Mcm2 deficiency results in short deletions allowing high resolution identification of genes contributing to lymphoblastic lymphoma

PubMed Central

Rusiniak, Michael E.; Kunnev, Dimiter; Freeland, Amy; Cady, Gillian K.; Pruitt, Steven C.

2011-01-01

Mini-chromosome maintenance (Mcm) proteins are part of the replication licensing complex that is loaded onto chromatin during the G1-phase of the cell cycle and required for initiation of DNA replication in the subsequent S-phase. Mcm proteins are typically loaded in excess of the number of locations that are utilized during S-phase. Nonetheless, partial depletion of Mcm proteins leads to cancers and stem cell deficiencies. Mcm2 deficient mice, on a 129Sv genetic background, display a high rate of thymic lymphoblastic lymphoma. Here array comparative genomic hybridization (aCGH) is utilized to characterize the genetic damage accruing in these tumors. The predominant events are deletions averaging less than 0.5 Mb, considerably shorter than observed in prior studies using alternative mouse lymphoma models or human tumors. Such deletions facilitate identification of specific genes and pathways responsible for the tumors. Mutations in many genes that have been implicated in human lymphomas are recapitulated in this mouse model. These features, and the fact that the mutation underlying the accelerated genetic damage does not target a specific gene or pathway a priori, are valuable features of this mouse model for identification of tumor suppressor genes. Genes affected in all tumors include Pten, Tcfe2a, Mbd3 and Setd1b. Notch1 and additional genes are affected in subsets of tumors. The high frequency of relatively short deletions is consistent with elevated recombination between nearby stalled replication forks in Mcm2 deficient mice. PMID:22158038

Ultrastructural study of Rift Valley fever virus in the mouse model

DOE Office of Scientific and Technical Information (OSTI.GOV)

Reed, Christopher; Steele, Keith E.; Honko, Anna

Detailed ultrastructural studies of Rift Valley fever virus (RVFV) in the mouse model are needed to develop and characterize a small animal model of RVF for the evaluation of potential vaccines and therapeutics. In this study, the ultrastructural features of RVFV infection in the mouse model were analyzed. The main changes in the liver included the presence of viral particles in hepatocytes and hepatic stem cells accompanied by hepatocyte apoptosis. However, viral particles were observed rarely in the liver; in contrast, particles were extremely abundant in the CNS. Despite extensive lymphocytolysis, direct evidence of viral replication was not observed inmore » the lymphoid tissue. These results correlate with the acute-onset hepatitis and delayed-onset encephalitis that are dominant features of severe human RVF, but suggest that host immune-mediated mechanisms contribute significantly to pathology. The results of this study expand our knowledge of RVFV-host interactions and further characterize the mouse model of RVF.« less

Co-localization of polar replication fork barriers and rRNA transcription terminators in mouse rDNA.

PubMed

López-estraño, C; Schvartzman, J B; Krimer, D B; Hernández, P

1998-03-27

We investigated the replication of the region where transcription terminates in mouse rDNA. It contains a replication fork barrier (RFB) that behaves in a polar manner, arresting only replication forks moving in the direction opposite to transcription. This RFB consists of several closely spaced fork arrest sites that co-localize with the transcription terminator elements, known as Sal boxes. Sal boxes are the target for mTTF-I (murine transcription termination factor I). These results suggest that both termination of rRNA transcription and replication fork arrest may share cis-acting as well as trans-acting factors. Copyright 1998 Academic Press Limited.

Dendritic spine dysgenesis in Autism Related Disorders

PubMed Central

Phillips, Mary; Pozzo-Miller, Lucas

2015-01-01

The activity-dependent structural and functional plasticity of dendritic spines has led to the long-standing belief that these neuronal compartments are the subcellular sites of learning and memory. Of relevance to human health, central neurons in several neuropsychiatric illnesses, including autism related disorders, have atypical numbers and morphologies of dendritic spines. These so-called dendritic spine dysgeneses found in individuals with autism related disorders are consistently replicated in experimental mouse models. Dendritic spine dysgenesis reflects the underlying synaptopathology that drives clinically relevant behavioral deficits in experimental mouse models, providing a platform for testing new therapeutic approaches. By examining molecular signaling pathways, synaptic deficits, and spine dysgenesis in experimental mouse models of autism related disorders we find strong evidence for mTOR to be a critical point of convergence and promising therapeutic target. PMID:25578949

The IL-1R/TLR signaling pathway is essential for efficient CD8+ T-cell responses against hepatitis B virus in the hydrodynamic injection mouse model.

PubMed

Ma, Zhiyong; Liu, Jia; Wu, Weimin; Zhang, Ejuan; Zhang, Xiaoyong; Li, Qian; Zelinskyy, Gennadiy; Buer, Jan; Dittmer, Ulf; Kirschning, Carsten J; Lu, Mengji

2017-12-01

The outcome of hepatitis B viral (HBV) infection is determined by the complex interactions between replicating HBV and the immune system. While the role of the adaptive immune system in the resolution of HBV infection has been studied extensively, the contribution of innate immune mechanisms remains to be defined. Here we examined the role of the interleukin-1 receptor/Toll-like receptor (IL-1R/TLR) signaling pathway in adaptive immune responses and viral clearance by exploring the HBV mouse model. Hydrodynamic injection with a replication-competent HBV genome was performed in wild-type mice (WT) and a panel of mouse strains lacking specific innate immunity component expression. We found higher levels of HBV protein production and replication in Tlr2 -/- , Tlr23479 -/- , 3d/Tlr24 -/- , Myd88/Trif -/- and Irak4 -/- mice, which was associated with reduced HBV-specific CD8 + T-cell responses in these mice. Importantly, HBV clearance was delayed for more than 2 weeks in 3d/Tlr24 -/- , Myd88/Trif -/- and Irak4 -/- mice compared to WT mice. HBV-specific CD8 + T-cell responses were functionally impaired for producing the cytokines IFN-γ, TNF-α and IL-2 in TLR signaling-deficient mice compared to WT mice. In conclusion, the IL-1R/TLR signaling pathway might contribute to controlling HBV infection by augmenting HBV-specific CD8 + T-cell responses.

An Immunocompetent Mouse Model of Zika Virus Infection.

PubMed

Gorman, Matthew J; Caine, Elizabeth A; Zaitsev, Konstantin; Begley, Matthew C; Weger-Lucarelli, James; Uccellini, Melissa B; Tripathi, Shashank; Morrison, Juliet; Yount, Boyd L; Dinnon, Kenneth H; Rückert, Claudia; Young, Michael C; Zhu, Zhe; Robertson, Shelly J; McNally, Kristin L; Ye, Jing; Cao, Bin; Mysorekar, Indira U; Ebel, Gregory D; Baric, Ralph S; Best, Sonja M; Artyomov, Maxim N; Garcia-Sastre, Adolfo; Diamond, Michael S

2018-05-09

Progress toward understanding Zika virus (ZIKV) pathogenesis is hindered by lack of immunocompetent small animal models, in part because ZIKV fails to effectively antagonize Stat2-dependent interferon (IFN) responses in mice. To address this limitation, we first passaged an African ZIKV strain (ZIKV-Dak-41525) through Rag1 -/- mice to obtain a mouse-adapted virus (ZIKV-Dak-MA) that was more virulent than ZIKV-Dak-41525 in mice treated with an anti-Ifnar1 antibody. A G18R substitution in NS4B was the genetic basis for the increased replication, and resulted in decreased IFN-β production, diminished IFN-stimulated gene expression, and the greater brain infection observed with ZIKV-Dak-MA. To generate a fully immunocompetent mouse model of ZIKV infection, human STAT2 was introduced into the mouse Stat2 locus (hSTAT2 KI). Subcutaneous inoculation of pregnant hSTAT2 KI mice with ZIKV-Dak-MA resulted in spread to the placenta and fetal brain. An immunocompetent mouse model of ZIKV infection may prove valuable for evaluating countermeasures to limit disease. Copyright © 2018 Elsevier Inc. All rights reserved.

Topical treatment of herpes simplex virus infection with enzymatically created siRNA swarm.

PubMed

Paavilainen, Henrik; Lehtinen, Jenni; Romanovskaya, Alesia; Nygårdas, Michaela; Bamford, Dennis H; Poranen, Minna M; Hukkanen, Veijo

2017-01-01

Herpes simplex virus (HSV) is a common human pathogen. Despite current antivirals, it causes a significant medical burden. Drug resistant strains exist and they are especially prevalent in immunocompromised patients and in HSV eye infections. New treatment modalities are needed. BALB/c mice were corneally infected with HSV and subsequently treated with a swarm of enzymatically created, Dicer-substrate small interfering RNA (siRNA) molecules that targeted the HSV gene UL29. Two infection models were used, one in which the infection was predominantly peripheral and another in which it spread to the central nervous system. Mouse survival, as well as viral spread, load, latency and peripheral shedding, was studied. The anti-HSV-UL29 siRNA swarm alleviated HSV infection symptoms, inhibited viral shedding and replication and had a favourable effect on mouse survival. Treatment with anti-HSV-UL29 siRNA swarm reduced symptoms and viral spread in HSV infection of mice and also inhibited local viral replication in mouse corneas.

PA-X protein decreases replication and pathogenicity of swine influenza virus in cultured cells and mouse models.

PubMed

Gong, Xiao-Qian; Sun, Ying-Feng; Ruan, Bao-Yang; Liu, Xiao-Min; Wang, Qi; Yang, Hai-Ming; Wang, Shuai-Yong; Zhang, Peng; Wang, Xiu-Hui; Shan, Tong-Ling; Tong, Wu; Zhou, Yan-Jun; Li, Guo-Xin; Zheng, Hao; Tong, Guang-Zhi; Yu, Hai

2017-06-01

Swine influenza viruses have been circulating in pigs throughout world and might be potential threats to human health. PA-X protein is a newly discovered protein produced from the PA gene by ribosomal frameshifting and the effects of PA-X on the 1918 H1N1, the pandemic 2009 H1N1, the highly pathogenic avian H5N1 and the avian H9N2 influenza viruses have been reported. However, the role of PA-X in the pathogenesis of swine influenza virus is still unknown. In this study, we rescued the H1N1 wild-type (WT) classical swine influenza virus (A/Swine/Guangdong/1/2011 (H1N1)) and H1N1 PA-X deficient virus containing mutations at the frameshift motif, and compared their replication properties and pathogenicity of swine influenza virus in vitro and in vivo. Our results show that the expression of PA-X inhibits virus replication and polymerase activity in cultured cells and decreases virulence in mouse models. Therefore, our study demonstrates that PA-X protein acts as a negative virulence regulator for classical H1N1 swine influenza virus and decreases virulence by inhibiting viral replication and polymerase activity, deepening our understanding of the pathogenesis of swine influenza virus. Copyright © 2017 Elsevier B.V. All rights reserved.

Cellular Chaperonin CCTγ Contributes to Rabies Virus Replication during Infection

PubMed Central

Zhang, Jinyang; Wu, Xiaopeng; Zan, Jie; Wu, Yongping; Ye, Chengjin; Ruan, Xizhen

2013-01-01

Rabies, as the oldest known infectious disease, remains a serious threat to public health worldwide. The eukaryotic cytosolic chaperonin TRiC/CCT complex facilitates the folding of proteins through ATP hydrolysis. Here, we investigated the expression, cellular localization, and function of neuronal CCTγ during neurotropic rabies virus (RABV) infection using mouse N2a cells as a model. Following RABV infection, 24 altered proteins were identified by using two-dimensional electrophoresis and mass spectrometry, including 20 upregulated proteins and 4 downregulated proteins. In mouse N2a cells infected with RABV or cotransfected with RABV genes encoding nucleoprotein (N) and phosphoprotein (P), confocal microscopy demonstrated that upregulated cellular CCTγ was colocalized with viral proteins N and P, which formed a hollow cricoid inclusion within the region around the nucleus. These inclusions, which correspond to Negri bodies (NBs), did not form in mouse N2a cells only expressing the viral protein N or P. Knockdown of CCTγ by lentivirus-mediated RNA interference led to significant inhibition of RABV replication. These results demonstrate that the complex consisting of viral proteins N and P recruits CCTγ to NBs and identify the chaperonin CCTγ as a host factor that facilitates intracellular RABV replication. This work illustrates how viruses can utilize cellular chaperonins and compartmentalization for their own benefit. PMID:23637400

Translational animal models of autism and neurodevelopmental disorders

PubMed Central

Crawley, Jacqueline N.

2012-01-01

Autism is a neurodevelopmental disorder whose diagnosis is based on three behavioral criteria: unusual reciprocal social interactions, deficits in communication, and stereotyped repetitive behaviors with restricted interests. A large number of de novo single gene mutations and chromosomal deletions are associated with autism spectrum disorders. Based on the strong genetic evidence, mice with targeted mutations in homologous genes have been generated as translational research tools. Mouse models of autism have revealed behavioral and biological outcomes of mutations in risk genes. The field is now poised to employ the most robust phenotypes in the most replicable mouse models for preclinical screening of novel therapeutics. PMID:23226954

A surgical approach appropriate for targeted cochlear gene therapy in the mouse.

PubMed

Jero, J; Tseng, C J; Mhatre, A N; Lalwani, A K

2001-01-01

Therapeutic manipulations of the mammalian cochlea, including cochlear gene transfer, have been predominantly studied using the guinea pig as the experimental model. With the significant developments in mouse genomics and the availability of mutant strains of mice with well-characterized hearing loss, the mouse justifiably will be the preferred animal model for therapeutic manipulations. However, the potential advantages of the mouse model have not been fully realized due to the surgical difficulty of accessing its small cochlea. This study describes a ventral approach, instead of the routinely used postauricular approach in other rodents, for accessing the mouse middle and inner ear, and its application in cochlear gene transfer. This ventral approach enabled rapid and direct delivery of liposome-transgene complex to the mouse inner ear while avoiding blood loss, facial nerve morbidity, and mortality. Transgene expression at 3 days was detected in Reissner's membrane, spiral limbus, spiral ligament, and spiral ganglion cells, in a pattern similar to that previously described in the guinea pig. The successful access and delivery of material to the mouse cochlea and the replication of gene expression seen in the guinea pig demonstrated in this study should promote the use of the mouse in future studies investigating targeted cochlear therapy.

Behavioral phenotypes of genetic mouse models of autism

PubMed Central

Kazdoba, T. M.; Leach, P. T.; Crawley, J. N.

2016-01-01

More than a hundred de novo single gene mutations and copy-number variants have been implicated in autism, each occurring in a small subset of cases. Mutant mouse models with syntenic mutations offer research tools to gain an understanding of the role of each gene in modulating biological and behavioral phenotypes relevant to autism. Knockout, knockin and transgenic mice incorporating risk gene mutations detected in autism spectrum disorder and comorbid neurodevelopmental disorders are now widely available. At present, autism spectrum disorder is diagnosed solely by behavioral criteria. We developed a constellation of mouse behavioral assays designed to maximize face validity to the types of social deficits and repetitive behaviors that are central to an autism diagnosis. Mouse behavioral assays for associated symptoms of autism, which include cognitive inflexibility, anxiety, hyperactivity, and unusual reactivity to sensory stimuli, are frequently included in the phenotypic analyses. Over the past 10 years, we and many other laboratories around the world have employed these and additional behavioral tests to phenotype a large number of mutant mouse models of autism. In this review, we highlight mouse models with mutations in genes that have been identified as risk genes for autism, which work through synaptic mechanisms and through the mTOR signaling pathway. Robust, replicated autism-relevant behavioral outcomes in a genetic mouse model lend credence to a causal role for specific gene contributions and downstream biological mechanisms in the etiology of autism. PMID:26403076

Dendritic spine dysgenesis in autism related disorders.

PubMed

Phillips, Mary; Pozzo-Miller, Lucas

2015-08-05

The activity-dependent structural and functional plasticity of dendritic spines has led to the long-standing belief that these neuronal compartments are the subcellular sites of learning and memory. Of relevance to human health, central neurons in several neuropsychiatric illnesses, including autism related disorders, have atypical numbers and morphologies of dendritic spines. These so-called dendritic spine dysgeneses found in individuals with autism related disorders are consistently replicated in experimental mouse models. Dendritic spine dysgenesis reflects the underlying synaptopathology that drives clinically relevant behavioral deficits in experimental mouse models, providing a platform for testing new therapeutic approaches. By examining molecular signaling pathways, synaptic deficits, and spine dysgenesis in experimental mouse models of autism related disorders we find strong evidence for mTOR to be a critical point of convergence and promising therapeutic target. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

MITOCHONDRIAL DISEASES PART II: MOUSE MODELS OF OXPHOS DEFICIENCIES CAUSED BY DEFECTS IN REGULATORY FACTORS AND OTHER COMPONENTS REQUIRED FOR MITOCHONDRIAL FUNCTION

PubMed Central

Iommarini, Luisa; Peralta, Susana; Torraco, Alessandra; Diaz, Francisca

2015-01-01

Mitochondrial disorders are defined as defects that affect the oxidative phosphorylation system (OXPHOS). They are characterized by a heterogeneous array of clinical presentations due in part to a wide variety of factors required for proper function of the components of the OXPHOS system. There is no cure for these disorders owing our poor knowledge of the pathogenic mechanisms of disease. To understand the mechanisms of human disease numerous mouse models have been developed in recent years. Here we summarize the features of several mouse models of mitochondrial diseases directly related to those factors affecting mtDNA maintenance, replication, transcription, translation as well to other proteins that are involved in mitochondrial dynamics and quality control which affect mitochondrial OXPHOS function without been intrinsic components of the system. We discuss how these models have contributed to our understanding of mitochondrial diseases and their pathogenic mechanisms. PMID:25640959

Neutrality of the canonical NF-kappaB-dependent pathway for human and murine cytomegalovirus transcription and replication in vitro.

PubMed

Benedict, Chris A; Angulo, Ana; Patterson, Ginelle; Ha, Sukwon; Huang, Huang; Messerle, Martin; Ware, Carl F; Ghazal, Peter

2004-01-01

Cytomegalovirus (CMV) is known to rapidly induce activation of nuclear factor kappaB (NF-kappaB) after infection of fibroblast and macrophage cells. NF-kappaB response elements are present in the enhancer region of the CMV major immediate-early promoter (MIEP), and activity of the MIEP is strongly upregulated by NF-kappaB in transient-transfection assays. Here we investigate whether the NF-kappaB-dependent pathway is required for initiating or potentiating human and murine CMV replication in vitro. We show that expression of a dominant negative mutant of the inhibitor of NF-kappaB-alpha (IkappaBalphaM) does not alter the replication kinetics of human or mouse CMV in cultured cells. In addition, mouse embryo fibroblasts genetically deficient for p65/RelA actually showed elevated levels of MCMV replication. Mutation of all NF-kappaB response elements within the enhancer of the MIEP in a recombinant mouse CMV containing the human MIEP (hMCMV-ES), which we have previously shown to replicate in murine fibroblasts with kinetics equivalent to that of wild-type mouse CMV, did not negatively affect replication in fibroblasts. Taken together, these data show that, for CMV replication in cultured fibroblasts activation of the canonical NF-kappaB pathway and binding of NF-kappaB to the MIEP are dispensable, and in the case of p65 may even interfere, thus uncovering a previously unrecognized level of complexity in the host regulatory network governing MIE gene expression in the context of a viral infection.

Expression of hepatitis B virus 1.3-fold genome plasmid in an SV40 T-antigen-immortalized mouse hepatic cell line

PubMed Central

Song, Xiu-Guang; Bian, Peng-Fei; Yu, Shu-Li; Zhao, Xiu-Hua; Xu, Wei; Bu, Xue-Hui; Li, Xia; Ma, Li-Xian

2013-01-01

AIM: To investigate the expression of the hepatitis B virus (HBV) 1.3-fold genome plasmid (pHBV1.3) in an immortalized mouse hepatic cell line induced by SV40 T-antigen (SV40T) expression. METHODS: Mouse hepatic cells were isolated from mouse liver tissue fragments from 3-5 d old Kunming mice by the direct collagenase digestion method and cultured in vitro. The pRSV-T plasmid was transfected into mouse hepatic cells to establish an SV40LT-immortalized mouse hepatic cell line. The SV40LT-immortalized mouse hepatic cells were identified and transfected with the pHBV1.3 plasmid. The levels of hepatitis B surface antigen (HBsAg) and hepatitis B e antigen (HBeAg) in the supernatant were determined by an electrochemiluminescence immunoassay at 24, 48, 72 and 96 h after transfection. The expressions of HBsAg and hepatitis B c antigen (HBcAg) in the cells were investigated by indirect immunofluorescence analysis. The presence of HBV DNA replication intermediates in the transfected cells and viral particles in the supernatant of the transfected cell cultures was monitored using the Southern hybridization assay and transmission electronic microscopy, respectively. RESULTS: The pRSV-T plasmid was used to immortalize mouse hepatocytes and an SV40LT-immortalized mouse hepatic cell line was successfully established. SV40LT-immortalized mouse hepatic cells have the same morphology and growth characteristics as primary mouse hepatic cells can be subcultured and produce albumin and cytokeratin-18 in vitro. Immortalized mouse hepatic cells did not show the characteristics of tumor cells, as alpha-fetoprotein levels were comparable (0.58 ± 0.37 vs 0.61 ± 0.31, P = 0.37). SV40LT-immortalized mouse hepatic cells were then transfected with the pHBV1.3 plasmid, and it was found that the HBV genome replicated in SV40LT-immortalized mouse hepatic cells. The levels of HBsAg and HBeAg continuously increased in the supernatant after the transfection of pHBV1.3, and began to decrease 72 h after transfection. The expressions of HBsAg and HBcAg were observed in the pHBV1.3-transfected cells. HBV DNA replication intermediates were also observed at 72 h after transfection, including relaxed circular DNA, double-stranded DNA and single-stranded DNA. Furthermore, a few 42 nm Dane particles, as well as many 22 nm subviral particles with a spherical or filamentous shape, were detected in the supernatant. CONCLUSION: SV40T expression can immortalize mouse hepatic cells, and the pHBV1.3-transfected SV40T-immortalized mouse hepatic cell line can be a new in vitro cell model. PMID:24307795

Expression of hepatitis B virus 1.3-fold genome plasmid in an SV40 T-antigen-immortalized mouse hepatic cell line.

PubMed

Song, Xiu-Guang; Bian, Peng-Fei; Yu, Shu-Li; Zhao, Xiu-Hua; Xu, Wei; Bu, Xue-Hui; Li, Xia; Ma, Li-Xian

2013-11-28

To investigate the expression of the hepatitis B virus (HBV) 1.3-fold genome plasmid (pHBV1.3) in an immortalized mouse hepatic cell line induced by SV40 T-antigen (SV40T) expression. Mouse hepatic cells were isolated from mouse liver tissue fragments from 3-5 d old Kunming mice by the direct collagenase digestion method and cultured in vitro. The pRSV-T plasmid was transfected into mouse hepatic cells to establish an SV40LT-immortalized mouse hepatic cell line. The SV40LT-immortalized mouse hepatic cells were identified and transfected with the pHBV1.3 plasmid. The levels of hepatitis B surface antigen (HBsAg) and hepatitis B e antigen (HBeAg) in the supernatant were determined by an electrochemiluminescence immunoassay at 24, 48, 72 and 96 h after transfection. The expressions of HBsAg and hepatitis B c antigen (HBcAg) in the cells were investigated by indirect immunofluorescence analysis. The presence of HBV DNA replication intermediates in the transfected cells and viral particles in the supernatant of the transfected cell cultures was monitored using the Southern hybridization assay and transmission electronic microscopy, respectively. The pRSV-T plasmid was used to immortalize mouse hepatocytes and an SV40LT-immortalized mouse hepatic cell line was successfully established. SV40LT-immortalized mouse hepatic cells have the same morphology and growth characteristics as primary mouse hepatic cells can be subcultured and produce albumin and cytokeratin-18 in vitro. Immortalized mouse hepatic cells did not show the characteristics of tumor cells, as alpha-fetoprotein levels were comparable (0.58 ± 0.37 vs 0.61 ± 0.31, P = 0.37). SV40LT-immortalized mouse hepatic cells were then transfected with the pHBV1.3 plasmid, and it was found that the HBV genome replicated in SV40LT-immortalized mouse hepatic cells. The levels of HBsAg and HBeAg continuously increased in the supernatant after the transfection of pHBV1.3, and began to decrease 72 h after transfection. The expressions of HBsAg and HBcAg were observed in the pHBV1.3-transfected cells. HBV DNA replication intermediates were also observed at 72 h after transfection, including relaxed circular DNA, double-stranded DNA and single-stranded DNA. Furthermore, a few 42 nm Dane particles, as well as many 22 nm subviral particles with a spherical or filamentous shape, were detected in the supernatant. SV40T expression can immortalize mouse hepatic cells, and the pHBV1.3-transfected SV40T-immortalized mouse hepatic cell line can be a new in vitro cell model.

Inhibition and recovery of the replication of depurinated parvovirus DNA in mouse fibroblasts

DOE Office of Scientific and Technical Information (OSTI.GOV)

Vos, J.M.; Avalosse, B.; Su, Z.Z.

Apurinic sites were introduced in the single-stranded DNA of parvovirus minute-virus-of-mice (MVM) and their effect on viral DNA synthesis was measured in mouse fibroblasts. Approximately one apurinic site per viral genome, is sufficient to block its replication in untreated cells. The exposure of host cells to a sublethal dose of UV-light 15 hours prior to virus infection, enhances their ability to support the replication of depurinated MVM. Cell preirradiation induces the apparent overcome of 10-15% of viral DNA replication blocks. These results indicate that apurinic sites prevent mammalian cells from replicating single-stranded DNA unless a recovery process is activated bymore » cell UV-irradiation.« less

Neutralizing inhibitors in the airways of naïve ferrets do not play a major role in modulating the virulence of H3 subtype influenza A viruses.

PubMed

Job, Emma R; Pizzolla, Angela; Nebl, Thomas; Short, Kirsty R; Deng, Yi-Mo; Carolan, Louise; Laurie, Karen L; Brooks, Andrew G; Reading, Patrick C

2016-07-01

Many insights regarding the pathogenesis of human influenza A virus (IAV) infections have come from studies in mice and ferrets. Surfactant protein (SP)-D is the major neutralizing inhibitor of IAV in mouse airway fluids and SP-D-resistant IAV mutants show enhanced virus replication and virulence in mice. Herein, we demonstrate that sialylated glycoproteins, rather than SP-D, represent the major neutralizing inhibitors against H3 subtype viruses in airway fluids from naïve ferrets. Moreover, while resistance to neutralizing inhibitors is a critical factor in modulating virus replication and disease in the mouse model, it does not appear to be so in the ferret model, as H3 mutants resistant to either SP-D or sialylated glycoproteins in ferret airway fluids did not show enhanced virulence in ferrets. These data have important implications for our understanding of pathogenesis and immunity to human IAV infections in these two widely used animal models of infection. Copyright © 2016. Published by Elsevier Inc.

Replication of DNA containing apurinic sites in human and mouse cells probed with parvoviruses MVM and H-1

DOE Office of Scientific and Technical Information (OSTI.GOV)

Vos, J.M.; Rommelaere, J.

We studied the effect of apurinic sites on DNA replication in mouse and human cells, using parvoviruses MVM (minute virus of mice) and H-1 as probes. Although apurinic sites are efficient blocks to the replication of these single-stranded DNA viruses in vivo, depurinated parvoviruses can be reactivated if host cells have been preexposed to a subtoxic dose of UV light. The target of this conditional reactivation process is the conversion of depurinated input DNA into double-stranded replicative forms; the concomitant increase in viral mutagenesis strongly suggests that apurinic sites can be bypassed in mammalian cells.

Cyclooxygenase activity is important for efficient replication of mouse hepatitis virus at an early stage of infection

PubMed Central

Raaben, Matthijs; Einerhand, Alexandra WC; Taminiau, Lucas JA; van Houdt, Michel; Bouma, Janneke; Raatgeep, Rolien H; Büller, Hans A; de Haan, Cornelis AM; Rossen, John WA

2007-01-01

Cyclooxygenases (COXs) play a significant role in many different viral infections with respect to replication and pathogenesis. Here we investigated the role of COXs in the mouse hepatitis coronavirus (MHV) infection cycle. Blocking COX activity by different inhibitors or by RNA interference affected MHV infection in different cells. The COX inhibitors reduced MHV infection at a post-binding step, but early in the replication cycle. Both viral RNA and viral protein synthesis were affected with subsequent loss of progeny virus production. Thus, COX activity appears to be required for efficient MHV replication, providing a potential target for anti-coronaviral therapy. PMID:17555580

Lgn1, a gene that determines susceptibility to Legionella pneumophila, maps to mouse chromosome 13

DOE Office of Scientific and Technical Information (OSTI.GOV)

Dietrich, W.F.; Damron, D.M.; Lander, E.S.

1995-04-10

The intracellular pathogen Legionella pneumophila is unable to replicate in macrophages derived from most inbred mouse strains. Here, we report the mapping of a gene, called Lgn1, that determines whether mouse macrophages are permissive for the intracellular replication of L. pneumophila. Although Lgn1 has been previously reported to map to mouse chromosome 15, we show here that it actually maps to chromosome 13, between D13Mit128 and D13Mit70. In the absence of any regional candidates for Lgn1, this map position will facilitate positional cloning attempts directed at this gene. 22 refs., 2 figs., 2 tabs.

Emotional Contagion is not Altered in Mice Prenatally Exposed to Poly (I:C) on Gestational Day 9.

PubMed

Gonzalez-Liencres, Cristina; Juckel, Georg; Esslinger, Manuela; Wachholz, Simone; Manitz, Marie-Pierre; Brüne, Martin; Friebe, Astrid

2016-01-01

Prenatal immune activation has been associated with increased risk of developing schizophrenia. The polyinosinic-polycytidylic acid (Poly(I:C)) mouse model replicates some of the endophenotype characteristic of this disorder but the social deficits observed in schizophrenia patients have not been well studied in this model. Therefore we aimed to investigate social behavior, in particular emotional contagion for pain, in this mouse model. We injected pregnant mouse dams with Poly(I:C) or saline (control) on gestation day 9 (GD9) and we evaluated their offspring in the pre-pulse inhibition (PPI) test at age 50-55 days old to confirm the reliability of our model. Mice were then evaluated in an emotional contagion test immediately followed by the light/dark test to explore post-test anxiety-like behavior at 10 weeks of age. In the emotional contagion test, an observer (prenatally exposed to Poly(I:C) or to saline) witnessed a familiar wild-type (WT) mouse (demonstrator) receiving electric foot shocks. Our results replicate the sensory gating impairments in the Poly(I:C) offspring but we only observed minor group differences in the social tasks. One of the differences we found was that demonstrators deposited fewer feces in the presence of control observers than of observers prenatally exposed to Poly(I:C), which we suggest could be due to the observers' behavior. We discuss the findings in the context of age, sex and day of prenatal injection, suggesting that Poly(I:C) on GD9 may be a valuable tool to assess other symptoms or symptom clusters of schizophrenia but perhaps not comprising the social domain.

Characterization of Barmah Forest virus pathogenesis in a mouse model.

PubMed

Herrero, Lara J; Lidbury, Brett A; Bettadapura, Jayaram; Jian, Peng; Herring, Belinda L; Hey-Cunningham, William J; Sheng, Kuo-Ching; Zakhary, Andrew; Mahalingam, Suresh

2014-10-01

Alphaviruses including Barmah Forest virus (BFV) and Ross River virus (RRV) cause arthritis, arthralgia and myalgia in humans. The rheumatic symptoms in human BFV infection are very similar to those of RRV. Although RRV disease has been studied extensively, little is known about the pathogenesis of BFV infection. We sought to establish a mouse model for BFV to facilitate our understanding of BFV infectivity, tropism and pathogenesis, and to identify key pathological and immunological mechanisms of BFV infection that may distinguish between infections with BFV and RRV. Here, to the best of our knowledge, we report the first study assessing the virulence and replication of several BFV isolates in a mouse model. We infected newborn Swiss outbred mice with BFV and established that the BFV2193 prototype was the most virulent strain. BFV2193 infection resulted in the highest mortality among all BFV variant isolates, comparable to that of RRV. In comparison with RRV, C57BL/6 mice infected with BFV showed delayed onset, moderate disease scores and early recovery of the disease. BFV replicated poorly in muscle and did not cause the severe myositis seen in RRV-infected mice. The mRNAs for the inflammatory mediators TNF-α, IL-6, CCL2 and arginase-1 were highly upregulated in RRV- but not BFV-infected muscle. To our knowledge, this is the first report of a mouse model of BFV infection, which we have used to demonstrate differences between BFV and RRV infections and to further understand disease pathogenesis. With an increasing number of BFV cases occurring annually, a better understanding of the disease mechanisms is essential for future therapeutic development. © 2014 The Authors.

Cohesin-SA1 deficiency drives aneuploidy and tumourigenesis in mice due to impaired replication of telomeres

PubMed Central

Remeseiro, Silvia; Cuadrado, Ana; Carretero, María; Martínez, Paula; Drosopoulos, William C; Cañamero, Marta; Schildkraut, Carl L; Blasco, María A; Losada, Ana

2012-01-01

Cohesin is a protein complex originally identified for its role in sister chromatid cohesion, although increasing evidence portrays it also as a major organizer of interphase chromatin. Vertebrate cohesin consists of Smc1, Smc3, Rad21/Scc1 and either stromal antigen 1 (SA1) or SA2. To explore the functional specificity of these two versions of cohesin and their relevance for embryonic development and cancer, we generated a mouse model deficient for SA1. Complete ablation of SA1 results in embryonic lethality, while heterozygous animals have shorter lifespan and earlier onset of tumourigenesis. SA1-null mouse embryonic fibroblasts show decreased proliferation and increased aneuploidy as a result of chromosome segregation defects. These defects are not caused by impaired centromeric cohesion, which depends on cohesin-SA2. Instead, they arise from defective telomere replication, which requires cohesion mediated specifically by cohesin-SA1. We propose a novel mechanism for aneuploidy generation that involves impaired telomere replication upon loss of cohesin-SA1, with clear implications in tumourigenesis. PMID:22415365

A CO-FISH assay to assess sister chromatid segregation patterns in mitosis of mouse embryonic stem cells.

PubMed

Sauer, Stephan; Burkett, Sandra S; Lewandoski, Mark; Klar, Amar J S

2013-05-01

Sister chromatids contain identical DNA sequence but are chiral with respect to both their helical handedness and their replication history. Emerging evidence from various model organisms suggests that certain stem cells segregate sister chromatids nonrandomly to either maintain genome integrity or to bias cellular differentiation in asymmetric cell divisions. Conventional methods for tracing of old vs. newly synthesized DNA strands generally lack resolution for individual chromosomes and employ halogenated thymidine analogs with profound cytotoxic effects on rapidly dividing cells. Here, we present a modified chromosome orientation fluorescence in situ hybridization (CO-FISH) assay, where identification of individual chromosomes and their replication history is achieved in subsequent hybridization steps with chromosome-specific DNA probes and PNA telomere probes. Importantly, we tackle the issue of BrdU cytotoxicity and show that our method is compatible with normal mouse ES cell biology, unlike a recently published related protocol. Results from our CO-FISH assay show that mitotic segregation of mouse chromosome 7 is random in ES cells, which contrasts previously published results from our laboratory and settles a controversy. Our straightforward protocol represents a useful resource for future studies on chromatid segregation patterns of in vitro-cultured cells from distinct model organisms.

Human Heart Mitochondrial DNA Is Organized in Complex Catenated Networks Containing Abundant Four-way Junctions and Replication Forks*

PubMed Central

Pohjoismäki, Jaakko L. O.; Goffart, Steffi; Tyynismaa, Henna; Willcox, Smaranda; Ide, Tomomi; Kang, Dongchon; Suomalainen, Anu; Karhunen, Pekka J.; Griffith, Jack D.; Holt, Ian J.; Jacobs, Howard T.

2009-01-01

Analysis of human heart mitochondrial DNA (mtDNA) by electron microscopy and agarose gel electrophoresis revealed a complete absence of the θ-type replication intermediates seen abundantly in mtDNA from all other tissues. Instead only Y- and X-junctional forms were detected after restriction digestion. Uncut heart mtDNA was organized in tangled complexes of up to 20 or more genome equivalents, which could be resolved to genomic monomers, dimers, and linear fragments by treatment with the decatenating enzyme topoisomerase IV plus the cruciform-cutting T7 endonuclease I. Human and mouse brain also contained a population of such mtDNA forms, which were absent, however, from mouse, rabbit, or pig heart. Overexpression in transgenic mice of two proteins involved in mtDNA replication, namely human mitochondrial transcription factor A or the mouse Twinkle DNA helicase, generated abundant four-way junctions in mtDNA of heart, brain, and skeletal muscle. The organization of mtDNA of human heart as well as of mouse and human brain in complex junctional networks replicating via a presumed non-θ mechanism is unprecedented in mammals. PMID:19525233

Reduction in open field activity in the absence of memory deficits in the AppNL-G-F knock-in mouse model of Alzheimer's disease.

PubMed

Whyte, Lauren S; Hemsley, Kim M; Lau, Adeline A; Hassiotis, Sofia; Saito, Takashi; Saido, Takaomi C; Hopwood, John J; Sargeant, Timothy J

2018-01-15

The recent development of knock-in mouse models of Alzheimer's disease provides distinct advantages over traditional transgenic mouse models that rely on over-expression of amyloid precursor protein. Two such knock-in models that have recently been widely adopted by Alzheimer's researchers are the App NL-F and App NL-G-F mice. This study aimed to further characterise the behavioural phenotype and amyloid plaque distribution of App NL-G-F/NL-G-F (C57BL/6J background) mice at six-months of age. An attempt to replicate a previous study that observed deficits in working memory in the Y-maze, showed no difference between App NL-G-F/NL-G-F and wild-type mice. Further assessment of these mice using the novel object recognition test and Morris water maze also revealed no differences between App NL-G-F/NL-G-F and wild-type mice. Despite a lack of demonstrated cognitive deficits, we report a reduction in locomotor/exploratory activity in an open field. Histological examination of App NL-G-F/NL-G-F mice showed widespread distribution of amyloid plaques at this age. We conclude that whilst at six-months of age, memory deficits are not sufficiently robust to be replicated in varying environments, amyloid plaque burden is significant in App NL-G-F/NL-G-F knock-in brain. Copyright © 2017 Elsevier B.V. All rights reserved.

The Complexity of Alcohol Drinking: Studies in Rodent Genetic Models

PubMed Central

Phillips, Tamara J.; Belknap, John K.

2012-01-01

Risk for alcohol dependence in humans has substantial genetic contributions. Successful rodent models generally attempt to address only selected features of the human diagnosis. Most such models target the phenotype of oral administration of alcohol solutions, usually consumption of or preference for an alcohol solution versus water. Data from rats and mice for more than 50 years have shown genetic influences on preference drinking and related phenotypes. This paper summarizes some key findings from that extensive literature. Much has been learned, including the genomic location and possible identity of several genes influencing preference drinking. We report new information from congenic lines confirming QTLs for drinking on mouse chromosomes 2 and 9. There are many strengths of the various phenotypic assays used to study drinking, but there are also some weaknesses. One major weakness, the lack of drinking excessively enough to become intoxicated, has recently been addressed with a new genetic animal model, mouse lines selectively bred for their high and intoxicating blood alcohol levels after a limited period of drinking in the circadian dark. We report here results from a second replicate of that selection and compare them with the first replicate. PMID:20552264

Zika virus transmission to mouse ear by mosquito bite: a laboratory model that replicates the natural transmission process.

PubMed

Secundino, Nagila Francinete Costa; Chaves, Barbara Aparecida; Orfano, Alessandra Silva; Silveira, Karine Renata Dias; Rodrigues, Nilton Barnabe; Campolina, Thais Bonifácio; Nacif-Pimenta, Rafael; Villegas, Luiz Eduardo Martinez; Silva, Breno Melo; Lacerda, Marcus Vinícius Guimarães; Norris, Douglas Eric; Pimenta, Paulo Filemon Paolucci

2017-07-20

Zika disease has transformed into a serious global health problem due to the rapid spread of the arbovirus and alarming severity including congenital complications, microcephaly and Guillain-Barré syndrome. Zika virus (ZIKV) is primarily transmitted to humans through the bite of an infective mosquito, with Aedes aegypti being the main vector. We successfully developed a ZIKV experimental transmission model by single infectious Ae. aegypti bite to a laboratory mouse using circulating Brazilian strains of both arbovirus and vector. Mosquitoes were orally infected and single Ae. aegypti were allowed to feed on mouse ears 14 days post-infection. Additionally, salivary gland (SG) homogenates from infected mosquitoes were intrathoracically inoculated into naïve Ae. aegypti. Mosquito and mouse tissue samples were cultured in C6/36 cells and processed by quantitative real-time PCR. A total of 26 Ae. aegypti were allowed to feed individually on mouse ears. Of these, 17 mosquitoes fed, all to full engorgement. The transmission rate of ZIKV by bite from these engorged mosquitoes to mouse ears was 100%. The amount of virus inoculated into the ears by bites ranged from 2 × 10 2 -2.1 × 10 10 ZIKV cDNA copies and was positively correlated with ZIKV cDNA quantified from SGs dissected from mosquitoes post-feeding. Replicating ZIKV was confirmed in macerated SGs (2.45 × 10 7 cDNA copies), mouse ear tissue (1.15 × 10 3 cDNA copies, and mosquitoes 14 days post-intrathoracic inoculation (1.49 × 10 7 cDNA copies) by cytopathic effect in C6/36 cell culture and qPCR. Our model illustrates successful transmission of ZIKV by an infectious mosquito bite to a live vertebrate host. This approach offers a comprehensive tool for evaluating the development of infection in and transmission from mosquitoes, and the vertebrate-ZIKV interaction and progression of infection following a natural transmission process.

Behavioral phenotypes of genetic mouse models of autism.

PubMed

Kazdoba, T M; Leach, P T; Crawley, J N

2016-01-01

More than a hundred de novo single gene mutations and copy-number variants have been implicated in autism, each occurring in a small subset of cases. Mutant mouse models with syntenic mutations offer research tools to gain an understanding of the role of each gene in modulating biological and behavioral phenotypes relevant to autism. Knockout, knockin and transgenic mice incorporating risk gene mutations detected in autism spectrum disorder and comorbid neurodevelopmental disorders are now widely available. At present, autism spectrum disorder is diagnosed solely by behavioral criteria. We developed a constellation of mouse behavioral assays designed to maximize face validity to the types of social deficits and repetitive behaviors that are central to an autism diagnosis. Mouse behavioral assays for associated symptoms of autism, which include cognitive inflexibility, anxiety, hyperactivity, and unusual reactivity to sensory stimuli, are frequently included in the phenotypic analyses. Over the past 10 years, we and many other laboratories around the world have employed these and additional behavioral tests to phenotype a large number of mutant mouse models of autism. In this review, we highlight mouse models with mutations in genes that have been identified as risk genes for autism, which work through synaptic mechanisms and through the mTOR signaling pathway. Robust, replicated autism-relevant behavioral outcomes in a genetic mouse model lend credence to a causal role for specific gene contributions and downstream biological mechanisms in the etiology of autism. © 2015 John Wiley & Sons Ltd and International Behavioural and Neural Genetics Society.

Immunocompetent syngeneic cotton rat tumor models for the assessment of replication-competent oncolytic adenovirus

DOE Office of Scientific and Technical Information (OSTI.GOV)

Steel, Jason C.; Morrison, Brian J.; Mannan, Poonam

Oncolytic adenoviruses as a treatment for cancer have demonstrated limited clinical activity. Contributing to this may be the relevance of preclinical animal models used to study these agents. Syngeneic mouse tumor models are generally non-permissive for adenoviral replication, whereas human tumor xenograft models exhibit attenuated immune responses to the vector. The cotton rat (Sigmodon hispidus) is susceptible to human adenovirus infection, permissive for viral replication and exhibits similar inflammatory pathology to humans with adenovirus replicating in the lungs, respiratory passages and cornea. We evaluated three transplantable tumorigenic cotton rat cell lines, CCRT, LCRT and VCRT as models for the studymore » of oncolytic adenoviruses. All three cells lines were readily infected with adenovirus type-5-based vectors and exhibited high levels of transgene expression. The cell lines supported viral replication demonstrated by the induction of cytopathogenic effect (CPE) in tissue culture, increase in virus particle numbers and assembly of virions seen on transmission electron microscopy. In vivo, LCRT and VCRT tumors demonstrated delayed growth after injection with replicating adenovirus. No in vivo antitumor activity was seen in CCRT tumors despite in vitro oncolysis. Adenovirus was also rapidly cleared from the CCRT tumors compared to LCRT and VCRT tumors. The effect observed with the different cotton rat tumor cell lines mimics the variable results of human clinical trials highlighting the potential relevance of this model for assessing the activity and toxicity of oncolytic adenoviruses.« less

Inhibition of Cell Division and DNA Replication Impair Mouse-Naïve Pluripotency Exit.

PubMed

Waisman, Ariel; Vazquez Echegaray, Camila; Solari, Claudia; Cosentino, María Soledad; Martyn, Iain; Deglincerti, Alessia; Ozair, Mohammad Zeeshan; Ruzo, Albert; Barañao, Lino; Miriuka, Santiago; Brivanlou, Ali; Guberman, Alejandra

2017-09-01

The cell cycle has gained attention as a key determinant for cell fate decisions, but the contribution of DNA replication and mitosis in stem cell differentiation has not been extensively studied. To understand if these processes act as "windows of opportunity" for changes in cell identity, we established synchronized cultures of mouse embryonic stem cells as they exit the ground state of pluripotency. We show that initial transcriptional changes in this transition do not require passage through mitosis and that conversion to primed pluripotency is linked to lineage priming in the G1 phase. Importantly, we demonstrate that impairment of DNA replication severely blocks transcriptional switch to primed pluripotency, even in the absence of p53 activity induced by the DNA damage response. Our data suggest an important role for DNA replication during mouse embryonic stem cell differentiation, which could shed light on why pluripotent cells are only receptive to differentiation signals during G1, that is, before the S phase. Copyright © 2017 Elsevier Ltd. All rights reserved.

A Comparative Encyclopedia of DNA Elements in the Mouse Genome

PubMed Central

Yue, Feng; Cheng, Yong; Breschi, Alessandra; Vierstra, Jeff; Wu, Weisheng; Ryba, Tyrone; Sandstrom, Richard; Ma, Zhihai; Davis, Carrie; Pope, Benjamin D.; Shen, Yin; Pervouchine, Dmitri D.; Djebali, Sarah; Thurman, Bob; Kaul, Rajinder; Rynes, Eric; Kirilusha, Anthony; Marinov, Georgi K.; Williams, Brian A.; Trout, Diane; Amrhein, Henry; Fisher-Aylor, Katherine; Antoshechkin, Igor; DeSalvo, Gilberto; See, Lei-Hoon; Fastuca, Meagan; Drenkow, Jorg; Zaleski, Chris; Dobin, Alex; Prieto, Pablo; Lagarde, Julien; Bussotti, Giovanni; Tanzer, Andrea; Denas, Olgert; Li, Kanwei; Bender, M. A.; Zhang, Miaohua; Byron, Rachel; Groudine, Mark T.; McCleary, David; Pham, Long; Ye, Zhen; Kuan, Samantha; Edsall, Lee; Wu, Yi-Chieh; Rasmussen, Matthew D.; Bansal, Mukul S.; Keller, Cheryl A.; Morrissey, Christapher S.; Mishra, Tejaswini; Jain, Deepti; Dogan, Nergiz; Harris, Robert S.; Cayting, Philip; Kawli, Trupti; Boyle, Alan P.; Euskirchen, Ghia; Kundaje, Anshul; Lin, Shin; Lin, Yiing; Jansen, Camden; Malladi, Venkat S.; Cline, Melissa S.; Erickson, Drew T.; Kirkup, Vanessa M; Learned, Katrina; Sloan, Cricket A.; Rosenbloom, Kate R.; de Sousa, Beatriz Lacerda; Beal, Kathryn; Pignatelli, Miguel; Flicek, Paul; Lian, Jin; Kahveci, Tamer; Lee, Dongwon; Kent, W. James; Santos, Miguel Ramalho; Herrero, Javier; Notredame, Cedric; Johnson, Audra; Vong, Shinny; Lee, Kristen; Bates, Daniel; Neri, Fidencio; Diegel, Morgan; Canfield, Theresa; Sabo, Peter J.; Wilken, Matthew S.; Reh, Thomas A.; Giste, Erika; Shafer, Anthony; Kutyavin, Tanya; Haugen, Eric; Dunn, Douglas; Reynolds, Alex P.; Neph, Shane; Humbert, Richard; Hansen, R. Scott; De Bruijn, Marella; Selleri, Licia; Rudensky, Alexander; Josefowicz, Steven; Samstein, Robert; Eichler, Evan E.; Orkin, Stuart H.; Levasseur, Dana; Papayannopoulou, Thalia; Chang, Kai-Hsin; Skoultchi, Arthur; Gosh, Srikanta; Disteche, Christine; Treuting, Piper; Wang, Yanli; Weiss, Mitchell J.; Blobel, Gerd A.; Good, Peter J.; Lowdon, Rebecca F.; Adams, Leslie B.; Zhou, Xiao-Qiao; Pazin, Michael J.; Feingold, Elise A.; Wold, Barbara; Taylor, James; Kellis, Manolis; Mortazavi, Ali; Weissman, Sherman M.; Stamatoyannopoulos, John; Snyder, Michael P.; Guigo, Roderic; Gingeras, Thomas R.; Gilbert, David M.; Hardison, Ross C.; Beer, Michael A.; Ren, Bing

2014-01-01

Summary As the premier model organism in biomedical research, the laboratory mouse shares the majority of protein-coding genes with humans, yet the two mammals differ in significant ways. To gain greater insights into both shared and species-specific transcriptional and cellular regulatory programs in the mouse, the Mouse ENCODE Consortium has mapped transcription, DNase I hypersensitivity, transcription factor binding, chromatin modifications, and replication domains throughout the mouse genome in diverse cell and tissue types. By comparing with the human genome, we not only confirm substantial conservation in the newly annotated potential functional sequences, but also find a large degree of divergence of other sequences involved in transcriptional regulation, chromatin state and higher order chromatin organization. Our results illuminate the wide range of evolutionary forces acting on genes and their regulatory regions, and provide a general resource for research into mammalian biology and mechanisms of human diseases. PMID:25409824

A comparative encyclopedia of DNA elements in the mouse genome.

PubMed

Yue, Feng; Cheng, Yong; Breschi, Alessandra; Vierstra, Jeff; Wu, Weisheng; Ryba, Tyrone; Sandstrom, Richard; Ma, Zhihai; Davis, Carrie; Pope, Benjamin D; Shen, Yin; Pervouchine, Dmitri D; Djebali, Sarah; Thurman, Robert E; Kaul, Rajinder; Rynes, Eric; Kirilusha, Anthony; Marinov, Georgi K; Williams, Brian A; Trout, Diane; Amrhein, Henry; Fisher-Aylor, Katherine; Antoshechkin, Igor; DeSalvo, Gilberto; See, Lei-Hoon; Fastuca, Meagan; Drenkow, Jorg; Zaleski, Chris; Dobin, Alex; Prieto, Pablo; Lagarde, Julien; Bussotti, Giovanni; Tanzer, Andrea; Denas, Olgert; Li, Kanwei; Bender, M A; Zhang, Miaohua; Byron, Rachel; Groudine, Mark T; McCleary, David; Pham, Long; Ye, Zhen; Kuan, Samantha; Edsall, Lee; Wu, Yi-Chieh; Rasmussen, Matthew D; Bansal, Mukul S; Kellis, Manolis; Keller, Cheryl A; Morrissey, Christapher S; Mishra, Tejaswini; Jain, Deepti; Dogan, Nergiz; Harris, Robert S; Cayting, Philip; Kawli, Trupti; Boyle, Alan P; Euskirchen, Ghia; Kundaje, Anshul; Lin, Shin; Lin, Yiing; Jansen, Camden; Malladi, Venkat S; Cline, Melissa S; Erickson, Drew T; Kirkup, Vanessa M; Learned, Katrina; Sloan, Cricket A; Rosenbloom, Kate R; Lacerda de Sousa, Beatriz; Beal, Kathryn; Pignatelli, Miguel; Flicek, Paul; Lian, Jin; Kahveci, Tamer; Lee, Dongwon; Kent, W James; Ramalho Santos, Miguel; Herrero, Javier; Notredame, Cedric; Johnson, Audra; Vong, Shinny; Lee, Kristen; Bates, Daniel; Neri, Fidencio; Diegel, Morgan; Canfield, Theresa; Sabo, Peter J; Wilken, Matthew S; Reh, Thomas A; Giste, Erika; Shafer, Anthony; Kutyavin, Tanya; Haugen, Eric; Dunn, Douglas; Reynolds, Alex P; Neph, Shane; Humbert, Richard; Hansen, R Scott; De Bruijn, Marella; Selleri, Licia; Rudensky, Alexander; Josefowicz, Steven; Samstein, Robert; Eichler, Evan E; Orkin, Stuart H; Levasseur, Dana; Papayannopoulou, Thalia; Chang, Kai-Hsin; Skoultchi, Arthur; Gosh, Srikanta; Disteche, Christine; Treuting, Piper; Wang, Yanli; Weiss, Mitchell J; Blobel, Gerd A; Cao, Xiaoyi; Zhong, Sheng; Wang, Ting; Good, Peter J; Lowdon, Rebecca F; Adams, Leslie B; Zhou, Xiao-Qiao; Pazin, Michael J; Feingold, Elise A; Wold, Barbara; Taylor, James; Mortazavi, Ali; Weissman, Sherman M; Stamatoyannopoulos, John A; Snyder, Michael P; Guigo, Roderic; Gingeras, Thomas R; Gilbert, David M; Hardison, Ross C; Beer, Michael A; Ren, Bing

2014-11-20

The laboratory mouse shares the majority of its protein-coding genes with humans, making it the premier model organism in biomedical research, yet the two mammals differ in significant ways. To gain greater insights into both shared and species-specific transcriptional and cellular regulatory programs in the mouse, the Mouse ENCODE Consortium has mapped transcription, DNase I hypersensitivity, transcription factor binding, chromatin modifications and replication domains throughout the mouse genome in diverse cell and tissue types. By comparing with the human genome, we not only confirm substantial conservation in the newly annotated potential functional sequences, but also find a large degree of divergence of sequences involved in transcriptional regulation, chromatin state and higher order chromatin organization. Our results illuminate the wide range of evolutionary forces acting on genes and their regulatory regions, and provide a general resource for research into mammalian biology and mechanisms of human diseases.

Rodent models in Down syndrome research: impact and future opportunities

PubMed Central

2017-01-01

ABSTRACT Down syndrome is caused by trisomy of chromosome 21. To date, a multiplicity of mouse models with Down-syndrome-related features has been developed to understand this complex human chromosomal disorder. These mouse models have been important for determining genotype-phenotype relationships and identification of dosage-sensitive genes involved in the pathophysiology of the condition, and in exploring the impact of the additional chromosome on the whole genome. Mouse models of Down syndrome have also been used to test therapeutic strategies. Here, we provide an overview of research in the last 15 years dedicated to the development and application of rodent models for Down syndrome. We also speculate on possible and probable future directions of research in this fast-moving field. As our understanding of the syndrome improves and genome engineering technologies evolve, it is necessary to coordinate efforts to make all Down syndrome models available to the community, to test therapeutics in models that replicate the whole trisomy and design new animal models to promote further discovery of potential therapeutic targets. PMID:28993310

Rodent models in Down syndrome research: impact and future opportunities.

PubMed

Herault, Yann; Delabar, Jean M; Fisher, Elizabeth M C; Tybulewicz, Victor L J; Yu, Eugene; Brault, Veronique

2017-10-01

Down syndrome is caused by trisomy of chromosome 21. To date, a multiplicity of mouse models with Down-syndrome-related features has been developed to understand this complex human chromosomal disorder. These mouse models have been important for determining genotype-phenotype relationships and identification of dosage-sensitive genes involved in the pathophysiology of the condition, and in exploring the impact of the additional chromosome on the whole genome. Mouse models of Down syndrome have also been used to test therapeutic strategies. Here, we provide an overview of research in the last 15 years dedicated to the development and application of rodent models for Down syndrome. We also speculate on possible and probable future directions of research in this fast-moving field. As our understanding of the syndrome improves and genome engineering technologies evolve, it is necessary to coordinate efforts to make all Down syndrome models available to the community, to test therapeutics in models that replicate the whole trisomy and design new animal models to promote further discovery of potential therapeutic targets. © 2017. Published by The Company of Biologists Ltd.

Poliomyelitis in transgenic mice expressing CD155 under the control of the Tage4 promoter after oral and parenteral poliovirus inoculation

PubMed Central

Khan, Shaukat; Toyoda, Hidemi; Linehan, Melissa; Iwasaki, Akiko; Nomoto, Akio; Bernhardt, Günter; Wimmer, Eckard

2014-01-01

An important step in poliovirus (PV) infection by the oral route in humans is replication of the virus in lymphatic tissues of the gastrointestinal (GI) tract, thought to be mainly in the Peyer’s patches of the small intestine. No immunocompetent transgenic (tg) mice that express human PV receptor (CD155) under the control of different promoters can be infected orally. The mouse orthologue of human CD155 is Tage4, a protein expressed at the surface of enterocytes and in the Peyer’s patches. We describe here the generation of a tg mouse model in which the Tage4 promoter was used to drive expression of the human PV receptor-coding region (Tage4-CD155tg mice). In this model, CD155 expression was observed by immunostaining in different regions in the Peyer’s patches but not in their germinal centres. Although a similar pattern of staining was observed between 3- and 6-week-old Tage4-CD155tg mice, poliomyelitis was only seen in the younger mice after PV infection by the oral route. When compared with TgPVR21 mice that expressed CD155 driven by its human promoter, 3-week-old Tage4-CD155tg mice were more susceptible to gut infection and paralysis following feeding with PV. Also, Tage4-CD155tg mice exhibited higher susceptibility to poliomyelitis after parenteral inoculation of PV. Remarkably, the LD50 after intracerebral inoculation of PV was similar in both CD155 tg mouse strains. The CD155 tg mouse model reported here, although moderately susceptible to oral infection, may be suitable to study mechanisms of PV replication in the gastrointestinal tract and to dissect important aspects of PV neuroinvasiveness. PMID:24784416

Poliomyelitis in transgenic mice expressing CD155 under the control of the Tage4 promoter after oral and parenteral poliovirus inoculation.

PubMed

Khan, Shaukat; Toyoda, Hidemi; Linehan, Melissa; Iwasaki, Akiko; Nomoto, Akio; Bernhardt, Günter; Cello, Jeronimo; Wimmer, Eckard

2014-08-01

An important step in poliovirus (PV) infection by the oral route in humans is replication of the virus in lymphatic tissues of the gastrointestinal (GI) tract, thought to be mainly in the Peyer's patches of the small intestine. No immunocompetent transgenic (tg) mice that express human PV receptor (CD155) under the control of different promoters can be infected orally. The mouse orthologue of human CD155 is Tage4, a protein expressed at the surface of enterocytes and in the Peyer's patches. We describe here the generation of a tg mouse model in which the Tage4 promoter was used to drive expression of the human PV receptor-coding region (Tage4-CD155tg mice). In this model, CD155 expression was observed by immunostaining in different regions in the Peyer's patches but not in their germinal centres. Although a similar pattern of staining was observed between 3- and 6-week-old Tage4-CD155tg mice, poliomyelitis was only seen in the younger mice after PV infection by the oral route. When compared with TgPVR21 mice that expressed CD155 driven by its human promoter, 3-week-old Tage4-CD155tg mice were more susceptible to gut infection and paralysis following feeding with PV. Also, Tage4-CD155tg mice exhibited higher susceptibility to poliomyelitis after parenteral inoculation of PV. Remarkably, the LD50 after intracerebral inoculation of PV was similar in both CD155 tg mouse strains. The CD155 tg mouse model reported here, although moderately susceptible to oral infection, may be suitable to study mechanisms of PV replication in the gastrointestinal tract and to dissect important aspects of PV neuroinvasiveness. © 2014 The Authors.

Pathogenesis of emerging severe fever with thrombocytopenia syndrome virus in C57/BL6 mouse model

PubMed Central

Jin, Cong; Liang, Mifang; Ning, Junyu; Gu, Wen; Jiang, Hong; Wu, Wei; Zhang, Fushun; Zhang, Quanfu; Zhu, Hua; Chen, Ting; Han, Ying; Zhang, Weilun; Zhang, Shuo; Wang, Qin; Sun, Lina; Liu, Qinzhi; Wang, Tao; Wei, Qiang; Wang, Shiwen; Deng, Ying; Qin, Chuan; Li, Dexin

2012-01-01

The discovery of an emerging viral disease, severe fever with thrombocytopenia syndrome (SFTS), caused by SFTS virus (SFTSV), has prompted the need to understand pathogenesis of SFTSV. We are unique in establishing an infectious model of SFTS in C57/BL6 mice, resulting in hallmark symptoms of thrombocytopenia and leukocytopenia. Viral RNA and histopathological changes were identified in the spleen, liver, and kidney. However, viral replication was only found in the spleen, which suggested the spleen to be the principle target organ of SFTSV. Moreover, the number of macrophages and platelets were largely increased in the spleen, and SFTSV colocalized with platelets in cytoplasm of macrophages in the red pulp of the spleen. In vitro cellular assays further revealed that SFTSV adhered to mouse platelets and facilitated the phagocytosis of platelets by mouse primary macrophages, which in combination with in vivo findings, suggests that SFTSV-induced thrombocytopenia is caused by clearance of circulating virus-bound platelets by splenic macrophages. Thus, this study has elucidated the pathogenic mechanisms of thrombocytopenia in a mouse model resembling human SFTS disease. PMID:22665769

Lack of protection against ebola virus from chloroquine in mice and hamsters.

PubMed

Falzarano, Darryl; Safronetz, David; Prescott, Joseph; Marzi, Andrea; Feldmann, Friederike; Feldmann, Heinz

2015-06-01

The antimalarial drug chloroquine has been suggested as a treatment for Ebola virus infection. Chloroquine inhibited virus replication in vitro, but only at cytotoxic concentrations. In mouse and hamster models, treatment did not improve survival. Chloroquine is not a promising treatment for Ebola. Efforts should be directed toward other drug classes.

A homolog of the variola virus B22 membrane protein contributes to ectromelia virus pathogenicity in the mouse footpad model.

PubMed

Reynolds, Sara E; Earl, Patricia L; Minai, Mahnaz; Moore, Ian; Moss, Bernard

2017-01-15

Most poxviruses encode a homolog of a ~200,000-kDa membrane protein originally identified in variola virus. We investigated the importance of the ectromelia virus (ECTV) homolog C15 in a natural infection model. In cultured mouse cells, the replication of a mutant virus with stop codons near the N-terminus (ECTV-C15Stop) was indistinguishable from a control virus (ECTV-C15Rev). However, for a range of doses injected into the footpads of BALB/c mice there was less mortality with the mutant. Similar virus loads were present at the site of infection with mutant or control virus whereas there was less ECTV-C15Stop in popliteal and inguinal lymph nodes, spleen and liver indicating decreased virus spread and replication. The latter results were supported by immunohistochemical analyses. Decreased spread was evidently due to immune modulatory activity of C15, rather than to an intrinsic viral function, as the survival of infected mice depended on CD4+ and CD8+ T cells. Published by Elsevier Inc.

SAMHD1 knockout mice: modeling retrovirus restriction in vivo.

PubMed

Wu, Li

2013-11-20

The host dNTP hydrolase SAMHD1 acts as a viral restriction factor to inhibit the replication of several retroviruses and DNA viruses in non-cycling human immune cells. However, understanding the physiological role of mammalian SAMHD1 has been elusive due to the lack of an animal model. Two recent studies reported the generation of samhd1 knockout mouse models for investigating the restriction of HIV-1 vectors and endogenous retroviruses in vivo. Both studies suggest that SAMHD1 is important for regulating the intracellular dNTP pool and the intrinsic immunity against retroviral infection, despite different outcomes of HIV-1 vector transduction in these mouse models. Here I discuss the significance of these new findings and the future directions in studying SAMHD1-mediated retroviral restriction.

In Vivo Regulation of Hepatitis B Virus Replication by Peroxisome Proliferators†

PubMed Central

Guidotti, Luca G.; Eggers, Carrie M.; Raney, Anneke K.; Chi, Susan Y.; Peters, Jeffrey M.; Gonzalez, Frank J.; McLachlan, Alan

1999-01-01

The role of the peroxisome proliferator-activated receptor α (PPARα) in regulating hepatitis B virus (HBV) transcription and replication in vivo was investigated in an HBV transgenic mouse model. Treatment of HBV transgenic mice with the peroxisome proliferators Wy-14,643 and clofibric acid resulted in a less than twofold increase in HBV transcription rates and steady-state levels of HBV RNAs in the livers of these mice. In male mice, this increase in transcription was associated with a 2- to 3-fold increase in replication intermediates, whereas in female mice it was associated with a 7- to 14-fold increase in replication intermediates. The observed increases in transcription and replication were dependent on PPARα. HBV transgenic mice lacking this nuclear hormone receptor showed similar levels of HBV transcripts and replication intermediates as untreated HBV transgenic mice expressing PPARα but failed to demonstrate alterations in either RNA or DNA synthesis in response to peroxisome proliferators. Therefore, it appears that very modest alterations in transcription can, under certain circumstances, result in relatively large increases in HBV replication in HBV transgenic mice. PMID:10559356

Autism-related neuroligin-3 mutation alters social behavior and spatial learning.

PubMed

Jaramillo, Thomas C; Liu, Shunan; Pettersen, Ami; Birnbaum, Shari G; Powell, Craig M

2014-04-01

Multiple candidate genes have been identified for autism spectrum disorders. While some of these genes reach genome-wide significance, others, such as the R451C point mutation in the synaptic cell adhesion molecule neuroligin-3, appear to be rare. Interestingly, two brothers with the same R451C point mutation in neuroligin-3 present clinically on seemingly disparate sides of the autism spectrum. These clinical findings suggest genetic background may play a role in modifying the penetrance of a particular autism-associated mutation. Animal models may contribute additional support for such mutations as functionally relevant and can provide mechanistic insights. Previously, in collaboration with the Südhof laboratory, we reported that mice with an R451C substitution in neuroligin-3 displayed social deficits and enhanced spatial learning. While some of these behavioral abnormalities have since been replicated independently in the Südhof laboratory, observations from the Crawley laboratory failed to replicate these findings in a similar neuroligin-3 mutant mouse model and suggested that genetic background may contribute to variation in observations across laboratories. Therefore, we sought to replicate our findings in the neuroligin-3 R451C point mutant knock-in mouse model (NL3R451C) in a different genetic background. We backcrossed our NL3R451C mouse line onto a 129S2/SvPasCrl genetic background and repeated a subset of our previous behavioral testing. NL3R451C mice on a 129S2/SvPasCrl displayed social deficits, enhanced spatial learning, and increased locomotor activity. These data extend our previous findings that NL3R451C mice exhibit autism-relevant behavioral abnormalities and further suggest that different genetic backgrounds can modify this behavioral phenotype through epistatic genetic interactions. © 2014 International Society for Autism Research, Wiley Periodicals, Inc.

Error-free replicative bypass of (6–4) photoproducts by DNA polymerase ζ in mouse and human cells

PubMed Central

Yoon, Jung-Hoon; Prakash, Louise; Prakash, Satya

2010-01-01

The ultraviolet (UV)-induced (6–4) pyrimidine–pyrimidone photoproduct [(6–4) PP] confers a large structural distortion in DNA. Here we examine in human cells the roles of translesion synthesis (TLS) DNA polymerases (Pols) in promoting replication through a (6–4) TT photoproduct carried on a duplex plasmid where bidirectional replication initiates from an origin of replication. We show that TLS contributes to a large fraction of lesion bypass and that it is mostly error-free. We find that, whereas Pol η and Pol ι provide alternate pathways for mutagenic TLS, surprisingly, Pol ζ functions independently of these Pols and in a predominantly error-free manner. We verify and extend these observations in mouse cells and conclude that, in human cells, TLS during replication can be markedly error-free even opposite a highly distorting DNA lesion. PMID:20080950

Glucose Induces Mouse β-Cell Proliferation via IRS2, MTOR, and Cyclin D2 but Not the Insulin Receptor

PubMed Central

Stamateris, Rachel E.; Sharma, Rohit B.; Kong, Yahui; Ebrahimpour, Pantea; Panday, Deepika; Ranganath, Pavana; Zou, Baobo; Levitt, Helena; Parambil, Nisha Abraham; O’Donnell, Christopher P.; García-Ocaña, Adolfo

2016-01-01

An important goal in diabetes research is to understand the processes that trigger endogenous β-cell proliferation. Hyperglycemia induces β-cell replication, but the mechanism remains debated. A prime candidate is insulin, which acts locally through the insulin receptor. Having previously developed an in vivo mouse hyperglycemia model, we tested whether glucose induces β-cell proliferation through insulin signaling. By using mice lacking insulin signaling intermediate insulin receptor substrate 2 (IRS2), we confirmed that hyperglycemia-induced β-cell proliferation requires IRS2 both in vivo and ex vivo. Of note, insulin receptor activation was not required for glucose-induced proliferation, and insulin itself was not sufficient to drive replication. Glucose and insulin caused similar acute signaling in mouse islets, but chronic signaling differed markedly, with mammalian target of rapamycin (MTOR) and extracellular signal–related kinase (ERK) activation by glucose and AKT activation by insulin. MTOR but not ERK activation was required for glucose-induced proliferation. Cyclin D2 was necessary for glucose-induced β-cell proliferation. Cyclin D2 expression was reduced when either IRS2 or MTOR signaling was lost, and restoring cyclin D2 expression rescued the proliferation defect. Human islets shared many of these regulatory pathways. Taken together, these results support a model in which IRS2, MTOR, and cyclin D2, but not the insulin receptor, mediate glucose-induced proliferation. PMID:26740601

FAM134B, the Selective Autophagy Receptor for Endoplasmic Reticulum Turnover, Inhibits Replication of Ebola Virus Strains Makona and Mayinga.

PubMed

Chiramel, Abhilash I; Dougherty, Jonathan D; Nair, Vinod; Robertson, Shelly J; Best, Sonja M

2016-10-15

Selective autophagy of the endoplasmic reticulum (termed ER-phagy) is controlled by members of the FAM134 reticulon protein family. Here we used mouse embryonic fibroblasts from mice deficient in FAM134B to examine the role of the ER in replication of historic (Mayinga) or contemporary (Makona GCO7) strains of Ebola virus (EBOV). Loss of FAM134B resulted in 1-2 log 10 higher production of infectious EBOV, which was associated with increased production of viral proteins GP and VP40 and greater accumulation of nucleocaspid lattices. In addition, only 10% of wild-type cells contained detectable nucleoprotein, whereas knockout of FAM134B resulted in 80% of cells positive for nucleoprotein. Together, these data suggest that FAM134B-dependent ER-phagy is an important limiting event in EBOV replication in mouse cells and may have implications for further development of antiviral therapeutics and murine models of infection. Published by Oxford University Press for the Infectious Diseases Society of America 2016. This work is written by (a) US Government employee(s) and is in the public domain in the US.

Cathepsin B & L are not required for ebola virus replication.

PubMed

Marzi, Andrea; Reinheckel, Thomas; Feldmann, Heinz

2012-01-01

Ebola virus (EBOV), family Filoviridae, emerged in 1976 on the African continent. Since then it caused several outbreaks of viral hemorrhagic fever in humans with case fatality rates up to 90% and remains a serious Public Health concern and biothreat pathogen. The most pathogenic and best-studied species is Zaire ebolavirus (ZEBOV). EBOV encodes one viral surface glycoprotein (GP), which is essential for replication, a determinant of pathogenicity and an important immunogen. GP mediates viral entry through interaction with cellular surface molecules, which results in the uptake of virus particles via macropinocytosis. Later in this pathway endosomal acidification activates the cysteine proteases Cathepsin B and L (CatB, CatL), which have been shown to cleave ZEBOV-GP leading to subsequent exposure of the putative receptor-binding and fusion domain and productive infection. We studied the effect of CatB and CatL on in vitro and in vivo replication of EBOV. Similar to previous findings, our results show an effect of CatB, but not CatL, on ZEBOV entry into cultured cells. Interestingly, cell entry by other EBOV species (Bundibugyo, Côte d'Ivoire, Reston and Sudan ebolavirus) was independent of CatB or CatL as was EBOV replication in general. To investigate whether CatB and CatL have a role in vivo during infection, we utilized the mouse model for ZEBOV. Wild-type (control), catB(-/-) and catL(-/-) mice were equally susceptible to lethal challenge with mouse-adapted ZEBOV with no difference in virus replication and time to death. In conclusion, our results show that CatB and CatL activity is not required for EBOV replication. Furthermore, EBOV glycoprotein cleavage seems to be mediated by an array of proteases making targeted therapeutic approaches difficult.

The replication of a mouse adapted SARS-CoV in a mouse cell line stably expressing the murine SARS-CoV receptor mACE2 efficiently induces the expression of proinflammatory cytokines

PubMed Central

Regla-Nava, Jose A.; Jimenez-Guardeño, Jose M.; Nieto-Torres, Jose L.; Gallagher, Thomas M.; Enjuanes, Luis; DeDiego, Marta L.

2013-01-01

Infection of conventional mice with a mouse adapted (MA15) severe acute respiratory syndrome (SARS) coronavirus (CoV) reproduces many aspects of human SARS such as pathological changes in lung, viremia, neutrophilia, and lethality. However, established mouse cell lines highly susceptible to mouse-adapted SARS-CoV infection are not available. In this work, efficiently transfectable mouse cell lines stably expressing the murine SARS-CoV receptor angiotensin converting enzyme 2 (ACE2) have been generated. These cells yielded high SARS-CoV-MA15 titers and also served as excellent tools for plaque assays. In addition, in these cell lines, SARS-CoV-MA15 induced the expression of proinflammatory cytokines and IFN-β, mimicking what has been observed in experimental animal models infected with SARS-CoV and SARS patients. These cell lines are valuable tools to perform in vitro studies in a mouse cell system that reflects the species used for in vivo studies of SARS-CoV-MA15 pathogenesis. PMID:23911968

Amino Acids 257 to 288 of Mouse p48 Control the Cooperation of Polyomavirus Large T Antigen, Replication Protein A, and DNA Polymerase α-Primase To Synthesize DNA In Vitro

PubMed Central

Kautz, Armin R.; Weisshart, Klaus; Schneider, Annerose; Grosse, Frank; Nasheuer, Heinz-Peter

2001-01-01

Although p48 is the most conserved subunit of mammalian DNA polymerase α-primase (pol-prim), the polypeptide is the major species-specific factor for mouse polyomavirus (PyV) DNA replication. Human and murine p48 contain two regions (A and B) that show significantly lower homology than the rest of the protein. Chimerical human-murine p48 was prepared and coexpressed with three wild-type subunits of pol-prim, and four subunit protein complexes were purified. All enzyme complexes synthesized DNA on single-stranded (ss) DNA and replicated simian virus 40 DNA. Although the recombinant protein complexes physically interacted with PyV T antigen (Tag), we determined that the murine region A mediates the species specificity of PyV DNA replication in vitro. More precisely, the nonconserved phenylalanine 262 of mouse p48 is crucial for this activity, and pol-prim with mutant p48, h-S262F, supports PyV DNA replication in vitro. DNA synthesis on RPA-bound ssDNA revealed that amino acid (aa) 262, aa 266, and aa 273 to 288 are involved in the functional cooperation of RPA, pol-prim, and PyV Tag. PMID:11507202

Inhibition of severe acute respiratory syndrome coronavirus replication in a lethal SARS-CoV BALB/c mouse model by stinging nettle lectin, Urtica dioica agglutinin.

PubMed

Kumaki, Yohichi; Wandersee, Miles K; Smith, Aaron J; Zhou, Yanchen; Simmons, Graham; Nelson, Nathan M; Bailey, Kevin W; Vest, Zachary G; Li, Joseph K-K; Chan, Paul Kay-Sheung; Smee, Donald F; Barnard, Dale L

2011-04-01

Urtica dioica agglutinin (UDA) is a small plant monomeric lectin, 8.7 kDa in size, with an N-acetylglucosamine specificity that inhibits viruses from Nidovirales in vitro. In the current study, we first examined the efficacy of UDA on the replication of different SARS-CoV strains in Vero 76 cells. UDA inhibited virus replication in a dose-dependent manner and reduced virus yields of the Urbani strain by 90% at 1.1 ± 0.4 μg/ml in Vero 76 cells. Then, UDA was tested for efficacy in a lethal SARS-CoV-infected BALB/c mouse model. BALB/c mice were infected with two LD50 (575 PFU) of virus for 4 h before the mice were treated intraperitoneally with UDA at 20, 10, 5 or 0 mg/kg/day for 4 days. Treatment with UDA at 5 mg/kg significantly protected the mice against a lethal infection with mouse-adapted SARS-CoV (p < 0.001), but did not significantly reduce virus lung titers. All virus-infected mice receiving UDA treatments were also significantly protected against weight loss (p < 0.001). UDA also effectively reduced lung pathology scores. At day 6 after virus exposure, all groups of mice receiving UDA had much lower lung weights than did the placebo-treated mice. Thus, our data suggest that UDA treatment of SARS infection in mice leads to a substantial therapeutic effect that protects mice against death and weight loss. Furthermore, the mode of action of UDA in vitro was further investigated using live SARS-CoV Urbani strain virus and retroviral particles pseudotyped with SARS-CoV spike (S). UDA specifically inhibited the replication of live SARS-CoV or SARS-CoV pseudotyped virus when added just before, but not after, adsorption. These data suggested that UDA likely inhibits SARS-CoV infection by targeting early stages of the replication cycle, namely, adsorption or penetration. In addition, we demonstrated that UDA neutralizes the virus infectivity, presumably by binding to the SARS-CoV spike (S) glycoprotein. Finally, the target molecule for the inhibition of virus replication was partially characterized. When UDA was exposed to N-acetylglucosamine and then UDA was added to cells just prior to adsorption, UDA did not inhibit the virus infection. These data support the conclusion that UDA might bind to N-acetylglucosamine-like residues present on the glycosylated envelope glycoproteins, thereby preventing virus attachment to cells. Copyright © 2011 Elsevier B.V. All rights reserved.

Inhibition of severe acute respiratory syndrome coronavirus replication in a lethal SARS-CoV BALB/c mouse model by stinging nettle lectin, Urtica dioica agglutinin

PubMed Central

Kumaki, Yohichi; Wandersee, Miles K.; Smith, Aaron J.; Zhou, Yanchen; Simmons, Graham; Nelson, Nathan M.; Bailey, Kevin W.; Vest, Zachary G.; Li, Joseph K.-K.; Chan, Paul Kay-Sheung; Smee, Donald F.; Barnard, Dale L.

2011-01-01

Urtica dioica agglutinin (UDA) is a small plant monomeric lectin, 8.7 kDa in size, with an N-acetylglucosamine specificity that inhibits viruses from Nidovirales in vitro. In the current study, we first examined the efficacy of UDA on the replication of different SARS-CoV strains in Vero 76 cells. UDA inhibited virus replication in a dose-dependent manner and reduced virus yields of the Urbani strain by 90% at 1.1 ± 0.4 µg/ml in Vero 76 cells. Then, UDA was tested for efficacy in a lethal SARS-CoV-infected BALB/c mouse model. BALB/c mice were infected with two LD50 (575 PFU) of virus for 4 hours before the mice were treated intraperitoneally with UDA at 20, 10, 5 or 0 mg/kg/day for 4 days. Treatment with UDA at 5 mg/kg significantly protected the mice against a lethal infection with mouse-adapted SARS-CoV (p<0.001), but did not significantly reduce virus lung titers. All virus-infected mice receiving UDA treatments were also significantly protected against weight loss (p<0.001). UDA also effectively reduced lung pathology scores. At day 6 after virus exposure, all groups of mice receiving UDA had much lower lung weights than did the placebo-treated mice. Thus, our data suggest that UDA treatment of SARS infection in mice leads to a substantial therapeutic effect that protects mice against death and weight loss. Furthermore, the mode of action of UDA in vitro was further investigated using live SARS-CoV Urbani strain virus and retroviral particles pseudotyped with SARS-CoV spike (S). UDA specifically inhibited the replication of live SARS-CoV or SARS-CoV pseudotyped virus when added just before, but not after, adsorption. These data suggested that UDA likely inhibits SARS-CoV infection by targeting early stages of the replication cycle, namely, adsorption or penetration. In addition, we demonstrated that UDA neutralizes the virus infectivity, presumably by binding to the SARS-CoV spike (S) glycoprotein. Finally, the target molecule for inhibition of virus replication was partially characterized. When UDA was exposed to N-acetylglucosamine and then UDA was added to cells just prior to adsorption, UDA did not inhibit the virus infection. These data support the conclusion that UDA might bind to N-acetylglucosamine-like residues present on the glycosylated envelope glycoproteins, thereby preventing virus attachment to cells. PMID:21338626

Peripheral re-localization of constitutive heterochromatin advances its replication timing and impairs maintenance of silencing marks.

PubMed

Heinz, Kathrin S; Casas-Delucchi, Corella S; Török, Timea; Cmarko, Dusan; Rapp, Alexander; Raska, Ivan; Cardoso, M Cristina

2018-05-10

The replication of the genome is a highly organized process, both spatially and temporally. Although a lot is known on the composition of the basic replication machinery, how its activity is regulated is mostly unknown. Several chromatin properties have been proposed as regulators, but a potential role of the nuclear DNA position remains unclear. We made use of the prominent structure and well-defined heterochromatic landscape of mouse pericentric chromosome domains as a well-studied example of late replicating constitutive heterochromatin. We established a method to manipulate its nuclear position and evaluated the effect on replication timing, DNA compaction and epigenetic composition. Using time-lapse microscopy, we observed that constitutive heterochromatin, known to replicate during late S-phase, was replicated in mid S-phase when repositioned to the nuclear periphery. Out-of-schedule replication resulted in deficient post-replicative maintenance of chromatin modifications, namely silencing marks. We propose that repositioned constitutive heterochromatin was activated in trans according to the domino model of origin firing by nearby (mid S) firing origins. In summary, our data provide, on the one hand, a novel approach to manipulate nuclear DNA position and, on the other hand, establish nuclear DNA position as a novel mechanism regulating DNA replication timing and epigenetic maintenance.

NYVAC vector modified by C7L viral gene insertion improves T cell immune responses and effectiveness against leishmaniasis.

PubMed

Sánchez-Sampedro, L; Mejías-Pérez, E; S Sorzano, Carlos Óscar; Nájera, J L; Esteban, M

2016-07-15

The NYVAC poxvirus vector is used as vaccine candidate for HIV and other diseases, although there is only limited experimental information on its immunogenicity and effectiveness for use against human pathogens. Here we defined the selective advantage of NYVAC vectors in a mouse model by comparing the immune responses and protection induced by vectors that express the LACK (Leishmania-activated C-kinase antigen), alone or with insertion of the viral host range gene C7L that allows the virus to replicate in human cells. Using DNA prime/virus boost protocols, we show that replication-competent NYVAC-LACK that expresses C7L (NYVAC-LACK-C7L) induced higher-magnitude polyfunctional CD8(+) and CD4(+) primary adaptive and effector memory T cell responses (IFNγ, TNFα, IL-2, CD107a) to LACK antigen than non-replicating NYVAC-LACK. Compared to NYVAC-LACK, the NYVAC-LACK-C7L-induced CD8(+) T cell population also showed higher proliferation when stimulated with LACK antigen. After a challenge by subcutaneous Leishmania major metacyclic promastigotes, NYVAC-LACK-C7L-vaccinated mouse groups showed greater protection than the NYVAC-LACK-vaccinated group. Our results indicate that the type and potency of immune responses induced by LACK-expressing NYVAC vectors is improved by insertion of the C7L gene, and that a replication-competent vector as a vaccine renders greater protection against a human pathogen than a non-replicating vector. Copyright © 2016 Elsevier B.V. All rights reserved.

Evaluation of synthetic vascular grafts in a mouse carotid grafting model.

PubMed

Chan, Alex H P; Tan, Richard P; Michael, Praveesuda L; Lee, Bob S L; Vanags, Laura Z; Ng, Martin K C; Bursill, Christina A; Wise, Steven G

2017-01-01

Current animal models for the evaluation of synthetic grafts are lacking many of the molecular tools and transgenic studies available to other branches of biology. A mouse model of vascular grafting would allow for the study of molecular mechanisms of graft failure, including in the context of clinically relevant disease states. In this study, we comprehensively characterise a sutureless grafting model which facilitates the evaluation of synthetic grafts in the mouse carotid artery. Using conduits electrospun from polycaprolactone (PCL) we show the gradual development of a significant neointima within 28 days, found to be greatest at the anastomoses. Histological analysis showed temporal increases in smooth muscle cell and collagen content within the neointima, demonstrating its maturation. Endothelialisation of the PCL grafts, assessed by scanning electron microscopy (SEM) analysis and CD31 staining, was near complete within 28 days, together replicating two critical aspects of graft performance. To further demonstrate the potential of this mouse model, we used longitudinal non-invasive tracking of bone-marrow mononuclear cells from a transgenic mouse strain with a dual reporter construct encoding both luciferase and green fluorescent protein (GFP). This enabled characterisation of mononuclear cell homing and engraftment to PCL using bioluminescence imaging and histological staining over time (7, 14 and 28 days). We observed peak luminescence at 7 days post-graft implantation that persisted until sacrifice at 28 days. Collectively, we have established and characterised a high-throughput model of grafting that allows for the evaluation of key clinical drivers of graft performance.

Pathogenesis, Transmissibility, and Ocular Tropism of a Highly Pathogenic Avian Influenza A (H7N3) Virus Associated with Human Conjunctivitis

PubMed Central

Belser, Jessica A.; Davis, C. Todd; Balish, Amanda; Edwards, Lindsay E.; Zeng, Hui; Maines, Taronna R.; Gustin, Kortney M.; Martínez, Irma López; Fasce, Rodrigo; Cox, Nancy J.; Katz, Jacqueline M.

