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Find video protocols related to scientific articles indexed in Pubmed.
Immune Complexes Inhibit IL-1 Secretion and Inflammasome Activation.
J. Immunol.
PUBLISHED: 10-15-2014
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IgG immune complexes have been shown to modify immune responses driven by APCs in either a pro- or anti-inflammatory direction depending upon the context of stimulation. However, the ability of immune complexes to modulate the inflammasome-dependent innate immune response is unknown. In this study, we show that IgG immune complexes suppress IL-1? and IL-1? secretion through inhibition of inflammasome activation. The mechanism by which this inhibition occurs is via immune complex ligation of activating Fc?Rs, resulting in prevention of both activation and assembly of the inflammasome complex in response to nucleotide-binding domain leucine-rich repeat (NLR) P3, NLRC4, or AIM2 agonists. In vivo, administration of Ag in the form of an immune complex during priming of the immune response inhibited resultant adaptive immune responses in an NLRP3-dependent model of allergic airway disease. Our data reveal an unexpected mechanism regulating CD4(+) T cell differentiation, by which immune complexes suppress inflammasome activation and the generation of IL-1? and IL-1? from APCs, which are critical for the Ag-driven differentiation of CD4(+) T cells.
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Characterization of a novel mouse model with genetic deletion of CD177.
Protein Cell
PUBLISHED: 09-01-2014
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Neutrophils play an essential role in the innate immune response to infection. Neutrophils migrate from the vasculature into the tissue in response to infection. Recently, a neutrophil cell surface receptor, CD177, was shown to help mediate neutrophil migration across the endothelium through interactions with PECAM1. We examined a publicly available gene array dataset of CD177 expression from human neutrophils following pulmonary endotoxin instillation. Among all 22,214 genes examined, CD177 mRNA was the most upregulated following endotoxin exposure. The high level of CD177 expression is also maintained in airspace neutrophils, suggesting a potential involvement of CD177 in neutrophil infiltration under infectious diseases. To determine the role of CD177 in neutrophils in vivo, we constructed a CD177-genetic knockout mouse model. The mice with homozygous deletion of CD177 have no discernible phenotype and no significant change in immune cells, other than decreased neutrophil counts in peripheral blood. We examined the role of CD177 in neutrophil accumulation using a skin infection model with Staphylococcus aureus. CD177 deletion reduced neutrophil counts in inflammatory skin caused by S. aureus. Mechanistically we found that CD177 deletion in mouse neutrophils has no significant impact in CXCL1/KC- or fMLP-induced migration, but led to significant cell death. Herein we established a novel genetic mouse model to study the role of CD177 and found that CD177 plays an important role in neutrophils.
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A Novel Animal Model for Locally Advanced Breast Cancer.
Ann. Surg. Oncol.
PUBLISHED: 06-21-2014
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Locally advanced breast cancer (LABC) poses complex management issues due to failure of response to chemotherapy and progression to local complications such as skin erosion, superinfection, and lymphedema. Most cell line and animal models are not adequate to study LABC.
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Preclinical validation of interleukin 6 as a therapeutic target in multiple myeloma.
Immunol. Res.
PUBLISHED: 05-22-2014
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Studies on the biologic and molecular genetic underpinnings of multiple myeloma (MM) have identified the pleiotropic, pro-inflammatory cytokine, interleukin-6 (IL-6), as a factor crucial to the growth, proliferation and survival of myeloma cells. IL-6 is also a potent stimulator of osteoclastogenesis and a sculptor of the tumor microenvironment in the bone marrow of patients with myeloma. This knowledge has engendered considerable interest in targeting IL-6 for therapeutic purposes, using a variety of antibody- and small-molecule-based therapies. However, despite the early recognition of the importance of IL-6 for myeloma and the steady progress in our knowledge of IL-6 in normal and malignant development of plasma cells, additional efforts will be required to translate the promise of IL-6 as a target for new myeloma therapies into significant clinical benefits for patients with myeloma. This review summarizes published research on the role of IL-6 in myeloma development and describes ongoing efforts by the University of Iowa Myeloma Multidisciplinary Oncology Group to develop new approaches to the design and testing of IL-6-targeted therapies and preventions of MM.
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PCB 126 perturbs hypoxia-induced HIF-1? activity and glucose consumption in human HepG2 cells.
Exp. Toxicol. Pathol.
PUBLISHED: 04-21-2014
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Aerobic organisms strongly depend on the availability of oxygen for respiration and countless other metabolic processes to maintain cellular homeostasis. Under certain conditions, the amount of available oxygen can be limited. To support survival in environments with limited oxygen supply, hypoxia-inducible factors (HIFs) reprogram vital components of cellular metabolism. HIF-1? is an important mediator of acute and adaptive responses to hypoxic stress. Interestingly, the heterodimeric partner required by HIF-1? to function as transcription factor, known as ARNT, is also an essential part of the aryl hydrocarbon receptor (AhR) transcription factor complex. Thus, via ARNT a crosstalk exists between these two pathways that might affect HIF-1?-mediated processes. In this study we sought to assess the effect of the AhR agonist PCB 126 on HIF-1? activity as well as on HIF-1?-regulated targets involved in cellular metabolism in human HepG2 cells. Our results show that PCB 126 reduced HIF-1? localization to the nucleus. Furthermore, in an in vivo setting, rats exposed to parenteral PCB 126 also displayed reduced hepatocyte nuclear localization of HIF-1?. Additionally, HepG2 cells exposed to PCB 126 displayed reduced hypoxia-regulated HRE-luciferase reporter gene expression as well as a reduction in glucose consumption in conditions of hypoxia. In summary, this study reveals that HIF-1?-regulated cellular metabolic processes are negatively affected by PCB 126 which might ultimately affect adaptive responses and cell survival in hypoxic environments.
