitinib , T-Lymphocytes, Regulatory/immunology
18.
J Gen Virol ; 98(10): 2520-2529, 2017 Oct.
Article in English | MEDLINE | ID: mdl-28942760

ABSTRACT

We report secondary cutaneous infections in the mouse papillomavirus (MmuPV1)/mouse model. Our previous study demonstrated that cutaneous MmuPV1 infection could spread to mucosal sites. Recently, we observed that mucosal infections could also spread to various cutaneous sites including the back, tail, muzzle and mammary tissues. The secondary site lesions were positive for viral DNA, viral capsid protein and viral particles as determined by in situ hybridization, immunohistochemistry and transmission electron microscopy analyses, respectively. We also demonstrated differential viral production and tumour growth at different secondarily infected skin sites. For example, fewer viral particles were detected in the least susceptible back tissues when compared with those in the infected muzzle and tail, although similar amounts of viral DNA were detected. Follow-up studies demonstrated that significantly lower amounts of viral DNA were packaged in the back lesions. Lavages harvested from the oral cavity and lower genital tracts were equally infectious at both cutaneous and mucosal sites, supporting the broad tissue tropism of this papillomavirus. Importantly, two secondary skin lesions on the forearms of two mice displayed a malignant phenotype at about 9.5 months post-primary infection. Therefore, MmuPV1 induces not only dysplasia at mucosal sites such as the vagina, anus and oral cavity but also skin carcinoma at cutaneous sites. These findings demonstrate that MmuPV1 mucosal infection can be spread to cutaneous sites and suggest that the model could serve a useful role in the study of the viral life cycle and pathogenesis of papillomavirus.

19.
Am J Physiol Regul Integr Comp Physiol ; 312(1): R146-R156, 2017 01 01.
Article in English | MEDLINE | ID: mdl-27834292

ABSTRACT

Cervical and high thoracic spinal cord injury (SCI) drastically impairs autonomic nervous system function. Individuals with SCI at thoracic spinal level 5 (T5) or higher often present cardiovascular disorders that include resting systemic arterial hypotension. Gastrointestinal (GI) tissues are critically dependent upon adequate blood flow and even brief periods of visceral hypoxia triggers GI dysmotility. The aim of this study was to test the hypothesis that T3-SCI induces visceral hypoperfusion, diminished postprandial vascular reflexes, and concomitant visceral inflammation. We measured in vivo systemic arterial blood pressure and superior mesenteric artery (SMA) and duodenal blood flow in anesthetized T3-SCI rats at 3 days and 3 wk postinjury either fasted or following enteral feeding of a liquid mixed-nutrient meal (Ensure). In separate cohorts of fasted T3-SCI rats, markers of intestinal inflammation were assayed by qRT-PCR. Our results show that T3-SCI rats displayed significantly reduced SMA blood flow under all experimental conditions (P < 0.05). Specifically, the anticipated elevation of SMA blood flow in response to duodenal nutrient infusion (postprandial hyperemia) was either delayed or absent after T3-SCI. The dysregulated SMA blood flow in acutely injured T3-SCI rats coincides with abnormal intestinal morphology and elevation of inflammatory markers, all of which resolve after 3 wk. Specifically, Icam1, Ccl2 (MCP-1), and Ccl3 (MIP-1α) were acutely elevated following T3-SCI. Our data suggest that arterial hypotension diminishes mesenteric blood flow necessary to meet mucosal demands at rest and during digestion. The resulting GI ischemia and low-grade inflammation may be an underlying pathology leading to GI dysfunction seen following acute T3-SCI.


Subject(s)
Cytokines/immunology , Duodenitis/etiology , Duodenitis/physiopathology , Mesenteric Artery, Superior/physiopathology , Spinal Cord Injuries/complications , Spinal Cord Injuries/physiopathology , Animals , Intestine, Small , Male , Rats , Rats, Wistar
20.
Vet Pathol ; 54(5): 851-854, 2017 09.
Article in English | MEDLINE | ID: mdl-28578627

ABSTRACT

Intensely eosinophilic and glassy intracytoplasmic inclusions were present in the neurons of the peripheral autonomic ganglia, Meissner's and Auerbach's plexus, and spinal ganglia in 20 aged white-nosed coatis ( Nasua narica, 7-19 years old) and in 4 of 7 brown-nosed coatis ( Nasua nasua, 2-21 years old) from multiple zoological institutions. Inclusions were single to numerous, sometimes distorting the cell. Pheochromocytomas were present in 5 of 16 white-nosed and 2 of 6 brown-nosed coatis, although no inclusions were present in the adrenal glands. Histochemically, immunohistochemically, and ultrastructurally, these inclusions were consistent with dense neurosecretory granules. Although similar inclusions have been reported sporadically in the adrenal medulla of humans and several other mammalian species as both incidental and pathologic findings, ganglionic inclusions reported herein appear to be unique and related to age in these species.


Subject(s)
Inclusion Bodies/ultrastructure , Procyonidae/anatomy & histology , Aging/pathology , Animals , Cytoplasm/ultrastructure , Female , Ganglia/ultrastructure , Hyalin/ultrastructure , Male , Neurons/ultrastructure
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