2013-01-01

H7 subtype influenza A viruses, responsible for numerous outbreaks in land-based poultry in Europe and the Americas, have caused over 100 cases of confirmed or presumed human infection over the last decade. The emergence of a highly pathogenic avian influenza H7N3 virus in poultry throughout the state of Jalisco, Mexico, resulting in two cases of human infection, prompted us to examine the virulence of this virus (A/Mexico/InDRE7218/2012 [MX/7218]) and related avian H7 subtype viruses in mouse and ferret models. Several high- and low-pathogenicity H7N3 and H7N9 viruses replicated efficiently in the respiratory tract of mice without prior adaptation following intranasal inoculation, but only MX/7218 virus caused lethal disease in this species. H7N3 and H7N9 viruses were also detected in the mouse eye following ocular inoculation. Virus from both H7N3 and H7N9 subtypes replicated efficiently in the upper and lower respiratory tracts of ferrets; however, only MX/7218 virus infection caused clinical signs and symptoms and was capable of transmission to naive ferrets in a direct-contact model. Similar to other highly pathogenic H7 viruses, MX/7218 replicated to high titers in human bronchial epithelial cells, yet it downregulated numerous genes related to NF-κB-mediated signaling transduction. These findings indicate that the recently isolated North American lineage H7 subtype virus associated with human conjunctivitis is capable of causing severe disease in mice and spreading to naive-contact ferrets, while concurrently retaining the ability to replicate within ocular tissue and allowing the eye to serve as a portal of entry. PMID:23487452

SEE locomotor behavior test discriminates C57BL/6J and DBA/2J mouse inbred strains across laboratories and protocol conditions.

PubMed

Kafkafi, Neri; Lipkind, Dina; Benjamini, Yoav; Mayo, Cheryl L; Elmer, Gregory I; Golani, Ilan

2003-06-01

Conventional tests of behavioral phenotyping frequently have difficulties differentiating certain genotypes and replicating these differences across laboratories and protocol conditions. This study explores the hypothesis that automated tests can be designed to quantify ethologically relevant behavior patterns that more readily characterize heritable and replicable phenotypes. It used SEE (Strategy for the Exploration of Exploration) to phenotype the locomotor behavior of the C57BL/6 and DBA/2 mouse inbred strains across 3 laboratories. The 2 genotypes differed in 15 different measures of behavior, none of which had a significant genotype-laboratory interaction. Within the same laboratory, most of these differences were replicated in additional experiments despite the test photoperiod phase being changed and saline being injected. Results suggest that well-designed tests may considerably enhance replicability across laboratories.

Affect-related related behaviors in mice selectively bred for high and low voluntary alcohol consumption

PubMed Central

Can, Adem; Grahame, Nicholas J.; Gould, Todd D.

2016-01-01

There is considerable evidence for the existence of comorbidity between alcohol-use disorders and depression in humans. One strategy to elucidate hereditary factors affecting the comorbidity of these disorders is to use genetic animal models, such as mouse lines selectively bred for voluntary ethanol consumption. We hypothesized that mice from lines that were bred for high-alcohol preference would manifest increased depression-like phenotypes compared to low-alcohol preferring mice. Mice that were bi-directionally selected and bred on the basis of their High-(HAP) or Low-Alcohol Preference (LAP) were tested in the open-field (OFT), dark-light box (DLB), forced swim (FST), and learned helplessness tests (LH). The study was conducted in two independently derived replicates. In the OFT, both HAP2 and HAP3 mice showed higher levels of general locomotion compared to LAP mice. However, only HAP2 mice spent more time in the center compared to LAP2 mice. In the DLB, there was a slightly higher anxiety-like phenotype in HAP mice. In both FST and LH, we observed higher depression-like behaviors in HAP mice compared to LAP mice, but this was limited to the Replicate 2 mice. Overall, we identified affect-related behavioral changes in mouse lines bred for high-alcohol preference. Notably, the Replicate 3 lines that showed fewer depression-like behaviors also manifest smaller differences in alcohol intake. These data suggest that there may be overlap between genes that predispose to excessive alcohol intake and those underlying affective-related behaviors in the mouse. PMID:21989731

Affect-related behaviors in mice selectively bred for high and low voluntary alcohol consumption.

PubMed

Can, Adem; Grahame, Nicholas J; Gould, Todd D

2012-03-01

There is considerable evidence for the existence of comorbidity between alcohol-use disorders and depression in humans. One strategy to elucidate hereditary factors affecting the comorbidity of these disorders is to use genetic animal models, such as mouse lines selectively bred for voluntary ethanol consumption. We hypothesized that mice from lines that were bred for high-alcohol preference would manifest increased depression-like phenotypes compared to low-alcohol preferring mice. Mice that were bi-directionally selected and bred on the basis of their High- (HAP) or Low-Alcohol Preference (LAP) were tested in the open-field (OFT), dark-light box (DLB), forced swim (FST), and learned helplessness tests (LH). The study was conducted in two independently derived replicates. In the OFT, both HAP2 and HAP3 mice showed higher levels of general locomotion compared to LAP mice. However, only HAP2 mice spent more time in the center compared to LAP2 mice. In the DLB, there was a slightly higher anxiety-like phenotype in HAP mice. In both FST and LH, we observed higher depression-like behaviors in HAP mice compared to LAP mice, but this was limited to the Replicate 2 mice. Overall, we identified affect-related behavioral changes in mouse lines bred for high-alcohol preference. Notably, the Replicate 3 lines that showed fewer depression-like behaviors also manifest smaller differences in alcohol intake. These data suggest that there may be overlap between genes that predispose to excessive alcohol intake and those underlying affect-related behaviors in the mouse.

Supraphysiologic control over HIV-1 replication mediated by CD8 T cells expressing a re-engineered CD4-based chimeric antigen receptor

PubMed Central

Richardson, Max W.; Ellebrecht, Christoph T.; Glover, Joshua A.; Secreto, Anthony J.; Kulikovskaya, Irina; Yi, Yanjie; Wang, Jianbin; Dufendach, Keith A.; Holmes, Michael C.; Collman, Ronald G.

2017-01-01

HIV is adept at avoiding naturally generated T cell responses; therefore, there is a need to develop HIV-specific T cells with greater potency for use in HIV cure strategies. Starting with a CD4-based chimeric antigen receptor (CAR) that was previously used without toxicity in clinical trials, we optimized the vector backbone, promoter, HIV targeting moiety, and transmembrane and signaling domains to determine which components augmented the ability of T cells to control HIV replication. This re-engineered CAR was at least 50-fold more potent in vitro at controlling HIV replication than the original CD4 CAR, or a TCR-based approach, and substantially better than broadly neutralizing antibody-based CARs. A humanized mouse model of HIV infection demonstrated that T cells expressing optimized CARs were superior at expanding in response to antigen, protecting CD4 T cells from infection, and reducing viral loads compared to T cells expressing the original, clinical trial CAR. Moreover, in a humanized mouse model of HIV treatment, CD4 CAR T cells containing the 4-1BB costimulatory domain controlled HIV spread after ART removal better than analogous CAR T cells containing the CD28 costimulatory domain. Together, these data indicate that potent HIV-specific T cells can be generated using improved CAR design and that CAR T cells could be important components of an HIV cure strategy. PMID:29023549

Temperature-dependent innate defense against the common cold virus limits viral replication at warm temperature in mouse airway cells.

PubMed

Foxman, Ellen F; Storer, James A; Fitzgerald, Megan E; Wasik, Bethany R; Hou, Lin; Zhao, Hongyu; Turner, Paul E; Pyle, Anna Marie; Iwasaki, Akiko

2015-01-20

Most isolates of human rhinovirus, the common cold virus, replicate more robustly at the cool temperatures found in the nasal cavity (33-35 °C) than at core body temperature (37 °C). To gain insight into the mechanism of temperature-dependent growth, we compared the transcriptional response of primary mouse airway epithelial cells infected with rhinovirus at 33 °C vs. 37 °C. Mouse airway cells infected with mouse-adapted rhinovirus 1B exhibited a striking enrichment in expression of antiviral defense response genes at 37 °C relative to 33 °C, which correlated with significantly higher expression levels of type I and type III IFN genes and IFN-stimulated genes (ISGs) at 37 °C. Temperature-dependent IFN induction in response to rhinovirus was dependent on the MAVS protein, a key signaling adaptor of the RIG-I-like receptors (RLRs). Stimulation of primary airway cells with the synthetic RLR ligand poly I:C led to greater IFN induction at 37 °C relative to 33 °C at early time points poststimulation and to a sustained increase in the induction of ISGs at 37 °C relative to 33 °C. Recombinant type I IFN also stimulated more robust induction of ISGs at 37 °C than at 33 °C. Genetic deficiency of MAVS or the type I IFN receptor in infected airway cells permitted higher levels of viral replication, particularly at 37 °C, and partially rescued the temperature-dependent growth phenotype. These findings demonstrate that in mouse airway cells, rhinovirus replicates preferentially at nasal cavity temperature due, in part, to a less efficient antiviral defense response of infected cells at cool temperature.

Genetically diverse CC-founder mouse strains replicate the human influenza gene expression signature.

PubMed

Elbahesh, Husni; Schughart, Klaus

2016-05-19

Influenza A viruses (IAV) are zoonotic pathogens that pose a major threat to human and animal health. Influenza virus disease severity is influenced by viral virulence factors as well as individual differences in host response. We analyzed gene expression changes in the blood of infected mice using a previously defined set of signature genes that was derived from changes in the blood transcriptome of IAV-infected human volunteers. We found that the human signature was reproduced well in the founder strains of the Collaborative Cross (CC) mice, thus demonstrating the relevance and importance of mouse experimental model systems for studying human influenza disease.

The replication of a mouse adapted SARS-CoV in a mouse cell line stably expressing the murine SARS-CoV receptor mACE2 efficiently induces the expression of proinflammatory cytokines.

PubMed

Regla-Nava, Jose A; Jimenez-Guardeño, Jose M; Nieto-Torres, Jose L; Gallagher, Thomas M; Enjuanes, Luis; DeDiego, Marta L

2013-11-01

Infection of conventional mice with a mouse adapted (MA15) severe acute respiratory syndrome (SARS) coronavirus (CoV) reproduces many aspects of human SARS such as pathological changes in lung, viremia, neutrophilia, and lethality. However, established mouse cell lines highly susceptible to mouse-adapted SARS-CoV infection are not available. In this work, efficiently transfectable mouse cell lines stably expressing the murine SARS-CoV receptor angiotensin converting enzyme 2 (ACE2) have been generated. These cells yielded high SARS-CoV-MA15 titers and also served as excellent tools for plaque assays. In addition, in these cell lines, SARS-CoV-MA15 induced the expression of proinflammatory cytokines and IFN-β, mimicking what has been observed in experimental animal models infected with SARS-CoV and SARS patients. These cell lines are valuable tools to perform in vitro studies in a mouse cell system that reflects the species used for in vivo studies of SARS-CoV-MA15 pathogenesis. Copyright © 2013 Elsevier B.V. All rights reserved.

Mouse Models for Drug Discovery. Can New Tools and Technology Improve Translational Power?

PubMed Central

Zuberi, Aamir; Lutz, Cathleen

2016-01-01

Abstract The use of mouse models in biomedical research and preclinical drug evaluation is on the rise. The advent of new molecular genome-altering technologies such as CRISPR/Cas9 allows for genetic mutations to be introduced into the germ line of a mouse faster and less expensively than previous methods. In addition, the rapid progress in the development and use of somatic transgenesis using viral vectors, as well as manipulations of gene expression with siRNAs and antisense oligonucleotides, allow for even greater exploration into genomics and systems biology. These technological advances come at a time when cost reductions in genome sequencing have led to the identification of pathogenic mutations in patient populations, providing unprecedented opportunities in the use of mice to model human disease. The ease of genetic engineering in mice also offers a potential paradigm shift in resource sharing and the speed by which models are made available in the public domain. Predictively, the knowledge alone that a model can be quickly remade will provide relief to resources encumbered by licensing and Material Transfer Agreements. For decades, mouse strains have provided an exquisite experimental tool to study the pathophysiology of the disease and assess therapeutic options in a genetically defined system. However, a major limitation of the mouse has been the limited genetic diversity associated with common laboratory mice. This has been overcome with the recent development of the Collaborative Cross and Diversity Outbred mice. These strains provide new tools capable of replicating genetic diversity to that approaching the diversity found in human populations. The Collaborative Cross and Diversity Outbred strains thus provide a means to observe and characterize toxicity or efficacy of new therapeutic drugs for a given population. The combination of traditional and contemporary mouse genome editing tools, along with the addition of genetic diversity in new modeling systems, are synergistic and serve to make the mouse a better model for biomedical research, enhancing the potential for preclinical drug discovery and personalized medicine. PMID:28053071

Macrophages sustain HIV replication in vivo independently of T cells.

PubMed

Honeycutt, Jenna B; Wahl, Angela; Baker, Caroline; Spagnuolo, Rae Ann; Foster, John; Zakharova, Oksana; Wietgrefe, Stephen; Caro-Vegas, Carolina; Madden, Victoria; Sharpe, Garrett; Haase, Ashley T; Eron, Joseph J; Garcia, J Victor

2016-04-01

Macrophages have long been considered to contribute to HIV infection of the CNS; however, a recent study has contradicted this early work and suggests that myeloid cells are not an in vivo source of virus production. Here, we addressed the role of macrophages in HIV infection by first analyzing monocytes isolated from viremic patients and patients undergoing antiretroviral treatment. We were unable to find viral DNA or viral outgrowth in monocytes isolated from peripheral blood. To determine whether tissue macrophages are productively infected, we used 3 different but complementary humanized mouse models. Two of these models (bone marrow/liver/thymus [BLT] mice and T cell-only mice [ToM]) have been previously described, and the third model was generated by reconstituting immunodeficient mice with human CD34+ hematopoietic stem cells that were devoid of human T cells (myeloid-only mice [MoM]) to specifically evaluate HIV replication in this population. Using MoM, we demonstrated that macrophages can sustain HIV replication in the absence of T cells; HIV-infected macrophages are distributed in various tissues including the brain; replication-competent virus can be rescued ex vivo from infected macrophages; and infected macrophages can establish de novo infection. Together, these results demonstrate that macrophages represent a genuine target for HIV infection in vivo that can sustain and transmit infection.

Macrophages sustain HIV replication in vivo independently of T cells

PubMed Central

Wahl, Angela; Baker, Caroline; Spagnuolo, Rae Ann; Foster, John; Zakharova, Oksana; Wietgrefe, Stephen; Caro-Vegas, Carolina; Sharpe, Garrett; Haase, Ashley T.; Eron, Joseph J.; Garcia, J. Victor

2016-01-01

Macrophages have long been considered to contribute to HIV infection of the CNS; however, a recent study has contradicted this early work and suggests that myeloid cells are not an in vivo source of virus production. Here, we addressed the role of macrophages in HIV infection by first analyzing monocytes isolated from viremic patients and patients undergoing antiretroviral treatment. We were unable to find viral DNA or viral outgrowth in monocytes isolated from peripheral blood. To determine whether tissue macrophages are productively infected, we used 3 different but complementary humanized mouse models. Two of these models (bone marrow/liver/thymus [BLT] mice and T cell–only mice [ToM]) have been previously described, and the third model was generated by reconstituting immunodeficient mice with human CD34+ hematopoietic stem cells that were devoid of human T cells (myeloid-only mice [MoM]) to specifically evaluate HIV replication in this population. Using MoM, we demonstrated that macrophages can sustain HIV replication in the absence of T cells; HIV-infected macrophages are distributed in various tissues including the brain; replication-competent virus can be rescued ex vivo from infected macrophages; and infected macrophages can establish de novo infection. Together, these results demonstrate that macrophages represent a genuine target for HIV infection in vivo that can sustain and transmit infection. PMID:26950420

Vaginal Exposure to Zika Virus during Pregnancy Leads to Fetal Brain Infection.

PubMed

Yockey, Laura J; Varela, Luis; Rakib, Tasfia; Khoury-Hanold, William; Fink, Susan L; Stutz, Bernardo; Szigeti-Buck, Klara; Van den Pol, Anthony; Lindenbach, Brett D; Horvath, Tamas L; Iwasaki, Akiko

2016-08-25

Zika virus (ZIKV) can be transmitted sexually between humans. However, it is unknown whether ZIKV replicates in the vagina and impacts the unborn fetus. Here, we establish a mouse model of vaginal ZIKV infection and demonstrate that, unlike other routes, ZIKV replicates within the genital mucosa even in wild-type (WT) mice. Mice lacking RNA sensors or transcription factors IRF3 and IRF7 resulted in higher levels of local viral replication. Furthermore, mice lacking the type I interferon (IFN) receptor (IFNAR) became viremic and died of infection after a high-dose vaginal ZIKV challenge. Notably, vaginal infection of pregnant dams during early pregnancy led to fetal growth restriction and infection of the fetal brain in WT mice. This was exacerbated in mice deficient in IFN pathways, leading to abortion. Our study highlights the vaginal tract as a highly susceptible site of ZIKV replication and illustrates the dire disease consequences during pregnancy. Copyright © 2016 Elsevier Inc. All rights reserved.

Resveratrol enhances HBV replication through activating Sirt1-PGC-1α-PPARα pathway.

PubMed

Shi, Yixian; Li, Yongjun; Huang, Chenjie; Ying, Lixiong; Xue, Jihua; Wu, Haicong; Chen, Zhi; Yang, Zhenggang

2016-04-21

The population of hepatitis B combined with a number of metabolic disorders is increasing significantly. Resveratrol (RSV) has been used as a preclinical drug for the treatment of the metabolic disorders. However, the impact of RSV on HBV replication remains unknown. In this study, the HBV-expressing hepatocelluar carcinoma cell line and mouse model created by hydrodynamic injection of viral DNA were used. We found that RSV activates Sirt1, which in turn deacetylates PGC-1α and subsequently increases the transcriptional activity of PPARα, leading to the enhanced HBV transcription and replication in vitro and in vivo. In addition, we found that this pathway is also required for fasting-induced HBV transcription. Taken together, this study identifies that RSV enhances HBV transcription and replication especially acting on the core promoter, which depends on Sirt1-PGC-1α-PPARα pathway. We conclude that RSV may exacerbate the progression of hepatitis B and that patients with hepatitis B infection should be cautious taking RSV as a dietary supplement.

ATM supports gammaherpesvirus replication by attenuating type I interferon pathway.

PubMed

Darrah, Eric J; Stoltz, Kyle P; Ledwith, Mitchell; Tarakanova, Vera L

2017-10-01

Ataxia-Telangiectasia mutated (ATM) kinase participates in multiple networks, including DNA damage response, oxidative stress, and mitophagy. ATM also supports replication of diverse DNA and RNA viruses. Gammaherpesviruses are prevalent cancer-associated viruses that benefit from ATM expression during replication. This proviral role of ATM had been ascribed to its signaling within the DNA damage response network; other functions of ATM have not been considered. In this study increased type I interferon (IFN) responses were observed in ATM deficient gammaherpesvirus-infected macrophages. Using a mouse model that combines ATM and type I IFN receptor deficiencies we show that increased type I IFN response in the absence of ATM fully accounts for the proviral role of ATM during gammaherpesvirus replication. Further, increased type I IFN response rendered ATM deficient macrophages more susceptible to antiviral effects of type II IFN. This study identifies attenuation of type I IFN responses as the primary mechanism underlying proviral function of ATM during gammaherpesvirus infection. Copyright © 2017 Elsevier Inc. All rights reserved.

Absence of Prenatal Forebrain Defects in the Dp(16)1Yey/+ Mouse Model of Down Syndrome

PubMed Central

Goodliffe, Joseph W.; Olmos-Serrano, Jose Luis; Aziz, Nadine M.; Pennings, Jeroen L.A.; Guedj, Faycal; Bianchi, Diana W.

2016-01-01

Studies in humans with Down syndrome (DS) show that alterations in fetal brain development are followed by postnatal deficits in neuronal numbers, synaptic plasticity, and cognitive and motor function. This same progression is replicated in several mouse models of DS. Dp(16)1Yey/+ (hereafter called Dp16) is a recently developed mouse model of DS in which the entire region of mouse chromosome 16 that is homologous to human chromosome 21 has been triplicated. As such, Dp16 mice may more closely reproduce neurodevelopmental changes occurring in humans with DS. Here, we present the first comprehensive cellular and behavioral study of the Dp16 forebrain from embryonic to adult stages. Unexpectedly, our results demonstrate that Dp16 mice do not have prenatal brain defects previously reported in human fetal neocortex and in the developing forebrains of other mouse models, including microcephaly, reduced neurogenesis, and abnormal cell proliferation. Nevertheless, we found impairments in postnatal developmental milestones, fewer inhibitory forebrain neurons, and deficits in motor and cognitive performance in Dp16 mice. Therefore, although this new model does not express prenatal morphological phenotypes associated with DS, abnormalities in the postnatal period appear sufficient to produce significant cognitive deficits in Dp16. SIGNIFICANCE STATEMENT Down syndrome (DS) leads to intellectual disability. Several mouse models have increased our understanding of the neuropathology of DS and are currently being used to test therapeutic strategies. A new mouse model that contains an expanded number of DS-related genes, known as Dp(16)1Yey/+ (Dp16), has been generated recently. We sought to determine whether the extended triplication creates a better phenocopy of DS-related brain pathologies. We measured embryonic development, forebrain maturation, and perinatal/adult behavior and revealed an absence of prenatal phenotypes in Dp16 fetal brain, but specific cellular and behavioral deficits after the first 2 postnatal weeks. These results uncover important differences in prenatal phenotype between Dp16 animals and humans with DS and other DS mouse models. PMID:26961948

Of Faeces and Sweat. How Much a Mouse is Willing to Run: Having a Hard Time Measuring Spontaneous Physical Activity in Different Mouse Sub-Strains.

PubMed

Coletti, Dario; Adamo, Sergio; Moresi, Viviana

2017-02-24

Invited Letter to the Editor. Physical activity has multiple beneficial effects in the physiology and pathology of the organism. In particular, we and other groups have shown that running counteracts cancer cachexia in both humans and rodents. The latter are prone to exercise in wheel-equipped cages even at advanced stages of cachexia. However, when we wanted to replicate the experimental model routinely used at the University of Rome in a different laboratory (i.e. at Paris 6 University), we had to struggle with puzzling results due to unpredicted mouse behavior. Here we report the experience and offer the explanation underlying these apparently irreproducible results. The original data are currently used for teaching purposes in undergraduate student classes of biological sciences.

Mouse Norovirus infection promotes autophagy induction to facilitate replication but prevents final autophagosome maturation

DOE Office of Scientific and Technical Information (OSTI.GOV)

O’Donnell, Tanya B.; Hyde, Jennifer L.; Mintern, Justine D.

Autophagy is a cellular process used to eliminate intracellular pathogens. Many viruses however are able to manipulate this cellular process for their own advantage. Here we demonstrate that Mouse Norovirus (MNV) infection induces autophagy but does not appear to utilise the autophagosomal membrane for establishment and formation of the viral replication complex. We have observed that MNV infection results in lipidation and recruitment of LC3 to the autophagosome membrane but prevents subsequent fusion of the autophagosomes with lysosomes, as SQSTM1 (an autophagy receptor) accumulates and Lysosome-Associated Membrane Protein1 is sequestered to the MNV replication complex (RC) rather than to autophagosomes.more » We have additionally observed that chemical modulation of autophagy differentially affects MNV replication. From this study we can conclude that MNV infection induces autophagy, however suppresses the final maturation step of this response, indicating that autophagy induction contributes to MNV replication independently of RC biogenesis. - Highlights: • MNV induces autophagy in infected murine macrophages. • MNV does not utilise autophagosomal membranes for replication. • The MNV-induced autophagosomes do not fuse with lysosomes. • MNV sequesters SQSTM1 to prevent autophagy degradation and turnover. • Chemical modulation of autophagy enhances MNV replication.« less

Use of Galleria mellonella as a model organism to study Legionella pneumophila infection.

PubMed

Harding, Clare R; Schroeder, Gunnar N; Collins, James W; Frankel, Gad

2013-11-22

Legionella pneumophila, the causative agent of a severe pneumonia named Legionnaires' disease, is an important human pathogen that infects and replicates within alveolar macrophages. Its virulence depends on the Dot/Icm type IV secretion system (T4SS), which is essential to establish a replication permissive vacuole known as the Legionella containing vacuole (LCV). L. pneumophila infection can be modeled in mice however most mouse strains are not permissive, leading to the search for novel infection models. We have recently shown that the larvae of the wax moth Galleria mellonella are suitable for investigation of L. pneumophila infection. G. mellonella is increasingly used as an infection model for human pathogens and a good correlation exists between virulence of several bacterial species in the insect and in mammalian models. A key component of the larvae's immune defenses are hemocytes, professional phagocytes, which take up and destroy invaders. L. pneumophila is able to infect, form a LCV and replicate within these cells. Here we demonstrate protocols for analyzing L. pneumophila virulence in the G. mellonella model, including how to grow infectious L. pneumophila, pretreat the larvae with inhibitors, infect the larvae and how to extract infected cells for quantification and immunofluorescence microscopy. We also describe how to quantify bacterial replication and fitness in competition assays. These approaches allow for the rapid screening of mutants to determine factors important in L. pneumophila virulence, describing a new tool to aid our understanding of this complex pathogen.

Chloroquine inhibited Ebola virus replication in vitro but failed to protect against infection and disease in the in vivo guinea pig model.

PubMed

Dowall, Stuart D; Bosworth, Andrew; Watson, Robert; Bewley, Kevin; Taylor, Irene; Rayner, Emma; Hunter, Laura; Pearson, Geoff; Easterbrook, Linda; Pitman, James; Hewson, Roger; Carroll, Miles W

2015-12-01

Ebola virus (EBOV) is highly pathogenic, with a predisposition to cause outbreaks in human populations accompanied by significant mortality. Owing to the lack of approved therapies, screening programmes of potentially efficacious drugs have been undertaken. One of these studies has demonstrated the possible utility of chloroquine against EBOV using pseudotyped assays. In mouse models of EBOV disease there are conflicting reports of the therapeutic effects of chloroquine. There are currently no reports of its efficacy using the larger and more stringent guinea pig model of infection. In this study we have shown that replication of live EBOV is impaired by chloroquine in vitro. However, no protective effects were observed in vivo when EBOV-infected guinea pigs were treated with chloroquine. These results advocate that chloroquine should not be considered as a treatment strategy for EBOV.

Characterizing the Relationship Between Blast Exposure and Mild TBI with Dynamic Modeling and Testing in a New Mouse Model

DTIC Science & Technology

2011-07-01

and lower surfaces by two steel plates. To represent an infinite MT, a 2 nm long model is constructed with symmetric boundary conditions on each...are 2 nm wide. The AFM tip with a radius of 20 nm is loaded with a distributed loading on the top surface. The base of the bottom steel plate is...constrained rigidly in all directions, again to replicate the AFM experiments. It should be noted that the axial faces have symmetric boundary

Depth-resolved analytical model and correction algorithm for photothermal optical coherence tomography

PubMed Central

Lapierre-Landry, Maryse; Tucker-Schwartz, Jason M.; Skala, Melissa C.

2016-01-01

Photothermal OCT (PT-OCT) is an emerging molecular imaging technique that occupies a spatial imaging regime between microscopy and whole body imaging. PT-OCT would benefit from a theoretical model to optimize imaging parameters and test image processing algorithms. We propose the first analytical PT-OCT model to replicate an experimental A-scan in homogeneous and layered samples. We also propose the PT-CLEAN algorithm to reduce phase-accumulation and shadowing, two artifacts found in PT-OCT images, and demonstrate it on phantoms and in vivo mouse tumors. PMID:27446693

Mouse embryonic stem cells have increased capacity for replication fork restart driven by the specific Filia-Floped protein complex.

PubMed

Zhao, Bo; Zhang, Weidao; Cun, Yixian; Li, Jingzheng; Liu, Yan; Gao, Jing; Zhu, Hongwen; Zhou, Hu; Zhang, Rugang; Zheng, Ping

2018-01-01

Pluripotent stem cells (PSCs) harbor constitutive DNA replication stress during their rapid proliferation and the consequent genome instability hampers their applications in regenerative medicine. It is therefore important to understand the regulatory mechanisms of replication stress response in PSCs. Here, we report that mouse embryonic stem cells (ESCs) are superior to differentiated cells in resolving replication stress. Specifically, ESCs utilize a unique Filia-Floped protein complex-dependent mechanism to efficiently promote the restart of stalled replication forks, therefore maintaining genomic stability. The ESC-specific Filia-Floped complex resides on replication forks under normal conditions. Replication stress stimulates their recruitment to stalling forks and the serine 151 residue of Filia is phosphorylated in an ATR-dependent manner. This modification enables the Filia-Floped complex to act as a functional scaffold, which then promotes the stalling fork restart through a dual mechanism: both enhancing recruitment of the replication fork restart protein, Blm, and stimulating ATR kinase activation. In the Blm pathway, the scaffolds recruit the E3 ubiquitin ligase, Trim25, to the stalled replication forks, and in turn Trim25 tethers and concentrates Blm at stalled replication forks through ubiquitination. In differentiated cells, the recruitment of the Trim25-Blm complex to replication forks and the activation of ATR signaling are much less robust due to lack of the ESC-specific Filia-Floped scaffold. Thus, our study reveals that ESCs utilize an additional and unique regulatory layer to efficiently promote the stalled fork restart and maintain genomic stability.

Replication of Salmonella enterica Serovar Typhimurium in Human Monocyte-Derived Macrophages

PubMed Central

Lathrop, Stephanie K.; Binder, Kelsey A.; Starr, Tregei; Cooper, Kendal G.; Chong, Audrey; Carmody, Aaron B.

2015-01-01

Salmonella enterica serovar Typhimurium is a common cause of food-borne gastrointestinal illness, but additionally it causes potentially fatal bacteremia in some immunocompromised patients. In mice, systemic spread and replication of the bacteria depend upon infection of and replication within macrophages, but replication in human macrophages is not widely reported or well studied. In order to assess the ability of Salmonella Typhimurium to replicate in human macrophages, we infected primary monocyte-derived macrophages (MDM) that had been differentiated under conditions known to generate different phenotypes. We found that replication in MDM depends greatly upon the phenotype of the cells, as M1-skewed macrophages did not allow replication, while M2a macrophages and macrophages differentiated with macrophage colony-stimulating factor (M-CSF) alone (termed M0) did. We describe how additional conditions that alter the macrophage phenotype or the gene expression of the bacteria affect the outcome of infection. In M0 MDM, the temporal expression of representative genes from Salmonella pathogenicity islands 1 and 2 (SPI1 and SPI2) and the importance of the PhoP/Q two-component regulatory system are similar to what has been shown in mouse macrophages. However, in contrast to mouse macrophages, where replication is SPI2 dependent, we observed early SPI2-independent replication in addition to later SPI2-dependent replication in M0 macrophages. Only SPI2-dependent replication was associated with death of the host cell at later time points. Altogether, our results reveal a very nuanced interaction between Salmonella and human macrophages. PMID:25895967

Replication of Salmonella enterica Serovar Typhimurium in Human Monocyte-Derived Macrophages.

PubMed

Lathrop, Stephanie K; Binder, Kelsey A; Starr, Tregei; Cooper, Kendal G; Chong, Audrey; Carmody, Aaron B; Steele-Mortimer, Olivia

2015-07-01

Salmonella enterica serovar Typhimurium is a common cause of food-borne gastrointestinal illness, but additionally it causes potentially fatal bacteremia in some immunocompromised patients. In mice, systemic spread and replication of the bacteria depend upon infection of and replication within macrophages, but replication in human macrophages is not widely reported or well studied. In order to assess the ability of Salmonella Typhimurium to replicate in human macrophages, we infected primary monocyte-derived macrophages (MDM) that had been differentiated under conditions known to generate different phenotypes. We found that replication in MDM depends greatly upon the phenotype of the cells, as M1-skewed macrophages did not allow replication, while M2a macrophages and macrophages differentiated with macrophage colony-stimulating factor (M-CSF) alone (termed M0) did. We describe how additional conditions that alter the macrophage phenotype or the gene expression of the bacteria affect the outcome of infection. In M0 MDM, the temporal expression of representative genes from Salmonella pathogenicity islands 1 and 2 (SPI1 and SPI2) and the importance of the PhoP/Q two-component regulatory system are similar to what has been shown in mouse macrophages. However, in contrast to mouse macrophages, where replication is SPI2 dependent, we observed early SPI2-independent replication in addition to later SPI2-dependent replication in M0 macrophages. Only SPI2-dependent replication was associated with death of the host cell at later time points. Altogether, our results reveal a very nuanced interaction between Salmonella and human macrophages. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Requirement of Sur2 for Efficient Replication of Mouse Adenovirus Type 1

PubMed Central

Fang, Lei; Stevens, Jennitte L.; Berk, Arnold J.; Spindler, Katherine R.

2004-01-01

Mouse adenovirus type 1 (MAV-1) early region 1A (E1A) encodes a virulence gene in viral infection of mice. To broaden our understanding of the functions of E1A in MAV-1 pathogenesis, an unbiased experimental approach, glutathione S-transferase (GST) pulldown, was used to screen for cellular proteins that interact with E1A protein. We identified mouse Sur2, a subunit of Mediator complex, as a protein that binds to MAV-1 E1A. The interaction between Sur2 and MAV-1 E1A was confirmed in virus-infected cells. Conserved region 3 (CR3) of MAV-1 E1A was mapped as the region required for Sur2-E1A interaction, as is the case for human adenovirus E1A. Although it has been proposed that human adenovirus E1A recruits the Mediator complex to transactivate transcription of viral early genes, Sur2 function in adenovirus replication has not been directly tested previously. Studies on the functions of Sur2 with mouse embryonic fibroblasts (MEFs) showed that there was a multiplicity-dependent growth defect of MAV-1 in Sur2−/− MEFs compared to Sur2+/+ MEFs. Comparison of the viral DNA and viral mRNA levels in Sur2+/+ and Sur2−/− MEFs confirmed that Sur2 was important for efficient viral replication. The viral replication defects in Sur2−/− MEFs appeared to be due at least in part to a defect in viral early gene transcription. PMID:15542641

Resistance to Rhabdoviridae Infection and Subversion of Antiviral Responses.

PubMed

Blondel, Danielle; Maarifi, Ghizlane; Nisole, Sébastien; Chelbi-Alix, Mounira K

2015-07-07

Interferon (IFN) treatment induces the expression of hundreds of IFN-stimulated genes (ISGs). However, only a selection of their products have been demonstrated to be responsible for the inhibition of rhabdovirus replication in cultured cells; and only a few have been shown to play a role in mediating the antiviral response in vivo using gene knockout mouse models. IFNs inhibit rhabdovirus replication at different stages via the induction of a variety of ISGs. This review will discuss how individual ISG products confer resistance to rhabdoviruses by blocking viral entry, degrading single stranded viral RNA, inhibiting viral translation or preventing release of virions from the cell. Furthermore, this review will highlight how these viruses counteract the host IFN system.

Prolonged diet induced obesity has minimal effects towards brain pathology in mouse model of cerebral amyloid angiopathy: implications for studying obesity-brain interactions in mice.

PubMed

Zhang, Le; Dasuri, Kalavathi; Fernandez-Kim, Sun-Ok; Bruce-Keller, Annadora J; Freeman, Linnea R; Pepping, Jennifer K; Beckett, Tina L; Murphy, M Paul; Keller, Jeffrey N

2013-09-01

Cerebral amyloid angiopathy (CAA) occurs in nearly every individual with Alzheimer's disease (AD) and Down's syndrome, and is the second largest cause of intracerebral hemorrhage. Mouse models of CAA have demonstrated evidence for increased gliosis contributing to CAA pathology. Nearly two thirds of Americans are overweight or obese, with little known about the effects of obesity on the brain, although increasingly the vasculature appears to be a principle target of obesity effects on the brain. In the current study we describe for the first time whether diet induced obesity (DIO) modulates glial reactivity, amyloid levels, and inflammatory signaling in a mouse model of CAA. In these studies we identify surprisingly that DIO does not significantly increase Aβ levels, astrocyte (GFAP) or microglial (IBA-1) gliosis in the CAA mice. However, within the hippocampal gyri a localized increase in reactive microglia were increased in the CA1 and stratum oriens relative to CAA mice on a control diet. DIO was observed to selectively increase IL-6 in CAA mice, with IL-1β and TNF-α not increased in CAA mice in response to DIO. Taken together, these data show that prolonged DIO has only modest effects towards Aβ in a mouse model of CAA, but appears to elevate some localized microglial reactivity within the hippocampal gyri and selective markers of inflammatory signaling. These data are consistent with the majority of the existing literature in other models of Aβ pathology, which surprisingly show a mixed profile of DIO effects towards pathological processes in mouse models of neurodegenerative disease. The importance for considering the potential impact of ceiling effects in pathology within mouse models of Aβ pathogenesis, and the current experimental limitations for DIO in mice to fully replicate metabolic dysfunction present in human obesity, are discussed. This article is part of a Special Issue entitled: Animal Models of Disease. Copyright © 2012. Published by Elsevier B.V.

Adaptive Immunity Restricts Replication of Novel Murine Astroviruses

PubMed Central

Yokoyama, Christine C.; Loh, Joy; Zhao, Guoyan; Stappenbeck, Thaddeus S.; Wang, David; Huang, Henry V.

2012-01-01

The mechanisms of astrovirus pathogenesis are largely unknown, in part due to a lack of a small-animal model of disease. Using shotgun sequencing and a custom analysis pipeline, we identified two novel astroviruses capable of infecting research mice, murine astrovirus (MuAstV) STL1 and STL2. Subsequent analysis revealed the presence of at least two additional viruses (MuAstV STL3 and STL4), suggestive of a diverse population of murine astroviruses in research mice. Complete genomic characterization and subsequent phylogenetic analysis showed that MuAstV STL1 to STL4 are members of the mamastrovirus genus and are likely members of a new mamastrovirus genogroup. Using Rag1−/− mice deficient in B and T cells, we demonstrate that adaptive immunity is required to control MuAstV infection. Furthermore, using Stat1−/− mice deficient in innate signaling, we demonstrate a role for the innate immune response in the control of MuAstV replication. Our results demonstrate that MuAstV STL permits the study of the mechanisms of astrovirus infection and host-pathogen interactions in a genetically manipulable small-animal model. Finally, we detected MuAstV in commercially available mice, suggesting that these viruses may be present in academic and commercial research mouse facilities, with possible implications for interpretation of data generated in current mouse models of disease. PMID:22951832

SerpinB1 Promotes Pancreatic β Cell Proliferation

DOE Office of Scientific and Technical Information (OSTI.GOV)

El Ouaamari, Abdelfattah; Dirice, Ercument; Gedeon, Nicholas

2016-01-01

Compensatory β-cell growth in response to insulin resistance is a common feature in diabetes. We recently reported that liver-derived factors participate in this compensatory response in the liver insulin receptor knockout (LIRKO) mouse, a model of significant islet hyperplasia. Here we show that serpinB1 is a liver-derived secretory protein that controls β-cell proliferation. SerpinB1 is abundant in the hepatocyte secretome and sera derived from LIRKO mice. SerpinB1 and small molecule compounds that partially mimic serpinB1 activity enhanced proliferation of zebrafish, mouse and human β-cells. We report that serpinB1-induced β-cell replication requires protease inhibition activity and mice lacking serpinB1 exhibit attenuatedmore » β-cell replication in response to insulin resistance. Finally, SerpinB1-treatment of islets modulated signaling proteins in growth and survival pathways such as MAPK, PKA and GSK3. Together, these data implicate SerpinB1 as a protein that can potentially be harnessed to enhance functional β-cell mass in patients with diabetes.« less

The Regenerative Response of Endogenous Neural Stem/Progenitor Cells to Traumatic Brain Injury

DTIC Science & Technology

2014-06-09

Genevieve M. Sullivan, Molecular and Cell Biology. 2014 Thesis directed by: Dr. Regina C. Armstrong, PhD, APG The complex pathological mechanisms ...treatments for TBI (83 ). Therefore it is necessary to investigate the complex pathological and molecular mechanisms that occur after heterogeneous...of cellular mechanisms that is not an option in other species with gyrencephalic brains. Therefore, even though a mouse model cannot fully replicate

Evaluation of stem cell reserve using serial bone marrow transplantation and competitive repopulation in a murine model of chronic hemolytic anemia

DOE Office of Scientific and Technical Information (OSTI.GOV)

Maggio-Price, L.; Wolf, N.S.; Priestley, G.V.

1988-09-01

Serial transplantation and competitive repopulation were used to evaluate any loss of self-replicative capacity of bone marrow stem cells in a mouse model with increased and persistent hemopoietic demands. Congenic marrows from old control and from young and old mice with hereditary spherocytic anemia (sphha/sphha) were serially transplanted at 35-day intervals into normal irradiated recipients. Old anemic marrow failed or reverted to recipient karyotype at a mean of 3.5 transplants, and young anemic marrow reverted at a mean of 4.0 transplants, whereas controls did so at a mean of 5.0 transplants. In a competitive assay in which a mixture ofmore » anemic and control marrow was transplanted, the anemic marrow persisted to 10 months following transplantation; anemic marrow repopulation was greater if anemic marrow sex matched with the host. It is possible that lifelong stress of severe anemia decreases stem cell reserve in the anemic sphha/sphha mouse marrow. However, marginal differences in serial transplantation number and the maintenance of anemic marrow in a competition assay would suggest that marrow stem cells, under prolonged stress, are capable of exhibiting good repopulating and self-replicating abilities.« less

Virus-encoded miRNAs in Ebola virus disease.

PubMed

Duy, Janice; Honko, Anna N; Altamura, Louis A; Bixler, Sandra L; Wollen-Roberts, Suzanne; Wauquier, Nadia; O'Hearn, Aileen; Mucker, Eric M; Johnson, Joshua C; Shamblin, Joshua D; Zelko, Justine; Botto, Miriam A; Bangura, James; Coomber, Moinya; Pitt, M Louise; Gonzalez, Jean-Paul; Schoepp, Randal J; Goff, Arthur J; Minogue, Timothy D

2018-04-24

Ebola virus (EBOV) is a negative-strand RNA virus that replicates in the cytoplasm and causes an often-fatal hemorrhagic fever. EBOV, like other viruses, can reportedly encode its own microRNAs (miRNAs) to subvert host immune defenses. miRNAs are short noncoding RNAs that can regulate gene expression by hybridizing to multiple mRNAs, and viral miRNAs can enhance viral replication and infectivity by regulating host or viral genes. To date, only one EBOV miRNA has been examined in human infection. Here, we assayed mouse, rhesus macaque, cynomolgus macaque, and human samples infected with three EBOV variants for twelve computationally predicted viral miRNAs using RT-qPCR. Ten miRNAs aligned to EBOV variants and were detectable in the four species during disease with several viral miRNAs showing presymptomatic amplification in animal models. miRNA abundances in both the mouse and nonhuman primate models mirrored the human cohort, with miR-1-5p, miR-1-3p, and miR-T3-3p consistently at the highest levels. These striking similarities in the most abundant miRNAs during infection with different EBOV variants and hosts indicate that these miRNAs are potential valuable diagnostic markers and key effectors of EBOV pathogenesis.

Analysis of HSV viral reactivation in explants of sensory neurons

PubMed Central

Turner, Anne-Marie W.; Kristie, Thomas M.

2014-01-01

As with all Herpesviruses, Herpes simplex virus (HSV) has both a lytic replication phase and a latency-reactivation cycle. During lytic replication, there is an ordered cascade of viral gene expression that leads to the synthesis of infectious viral progeny. In contrast, latency is characterized by the lack of significant lytic gene expression and the absence of infectious virus. Reactivation from latency is characterized by the re-entry of the virus into the lytic replication cycle and the production of recurrent disease. This unit describes the establishment of the mouse sensory neuron model of HSV-1 latency-reactivation as a useful in vivo system for the analysis of mechanisms involved in latency and reactivation. Assays including the determination of viral yields, immunohistochemical/immunofluorescent detection of viral antigens, and mRNA quantitation are used in experiments designed to investigate the network of cellular and viral proteins regulating HSV-1 lytic infection, latency, and reactivation. PMID:25367271

Immunization against Genital Herpes with a Vaccine Virus That has Defects in Productive and Latent Infection

NASA Astrophysics Data System (ADS)

da Costa, Xavier J.; Jones, Cheryl A.; Knipe, David M.

1999-06-01

An effective vaccine for genital herpes has been difficult to achieve because of the limited efficacy of subunit vaccines and the safety concerns about live viruses. As an alternative approach, mutant herpes simplex virus strains that are replication-defective can induce protective immunity. To increase the level of safety and to prove that replication was not needed for immunization, we constructed a mutant herpes simplex virus 2 strain containing two deletion mutations, each of which eliminated viral replication. The double-mutant virus induces protective immunity that can reduce acute viral shedding and latent infection in a mouse genital model, but importantly, the double-mutant virus shows a phenotypic defect in latent infection. This herpes vaccine strain, which is immunogenic but has defects in both productive and latent infection, provides a paradigm for the design of vaccines and vaccine vectors for other sexually transmitted diseases, such as AIDS.

Cyclooxygenase-2 facilitates dengue virus replication and serves as a potential target for developing antiviral agents.

PubMed

Lin, Chun-Kuang; Tseng, Chin-Kai; Wu, Yu-Hsuan; Liaw, Chih-Chuang; Lin, Chun-Yu; Huang, Chung-Hao; Chen, Yen-Hsu; Lee, Jin-Ching

2017-03-20

Cyclooxygenase-2 (COX-2) is one of the important mediators of inflammation in response to viral infection, and it contributes to viral replication, for example, cytomegalovirus or hepatitis C virus replication. The role of COX-2 in dengue virus (DENV) replication remains unclear. In the present study, we observed an increased level of COX-2 in patients with dengue fever compared with healthy donors. Consistent with the clinical data, an elevated level of COX-2 expression was also observed in DENV-infected ICR suckling mice. Using cell-based experiments, we revealed that DENV-2 infection significantly induced COX-2 expression and prostaglandin E 2 (PGE 2 ) production in human hepatoma Huh-7 cells. The exogenous expression of COX-2 or PGE 2 treatment dose-dependently enhanced DENV-2 replication. In contrast, COX-2 gene silencing and catalytic inhibition sufficiently suppressed DENV-2 replication. In an ICR suckling mouse model, we identified that the COX-2 inhibitor NS398 protected mice from succumbing to life-threatening DENV-2 infection. By using COX-2 promoter-based analysis and specific inhibitors against signaling molecules, we identified that NF-κB and MAPK/JNK are critical factors for DENV-2-induced COX-2 expression and viral replication. Altogether, our results reveal that COX-2 is an important factor for DENV replication and can serve as a potential target for developing therapeutic agents against DENV infection.

Repeatability of Mice Consumption Discrimination of Wheat (Triticum aestivum L.) Varieties across Field Experiments and Mouse Cohorts.

PubMed

Kiszonas, Alecia M; Fuerst, E Patrick; Morris, Craig F

2015-07-01

Whole grain wheat (Triticum aestivum L.) foods can provide critical nutrients for health and nutrition in the human diet. Potential flavor differences among varieties can be examined using consumption discrimination of the house mouse (Mus musculus L.) as a model system. This study examines consistency and repeatability of the mouse model and potentially, wheat grain flavor. A single elimination tournament design was used to measure relative consumption preference for hard red spring and hard white spring varieties across all 3 experiments in combination with 2 mouse cohorts. Fifteen replicate mice were used in 24-h trials to examine differences in preference among paired wheat varieties until an overall "winner" was established as the most highly preferred variety of wheat. In all 3 experiment-cohort combinations, the same varieties were preferred as the "winner" of both the hard red spring and hard white spring wheat varieties, Hollis and BR 7030, respectively. Despite the consistent preference for these varieties across experiments, the degree (magnitude) to which the mice preferred these varieties varied across experiments. For the hard white spring wheat varieties, the small number of varieties and confounding effects of experiment and cohort limited our ability to accurately gauge repeatability. Conversely, for the hard red spring wheat varieties, consumption preferences were consistent across experiments and mice cohorts. The single-elimination tournament model was effective in providing repeatable results in an effort to more fully understand the mouse model system and possible flavor differences among wheat varieties. The mouse model system used here is effective in identifying wheat varieties that may be more or less desirable to humans in whole wheat foods. The system identifies consistent differences across different mouse cohorts and crop years. Published 2015. This article is a U.S. Government work and is in the public domain in the USA.

Dengue virus requires the CC-chemokine receptor CCR5 for replication and infection development.

PubMed

Marques, Rafael E; Guabiraba, Rodrigo; Del Sarto, Juliana L; Rocha, Rebeca F; Queiroz, Ana Luiza; Cisalpino, Daniel; Marques, Pedro E; Pacca, Carolina C; Fagundes, Caio T; Menezes, Gustavo B; Nogueira, Maurício L; Souza, Danielle G; Teixeira, Mauro M

2015-08-01

Dengue is a mosquito-borne disease that affects millions of people worldwide yearly. Currently, there is no vaccine or specific treatment available. Further investigation on dengue pathogenesis is required to better understand the disease and to identify potential therapeutic targets. The chemokine system has been implicated in dengue pathogenesis, although the specific role of chemokines and their receptors remains elusive. Here we describe the role of the CC-chemokine receptor CCR5 in Dengue virus (DENV-2) infection. In vitro experiments showed that CCR5 is a host factor required for DENV-2 replication in human and mouse macrophages. DENV-2 infection induces the expression of CCR5 ligands. Incubation with an antagonist prevents CCR5 activation and reduces DENV-2 positive-stranded (+) RNA inside macrophages. Using an immunocompetent mouse model of DENV-2 infection we found that CCR5(-/-) mice were resistant to lethal infection, presenting at least 100-fold reduction of viral load in target organs and significant reduction in disease severity. This phenotype was reproduced in wild-type mice treated with CCR5-blocking compounds. Therefore, CCR5 is a host factor required for DENV-2 replication and disease development. Targeting CCR5 might represent a therapeutic strategy for dengue fever. These data bring new insights on the association between viral infections and the chemokine receptor CCR5. © 2015 John Wiley & Sons Ltd.

Efficacy of Favipiravir Alone and in Combination With Ribavirin in a Lethal, Immunocompetent Mouse Model of Lassa Fever.

PubMed

Oestereich, Lisa; Rieger, Toni; Lüdtke, Anja; Ruibal, Paula; Wurr, Stephanie; Pallasch, Elisa; Bockholt, Sabrina; Krasemann, Susanne; Muñoz-Fontela, César; Günther, Stephan

2016-03-15

We studied the therapeutic potential of favipiravir (T-705) for Lassa fever, both alone and in combination with ribavirin. Favipiravir suppressed Lassa virus replication in cell culture by 5 log10 units. In a novel lethal mouse model, it lowered the viremia level and the virus load in organs and normalized levels of cell-damage markers. Treatment with 300 mg/kg per day, commenced 4 days after infection, when the viremia level had reached 4 log10 virus particles/mL, rescued 100% of Lassa virus-infected mice. We found a synergistic interaction between favipiravir and ribavirin in vitro and an increased survival rate and extended survival time when combining suboptimal doses in vivo. © The Author 2015. Published by Oxford University Press for the Infectious Diseases Society of America.

Infection of Mouse Macrophages by Seasonal Influenza Viruses Can Be Restricted at the Level of Virus Entry and at a Late Stage in the Virus Life Cycle

PubMed Central

Londrigan, Sarah L.; Short, Kirsty R.; Ma, Joel; Gillespie, Leah; Rockman, Steven P.; Brooks, Andrew G.

2015-01-01

ABSTRACT Airway epithelial cells are susceptible to infection with seasonal influenza A viruses (IAV), resulting in productive virus replication and release. Macrophages (MΦ) are also permissive to IAV infection; however, virus replication is abortive. Currently, it is unclear how productive infection of MΦ is impaired or the extent to which seasonal IAV replicate in MΦ. Herein, we compared mouse MΦ and epithelial cells for their ability to support genomic replication and transcription, synthesis of viral proteins, assembly of virions, and release of infectious progeny following exposure to genetically defined IAV. We confirm that seasonal IAV differ in their ability to utilize cell surface receptors for infectious entry and that this represents one level of virus restriction. Following virus entry, we demonstrate synthesis of all eight segments of genomic viral RNA (vRNA) and mRNA, as well as seven distinct IAV proteins, in IAV-infected mouse MΦ. Although newly synthesized hemagglutinin (HA) and neuraminidase (NA) glycoproteins are incorporated into the plasma membrane and expressed at the cell surface, electron microscopy confirmed that virus assembly was defective in IAV-infected MΦ, defining a second level of restriction late in the virus life cycle. IMPORTANCE Seasonal influenza A viruses (IAV) and highly pathogenic avian influenza viruses (HPAI) infect macrophages, but only HPAI replicate productively in these cells. Herein, we demonstrate that impaired virus uptake into macrophages represents one level of restriction limiting infection by seasonal IAV. Following uptake, seasonal IAV do not complete productive replication in macrophages, representing a second level of restriction. Using murine macrophages, we demonstrate that productive infection is blocked late in the virus life cycle, such that virus assembly is defective and newly synthesized virions are not released. These studies represent an important step toward identifying host-encoded factors that block replication of seasonal IAV, but not HPAI, in macrophages. PMID:26423941

Infection of Mouse Macrophages by Seasonal Influenza Viruses Can Be Restricted at the Level of Virus Entry and at a Late Stage in the Virus Life Cycle.

PubMed

Londrigan, Sarah L; Short, Kirsty R; Ma, Joel; Gillespie, Leah; Rockman, Steven P; Brooks, Andrew G; Reading, Patrick C

2015-12-01

Airway epithelial cells are susceptible to infection with seasonal influenza A viruses (IAV), resulting in productive virus replication and release. Macrophages (MΦ) are also permissive to IAV infection; however, virus replication is abortive. Currently, it is unclear how productive infection of MΦ is impaired or the extent to which seasonal IAV replicate in MΦ. Herein, we compared mouse MΦ and epithelial cells for their ability to support genomic replication and transcription, synthesis of viral proteins, assembly of virions, and release of infectious progeny following exposure to genetically defined IAV. We confirm that seasonal IAV differ in their ability to utilize cell surface receptors for infectious entry and that this represents one level of virus restriction. Following virus entry, we demonstrate synthesis of all eight segments of genomic viral RNA (vRNA) and mRNA, as well as seven distinct IAV proteins, in IAV-infected mouse MΦ. Although newly synthesized hemagglutinin (HA) and neuraminidase (NA) glycoproteins are incorporated into the plasma membrane and expressed at the cell surface, electron microscopy confirmed that virus assembly was defective in IAV-infected MΦ, defining a second level of restriction late in the virus life cycle. Seasonal influenza A viruses (IAV) and highly pathogenic avian influenza viruses (HPAI) infect macrophages, but only HPAI replicate productively in these cells. Herein, we demonstrate that impaired virus uptake into macrophages represents one level of restriction limiting infection by seasonal IAV. Following uptake, seasonal IAV do not complete productive replication in macrophages, representing a second level of restriction. Using murine macrophages, we demonstrate that productive infection is blocked late in the virus life cycle, such that virus assembly is defective and newly synthesized virions are not released. These studies represent an important step toward identifying host-encoded factors that block replication of seasonal IAV, but not HPAI, in macrophages. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Primary Role for Toll-Like Receptor-Driven Tumor Necrosis Factor Rather than Cytosolic Immune Detection in Restricting Coxiella burnetii Phase II Replication within Mouse Macrophages

PubMed Central

Bradley, William P.; Boyer, Mark A.; Nguyen, Hieu T.; Birdwell, L. Dillon; Yu, Janet; Ribeiro, Juliana M.; Roy, Craig R.

2016-01-01

Coxiella burnetii replicates within permissive host cells by employing a Dot/Icm type IV secretion system (T4SS) to translocate effector proteins that direct the formation of a parasitophorous vacuole. C57BL/6 mouse macrophages restrict the intracellular replication of the C. burnetii Nine Mile phase II (NMII) strain. However, eliminating Toll-like receptor 2 (TLR2) permits bacterial replication, indicating that the restriction of bacterial replication is immune mediated. Here, we examined whether additional innate immune pathways are employed by C57BL/6 macrophages to sense and restrict NMII replication. In addition to the known role of TLR2 in detecting and restricting NMII infection, we found that TLR4 also contributes to cytokine responses but is not required to restrict bacterial replication. Furthermore, the TLR signaling adaptors MyD88 and Trif are required for cytokine responses and restricting bacterial replication. The C. burnetii NMII T4SS translocates bacterial products into C57BL/6 macrophages. However, there was little evidence of cytosolic immune sensing of NMII, as there was a lack of inflammasome activation, T4SS-dependent cytokine responses, and robust type I interferon (IFN) production, and these pathways were not required to restrict bacterial replication. Instead, endogenous tumor necrosis factor (TNF) produced upon TLR sensing of C. burnetii NMII was required to control bacterial replication. Therefore, our findings indicate a primary role for TNF produced upon immune detection of C. burnetii NMII by TLRs, rather than cytosolic PRRs, in enabling C57BL/6 macrophages to restrict bacterial replication. PMID:26787725

Topologically associating domains are stable units of replication-timing regulation.

PubMed

Pope, Benjamin D; Ryba, Tyrone; Dileep, Vishnu; Yue, Feng; Wu, Weisheng; Denas, Olgert; Vera, Daniel L; Wang, Yanli; Hansen, R Scott; Canfield, Theresa K; Thurman, Robert E; Cheng, Yong; Gülsoy, Günhan; Dennis, Jonathan H; Snyder, Michael P; Stamatoyannopoulos, John A; Taylor, James; Hardison, Ross C; Kahveci, Tamer; Ren, Bing; Gilbert, David M

2014-11-20

Eukaryotic chromosomes replicate in a temporal order known as the replication-timing program. In mammals, replication timing is cell-type-specific with at least half the genome switching replication timing during development, primarily in units of 400-800 kilobases ('replication domains'), whose positions are preserved in different cell types, conserved between species, and appear to confine long-range effects of chromosome rearrangements. Early and late replication correlate, respectively, with open and closed three-dimensional chromatin compartments identified by high-resolution chromosome conformation capture (Hi-C), and, to a lesser extent, late replication correlates with lamina-associated domains (LADs). Recent Hi-C mapping has unveiled substructure within chromatin compartments called topologically associating domains (TADs) that are largely conserved in their positions between cell types and are similar in size to replication domains. However, TADs can be further sub-stratified into smaller domains, challenging the significance of structures at any particular scale. Moreover, attempts to reconcile TADs and LADs to replication-timing data have not revealed a common, underlying domain structure. Here we localize boundaries of replication domains to the early-replicating border of replication-timing transitions and map their positions in 18 human and 13 mouse cell types. We demonstrate that, collectively, replication domain boundaries share a near one-to-one correlation with TAD boundaries, whereas within a cell type, adjacent TADs that replicate at similar times obscure replication domain boundaries, largely accounting for the previously reported lack of alignment. Moreover, cell-type-specific replication timing of TADs partitions the genome into two large-scale sub-nuclear compartments revealing that replication-timing transitions are indistinguishable from late-replicating regions in chromatin composition and lamina association and accounting for the reduced correlation of replication timing to LADs and heterochromatin. Our results reconcile cell-type-specific sub-nuclear compartmentalization and replication timing with developmentally stable structural domains and offer a unified model for large-scale chromosome structure and function.

Selective efficacy of zoledronic acid on metastasis in a patient-derived orthotopic xenograph (PDOX) nude-mouse model of human pancreatic cancer.

PubMed

Hiroshima, Yukihiko; Maawy, Ali A; Katz, Matthew H G; Fleming, Jason B; Bouvet, Michael; Endo, Itaru; Hoffman, Robert M

2015-03-01

Patient-derived orthotopic xenograft (PDOX) nude-mouse models replicate the behavior of clinical cancer, including metastasis. The objective of the study was to determine the efficacy of zoledronic acid (ZA) on metastasis of a patient-derived orthotopic xenograft (PDOX) nude-mouse model of pancreatic cancer. In the present study, we examined the efficacy of ZA on pancreatic cancer growth and metastasis in a PDOX nude-mouse model. ZA monotherapy did not significantly suppress primary tumor growth. However, the primary tumor weight of gemcitabine (GEM) and combination GEM + ZA-treated mice was significantly decreased compared to the control group (GEM: P = 0.003; GEM + ZA: P = 0.002). The primary tumor weight of GEM + ZA-treated mice was significantly decreased compared to GEM-treated mice (P = 0.016). The metastasis weight decreased in ZA- or GEM-treated mice compared to the control group (ZA: P = 0.009; GEM: P = 0.007. No metastasis was detected in combination GEM + ZA-treated mice compared to the control group (GEM + ZA; P = 0.005). The results of the present study indicate that ZA can selectively target metastasis in a pancreatic cancer PDOX model and that the combination of ZA and GEM should be evaluated clinically in the near future for this highly treatment-resistant disease. © 2014 Wiley Periodicals, Inc.

Human-Specific Adaptations in Vpu Conferring Anti-tetherin Activity Are Critical for Efficient Early HIV-1 Replication In Vivo.

PubMed

Yamada, Eri; Nakaoka, Shinji; Klein, Lukas; Reith, Elisabeth; Langer, Simon; Hopfensperger, Kristina; Iwami, Shingo; Schreiber, Gideon; Kirchhoff, Frank; Koyanagi, Yoshio; Sauter, Daniel; Sato, Kei

2018-01-10

The HIV-1-encoded accessory protein Vpu exerts several immunomodulatory functions, including counteraction of the host restriction factor tetherin, downmodulation of CD4, and inhibition of NF-κB activity to facilitate HIV-1 infection. However, the relative contribution of individual Vpu functions to HIV-1 infection in vivo remained unclear. Here, we used a humanized mouse model and HIV-1 strains with selective mutations in vpu to demonstrate that the anti-tetherin activity of Vpu is a prerequisite for efficient viral spread during the early phase of infection. Mathematical modeling and gain-of-function mutations in SIVcpz, the simian precursor of pandemic HIV-1, corroborate this finding. Blockage of interferon signaling combined with transcriptome analyses revealed that basal tetherin levels are sufficient to control viral replication. These results establish tetherin as a key effector of the intrinsic immune defense against HIV-1, and they demonstrate that Vpu-mediated tetherin antagonism is critical for efficient viral spread during the initial phase of HIV-1 replication. Copyright © 2017 Elsevier Inc. All rights reserved.

Verification of immune response optimality through cybernetic modeling.

PubMed

Batt, B C; Kompala, D S

1990-02-09

An immune response cascade that is T cell independent begins with the stimulation of virgin lymphocytes by antigen to differentiate into large lymphocytes. These immune cells can either replicate themselves or differentiate into plasma cells or memory cells. Plasma cells produce antibody at a specific rate up to two orders of magnitude greater than large lymphocytes. However, plasma cells have short life-spans and cannot replicate. Memory cells produce only surface antibody, but in the event of a subsequent infection by the same antigen, memory cells revert rapidly to large lymphocytes. Immunologic memory is maintained throughout the organism's lifetime. Many immunologists believe that the optimal response strategy calls for large lymphocytes to replicate first, then differentiate into plasma cells and when the antigen has been nearly eliminated, they form memory cells. A mathematical model incorporating the concept of cybernetics has been developed to study the optimality of the immune response. Derived from the matching law of microeconomics, cybernetic variables control the allocation of large lymphocytes to maximize the instantaneous antibody production rate at any time during the response in order to most efficiently inactivate the antigen. A mouse is selected as the model organism and bacteria as the replicating antigen. In addition to verifying the optimal switching strategy, results showing how the immune response is affected by antigen growth rate, initial antigen concentration, and the number of antibodies required to eliminate an antigen are included.

Evaluation of the immunomodulatory and antiviral effects of the cytokine combination IFN-α and IL-7 in the lymphocytic choriomeningitis virus and Friend retrovirus mouse infection models.

PubMed

Audigé, Annette; Hofer, Ursula; Dittmer, Ulf; van den Broek, Maries; Speck, Roberto F

2011-10-01

Existing therapies for chronic viral infections are still suboptimal or have considerable side effects, so new therapeutic strategies need to be developed. One option is to boost the host's immune response with cytokines. We have recently shown in an acute ex vivo HIV infection model that co-administration of interferon (IFN)-α and interleukin (IL)-7 allows us to combine the potent anti-HIV activity of IFN-α with the beneficial effects of IL-7 on T-cell survival and function. Here we evaluated the effect of combining IFN-α and IL-7 on viral replication in vivo in the chronic lymphocytic choriomeningitis virus (LCMV) and acute Friend retrovirus (FV) infection models. In the chronic LCMV model, cytokine treatment was started during the early replication phase (i.e., on day 7 post-infection [pi]). Under the experimental conditions used, exogenous IFN-α inhibited FV replication, but had no effect on viral replication in the LCMV model. There was no therapeutic benefit of IL-7 either alone or in combination with IFN-α in either of the two infection models. In the LCMV model, dose-dependent effects of the cytokine combination on T-cell phenotype/function were observed. It is possible that these effects would translate into antiviral activity in re-challenged mice. It is also possible that another type of IFN-α/β or induction of endogenous IFN-α/β alone or in combination with IL-7 would have antiviral activity in the LCMV model. Furthermore, we cannot exclude that some effect on viral titers would have been seen at later time points not investigated here (i.e., beyond day 34 pi). Finally, IFN-α/IL-7 may inhibit the replication of other viruses. Thus it might be worth testing these cytokines in other in vivo models of chronic viral infections.

Transforming growth factor β-activated kinase 1 transcriptionally suppresses hepatitis B virus replication.

PubMed

Pang, Jinke; Zhang, Geng; Lin, Yong; Xie, Zhanglian; Liu, Hongyan; Tang, Libo; Lu, Mengji; Yan, Ran; Guo, Haitao; Sun, Jian; Hou, Jinlin; Zhang, Xiaoyong

2017-01-03

Hepatitis B Virus (HBV) replication in hepatocytes is restricted by the host innate immune system and related intracellular signaling pathways. Transforming growth factor β-activated kinase 1 (TAK1) is a key mediator of toll-like receptors and pro-inflammatory cytokine signaling pathways. Here, we report that silencing or inhibition of endogenous TAK1 in hepatoma cell lines leads to an upregulation of HBV replication, transcription, and antigen expression. In contrast, overexpression of TAK1 significantly suppresses HBV replication, while an enzymatically inactive form of TAK1 exerts no effect. By screening TAK1-associated signaling pathways with inhibitors and siRNAs, we found that the MAPK-JNK pathway was involved in TAK1-mediated HBV suppression. Moreover, TAK1 knockdown or JNK pathway inhibition induced the expression of farnesoid X receptor α, a transcription factor that upregulates HBV transcription. Finally, ectopic expression of TAK1 in a HBV hydrodynamic injection mouse model resulted in lower levels of HBV DNA and antigens in both liver and serum. In conclusion, our data suggest that TAK1 inhibits HBV primarily at viral transcription level through activation of MAPK-JNK pathway, thus TAK1 represents an intrinsic host restriction factor for HBV replication in hepatocytes.

Inadequate satellite cell replication compromises muscle regrowth following postnatal nutrient restriction

USDA-ARS?s Scientific Manuscript database

Perinatal growth impairment permanently compromises skeletal muscle mass. The present study assessed the contribution of muscle satellite cell replicative capacity to this deficit. Mouse dams were fed either a low protein (LP, n=7) or control (C, n=6) diet during lactation. Pups were weaned at 21 d ...

Identification of HIV-1 determinants for replication in vivo.

PubMed

Su, L; Kaneshima, H; Bonyhadi, M L; Lee, R; Auten, J; Wolf, A; Du, B; Rabin, L; Hahn, B H; Terwilliger, E; Mccune, J M

1997-01-06

Pathogenic organisms are frequently attenuated after long-term culture in vitro. The mechanisms of the attenuation process are not clear, but probably involve mutations of functions required for replication and pathogenicity in vivo. To identify these functions, a direct comparison must be made between attenuated genomes and those that remain pathogenic in vivo. In this study, we used the heterochimeric SCID-hu Thy/Liv mouse as an in vivo model to define human immunodeficiency virus type 1 (HIV-1) determinants which are uniquely required for replication in vivo. The Lai/IIIB isolate and its associated infectious molecular clones (e.g., HXB2) were found to infect T cell lines but failed to replicate in the SCID-hu Thy/Liv model. When a lab worker was accidentally infected by Lai/IIIB, however, HIV-1 was isolated only from infection of primary PBMC, and not from infection of T cell lines. We hypothesized that the lab worker was exposed to a heterogeneous viral stock which had been attenuated by passage in immortalized T cell lines. Either a rare family member from this stock was selected for in vivo replication or, alternatively, an attenuated genotype dominant in vitro may have reverted to become more infectious in vivo. To address this hypothesis, we have used the SCID-hu Thy/Liv model to study the replication of HXB2 and of HXB2 recombinant viruses with HIV-1 fragments isolated from the infected lab worker. HXB2 showed no or very low levels of replication in the Thy/Liv organ. Replacement of its subgenomic fragment encoding the envelope gene with a corresponding fragment from the lab worker isolate generated a recombinant virus (HXB2/LW) which replicated actively in SCID-hu mice. The NEF mutation in the HXB2 genome is still present in HXB2/LW. Thus, the LW sequences encode HIV-1 determinants which enhance HIV replication in vivo in a NEF-independent mechanism. The specific determinants have been mapped to the V1-V3 regions of the HIV-1 genome. Six unique mutations in the V3 loop region of HXB2/LW have been identified which contribute to the increased replication in vivo.

RTEL1 contributes to DNA replication and repair and telomere maintenance.

PubMed

Uringa, Evert-Jan; Lisaingo, Kathleen; Pickett, Hilda A; Brind'Amour, Julie; Rohde, Jan-Hendrik; Zelensky, Alex; Essers, Jeroen; Lansdorp, Peter M

2012-07-01

Telomere maintenance and DNA repair are important processes that protect the genome against instability. mRtel1, an essential helicase, is a dominant factor setting telomere length in mice. In addition, mRtel1 is involved in DNA double-strand break repair. The role of mRtel1 in telomere maintenance and genome stability is poorly understood. Therefore we used mRtel1-deficient mouse embryonic stem cells to examine the function of mRtel1 in replication, DNA repair, recombination, and telomere maintenance. mRtel1-deficient mouse embryonic stem cells showed sensitivity to a range of DNA-damaging agents, highlighting its role in replication and genome maintenance. Deletion of mRtel1 increased the frequency of sister chromatid exchange events and suppressed gene replacement, demonstrating the involvement of the protein in homologous recombination. mRtel1 localized transiently at telomeres and is needed for efficient telomere replication. Of interest, in the absence of mRtel1, telomeres in embryonic stem cells appeared relatively stable in length, suggesting that mRtel1 is required to allow extension by telomerase. We propose that mRtel1 is a key protein for DNA replication, recombination, and repair and efficient elongation of telomeres by telomerase.

Mouse Models for Drug Discovery. Can New Tools and Technology Improve Translational Power?

PubMed

Zuberi, Aamir; Lutz, Cathleen

2016-12-01

The use of mouse models in biomedical research and preclinical drug evaluation is on the rise. The advent of new molecular genome-altering technologies such as CRISPR/Cas9 allows for genetic mutations to be introduced into the germ line of a mouse faster and less expensively than previous methods. In addition, the rapid progress in the development and use of somatic transgenesis using viral vectors, as well as manipulations of gene expression with siRNAs and antisense oligonucleotides, allow for even greater exploration into genomics and systems biology. These technological advances come at a time when cost reductions in genome sequencing have led to the identification of pathogenic mutations in patient populations, providing unprecedented opportunities in the use of mice to model human disease. The ease of genetic engineering in mice also offers a potential paradigm shift in resource sharing and the speed by which models are made available in the public domain. Predictively, the knowledge alone that a model can be quickly remade will provide relief to resources encumbered by licensing and Material Transfer Agreements. For decades, mouse strains have provided an exquisite experimental tool to study the pathophysiology of the disease and assess therapeutic options in a genetically defined system. However, a major limitation of the mouse has been the limited genetic diversity associated with common laboratory mice. This has been overcome with the recent development of the Collaborative Cross and Diversity Outbred mice. These strains provide new tools capable of replicating genetic diversity to that approaching the diversity found in human populations. The Collaborative Cross and Diversity Outbred strains thus provide a means to observe and characterize toxicity or efficacy of new therapeutic drugs for a given population. The combination of traditional and contemporary mouse genome editing tools, along with the addition of genetic diversity in new modeling systems, are synergistic and serve to make the mouse a better model for biomedical research, enhancing the potential for preclinical drug discovery and personalized medicine. © The Author 2016. Published by Oxford University Press.

Sequences in Influenza A Virus PB2 Protein That Determine Productive Infection for an Avian Influenza Virus in Mouse and Human Cell Lines

PubMed Central

Yao, Yongxiu; Mingay, Louise J.; McCauley, John W.; Barclay, Wendy S.

2001-01-01

Reverse genetics was used to analyze the host range of two avian influenza viruses which differ in their ability to replicate in mouse and human cells in culture. Engineered viruses carrying sequences encoding amino acids 362 to 581 of PB2 from a host range variant productively infect mouse and human cells. PMID:11333926

Leflunomide/teriflunomide inhibit Epstein-Barr virus (EBV)- induced lymphoproliferative disease and lytic viral replication.

PubMed

Bilger, Andrea; Plowshay, Julie; Ma, Shidong; Nawandar, Dhananjay; Barlow, Elizabeth A; Romero-Masters, James C; Bristol, Jillian A; Li, Zhe; Tsai, Ming-Han; Delecluse, Henri-Jacques; Kenney, Shannon C

2017-07-04

EBV infection causes mononucleosis and is associated with specific subsets of B cell lymphomas. Immunosuppressed patients such as organ transplant recipients are particularly susceptible to EBV-induced lymphoproliferative disease (LPD), which can be fatal. Leflunomide (a drug used to treat rheumatoid arthritis) and its active metabolite teriflunomide (used to treat multiple sclerosis) inhibit de novo pyrimidine synthesis by targeting the cellular dihydroorotate dehydrogenase, thereby decreasing T cell proliferation. Leflunomide also inhibits the replication of cytomegalovirus and BK virus via both "on target" and "off target" mechanisms and is increasingly used to treat these viruses in organ transplant recipients. However, whether leflunomide/teriflunomide block EBV replication or inhibit EBV-mediated B cell transformation is currently unknown. We show that teriflunomide inhibits cellular proliferation, and promotes apoptosis, in EBV-transformed B cells in vitro at a clinically relevant dose. In addition, teriflunomide prevents the development of EBV-induced lymphomas in both a humanized mouse model and a xenograft model. Furthermore, teriflunomide inhibits lytic EBV infection in vitro both by preventing the initial steps of lytic viral reactivation, and by blocking lytic viral DNA replication. Leflunomide/teriflunomide might therefore be clinically useful for preventing EBV-induced LPD in patients who have high EBV loads yet require continued immunosuppression.

Progress toward generating a ferret model of cystic fibrosis by somatic cell nuclear transfer

PubMed Central

Li, Ziyi; Engelhardt, John F

2003-01-01

Mammalian cloning by nuclear transfer from somatic cells has created new opportunities to generate animal models of genetic diseases in species other than mice. Although genetic mouse models play a critical role in basic and applied research for numerous diseases, often mouse models do not adequately reproduce the human disease phenotype. Cystic fibrosis (CF) is one such disease. Targeted ablation of the cystic fibrosis transmembrane conductance regulator (CFTR) gene in mice does not adequately replicate spontaneous bacterial infections observed in the human CF lung. Hence, several laboratories are pursuing alternative animal models of CF in larger species such as the pig, sheep, rabbits, and ferrets. Our laboratory has focused on developing the ferret as a CF animal model. Over the past few years, we have investigated several experimental parameters required for gene targeting and nuclear transfer (NT) cloning in the ferret using somatic cells. In this review, we will discuss our progress and the hurdles to NT cloning and gene-targeting that accompany efforts to generate animal models of genetic diseases in species such as the ferret. PMID:14613541

Resistance to Rhabdoviridae Infection and Subversion of Antiviral Responses

PubMed Central

Blondel, Danielle; Maarifi, Ghizlane; Nisole, Sébastien; Chelbi-Alix, Mounira K.

2015-01-01

Interferon (IFN) treatment induces the expression of hundreds of IFN-stimulated genes (ISGs). However, only a selection of their products have been demonstrated to be responsible for the inhibition of rhabdovirus replication in cultured cells; and only a few have been shown to play a role in mediating the antiviral response in vivo using gene knockout mouse models. IFNs inhibit rhabdovirus  replication at different stages via the induction of a variety of ISGs. This review will discuss how individual ISG products confer resistance to rhabdoviruses by blocking viral entry, degrading single stranded viral RNA, inhibiting viral translation or preventing release of virions from the cell. Furthermore, this review will highlight how these viruses counteract the host IFN system. PMID:26198243

In vitro models of viral-induced congenital deafness.

PubMed

Davis, G L

1981-10-01

Cytomegalovirus (CMV) infects 1 to 2 percent of liveborn infants in the United States and causes varying degrees of perceptive hearing loss. There are eight reported pathologic studies of temporal bones in CMV-infected neonates. Viral replication occurs in nonsensory endolabyrinthine epithelium, but viral antigen is also found in the organ of Corti and spiral ganglion neurons, and CMV has been cultured from perilymph. Further clinicopathologic correlation is frustrated, since the inner ear cannot be biopsied during life, and the number of temporal bones available for study is limited, owing to the decrease in the number of autopsies being performed. Inoculation of CMV into newborn mice, and extracorporeal preparations of mouse and guinea pig fetal inner ears, either in organ culture or as grafts on chick chorioallantoic membranes, yields viral perilabyrinthitis. The different ultrastructural appearances of CMV replicating in epithelial and mesenchymal cells show that animal CMV replicates in mesenchymal cells and human CMV replicates in epithelial cells of the inner ear. These different ultrastructural patterns indicate that the chromophobe (transitional) cells of the stria vascularis of the guinea pig are of mesenchymal origin.

Inhibition of dengue virus replication by novel inhibitors of RNA-dependent RNA polymerase and protease activities.

PubMed

Pelliccia, Sveva; Wu, Yu-Hsuan; Coluccia, Antonio; La Regina, Giuseppe; Tseng, Chin-Kai; Famiglini, Valeria; Masci, Domiziana; Hiscott, John; Lee, Jin-Ching; Silvestri, Romano

2017-12-01

Dengue virus (DENV) is the leading mosquito-transmitted viral infection in the world. With more than 390 million new infections annually, and up to 1 million clinical cases with severe disease manifestations, there continues to be a need to develop new antiviral agents against dengue infection. In addition, there is no approved anti-DENV agents for treating DENV-infected patients. In the present study, we identified new compounds with anti-DENV replication activity by targeting viral replication enzymes - NS5, RNA-dependent RNA polymerase (RdRp) and NS3 protease, using cell-based reporter assay. Subsequently, we performed an enzyme-based assay to clarify the action of these compounds against DENV RdRp or NS3 protease activity. Moreover, these compounds exhibited anti-DENV activity in vivo in the ICR-suckling DENV-infected mouse model. Combination drug treatment exhibited a synergistic inhibition of DENV replication. These results describe novel prototypical small anti-DENV molecules for further development through compound modification and provide potential antivirals for treating DENV infection and DENV-related diseases.

Mouse infection by Legionella, a model to analyze autophagy

PubMed Central

Dubuisson, Jean-François; Swanson, Michele S.

2006-01-01

Summary Autophagy is a conserved membrane traffic pathway that equips eukaryotic cells to capture cytoplasmic components within a double-membrane vacuole, or autophagosome, for delivery to lysosomes. Although best known as a mechanism to survive starvation, autophagy is now recognized to combat infection by a variety of microbes.1–3 Not surprisingly, to establish a replication niche in host cells, some intracellular pathogens have acquired mechanisms either to evade or subvert the autophagic pathway. Because they are amenable to genetic manipulation, these microbes can be exploited as experimental tools to investigate the contribution of autophagy to immunity. Here we discuss the mouse macrophage response to L. pneumophila, the facultative intracellular bacterium responsible for an acute form of pneumonia, Legionnaire’s disease. PMID:16874080

Cyclooxygenase‐2 facilitates dengue virus replication and serves as a potential target for developing antiviral agents

PubMed Central

Lin, Chun-Kuang; Tseng, Chin-Kai; Wu, Yu-Hsuan; Liaw, Chih-Chuang; Lin, Chun-Yu; Huang, Chung-Hao; Chen, Yen-Hsu; Lee, Jin-Ching

2017-01-01

Cyclooxygenase-2 (COX-2) is one of the important mediators of inflammation in response to viral infection, and it contributes to viral replication, for example, cytomegalovirus or hepatitis C virus replication. The role of COX-2 in dengue virus (DENV) replication remains unclear. In the present study, we observed an increased level of COX-2 in patients with dengue fever compared with healthy donors. Consistent with the clinical data, an elevated level of COX-2 expression was also observed in DENV-infected ICR suckling mice. Using cell-based experiments, we revealed that DENV-2 infection significantly induced COX-2 expression and prostaglandin E2 (PGE2) production in human hepatoma Huh-7 cells. The exogenous expression of COX-2 or PGE2 treatment dose-dependently enhanced DENV-2 replication. In contrast, COX-2 gene silencing and catalytic inhibition sufficiently suppressed DENV-2 replication. In an ICR suckling mouse model, we identified that the COX-2 inhibitor NS398 protected mice from succumbing to life-threatening DENV-2 infection. By using COX-2 promoter-based analysis and specific inhibitors against signaling molecules, we identified that NF-κB and MAPK/JNK are critical factors for DENV-2-induced COX-2 expression and viral replication. Altogether, our results reveal that COX-2 is an important factor for DENV replication and can serve as a potential target for developing therapeutic agents against DENV infection. PMID:28317866

Single-cell tracking of flavivirus RNA uncovers species-specific interactions with the immune system dictating disease outcome

PubMed Central

Douam, Florian; Hrebikova, Gabriela; Albrecht, Yentli E. Soto; Sellau, Julie; Sharon, Yael; Ding, Qiang; Ploss, Alexander

2017-01-01

Positive-sense RNA viruses pose increasing health and economic concerns worldwide. Our limited understanding of how these viruses interact with their host and how these processes lead to virulence and disease seriously hampers the development of anti-viral strategies. Here, we demonstrate the tracking of (+) and (−) sense viral RNA at single-cell resolution within complex subsets of the human and murine immune system in different mouse models. Our results provide insights into how a prototypic flavivirus, yellow fever virus (YFV-17D), differentially interacts with murine and human hematopoietic cells in these mouse models and how these dynamics influence distinct outcomes of infection. We detect (−) YFV-17D RNA in specific secondary lymphoid compartments and cell subsets not previously recognized as permissive for YFV replication, and we highlight potential virus–host interaction events that could be pivotal in regulating flavivirus virulence and attenuation. PMID:28290449

A Murine Model for Escherichia coli Urinary Tract Infection.

PubMed

Hannan, Thomas J; Hunstad, David A

2016-01-01

Urinary tract infections (UTI) are among the most common bacterial infections of humans. The mouse provides an excellent and tractable model system for cystitis and pyelonephritis caused by Escherichia coli and other uropathogens. Using a well-established model of experimental cystitis in which the bladders of female mice are infected via transurethral catheterization, the molecular details of the pathogenesis of bacterial cystitis have been substantially illuminated in the last decade. Uropathogenic E. coli attach to bladder epithelium (both in human and mouse) via adhesive type 1 pili, establish a replicative niche within epithelial cell cytoplasm, and form intracellular bacterial communities that are protected from antibiotic effects and immune clearance. The use of different inbred and mutant mouse strains offers the opportunity to study outcomes of infection, including resolution, formation of quiescent intracellular bacterial reservoirs, chronic bacterial cystitis, and recurrent infections. Urine, bladder, and kidney tissues can be analyzed by bacterial culture, histology, immunohistochemistry, immunofluorescent and confocal microscopy, electron microscopy, and flow cytometry, while a broad array of soluble markers (e.g., cytokines) can also be profiled in serum, urine, and tissue homogenates by ELISA, Western blotting, multiplex bead array, and other approaches. This model promises to afford continued opportunity for discovery of pathogenic mechanisms and evaluation of therapeutic and preventive strategies for acute, chronic, and recurrent UTI.

A Mouse Model of Acinetobacter baumannii-Associated Pneumonia Using a Clinically Isolated Hypervirulent Strain

PubMed Central

Harris, Greg; Kuo Lee, Rhonda; Lam, Christopher K.; Kanzaki, Gregory; Patel, Girishchandra B.; Xu, H. Howard

2013-01-01

Acinetobacter baumannii is an important emerging pathogen in health care-acquired infections and is responsible for severe nosocomial and community-acquired pneumonia. Currently available mouse models of A. baumannii pneumonia show poor colonization with little to no extrapulmonary dissemination. Here, we describe a mouse model of A. baumannii pneumonia using a clinical isolate (LAC-4 strain) that reliably reproduces the most relevant features of human pulmonary A. baumannii infection and pathology. Using this model, we have shown that LAC-4 infection induced rapid bacterial replication in the lungs, significant extrapulmonary dissemination, and severe bacteremia by 24 h postintranasal inoculation. Infected mice showed severe bronchopneumonia and dilatation and inflammatory cell infiltration in the perivascular space. More significantly, 100% of C57BL/6 and BALB/c mice succumbed to 108 CFU of LAC-4 inoculation within 48 h. When this model was used to assess the efficacy of antimicrobials, all mice treated with imipenem and tigecycline survived a lethal intranasal challenge, with minimal clinical signs and body weight loss. Moreover, intranasal immunization of mice with formalin-fixed LAC-4 protected 40% of mice from a lethal (100× 100% lethal dose) intraperitoneal challenge. Thus, this model offers a reproducible acute course of A. baumannii pneumonia without requiring additional manipulation of host immune status, which will facilitate the development of therapeutic agents and vaccines against A. baumannii pneumonia in humans. PMID:23689726

Multiplication of infectious hematopoietic necrosis virus in rainbow trout following immersion infection: whole-body assay and immunohistochemistry

USGS Publications Warehouse

Yamamoto, T.; Batts, W.N.; Arakawa, C.K.; Winton, J.R.

1990-01-01

The sites of replication of infectious hematopoietic necrosis virus (IHNV) in infected tissues were detected in fingerling rainbow trout Oncorhynchus mykiss by in situ histologic techniques following immersion infection. Virus antigens in tissues were detected by a neutralizing mouse monoclonal antibody and a one-step anti-mouse biotin-streptavidin conjugated to horseradish peroxidase. The efficiency of infection and virulence of the virus determined by mortality rates showed high virulence of the selected IHNV isolates, and viral replication in individual fish showed that virus content of the fish increased rapidly from the second day to the seventh day postinfection. The earliest viral lesions following infection were detected in the epidermis of the pectoral fins, opercula, and ventral surface of the body. Virus lesions became evident in kidneys on the third day. By the fifth day, when there was a significant increase in virus titer, foci of viral replication were detected in gill tissue and in the anterior internal tissues below the epidermis. Subsequently, extensive virus replication and tissue destruction were observed in the spleen, dorsal adipose tissues, ventricle, and pseudobranch. Replication in the liver, the muscularis layers of the digestive tract, and the general body musculature followed later. These infection experiments indicated that the epidermis and gills of fish constitute important sites of early IHNV replication.