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The tick salivary protein sialostatin L2 inhibits caspase-1-mediated inflammation during Anaplasma phagocytophilum infection.
Infect. Immun.
PUBLISHED: 03-31-2014
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Saliva from arthropod vectors facilitates blood feeding by altering host inflammation. Whether arthropod saliva counters inflammasome signaling, a protein scaffold that regulates the activity of caspase-1 and cleavage of interleukin-1? (IL-1?) and IL-18 into mature molecules, remains elusive. In this study, we provide evidence that a tick salivary protein, sialostatin L2, inhibits inflammasome formation during pathogen infection. We show that sialostatin L2 targets caspase-1 activity during host stimulation with the rickettsial agent Anaplasma phagocytophilum. A. phagocytophilum causes macrophage activation and hemophagocytic syndrome features. The effect of sialostatin L2 in macrophages was not due to direct caspase-1 enzymatic inhibition, and it did not rely on nuclear factor ?B or cathepsin L signaling. Reactive oxygen species from NADPH oxidase and the Loop2 domain of sialostatin L2 were important for the regulatory process. Altogether, our data expand the knowledge of immunoregulatory pathways of tick salivary proteins and unveil an important finding in inflammasome biology.
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A new model of LMP1-MYC interaction in B cell lymphoma.
Leuk. Lymphoma
PUBLISHED: 03-11-2014
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Epstein-Barr virus (EBV) is associated with aggressive B cell lymphomas (BCLs). Latent membrane protein 1 (LMP1) of EBV is an oncogenic protein required for EBV B cell transformation. However, LMP1 is a weak oncogene in mice. Mice expressing Myc inserted 5' of the E? enhancer (iMyc(E?)), mimicking the t(8;14) translocation of endemic Burkitt lymphoma, develop delayed onset BCLs. To investigate potential cooperation between LMP1 and oncogenic MYC, we produced mice expressing the LMP1 signaling domain via a hybrid CD40-LMP1 transgene (mCD40-LMP1), and the dysregulated MYC protein of aggressive EBV+ BCLs. mCD40-LMP1/iMyc(E?) mice trended toward earlier BCL onset. BCLs from mCD40-LMP1/iMyc(E?) mice expressed LMP1 and were transplantable into immunocompetent recipients. iMyc(E?) and mCD40-LMP1/iMyc(E?) mice developed BCLs with similar immunophenotypes. LMP1 signaling was intact in BCLs as shown by inducible interleukin-6. Additionally, LMP1 signaling to tumor cells induced the two isoforms of Pim1, a constitutively active prosurvival kinase implicated in lymphomagenesis.
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Gastrointestinal pathology in juvenile and adult CFTR-knockout ferrets.
Am. J. Pathol.
PUBLISHED: 01-27-2014
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Cystic fibrosis (CF) is a multiorgan disease caused by loss of a functional cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel in many epithelia of the body. Here we report the pathology observed in the gastrointestinal organs of juvenile to adult CFTR-knockout ferrets. CF gastrointestinal manifestations included gastric ulceration, intestinal bacterial overgrowth with villous atrophy, and rectal prolapse. Metagenomic phylogenetic analysis of fecal microbiota by deep sequencing revealed considerable genotype-independent microbial diversity between animals, with the majority of taxa overlapping between CF and non-CF pairs. CF hepatic manifestations were variable, but included steatosis, necrosis, biliary hyperplasia, and biliary fibrosis. Gallbladder cystic mucosal hyperplasia was commonly found in 67% of CF animals. The majority of CF animals (85%) had pancreatic abnormalities, including extensive fibrosis, loss of exocrine pancreas, and islet disorganization. Interestingly, 2 of 13 CF animals retained predominantly normal pancreatic histology (84% to 94%) at time of death. Fecal elastase-1 levels from these CF animals were similar to non-CF controls, whereas all other CF animals evaluated were pancreatic insufficient (<2 ?g elastase-1 per gram of feces). These findings suggest that genetic factors likely influence the extent of exocrine pancreas disease in CF ferrets and have implications for the etiology of pancreatic sufficiency in CF patients. In summary, these studies demonstrate that the CF ferret model develops gastrointestinal pathology similar to CF patients.
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Inflammasome-independent IL-1? mediates autoinflammatory disease in Pstpip2-deficient mice.
Proc. Natl. Acad. Sci. U.S.A.