An assessment of the effects of serotonin 6 (5-HT6) receptor antagonists in rodent models of learning.

PubMed

Lindner, Mark D; Hodges, Donald B; Hogan, John B; Orie, Anitra F; Corsa, Jason A; Barten, Donna M; Polson, Craig; Robertson, Barbara J; Guss, Valerie L; Gillman, Kevin W; Starrett, John E; Gribkoff, Valentin K

2003-11-01

Antagonists of serotonin 6 (5-HT6) receptors have been reported to enhance cognition in animal models of learning, although this finding has not been universal. We have assessed the therapeutic potential of the specific 5-HT6 receptor antagonists 4-amino-N-(2,6-bis-methylamino-pyrimidin-4-yl)-benzenesulfonamide (Ro 04-6790) and 5-chloro-N-(4-methoxy-3-piperazin-1-yl-phenyl)-3-methyl-2-benzothiophenesulfonamide (SB-271046) in rodent models of cognitive function. Although mice express the 5-HT6 receptor and the function of this receptor has been investigated in mice, all reports of activity with 5-HT6 receptor antagonists have used rat models. In the present study, receptor binding revealed that the pharmacological properties of the mouse receptor are different from the rat and human receptor: Ro 04-6790 does not bind to the mouse 5-HT6 receptor, so all in vivo testing included in the present report was conducted in rats. We replicated previous reports that 5-HT6 receptor antagonists produce a stretching syndrome previously shown to be mediated through cholinergic mechanisms, but Ro 04-6790 and SB-271046 failed to attenuate scopolamine-induced deficits in a test of contextual fear conditioning. We also failed to replicate the significant effects reported previously in both an autoshaping task and in a version of the Morris water maze. The results of our experiments are not consistent with previous reports that suggested that 5-HT6 antagonists might have therapeutic potential for cognitive disorders.

A mouse model for pain and neuroplastic changes associated with pancreatic ductal adenocarcinoma.

PubMed

Selvaraj, Deepitha; Hirth, Michael; Gandla, Jagadeesh; Kuner, Rohini

2017-08-01

Pancreatic ductal adenocarcinoma (PDAC) continues to be one of the deadliest human malignancies and is associated with excruciating pain, which is a serious complication and severely impacts the quality of life in patients. In human patients, poor survival prognosis is linked to remarkable remodeling of intrapancreatic nerves, which, in turn, is correlated to increased pain intensity. Understanding mechanisms underlying pain associated with PDAC has been hampered by the lack of animal models which replicate all germane aspects of the disease and importantly, enable analyses of pain associated with PDAC. In this study, we describe an immunocompetent orthotopic mouse model of PDAC involving intrapancreatic growth of K8484 mouse PDAC cells, which reliably exhibits a large number of key characteristics of human PDAC, including its unique histopathology and neuroplastic changes. We observed that tumor-bearing mice demonstrated significant abdominal mechanical hypersensitivity to von Frey stimuli as well as on-going pain in the conditioned place preference paradigm. Moreover, a myriad of other behavioral tests revealed that indicators of overall well-being were significantly reduced in tumor-bearing mice as compared to sham mice. Morphological and immunohistochemical analyses revealed structural remodeling in several different types of sensory and autonomic nerve fibers. Finally, perineural invasion of tumor cells, a cardinal manifestation in human PDAC, was also observed in our orthotopic mouse model. Thus, we describe a validated tumor model for quantitatively testing hypersensitivity and pain in PDAC, which lays a crucial basis for interrogating tumor-nerve interactions and the molecular and cellular mechanisms underlying pain in PDAC.

An epigenetic state associated with areas of gene duplication

PubMed Central

Gimelbrant, Alexander A.; Chess, Andrew

2006-01-01

Asynchronous DNA replication is an epigenetically determined feature found in all cases of monoallelic expression, including genomic imprinting, X-inactivation, and random monoallelic expression of autosomal genes such as immunoglobulins and olfactory receptor genes. Most genes of the latter class were identified in experiments focused on genes functioning in the chemosensory and immune systems. We performed an unbiased survey of asynchronous replication in the mouse genome, excluding known asynchronously replicated genes. Fully 10% (eight of 80) of the genes tested exhibited asynchronous replication. A common feature of the newly identified asynchronously replicated areas is their proximity to areas of tandem gene duplication. Testing of other clustered areas supported the idea that such regions are enriched with asynchronously replicated genes. PMID:16687731

The Dot/Icm effector SdhA is necessary for virulence of Legionella pneumophila in Galleria mellonella and A/J mice.

PubMed

Harding, Clare R; Stoneham, Charlotte A; Schuelein, Ralf; Newton, Hayley; Oates, Clare V; Hartland, Elizabeth L; Schroeder, Gunnar N; Frankel, Gad

2013-07-01

Legionella pneumophila is an intracellular bacterium that resides within amoebae and macrophages in a specialized compartment termed the Legionella-containing vacuole (LCV). As well as providing an intracellular niche for replication, the LCV helps to prevent the release of bacterial components into the cytoplasm. Recognition of these components as danger signals by the host activates immune responses leading to clearance of the bacterium. Here, we examined the role of two important virulence factors of L. pneumophila, the potent danger signal flagellin and the translocated Dot/Icm type IVB secretion system effector SdhA, which is crucial to maintain LCV integrity, in the Galleria mellonella infection model. We demonstrate that flagellin expression does not contribute to virulence, replication, or induction of clearance mechanisms. Conversely, SdhA expression is important for virulence. We found that in the absence of SdhA, the LCV in hemocytes showed signs of instability and leakage. Furthermore, in contrast to wild-type L. pneumophila, a ΔsdhA mutant caused a transient depletion of hemocytes and reduced mortality. Analysis of the ΔsdhA mutant in the A/J mouse model also showed a significant replication defect. Together, our data underline the crucial importance of SdhA in infection across different model organisms.

A doxycycline-dependent human immunodeficiency virus type 1 replicates in vivo without inducing CD4+ T-cell depletion

PubMed Central

Legrand, Nicolas; van der Velden, Gisela J.; Fang, Raphaël Ho Tsong; Douaisi, Marc; Weijer, Kees; Das, Atze T.; Blom, Bianca; Uittenbogaart, Christel H.; Berkhout, Ben

2012-01-01

A novel genetic approach for the control of virus replication was used for the design of a conditionally replicating human immunodeficiency virus (HIV) variant, HIV-rtTA. HIV-rtTA gene expression and virus replication are strictly dependent on the presence of a non-toxic effector molecule, doxycycline (dox), and thus can be turned on and off at will in a graded and reversible manner. The in vivo replication capacity, pathogenicity and genetic stability of this HIV-rtTA variant were evaluated in a humanized mouse model of haematopoiesis that harbours lymphoid and myeloid components of the human immune system (HIS). Infection of dox-fed BALB Rag/γc HIS (BRG-HIS) mice with HIV-rtTA led to the establishment of a productive infection without CD4+ T-cell depletion. The virus did not show any sign of escape from dox control for up to 10 weeks after the onset of infection. No reversion towards a functional Tat–transactivating responsive (TAR) RNA element axis was observed, confirming the genetic stability of the HIV-rtTA variant in vivo. These results demonstrate the proof of concept that HIV-rtTA replicates efficiently in vivo. HIV-rtTA is a promising tool for fundamental research to study virus–host interactions in vivo in a controlled fashion. PMID:22647372

Associative DNA methylation changes in children with prenatal alcohol exposure.

PubMed

Laufer, Benjamin I; Kapalanga, Joachim; Castellani, Christina A; Diehl, Eric J; Yan, Liying; Singh, Shiva M

2015-01-01

Prenatal alcohol exposure (PAE) can cause fetal alcohol spectrum disorders (FASD). Previously, we assessed PAE in brain tissue from mouse models, however whether these changes are present in humans remains unknown. In this report, we show some identical changes in DNA methylation in the buccal swabs of six children with FASD using the 450K array. The changes occur in genes related to protocadherins, glutamatergic synapses, and hippo signaling. The results were found to be similar in another heterogeneous replication group of six FASD children. The replicated results suggest that children born with FASD have unique DNA methylation defects that can be influenced by sex and medication exposure. Ultimately, with future clinical development, assessment of DNA methylation from buccal swabs can provide a novel strategy for the diagnosis of FASD.

Topologically-associating domains are stable units of replication-timing regulation

PubMed Central

Pope, Benjamin D.; Ryba, Tyrone; Dileep, Vishnu; Yue, Feng; Wu, Weisheng; Denas, Olgert; Vera, Daniel L.; Wang, Yanli; Hansen, R. Scott; Canfield, Theresa K.; Thurman, Robert E.; Cheng, Yong; Gülsoy, Günhan; Dennis, Jonathan H.; Snyder, Michael P.; Stamatoyannopoulos, John A.; Taylor, James; Hardison, Ross C.; Kahveci, Tamer; Ren, Bing; Gilbert, David M.

2014-01-01

Summary Eukaryotic chromosomes replicate in a temporal order known as the replication-timing program1. During mammalian development, at least half the genome changes replication timing, primarily in units of 400–800 kb (“replication domains”; RDs), whose positions are preserved in different cell types, conserved between species, and appear to confine long-range effects of chromosome rearrangements2–7. Early and late replication correlate strongly with open and closed chromatin compartments identified by high-resolution chromosome conformation capture (Hi-C), and, to a lesser extent, lamina-associated domains (LADs)4,5,8,9. Recent Hi-C mapping has unveiled a substructure of topologically-associating domains (TADs) that are largely conserved in their positions between cell types and are similar in size to RDs8,10. However, TADs can be further sub-stratified into smaller domains, challenging the significance of structures at any particular scale11,12. Moreover, attempts to reconcile TADs and LADs to replication-timing data have not revealed a common, underlying domain structure8,9,13. Here, we localize boundaries of RDs to the early-replicating border of replication-timing transitions and map their positions in 18 human and 13 mouse cell types. We demonstrate that, collectively, RD boundaries share a near one-to-one correlation with TAD boundaries, whereas within a cell type, adjacent TADs that replicate at similar times obscure RD boundaries, largely accounting for the previously reported lack of alignment. Moreover, cell-type specific replication timing of TADs partitions the genome into two large-scale sub-nuclear compartments revealing that replication-timing transitions are indistinguishable from late-replicating regions in chromatin composition and lamina association and accounting for the reduced correlation of replication timing to LADs and heterochromatin. Our results reconcile cell type specific sub-nuclear compartmentalization with developmentally stable chromosome domains and offer a unified model for large-scale chromosome structure and function. PMID:25409831

Estimating replicate time shifts using Gaussian process regression

PubMed Central

Liu, Qiang; Andersen, Bogi; Smyth, Padhraic; Ihler, Alexander

2010-01-01

Motivation: Time-course gene expression datasets provide important insights into dynamic aspects of biological processes, such as circadian rhythms, cell cycle and organ development. In a typical microarray time-course experiment, measurements are obtained at each time point from multiple replicate samples. Accurately recovering the gene expression patterns from experimental observations is made challenging by both measurement noise and variation among replicates' rates of development. Prior work on this topic has focused on inference of expression patterns assuming that the replicate times are synchronized. We develop a statistical approach that simultaneously infers both (i) the underlying (hidden) expression profile for each gene, as well as (ii) the biological time for each individual replicate. Our approach is based on Gaussian process regression (GPR) combined with a probabilistic model that accounts for uncertainty about the biological development time of each replicate. Results: We apply GPR with uncertain measurement times to a microarray dataset of mRNA expression for the hair-growth cycle in mouse back skin, predicting both profile shapes and biological times for each replicate. The predicted time shifts show high consistency with independently obtained morphological estimates of relative development. We also show that the method systematically reduces prediction error on out-of-sample data, significantly reducing the mean squared error in a cross-validation study. Availability: Matlab code for GPR with uncertain time shifts is available at http://sli.ics.uci.edu/Code/GPRTimeshift/ Contact: ihler@ics.uci.edu PMID:20147305

A statistical analysis of murine incisional and excisional acute wound models.

PubMed

Ansell, David M; Campbell, Laura; Thomason, Helen A; Brass, Andrew; Hardman, Matthew J

2014-01-01

Mice represent the most commonly used species for preclinical in vivo research. While incisional and excisional acute murine wound models are both frequently employed, there is little agreement on which model is optimum. Moreover, current lack of standardization of wounding procedure, analysis time point(s), method of assessment, and the use of individual wounds vs. individual animals as replicates makes it difficult to compare across studies. Here we have profiled secondary intention healing of incisional and excisional wounds within the same animal, assessing multiple parameters to determine the optimal methodology for future studies. We report that histology provides the least variable assessment of healing. Furthermore, histology alone (not planimetry) is able to detect accelerated healing in a castrated mouse model. Perhaps most importantly, we find virtually no correlation between wounds within the same animal, suggesting that use of wound (not animal) biological replicates is perfectly acceptable. Overall, these findings should guide and refine future studies, increasing the likelihood of detecting novel phenotypes while reducing the numbers of animals required for experimentation. © 2014 by the Wound Healing Society.

A statistical analysis of murine incisional and excisional acute wound models

PubMed Central

Ansell, David M; Campbell, Laura; Thomason, Helen A; Brass, Andrew; Hardman, Matthew J

2014-01-01

Mice represent the most commonly used species for preclinical in vivo research. While incisional and excisional acute murine wound models are both frequently employed, there is little agreement on which model is optimum. Moreover, current lack of standardization of wounding procedure, analysis time point(s), method of assessment, and the use of individual wounds vs. individual animals as replicates makes it difficult to compare across studies. Here we have profiled secondary intention healing of incisional and excisional wounds within the same animal, assessing multiple parameters to determine the optimal methodology for future studies. We report that histology provides the least variable assessment of healing. Furthermore, histology alone (not planimetry) is able to detect accelerated healing in a castrated mouse model. Perhaps most importantly, we find virtually no correlation between wounds within the same animal, suggesting that use of wound (not animal) biological replicates is perfectly acceptable. Overall, these findings should guide and refine future studies, increasing the likelihood of detecting novel phenotypes while reducing the numbers of animals required for experimentation. PMID:24635179

Chloroquine, an Endocytosis Blocking Agent, Inhibits Zika Virus Infection in Different Cell Models

PubMed Central

Delvecchio, Rodrigo; Higa, Luiza M.; Pezzuto, Paula; Valadão, Ana Luiza; Garcez, Patrícia P.; Monteiro, Fábio L.; Loiola, Erick C.; Dias, André A.; Silva, Fábio J. M.; Aliota, Matthew T.; Caine, Elizabeth A.; Osorio, Jorge E.; Bellio, Maria; O’Connor, David H.; Rehen, Stevens; de Aguiar, Renato Santana; Savarino, Andrea; Campanati, Loraine; Tanuri, Amilcar

2016-01-01

Zika virus (ZIKV) infection in utero might lead to microcephaly and other congenital defects. Since no specific therapy is available thus far, there is an urgent need for the discovery of agents capable of inhibiting its viral replication and deleterious effects. Chloroquine is widely used as an antimalarial drug, anti-inflammatory agent, and it also shows antiviral activity against several viruses. Here we show that chloroquine exhibits antiviral activity against ZIKV in Vero cells, human brain microvascular endothelial cells, human neural stem cells, and mouse neurospheres. We demonstrate that chloroquine reduces the number of ZIKV-infected cells in vitro, and inhibits virus production and cell death promoted by ZIKV infection without cytotoxic effects. In addition, chloroquine treatment partially reveres morphological changes induced by ZIKV infection in mouse neurospheres. PMID:27916837

Chloroquine, an Endocytosis Blocking Agent, Inhibits Zika Virus Infection in Different Cell Models.

PubMed

Delvecchio, Rodrigo; Higa, Luiza M; Pezzuto, Paula; Valadão, Ana Luiza; Garcez, Patrícia P; Monteiro, Fábio L; Loiola, Erick C; Dias, André A; Silva, Fábio J M; Aliota, Matthew T; Caine, Elizabeth A; Osorio, Jorge E; Bellio, Maria; O'Connor, David H; Rehen, Stevens; de Aguiar, Renato Santana; Savarino, Andrea; Campanati, Loraine; Tanuri, Amilcar

2016-11-29

Zika virus (ZIKV) infection in utero might lead to microcephaly and other congenital defects. Since no specific therapy is available thus far, there is an urgent need for the discovery of agents capable of inhibiting its viral replication and deleterious effects. Chloroquine is widely used as an antimalarial drug, anti-inflammatory agent, and it also shows antiviral activity against several viruses. Here we show that chloroquine exhibits antiviral activity against ZIKV in Vero cells, human brain microvascular endothelial cells, human neural stem cells, and mouse neurospheres. We demonstrate that chloroquine reduces the number of ZIKV-infected cells in vitro, and inhibits virus production and cell death promoted by ZIKV infection without cytotoxic effects. In addition, chloroquine treatment partially reveres morphological changes induced by ZIKV infection in mouse neurospheres.

The Histoplasma capsulatum Vacuolar ATPase is Required for Iron Homeostasis, Intracellular Replication in Macrophages, and Virulence in a Murine Model of Histoplasmosis

PubMed Central

Hilty, Jeremy; Smulian, A. George; Newman, Simon L.

2008-01-01

Summary Histoplasma capsulatum is a dimorphic fungal pathogen that survives and replicates within macrophages (Mϕ). To identify specific genes required for intracellular survival, we utilized Agrobacterium tumefaciens-mediated mutagenesis, and screened for H. capsulatum insertional mutants that were unable to survive in human Mϕ. One colony was identified that had an insertion within VMA1, the catalytic subunit A of the vacuolar ATPase (V-ATPase). The vma1 mutant (vma1::HPH) grew normally on iron replete medium, but not on iron deficient media. On iron deficient medium, the growth of the vma1 mutant was restored in the presence of wild type (WT) H. capsulatum yeasts, or the hydroxamate siderophore, rhodotorulic acid. However, the inability to replicate within Mϕ was only partially restored by the addition of exogenous iron. The vma1::HPH mutant also did not grow as a mold at 28°C. Complementation of the mutant (vma/VMA1) restored its ability to replicate in Mϕ, grow on iron poor medium, and grow as a mold at 28°C. The vma1::HPH mutant was avirulent in a mouse model of histoplasmosis, whereas the vma1/VMA1 strain was as pathogenic as WT yeasts. These studies demonstrate the importance of V-ATPase function in the pathogenicity of H. capsulatum, in iron homeostasis, and in fungal dimorphism. PMID:18699866

Inhibition of Hepatitis C Virus Replication by Intracellular Delivery of Multiple siRNAs by Nanosomes

PubMed Central

Chandra, Partha K; Kundu, Anup K; Hazari, Sidhartha; Chandra, Sruti; Bao, Lili; Ooms, Tara; Morris, Gilbert F; Wu, Tong; Mandal, Tarun K; Dash, Srikanta

2012-01-01

Sustained antiviral responses of chronic hepatitis C virus (HCV) infection have improved recently by the use of direct-acting antiviral agents along with interferon (IFN)-α and ribavirin. However, the emergence of drug-resistant variants is expected to be a major problem. We describe here a novel combinatorial small interfering RNA (siRNA) nanosome-based antiviral approach to clear HCV infection. Multiple siRNAs targeted to the highly conserved 5′-untranslated region (UTR) of the HCV genome were synthesized and encapsulated into lipid nanoparticles called nanosomes. We show that siRNA can be repeatedly delivered to 100% of cells in culture using nanosomes without toxicity. Six siRNAs dramatically reduced HCV replication in both the replicon and infectious cell culture model. Repeated treatments with two siRNAs were better than a single siRNA treatment in minimizing the development of an escape mutant, resulting in rapid inhibition of viral replication. Systemic administration of combinatorial siRNA-nanosomes is well tolerated in BALB/c mice without liver injury or histological toxicity. As a proof-of-principle, we showed that systemic injections of siRNA nanosomes significantly reduced HCV replication in a liver tumor-xenotransplant mouse model of HCV. Our results indicate that systemic delivery of combinatorial siRNA nanosomes can be used to minimize the development of escape mutants and inhibition of HCV infection. PMID:22617108

Diversity, Replication, Pathogenicity and Cell Biology of Crimean Congo Hemorrhagic Fever Virus

DTIC Science & Technology

2010-10-01

in CCHFV pathogenesis or whether it is even O glycosylated. A third unusual feature is * Corresponding author. Mailing address: Department of Microbi ...CA), followed by Western blot analysis with mouse anti-V5 (Invitrogen) as the primary antibody and sheep anti-mouse horseradish peroxidase conjugate as...250) or with mouse anti-V5 MAb (diluted 1:500) (Invitrogen) in PBS containing 0.5 mM MgCl2 and 4% fetal bovine serum. In addition, TGN46, a sheep

Cholinergic degeneration and memory loss delayed by vitamin E in a Down syndrome mouse model

PubMed Central

Lockrow, Jason; Prakasam, Annamalai; Huang, Peng; Bimonte-Nelson, Heather; Sambamurti, Kumar; Granholm, Ann-Charlotte

2009-01-01

Down syndrome (DS) individuals develop several neuropathological hallmarks seen in Alzheimer's disease, including cognitive decline and the early loss of cholinergic markers in the basal forebrain. These deficits are replicated in the Ts65Dn mouse, which contains a partial trisomy of murine chromosome 16, the orthologous genetic segment to human chromosome 21. Oxidative stress levels are elevated early in DS, and may contribute to the neurodegeneration seen in these individuals. We evaluated oxidative stress in Ts65Dn mice, and assessed the efficacy of long-term antioxidant supplementation on memory and basal forebrain pathology. We report that oxidative stress was elevated in the adult Ts65Dn brain, and that supplementation with the antioxidant vitamin E effectively reduced these markers. Also, Ts65Dn mice receiving vitamin E exhibited improved performance on a spatial working memory task and showed an attenuation of cholinergic neuron pathology in the basal forebrain. This study provides evidence that vitamin E delays onset of cognitive and morphological abnormalities in a mouse model of DS, and may represent a safe and effective treatment early in the progression of DS neuropathology. PMID:19135442

Establishment of a mouse model for the complete mosquito-mediated transmission cycle of Zika virus.

PubMed

Kuo, Yi-Ping; Tsai, Kuen-Nan; Luo, Yin-Chiu; Chung, Pei-Jung; Su, Yu-Wen; Teng, Yu; Wu, Ming-Sian; Lin, Yu-Feng; Lai, Chao-Yang; Chuang, Tsung-Hsien; Dai, Shih-Syong; Tseng, Fan-Chen; Hsieh, Cheng-Han; Tsai, De-Jiun; Tsai, Wan-Ting; Chen, Chun-Hong; Yu, Guann-Yi

2018-04-01

Zika virus (ZIKV) is primarily transmitted by Aedes mosquitoes in the subgenus Stegomyia but can also be transmitted sexually and vertically in humans. STAT1 is an important downstream factor that mediates type I and II interferon signaling. In the current study, we showed that mice with STAT1 knockout (Stat1-/-) were highly susceptible to ZIKV infection. As low as 5 plaque-forming units of ZIKV could cause viremia and death in Stat1-/- mice. ZIKV replication was initially detected in the spleen but subsequently spread to the brain with concomitant reduction of the virus in the spleen in the infected mice. Furthermore, ZIKV could be transmitted from mosquitoes to Stat1-/- mice back to mosquitoes and then to naïve Stat1-/- mice. The 50% mosquito infectious dose of viremic Stat1-/- mouse blood was close to 810 focus-forming units (ffu)/ml. Our further studies indicated that the activation of macrophages and conventional dendritic cells were likely critical for the resolution of ZIKV infection. The newly developed mouse and mosquito transmission models for ZIKV infection will be useful for the evaluation of antiviral drugs targeting the virus, vector, and host.

Histone deacetylase inhibitors suppress RSV infection and alleviate virus-induced airway inflammation

PubMed Central

Feng, Qiuqin; Su, Zhonglan; Song, Shiyu; Xu, Hui; Zhang, Bin; Yi, Long; Tian, Man; Wang, Hongwei

2016-01-01

Respiratory syncytial virus (RSV) is the leading cause of lower respiratory tract infections in infants and young children. However, the majority of RSV-infected patients only show mild symptoms. Different severities of infection and responses among the RSV-infected population indicate that epigenetic regulation as well as personal genetic background may affect RSV infectivity. Histone deacetylase (HDAC) is an important epigenetic regulator in lung diseases. The present study aimed to explore the possible connection between HDAC expression and RSV-induced lung inflammation. To address this question, RSV-infected airway epithelial cells (BEAS-2B) were prepared and a mouse model of RSV infection was established, and then treated with various concentrations of HDAC inhibitors (HDACis), namely trichostatin A (TSA) and suberoylanilide hydroxamic acid (SAHA). Viral replication and markers of virus-induced airway inflammation or oxidative stress were assessed. The activation of the nuclear factor-κB (NF-κB), cyclo-oxygenase-2 (COX-2), mitogen-activated protein kinase (MAPK) and signal transducer and activator of transcription 3 (STAT3) signaling pathways was evaluated by western blot analysis. Our results showed that RSV infection in airway epithelial cells (AECs) significantly decreased histone acetylation levels by altering HDAC2 expression. The treatment of RSV-infected AECs with HDACis significantly restricted RSV replication by upregulating the interferon-α (IFN-α) related signaling pathways. The treatment of RSV-infected AECs with HDACis also significantly inhibited RSV-induced pro-inflammatory cytokine release [interleukin (IL)-6 and IL-8] and oxidative stress-related molecule production [malondialdehyde (MDA), and nitrogen monoxide (NO)]. The activation of NF-κB, COX-2, MAPK and Stat3, which orchestrate pro-inflammatory gene expression and oxidative stress injury, was also significantly inhibited. Our in vivo study using a mouse model of RSV infection validated these results. Treatment with HDACis alleviated airway inflammation and reduced in vivo RSV replication. Our data demonstrated that RSV reduced histone acetylation by enhancing HDAC2 expression. Treatment with HDACis (TSA/SAHA) significantly inhibited RSV replication and decreased RSV-induced airway inflammation and oxidative stress. Therefore, the inhibition of HDACs represents a novel therapeutic approach in modulating RSV-induced lung disease. PMID:27460781

Complete Plasmodium falciparum liver-stage development in liver-chimeric mice

PubMed Central

Vaughan, Ashley M.; Mikolajczak, Sebastian A.; Wilson, Elizabeth M.; Grompe, Markus; Kaushansky, Alexis; Camargo, Nelly; Bial, John; Ploss, Alexander; Kappe, Stefan H.I.

2012-01-01

Plasmodium falciparum, which causes the most lethal form of human malaria, replicates in the host liver during the initial stage of infection. However, in vivo malaria liver-stage (LS) studies in humans are virtually impossible, and in vitro models of LS development do not reconstitute relevant parasite growth conditions. To overcome these obstacles, we have adopted a robust mouse model for the study of P. falciparum LS in vivo: the immunocompromised and fumarylacetoacetate hydrolase–deficient mouse (Fah–/–, Rag2–/–, Il2rg–/–, termed the FRG mouse) engrafted with human hepatocytes (FRG huHep). FRG huHep mice supported vigorous, quantifiable P. falciparum LS development that culminated in complete maturation of LS at approximately 7 days after infection, providing a relevant model for LS development in humans. The infections allowed observations of previously unknown expression of proteins in LS, including P. falciparum translocon of exported proteins 150 (PTEX150) and exported protein-2 (EXP-2), components of a known parasite protein export machinery. LS schizonts exhibited exoerythrocytic merozoite formation and merosome release. Furthermore, FRG mice backcrossed to the NOD background and repopulated with huHeps and human red blood cells supported reproducible transition from LS infection to blood-stage infection. Thus, these mice constitute reliable models to study human LS directly in vivo and demonstrate utility for studies of LS–to–blood-stage transition of a human malaria parasite. PMID:22996664

Complete Plasmodium falciparum liver-stage development in liver-chimeric mice.

PubMed

Vaughan, Ashley M; Mikolajczak, Sebastian A; Wilson, Elizabeth M; Grompe, Markus; Kaushansky, Alexis; Camargo, Nelly; Bial, John; Ploss, Alexander; Kappe, Stefan H I

2012-10-01

Plasmodium falciparum, which causes the most lethal form of human malaria, replicates in the host liver during the initial stage of infection. However, in vivo malaria liver-stage (LS) studies in humans are virtually impossible, and in vitro models of LS development do not reconstitute relevant parasite growth conditions. To overcome these obstacles, we have adopted a robust mouse model for the study of P. falciparum LS in vivo: the immunocompromised and fumarylacetoacetate hydrolase-deficient mouse (Fah-/-, Rag2-/-, Il2rg-/-, termed the FRG mouse) engrafted with human hepatocytes (FRG huHep). FRG huHep mice supported vigorous, quantifiable P. falciparum LS development that culminated in complete maturation of LS at approximately 7 days after infection, providing a relevant model for LS development in humans. The infections allowed observations of previously unknown expression of proteins in LS, including P. falciparum translocon of exported proteins 150 (PTEX150) and exported protein-2 (EXP-2), components of a known parasite protein export machinery. LS schizonts exhibited exoerythrocytic merozoite formation and merosome release. Furthermore, FRG mice backcrossed to the NOD background and repopulated with huHeps and human red blood cells supported reproducible transition from LS infection to blood-stage infection. Thus, these mice constitute reliable models to study human LS directly in vivo and demonstrate utility for studies of LS-to-blood-stage transition of a human malaria parasite.

Nuclear Architecture Organized by Rif1 Underpins the Replication-Timing Program

PubMed Central

Foti, Rossana; Gnan, Stefano; Cornacchia, Daniela; Dileep, Vishnu; Bulut-Karslioglu, Aydan; Diehl, Sarah; Buness, Andreas; Klein, Felix A.; Huber, Wolfgang; Johnstone, Ewan; Loos, Remco; Bertone, Paul; Gilbert, David M.; Manke, Thomas; Jenuwein, Thomas; Buonomo, Sara C.B.

2016-01-01

Summary DNA replication is temporally and spatially organized in all eukaryotes, yet the molecular control and biological function of the replication-timing program are unclear. Rif1 is required for normal genome-wide regulation of replication timing, but its molecular function is poorly understood. Here we show that in mouse embryonic stem cells, Rif1 coats late-replicating domains and, with Lamin B1, identifies most of the late-replicating genome. Rif1 is an essential determinant of replication timing of non-Lamin B1-bound late domains. We further demonstrate that Rif1 defines and restricts the interactions between replication-timing domains during the G1 phase, thereby revealing a function of Rif1 as organizer of nuclear architecture. Rif1 loss affects both number and replication-timing specificity of the interactions between replication-timing domains. In addition, during the S phase, Rif1 ensures that replication of interacting domains is temporally coordinated. In summary, our study identifies Rif1 as the molecular link between nuclear architecture and replication-timing establishment in mammals. PMID:26725008

The Development of Afterlife Beliefs in Religiously and Secularly Schooled Children

ERIC Educational Resources Information Center

Bering, Jesse M.; Blasi, Carlos Hernandez; Bjorklund, David F.

2005-01-01

Children aged from 4;10 to 12;9 attending either a Catholic school or a public, secular school in an eastern Spanish city observed a puppet show in which a mouse was eaten by an alligator. Children were then asked questions about the dead mouse's biological and psychological functioning. The pattern of results generally replicated that obtained…

eIF4E Threshold Levels Differ in Governing Normal and Neoplastic Expansion of Mammary Stem and Luminal Progenitor cells

PubMed Central

Avdulov, Svetlana; Herrera, Jeremy; Smith, Karen; Peterson, Mark; Gomez-Garcia, Jose R.; Beadnell, Thomas C.; Schwertfeger, Kathryn L.; Benyumov, Alexey O.; Manivel, J. Carlos; Li, Shunan; Bielinsky, Anja-Katrin; Yee, Douglas; Bitterman, Peter B.; Polunovsky, Vitaly A.

2015-01-01

Translation initiation factor eIF4E mediates normal cell proliferation, yet induces tumorigenesis when overexpressed. The mechanisms by which eIF4E directs such distinct biological outputs remains unknown. We found that mouse mammary morphogenesis during pregnancy and lactation is accompanied by increased cap-binding capability of eIF4E and activation of the eIF4E-dependent translational apparatus, but only subtle oscillations in eIF4E abundance. Using a transgenic mouse model engineered so that lactogenic hormones stimulate a sustained increase in eIF4E abundance in stem/progenitor cells of lactogenic mammary epithelium during successive pregnancy/lactation cycles, eIF4E overexpression increased cell self-renewal, triggered DNA replication stress, and induced formation of pre-malignant and malignant lesions. Using complementary in vivo and ex vivo approaches, we found that increasing eIF4E levels rescued cells harboring oncogenic c-Myc or H-RasV12 from DNA replication stress and oncogene-induced replication catastrophe. Our findings indicate that distinct threshold levels of eIF4E govern its biological output in lactating mammary glands, and that eIF4E overexpression in the context of stem/progenitor cell population expansion can initiate malignant transformation by enabling cells to evade DNA damage checkpoints activated by oncogenic stimuli. Maintaining eIF4E levels below its pro-neoplastic threshold is an important anticancer defense in normal cells, with important implications for understanding pregnancy-associated breast cancer. PMID:25524901

Dynamics of success and failure in phage and antibiotic therapy in experimental infections.

PubMed

Bull, J J; Levin, Bruce R; DeRouin, Terry; Walker, Nina; Bloch, Craig A

2002-11-26

In 1982 Smith and Huggins showed that bacteriophages could be at least as effective as antibiotics in preventing mortality from experimental infections with a capsulated E. coli (K1) in mice. Phages that required the K1 capsule for infection were more effective than phages that did not require this capsule, but the efficacies of phages and antibiotics in preventing mortality both declined with time between infection and treatment, becoming virtually ineffective within 16 hours. We develop quantitative microbiological procedures that (1) explore the in vivo processes responsible for the efficacy of phage and antibiotic treatment protocols in experimental infections (the Resistance Competition Assay, or RCA), and (2) survey the therapeutic potential of phages in vitro (the Phage Replication Assay or PRA). We illustrate the application and utility of these methods in a repetition of Smith and Huggins' experiments, using the E. coli K1 mouse thigh infection model, and applying treatments of phages or streptomycin. 1) The Smith and Huggins phage and antibiotic therapy results are quantitatively and qualitatively robust. (2) Our RCA values reflect the microbiological efficacies of the different phages and of streptomycin in preventing mortality, and reflect the decline in their efficacy with a delay in treatment. These results show specifically that bacteria become refractory to treatment over the term of infection. (3) The K1-specific and non-specific phages had similar replication rates on bacteria grown in broth (based on the PRA), but the K1-specific phage had markedly greater replication rates in mouse serum.

eIF4E threshold levels differ in governing normal and neoplastic expansion of mammary stem and luminal progenitor cells.

PubMed

Avdulov, Svetlana; Herrera, Jeremy; Smith, Karen; Peterson, Mark; Gomez-Garcia, Jose R; Beadnell, Thomas C; Schwertfeger, Kathryn L; Benyumov, Alexey O; Manivel, J Carlos; Li, Shunan; Bielinsky, Anja-Katrin; Yee, Douglas; Bitterman, Peter B; Polunovsky, Vitaly A

2015-02-15

Translation initiation factor eIF4E mediates normal cell proliferation, yet induces tumorigenesis when overexpressed. The mechanisms by which eIF4E directs such distinct biologic outputs remain unknown. We found that mouse mammary morphogenesis during pregnancy and lactation is accompanied by increased cap-binding capability of eIF4E and activation of the eIF4E-dependent translational apparatus, but only subtle oscillations in eIF4E abundance. Using a transgenic mouse model engineered so that lactogenic hormones stimulate a sustained increase in eIF4E abundance in stem/progenitor cells of lactogenic mammary epithelium during successive pregnancy/lactation cycles, eIF4E overexpression increased self-renewal, triggered DNA replication stress, and induced formation of premalignant and malignant lesions. Using complementary in vivo and ex vivo approaches, we found that increasing eIF4E levels rescued cells harboring oncogenic c-Myc or H-RasV12 from DNA replication stress and oncogene-induced replication catastrophe. Our findings indicate that distinct threshold levels of eIF4E govern its biologic output in lactating mammary glands and that eIF4E overexpression in the context of stem/progenitor cell population expansion can initiate malignant transformation by enabling cells to evade DNA damage checkpoints activated by oncogenic stimuli. Maintaining eIF4E levels below its proneoplastic threshold is an important anticancer defense in normal cells, with important implications for understanding pregnancy-associated breast cancer. ©2014 American Association for Cancer Research.

Linker Histone Phosphorylation Regulates Global Timing of Replication Origin Firing*S⃞

PubMed Central

Thiriet, Christophe; Hayes, Jeffrey J.

2009-01-01

Despite the presence of linker histone in all eukaryotes, the primary function(s) of this histone have been difficult to clarify. Knock-out experiments indicate that H1s play a role in regulation of only a small subset of genes but are an essential component in mouse development. Here, we show that linker histone (H1) is involved in the global regulation of DNA replication in Physarum polycephalum. We find that genomic DNA of H1 knock-down cells is more rapidly replicated, an effect due at least in part to disruption of the native timing of replication fork firing. Immunoprecipitation experiments demonstrate that H1 is transiently lost from replicating chromatin via a process facilitated by phosphorylation. Our results suggest that linker histones generate a chromatin environment refractory to replication and that their transient removal via protein phosphorylation during S phase is a critical step in the epigenetic regulation of replication timing. PMID:19015270

What have we learned from brucellosis in the mouse model?

PubMed Central

2012-01-01

Brucellosis is a zoonosis caused by Brucella species. Brucellosis research in natural hosts is often precluded by practical, economical and ethical reasons and mice are widely used. However, mice are not natural Brucella hosts and the course of murine brucellosis depends on bacterial strain virulence, dose and inoculation route as well as breed, genetic background, age, sex and physiological statu of mice. Therefore, meaningful experiments require a definition of these variables. Brucella spleen replication profiles are highly reproducible and course in four phases: i), onset or spleen colonization (first 48 h); ii), acute phase, from the third day to the time when bacteria reach maximal numbers; iii), chronic steady phase, where bacterial numbers plateaus; and iv), chronic declining phase, during which brucellae are eliminated. This pattern displays clear physiopathological signs and is sensitive to small virulence variations, making possible to assess attenuation when fully virulent bacteria are used as controls. Similarly, immunity studies using mice with known defects are possible. Mutations affecting INF-γ, TLR9, Myd88, Tγδ and TNF-β favor Brucella replication; whereas IL-1β, IL-18, TLR4, TLR5, TLR2, NOD1, NOD2, GM-CSF, IL/17r, Rip2, TRIF, NK or Nramp1 deficiencies have no noticeable effects. Splenomegaly development is also useful: it correlates with IFN-γ and IL-12 levels and with Brucella strain virulence. The genetic background is also important: Brucella-resistant mice (C57BL) yield lower splenic bacterial replication and less splenomegaly than susceptible breeds. When inoculum is increased, a saturating dose above which bacterial numbers per organ do not augment, is reached. Unlike many gram-negative bacteria, lethal doses are large (≥ 108 bacteria/mouse) and normally higher than the saturating dose. Persistence is a useful virulence/attenuation index and is used in vaccine (Residual Virulence) quality control. Vaccine candidates are also often tested in mice by determining splenic Brucella numbers after challenging with appropriate virulent brucellae doses at precise post-vaccination times. Since most live or killed Brucella vaccines provide some protection in mice, controls immunized with reference vaccines (S19 or Rev1) are critical. Finally, mice have been successfully used to evaluate brucellosis therapies. It is concluded that, when used properly, the mouse is a valuable brucellosis model. PMID:22500859

Model-based risk assessment and public health analysis to prevent Lyme disease

PubMed Central

Sabounchi, Nasim S.; Roome, Amanda; Spathis, Rita; Garruto, Ralph M.

2017-01-01

The number of Lyme disease (LD) cases in the northeastern United States has been dramatically increasing with over 300 000 new cases each year. This is due to numerous factors interacting over time including low public awareness of LD, risk behaviours and clothing choices, ecological and climatic factors, an increase in rodents within ecologically fragmented peri-urban built environments and an increase in tick density and infectivity in such environments. We have used a system dynamics (SD) approach to develop a simulation tool to evaluate the significance of risk factors in replicating historical trends of LD cases, and to investigate the influence of different interventions, such as increasing awareness, controlling clothing risk and reducing mouse populations, in reducing LD risk. The model accurately replicates historical trends of LD cases. Among several interventions tested using the simulation model, increasing public awareness most significantly reduces the number of LD cases. This model provides recommendations for LD prevention, including further educational programmes to raise awareness and control behavioural risk. This model has the potential to be used by the public health community to assess the risk of exposure to LD. PMID:29291075

Mycobacterium-Host Cell Relationships in Granulomatous Lesions in a Mouse Model of Latent Tuberculous Infection

PubMed Central

2015-01-01

Tuberculosis (TB) is a dangerous infectious disease characterized by a tight interplay between mycobacteria and host cells in granulomatous lesions (granulomas) during the latent, asymptomatic stage of infection. Mycobacterium-host cell relationships were analyzed in granulomas obtained from various organs of BALB/c mice with chronic TB infection caused by in vivo exposure to the Bacillus Calmette-Guérin (BCG) vaccine. Acid-fast BCG-mycobacteria were found to be morphologically and functionally heterogeneous (in size, shape, and replication rates in colonies) in granuloma macrophages, dendritic cells, and multinucleate Langhans giant cells. Cord formation by BCG-mycobacteria in granuloma cells has been observed. Granuloma macrophages retained their ability to ingest damaged lymphocytes and thrombocytes in the phagosomes; however, their ability to destroy BCG-mycobacteria contained in these cells was compromised. No colocalization of BCG-mycobacteria and the LysoTracker dye was observed in the mouse cells. Various relationships between granuloma cells and BCG-mycobacteria were observed in different mice belonging to the same line. Several mice totally eliminated mycobacterial infection. Granulomas in the other mice had mycobacteria actively replicating in cells of different types and forming cords, which is an indicator of mycobacterial virulence and, probably, a marker of the activation of tuberculous infection in animals. PMID:26064970

Mycobacterium-Host Cell Relationships in Granulomatous Lesions in a Mouse Model of Latent Tuberculous Infection.

PubMed

Ufimtseva, Elena

2015-01-01

Tuberculosis (TB) is a dangerous infectious disease characterized by a tight interplay between mycobacteria and host cells in granulomatous lesions (granulomas) during the latent, asymptomatic stage of infection. Mycobacterium-host cell relationships were analyzed in granulomas obtained from various organs of BALB/c mice with chronic TB infection caused by in vivo exposure to the Bacillus Calmette-Guérin (BCG) vaccine. Acid-fast BCG-mycobacteria were found to be morphologically and functionally heterogeneous (in size, shape, and replication rates in colonies) in granuloma macrophages, dendritic cells, and multinucleate Langhans giant cells. Cord formation by BCG-mycobacteria in granuloma cells has been observed. Granuloma macrophages retained their ability to ingest damaged lymphocytes and thrombocytes in the phagosomes; however, their ability to destroy BCG-mycobacteria contained in these cells was compromised. No colocalization of BCG-mycobacteria and the LysoTracker dye was observed in the mouse cells. Various relationships between granuloma cells and BCG-mycobacteria were observed in different mice belonging to the same line. Several mice totally eliminated mycobacterial infection. Granulomas in the other mice had mycobacteria actively replicating in cells of different types and forming cords, which is an indicator of mycobacterial virulence and, probably, a marker of the activation of tuberculous infection in animals.

Activation of chronic toxoplasmosis by transportation stress in a mouse model.

PubMed

Shen, Bang; Yuan, Yuan; Cheng, Jianxi; Pan, Ming; Xia, Ningbo; Zhang, Weichao; Wang, Yifan; Zhou, Yanqin; Zhao, Junlong

2016-12-27

Toxoplasma gondii is an obligate intracellular parasite infecting 25% of the world population and enormous number of animals. It can exist in two forms in intermediate hosts: the fast replicating tachyzoites responsible for acute infection and the slowly replicating bradyzoites responsible for life-long chronic infection. The interconversion between tachyzoites and bradyzoites plays critical roles in the transmission and pathogenesis of T. gondii. However, the molecular mechanisms that govern the interconversion are largely unknown. In this study, we established a chronic infection model in mice and examined the impact of transportation stress on the status of chronic infection. Our results demonstrated that, treating chronically infected mice with conditions mimicking transportation stress reduced the levels of several key cytokines that restrict the infection at chronic stage. Increased expression of the tachyzoite specific gene SAG1 (surface antigen 1) was detected in brain cysts of stress treated mice, indicating activation and conversion of bradyzoites to tachyzoites. Using this model, we identified fifteen toxoplasmic proteins that had significant abundance changes during stress induced cysts reactivation. These proteins serve as a basis for further investigation of the mechanisms governing bradyzoite conversion.

Innate Immune Sensing and Response to Influenza

PubMed Central

Pulendran, Bali; Maddur, Mohan S.

2015-01-01

Influenza viruses pose a substantial threat to human and animal health worldwide. Recent studies in mouse models have revealed an indispensable role for the innate immune system in defense against influenza virus. Recognition of the virus by innate immune receptors in a multitude of cell types activates intricate signaling networks, functioning to restrict viral replication. Downstream effector mechanisms include activation of innate immune cells and, induction and regulation of adaptive immunity. However, uncontrolled innate responses are associated with exaggerated disease, especially in pandemic influenza virus infection. Despite advances in the understanding of innate response to influenza in the mouse model, there is a large knowledge gap in humans, particularly in immunocom-promised groups such as infants and the elderly. We propose here, the need for further studies in humans to decipher the role of innate immunity to influenza virus, particularly at the site of infection. These studies will complement the existing work in mice and facilitate the quest to design improved vaccines and therapeutic strategies against influenza. PMID:25078919

Innate immune sensing and response to influenza.

PubMed

Pulendran, Bali; Maddur, Mohan S

2015-01-01

Influenza viruses pose a substantial threat to human and animal health worldwide. Recent studies in mouse models have revealed an indispensable role for the innate immune system in defense against influenza virus. Recognition of the virus by innate immune receptors in a multitude of cell types activates intricate signaling networks, functioning to restrict viral replication. Downstream effector mechanisms include activation of innate immune cells and, induction and regulation of adaptive immunity. However, uncontrolled innate responses are associated with exaggerated disease, especially in pandemic influenza virus infection. Despite advances in the understanding of innate response to influenza in the mouse model, there is a large knowledge gap in humans, particularly in immunocompromised groups such as infants and the elderly. We propose here, the need for further studies in humans to decipher the role of innate immunity to influenza virus, particularly at the site of infection. These studies will complement the existing work in mice and facilitate the quest to design improved vaccines and therapeutic strategies against influenza.

Boosting ATM activity alleviates aging and extends lifespan in a mouse model of progeria

PubMed Central

Peng, Linyuan; Tang, Xiaolong; Meng, Fanbiao; Ao, Ying; Zhou, Mingyan; Wang, Ming; Cao, Xinyue; Qin, Baoming; Wang, Zimei; Zhou, Zhongjun; Wang, Guangming; Gao, Zhengliang; Xu, Jun

2018-01-01

DNA damage accumulates with age (Lombard et al., 2005). However, whether and how robust DNA repair machinery promotes longevity is elusive. Here, we demonstrate that ATM-centered DNA damage response (DDR) progressively declines with senescence and age, while low dose of chloroquine (CQ) activates ATM, promotes DNA damage clearance, rescues age-related metabolic shift, and prolongs replicative lifespan. Molecularly, ATM phosphorylates SIRT6 deacetylase and thus prevents MDM2-mediated ubiquitination and proteasomal degradation. Extra copies of Sirt6 extend lifespan in Atm-/- mice, with restored metabolic homeostasis. Moreover, the treatment with CQ remarkably extends lifespan of Caenorhabditis elegans, but not the ATM-1 mutants. In a progeria mouse model with low DNA repair capacity, long-term administration of CQ ameliorates premature aging features and extends lifespan. Thus, our data highlights a pro-longevity role of ATM, for the first time establishing direct causal links between robust DNA repair machinery and longevity, and providing therapeutic strategy for progeria and age-related metabolic diseases. PMID:29717979

Boosting ATM activity alleviates aging and extends lifespan in a mouse model of progeria.

PubMed

Qian, Minxian; Liu, Zuojun; Peng, Linyuan; Tang, Xiaolong; Meng, Fanbiao; Ao, Ying; Zhou, Mingyan; Wang, Ming; Cao, Xinyue; Qin, Baoming; Wang, Zimei; Zhou, Zhongjun; Wang, Guangming; Gao, Zhengliang; Xu, Jun; Liu, Baohua

2018-05-02

DNA damage accumulates with age (Lombard et al., 2005). However, whether and how robust DNA repair machinery promotes longevity is elusive. Here, we demonstrate that ATM-centered DNA damage response (DDR) progressively declines with senescence and age, while low dose of chloroquine (CQ) activates ATM, promotes DNA damage clearance, rescues age-related metabolic shift, and prolongs replicative lifespan. Molecularly, ATM phosphorylates SIRT6 deacetylase and thus prevents MDM2-mediated ubiquitination and proteasomal degradation. Extra copies of Sirt6 extend lifespan in Atm-/- mice, with restored metabolic homeostasis. Moreover, the treatment with CQ remarkably extends lifespan of Caenorhabditis elegans , but not the ATM-1 mutants. In a progeria mouse model with low DNA repair capacity, long-term administration of CQ ameliorates premature aging features and extends lifespan. Thus, our data highlights a pro-longevity role of ATM, for the first time establishing direct causal links between robust DNA repair machinery and longevity, and providing therapeutic strategy for progeria and age-related metabolic diseases. © 2018, Qian et al.

Lack of evidence that the XqYq pairing tips at meiosis in the mouse show hypersensitivity to DNAse I.

PubMed

Separovic, E R; Chandley, A C

1987-01-01

In situ nick translation procedures have been applied to meiotic metaphase I divisions of the normal and XY, Sxr mouse. Unlike in man, where the pairing tips of the XY bivalent show a special sensitivity to DNAse I nicking, no such sensitivity can be detected for either of these types of mouse. Hypersensitivity in the D-band equivalent region of the X chromosome does, however, exist, this site being early replicating in somatic cells and housing the X inactivation centre (Xce).

Investigating the Role of Helicobacter pylori PriA Protein.

PubMed

Singh, Aparna; Blaskovic, Dusan; Joo, Jungsoo; Yang, Zhen; Jackson, Sharon H; Coleman, William G; Yan, Ming

2016-08-01

In bacteria, PriA protein, a conserved DEXH-type DNA helicase, plays a central role in replication restart at stalled replication forks. Its unique DNA binding property allows it to recognize and stabilize stalled forks and the structures derived from them. PriA plays a very critical role in replication fork stabilization and DNA repair in E. coli and N. gonorrhoeae. In our in vivo expression technology screen, priA gene was induced in vivo when Helicobacter pylori infects mouse stomach. We decided to elucidate the role of H. pylori PriA protein in survival in mouse stomach, survival in gastric epithelial cells and macrophage cells, DNA repair, acid stress, and oxidative stress. The priA null mutant strain was unable to colonize mice stomach mucosa after long-term infections. Mouse colonization was observed after 1 week of infection, but the levels were much lower than the wild-type HpSS1 strain. PriA protein was found to be important for intracellular survival of epithelial cell-/macrophage cell-ingested H. pylori. Also, a priA null mutant was more sensitive to DNA-damaging agents and was much more sensitive to acid and oxidative stress as compared to the wild-type strain. These data suggest that the PriA protein is needed for survival and persistence of H. pylori in mice stomach mucosa. © 2016 John Wiley & Sons Ltd.

An ear punch model for studying the effect of radiation on wound healing.

PubMed

Deoliveira, Divino; Jiao, Yiqun; Ross, Joel R; Corbin, Kayla; Xiao, Qizhen; Toncheva, Greta; Anderson-Evans, Colin; Yoshizumi, Terry T; Chen, Benny J; Chao, Nelson J

2011-08-01

Radiation and wound combined injury represents a major clinical challenge because of the synergistic interactions that lead to higher morbidity and mortality than either insult would produce singly. The purpose of this study was to develop a mouse ear punch model to study the physiological mechanisms underlying radiation effects on healing wounds. Surgical wounds were induced by a 2 mm surgical punch in the ear pinnae of MRL/MpJ mice. Photographs of the wounds were taken and the sizes of the ear punch wounds were quantified by image analysis. Local radiation to the ear was delivered by orthovoltage X-ray irradiator using a specially constructed jig that shields the other parts of body. Using this model, we demonstrated that local radiation to the wound area significantly delayed the healing of ear punch wounds in a dose-dependent fashion. The addition of sublethal whole body irradiation (7 Gy) further delayed the healing of ear punch wounds. These results were replicated in C57BL/6 mice; however, wound healing in MRL/MpJ mice was accelerated. These data indicate that the mouse ear punch model is a valuable model to study radiation and wound combined injury.

MITA/STING and Its Alternative Splicing Isoform MRP Restrict Hepatitis B Virus Replication

PubMed Central

Liu, Shuhui; Zhao, Kaitao; Su, Xi; Lu, Lu; Zhao, He; Zhang, Xianwen; Wang, Yun; Wu, Chunchen; Chen, Jizheng; Zhou, Yuan; Hu, Xue; Wang, Yanyi; Lu, Mengji; Chen, Xinwen; Pei, Rongjuan

2017-01-01

An efficient clearance of hepatitis B virus (HBV) requires the coordinated work of both the innate and adaptive immune responses. MITA/STING, an adapter protein of the innate immune signaling pathways, plays a key role in regulating innate and adaptive immune responses to DNA virus infection. Previously, we identified an alternatively spliced isoform of MITA/STING, called MITA-related protein (MRP), and found that MRP could specifically block MITA-mediated interferon (IFN) induction while retaining the ability to activate NF-κB. Here, we asked whether MITA/STING and MRP were able to control the HBV replication. Both MITA/STING and MRP significantly inhibited HBV replication in vitro. MITA overexpression stimulated IRF3-IFN pathway; while MRP overexpression activated NF-κB pathway, suggesting these two isoforms may inhibit HBV replication through different ways. Using a hydrodynamic injection (HI) mouse model, we found that HBV replication was reduced following MITA/STING and MRP expression vectors in mice and was enhanced by the knockout of MITA/STING (MITA/STING-/-). The HBV specific humoral and CD8+ T cell responses were impaired in MITA/STING deficient mice, suggesting the participation of MITA/STING in the initiation of host adaptive immune responses. In summary, our data suggest that MITA/STING and MRP contribute to HBV control via modulation of the innate and adaptive responses. PMID:28056087

MITA/STING and Its Alternative Splicing Isoform MRP Restrict Hepatitis B Virus Replication.

PubMed

Liu, Shuhui; Zhao, Kaitao; Su, Xi; Lu, Lu; Zhao, He; Zhang, Xianwen; Wang, Yun; Wu, Chunchen; Chen, Jizheng; Zhou, Yuan; Hu, Xue; Wang, Yanyi; Lu, Mengji; Chen, Xinwen; Pei, Rongjuan

2017-01-01

An efficient clearance of hepatitis B virus (HBV) requires the coordinated work of both the innate and adaptive immune responses. MITA/STING, an adapter protein of the innate immune signaling pathways, plays a key role in regulating innate and adaptive immune responses to DNA virus infection. Previously, we identified an alternatively spliced isoform of MITA/STING, called MITA-related protein (MRP), and found that MRP could specifically block MITA-mediated interferon (IFN) induction while retaining the ability to activate NF-κB. Here, we asked whether MITA/STING and MRP were able to control the HBV replication. Both MITA/STING and MRP significantly inhibited HBV replication in vitro. MITA overexpression stimulated IRF3-IFN pathway; while MRP overexpression activated NF-κB pathway, suggesting these two isoforms may inhibit HBV replication through different ways. Using a hydrodynamic injection (HI) mouse model, we found that HBV replication was reduced following MITA/STING and MRP expression vectors in mice and was enhanced by the knockout of MITA/STING (MITA/STING-/-). The HBV specific humoral and CD8+ T cell responses were impaired in MITA/STING deficient mice, suggesting the participation of MITA/STING in the initiation of host adaptive immune responses. In summary, our data suggest that MITA/STING and MRP contribute to HBV control via modulation of the innate and adaptive responses.

Dormancy in a model of murine B cell lymphoma.

PubMed

Uhr, J W; Marches, R

2001-08-01

A B cell lymphoma model of dormancy in mice was established by prior immunization to the B cell membrane immunoglobulin idiotype. The antibody to the idiotype was the major factor in inducing and maintaining dormancy and acted primarily as an agonist rather than via effector functions. CD8+ T cells synergized with anti-Id in inducing dormancy by secreting IFN-gamma. Cycling in the dormant population was reduced 3-5 fold, but each mouse contained approximately 10(6) tumor cells in its spleen, some of which were cycling, during the 1.5 years of observation. Thus, replication is balanced by cell death. Copyright 2001 Academic Press.

“Humanized mice for HIV and AIDS research”

PubMed Central

Garcia, J. Victor

2016-01-01

HIV has a very limited species tropism that prevents the use of most conventional small animal models for AIDS research. The in vivo analysis of HIV/AIDS has benefited extensively from novel chimeric animal models that accurately recapitulate key aspects of the human condition. Specifically, immunodeficient mice that are systemically repopulated with human hematolymphoid cells offer a viable alternative for the study of a multitude of highly relevant aspects of HIV replication, pathogenesis, therapy, transmission, prevention, and eradication. This article summarizes some of the multiple contributions that humanized mouse models of HIV infection have made to the field of AIDS research. These models have proven to be highly informative and hold great potential for accelerating multiple aspects of HIV research in the future. PMID:27447446

Influenza-Induced Inflammation Drives Pneumococcal Otitis Media

PubMed Central

Reading, Patrick C.; Brown, Lorena E.; Pedersen, John; Gilbertson, Brad; Job, Emma R.; Edenborough, Kathryn M.; Habets, Marrit N.; Zomer, Aldert; Hermans, Peter W. M.; Diavatopoulos, Dimitri A.; Wijburg, Odilia L.

2013-01-01

Influenza A virus (IAV) predisposes individuals to secondary infections with the bacterium Streptococcus pneumoniae (the pneumococcus). Infections may manifest as pneumonia, sepsis, meningitis, or otitis media (OM). It remains controversial as to whether secondary pneumococcal disease is due to the induction of an aberrant immune response or IAV-induced immunosuppression. Moreover, as the majority of studies have been performed in the context of pneumococcal pneumonia, it remains unclear how far these findings can be extrapolated to other pneumococcal disease phenotypes such as OM. Here, we used an infant mouse model, human middle ear epithelial cells, and a series of reverse-engineered influenza viruses to investigate how IAV promotes bacterial OM. Our data suggest that the influenza virus HA facilitates disease by inducing a proinflammatory response in the middle ear cavity in a replication-dependent manner. Importantly, our findings suggest that it is the inflammatory response to IAV infection that mediates pneumococcal replication. This study thus provides the first evidence that inflammation drives pneumococcal replication in the middle ear cavity, which may have important implications for the treatment of pneumococcal OM. PMID:23319557

Sensitive luminescent reporter viruses reveal appreciable release of hepatitis C virus NS5A protein into the extracellular environment.

PubMed

Eyre, Nicholas S; Aloia, Amanda L; Joyce, Michael A; Chulanetra, Monrat; Tyrrell, D Lorne; Beard, Michael R

2017-07-01

The HCV NS5A protein is essential for viral RNA replication and virus particle assembly. To study the viral replication cycle and NS5A biology we generated an infectious HCV construct with a NanoLuciferase (NLuc) insertion within NS5A. Surprisingly, beyond its utility as a sensitive reporter of cytoplasmic viral RNA replication, we also observed strong luminescence in cell culture fluids. Further analysis using assembly-defective viruses and subgenomic replicons revealed that infectious virus production was not required for extracellular NS5A-NLuc activity but was associated with enrichment of extracellular NS5A-NLuc in intermediate-density fractions similar to those of exosomes and virus particles. Additionally, BRET analysis indicated that intracellular and extracellular forms of NS5A may adopt differing conformations. Importantly, infection studies using a human liver chimeric mouse model confirmed robust infection in vivo and ready detection of NLuc activity in serum. We hypothesise that the presence of NS5A in extracellular fluids contributes to HCV pathogenesis. Copyright © 2017 Elsevier Inc. All rights reserved.

Lentiviral-mediated gene therapy results in sustained expression of β-glucuronidase for up to 12 months in the gus(mps/mps) and up to 18 months in the gus(tm(L175F)Sly) mouse models of mucopolysaccharidosis type VII.

PubMed

Derrick-Roberts, Ainslie L K; Pyragius, Carmen E; Kaidonis, Xenia M; Jackson, Matilda R; Anson, Donald S; Byers, Sharon

2014-09-01

A number of mucopolysaccharidosis type VII (MPS VII) mouse models with different levels of residual enzyme activity have been created replicating the range of clinical phenotypes observed in human MPS VII patients. In this study, a lentivirus encoding murine β-glucuronidase was administered intravenously at birth to both the severe (Gus(mps/mps) strain) and attenuated (Gus(tm(L175F)Sly) strain) mouse models of MPS VII. Circulating enzyme levels were normalized in the Gus(mps/mps) mice and were 3.5-fold higher than normal in the Gus(tm(L175F)Sly) mouse 12 and 18 months after administration. Tissue β-glucuronidase activity increased over untreated levels in all tissues evaluated in both strains at 12 months, and the elevated level was maintained in Gus(tm(L175F)Sly) tissues at 18 months. These elevated enzyme levels reduced glycosaminoglycan storage in the liver, spleen, kidney, and heart in both models. Bone mineral volume decreased toward normal in both models after 12 months of therapy and after 18 months in the Gus(tm(L175F)Sly) mouse. Open-field exploration was improved in 18-month-old treated Gus(tm(L175F)Sly) mice, while spatial learning improved in both 12- and 18-month-old treated Gus(tm(L175F)Sly) mice. Overall, neonatal administration of lentiviral gene therapy resulted in sustained enzyme expression for up to 18 months in murine models of MPS VII. Significant improvements in biochemistry and enzymology as well as functional improvement of bone and behavior deficits in the Gus(tm(L175F)Sly) model were observed. Therapy significantly increased the lifespan of Gus(mps/mps) mice, with 12 months being the longest reported lentiviral treatment for this strain. It is important to assess the long-term outcome on enzyme levels and effect on pathology for lentiviral gene therapy to be a potential therapy for MPS patients.

Multicolor Fluorescence Imaging of Traumatic Brain Injury in a Cryolesion Mouse Model

PubMed Central

2012-01-01

Traumatic brain injury is characterized by initial tissue damage, which then can lead to secondary processes such as cell death and blood-brain-barrier disruption. Clinical and preclinical studies of traumatic brain injury typically employ anatomical imaging techniques and there is a need for new molecular imaging methods that provide complementary biochemical information. Here, we assess the ability of a targeted, near-infrared fluorescent probe, named PSS-794, to detect cell death in a brain cryolesion mouse model that replicates certain features of traumatic brain injury. In short, the model involves brief contact of a cold rod to the head of a living, anesthetized mouse. Using noninvasive whole-body fluorescence imaging, PSS-794 permitted visualization of the cryolesion in the living animal. Ex vivo imaging and histological analysis confirmed PSS-794 localization to site of brain cell death. The nontargeted, deep-red Tracer-653 was validated as a tracer dye for monitoring blood-brain-barrier disruption, and a binary mixture of PSS-794 and Tracer-653 was employed for multicolor imaging of cell death and blood-brain-barrier permeability in a single animal. The imaging data indicates that at 3 days after brain cryoinjury the amount of cell death had decreased significantly, but the integrity of the blood-brain-barrier was still impaired; at 7 days, the blood-brain-barrier was still three times more permeable than before cryoinjury. PMID:22860222

Oncolytic adenovirus that overproduces ADP and replicates selectively in tumors due to hTERT promoter-regulated E4 gene expression.

PubMed

Kuppuswamy, M; Spencer, J F; Doronin, K; Tollefson, A E; Wold, W S M; Toth, K

2005-11-01

We have constructed a novel oncolytic adenovirus (Ad) vector, named VRX-011, in which the replication of the vector is targeted to cancer cells by the replacement of the wild-type Ad E4 promoter with the human telomerase reverse transcriptase (hTERT) promoter. Genes in the Ad E4 transcription unit are essential for Ad replication; therefore, VRX-011 will grow efficiently only in cells in which the hTERT promoter is active, that is, in a wide range of cancer and immortalized cells but not in most somatic cells. Consistent with these expectations, VRX-011 replicated efficiently in all cancer cell lines examined, while its growth was restricted in various primary and normal cells. VRX-011 overexpresses ADP (also known as E3-11.6K), an Ad protein required for efficient cell lysis and release of virions from cells at late stages of infection. This overexpression enhances cell-to-cell spread and could significantly increase antitumor efficacy. In a xenograft model in nude mice, both intratumoral and intravenous administration of VRX-011 effectively suppressed the growth of subcutaneous Hep3B human liver tumors. Also, intravenous delivery of VRX-011 greatly reduced the number and size of A549 human lung cancer cell nodules in a disseminated lung tumor model in nude mice. Importantly, tail vein administration of different doses of VRX-011 in C57BL/6 mice showed minimal liver toxicity. Considering its broad range of lytic replication in cancer cells, its attenuated phenotype in primary cells, its efficacy in suppressing xenografts, and its low toxicity in mouse liver, VRX-011 is a promising candidate for further evaluation as an anticancer therapeutic.

Saquinavir-mediated inhibition of human immunodeficiency virus (HIV) infection in SCID mice implanted with human fetal thymus and liver tissue: an in vivo model for evaluating the effect of drug therapy on HIV infection in lymphoid tissues.

PubMed Central

Pettoello-Mantovani, M; Kollmann, T R; Raker, C; Kim, A; Yurasov, S; Tudor, R; Wiltshire, H; Goldstein, H

1997-01-01

Treatment with protease inhibitors alone or in combination with inhibitors of reverse transcriptase potently suppresses levels of human immunodeficiency virus (HIV) RNA in plasma and thereby may significantly delay the progression of HIV-mediated disease. To investigate the effect of treatment with the protease inhibitor saquinavir on HIV replication in the lymphoid tissues, we used a SCID-hu mouse model that we developed, in which human thymic and liver tissues (hu-thy/liv) were implanted under both kidney capsules in SCID mice (thy/liv-SCID-hu mice). These mice are populated in the periphery with large numbers of human T cells and develop disseminated HIV infection after intraimplant injection. thy/liv-SCID-hu mice with established HIV infection that were treated for 1 month with saquinavir had a significantly lower viral load present in the implanted hu-thy/liv and mouse spleen than did the untreated HIV-infected thy/liv-SCID-hu mice. To examine the capacity of acute treatment with saquinavir to prevent HIV infection, some thy/liv-SCID-hu mice were inoculated with HIV and then immediately started on saquinavir. Although treated mice had markedly lower viral loads in the thy/liv implants and spleens, HIV infection was not completely prevented. Thus, the effect of antiviral therapy on HIV infection in the major site of HIV replication, the lymphoid tissues, can be readily evaluated in our thy/liv-SCID-hu mice. These mice should prove to be a useful model for determining the in vivo effectiveness of different therapeutic interventions on acute and chronic HIV infection. PMID:9303378

Cyclosporin A Inhibits Rotavirus Replication and Restores Interferon-Beta Signaling Pathway In Vitro and In Vivo

PubMed Central

He, Haiyang; Wu, Yuzhang

2013-01-01

Rotavirus (RV) is the most common cause of severe diarrhea among infants and young children. Currently, there is no specific drug available against rotavirus, largely due to the lack of an ideal target molecule which has hampered drug development. Our previous studies have revealed that cyclosporin A (CsA) might be potentially useful as an anti-RV drug. We therefore used both cellular and mouse models to study the immunological safety and effectiveness of CsA as an anti-RV drug. We found that CsA treatment of HT-29 cells before, during, and after viral infection efficiently inhibited Wa strain RV replication and restored IFN-β expression in a HT-29 cell line model. Exploring the underlying mechanisms showed that CsA promoted Interferon Regulatory Factor-5 (IRF-5) expression (a key positive regulator of the type I IFN signaling pathway), but not IRF-1, IRF-3, or IRF-7. Additionally, CsA inhibited SOCS-1 expression (the key negative regulator of IFN-α/β), but not SOCS-2 or SOCS-3. The antiviral effect of CsA was confirmed in an RV-infected neonatal mouse model by evaluation of antigen clearance and assessment of changes in intestinal tissue pathology. Also, no differences in T cell frequency or proliferation between the CsA- and vehicle-treated groups were observed. Thus, both our in vitro and in vivo findings suggest that CsA, through modulating the expression of key regulators in IFN signaling pathway, promote type I IFN-based intracellular innate immunity in RV host cells. These findings suggest that CsA may be a useful candidate to develop a new anti-RV strategy, although further evaluation and characterization of CsA on RV-induced diarrhea are warranted. PMID:23990993

Antibodies Mediate Formation of Neutrophil Extracellular Traps in the Middle Ear and Facilitate Secondary Pneumococcal Otitis Media

PubMed Central

Short, Kirsty R.; von Köckritz-Blickwede, Maren; Langereis, Jeroen D.; Chew, Keng Yih; Job, Emma R.; Armitage, Charles W.; Hatcher, Brandon; Fujihashi, Kohtaro; Reading, Patrick C.; Hermans, Peter W.

2014-01-01

Otitis media (OM) (a middle ear infection) is a common childhood illness that can leave some children with permanent hearing loss. OM can arise following infection with a variety of different pathogens, including a coinfection with influenza A virus (IAV) and Streptococcus pneumoniae (the pneumococcus). We and others have demonstrated that coinfection with IAV facilitates the replication of pneumococci in the middle ear. Specifically, we used a mouse model of OM to show that IAV facilitates the outgrowth of S. pneumoniae in the middle ear by inducing middle ear inflammation. Here, we seek to understand how the host inflammatory response facilitates bacterial outgrowth in the middle ear. Using B cell-deficient infant mice, we show that antibodies play a crucial role in facilitating pneumococcal replication. We subsequently show that this is due to antibody-dependent neutrophil extracellular trap (NET) formation in the middle ear, which, instead of clearing the infection, allows the bacteria to replicate. We further demonstrate the importance of these NETs as a potential therapeutic target through the transtympanic administration of a DNase, which effectively reduces the bacterial load in the middle ear. Taken together, these data provide novel insight into how pneumococci are able to replicate in the middle ear cavity and induce disease. PMID:24191297

Comparative analysis of European bat lyssavirus 1 pathogenicity in the mouse model.

PubMed

Eggerbauer, Elisa; Pfaff, Florian; Finke, Stefan; Höper, Dirk; Beer, Martin; Mettenleiter, Thomas C; Nolden, Tobias; Teifke, Jens-Peter; Müller, Thomas; Freuling, Conrad M

2017-06-01

European bat lyssavirus 1 is responsible for most bat rabies cases in Europe. Although EBLV-1 isolates display a high degree of sequence identity, different sublineages exist. In individual isolates various insertions and deletions have been identified, with unknown impact on viral replication and pathogenicity. In order to assess whether different genetic features of EBLV-1 isolates correlate with phenotypic changes, different EBLV-1 variants were compared for pathogenicity in the mouse model. Groups of three mice were infected intracranially (i.c.) with 102 TCID50/ml and groups of six mice were infected intramuscularly (i.m.) with 105 TCID50/ml and 102 TCID50/ml as well as intranasally (i.n.) with 102 TCID50/ml. Significant differences in survival following i.m. inoculation with low doses as well as i.n. inoculation were observed. Also, striking variations in incubation periods following i.c. inoculation and i.m. inoculation with high doses were seen. Hereby, the clinical picture differed between general symptoms, spasms and aggressiveness depending on the inoculation route. Immunohistochemistry of mouse brains showed that the virus distribution in the brain depended on the inoculation route. In conclusion, different EBLV-1 isolates differ in pathogenicity indicating variation which is not reflected in studies of single isolates.

Comparative analysis of European bat lyssavirus 1 pathogenicity in the mouse model

PubMed Central

Eggerbauer, Elisa; Pfaff, Florian; Finke, Stefan; Höper, Dirk; Beer, Martin; Mettenleiter, Thomas C.; Nolden, Tobias; Teifke, Jens-Peter; Müller, Thomas

2017-01-01

European bat lyssavirus 1 is responsible for most bat rabies cases in Europe. Although EBLV-1 isolates display a high degree of sequence identity, different sublineages exist. In individual isolates various insertions and deletions have been identified, with unknown impact on viral replication and pathogenicity. In order to assess whether different genetic features of EBLV-1 isolates correlate with phenotypic changes, different EBLV-1 variants were compared for pathogenicity in the mouse model. Groups of three mice were infected intracranially (i.c.) with 102 TCID50/ml and groups of six mice were infected intramuscularly (i.m.) with 105 TCID50/ml and 102 TCID50/ml as well as intranasally (i.n.) with 102 TCID50/ml. Significant differences in survival following i.m. inoculation with low doses as well as i.n. inoculation were observed. Also, striking variations in incubation periods following i.c. inoculation and i.m. inoculation with high doses were seen. Hereby, the clinical picture differed between general symptoms, spasms and aggressiveness depending on the inoculation route. Immunohistochemistry of mouse brains showed that the virus distribution in the brain depended on the inoculation route. In conclusion, different EBLV-1 isolates differ in pathogenicity indicating variation which is not reflected in studies of single isolates. PMID:28628617

Sensitization to radiation and alkylating agents by inhibitors of poly(ADP-ribose) polymerase is enhanced in cells deficient in DNA double-strand break repair.

PubMed

Löser, Dana A; Shibata, Atsushi; Shibata, Akiko K; Woodbine, Lisa J; Jeggo, Penny A; Chalmers, Anthony J

2010-06-01

As single agents, chemical inhibitors of poly(ADP-ribose) polymerase (PARP) are nontoxic and have clinical efficacy against BRCA1- and BRCA2-deficient tumors. PARP inhibitors also enhance the cytotoxicity of ionizing radiation and alkylating agents but will only improve clinical outcomes if tumor sensitization exceeds effects on normal tissues. It is unclear how tumor DNA repair proficiency affects the degree of sensitization. We have previously shown that the radiosensitizing effect of PARP inhibition requires DNA replication and will therefore affect rapidly proliferating tumors more than normal tissues. Because many tumors exhibit defective DNA repair, we investigated the impact of double-strand break (DSB) repair integrity on the sensitizing effects of the PARP inhibitor olaparib. Sensitization to ionizing radiation and the alkylating agent methylmethane sulfonate was enhanced in DSB repair-deficient cells. In Artemis(-/-) and ATM(-/-) mouse embryo fibroblasts, sensitization was replication dependent and associated with defective repair of replication-associated damage. Radiosensitization of Ligase IV(-/-) mouse embryo fibroblasts was independent of DNA replication and is explained by inhibition of "alternative" end joining. After methylmethane sulfonate treatment, PARP inhibition promoted replication-independent accumulation of DSB, repair of which required Ligase IV. Our findings predict that the sensitizing effects of PARP inhibitors will be more pronounced in rapidly dividing and/or DNA repair defective tumors than normal tissues and show their potential to enhance the therapeutic ratio achieved by conventional DNA-damaging agents.

PrPC Governs Susceptibility to Prion Strains in Bank Vole, While Other Host Factors Modulate Strain Features

PubMed Central

Espinosa, J. C.; Nonno, R.; Di Bari, M.; Aguilar-Calvo, P.; Pirisinu, L.; Fernández-Borges, N.; Vanni, I.; Vaccari, G.; Marín-Moreno, A.; Frassanito, P.; Lorenzo, P.; Agrimi, U.

2016-01-01

ABSTRACT Bank vole is a rodent species that shows differential susceptibility to the experimental transmission of different prion strains. In this work, the transmission features of a panel of diverse prions with distinct origins were assayed both in bank vole expressing methionine at codon 109 (Bv109M) and in transgenic mice expressing physiological levels of bank vole PrPC (the BvPrP-Tg407 mouse line). This work is the first systematic comparison of the transmission features of a collection of prion isolates, representing a panel of diverse prion strains, in a transgenic-mouse model and in its natural counterpart. The results showed very similar transmission properties in both the natural species and the transgenic-mouse model, demonstrating the key role of the PrP amino acid sequence in prion transmission susceptibility. However, differences in the PrPSc types propagated by Bv109M and BvPrP-Tg407 suggest that host factors other than PrPC modulate prion strain features. IMPORTANCE The differential susceptibility of bank voles to prion strains can be modeled in transgenic mice, suggesting that this selective susceptibility is controlled by the vole PrP sequence alone rather than by other species-specific factors. Differences in the phenotypes observed after prion transmissions in bank voles and in the transgenic mice suggest that host factors other than the PrPC sequence may affect the selection of the substrain replicating in the animal model. PMID:27654300

Generation of influenza A viruses as live but replication-incompetent virus vaccines.

PubMed

Si, Longlong; Xu, Huan; Zhou, Xueying; Zhang, Ziwei; Tian, Zhenyu; Wang, Yan; Wu, Yiming; Zhang, Bo; Niu, Zhenlan; Zhang, Chuanling; Fu, Ge; Xiao, Sulong; Xia, Qing; Zhang, Lihe; Zhou, Demin

2016-12-02

The conversion of life-threatening viruses into live but avirulent vaccines represents a revolution in vaccinology. In a proof-of-principle study, we expanded the genetic code of the genome of influenza A virus via a transgenic cell line containing orthogonal translation machinery. This generated premature termination codon (PTC)-harboring viruses that exerted full infectivity but were replication-incompetent in conventional cells. Genome-wide optimization of the sites for incorporation of multiple PTCs resulted in highly reproductive and genetically stable progeny viruses in transgenic cells. In mouse, ferret, and guinea pig models, vaccination with PTC viruses elicited robust humoral, mucosal, and T cell-mediated immunity against antigenically distinct influenza viruses and even neutralized existing infecting strains. The methods presented here may become a general approach for generating live virus vaccines that can be adapted to almost any virus. Copyright © 2016, American Association for the Advancement of Science.

A systematic approach to the development of a safe live attenuated Zika vaccine.

PubMed

Kwek, Swee Sen; Watanabe, Satoru; Chan, Kuan Rong; Ong, Eugenia Z; Tan, Hwee Cheng; Ng, Wy Ching; Nguyen, Mien T X; Gan, Esther S; Zhang, Summer L; Chan, Kitti W K; Tan, Jun Hao; Sessions, October M; Manuel, Menchie; Pompon, Julien; Chua, Camillus; Hazirah, Sharifah; Tryggvason, Karl; Vasudevan, Subhash G; Ooi, Eng Eong

2018-03-12

Zika virus (ZIKV) is a flavivirus that can cause congenital disease and requires development of an effective long-term preventative strategy. A replicative ZIKV vaccine with properties similar to the yellow fever 17D (YF17D) live-attenuated vaccine (LAV) would be advantageous, as a single dose of YF17D produces lifelong immunity. However, a replicative ZIKV vaccine must also be safe from causing persistent organ infections. Here we report an approach to ZIKV LAV development. We identify a ZIKV variant that produces small plaques due to interferon (IFN)-restricted viral propagation and displays attenuated infection of endothelial cells. We show that these properties collectively reduce the risk of organ infections and vertical transmission in a mouse model but remain sufficiently immunogenic to prevent wild-type ZIKV infection. Our findings suggest a strategy for the development of a safe but efficacious ZIKV LAV.

Evidence for a Stable Intermediate in Leukemia Virus Activation in AKR Mouse Embryo Cells

PubMed Central

Ihle, James N.; Kenney, Francis T.; Tennant, Raymond W.

1974-01-01

Analysis of the requirement for serum in the activation of the endogenous leukemia virus expression in AKR mouse embryo cells by 5-iododeoxyuridine shows that activation can be dissociated into two discrete serum-dependent events. The first involves incorporation of 5-iododeoxyuridine into DNA and results in the formation of a stable “activation intermediate” resembling the provirus formed during infection of stationary mouse embryo cells with exogenous leukemia virus. The second event, resulting in expression of the activation intermediate as synthesis of virus proteins, requires DNA replication but not 5-iododeoxyuridine. PMID:4604455

A Chimeric Humanized Mouse Model by Engrafting the Human Induced Pluripotent Stem Cell-Derived Hepatocyte-Like Cell for the Chronic Hepatitis B Virus Infection

PubMed Central

Yuan, Lunzhi; Liu, Xuan; Zhang, Liang; Li, Xiaoling; Zhang, Yali; Wu, Kun; Chen, Yao; Cao, Jiali; Hou, Wangheng; Zhang, Jun; Zhu, Hua; Yuan, Quan; Tang, Qiyi; Cheng, Tong; Xia, Ningshao

2018-01-01

Humanized mouse model generated by grafting primary human hepatocytes (PHHs) to immunodeficient mouse has contributed invaluably to understanding the pathogenesis of hepatitis B virus (HBV). However, the source of PHHs is limited, which necessitates the search for alternatives. Recently, hepatocyte-like cells (HLCs) generated from human induced pluripotent stem cells (hiPSCs) have been used for in vitro HBV infection. Herein, we developed a robust human liver chimeric animal model to study in vivo HBV infection by engrafting the hiPSC-HLCs to Fah-/-Rag2-/-IL-2Rγc-/- SCID (FRGS) mice. After being optimized by a small molecule, XMU-MP-1, the hiPSC-HLCs engrafted FRGS (hHLC-FRGS) mice displayed approximately 40% liver chimerism at week 6 after engraftment and maintained at this level for at least 14 weeks. Viremia and HBV infection markers include antigens, RNA, DNA, and covalently closed circular DNA were detectable in HBV infected hHLC-FRGS mice. Furthermore, hiPSC-HLCs and hHLC-FRGS mice were successfully used to evaluate different antivirals. Therefore, we established a humanized mouse model for not only investigating HBV pathogenesis but also testing the effects of the anti-HBV drugs. Highlights:    (1) The implanted hiPSC-HLCs established a long-term chimerism in FRGS mice liver.    (2) hHLC-FRGS mice are adequate to support chronic HBV infection with a full viral life cycle.    (3) hiPSC-HLCs and hHLC-FRGS mice are useful tools for evaluation of antivirals against HBV infection in vitro and in vivo. Research in Context  To overcome the disadvantages of using primary human hepatocytes, we induced human pluripotent stem cells to hepatocyte-like cells (hiPSC-HLCs) that developed the capability to express important liver functional markers and critical host factors for HBV infection. The hiPSC-HLCs were permissive for the HBV infection and supported a full HBV replication. The hiPSC-HLCs were then engrafted to immunodeficient mouse to establish a chimeric liver mouse model, which was capable of supporting HBV infection in vivo and evaluating the effects of antiviral drugs. Our results shed light into improving the cellular and animal models for studying HBV and other hepatotropic viruses. PMID:29867819

Electron Microscopic Analysis of the Effects of Psoralen and Interferon on Replicative Intermediates Formed During Encephalomyocarditis Virus Infection of Mouse L Cells

DTIC Science & Technology

1987-08-24

became possible with the development of practical methods for the propagation of poliovirus in cultured cells, the development of plaque assays...definition of the essential nutrients in cultured cells, and the purification, crystalization and physicochemical characterization of poliovirus ...Levintow, 1974). Information on viral replication of the most conunonly studied picornavirus, human poliovirus , was reviewed by Darnell and Eagle (1960

Mammalian DNA enriched for replication origins is enriched for snap-back sequences.

PubMed

Zannis-Hadjopoulos, M; Kaufmann, G; Martin, R G

1984-11-15

Using the instability of replication loops as a method for the isolation of double-stranded nascent DNA, extruded DNA enriched for replication origins was obtained and denatured. Snap-back DNA, single-stranded DNA with inverted repeats (palindromic sequences), reassociates rapidly into stem-loop structures with zero-order kinetics when conditions are changed from denaturing to renaturing, and can be assayed by chromatography on hydroxyapatite. Origin-enriched nascent DNA strands from mouse, rat and monkey cells growing either synchronously or asynchronously were purified and assayed for the presence of snap-back sequences. The results show that origin-enriched DNA is also enriched for snap-back sequences, implying that some origins for mammalian DNA replication contain or lie near palindromic sequences.

Brucella discriminates between mouse dendritic cell subsets upon in vitro infection.

PubMed

Papadopoulos, Alexia; Gagnaire, Aurélie; Degos, Clara; de Chastellier, Chantal; Gorvel, Jean-Pierre

2016-01-01

Brucella is a Gram-negative bacterium responsible for brucellosis, a worldwide re-emerging zoonosis. Brucella has been shown to infect and replicate within Granulocyte macrophage colony-stimulating factor (GMCSF) in vitro grown bone marrow-derived dendritic cells (BMDC). In this cell model, Brucella can efficiently control BMDC maturation. However, it has been shown that Brucella infection in vivo induces spleen dendritic cells (DC) migration and maturation. As DCs form a complex network composed by several subpopulations, differences observed may be due to different interactions between Brucella and DC subsets. Here, we compare Brucella interaction with several in vitro BMDC models. The present study shows that Brucella is capable of replicating in all the BMDC models tested with a high infection rate at early time points in GMCSF-IL15 DCs and Flt3l DCs. GMCSF-IL15 DCs and Flt3l DCs are more activated than the other studied DC models and consequently intracellular bacteria are not efficiently targeted to the ER replicative niche. Interestingly, GMCSF-DC and GMCSF-Flt3l DC response to infection is comparable. However, the key difference between these 2 models concerns IL10 secretion by GMCSF DCs observed at 48 h post-infection. IL10 secretion can explain the weak secretion of IL12p70 and TNFα in the GMCSF-DC model and the low level of maturation observed when compared to GMCSF-IL15 DCs and Flt3l DCs. These models provide good tools to understand how Brucella induce DC maturation in vivo and may lead to new therapeutic design using DCs as cellular vaccines capable of enhancing immune response against pathogens.

Brain growth trajectories in mouse strains with central and peripheral serotonin differences: relevance to autism models.

PubMed

Flood, Z C; Engel, D L J; Simon, C C; Negherbon, K R; Murphy, L J; Tamavimok, W; Anderson, G M; Janušonis, S

2012-05-17

The genetic heterogeneity of autism spectrum disorders (ASDs) suggests that their underlying neurobiology involves dysfunction at the neural network level. Understanding these neural networks will require a major collaborative effort and will depend on validated and widely accepted animal models. Many mouse models have been proposed in autism research, but the assessment of their validity often has been limited to measuring social interactions. However, two other well-replicated findings have been reported in ASDs: transient brain overgrowth in early postnatal life and elevated 5-HT (serotonin) levels in blood platelets (platelet hyperserotonemia). We examined two inbred mouse strains (C57BL/6 and BALB/c) with respect to these phenomena. The BALB/c strain is less social and exhibits some other autistic-like behaviors. In addition, it has a lower 5-HT synthesis rate in the central nervous system due to a single-nucleotide polymorphism in the tryptophan hydroxylase 2 (Tph2) gene. The postnatal growth of brain mass was analyzed with mixed-effects models that included litter effects. The volume of the hippocampal complex and the thickness of the somatosensory cortex were measured in 3D-brain reconstructions from serial sections. The postnatal whole-blood 5-HT levels were assessed with high-performance liquid chromatography. With respect to the BALB/c strain, the C57BL/6 strain showed transient brain overgrowth and persistent blood hyperserotonemia. The hippocampal volume was permanently enlarged in the C57BL/6 strain, with no change in the adult brain mass. These results indicate that, in mice, autistic-like shifts in the brain and periphery may be associated with less autistic-like behaviors. Importantly, they suggest that consistency among behavioral, anatomical, and physiological measures may expedite the validation of new and previously proposed mouse models of autism, and that the construct validity of models should be demonstrated when these measures are inconsistent. Copyright © 2012 IBRO. Published by Elsevier Ltd. All rights reserved.

Intelligence in the brain: a theory of how it works and how to build it.

PubMed

Werbos, Paul J

2009-04-01

This paper presents a theory of how general-purpose learning-based intelligence is achieved in the mammal brain, and how we can replicate it. It reviews four generations of ever more powerful general-purpose learning designs in Adaptive, Approximate Dynamic Programming (ADP), which includes reinforcement learning as a special case. It reviews empirical results which fit the theory, and suggests important new directions for research, within the scope of NSF's recent initiative on Cognitive Optimization and Prediction. The appendices suggest possible connections to the realms of human subjective experience, comparative cognitive neuroscience, and new challenges in electric power. The major challenge before us today in mathematical neural networks is to replicate the "mouse level", but the paper does contain a few thoughts about building, understanding and nourishing levels of general intelligence beyond the mouse.

Molecular Interactions in the Replication of Mouse Hepatitis Virus

DTIC Science & Technology

1987-05-08

viruses of vertebrate species which cause a variety of acute 1 2 Figure 1. Mouse hepatitis virus , strain A59. (a) Negative stain of a virion...coronaviruses and the human corona - virus 229E (Weiss and Leibowitz, 1981). Antigenic relationships. Coronaviruses fall into two main groups...rate zonal and isopycnic centrifugation on density gradients. The density of corona - viruses in sucrose is 1.16 to 1.18 g/ml. Virions contain RNA

Histone deacetylase inhibitors suppress RSV infection and alleviate virus-induced airway inflammation.

PubMed

Feng, Qiuqin; Su, Zhonglan; Song, Shiyu; Χu, Hui; Zhang, Bin; Yi, Long; Tian, Man; Wang, Hongwei

2016-09-01

Respiratory syncytial virus (RSV) is the leading cause of lower respiratory tract infections in infants and young children. However, the majority of RSV-infected patients only show mild symptoms. Different severities of infection and responses among the RSV-infected population indicate that epigenetic regulation as well as personal genetic background may affect RSV infectivity. Histone deacetylase (HDAC) is an important epigenetic regulator in lung diseases. The present study aimed to explore the possible connection between HDAC expression and RSV-induced lung inflammation. To address this question, RSV-infected airway epithelial cells (BEAS‑2B) were prepared and a mouse model of RSV infection was established, and then treated with various concentrations of HDAC inhibitors (HDACis), namely trichostatin A (TSA) and suberoylanilide hydroxamic acid (SAHA). Viral replication and markers of virus-induced airway inflammation or oxidative stress were assessed. The activation of the nuclear factor-κB (NF-κB), cyclo-oxygenase-2 (COX-2), mitogen-activated protein kinase (MAPK) and signal transducer and activator of transcription 3 (STAT3) signaling pathways was evaluated by western blot analysis. Our results showed that RSV infection in airway epithelial cells (AECs) significantly decreased histone acetylation levels by altering HDAC2 expression. The treatment of RSV-infected AECs with HDACis significantly restricted RSV replication by upregulating the interferon-α (IFN-α) related signaling pathways. The treatment of RSV-infected AECs with HDACis also significantly inhibited RSV-induced pro-inflammatory cytokine release [interleukin (IL)-6 and IL-8] and oxidative stress-related molecule production [malondialdehyde (MDA), and nitrogen monoxide (NO)]. The activation of NF-κB, COX-2, MAPK and Stat3, which orchestrate pro‑inflammatory gene expression and oxidative stress injury, was also significantly inhibited. Our in vivo study using a mouse model of RSV infection validated these results. Treatment with HDACis alleviated airway inflammation and reduced in vivo RSV replication. Our data demonstrated that RSV reduced histone acetylation by enhancing HDAC2 expression. Treatment with HDACis (TSA/SAHA) significantly inhibited RSV replication and decreased RSV-induced airway inflammation and oxidative stress. Therefore, the inhibition of HDACs represents a novel therapeutic approach in modulating RSV-induced lung disease.

T cells establish and maintain CNS viral infection in HIV-infected humanized mice.

PubMed

Honeycutt, Jenna B; Liao, Baolin; Nixon, Christopher C; Cleary, Rachel A; Thayer, William O; Birath, Shayla L; Swanson, Michael D; Sheridan, Patricia; Zakharova, Oksana; Prince, Francesca; Kuruc, JoAnn; Gay, Cynthia L; Evans, Chris; Eron, Joseph J; Wahl, Angela; Garcia, J Victor

2018-06-04

The human brain is an important site of HIV replication and persistence during antiretroviral therapy (ART). Direct evaluation of HIV infection in the brains of otherwise healthy individuals is not feasible; therefore, we performed a large-scale study of bone marrow/liver/thymus (BLT) humanized mice as an in vivo model to study HIV infection in the brain. Human immune cells, including CD4+ T cells and macrophages, were present throughout the BLT mouse brain. HIV DNA, HIV RNA, and/or p24+ cells were observed in the brains of HIV-infected animals, regardless of the HIV isolate used. HIV infection resulted in decreased numbers of CD4+ T cells, increased numbers of CD8+ T cells, and a decreased CD4+/CD8+ T cell ratio in the brain. Using humanized T cell-only mice (ToM), we demonstrated that T cells establish and maintain HIV infection of the brain in the complete absence of human myeloid cells. HIV infection of ToM resulted in CD4+ T cell depletion and a reduced CD4+/CD8+ T cell ratio. ART significantly reduced HIV levels in the BLT mouse brain, and the immune cell populations present were indistinguishable from those of uninfected controls, which demonstrated the effectiveness of ART in controlling HIV replication in the CNS and returning cellular homeostasis to a pre-HIV state.

The new strains Brucella inopinata BO1 and Brucella species 83-210 behave biologically like classic infectious Brucella species and cause death in murine models of infection.

PubMed

Jiménez de Bagüés, María P; Iturralde, María; Arias, Maykel A; Pardo, Julián; Cloeckaert, Axel; Zygmunt, Michel S

2014-08-01

Recently, novel atypical Brucella strains isolated from humans and wild rodents have been reported. They are phenotypically close to Ochrobactrum species but belong to the genus Brucella, based on genetic relatedness, although genetic diversity is higher among the atypical Brucella strains than between the classic species. They were classified within or close to the novel species Brucella inopinata. However, with the exception of Brucella microti, the virulence of these novel strains has not been investigated in experimental models of infection. The type species B. inopinata strain BO1 (isolated from a human) and Brucella species strain 83-210 (isolated from a wild Australian rodent) were investigated. A classic infectious Brucella reference strain, B. suis 1330, was also used. BALB/c, C57BL/6, and CD1 mice models and C57BL/6 mouse bone-marrow-derived macrophages (BMDMs) were used as infection models. Strains BO1 and 83-210 behaved similarly to reference strain 1330 in all mouse infection models: there were similar growth curves in spleens and livers of mice and similar intracellular replication rates in BMDMs. However, unlike strain 1330, strains BO1 and 83-210 showed lethality in the 3 mouse models. The novel atypical Brucella strains of this study behave like classic intracellular Brucella pathogens. In addition, they cause death in murine models of infection, as previously published for B. microti, another recently described environmental and wildlife species. © The Author 2014. Published by Oxford University Press on behalf of the Infectious Diseases Society of America. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

Genetically engineered mouse models for functional studies of SKP1-CUL1-F-box-protein (SCF) E3 ubiquitin ligases.

PubMed

Zhou, Weihua; Wei, Wenyi; Sun, Yi

2013-05-01

The SCF (SKP1 (S-phase-kinase-associated protein 1), Cullin-1, F-box protein) E3 ubiquitin ligases, the founding member of Cullin-RING ligases (CRLs), are the largest family of E3 ubiquitin ligases in mammals. Each individual SCF E3 ligase consists of one adaptor protein SKP1, one scaffold protein cullin-1 (the first family member of the eight cullins), one F-box protein out of 69 family members, and one out of two RING (Really Interesting New Gene) family proteins RBX1/ROC1 or RBX2/ROC2/SAG/RNF7. Various combinations of these four components construct a large number of SCF E3s that promote the degradation of many key regulatory proteins in cell-context, temporally, and spatially dependent manners, thus controlling precisely numerous important cellular processes, including cell cycle progression, apoptosis, gene transcription, signal transduction, DNA replication, maintenance of genome integrity, and tumorigenesis. To understand how the SCF E3 ligases regulate these cellular processes and embryonic development under in vivo physiological conditions, a number of mouse models with transgenic (Tg) expression or targeted deletion of components of SCF have been established and characterized. In this review, we will provide a brief introduction to the ubiquitin-proteasome system (UPS) and the SCF E3 ubiquitin ligases, followed by a comprehensive overview on the existing Tg and knockout (KO) mouse models of the SCF E3s, and discuss the role of each component in mouse embryogenesis, cell proliferation, apoptosis, carcinogenesis, as well as other pathogenic processes associated with human diseases. We will end with a brief discussion on the future directions of this research area and the potential applications of the knowledge gained to more effective therapeutic interventions of human diseases.

Repetitive Self-Grooming Behavior in the BTBR Mouse Model of Autism is Blocked by the mGluR5 Antagonist MPEP

PubMed Central

Silverman, Jill L; Tolu, Seda S; Barkan, Charlotte L; Crawley, Jacqueline N

2010-01-01

Autism is a neurodevelopmental disorder characterized by abnormal reciprocal social interactions, communication deficits, and repetitive behaviors with restricted interests. BTBR T+tf/J (BTBR) is an inbred mouse strain that shows robust behavioral phenotypes with analogies to all three of the diagnostic symptoms of autism, including well-replicated deficits in reciprocal social interactions and social approach, unusual patterns of ultrasonic vocalization, and high levels of repetitive self-grooming. These phenotypes offer straightforward behavioral assays for translational investigations of pharmacological compounds. Two suggested treatments for autism were evaluated in the BTBR mouse model. Methyl-6-phenylethynyl-pyridine (MPEP), an antagonist of the mGluR5 metabotropic glutamate receptor, blocks aberrant phenotypes in the Fmr1 mouse model of Fragile X, a comorbid neurodevelopmental disorder with autistic features. Risperidone has been approved by the United States Food and Drug Administration for the treatment of irritability, tantrums, and self-injurious behavior in autistic individuals. We evaluated the actions of MPEP and risperidone on two BTBR phenotypes, low sociability and high repetitive self-grooming. Open field activity served as an independent control for non-social exploratory activity and motor functions. C57BL/6J (B6), an inbred strain with high sociability and low self-grooming, served as the strain control. MPEP significantly reduced repetitive self-grooming in BTBR, at doses that had no sedating effects on open field activity. Risperidone reduced repetitive self-grooming in BTBR, but only at doses that induced sedation in both strains. No overall improvements in sociability were detected in BTBR after treatment with either MPEP or risperidone. Our findings suggest that antagonists of mGluR5 receptors may have selective therapeutic efficacy in treating repetitive behaviors in autism. PMID:20032969

An ear punch model for studying the effect of radiation on wound healing

PubMed Central

DeOLIVEIRA, DIVINO; JIAO, YIQUN; ROSS, JOEL R.; CORBIN, KAYLA; XIAO, QIZHEN; TONCHEVA, GRETA; ANDERSON-EVANS, COLIN; YOSHIZUMI, TERRY T.; CHEN, BENNY J.; CHAO, NELSON J.

2011-01-01

Purpose Radiation and wound combined injury represents a major clinical challenge because of the synergistic interactions that lead to higher morbidity and mortality than either insult would produce singly. The purpose of this study was to develop a mouse ear punch model to study the physiological mechanisms underlying radiation effects on healing wounds. Materials and methods Surgical wounds were induced by a 2 mm surgical punch in the ear pinnae of MRL/MpJ mice. Photographs of the wounds were taken and the sizes of the ear punch wounds were quantified by image analysis. Local radiation to the ear was delivered by orthovoltage X-ray irradiator using a specially constructed jig that shields the other parts of body. Results Using this model, we demonstrated that local radiation to the wound area significantly delayed the healing of ear punch wounds in a dose-dependent fashion. The addition of sublethal whole body irradiation (7 Gy) further delayed the healing of ear punch wounds. These results were replicated in C57BL/6 mice; however, wound healing in MRL/MpJ mice was accelerated. Conclusions These data indicate that the mouse ear punch model is a valuable model to study radiation and wound combined injury. PMID:21480768

Amplified and persistent immune responses generated by single-cycle replicating adenovirus vaccines.

PubMed

Crosby, Catherine M; Nehete, Pramod; Sastry, K Jagannadha; Barry, Michael A

2015-01-01

Replication-competent adenoviral (RC-Ad) vectors generate exceptionally strong gene-based vaccine responses by amplifying the antigen transgenes they carry. While they are potent, they also risk causing adenovirus infections. More common replication-defective Ad (RD-Ad) vectors with deletions of E1 avoid this risk but do not replicate their transgene and generate markedly weaker vaccine responses. To amplify vaccine transgenes while avoiding production of infectious progeny viruses, we engineered "single-cycle" adenovirus (SC-Ad) vectors by deleting the gene for IIIa capsid cement protein of lower-seroprevalence adenovirus serotype 6. In mouse, human, hamster, and macaque cells, SC-Ad6 still replicated its genome but prevented genome packaging and virion maturation. When used for mucosal intranasal immunization of Syrian hamsters, both SC-Ad and RC-Ad expressed transgenes at levels hundreds of times higher than that of RD-Ad. Surprisingly, SC-Ad, but not RC-Ad, generated higher levels of transgene-specific antibody than RD-Ad, which notably climbed in serum and vaginal wash samples over 12 weeks after single mucosal immunization. When RD-Ad and SC-Ad were tested by single sublingual immunization in rhesus macaques, SC-Ad generated higher gamma interferon (IFN-γ) responses and higher transgene-specific serum antibody levels. These data suggest that SC-Ad vectors may have utility as mucosal vaccines. This work illustrates the utility of our recently developed single-cycle adenovirus (SC-Ad6) vector as a new vaccine platform. Replication-defective (RD-Ad6) vectors produce low levels of transgene protein, which leads to minimal antibody responses in vivo. This study shows that replicating SC-Ad6 produces higher levels of luciferase and induces higher levels of green fluorescent protein (GFP)-specific antibodies than RD in a permissive Syrian hamster model. Surprisingly, although a replication-competent (RC-Ad6) vector produces more luciferase than SC-Ad6, it does not elicit comparable levels of anti-GFP antibodies in permissive hamsters. When tested in the larger rhesus macaque model, SC-Ad6 induces higher transgene-specific antibody and T cell responses. Together, these data suggest that SC-Ad6 could be a more effective platform for developing vaccines against more relevant antigens. This could be especially beneficial for developing vaccines for pathogens for which traditional replication-defective adenovirus vectors have not been effective. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

T cell mediated suppression of neurotropic coronavirus replication in neural precursor cells

PubMed Central

Plaisted, Warren C.; Weinger, Jason G.; Walsh, Craig M.; Lane, Thomas E.

2014-01-01

Neural precursor cells (NPCs) are the subject of intense investigation for their potential to treat neurodegenerative disorders, yet the consequences of neuroinvasive virus infection of NPCs remain unclear. This study demonstrates that NPCs support replication following infection by the neurotropic JHM strain of mouse hepatitis virus (JHMV). JHMV infection leads to increased cell death and dampens IFN-γ-induced MHC class II expression. Importantly, cytokines secreted by CD4+ T cells inhibit JHMV replication in NPCs, and CD8+ T cells specifically target viral peptide-pulsed NPCs for lysis. Furthermore, treatment with IFN-γ inhibits JHMV replication in a dose-dependent manner. Together, these findings suggest that T cells play a critical role in controlling replication of a neurotropic virus in NPCs, a finding which has important implications when considering immune modulation for NPC-based therapies for treatment of human neurologic diseases. PMID:24418558

RTEL1 is a replisome-associated helicase that promotes telomere and genome-wide replication.

PubMed

Vannier, Jean-Baptiste; Sandhu, Sumit; Petalcorin, Mark I R; Wu, Xiaoli; Nabi, Zinnatun; Ding, Hao; Boulton, Simon J

2013-10-11

Regulator of telomere length 1 (RTEL1) is an essential DNA helicase that disassembles telomere loops (T loops) and suppresses telomere fragility to maintain the integrity of chromosome ends. We established that RTEL1 also associates with the replisome through binding to proliferating cell nuclear antigen (PCNA). Mouse cells disrupted for the RTEL1-PCNA interaction (PIP mutant) exhibited accelerated senescence, replication fork instability, reduced replication fork extension rates, and increased origin usage. Although T-loop disassembly at telomeres was unaffected in the mutant cells, telomere replication was compromised, leading to fragile sites at telomeres. RTEL1-PIP mutant mice were viable, but loss of the RTEL1-PCNA interaction accelerated the onset of tumorigenesis in p53-deficient mice. We propose that RTEL1 plays a critical role in both telomere and genome-wide replication, which is crucial for genetic stability and tumor avoidance.

Genome Wide Identification of SARS-CoV Susceptibility Loci Using the Collaborative Cross

PubMed Central

Gralinski, Lisa E.; Ferris, Martin T.; Aylor, David L.; Whitmore, Alan C.; Green, Richard; Frieman, Matthew B.; Deming, Damon; Menachery, Vineet D.; Miller, Darla R.; Buus, Ryan J.; Bell, Timothy A.; Churchill, Gary A.; Threadgill, David W.; Katze, Michael G.; McMillan, Leonard; Valdar, William; Heise, Mark T.; Pardo-Manuel de Villena, Fernando; Baric, Ralph S.

2015-01-01

New systems genetics approaches are needed to rapidly identify host genes and genetic networks that regulate complex disease outcomes. Using genetically diverse animals from incipient lines of the Collaborative Cross mouse panel, we demonstrate a greatly expanded range of phenotypes relative to classical mouse models of SARS-CoV infection including lung pathology, weight loss and viral titer. Genetic mapping revealed several loci contributing to differential disease responses, including an 8.5Mb locus associated with vascular cuffing on chromosome 3 that contained 23 genes and 13 noncoding RNAs. Integrating phenotypic and genetic data narrowed this region to a single gene, Trim55, an E3 ubiquitin ligase with a role in muscle fiber maintenance. Lung pathology and transcriptomic data from mice genetically deficient in Trim55 were used to validate its role in SARS-CoV-induced vascular cuffing and inflammation. These data establish the Collaborative Cross platform as a powerful genetic resource for uncovering genetic contributions of complex traits in microbial disease severity, inflammation and virus replication in models of outbred populations. PMID:26452100

Relationship between Immunity to Borrelia burgdorferi Outer-surface Protein A (OspA) and Lyme Arthritis

PubMed Central

Drouin, Elise E.; Glickstein, Lisa J.

2011-01-01

Antibiotic-refractory Lyme arthritis may result from Borrelia burgdorferi–induced autoimmunity in affected joints. Such patients usually have certain HLA-DRB1 molecules that bind an epitope of B. burgdorferi outer-surface protein A (OspA163–175), and cellular and humoral immune responses to OspA are greater in patients with antibiotic-refractory arthritis than in those with antibiotic-responsive arthritis. Recent work in a mouse model suggests that, during B. burgdorferi infection, OspA in genetically susceptible individuals stimulates a particularly strong TH1 response, which may be one of several factors that can help set the stage for a putative autoimmune response in affected joints. However, vaccination with OspA did not induce arthritis in this mouse model, and case and control comparisons in human vaccine trials did not show an increased frequency of arthritis among OspA-vaccinated individuals. Thus, a vaccine-induced immune response to OspA does not replicate the sequence of events needed in the natural infection to induce antibiotic-refractory Lyme arthritis. PMID:21217173

Expression quantitative trait loci: replication, tissue- and sex-specificity in mice.

PubMed

van Nas, Atila; Ingram-Drake, Leslie; Sinsheimer, Janet S; Wang, Susanna S; Schadt, Eric E; Drake, Thomas; Lusis, Aldons J

2010-07-01

By treating the transcript abundance as a quantitative trait, gene expression can be mapped to local or distant genomic regions relative to the gene encoding the transcript. Local expression quantitative trait loci (eQTL) generally act in cis (that is, control the expression of only the contiguous structural gene), whereas distal eQTL act in trans. Distal eQTL are more difficult to identify with certainty due to the fact that significant thresholds are very high since all regions of the genome must be tested, and confounding factors such as batch effects can produce false positives. Here, we compare findings from two large genetic crosses between mouse strains C3H/HeJ and C57BL/6J to evaluate the reliability of distal eQTL detection, including "hotspots" influencing the expression of multiple genes in trans. We found that >63% of local eQTL and >18% of distal eQTL were replicable at a threshold of LOD > 4.3 between crosses and 76% of local and >24% of distal eQTL at a threshold of LOD > 6. Additionally, at LOD > 4.3 four tissues studied (adipose, brain, liver, and muscle) exhibited >50% preservation of local eQTL and >17% preservation of distal eQTL. We observed replicated distal eQTL hotspots between the crosses on chromosomes 9 and 17. Finally, >69% of local eQTL and >10% of distal eQTL were preserved in most tissues between sexes. We conclude that most local eQTL are highly replicable between mouse crosses, tissues, and sex as compared to distal eQTL, which exhibited modest replicability.

A Recombinant Adenovirus Expressing Ovine Interferon Tau Prevents Influenza Virus-Induced Lethality in Mice.

PubMed

Martín, V; Pascual, E; Avia, M; Rangel, G; de Molina, A; Alejo, A; Sevilla, N

2016-01-06

Ovine interferon tau (IFN-τ) is a unique type I interferon with low toxicity and a broad host range in vivo. We report the generation of a nonreplicative recombinant adenovirus expressing biologically active IFN-τ. Using the B6.A2G-Mx1 mouse model, we showed that single-dose intranasal administration of recombinant Ad5-IFN-τ can effectively prevent lethality and disease induced by highly virulent hv-PR8 influenza virus by activating the interferon response and preventing viral replication. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Inferring cortical function in the mouse visual system through large-scale systems neuroscience.

PubMed

Hawrylycz, Michael; Anastassiou, Costas; Arkhipov, Anton; Berg, Jim; Buice, Michael; Cain, Nicholas; Gouwens, Nathan W; Gratiy, Sergey; Iyer, Ramakrishnan; Lee, Jung Hoon; Mihalas, Stefan; Mitelut, Catalin; Olsen, Shawn; Reid, R Clay; Teeter, Corinne; de Vries, Saskia; Waters, Jack; Zeng, Hongkui; Koch, Christof

2016-07-05

The scientific mission of the Project MindScope is to understand neocortex, the part of the mammalian brain that gives rise to perception, memory, intelligence, and consciousness. We seek to quantitatively evaluate the hypothesis that neocortex is a relatively homogeneous tissue, with smaller functional modules that perform a common computational function replicated across regions. We here focus on the mouse as a mammalian model organism with genetics, physiology, and behavior that can be readily studied and manipulated in the laboratory. We seek to describe the operation of cortical circuitry at the computational level by comprehensively cataloging and characterizing its cellular building blocks along with their dynamics and their cell type-specific connectivities. The project is also building large-scale experimental platforms (i.e., brain observatories) to record the activity of large populations of cortical neurons in behaving mice subject to visual stimuli. A primary goal is to understand the series of operations from visual input in the retina to behavior by observing and modeling the physical transformations of signals in the corticothalamic system. We here focus on the contribution that computer modeling and theory make to this long-term effort.

Primer retention owing to the absence of RNase H1 is catastrophic for mitochondrial DNA replication.

PubMed

Holmes, J Bradley; Akman, Gokhan; Wood, Stuart R; Sakhuja, Kiran; Cerritelli, Susana M; Moss, Chloe; Bowmaker, Mark R; Jacobs, Howard T; Crouch, Robert J; Holt, Ian J

2015-07-28

Encoding ribonuclease H1 (RNase H1) degrades RNA hybridized to DNA, and its function is essential for mitochondrial DNA maintenance in the developing mouse. Here we define the role of RNase H1 in mitochondrial DNA replication. Analysis of replicating mitochondrial DNA in embryonic fibroblasts lacking RNase H1 reveals retention of three primers in the major noncoding region (NCR) and one at the prominent lagging-strand initiation site termed Ori-L. Primer retention does not lead immediately to depletion, as the persistent RNA is fully incorporated in mitochondrial DNA. However, the retained primers present an obstacle to the mitochondrial DNA polymerase γ in subsequent rounds of replication and lead to the catastrophic generation of a double-strand break at the origin when the resulting gapped molecules are copied. Hence, the essential role of RNase H1 in mitochondrial DNA replication is the removal of primers at the origin of replication.

Interferon lambda (IFN-λ) efficiently blocks norovirus transmission in a mouse model.

PubMed

Rocha-Pereira, Joana; Jacobs, Sophie; Noppen, Sam; Verbeken, Eric; Michiels, Thomas; Neyts, Johan

2018-01-01

Human noroviruses are highly efficient in person to person transmission thus associated with explosive outbreaks of acute gastroenteritis. Outbreak control is limited to disinfection and isolation measures. Strategies to control the spread of noroviruses should be developed and models to study norovirus transmission will greatly facilitate this. Here, a mouse-to-mouse transmission model, in which mice develop acute murine norovirus (MNV)-induced diarrhea, was used to explore the role of interferon lambda (IFN-λ) in the control of a norovirus infection. Sentinel AG129 mice [deficient in IFN-α/β and IFN-γ receptors] that were co-housed with MNV-infected mice shedding high amounts of virus in their stool, developed a MNV-infection with associated diarrhea. Inoculation of such sentinel mice with an IFN-λ expression plasmid resulted in the production of circulating IFN-λ and upregulation of the expression of IFN-stimulated genes (ISGs) of the gut. Injection of the IFN-λ-expressing plasmid to sentinels prevents MNV-induced disease upon exposure to MNV-infected mice, as well as MNV replication in the small intestine, the associated signs of inflammation and the mounting of a specific IgG-based immune response. This demonstrates that IFN-λ can alone mediate protection against transmission of norovirus. The development of a simple delivery method for IFN-λ could be explored as a strategy to control norovirus outbreaks and protect vulnerable populations such as the elderly and immunocompromised. Copyright © 2017. Published by Elsevier B.V.

A novel type of cellular senescence that can be enhanced in mouse models and human tumor xenografts to suppress prostate tumorigenesis

PubMed Central

Alimonti, Andrea; Nardella, Caterina; Chen, Zhenbang; Clohessy, John G.; Carracedo, Arkaitz; Trotman, Lloyd C.; Cheng, Ke; Varmeh, Shohreh; Kozma, Sara C.; Thomas, George; Rosivatz, Erika; Woscholski, Rudiger; Cognetti, Francesco; Scher, Howard I.; Pandolfi, Pier Paolo

2010-01-01

Irreversible cell growth arrest, a process termed cellular senescence, is emerging as an intrinsic tumor suppressive mechanism. Oncogene-induced senescence is thought to be invariably preceded by hyperproliferation, aberrant replication, and activation of a DNA damage checkpoint response (DDR), rendering therapeutic enhancement of this process unsuitable for cancer treatment. We previously demonstrated in a mouse model of prostate cancer that inactivation of the tumor suppressor phosphatase and tensin homolog deleted on chromosome 10 (Pten) elicits a senescence response that opposes tumorigenesis. Here, we show that Pten-loss–induced cellular senescence (PICS) represents a senescence response that is distinct from oncogene-induced senescence and can be targeted for cancer therapy. Using mouse embryonic fibroblasts, we determined that PICS occurs rapidly after Pten inactivation, in the absence of cellular proliferation and DDR. Further, we found that PICS is associated with enhanced p53 translation. Consistent with these data, we showed that in mice p53-stabilizing drugs potentiated PICS and its tumor suppressive potential. Importantly, we demonstrated that pharmacological inhibition of PTEN drives senescence and inhibits tumorigenesis in vivo in a human xenograft model of prostate cancer. Taken together, our data identify a type of cellular senescence that can be triggered in nonproliferating cells in the absence of DNA damage, which we believe will be useful for developing a “pro-senescence” approach for cancer prevention and therapy. PMID:20197621

Crimean-Congo haemorrhagic fever virus replication in adult Hyalomma truncatum and Amblyomma variegatum ticks.

PubMed

Gonzalez, J P; Cornet, J P; Wilson, M L; Camicas, J L

1991-01-01

The kinetics of the replication of the Crimean-Congo haemorrhagic fever virus (CCHFV) was studied in intra-anally inoculated adult Hyalomma truncatum and Amblyomma variegatum ticks. The virus was re-isolated by suckling mouse inoculation and revealed by antigen capture with ground ticks and indirect immunofluorescence of haemolymph. The virus was detected in ticks in the first hours post-inoculation (p.i.) and its replication was observed from 36 h p.i. onwards. Virus titre reached a maximum within 3-5 days then decreased slowly to a level of at 2 log LD50/ml for several months until the end of observations. Several specific, non-identified factors seem to favour CCHFV replication in H. truncatum. Long-term virus persistence seems to occur in CCHFV-infected adult ticks.

The Nucleocapsid Protein of Coronaviruses Acts as a Viral Suppressor of RNA Silencing in Mammalian Cells.

PubMed

Cui, Lei; Wang, Haiying; Ji, Yanxi; Yang, Jie; Xu, Shan; Huang, Xingyu; Wang, Zidao; Qin, Lei; Tien, Po; Zhou, Xi; Guo, Deyin; Chen, Yu

2015-09-01

RNA interference (RNAi) is a process of eukaryotic posttranscriptional gene silencing that functions in antiviral immunity in plants, nematodes, and insects. However, recent studies provided strong supports that RNAi also plays a role in antiviral mechanism in mammalian cells. To combat RNAi-mediated antiviral responses, many viruses encode viral suppressors of RNA silencing (VSR) to facilitate their replication. VSRs have been widely studied for plant and insect viruses, but only a few have been defined for mammalian viruses currently. We identified a novel VSR from coronaviruses, a group of medically important mammalian viruses including Severe acute respiratory syndrome coronavirus (SARS-CoV), and showed that the nucleocapsid protein (N protein) of coronaviruses suppresses RNAi triggered by either short hairpin RNAs or small interfering RNAs in mammalian cells. Mouse hepatitis virus (MHV) is closely related to SARS-CoV in the family Coronaviridae and was used as a coronavirus replication model. The replication of MHV increased when the N proteins were expressed in trans, while knockdown of Dicer1 or Ago2 transcripts facilitated the MHV replication in mammalian cells. These results support the hypothesis that RNAi is a part of the antiviral immunity responses in mammalian cells. IMPORTANCE RNAi has been well known to play important antiviral roles from plants to invertebrates. However, recent studies provided strong supports that RNAi is also involved in antiviral response in mammalian cells. An important indication for RNAi-mediated antiviral activity in mammals is the fact that a number of mammalian viruses encode potent suppressors of RNA silencing. Our results demonstrate that coronavirus N protein could function as a VSR through its double-stranded RNA binding activity. Mutational analysis of N protein allowed us to find out the critical residues for the VSR activity. Using the MHV-A59 as the coronavirus replication model, we showed that ectopic expression of SARS-CoV N protein could promote MHV replication in RNAi-active cells but not in RNAi-depleted cells. These results indicate that coronaviruses encode a VSR that functions in the replication cycle and provide further evidence to support that RNAi-mediated antiviral response exists in mammalian cells.

Repurposing Cytarabine for Treating Primary Effusion Lymphoma by Targeting Kaposi’s Sarcoma-Associated Herpesvirus Latent and Lytic Replications

PubMed Central

Gruffaz, Marion; Zhou, Shenghua; Vasan, Karthik; Rushing, Teresa; Michael, Qing Liu; Lu, Chu

2018-01-01

ABSTRACT Oncogenic Kaposi’s sarcoma-associated herpesvirus (KSHV) is etiologically linked to primary effusion lymphoma (PEL), an aggressive and nontreatable malignancy commonly found in AIDS patients. In this study, we performed a high-throughput screening of 3,731 characterized compounds and identified cytarabine, approved by the FDA for treating numerous types of cancer, as a potent inhibitor of KSHV-induced PEL. We showed the high efficacy of cytarabine in the growth inhibition of various PEL cells by inducing cell cycle arrest and apoptosis. Cytarabine inhibited host DNA and RNA syntheses and therefore induced cellular cytotoxicity. Furthermore, cytarabine inhibited viral DNA and RNA syntheses and induced the rapid degradation of KSHV major latent protein LANA (latency-associated nuclear antigen), leading to the suppression of KSHV latent replication. Importantly, cytarabine effectively inhibited active KSHV replication and virion production in PEL cells. Finally, cytarabine treatments not only effectively inhibited the initiation and progression of PEL tumors but also induced regression of grown PEL tumors in a xenograft mouse model. Altogether, our study has identified cytarabine as a novel therapeutic agent for treating PEL as well as eliminating KSHV persistent infection. PMID:29739902

Tumor-specific cytolysis caused by an E1B55K-attenuated adenovirus in nasopharyngeal carcinoma is augmented by cisplatin.

PubMed

Liu, Ran-Yi; Peng, Ji-Lin; Li, Yong-Qiang; Huang, Bi-Jun; Lin, Huan-Xin; Zhou, Ling; Luo, Hui-Ling; Huang, Wenlin

2013-12-01

An E1B55K-attenuated adenovirus, dl1520, has been shown to replicate selectively in and lyse tumor cells. In this study, the antitumor activities of dl1520, alone or in combination with the chemotherapeutic agent cisplatin, were investigated in nasopharyngeal carcinoma (NPC) cells. The results demonstrated that dl1520 replicated in and destroyed NPC cells, and induced apoptosis in vitro. In a nude mouse xenograft model, dl1520 significantly inhibited the growth of NPC cell xenografts, and the viral replication was associated with tumor regression. Importantly, the antitumor activity of dl1520 was augmented by the addition of cisplatin both in vitro and in vivo, showing that dl1520 and cisplatin have a synergistic anti-NPC effect. These data suggest that dl1520 exerts an efficient anti-NPC activity through oncolysis and the induction of apoptosis, which is enhanced synergistically by cisplatin. These findings indicate that oncolytic viral therapeutics using the E1B55K-attenuated adenovirus dl1520 could be promising in the comprehensive treatment of NPC, especially in combination with platinum-based chemotherapy. Copyright © 2013 Wiley Periodicals, Inc.

A poliovirus-induced cytoplasmic membrane complex is exploited by the RNA polymerase of superinfecting Mouse Elberfeld (ME) virus.

PubMed

Zeichhardt, H; Habermehl, K O; Wetz, K

1983-04-01

The preexistence of a cytoplasmic membrane complex in HEp-2 cells, induced by poliovirus when inhibited in its reproduction by guanidine, was a prerequisite for accelerated reproduction of superinfecting Mouse Elberfeld (ME) virus. Guanidine-inhibited poliovirus induced a membrane complex of 470S that was successively modified into a faster sedimenting membrane complex (up to 700S) by superinfecting ME virus and exploited for ME virus reproduction. The modified membrane complex was the site for ME virus-specific RNA polymerization characterized by the existence of in vivo and in vitro activity of ME virus RNA polymerase associated with the modified membrane complex. Proof of membrane-bound RNA polymerase and newly synthesized ME virus RNA including replicative intermediate led to the conclusion that superinfecting ME virus exploits the 'poliovirus/guanidine'-induced complex as the site of action of its replication complex.

Identification of Novel Kaposi's Sarcoma-Associated Herpesvirus Orf50 Transcripts: Discovery of New RTA Isoforms with Variable Transactivation Potential

PubMed Central

Wakeman, Brian S.; Izumiya, Yoshihiro

2016-01-01

ABSTRACT Kaposi's sarcoma-associated herpesvirus (KSHV) is a gammaherpesvirus that has been associated with primary effusion lymphoma and multicentric Castleman's disease, as well as its namesake Kaposi's sarcoma. As a gammaherpesvirus, KSHV is able to acutely replicate, enter latency, and reactivate from this latent state. A key protein involved in both acute replication and reactivation from latency is the replication and transcriptional activator (RTA) encoded by the gene Orf50. RTA is a known transactivator of multiple viral genes, allowing it to control the switch between latency and virus replication. We report here the identification of six alternatively spliced Orf50 transcripts that are generated from four distinct promoters. These newly identified promoters are shown to be transcriptionally active in 293T (embryonic kidney), Vero (African-green monkey kidney epithelial), 3T12 (mouse fibroblast), and RAW 264.7 (mouse macrophage) cell lines. Notably, the newly identified Orf50 transcripts are predicted to encode four different isoforms of the RTA which differ by 6 to 10 residues at the amino terminus of the protein. We show the global viral transactivation potential of all four RTA isoforms and demonstrate that all isoforms can transcriptionally activate an array of KSHV promoters to various levels. The pattern of transcriptional activation appears to support a transcriptional interference model within the Orf50 region, where silencing of previously expressed isoforms by transcription initiation from upstream Orf50 promoters has the potential to modulate the pattern of viral gene activation. IMPORTANCE Gammaherpesviruses are associated with the development of lymphomas and lymphoproliferative diseases, as well as several other types of cancer. The human gammaherpesvirus, Kaposi's sarcoma-associated herpesvirus (KSHV), is tightly associated with the development of Kaposi's sarcoma and multicentric Castleman's disease, as well as a rare form of B cell lymphoma (primary effusion lymphoma) primarily observed in HIV-infected individuals. RTA is an essential viral gene product involved in the initiation of gammaherpesvirus replication and is conserved among all known gammaherpesviruses. We show here for KSHV that transcription of the gene encoding RTA is complex and leads to the expression of several isoforms of RTA with distinct functions. This observed complexity in KSHV RTA expression and function likely plays a critical role in the regulation of downstream viral and cellular gene expression, leading to the efficient production of mature virions. PMID:27795414

Human hepatocytes support the hypertrophic but not the hyperplastic response to the murine nongenotoxic hepatocarcinogen sodium phenobarbital in an in vivo study using a chimeric mouse with humanized liver.

PubMed

Yamada, Tomoya; Okuda, Yu; Kushida, Masahiko; Sumida, Kayo; Takeuchi, Hayato; Nagahori, Hirohisa; Fukuda, Takako; Lake, Brian G; Cohen, Samuel M; Kawamura, Satoshi

2014-11-01

High doses of sodium phenobarbital (NaPB), a constitutive androstane receptor (CAR) activator, have been shown to produce hepatocellular tumors in rodents by a mitogenic mode of action (MOA) involving CAR activation. The effect of 1-week dietary treatment with NaPB on liver weight and histopathology, hepatic CYP2B enzyme activity and CYP2B/3A mRNA expression, replicative DNA synthesis and selected genes related to cell proliferation, and functional transcriptomic and metabolomic analyses was studied in male CD-1 mice, Wistar Hannover (WH) rats, and chimeric mice with human hepatocytes. The treatment of chimeric mice with 1000-1500-ppm NaPB resulted in plasma levels around 3-5-fold higher than those observed in human subjects given therapeutic doses of NaPB. NaPB produced dose-dependent increases in hepatic CYP2B activity and CYP2B/3A mRNA levels in all animal models. Integrated functional metabolomic and transcriptomic analyses demonstrated that the responses to NaPB in the human liver were clearly different from those in rodents. Although NaPB produced a dose-dependent increase in hepatocyte replicative DNA synthesis in CD-1 mice and WH rats, no increase in replicative DNA synthesis was observed in human hepatocyte-originated areas of chimeric mice. In addition, treatment with NaPB had no effect on Ki-67, PCNA, GADD45β, and MDM2 mRNA expression in chimeric mice, whereas significant increases were observed in CD-1 mice and/or WH rats. However, increases in hepatocyte replicative DNA synthesis were observed in chimeric mice both in vivo and in vitro after treatment epidermal growth factor. Thus, although NaPB could activate CAR in both rodent and human hepatocytes, NaPB did not increase replicative DNA synthesis in human hepatocytes of chimeric mice, whereas it was mitogenic to rat and mouse hepatocytes. As human hepatocytes are refractory to the mitogenic effects of NaPB, the MOA for NaPB-induced rodent liver tumor formation is thus not relevant for humans. © The Author 2014. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For permissions, please email: journals.permissions@oup.com.

[Development of a hepatitis B virus carrier transgenic mice model].

PubMed

Caner, Müge; Arat, Sezen; Bircan, Rifat

2008-01-01

The studies for the development of transgenic mice models which provide important profits for the studies concerning immunopathogenesis of hepatitis B virus (HBV) infections are in progress since 20 years. For this purpose different lineages bearing whole HBV genome or selected viral genes have been developed and their usage in clarifying the HBV replication and pathogenesis mechanisms have been emphasized. The aim of this study was to develop and breed a HBV carrier mice model. In the study the full HBV genome has been transferred to mouse embryos by microinjection procedure. Following transgenic manipulation, the HBV carriers among the daughter mice have been detected by molecular methods in which HBV-DNA replication and expression have been shown. The manipulations for transgene transfers have been performed in TUBITAK Marmara Research Center Transgene Laboratory, Gebze, Istanbul. The HBV-DNA carrier mice have been demonstrated by polymerase chain reaction (PCR) using the DNA samples obtained from tail tissues and also by dot-blot hybridization of the mice sera. Integrated HBV-DNA has been detected by applying in-situ hybridization to the liver tissue sections. HBV-DNA expression has been shown by reverse transcriptase PCR method with total RNA molecules that have been isolated from the liver tissues of the HBV-DNA carrier mice. HBsAg has been detected in the liver by immunohistochemical method, and HBsAg and HBeAg have additionally been demonstrated by ELISA. HBV genome, expression of the genome and the expression products have been determined in approximately 10% of the mice of which HBV-DNA have been transferred. By inbreeding heterozygote carrier mice, homozygote HBV transgenic mice line have been obtained. These HBV transgenic mice are the first lineages developed in our country. It is hopefully thought that this HBV carrier transgenic mouse model may contribute to the studies on the pathogenesis of HBV infections which are important health problems in the world as well as in Turkey.

Mouse model for the Rift Valley fever virus MP12 strain infection.

PubMed

Lang, Yuekun; Henningson, Jamie; Jasperson, Dane; Li, Yonghai; Lee, Jinhwa; Ma, Jingjiao; Li, Yuhao; Cao, Nan; Liu, Haixia; Wilson, William; Richt, Juergen; Ruder, Mark; McVey, Scott; Ma, Wenjun

2016-11-15

Rift Valley fever virus (RVFV), a Category A pathogen and select agent, is the causative agent of Rift Valley fever. To date, no fully licensed vaccine is available in the U.S. for human or animal use and effective antiviral drugs have not been identified. The RVFV MP12 strain is conditionally licensed for use for veterinary purposes in the U.S. which was excluded from the select agent rule of Health and Human Services and the U.S. Department of Agriculture. The MP12 vaccine strain is commonly used in BSL-2 laboratories that is generally not virulent in mice. To establish a small animal model that can be used in a BSL-2 facility for antiviral drug development, we investigated susceptibility of six mouse strains (129S6/SvEv, STAT-1 KO, 129S1/SvlmJ, C57BL/6J, NZW/LacJ, BALB/c) to the MP12 virus infection via an intranasal inoculation route. Severe weight loss, obvious clinical and neurologic signs, and 50% mortality was observed in the STAT-1 KO mice, whereas the other 5 mouse strains did not display obvious and/or severe disease. Virus replication and histopathological lesions were detected in brain and liver of MP12-infected STAT-1 KO mice that developed the acute-onset hepatitis and delayed-onset encephalitis. In conclusion, the STAT-1 KO mouse strain is susceptible to MP12 virus infection, indicating that it can be used to investigate RVFV antivirals in a BSL-2 environment. Copyright © 2016 Elsevier B.V. All rights reserved.

Ectromelia virus lacking the E3L ortholog is replication-defective and nonpathogenic but does induce protective immunity in a mouse strain susceptible to lethal mousepox.

PubMed

Frey, Tiffany R; Forsyth, Katherine S; Sheehan, Maura M; De Haven, Brian C; Pevarnik, Julia G; Hand, Erin S; Pizzorno, Marie C; Eisenlohr, Laurence C; Hersperger, Adam R

2018-05-01

All known orthopoxviruses, including ectromelia virus (ECTV), contain a gene in the E3L family. The protein product of this gene, E3, is a double-stranded RNA-binding protein. It can impact host range and is used by orthopoxviruses to combat cellular defense pathways, such as PKR and RNase L. In this work, we constructed an ECTV mutant with a targeted disruption of the E3L open reading frame (ECTVΔE3L). Infection with this virus resulted in an abortive replication cycle in all cell lines tested. We detected limited transcription of late genes but no significant translation of these mRNAs. Notably, the replication defects of ECTVΔE3L were rescued in human and mouse cells lacking PKR. ECTVΔE3L was nonpathogenic in BALB/c mice, a strain susceptible to lethal mousepox disease. However, infection with ECTVΔE3L induced protective immunity upon subsequent challenge with wild-type virus. In summary, E3L is an essential gene for ECTV. Copyright © 2018 Elsevier Inc. All rights reserved.

Identification of genetic determinants of a tick-borne flavivirus associated with host-specific adaptation and pathogenicity.

PubMed

Mitzel, Dana N; Best, Sonja M; Masnick, Max F; Porcella, Stephen F; Wolfinbarger, James B; Bloom, Marshall E

2008-11-25

Tick-borne flaviviruses are maintained in nature in an enzootic cycle involving a tick vector and a vertebrate host. Thus, the virus replicates in two disparate hosts, each providing selective pressures that can influence virus replication and pathogenicity. To identify viral determinants associated with replication in the individual hosts, plaque purified Langat virus (TP21pp) was adapted to growth in mouse or tick cell lines to generate two virus variants, MNBp20 and ISEp20, respectively. Virus adaptation to mouse cells resulted in four amino acid changes in MNBp20 relative to TP21pp, occurring in E, NS4A and NS4B. A comparison between TP21pp and ISEp20 revealed three amino acid modifications in M, NS3 and NS4A of ISEp20. ISEp20, but not MNBp20, was attenuated following intraperitoneal inoculation of mice. Following isolation from mice brains, additional mutations reproducibly emerged in E and NS3 of ISEp20 that were possibly compensatory for the initial adaptation to tick cells. Thus, our data implicate a role for E, M, NS3, NS4A and NS4B in host adaptation and pathogenicity of tick-borne flaviviruses.

Nucleoprotein Complexes Containing Replicating Simian Virus 40 DNA: Comparison with Polyoma Nucleoprotein Complexes

PubMed Central

Hall, Mark R.; Meinke, William; Goldstein, David A.

1973-01-01

Procedures for isolating nucleoprotein complexes containing replicating polyoma DNA from infected mouse cells were used to prepare short-lived nucleoprotein complexes (r-SV40 complexes) containing replicating simian virus 40 (SV40) DNA from infected monkey cells. Like the polyoma complexes, r-SV40 complexes were only partially released from nuclei by cell lysis but could be extracted from nuclei by prolonged treatment with solutions containing Triton X-100. r-SV40 complexes sedimented faster than complexes containing SV40 supercoiled DNA (SV40 complex) in sucrose gradients, and both types of SV40 nucleoprotein complexes sedimented ahead of polyoma complexes containing supercoiled polyoma DNA (py complex). The sedimentation rates of py complex and SV40 complex were 56 and 61S, respectively, based on the sedimentation rate of the mouse large ribosomal subunit as a marker. r-SV40 complexes sedimented as multiple peaks between 56 and 75S. Sedimentation and buoyant density measurements indicated that protein is bound to all forms of SV40 DNA at about the same ratio of protein to DNA (1-2/1) as was reported for polyoma nucleoproteins. PMID:4359958

Fecal Microbiota and Metabolome in a Mouse Model of Spontaneous Chronic Colitis: Relevance to Human Inflammatory Bowel Disease.

PubMed

Robinson, Ainsley M; Gondalia, Shakuntla V; Karpe, Avinash V; Eri, Rajaraman; Beale, David J; Morrison, Paul D; Palombo, Enzo A; Nurgali, Kulmira

2016-12-01

Dysbiosis of the gut microbiota may be involved in the pathogenesis of inflammatory bowel disease (IBD). However, the mechanisms underlying the role of the intestinal microbiome and metabolome in IBD onset and its alteration during active treatment and recovery remain unknown. Animal models of chronic intestinal inflammation with similar microbial and metabolomic profiles would enable investigation of these mechanisms and development of more effective treatments. Recently, the Winnie mouse model of colitis closely representing the clinical symptoms and characteristics of human IBD has been developed. In this study, we have analyzed fecal microbial and metabolomic profiles in Winnie mice and discussed their relevance to human IBD. The 16S rRNA gene was sequenced from fecal DNA of Winnie and C57BL/6 mice to define operational taxonomic units at ≥97% similarity threshold. Metabolomic profiling of the same fecal samples was performed by gas chromatography-mass spectrometry. Composition of the dominant microbiota was disturbed, and prominent differences were evident at all levels of the intestinal microbiome in fecal samples from Winnie mice, similar to observations in patients with IBD. Metabolomic profiling revealed that chronic colitis in Winnie mice upregulated production of metabolites and altered several metabolic pathways, mostly affecting amino acid synthesis and breakdown of monosaccharides to short chain fatty acids. Significant dysbiosis in the Winnie mouse gut replicates many changes observed in patients with IBD. These results provide justification for the suitability of this model to investigate mechanisms underlying the role of intestinal microbiota and metabolome in the pathophysiology of IBD.

PrPC Governs Susceptibility to Prion Strains in Bank Vole, While Other Host Factors Modulate Strain Features.

PubMed

Espinosa, J C; Nonno, R; Di Bari, M; Aguilar-Calvo, P; Pirisinu, L; Fernández-Borges, N; Vanni, I; Vaccari, G; Marín-Moreno, A; Frassanito, P; Lorenzo, P; Agrimi, U; Torres, J M

2016-12-01

Bank vole is a rodent species that shows differential susceptibility to the experimental transmission of different prion strains. In this work, the transmission features of a panel of diverse prions with distinct origins were assayed both in bank vole expressing methionine at codon 109 (Bv109M) and in transgenic mice expressing physiological levels of bank vole PrP C (the BvPrP-Tg407 mouse line). This work is the first systematic comparison of the transmission features of a collection of prion isolates, representing a panel of diverse prion strains, in a transgenic-mouse model and in its natural counterpart. The results showed very similar transmission properties in both the natural species and the transgenic-mouse model, demonstrating the key role of the PrP amino acid sequence in prion transmission susceptibility. However, differences in the PrP Sc types propagated by Bv109M and BvPrP-Tg407 suggest that host factors other than PrP C modulate prion strain features. The differential susceptibility of bank voles to prion strains can be modeled in transgenic mice, suggesting that this selective susceptibility is controlled by the vole PrP sequence alone rather than by other species-specific factors. Differences in the phenotypes observed after prion transmissions in bank voles and in the transgenic mice suggest that host factors other than the PrP C sequence may affect the selection of the substrain replicating in the animal model. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Correlations of Behavioral Deficits with Brain Pathology Assessed through Longitudinal MRI and Histopathology in the HdhQ150/Q150 Mouse Model of Huntington’s Disease

PubMed Central

Rattray, Ivan; Smith, Edward J.; Crum, William R.; Walker, Thomas A.; Gale, Richard; Bates, Gillian P.

2017-01-01

A variety of mouse models have been developed that express mutant huntingtin (mHTT) leading to aggregates and inclusions that model the molecular pathology observed in Huntington’s disease. Here we show that although homozygous HdhQ150 knock-in mice developed motor impairments (rotarod, locomotor activity, grip strength) by 36 weeks of age, cognitive dysfunction (swimming T maze, fear conditioning, odor discrimination, social interaction) was not evident by 94 weeks. Concomitant to behavioral assessments, T2-weighted MRI volume measurements indicated a slower striatal growth with a significant difference between wild type (WT) and HdhQ150 mice being present even at 15 weeks. Indeed, MRI indicated significant volumetric changes prior to the emergence of the “clinical horizon” of motor impairments at 36 weeks of age. A striatal decrease of 27% was observed over 94 weeks with cortex (12%) and hippocampus (21%) also indicating significant atrophy. A hypothesis-free analysis using tensor-based morphometry highlighted further regions undergoing atrophy by contrasting brain growth and regional neurodegeneration. Histology revealed the widespread presence of mHTT aggregates and cellular inclusions. However, there was little evidence of correlations between these outcome measures, potentially indicating that other factors are important in the causal cascade linking the molecular pathology to the emergence of behavioral impairments. In conclusion, the HdhQ150 mouse model replicates many aspects of the human condition, including an extended pre-manifest period prior to the emergence of motor impairments. PMID:28099507

Replication and pathogenic potential of influenza A virus subtypes H3, H7, and H15 from free-range ducks in Bangladesh in mammals.

PubMed

El-Shesheny, Rabeh; Feeroz, Mohammed M; Krauss, Scott; Vogel, Peter; McKenzie, Pamela; Webby, Richard J; Webster, Robert G

2018-04-25

Surveillance of wild aquatic birds and free-range domestic ducks in the Tanguar Haor wetlands in Bangladesh has identified influenza virus subtypes H3N6, H7N1, H7N5, H7N9, and H15N9. Molecular characterization of these viruses indicates their contribution to the genesis of new genotypes of H5N1 influenza viruses from clade 2.3.2.1a that are dominant in poultry markets in Bangladesh as well as to the genesis of the highly pathogenic H5N8 virus currently causing disease outbreaks in domestic poultry in Europe and the Middle East. Therefore, we studied the antigenicity, replication, and pathogenicity of influenza viruses isolated from Tanguar Haor in the DBA/2J mouse model. All viruses replicated in the lung without prior mammalian adaptation, and H7N1 and H7N9 viruses caused 100% and 60% mortality, respectively. H7N5 viruses replicated only in the lungs, whereas H7N1 and H7N9 viruses also replicated in the heart, liver, and brain. Replication and transmission studies in mallard ducks showed that H7N1 and H7N9 viruses replicated in ducks without clinical signs of disease and shed at high titers from the cloaca of infected and contact ducks, which could facilitate virus transmission and spread. Our results indicate that H7 avian influenza viruses from free-range ducks can replicate in mammals, cause severe disease, and be efficiently transmitted to contact ducks. Our study highlights the role of free-range ducks in the spread of influenza viruses to other species in live poultry markets and the potential for these viruses to infect and cause disease in mammals.

Inhibition of hepatitis B virus replication in vivo using lipoplexes containing altritol-modified antiviral siRNAs

PubMed Central

Ely, Abdullah; ul Islam, Rafique; Barichievy, Samantha; Bloom, Kristie; Weinberg, Marc S; van Otterlo, Willem AL; de Koning, Charles B; Salazar, Felix; Marion, Patricia; Roesch, Eric B; LeMaitre, Marc; Herdewijn, Piet

2010-01-01

Chronic infection with the hepatitis B virus (HBV) occurs in approximately 6% of the world's population and carriers of the virus are at risk for complicating hepatocellular carcinoma. Current treatment options have limited efficacy and chronic HBV infection is likely to remain a significant global medical problem for many years to come. Silencing HBV gene expression by harnessing RNA interference (RNAi) presents an attractive option for development of novel and effective anti HBV agents. However, despite significant and rapid progress, further refinement of existing technologies is necessary before clinical application of RNAi-based HBV therapies is realized. Limiting off target effects, improvement of delivery efficiency, dose regulation and preventing reactivation of viral replication are some of the hurdles that need to be overcome. To address this, we assessed the usefulness of the recently described class of altritol-containing synthetic siRNAs (ANA siRNAs), which were administered as lipoplexes and tested in vivo in a stringent HBV transgenic mouse model. Our observations show that ANA siRNAs are capable of silencing of HBV replication in vivo. Importantly, non specific immunostimulation was observed with unmodified siRNAs and this undesirable effect was significantly attenuated by ANA modification. Inhibition of HBV replication of approximately 50% was achieved without evidence for induction of toxicity. These results augur well for future application of ANA siRNA therapeutic lipoplexes. PMID:21687523

Combination Kinase Inhibitor Treatment Suppresses Rift Valley Fever Virus Replication.

PubMed

Bell, Todd M; Espina, Virginia; Lundberg, Lindsay; Pinkham, Chelsea; Brahms, Ashwini; Carey, Brian D; Lin, Shih-Chao; Dahal, Bibha; Woodson, Caitlin; de la Fuente, Cynthia; Liotta, Lance A; Bailey, Charles L; Kehn-Hall, Kylene

2018-04-13

Viruses must parasitize host cell translational machinery in order to make proteins for viral progeny. In this study, we sought to use this signal transduction conduit against them by inhibiting multiple kinases that influence translation. Previous work indicated that several kinases involved in translation, including p70 S6K, p90RSK, ERK, and p38 MAPK, are phosphorylated following Rift Valley fever virus (RVFV) infection. Furthermore, inhibiting p70 S6K through treatment with the FDA approved drug rapamycin prevents RVFV pathogenesis in a mouse model of infection. We hypothesized that inhibiting either p70 S6K, p90RSK, or p90RSK’s upstream kinases, ERK and p38 MAPK, would decrease translation and subsequent viral replication. Treatment with the p70 S6K inhibitor PF-4708671 resulted in decreased phosphorylation of translational proteins and reduced RVFV titers. In contrast, treatment with the p90RSK inhibitor BI-D1870, p38MAPK inhibitor SB203580, or the ERK inhibitor PD0325901 alone had minimal influence on RVFV titers. The combination of PF-4708671 and BI-D1870 treatment resulted in robust inhibition of RVFV replication. Likewise, a synergistic inhibition of RVFV replication was observed with p38MAPK inhibitor SB203580 or the ERK inhibitor PD0325901 combined with rapamycin treatment. These findings serve as a proof of concept regarding combination kinase inhibitor treatment for RVFV infection.

Combination Kinase Inhibitor Treatment Suppresses Rift Valley Fever Virus Replication

PubMed Central

Bell, Todd M.; Espina, Virginia; Lundberg, Lindsay; Pinkham, Chelsea; Brahms, Ashwini; Dahal, Bibha; Woodson, Caitlin; de la Fuente, Cynthia; Liotta, Lance A.; Bailey, Charles L.

2018-01-01

Viruses must parasitize host cell translational machinery in order to make proteins for viral progeny. In this study, we sought to use this signal transduction conduit against them by inhibiting multiple kinases that influence translation. Previous work indicated that several kinases involved in translation, including p70 S6K, p90RSK, ERK, and p38 MAPK, are phosphorylated following Rift Valley fever virus (RVFV) infection. Furthermore, inhibiting p70 S6K through treatment with the FDA approved drug rapamycin prevents RVFV pathogenesis in a mouse model of infection. We hypothesized that inhibiting either p70 S6K, p90RSK, or p90RSK’s upstream kinases, ERK and p38 MAPK, would decrease translation and subsequent viral replication. Treatment with the p70 S6K inhibitor PF-4708671 resulted in decreased phosphorylation of translational proteins and reduced RVFV titers. In contrast, treatment with the p90RSK inhibitor BI-D1870, p38MAPK inhibitor SB203580, or the ERK inhibitor PD0325901 alone had minimal influence on RVFV titers. The combination of PF-4708671 and BI-D1870 treatment resulted in robust inhibition of RVFV replication. Likewise, a synergistic inhibition of RVFV replication was observed with p38MAPK inhibitor SB203580 or the ERK inhibitor PD0325901 combined with rapamycin treatment. These findings serve as a proof of concept regarding combination kinase inhibitor treatment for RVFV infection. PMID:29652799

Melanoma cultures show different susceptibility towards E1A-, E1B-19 kDa- and fiber-modified replication-competent adenoviruses.

PubMed

Schmitz, M; Graf, C; Gut, T; Sirena, D; Peter, I; Dummer, R; Greber, U F; Hemmi, S

2006-06-01

Replicating adenovirus (Ad) vectors with tumour tissue specificity hold great promise for treatment of cancer. We have recently constructed a conditionally replicating Ad5 AdDeltaEP-TETP inducing tumour regression in a xenograft mouse model. For further improvement of this vector, we introduced four genetic modifications and analysed the viral cytotoxicity in a large panel of melanoma cell lines and patient-derived melanoma cells. (1) The antiapoptotic gene E1B-19 kDa (Delta19 mutant) was deleted increasing the cytolytic activity in 18 of 21 melanoma cells. (2) Introduction of the E1A 122-129 deletion (Delta24 mutant), suggested to attenuate viral replication in cell cycle-arrested cells, did not abrogate this activity and increased the cytolytic activity in two of 21 melanoma cells. (3) We inserted an RGD sequence into the fiber to extend viral tropism to alphav integrin-expressing cells, and (4) swapped the fiber with the Ad35 fiber (F35) enhancing the tropism to malignant melanoma cells expressing CD46. The RGD-fiber modification strongly increased cytolysis in all of the 11 CAR-low melanoma cells. The F35 fiber-chimeric vector boosted the cytotoxicity in nine of 11 cells. Our results show that rational engineering additively enhances the cytolytic potential of Ad vectors, a prerequisite for the development of patient-customized viral therapies.

Time-controllable Nkcc1 knockdown replicates reversible hearing loss in postnatal mice.

PubMed

Watabe, Takahisa; Xu, Ming; Watanabe, Miho; Nabekura, Junichi; Higuchi, Taiga; Hori, Karin; Sato, Mitsuo P; Nin, Fumiaki; Hibino, Hiroshi; Ogawa, Kaoru; Masuda, Masatsugu; Tanaka, Kenji F

2017-10-19

Identification of the causal effects of specific proteins on recurrent and partially reversible hearing loss has been difficult because of the lack of an animal model that provides reversible gene knockdown. We have developed the transgenic mouse line Actin-tTS::Nkcc1 tetO/tetO for manipulatable expression of the cochlear K + circulation protein, NKCC1. Nkcc1 transcription was blocked by the binding of a tetracycline-dependent transcriptional silencer to the tetracycline operator sequences inserted upstream of the Nkcc1 translation initiation site. Administration of the tetracycline derivative doxycycline reversibly regulated Nkcc1 knockdown. Progeny from pregnant/lactating mothers fed doxycycline-free chow from embryonic day 0 showed strong suppression of Nkcc1 expression (~90% downregulation) and Nkcc1 null phenotypes at postnatal day 35 (P35). P35 transgenic mice from mothers fed doxycycline-free chow starting at P0 (delivery) showed weaker suppression of Nkcc1 expression (~70% downregulation) and less hearing loss with mild cochlear structural changes. Treatment of these mice at P35 with doxycycline for 2 weeks reactivated Nkcc1 transcription to control levels and improved hearing level at high frequency; i.e., these doxycycline-treated mice exhibited partially reversible hearing loss. Thus, development of the Actin-tTS::Nkcc1 tetO/tetO transgenic mouse line provides a mouse model for the study of variable hearing loss through reversible knockdown of Nkcc1.

Targeting the kinase activities of ATR and ATM exhibits antitumoral activity in mouse models of MLL-rearranged AML.

PubMed

Morgado-Palacin, Isabel; Day, Amanda; Murga, Matilde; Lafarga, Vanesa; Anton, Marta Elena; Tubbs, Anthony; Chen, Hua Tang; Ergan, Aysegul; Anderson, Rhonda; Bhandoola, Avinash; Pike, Kurt G; Barlaam, Bernard; Cadogan, Elaine; Wang, Xi; Pierce, Andrew J; Hubbard, Chad; Armstrong, Scott A; Nussenzweig, André; Fernandez-Capetillo, Oscar

2016-09-13

Among the various subtypes of acute myeloid leukemia (AML), those with chromosomal rearrangements of the MLL oncogene (AML-MLL) have a poor prognosis. AML-MLL tumor cells are resistant to current genotoxic therapies because of an attenuated response by p53, a protein that induces cell cycle arrest and apoptosis in response to DNA damage. In addition to chemicals that damage DNA, efforts have focused on targeting DNA repair enzymes as a general chemotherapeutic approach to cancer treatment. Here, we found that inhibition of the kinase ATR, which is the primary sensor of DNA replication stress, induced chromosomal breakage and death of mouse AML(MLL) cells (with an MLL-ENL fusion and a constitutively active N-RAS independently of p53. Moreover, ATR inhibition as a single agent exhibited antitumoral activity, both reducing tumor burden after establishment and preventing tumors from growing, in an immunocompetent allograft mouse model of AML(MLL) and in xenografts of a human AML-MLL cell line. We also found that inhibition of ATM, a kinase that senses DNA double-strand breaks, also promoted the survival of the AML(MLL) mice. Collectively, these data indicated that ATR or ATM inhibition represent potential therapeutic strategies for the treatment of AML, especially MLL-driven leukemias. Copyright © 2016, American Association for the Advancement of Science.

Mitochondrial transcription: Lessons from mouse models

PubMed Central

Peralta, Susana; Wang, Xiao; Moraes, Carlos T.

2012-01-01

Mammalian mitochondrial DNA (mtDNA) is a circular double-stranded DNA genome of ∼ 16.5 kilobase pairs (kb) that encodes 13 catalytic proteins of the ATP-producing oxidative phosphorylation system (OXPHOS), and the rRNAs and tRNAs required for the translation of the mtDNA transcripts. All the components needed for transcription and replication of the mtDNA are, therefore, encoded in the nuclear genome, as are the remaining components of the OXPHOS system and the mitochondrial translation machinery. Regulation of mtDNA gene expression is very important for modulating the OXPHOS capacity in response to metabolic requirements and in pathological processes. The combination of in vitro and in vivo studies has allowed the identification of the core machinery required for basal mtDNA transcription in mammals and a few proteins that regulate mtDNA transcription. Specifically, the generation of knockout mouse strains in the last several years, has been key to understanding the basis of mtDNA transcription in vivo. However, it is well accepted that many components of the transcription machinery are still unknown and little is known about mtDNA gene expression regulation under different metabolic requirements or disease processes. In this review we will focus on how the creation of knockout mouse models and the study of their phenotypes have contributed to the understanding of mitochondrial transcription in mammals. PMID:22120174

Herpes simplex virus serotype and entry receptor availability alter CNS disease in a mouse model of neonatal HSV.

PubMed

Kopp, Sarah J; Ranaivo, Hantamalala R; Wilcox, Douglas R; Karaba, Andrew H; Wainwright, Mark S; Muller, William J

2014-12-01

Outcomes of neonates with herpes simplex virus (HSV) encephalitis are worse after infection with HSV-2 when compared with HSV-1. The proteins herpes virus entry mediator (HVEM) and nectin-1 mediate HSV entry into susceptible cells. Prior studies have shown receptor-dependent differences in pathogenesis that depend on route of inoculation and host developmental age. We investigated serotype-related differences in HSV disease and their relationship to entry receptor availability in a mouse model of encephalitis. Mortality was attenuated in 7-d-old, wild-type (WT) mice inoculated with HSV-1(F) when compared with HSV-2(333). No serotype-specific differences were seen after inoculation of adult mice. HSV-1 pathogenesis was also attenuated relative to HSV-2 in newborn but not adult mice lacking HVEM or nectin-1. HSV-2 requires nectin-1 for encephalitis in adult but not newborn mice; in contrast, nectin-1 was important for HSV-1 pathogenesis in both age groups. Early viral replication was independent of age, viral serotype, or mouse genotype, suggesting host responses influence outcomes. In this regard, significantly greater amounts of inflammatory mediators were detected in brain homogenates from WT newborns 2 d after infection compared with adults and receptor-knockout newborns. Dysregulation of inflammatory responses induced by infection may influence the severity of HSV encephalitis.

Impact of radiation therapy on the oncolytic adenovirus dl520: implications on the treatment of glioblastoma.

PubMed

Bieler, Alexa; Mantwill, Klaus; Holzmüller, Regina; Jürchott, Karsten; Kaszubiak, Alexander; Stärk, Sybille; Glockzin, Gabriel; Lage, Hermann; Grosu, Anca-Ligia; Gansbacher, Bernd; Holm, Per Sonne

2008-03-01

Viral oncolytic therapy is emerging as a new form of anticancer therapy and has shown promising preclinical results, especially in combination with radio- and chemotherapy. We recently reported that nuclear localization of the human transcription factor YB-1 in multidrug-resistant cells facilitates E1-independent adenoviral replication. The aim of this study was to evaluate the combined treatment of the conditionally-replicating adenovirus dl520 and radiotherapy in glioma cell lines in vitro and in human tumor xenografts. Furthermore, the dependency of YB-1 on dl520 replication was verified by shRNA directed down regulation of YB-1. Localization of YB-1 was determined by immunostaining. Glioma cell lines LN-18, U373 and U87 were infected with dl520. Induction of cytopathic effect (CPE), viral replication, viral yield and viral release were determined after viral infection, radiation therapy and the combination of both treatment modalities. The capacity of treatments alone or combined to induce tumor growth inhibition of subcutaneous U373 tumors was tested also in nude mice. Quantitative real-time PCR demonstrated that the shRNA-mediated down regulation of YB-1 is leading to a dramatic decrease in adenoviral replication of dl520. Immunostaining analysis showed that the YB-1 protein was predominantly located in the cytoplasm in the perinuclear space and less abundant in the nucleus. After irradiation we found an increase of nuclear YB-1. The addition of radiotherapy increased the oncolytic effect of dl520 with enhanced viral replication, viral yield and viral release. The oncolytic activity of dl520 plus radiation inhibited the growth of subcutaneous U373 tumors in a xenograft mouse model. Radiation mediated increase of nuclear YB-1 in glioma cells enhanced the oncolytic potential of adenovirus dl520.

Replication-Competent Controlled Herpes Simplex Virus

PubMed Central

Bloom, David C.; Feller, Joyce; McAnany, Peterjon; Vilaboa, Nuria

2015-01-01

ABSTRACT We present the development and characterization of a replication-competent controlled herpes simplex virus 1 (HSV-1). Replication-essential ICP4 and ICP8 genes of HSV-1 wild-type strain 17syn+ were brought under the control of a dually responsive gene switch. The gene switch comprises (i) a transactivator that is activated by a narrow class of antiprogestins, including mifepristone and ulipristal, and whose expression is mediated by a promoter cassette that comprises an HSP70B promoter and a transactivator-responsive promoter and (ii) transactivator-responsive promoters that drive the ICP4 and ICP8 genes. Single-step growth experiments in different cell lines demonstrated that replication of the recombinant virus, HSV-GS3, is strictly dependent on an activating treatment consisting of administration of a supraphysiological heat dose in the presence of an antiprogestin. The replication-competent controlled virus replicates with an efficiency approaching that of the wild-type virus from which it was derived. Essentially no replication occurs in the absence of activating treatment or if HSV-GS3-infected cells are exposed only to heat or antiprogestin. These findings were corroborated by measurements of amounts of viral DNA and transcripts of the regulated ICP4 gene and the glycoprotein C (gC) late gene, which was not regulated. Similar findings were made in experiments with a mouse footpad infection model. IMPORTANCE The alphaherpesviruses have long been considered vectors for recombinant vaccines and oncolytic therapies. The traditional approach uses vector backbones containing attenuating mutations that restrict replication to ensure safety. The shortcoming of this approach is that the attenuating mutations tend to limit both the immune presentation and oncolytic properties of these vectors. HSV-GS3 represents a novel type of vector that, when activated, replicates with the efficiency of a nonattenuated virus and whose safety is derived from deliberate, stringent regulation of multiple replication-essential genes. By directing activating heat to the region of virus administration, replication is strictly confined to infected cells within this region. The requirement for antiprogestin provides an additional level of safety, ensuring that virus replication cannot be triggered inadvertently. Replication-competent controlled vectors such as HSV-GS3 may have the potential to be superior to conventional attenuated HSV vaccine and oncolytic vectors without sacrificing safety. PMID:26269179

Preneoplastic lesion growth driven by the death of adjacent normal stem cells

PubMed Central

Chao, Dennis L.; Eck, J. Thomas; Brash, Douglas E.; Maley, Carlo C.; Luebeck, E. Georg

2008-01-01

Clonal expansion of premalignant lesions is an important step in the progression to cancer. This process is commonly considered to be a consequence of sustaining a proliferative mutation. Here, we investigate whether the growth trajectory of clones can be better described by a model in which clone growth does not depend on a proliferative advantage. We developed a simple computer model of clonal expansion in an epithelium in which mutant clones can only colonize space left unoccupied by the death of adjacent normal stem cells. In this model, competition for space occurs along the frontier between mutant and normal territories, and both the shapes and the growth rates of lesions are governed by the differences between mutant and normal cells' replication or apoptosis rates. The behavior of this model of clonal expansion along a mutant clone's frontier, when apoptosis of both normal and mutant cells is included, matches the growth of UVB-induced p53-mutant clones in mouse dorsal epidermis better than a standard exponential growth model that does not include tissue architecture. The model predicts precancer cell mutation and death rates that agree with biological observations. These results support the hypothesis that clonal expansion of premalignant lesions can be driven by agents, such as ionizing or nonionizing radiation, that cause cell killing but do not directly stimulate cell replication. PMID:18815380

APOBEC3D and APOBEC3F potently promote HIV-1 diversification and evolution in humanized mouse model.

PubMed

Sato, Kei; Takeuchi, Junko S; Misawa, Naoko; Izumi, Taisuke; Kobayashi, Tomoko; Kimura, Yuichi; Iwami, Shingo; Takaori-Kondo, Akifumi; Hu, Wei-Shau; Aihara, Kazuyuki; Ito, Mamoru; An, Dong Sung; Pathak, Vinay K; Koyanagi, Yoshio

2014-10-01

Several APOBEC3 proteins, particularly APOBEC3D, APOBEC3F, and APOBEC3G, induce G-to-A hypermutations in HIV-1 genome, and abrogate viral replication in experimental systems, but their relative contributions to controlling viral replication and viral genetic variation in vivo have not been elucidated. On the other hand, an HIV-1-encoded protein, Vif, can degrade these APOBEC3 proteins via a ubiquitin/proteasome pathway. Although APOBEC3 proteins have been widely considered as potent restriction factors against HIV-1, it remains unclear which endogenous APOBEC3 protein(s) affect HIV-1 propagation in vivo. Here we use a humanized mouse model and HIV-1 with mutations in Vif motifs that are responsible for specific APOBEC3 interactions, DRMR/AAAA (4A) or YRHHY/AAAAA (5A), and demonstrate that endogenous APOBEC3D/F and APOBEC3G exert strong anti-HIV-1 activity in vivo. We also show that the growth kinetics of 4A HIV-1 negatively correlated with the expression level of APOBEC3F. Moreover, single genome sequencing analyses of viral RNA in plasma of infected mice reveal that 4A HIV-1 is specifically and significantly diversified. Furthermore, a mutated virus that is capable of using both CCR5 and CXCR4 as entry coreceptor is specifically detected in 4A HIV-1-infected mice. Taken together, our results demonstrate that APOBEC3D/F and APOBEC3G fundamentally work as restriction factors against HIV-1 in vivo, but at the same time, that APOBEC3D and APOBEC3F are capable of promoting viral diversification and evolution in vivo.

Development of Eczema Vaccinatum in Atopic Mouse Models and Efficacy of MVA Vaccination against Lethal Poxviral Infection

PubMed Central

Knitlova, Jarmila; Hajkova, Vera; Voska, Ludek; Elsterova, Jana; Obrova, Barbora; Melkova, Zora

2014-01-01

Smallpox vaccine based on live, replicating vaccinia virus (VACV) is associated with several potentially serious and deadly complications. Consequently, a new generation of vaccine based on non-replicating Modified vaccinia virus Ankara (MVA) has been under clinical development. MVA seems to induce good immune responses in blood tests, but it is impossible to test its efficacy in vivo in human. One of the serious complications of the replicating vaccine is eczema vaccinatum (EV) occurring in individuals with atopic dermatitis (AD), thus excluding them from all preventive vaccination schemes. In this study, we first characterized and compared development of eczema vaccinatum in different mouse strains. Nc/Nga, Balb/c and C57Bl/6J mice were epicutaneously sensitized with ovalbumin (OVA) or saline control to induce signs of atopic dermatitis and subsequently trans-dermally (t.d.) immunized with VACV strain Western Reserve (WR). Large primary lesions occurred in both mock- and OVA-sensitized Nc/Nga mice, while they remained small in Balb/c and C57Bl/6J mice. Satellite lesions developed in both mock- and OVA-sensitized Nc/Nga and in OVA-sensitized Balb/c mice with the rate 40–50%. Presence of mastocytes and eosinophils was the highest in Nc/Nga mice. Consequently, we have chosen Nc/Nga mice as a model of AD/EV and tested efficacy of MVA and Dryvax vaccinations against a lethal intra-nasal (i.n.) challenge with WR, the surrogate of smallpox. Inoculation of MVA intra-muscularly (i.m.) or t.d. resulted in no lesions, while inoculation of Dryvax t.d. yielded large primary and many satellite lesions similar to WR. Eighty three and 92% of mice vaccinated with a single dose of MVA i.m. or t.d., respectively, survived a lethal i.n. challenge with WR without any serious illness, while all Dryvax-vaccinated animals survived. This is the first formal prove of protective immunity against a lethal poxvirus challenge induced by vaccination with MVA in an atopic organism. PMID:25486419

A Simple Mouse Model for the Study of Human Immunodeficiency Virus.

PubMed

Kim, Kang Chang; Choi, Byeong-Sun; Kim, Kyung-Chang; Park, Ki Hoon; Lee, Hee Jung; Cho, Young Keol; Kim, Sang Il; Kim, Sung Soon; Oh, Yu-Kyoung; Kim, Young Bong

2016-02-01

Humanized mouse models derived from immune-deficient mice have been the primary tool for studies of human infectious viruses, such as human immunodeficiency virus (HIV). However, the current protocol for constructing humanized mice requires elaborate procedures and complicated techniques, limiting the supply of such mice for viral studies. Here, we report a convenient method for constructing a simple HIV-1 mouse model. Without prior irradiation, NOD/SCID/IL2Rγ-null (NSG) mice were intraperitoneally injected with 1 × 10(7) adult human peripheral blood mononuclear cells (hu-PBMCs). Four weeks after PBMC inoculation, human CD45(+) cells, and CD3(+)CD4(+) and CD3(+)CD8(+) T cells were detected in peripheral blood, lymph nodes, spleen, and liver, whereas human CD19(+) cells were observed in lymph nodes and spleen. To examine the usefulness of hu-PBMC-inoculated NSG (hu-PBMC-NSG) mice as an HIV-1 infection model, we intravenously injected these mice with dual-tropic HIV-1DH12 and X4-tropic HIV-1NL4-3 strains. HIV-1-infected hu-PBMC-NSG mice showed significantly lower human CD4(+) T cell counts and high HIV viral loads in the peripheral blood compared with noninfected hu-PBMC-NSG mice. Following highly active antiretroviral therapy (HAART) and neutralizing antibody treatment, HIV-1 replication was significantly suppressed in HIV-1-infected hu-PBMC-NSG mice without detectable viremia or CD4(+) T cell depletion. Moreover, the numbers of human T cells were maintained in hu-PBMC-NSG mice for at least 10 weeks. Taken together, our results suggest that hu-PBMC-NSG mice may serve as a relevant HIV-1 infection and pathogenesis model that could facilitate in vivo studies of HIV-1 infection and candidate HIV-1 protective drugs.

Involvement of GABA Transporters in Atropine-Treated Myopic Retina As Revealed by iTRAQ Quantitative Proteomics

PubMed Central

2015-01-01

Atropine, a muscarinic antagonist, is known to inhibit myopia progression in several animal models and humans. However, the mode of action is not established yet. In this study, we compared quantitative iTRAQ proteomic analysis in the retinas collected from control and lens-induced myopic (LIM) mouse eyes treated with atropine. The myopic group received a (−15D) spectacle lens over the right eye on postnatal day 10 with or without atropine eye drops starting on postnatal day 24. Axial length was measured by optical low coherence interferometry (OLCI), AC-Master, and refraction was measured by automated infrared photorefractor at postnatal 24, 38, and 52 days. Retinal tissue samples were pooled from six eyes for each group. The experiments were repeated twice, and technical replicates were also performed for liquid chromatography–tandem mass spectrometry (LC–MS/MS) analysis. MetaCore was used to perform protein profiling for pathway analysis. We identified a total of 3882 unique proteins with <1% FDR by analyzing the samples in replicates for two independent experiments. This is the largest number of mouse retina proteome reported to date. Thirty proteins were found to be up-regulated (ratio for myopia/control > global mean ratio + 1 standard deviation), and 28 proteins were down-regulated (ratio for myopia/control < global mean ratio - 1 standard deviation) in myopic eyes as compared with control retinas. Pathway analysis using MetaCore revealed regulation of γ-aminobutyric acid (GABA) levels in the myopic eyes. Detailed analysis of the quantitative proteomics data showed that the levels of GABA transporter 1 (GAT-1) were elevated in myopic retina and significantly reduced after atropine treatment. These results were further validated with immunohistochemistry and Western blot analysis. In conclusion, this study provides a comprehensive quantitative proteomic analysis of atropine-treated mouse retina and suggests the involvement of GABAergic signaling in the antimyopic effects of atropine in mouse eyes. The GABAergic transmission in the neural retina plays a pivotal role in the maintenance of axial eye growth in mammals. PMID:25211393

Development, validation and characterization of a novel mouse model of Adynamic Bone Disease (ABD).

PubMed

Ng, Adeline H; Willett, Thomas L; Alman, Benjamin A; Grynpas, Marc D

2014-11-01

The etiology of Adynamic Bone Disease (ABD) is poorly understood but the hallmark of ABD is a lack of bone turnover. ABD occurs in renal osteodystrophy (ROD) and is suspected to occur in elderly patients on long-term anti-resorptive therapy. A major clinical concern of ABD is diminished bone quality and an increased fracture risk. To our knowledge, experimental animal models for ABD other than ROD-ABD have not been developed or studied. The objectives of this study were to develop a mouse model of ABD without the complications of renal ablation, and to characterize changes in bone quality in ABD relative to controls. To re-create the adynamic bone condition, 4-month old female Col2.3Δtk mice were treated with ganciclovir to specifically ablate osteoblasts, and pamidronate was used to inhibit osteoclastic resorption. Four groups of animals were used to characterize bone quality in ABD: Normal bone controls, No Formation controls, No Resorption controls, and an Adynamic group. After a 6-week treatment period, the animals were sacrificed and the bones were harvested for analyses. Bone quality assessments were conducted using established techniques including bone histology, quantitative backscattered electron imaging (qBEI), dual energy X-ray absorptiometry (DXA), microcomputed tomography (microCT), and biomechanical testing. Histomorphometry confirmed osteoblast-related hallmarks of ABD in our mouse model. Bone formation was near complete suppression in the No Formation and Adynamic specimens. Inhibition of bone resorption in the Adynamic group was confirmed by tartrate-resistant acid phosphatase (TRAP) stain. Normal bone mineral density and architecture were maintained in the Adynamic group, whereas the No Formation group showed a reduction in bone mineral content and trabecular thickness relative to the Adynamic group. As expected, the No Formation group had a more hypomineralized profile and the Adynamic group had a higher mean mineralization profile that is similar to suppressed bone turnover in human. This data confirms successful replication of the adynamic bone condition in a mouse without the complication of renal ablation. Our approach is the first model of ABD that uses pharmacological manipulation in a transgenic mouse to mimic the bone cellular dynamics observed in the human ABD condition. We plan to use our mouse model to investigate the adynamic bone condition in aging and to study changes to bone quality and fracture risk as a consequence of over-suppressed bone turnover. Copyright © 2014 Elsevier Inc. All rights reserved.

Long-Term Culture of Genome-Stable Bipotent Stem Cells from Adult Human Liver

PubMed Central

Huch, Meritxell; Gehart, Helmuth; van Boxtel, Ruben; Hamer, Karien; Blokzijl, Francis; Verstegen, Monique M.A.; Ellis, Ewa; van Wenum, Martien; Fuchs, Sabine A.; de Ligt, Joep; van de Wetering, Marc; Sasaki, Nobuo; Boers, Susanne J.; Kemperman, Hans; de Jonge, Jeroen; Ijzermans, Jan N.M.; Nieuwenhuis, Edward E.S.; Hoekstra, Ruurdtje; Strom, Stephen; Vries, Robert R.G.; van der Laan, Luc J.W.; Cuppen, Edwin; Clevers, Hans

2015-01-01

Summary Despite the enormous replication potential of the human liver, there are currently no culture systems available that sustain hepatocyte replication and/or function in vitro. We have shown previously that single mouse Lgr5+ liver stem cells can be expanded as epithelial organoids in vitro and can be differentiated into functional hepatocytes in vitro and in vivo. We now describe conditions allowing long-term expansion of adult bile duct-derived bipotent progenitor cells from human liver. The expanded cells are highly stable at the chromosome and structural level, while single base changes occur at very low rates. The cells can readily be converted into functional hepatocytes in vitro and upon transplantation in vivo. Organoids from α1-antitrypsin deficiency and Alagille syndrome patients mirror the in vivo pathology. Clonal long-term expansion of primary adult liver stem cells opens up experimental avenues for disease modeling, toxicology studies, regenerative medicine, and gene therapy. PMID:25533785

Replication of CMV in the gut of HIV-infected individuals and epithelial barrier dysfunction

PubMed Central

Somsouk, Ma; Hunt, Peter W.