PUBLISHED: 01-06-2014
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Chronic recurrent multifocal osteomyelitis (CRMO) is a human autoinflammatory disorder that primarily affects bone. Missense mutation (L98P) of proline-serine-threonine phosphatase-interacting protein 2 (Pstpip2) in mice leads to a disease that is phenotypically similar to CRMO called chronic multifocal osteomyelitis (cmo). Here we show that deficiency of IL-1RI in cmo mice resulted in a significant reduction in the time to onset of disease as well as the degree of bone pathology. Additionally, the proinflammatory cytokine IL-1?, but not IL-1?, played a critical role in the pathology observed in cmo mice. In contrast, disease in cmo mice was found to be independent of the nucleotide-binding domain, leucine-rich repeat-containing family, pyrin domain-containing 3 (NLRP3) inflammasome as well as caspase-1. Neutrophils, but not bone marrow-derived macrophages, from cmo mice secreted increased IL-1? in response to ATP, silica, and Pseudomonas aeruginosa compared with neutrophils from WT mice. This aberrant neutrophil response was sensitive to inhibition by serine protease inhibitors. These results demonstrate an inflammasome-independent role for IL-1? in disease progression of cmo and implicate neutrophils and neutrophil serine proteases in disease pathogenesis. These data provide a rationale for directly targeting IL-1RI or IL-1? as a therapeutic strategy in CRMO.
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Functional validation of a human CAPN5 exome variant by lentiviral transduction into mouse retina.
Hum. Mol. Genet.
PUBLISHED: 12-30-2013
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Exome sequencing indicated that the gene encoding the calpain-5 protease, CAPN5, is the likely cause of retinal degeneration and autoimmune uveitis in human patients with autosomal dominant neovascular inflammatory vitreoretinopathy (ADNIV, OMIM #193235). To explore the mechanism of ADNIV, a human CAPN5 disease allele was expressed in mouse retinas with a lentiviral vector created to express either the wild-type human (h) CAPN5 or the ADNIV mutant hCAPN5-R243 L allele under a rhodopsin promoter with tandem green fluorescent protein (GFP) expression. Vectors were injected into the subretinal space of perinatal mice. Mouse phenotypes were analyzed using electroretinography, histology, and inflammatory gene expression profiling. Mouse calpain-5 showed high homology to its human ortholog with over 98% sequence identity that includes the ADNIV mutant residue. Calpain-5 protein was expressed in the inner and outer segments of the photoreceptors and in the outer plexiform layer. Expression of the hCAPN5-R243 L allele caused loss of the electroretinogram b-wave, photoreceptor degeneration, and induction of immune cell infiltration and inflammatory genes in the retina, recapitulating major features of the ADNIV phenotype. Intraocular neovascularization and fibrosis was not observed during the study period. Our study shows that expression of the hCAPN5-R243 L disease allele elicits an ADNIV-like disease in mice. It further suggests that ADNIV is due to CAPN5 gain-of-function rather than haploinsufficiency, and retinal expression may be sufficient to generate an autoimmune response. Genetic models of ADNIV in the mouse can be used to explore protease mechanisms in retinal degeneration and inflammation as well as therapeutic testing in pre-clinical models.
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Synthetic embryonic lethality upon deletion of the ER cochaperone p58(IPK) and the ER stress sensor ATF6?.
Biochem. Biophys. Res. Commun.
PUBLISHED: 11-09-2013
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The unfolded protein response (UPR) is activated as a consequence of alterations to ER homeostasis. It upregulates a group of ER chaperones and cochaperones, as well as other genes that improve protein processing within the secretory pathway. The UPR effector ATF6? augments-but is not essential for-maximal induction of ER chaperones during stress, yet its role, if any, in protecting cellular function during normal development and physiology is unknown. A systematic analysis of multiple tissues from Atf6?-/- mice revealed that all tissues examined were grossly insensitive to loss of ATF6?. However, combined deletion of ATF6? and the ER cochaperone p58(IPK) resulted in synthetic embryonic lethality. These findings reveal for the first time that an intact UPR can compensate for the genetic impairment of protein folding in the ER in vivo. The also expose a role for p58(IPK) in normal embryonic development.
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Superoxide Mediates Acute Liver Injury in Irradiated Mice Lacking Sirtuin 3.
Antioxid. Redox Signal.
PUBLISHED: 09-28-2013
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Abstract Aims: This study determined whether acute radiation-induced liver injury seen in Sirtuin3(-/-) mice after exposure to Cs-137 ?-rays was mediated by superoxide anion (O2(•-)). Results: Male wild-type (WT) and SIRT3(-/-) mice were given 2×2?Gy whole-body radiation doses separated by 24?h and livers were harvested 20?h after the second dose. Ex vivo measurements in fresh frozen liver sections demonstrated 50% increases in dihydroethidium oxidation from SIRT3(-/-) animals, relative to WT animals, before irradiation, but this increase was not detected 20?h after radiation exposure. In addition, irradiated livers from SIRT3(-/-) animals showed significant hydropic degeneration, loss of MitoTracker Green FM staining, increased immunohistochemical staining for 3-nitrotyrosine, loss of Ki67 staining, and increased mitochondrial localization of p53. These parameters of radiation-induced injury were significantly attenuated by an intraperitoneal injection of 2?mg/kg of the highly specific superoxide dismutase mimic, GC4401, 30?min before each fraction. Innovation: Sirtuin 3 (SIRT3) is believed to regulate mitochondrial oxidative metabolism and antioxidant defenses in response to acute radiation-induced liver injury. This work provides strong evidence for the causal role of O2(•-) in the liver injury process initiated by whole-body irradiation in SIRT3(-/-) mice. Conclusion: These results support the hypothesis that O2(•-) mediates acute liver injury in SIRT3(-/-) animals exposed to whole-body ?-radiation and suggest that GC4401 could be used as a radio-protective compound in vivo. Antioxid. Redox Signal. 00, 000-000.