2017-01-01

Although invasive cytomegalovirus (CMV) disease is uncommon in the era of antiretroviral therapy (ART), asymptomatic CMV coinfection is nearly ubiquitous in HIV infected individuals. While microbial translocation and gut epithelial barrier dysfunction may promote persistent immune activation in treated HIV infection, potentially contributing to morbidity and mortality, it has been unclear whether CMV replication in individuals with no symptoms of CMV disease might play a role in this process. We hypothesized that persistent CMV replication in the intestinal epithelium of HIV/CMV-coinfected individuals impairs gut epithelial barrier function. Using a combination of state-of-the-art in situ hybridization technology (RNAscope) and immunohistochemistry, we detected CMV DNA and proteins and evidence of intestinal damage in rectosigmoid samples from CMV-positive individuals with both untreated and ART-suppressed HIV infection. Two different model systems, primary human intestinal cells differentiated in vitro to form polarized monolayers and a humanized mouse model of human gut, together demonstrated that intestinal epithelial cells are fully permissive to CMV replication. Independent of HIV, CMV disrupted tight junctions of polarized intestinal cells, significantly reducing transepithelial electrical resistance, a measure of monolayer integrity, and enhancing transepithelial permeability. The effect of CMV infection on the intestinal epithelium is mediated, at least in part, by the CMV-induced proinflammatory cytokine IL-6. Furthermore, letermovir, a novel anti-CMV drug, dampened the effects of CMV on the epithelium. Together, our data strongly suggest that CMV can disrupt epithelial junctions, leading to bacterial translocation and chronic inflammation in the gut and that CMV could serve as a target for therapeutic intervention to prevent or treat gut epithelial barrier dysfunction during HIV infection. PMID:28241080

The Cytoplasmic C-Tail of the Mouse Cytomegalovirus 7 Transmembrane Receptor Homologue, M78, Regulates Endocytosis of the Receptor and Modulates Virus Replication in Different Cell Types

PubMed Central

2016-01-01

Virus homologues of seven-transmembrane receptors (7TMR) are encoded by all beta- and gammaherpesviruses, suggesting important functional roles. M78 of mouse cytomegalovirus (MCMV) is representative of a family of 7TMR conserved in all betaherpesviruses. M78 family members have been found to exhibit cell-type specific effects upon virus replication in tissue culture and to affect virus pathogenesis in vivo. We reported previously that M78, for which no ligands are known, undergoes rapid, constitutive endocytosis. In this study, we have investigated the role of the M78 cytoplasmic C-tail in mediating endocytosis and consequences of C-tail deletion upon replication and pathogenesis. Mutations of M78 (C-tail truncations or point mutations) and CCR5-M78 chimeras identified two distinct regions affecting endocytosis. The first was a classical acidic di-leucine motif (DDxxxLL), located close to the C-terminus. The second region, the activity of which was suppressed by downstream sequences, included the putative 8th helix, located close to the 7th transmembrane domain. A recombinant MCMV expressing an endocytosis-deficient M78, lacking most of the C-tail (M78_CΔ155), had a cell-type specific replication phenotype. M78_CΔ155 had restricted replication in bone marrow macrophages, indistinguishable from an M78-null recombinant. In contrast, M78_CΔ155 replicated normally or with enhanced titres to wild type virus in other tested cell-types, whereas M78-null was attenuated. Distinct phenotypes for M78_CΔ155 and M78-null suggest that the C-tail deletion resulted in M78 dysfunction, rather than complete loss of function; furthermore, they highlight a cell-type specific role of M78 during replication. Infection of mice (intranasal) demonstrated that M78_CΔ155, similar to M78-null, was cleared more rapidly from the lungs than wild type virus and was severely attenuated for replication in salivary glands. It may be speculated that attenuation of both M78_CΔ155 and M78-null for replication in macrophages may have contributed to their similar pathogenic phenotypes. PMID:27760189

The Canonical Immediate Early 3 Gene Product pIE611 of Mouse Cytomegalovirus Is Dispensable for Viral Replication but Mediates Transcriptional and Posttranscriptional Regulation of Viral Gene Products.

PubMed

Rattay, Stephanie; Trilling, Mirko; Megger, Dominik A; Sitek, Barbara; Meyer, Helmut E; Hengel, Hartmut; Le-Trilling, Vu Thuy Khanh

2015-08-01

Transcription of mouse cytomegalovirus (MCMV) immediate early ie1 and ie3 is controlled by the major immediate early promoter/enhancer (MIEP) and requires differential splicing. Based on complete loss of genome replication of an MCMV mutant carrying a deletion of the ie3-specific exon 5, the multifunctional IE3 protein (611 amino acids; pIE611) is considered essential for viral replication. Our analysis of ie3 transcription resulted in the identification of novel ie3 isoforms derived from alternatively spliced ie3 transcripts. Construction of an IE3-hemagglutinin (IE3-HA) virus by insertion of an in-frame HA epitope sequence allowed detection of the IE3 isoforms in infected cells, verifying that the newly identified transcripts code for proteins. This prompted the construction of an MCMV mutant lacking ie611 but retaining the coding capacity for the newly identified isoforms ie453 and ie310. Using Δie611 MCMV, we demonstrated the dispensability of the canonical ie3 gene product pIE611 for viral replication. To determine the role of pIE611 for viral gene expression during MCMV infection in an unbiased global approach, we used label-free quantitative mass spectrometry to delineate pIE611-dependent changes of the MCMV proteome. Interestingly, further analysis revealed transcriptional as well as posttranscriptional regulation of MCMV gene products by pIE611. Cytomegaloviruses are pathogenic betaherpesviruses persisting in a lifelong latency from which reactivation can occur under conditions of immunosuppression, immunoimmaturity, or inflammation. The switch from latency to reactivation requires expression of immediate early genes. Therefore, understanding of immediate early gene regulation might add insights into viral pathogenesis. The mouse cytomegalovirus (MCMV) immediate early 3 protein (611 amino acids; pIE611) is considered essential for viral replication. The identification of novel protein isoforms derived from alternatively spliced ie3 transcripts prompted the construction of an MCMV mutant lacking ie611 but retaining the coding capacity for the newly identified isoforms ie453 and ie310. Using Δie611 MCMV, we demonstrated the dispensability of the canonical ie3 gene product pIE611 for viral replication and delineated pIE611-dependent changes of the MCMV proteome. Our findings have fundamental implications for the interpretation of earlier studies on pIE3 functions and highlight the complex orchestration of MCMV gene regulation. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Eco-HAB as a fully automated and ecologically relevant assessment of social impairments in mouse models of autism.

PubMed

Puścian, Alicja; Łęski, Szymon; Kasprowicz, Grzegorz; Winiarski, Maciej; Borowska, Joanna; Nikolaev, Tomasz; Boguszewski, Paweł M; Lipp, Hans-Peter; Knapska, Ewelina

2016-10-12

Eco-HAB is an open source, RFID-based system for automated measurement and analysis of social preference and in-cohort sociability in mice. The system closely follows murine ethology. It requires no contact between a human experimenter and tested animals, overcoming the confounding factors that lead to irreproducible assessment of murine social behavior between laboratories. In Eco-HAB, group-housed animals live in a spacious, four-compartment apparatus with shadowed areas and narrow tunnels, resembling natural burrows. Eco-HAB allows for assessment of the tendency of mice to voluntarily spend time together in ethologically relevant mouse group sizes. Custom-made software for automated tracking, data extraction, and analysis enables quick evaluation of social impairments. The developed protocols and standardized behavioral measures demonstrate high replicability. Unlike classic three-chambered sociability tests, Eco-HAB provides measurements of spontaneous, ecologically relevant social behaviors in group-housed animals. Results are obtained faster, with less manpower, and without confounding factors.

POLD3 is haploinsufficient for DNA replication in mice

PubMed Central

Murga, Matilde; Lecona, Emilio; Kamileri, Irene; Díaz, Marcos; Lugli, Natalia; Sotiriou, Sotirios K.; Anton, Marta E.; Méndez, Juan; Halazonetis, Thanos D.; Fernandez-Capetillo, Oscar

2016-01-01

Summary The Pold3 gene encodes a subunit of the Polδ DNA polymerase complex. Pold3 orthologues are not essential in Saccharomyces cerevisiae or chicken DT40 cells, but the Schizzosaccharomyces pombe orthologue is essential. POLD3 also has a specialized role in the repair of broken replication forks, suggesting that POLD3 activity could be particularly relevant for cancer cells enduring high levels of DNA replication stress. We report here that POLD3 is essential for mouse development and is also required for viability in adult animals. Strikingly, even Pold3+/- mice were born at sub-Mendelian ratios and, of those born, some presented hydrocephaly and had a reduced lifespan. In cells, POLD3 deficiency led to replication stress and cell death, which were aggravated by expression of activated oncogenes. Finally, we show that Pold3 deletion destabilizes all members of the Polδ complex, explaining its major role in DNA replication and the severe impact of its deficiency. PMID:27524497

Minichromosome maintenance helicase paralog MCM9 is dispensible for DNA replication but functions in germ-line stem cells and tumor suppression.

PubMed

Hartford, Suzanne A; Luo, Yunhai; Southard, Teresa L; Min, Irene M; Lis, John T; Schimenti, John C

2011-10-25

Effective DNA replication is critical to the health and reproductive success of organisms. The six MCM2-7 proteins, which form the replicative helicase, are essential for high-fidelity replication of the genome. Many eukaryotes have a divergent paralog, MCM9, that was reported to be essential for loading MCM2-7 onto replication origins in the Xenopus oocyte extract system. To address the in vivo role of mammalian MCM9, we created and analyzed the phenotypes of mice with various mutations in Mcm9 and an intronic DNA replication-related gene Asf1a. Ablation of Mcm9 was compatible with cell proliferation and mouse viability, showing that it is nonessential for MCM2-7 loading or DNA replication. Mcm9 mutants underwent p53-independent embryonic germ-cell depletion in both sexes, with males also exhibiting defective spermatogonial stem-cell renewal. MCM9-deficient cells had elevated genomic instability and defective cell cycle reentry following replication stress, and mutant animals were prone to sex-specific cancers, most notably hepatocellular carcinoma in males. The phenotypes of mutant mice and cells suggest that MCM9 evolved a specialized but nonessential role in DNA replication or replication-linked quality-control mechanisms that are especially important for germ-line stem cells, and also for tumor suppression and genome maintenance in the soma.

Neuroprotective Effects of Psychotropic Drugs in Huntington’s Disease

PubMed Central

Lauterbach, Edward C.

2013-01-01

Psychotropics (antipsychotics, mood stabilizers, antidepressants, anxiolytics, etc.) are commonly prescribed to treat Huntington’s disease (HD). In HD preclinical models, while no psychotropic has convincingly affected huntingtin gene, HD modifying gene, or huntingtin protein expression, psychotropic neuroprotective effects include upregulated huntingtin autophagy (lithium), histone acetylation (lithium, valproate, lamotrigine), miR-222 (lithium-plus-valproate), mitochondrial protection (haloperidol, trifluoperazine, imipramine, desipramine, nortriptyline, maprotiline, trazodone, sertraline, venlafaxine, melatonin), neurogenesis (lithium, valproate, fluoxetine, sertraline), and BDNF (lithium, valproate, sertraline) and downregulated AP-1 DNA binding (lithium), p53 (lithium), huntingtin aggregation (antipsychotics, lithium), and apoptosis (trifluoperazine, loxapine, lithium, desipramine, nortriptyline, maprotiline, cyproheptadine, melatonin). In HD live mouse models, delayed disease onset (nortriptyline, melatonin), striatal preservation (haloperidol, tetrabenazine, lithium, sertraline), memory preservation (imipramine, trazodone, fluoxetine, sertraline, venlafaxine), motor improvement (tetrabenazine, lithium, valproate, imipramine, nortriptyline, trazodone, sertraline, venlafaxine), and extended survival (lithium, valproate, sertraline, melatonin) have been documented. Upregulated CREB binding protein (CBP; valproate, dextromethorphan) and downregulated histone deacetylase (HDAC; valproate) await demonstration in HD models. Most preclinical findings await replication and their limitations are reviewed. The most promising findings involve replicated striatal neuroprotection and phenotypic disease modification in transgenic mice for tetrabenazine and for sertraline. Clinical data consist of an uncontrolled lithium case series (n = 3) suggesting non-progression and a primarily negative double-blind, placebo-controlled clinical trial of lamotrigine. PMID:24248060

Analysis of response to 20 generations of selection for body composition in mice: fit to infinitesimal model assumptions

PubMed Central

Martinez, Victor; Bünger, Lutz; Hill, William G

2000-01-01

Data were analysed from a divergent selection experiment for an indicator of body composition in the mouse, the ratio of gonadal fat pad to body weight (GFPR). Lines were selected for 20 generations for fat (F), lean (L) or were unselected (C), with three replicates of each. Selection was within full-sib families, 16 families per replicate for the first seven generations, eight subsequently. At generation 20, GFPR in the F lines was twice and in the L lines half that of C. A log transformation removed both asymmetry of response and heterogeneity of variance among lines, and so was used throughout. Estimates of genetic variance and heritability (approximately 50%) obtained using REML with an animal model were very similar, whether estimated from the first few generations of selection, or from all 20 generations, or from late generations having fitted pedigree. The estimates were also similar when estimated from selected or control lines. Estimates from REML also agreed with estimates of realised heritability. The results all accord with expectations under the infinitesimal model, despite the four-fold changes in mean. Relaxed selection lines, derived from generation 20, showed little regression in fatness after 40 generations without selection. PMID:14736404

Standardized Profiling of The Membrane-Enriched Proteome of Mouse Dorsal Root Ganglia (DRG) Provides Novel Insights Into Chronic Pain.

PubMed

Rouwette, Tom; Sondermann, Julia; Avenali, Luca; Gomez-Varela, David; Schmidt, Manuela

2016-06-01

Chronic pain is a complex disease with limited treatment options. Several profiling efforts have been employed with the aim to dissect its molecular underpinnings. However, generated results are often inconsistent and nonoverlapping, which is largely because of inherent technical constraints. Emerging data-independent acquisition (DIA)-mass spectrometry (MS) has the potential to provide unbiased, reproducible and quantitative proteome maps - a prerequisite for standardization among experiments. Here, we designed a DIA-based proteomics workflow to profile changes in the abundance of dorsal root ganglia (DRG) proteins in two mouse models of chronic pain, inflammatory and neuropathic. We generated a DRG-specific spectral library containing 3067 DRG proteins, which enables their standardized quantification by means of DIA-MS in any laboratory. Using this resource, we profiled 2526 DRG proteins in each biological replicate of both chronic pain models and respective controls with unprecedented reproducibility. We detected numerous differentially regulated proteins, the majority of which exhibited pain model-specificity. Our approach recapitulates known biology and discovers dozens of proteins that have not been characterized in the somatosensory system before. Functional validation experiments and analysis of mouse pain behaviors demonstrate that indeed meaningful protein alterations were discovered. These results illustrate how the application of DIA-MS can open new avenues to achieve the long-awaited standardization in the molecular dissection of pathologies of the somatosensory system. Therefore, our findings provide a valuable framework to qualitatively extend our understanding of chronic pain and somatosensation. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Evaluation of a mouse model for the West Nile virus group for the purpose of determining viral pathotypes.

PubMed

Bingham, John; Payne, Jean; Harper, Jennifer; Frazer, Leah; Eastwood, Sarah; Wilson, Susanne; Lowther, Sue; Lunt, Ross; Warner, Simone; Carr, Mary; Hall, Roy A; Durr, Peter A

2014-06-01

West Nile virus (WNV; family Flaviviridae; genus Flavivirus) group members are an important cause of viral meningoencephalitis in some areas of the world. They exhibit marked variation in pathogenicity, with some viral lineages (such as those from North America) causing high prevalence of severe neurological disease, whilst others (such as Australian Kunjin virus) rarely cause disease. The aim of this study was to characterize WNV disease in a mouse model and to elucidate the pathogenetic features that distinguish disease variation. Tenfold dilutions of five WNV strains (New York 1999, MRM16 and three horse isolates of WNV-Kunjin: Boort and two isolates from the 2011 Australian outbreak) were inoculated into mice by the intraperitoneal route. All isolates induced meningoencephalitis in different proportions of infected mice. WNVNY99 was the most pathogenic, the three horse isolates were of intermediate pathogenicity and WNVKUNV-MRM16 was the least, causing mostly asymptomatic disease with seroconversion. Infectivity, but not pathogenicity, was related to challenge dose. Using cluster analysis of the recorded clinical signs, histopathological lesions and antigen distribution scores, the cases could be classified into groups corresponding to disease severity. Metrics that were important in determining pathotype included neurological signs (paralysis and seizures), meningoencephalitis, brain antigen scores and replication in extra-neural tissues. Whereas all mice infected with WNVNY99 had extra-neural antigen, those infected with the WNV-Kunjin viruses only occasionally had antigen outside the nervous system. We conclude that the mouse model could be a useful tool for the assessment of pathotype for WNVs. © 2014 CSIRO.

Investigation of the mechanism of action of alemtuzumab in a human CD52 transgenic mouse model

PubMed Central

Hu, Yanping; Turner, Michael J; Shields, Jacqueline; Gale, Matthew S; Hutto, Elizabeth; Roberts, Bruce L; Siders, William M; Kaplan, Johanne M

2009-01-01

Alemtuzumab is a humanized monoclonal antibody against CD52, an antigen found on the surface of normal and malignant lymphocytes. It is approved for the treatment of B-cell chronic lymphocytic leukaemia and is undergoing Phase III clinical trials for the treatment of multiple sclerosis. The exact mechanism by which alemtuzumab mediates its biological effects in vivo is not clearly defined and mechanism of action studies have been hampered by the lack of cross-reactivity between human and mouse CD52. To address this issue, a transgenic mouse expressing human CD52 (hCD52) was created. Transgenic mice did not display any phenotypic abnormalities and were able to mount normal immune responses. The tissue distribution of hCD52 and the level of expression by various immune cell populations were comparable to those seen in humans. Treatment with alemtuzumab replicated the transient increase in serum cytokines and depletion of peripheral blood lymphocytes observed in humans. Lymphocyte depletion was not as profound in lymphoid organs, providing a possible explanation for the relatively low incidence of infection in alemtuzumab-treated patients. Interestingly, both lymphocyte depletion and cytokine induction by alemtuzumab were largely independent of complement and appeared to be mediated by neutrophils and natural killer cells because removal of these populations with antibodies to Gr-1 or asialo-GM-1, respectively, strongly inhibited the activity of alemtuzumab whereas removal of complement by treatment with cobra venom factor had no impact. The hCD52 transgenic mouse appears to be a useful model and has provided evidence for the previously uncharacterized involvement of neutrophils in the activity of alemtuzumab. PMID:19740383

Resistance to Two Heterologous Neurotropic Oncolytic Viruses, Semliki Forest Virus and Vaccinia Virus, in Experimental Glioma

PubMed Central

Le Boeuf, Fabrice; Lemay, Chantal; De Silva, Naomi; Diallo, Jean-Simon; Cox, Julie; Becker, Michelle; Choi, Youngmin; Ananth, Abhirami; Sellers, Clara; Breton, Sophie; Roy, Dominic; Falls, Theresa; Brun, Jan; Hemminki, Akseli; Hinkkanen, Ari; Bell, John C.

2013-01-01

Attenuated Semliki Forest virus (SFV) may be suitable for targeting malignant glioma due to its natural neurotropism, but its replication in brain tumor cells may be restricted by innate antiviral defenses. We attempted to facilitate SFV replication in glioma cells by combining it with vaccinia virus, which is capable of antagonizing such defenses. Surprisingly, we found parenchymal mouse brain tumors to be refractory to both viruses. Also, vaccinia virus appears to be sensitive to SFV-induced antiviral interference. PMID:23221568

2′-5′-Oligoadenylate Synthetase-Like Protein Inhibits Respiratory Syncytial Virus Replication and Is Targeted by the Viral Nonstructural Protein 1

PubMed Central

Dhar, Jayeeta; Cuevas, Rolando A.; Goswami, Ramansu; Zhu, Jianzhong

2015-01-01

2′-5′-Oligoadenylate synthetase-like protein (OASL) is an interferon-inducible antiviral protein. Here we describe differential inhibitory activities of human OASL and the two mouse OASL homologs against respiratory syncytial virus (RSV) replication. Interestingly, nonstructural protein 1 (NS1) of RSV promoted proteasome-dependent degradation of specific OASL isoforms. We conclude that OASL acts as a cellular antiviral protein and that RSV NS1 suppresses this function to evade cellular innate immunity and allow virus growth. PMID:26178980

Development of a Sox2 reporter system modeling cellular heterogeneity in glioma.

PubMed

Stoltz, Kevin; Sinyuk, Maksim; Hale, James S; Wu, Qiulian; Otvos, Balint; Walker, Kiera; Vasanji, Amit; Rich, Jeremy N; Hjelmeland, Anita B; Lathia, Justin D

2015-03-01

Malignant gliomas are complex systems containing a number of factors that drive tumor initiation and progression, including genetic aberrations that lead to extensive cellular heterogeneity within the neoplastic compartment. Mouse models recapitulate these genetic aberrations, but readily observable heterogeneity remains challenging. To interrogate cellular heterogeneity in mouse glioma models, we utilized a replication-competent avian sarcoma-leukosis virus long terminal repeat with splice acceptor/tumor virus A (RCAS-tva) system to generate spontaneous mouse gliomas that contained a Sox2-enhanced green fluorescent protein (EGFP) reporter. Glial fibrillary acidic protein-tva mice were crossed with Sox2-EGFP mice, and tumors were initiated that contained a subpopulation of Sox2-EGFP-high cells enriched for tumor-initiating cell properties such as self-renewal, multilineage differentiation potential, and perivascular localization. Following implantation into recipient mice, Sox2-EGFP-high cells generated tumors containing Sox2-EGFP-high and Sox2-EGFP-low cells. Kinomic analysis of Sox2-EGFP-high cells revealed activation of known glioma signaling pathways that are strongly correlated with patient survival including platelet-derived growth factor receptor beta, phosphoinositide-3 kinase, and vascular endothelial growth factor. Our functional analysis identified active feline sarcoma (Fes) signaling in Sox2-EGFP-high cells. Fes negatively correlated with glioma patient survival and was coexpressed with Sox2-positive cells in glioma xenografts and primary patient-derived tissue. Our RCAS-tva/Sox2-EGFP model will empower closer examination of cellular heterogeneity and will be useful for identifying novel glioma pathways as well as testing preclinical treatment efficacy. © The Author(s) 2014. Published by Oxford University Press on behalf of the Society for Neuro-Oncology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Characteristic features of intracellular pathogenic Leptospira in infected murine macrophages.

PubMed

Toma, Claudia; Okura, Nobuhiko; Takayama, Chitoshi; Suzuki, Toshihiko

2011-11-01

Leptospira interrogans is a spirochaete responsible for a zoonotic disease known as leptospirosis. Leptospires are able to penetrate the abraded skin and mucous membranes and rapidly disseminate to target organs such as the liver, lungs and kidneys. How this pathogen escape from innate immune cells and spread to target organs remains poorly understood. In this paper, the intracellular trafficking undertaken by non-pathogenic Leptospira biflexa and pathogenic L. interrogans in mouse bone marrow-derived macrophages was compared. The delayed in the clearance of L. interrogans was observed. Furthermore, the acquisition of lysosomal markers by L. interrogans-containing phagosomes lagged behind that of L. biflexa-containing phagosomes, and although bone marrow-derived macrophages could degrade L. biflexa as well as L. interrogans, a population of L. interrogans was able to survive and replicate. Intact leptospires were found within vacuoles at 24 h post infection, suggesting that bacterial replication occurs within a membrane-bound compartment. In contrast, L. biflexa were completely degraded at 24 h post infection. Furthermore, L. interrogans but not L. biflexa, were released to the extracellular milieu. These results suggest that pathogenic leptospires are able to survive, replicate and exit from mouse macrophages, enabling their eventual spread to target organs. © 2011 Blackwell Publishing Ltd.

Biochemical Evaluation of the Inhibition Properties of Favipiravir and 2′-C-Methyl-Cytidine Triphosphates against Human and Mouse Norovirus RNA Polymerases

PubMed Central

Tucker, Kathryn; Lin, Xiaoyan; Kao, C. Cheng; Shaw, Ken; Tan, Hua; Symons, Julian; Behera, Ishani; Rajwanshi, Vivek K.; Dyatkina, Natalia; Wang, Guangyi; Beigelman, Leo

2015-01-01

Norovirus (NoV) is a positive-sense single-stranded RNA virus that causes acute gastroenteritis and is responsible for 200,000 deaths per year worldwide. No effective vaccine or treatment is available. Recent studies have shown that the nucleoside analogs favipiravir (T-705) and 2′-C-methyl-cytidine (2CM-C) inhibit NoV replication in vitro and in animal models, but their precise mechanism of action is unknown. We evaluated the molecular interactions between nucleoside triphosphates and NoV RNA-dependent RNA polymerase (NoVpol), the enzyme responsible for replication and transcription of NoV genomic RNA. We found that T-705 ribonucleoside triphosphate (RTP) and 2CM-C triphosphate (2CM-CTP) equally inhibited human and mouse NoVpol activities at concentrations resulting in 50% of maximum inhibition (IC50s) in the low micromolar range. 2CM-CTP inhibited the viral polymerases by competing directly with natural CTP during primer elongation, whereas T-705 RTP competed mostly with ATP and GTP at the initiation and elongation steps. Incorporation of 2CM-CTP into viral RNA blocked subsequent RNA synthesis, whereas T-705 RTP did not cause immediate chain termination of NoVpol. 2CM-CTP and T-705 RTP displayed low levels of enzyme selectivity, as they were both recognized as substrates by human mitochondrial RNA polymerase. The level of discrimination by the human enzyme was increased with a novel analog of T-705 RTP containing a 2′-C-methyl substitution. Collectively, our data suggest that 2CM-C inhibits replication of NoV by acting as a classic chain terminator, while T-705 may inhibit the virus by multiple mechanisms of action. Understanding the precise mechanism of action of anti-NoV compounds could provide a rational basis for optimizing their inhibition potencies and selectivities. PMID:26392512

Geminin deficiency enhances survival in a murine medulloblastoma model by inducing apoptosis of preneoplastic granule neuron precursors

PubMed Central

Sankar, Savita; Patterson, Ethan; Lewis, Emily M.; Waller, Laura E.; Tong, Caili; Dearborn, Joshua; Wozniak, David; Rubin, Joshua B.; Kroll, Kristen L.

2017-01-01

Medulloblastoma is the most common malignant brain cancer of childhood. Further understanding of tumorigenic mechanisms may define new therapeutic targets. Geminin maintains genome fidelity by controlling re-initiation of DNA replication within a cell cycle. In some contexts, Geminin inhibition induces cancer-selective cell cycle arrest and apoptosis and/or sensitizes cancer cells to Topoisomerase IIα inhibitors such as etoposide, which is used in combination chemotherapies for medulloblastoma. However, Geminin's potential role in medulloblastoma tumorigenesis remained undefined. Here, we found that Geminin is highly expressed in human and mouse medulloblastomas and in murine granule neuron precursor (GNP) cells during cerebellar development. Conditional Geminin loss significantly enhanced survival in the SmoA1 mouse medulloblastoma model. Geminin loss in this model also reduced numbers of preneoplastic GNPs persisting at one postnatal month, while at two postnatal weeks these cells exhibited an elevated DNA damage response and apoptosis. Geminin knockdown likewise impaired human medulloblastoma cell growth, activating G2 checkpoint and DNA damage response pathways, triggering spontaneous apoptosis, and enhancing G2 accumulation of cells in response to etoposide treatment. Together, these data suggest preneoplastic and cancer cell-selective roles for Geminin in medulloblastoma, and suggest that targeting Geminin may impair tumor growth and enhance responsiveness to Topoisomerase IIα-directed chemotherapies. PMID:29234490

Rapid Pathogen-Induced Apoptosis: A Mechanism Used by Dendritic Cells to Limit Intracellular Replication of Legionella pneumophila

PubMed Central

Nogueira, Catarina V.; Lindsten, Tullia; Jamieson, Amanda M.; Case, Christopher L.; Shin, Sunny; Thompson, Craig B.; Roy, Craig R.

2009-01-01

Dendritic cells (DCs) are specialized phagocytes that internalize exogenous antigens and microbes at peripheral sites, and then migrate to lymphatic organs to display foreign peptides to naïve T cells. There are several examples where DCs have been shown to be more efficient at restricting the intracellular replication of pathogens compared to macrophages, a property that could prevent DCs from enhancing pathogen dissemination. To understand DC responses to pathogens, we investigated the mechanisms by which mouse DCs are able to restrict replication of the intracellular pathogen Legionella pneumophila. We show that both DCs and macrophages have the ability to interfere with L. pneumophila replication through a cell death pathway mediated by caspase-1 and Naip5. L. pneumophila that avoided Naip5-dependent responses, however, showed robust replication in macrophages but remained unable to replicate in DCs. Apoptotic cell death mediated by caspase-3 was found to occur much earlier in DCs following infection by L. pneumophila compared to macrophages infected similarly. Eliminating the pro-apoptotic proteins Bax and Bak or overproducing the anti-apoptotic protein Bcl-2 were both found to restore L. pneumophila replication in DCs. Thus, DCs have a microbial response pathway that rapidly activates apoptosis to limit pathogen replication. PMID:19521510

Encapsulated Stem Cells Loaded With Hyaluronidase-expressing Oncolytic Virus for Brain Tumor Therapy

PubMed Central

Martinez-Quintanilla, Jordi; He, Derek; Wakimoto, Hiroaki; Alemany, Ramon; Shah, Khalid

2015-01-01

Despite the proven safety of oncolytic viruses (OV) in clinical trials for glioblastoma (GBM), their efficacy has been hindered by suboptimal spreading within the tumor. We show that hyaluronan or hyaluronic acid (HA), an important component of extracellular matrix (ECM), is highly expressed in a majority of tumor xenografts established from patient-derived GBM lines that present both invasive and nodular phenotypes. Intratumoral injection of a conditionally replicating adenovirus expressing soluble hyaluronidase (ICOVIR17) into nodular GBM, mediated HA degradation and enhanced viral spread, resulting in a significant antitumor effect and mice survival. In an effort to translate OV-based therapeutics into clinical settings, we encapsulated human adipose-derived mesenchymal stem cells (MSC) loaded with ICOVIR17 in biocompatible synthetic extracellular matrix (sECM) and tested their efficacy in a clinically relevant mouse model of GBM resection. Compared with direct injection of ICOVIR17, sECM-MSC loaded with ICOVIR17 resulted in a significant decrease in tumor regrowth and increased mice survival. This is the first report of its kind revealing the expression of HA in GBM and the role of OV-mediated HA targeting in clinically relevant mouse model of GBM resection and thus has clinical implications. PMID:25352242

Lithium rescues synaptic plasticity and memory in Down syndrome mice

PubMed Central

Contestabile, Andrea; Greco, Barbara; Ghezzi, Diego; Tucci, Valter; Benfenati, Fabio; Gasparini, Laura

2012-01-01

Down syndrome (DS) patients exhibit abnormalities of hippocampal-dependent explicit memory, a feature that is replicated in relevant mouse models of the disease. Adult hippocampal neurogenesis, which is impaired in DS and other neuropsychiatric diseases, plays a key role in hippocampal circuit plasticity and has been implicated in learning and memory. However, it remains unknown whether increasing adult neurogenesis improves hippocampal plasticity and behavioral performance in the multifactorial context of DS. We report that, in the Ts65Dn mouse model of DS, chronic administration of lithium, a clinically used mood stabilizer, promoted the proliferation of neuronal precursor cells through the pharmacological activation of the Wnt/β-catenin pathway and restored adult neurogenesis in the hippocampal dentate gyrus (DG) to physiological levels. The restoration of adult neurogenesis completely rescued the synaptic plasticity of newborn neurons in the DG and led to the full recovery of behavioral performance in fear conditioning, object location, and novel object recognition tests. These findings indicate that reestablishing a functional population of hippocampal newborn neurons in adult DS mice rescues hippocampal plasticity and memory and implicate adult neurogenesis as a promising therapeutic target to alleviate cognitive deficits in DS patients. PMID:23202733

Clinical Implications of Basic Science Discoveries: Microchimerism Finds a Major Role in Reproductive Success; but Does It Also Contribute to Transplant Success?

PubMed

Burlingham, W J

2016-10-01

Conventional wisdom argues against inbreeding, to maintain hybrid vigor and increase MHC diversity in response to pathogens. A recent report from the laboratory of Sing-Sing Way uses a mouse model to test a hypothesis put forward by Ray D. Owen more than 60 years ago: that a certain amount of inbreeding is a good thing. Owen proposed that antigens not inherited from the mother (noninherited maternal antigens), when replicated on the mate of the daughter, could protect the latter's developing child from fetal wastage due to immune attack during her pregnancy. Kinder et al use elegant mouse breeding models and MHC class II peptide tetramers to show that Owen's hypothesis, based only on humoral (anti-Rh IgG) data and a small sample size, was indeed correct. The mediators of this cross-generational protection turn out to be a special kind of Foxp3 + T regulatory cell, the development of which requires the persistence of maternal microchimerism into adulthood. The implications of this discovery for the role of microchimerism in tolerance to transplants are discussed. © Copyright 2016 The American Society of Transplantation and the American Society of Transplant Surgeons.

Partial hepatic resistance to IL-6-induced inflammation develops in type 2 diabetic mice, while the anti-inflammatory effect of AMPK is maintained.

PubMed

Cansby, Emmelie; Nerstedt, Annika; Amrutkar, Manoj; Durán, Esther Nuñez; Smith, Ulf; Mahlapuu, Margit

2014-08-05

Interleukin-6 (IL-6) induces hepatic inflammation and insulin resistance, and therapeutic strategies to counteract the IL-6 action in liver are of high interest. In this study, we demonstrate that acute treatment with AMP-activated protein kinase (AMPK) agonists AICAR and metformin efficiently repressed IL-6-induced hepatic proinflammatory gene expression and activation of STAT3 in a mouse model of diet-induced type 2 diabetes, bringing it back to basal nonstimulated level. Surprisingly, the inflammatory response in liver induced by IL-6 administration in vivo was markedly blunted in the mice fed a high-fat diet, compared to lean chow-fed controls, while this difference was not replicated in vitro in primary hepatocytes derived from these two groups of mice. In summary, our work reveals that partial hepatic IL-6 resistance develops in the mouse model of type 2 diabetes, while the anti-inflammatory action of AMPK is maintained. Systemic factors, rather than differences in intracellular IL-6 receptor signaling, are likely mediating the relative impairment in IL-6 effect. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

Salmonella exploits the host endolysosomal tethering factor HOPS complex to promote its intravacuolar replication

PubMed Central

Sindhwani, Aastha; Kaur, Harmeet; Tuli, Amit

2017-01-01

Salmonella enterica serovar typhimurium extensively remodels the host late endocytic compartments to establish its vacuolar niche within the host cells conducive for its replication, also known as the Salmonella-containing vacuole (SCV). By maintaining a prolonged interaction with late endosomes and lysosomes of the host cells in the form of interconnected network of tubules (Salmonella-induced filaments or SIFs), Salmonella gains access to both membrane and fluid-phase cargo from these compartments. This is essential for maintaining SCV membrane integrity and for bacterial intravacuolar nutrition. Here, we have identified the multisubunit lysosomal tethering factor—HOPS (HOmotypic fusion and Protein Sorting) complex as a crucial host factor facilitating delivery of late endosomal and lysosomal content to SCVs, providing membrane for SIF formation, and nutrients for intravacuolar bacterial replication. Accordingly, depletion of HOPS subunits significantly reduced the bacterial load in non-phagocytic and phagocytic cells as well as in a mouse model of Salmonella infection. We found that Salmonella effector SifA in complex with its binding partner; SKIP, interacts with HOPS subunit Vps39 and mediates recruitment of this tethering factor to SCV compartments. The lysosomal small GTPase Arl8b that binds to, and promotes membrane localization of Vps41 (and other HOPS subunits) was also required for HOPS recruitment to SCVs and SIFs. Our findings suggest that Salmonella recruits the host late endosomal and lysosomal membrane fusion machinery to its vacuolar niche for access to host membrane and nutrients, ensuring its intracellular survival and replication. PMID:29084291

Fucoidan from Fucus vesiculosus suppresses hepatitis B virus replication by enhancing extracellular signal-regulated Kinase activation.

PubMed

Li, Huifang; Li, Junru; Tang, Yuan; Lin, Lin; Xie, Zhanglian; Zhou, Jia; Zhang, Liyun; Zhang, Xiaoyong; Zhao, Xiaoshan; Chen, Zhengliang; Zuo, Daming

2017-09-16

Hepatitis B virus (HBV) infection is a serious public health problem leading to cirrhosis and hepatocellular carcinoma. As the clinical utility of current therapies is limited, the development of new therapeutic approaches for the prevention and treatment of HBV infection is imperative. Fucoidan is a natural sulfated polysaccharide that extracted from different species of brown seaweed, which was reported to exhibit various bioactivities. However, it remains unclear whether fucoidan influences HBV replication or not. The HBV-infected mouse model was established by hydrodynamic injection of HBV replicative plasmid, and the mice were treated with saline or fucoidan respectively. Besides, we also tested the inhibitory effect of fucoidan against HBV infection in HBV-transfected cell lines. The result showed that fucoidan from Fucus vesiculosus decreased serum HBV DNA, HBsAg and HBeAg levels and hepatic HBcAg expression in HBV-infected mice. Moreover, fucoidan treatment also suppressed intracellular HBcAg expression and the secretion of the HBV DNA as well as HBsAg and HBeAg in HBV-expressing cells. Furthermore, we proved that the inhibitory activity by fucoidan was due to the activation of the extracellular signal-regulated kinase (ERK) pathway and the subsequent production of type I interferon. Using specific inhibitor of ERK pathway abrogated the fucoidan-mediated inhibition of HBV replication. This study highlights that fucoidan might be served as an alternative therapeutic approach for the treatment of HBV infection.

An essential role for Ink4 and Cip/Kip cell-cycle inhibitors in preventing replicative stress.

PubMed

Quereda, V; Porlan, E; Cañamero, M; Dubus, P; Malumbres, M

2016-03-01

Cell-cycle inhibitors of the Ink4 and Cip/Kip families are involved in cellular senescence and tumor suppression. These inhibitors are individually dispensable for the cell cycle and inactivation of specific family members results in increased proliferation and enhanced susceptibility to tumor development. We have now analyzed the consequences of eliminating a substantial part of the cell-cycle inhibitory activity in the cell by generating a mouse model, which combines the absence of both p21(Cip1) and p27(Kip1) proteins with the endogenous expression of a Cdk4 R24C mutant insensitive to Ink4 inhibitors. Pairwise combination of Cdk4 R24C, p21-null and p27-null alleles results in frequent hyperplasias and tumors, mainly in cells of endocrine origin such as pituitary cells and in mesenchymal tissues. Interestingly, complete abrogation of p21(Cip1) and p27(Kip1) in Cdk4 R24C mutant mice results in a different phenotype characterized by perinatal death accompanied by general hypoplasia in most tissues. This phenotype correlates with increased replicative stress in developing tissues such as the nervous system and subsequent apoptotic cell death. Partial inhibition of Cdk4/6 rescues replicative stress signaling as well as p53 induction in the absence of cell-cycle inhibitors. We conclude that one of the major physiological activities of cell-cycle inhibitors is to prevent replicative stress during development.

Analysis of Host Range Restriction Determinants in the Rabbit Model: Comparison of Homologous and Heterologous Rotavirus Infections

PubMed Central

Ciarlet, Max; Estes, Mary K.; Barone, Christopher; Ramig, Robert F.; Conner, Margaret E.

1998-01-01

The main limitation of both the rabbit and mouse models of rotavirus infection is that human rotavirus (HRV) strains do not replicate efficiently in either animal. The identification of individual genes necessary for conferring replication competence in a heterologous host is important to an understanding of the host range restriction of rotavirus infections. We recently reported the identification of the P type of the spike protein VP4 of four lapine rotavirus strains as being P[14]. To determine whether VP4 is involved in host range restriction in rabbits, we evaluated infection in rotavirus antibody-free rabbits inoculated orally with two P[14] HRVs, PA169 (G6) and HAL1166 (G8), and with several other HRV strains and animal rotavirus strains of different P and G types. We also evaluated whether the parental rhesus rotavirus (RRV) (P5B[3], G3) and the derived RRV-HRV reassortant candidate vaccine strains RRV × D (G1), RRV × DS-1 (G2), and RRV × ST3 (G4) would productively infect rabbits. Based on virus shedding, limited replication was observed with the P[14] HRV strains and with the SA11 Cl3 (P[2], G3) and SA11 4F (P6[1], G3) animal rotavirus strains, compared to the homologous ALA strain (P[14], G3). However, even limited infection provided complete protection from rotavirus infection when rabbits were challenged orally 28 days postinoculation (DPI) with 103 50% infective doses of ALA rabbit rotavirus. Other HRVs did not productively infect rabbits and provided no significant protection from challenge, in spite of occasional seroconversion. Simian RRV replicated as efficiently as lapine ALA rotavirus in rabbits and provided complete protection from ALA challenge. Live attenuated RRV reassortant vaccine strains resulted in no, limited, or productive infection of rabbits, but all rabbits were completely protected from heterotypic ALA challenge. The altered replication efficiency of the reassortants in rabbits suggests a role for VP7 in host range restriction. Also, our results suggest that VP4 may be involved in, but is not exclusively responsible for, host range restriction in the rabbit model. The replication efficiency of rotavirus in rabbits also is not controlled by the product of gene 5 (NSP1) alone, since a reassortant rotavirus with ALA gene 5 and all other genes from SA11 was more severely replication restricted than either parental rotavirus strain. PMID:9499095

Reverse genetics in high throughput: rapid generation of complete negative strand RNA virus cDNA clones and recombinant viruses thereof.

PubMed

Nolden, T; Pfaff, F; Nemitz, S; Freuling, C M; Höper, D; Müller, T; Finke, Stefan

2016-04-05

Reverse genetics approaches are indispensable tools for proof of concepts in virus replication and pathogenesis. For negative strand RNA viruses (NSVs) the limited number of infectious cDNA clones represents a bottleneck as clones are often generated from cell culture adapted or attenuated viruses, with limited potential for pathogenesis research. We developed a system in which cDNA copies of complete NSV genomes were directly cloned into reverse genetics vectors by linear-to-linear RedE/T recombination. Rapid cloning of multiple rabies virus (RABV) full length genomes and identification of clones identical to field virus consensus sequence confirmed the approache's reliability. Recombinant viruses were recovered from field virus cDNA clones. Similar growth kinetics of parental and recombinant viruses, preservation of field virus characters in cell type specific replication and virulence in the mouse model were confirmed. Reduced titers after reporter gene insertion indicated that the low level of field virus replication is affected by gene insertions. The flexibility of the strategy was demonstrated by cloning multiple copies of an orthobunyavirus L genome segment. This important step in reverse genetics technology development opens novel avenues for the analysis of virus variability combined with phenotypical characterization of recombinant viruses at a clonal level.

Coexisting cholinergic and parahippocampal degeneration: a key to memory loss in dementia and a challenge for transgenic models?

PubMed

Cassel, Jean-Christophe; Mathis, Chantal; Majchrzak, Monique; Moreau, Pierre-Henri; Dalrymple-Alford, John C

2008-01-01

One century after Alzheimer's initial report, a variety of animal models of Alzheimer's disease (AD) are being used to mimic one or more pathological signs viewed as critical for the evolution of cognitive decline in dementia. Among the most common are, (a) traditional lesion models aimed at reproducing the degeneration of one of two key brain regions affected in AD, namely the cholinergic basal forebrain (CBF) and the transentorhinal region, and (b) transgenic mouse models aimed at reproducing AD histopathological hallmarks, namely amyloid plaques and neurofibrillary tangles. These models have provided valuable insights into the development and consequences of the pathology, but they have not consistently reproduced the severity of memory deficits exhibited in AD. The reasons for this lack of correspondence with the severity of expected deficits may include the limited replication of multiple neuropathology in potentially key brain regions. A recent lesion model in the rat found that severe memory impairment was obtained only when the two traditional lesions were combined together (i.e. conjoint CBF and entorhinal cortex lesions), indicative of a dramatic impact on cognitive function when there is coexisting, rather than isolated, damage in these two brain regions. It is proposed that combining AD transgenic mouse models with additional experimental damage to both the CBF and entorhinal regions might provide a unique opportunity to further understand the evolution of the disease and improve treatments of severe cognitive dysfunction in neurodegenerative dementias. (c) 2008 S. Karger AG, Basel

HUMAN ALVEOLAR AND PERITONEAL MACROPHAGES MEDIATE FUNGISTASIS INDEPENDENTLY OF L-ARGININE OXIDATION TO NITRITE OR NITRATE

EPA Science Inventory

Human alveolar macrophages (HAM) from 28 normal volunteers were found to inhibit replication of Cryptoccous neoformans. onditions under which fungistasis occurred were different than those required for mouse peritoneal macrophage-mediated fungi stasis. nhibition of fungal replica...

Low-level mitochondrial heteroplasmy modulates DNA replication, glucose metabolism and lifespan in mice.

PubMed

Hirose, Misa; Schilf, Paul; Gupta, Yask; Zarse, Kim; Künstner, Axel; Fähnrich, Anke; Busch, Hauke; Yin, Junping; Wright, Marvin N; Ziegler, Andreas; Vallier, Marie; Belheouane, Meriem; Baines, John F; Tautz, Diethard; Johann, Kornelia; Oelkrug, Rebecca; Mittag, Jens; Lehnert, Hendrik; Othman, Alaa; Jöhren, Olaf; Schwaninger, Markus; Prehn, Cornelia; Adamski, Jerzy; Shima, Kensuke; Rupp, Jan; Häsler, Robert; Fuellen, Georg; Köhling, Rüdiger; Ristow, Michael; Ibrahim, Saleh M

2018-04-12

Mutations in mitochondrial DNA (mtDNA) lead to heteroplasmy, i.e., the intracellular coexistence of wild-type and mutant mtDNA strands, which impact a wide spectrum of diseases but also physiological processes, including endurance exercise performance in athletes. However, the phenotypic consequences of limited levels of naturally arising heteroplasmy have not been experimentally studied to date. We hence generated a conplastic mouse strain carrying the mitochondrial genome of an AKR/J mouse strain (B6-mt AKR ) in a C57BL/6 J nuclear genomic background, leading to >20% heteroplasmy in the origin of light-strand DNA replication (OriL). These conplastic mice demonstrate a shorter lifespan as well as dysregulation of multiple metabolic pathways, culminating in impaired glucose metabolism, compared to that of wild-type C57BL/6 J mice carrying lower levels of heteroplasmy. Our results indicate that physiologically relevant differences in mtDNA heteroplasmy levels at a single, functionally important site impair the metabolic health and lifespan in mice.

Combining Zebrafish and Mouse Models to Test the Function of Deubiquitinating Enzyme (Dubs) Genes in Development: Role of USP45 in the Retina.

PubMed

Toulis, Vasileios; Garanto, Alejandro; Marfany, Gemma

2016-01-01

Ubiquitination is a dynamic and reversible posttranslational modification. Much effort has been devoted to characterize the function of ubiquitin pathway genes in the cell context, but much less is known on their functional role in the development and maintenance of organs and tissues in the organism. In fact, several ubiquitin ligases and deubiquitinating enzymes (DUBs) are implicated in human pathological disorders, from cancer to neurodegeneration. The aim of our work is to explore the relevance of DUBs in retinal function in health and disease, particularly since some genes related to the ubiquitin or SUMO pathways cause retinal dystrophies, a group of rare diseases that affect 1:3000 individuals worldwide. We propose zebrafish as an extremely useful and informative genetic model to characterize the function of any particular gene in the retina, and thus complement the expression data from mouse. A preliminary characterization of gene expression in mouse retinas (RT-PCR and in situ hybridization) was performed to select particularly interesting genes, and we later replicated the experiments in zebrafish. As a proof of concept, we selected ups45 to be knocked down by morpholino injection in zebrafish embryos. Morphant phenotypic analysis showed moderate to severe eye morphological defects, with a defective formation of the retinal structures, therefore supporting the relevance of DUBs in the formation and differentiation of the vertebrate retina, and suggesting that genes encoding ubiquitin pathway enzymes are good candidates for causing hereditary retinal dystrophies.

T2N as a new tool for robust electrophysiological modeling demonstrated for mature and adult-born dentate granule cells

PubMed Central

Mongiat, Lucas Alberto; Schwarzacher, Stephan Wolfgang

2017-01-01

Compartmental models are the theoretical tool of choice for understanding single neuron computations. However, many models are incomplete, built ad hoc and require tuning for each novel condition rendering them of limited usability. Here, we present T2N, a powerful interface to control NEURON with Matlab and TREES toolbox, which supports generating models stable over a broad range of reconstructed and synthetic morphologies. We illustrate this for a novel, highly detailed active model of dentate granule cells (GCs) replicating a wide palette of experiments from various labs. By implementing known differences in ion channel composition and morphology, our model reproduces data from mouse or rat, mature or adult-born GCs as well as pharmacological interventions and epileptic conditions. This work sets a new benchmark for detailed compartmental modeling. T2N is suitable for creating robust models useful for large-scale networks that could lead to novel predictions. We discuss possible T2N application in degeneracy studies. PMID:29165247

Ferret and pig models of cystic fibrosis: prospects and promise for gene therapy.

PubMed

Yan, Ziying; Stewart, Zoe A; Sinn, Patrick L; Olsen, John C; Hu, Jim; McCray, Paul B; Engelhardt, John F

2015-03-01

Large animal models of genetic diseases are rapidly becoming integral to biomedical research as technologies to manipulate the mammalian genome improve. The creation of cystic fibrosis (CF) ferrets and pigs is an example of such progress in animal modeling, with the disease phenotypes in the ferret and pig models more reflective of human CF disease than mouse models. The ferret and pig CF models also provide unique opportunities to develop and assess the effectiveness of gene and cell therapies to treat affected organs. In this review, we examine the organ disease phenotypes in these new CF models and the opportunities to test gene therapies at various stages of disease progression in affected organs. We then discuss the progress in developing recombinant replication-defective adenoviral, adeno-associated viral, and lentiviral vectors to target genes to the lung and pancreas in ferrets and pigs, the two most affected organs in CF. Through this review, we hope to convey the potential of these new animal models for developing CF gene and cell therapies.

Specific excitatory connectivity for feature integration in mouse primary visual cortex

PubMed Central

Molina-Luna, Patricia; Roth, Morgane M.

2017-01-01

Local excitatory connections in mouse primary visual cortex (V1) are stronger and more prevalent between neurons that share similar functional response features. However, the details of how functional rules for local connectivity shape neuronal responses in V1 remain unknown. We hypothesised that complex responses to visual stimuli may arise as a consequence of rules for selective excitatory connectivity within the local network in the superficial layers of mouse V1. In mouse V1 many neurons respond to overlapping grating stimuli (plaid stimuli) with highly selective and facilitatory responses, which are not simply predicted by responses to single gratings presented alone. This complexity is surprising, since excitatory neurons in V1 are considered to be mainly tuned to single preferred orientations. Here we examined the consequences for visual processing of two alternative connectivity schemes: in the first case, local connections are aligned with visual properties inherited from feedforward input (a ‘like-to-like’ scheme specifically connecting neurons that share similar preferred orientations); in the second case, local connections group neurons into excitatory subnetworks that combine and amplify multiple feedforward visual properties (a ‘feature binding’ scheme). By comparing predictions from large scale computational models with in vivo recordings of visual representations in mouse V1, we found that responses to plaid stimuli were best explained by assuming feature binding connectivity. Unlike under the like-to-like scheme, selective amplification within feature-binding excitatory subnetworks replicated experimentally observed facilitatory responses to plaid stimuli; explained selective plaid responses not predicted by grating selectivity; and was consistent with broad anatomical selectivity observed in mouse V1. Our results show that visual feature binding can occur through local recurrent mechanisms without requiring feedforward convergence, and that such a mechanism is consistent with visual responses and cortical anatomy in mouse V1. PMID:29240769

Streptozocin-induced diabetic mouse model of urinary tract infection.

PubMed

Rosen, David A; Hung, Chia-Suei; Kline, Kimberly A; Hultgren, Scott J

2008-09-01

Diabetics have a higher incidence of urinary tract infection (UTI), are infected with a broader range of uropathogens, and more commonly develop serious UTI sequelae than nondiabetics. To better study UTI in the diabetic host, we created and characterized a murine model of diabetic UTI using the pancreatic islet beta-cell toxin streptozocin in C3H/HeN, C3H/HeJ, and C57BL/6 mouse backgrounds. Intraperitoneal injections of streptozocin were used to initiate diabetes in healthy mouse backgrounds, as defined by consecutive blood glucose levels of >250 mg/dl. UTIs caused by uropathogenic Escherichia coli (UTI89), Klebsiella pneumoniae (TOP52 1721), and Enterococcus faecalis (0852) were studied, and diabetic mice were found to be considerably more susceptible to infection. All three uropathogens produced significantly higher bladder and kidney titers than buffer-treated controls. Uropathogens did not have as large an advantage in the Toll-like receptor 4-defective C3H/HeJ diabetic mouse, arguing that the dramatic increase in colonization seen in C3H/HeN diabetic mice may partially be due to diabetic-induced defects in innate immunity. Competition experiments demonstrated that E. coli had a significant advantage over K. pneumoniae in the bladders of healthy mice and less of an advantage in diabetic bladders. In the kidneys, K. pneumoniae outcompeted E. coli in healthy mice but in diabetic mice E. coli outcompeted K. pneumoniae and caused severe pyelonephritis. Diabetic kidneys contained renal tubules laden with communities of E. coli UTI89 bacteria within an extracellular-matrix material. Diabetic mice also had glucosuria, which may enhance bacterial replication in the urinary tract. These data support that this murine diabetic UTI model is consistent with known characteristics of human diabetic UTI and can provide a powerful tool for dissecting this infection in the multifactorial setting of diabetes.

Replication and discovery of musculoskeletal QTLs in LG/J and SM/J advanced intercross lines.

PubMed

Hernandez Cordero, Ana I; Carbonetto, Peter; Riboni Verri, Gioia; Gregory, Jennifer S; Vandenbergh, David J; P Gyekis, Joseph; Blizard, David A; Lionikas, Arimantas

2018-02-01

The genetics underlying variation in health-related musculoskeletal phenotypes can be investigated in a mouse model. Quantitative trait loci (QTLs) affecting musculoskeletal traits in the LG/J and SM/J strain lineage remain to be refined and corroborated. The aim of this study was to map muscle and bone traits in males (n = 506) of the 50th filial generation of advanced intercross lines (LG/SM AIL) derived from the two strains. Genetic contribution to variation in all musculoskeletal traits was confirmed; the SNP heritability of muscle mass ranged between 0.46 and 0.56; and the SNP heritability of tibia length was 0.40. We used two analytical software, GEMMA and QTLRel, to map the underlying QTLs. GEMMA required substantially less computation and recovered all the QTLs identified by QTLRel. Seven significant QTLs were identified for muscle weight (Chr 1, 7, 11, 12, 13, 15, and 16), and two for tibia length, (Chr 1 and 13). Each QTL explained 4-5% of phenotypic variation. One muscle and both bone loci replicated previous findings; the remaining six were novel. Positional candidates for the replicated QTLs were prioritized based on in silico analyses and gene expression in muscle tissue. In summary, we replicated existing QTLs and identified novel QTLs affecting muscle weight, and replicated bone length QTLs in LG/SM AIL males. Heritability estimates substantially exceed the cumulative effect of the QTLs, hence a richer genetic architecture contributing to muscle and bone variability could be uncovered with a larger sample size. © 2018 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of The Physiological Society and the American Physiological Society.

Identification of Novel Kaposi's Sarcoma-Associated Herpesvirus Orf50 Transcripts: Discovery of New RTA Isoforms with Variable Transactivation Potential.

PubMed

Wakeman, Brian S; Izumiya, Yoshihiro; Speck, Samuel H

2017-01-01

Kaposi's sarcoma-associated herpesvirus (KSHV) is a gammaherpesvirus that has been associated with primary effusion lymphoma and multicentric Castleman's disease, as well as its namesake Kaposi's sarcoma. As a gammaherpesvirus, KSHV is able to acutely replicate, enter latency, and reactivate from this latent state. A key protein involved in both acute replication and reactivation from latency is the replication and transcriptional activator (RTA) encoded by the gene Orf50 RTA is a known transactivator of multiple viral genes, allowing it to control the switch between latency and virus replication. We report here the identification of six alternatively spliced Orf50 transcripts that are generated from four distinct promoters. These newly identified promoters are shown to be transcriptionally active in 293T (embryonic kidney), Vero (African-green monkey kidney epithelial), 3T12 (mouse fibroblast), and RAW 264.7 (mouse macrophage) cell lines. Notably, the newly identified Orf50 transcripts are predicted to encode four different isoforms of the RTA which differ by 6 to 10 residues at the amino terminus of the protein. We show the global viral transactivation potential of all four RTA isoforms and demonstrate that all isoforms can transcriptionally activate an array of KSHV promoters to various levels. The pattern of transcriptional activation appears to support a transcriptional interference model within the Orf50 region, where silencing of previously expressed isoforms by transcription initiation from upstream Orf50 promoters has the potential to modulate the pattern of viral gene activation. Gammaherpesviruses are associated with the development of lymphomas and lymphoproliferative diseases, as well as several other types of cancer. The human gammaherpesvirus, Kaposi's sarcoma-associated herpesvirus (KSHV), is tightly associated with the development of Kaposi's sarcoma and multicentric Castleman's disease, as well as a rare form of B cell lymphoma (primary effusion lymphoma) primarily observed in HIV-infected individuals. RTA is an essential viral gene product involved in the initiation of gammaherpesvirus replication and is conserved among all known gammaherpesviruses. We show here for KSHV that transcription of the gene encoding RTA is complex and leads to the expression of several isoforms of RTA with distinct functions. This observed complexity in KSHV RTA expression and function likely plays a critical role in the regulation of downstream viral and cellular gene expression, leading to the efficient production of mature virions. Copyright © 2016 American Society for Microbiology.

Dissection of complex adult traits in a mouse synthetic population.

PubMed

Burke, David T; Kozloff, Kenneth M; Chen, Shu; West, Joshua L; Wilkowski, Jodi M; Goldstein, Steven A; Miller, Richard A; Galecki, Andrzej T

2012-08-01

Finding the causative genetic variations that underlie complex adult traits is a significant experimental challenge. The unbiased search strategy of genome-wide association (GWAS) has been used extensively in recent human population studies. These efforts, however, typically find only a minor fraction of the genetic loci that are predicted to affect variation. As an experimental model for the analysis of adult polygenic traits, we measured a mouse population for multiple phenotypes and conducted a genome-wide search for effector loci. Complex adult phenotypes, related to body size and bone structure, were measured as component phenotypes, and each subphenotype was associated with a genomic spectrum of candidate effector loci. The strategy successfully detected several loci for the phenotypes, at genome-wide significance, using a single, modest-sized population (N = 505). The effector loci each explain 2%-10% of the measured trait variation and, taken together, the loci can account for over 25% of a trait's total population variation. A replicate population (N = 378) was used to confirm initially observed loci for one trait (femur length), and, when the two groups were merged, the combined population demonstrated increased power to detect loci. In contrast to human population studies, our mouse genome-wide searches find loci that individually explain a larger fraction of the observed variation. Also, the additive effects of our detected mouse loci more closely match the predicted genetic component of variation. The genetic loci discovered are logical candidates for components of the genetic networks having evolutionary conservation with human biology.

Identification of Gene Markers for Activation of the Nuclear Receptor Pregnane X Receptor

EPA Science Inventory

Many environmentally-relevant chemicals and drugs activate the nuclear receptor pregnane X receptor (PXR). Activation of PXR in the mouse liver can lead to increases in liver weight in part through increased hepatocyte replication similar to chemicals that activate other nuclear ...

La Crosse virus infectivity, pathogenesis, and immunogenicity in mice and monkeys

PubMed Central

Bennett, Richard S; Cress, Christina M; Ward, Jerrold M; Firestone, Cai-Yen; Murphy, Brian R; Whitehead, Stephen S

2008-01-01

Background La Crosse virus (LACV), family Bunyaviridae, was first identified as a human pathogen in 1960 after its isolation from a 4 year-old girl with fatal encephalitis in La Crosse, Wisconsin. LACV is a major cause of pediatric encephalitis in North America and infects up to 300,000 persons each year of which 70–130 result in severe disease of the central nervous system (CNS). As an initial step in the establishment of useful animal models to support vaccine development, we examined LACV infectivity, pathogenesis, and immunogenicity in both weanling mice and rhesus monkeys. Results Following intraperitoneal inoculation of mice, LACV replicated in various organs before reaching the CNS where it replicates to high titer causing death from neurological disease. The peripheral site where LACV replicates to highest titer is the nasal turbinates, and, presumably, LACV can enter the CNS via the olfactory neurons from nasal olfactory epithelium. The mouse infectious dose50 and lethal dose50 was similar for LACV administered either intranasally or intraperitoneally. LACV was highly infectious for rhesus monkeys and infected 100% of the animals at 10 PFU. However, the infection was asymptomatic, and the monkeys developed a strong neutralizing antibody response. Conclusion In mice, LACV likely gains access to the CNS via the blood stream or via olfactory neurons. The ability to efficiently infect mice intranasally raises the possibility that LACV might use this route to infect its natural hosts. Rhesus monkeys are susceptible to LACV infection and develop strong neutralizing antibody responses after inoculation with as little as 10 PFU. Mice and rhesus monkeys are useful animal models for LACV vaccine immunologic testing although the rhesus monkey model is not optimal. PMID:18267012

Low-fidelity Venezuelan equine encephalitis virus polymerase mutants to improve live-attenuated vaccine safety and efficacy

PubMed Central

Kautz, Tiffany F; Guerbois, Mathilde; Khanipov, Kamil; Yun, Ruimei; Warmbrod, Kelsey L; Fofanov, Yuriy; Weaver, Scott C; Forrester, Naomi L

2018-01-01

Abstract During RNA virus replication, there is the potential to incorporate mutations that affect virulence or pathogenesis. For live-attenuated vaccines, this has implications for stability, as replication may result in mutations that either restore the wild-type phenotype via reversion or compensate for the attenuating mutations by increasing virulence (pseudoreversion). Recent studies have demonstrated that altering the mutation rate of an RNA virus is an effective attenuation tool. To validate the safety of low-fidelity mutations to increase vaccine attenuation, several mutations in the RNA-dependent RNA-polymerase (RdRp) were tested in the live-attenuated Venezuelan equine encephalitis virus vaccine strain, TC-83. Next generation sequencing after passage in the presence of mutagens revealed a mutant containing three mutations in the RdRp, TC-83 3x, to have decreased replication fidelity, while a second mutant, TC-83 4x displayed no change in fidelity, but shared many phenotypic characteristics with TC-83 3x. Both mutants exhibited increased, albeit inconsistent attenuation in an infant mouse model, as well as increased immunogenicity and complete protection against lethal challenge of an adult murine model compared with the parent TC-83. During serial passaging in a highly permissive model, the mutants increased in virulence but remained less virulent than the parent TC-83. These results suggest that the incorporation of low-fidelity mutations into the RdRp of live-attenuated vaccines for RNA viruses can confer increased immunogenicity whilst showing some evidence of increased attenuation. However, while in theory such constructs may result in more effective vaccines, the instability of the vaccine phenotype decreases the likelihood of this being an effective vaccine strategy. PMID:29593882

IFITM3 restricts the morbidity and mortality associated with influenza.

PubMed

Everitt, Aaron R; Clare, Simon; Pertel, Thomas; John, Sinu P; Wash, Rachael S; Smith, Sarah E; Chin, Christopher R; Feeley, Eric M; Sims, Jennifer S; Adams, David J; Wise, Helen M; Kane, Leanne; Goulding, David; Digard, Paul; Anttila, Verneri; Baillie, J Kenneth; Walsh, Tim S; Hume, David A; Palotie, Aarno; Xue, Yali; Colonna, Vincenza; Tyler-Smith, Chris; Dunning, Jake; Gordon, Stephen B; Smyth, Rosalind L; Openshaw, Peter J; Dougan, Gordon; Brass, Abraham L; Kellam, Paul

2012-03-25

The 2009 H1N1 influenza pandemic showed the speed with which a novel respiratory virus can spread and the ability of a generally mild infection to induce severe morbidity and mortality in a subset of the population. Recent in vitro studies show that the interferon-inducible transmembrane (IFITM) protein family members potently restrict the replication of multiple pathogenic viruses. Both the magnitude and breadth of the IFITM proteins' in vitro effects suggest that they are critical for intrinsic resistance to such viruses, including influenza viruses. Using a knockout mouse model, we now test this hypothesis directly and find that IFITM3 is essential for defending the host against influenza A virus in vivo. Mice lacking Ifitm3 display fulminant viral pneumonia when challenged with a normally low-pathogenicity influenza virus, mirroring the destruction inflicted by the highly pathogenic 1918 'Spanish' influenza. Similar increased viral replication is seen in vitro, with protection rescued by the re-introduction of Ifitm3. To test the role of IFITM3 in human influenza virus infection, we assessed the IFITM3 alleles of individuals hospitalized with seasonal or pandemic influenza H1N1/09 viruses. We find that a statistically significant number of hospitalized subjects show enrichment for a minor IFITM3 allele (SNP rs12252-C) that alters a splice acceptor site, and functional assays show the minor CC genotype IFITM3 has reduced influenza virus restriction in vitro. Together these data reveal that the action of a single intrinsic immune effector, IFITM3, profoundly alters the course of influenza virus infection in mouse and humans.

Iron overload favors the elimination of Leishmania infantum from mouse tissues through interaction with reactive oxygen and nitrogen species.

PubMed

Vale-Costa, Sílvia; Gomes-Pereira, Sandra; Teixeira, Carlos Miguel; Rosa, Gustavo; Rodrigues, Pedro Nuno; Tomás, Ana; Appelberg, Rui; Gomes, Maria Salomé

2013-01-01

Iron plays a central role in host-parasite interactions, since both intervenients need iron for survival and growth, but are sensitive to iron-mediated toxicity. The host's iron overload is often associated with susceptibility to infection. However, it has been previously reported that iron overload prevented the growth of Leishmania major, an agent of cutaneous leishmaniasis, in BALB/c mice. In order to further clarify the impact of iron modulation on the growth of Leishmania in vivo, we studied the effects of iron supplementation or deprivation on the growth of L. infantum, the causative agent of Mediterranean visceral leishmaniasis, in the mouse model. We found that dietary iron deficiency did not affect the protozoan growth, whereas iron overload decreased its replication in the liver and spleen of a susceptible mouse strain. The fact that the iron-induced inhibitory effect could not be seen in mice deficient in NADPH dependent oxidase or nitric oxide synthase 2 suggests that iron eliminates L. infantum in vivo through the interaction with reactive oxygen and nitrogen species. Iron overload did not significantly alter the mouse adaptive immune response against L. infantum. Furthermore, the inhibitory action of iron towards L. infantum was also observed, in a dose dependent manner, in axenic cultures of promastigotes and amastigotes. Importantly, high iron concentrations were needed to achieve such effects. In conclusion, externally added iron synergizes with the host's oxidative mechanisms of defense in eliminating L. infantum from mouse tissues. Additionally, the direct toxicity of iron against Leishmania suggests a potential use of this metal as a therapeutic tool or the further exploration of iron anti-parasitic mechanisms for the design of new drugs.

Oleate induces KATP channel-dependent hyperpolarization in mouse hypothalamic glucose-excited neurons without altering cellular energy charge.

PubMed

Dadak, Selma; Beall, Craig; Vlachaki Walker, Julia M; Soutar, Marc P M; McCrimmon, Rory J; Ashford, Michael L J

2017-03-27

The unsaturated fatty acid, oleate exhibits anorexigenic properties reducing food intake and hepatic glucose output. However, its mechanism of action in the hypothalamus has not been fully determined. This study investigated the effects of oleate and glucose on GT1-7 mouse hypothalamic cells (a model of glucose-excited (GE) neurons) and mouse arcuate nucleus (ARC) neurons. Whole-cell and perforated patch-clamp recordings, immunoblotting and cell energy status measures were used to investigate oleate- and glucose-sensing properties of mouse hypothalamic neurons. Oleate or lowered glucose concentration caused hyperpolarization and inhibition of firing of GT1-7 cells by the activation of ATP-sensitive K + channels (K ATP ). This effect of oleate was not dependent on fatty acid oxidation or raised AMP-activated protein kinase activity or prevented by the presence of the UCP2 inhibitor genipin. Oleate did not alter intracellular calcium, indicating that CD36/fatty acid translocase may not play a role. However, oleate activation of K ATP may require ATP metabolism. The short-chain fatty acid octanoate was unable to replicate the actions of oleate on GT1-7 cells. Although oleate decreased GT1-7 cell mitochondrial membrane potential there was no change in total cellular ATP or ATP/ADP ratios. Perforated patch and whole-cell recordings from mouse hypothalamic slices demonstrated that oleate hyperpolarized a subpopulation of ARC GE neurons by K ATP activation. Additionally, in a separate small population of ARC neurons, oleate application or lowered glucose concentration caused membrane depolarization. In conclusion, oleate induces K ATP- dependent hyperpolarization and inhibition of firing of a subgroup of GE hypothalamic neurons without altering cellular energy charge. Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.

Iron Overload Favors the Elimination of Leishmania infantum from Mouse Tissues through Interaction with Reactive Oxygen and Nitrogen Species

PubMed Central

Vale-Costa, Sílvia; Gomes-Pereira, Sandra; Teixeira, Carlos Miguel; Rosa, Gustavo; Rodrigues, Pedro Nuno; Tomás, Ana; Appelberg, Rui; Gomes, Maria Salomé

2013-01-01

Iron plays a central role in host-parasite interactions, since both intervenients need iron for survival and growth, but are sensitive to iron-mediated toxicity. The host's iron overload is often associated with susceptibility to infection. However, it has been previously reported that iron overload prevented the growth of Leishmania major, an agent of cutaneous leishmaniasis, in BALB/c mice. In order to further clarify the impact of iron modulation on the growth of Leishmania in vivo, we studied the effects of iron supplementation or deprivation on the growth of L. infantum, the causative agent of Mediterranean visceral leishmaniasis, in the mouse model. We found that dietary iron deficiency did not affect the protozoan growth, whereas iron overload decreased its replication in the liver and spleen of a susceptible mouse strain. The fact that the iron-induced inhibitory effect could not be seen in mice deficient in NADPH dependent oxidase or nitric oxide synthase 2 suggests that iron eliminates L. infantum in vivo through the interaction with reactive oxygen and nitrogen species. Iron overload did not significantly alter the mouse adaptive immune response against L. infantum. Furthermore, the inhibitory action of iron towards L. infantum was also observed, in a dose dependent manner, in axenic cultures of promastigotes and amastigotes. Importantly, high iron concentrations were needed to achieve such effects. In conclusion, externally added iron synergizes with the host's oxidative mechanisms of defense in eliminating L. infantum from mouse tissues. Additionally, the direct toxicity of iron against Leishmania suggests a potential use of this metal as a therapeutic tool or the further exploration of iron anti-parasitic mechanisms for the design of new drugs. PMID:23459556

2'-5'-Oligoadenylate Synthetase-Like Protein Inhibits Respiratory Syncytial Virus Replication and Is Targeted by the Viral Nonstructural Protein 1.

PubMed

Dhar, Jayeeta; Cuevas, Rolando A; Goswami, Ramansu; Zhu, Jianzhong; Sarkar, Saumendra N; Barik, Sailen

2015-10-01

2'-5'-Oligoadenylate synthetase-like protein (OASL) is an interferon-inducible antiviral protein. Here we describe differential inhibitory activities of human OASL and the two mouse OASL homologs against respiratory syncytial virus (RSV) replication. Interestingly, nonstructural protein 1 (NS1) of RSV promoted proteasome-dependent degradation of specific OASL isoforms. We conclude that OASL acts as a cellular antiviral protein and that RSV NS1 suppresses this function to evade cellular innate immunity and allow virus growth. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

The Stress Granule Component TIA-1 Binds Tick-Borne Encephalitis Virus RNA and Is Recruited to Perinuclear Sites of Viral Replication To Inhibit Viral Translation

PubMed Central

Albornoz, Amelina; Carletti, Tea; Corazza, Gianmarco

2014-01-01

ABSTRACT Flaviviruses are a major cause of disease in humans and animals worldwide. Tick-borne encephalitis virus (TBEV) is the most important arthropod-borne flavivirus endemic in Europe and is the etiological agent of tick-borne encephalitis, a potentially fatal infection of the central nervous system. However, the contributions of host proteins during TBEV infection are poorly understood. In this work, we investigate the cellular protein TIA-1 and its cognate factor TIAR, which are stress-induced RNA-binding proteins involved in the repression of initiation of translation of cellular mRNAs and in the formation of stress granules. We show that TIA-1 and TIAR interact with viral RNA in TBEV-infected cells. During TBEV infection, cytoplasmic TIA-1 and TIAR are recruited at sites of viral replication with concomitant depletion from stress granules. This effect is specific, since G3BP1, another component of these cytoplasmic structures, remains localized to stress granules. Moreover, heat shock induction of stress granules containing TIA-1, but not G3BP1, is inhibited in TBEV-infected cells. Infection of cells depleted of TIA-1 or TIAR by small interfering RNA (siRNA) or TIA-1−/− mouse fibroblasts, leads to a significant increase in TBEV extracellular infectivity. Interestingly, TIAR−/− fibroblasts show the opposite effect on TBEV infection, and this phenotype appears to be related to an excess of TIA-1 in these cells. Taking advantage of a TBE-luciferase replicon system, we also observed increased luciferase activity in TIA-1−/− mouse fibroblasts at early time points, consistent with TIA-1-mediated inhibition at the level of the first round of viral translation. These results indicate that, in response to TBEV infection, TIA-1 is recruited to sites of virus replication to bind TBEV RNA and modulate viral translation independently of stress granule (SG) formation. IMPORTANCE This study (i) extends previous work that showed TIA-1/TIAR recruitment at sites of flavivirus replication, (ii) demonstrates that TIAR behaves like TIA-1 as an inhibitor of viral replication using an RNA interference (RNAi) approach in human cells that contradicts the previous hypothesis based on mouse embryonic fibroblast (MEF) knockouts only, (iii) demonstrates that tick-borne encephalitis virus (TBEV) is capable of inducing bona fide G3BP1/eIF3/eIF4B-positive stress granules, (iv) demonstrates a differential phenotype of stress response proteins following viral infection, and (v) implicates TIA-1 in viral translation and as a modulator of TBEV replication. PMID:24696465

The stress granule component TIA-1 binds tick-borne encephalitis virus RNA and is recruited to perinuclear sites of viral replication to inhibit viral translation.

PubMed

Albornoz, Amelina; Carletti, Tea; Corazza, Gianmarco; Marcello, Alessandro

2014-06-01

Flaviviruses are a major cause of disease in humans and animals worldwide. Tick-borne encephalitis virus (TBEV) is the most important arthropod-borne flavivirus endemic in Europe and is the etiological agent of tick-borne encephalitis, a potentially fatal infection of the central nervous system. However, the contributions of host proteins during TBEV infection are poorly understood. In this work, we investigate the cellular protein TIA-1 and its cognate factor TIAR, which are stress-induced RNA-binding proteins involved in the repression of initiation of translation of cellular mRNAs and in the formation of stress granules. We show that TIA-1 and TIAR interact with viral RNA in TBEV-infected cells. During TBEV infection, cytoplasmic TIA-1 and TIAR are recruited at sites of viral replication with concomitant depletion from stress granules. This effect is specific, since G3BP1, another component of these cytoplasmic structures, remains localized to stress granules. Moreover, heat shock induction of stress granules containing TIA-1, but not G3BP1, is inhibited in TBEV-infected cells. Infection of cells depleted of TIA-1 or TIAR by small interfering RNA (siRNA) or TIA-1(-/-) mouse fibroblasts, leads to a significant increase in TBEV extracellular infectivity. Interestingly, TIAR(-/-) fibroblasts show the opposite effect on TBEV infection, and this phenotype appears to be related to an excess of TIA-1 in these cells. Taking advantage of a TBE-luciferase replicon system, we also observed increased luciferase activity in TIA-1(-/-) mouse fibroblasts at early time points, consistent with TIA-1-mediated inhibition at the level of the first round of viral translation. These results indicate that, in response to TBEV infection, TIA-1 is recruited to sites of virus replication to bind TBEV RNA and modulate viral translation independently of stress granule (SG) formation. This study (i) extends previous work that showed TIA-1/TIAR recruitment at sites of flavivirus replication, (ii) demonstrates that TIAR behaves like TIA-1 as an inhibitor of viral replication using an RNA interference (RNAi) approach in human cells that contradicts the previous hypothesis based on mouse embryonic fibroblast (MEF) knockouts only, (iii) demonstrates that tick-borne encephalitis virus (TBEV) is capable of inducing bona fide G3BP1/eIF3/eIF4B-positive stress granules, (iv) demonstrates a differential phenotype of stress response proteins following viral infection, and (v) implicates TIA-1 in viral translation and as a modulator of TBEV replication. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

New insights into replication origin characteristics in metazoans

PubMed Central

Puy, Aurore; Rialle, Stéphanie; Kaplan, Noam; Segal, Eran

2012-01-01

We recently reported the identification and characterization of DNA replication origins (Oris) in metazoan cell lines. Here, we describe additional bioinformatic analyses showing that the previously identified GC-rich sequence elements form origin G-rich repeated elements (OGREs) that are present in 67% to 90% of the DNA replication origins from Drosophila to human cells, respectively. Our analyses also show that initiation of DNA synthesis takes place precisely at 160 bp (Drosophila) and 280 bp (mouse) from the OGRE. We also found that in most CpG islands, an OGRE is positioned in opposite orientation on each of the two DNA strands and detected two sites of initiation of DNA synthesis upstream or downstream of each OGRE. Conversely, Oris not associated with CpG islands have a single initiation site. OGRE density along chromosomes correlated with previously published replication timing data. Ori sequences centered on the OGRE are also predicted to have high intrinsic nucleosome occupancy. Finally, OGREs predict G-quadruplex structures at Oris that might be structural elements controlling the choice or activation of replication origins. PMID:22373526

Pathogenesis, Transmissibility, and Tropism of a Highly Pathogenic Avian Influenza A(H7N7) Virus Associated With Human Conjunctivitis in Italy, 2013.

PubMed

Belser, Jessica A; Creager, Hannah M; Zeng, Hui; Maines, Taronna R; Tumpey, Terrence M

2017-09-15

H7 subtype influenza viruses represent a persistent public health threat because of their continued detection in poultry and ability to cause human infection. An outbreak of highly pathogenic avian influenza H7N7 virus in Italy during 2013 resulted in 3 cases of human conjunctivitis. We determined the pathogenicity and transmissibility of influenza A/Italy/3/2013 virus in mouse and ferret models and examined the replication kinetics of this virus in several human epithelial cell types. The moderate virulence observed in mammalian models and the capacity for transmission in a direct contact model underscore the need for continued study of H7 subtype viruses. Published by Oxford University Press for the Infectious Diseases Society of America 2017. This work is written by (a) US Government employee(s) and is in the public domain in the US.

RipA, a Cytoplasmic Membrane Protein Conserved among Francisella Species, Is Required for Intracellular Survival▿

PubMed Central

Fuller, James R.; Craven, Robin R.; Hall, Joshua D.; Kijek, Todd M.; Taft-Benz, Sharon; Kawula, Thomas H.

2008-01-01

Francisella tularensis is a highly virulent bacterial pathogen that invades and replicates within numerous host cell types, including macrophages and epithelial cells. In an effort to better understand this process, we screened a transposon insertion library of the F. tularensis live vaccine strain (LVS) for mutant strains that invaded but failed to replicate within alveolar epithelial cell lines. One such strain isolated from this screen contained an insertion in the gene FTL_1914, which is conserved among all sequenced Francisella species yet lacks significant homology to any gene with known function. A deletion strain lacking FTL_1914 was constructed. This strain did not replicate in either epithelial or macrophage-like cells, and intracellular replication was restored by the wild-type allele in trans. Based on the deletion mutant phenotype, FTL_1914 was termed ripA (required for intracellular proliferation, factor A). Following uptake by J774.A1 cells, F. tularensis LVS ΔripA colocalized with LAMP-1 then escaped the phagosome at the same rate and frequency as wild-type LVS-infected cells. Electron micrographs of the F. tularensis LVS ΔripA mutant demonstrated the reentry of the mutant bacteria into double membrane vacuoles characteristic of autophagosomes in a process that was not dependent on replication. The F. tularensis LVS ΔripA mutant was significantly impaired in its ability to persist in the lung and in its capacity to disseminate and colonize the liver and spleen in a mouse model of pulmonary tularemia. The RipA protein was expressed during growth in laboratory media and localized to the cytoplasmic membrane. Thus, RipA is a cytoplasmic membrane protein conserved among Francisella species that is required for intracellular replication within the host cell cytoplasm as well as disease progression, dissemination, and virulence. PMID:18765722

Rosa26-GFP Direct Repeat (RaDR-GFP) Mice Reveal Tissue- and Age-Dependence of Homologous Recombination in Mammals In Vivo

PubMed Central

Kay, Jennifer E.; Na, Li; Rowland, Elizabeth A.; Winther, Kelly E.; Chow, Danielle N.; Kimoto, Takafumi; Matsuguchi, Tetsuya; Jonnalagadda, Vidya S.; Maklakova, Vilena I.; Singh, Vijay R.; Wadduwage, Dushan N.; Rajapakse, Jagath; So, Peter T. C.; Collier, Lara S.; Engelward, Bevin P.

2014-01-01

Homologous recombination (HR) is critical for the repair of double strand breaks and broken replication forks. Although HR is mostly error free, inherent or environmental conditions that either suppress or induce HR cause genomic instability. Despite its importance in carcinogenesis, due to limitations in our ability to detect HR in vivo, little is known about HR in mammalian tissues. Here, we describe a mouse model in which a direct repeat HR substrate is targeted to the ubiquitously expressed Rosa26 locus. In the Rosa26 Direct Repeat-GFP (RaDR-GFP) mice, HR between two truncated EGFP expression cassettes can yield a fluorescent signal. In-house image analysis software provides a rapid method for quantifying recombination events within intact tissues, and the frequency of recombinant cells can be evaluated by flow cytometry. A comparison among 11 tissues shows that the frequency of recombinant cells varies by more than two orders of magnitude among tissues, wherein HR in the brain is the lowest. Additionally, de novo recombination events accumulate with age in the colon, showing that this mouse model can be used to study the impact of chronic exposures on genomic stability. Exposure to N-methyl-N-nitrosourea, an alkylating agent similar to the cancer chemotherapeutic temozolomide, shows that the colon, liver and pancreas are susceptible to DNA damage-induced HR. Finally, histological analysis of the underlying cell types reveals that pancreatic acinar cells and liver hepatocytes undergo HR and also that HR can be specifically detected in colonic somatic stem cells. Taken together, the RaDR-GFP mouse model provides new understanding of how tissue and age impact susceptibility to HR, and enables future studies of genetic, environmental and physiological factors that modulate HR in mammals. PMID:24901438

SARS-like cluster of circulating bat coronavirus pose threat for human emergence

PubMed Central

Menachery, Vineet D.; Yount, Boyd L.; Debbink, Kari; Agnihothram, Sudhakar; Gralinski, Lisa E.; Plante, Jessica A.; Graham, Rachel L.; Scobey, Trevor; Ge, Xing-Yi; Donaldson, Eric F.; Randell, Scott H.; Lanzavecchia, Antonio; Marasco, Wayne A.; Shi, Zhengli-Li; Baric, Ralph S.

2016-01-01

The emergence of Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV) and Middle East Respiratory Syndrome (MERS)-CoV underscores the threat of cross-species transmission events leading to outbreaks in humans. In this study, we examine the disease potential for SARS-like CoVs currently circulating in Chinese horseshoe bat populations. Utilizing the SARS-CoV infectious clone, we generated and characterized a chimeric virus expressing the spike of bat coronavirus SHC014 in a mouse adapted SARS-CoV backbone. The results indicate that group 2b viruses encoding the SHC014 spike in a wild type backbone can efficiently utilize multiple ACE2 receptor orthologs, replicate efficiently in primary human airway cells, and achieve in vitro titers equivalent to epidemic strains of SARS-CoV. Additionally, in vivo experiments demonstrate replication of the chimeric virus in mouse lung with notable pathogenesis. Evaluation of available SARS-based immune-therapeutic and prophylactic modalities revealed poor efficacy; both monoclonal antibody and vaccine approaches failed to neutralize and protect from CoVs utilizing the novel spike protein. Importantly, based on these findings, we synthetically rederived an infectious full length SHC014 recombinant virus and demonstrate robust viral replication both in vitro and in vivo. Together, the work highlights a continued risk of SARS-CoV reemergence from viruses currently circulating in bat populations. PMID:26552008

Maternal DCAF2 is crucial for maintenance of genome stability during the first cell cycle in mice.

PubMed

Xu, Yi-Wen; Cao, Lan-Rui; Wang, Min; Xu, Ying; Wu, Xin; Liu, Junping; Tong, Chao; Fan, Heng-Yu

2017-10-01

Precise regulation of DNA replication and genome integrity is crucial for gametogenesis and early embryogenesis. Cullin ring-finger ubiquitin ligase 4 (CRL4) has multiple functions in the maintenance of germ cell survival, oocyte meiotic maturation, and maternal-zygotic transition in mammals. DDB1-cullin-4-associated factor-2 (DCAF2, also known as DTL or CDT2) is an evolutionarily conserved substrate receptor of CRL4. To determine whether DCAF2 is a key CRL4 substrate adaptor in mammalian oocytes, we generated a novel mouse strain that carries a Dcaf2 allele flanked by l oxP sequences, and specifically deleted Dcaf2 in oocytes. Dcaf2 knockout in mouse oocytes leads to female infertility. Although Dcaf2 -null oocytes were able to develop and mature normally, the embryos derived from them were arrested at one- to two-cell stage, owing to prolonged DNA replication and accumulation of massive DNA damage. These results indicate that DCAF2 is a previously unrecognized maternal factor that safeguards zygotic genome stability. Maternal DCAF2 protein is crucial for prevention of DNA re-replication in the first and unique mitotic cell cycle of the zygote.This article has an associated First Person interview with the first author of the paper. © 2017. Published by The Company of Biologists Ltd.