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CaMKII is essential for the proasthmatic effects of oxidation.
Sci Transl Med
PUBLISHED: 07-26-2013
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Increased reactive oxygen species (ROS) contribute to asthma, but little is known about the molecular mechanisms connecting increased ROS with characteristic features of asthma. We show that enhanced oxidative activation of the Ca(2+)/calmodulin-dependent protein kinase (ox-CaMKII) in bronchial epithelium positively correlates with asthma severity and that epithelial ox-CaMKII increases in response to inhaled allergens in patients. We used mouse models of allergic airway disease induced by ovalbumin (OVA) or Aspergillus fumigatus (Asp) and found that bronchial epithelial ox-CaMKII was required to increase a ROS- and picrotoxin-sensitive Cl(-) current (ICl) and MUC5AC expression, upstream events in asthma progression. Allergen challenge increased epithelial ROS by activating NADPH oxidases. Mice lacking functional NADPH oxidases due to knockout of p47 and mice with epithelial-targeted transgenic expression of a CaMKII inhibitory peptide or wild-type mice treated with inhaled KN-93, an experimental small-molecule CaMKII antagonist, were protected against increases in ICl, MUC5AC expression, and airway hyperreactivity to inhaled methacholine. Our findings support the view that CaMKII is a ROS-responsive, pluripotent proasthmatic signal and provide proof-of-concept evidence that CaMKII is a therapeutic target in asthma.
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Mitochondrial cardiolipin is required for Nlrp3 inflammasome activation.
Immunity
PUBLISHED: 05-03-2013
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Nlrp3 inflammasome activation occurs in response to numerous agonists but the specific mechanism by which this takes place remains unclear. All previously evaluated activators of the Nlrp3 inflammasome induce the generation of mitochondrial reactive oxygen species (ROS), suggesting a model in which ROS is a required upstream mediator of Nlrp3 inflammasome activation. Here we have identified the oxazolidinone antibiotic linezolid as a Nlrp3 agonist that activates the Nlrp3 inflammasome independently of ROS. The pathways for ROS-dependent and ROS-independent Nlrp3 activation converged upon mitochondrial dysfunction and specifically the mitochondrial lipid cardiolipin. Cardiolipin bound to Nlrp3 directly and interference with cardiolipin synthesis specifically inhibited Nlrp3 inflammasome activation. Together these data suggest that mitochondria play a critical role in the activation of the Nlrp3 inflammasome through the direct binding of Nlrp3 to cardiolipin.
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Does dietary copper supplementation enhance or diminish PCB126 toxicity in the rodent liver?
Chem. Res. Toxicol.
PUBLISHED: 04-15-2013
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Copper is essential for the function of the mitochondrial electron transport chain and several antioxidant proteins. However, in its free form copper can participate in Fenton-like reactions that produce reactive hydroxyl radicals. Aryl-hydrocarbon receptor (AhR) agonists, including the most potent polychlorinated biphenyl (PCB) congener, 3,3,4,4,5-pentachlorobiphenyl (PCB126), increase copper levels in rodent livers. This is accompanied by biochemical and toxic changes. To assess the involvement of copper in PCB toxicity, male Sprague-Dawley rats were fed an AIN-93G diet with differing dietary copper levels: low (2 ppm), adequate (6 ppm), and high (10 ppm). After three weeks, rats from each group were given a single ip injection of corn oil (control), 1, or 5 ?mol/kg body weight PCB126. Two weeks following injections, biochemical and morphological markers of hepatic toxicity, trace metal status, and hepatic gene expression of metalloproteins were evaluated. Increasing dietary copper was associated with elevated tissue levels of copper and ceruloplasmin. In the livers of PCB126-treated rats, the hallmark signs of AhR activation were present, including increased cytochrome P450 and lipid levels and decreased glutathione. In addition, a doubling of hepatic copper levels was seen, and overall metal homeostasis was disturbed, resulting in decreased hepatic selenium, manganese, zinc, and iron. Expression of key metalloproteins was either decreased (cytochrome c oxidase), unchanged (ceruloplasmin and CuZnSOD), or increased (tyrosinase and metallothioneins 1 and 2) with exposure to PCB126. Increases in metallothionein may contribute/reflect the increased copper seen. Alterations in dietary copper did not amplify or abrogate the hepatic toxicity of PCB126. PCB126 toxicity, i.e., oxidative stress and steatosis, is clearly associated with disturbed metal homeostasis. Understanding the mechanisms of this disturbance may provide tools to prevent liver toxicity by other AhR agonists.
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Adaptive immunity does not strongly suppress spontaneous tumors in a Sleeping Beauty model of cancer.