["In vitro" interactions between influenza virus and mouse lung alveolar macrophages (author's transl)].

PubMed

Lemercier, G; Mavet, S; Burckhart, M F; Fontanges, R

1979-01-01

Interactions between influenza virus A/PR/8/34 (H0N1) and Balb/c mouse lung alveolar macrophages have been studied in vitro. One day after initiation of alveolar macrophage culture in 35 mm Falcon dishes, the virus suspension was allowed to adsorb to the cells for 1 h. Detachment of cells from the plastic substrate, morphological changes in adherent cells and decreased phagocytosis of heat-killed Candida albicans occured slowly as compared to control cultures. These facts appeared to be directly correlated to the concentration of viruses in the inoculum. Data yielded by virus titrations, electron microscopy and immunofluorescence suggest that mouse lung alveolar macrophages are able to take up a large amount of viral particles and inhibit their replication, allowing only an abortive viral cycle.

Comparison of the effects of sodium phenobarbital in wild type and humanized constitutive androstane receptor (CAR)/pregnane X receptor (PXR) mice and in cultured mouse, rat and human hepatocytes.

PubMed

Haines, Corinne; Elcombe, Barbara M; Chatham, Lynsey R; Vardy, Audrey; Higgins, Larry G; Elcombe, Clifford R; Lake, Brian G

2018-03-01

Phenobarbital (PB), a constitutive androstane receptor (CAR) activator, produces liver tumours in rodents by a mitogenic mode of action involving CAR activation. In this study, the hepatic effects of sodium phenobarbital (NaPB) were compared in male C57BL/6J wild type (WT) mice and in humanized mice, where both the mouse CAR and pregnane X receptor (PXR) have been replaced by their human counterparts (hCAR/hPXR mice). Investigations were also performed in cultured male C57BL/6J and CD-1 mouse, male Sprague-Dawley rat and male and female human hepatocytes. The treatment of WT and hCAR/hPXR mice with 186-984 ppm NaPB in the diet for 7 days resulted in increased relative liver weight, hypertrophy and induction of cytochrome P450 (CYP) enzyme activities. Treatment with NaPB also produced dose-dependent increases in hepatocyte replicative DNA synthesis (RDS), with the effect being more marked in WT than in hCAR/hPXR mice. While the treatment of cultured C57BL/6J and CD-1 mouse, Sprague-Dawley rat and human hepatocytes with 100 and/or 1000 μM NaPB for 4 days induced CYP enzyme activities, increased RDS was only observed in mouse and rat hepatocytes. However, as a positive control, epidermal growth factor increased RDS in hepatocytes from all three species. In summary, although human hepatocytes are refractory to the mitogenic effects of NaPB, treatment with NaPB induced RDS in vivo in hCAR/hPXR mice, which is presumably due to the human CAR and PXR receptors operating in a mouse hepatocyte regulatory environment. As the response of the hCAR/hPXR mouse to the CAR activator NaPB differs markedly from that of human hepatocytes, the hCAR/hPXR mouse is thus not a suitable animal model for studies on the hepatic effects of nongenotoxic rodent CAR activators. Copyright © 2018 Elsevier B.V. All rights reserved.

Targeting human respiratory syncytial virus transcription anti-termination factor M2-1 to inhibit in vivo viral replication

PubMed Central

Bailly, B.; Richard, C.-A.; Sharma, G.; Wang, L.; Johansen, L.; Cao, J.; Pendharkar, V.; Sharma, D.-C.; Galloux, M.; Wang, Y.; Cui, R.; Zou, G.; Guillon, P.; von Itzstein, M.; Eléouët, J.-F.; Altmeyer, R.

2016-01-01

Human respiratory syncytial virus (hRSV) is a leading cause of acute lower respiratory tract infection in infants, elderly and immunocompromised individuals. To date, no specific antiviral drug is available to treat or prevent this disease. Here, we report that the Smoothened receptor (Smo) antagonist cyclopamine acts as a potent and selective inhibitor of in vitro and in vivo hRSV replication. Cyclopamine inhibits hRSV through a novel, Smo-independent mechanism. It specifically impairs the function of the hRSV RNA-dependent RNA polymerase complex notably by reducing expression levels of the viral anti-termination factor M2-1. The relevance of these findings is corroborated by the demonstration that a single R151K mutation in M2-1 is sufficient to confer virus resistance to cyclopamine in vitro and that cyclopamine is able to reduce virus titers in a mouse model of hRSV infection. The results of our study open a novel avenue for the development of future therapies against hRSV infection. PMID:27194388

Targeting the centriolar replication factor STIL synergizes with DNA damaging agents for treatment of ovarian cancer

PubMed Central

Rabinowicz, Noa; Mangala, Lingegowda S.; Brown, Kevin R.; Checa-Rodriguez, Cintia; Castiel, Asher; Moskovich, Oren; Zarfati, Giulia; Trakhtenbrot, Luba; Levy-Barda, Adva; Jiang, Dahai; Rodriguez-Aguayo, Cristian; Pradeep, Sunila; van Praag, Yael; Lopez-Berestein, Gabriel; David, Ahuvit; Novikov, Ilya; Huertas, Pablo; Rottapel, Robert; Sood, Anil K.; Izraeli, Shai

2017-01-01

Advanced ovarian cancer is an incurable disease. Thus, novel therapies are required. We wished to identify new therapeutic targets for ovarian cancer. ShRNA screen performed in 42 ovarian cancer cell lines identified the centriolar replication factor STIL as an essential gene for ovarian cancer cells. This was verified in-vivo in orthotopic human ovarian cancer mouse models. STIL depletion by administration of siRNA in neutral liposomes resulted in robust anti-tumor effect that was further enhanced in combination with cisplatin. Consistent with this finding, STIL depletion enhanced the extent of DNA double strand breaks caused by DNA damaging agents. This was associated with centrosomal depletion, ongoing genomic instability and enhanced formation of micronuclei. Interestingly, the ongoing DNA damage was not associated with reduced DNA repair. Indeed, we observed that depletion of STIL enhanced canonical homologous recombination repair and increased BRCA1 and RAD51 foci in response to DNA double strand breaks. Thus, inhibition of STIL significantly enhances the efficacy of DNA damaging chemotherapeutic drugs in treatment of ovarian cancer. PMID:28423708

Mechanisms of formation and accumulation of mitochondrial DNA deletions in aging neurons.

PubMed

Fukui, Hirokazu; Moraes, Carlos T

2009-03-15

Age-dependent accumulation of partially deleted mitochondrial DNA (DeltamtDNA) has been suggested to contribute to aging and the development of age-associated diseases including Parkinson's disease. However, the molecular mechanisms underlying the generation and accumulation of DeltamtDNA have not been addressed in vivo. In this study, we have developed a mouse model expressing an inducible mitochondria-targeted restriction endonuclease (PstI). Using this system, we could trigger mtDNA double-strand breaks (DSBs) in adult neurons. We found that this transient event leads to the generation of a family of DeltamtDNA with features that closely resemble naturally-occurring mtDNA deletions. The formation of these deleted species is likely to be mediated by yet uncharacterized DNA repairing machineries that participate in homologous recombination and non-homologous end-joining. Furthermore, we obtained in vivo evidence that DeltamtDNAs with larger deletions accumulate faster than those with smaller deletions, implying a replicative advantage of smaller mtDNAs. These findings identify DSB, DNA repair systems and replicative advantage as likely mechanisms underlying the generation and age-associated accumulation of DeltamtDNA in mammalian neurons.

Targeting the centriolar replication factor STIL synergizes with DNA damaging agents for treatment of ovarian cancer.

PubMed

Rabinowicz, Noa; Mangala, Lingegowda S; Brown, Kevin R; Checa-Rodriguez, Cintia; Castiel, Asher; Moskovich, Oren; Zarfati, Giulia; Trakhtenbrot, Luba; Levy-Barda, Adva; Jiang, Dahai; Rodriguez-Aguayo, Cristian; Pradeep, Sunila; van Praag, Yael; Lopez-Berestein, Gabriel; David, Ahuvit; Novikov, Ilya; Huertas, Pablo; Rottapel, Robert; Sood, Anil K; Izraeli, Shai

2017-04-18

Advanced ovarian cancer is an incurable disease. Thus, novel therapies are required. We wished to identify new therapeutic targets for ovarian cancer. ShRNA screen performed in 42 ovarian cancer cell lines identified the centriolar replication factor STIL as an essential gene for ovarian cancer cells. This was verified in-vivo in orthotopic human ovarian cancer mouse models. STIL depletion by administration of siRNA in neutral liposomes resulted in robust anti-tumor effect that was further enhanced in combination with cisplatin. Consistent with this finding, STIL depletion enhanced the extent of DNA double strand breaks caused by DNA damaging agents. This was associated with centrosomal depletion, ongoing genomic instability and enhanced formation of micronuclei. Interestingly, the ongoing DNA damage was not associated with reduced DNA repair. Indeed, we observed that depletion of STIL enhanced canonical homologous recombination repair and increased BRCA1 and RAD51 foci in response to DNA double strand breaks. Thus, inhibition of STIL significantly enhances the efficacy of DNA damaging chemotherapeutic drugs in treatment of ovarian cancer.

STUDIES ON PNEUMONIA VIRUS OF MICE (PVM) IN CELL CULTURE

PubMed Central

Harter, Donald H.; Choppin, Purnell W.

1967-01-01

Pneumonia virus of mice (PVM) has been serially propagated in a line of baby hamster kidney (BHK21) cells. A maximum titer of 6.3 x 106 TCID50 per ml was obtained, and there was little variation in yield on serial passage. PVM grown in BHK21 cells was antigenically similar to virus obtained from the mouse lung, but was somewhat less virulent for the mouse after 10 serial passages in these cells. Virus produced by BHK21 cells agglutinated mouse erythrocytes without prior heating or other treatment. Sedimentation of PVM in the ultracentrifuge or precipitation by ammonium sulfate resulted in a loss in infectivity but an increase in hemagglutinating activity, presumably due to disruption of the virus particle. In a potassium tartrate density gradient, the major portion of infective virus sedimented at a density of approximately 1.15, and noninfective hemagglutinin, at a density of approximately 1.13. Stock virus preparations appear to contain a large amount of noninfective hemagglutinin. The replication of PVM was not inhibited by 5-fluoro-2'-deoxyuridine, 5-bromo-2'-deoxyuridine, or 5-iodo-2'-deoxyuridine. Infected cells contained eosinophilic cytoplasmic inclusions which showed the acridine orange staining characteristic of single-stranded RNA. Foci of viral antigen were observed in the cytoplasm of infected cells by fluorescent antibody staining. The results suggest that PVM is an RNA virus that replicates in the cytoplasm. PMID:4165740

Animal and Human Tissue Models of Vertical Listeria monocytogenes Transmission and Implications for Other Pregnancy-Associated Infections.

PubMed

Lowe, David E; Robbins, Jennifer R; Bakardjiev, Anna I

2018-06-01

Intrauterine infections lead to serious complications for mother and fetus, including preterm birth, maternal and fetal death, and neurological sequelae in the surviving offspring. Improving maternal and child heath is a global priority. Yet, the development of strategies to prevent and treat pregnancy-related diseases has lagged behind progress made in other medical fields. One of the challenges is finding tractable model systems that replicate the human maternal-fetal interface. Animal models offer the ability to study pathogenesis and host defenses in vivo However, the anatomy of the maternal-fetal interface is highly divergent across species. While many tools are available to study host responses in the pregnant mouse model, other animals have placentas that are more similar to that of humans. Here we describe new developments in animal and human tissue models to investigate the pathogenesis of listeriosis at the maternal-fetal interface. We highlight gaps in existing knowledge and make recommendations on how they can be filled. Copyright © 2018 American Society for Microbiology.

The efficacy of combination therapy with oncolytic herpes simplex virus HF10 and dacarbazine in a mouse melanoma model.

PubMed

Tanaka, Rui; Goshima, Fumi; Esaki, Shinichi; Sato, Yoshitaka; Murata, Takayuki; Nishiyama, Yukihiro; Watanabe, Daisuke; Kimura, Hiroshi

2017-01-01

Advanced melanoma has long been treated with chemotherapy using cytotoxic agents like dacarbazine (DTIC), but overall survival rates with these drugs have been generally low. Recently, immunoregulatory monoclonal antibodies and molecularly targeted therapy with a BRAF inhibitor and/or a MEK inhibitor, have been used to treat malignant melanoma and have improved the survival rate of patients with advanced melanoma. However, high prices of these drugs are problematic. In this study, we evaluated the oncolytic efficacy of HF10, an attenuated, replication-competent HSV, with DTIC in immunocompetent mice model of malignant melanoma. For in vitro studies, cytotoxicity assays were conducted in clone M3 mouse melanoma cells. For the in vivo studies, subcutaneous melanoma models were prepared in DBA/2 mice with clone M3 cells, and then HF10 was intratumorally inoculated with/without intraperitoneal DTIC injection. The efficacy of the therapies was evaluated by survival, growth of subcutaneous tumor, and histopathological and immunological analyses. Both HF10 infection and DTIC treatment showed cytotoxic effects in melanoma cells, but combination treatment with HF10 and DTIC showed a rapid and strong cytotoxic effect compared with monotherapy. In the subcutaneous melanoma model, intratumoral HF10 inoculation significantly inhibited tumor growth. HF10 also inhibited the growth of non-inoculated contralateral tumors when it was injected into the ipsilateral tumors of mice. In histologic and immunohistochemical analysis, tumor lysis and inflammatory cell infiltration were observed after intratumoral HF10 inoculation. When mice were treated with HF10 and DTIC, the combination therapy induced a robust systemic anti-tumor immune response and prolonged survival. IFN-γ secretion from splenocytes of the HF10-DTIC combination therapy group showed more IFN-γ secretion than did the other groups. These data showed the efficacy of HF10 and DTIC combination therapy in a mouse melanoma model.

The efficacy of combination therapy with oncolytic herpes simplex virus HF10 and dacarbazine in a mouse melanoma model

PubMed Central

Tanaka, Rui; Goshima, Fumi; Esaki, Shinichi; Sato, Yoshitaka; Murata, Takayuki; Nishiyama, Yukihiro; Watanabe, Daisuke; Kimura, Hiroshi

2017-01-01

Advanced melanoma has long been treated with chemotherapy using cytotoxic agents like dacarbazine (DTIC), but overall survival rates with these drugs have been generally low. Recently, immunoregulatory monoclonal antibodies and molecularly targeted therapy with a BRAF inhibitor and/or a MEK inhibitor, have been used to treat malignant melanoma and have improved the survival rate of patients with advanced melanoma. However, high prices of these drugs are problematic. In this study, we evaluated the oncolytic efficacy of HF10, an attenuated, replication-competent HSV, with DTIC in immunocompetent mice model of malignant melanoma. For in vitro studies, cytotoxicity assays were conducted in clone M3 mouse melanoma cells. For the in vivo studies, subcutaneous melanoma models were prepared in DBA/2 mice with clone M3 cells, and then HF10 was intratumorally inoculated with/without intraperitoneal DTIC injection. The efficacy of the therapies was evaluated by survival, growth of subcutaneous tumor, and histopathological and immunological analyses. Both HF10 infection and DTIC treatment showed cytotoxic effects in melanoma cells, but combination treatment with HF10 and DTIC showed a rapid and strong cytotoxic effect compared with monotherapy. In the subcutaneous melanoma model, intratumoral HF10 inoculation significantly inhibited tumor growth. HF10 also inhibited the growth of non-inoculated contralateral tumors when it was injected into the ipsilateral tumors of mice. In histologic and immunohistochemical analysis, tumor lysis and inflammatory cell infiltration were observed after intratumoral HF10 inoculation. When mice were treated with HF10 and DTIC, the combination therapy induced a robust systemic anti-tumor immune response and prolonged survival. IFN-γ secretion from splenocytes of the HF10-DTIC combination therapy group showed more IFN-γ secretion than did the other groups. These data showed the efficacy of HF10 and DTIC combination therapy in a mouse melanoma model. PMID:28861325

Intracranial AAV-IFN-β gene therapy eliminates invasive xenograft glioblastoma and improves survival in orthotopic syngeneic murine model.

PubMed

GuhaSarkar, Dwijit; Neiswender, James; Su, Qin; Gao, Guangping; Sena-Esteves, Miguel

2017-02-01

The highly invasive property of glioblastoma (GBM) cells and genetic heterogeneity are largely responsible for tumor recurrence after the current standard-of-care treatment and thus a direct cause of death. Previously, we have shown that intracranial interferon-beta (IFN-β) gene therapy by locally administered adeno-associated viral vectors (AAV) successfully treats noninvasive orthotopic glioblastoma models. Here, we extend these findings by testing this approach in invasive human GBM xenograft and syngeneic mouse models. First, we show that a single intracranial injection of AAV encoding human IFN-β eliminates invasive human GBM8 tumors and promotes long-term survival. Next, we screened five AAV-IFN-β vectors with different promoters to drive safe expression of mouse IFN-β in the brain in the context of syngeneic GL261 tumors. Two AAV-IFN-β vectors were excluded due to safety concerns, but therapeutic studies with the other three vectors showed extensive tumor cell death, activation of microglia surrounding the tumors, and a 56% increase in median survival of the animals treated with AAV/P2-Int-mIFN-β vector. We also assessed the therapeutic effect of combining AAV-IFN-β therapy with temozolomide (TMZ). As TMZ affects DNA replication, an event that is crucial for second-strand DNA synthesis of single-stranded AAV vectors before active transcription, we tested two TMZ treatment regimens. Treatment with TMZ prior to AAV-IFN-β abrogated any benefit from the latter, while the reverse order of treatment doubled the median survival compared to controls. These studies demonstrate the therapeutic potential of intracranial AAV-IFN-β therapy in a highly migratory GBM model as well as in a syngeneic mouse model and that combination with TMZ is likely to enhance its antitumor potency. © 2016 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.

Receptor specificity of the influenza virus hemagglutinin modulates sensitivity to soluble collectins of the innate immune system and virulence in mice.

PubMed

Tate, Michelle D; Brooks, Andrew G; Reading, Patrick C

2011-04-25

The hemagglutinin (HA) glycoprotein of influenza virus binds to cell surface sialic acid (SA) to initiate infection. In this study, a mutant of influenza A virus strain BJx109 (H3N2) was plaque-purified from the lungs of virus-infected mice that had been depleted of airway macrophages. Sequence analysis identified a single amino acid substitution (S186I) in the vicinity of the receptor-binding site of HA. This substitution was associated with enhanced binding to α(2,3)-Gal-linked SA and an increased ability to infect murine airway epithelial cells. Mutant viruses were less sensitive to neutralization by mouse airway fluids and less efficient in their ability to infect murine macrophages. Moreover, infection of mice with viruses bearing the S186I substitution led to severe disease, characterized by enhanced virus replication, lung pathology and pulmonary edema. Together, these studies confirm that residue 186 of H3 subtype viruses is a critical determinant of virulence in a mouse model of influenza infection. Copyright © 2010 Elsevier Inc. All rights reserved.

Eco-HAB as a fully automated and ecologically relevant assessment of social impairments in mouse models of autism

PubMed Central

Puścian, Alicja; Łęski, Szymon; Kasprowicz, Grzegorz; Winiarski, Maciej; Borowska, Joanna; Nikolaev, Tomasz; Boguszewski, Paweł M; Lipp, Hans-Peter; Knapska, Ewelina

2016-01-01

Eco-HAB is an open source, RFID-based system for automated measurement and analysis of social preference and in-cohort sociability in mice. The system closely follows murine ethology. It requires no contact between a human experimenter and tested animals, overcoming the confounding factors that lead to irreproducible assessment of murine social behavior between laboratories. In Eco-HAB, group-housed animals live in a spacious, four-compartment apparatus with shadowed areas and narrow tunnels, resembling natural burrows. Eco-HAB allows for assessment of the tendency of mice to voluntarily spend time together in ethologically relevant mouse group sizes. Custom-made software for automated tracking, data extraction, and analysis enables quick evaluation of social impairments. The developed protocols and standardized behavioral measures demonstrate high replicability. Unlike classic three-chambered sociability tests, Eco-HAB provides measurements of spontaneous, ecologically relevant social behaviors in group-housed animals. Results are obtained faster, with less manpower, and without confounding factors. DOI: http://dx.doi.org/10.7554/eLife.19532.001 PMID:27731798

Syrian Hamster as an Animal Model for the Study of Human Influenza Virus Infection.

PubMed

Iwatsuki-Horimoto, Kiyoko; Nakajima, Noriko; Ichiko, Yurie; Sakai-Tagawa, Yuko; Noda, Takeshi; Hasegawa, Hideki; Kawaoka, Yoshihiro

2018-02-15

Ferrets and mice are frequently used as animal models for influenza research. However, ferrets are demanding in terms of housing space and handling, whereas mice are not naturally susceptible to infection with human influenza A or B viruses. Therefore, prior adaptation of human viruses is required for their use in mice. In addition, there are no mouse-adapted variants of the recent H3N2 viruses, because these viruses do not replicate well in mice. In this study, we investigated the susceptibility of Syrian hamsters to influenza viruses with a view to using the hamster model as an alternative to the mouse model. We found that hamsters are sensitive to influenza viruses, including the recent H3N2 viruses, without adaptation. Although the hamsters did not show weight loss or clinical signs of H3N2 virus infection, we observed pathogenic effects in the respiratory tracts of the infected animals. All of the H3N2 viruses tested replicated in the respiratory organs of the hamsters, and some of them were detected in the nasal washes of infected animals. Moreover, a 2009 pandemic (pdm09) virus and a seasonal H1N1 virus, as well as one of the two H3N2 viruses, but not a type B virus, were transmissible by the airborne route in these hamsters. Hamsters thus have the potential to be a small-animal model for the study of influenza virus infection, including studies of the pathogenicity of H3N2 viruses and other strains, as well as for use in H1N1 virus transmission studies. IMPORTANCE We found that Syrian hamsters are susceptible to human influenza viruses, including the recent H3N2 viruses, without adaptation. We also found that a pdm09 virus and a seasonal H1N1 virus, as well as one of the H3N2 viruses, but not a type B virus tested, are transmitted by the airborne route in these hamsters. Syrian hamsters thus have the potential to be used as a small-animal model for the study of human influenza viruses. Copyright © 2018 American Society for Microbiology.

Phenobarbital induces cell cycle transcriptional responses in mouse liver humanized for constitutive androstane and pregnane x receptors.

PubMed

Luisier, Raphaëlle; Lempiäinen, Harri; Scherbichler, Nina; Braeuning, Albert; Geissler, Miriam; Dubost, Valerie; Müller, Arne; Scheer, Nico; Chibout, Salah-Dine; Hara, Hisanori; Picard, Frank; Theil, Diethilde; Couttet, Philippe; Vitobello, Antonio; Grenet, Olivier; Grasl-Kraupp, Bettina; Ellinger-Ziegelbauer, Heidrun; Thomson, John P; Meehan, Richard R; Elcombe, Clifford R; Henderson, Colin J; Wolf, C Roland; Schwarz, Michael; Moulin, Pierre; Terranova, Rémi; Moggs, Jonathan G

2014-06-01

The constitutive androstane receptor (CAR) and the pregnane X receptor (PXR) are closely related nuclear receptors involved in drug metabolism and play important roles in the mechanism of phenobarbital (PB)-induced rodent nongenotoxic hepatocarcinogenesis. Here, we have used a humanized CAR/PXR mouse model to examine potential species differences in receptor-dependent mechanisms underlying liver tissue molecular responses to PB. Early and late transcriptomic responses to sustained PB exposure were investigated in liver tissue from double knock-out CAR and PXR (CAR(KO)-PXR(KO)), double humanized CAR and PXR (CAR(h)-PXR(h)), and wild-type C57BL/6 mice. Wild-type and CAR(h)-PXR(h) mouse livers exhibited temporally and quantitatively similar transcriptional responses during 91 days of PB exposure including the sustained induction of the xenobiotic response gene Cyp2b10, the Wnt signaling inhibitor Wisp1, and noncoding RNA biomarkers from the Dlk1-Dio3 locus. Transient induction of DNA replication (Hells, Mcm6, and Esco2) and mitotic genes (Ccnb2, Cdc20, and Cdk1) and the proliferation-related nuclear antigen Mki67 were observed with peak expression occurring between 1 and 7 days PB exposure. All these transcriptional responses were absent in CAR(KO)-PXR(KO) mouse livers and largely reversible in wild-type and CAR(h)-PXR(h) mouse livers following 91 days of PB exposure and a subsequent 4-week recovery period. Furthermore, PB-mediated upregulation of the noncoding RNA Meg3, which has recently been associated with cellular pluripotency, exhibited a similar dose response and perivenous hepatocyte-specific localization in both wild-type and CAR(h)-PXR(h) mice. Thus, mouse livers coexpressing human CAR and PXR support both the xenobiotic metabolizing and the proliferative transcriptional responses following exposure to PB.

Pathogenesis and Transmission Assessments of Two H7N8 Influenza A Viruses Recently Isolated from Turkey Farms in Indiana Using Mouse and Ferret Models.

PubMed

Sun, Xiangjie; Belser, Jessica A; Pulit-Penaloza, Joanna A; Zeng, Hui; Lewis, Amanda; Shieh, Wun-Ju; Tumpey, Terrence M; Maines, Taronna R

2016-12-01

Avian influenza A H7 viruses have caused multiple outbreaks in domestic poultry throughout North America, resulting in occasional infections of humans in close contact with affected birds. In early 2016, the presence of H7N8 highly pathogenic avian influenza (HPAI) viruses and closely related H7N8 low-pathogenic avian influenza (LPAI) viruses was confirmed in commercial turkey farms in Indiana. These H7N8 viruses represent the first isolation of this subtype in domestic poultry in North America, and their virulence in mammalian hosts and the potential risk for human infection are largely unknown. In this study, we assessed the ability of H7N8 HPAI and LPAI viruses to replicate in vitro in human airway cells and in vivo in mouse and ferret models. Both H7N8 viruses replicated efficiently in vitro and in vivo, but they exhibited substantial differences in disease severity in mammals. In mice, while the H7N8 LPAI virus largely remained avirulent, the H7N8 HPAI virus exhibited greater infectivity, virulence, and lethality. Both H7N8 viruses replicated similarly in ferrets, but only the H7N8 HPAI virus caused moderate weight loss, lethargy, and mortality. The H7N8 LPAI virus displayed limited transmissibility in ferrets placed in direct contact with an inoculated animal, while no transmission of H7N8 HPAI virus was detected. Our results indicate that the H7N8 avian influenza viruses from Indiana are able to replicate in mammals and cause severe disease but with limited transmission. The recent appearance of H7N8 viruses in domestic poultry highlights the need for continued influenza surveillance in wild birds and close monitoring of the potential risk to human health. H7 influenza viruses circulate in wild birds in the United States, but when the virus emerges in domestic poultry populations, the frequency of human exposure and the potential for human infections increases. An H7N8 highly pathogenic avian influenza (HPAI) virus and an H7N8 low-pathogenic avian influenza (LPAI) virus were recently isolated from commercial turkey farms in Indiana. To determine the risk that these influenza viruses pose to humans, we assessed their pathogenesis and transmission in vitro and in mammalian models. We found that the H7N8 HPAI virus exhibited enhanced virulence, and although transmission was only observed with the H7N8 LPAI virus, the ability of this H7 virus to transmit in a mammalian host and quickly evolve to a more virulent strain is cause for concern. Our findings offer important insight into the potential for emerging H7 avian influenza viruses to acquire the ability to cause disease and transmit among mammals. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Pathogenesis and Transmission Assessments of Two H7N8 Influenza A Viruses Recently Isolated from Turkey Farms in Indiana Using Mouse and Ferret Models

PubMed Central

Sun, Xiangjie; Belser, Jessica A.; Pulit-Penaloza, Joanna A.; Zeng, Hui; Lewis, Amanda; Shieh, Wun-Ju; Tumpey, Terrence M.

2016-01-01

ABSTRACT Avian influenza A H7 viruses have caused multiple outbreaks in domestic poultry throughout North America, resulting in occasional infections of humans in close contact with affected birds. In early 2016, the presence of H7N8 highly pathogenic avian influenza (HPAI) viruses and closely related H7N8 low-pathogenic avian influenza (LPAI) viruses was confirmed in commercial turkey farms in Indiana. These H7N8 viruses represent the first isolation of this subtype in domestic poultry in North America, and their virulence in mammalian hosts and the potential risk for human infection are largely unknown. In this study, we assessed the ability of H7N8 HPAI and LPAI viruses to replicate in vitro in human airway cells and in vivo in mouse and ferret models. Both H7N8 viruses replicated efficiently in vitro and in vivo, but they exhibited substantial differences in disease severity in mammals. In mice, while the H7N8 LPAI virus largely remained avirulent, the H7N8 HPAI virus exhibited greater infectivity, virulence, and lethality. Both H7N8 viruses replicated similarly in ferrets, but only the H7N8 HPAI virus caused moderate weight loss, lethargy, and mortality. The H7N8 LPAI virus displayed limited transmissibility in ferrets placed in direct contact with an inoculated animal, while no transmission of H7N8 HPAI virus was detected. Our results indicate that the H7N8 avian influenza viruses from Indiana are able to replicate in mammals and cause severe disease but with limited transmission. The recent appearance of H7N8 viruses in domestic poultry highlights the need for continued influenza surveillance in wild birds and close monitoring of the potential risk to human health. IMPORTANCE H7 influenza viruses circulate in wild birds in the United States, but when the virus emerges in domestic poultry populations, the frequency of human exposure and the potential for human infections increases. An H7N8 highly pathogenic avian influenza (HPAI) virus and an H7N8 low-pathogenic avian influenza (LPAI) virus were recently isolated from commercial turkey farms in Indiana. To determine the risk that these influenza viruses pose to humans, we assessed their pathogenesis and transmission in vitro and in mammalian models. We found that the H7N8 HPAI virus exhibited enhanced virulence, and although transmission was only observed with the H7N8 LPAI virus, the ability of this H7 virus to transmit in a mammalian host and quickly evolve to a more virulent strain is cause for concern. Our findings offer important insight into the potential for emerging H7 avian influenza viruses to acquire the ability to cause disease and transmit among mammals. PMID:27681133

Early increase and late decrease of purkinje cell dendritic spine density in prion-infected organotypic mouse cerebellar cultures.

PubMed

Campeau, Jody L; Wu, Gengshu; Bell, John R; Rasmussen, Jay; Sim, Valerie L

2013-01-01

Prion diseases are infectious neurodegenerative diseases associated with the accumulation of protease-resistant prion protein, neuronal loss, spongiform change and astrogliosis. In the mouse model, the loss of dendritic spines is one of the earliest pathological changes observed in vivo, occurring 4-5 weeks after the first detection of protease-resistant prion protein in the brain. While there are cell culture models of prion infection, most do not recapitulate the neuropathology seen in vivo. Only the recently developed prion organotypic slice culture assay has been reported to undergo neuronal loss and the development of some aspects of prion pathology, namely small vacuolar degeneration and tubulovesicular bodies. Given the rapid replication of prions in this system, with protease-resistant prion protein detectable by 21 days, we investigated whether the dendritic spine loss and altered dendritic morphology seen in prion disease might also develop within the lifetime of this culture system. Indeed, six weeks after first detection of protease-resistant prion protein in tga20 mouse cerebellar slice cultures infected with RML prion strain, we found a statistically significant loss of Purkinje cell dendritic spines and altered dendritic morphology in infected cultures, analogous to that seen in vivo. In addition, we found a transient but statistically significant increase in Purkinje cell dendritic spine density during infection, at the time when protease-resistant prion protein was first detectable in culture. Our findings support the use of this slice culture system as one which recapitulates prion disease pathology and one which may facilitate study of the earliest stages of prion disease pathogenesis.

Prion Replication Elicits Cytopathic Changes in Differentiated Neurosphere Cultures

PubMed Central

Iwamaru, Yoshifumi; Takenouchi, Takato; Imamura, Morikazu; Shimizu, Yoshihisa; Miyazawa, Kohtaro; Mohri, Shirou; Yokoyama, Takashi

2013-01-01

The molecular mechanisms of prion-induced cytotoxicity remain largely obscure. Currently, only a few cell culture models have exhibited the cytopathic changes associated with prion infection. In this study, we introduced a cell culture model based on differentiated neurosphere cultures isolated from the brains of neonatal prion protein (PrP)-null mice and transgenic mice expressing murine PrP (dNP0 and dNP20 cultures). Upon exposure to mouse Chandler prions, dNP20 cultures supported the de novo formation of abnormal PrP and the resulting infectivity, as assessed by bioassays. Furthermore, this culture was susceptible to various prion strains, including mouse-adapted scrapie, bovine spongiform encephalopathy, and Gerstmann-Sträussler-Scheinker syndrome prions. Importantly, a subset of the cells in the infected culture that was mainly composed of astrocyte lineage cells consistently displayed late-occurring, progressive signs of cytotoxicity as evidenced by morphological alterations, decreased cell viability, and increased lactate dehydrogenase release. These signs of cytotoxicity were not observed in infected dNP0 cultures, suggesting the requirement of endogenous PrP expression for prion-induced cytotoxicity. Degenerated cells positive for glial fibrillary acidic protein accumulated abnormal PrP and exhibited features of apoptotic death as assessed by active caspase-3 and terminal deoxynucleotidyltransferase nick-end staining. Furthermore, caspase inhibition provided partial protection from prion-mediated cell death. These results suggest that differentiated neurosphere cultures can provide an in vitro bioassay for mouse prions and permit the study of the molecular basis for prion-induced cytotoxicity at the cellular level. PMID:23740992

Low-oxygen tensions found in Salmonella-infected gut tissue boost Salmonella replication in macrophages by impairing antimicrobial activity and augmenting Salmonella virulence.

PubMed

Jennewein, Jonas; Matuszak, Jasmin; Walter, Steffi; Felmy, Boas; Gendera, Kathrin; Schatz, Valentin; Nowottny, Monika; Liebsch, Gregor; Hensel, Michael; Hardt, Wolf-Dietrich; Gerlach, Roman G; Jantsch, Jonathan

2015-12-01

In Salmonella infection, the Salmonella pathogenicity island-2 (SPI-2)-encoded type three secretion system (T3SS2) is of key importance for systemic disease and survival in host cells. For instance, in the streptomycin-pretreated mouse model SPI-2-dependent Salmonella replication in lamina propria CD11c(-)CXCR1(-) monocytic phagocytes/macrophages (MΦ) is required for the development of colitis. In addition, containment of intracellular Salmonella in the gut critically depends on the antimicrobial effects of the phagocyte NADPH oxidase (PHOX), and possibly type 2 nitric oxide synthase (NOS2). For both antimicrobial enzyme complexes, oxygen is an essential substrate. However, the amount of available oxygen upon enteroinvasive Salmonella infection in the gut tissue and its impact on Salmonella-MΦ interactions was unknown. Therefore, we measured the gut tissue oxygen levels in a model of Salmonella enterocolitis using luminescence two-dimensional in vivo oxygen imaging. We found that gut tissue oxygen levels dropped from ∼78 Torr (∼11% O2) to values of ∼16 Torr (∼2% O2) during infection. Because in vivo virulence of Salmonella depends on the Salmonella survival in MΦ, Salmonella-MΦ interaction was analysed under such low oxygen values. These experiments revealed an increased intracellular replication and survival of wild-type and t3ss2 non-expressing Salmonella. These findings were paralleled by blunted nitric oxide and reactive oxygen species (ROS) production and reduced Salmonella ROS perception. In addition, hypoxia enhanced SPI-2 transcription and translocation of SPI-2-encoded virulence protein. Neither pharmacological blockade of PHOX and NOS2 nor impairment of T3SS2 virulence function alone mimicked the effect of hypoxia on Salmonella replication under normoxic conditions. However, if t3ss2 non-expressing Salmonella were used, hypoxia did not further enhance Salmonella recovery in a PHOX and NOS2-deficient situation. Hence, these data suggest that hypoxia-induced impairment of antimicrobial activity and Salmonella virulence cooperate to allow for enhanced Salmonella replication in MΦ. © 2015 John Wiley & Sons Ltd.

Pre-clinical efficacy and safety of experimental vaccines based on non-replicating vaccinia vectors against yellow fever.

PubMed

Schäfer, Birgit; Holzer, Georg W; Joachimsthaler, Alexandra; Coulibaly, Sogue; Schwendinger, Michael; Crowe, Brian A; Kreil, Thomas R; Barrett, P Noel; Falkner, Falko G

2011-01-01

Currently existing yellow fever (YF) vaccines are based on the live attenuated yellow fever virus 17D strain (YFV-17D). Although, a good safety profile was historically attributed to the 17D vaccine, serious adverse events have been reported, making the development of a safer, more modern vaccine desirable. A gene encoding the precursor of the membrane and envelope (prME) protein of the YFV-17D strain was inserted into the non-replicating modified vaccinia virus Ankara and into the D4R-defective vaccinia virus. Candidate vaccines based on the recombinant vaccinia viruses were assessed for immunogenicity and protection in a mouse model and compared to the commercial YFV-17D vaccine. The recombinant live vaccines induced γ-interferon-secreting CD4- and functionally active CD8-T cells, and conferred full protection against lethal challenge already after a single low immunization dose of 10(5) TCID(50). Surprisingly, pre-existing immunity against wild-type vaccinia virus did not negatively influence protection. Unlike the classical 17D vaccine, the vaccinia virus-based vaccines did not cause mortality following intracerebral administration in mice, demonstrating better safety profiles. The non-replicating recombinant YF candidate live vaccines induced a broad immune response after single dose administration, were effective even in the presence of a pre-existing immunity against vaccinia virus and demonstrated an excellent safety profile in mice.

[Methods for increasing the immunogenicity of vaccines].

PubMed

Kündig, T M

2000-09-14

In the past years, enormous efforts have been undertaken to develop vaccine strategies against cancer. The aim is to have the immune system generate what are called killer cells that can specifically recognize the tumor. The surface of tumor cells contains MHC/HLA antigens which present short-chain peptides of tumor specific antigens. A large number of these oligopeptide antigens have been characterized in recent years. They are now available for use as tumor-specific vaccines. The problem is, however, that the immune response of producing T killer cells is very inefficient when these oligopeptide antigens are injected. As the physiological function of these killer cells virus-infected cells, a process associated with substantial tissue damage, the immune system has learned to use these killer cells with reticence over the course of evolution, in other words, when the life of the host is threatened. This does not happen until pathogens start to spread via lymphogenous or hematogenous pathways. And then it takes a certain amount of time after the invader is present for replication to take place. Since the oligopeptide antigens used as vaccines have a very short half-life in the tissue, not enough of them get to the lymph nodes and stay there for enough time to efficiently induce an immune response. Using a mouse model, we were able to show that the efficiency of the vaccine can be increased a million-fold by directly injecting the vaccine into a lymph node or the spleen which imitates lymphogenous or hematogenous spread. The efficiency of the "inactivated vaccine" can be enhanced even more by continuous administration of the vaccine over several days, simulating an especially dangerous virus replication. The evidence gathered in this mouse model was transferred to a clinical trial. The melanoma-specific inactivated vaccine is infused directly into a lymph node of tumor patients. The infusion is continued for several days. Booster vaccines are given every two weeks.

Percutaneous Vaccination as an Effective Method of Delivery of MVA and MVA-Vectored Vaccines.

PubMed

Meseda, Clement A; Atukorale, Vajini; Kuhn, Jordan; Schmeisser, Falko; Weir, Jerry P

2016-01-01

The robustness of immune responses to an antigen could be dictated by the route of vaccine inoculation. Traditional smallpox vaccines, essentially vaccinia virus strains, that were used in the eradication of smallpox were administered by percutaneous inoculation (skin scarification). The modified vaccinia virus Ankara is licensed as a smallpox vaccine in Europe and Canada and currently undergoing clinical development in the United States. MVA is also being investigated as a vector for the delivery of heterologous genes for prophylactic or therapeutic immunization. Since MVA is replication-deficient, MVA and MVA-vectored vaccines are often inoculated through the intramuscular, intradermal or subcutaneous routes. Vaccine inoculation via the intramuscular, intradermal or subcutaneous routes requires the use of injection needles, and an estimated 10 to 20% of the population of the United States has needle phobia. Following an observation in our laboratory that a replication-deficient recombinant vaccinia virus derived from the New York City Board of Health strain elicited protective immune responses in a mouse model upon inoculation by tail scarification, we investigated whether MVA and MVA recombinants can elicit protective responses following percutaneous administration in mouse models. Our data suggest that MVA administered by percutaneous inoculation, elicited vaccinia-specific antibody responses, and protected mice from lethal vaccinia virus challenge, at levels comparable to or better than subcutaneous or intramuscular inoculation. High titers of specific neutralizing antibodies were elicited in mice inoculated with a recombinant MVA expressing the herpes simplex type 2 glycoprotein D after scarification. Similarly, a recombinant MVA expressing the hemagglutinin of attenuated influenza virus rgA/Viet Nam/1203/2004 (H5N1) elicited protective immune responses when administered at low doses by scarification. Taken together, our data suggest that MVA and MVA-vectored vaccines inoculated by scarification can elicit protective immune responses that are comparable to subcutaneous vaccination, and may allow for antigen sparing when vaccine supply is limited.

Rice bran extracts inhibit invasion and intracellular replication of Salmonella typhimurium in mouse and porcine intestinal epithelial cells

USDA-ARS?s Scientific Manuscript database

Dietary rice bran supplementation has been shown to inhibit Salmonella fecal shedding in animals. The aim of this study was to determine if bran extracts from two distinct rice varieties, Lijiangxintuanheigu (LTH) and Sanhuangzhan-2 (SHZ-2), differentially inhibit Salmonella enterica serover Typhimu...

Understanding the Etiology of Tuberous Sclerosis Complex

DTIC Science & Technology

2012-07-01

catalog #4856), mouse anti-NeuN (1:500; Millipore), GFAP (1:100, DAKO) and DCX (1:500, Santa Cruz Biotechnology). Each staining was replicated in slices...Tramontin, A.D., Quinones-Hinojosa, A., Barbaro, N.M., Gupta, N., Kunwar, S., Lawton, M.T., McDermott, M.W., Parsa, A.T., Manuel -Garcia, V.J. et al

Differential expression of growth factors at the cellular level in virus-infected brain

PubMed Central

Prosniak, Mikhail; Zborek, Anna; Scott, Gwen S.; Roy, Anirban; Phares, Timothy W.; Koprowski, Hilary; Hooper, D. Craig

2003-01-01

The contribution of host factors to rabies virus (RV) transcription/replication and axonal/transsynaptic spread is largely unknown. We previously identified several host genes that are up-regulated in the mouse brain during RV infection, including neuroleukin, which is involved in neuronal growth and survival, cell motility, and differentiation, and fibroblast growth factor homologous factor 4 (FHF4), which has been implicated in limb and nervous system development. In this study, we used real-time quantitative RT-PCR to assess the expression of mRNAs specific for neuroleukin, the two isoforms of FHF4 (FHF4-1a and -1b) encoded by the FHF4 gene, and N protein of RV in neurons and astrocytes isolated by laser capture microdissection from mouse brains infected with the laboratory-adapted RV strain CVS-N2c or with a street RV of silver-haired bat origin. Differences in the gene expression patterns suggest that the capacity of RV strains to infect nonneuronal cells and differentially modulate host gene expression may be important in virus replication and spread in the CNS. PMID:12736376

Dengue Virus Modulates the Unfolded Protein Response in a Time-dependent Manner*

PubMed Central

Peña, José; Harris, Eva

2011-01-01

Flaviviruses, such as dengue virus (DENV), depend on the host endoplasmic reticulum for translation, replication, and packaging of their genomes. Here we report that DENV-2 infection modulates the unfolded protein response in a time-dependent manner. We show that early DENV-2 infection triggers and then suppresses PERK-mediated eIF2α phosphorylation and that in mid and late DENV-2 infection, the IRE1-XBP1 and ATF6 pathways are activated, respectively. Activation of IRE1-XBP1 correlated with induction of downstream targets GRP78, CHOP, and GADD34. Furthermore, induction of CHOP did not induce apoptotic markers, such as suppression of anti-apoptotic protein Bcl-2, activation of caspase-9 or caspase-3, and cleavage of poly(ADP-ribose) polymerase. Finally, we show that DENV-2 replication is affected in PERK−/− and IRE1−/− mouse embryo fibroblasts when compared with wild-type mouse embryo fibroblasts. These results demonstrate that time-dependent activation of the unfolded protein response by DENV-2 can override inhibition of translation, prevent apoptosis, and prolong the viral life cycle. PMID:21385877

Immunogenicity and protective efficacy of rotavirus VP8* fused to cholera toxin B subunit in a mouse model.

PubMed

Xue, Miaoge; Yu, Linqi; Jia, Lianzhi; Li, Yijian; Zeng, Yuanjun; Li, Tingdong; Ge, Shengxiang; Xia, Ningshao

2016-11-01

In attempts to develop recombinant subunit vaccines against rotavirus disease, it was previously shown that the N-terminal truncated VP8* protein, VP8-1 (aa26-231), is a good vaccine candidate when used for immunization in combination with Freund's adjuvant. However, this protein stimulated only weak immune response when aluminum hydroxide was used as an adjuvant. In this study, the nontoxic B subunit of cholera toxin (CTB) was employed as intra-molecular adjuvant to improve the immunogenicity of VP8-1. Both, the N-terminal and C-terminal fusion proteins, were purified to homogeneity, at which stage they formed pentamers, and showed significantly higher immunogenicity and protective efficacy than a VP8-1/aluminum hydroxide mixture in a mouse model. Compared to VP8-1-CTB, CTB-VP8-1 showed higher binding activity to both, GM1 and the conformation sensitive neutralizing monoclonal antibodies specific to VP8. More importantly, CTB-VP8-1 elicited higher titers of neutralizing antibodies and conferred higher protective efficacy than VP8-1-CTB. Therefore, the protein CTB-VP8-1, with enhanced immunogenicity and immunoprotectivity, could be considered as a viable candidate for further development of an alternative, replication-incompetent, parenterally administered vaccine against rotavirus disease.

Effective Antimicrobial Regimens for Use in Humans for Therapy of Bacillus anthracis Infections and Postexposure Prophylaxis†

PubMed Central

Deziel, Mark R.; Heine, Henry; Louie, Arnold; Kao, Mark; Byrne, William R.; Basset, Jennifer; Miller, Lynda; Bush, Karen; Kelly, Michael; Drusano, G. L.

2005-01-01

Expanded options for treatments directed against pathogens that can be used for bioterrorism are urgently needed. Treatment regimens directed against such pathogens can be identified only by using data derived from in vitro and animal studies. It is crucial that these studies reliably predict the efficacy of proposed treatments in humans. The objective of this study was to identify a levofloxacin treatment regimen that will serve as an effective therapy for Bacillus anthracis infections and postexposure prophylaxis. An in vitro hollow-fiber infection model that replicates the pharmacokinetic profile of levofloxacin observed in humans (half-life [t1/2], 7.5 h) or in animals, such as the mouse or the rhesus monkey (t1/2, ∼2 h), was used to evaluate a proposed indication for levofloxacin (500 mg once daily) for the treatment of Bacillus anthracis infections. The results obtained with the in vitro model served as the basis for the doses and the dose schedules that were evaluated in the mouse inhalational anthrax model. The effects of levofloxacin and ciprofloxacin treatment were compared to those of no treatment (untreated controls). The main outcome measure in the in vitro hollow-fiber infection model was a persistent reduction of culture density (≥4 log10 reduction) and prevention of the emergence of levofloxacin-resistant organisms. In the mouse inhalational anthrax model the main outcome measure was survival. The results indicated that levofloxacin given once daily with simulated human pharmacokinetics effectively sterilized Bacillus anthracis cultures. By using a simulated animal pharmacokinetic profile, a once-daily dosing regimen that provided a human-equivalent exposure failed to sterilize the cultures. Dosing regimens that “partially humanized” levofloxacin exposures within the constraints of animal pharmacokinetics reproduced the antimicrobial efficacy seen with human pharmacokinetics. In a mouse inhalational anthrax model, once-daily dosing was significantly inferior (survival end point) to regimens of dosing every 12 h or every 6 h with identical total daily levofloxacin doses. These results demonstrate the predictive value of the in vitro hollow-fiber infection model with respect to the success or the failure of treatment regimens in animals. Furthermore, the model permits the evaluation of treatment regimens that “humanize” antibiotic exposures in animal models, enhancing the confidence with which animal models may be used to reliably predict the efficacies of proposed antibiotic treatments in humans in situations (e.g., the release of pathogens as agents of bioterrorism or emerging infectious diseases) where human trials cannot be performed. A treatment regimen effective in rhesus monkeys was identified. PMID:16304178

New Experimental Models of Diabetic Nephropathy in Mice Models of Type 2 Diabetes: Efforts to Replicate Human Nephropathy

PubMed Central

Soler, María José; Riera, Marta; Batlle, Daniel

2012-01-01

Diabetic nephropathy (DN) is the leading cause of end-stage renal disease. The use of experimental models of DN has provided valuable information regarding many aspects of DN, including pathophysiology, progression, implicated genes, and new therapeutic strategies. A large number of mouse models of diabetes have been identified and their kidney disease was characterized to various degrees. Most experimental models of type 2 DN are helpful in studying early stages of DN, but these models have not been able to reproduce the characteristic features of more advanced DN in humans such as nodules in the glomerular tuft or glomerulosclerosis. The generation of new experimental models of DN created by crossing, knockdown, or knockin of genes continues to provide improved tools for studying DN. These models provide an opportunity to search for new mechanisms involving the development of DN, but their shortcomings should be recognized as well. Moreover, it is important to recognize that the genetic background has a substantial effect on the susceptibility to diabetes and kidney disease development in the various models of diabetes. PMID:22461787

The circadian clock controls sunburn apoptosis and erythema in mouse skin.

PubMed

Gaddameedhi, Shobhan; Selby, Christopher P; Kemp, Michael G; Ye, Rui; Sancar, Aziz

2015-04-01

Epidemiological studies of humans and experimental studies with mouse models suggest that sunburn resulting from exposure to excessive UV light and damage to DNA confers an increased risk for melanoma and non-melanoma skin cancer. Previous reports have shown that both nucleotide excision repair, which is the sole pathway in humans for removing UV photoproducts, and DNA replication are regulated by the circadian clock in mouse skin. Furthermore, the timing of UV exposure during the circadian cycle has been shown to affect skin carcinogenesis in mice. Because sunburn and skin cancer are causally related, we investigated UV-induced sunburn apoptosis and erythema in mouse skin as a function of circadian time. Interestingly, we observed that sunburn apoptosis, inflammatory cytokine induction, and erythema were maximal following an acute early-morning exposure to UV and minimal following an afternoon exposure. Early-morning exposure to UV also produced maximal activation of ataxia telangiectasia mutated and Rad3-related (Atr)-mediated DNA damage checkpoint signaling, including activation of the tumor suppressor p53, which is known to control the process of sunburn apoptosis. These data provide early evidence that the circadian clock has an important role in the erythemal response in UV-irradiated skin. The early morning is when DNA repair is at a minimum, and thus the acute responses likely are associated with unrepaired DNA damage. The prior report that mice are more susceptible to skin cancer induction following chronic irradiation in the AM, when p53 levels are maximally induced, is discussed in terms of the mutational inactivation of p53 during chronic irradiation.

The Circadian Clock Controls Sunburn Apoptosis and Erythema in Mouse Skin

PubMed Central

Gaddameedhi, Shobhan; Selby, Christopher P.; Kemp, Michael G.; Ye, Rui; Sancar, Aziz

2014-01-01

Epidemiological studies of humans and experimental studies with mouse models suggest that sunburn resulting from exposure to excessive UV light and damage to DNA confers an increased risk for melanoma and non-melanoma skin cancer. Previous reports have shown that both nucleotide excision repair, which is the sole pathway in humans for removing UV photoproducts, and DNA replication, are regulated by the circadian clock in mouse skin. Furthermore, the timing of UV exposure during the circadian cycle has been shown to affect skin carcinogenesis in mice. Because sunburn and skin cancer are causally related, we investigated UV-induced sunburn apoptosis and erythema in mouse skin as a function of circadian time. Interestingly, we observed that sunburn apoptosis, inflammatory cytokine induction, and erythema were maximal following an acute early morning exposure to UV and minimal following an afternoon exposure. Early morning exposure to UV also produced maximal activation of Atr-mediated DNA damage checkpoint signaling including activation of the tumor suppressor p53, which is known to control the process of sunburn apoptosis. To our knowledge these data provide the first evidence that the circadian clock plays an important role in the erythemal response in UV-irradiated skin. The early morning is when DNA repair is at a minimum, thus the acute responses likely are associated with unrepaired DNA damage. The prior report that mice are more susceptible to skin cancer induction following chronic irradiation in the AM, when p53 levels are maximally induced, is discussed in terms of the mutational inactivation of p53 during chronic irradiation. PMID:25431853

The Mammalian Cell Cycle Regulates Parvovirus Nuclear Capsid Assembly

PubMed Central

Riolobos, Laura; Domínguez, Carlos; Kann, Michael; Almendral, José M.

2015-01-01

It is unknown whether the mammalian cell cycle could impact the assembly of viruses maturing in the nucleus. We addressed this question using MVM, a reference member of the icosahedral ssDNA nuclear parvoviruses, which requires cell proliferation to infect by mechanisms partly understood. Constitutively expressed MVM capsid subunits (VPs) accumulated in the cytoplasm of mouse and human fibroblasts synchronized at G0, G1, and G1/S transition. Upon arrest release, VPs translocated to the nucleus as cells entered S phase, at efficiencies relying on cell origin and arrest method, and immediately assembled into capsids. In synchronously infected cells, the consecutive virus life cycle steps (gene expression, proteins nuclear translocation, capsid assembly, genome replication and encapsidation) proceeded tightly coupled to cell cycle progression from G0/G1 through S into G2 phase. However, a DNA synthesis stress caused by thymidine irreversibly disrupted virus life cycle, as VPs became increasingly retained in the cytoplasm hours post-stress, forming empty capsids in mouse fibroblasts, thereby impairing encapsidation of the nuclear viral DNA replicative intermediates. Synchronously infected cells subjected to density-arrest signals while traversing early S phase also blocked VPs transport, resulting in a similar misplaced cytoplasmic capsid assembly in mouse fibroblasts. In contrast, thymidine and density arrest signals deregulating virus assembly neither perturbed nuclear translocation of the NS1 protein nor viral genome replication occurring under S/G2 cycle arrest. An underlying mechanism of cell cycle control was identified in the nuclear translocation of phosphorylated VPs trimeric assembly intermediates, which accessed a non-conserved route distinct from the importin α2/β1 and transportin pathways. The exquisite cell cycle-dependence of parvovirus nuclear capsid assembly conforms a novel paradigm of time and functional coupling between cellular and virus life cycles. This junction may determine the characteristic parvovirus tropism for proliferative and cancer cells, and its disturbance could critically contribute to persistence in host tissues. PMID:26067441

Replication and Immunogenicity of Swine, Equine, and Avian H3 Subtype Influenza Viruses in Mice and Ferrets

PubMed Central

Baz, Mariana; Paskel, Myeisha; Matsuoka, Yumiko; Zengel, James; Cheng, Xing; Jin, Hong

2013-01-01

Since it is difficult to predict which influenza virus subtype will cause an influenza pandemic, it is important to prepare influenza virus vaccines against different subtypes and evaluate the safety and immunogenicity of candidate vaccines in preclinical and clinical studies prior to a pandemic. In addition to infecting humans, H3 influenza viruses commonly infect pigs, horses, and avian species. We selected 11 swine, equine, and avian H3 influenza viruses and evaluated their kinetics of replication and ability to induce a broadly cross-reactive antibody response in mice and ferrets. The swine and equine viruses replicated well in the upper respiratory tract of mice. With the exception of one avian virus that replicated poorly in the lower respiratory tract, all of the viruses replicated in mouse lungs. In ferrets, all of the viruses replicated well in the upper respiratory tract, but the equine viruses replicated poorly in the lungs. Extrapulmonary spread was not observed in either mice or ferrets. No single virus elicited antibodies that cross-reacted with viruses from all three animal sources. Avian and equine H3 viruses elicited broadly cross-reactive antibodies against heterologous viruses isolated from the same or other species, but the swine viruses did not. We selected an equine and an avian H3 influenza virus for further development as vaccines. PMID:23576512

Application of the microfluidic-assisted replication track analysis to measure DNA repair in human and mouse cells.

PubMed

Welcsh, Piri; Kehrli, Keffy; Lazarchuk, Pavlo; Ladiges, Warren; Sidorova, Julia

2016-10-01

Functional studies of the roles that DNA helicases play in human cells have benefited immensely from DNA fiber (or single molecule) technologies, which enable us to discern minute differences in behaviors of individual replication forks in genomic DNA in vivo. DNA fiber technologies are a group of methods that use different approaches to unravel and stretch genomic DNA to its contour length, and display it on a glass surface in order to immuno-stain nucleoside analog incorporation into DNA to reveal tracks (or tracts) of replication. We have previously adopted a microfluidic approach to DNA stretching and used it to analyze DNA replication. This method was introduced under the moniker maRTA or microfluidic-assisted Replication Track Analysis, and we have since used it to analyze roles of the RECQ helicases WRN and BLM, and other proteins in normal and perturbed replication. Here we describe a novel application of maRTA to detect and measure repair of DNA damage produced by three different agents relevant to etiology or therapy of cancer: methyl-methanesulfonate, UV irradiation, and mitomycin C. Moreover, we demonstrate the utility of this method by analyzing DNA repair in cells with reduced levels of WRN or of the base excision repair protein XRCC1. Copyright © 2016 Elsevier Inc. All rights reserved.

Replication domains are self-interacting structural chromatin units of human chromosomes

NASA Astrophysics Data System (ADS)

Arneodo, Alain

2011-03-01

In higher eukaryotes, the absence of specific sequence motifs marking the origins of replication has been a serious hindrance to the understanding of the mechanisms that regulate the initiation and the maintenance of the replication program in different cell types. In silico analysis of nucleotide compositional skew has predicted the existence, in the germline, of replication N-domains bordered by putative replication origins and where the skew decreases rather linearly as the signature of a progressive inversion of the average fork polarity. Here, from the demonstration that the average fork polarity can be directly extracted from the derivative of replication timing profiles, we develop a wavelet-based pattern recognition methodology to delineate replication U-domains where the replication timing profile is shaped as a U and its derivative as a N. Replication U-domains are robustly found in seven cell lines as covering a significant portion (40-50%) of the human genome where the replication timing data actually displays some plasticity between cell lines. The early replication initiation zones at U-domains borders are found to be hypersensitive to DNase I cleavage, to be associated with transcriptional activity and to present a significant enrichment in insular-binding proteins CTCF, the hallmark of an open chromatin structure. A comparative analysis of genome-wide chromatin interaction (HiC) data shows that replication-U domains correspond to self-interacting structural high order chromatin units of megabase characteristic size. Taken together, these findings provide evidence that the epigenetic compartmentalization of the human genome into autonomous replication U-domains comes along with an extensive remodelling of the threedimensional chromosome architecture during development or in specific diseases. The observed cell specific conservation of the replication timing between the human and mouse genomes strongly suggests that this chromosome organization into self-interacting structural and functional units is a general feature of mammalian organisms.

Flea-borne transmission model to evaluate vaccine efficacy against naturally acquired bubonic plague.

PubMed

Jarrett, Clayton O; Sebbane, Florent; Adamovicz, Jeffrey J; Andrews, Gerard P; Hinnebusch, B Joseph

2004-04-01

A flea-to-mouse transmission model was developed for use in testing new candidate vaccines for the ability to protect against flea-borne plague. The model was used to evaluate a recombinant fusion protein vaccine consisting of the Yersinia pestis F1 and V antigens. After one to three challenges with Y. pestis-infected fleas, 14 of 15 unvaccinated control mice developed plague, with an average septicemia level of 9.2 x 10(8) Y. pestis CFU/ml. None of 15 vaccinated mice developed the disease after similar challenges, and serological testing indicated that transmitted bacteria were eliminated by the immune system before extensive replication and systemic infection could occur. The transmission and development of disease in control mice correlated with the number of bites by blocked fleas but not with the total number of fleabites. The model provides a means to directly assess the efficacy of new vaccines to prevent naturally acquired bubonic plague and to study events at the vector-host interface that lead to dissemination and disease.

Ferret and Pig Models of Cystic Fibrosis: Prospects and Promise for Gene Therapy

PubMed Central

Yan, Ziying; Stewart, Zoe A.; Sinn, Patrick L.; Olsen, John C.; Hu, Jim; McCray, Paul B.

2015-01-01

Abstract Large animal models of genetic diseases are rapidly becoming integral to biomedical research as technologies to manipulate the mammalian genome improve. The creation of cystic fibrosis (CF) ferrets and pigs is an example of such progress in animal modeling, with the disease phenotypes in the ferret and pig models more reflective of human CF disease than mouse models. The ferret and pig CF models also provide unique opportunities to develop and assess the effectiveness of gene and cell therapies to treat affected organs. In this review, we examine the organ disease phenotypes in these new CF models and the opportunities to test gene therapies at various stages of disease progression in affected organs. We then discuss the progress in developing recombinant replication-defective adenoviral, adeno-associated viral, and lentiviral vectors to target genes to the lung and pancreas in ferrets and pigs, the two most affected organs in CF. Through this review, we hope to convey the potential of these new animal models for developing CF gene and cell therapies. PMID:25675143

Small Animal Models for Evaluating Filovirus Countermeasures.

PubMed

Banadyga, Logan; Wong, Gary; Qiu, Xiangguo

2018-05-11

The development of novel therapeutics and vaccines to treat or prevent disease caused by filoviruses, such as Ebola and Marburg viruses, depends on the availability of animal models that faithfully recapitulate clinical hallmarks of disease as it is observed in humans. In particular, small animal models (such as mice and guinea pigs) are historically and frequently used for the primary evaluation of antiviral countermeasures, prior to testing in nonhuman primates, which represent the gold-standard filovirus animal model. In the past several years, however, the filovirus field has witnessed the continued refinement of the mouse and guinea pig models of disease, as well as the introduction of the hamster and ferret models. We now have small animal models for most human-pathogenic filoviruses, many of which are susceptible to wild type virus and demonstrate key features of disease, including robust virus replication, coagulopathy, and immune system dysfunction. Although none of these small animal model systems perfectly recapitulates Ebola virus disease or Marburg virus disease on its own, collectively they offer a nearly complete set of tools in which to carry out the preclinical development of novel antiviral drugs.

Interaction of primary mast cells with Borrelia burgdorferi (sensu stricto): role in transmission and dissemination in C57BL/6 mice.

PubMed

Bernard, Quentin; Wang, Zhenping; Di Nardo, Anna; Boulanger, Nathalie

2017-06-27

Borrelia burgdorferi (sensu lato), the causative agent of Lyme borreliosis is a bacterium transmitted by hard ticks, Ixodes spp. Bacteria are injected into the host skin during the tick blood meal with tick saliva. There, Borrelia and saliva interact together with skin cells such as keratinocytes, fibroblasts, mast cells and other specific immune cells before disseminating to target organs. To study the role of mast cells in the transmission of Lyme borreliosis, we isolated mouse primary mast cells from bone marrow and incubated them in the presence of Borrelia burgdorferi (sensu stricto) and tick salivary gland extract. We further analyzed their potential role in vivo, in a mouse model of deficient in mast cells (Kit wsh-/- mice). To our knowledge, we report here for the first time the bacteria ability to induce the inflammatory response of mouse primary mast cells. We show that OspC, a major surface lipoprotein involved in the early transmission of Borrelia, induces the degranulation of primary mast cells but has a limited effect on the overall inflammatory response of these cells. In contrast, whole bacteria have an opposite effect. We also show that mast cell activation is significantly inhibited by tick salivary gland extract. Finally, we demonstrate that mast cells are likely not the only host cells involved in the early transmission and dissemination of Borrelia since the use of mast cell deficient Kit wsh-/- mice shows a limited impact on these two processes in the context of this mouse genetic background. The absence of mast cells did not change the replication rate of Borrelia in the skin. However, in the absence of mast cells, Borrelia dissemination to the joints was faster. Mast cells do not control skin bacterial proliferation during primary infection and the establishment of the primary infection, as shown in the C57BL/6 mouse model studied. Nevertheless, the Borrelia induced cytotokine modulation on mast cells might be involved in long term and/or repeated infections and protect from Lyme borreliosis due to the development of a hypersensitivity to tick saliva.

Inhibition of hepatitis B virus replication via HBV DNA cleavage by Cas9 from Staphylococcus aureus.

PubMed

Liu, Yu; Zhao, Miaoxian; Gong, Mingxing; Xu, Ying; Xie, Cantao; Deng, Haohui; Li, Xueying; Wu, Hongkai; Wang, Zhanhui

2018-04-01

Chronic hepatitis B virus (HBV) infection is difficult to cure due to the presence of covalently closed circular DNA (cccDNA). Accumulating evidence indicates that the CRISPR/Cas9 system effectively disrupts HBV genome, including cccDNA, in vitro and in vivo. However, efficient delivery of CRISPR/Cas9 system to the liver or hepatocytes using an adeno-associated virus (AAV) vector remains challenging due to the large size of Cas9 from Streptococcus pyogenes (Sp). The recently identified Cas9 protein from Staphylococcus aureus (Sa) is smaller than SpCas9 and thus is able to be packaged into the AAV vector. To examine the efficacy of SaCas9 system on HBV genome destruction, we designed 5 guide RNAs (gRNAs) that targeted different HBV genotypes, 3 of which were shown to be effective. The SaCas9 system significantly reduced HBV antigen expression, as well as pgRNA and cccDNA levels, in Huh7, HepG2.2.15 and HepAD38 cells. The dual expression of gRNAs/SaCas9 in these cell lines resulted in more efficient HBV genome cleavage. In the mouse model, hydrodynamic injection of gRNA/SaCas9 plasmids resulted in significantly lower levels of HBV protein expression. We also delivered the SaCas9 system into mice with persistent HBV replication using an AAV vector. Both the AAV vector and the mRNA of Cas9 could be detected in the C3H mouse liver cells. Decreased hepatitis B surface antigen (HBsAg), HBV DNA and pgRNA levels were observed when a higher titer of AAV was injected, although this decrease was not significantly different from the control. In summary, the SaCas9 system accurately and efficiently targeted the HBV genome and inhibited HBV replication both in vitro and in vivo. The system was delivered by an AAV vector and maybe used as a novel therapeutic strategy against chronic HBV infection. Copyright © 2018 Elsevier B.V. All rights reserved.

Latency Entry of Herpes Simplex Virus 1 Is Determined by the Interaction of Its Genome with the Nuclear Environment

PubMed Central

Cohen, Camille; Streichenberger, Nathalie; Texier, Pascale; Takissian, Julie; Rousseau, Antoine; Poccardi, Nolwenn; Welsch, Jérémy; Corpet, Armelle; Schaeffer, Laurent; Labetoulle, Marc; Lomonte, Patrick

2016-01-01

Herpes simplex virus 1 (HSV-1) establishes latency in trigeminal ganglia (TG) sensory neurons of infected individuals. The commitment of infected neurons toward the viral lytic or latent transcriptional program is likely to depend on both viral and cellular factors, and to differ among individual neurons. In this study, we used a mouse model of HSV-1 infection to investigate the relationship between viral genomes and the nuclear environment in terms of the establishment of latency. During acute infection, viral genomes show two major patterns: replication compartments or multiple spots distributed in the nucleoplasm (namely “multiple-acute”). Viral genomes in the “multiple-acute” pattern are systematically associated with the promyelocytic leukemia (PML) protein in structures designated viral DNA-containing PML nuclear bodies (vDCP-NBs). To investigate the viral and cellular features that favor the acquisition of the latency-associated viral genome patterns, we infected mouse primary TG neurons from wild type (wt) mice or knock-out mice for type 1 interferon (IFN) receptor with wt or a mutant HSV-1, which is unable to replicate due to the synthesis of a non-functional ICP4, the major virus transactivator. We found that the inability of the virus to initiate the lytic program combined to its inability to synthesize a functional ICP0, are the two viral features leading to the formation of vDCP-NBs. The formation of the “multiple-latency” pattern is favored by the type 1 IFN signaling pathway in the context of neurons infected by a virus able to replicate through the expression of a functional ICP4 but unable to express functional VP16 and ICP0. Analyses of TGs harvested from HSV-1 latently infected humans showed that viral genomes and PML occupy similar nuclear areas in infected neurons, eventually forming vDCP-NB-like structures. Overall our study designates PML protein and PML-NBs to be major cellular components involved in the control of HSV-1 latency, probably during the entire life of an individual. PMID:27618691

Immune response of mice to non-adapted avian influenza A virus.

PubMed

Stropkovská, A; Mikušková, T; Bobišová, Z; Košík, I; Mucha, V; Kostolanský, F; Varečková, E

2015-12-01

Human infections with avian influenza A viruses (IAVs) without or with clinical symptoms of disease were recently reported from several continents, mainly in high risk groups of people, who came into the contact with infected domestic birds or poultry. It was shown that avian IAVs are able to infect humans directly without previous adaptation, however, their ability to replicate and to cause a disease in this new host can differ. No spread of these avian IAVs among humans has been documented until now, except for one case described in Netherlands in the February of 2003 in people directly involved in handling IAV (H7N7)-infected poultry. The aim of our work was to examine whether a low pathogenic avian IAV can induce a virus-specific immune response of biological relevancy, in spite of its restricted replication in mammals. As a model we used a low pathogenic virus A/Duck/Czechoslovakia/1956 (H4N6) (A/Duck), which replicated well in MDCK cells and produced plaques on cell monolayers, but was unable to replicate productively in mouse lungs. We examined how the immune system of mice responds to the intranasal application of this non-adapted avian virus. Though we did not prove the infectious virus in lungs of mice following A/Duck application even after its multiple passaging in mice, we detected virus-specific vRNA till day 8 post infection. Moreover, we detected virus-specific mRNA and de novo synthesized viral nucleoprotein (NP) and membrane protein (M1) in lungs of mice on day 2 and 4 after exposure to A/Duck. Virus-specific antibodies in sera of these mice were detectable by ELISA already after a single intranasal dose of A/Duck virus. Not only antibodies specific to the surface glycoprotein hemagglutinin (HA) were induced, but also antibodies specific to the NP and M1 of IAV were detected by Western blot and their titers increased after the second exposure of mice to this virus. Importantly, antibodies neutralizing virus A/Duck were proved in mouse immune sera after the second dose of virus and a slight increase of mRNA expression of immune mediators tumor necrosis factor alpha (TNF-α) and IP10 has been observed in lungs of these mice 48 hr after the infection. These observations correspond to the limited replication ability of the virus in mice and provided an important information about its ability to induce virus-specific antibodies, including those neutralizing virus, even without the previous virus adaptation to the new mammalian host. Such antibodies could consequently influence the immune potential of exposed individuals and their defensive capability against the newly emerged, even more virulent IAV.

Roles of the first and second round of DNA replication in the regulation of zygotic gene activation in mice.

PubMed

Sonehara, Hiroki; Nagata, Masao; Aoki, Fugaku

2008-10-01

In the mouse embryo, expression of zygotic genes starts in the S/G2 phase of the 1-cell stage and greatly increases during the 2-cell stage. Although the timing of zygotic gene activation (ZGA) is thus established, the mechanism regulating ZGA is poorly understood. Previous studies using reporter genes have suggested that a transcriptionally repressive state is established during the 2-cell stage and that the first and second rounds of DNA replication are involved in this process. To further elucidate the respective roles of the two rounds of DNA replication in ZGA, we analyzed the expression of four ZGA genes (hsp70.1, eif-1a, muerv and zscan4d) in embryos whose DNA replication was inhibited by treatment with aphidicolin, an inhibitor of DNA polymerase. Inhibiting the first round increased the expression levels of hsp70.1, eif-1a and zscan4d but decreased that of muerv, while inhibiting the second round increased the expression levels of all four genes. These results suggest that the transcriptionally repressive state seems to be established after the second round of DNA replication.

Doxycycline Inducible Melanogenic Vaccinia Virus as Theranostic Anti-Cancer Agent.

PubMed

Kirscher, Lorenz; Deán-Ben, Xosé Luis; Scadeng, Miriam; Zaremba, Angelika; Zhang, Qian; Kober, Christina; Fehm, Thomas Felix; Razansky, Daniel; Ntziachristos, Vasilis; Stritzker, Jochen; Szalay, Aladar A

2015-01-01

We reported earlier the diagnostic potential of a melanogenic vaccinia virus based system in magnetic resonance (MRI) and optoacoustic deep tissue imaging (MSOT). Since melanin overproduction lead to attenuated virus replication, we constructed a novel recombinant vaccinia virus strain (rVACV), GLV-1h462, which expressed the key enzyme of melanogenesis (tyrosinase) under the control of an inducible promoter-system. In this study melanin production was detected after exogenous addition of doxycycline in two different tumor xenograft mouse models. Furthermore, it was confirmed that this novel vaccinia virus strain still facilitated signal enhancement as detected by MRI and optoacoustic tomography. At the same time we demonstrated an enhanced oncolytic potential compared to the constitutively melanin synthesizing rVACV system.

Preclinical Activity of VX-787, a First-in-Class, Orally Bioavailable Inhibitor of the Influenza Virus Polymerase PB2 Subunit

PubMed Central

Byrn, Randal A.; Jones, Steven M.; Bennett, Hamilton B.; Bral, Chris; Clark, Michael P.; Jacobs, Marc D.; Kwong, Ann D.; Ledeboer, Mark W.; Leeman, Joshua R.; McNeil, Colleen F.; Murcko, Mark A.; Nezami, Azin; Perola, Emanuele; Rijnbrand, Rene; Saxena, Kumkum; Tsai, Alice W.; Zhou, Yi

2014-01-01

VX-787 is a novel inhibitor of influenza virus replication that blocks the PB2 cap-snatching activity of the influenza viral polymerase complex. Viral genetics and X-ray crystallography studies provide support for the idea that VX-787 occupies the 7-methyl GTP (m7GTP) cap-binding site of PB2. VX-787 binds the cap-binding domain of the PB2 subunit with a KD (dissociation constant) of 24 nM as determined by isothermal titration calorimetry (ITC). The cell-based EC50 (the concentration of compound that ensures 50% cell viability of an uninfected control) for VX-787 is 1.6 nM in a cytopathic effect (CPE) assay, with a similar EC50 in a viral RNA replication assay. VX-787 is active against a diverse panel of influenza A virus strains, including H1N1pdm09 and H5N1 strains, as well as strains with reduced susceptibility to neuraminidase inhibitors (NAIs). VX-787 was highly efficacious in both prophylaxis and treatment models of mouse influenza and was superior to the neuraminidase inhibitor, oseltamivir, including in delayed-start-to-treat experiments, with 100% survival at up to 96 h postinfection and partial survival in groups where the initiation of therapy was delayed up to 120 h postinfection. At different doses, VX-787 showed a 1-log to >5-log reduction in viral load (relative to vehicle controls) in mouse lungs. Overall, these favorable findings validate the PB2 subunit of the viral polymerase as a drug target for influenza therapy and support the continued development of VX-787 as a novel antiviral agent for the treatment of influenza infection. PMID:25547360

Preclinical activity of VX-787, a first-in-class, orally bioavailable inhibitor of the influenza virus polymerase PB2 subunit.

PubMed

Byrn, Randal A; Jones, Steven M; Bennett, Hamilton B; Bral, Chris; Clark, Michael P; Jacobs, Marc D; Kwong, Ann D; Ledeboer, Mark W; Leeman, Joshua R; McNeil, Colleen F; Murcko, Mark A; Nezami, Azin; Perola, Emanuele; Rijnbrand, Rene; Saxena, Kumkum; Tsai, Alice W; Zhou, Yi; Charifson, Paul S

2015-03-01

VX-787 is a novel inhibitor of influenza virus replication that blocks the PB2 cap-snatching activity of the influenza viral polymerase complex. Viral genetics and X-ray crystallography studies provide support for the idea that VX-787 occupies the 7-methyl GTP (m(7)GTP) cap-binding site of PB2. VX-787 binds the cap-binding domain of the PB2 subunit with a KD (dissociation constant) of 24 nM as determined by isothermal titration calorimetry (ITC). The cell-based EC50 (the concentration of compound that ensures 50% cell viability of an uninfected control) for VX-787 is 1.6 nM in a cytopathic effect (CPE) assay, with a similar EC50 in a viral RNA replication assay. VX-787 is active against a diverse panel of influenza A virus strains, including H1N1pdm09 and H5N1 strains, as well as strains with reduced susceptibility to neuraminidase inhibitors (NAIs). VX-787 was highly efficacious in both prophylaxis and treatment models of mouse influenza and was superior to the neuraminidase inhibitor, oseltamivir, including in delayed-start-to-treat experiments, with 100% survival at up to 96 h postinfection and partial survival in groups where the initiation of therapy was delayed up to 120 h postinfection. At different doses, VX-787 showed a 1-log to >5-log reduction in viral load (relative to vehicle controls) in mouse lungs. Overall, these favorable findings validate the PB2 subunit of the viral polymerase as a drug target for influenza therapy and support the continued development of VX-787 as a novel antiviral agent for the treatment of influenza infection. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

LuIII Parvovirus Selectively and Efficiently Targets, Replicates in, and Kills Human Glioma Cells

PubMed Central

Paglino, Justin C.; Ozduman, Koray

2012-01-01

Because productive infection by parvoviruses requires cell division and is enhanced by oncogenic transformation, some parvoviruses may have potential utility in killing cancer cells. To identify the parvovirus(es) with the optimal oncolytic effect against human glioblastomas, we screened 12 parvoviruses at a high multiplicity of infection (MOI). MVMi, MVMc, MVM-G17, tumor virus X (TVX), canine parvovirus (CPV), porcine parvovirus (PPV), rat parvovirus 1A (RPV1A), and H-3 were relatively ineffective. The four viruses with the greatest oncolytic activity, LuIII, H-1, MVMp, and MVM-G52, were tested for the ability, at a low MOI, to progressively infect the culture over time, causing cell death at a rate higher than that of cell proliferation. LuIII alone was effective in all five human glioblastomas tested. H-1 progressively infected only two of five; MVMp and MVM-G52 were ineffective in all five. To investigate the underlying mechanism of LuIII's phenotype, we used recombinant parvoviruses with the LuIII capsid replacing the MVMp capsid or with molecular alteration of the P4 promoter. The LuIII capsid enhanced efficient replication and oncolysis in MO59J gliomas cells; other gliomas tested required the entire LuIII genome to exhibit enhanced infection. LuIII selectively infected glioma cells over normal glial cells in vitro. In mouse models, human glioblastoma xenografts were selectively infected by LuIII when administered intratumorally; LuIII reduced tumor growth by 75%. LuIII also had the capacity to selectively infect subcutaneous or intracranial gliomas after intravenous inoculation. Intravenous or intracranial LuIII caused no adverse effects. Intracranial LuIII caused no infection of mature mouse neurons or glia in vivo but showed a modest infection of developing neurons. PMID:22553327

LuIII parvovirus selectively and efficiently targets, replicates in, and kills human glioma cells.

PubMed

Paglino, Justin C; Ozduman, Koray; van den Pol, Anthony N

2012-07-01

Because productive infection by parvoviruses requires cell division and is enhanced by oncogenic transformation, some parvoviruses may have potential utility in killing cancer cells. To identify the parvovirus(es) with the optimal oncolytic effect against human glioblastomas, we screened 12 parvoviruses at a high multiplicity of infection (MOI). MVMi, MVMc, MVM-G17, tumor virus X (TVX), canine parvovirus (CPV), porcine parvovirus (PPV), rat parvovirus 1A (RPV1A), and H-3 were relatively ineffective. The four viruses with the greatest oncolytic activity, LuIII, H-1, MVMp, and MVM-G52, were tested for the ability, at a low MOI, to progressively infect the culture over time, causing cell death at a rate higher than that of cell proliferation. LuIII alone was effective in all five human glioblastomas tested. H-1 progressively infected only two of five; MVMp and MVM-G52 were ineffective in all five. To investigate the underlying mechanism of LuIII's phenotype, we used recombinant parvoviruses with the LuIII capsid replacing the MVMp capsid or with molecular alteration of the P4 promoter. The LuIII capsid enhanced efficient replication and oncolysis in MO59J gliomas cells; other gliomas tested required the entire LuIII genome to exhibit enhanced infection. LuIII selectively infected glioma cells over normal glial cells in vitro. In mouse models, human glioblastoma xenografts were selectively infected by LuIII when administered intratumorally; LuIII reduced tumor growth by 75%. LuIII also had the capacity to selectively infect subcutaneous or intracranial gliomas after intravenous inoculation. Intravenous or intracranial LuIII caused no adverse effects. Intracranial LuIII caused no infection of mature mouse neurons or glia in vivo but showed a modest infection of developing neurons.

Detection of neuroendocrine tumors using promoter-specific secreted Gaussia luciferase.

PubMed

Tseng, Alan Wei-Shun; Akerstrom, Victoria; Chen, Chiachen; Breslin, Mary B; Lan, Michael S

2016-01-01

Accurate detection of neuroendocrine (NE) tumors is critically important for better prognosis and treatment outcomes in patients. To demonstrate the efficacy of using an adenoviral vector for the detection of NE tumors, we have constructed a pair of adenoviral vectors which, in combination, can conditionally replicate and release Gaussia luciferase into the circulation after infecting the NE tumors. The expression of these two vectors is regulated upstream by an INSM1-promoter (insulinoma-associated-1) that is specifically active in NE tumors and developing NE tissues, but silenced in normal adult tissues. In order to retain the tumor-specificity of the INSM1 promoter, we have modified the promoter using the core insulator sequence from the chicken β-globin HS4 insulator and the neuronal restrictive silencing element (NRSE). This modified INSM1-promoter can retain NE tumor specificity in an adenoviral construct while driving a mutated adenovirus E1A gene (∆24E1A), the Metridia, or Gaussia luciferase gene. The in vitro cell line and mouse xenograft human tumor studies revealed the NE specificity of the INSM1-promoter in NE lung cancer, neuroblastoma, medulloblastoma, retinoblastoma, and insulinoma. When we combined the INSM1-promoter driven Gaussia luciferase with ∆24E1A, the co-infected NE tumor secreted higher levels of Gaussia luciferase as compared to the INSM1p-Gaussia virus alone. In a mouse subcutaneous xenograft tumor model, the combination viruses secreted detectable level of Gaussia luciferase after infecting an INSM1-positive NE lung tumor for ≥12 days. Therefore, the INSM1-promoter specific conditional replicating adenovirus represents a sensitive diagnostic tool to aid clinicians in the detection of NE tumors.

A SARS-like cluster of circulating bat coronaviruses shows potential for human emergence.

PubMed

Menachery, Vineet D; Yount, Boyd L; Debbink, Kari; Agnihothram, Sudhakar; Gralinski, Lisa E; Plante, Jessica A; Graham, Rachel L; Scobey, Trevor; Ge, Xing-Yi; Donaldson, Eric F; Randell, Scott H; Lanzavecchia, Antonio; Marasco, Wayne A; Shi, Zhengli-Li; Baric, Ralph S

2015-12-01

The emergence of severe acute respiratory syndrome coronavirus (SARS-CoV) and Middle East respiratory syndrome (MERS)-CoV underscores the threat of cross-species transmission events leading to outbreaks in humans. Here we examine the disease potential of a SARS-like virus, SHC014-CoV, which is currently circulating in Chinese horseshoe bat populations. Using the SARS-CoV reverse genetics system, we generated and characterized a chimeric virus expressing the spike of bat coronavirus SHC014 in a mouse-adapted SARS-CoV backbone. The results indicate that group 2b viruses encoding the SHC014 spike in a wild-type backbone can efficiently use multiple orthologs of the SARS receptor human angiotensin converting enzyme II (ACE2), replicate efficiently in primary human airway cells and achieve in vitro titers equivalent to epidemic strains of SARS-CoV. Additionally, in vivo experiments demonstrate replication of the chimeric virus in mouse lung with notable pathogenesis. Evaluation of available SARS-based immune-therapeutic and prophylactic modalities revealed poor efficacy; both monoclonal antibody and vaccine approaches failed to neutralize and protect from infection with CoVs using the novel spike protein. On the basis of these findings, we synthetically re-derived an infectious full-length SHC014 recombinant virus and demonstrate robust viral replication both in vitro and in vivo. Our work suggests a potential risk of SARS-CoV re-emergence from viruses currently circulating in bat populations.

MicroRNA-33 promotes the replicative senescence of mouse embryonic fibroblasts by suppressing CDK6

DOE Office of Scientific and Technical Information (OSTI.GOV)

Xu, Shun; Huang, Haijiao; Li, Nanhong

2016-05-13

MicroRNAs are a large class of tiny noncoding RNAs, which have emerged as critical regulators of gene expression, and thus are involved in multiple cellular processes, including cellular senescence. MicroRNA-33 has previously been established to exert crucial effect on cell proliferation, lipid metabolism and cholesterol metabolism. Nonetheless, the association between microRNA-33 and cellular senescence and its underlying molecular mechanism are far to be elucidated. The present study has attempted to probe into the effect of microRNA-33 on MEFs senescence. Our data unveiled that microRNA-33 was dramatically down-regulated in senescent MEFs compared to the young MEFs, and ectopic expression of microRNA-33more » promoted MEFs senescence, while knock-down of microRNA-33 exhibited a protective effect against senescence phenotype. Moreover, we verified CDK6 as a direct target of microRNA-33 in mouse. Silencing of CDK6 induced the premature senescence phenotype of MEFs similarly as microRNA-33, while enforced expression of CDK6 significantly reverse the senescence-induction effect of microRNA-33. Taken together, our results suggested that microRNA-33 enhanced the replicative senescence of MEFs potentially by suppressing CDK6 expression. -- Highlights: •MicroRNA-33 was dramatically down-regulated in senescent MEF cells. •Altered expression of microRNA-33 exerted a critical role in MEFs senescence. •MicroRNA-33 promoted the replicative senescence of MEFs via targeting of CDK6.« less

Challenges in identifying and determining the impacts of infection with pestiviruses on the herd health of free ranging cervid populations

USDA-ARS?s Scientific Manuscript database

Although most commonly associated with the infection of domestic livestock, the replication of pestiviruses, in particular bovine viral diarrhea virus (BVDV), occurs in a wide range of free ranging cervids including white-tailed deer, mule deer, fallow deer, elk, red deer, roe deer, eland and moused...

"Isogaba Maware": quality control of genome DNA by checkpoints.