J. Immunol.
PUBLISHED: 03-08-2013
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The tumor immunosurveillance hypothesis describes a process by which the immune system recognizes and suppresses the growth of transformed cancer cells. A variety of epidemiological and experimental evidence supports this hypothesis. Nevertheless, there are a number of conflicting reports regarding the degree of immune protection conferred, the immune cell types responsible for protection, and the potential contributions of immunosuppressive therapies to tumor induction. The purpose of this study was to determine whether the adaptive immune system actively suppresses tumorigenesis in a Sleeping Beauty (SB) mouse model of cancer. SB transposon mutagenesis was performed in either a wild-type or immunocompromised (Rag2-null) background. Tumor latency and multiplicity were remarkably similar in both immune cohorts, suggesting that the adaptive immune system is not efficiently suppressing tumor formation in our model. Exceptions included skin tumors, which displayed increased multiplicity in wild-type animals, and leukemias, which developed with shorter latency in immune-deficient mice. Overall tumor distribution was also altered such that tumors affecting the gastrointestinal tract were more frequent and hemangiosarcomas were less frequent in immune-deficient mice compared with wild-type mice. Finally, genetic profiling of transposon-induced mutations identified significant differences in mutation prevalence for a number of genes, including Uba1. Taken together, these results indicate that B and T cells function to shape the genetic profile of tumors in various tumor types, despite being ineffective at clearing SB-induced tumors. To our knowledge, this study represents the first forward genetic screen designed to examine tumor immunosurveillance mechanisms.
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Identification of candidate B-lymphoma genes by cross-species gene expression profiling.
PLoS ONE
PUBLISHED: 01-01-2013
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Comparative genome-wide expression profiling of malignant tumor counterparts across the human-mouse species barrier has a successful track record as a gene discovery tool in liver, breast, lung, prostate and other cancers, but has been largely neglected in studies on neoplasms of mature B-lymphocytes such as diffuse large B cell lymphoma (DLBCL) and Burkitt lymphoma (BL). We used global gene expression profiles of DLBCL-like tumors that arose spontaneously in Myc-transgenic C57BL/6 mice as a phylogenetically conserved filter for analyzing the human DLBCL transcriptome. The human and mouse lymphomas were found to have 60 concordantly deregulated genes in common, including 8 genes that Cox hazard regression analysis associated with overall survival in a published landmark dataset of DLBCL. Genetic network analysis of the 60 genes followed by biological validation studies indicate FOXM1 as a candidate DLBCL and BL gene, supporting a number of studies contending that FOXM1 is a therapeutic target in mature B cell tumors. Our findings demonstrate the value of the "mouse filter" for genomic studies of human B-lineage neoplasms for which a vast knowledge base already exists.
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Characterization of regulatory dendritic cells that mitigate acute graft-versus-host disease in older mice following allogeneic bone marrow transplantation.
PLoS ONE
PUBLISHED: 01-01-2013
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Despite improvements in human leukocyte antigen matching and pharmacologic prophylaxis, acute graft-versus-host disease (GVHD) is often a fatal complication following hematopoietic stem cell transplant (HSCT). Older HSCT recipients experience significantly increased morbidity and mortality compared to young recipients. Prophylaxis with syngeneic regulatory dendritic cells (DCreg) in young bone marrow transplanted (BMT) mice has been shown to decrease GVHD-associated mortality. To evaluate this approach in older BMT recipients, young (3-4 months) and older (14-18 months) DCreg were generated using GM-CSF, IL-10, and TGF?. Analysis of young versus older DCreg following culture revealed no differences in phenotype. The efficacy of DCreg treatment in older BMT mice was evaluated in a BALB/c?C57Bl/6 model of GVHD; on day 2 post-BMT (d +2), mice received syngeneic, age-matched DCreg. Although older DCreg-treated BMT mice showed decreased morbidity and mortality compared to untreated BMT mice (all of which died), there was a small but significant decrease in the survival of older DCreg-treated BMT mice (75% survival) compared to young DCreg-treated BMT mice (90% survival). To investigate differences between dendritic cells (DC) in young and older DCreg-treated BMT mice that may play a role in DCreg function in vivo, DC phenotypes were assessed following DCreg adoptive transfer. Transferred DCreg identified in older DCreg-treated BMT mice at d +3 showed significantly lower expression of PD-L1 and PIR B compared to DCreg from young DCreg-treated BMT mice. In addition, donor DC identified in d +21 DCreg-treated BMT mice displayed increased inhibitory molecule and decreased co-stimulatory molecule expression compared to d +3, suggesting induction of a regulatory phenotype on the donor DC. In conclusion, these data indicate DCreg treatment is effective in the modulation of GVHD in older BMT recipients and provide evidence for inhibitory pathways that DCreg and donor DC may utilize to induce and maintain tolerance to GVHD.
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Exogenous administration of vascular endothelial growth factor prior to human respiratory syncytial virus a2 infection reduces pulmonary pathology in neonatal lambs and alters epithelial innate immune responses.
Exp. Lung Res.
PUBLISHED: 02-11-2011
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Human respiratory syncytial virus (RSV) affects thousands of children every year. Vascular endothelial growth factor (VEGF) is a regulator of vasculogenesis, pulmonary maturation, and immunity. In order to test the extent to which VEGF may alter RSV infection, 4 groups of lambs received either human recombinant VEGF (rhVEGF) or phosphate-buffered saline (PBS) pretreatment followed by inoculation with human RSV strain A2 or sterile medium. Lambs in each group were sacrificed at 2, 4, and 6 days post infection. Expression of surfactant protein-A (SP-A), surfactant protein-D (SP-D), sheep ?-defensin-1 (SBD-1), tumor necrosis factor ? (TNF?), interleukin (IL)-6, IL-8, interferon ?, and endogenous VEGF were measured to determine effect of rhVEGF pretreatment. RSV lambs pretreated with rhVEGF had reduced viral mRNA and decreased pulmonary pathology at day 6. Pretreatment with rhVEGF increased mRNA expression of SP-A, SBD-1, and TNF?, with alteration of expression in RSV lambs. Endogenous VEGF mRNA levels were increased at day 2 regardless of pretreatment. Pretreatment with rhVEGF increased pulmonary cellular proliferation in RSV lambs at day 4 post infection. Overall, these results suggest that pretreatment with rhVEGF protein may have therapeutic potential to decrease RSV viral load, decrease pulmonary lesion severity, and alter both epithelial innate immune responses and epithelial cell proliferation.
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Respiratory syncytial virus is associated with an inflammatory response in lungs and architectural remodeling of lung-draining lymph nodes of newborn lambs.
Am. J. Physiol. Lung Cell Mol. Physiol.
PUBLISHED: 10-08-2010
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Respiratory syncytial virus (RSV) is the leading cause of lower respiratory tract infection in children worldwide. The understanding of neonatal RSV pathogenesis depends on using an animal model that reproduces neonatal RSV disease. Previous studies from us and others demonstrated that the neonatal lamb model resembles human neonatal RSV infection. Here, we provide an extensive and detailed characterization of the histopathology, viral load, cellular infiltration, and cytokine production in lungs and tracheobronchial lymph nodes of lambs inoculated with human RSV strain A2 over the course of infection. In the lung, RSV titers were low at day 3 postinfection, increased significantly by day 6, and decreased to baseline levels at day 14. Infection in the lung was associated with an accumulation of macrophages, CD4(+) and CD8(+) T cells, and a transcriptional response of genes involved in inflammation, chemotaxis, and interferon response, characterized by increased IFN?, IL-8, MCP-1, and PD-L1, and decreased IFN?, IL-10, and TGF-?. Laser capture microdissection studies determined that lung macrophage-enriched populations were the source of MCP-1 but not IL-8. Immunoreactivity to caspase 3 occurred within bronchioles and alveoli of day 6-infected lambs. In lung-draining lymph nodes, RSV induced lymphoid hyperplasia, suggesting an ability of RSV to enhance lymphocytic proliferation and differentiation pathways. This study suggests that, in lambs with moderate clinical disease, RSV enhances the activation of caspase cell death and Th1-skewed inflammatory pathways, and complements previous observations that emphasize the role of inflammation in the pathogenesis of RSV disease.
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Sirt3-mediated deacetylation of evolutionarily conserved lysine 122 regulates MnSOD activity in response to stress.
Mol. Cell
PUBLISHED: 07-13-2010
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Genetic deletion of the mitochondrial deacetylase sirtuin-3 (Sirt3) results in increased mitochondrial superoxide, a tumor-permissive environment, and mammary tumor development. MnSOD contains a nutrient- and ionizing radiation (IR)-dependent reversible acetyl-lysine that is hyperacetylated in Sirt3?/? livers at 3 months of age. Livers of Sirt3?/? mice exhibit decreased MnSOD activity, but not immunoreactive protein, relative to wild-type livers. Reintroduction of wild-type but not deacetylation null Sirt3 into Sirt3?/? MEFs deacetylated lysine and restored MnSOD activity. Site-directed mutagenesis of MnSOD lysine 122 to an arginine, mimicking deacetylation (lenti-MnSOD(K122-R)), increased MnSOD activity when expressed in MnSOD?/? MEFs, suggesting acetylation directly regulates function. Furthermore, infection of Sirt3?/? MEFs with lenti-MnSOD(K122-R) inhibited in vitro immortalization by an oncogene (Ras), inhibited IR-induced genomic instability, and decreased mitochondrial superoxide. Finally, IR was unable to induce MnSOD deacetylation or activity in Sirt3?/? livers, and these irradiated livers displayed significant IR-induced cell damage and microvacuolization in their hepatocytes.
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Idiopathic eosinophilic meningoencephalomyelitis in a Rottweiler dog.
J. Vet. Diagn. Invest.
PUBLISHED: 07-13-2010
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A 6-month-old, female, intact Rottweiler dog was presented to the Iowa State University Veterinary Teaching Hospital for a progressive history of abnormal behavior and generalized ataxia. At necropsy, there was eosinophilic infiltration of the brain and spinal cord, most severe in the medulla oblongata, cerebellum, and cervical spinal cord. Infiltrates of eosinophils were also present in the liver and small intestines. The dog was diagnosed with idiopathic eosinophilic meningoencephalomyelitis based on cerebrospinal fluid analysis, histopathology, and special stains to exclude etiologic agents.
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Differential expression of cytokine transcripts in neonatal and adult ovine alveolar macrophages in response to respiratory syncytial virus or toll-like receptor ligation.
Vet. Immunol. Immunopathol.