PubMed

Kitazono, A; Matsumoto, T

1998-05-01

Checkpoints maintain the interdependency of cell cycle events by permitting the onset of an event only after the completion of the preceding event. The DNA replication checkpoint induces a cell cycle arrest until the completion of the DNA replication. Similarly, the DNA damage checkpoint arrests cell cycle progression if DNA repair is incomplete. A number of genes that play a role in the two checkpoints have been identified through genetic studies in yeasts, and their homologues have been found in fly, mouse, and human. They form signaling cascades activated by a DNA replication block or DNA damage and subsequently generate the negative constraints on cell cycle regulators. The failure of these signaling cascades results in producing offspring that carry mutations or that lack a portion of the genome. In humans, defects in the checkpoints are often associated with cancer-prone diseases. Focusing mainly on the studies in budding and fission yeasts, we summarize the recent progress.

Inhibition of adenovirus 5 replication in COS-1 cells by antisense RNAs against the viral E1a region.

PubMed

Miroshnichenko, O I; Ponomareva, T I; Tikchonenko, T I

1989-12-07

To study the effect of antisense E1a RNA (asRNA) on adenovirus development, two types of adenovirus 5 E1a antisense constructs have been engineered. One was complementary to the viral DNA region [nucleotide (nt) positions 500-720] regulated by the metallothionein-I promoter, and the other was complementary to the DNA regions (nt positions 630-1570) under control of the long terminal repeat Moloney mouse leukosis virus promoter. Both asRNA constructs were cloned into a plasmid containing the simian virus 40 origin of replication, the gene controlling geneticin (G418) resistance (G418R), and other regulatory elements. The COS-1 cells, which contained up to 100 copies of the engineered plasmids, synthesized antiviral asRNAs, which provided 71 to over 95% inhibition of adenoviral replication, in comparison to the control cells not synthesizing asRNAs.

Modification of Three Amino Acids in Sodium Taurocholate Cotransporting Polypeptide Renders Mice Susceptible to Infection with Hepatitis D Virus In Vivo

PubMed Central

He, Wenhui; Cao, Zhiliang; Mao, Fengfeng; Ren, Bijie; Li, Yunfei; Li, Dan; Li, Huiyu; Peng, Bo; Yan, Huan; Qi, Yonghe; Sun, Yinyan; Wang, Fengchao

2016-01-01

ABSTRACT Sodium taurocholate cotransporting polypeptide (NTCP) was identified as a functional receptor for hepatitis D virus (HDV) and its helper hepatitis B virus (HBV). In cultured cell lines, HDV infection through mouse NTCP is restricted by residues 84 to 87 of the receptor. This study shows that mice with these three amino acids altered their corresponding human residues (H84R, T86K, and S87N) in endogenous mouse NTCP support de novo HDV infection in vivo. HDV infection was documented by the presence of replicative forms of HDV RNA and HDV proteins in liver cells at day 6 after viral inoculation. Monoclonal antibody specifically binding to the motif centered on K86 in NTCP partially inhibited HDV infection. These studies demonstrated specific interaction between the receptor and the viral envelopes in vivo and established a novel mouse model with minimal genetic manipulation for studying HDV infection. The model will also be useful for evaluating entry inhibitors against HDV and its helper HBV. IMPORTANCE NTCP was identified as a functional receptor for both HDV and HBV in cell cultures. We recently showed that neonatal C57BL/6 transgenic (Tg) mice exogenously expressing human NTCP (hNTCP-Tg) in liver support transient HDV infection. In this study, we introduced alterations of three amino acids in the endogenous NTCP of FVB mice through genome editing. The mice with the humanized NTCP residues (H84R, T86K, and S87N) are susceptible to HDV infection, and the infection can be established in both neonatal and adult mice with this editing. We also developed a monoclonal antibody specifically targeting the region of NTCP centered on lysine residue 86, and it can differentiate the modified mouse NTCP from that of the wild type and partially inhibited HDV infection. These studies shed new light on NTCP-mediated HDV infection in vivo, and the NTCP-modified mice provide a useful animal model for studying HDV infection and evaluating antivirals against the infection. PMID:27466423

Comparative study of murid gammaherpesvirus 4 infection in mice and in a natural host, bank voles.

PubMed

François, Sylvie; Vidick, Sarah; Sarlet, Michaël; Michaux, Johan; Koteja, Pawel; Desmecht, Daniel; Stevenson, Philip G; Vanderplasschen, Alain; Gillet, Laurent

2010-10-01

Gammaherpesviruses are archetypal pathogenic persistent viruses. The known human gammaherpesviruses (Epstein-Barr virus and Kaposi's sarcoma-associated herpesvirus) are host-specific and therefore lack a convenient in vivo infection model. This makes related animal gammaherpesviruses an important source of information. Infection by murid herpesvirus 4 (MuHV-4), a virus originally isolated from bank voles (Myodes glareolus), was studied here. MuHV-4 infection of inbred laboratory mouse strains (Mus musculus) is commonly used as a general model of gammaherpesvirus pathogenesis. However, MuHV-4 has not been isolated from house mice, and no systematic comparison has been made between experimental MuHV-4 infections of mice and bank voles. This study therefore characterized MuHV-4 (strain MHV-68) infection of bank voles through global luciferase imaging and classical virological methods. As in mice, intranasal virus inoculation led to productive replication in bank vole lungs, accompanied by massive cellular infiltrates. However, the extent of lytic virus replication was approximately 1000-fold lower in bank voles than in mice. Peak latency titres in lymphoid tissue were also lower, although latency was still established. Finally, virus transmission was tested between animals maintained in captivity. However, as observed in mice, MuHV-4 was not transmitted between voles under these conditions. In conclusion, this study revealed that, despite quantitative differences, replication and the latency sites of MuHV-4 are comparable in bank voles and mice. Therefore, it appears that, so far, Mus musculus represents a suitable host for studying gammaherpesvirus pathogenesis with MuHV-4. Establishing transmission conditions in captivity will be a vital step for further research in this field.

Neural networks and the experience and cultivation of mind.

PubMed

Werbos, Paul J

2012-08-01

Hard core neural network research includes development of mathematical models of cognitive prediction and optimization aimed at dual use, both as models of what we see in brain circuits and behavior, and as useful general-purpose engineering technology. The pathway and principles now exist to let us someday replicate learning abilities as elevated as what we see in the brain of the mouse-but how can this help us today in understanding and maximizing the much greater potential of the human mind, as addressed by many schools of thought all over the world for centuries? This paper discusses how we might use what we have learned at a lower level to better illuminate key phenomena in first person and clinical human experience such as Freud's "psychic energy", the role of traumatic experience, the interpretation of dreams, creativity, the cultivation of sanity and sensitivity, and the biological foundations of language. Published by Elsevier Ltd.

Influenza Infects Lung Microvascular Endothelium Leading to Microvascular Leak: Role of Apoptosis and Claudin-5

PubMed Central

Armstrong, Susan M.; Wang, Changsen; Tigdi, Jayesh; Si, Xiaoe; Dumpit, Carlo; Charles, Steffany; Gamage, Asela; Moraes, Theo J.; Lee, Warren L.

2012-01-01

Severe influenza infections are complicated by acute lung injury, a syndrome of pulmonary microvascular leak. The pathogenesis of this complication is unclear. We hypothesized that human influenza could directly infect the lung microvascular endothelium, leading to loss of endothelial barrier function. We infected human lung microvascular endothelium with both clinical and laboratory strains of human influenza. Permeability of endothelial monolayers was assessed by spectrofluorimetry and by measurement of the transendothelial electrical resistance. We determined the molecular mechanisms of flu-induced endothelial permeability and developed a mouse model of severe influenza. We found that both clinical and laboratory strains of human influenza can infect and replicate in human pulmonary microvascular endothelium, leading to a marked increase in permeability. This was caused by apoptosis of the lung endothelium, since inhibition of caspases greatly attenuated influenza-induced endothelial leak. Remarkably, replication-deficient virus also caused a significant degree of endothelial permeability, despite displaying no cytotoxic effects to the endothelium. Instead, replication-deficient virus induced degradation of the tight junction protein claudin-5; the adherens junction protein VE-cadherin and the actin cytoskeleton were unaffected. Over-expression of claudin-5 was sufficient to prevent replication-deficient virus-induced permeability. The barrier-protective agent formoterol was able to markedly attenuate flu-induced leak in association with dose-dependent induction of claudin-5. Finally, mice infected with human influenza developed pulmonary edema that was abrogated by parenteral treatment with formoterol. Thus, we describe two distinct mechanisms by which human influenza can induce pulmonary microvascular leak. Our findings have implications for the pathogenesis and treatment of acute lung injury from severe influenza. PMID:23115643

Gamma-interferon exerts a critical early restriction on replication and dissemination of yellow fever virus vaccine strain 17D-204.

PubMed

Lam, L K Metthew; Watson, Alan M; Ryman, Kate D; Klimstra, William B

2018-01-01

Live attenuated viruses are historically among the most effective viral vaccines. Development of a safe vaccine requires the virus to be less virulent, a phenotype that is historically arrived by empirical evaluation often leaving the mechanisms of attenuation unknown. The yellow fever virus 17D live attenuated vaccine strain has been developed as a delivery vector for heterologous antigens; however, the mechanisms of attenuation remain elusive. The successful and safe progress of 17D as a vaccine vector and the development of live attenuated vaccines (LAVs) to related flaviviruses requires an understanding of the molecular mechanisms leading to attenuation. Using subcutaneous infection of interferon-deficient mouse models of wild type yellow fever virus (WT YFV) pathogenesis and 17D-mediated immunity, we found that, in the absence of type I IFN (IFN-α/β), type II interferon (IFN-γ) restricted 17D replication, but not that of WT YFV, by 1-2 days post-infection. In this context, IFN-γ responses protected 17D-infected animals from mortality, largely restricted the virus to lymphoid organs, and eliminated viscerotropic disease signs such as steatosis in the liver and inflammatory cell infiltration into the spleen. However, WT YFV caused a disseminated infection, gross liver pathology, and rapid death of the animals. In vitro, IFN-γ treatment of myeloid cells suppressed the replication of 17D significantly more than that of WT YFV, suggesting a direct differential effect on 17D virus replication. Together these data indicate that an important mechanism of 17D attenuation in vivo is increased sensitivity to IFN-γ stimulated responses elicited early after infection.

Enhancing the antiviral potency of ER α-glucosidase inhibitor IHVR-19029 against hemorrhagic fever viruses in vitro and in vivo.

PubMed

Ma, Julia; Zhang, Xuexiang; Soloveva, Veronica; Warren, Travis; Guo, Fang; Wu, Shuo; Lu, Huagang; Guo, Jia; Su, Qing; Shen, Helen; Solon, Eric; Comunale, Mary Ann; Mehta, Anand; Guo, Ju-Tao; Bavari, Sina; Du, Yanming; Block, Timothy M; Chang, Jinhong

2018-02-01

Targeting host functions essential for viral replication has been considered as a broad spectrum and resistance-refractory antiviral approach. However, only a few host functions have, thus far, been validated as broad-spectrum antiviral targets in vivo. ER α-glucosidases I and II have been demonstrated to be essential for the morphogenesis of many enveloped viruses, including members from four families of viruses causing hemorrhagic fever. In vivo antiviral efficacy of various iminosugar-based ER α-glucosidase inhibitors has been reported in animals infected with Dengue, Japanese encephalitis, Ebola, Marburg and influenza viruses. Herein, we established Huh7.5-derived cell lines with ER α-glucosidase I or II knockout using CRISPR/Cas9 and demonstrated that the replication of Dengue, Yellow fever and Zika viruses was reduced by only 1-2 logs in the knockout cell lines. The results clearly indicate that only a partial suppression of viral replication can possibly be achieved with a complete inhibition of ER-α-glucosidases I or II by their inhibitors. We therefore explore to improve the antiviral efficacy of a lead iminosugar IHVR-19029 through combination with another broad-spectrum antiviral agent, favipiravir (T-705). Indeed, combination of IHVR-19029 and T-705 synergistically inhibited the replication of Yellow fever and Ebola viruses in cultured cells. Moreover, in a mouse model of Ebola virus infection, combination of sub-optimal doses of IHVR-19029 and T-705 significantly increased the survival rate of infected animals. We have thus proved the concept of combinational therapeutic strategy for the treatment of viral hemorrhagic fevers with broad spectrum host- and viral- targeting antiviral agents. Copyright © 2017 Elsevier B.V. All rights reserved.

Plasmacytoid Dendritic Cells Suppress HIV-1 Replication but Contribute to HIV-1 Induced Immunopathogenesis in Humanized Mice

PubMed Central

Li, Guangming; Cheng, Menglan; Nunoya, Jun-ichi; Cheng, Liang; Guo, Haitao; Yu, Haisheng; Liu, Yong-jun; Su, Lishan; Zhang, Liguo

2014-01-01

The role of plasmacytoid dendritic cells (pDC) in human immunodeficiency virus type 1 (HIV-1) infection and pathogenesis remains unclear. HIV-1 infection in the humanized mouse model leads to persistent HIV-1 infection and immunopathogenesis, including type I interferons (IFN-I) induction, immune-activation and depletion of human leukocytes, including CD4 T cells. We developed a monoclonal antibody that specifically depletes human pDC in all lymphoid organs in humanized mice. When pDC were depleted prior to HIV-1 infection, the induction of IFN-I and interferon-stimulated genes (ISGs) were abolished during acute HIV-1 infection with either a highly pathogenic CCR5/CXCR4-dual tropic HIV-1 or a standard CCR5-tropic HIV-1 isolate. Consistent with the anti-viral role of IFN-I, HIV-1 replication was significantly up-regulated in pDC-depleted mice. Interestingly, the cell death induced by the highly pathogenic HIV-1 isolate was severely reduced in pDC-depleted mice. During chronic HIV-1 infection, depletion of pDC also severely reduced the induction of IFN-I and ISGs, associated with elevated HIV-1 replication. Surprisingly, HIV-1 induced depletion of human immune cells including T cells in lymphoid organs, but not the blood, was reduced in spite of the increased viral replication. The increased cell number in lymphoid organs was associated with a reduced level of HIV-induced cell death in human leukocytes including CD4 T cells. We conclude that pDC play opposing roles in suppressing HIV-1 replication and in promoting HIV-1 induced immunopathogenesis. These findings suggest that pDC-depletion and IFN-I blockade will provide novel strategies for treating those HIV-1 immune non-responsive patients with persistent immune activation despite effective anti-retrovirus treatment. PMID:25077616

A53T-α-synuclein overexpression in murine locus coeruleus induces Parkinson's disease-like pathology in neurons and glia.

PubMed

Henrich, Martin Timo; Geibl, Fanni Fruzsina; Lee, Bolam; Chiu, Wei-Hua; Koprich, James Benjamin; Brotchie, Jonathan Michael; Timmermann, Lars; Decher, Niels; Matschke, Lina Anita; Oertel, Wolfgang Hermann

2018-05-10

Degeneration of noradrenergic locus coeruleus neurons occurs during the prodromal phase of Parkinson's disease and contributes to a variety of non-motor symptoms, e.g. depression, anxiety and REM sleep behavior disorder. This study was designed to establish the first locus coeruleus α-synucleinopathy mouse model, which should provide sufficient information about the time-course of noradrenergic neurodegeneration, replicate cardinal histopathological features of the human Parkinson's disease neuropathology and finally lead to robust histological markers, which are sufficient to assess the pathological changes in a quantitative and qualitative way. We show that targeted viral vector-mediated overexpression of human mutant A53T-α-synuclein in vivo in locus coeruleus neurons of wild-type mice resulted in progressive noradrenergic neurodegeneration over a time frame of 9 weeks. Observed neuronal cell loss was accompanied by progressive α-synuclein phosphorylation, formation of proteinase K-resistant α-synuclein-aggregates, accumulation of Ubi-1- and p62-positive inclusions in microglia and induction of progressive micro- and astrogliosis. Apart from this local pathology, abundant α-synuclein-positive axons were found in locus coeruleus output regions, indicating rapid anterograde axonal transport of A53T-α-synuclein. Taken together, we present the first model of α-synucleinopathy in the murine locus coeruleus, replicating essential morphological features of human Parkinson's disease pathology. This new model may contribute to the research on prodromal Parkinson's disease, in respect to pathophysiology and the development of disease-modifying therapy.

TRIM79α, an interferon-stimulated gene product, restricts tick-borne encephalitis virus replication by degrading the viral RNA polymerase

PubMed Central

Taylor, R. Travis; Lubick, Kirk J.; Robertson, Shelly J.; Broughton, James P.; Bloom, Marshall E.; Bresnahan, Wade A.; Best, Sonja M.

2011-01-01

In response to virus infection, type I interferons (IFNs) induce several genes, most of whose functions are largely unknown. Here we show that the tripartite motif (TRIM) protein, TRIM79α, is an IFN-stimulated gene (ISG) product that specifically targets tick-borne encephalitis virus (TBEV), a Flavivirus that causes encephalitides in humans. TRIM79α restricts TBEV replication by mediating lysosome-dependent degradation of the flavivirus NS5 protein, an RNA-dependent RNA polymerase essential for virus replication. NS5 degradation was specific to tick-borne flaviviruses as TRIM79α did not recognize NS5 from West Nile virus (WNV) or inhibit WNV replication. In the absence of TRIM79α, IFN-β was less effective in inhibiting tick-borne flavivirus infection of mouse macrophages, highlighting the importance of a single virus-specific ISG in establishing an antiviral state. The specificity of TRIM79α for TBEV reveals a remarkable ability of the innate IFN response to discriminate between closely related flaviviruses. PMID:21925107

Role of zinc-finger anti-viral protein in host defense against Sindbis virus

PubMed Central

Kozaki, Tatsuya; Takahama, Michihiro; Misawa, Takuma; Matsuura, Yoshiharu; Saitoh, Tatsuya

2015-01-01

Accumulating evidence indicates that type I interferon (IFN) mediates the host protective response to RNA viruses. However, the anti-viral effector molecules involved in this response have not been fully identified. Here, we show that zinc-finger anti-viral protein (ZAP), an IFN-inducible gene, plays a critical role in the elimination of Sindbis virus (SINV) in vitro and in vivo. The loss of ZAP greatly enhances the replication of SINV but does not inhibit type I IFN production in primary mouse embryonic fibroblasts (MEFs). ZAP binds and destabilizes SINV RNA, thereby suppressing the replication of SINV. Type I IFN fails to suppress SINV replication in ZAP-deficient MEFs, whereas the ectopic expression of ZAP is sufficient to suppress the replication of SINV in MEFs lacking the expression of type I IFN and the IFN-inducible genes. ZAP-deficient mice are highly susceptible to SINV infection, although they produce sufficient amounts of type I IFN. Therefore, ZAP is an RNA-sensing anti-viral effector molecule that mediates the type-I-IFN-dependent host defense against SINV. PMID:25758257

Replicating Health Economic Models: Firm Foundations or a House of Cards?

PubMed

Bermejo, Inigo; Tappenden, Paul; Youn, Ji-Hee

2017-11-01

Health economic evaluation is a framework for the comparative analysis of the incremental health gains and costs associated with competing decision alternatives. The process of developing health economic models is usually complex, financially expensive and time-consuming. For these reasons, model development is sometimes based on previous model-based analyses; this endeavour is usually referred to as model replication. Such model replication activity may involve the comprehensive reproduction of an existing model or 'borrowing' all or part of a previously developed model structure. Generally speaking, the replication of an existing model may require substantially less effort than developing a new de novo model by bypassing, or undertaking in only a perfunctory manner, certain aspects of model development such as the development of a complete conceptual model and/or comprehensive literature searching for model parameters. A further motivation for model replication may be to draw on the credibility or prestige of previous analyses that have been published and/or used to inform decision making. The acceptability and appropriateness of replicating models depends on the decision-making context: there exists a trade-off between the 'savings' afforded by model replication and the potential 'costs' associated with reduced model credibility due to the omission of certain stages of model development. This paper provides an overview of the different levels of, and motivations for, replicating health economic models, and discusses the advantages, disadvantages and caveats associated with this type of modelling activity. Irrespective of whether replicated models should be considered appropriate or not, complete replicability is generally accepted as a desirable property of health economic models, as reflected in critical appraisal checklists and good practice guidelines. To this end, the feasibility of comprehensive model replication is explored empirically across a small number of recent case studies. Recommendations are put forward for improving reporting standards to enhance comprehensive model replicability.

An acute toxicology study with INGN 007, an oncolytic adenovirus vector, in mice and permissive Syrian hamsters; comparisons with wild-type Ad5 and a replication-defective adenovirus vector

PubMed Central

Lichtenstein, DL; Spencer, JF; Doronin, K; Patra, D; Meyer, JM; Shashkova, EV; Kuppuswamy, M; Dhar, D; Thomas, MA; Tollefson, AE; Zumstein, LA; Wold, WSM; Toth, K

2012-01-01

Oncolytic (replication-competent) adenoviruses as anticancer agents provide new, promising tools to fight cancer. In support of a Phase I clinical trial, here we report safety data with INGN 007 (VRX-007), an oncolytic adenovirus with increased anti-tumor efficacy due to overexpression of the adenovirus-encoded ADP protein. Wild-type adenovirus type 5 (Ad5) and a replication-defective version of Ad5 were also studied as controls. A parallel study investigating the biodistribution of these viruses is described elsewhere in this issue. The toxicology experiments were conducted in two species, the Syrian hamster, which is permissive for INGN 007 and Ad5 replication and the poorly permissive mouse. The studies demonstrated that the safety profile of INGN 007 is similar to Ad5. Both viruses caused transient liver damage upon intravenous injection that resolved by 28 days post-infection. The No-Observable-Adverse-Effect-Level (NOAEL) for INGN 007 in hamsters was 3 × 1010 viral particles per kg. In hamsters, the replication-defective vector caused less toxicity, indicating that replication of Ad vectors in the host is an important factor in pathogenesis. With mice, INGN 007 and Ad5 caused toxicity comparable to the replication-defective adenovirus vector. Partially based on these results, the FDA granted permission to enter into a Phase I clinical trial with INGN 007. PMID:19197324

An acute toxicology study with INGN 007, an oncolytic adenovirus vector, in mice and permissive Syrian hamsters; comparisons with wild-type Ad5 and a replication-defective adenovirus vector.

PubMed

Lichtenstein, D L; Spencer, J F; Doronin, K; Patra, D; Meyer, J M; Shashkova, E V; Kuppuswamy, M; Dhar, D; Thomas, M A; Tollefson, A E; Zumstein, L A; Wold, W S M; Toth, K

2009-08-01

Oncolytic (replication-competent) adenoviruses as anticancer agents provide new, promising tools to fight cancer. In support of a Phase I clinical trial, here we report safety data with INGN 007 (VRX-007), an oncolytic adenovirus with increased anti-tumor efficacy due to overexpression of the adenovirus-encoded ADP protein. Wild-type adenovirus type 5 (Ad5) and a replication-defective version of Ad5 were also studied as controls. A parallel study investigating the biodistribution of these viruses is described elsewhere in this issue. The toxicology experiments were conducted in two species, the Syrian hamster, which is permissive for INGN 007 and Ad5 replication and the poorly permissive mouse. The studies demonstrated that the safety profile of INGN 007 is similar to Ad5. Both viruses caused transient liver damage upon intravenous injection that resolved by 28 days post-infection. The No-Observable-Adverse-Effect-Level (NOAEL) for INGN 007 in hamsters was 3 x 10(10) viral particles per kg. In hamsters, the replication-defective vector caused less toxicity, indicating that replication of Ad vectors in the host is an important factor in pathogenesis. With mice, INGN 007 and Ad5 caused toxicity comparable to the replication-defective adenovirus vector. Partially based on these results, the FDA granted permission to enter into a Phase I clinical trial with INGN 007.

Protein domains connect cell cycle stimulation directly to initiation of DNA replication.

PubMed Central

Gjørup, O V; Rose, P E; Holman, P S; Bockus, B J; Schaffhausen, B S

1994-01-01

Polyoma large T antigen (LT) is the only viral gene product required for viral DNA replication. LT can be divided into two domains, one N-terminal (NT) spanning residues 1-260 and one C-terminal (CT) comprising approximately residues 264-785. NT is known to immortalize primary cells in a manner dependent on binding of pRB/p107. Here a CT construct comprising residues 264-785 was shown to have independent function in DNA replication. CT is entirely sufficient for driving viral DNA replication in vivo in growing mouse cells at a level approaching that of full-length LT. In contrast, CT is strikingly deficient for replication in serum-starved cells. However, this deficiency can be complemented by coexpression of NT. BrdUrd incorporation in transfected, starved cells showed that NT was sufficient for inducing S phase, suggesting a mechanism for complementation. By contrast, CT was unable to induce S phase when tested in the same assay. NT also promotes phosphorylation of sites in CT that are likely to be important for replication. Other DNA tumor virus gene products such as adenovirus E1A 12S and human papillomavirus 16 E7 could also complement CT for replication. Although NT, E1A 12S, and E7 all bind the retinoblastoma gene product (pRB) and p107, genetic analysis demonstrates an additional function, independent of that binding, is responsible for complementation. Images PMID:7991595

Spatio-temporal re-organization of replication foci accompanies replication domain consolidation during human pluripotent stem cell lineage specification

PubMed Central

Wilson, Korey A.; Elefanty, Andrew G.; Stanley, Edouard G.; Gilbert, David M.

2016-01-01

ABSTRACT Lineage specification of both mouse and human pluripotent stem cells (PSCs) is accompanied by spatial consolidation of chromosome domains and temporal consolidation of their replication timing. Replication timing and chromatin organization are both established during G1 phase at the timing decision point (TDP). Here, we have developed live cell imaging tools to track spatio-temporal replication domain consolidation during differentiation. First, we demonstrate that the fluorescence ubiquitination cell cycle indicator (Fucci) system is incapable of demarcating G1/S or G2/M cell cycle transitions. Instead, we employ a combination of fluorescent PCNA to monitor S phase progression, cytokinesis to demarcate mitosis, and fluorescent nucleotides to label early and late replication foci and track their 3D organization into sub-nuclear chromatin compartments throughout all cell cycle transitions. We find that, as human PSCs differentiate, the length of S phase devoted to replication of spatially clustered replication foci increases, coincident with global compartmentalization of domains into temporally clustered blocks of chromatin. Importantly, re-localization and anchorage of domains was completed prior to the onset of S phase, even in the context of an abbreviated PSC G1 phase. This approach can also be employed to investigate cell fate transitions in single PSCs, which could be seen to differentiate preferentially from G1 phase. Together, our results establish real-time, live-cell imaging methods for tracking cell cycle transitions during human PSC differentiation that can be applied to study chromosome domain consolidation and other aspects of lineage specification. PMID:27433885

Genomic pathways modulated by Twist in breast cancer.

PubMed

Vesuna, Farhad; Bergman, Yehudit; Raman, Venu

2017-01-13

The basic helix-loop-helix transcription factor TWIST1 (Twist) is involved in embryonic cell lineage determination and mesodermal differentiation. There is evidence to indicate that Twist expression plays a role in breast tumor formation and metastasis, but the role of Twist in dysregulating pathways that drive the metastatic cascade is unclear. Moreover, many of the genes and pathways dysregulated by Twist in cell lines and mouse models have not been validated against data obtained from larger, independant datasets of breast cancer patients. We over-expressed the human Twist gene in non-metastatic MCF-7 breast cancer cells to generate the estrogen-independent metastatic breast cancer cell line MCF-7/Twist. These cells were inoculated in the mammary fat pad of female severe compromised immunodeficient mice, which subsequently formed xenograft tumors that metastasized to the lungs. Microarray data was collected from both in vitro (MCF-7 and MCF-7/Twist cell lines) and in vivo (primary tumors and lung metastases) models of Twist expression. Our data was compared to several gene datasets of various subtypes, classes, and grades of human breast cancers. Our data establishes a Twist over-expressing mouse model of breast cancer, which metastasizes to the lung and replicates some of the ontogeny of human breast cancer progression. Gene profiling data, following Twist expression, exhibited novel metastasis driver genes as well as cellular maintenance genes that were synonymous with the metastatic process. We demonstrated that the genes and pathways altered in the transgenic cell line and metastatic animal models parallel many of the dysregulated gene pathways observed in human breast cancers. Analogous gene expression patterns were observed in both in vitro and in vivo Twist preclinical models of breast cancer metastasis and breast cancer patient datasets supporting the functional role of Twist in promoting breast cancer metastasis. The data suggests that genetic dysregulation of Twist at the cellular level drives alterations in gene pathways in the Twist metastatic mouse model which are comparable to changes seen in human breast cancers. Lastly, we have identified novel genes and pathways that could be further investigated as targets for drugs to treat metastatic breast cancer.

Systematic mutagenesis of genes encoding predicted autotransported proteins of Burkholderia pseudomallei identifies factors mediating virulence in mice, net intracellular replication and a novel protein conferring serum resistance.

PubMed

Lazar Adler, Natalie R; Stevens, Mark P; Dean, Rachel E; Saint, Richard J; Pankhania, Depesh; Prior, Joann L; Atkins, Timothy P; Kessler, Bianca; Nithichanon, Arnone; Lertmemongkolchai, Ganjana; Galyov, Edouard E

2015-01-01

Burkholderia pseudomallei is the causative agent of the severe tropical disease melioidosis, which commonly presents as sepsis. The B. pseudomallei K96243 genome encodes eleven predicted autotransporters, a diverse family of secreted and outer membrane proteins often associated with virulence. In a systematic study of these autotransporters, we constructed insertion mutants in each gene predicted to encode an autotransporter and assessed them for three pathogenesis-associated phenotypes: virulence in the BALB/c intra-peritoneal mouse melioidosis model, net intracellular replication in J774.2 murine macrophage-like cells and survival in 45% (v/v) normal human serum. From the complete repertoire of eleven autotransporter mutants, we identified eight mutants which exhibited an increase in median lethal dose of 1 to 2-log10 compared to the isogenic parent strain (bcaA, boaA, boaB, bpaA, bpaC, bpaE, bpaF and bimA). Four mutants, all demonstrating attenuation for virulence, exhibited reduced net intracellular replication in J774.2 macrophage-like cells (bimA, boaB, bpaC and bpaE). A single mutant (bpaC) was identified that exhibited significantly reduced serum survival compared to wild-type. The bpaC mutant, which demonstrated attenuation for virulence and net intracellular replication, was sensitive to complement-mediated killing via the classical and/or lectin pathway. Serum resistance was rescued by in trans complementation. Subsequently, we expressed recombinant proteins of the passenger domain of four predicted autotransporters representing each of the phenotypic groups identified: those attenuated for virulence (BcaA), those attenuated for virulence and net intracellular replication (BpaE), the BpaC mutant with defects in virulence, net intracellular replication and serum resistance and those displaying wild-type phenotypes (BatA). Only BcaA and BpaE elicited a strong IFN-γ response in a restimulation assay using whole blood from seropositive donors and were recognised by seropositive human sera from the endemic area. To conclude, several predicted autotransporters contribute to B. pseudomallei virulence and BpaC may do so by conferring resistance against complement-mediated killing.

3D replicon distributions arise from stochastic initiation and domino-like DNA replication progression.

PubMed

Löb, D; Lengert, N; Chagin, V O; Reinhart, M; Casas-Delucchi, C S; Cardoso, M C; Drossel, B

2016-04-07

DNA replication dynamics in cells from higher eukaryotes follows very complex but highly efficient mechanisms. However, the principles behind initiation of potential replication origins and emergence of typical patterns of nuclear replication sites remain unclear. Here, we propose a comprehensive model of DNA replication in human cells that is based on stochastic, proximity-induced replication initiation. Critical model features are: spontaneous stochastic firing of individual origins in euchromatin and facultative heterochromatin, inhibition of firing at distances below the size of chromatin loops and a domino-like effect by which replication forks induce firing of nearby origins. The model reproduces the empirical temporal and chromatin-related properties of DNA replication in human cells. We advance the one-dimensional DNA replication model to a spatial model by taking into account chromatin folding in the nucleus, and we are able to reproduce the spatial and temporal characteristics of the replication foci distribution throughout S-phase.

Variations in immunohistochemical preservation of proteins in a mummification model

PubMed Central

Metcalfe, Ryan; Freemont, Tony

2012-01-01

Immunohistochemistry is an important tool in the investigation of ancient mummified remains because of its ability not only to detect proteins but also to isolate their location to specific tissues and thereby improve confidence that the results are genuine. A mouse model of Egyptian mummification has been used to demonstrate that the survival of proteins, judged by the retention of immunohistochemical staining, varies markedly. Some survive the process well, whereas others become barely detectable despite the morphology of the tissue being excellently preserved. The results obtained show that protein preservation is multi-factorial, with tissue type and degradation, and the properties of the protein itself all having significant effects. Proteins forming large, multi-subunit complexes such as collagen IV appear to be more resistant to degradation than those that do not, such as S-100. Although modern modelling studies cannot replicate the full extent of degradative processes and taphonomic changes experienced by real mummies, the results obtained can be useful for guiding research that requires ancient tissues. PMID:22050406

Severe Acute Respiratory Syndrome Coronavirus Replication Is Severely Impaired by MG132 due to Proteasome-Independent Inhibition of M-Calpain

PubMed Central

Schneider, Martha; Ackermann, Kerstin; Stuart, Melissa; Wex, Claudia; Protzer, Ulrike; Schätzl, Hermann M.

2012-01-01

The ubiquitin-proteasome system (UPS) is involved in the replication of a broad range of viruses. Since replication of the murine hepatitis virus (MHV) is impaired upon proteasomal inhibition, the relevance of the UPS for the replication of the severe acute respiratory syndrome coronavirus (SARS-CoV) was investigated in this study. We demonstrate that the proteasomal inhibitor MG132 strongly inhibits SARS-CoV replication by interfering with early steps of the viral life cycle. Surprisingly, other proteasomal inhibitors (e.g., lactacystin and bortezomib) only marginally affected viral replication, indicating that the effect of MG132 is independent of proteasomal impairment. Induction of autophagy by MG132 treatment was excluded from playing a role, and no changes in SARS-CoV titers were observed during infection of wild-type or autophagy-deficient ATG5−/− mouse embryonic fibroblasts overexpressing the human SARS-CoV receptor, angiotensin-converting enzyme 2 (ACE2). Since MG132 also inhibits the cysteine protease m-calpain, we addressed the role of calpains in the early SARS-CoV life cycle using calpain inhibitors III (MDL28170) and VI (SJA6017). In fact, m-calpain inhibition with MDL28170 resulted in an even more pronounced inhibition of SARS-CoV replication (>7 orders of magnitude) than did MG132. Additional m-calpain knockdown experiments confirmed the dependence of SARS-CoV replication on the activity of the cysteine protease m-calpain. Taken together, we provide strong experimental evidence that SARS-CoV has unique replication requirements which are independent of functional UPS or autophagy pathways compared to other coronaviruses. Additionally, this work highlights an important role for m-calpain during early steps of the SARS-CoV life cycle. PMID:22787216

Hepatitis B virus core antigen determines viral persistence in a C57BL/6 mouse model.

PubMed

Lin, Yi-Jiun; Huang, Li-Rung; Yang, Hung-Chih; Tzeng, Horng-Tay; Hsu, Ping-Ning; Wu, Hui-Lin; Chen, Pei-Jer; Chen, Ding-Shinn

2010-05-18

We recently developed a mouse model of hepatitis B virus (HBV) persistence, in which a single i.v. hydrodynamic injection of HBV DNA to C57BL/6 mice allows HBV replication and induces a partial immune response, so that about 20-30% of the mice carry HBV for more than 6 months. The model was used to identify the viral antigen crucial for HBV persistence. We knocked out individual HBV genes by introducing a premature termination codon to the HBV core, HBeAg, HBx, and polymerase ORFs. The specific-gene-deficient HBV mutants were hydrodynamically injected into mice and the HBV profiles of the mice were monitored. About 90% of the mice that received the HBcAg-mutated HBV plasmid exhibited high levels of hepatitis B surface antigenemia and maintained HBsAg expression for more than 6 months after injection. To map the region of HBcAg essential for viral clearance, we constructed a set of serial HBcAg deletion mutants for hydrodynamic injection. We localized the essential region of HBcAg to the carboxyl terminus, specifically to the 10 terminal amino acids (HBcAg176-185). The majority of mice receiving this HBV mutant DNA did not elicit a proper HBcAg-specific IFN-gamma response and expressed HBV virions for 6 months. These results indicate that the immune response triggered in mice by HBcAg during exposure to HBV is important in determining HBV persistence.

Defective prolactin signaling impairs pancreatic β-cell development during the perinatal period

PubMed Central

Auffret, Julien; Freemark, Michael; Carré, Nadège; Mathieu, Yves; Tourrel-Cuzin, Cécile; Lombès, Marc; Movassat, Jamileh

2013-01-01

Prolactin (PRL) and placental lactogens stimulate β-cell replication and insulin production in pancreatic islets and insulinoma cells through binding to the PRL receptor (PRLR). However, the contribution of PRLR signaling to β-cell ontogeny and function in perinatal life and the effects of the lactogens on adaptive islet growth are poorly understood. We provide evidence that expansion of β-cell mass during both embryogenesis and the postnatal period is impaired in the PRLR−/− mouse model. PRLR−/− newborns display a 30% reduction of β-cell mass, consistent with reduced proliferation index at E18.5. PRL stimulates leucine incorporation and S6 kinase phosphorylation in INS-1 cells, supporting a role for β-cell mTOR signaling in PRL action. Interestingly, a defect in the development of acini is also observed in absence of PRLR signaling, with a sharp decline in cellular size in both endocrine and exocrine compartments. Of note, a decrease in levels of IGF-II, a PRL target, in the Goto-Kakizaki (GK) rat, a spontaneous model of type 2 diabetes, is associated with a lack of PRL-mediated β-cell proliferation in embryonic pancreatic buds. Reduced pancreatic IGF-II expression in both rat and mouse models suggests that this factor may constitute a molecular link between PRL signaling and cell ontogenesis. Together, these results provide evidence that PRL signaling is essential for pancreas ontogenesis during the critical perinatal window responsible for establishing functional β-cell reserve. PMID:24064341

Altered development of the brain after focal herpesvirus infection of the central nervous system.

PubMed

Koontz, Thad; Bralic, Marina; Tomac, Jelena; Pernjak-Pugel, Ester; Bantug, Glen; Jonjic, Stipan; Britt, William J

2008-02-18

Human cytomegalovirus infection of the developing central nervous system (CNS) is a major cause of neurological damage in newborn infants and children. To investigate the pathogenesis of this human infection, we developed a mouse model of infection in the developing CNS. Intraperitoneal inoculation of newborn animals with murine cytomegalovirus resulted in virus replication in the liver followed by virus spread to the brain. Virus infection of the CNS was associated with the induction of inflammatory responses, including the induction of a large number of interferon-stimulated genes and histological evidence of focal encephalitis with recruitment of mononuclear cells to foci containing virus-infected cells. The morphogenesis of the cerebellum was delayed in infected animals. The defects in cerebellar development in infected animals were generalized and, although correlated temporally with virus replication and CNS inflammation, spatially unrelated to foci of virus-infected cells. Specific defects included decreased granular neuron proliferation and migration, expression of differentiation markers, and activation of neurotrophin receptors. These findings suggested that in the developing CNS, focal virus infection and induction of inflammatory responses in resident and infiltrating mononuclear cells resulted in delayed cerebellar morphogenesis.

Altered development of the brain after focal herpesvirus infection of the central nervous system

PubMed Central

Koontz, Thad; Bralic, Marina; Tomac, Jelena; Pernjak-Pugel, Ester; Bantug, Glen; Jonjic, Stipan; Britt, William J.

2008-01-01

Human cytomegalovirus infection of the developing central nervous system (CNS) is a major cause of neurological damage in newborn infants and children. To investigate the pathogenesis of this human infection, we developed a mouse model of infection in the developing CNS. Intraperitoneal inoculation of newborn animals with murine cytomegalovirus resulted in virus replication in the liver followed by virus spread to the brain. Virus infection of the CNS was associated with the induction of inflammatory responses, including the induction of a large number of interferon-stimulated genes and histological evidence of focal encephalitis with recruitment of mononuclear cells to foci containing virus-infected cells. The morphogenesis of the cerebellum was delayed in infected animals. The defects in cerebellar development in infected animals were generalized and, although correlated temporally with virus replication and CNS inflammation, spatially unrelated to foci of virus-infected cells. Specific defects included decreased granular neuron proliferation and migration, expression of differentiation markers, and activation of neurotrophin receptors. These findings suggested that in the developing CNS, focal virus infection and induction of inflammatory responses in resident and infiltrating mononuclear cells resulted in delayed cerebellar morphogenesis. PMID:18268036

Pneumonia Virus of Mice Severe Respiratory Virus Infection in a Natural Host

PubMed Central

Rosenberg, Helene F.; Domachowske, Joseph B.

2008-01-01

Pneumonia virus of mice (PVM; family Paramyxoviridae, genus Pneumovirus) is a natural mouse pathogen that is closely related to the human and bovine respiratory syncytial viruses. Among the prominent features of this infection, robust replication of PVM takes place in bronchial epithelial cells in response to a minimal virus inoculum. Virus replication in situ results in local production of proinflammatory cytokines (MIP-1α, MIP-2, MCP-1 and IFNγ) and granulocyte recruitment to the lung. If left unchecked, PVM infection and the ensuing inflammatory response ultimately lead to pulmonary edema, respiratory compromise and death. In this review, we consider the recent studies using the PVM model that have provided important insights into the role of the inflammatory response in the pathogenesis of severe respiratory virus infection. We also highlight several works that have elucidated acquired immune responses to this pathogen, including T cell responses and the development of humoral immunity. Finally, we consider several immunomodulatory strategies that have been used successfully to reduce morbidity and mortality when administered to PVM infected, symptomatic mice, and thus hold promise as realistic therapeutic strategies for severe respiratory virus infections in human subjects. PMID:18471897

Mouse-adapted MERS coronavirus causes lethal lung disease in human DPP4 knockin mice.

PubMed

Li, Kun; Wohlford-Lenane, Christine L; Channappanavar, Rudragouda; Park, Jung-Eun; Earnest, James T; Bair, Thomas B; Bates, Amber M; Brogden, Kim A; Flaherty, Heather A; Gallagher, Tom; Meyerholz, David K; Perlman, Stanley; McCray, Paul B

2017-04-11

The Middle East respiratory syndrome (MERS) emerged in Saudi Arabia in 2012, caused by a zoonotically transmitted coronavirus (CoV). Over 1,900 cases have been reported to date, with ∼36% fatality rate. Lack of autopsies from MERS cases has hindered understanding of MERS-CoV pathogenesis. A small animal model that develops progressive pulmonary manifestations when infected with MERS-CoV would advance the field. As mice are restricted to infection at the level of DPP4, the MERS-CoV receptor, we generated mice with humanized exons 10-12 of the mouse Dpp4 locus. Upon inoculation with MERS-CoV, human DPP4 knockin (KI) mice supported virus replication in the lungs, but developed no illness. After 30 serial passages through the lungs of KI mice, a mouse-adapted virus emerged (MERS MA ) that grew in lungs to over 100 times higher titers than the starting virus. A plaque-purified MERS MA clone caused weight loss and fatal infection. Virus antigen was observed in airway epithelia, pneumocytes, and macrophages. Pathologic findings included diffuse alveolar damage with pulmonary edema and hyaline membrane formation associated with accumulation of activated inflammatory monocyte-macrophages and neutrophils in the lungs. Relative to the parental MERS-CoV, MERS MA viruses contained 13-22 mutations, including several within the spike (S) glycoprotein gene. S-protein mutations sensitized viruses to entry-activating serine proteases and conferred more rapid entry kinetics. Recombinant MERS MA bearing mutant S proteins were more virulent than the parental virus in hDPP4 KI mice. The hDPP4 KI mouse and the MERS MA provide tools to investigate disease causes and develop new therapies.

The systemic delivery of an oncolytic adenovirus expressing decorin inhibits bone metastasis in a mouse model of human prostate cancer

DOE PAGES

Xu, Weidong; Neill, Thomas; Yang, Yuefeng; ...

2014-12-11

In an effort to develop a new therapy for prostate cancer bone metastases, we have created Ad.dcn, a recombinant oncolytic adenovirus carrying the human decorin gene. Infection of PC-3 and DU-145, the human prostate tumor cells, with Ad.dcn or a non-replicating adenovirus Ad(E1-).dcn resulted in decorin expression; Ad.dcn produced high viral titers and cytotoxicity in human prostate tumor cells. Adenoviral-mediated decorin expression inhibited Met, the Wnt/β- catenin signaling axis, vascular endothelial growth factor A, reduced mitochondrial DNA levels, and inhibited tumor cell migration. To examine the anti-tumor response of Ad.dcn, PC-3-luc cells were inoculated in the left heart ventricle tomore » establish bone metastases in nude mice. Ad.dcn, in conjunction with control replicating and non-replicating vectors were injected via tail vein. The real-time monitoring of mice, once a week, by bioluminescence imaging and X-ray radiography showed that Ad.dcn produced significant inhibition of skeletal metastases. Analyses of the mice at the terminal time point indicated a significant reduction in the tumor burden, osteoclast number, serum TRACP 5b levels, osteocalcin levels, hypercalcemia, inhibition of cancer cachexia, and an increase in the animal survival. Finally, based on these studies, we believe that Ad.dcn can be developed as a potential new therapy for prostate cancer bone metastasis.« less

Genome-wide association study using extreme truncate selection identifies novel genes affecting bone mineral density and fracture risk.

PubMed

Duncan, Emma L; Danoy, Patrick; Kemp, John P; Leo, Paul J; McCloskey, Eugene; Nicholson, Geoffrey C; Eastell, Richard; Prince, Richard L; Eisman, John A; Jones, Graeme; Sambrook, Philip N; Reid, Ian R; Dennison, Elaine M; Wark, John; Richards, J Brent; Uitterlinden, Andre G; Spector, Tim D; Esapa, Chris; Cox, Roger D; Brown, Steve D M; Thakker, Rajesh V; Addison, Kathryn A; Bradbury, Linda A; Center, Jacqueline R; Cooper, Cyrus; Cremin, Catherine; Estrada, Karol; Felsenberg, Dieter; Glüer, Claus-C; Hadler, Johanna; Henry, Margaret J; Hofman, Albert; Kotowicz, Mark A; Makovey, Joanna; Nguyen, Sing C; Nguyen, Tuan V; Pasco, Julie A; Pryce, Karena; Reid, David M; Rivadeneira, Fernando; Roux, Christian; Stefansson, Kari; Styrkarsdottir, Unnur; Thorleifsson, Gudmar; Tichawangana, Rumbidzai; Evans, David M; Brown, Matthew A

2011-04-01

Osteoporotic fracture is a major cause of morbidity and mortality worldwide. Low bone mineral density (BMD) is a major predisposing factor to fracture and is known to be highly heritable. Site-, gender-, and age-specific genetic effects on BMD are thought to be significant, but have largely not been considered in the design of genome-wide association studies (GWAS) of BMD to date. We report here a GWAS using a novel study design focusing on women of a specific age (postmenopausal women, age 55-85 years), with either extreme high or low hip BMD (age- and gender-adjusted BMD z-scores of +1.5 to +4.0, n = 1055, or -4.0 to -1.5, n = 900), with replication in cohorts of women drawn from the general population (n = 20,898). The study replicates 21 of 26 known BMD-associated genes. Additionally, we report suggestive association of a further six new genetic associations in or around the genes CLCN7, GALNT3, IBSP, LTBP3, RSPO3, and SOX4, with replication in two independent datasets. A novel mouse model with a loss-of-function mutation in GALNT3 is also reported, which has high bone mass, supporting the involvement of this gene in BMD determination. In addition to identifying further genes associated with BMD, this study confirms the efficiency of extreme-truncate selection designs for quantitative trait association studies.

Genome-Wide Association Study Using Extreme Truncate Selection Identifies Novel Genes Affecting Bone Mineral Density and Fracture Risk

PubMed Central

Duncan, Emma L.; Danoy, Patrick; Kemp, John P.; Leo, Paul J.; McCloskey, Eugene; Nicholson, Geoffrey C.; Eastell, Richard; Prince, Richard L.; Eisman, John A.; Jones, Graeme; Sambrook, Philip N.; Reid, Ian R.; Dennison, Elaine M.; Wark, John; Richards, J. Brent; Uitterlinden, Andre G.; Spector, Tim D.; Esapa, Chris; Cox, Roger D.; Brown, Steve D. M.; Thakker, Rajesh V.; Addison, Kathryn A.; Bradbury, Linda A.; Center, Jacqueline R.; Cooper, Cyrus; Cremin, Catherine; Estrada, Karol; Felsenberg, Dieter; Glüer, Claus-C.; Hadler, Johanna; Henry, Margaret J.; Hofman, Albert; Kotowicz, Mark A.; Makovey, Joanna; Nguyen, Sing C.; Nguyen, Tuan V.; Pasco, Julie A.; Pryce, Karena; Reid, David M.; Rivadeneira, Fernando; Roux, Christian; Stefansson, Kari; Styrkarsdottir, Unnur; Thorleifsson, Gudmar; Tichawangana, Rumbidzai; Evans, David M.; Brown, Matthew A.

2011-01-01

Osteoporotic fracture is a major cause of morbidity and mortality worldwide. Low bone mineral density (BMD) is a major predisposing factor to fracture and is known to be highly heritable. Site-, gender-, and age-specific genetic effects on BMD are thought to be significant, but have largely not been considered in the design of genome-wide association studies (GWAS) of BMD to date. We report here a GWAS using a novel study design focusing on women of a specific age (postmenopausal women, age 55–85 years), with either extreme high or low hip BMD (age- and gender-adjusted BMD z-scores of +1.5 to +4.0, n = 1055, or −4.0 to −1.5, n = 900), with replication in cohorts of women drawn from the general population (n = 20,898). The study replicates 21 of 26 known BMD–associated genes. Additionally, we report suggestive association of a further six new genetic associations in or around the genes CLCN7, GALNT3, IBSP, LTBP3, RSPO3, and SOX4, with replication in two independent datasets. A novel mouse model with a loss-of-function mutation in GALNT3 is also reported, which has high bone mass, supporting the involvement of this gene in BMD determination. In addition to identifying further genes associated with BMD, this study confirms the efficiency of extreme-truncate selection designs for quantitative trait association studies. PMID:21533022

Replication-Competent Influenza A and B Viruses Expressing a Fluorescent Dynamic Timer Protein for In Vitro and In Vivo Studies

PubMed Central

Baker, Steven F.; Perez, Daniel R.; Martínez-Sobrido, Luis

2016-01-01

Influenza A and B viruses (IAV and IBV, respectively) cause annual seasonal human respiratory disease epidemics. In addition, IAVs have been implicated in occasional pandemics with inordinate health and economic consequences. Studying influenza viruses in vitro or in vivo requires the use of laborious secondary methodologies to identify infected cells. To circumvent this requirement, replication-competent infectious influenza viruses expressing an easily traceable fluorescent reporter protein can be used. Timer is a fluorescent protein that undergoes a time-dependent color emission conversion from green to red. The rate of spectral change is independent of Timer protein concentration and can be used to chronologically measure the duration of its expression. Here, we describe the generation of replication-competent IAV and IBV where the viral non-structural protein 1 (NS1) was fused to the fluorescent dynamic Timer protein. Timer-expressing IAV and IBV displayed similar plaque phenotypes and growth kinetics to wild-type viruses in tissue culture. Within infected cells, Timer’s spectral shift can be used to measure the rate and cell-to-cell spread of infection using fluorescent microscopy, plate readers, or flow cytometry. The progression of Timer-expressing IAV infection was also evaluated in a mouse model, demonstrating the feasibility to characterize IAV cell-to-cell infections in vivo. By providing the ability to chronologically track viral spread, Timer-expressing influenza viruses are an excellent option to evaluate the in vitro and in vivo dynamics of viral infection. PMID:26809059

Replication-Competent Influenza A and B Viruses Expressing a Fluorescent Dynamic Timer Protein for In Vitro and In Vivo Studies.

PubMed

Breen, Michael; Nogales, Aitor; Baker, Steven F; Perez, Daniel R; Martínez-Sobrido, Luis

2016-01-01

Influenza A and B viruses (IAV and IBV, respectively) cause annual seasonal human respiratory disease epidemics. In addition, IAVs have been implicated in occasional pandemics with inordinate health and economic consequences. Studying influenza viruses in vitro or in vivo requires the use of laborious secondary methodologies to identify infected cells. To circumvent this requirement, replication-competent infectious influenza viruses expressing an easily traceable fluorescent reporter protein can be used. Timer is a fluorescent protein that undergoes a time-dependent color emission conversion from green to red. The rate of spectral change is independent of Timer protein concentration and can be used to chronologically measure the duration of its expression. Here, we describe the generation of replication-competent IAV and IBV where the viral non-structural protein 1 (NS1) was fused to the fluorescent dynamic Timer protein. Timer-expressing IAV and IBV displayed similar plaque phenotypes and growth kinetics to wild-type viruses in tissue culture. Within infected cells, Timer's spectral shift can be used to measure the rate and cell-to-cell spread of infection using fluorescent microscopy, plate readers, or flow cytometry. The progression of Timer-expressing IAV infection was also evaluated in a mouse model, demonstrating the feasibility to characterize IAV cell-to-cell infections in vivo. By providing the ability to chronologically track viral spread, Timer-expressing influenza viruses are an excellent option to evaluate the in vitro and in vivo dynamics of viral infection.

Pre-Clinical Efficacy and Safety of Experimental Vaccines Based on Non-Replicating Vaccinia Vectors against Yellow Fever

PubMed Central

Schäfer, Birgit; Holzer, Georg W.; Joachimsthaler, Alexandra; Coulibaly, Sogue; Schwendinger, Michael; Crowe, Brian A.; Kreil, Thomas R.; Barrett, P. Noel; Falkner, Falko G.

2011-01-01

Background Currently existing yellow fever (YF) vaccines are based on the live attenuated yellow fever virus 17D strain (YFV-17D). Although, a good safety profile was historically attributed to the 17D vaccine, serious adverse events have been reported, making the development of a safer, more modern vaccine desirable. Methodology/Principal Findings A gene encoding the precursor of the membrane and envelope (prME) protein of the YFV-17D strain was inserted into the non-replicating modified vaccinia virus Ankara and into the D4R-defective vaccinia virus. Candidate vaccines based on the recombinant vaccinia viruses were assessed for immunogenicity and protection in a mouse model and compared to the commercial YFV-17D vaccine. The recombinant live vaccines induced γ-interferon-secreting CD4- and functionally active CD8-T cells, and conferred full protection against lethal challenge already after a single low immunization dose of 105 TCID50. Surprisingly, pre-existing immunity against wild-type vaccinia virus did not negatively influence protection. Unlike the classical 17D vaccine, the vaccinia virus-based vaccines did not cause mortality following intracerebral administration in mice, demonstrating better safety profiles. Conclusions/Significance The non-replicating recombinant YF candidate live vaccines induced a broad immune response after single dose administration, were effective even in the presence of a pre-existing immunity against vaccinia virus and demonstrated an excellent safety profile in mice. PMID:21931732

Live or let die: manipulation of cellular suicide programs by murine cytomegalovirus.

PubMed

Handke, Wiebke; Krause, Eva; Brune, Wolfram

2012-11-01

Cytomegaloviruses (CMVs) are large double-stranded DNA viruses that replicate slowly and cause life-long persisting infections in their hosts. To achieve this, the CMVs had to evolve numerous countermeasures against innate and adaptive immune responses. Induction of programmed cell death is one important host defense mechanism against intracellular pathogens such as viruses. For a multicellular organism, it is advantageous to let infected cells die in order to thwart viral replication and dissemination. For a virus, by contrast, it is better to inhibit cell death and keep infected cells alive until the viral replication cycle has been completed. As a matter of fact, the CMVs encode a number of proteins devoted to interfering with different forms of programmed cell death: apoptosis and necroptosis. In this review, we summarize the known functions of the four best characterized cell death inhibitors of murine cytomegalovirus (MCMV), which are encoded by open reading frames, M36, m38.5, m41.1, and M45. The viral proteins interact with key molecules within different cell death pathways, namely caspase-8, Bax, Bak, and RIP1/RIP3. In addition, we discuss which events during MCMV infection might trigger apoptosis or necrosis and how MCMV's countermeasures compare to those of other herpesviruses. Since both, MCMV and its natural host, are amenable to genetic manipulation, the mouse model for CMV infection provides a particularly suitable system to study mechanisms of cell death induction and inhibition.

Vectors for Treatment of Metastatic Breast Cancer

DTIC Science & Technology

2005-08-01

from human carcinomas of the breast, ovary and mouse breast cancer. In this study, in vitro cytotoxicity tests were carried out with the replication...injection of dendritic cells engineered to secrete interleukin-12 by recombinant adenovirus in patients with metastatic gastrointestinal carcinomas . J...see Figure IC) in carcinomas of the breast, lung, prostate, ovary, cervix, endometrium, esophagous, stomach and colon is associated with metastasis

Cell migration is another player of the minute virus of mice infection

DOE Office of Scientific and Technical Information (OSTI.GOV)

Garcin, Pierre O.; Panté, Nelly, E-mail: pante@zoology.ubc.ca

2014-11-15

The parvovirus minute virus of mice, prototype strain (MVMp), preferentially infects and kills cancer cells. This intrinsic MVMp oncotropism may depend in part on the early stages of MVMp infection. To test this hypothesis, we investigated the early events of MVMp infection in mouse LA9 fibroblasts and a highly invasive mouse mammary tumor cell line derived from polyomavirus middle T antigen-mediated transformation. Using a combination of fluorescence and electron microscopy, we found that various parameters of the cell migration process affect MVMp infection. We show that, after binding to the plasma membrane, MVMp particles rapidly cluster at the leading edgemore » of migrating cells, which exhibit higher levels of MVMp uptake than non-motile cells. Moreover, promoting cell migration on a fibronectin matrix increased MVMp infection, and induction of epithelial–mesenchymal transition allowed MVMp replication in non-permissive epithelial cells. Hence, we propose that cell migration influences the early stages of MVMp infection. - Highlights: • We document early steps of MVMp infection. • We report that a fibronectin matrix promotes MVMp infection. • We show that cellular migration plays a role in MVMp uptake. • We show that epithelial–mesenchymal transition allows MVMp replication.« less

Reward-Related Behavioral Paradigms for Addiction Research in the Mouse: Performance of Common Inbred Strains

PubMed Central

Feyder, Michael; Brigman, Jonathan L.; Crombag, Hans S.; Saksida, Lisa M.; Bussey, Timothy J.; Holmes, Andrew

2011-01-01

The mouse has emerged as a uniquely valuable species for studying the molecular and genetic basis of complex behaviors and modeling neuropsychiatric disease states. While valid and reliable preclinical assays for reward-related behaviors are critical to understanding addiction-related processes, and various behavioral procedures have been developed and characterized in rats and primates, there have been relatively few studies using operant-based addiction-relevant behavioral paradigms in the mouse. Here we describe the performance of the C57BL/6J inbred mouse strain on three major reward-related paradigms, and replicate the same procedures in two other commonly used inbred strains (DBA/2J, BALB/cJ). We examined Pavlovian-instrumental transfer (PIT) by measuring the ability of an auditory cue associated with food reward to promote an instrumental (lever press) response. In a separate experiment, we assessed the acquisition and extinction of a simple stimulus-reward instrumental behavior on a touchscreen-based task. Reinstatement of this behavior was then examined following either continuous exposure to cues (conditioned reinforcers, CRs) associated with reward, brief reward and CR exposure, or brief reward exposure followed by continuous CR exposure. The third paradigm examined sensitivity of an instrumental (lever press) response to devaluation of food reward (a probe for outcome insensitive, habitual behavior) by repeated pairing with malaise. Results showed that C57BL/6J mice displayed robust PIT, as well as clear extinction and reinstatement, but were insensitive to reinforcer devaluation. DBA/2J mice showed good PIT and (rewarded) reinstatement, but were slow to extinguish and did not show reinforcer devaluation or significant CR-reinstatement. BALB/cJ mice also displayed good PIT, extinction and reinstatement, and retained instrumental responding following devaluation, but, unlike the other strains, demonstrated reduced Pavlovian approach behavior (food magazine head entries). Overall, these assays provide robust paradigms for future studies using the mouse to elucidate the neural, molecular and genetic factors underpinning reward-related behaviors relevant to addiction research. PMID:21249214

Reward-related behavioral paradigms for addiction research in the mouse: performance of common inbred strains.

PubMed

Lederle, Lauren; Weber, Susanna; Wright, Tara; Feyder, Michael; Brigman, Jonathan L; Crombag, Hans S; Saksida, Lisa M; Bussey, Timothy J; Holmes, Andrew

2011-01-10

The mouse has emerged as a uniquely valuable species for studying the molecular and genetic basis of complex behaviors and modeling neuropsychiatric disease states. While valid and reliable preclinical assays for reward-related behaviors are critical to understanding addiction-related processes, and various behavioral procedures have been developed and characterized in rats and primates, there have been relatively few studies using operant-based addiction-relevant behavioral paradigms in the mouse. Here we describe the performance of the C57BL/6J inbred mouse strain on three major reward-related paradigms, and replicate the same procedures in two other commonly used inbred strains (DBA/2J, BALB/cJ). We examined Pavlovian-instrumental transfer (PIT) by measuring the ability of an auditory cue associated with food reward to promote an instrumental (lever press) response. In a separate experiment, we assessed the acquisition and extinction of a simple stimulus-reward instrumental behavior on a touch screen based task. Reinstatement of this behavior was then examined following either continuous exposure to cues (conditioned reinforcers, CRs) associated with reward, brief reward and CR exposure, or brief reward exposure followed by continuous CR exposure. The third paradigm examined sensitivity of an instrumental (lever press) response to devaluation of food reward (a probe for outcome insensitive, habitual behavior) by repeated pairing with malaise. Results showed that C57BL/6J mice displayed robust PIT, as well as clear extinction and reinstatement, but were insensitive to reinforcer devaluation. DBA/2J mice showed good PIT and (rewarded) reinstatement, but were slow to extinguish and did not show reinforcer devaluation or significant CR-reinstatement. BALB/cJ mice also displayed good PIT, extinction and reinstatement, and retained instrumental responding following devaluation, but, unlike the other strains, demonstrated reduced Pavlovian approach behavior (food magazine head entries). Overall, these assays provide robust paradigms for future studies using the mouse to elucidate the neural, molecular and genetic factors underpinning reward-related behaviors relevant to addiction research.

Multiple adaptive amino acid substitutions increase the virulence of a wild waterfowl-origin reassortant H5N8 avian influenza virus in mice.

PubMed

Yu, Zhijun; Cheng, Kaihui; Sun, Weiyang; Zhang, Xinghai; Xia, Xianzhu; Gao, Yuwei

2018-01-15

A novel H5N8 highly pathogenic avian influenza virus (HPAIV) caused poultry outbreaks in the Republic of Korea in 2014. The novel H5N8 HPAIV has spread to Asia, Europe, and North America and caused great public concern from then on. Here, we generated mouse-adapted variants of a wild waterfowl-origin H5N8 HPAIV to identify adaptive mutants that confer enhanced pathogenicity in mammals. The mouse lethal doses (MLD 50 ) of the mouse-adapted variants were reduced 31623-fold compared to the wild-type (WT) virus. Mouse-adapted variants displayed enhanced replication in vitro and in vivo, and expanded tissue tropism in mice. Sequence analysis revealed four amino acid substitutions in the PB2 (E627K), PA (F35S), HA (R227H), and NA (I462V) proteins. These data suggest that multiple amino acid substitutions collaboratively increase the virulence of a wild bird-origin reassortant H5N8 HPAIV and cause severe disease in mice. Copyright © 2017 Elsevier B.V. All rights reserved.

Unique nonstructural proteins of Pneumonia Virus of Mice (PVM) promote degradation of interferon (IFN) pathway components and IFN-stimulated gene proteins.

PubMed

Dhar, Jayeeta; Barik, Sailen

2016-12-01

Pneumonia Virus of Mice (PVM) is the only virus that shares the Pneumovirus genus of the Paramyxoviridae family with Respiratory Syncytial Virus (RSV). A deadly mouse pathogen, PVM has the potential to serve as a robust animal model of RSV infection, since human RSV does not fully replicate the human pathology in mice. Like RSV, PVM also encodes two nonstructural proteins that have been implicated to suppress the IFN pathway, but surprisingly, they exhibit no sequence similarity with their RSV equivalents. The molecular mechanism of PVM NS function, therefore, remains unknown. Here, we show that recombinant PVM NS proteins degrade the mouse counterparts of the IFN pathway components. Proteasomal degradation appears to be mediated by ubiquitination promoted by PVM NS proteins. Interestingly, NS proteins of PVM lowered the levels of several ISG (IFN-stimulated gene) proteins as well. These results provide a molecular foundation for the mechanisms by which PVM efficiently subverts the IFN response of the murine cell. They also reveal that in spite of their high sequence dissimilarity, the two pneumoviral NS proteins are functionally and mechanistically similar.

Early Detection of Apathetic Phenotypes in Huntington's Disease Knock-in Mice Using Open Source Tools.

PubMed

Minnig, Shawn; Bragg, Robert M; Tiwana, Hardeep S; Solem, Wes T; Hovander, William S; Vik, Eva-Mari S; Hamilton, Madeline; Legg, Samuel R W; Shuttleworth, Dominic D; Coffey, Sydney R; Cantle, Jeffrey P; Carroll, Jeffrey B

2018-02-02

Apathy is one of the most prevalent and progressive psychiatric symptoms in Huntington's disease (HD) patients. However, preclinical work in HD mouse models tends to focus on molecular and motor, rather than affective, phenotypes. Measuring behavior in mice often produces noisy data and requires large cohorts to detect phenotypic rescue with appropriate power. The operant equipment necessary for measuring affective phenotypes is typically expensive, proprietary to commercial entities, and bulky which can render adequately sized mouse cohorts as cost-prohibitive. Thus, we describe here a home-built, open-source alternative to commercial hardware that is reliable, scalable, and reproducible. Using off-the-shelf hardware, we adapted and built several of the rodent operant buckets (ROBucket) to test Htt Q111/+ mice for attention deficits in fixed ratio (FR) and progressive ratio (PR) tasks. We find that, despite normal performance in reward attainment in the FR task, Htt Q111/+ mice exhibit reduced PR performance at 9-11 months of age, suggesting motivational deficits. We replicated this in two independent cohorts, demonstrating the reliability and utility of both the apathetic phenotype, and these ROBuckets, for preclinical HD studies.

Studying skin tumourigenesis and progression in immunocompetent hairless SKH1-hr mice using chronic 7,12-dimethylbenz(a)anthracene topical applications to develop a useful experimental skin cancer model.

PubMed

Thomas, Giju; Tuk, Bastiaan; Song, Ji-Ying; Truong, Hoa; Gerritsen, Hans C; de Gruijl, Frank R; Sterenborg, Henricus J C M

2017-02-01

Previous studies have established that 7,12-dimethylbenz(a)anthracene (DMBA) can initiate skin tumourigenesis in conventional furred mouse models by acting on hair follicle stem cells. However, further cancer progression depends on repeated applications of tumour promoter agents. This study evaluated the timeline involved in skin tumourigenesis and progression in immunocompetent hairless SKH1-hr mice with dysfunctional hair follicles using only DMBA with no additional tumour promoter agents. The results showed that topical application of 30 µg (117 nmol) of DMBA over the back and flank regions of the mouse once a week and 15 µg (58.5 nmol) twice a week produced skin tumours after 7-8 weeks. However, by week 14 a heavy benign tumour load required the mice to be euthanized. Lowering the DMBA dose to 15 µg (58.5 nmol) once a week produced tumours more slowly and allowed the mice to be studied for a longer period to week 23. This low-dose DMBA regimen yielded a high percentage of malignant tumours (58.8%) after 23 weekly applications. Additionally DMBA-treated skin showed an increase in mean epidermal thickness in comparison to untreated and acetone-treated skin. Despite the aberrant hair follicles in SKH1-hr mice, this chemically driven skin cancer model in hairless mice can serve as a suitable alternative to the ultraviolet-induced skin cancer models and can be reliably replicated as demonstrated by both the pilot and main experiments.

Pathogen reduction by ultraviolet C light effectively inactivates human white blood cells in platelet products.

PubMed

Pohler, Petra; Müller, Meike; Winkler, Carla; Schaudien, Dirk; Sewald, Katherina; Müller, Thomas H; Seltsam, Axel

2015-02-01

Residual white blood cells (WBCs) in cellular blood components induce a variety of adverse immune events, including nonhemolytic febrile transfusion reactions, alloimmunization to HLA antigens, and transfusion-associated graft-versus-host disease (TA-GVHD). Pathogen reduction (PR) methods such as the ultraviolet C (UVC) light-based THERAFLEX UV-Platelets system were developed to reduce the risk of transfusion-transmitted infection. As UVC light targets nucleic acids, it interferes with the replication of both pathogens and WBCs. This preclinical study aimed to evaluate the ability of UVC light to inactivate contaminating WBCs in platelet concentrates (PCs). The in vitro and in vivo function of WBCs from UVC-treated PCs was compared to that of WBCs from gamma-irradiated and untreated PCs by measuring cell viability, proliferation, cytokine secretion, antigen presentation in vitro, and xenogeneic GVHD responses in a humanized mouse model. UVC light was at least as effective as gamma irradiation in preventing GVHD in the mouse model. It was more effective in suppressing T-cell proliferation (>5-log reduction in the limiting dilution assay), cytokine secretion, and antigen presentation than gamma irradiation. The THERAFLEX UV-Platelets (MacoPharma) PR system can substitute gamma irradiation for TA-GVHD prophylaxis in platelet (PLT) transfusion. Moreover, UVC treatment achieves suppression of antigen presentation and inhibition of cytokine accumulation during storage of PCs, which has potential benefits for transfusion recipients. © 2014 AABB.

[Herpes simplex virus-mediated RNA interference targeting vesicular glutamate transporter 3 attenuates tactile allodynia in mice].

PubMed

Liu, Jie-Qiong; Li, Chen-Hong; Luo, Qiong; Yin, Ping-Ping; Lei, Tao; Luo, Fang

2016-11-20

To construct a replication-deficient herpes simplex virus (HSV-1) for delivering a short hairpin RNA (shRNA) targeting vesicular glutamate transporter 3 (VGLUT3) and observe its effect in alleviating allodynia in mice. The recombinant HSV-1 vector carrying the shRNA targeting Vglut3 (HSV-1-shvglut3) was constructed and inoculated in the sciatic nerve in a mouse model of mechanical allodynia to test its analgesia effect. Mechanical allodynia and heat hypersensitivity of the mice were tested by von Frey filaments and Hargreaves' test, respectively. VGLUT3 expression in the dorsal root ganglion (DRG) was evaluated by immunohistochemistry and Western blotting. Following inoculation in the sciatic nerve, the HSV vector HSV-1-shvglut3 was retrogradely transported to the DRG. Mechanical withdraw thresholds of the mouse models receiving HSV-1-shvglut3 inoculation were reversed to nearly the baseline level, and VGLUT3 expression in the DRG was down-regulated 2 weeks after vector inoculation. The analgesic effect lasted for over 2 weeks in these mice without obvious systematic side effects or changes in heat hypersensitivity threshold. Vglut3 in the DRG is a promising therapeutic target for alleviating mechanical allodynia, and HSV-1 vector-mediated RNA interference is safe and efficient for inducing long-lasting analgesia after peripheral inoculation of the vector.

Specific inhibition of NLRP3 in chikungunya disease reveals a role for inflammasomes in alphavirus-induced inflammation.

PubMed

Chen, Weiqiang; Foo, Suan-Sin; Zaid, Ali; Teng, Terk-Shin; Herrero, Lara J; Wolf, Stefan; Tharmarajah, Kothila; Vu, Luan D; van Vreden, Caryn; Taylor, Adam; Freitas, Joseph R; Li, Rachel W; Woodruff, Trent M; Gordon, Richard; Ojcius, David M; Nakaya, Helder I; Kanneganti, Thirumala-Devi; O'Neill, Luke A J; Robertson, Avril A B; King, Nicholas J; Suhrbier, Andreas; Cooper, Matthew A; Ng, Lisa F P; Mahalingam, Suresh

2017-10-01

Mosquito-borne viruses can cause severe inflammatory diseases and there are limited therapeutic solutions targeted specifically at virus-induced inflammation. Chikungunya virus (CHIKV), a re-emerging alphavirus responsible for several outbreaks worldwide in the past decade, causes debilitating joint inflammation and severe pain. Here, we show that CHIKV infection activates the NLRP3 inflammasome in humans and mice. Peripheral blood mononuclear cells isolated from CHIKV-infected patients showed elevated NLRP3, caspase-1 and interleukin-18 messenger RNA expression and, using a mouse model of CHIKV infection, we found that high NLRP3 expression was associated with peak inflammatory symptoms. Inhibition of NLRP3 activation using the small-molecule inhibitor MCC950 resulted in reduced CHIKV-induced inflammation and abrogated osteoclastogenic bone loss and myositis, but did not affect in vivo viral replication. Mice treated with MCC950 displayed lower expression levels of the cytokines interleukin-6, chemokine ligand 2 and tumour necrosis factor in joint tissue. Interestingly, MCC950 treatment abrogated disease signs in mice infected with a related arthritogenic alphavirus, Ross River virus, but not in mice infected with West Nile virus-a flavivirus. Here, using mouse models of alphavirus-induced musculoskeletal disease, we demonstrate that NLRP3 inhibition in vivo can reduce inflammatory pathology and that further development of therapeutic solutions targeting inflammasome function could help treat arboviral diseases.

Dynamics of Tissue-Specific CD8+ T Cell Responses during West Nile Virus Infection.

PubMed

Aguilar-Valenzuela, Renan; Netland, Jason; Seo, Young-Jin; Bevan, Michael J; Grakoui, Arash; Suthar, Mehul S

2018-05-15

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. Copyright © 2018 American Society for Microbiology.

Identification of novel inhibitors of non-replicating Mycobacterium tuberculosis using a carbon starvation model

PubMed Central

Grant, Sarah Schmidt; Kawate, Tomohiko; Nag, Partha P.; Silvis, Melanie R.; Gordon, Katherine; Stanley, Sarah A.; Kazyanskaya, Ed; Nietupski, Ray; Golas, Aaron; Fitzgerald, Michael; Cho, Sanghyun; Franzblau, Scott G.; Hung, Deborah T.

2013-01-01

During Mycobacterium tuberculosis infection, a population of bacteria is thought to exist in a non-replicating state, refractory to antibiotics, which may contribute to the need for prolonged antibiotic therapy. The identification of inhibitors of the non-replicating state provides tools that can be used to probe this hypothesis and the physiology of this state. The development of such inhibitors also has the potential to shorten the duration of antibiotic therapy required. Here we describe the development of a novel non-replicating assay amenable to high-throughput chemical screening coupled with secondary assays that use carbon starvation as the in vitro model. Together these assays identify compounds with activity against replicating and non-replicating M. tuberculosis as well as compounds that inhibit the transition from non-replicating to replicating stages of growth. Using these assays we successfully screened over 300,000 compounds and identified 786 inhibitors of non-replicating M. tuberculosis. In order to understand the relationship among different non-replicating models, we teste 52 of these molecules in a hypoxia model and four different chemical scaffolds in a stochastic persist model and a streptomycin dependent model. We found that compounds display varying levels of activity in different models for the non-replicating state, suggesting important differences in bacterial physiology between models. Therefore, chemical tools identified in this assay may be useful for determining the relevance of different non-replicating in vitro models to in vivo M. tuberculosis infection. Given our current limited understanding, molecules that are active across multiple models may represent more promising candidates for further development. PMID:23898841

Cognition in people with Prader-Willi syndrome: Insights into genetic influences on cognitive and social development.

PubMed

Whittington, Joyce; Holland, Anthony

2017-01-01

We present a mini-review of cognition in Prader-Willi syndrome. Studies cited include findings on general ability (IQ), IQ correlates with family members, strengths and weaknesses in cognitive profiles in genetic subtypes, attainment in literacy and numeracy, language, comprehension, modality preferences, executive functions, and social cognition. The latter includes investigations of theory of mind, emotion recognition, face processing and knowledge of social norms. Results from research on mouse models and brain imaging studies relevant to cognition are briefly discussed. The importance of these studies to understanding and managing education and behaviour in PWS and the limitations of the studies in terms of small numbers, non-representativeness, and lack of replication is also touched upon. Copyright © 2016 Elsevier Ltd. All rights reserved.

Locomotor activity, emotionality, sensori-motor gating, learning and memory in the APPswe/PS1dE9 mouse model of Alzheimer's disease.

PubMed

O'Leary, Timothy P; Hussin, Ahmed T; Gunn, Rhian K; Brown, Richard E

2018-06-02

The APPswe/PS1dE9 mouse (line 85) is a double transgenic model of Alzheimer's disease (AD) with familial amyloid precursor protein and presenilin-1 mutations. These mice develop age-related behavioral changes reflective of the neuropsychiatric symptoms (altered anxiety-like behaviour, hyperactivity) and cognitive dysfunction (impaired learning and memory) observed in AD. The APPswe/PS1dE9 mouse has been used to examine the efficacy of therapeutic interventions on behaviour, despite previous difficulties in replicating behavioural phenotypes. Therefore, the purpose of this study was to establish the reliability of these phenotypes by further characterizing the behaviour of male APPswe/PS1dE9 and wild-type mice between 7 and 14 months of age. Mice were tested on the open-field over 5-days to examine emotionality, locomotor activity and inter-session habituation. Mice were also tested on the repeated-reversal water maze task and spontaneous alternation on the Y-maze to assess working memory. Sensori-motor gating was examined with acoustic startle and pre-pulse inhibition. Lastly contextual and cued (trace) memory was assessed with fear conditioning. The results show that among non-cognitive behaviours, APPswe/PS1dE9 mice have normal locomotor activity, anxiety-like behavior, habituation and sensori-motor gating. However, APPswe/PS1dE9 mice show impaired working memory on the repeated-reversal water-maze and impaired memory in contextual but not trace-cued fear conditioning. These results indicate that the APPswe/PS1dE9 (line 85) mice have deficits in some types of hippocampal-dependent learning and memory and, at the ages tested, APPswe/PS1dE9 mice model cognitive dysfunction but not neuropsychiatric symptoms. Copyright © 2018. Published by Elsevier Inc.

MicroRNA-21 drives severe, steroid-insensitive experimental asthma by amplifying phosphoinositide 3-kinase-mediated suppression of histone deacetylase 2.

PubMed

Kim, Richard Y; Horvat, Jay C; Pinkerton, James W; Starkey, Malcolm R; Essilfie, Ama T; Mayall, Jemma R; Nair, Prema M; Hansbro, Nicole G; Jones, Bernadette; Haw, Tatt Jhong; Sunkara, Krishna P; Nguyen, Thi Hiep; Jarnicki, Andrew G; Keely, Simon; Mattes, Joerg; Adcock, Ian M; Foster, Paul S; Hansbro, Philip M

2017-02-01

Severe steroid-insensitive asthma is a substantial clinical problem. Effective treatments are urgently required, however, their development is hampered by a lack of understanding of the mechanisms of disease pathogenesis. Steroid-insensitive asthma is associated with respiratory tract infections and noneosinophilic endotypes, including neutrophilic forms of disease. However, steroid-insensitive patients with eosinophil-enriched inflammation have also been described. The mechanisms that underpin infection-induced, severe steroid-insensitive asthma can be elucidated by using mouse models of disease. We sought to develop representative mouse models of severe, steroid-insensitive asthma and to use them to identify pathogenic mechanisms and investigate new treatment approaches. Novel mouse models of Chlamydia, Haemophilus influenzae, influenza, and respiratory syncytial virus respiratory tract infections and ovalbumin-induced, severe, steroid-insensitive allergic airway disease (SSIAAD) in BALB/c mice were developed and interrogated. Infection induced increases in the levels of microRNA (miRNA)-21 (miR-21) expression in the lung during SSIAAD, whereas expression of the miR-21 target phosphatase and tensin homolog was reduced. This was associated with an increase in levels of phosphorylated Akt, an indicator of phosphoinositide 3-kinase (PI3K) activity, and decreased nuclear histone deacetylase (HDAC)2 levels. Treatment with an miR-21-specific antagomir (Ant-21) increased phosphatase and tensin homolog levels. Treatment with Ant-21, or the pan-PI3K inhibitor LY294002, reduced PI3K activity and restored HDAC2 levels. This led to suppression of airway hyperresponsiveness and restored steroid sensitivity to allergic airway disease. These observations were replicated with SSIAAD associated with 4 different pathogens. We identify a previously unrecognized role for an miR-21/PI3K/HDAC2 axis in SSIAAD. Our data highlight miR-21 as a novel therapeutic target for the treatment of this form of asthma. Copyright © 2016 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.

A switch between DNA polymerases δ and λ promotes error-free bypass of 8-oxo-G lesions

PubMed Central

Markkanen, Enni; Castrec, Benoît; Villani, Giuseppe; Hübscher, Ulrich

2012-01-01

7,8-Dihydro-8-oxoguanine (8-oxo-G) is a highly abundant and mutagenic lesion. Replicative DNA polymerases (pols) are slowed down at 8-oxo-G and insert both correct cytosine (C) and incorrect adenine (A) opposite 8-oxo-G, but they preferentially extend A:8-oxo-G mispairs. Nevertheless, 8-oxo-G bypass is fairly accurate in vivo. Thus, the question how correct bypass of 8-oxo-G lesions is accomplished despite the poor extension of C:8-oxo-G base pairs by replicative pols remains unanswered. Here we show that replicative pol δ pauses in front of 8-oxo-G and displays difficulties extending from correct C:8-oxo-G in contrast to extension from incorrect A:8-oxo-G. This leads to stalling of pol δ at 8-oxo-G after incorporation of correct C. This stalling at C:8-oxo-G can be overcome by a switch from pol δ to pols λ, β, or η, all of which are able to assist pol δ in 8-oxo-G bypass by translesion synthesis (TLS). Importantly, however, only pol λ selectively catalyzes the correct TLS past 8-oxo-G, whereas pols β and η show no selectivity and even preferentially enhance incorrect TLS. The selectivity of pol λ to promote the correct bypass depends on its N-terminal domain. Furthermore, pol λ−/− mouse embryonic fibroblast extracts display reduced 8-oxo-G TLS. Finally, the correct bypass of 8-oxo-G in gapped plasmids in mouse embryonic fibroblasts and HeLa cells is promoted in the presence of pol λ. Our findings suggest that even though 8-oxo-G is not a blocking lesion per se, correct replication over 8-oxo-G is promoted by a pol switch between pols δ and λ. PMID:23175785

ISG15 Functions as an Interferon-Mediated Antiviral Effector Early in the Murine Norovirus Life Cycle

PubMed Central

Rodriguez, Marisela R.; Monte, Kristen; Thackray, Larissa B.

2014-01-01

ABSTRACT Human noroviruses (HuNoV) are the leading cause of nonbacterial gastroenteritis worldwide. Similar to HuNoV, murine noroviruses (MNV) are enteric pathogens spread via the fecal-oral route and have been isolated from numerous mouse facilities worldwide. Type I and type II interferons (IFN) restrict MNV-1 replication; however, the antiviral effectors impacting MNV-1 downstream of IFN signaling are largely unknown. Studies using dendritic cells, macrophages, and mice deficient in free and conjugated forms of interferon-stimulated gene 15 (ISG15) revealed that ISG15 conjugation contributes to protection against MNV-1 both in vitro and in vivo. ISG15 inhibited a step early in the viral life cycle upstream of viral genome transcription. Directly transfecting MNV-1 RNA into IFN-stimulated mouse embryonic fibroblasts (MEFs) and bone marrow-derived dendritic cells (BMDC) lacking ISG15 conjugates bypassed the antiviral activity of ISG15, further suggesting that ISG15 conjugates restrict the MNV-1 life cycle at the viral entry/uncoating step. These results identify ISG15 as the first type I IFN effector regulating MNV-1 infection both in vitro and in vivo and for the first time implicate the ISG15 pathway in the regulation of early stages of MNV-1 replication. IMPORTANCE Type I IFNs are important in controlling murine norovirus 1 (MNV-1) infections; however, the proteins induced by IFNs that restrict viral growth are largely unknown. This report reveals that interferon-stimulated gene 15 (ISG15) mitigates MNV-1 replication both in vitro and in vivo. In addition, it shows that ISG15 inhibits MNV-1 replication by targeting an early step in the viral life cycle, MNV-1 entry and/or uncoating. These results identify ISG15 as the first type I IFN effector regulating MNV-1 infection both in vitro and in vivo and for the first time implicate the ISG15 pathway in the regulation of viral entry/uncoating. PMID:24899198

3D replicon distributions arise from stochastic initiation and domino-like DNA replication progression

PubMed Central

Löb, D.; Lengert, N.; Chagin, V. O.; Reinhart, M.; Casas-Delucchi, C. S.; Cardoso, M. C.; Drossel, B.

2016-01-01

DNA replication dynamics in cells from higher eukaryotes follows very complex but highly efficient mechanisms. However, the principles behind initiation of potential replication origins and emergence of typical patterns of nuclear replication sites remain unclear. Here, we propose a comprehensive model of DNA replication in human cells that is based on stochastic, proximity-induced replication initiation. Critical model features are: spontaneous stochastic firing of individual origins in euchromatin and facultative heterochromatin, inhibition of firing at distances below the size of chromatin loops and a domino-like effect by which replication forks induce firing of nearby origins. The model reproduces the empirical temporal and chromatin-related properties of DNA replication in human cells. We advance the one-dimensional DNA replication model to a spatial model by taking into account chromatin folding in the nucleus, and we are able to reproduce the spatial and temporal characteristics of the replication foci distribution throughout S-phase. PMID:27052359

Chimeric elk/mouse prion proteins in transgenic mice.