PUBLISHED: 01-29-2010
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Alveolar macrophages (AMvarphis) secrete regulatory molecules that are believed to be critical in maintaining normal lung homeostasis. However, in response to activating signals, AMvarphis have been shown to become highly phagocytic cells capable of secreting significant levels of pro-inflammatory cytokines. There is evidence to suggest that susceptibility of Mvarphi subpopulations to viral infection, and their subsequent cytokine/chemokine response, is dependent on age of the host. In the present study, we compared bovine respiratory syncytial virus (BRSV) replication and induction of cytokine responses in neonatal ovine AMvarphis to those cells isolated from adult animals. While neonatal AMvarphis could be infected with BRSV, viral replication was limited as previously shown for AMvarphis from mature animals. Interestingly, following BRSV infection, peak mRNA levels of IL-1beta and IL-8 in neonatal AMvarphi were several fold higher than levels induced in adult AMvarphis. In addition, peak mRNA expression for the cytokines examined occurred at earlier time points in neonatal AMvarphis compared to adult AMvarphis. However, the data indicated that viral replication was not required for the induction of specific cytokines in either neonatal or adult AMvarphis. TLR3 and TLR4 agonists induced significantly higher levels of cytokine transcripts than BRSV in both neonatal and adult AMvarphis. It was recently proposed that immaturity of the neonatal immune system extends from production of pro-inflammatory cytokines to regulation of such responses. Differential regulation of cytokines in neonatal AMvarphis compared to adult AMvarphis in response to RSV could be a contributory factor to more severe clinical episodes seen in neonates.
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Human respiratory syncytial virus A2 strain replicates and induces innate immune responses by respiratory epithelia of neonatal lambs.
Int J Exp Pathol
PUBLISHED: 08-08-2009
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Human respiratory syncytial virus (hRSV) is a pneumovirus that causes significant respiratory disease in premature and full-term infants. It was our hypothesis that a common strain of RSV, strain A2, would infect, cause pulmonary pathology, and alter respiratory epithelial innate immune responses in neonatal lambs similarly to RSV infection in human neonates. Newborn lambs between 2 and 3 days of age were inoculated intrabronchially with RSV strain A2. The lambs were sacrificed at days 3, 6, and 14 days postinoculation. Pulmonary lesions in the 6-day postinoculation group were typical of RSV infection including bronchiolitis with neutrophils and mild peribronchiolar interstitial pneumonia. RSV mRNA and antigen were detected by qPCR and immunohistochemistry, respectively with peak mRNA levels and antigen at day 6. Expression of surfactant proteins A and D, sheep beta-defensin-1 and thyroid transcription factor-1 mRNA were also assessed by real-time qPCR. There was a significant increase in surfactant A and D mRNA expression in RSV-infected animals at day 6 postinoculation. There were no significant changes in sheep beta-defensin-1 and thyroid transcription factor-1 mRNA expression. This study shows that neonatal lambs can be infected with RSV strain A2 and the pulmonary pathology mimics that of RSV infection in human infants thereby making the neonatal lamb a useful animal model to study disease pathogenesis and therapeutics. RSV infection induces increased expression of surfactant proteins A and D in lambs, which may also be an important feature of infection in newborn infants.
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Cutting edge: Nlrp10 is essential for protective antifungal adaptive immunity against Candida albicans.
J. Immunol.
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Nucleotide-binding domain leucine-rich repeat containing receptors (NLRs) are cytosolic receptors that initiate immune responses to sterile and infectious insults to the host. Studies demonstrated that Nlrp3 is critical for the control of Candida albicans infections and in the generation of antifungal Th17 responses. In this article, we show that the NLR family member Nlrp10 also plays a unique role in the control of disseminated C. albicans infection in vivo. Nlrp10-deficient mice had increased susceptibility to disseminated candidiasis, as indicated by decreased survival and increased fungal burdens. In contrast to Nlrp3, Nlrp10 deficiency did not affect innate proinflammatory cytokine production from macrophages and dendritic cells challenged with C. albicans. However, Nlrp10-deficient mice displayed a profound defect in Candida-specific Th1 and Th17 responses. These results demonstrate a novel role for Nlrp10 in the generation of adaptive immune responses to fungal infection.
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Abnormal endocrine pancreas function at birth in cystic fibrosis ferrets.
J. Clin. Invest.
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Diabetes is a common comorbidity in cystic fibrosis (CF) that worsens prognosis. The lack of an animal model for CF-related diabetes (CFRD) has made it difficult to dissect how the onset of pancreatic pathology influences the emergence of CFRD. We evaluated the structure and function of the neonatal CF endocrine pancreas using a new CFTR-knockout ferret model. Although CF kits are born with only mild exocrine pancreas disease, progressive exocrine and endocrine pancreatic loss during the first months of life was associated with pancreatic inflammation, spontaneous hyperglycemia, and glucose intolerance. Interestingly, prior to major exocrine pancreas disease, CF kits demonstrated significant abnormalities in blood glucose and insulin regulation, including diminished first-phase and accentuated peak insulin secretion in response to glucose, elevated peak glucose levels following glucose challenge, and variably elevated insulin and C-peptide levels in the nonfasted state. Although there was no difference in lobular insulin and glucagon expression between genotypes at birth, significant alterations in the frequencies of small and large islets were observed. Newborn cultured CF islets demonstrated dysregulated glucose-dependent insulin secretion in comparison to controls, suggesting intrinsic abnormalities in CF islets. These findings demonstrate that early abnormalities exist in the regulation of insulin secretion by the CF endocrine pancreas.
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Genetically modified species in research: Opportunities and challenges for the histology core laboratory.
J Histotechnol
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Translational research using animal models has traditionally involved genetically modified rodents; however there is increasing use of other novel genetically engineered species. As histology laboratories interface with researchers studying these novel species there will be many situations in which protocols will need to be adapted to the species, model and research goals. This paper gives examples of protocol adaptations to meet research needs and addresses common considerations that should be addressed for all research tissues submitted to the histotechnology laboratory. Positioning the histotechnologist, as well as the investigator, to meet the challenges associated with novel research models will help maximize research efficacy and quality.
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Successful Integration of the Histology Core Laboratory in Translational Research.
J Histotechnol
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In a diagnostic setting, the histology laboratory is a key resource for production of quality tissues so the pathologist can make an appropriate diagnosis. In a research setting, the histology laboratory is a valuable resource in providing an excellent quality product for publications and grants for the investigator. Optimal collaboration with research investigators requires that histotechnologists recognize the diverse challenges and opportunities in research. This paper emphasizes the importance of positive interaction with researchers, optimizing professional service for these clients and recognizing key services of histology laboratories in a research setting to maximize success.
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N-acetylcysteine (NAC) diminishes the severity of PCB 126-induced fatty liver in male rodents.
Toxicology
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Potent aryl hydrocarbon receptor agonists like PCB 126 (3,3,4,4,5-pentachlorobiphenyl) cause oxidative stress and liver pathology, including fatty liver. Our question was whether dietary supplementation with N-acetylcysteine (NAC), an antioxidant, can prevent these adverse changes. Male Sprague-Dawley rats were fed a standard AIN-93G diet (sufficient in cysteine) or a modified diet supplemented with 1.0% NAC. After one week, rats on each diet were exposed to 0, 1, or 5?mol/kg body weight PCB 126 by i.p. injection (6 rats per group) and euthanized two weeks later. PCB-treatment caused a dose-dependent reduction in growth, feed consumption, relative thymus weight, total glutathione and glutathione disulfide (GSSG), while relative liver weight, glutathione transferase activity and hepatic lipid content were dose-dependently increased with PCB dose. Histologic examination of liver tissue showed PCB 126-induced hepatocellular steatosis with dose dependent increase in lipid deposition and distribution. Dietary NAC resulted in a reduction in hepatocellular lipid in both PCB groups. This effect was confirmed by gravimetric analysis of extracted lipids. Expression of CD36, a scavenger receptor involved in regulating hepatic fatty acid uptake, was reduced with high dose PCB treatment but unaltered in PCB-treated rats on NAC-supplemented diet. These results demonstrate that NAC has a protective effect against hepatic lipid accumulation in rats exposed to PCB 126. The mechanism of this protective effect appears to be independent of NAC as a source of cysteine/precursor of glutathione.
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Lung Phenotype of Juvenile and Adult CFTR-Knockout Ferrets.
Am. J. Respir. Cell Mol. Biol.
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Chronic bacterial lung infections in cystic fibrosis (CF) are caused by defects in the CFTR chloride channel. Previously, we described that newborn CFTR-knockout ferrets rapidly develop lung infections within the first week of life. Here we report a more slowly progressing lung bacterial colonization phenotype observed in juvenile to adult CF ferrets reared on a layered antibiotic regimen. Even on antibiotics, CF ferrets were still very susceptible to bacterial lung infection. The severity of lung histopathology ranged from mild to severe and variably included mucus obstruction of the airways and submucosal glands, air trapping, atelectasis, bronchopneumonia, and interstitial pneumonia. In all CF lungs, significant numbers of bacteria were detected and impaired tracheal mucociliary clearance was observed. Although Streptococcus, Staphylococcus, and Enterococcus were observed most frequently in the lungs of CF animals, each animal displayed a predominant bacterial species that accounted for >50% of the culturable bacteria with no one bacterial taxon predominating in all animals. MALDI-TOF MS fingerprinting was used to quantify lung bacteria in ten CF animals and demonstrated Streptococcus, Staphylococcus, Enterococcus, or Escherichia as the most abundant genera. Interestingly, there was significant overlap in the types of bacteria observed in the lung and intestine of a given CF animal, including bacterial taxa unique to the lung and gut of each CF animal analyzed. These findings demonstrate that CF ferrets develop lung disease during the juvenile and adult stages that is similar to CF patients and suggest that enteric bacterial flora may seed the CF ferret lung.
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JoVE Visualize is a tool created to match the last 5 years of PubMed publications to methods in JoVE's video library.

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In developing our video relationships, we compare around 5 million PubMed articles to our library of over 4,500 methods videos. In some cases the language used in the PubMed abstracts makes matching that content to a JoVE video difficult. In other cases, there happens not to be any content in our video library that is relevant to the topic of a given abstract. In these cases, our algorithms are trying their best to display videos with relevant content, which can sometimes result in matched videos with only a slight relation.