PubMed

Tamgüney, Gültekin; Giles, Kurt; Oehler, Abby; Johnson, Natrina L; DeArmond, Stephen J; Prusiner, Stanley B

2013-02-01

Chronic wasting disease (CWD) of deer and elk is a highly communicable neurodegenerative disorder caused by prions. Investigations of CWD are hampered by slow bioassays in transgenic (Tg) mice. Towards the development of Tg mice that will be more susceptible to CWD prions, we created a series of chimeric elk/mouse transgenes that encode the N terminus of elk PrP (ElkPrP) up to residue Y168 and the C terminus of mouse PrP (MoPrP) beyond residue 169 (mouse numbering), designated Elk3M(SNIVVK). Between codons 169 and 219, six residues distinguish ElkPrP from MoPrP: N169S, T173N, V183I, I202V, I214V and R219K. Using chimeric elk/mouse PrP constructs, we generated 12 Tg mouse lines and determined incubation times after intracerebral inoculation with the mouse-passaged RML scrapie or Elk1P CWD prions. Unexpectedly, one Tg mouse line expressing Elk3M(SNIVVK) exhibited incubation times of <70 days when inoculated with RML prions; a second line had incubation times of <90 days. In contrast, mice expressing full-length ElkPrP had incubation periods of >250 days for RML prions. Tg(Elk3M,SNIVVK) mice were less susceptible to CWD prions than Tg(ElkPrP) mice. Changing three C-terminal mouse residues (202, 214 and 219) to those of elk doubled the incubation time for mouse RML prions and rendered the mice resistant to Elk1P CWD prions. Mutating an additional two residues from mouse to elk at codons 169 and 173 increased the incubation times for mouse prions to >300 days, but made the mice susceptible to CWD prions. Our findings highlight the role of C-terminal residues in PrP that control the susceptibility and replication of prions.

Replication of surface features from a master model to an amorphous metallic article

DOEpatents

Johnson, William L.; Bakke, Eric; Peker, Atakan

1999-01-01

The surface features of an article are replicated by preparing a master model having a preselected surface feature thereon which is to be replicated, and replicating the preselected surface feature of the master model. The replication is accomplished by providing a piece of a bulk-solidifying amorphous metallic alloy, contacting the piece of the bulk-solidifying amorphous metallic alloy to the surface of the master model at an elevated replication temperature to transfer a negative copy of the preselected surface feature of the master model to the piece, and separating the piece having the negative copy of the preselected surface feature from the master model.

The Checkpoint Kinase 1 Inhibitor Prexasertib Induces Regression of Preclinical Models of Human Neuroblastoma.

PubMed

Lowery, Caitlin D; VanWye, Alle B; Dowless, Michele; Blosser, Wayne; Falcon, Beverly L; Stewart, Julie; Stephens, Jennifer; Beckmann, Richard P; Bence Lin, Aimee; Stancato, Louis F

2017-08-01

Purpose: Checkpoint kinase 1 (CHK1) is a key regulator of the DNA damage response and a mediator of replication stress through modulation of replication fork licensing and activation of S and G 2 -M cell-cycle checkpoints. We evaluated prexasertib (LY2606368), a small-molecule CHK1 inhibitor currently in clinical testing, in multiple preclinical models of pediatric cancer. Following an initial assessment of prexasertib activity, this study focused on the preclinical models of neuroblastoma. Experimental Design: We evaluated the antiproliferative activity of prexasertib in a panel of cancer cell lines; neuroblastoma cell lines were among the most sensitive. Subsequent Western blot and immunofluorescence analyses measured DNA damage and DNA repair protein activation. Prexasertib was investigated in several cell line-derived xenograft mouse models of neuroblastoma. Results: Within 24 hours, single-agent prexasertib promoted γH2AX-positive double-strand DNA breaks and phosphorylation of DNA damage sensors ATM and DNA-PKcs, leading to neuroblastoma cell death. Knockdown of CHK1 and/or CHK2 by siRNA verified that the double-strand DNA breaks and cell death elicited by prexasertib were due to specific CHK1 inhibition. Neuroblastoma xenografts rapidly regressed following prexasertib administration, independent of starting tumor volume. Decreased Ki67 and increased immunostaining of endothelial and pericyte markers were observed in xenografts after only 6 days of exposure to prexasertib, potentially indicating a swift reduction in tumor volume and/or a direct effect on tumor vasculature. Conclusions: Overall, these data demonstrate that prexasertib is a specific inhibitor of CHK1 in neuroblastoma and leads to DNA damage and cell death in preclinical models of this devastating pediatric malignancy. Clin Cancer Res; 23(15); 4354-63. ©2017 AACR . ©2017 American Association for Cancer Research.

Long-term expansion of epithelial organoids from human colon, adenoma, adenocarcinoma, and Barrett's epithelium.

PubMed

Sato, Toshiro; Stange, Daniel E; Ferrante, Marc; Vries, Robert G J; Van Es, Johan H; Van den Brink, Stieneke; Van Houdt, Winan J; Pronk, Apollo; Van Gorp, Joost; Siersema, Peter D; Clevers, Hans

2011-11-01

We previously established long-term culture conditions under which single crypts or stem cells derived from mouse small intestine expand over long periods. The expanding crypts undergo multiple crypt fission events, simultaneously generating villus-like epithelial domains that contain all differentiated types of cells. We have adapted the culture conditions to grow similar epithelial organoids from mouse colon and human small intestine and colon. Based on the mouse small intestinal culture system, we optimized the mouse and human colon culture systems. Addition of Wnt3A to the combination of growth factors applied to mouse colon crypts allowed them to expand indefinitely. Addition of nicotinamide, along with a small molecule inhibitor of Alk and an inhibitor of p38, were required for long-term culture of human small intestine and colon tissues. The culture system also allowed growth of mouse Apc-deficient adenomas, human colorectal cancer cells, and human metaplastic epithelia from regions of Barrett's esophagus. We developed a technology that can be used to study infected, inflammatory, or neoplastic tissues from the human gastrointestinal tract. These tools might have applications in regenerative biology through ex vivo expansion of the intestinal epithelia. Studies of these cultures indicate that there is no inherent restriction in the replicative potential of adult stem cells (or a Hayflick limit) ex vivo. Copyright © 2011 AGA Institute. Published by Elsevier Inc. All rights reserved.

Surface expression of an immunodominant malaria protein B cell epitope by yellow fever virus.

PubMed

Bonaldo, Myrna C; Garratt, Richard C; Caufour, Philippe S; Freire, Marcos S; Rodrigues, Mauricio M; Nussenzweig, Ruth S; Galler, Ricardo

2002-01-25

The yellow fever 17D virus (YF17D) has several characteristics that are desirable for the development of new, live attenuated vaccines. We approached its development as a vector for heterologous antigens by studying the expression of a humoral epitope at the surface of the E protein based on the results of modelling its three-dimensional structure. This model indicated that the most promising insertion site is between beta-strands f and g, a site that is exposed at the external surface of the virus. The large deletion of six residues from the fg loop of the E protein from yellow fever virus, compared to tick-born encephalitis virus, leaves space at the dimer interface for a large insertion without creating steric hindrance. We have tested this hypothesis by inserting a model humoral epitope from the circumsporozoite protein of Plasmodium falciparum consisting of triple NANP repeats. Recombinant virus (17D/8) expressing this insertion flanked by two glycine residues at each end, is specifically neutralized by a monoclonal antibody to the model epitope. Furthermore, mouse antibodies raised to the recombinant virus recognize the parasite protein in an ELISA assay. Serial passage analysis confirmed the genetic stability of the insertion made in the viral genome and the resulting 17D/8 virus is significantly more attenuated in mouse neurovirulence tests than the 17DD vaccine. The fg loop belongs to the dimerization domain of the E protein and lies at the interface between monomers. This domain undergoes a low pH transition, which is related to the fusion of the viral envelope to the endosome membrane. It is conceivable that a slower rate of fusion, resulting from the insertion close to the dimer interface, may delay the onset of virus production and thereby lead to a milder infection of the host. This would account for the more attenuated phenotype of the recombinant virus in the mouse model and lower extent of replication in cultured cells. The vectorial capacity of the yellow fever virus is being further explored for the expression and presentation of other epitopes, including those mediating T-cell responses. Copyright 2002 Academic Press.

Chlamydia muridarum evades growth restriction by the IFN-gamma-inducible host resistance factor Irgb10.

PubMed

Coers, Jörn; Bernstein-Hanley, Isaac; Grotsky, David; Parvanova, Iana; Howard, Jonathan C; Taylor, Gregory A; Dietrich, William F; Starnbach, Michael N

2008-05-01

Chlamydiae are obligate intracellular bacterial pathogens that exhibit a broad range of host tropism. Differences in host tropism between Chlamydia species have been linked to host variations in IFN-gamma-mediated immune responses. In mouse cells, IFN-gamma can effectively restrict growth of the human pathogen Chlamydia trachomatis but fails to control growth of the closely related mouse pathogen Chlamydia muridarum. The ability of mouse cells to resist C. trachomatis replication is largely dependent on the induction of a family of IFN-gamma-inducible GTPases called immunity-related GTPases or IRGs. In this study we demonstrate that C. muridarum can specifically evade IRG-mediated host resistance. It has previously been suggested that C. muridarum inactivates the IRG protein Irga6 (Iigp1) to dampen the murine immune response. However, we show that Irga6 is dispensable for the control of C. trachomatis replication. Instead, an effective IFN-gamma response to C. trachomatis requires the IRG proteins Irgm1 (Lrg47), Irgm3 (Igtp), and Irgb10. Ectopic expression of Irgb10 in the absence of IFN-gamma is sufficient to reduce intracellular growth of C. trachomatis but fails to restrict growth of C. muridarum, indicating that C. muridarum can specifically evade Irgb10-driven host responses. Importantly, we find that Irgb10 protein intimately associates with inclusions harboring C. trachomatis but is absent from inclusions formed by C. muridarum. These data suggest that C. muridarum has evolved a mechanism to escape the murine IFN-gamma response by restricting access of Irgb10 and possibly other IRG proteins to the inclusion.

Virology Interest Group Seminar | Center for Cancer Research

Cancer.gov

Virology Interest Group Seminar.  September 7th, Building 50, Room 2328 from 3:00 until 4:00.   We will have two presenters. Dr. Vladimir Majerciak: The full transcription map of mouse papillomavirus type 1 (MmuPV1), Tumor Virus RNA Biology Section, RNA Biology Laboratory, NCI Dr. Zhi-Ming Zheng: Viral DNA replication regulates HPV18 transcription and gene expression, Tumor

Divergent Requirement for a DNA Repair Enzyme during Enterovirus Infections

PubMed Central

Maciejewski, Sonia; Nguyen, Joseph H. C.; Gómez-Herreros, Fernando; Cortés-Ledesma, Felipe; Caldecott, Keith W.

2015-01-01

ABSTRACT Viruses of the Enterovirus genus of picornaviruses, including poliovirus, coxsackievirus B3 (CVB3), and human rhinovirus, commandeer the functions of host cell proteins to aid in the replication of their small viral genomic RNAs during infection. One of these host proteins is a cellular DNA repair enzyme known as 5′ tyrosyl-DNA phosphodiesterase 2 (TDP2). TDP2 was previously demonstrated to mediate the cleavage of a unique covalent linkage between a viral protein (VPg) and the 5′ end of picornavirus RNAs. Although VPg is absent from actively translating poliovirus mRNAs, the removal of VPg is not required for the in vitro translation and replication of the RNA. However, TDP2 appears to be excluded from replication and encapsidation sites during peak times of poliovirus infection of HeLa cells, suggesting a role for TDP2 during the viral replication cycle. Using a mouse embryonic fibroblast cell line lacking TDP2, we found that TDP2 is differentially required among enteroviruses. Our single-cycle viral growth analysis shows that CVB3 replication has a greater dependency on TDP2 than does poliovirus or human rhinovirus replication. During infection, CVB3 protein accumulation is undetectable (by Western blot analysis) in the absence of TDP2, whereas poliovirus protein accumulation is reduced but still detectable. Using an infectious CVB3 RNA with a reporter, CVB3 RNA could still be replicated in the absence of TDP2 following transfection, albeit at reduced levels. Overall, these results indicate that TDP2 potentiates viral replication during enterovirus infections of cultured cells, making TDP2 a potential target for antiviral development for picornavirus infections. PMID:26715620

Modulation of human beta-defensin-1 (hBD-1) in plasmacytoid dendritic cells (PDC), monocytes, and epithelial cells by influenza virus, Herpes simplex virus, and Sendai virus and its possible role in innate immunity.

PubMed

Ryan, Lisa K; Dai, Jihong; Yin, Zhiwei; Megjugorac, Nicholas; Uhlhorn, Victoria; Yim, Sunghan; Schwartz, Kyell D; Abrahams, Joshua M; Diamond, Gill; Fitzgerald-Bocarsly, Patricia

2011-08-01

hBD comprise a family of antimicrobial peptides that plays a role in bridging the innate and adaptive immune responses to infection. The expression of hBD-2 increases upon stimulation of numerous cell types with LPS and proinflammatory cytokines. In contrast, hBD-1 remains constitutively expressed in most cells in spite of cytokine or LPS stimulation; however, its presence in human PDC suggests it plays a role in viral host defense. To examine this, we characterized the expression of hBD-1 in innate immune cells in response to viral challenge. PDC and monocytes increased production of hBD-1 peptide and mRNA as early as 2 h following infection of purified cells and PBMCs with PR8, HSV-1, and Sendai virus. However, treatment of primary NHBE cells with influenza resulted in a 50% decrease in hBD-1 mRNA levels, as measured by qRT-PCR at 3 h following infection. A similar inhibition occurred with HSV-1 challenge of human gingival epithelial cells. Studies with HSV-1 showed that replication occurred in epithelial cells but not in PDC. Together, these results suggest that hBD-1 may play a role in preventing viral replication in immune cells. To test this, we infected C57BL/6 WT mice and mBD-1((-/-)) mice with mouse-adapted HK18 (300 PFU/mouse). mBD-1((-/-)) mice lost weight earlier and died sooner than WT mice (P=0.0276), suggesting that BD-1 plays a role in early innate immune responses against influenza in vivo. However, lung virus titers were equal between the two mouse strains. Histopathology showed a greater inflammatory influx in the lungs of mBD-1((-/-)) mice at Day 3 postinfection compared with WT C57BL/6 mice. The results suggest that BD-1 protects mice from influenza pathogenesis with a mechanism other than inhibition of viral replication.

RIPK1 and PGAM5 Control Leishmania Replication through Distinct Mechanisms.

PubMed

Farias Luz, Nivea; Balaji, Sakthi; Okuda, Kendi; Barreto, Aline Silva; Bertin, John; Gough, Peter J; Gazzinelli, Ricardo; Almeida, Roque P; Bozza, Marcelo T; Borges, Valeria M; Chan, Francis Ka-Ming

2016-06-15

Leishmaniasis is an important parasitic disease found in the tropics and subtropics. Cutaneous and visceral leishmaniasis affect an estimated 1.5 million people worldwide. Despite its human health relevance, relatively little is known about the cell death pathways that control Leishmania replication in the host. Necroptosis is a recently identified form of cell death with potent antiviral effects. Receptor interacting protein kinase 1 (RIPK1) is a critical kinase that mediates necroptosis downstream of death receptors and TLRs. Heme, a product of hemoglobin catabolism during certain intracellular pathogen infections, is also a potent inducer of macrophage necroptosis. We found that human visceral leishmaniasis patients exhibit elevated serum levels of heme. Therefore, we examined the impact of heme and necroptosis on Leishmania replication. Indeed, heme potently inhibited Leishmania replication in bone marrow-derived macrophages. Moreover, we found that inhibition of RIPK1 kinase activity also enhanced parasite replication in the absence of heme. We further found that the mitochondrial phosphatase phosphoglycerate mutase family member 5 (PGAM5), a putative downstream effector of RIPK1, was also required for inhibition of Leishmania replication. In mouse infection, both PGAM5 and RIPK1 kinase activity are required for IL-1β expression in response to Leishmania However, PGAM5, but not RIPK1 kinase activity, was directly responsible for Leishmania-induced IL-1β secretion and NO production in bone marrow-derived macrophages. Collectively, these results revealed that RIPK1 and PGAM5 function independently to exert optimal control of Leishmania replication in the host. Copyright © 2016 by The American Association of Immunologists, Inc.

Characterization of conserved arginine residues on Cdt1 that affect licensing activity and interaction with Geminin or Mcm complex.

PubMed

You, Zhiying; Ode, Koji L; Shindo, Mayumi; Takisawa, Haruhiko; Masai, Hisao

2016-05-02

All organisms ensure once and only once replication during S phase through a process called replication licensing. Cdt1 is a key component and crucial loading factor of Mcm complex, which is a central component for the eukaryotic replicative helicase. In higher eukaryotes, timely inhibition of Cdt1 by Geminin is essential to prevent rereplication. Here, we address the mechanism of DNA licensing using purified Cdt1, Mcm and Geminin proteins in combination with replication in Xenopus egg extracts. We mutagenized the 223th arginine of mouse Cdt1 (mCdt1) to cysteine or serine (R-S or R-C, respectively) and 342nd and 346th arginines constituting an arginine finger-like structure to alanine (RR-AA). The RR-AA mutant of Cdt1 could not only rescue the DNA replication activity in Cdt1-depleted extracts but also its specific activity for DNA replication and licensing was significantly increased compared to the wild-type protein. In contrast, the R223 mutants were partially defective in rescue of DNA replication and licensing. Biochemical analyses of these mutant Cdt1 proteins indicated that the RR-AA mutation disabled its functional interaction with Geminin, while R223 mutations resulted in ablation in interaction with the Mcm2∼7 complex. Intriguingly, the R223 mutants are more susceptible to the phosphorylation-induced inactivation or chromatin dissociation. Our results show that conserved arginine residues play critical roles in interaction with Geminin and Mcm that are crucial for proper conformation of the complexes and its licensing activity.

The resolving power of in vitro genotoxicity assays for cigarette smoke particulate matter.

PubMed

Scott, K; Saul, J; Crooks, I; Camacho, O M; Dillon, D; Meredith, C

2013-06-01

In vitro genotoxicity assays are often used to compare tobacco smoke particulate matter (PM) from different cigarettes. The quantitative aspect of the comparisons requires appropriate statistical methods and replication levels, to support the interpretation in terms of power and significance. This paper recommends a uniform statistical analysis for the Ames test, mouse lymphoma mammalian cell mutation assay (MLA) and the in vitro micronucleus test (IVMNT); involving a hierarchical decision process with respect to slope, fixed effect and single dose comparisons. With these methods, replication levels of 5 (Ames test TA98), 4 (Ames test TA100), 10 (Ames test TA1537), 6 (MLA) and 4 (IVMNT) resolved a 30% difference in PM genotoxicity. Copyright © 2013 Elsevier Ltd. All rights reserved.

Intracerebral Inoculation of Mouse-Passaged Saffold Virus Type 3 Affects Cerebellar Development in Neonatal Mice

PubMed Central

Kotani, Osamu; Suzuki, Tadaki; Yokoyama, Masaru; Iwata-Yoshikawa, Naoko; Nakajima, Noriko; Sato, Hironori; Hasegawa, Hideki; Taguchi, Fumihiro; Shimizu, Hiroyuki

2016-01-01

ABSTRACT Saffold virus (SAFV), a human cardiovirus, is occasionally detected in infants with neurological disorders, including meningitis and cerebellitis. We recently reported that SAFV type 3 isolates infect cerebellar glial cells, but not large neurons, in mice. However, the impact of this infection remained unclear. Here, we determined the neuropathogenesis of SAFV type 3 in the cerebella of neonatal ddY mice by using SAFV passaged in the cerebella of neonatal BALB/c mice. The virus titer in the cerebellum increased following the inoculation of each of five passaged strains. The fifth passaged strain harbored amino acid substitutions in the VP2 (H160R and Q239R) and VP3 (K62M) capsid proteins. Molecular modeling of the capsid proteins suggested that the VP2-H160R and VP3-K62M mutations alter the structural dynamics of the receptor binding surface via the formation of a novel hydrophobic interaction between the VP2 puff B and VP3 knob regions. Compared with the original strain, the passaged strain showed altered growth characteristics in human-derived astroglial cell lines and greater replication in the brains of neonatal mice. In addition, the passaged strain was more neurovirulent than the original strain, while both strains infected astroglial and neural progenitor cells in the mouse brain. Intracerebral inoculation of either the original or the passaged strain affected brain Purkinje cell dendrites, and a high titer of the passaged strain induced cerebellar hypoplasia in neonatal mice. Thus, infection by mouse-passaged SAFV affected cerebellar development in neonatal mice. This animal model contributes to the understanding of the neuropathogenicity of SAFV infections in infants. IMPORTANCE Saffold virus (SAFV) is a candidate neuropathogenic agent in infants and children, but the neuropathogenicity of the virus has not been fully elucidated. Recently, we evaluated the pathogenicity of two clinical SAFV isolates in mice. Similar to other neurotropic picornaviruses, these isolates showed mild infectivity of glial and neural progenitor cells, but not of large neurons, in the cerebellum. However, the outcome of this viral infection in the cerebellum has not been clarified. Here, we examined the tropism of SAFV in the cerebellum. We obtained an in vivo-passaged strain from the cerebella of neonatal mice and examined its genome and its neurovirulence in the neonatal mouse brain. The passaged virus showed high infectivity and neurovirulence in the brain, especially the cerebellum, and affected cerebellar development. This unique neonatal mouse model will be helpful for elucidating the neuropathogenesis of SAFV infections occurring early in life. PMID:27581974

Comparison of the virulence of three H3N2 canine influenza virus isolates from Korea and China in mouse and Guinea pig models.

PubMed

Xie, Xing; Na, Woonsung; Kang, Aram; Yeom, Minjoo; Yuk, Heejun; Moon, Hyoungjoon; Kim, Sung-Jae; Kim, Hyun-Woo; Kim, Jeong-Ki; Pang, Maoda; Wang, Yongshan; Liu, Yongjie; Song, Daesub

2018-05-02

Avian-origin H3N2 canine influenza virus (CIV) has been the most common subtype in Korea and China since 2007. Here, we compared the pathogenicity and transmissibility of three H3N2 CIV strains [Chinese CIV (JS/10), Korean CIV (KR/07), and Korean recombinant CIV between the classic H3N2 CIV and the pandemic H1N1 virus (MV/12)] in BALB/c mouse and guinea pig models. The pandemic H1N1 (CA/09) strain served as the control. BALB/c mice infected with H1N1 had high mortality and obvious body weight loss, whereas no overt disease symptoms were observed in mice inoculated with H3N2 CIV strains. The viral titers were higher in the group MV/12 than those in groups JS/10 and KR/07, while the mice infected with JS/10 showed higher viral titers in all tissues (except for the lung) than the mice infected with KR/07. The data obtained in guinea pigs also demonstrated that group MV/12 presented the highest loads in most of the tissues, followed by group JS/10 and KR/07. Also, direct contact transmissions of all the three CIV strains could be observed in guinea pigs, and for the inoculated and the contact groups, the viral titer of group MV/12 and KR/07 was higher than that of group JS/10 in nasal swabs. These findings indicated that the matrix (M) gene obtained from the pandemic H1N1 may enhance viral replication of classic H3N2 CIV; JS/10 has stronger viral replication ability in tissues as compared to KR/07, whereas KR/07 infected guinea pigs have more viral shedding than JS/10 infected guinea pigs. There exists a discrepancy in pathobiology among CIV isolates. Reverse genetics regarding the genomes of CIV isolates will be helpful to further explain the virus characteristics.

Parameters of Mosquito-Enhanced West Nile Virus Infection.

PubMed

Moser, Lindsey A; Lim, Pei-Yin; Styer, Linda M; Kramer, Laura D; Bernard, Kristen A

2016-01-01

The arthropod-borne West Nile virus (WNV) emerged in New York State in 1999 and quickly spread throughout the United States. Transmission is maintained in an enzootic cycle in which infected mosquitoes transmit the virus to susceptible hosts during probing and feeding. Arthropod-derived components within the viral inoculum are increasingly acknowledged to play a role in infection of vertebrate hosts. We previously showed that Culex tarsalis mosquito saliva and salivary gland extract (SGE) enhance the in vivo replication of WNV. Here, we characterized the effective dose, timing, and proximity of saliva and SGE administration necessary for enhancement of WNV viremia using a mouse model. Mosquito saliva and SGE enhanced viremia in a dose-dependent manner, and a single mosquito bite or as little as 0.01 μg of SGE was effective at enhancing viremia, suggesting a potent active salivary factor. Viremia was enhanced when SGE was injected in the same location as virus inoculation from 24 h before virus inoculation through 12 h after virus inoculation. These results were confirmed with mosquito saliva deposited by uninfected mosquitoes. When salivary treatment and virus inoculation were spatially separated, viremia was not enhanced. In summary, the effects of mosquito saliva and SGE were potent, long lasting, and localized, and these studies have implications for virus transmission in nature, where vertebrate hosts are fed upon by both infected and uninfected mosquitoes over time. Furthermore, our model provides a robust system to identify the salivary factor(s) responsible for enhancement of WNV replication. Mosquito-borne viruses are a significant class of agents causing emerging infectious diseases. WNV has caused over 18,000 cases of neuroinvasive disease in the United States since its emergence. We have shown that Culex tarsalis mosquito saliva and SGE enhance the replication of WNV. We now demonstrate that saliva and SGE have potent, long-lasting, and localized effects. Our model provides a robust system to identify the salivary factor(s) and characterize the mechanism responsible for enhancement of WNV replication. These studies could lead to the identification of novel prophylactic or treatment options useful in limiting the spread of WNV, other mosquito-borne viruses, and the diseases that they cause. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

HIV-1 infection, response to treatment and establishment of viral latency in a novel humanized T cell-only mouse (TOM) model.

PubMed

Honeycutt, Jenna B; Wahl, Angela; Archin, Nancie; Choudhary, Shailesh; Margolis, David; Garcia, J Victor

2013-10-24

The major targets of HIV infection in humans are CD4⁺ T cells. CD4⁺ T cell depletion is a hallmark of AIDS. Previously, the SCID-hu thy/liv model was used to study the effect of HIV on thymopoeisis in vivo. However, these mice did not develop high levels of peripheral T cell reconstitution and required invasive surgery for infection and analysis. Here, we describe a novel variant of this model in which thy/liv implantation results in systemic reconstitution with human T cells in the absence of any other human hematopoietic lineages. NOD/SCID-hu thy/liv and NSG-hu thy/liv mice were created by implanting human fetal thymus and liver tissues under the kidney capsule of either NOD/SCID or NSG mice. In contrast to NOD/SCID-hu thy/liv mice that show little or no human cells in peripheral blood or tissues, substantial systemic human reconstitution occurs in NSG-hu thy/liv. These mice are exclusively reconstituted with human T cells (i.e. T-cell only mice or TOM). Despite substantial levels of human T cells no signs of graft-versus-host disease (GVHD) were noted in these mice over a period of 14 months. TOM are readily infected after parenteral exposure to HIV-1. HIV replication is sustained in peripheral blood at high levels and results in modest reduction of CD4⁺ T cells. HIV-1 replication in TOM responds to daily administration of combination antiretroviral therapy (ART) resulting in strong suppression of virus replication as determined by undetectable viral load in plasma. Latently HIV infected resting CD4⁺ T cells can be isolated from suppressed mice that can be induced to express HIV ex-vivo upon activation demonstrating the establishment of latency in vivo. NSG-hu thy/liv mice are systemically reconstituted with human T cells. No other human lymphoid lineages are present in these mice (i.e. monocytes/macrophages, B cells and DC are all absent). These T cell only mice do not develop GVHD, are susceptible to HIV-1 infection and can efficiently maintain virus replication. HIV infected TOM undergoing ART harbor latently infected, resting CD4+ T cells.

Using Model Replication to Improve the Reliability of Agent-Based Models

NASA Astrophysics Data System (ADS)

Zhong, Wei; Kim, Yushim

The basic presupposition of model replication activities for a computational model such as an agent-based model (ABM) is that, as a robust and reliable tool, it must be replicable in other computing settings. This assumption has recently gained attention in the community of artificial society and simulation due to the challenges of model verification and validation. Illustrating the replication of an ABM representing fraudulent behavior in a public service delivery system originally developed in the Java-based MASON toolkit for NetLogo by a different author, this paper exemplifies how model replication exercises provide unique opportunities for model verification and validation process. At the same time, it helps accumulate best practices and patterns of model replication and contributes to the agenda of developing a standard methodological protocol for agent-based social simulation.

Large transcription units unify copy number variants and common fragile sites arising under replication stress.

PubMed

Wilson, Thomas E; Arlt, Martin F; Park, So Hae; Rajendran, Sountharia; Paulsen, Michelle; Ljungman, Mats; Glover, Thomas W

2015-02-01

Copy number variants (CNVs) resulting from genomic deletions and duplications and common fragile sites (CFSs) seen as breaks on metaphase chromosomes are distinct forms of structural chromosome instability precipitated by replication inhibition. Although they share a common induction mechanism, it is not known how CNVs and CFSs are related or why some genomic loci are much more prone to their occurrence. Here we compare large sets of de novo CNVs and CFSs in several experimental cell systems to each other and to overlapping genomic features. We first show that CNV hotpots and CFSs occurred at the same human loci within a given cultured cell line. Bru-seq nascent RNA sequencing further demonstrated that although genomic regions with low CNV frequencies were enriched in transcribed genes, the CNV hotpots that matched CFSs specifically corresponded to the largest active transcription units in both human and mouse cells. Consistently, active transcription units >1 Mb were robust cell-type-specific predictors of induced CNV hotspots and CFS loci. Unlike most transcribed genes, these very large transcription units replicated late and organized deletion and duplication CNVs into their transcribed and flanking regions, respectively, supporting a role for transcription in replication-dependent lesion formation. These results indicate that active large transcription units drive extreme locus- and cell-type-specific genomic instability under replication stress, resulting in both CNVs and CFSs as different manifestations of perturbed replication dynamics. © 2015 Wilson et al.; Published by Cold Spring Harbor Laboratory Press.

A new mechanism of interferon's antiviral action: Induction of autophagy, essential for paramyxovirus replication, is inhibited by the interferon stimulated gene, TDRD7.

PubMed

Subramanian, Gayatri; Kuzmanovic, Teodora; Zhang, Ying; Peter, Cara Beate; Veleeparambil, Manoj; Chakravarti, Ritu; Sen, Ganes C; Chattopadhyay, Saurabh

2018-01-01

The interferon (IFN) system represents the first line of defense against a wide range of viruses. Virus infection rapidly triggers the transcriptional induction of IFN-β and IFN Stimulated Genes (ISGs), whose protein products act as viral restriction factors by interfering with specific stages of virus life cycle, such as entry, transcription, translation, genome replication, assembly and egress. Here, we report a new mode of action of an ISG, IFN-induced TDRD7 (tudor domain containing 7) inhibited paramyxovirus replication by inhibiting autophagy. TDRD7 was identified as an antiviral gene by a high throughput screen of an ISG shRNA library for blocking IFN's protective effect against Sendai virus (SeV) replication. The antiviral activity of TDRD7 against SeV, human parainfluenza virus 3 and respiratory syncytial virus was confirmed by its genetic ablation or ectopic expression in several types of mouse and human cells. TDRD7's antiviral action was mediated by its ability to inhibit autophagy, a cellular catabolic process which was robustly induced by SeV infection and required for its replication. Mechanistic investigation revealed that TDRD7 interfered with the activation of AMP-dependent kinase (AMPK), an enzyme required for initiating autophagy. AMPK activity was required for efficient replication of several paramyxoviruses, as demonstrated by its genetic ablation or inhibition of its activity by TDRD7 or chemical inhibitors. Therefore, our study has identified a new antiviral ISG with a new mode of action.

Large transcription units unify copy number variants and common fragile sites arising under replication stress

PubMed Central

Park, So Hae; Rajendran, Sountharia; Paulsen, Michelle; Ljungman, Mats; Glover, Thomas W.

2015-01-01

Copy number variants (CNVs) resulting from genomic deletions and duplications and common fragile sites (CFSs) seen as breaks on metaphase chromosomes are distinct forms of structural chromosome instability precipitated by replication inhibition. Although they share a common induction mechanism, it is not known how CNVs and CFSs are related or why some genomic loci are much more prone to their occurrence. Here we compare large sets of de novo CNVs and CFSs in several experimental cell systems to each other and to overlapping genomic features. We first show that CNV hotpots and CFSs occurred at the same human loci within a given cultured cell line. Bru-seq nascent RNA sequencing further demonstrated that although genomic regions with low CNV frequencies were enriched in transcribed genes, the CNV hotpots that matched CFSs specifically corresponded to the largest active transcription units in both human and mouse cells. Consistently, active transcription units >1 Mb were robust cell-type-specific predictors of induced CNV hotspots and CFS loci. Unlike most transcribed genes, these very large transcription units replicated late and organized deletion and duplication CNVs into their transcribed and flanking regions, respectively, supporting a role for transcription in replication-dependent lesion formation. These results indicate that active large transcription units drive extreme locus- and cell-type-specific genomic instability under replication stress, resulting in both CNVs and CFSs as different manifestations of perturbed replication dynamics. PMID:25373142

Rapid acquisition adaptive amino acid substitutions involved in the virulence enhancement of an H1N2 avian influenza virus in mice.

PubMed

Yu, Zhijun; Sun, Weiyang; Zhang, Xinghai; Cheng, Kaihui; Zhao, Chuqi; Xia, Xianzhu; Gao, Yuwei

2017-08-01

Although H1N2 avian influenza virus (AIV) only infect birds, documented cases of swine infection with H1N2 influenza viruses suggest this subtype AIV may pose a potential threat to mammals. Here, we generated mouse-adapted variants of a H1N2 AIV to identify adaptive changes that increased virulence in mammals. MLD 50 of the variants were reduced >1000-fold compared to the parental virus. Variants displayed enhanced replication in vitro and in vivo, and replicate in extrapulmonary organs. These data show that enhanced replication capacity and expanded tissue tropism may increase the virulence of H1N2 AIV in mice. Sequence analysis revealed multiple amino acid substitutions in the PB2 (L134H, I647L, and D701N), HA (G228S), and M1 (D231N) proteins. These results indicate that H1N2 AIV can rapidly acquire adaptive amino acid substitutions in mammalian hosts, and these amino acid substitutions collaboratively enhance the ability of H1N2 AIV to replicate and cause severe disease in mammals. Copyright © 2017 Elsevier B.V. All rights reserved.

Efficacy of SCH27899 in an Animal Model of Legionnaires' Disease Using Immunocompromised A/J Mice

PubMed Central

Brieland, Joan K.; Loebenberg, David; Menzel, Fred; Hare, Roberta S.

2000-01-01

The efficacy of SCH27899, a new everninomicin antibiotic, against replicative Legionella pneumophila lung infections in an immunocompromised host was evaluated using a murine model of Legionnaires' disease. A/J mice were immunocompromised with cortisone acetate and inoculated intratracheally with L. pneumophila serogroup 1 (105 CFU per mouse). At 24 h postinoculation, mice were administered either SCH27899 (6 to 60 mg/kg [MPK] intravenously) or a placebo once daily for 5 days, and mortality and intrapulmonary growth of L. pneumophila were assessed. In the absence of SCH27899, there was 100% mortality in L. pneumophila-infected mice, with exponential intrapulmonary growth of the bacteria. In contrast, administration of SCH27899 at a dose of ≥30 MPK resulted in ≥90% survival of infected mice, which was associated with inhibition of intrapulmonary growth of L. pneumophila. In subsequent studies, the efficacy of SCH27899 was compared to ofloxacin (OFX) and azithromycin (AZI). Administration of SCH27899, OFX, or AZI at a dose of ≥30 MPK once daily for 5 days resulted in ≥85% survival of infected mice and inhibition of intrapulmonary growth of the bacteria. However, L. pneumophila CFU were recovered in lung homogenates following cessation of therapy with all three antibiotics. These studies demonstrate that SCH27899 effectively prevents fatal replicative L. pneumophila lung infection in immunocompromised A/J mice by inhibition of intrapulmonary growth of the bacteria. However, in this murine model of pulmonary legionellosis, SCH27899, like OFX and AZI, was bacteriostatic. PMID:10770771

The effect of inhibition of PP1 and TNFα signaling on pathogenesis of SARS coronavirus.

PubMed

McDermott, Jason E; Mitchell, Hugh D; Gralinski, Lisa E; Eisfeld, Amie J; Josset, Laurence; Bankhead, Armand; Neumann, Gabriele; Tilton, Susan C; Schäfer, Alexandra; Li, Chengjun; Fan, Shufang; McWeeney, Shannon; Baric, Ralph S; Katze, Michael G; Waters, Katrina M

2016-09-23

The complex interplay between viral replication and host immune response during infection remains poorly understood. While many viruses are known to employ anti-immune strategies to facilitate their replication, highly pathogenic virus infections can also cause an excessive immune response that exacerbates, rather than reduces pathogenicity. To investigate this dichotomy in severe acute respiratory syndrome coronavirus (SARS-CoV), we developed a transcriptional network model of SARS-CoV infection in mice and used the model to prioritize candidate regulatory targets for further investigation. We validated our predictions in 18 different knockout (KO) mouse strains, showing that network topology provides significant predictive power to identify genes that are important for viral infection. We identified a novel player in the immune response to virus infection, Kepi, an inhibitory subunit of the protein phosphatase 1 (PP1) complex, which protects against SARS-CoV pathogenesis. We also found that receptors for the proinflammatory cytokine tumor necrosis factor alpha (TNFα) promote pathogenesis, presumably through excessive inflammation. The current study provides validation of network modeling approaches for identifying important players in virus infection pathogenesis, and a step forward in understanding the host response to an important infectious disease. The results presented here suggest the role of Kepi in the host response to SARS-CoV, as well as inflammatory activity driving pathogenesis through TNFα signaling in SARS-CoV infections. Though we have reported the utility of this approach in bacterial and cell culture studies previously, this is the first comprehensive study to confirm that network topology can be used to predict phenotypes in mice with experimental validation.

BinQuasi: a peak detection method for ChIP-sequencing data with biological replicates.

PubMed

Goren, Emily; Liu, Peng; Wang, Chao; Wang, Chong

2018-04-19

ChIP-seq experiments that are aimed at detecting DNA-protein interactions require biological replication to draw inferential conclusions, however there is no current consensus on how to analyze ChIP-seq data with biological replicates. Very few methodologies exist for the joint analysis of replicated ChIP-seq data, with approaches ranging from combining the results of analyzing replicates individually to joint modeling of all replicates. Combining the results of individual replicates analyzed separately can lead to reduced peak classification performance compared to joint modeling. Currently available methods for joint analysis may fail to control the false discovery rate at the nominal level. We propose BinQuasi, a peak caller for replicated ChIP-seq data, that jointly models biological replicates using a generalized linear model framework and employs a one-sided quasi-likelihood ratio test to detect peaks. When applied to simulated data and real datasets, BinQuasi performs favorably compared to existing methods, including better control of false discovery rate than existing joint modeling approaches. BinQuasi offers a flexible approach to joint modeling of replicated ChIP-seq data which is preferable to combining the results of replicates analyzed individually. Source code is freely available for download at https://cran.r-project.org/package=BinQuasi, implemented in R. pliu@iastate.edu or egoren@iastate.edu. Supplementary material is available at Bioinformatics online.

Insulin-like growth factor-I extends in vitro replicative life span of skeletal muscle satellite cells by enhancing G1/S cell cycle progression via the activation of phosphatidylinositol 3'-kinase/Akt signaling pathway

NASA Technical Reports Server (NTRS)

Chakravarthy, M. V.; Abraha, T. W.; Schwartz, R. J.; Fiorotto, M. L.; Booth, F. W.

2000-01-01

Interest is growing in methods to extend replicative life span of non-immortalized stem cells. Using the insulin-like growth factor I (IGF-I) transgenic mouse in which the IGF-I transgene is expressed during skeletal muscle development and maturation prior to isolation and during culture of satellite cells (the myogenic stem cells of mature skeletal muscle fibers) as a model system, we elucidated the underlying molecular mechanisms of IGF-I-mediated enhancement of proliferative potential of these cells. Satellite cells from IGF-I transgenic muscles achieved at least five additional population doublings above the maximum that was attained by wild type satellite cells. This IGF-I-induced increase in proliferative potential was mediated via activation of the phosphatidylinositol 3'-kinase/Akt pathway, independent of mitogen-activated protein kinase activity, facilitating G(1)/S cell cycle progression via a down-regulation of p27(Kip1). Adenovirally mediated ectopic overexpression of p27(Kip1) in exponentially growing IGF-I transgenic satellite cells reversed the increase in cyclin E-cdk2 kinase activity, pRb phosphorylation, and cyclin A protein abundance, thereby implicating an important role for p27(Kip1) in promoting satellite cell senescence. These observations provide a more complete dissection of molecular events by which increased local expression of a growth factor in mature skeletal muscle fibers extends replicative life span of primary stem cells than previously known.

Regulation and Modulation of Human DNA Polymerase δ Activity and Function

PubMed Central

Wang, Xiaoxiao; Zhang, Sufang; Zhang, Zhongtao; Lee, Ernest Y. C.

2017-01-01

This review focuses on the regulation and modulation of human DNA polymerase δ (Pol δ). The emphasis is on the mechanisms that regulate the activity and properties of Pol δ in DNA repair and replication. The areas covered are the degradation of the p12 subunit of Pol δ, which converts it from a heterotetramer (Pol δ4) to a heterotrimer (Pol δ3), in response to DNA damage and also during the cell cycle. The biochemical mechanisms that lead to degradation of p12 are reviewed, as well as the properties of Pol δ4 and Pol δ3 that provide insights into their functions in DNA replication and repair. The second focus of the review involves the functions of two Pol δ binding proteins, polymerase delta interaction protein 46 (PDIP46) and polymerase delta interaction protein 38 (PDIP38), both of which are multi-functional proteins. PDIP46 is a novel activator of Pol δ4, and the impact of this function is discussed in relation to its potential roles in DNA replication. Several new models for the roles of Pol δ3 and Pol δ4 in leading and lagging strand DNA synthesis that integrate a role for PDIP46 are presented. PDIP38 has multiple cellular localizations including the mitochondria, the spliceosomes and the nucleus. It has been implicated in a number of cellular functions, including the regulation of specialized DNA polymerases, mitosis, the DNA damage response, mouse double minute 2 homolog (Mdm2) alternative splicing and the regulation of the NADPH oxidase 4 (Nox4). PMID:28737709

Lucidone suppresses dengue viral replication through the induction of heme oxygenase-1.

PubMed

Chen, Wei-Chun; Tseng, Chin-Kai; Lin, Chun-Kuang; Wang, Shen-Nien; Wang, Wen-Hung; Hsu, Shih-Hsien; Wu, Yu-Hsuan; Hung, Ling-Chien; Chen, Yen-Hsu; Lee, Jin-Ching

2018-01-01

Dengue virus (DENV) infection causes life-threatening diseases such as dengue hemorrhagic fever and dengue shock syndrome. Currently, there is no effective therapeutic agent or vaccine against DENV infection; hence, there is an urgent need to discover anti-DENV agents. The potential therapeutic efficacy of lucidone was first evaluated in vivo using a DENV-infected Institute of Cancer Research (ICR) suckling mouse model by monitoring body weight, clinical score, survival rate, and viral titer. We found that lucidone effectively protected mice from DENV infection by sustaining survival rate and reducing viral titers in DENV-infected ICR suckling mice. Then, the anti-DENV activity of lucidone was confirmed by western blotting and quantitative-reverse-transcription-polymerase chain reaction analysis, with an EC 50 value of 25 ± 3 μM. Lucidone significantly induced heme oxygenase-1 (HO-1) production against DENV replication by inhibiting DENV NS2B/3 protease activity to induce the DENV-suppressed antiviral interferon response. The inhibitory effect of lucidone on DENV replication was attenuated by silencing of HO-1 gene expression or blocking HO-1 activity. In addition, lucidone-stimulated nuclear factor erythroid 2-related factor 2 (Nrf2), which is involved in transactivation of HO-1 expression for its anti-DENV activity. Taken together, the mechanistic investigations revealed that lucidone exhibits significant anti-DENV activity in in vivo and in vitro by inducing Nrf2-mediated HO-1 expression, leading to blockage of viral protease activity to induce the anti-viral interferon (IFN) response. These results suggest that lucidone is a promising candidate for drug development.

Ssb1 and Ssb2 cooperate to regulate mouse hematopoietic stem and progenitor cells by resolving replicative stress.

PubMed

Shi, Wei; Vu, Therese; Boucher, Didier; Biernacka, Anna; Nde, Jules; Pandita, Raj K; Straube, Jasmin; Boyle, Glen M; Al-Ejeh, Fares; Nag, Purba; Jeffery, Jessie; Harris, Janelle L; Bain, Amanda L; Grzelak, Marta; Skrzypczak, Magdalena; Mitra, Abhishek; Dojer, Norbert; Crosetto, Nicola; Cloonan, Nicole; Becherel, Olivier J; Finnie, John; Skaar, Jeffrey R; Walkley, Carl R; Pandita, Tej K; Rowicka, Maga; Ginalski, Krzysztof; Lane, Steven W; Khanna, Kum Kum

2017-05-04

Hematopoietic stem and progenitor cells (HSPCs) are vulnerable to endogenous damage and defects in DNA repair can limit their function. The 2 single-stranded DNA (ssDNA) binding proteins SSB1 and SSB2 are crucial regulators of the DNA damage response; however, their overlapping roles during normal physiology are incompletely understood. We generated mice in which both Ssb1 and Ssb2 were constitutively or conditionally deleted. Constitutive Ssb1/Ssb2 double knockout (DKO) caused early embryonic lethality, whereas conditional Ssb1/Ssb2 double knockout (cDKO) in adult mice resulted in acute lethality due to bone marrow failure and intestinal atrophy featuring stem and progenitor cell depletion, a phenotype unexpected from the previously reported single knockout models of Ssb1 or Ssb2 Mechanistically, cDKO HSPCs showed altered replication fork dynamics, massive accumulation of DNA damage, genome-wide double-strand breaks enriched at Ssb-binding regions and CpG islands, together with the accumulation of R -loops and cytosolic ssDNA. Transcriptional profiling of cDKO HSPCs revealed the activation of p53 and interferon (IFN) pathways, which enforced cell cycling in quiescent HSPCs, resulting in their apoptotic death. The rapid cell death phenotype was reproducible in in vitro cultured cDKO-hematopoietic stem cells, which were significantly rescued by nucleotide supplementation or after depletion of p53. Collectively, Ssb1 and Ssb2 control crucial aspects of HSPC function, including proliferation and survival in vivo by resolving replicative stress to maintain genomic stability. © 2017 by The American Society of Hematology.

Ssb1 and Ssb2 cooperate to regulate mouse hematopoietic stem and progenitor cells by resolving replicative stress

PubMed Central

Vu, Therese; Boucher, Didier; Biernacka, Anna; Nde, Jules; Pandita, Raj K.; Straube, Jasmin; Boyle, Glen M.; Al-Ejeh, Fares; Jeffery, Jessie; Harris, Janelle L.; Bain, Amanda L.; Grzelak, Marta; Skrzypczak, Magdalena; Mitra, Abhishek; Dojer, Norbert; Crosetto, Nicola; Cloonan, Nicole; Becherel, Olivier J.; Finnie, John; Skaar, Jeffrey R.; Walkley, Carl R.; Pandita, Tej K.; Rowicka, Maga; Ginalski, Krzysztof

2017-01-01

Hematopoietic stem and progenitor cells (HSPCs) are vulnerable to endogenous damage and defects in DNA repair can limit their function. The 2 single-stranded DNA (ssDNA) binding proteins SSB1 and SSB2 are crucial regulators of the DNA damage response; however, their overlapping roles during normal physiology are incompletely understood. We generated mice in which both Ssb1 and Ssb2 were constitutively or conditionally deleted. Constitutive Ssb1/Ssb2 double knockout (DKO) caused early embryonic lethality, whereas conditional Ssb1/Ssb2 double knockout (cDKO) in adult mice resulted in acute lethality due to bone marrow failure and intestinal atrophy featuring stem and progenitor cell depletion, a phenotype unexpected from the previously reported single knockout models of Ssb1 or Ssb2. Mechanistically, cDKO HSPCs showed altered replication fork dynamics, massive accumulation of DNA damage, genome-wide double-strand breaks enriched at Ssb-binding regions and CpG islands, together with the accumulation of R-loops and cytosolic ssDNA. Transcriptional profiling of cDKO HSPCs revealed the activation of p53 and interferon (IFN) pathways, which enforced cell cycling in quiescent HSPCs, resulting in their apoptotic death. The rapid cell death phenotype was reproducible in in vitro cultured cDKO-hematopoietic stem cells, which were significantly rescued by nucleotide supplementation or after depletion of p53. Collectively, Ssb1 and Ssb2 control crucial aspects of HSPC function, including proliferation and survival in vivo by resolving replicative stress to maintain genomic stability. PMID:28270450

A Single Mutation at Position 190 in Hemagglutinin Enhances Binding Affinity for Human Type Sialic Acid Receptor and Replication of H9N2 Avian Influenza Virus in Mice

PubMed Central

Teng, Qiaoyang; Xu, Dawei; Shen, Weixia; Liu, Qinfang; Rong, Guangyu; Li, Xuesong; Yan, Liping; Yang, Jianmei; Chen, Hongjun; Yu, Hai

2016-01-01

ABSTRACT H9N2 avian influenza virus (AIV) has an extended host range, but the molecular basis underlying H9N2 AIV transmission to mammals remains unclear. We isolated more than 900 H9N2 AIVs in our 3-year surveillance in live bird markets in China from 2009 to 2012. Thirty-seven representative isolates were selected for further detailed characterization. These isolates were categorized into 8 genotypes (B64 to B71) and formed a distinct antigenic subgroup. Three isolates belonging to genotype B69, which is a predominant genotype circulating in China, replicated efficiently in mice, while the viruses tested in parallel in other genotypes replicated poorly, although they, like the three B69 isolates, have a leucine at position 226 in the hemagglutinin (HA) receptor binding site, which is critical for binding human type sialic acid receptors. Further molecular and single mutation analysis revealed that a valine (V) residue at position 190 in HA is responsible for efficient replication of these H9N2 viruses in mice. The 190V in HA does not affect virus receptor binding specificity but enhances binding affinity to human cells and lung tissues from mouse and humans. All these data indicate that the 190V in HA is one of the important determinants for H9N2 AIVs to cross the species barrier to infect mammals despite multiple genes conferring adaptation and replication of H9N2 viruses in mammals. Our findings provide novel insights on understanding host range expansion of H9N2 AIVs. IMPORTANCE Influenza virus hemagglutinin (HA) is responsible for binding to host cell receptors and therefore influences the viral host range and pathogenicity in different species. We showed that the H9N2 avian influenza viruses harboring 190V in the HA exhibit enhanced virus replication in mice. Further studies demonstrate that 190V in the HA does not change virus receptor binding specificity but enhances virus binding affinity of the H9N2 virus to human cells and attachment to lung tissues from humans and mouse. Our findings suggest that more attention should be given to the H9N2 AIVs with HA-190V during surveillance due to their potential threat to mammals, including humans. PMID:27558420

A Single Mutation at Position 190 in Hemagglutinin Enhances Binding Affinity for Human Type Sialic Acid Receptor and Replication of H9N2 Avian Influenza Virus in Mice.

PubMed

Teng, Qiaoyang; Xu, Dawei; Shen, Weixia; Liu, Qinfang; Rong, Guangyu; Li, Xuesong; Yan, Liping; Yang, Jianmei; Chen, Hongjun; Yu, Hai; Ma, Wenjun; Li, Zejun

2016-11-01

H9N2 avian influenza virus (AIV) has an extended host range, but the molecular basis underlying H9N2 AIV transmission to mammals remains unclear. We isolated more than 900 H9N2 AIVs in our 3-year surveillance in live bird markets in China from 2009 to 2012. Thirty-seven representative isolates were selected for further detailed characterization. These isolates were categorized into 8 genotypes (B64 to B71) and formed a distinct antigenic subgroup. Three isolates belonging to genotype B69, which is a predominant genotype circulating in China, replicated efficiently in mice, while the viruses tested in parallel in other genotypes replicated poorly, although they, like the three B69 isolates, have a leucine at position 226 in the hemagglutinin (HA) receptor binding site, which is critical for binding human type sialic acid receptors. Further molecular and single mutation analysis revealed that a valine (V) residue at position 190 in HA is responsible for efficient replication of these H9N2 viruses in mice. The 190V in HA does not affect virus receptor binding specificity but enhances binding affinity to human cells and lung tissues from mouse and humans. All these data indicate that the 190V in HA is one of the important determinants for H9N2 AIVs to cross the species barrier to infect mammals despite multiple genes conferring adaptation and replication of H9N2 viruses in mammals. Our findings provide novel insights on understanding host range expansion of H9N2 AIVs. Influenza virus hemagglutinin (HA) is responsible for binding to host cell receptors and therefore influences the viral host range and pathogenicity in different species. We showed that the H9N2 avian influenza viruses harboring 190V in the HA exhibit enhanced virus replication in mice. Further studies demonstrate that 190V in the HA does not change virus receptor binding specificity but enhances virus binding affinity of the H9N2 virus to human cells and attachment to lung tissues from humans and mouse. Our findings suggest that more attention should be given to the H9N2 AIVs with HA-190V during surveillance due to their potential threat to mammals, including humans. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Secreted Gaussia princeps luciferase as a reporter of Escherichia coli replication in a mouse tissue cage model of infection.

PubMed

Liu, Mingyu; Blinn, Christina; McLeod, Sarah M; Wiseman, John W; Newman, Joseph V; Fisher, Stewart L; Walkup, Grant K

2014-01-01

Measurement of bacterial burden in animal infection models is a key component for both bacterial pathogenesis studies and therapeutic agent research. The traditional quantification means for in vivo bacterial burden requires frequent animal sacrifice and enumerating colony forming units (CFU) recovered from infection loci. To address these issues, researchers have developed a variety of luciferase-expressing bacterial reporter strains to enable bacterial detection in living animals. To date, all such luciferase-based bacterial reporters are in cell-associated form. Production of luciferase-secreting recombinant bacteria could provide the advantage of reporting CFU from both infection loci themselves and remote sampling (eg. body fluid and plasma). Toward this end, we have genetically manipulated a pathogenic Escherichia coli (E. coli) strain, ATCC25922, to secrete the marine copepod Gaussia princeps luciferase (Gluc), and assessed the use of Gluc as both an in situ and ex situ reporter for bacterial burden in mouse tissue cage infections. The E. coli expressing Gluc demonstrates in vivo imaging of bacteria in a tissue cage model of infection. Furthermore, secreted Gluc activity and bacterial CFUs recovered from tissue cage fluid (TCF) are correlated along 18 days of infection. Importantly, secreted Gluc can also be detected in plasma samples and serve as an ex situ indicator for the established tissue cage infection, once high bacterial burdens are achieved. We have demonstrated that Gluc from marine eukaryotes can be stably expressed and secreted by pathogenic E. coli in vivo to enable a facile tool for longitudinal evaluation of persistent bacterial infection.

Trace: a high-throughput tomographic reconstruction engine for large-scale datasets

DOE PAGES

Bicer, Tekin; Gursoy, Doga; Andrade, Vincent De; ...

2017-01-28

Here, synchrotron light source and detector technologies enable scientists to perform advanced experiments. These scientific instruments and experiments produce data at such scale and complexity that large-scale computation is required to unleash their full power. One of the widely used data acquisition technique at light sources is Computed Tomography, which can generate tens of GB/s depending on x-ray range. A large-scale tomographic dataset, such as mouse brain, may require hours of computation time with a medium size workstation. In this paper, we present Trace, a data-intensive computing middleware we developed for implementation and parallelization of iterative tomographic reconstruction algorithms. Tracemore » provides fine-grained reconstruction of tomography datasets using both (thread level) shared memory and (process level) distributed memory parallelization. Trace utilizes a special data structure called replicated reconstruction object to maximize application performance. We also present the optimizations we have done on the replicated reconstruction objects and evaluate them using a shale and a mouse brain sinogram. Our experimental evaluations show that the applied optimizations and parallelization techniques can provide 158x speedup (using 32 compute nodes) over single core configuration, which decreases the reconstruction time of a sinogram (with 4501 projections and 22400 detector resolution) from 12.5 hours to less than 5 minutes per iteration.« less

Nucleic Acid Chaperone Activity of the ORF1 Protein from the Mouse LINE-1 Retrotransposon

PubMed Central

Martin, Sandra L.; Bushman, Frederic D.

2001-01-01

Non-LTR retrotransposons such as L1 elements are major components of the mammalian genome, but their mechanism of replication is incompletely understood. Like retroviruses and LTR-containing retrotransposons, non-LTR retrotransposons replicate by reverse transcription of an RNA intermediate. The details of cDNA priming and integration, however, differ between these two classes. In retroviruses, the nucleocapsid (NC) protein has been shown to assist reverse transcription by acting as a “nucleic acid chaperone,” promoting the formation of the most stable duplexes between nucleic acid molecules. A protein-coding region with an NC-like sequence is present in most non-LTR retrotransposons, but no such sequence is evident in mammalian L1 elements or other members of its class. Here we investigated the ORF1 protein from mouse L1 and found that it does in fact display nucleic acid chaperone activities in vitro. L1 ORF1p (i) promoted annealing of complementary DNA strands, (ii) facilitated strand exchange to form the most stable hybrids in competitive displacement assays, and (iii) facilitated melting of an imperfect duplex but stabilized perfect duplexes. These findings suggest a role for L1 ORF1p in mediating nucleic acid strand transfer steps during L1 reverse transcription. PMID:11134335

Trace: a high-throughput tomographic reconstruction engine for large-scale datasets

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bicer, Tekin; Gursoy, Doga; Andrade, Vincent De

Here, synchrotron light source and detector technologies enable scientists to perform advanced experiments. These scientific instruments and experiments produce data at such scale and complexity that large-scale computation is required to unleash their full power. One of the widely used data acquisition technique at light sources is Computed Tomography, which can generate tens of GB/s depending on x-ray range. A large-scale tomographic dataset, such as mouse brain, may require hours of computation time with a medium size workstation. In this paper, we present Trace, a data-intensive computing middleware we developed for implementation and parallelization of iterative tomographic reconstruction algorithms. Tracemore » provides fine-grained reconstruction of tomography datasets using both (thread level) shared memory and (process level) distributed memory parallelization. Trace utilizes a special data structure called replicated reconstruction object to maximize application performance. We also present the optimizations we have done on the replicated reconstruction objects and evaluate them using a shale and a mouse brain sinogram. Our experimental evaluations show that the applied optimizations and parallelization techniques can provide 158x speedup (using 32 compute nodes) over single core configuration, which decreases the reconstruction time of a sinogram (with 4501 projections and 22400 detector resolution) from 12.5 hours to less than 5 minutes per iteration.« less

Half brain irradiation in a murine model of breast cancer brain metastasis: magnetic resonance imaging and histological assessments of dose-response.

PubMed

Zarghami, Niloufar; Murrell, Donna H; Jensen, Michael D; Dick, Frederick A; Chambers, Ann F; Foster, Paula J; Wong, Eugene

2018-06-01

Brain metastasis is becoming increasingly prevalent in breast cancer due to improved extra-cranial disease control. With emerging availability of modern image-guided radiation platforms, mouse models of brain metastases and small animal magnetic resonance imaging (MRI), we examined brain metastases' responses from radiotherapy in the pre-clinical setting. In this study, we employed half brain irradiation to reduce inter-subject variability in metastases dose-response evaluations. Half brain irradiation was performed on a micro-CT/RT system in a human breast cancer (MDA-MB-231-BR) brain metastasis mouse model. Radiation induced DNA double stranded breaks in tumors and normal mouse brain tissue were quantified using γ-H2AX immunohistochemistry at 30 min (acute) and 11 days (longitudinal) after half-brain treatment for doses of 8, 16 and 24 Gy. In addition, tumor responses were assessed volumetrically with in-vivo longitudinal MRI and histologically for tumor cell density and nuclear size. In the acute setting, γ-H2AX staining in tumors saturated at higher doses while normal mouse brain tissue continued to increase linearly in the phosphorylation of H2AX. While γ-H2AX fluorescence intensities returned to the background level in the brain 11 days after treatment, the residual γ-H2AX phosphorylation in the radiated tumors remained elevated compared to un-irradiated contralateral tumors. With radiation, MRI-derived relative tumor growth was significantly reduced compared to the un-irradiated side. While there was no difference in MRI tumor volume growth between 16 and 24 Gy, there was a significant reduction in tumor cell density from histology with increasing dose. In the longitudinal study, nuclear size in the residual tumor cells increased significantly as the radiation dose was increased. Radiation damages to the DNAs in the normal brain parenchyma are resolved over time, but remain unrepaired in the treated tumors. Furthermore, there is a radiation dose response in nuclear size of surviving tumor cells. Increase in nuclear size together with unrepaired DNA damage indicated that the surviving tumor cells post radiation had continued to progress in the cell cycle with DNA replication, but failed cytokinesis. Half brain irradiation provides efficient evaluation of dose-response for cancer cell lines, a pre-requisite to perform experiments to understand radio-resistance in brain metastases.

Finding mouse models of human lymphomas and leukemia's using the Jackson laboratory mouse tumor biology database.

PubMed

Begley, Dale A; Sundberg, John P; Krupke, Debra M; Neuhauser, Steven B; Bult, Carol J; Eppig, Janan T; Morse, Herbert C; Ward, Jerrold M

2015-12-01

Many mouse models have been created to study hematopoietic cancer types. There are over thirty hematopoietic tumor types and subtypes, both human and mouse, with various origins, characteristics and clinical prognoses. Determining the specific type of hematopoietic lesion produced in a mouse model and identifying mouse models that correspond to the human subtypes of these lesions has been a continuing challenge for the scientific community. The Mouse Tumor Biology Database (MTB; http://tumor.informatics.jax.org) is designed to facilitate use of mouse models of human cancer by providing detailed histopathologic and molecular information on lymphoma subtypes, including expertly annotated, on line, whole slide scans, and providing a repository for storing information on and querying these data for specific lymphoma models. Copyright © 2015 Elsevier Inc. All rights reserved.

Construction and production of oncotropic vectors, derived from MVM(p), that share reduced sequence homology with helper plasmids.

PubMed

Clément, Nathalie; Velu, Thierry; Brandenburger, Annick

2002-09-01

The production of currently available vectors derived from autonomous parvoviruses requires the expression of capsid proteins in trans, from helper sequences. Cotransfection of a helper plasmid always generates significant amounts of replication-competent virus (RCV) that can be reduced by the integration of helper sequences into a packaging cell line. Although stocks of minute virus of mice (MVM)-based vectors with no detectable RCV could be produced by transfection into packaging cells; the latter appear after one or two rounds of replication, precluding further amplification of the vector stock. Indeed, once RCVs become detectable, they are efficiently amplified and rapidly take over the culture. Theoretically RCV-free vector stocks could be produced if all homology between vector and helper DNA is eliminated, thus preventing homologous recombination. We constructed new vectors based on the structure of spontaneously occurring defective particles of MVM. Based on published observations related to the size of vectors and the sequence of the viral origin of replication, these vectors were modified by the insertion of foreign DNA sequences downstream of the transgene and by the introduction of a consensus NS-1 nick site near the origin of replication to optimize their production. In one of the vectors the inserted fragment of mouse genomic DNA had a synergistic effect with the modified origin of replication in increasing vector production.

Mouse models rarely mimic the transcriptome of human neurodegenerative diseases: A systematic bioinformatics-based critique of preclinical models.

PubMed

Burns, Terry C; Li, Matthew D; Mehta, Swapnil; Awad, Ahmed J; Morgan, Alexander A

2015-07-15

Translational research for neurodegenerative disease depends intimately upon animal models. Unfortunately, promising therapies developed using mouse models mostly fail in clinical trials, highlighting uncertainty about how well mouse models mimic human neurodegenerative disease at the molecular level. We compared the transcriptional signature of neurodegeneration in mouse models of Alzheimer׳s disease (AD), Parkinson׳s disease (PD), Huntington׳s disease (HD) and amyotrophic lateral sclerosis (ALS) to human disease. In contrast to aging, which demonstrated a conserved transcriptome between humans and mice, only 3 of 19 animal models showed significant enrichment for gene sets comprising the most dysregulated up- and down-regulated human genes. Spearman׳s correlation analysis revealed even healthy human aging to be more closely related to human neurodegeneration than any mouse model of AD, PD, ALS or HD. Remarkably, mouse models frequently upregulated stress response genes that were consistently downregulated in human diseases. Among potential alternate models of neurodegeneration, mouse prion disease outperformed all other disease-specific models. Even among the best available animal models, conserved differences between mouse and human transcriptomes were found across multiple animal model versus human disease comparisons, surprisingly, even including aging. Relative to mouse models, mouse disease signatures demonstrated consistent trends toward preserved mitochondrial function protein catabolism, DNA repair responses, and chromatin maintenance. These findings suggest a more complex and multifactorial pathophysiology in human neurodegeneration than is captured through standard animal models, and suggest that even among conserved physiological processes such as aging, mice are less prone to exhibit neurodegeneration-like changes. This work may help explain the poor track record of mouse-based translational therapies for neurodegeneration and provides a path forward to critically evaluate and improve animal models of human disease. Copyright © 2015 Elsevier B.V. All rights reserved.

TWEAK induces liver progenitor cell proliferation

PubMed Central

Jakubowski, Aniela; Ambrose, Christine; Parr, Michael; Lincecum, John M.; Wang, Monica Z.; Zheng, Timothy S.; Browning, Beth; Michaelson, Jennifer S.; Baestcher, Manfred; Wang, Bruce; Bissell, D. Montgomery; Burkly, Linda C.

2005-01-01

Progenitor (“oval”) cell expansion accompanies many forms of liver injury, including alcohol toxicity and submassive parenchymal necrosis as well as experimental injury models featuring blocked hepatocyte replication. Oval cells can potentially become either hepatocytes or biliary epithelial cells and may be critical to liver regeneration, particularly when hepatocyte replication is impaired. The regulation of oval cell proliferation is incompletely understood. Herein we present evidence that a TNF family member called TWEAK (TNF-like weak inducer of apoptosis) stimulates oval cell proliferation in mouse liver through its receptor Fn14. TWEAK has no effect on mature hepatocytes and thus appears to be selective for oval cells. Transgenic mice overexpressing TWEAK in hepatocytes exhibit periportal oval cell hyperplasia. A similar phenotype was obtained in adult wild-type mice, but not Fn14-null mice, by administering TWEAK-expressing adenovirus. Oval cell expansion induced by 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) was significantly reduced in Fn14-null mice as well as in adult wild-type mice with a blocking anti-TWEAK mAb. Importantly, TWEAK stimulated the proliferation of an oval cell culture model. Finally, we show increased Fn14 expression in chronic hepatitis C and other human liver diseases relative to its expression in normal liver, which suggests a role for the TWEAK/Fn14 pathway in human liver injury. We conclude that TWEAK has a selective mitogenic effect for liver oval cells that distinguishes it from other previously described growth factors. PMID:16110324

Cell-penetrating peptide-morpholino conjugates alter pre-mRNA splicing of DMD (Duchenne muscular dystrophy) and inhibit murine coronavirus replication in vivo.

PubMed

Moulton, H M; Fletcher, S; Neuman, B W; McClorey, G; Stein, D A; Abes, S; Wilton, S D; Buchmeier, M J; Lebleu, B; Iversen, P L

2007-08-01

The cellular uptake of PMOs (phosphorodiamidate morpholino oligomers) can be enhanced by their conjugation to arginine-rich CPPs (cell-penetrating peptides). Here, we discuss our recent findings regarding (R-Ahx-R)(4)AhxB (Ahx is 6-aminohexanoic acid and B is beta-alanine) CPP-PMO conjugates in DMD (Duchenne muscular dystrophy) and murine coronavirus research. An (R-Ahx-R)(4)AhxB-PMO conjugate was the most effective compound in inducing the correction of mutant dystrophin transcripts in myoblasts derived from a canine model of DMD. Similarly, normal levels of dystrophin expression were restored in the diaphragms of mdx mice, with treatment starting at the neonatal stage, and protein was still detecTable 22 weeks after the last dose of an (R-Ahx-R)(4)AhxB-PMO conjugate. Effects of length, linkage and carbohydrate modification of this CPP on the delivery of a PMO were investigated in a coronavirus mouse model. An (R-Ahx-R)(4)AhxB-PMO conjugate effectively inhibited viral replication, in comparison with other peptides conjugated to the same PMO. Shortening the CPP length, modifying it with a mannosylated serine moiety or replacing it with the R(9)F(2) CPP significantly decreased the efficacy of the resulting PPMO (CPP-PMO conjugate). We attribute the success of this CPP to its stability in serum and its capacity to transport PMO to RNA targets in a manner superior to that of poly-arginine CPPs.

XMRV Discovery and Prostate Cancer-Related Research.

PubMed

Kang, David E; Lee, Michael C; Das Gupta, Jaydip; Klein, Eric A; Silverman, Robert H

2011-01-01

Xenotropic murine leukemia virus-related virus (XMRV) was first reported in 2006 in a study of human prostate cancer patients with genetic variants of the antiviral enzyme, RNase L. Subsequent investigations in North America, Europe, Asia, and Africa have either observed or failed to detect XMRV in patients (prostate cancer, chronic fatigue syndrome-myalgic encephalomyelitis (CFS-ME), and immunosuppressed with respiratory tract infections) or normal, healthy, control individuals. The principal confounding factors are the near ubiquitous presence of mouse-derived reagents, antibodies and cells, and often XMRV itself, in laboratories. XMRV infects and replicates well in many human cell lines, but especially in certain prostate cancer cell lines. XMRV also traffics to prostate in a nonhuman primate model of infection. Here, we will review the discovery of XMRV and then focus on prostate cancer-related research involving this intriguing virus.

Nucleoside reverse transcriptase inhibitors possess intrinsic anti-inflammatory activity

PubMed Central

Fowler, Benjamin J.; Gelfand, Bradley D.; Kim, Younghee; Kerur, Nagaraj; Tarallo, Valeria; Hirano, Yoshio; Amarnath, Shoba; Fowler, Daniel H.; Radwan, Marta; Young, Mark T.; Pittman, Keir; Kubes, Paul; Agarwal, Hitesh K.; Parang, Keykavous A.; Hinton, David R.; Bastos-Carvalho, Ana; Li, Shengjian; Yasuma, Tetsuhiro; Mizutani, Takeshi; Yasuma, Reo; Wright, Charles; Ambati, Jayakrishna

2014-01-01

Nucleoside reverse transcriptase inhibitors (NRTIs) are mainstay therapeutics for HIV that block retrovirus replication. Alu (an endogenous retroelement that also requires reverse transcriptase for its life cycle)-derived RNAs activate P2X7 and the NLRP3 inflammasome to cause cell death of the retinal pigment epithelium (RPE) in geographic atrophy, a type of age-related macular degeneration. We found that NRTIs inhibit P2X7-mediated NLRP3 inflammasome activation independent of reverse transcriptase inhibition. Multiple approved and clinically relevant NRTIs prevented caspase-1 activation, the effector of the NLRP3 inflammasome, induced by Alu RNA. NRTIs were efficacious in mouse models of geographic atrophy, choroidal neovascularization, graft-versus-host disease (GVHD), and sterile liver inflammation. Our findings suggest that NRTIs are ripe for drug repurposing in P2X7-driven diseases. PMID:25414314

Evaluation of multispectral optoacoustic tomography (MSOT) performance in phantoms and in vivo

NASA Astrophysics Data System (ADS)

Joseph, James; Tomaszewski, Michal; Morgan, Fiona J. E.; Bohndiek, Sarah E.

2015-03-01

MultiSpectral optoacoustic tomography (MSOT) is an emerging modality that combines the high contrast of optical imaging with the spatial resolution and penetration depth of ultrasound, to provide detailed images of hemoglobin concentration and oxygenation. To facilitate accurate determination of changes in the vascularity and oxygenation of a biological tissue over time, a tumor in response to cancer therapy for example, an extensive study of stability and reproducibility of a small animal MSOT system has been performed. Investigations were first made with a stable phantom imaged repeatedly over time scales of hours, days and months to evaluate the reproducibility of the system over time. We found that the small animal MSOT system exhibited excellent reproducibility with a coefficient of variation (COV) in the measured MSOT signals of less than 8% over the course of 30 days and within 1.5% over a single day. Experiments performed in vivo demonstrated the potential for measurement of oxyhemoglobin over time in a realistic experimental setting. The effect of breathing medical air or oxygen under conditions of fixed respiration rate and body temperature within normal organs, including the spleen and kidneys, were investigated. The COV for oxyhemoglobin signals retrieved from spectral unmixing was assessed within both biological (different mouse) and imaging (different scan) replicates. As expected, biological replicates produced a large COV (up to 40% within the spleen) compared to imaging replicates within a single mouse (up to 10% within the spleen). Furthermore, no significant difference was found between data acquired by different operators. The data presented here suggest that MSOT is highly reproducible for both phantom and in vivo imaging, hence could reliably detect changes in oxygenation occurring in living subjects.

Allele-specific control of replication timing and genome organization during development.

PubMed

Rivera-Mulia, Juan Carlos; Dimond, Andrew; Vera, Daniel; Trevilla-Garcia, Claudia; Sasaki, Takayo; Zimmerman, Jared; Dupont, Catherine; Gribnau, Joost; Fraser, Peter; Gilbert, David M

2018-05-07

DNA replication occurs in a defined temporal order known as the replication-timing (RT) program. RT is regulated during development in discrete chromosomal units, coordinated with transcriptional activity and 3D genome organization. Here, we derived distinct cell types from F1 hybrid musculus X castaneus mouse crosses and exploited the high single nucleotide polymorphism (SNP) density to characterize allelic differences in RT (Repli-seq), genome organization (Hi-C and promoter-capture Hi-C), gene expression (total nuclear RNA-seq) and chromatin accessibility (ATAC-seq). We also present HARP: a new computational tool for sorting SNPs in phased genomes to efficiently measure allele-specific genome-wide data. Analysis of six different hybrid mESC clones with different genomes (C57BL/6, 129/sv and CAST/Ei), parental configurations and gender revealed significant RT asynchrony between alleles across ~12% of the autosomal genome linked to sub-species genomes but not to parental origin, growth conditions or gender. RT asynchrony in mESCs strongly correlated with changes in Hi-C compartments between alleles but not SNP density, gene expression, imprinting or chromatin accessibility. We then tracked mESC RT asynchronous regions during development by analyzing differentiated cell types including extraembryonic endoderm stem (XEN) cells, 4 male and female primary mouse embryonic fibroblasts (MEFs) and neural precursor cells (NPCs) differentiated in vitro from mESCs with opposite parental configurations. We found that RT asynchrony and allelic discordance in Hi-C compartments seen in mESCs was largely lost in all differentiated cell types, coordinated with a more uniform Hi-C compartment arrangement, suggesting that genome organization of homologues converges to similar folding patterns during cell fate commitment. Published by Cold Spring Harbor Laboratory Press.

2009 pandemic H1N1 influenza virus elicits similar clinical course but differential host transcriptional response in mouse, macaque, and swine infection models

PubMed Central

2012-01-01

Background The 2009 pandemic H1N1 influenza virus emerged in swine and quickly became a major global health threat. In mouse, non human primate, and swine infection models, the pH1N1 virus efficiently replicates in the lung and induces pro-inflammatory host responses; however, whether similar or different cellular pathways were impacted by pH1N1 virus across independent infection models remains to be further defined. To address this we have performed a comparative transcriptomic analysis of acute phase responses to a single pH1N1 influenza virus, A/California/04/2009 (CA04), in the lung of mice, macaques and swine. Results Despite similarities in the clinical course, we observed differences in inflammatory molecules elicited, and the kinetics of their gene expression changes across all three species. We found genes associated with the retinoid X receptor (RXR) signaling pathway known to control pro-inflammatory and metabolic processes that were differentially regulated during infection in each species, though the heterodimeric RXR partner, pathway associated signaling molecules, and gene expression patterns varied among the three species. Conclusions By comparing transcriptional changes in the context of clinical and virological measures, we identified differences in the host transcriptional response to pH1N1 virus across independent models of acute infection. Antiviral resistance and the emergence of new influenza viruses have placed more focus on developing drugs that target the immune system. Underlying overt clinical disease are molecular events that suggest therapeutic targets identified in one host may not be appropriate in another. PMID:23153050

Clinically isolated enterovirus A71 subgenogroup C4 strain with lethal pathogenicity in 14-day-old mice and the application as an EV-A71 mouse infection model.

PubMed

Xu, Yi; Ma, Shuzhi; Zhu, Limeng; Huang, Zhiqiu; Chen, Liyun; Xu, Yuhua; Yin, Haibin; Peng, Tao; Wang, Yi

2017-01-01

The Enterovirus A71 (EV-A71) subgenogroup C4 is prevalent in China. EV-A71 causes hand, foot and mouth disease (HFMD) in children and may lead to severe neurological diseases. The development of antiviral and protective vaccines against EV-A71 is significantly hindered by the lack of suitable animal models to recapitulate human neurological symptoms. In this study, GZ-CII, a highly virulent EV-A71 subgenogroup C4 strain, was isolated from hospitalized children with HFMD. Intraperitoneal infections of GZ-CII resulted in progressive neurological disease in mice as old as 14 days. Administration of an inactivated EV-A71 vaccine or an anti-EV-A71 immune serum protected the mice against the GZ-CII infection. This demonstrated that a mouse model with EV-A71 GZ-CII could be used to evaluate potential vaccine candidates and therapeutics for subgenogroup C4. Comparing the genome sequence of GZ-CII with that of the avirulent EV-A71 subgenogroup C4 strain revealed unique mutations in GZ-CII. When mutation VP2-K149I was introduced into the nonpathogenic EV-A71 subgenogroup C4 strain, the variant similar to GZ-CII significantly increased viral replication and virulence in mice. These results indicated that the VP2-K149I mutation played an important role in enhancing the virulence of the EV-A71 subgenogroup C4 strain in mice, and that mice infected with the GZ-CII strain are a promising model for evaluating vaccines and therapeutics against the EV-A71 subgenogroup C4. Copyright © 2016 Elsevier B.V. All rights reserved.

Obeticholic acid raises LDL-cholesterol and reduces HDL-cholesterol in the Diet-Induced NASH (DIN) hamster model.

PubMed

Briand, François; Brousseau, Emmanuel; Quinsat, Marjolaine; Burcelin, Rémy; Sulpice, Thierry

2018-01-05

The use of rat and mouse models limits the translation to humans for developing novel drugs targeting nonalcoholic steatohepatitis (NASH). Obeticholic acid (OCA) illustrates this limitation since its dyslipidemic effect in humans cannot be observed in these rodents. Conversely, Golden Syrian hamsters have a lipoprotein metabolism mimicking human dyslipidemia since it does express the cholesteryl ester transfer protein (CETP). We therefore developed a Diet-Induced NASH (DIN) hamster model and evaluated the impact of OCA. Compared with chow fed controls, hamsters fed for 20 weeks with a free-choice (FC) diet, developed obesity, insulin resistance, dyslipidemia and NASH (microvesicular steatosis, inflammation, hepatocyte ballooning and perisinusoidal to bridging fibrosis). After 20 weeks of diet, FC fed hamsters were treated without or with obeticholic acid (15mg/kg/day) for 5 weeks. Although a non-significant trend towards higher dietary caloric intake was observed, OCA significantly lowered body weight after 5 weeks of treatment. OCA significantly increased CETP activity and LDL-C levels by 20% and 27%, and reduced HDL-C levels by 20%. OCA blunted hepatic gene expression of Cyp7a1 and Cyp8b1 and reduced fecal bile acids mass excretion by 64% (P < 0.05). Hamsters treated with OCA showed a trend towards higher scavenger receptor Class B type I (SR-BI) and lower LDL-receptor hepatic protein expression. OCA reduced NAS score for inflammation (P < 0.01) and total NAS score, although not significantly. Compared to mouse and rat models, the DIN hamster replicates benefits and side effects of OCA as observed in humans, and should be useful for evaluating novel drugs targeting NASH. Copyright © 2017 Elsevier B.V. All rights reserved.

Attenuated mutant strain of Salmonella Typhimurium lacking the ZnuABC transporter contrasts tumor growth promoting anti-cancer immune response.

PubMed

Chirullo, Barbara; Ammendola, Serena; Leonardi, Leonardo; Falcini, Roberto; Petrucci, Paola; Pistoia, Claudia; Vendetti, Silvia; Battistoni, Andrea; Pasquali, Paolo

2015-07-10

Salmonella Typhimurium has been shown to be highly effective as antitumor agent. The aim of this study was to investigate the tumor targeting efficacy and the mechanism of action of a specific attenuated mutant strain of Salmonella Typhimurium (STM) devoid of the whole operon coding for the high-affinity zinc transporter ZnuABC, which is required for bacterial growth in environments poor in zinc and for conferring full virulence to different Gram-negative pathogens.We showed that STM is able to penetrate and replicate into tumor cells in in vitro and in vivo models. The subcutaneous administration of STM in mammary adenocarcinoma mouse model led to both reduction of tumor growth and increase in life expectancy of STM treated mice. Moreover, investigating the potential mechanism behind the favorable clinical outcomes, we provide evidence that STM stimulates a potent inflammatory response and a specific immune pattern, recruiting a large number of innate and adaptive immune cells capable to contrast the immunosuppressive environment generated by tumors.

Plasmodium vivax liver stage development and hypnozoite persistence in human liver-chimeric mice

PubMed Central

Mikolajczak, Sebastian A.; Vaughan, Ashley M.; Kangwanrangsan, Niwat; Roobsoong, Wanlapa; Fishbaugher, Matthew; Yimamnuaychok, Narathatai; Rezakhani, Nastaran; Lakshmanan, Viswanathan; Singh, Naresh; Kaushansky, Alexis; Camargo, Nelly; Baldwin, Michael; Lindner, Scott E.; Adams, John H.; Prachumsri, Jetsumon; Kappe, Stefan H.I.

2017-01-01

Plasmodium vivax malaria is characterized by periodic relapses of symptomatic blood stage parasite infections likely initiated by activation of dormant liver stage parasites -hypnozoites. The lack of tractable animal models for P. vivax constitutes a severe obstacle to investigate this unique aspect of its biology and to test drug efficacy against liver stages. We show that the FRG KO huHep liver-humanized mice support P. vivax sporozoite infection, development of liver stages, and the formation of small non-replicating hypnozoites. Cellular characterization of P. vivax liver stage development in vivo demonstrates complete maturation into infectious exo-erythrocytic merozoites and continuing persistence of hypnozoites. Primaquine prophylaxis or treatment prevents and eliminates liver stage infection. Thus, the P. vivax/FRG KO huHep mouse infection model constitutes an important new tool to investigate the biology of liver stage development and dormancy and might aid in the discovery of new drugs for the prevention of relapsing malaria. PMID:25800544

Hepatic Proteomic Analysis Revealed Altered Metabolic Pathways in Insulin Resistant Akt1+/-/Akt2-/-Mice

PubMed Central

Pedersen, Brian A; Wang, Weiwen; Taylor, Jared F; Khattab, Omar S; Chen, Yu-Han; Edwards, Robert A; Yazdi, Puya G; Wang, Ping H

2015-01-01

Objective The aim of this study was to identify liver proteome changes in a mouse model of severe insulin resistance and markedly decreased leptin levels. Methods Two-dimensional differential gel electrophoresis was utilized to identify liver proteome changes in AKT1+/-/AKT2-/- mice. Proteins with altered levels were identified with tandem mass spectrometry. Ingenuity Pathway analysis was performed for the interpretation of the biological significance of the observed proteomic changes. Results 11 proteins were identified from 2 biological replicates to be differentially expressed by a ratio of at least 1.3 between age-matched insulin resistant (Akt1+/-/Akt2-/-) and wild type mice. Albumin and mitochondrial ornithine aminotransferase were detected from multiple spots, which suggest post-translational modifications. Enzymes of the urea cycle were common members of top regulated pathways. Conclusion Our results help to unveil the regulation of the liver proteome underlying altered metabolism in an animal model of severe insulin resistance. PMID:26455965

Kinase-dead ATM protein is highly oncogenic and can be preferentially targeted by Topo-isomerase I inhibitors.

PubMed

Yamamoto, Kenta; Wang, Jiguang; Sprinzen, Lisa; Xu, Jun; Haddock, Christopher J; Li, Chen; Lee, Brian J; Loredan, Denis G; Jiang, Wenxia; Vindigni, Alessandro; Wang, Dong; Rabadan, Raul; Zha, Shan

2016-06-15

Missense mutations in ATM kinase, a master regulator of DNA damage responses, are found in many cancers, but their impact on ATM function and implications for cancer therapy are largely unknown. Here we report that 72% of cancer-associated ATM mutations are missense mutations that are enriched around the kinase domain. Expression of kinase-dead ATM (Atm(KD/-)) is more oncogenic than loss of ATM (Atm(-/-)) in mouse models, leading to earlier and more frequent lymphomas with Pten deletions. Kinase-dead ATM protein (Atm-KD), but not loss of ATM (Atm-null), prevents replication-dependent removal of Topo-isomerase I-DNA adducts at the step of strand cleavage, leading to severe genomic instability and hypersensitivity to Topo-isomerase I inhibitors. Correspondingly, Topo-isomerase I inhibitors effectively and preferentially eliminate Atm(KD/-), but not Atm-proficientor Atm(-/-) leukemia in animal models. These findings identify ATM kinase-domain missense mutations as a potent oncogenic event and a biomarker for Topo-isomerase I inhibitor based therapy.

MicroRNA-302a suppresses influenza A virus-stimulated interferon regulatory factor-5 expression and cytokine storm induction.

PubMed

Chen, Xueyuan; Zhou, Li; Peng, Nanfang; Yu, Haisheng; Li, Mengqi; Cao, Zhongying; Lin, Yong; Wang, Xueyu; Li, Qian; Wang, Jun; She, Yinglong; Zhu, Chengliang; Lu, Mengji; Zhu, Ying; Liu, Shi

2017-12-29

During influenza A virus (IAV) infection, cytokine storms play a vital and critical role in clinical outcomes. We have previously reported that microRNA (miR)-302c regulates IAV-induced IFN expression by targeting the 3'-UTR of nuclear factor κB (NF-κB)-inducing kinase. In the current study, we found that miR-302a, another member of the miR-302 cluster, controls the IAV-induced cytokine storm. According to results from cell-based and knockout mouse models, IAV induces a cytokine storm via interferon regulatory factor-5 (IRF-5). We also found that IAV infection up-regulates IRF-5 expression and that IRF-5 in turn promotes IAV replication. Furthermore, we observed that IRF-5 is a direct target of miR-302a, which down-regulated IRF-5 expression by binding its 3'-UTR. Moreover, IAV increased IRF-5 expression by down-regulating miR-302a expression. Interestingly, miR-302a inhibited IAV replication. In IAV-infected patients, miR-302a expression was down-regulated, whereas IRF-5 expression was up-regulated. Taken together, our work uncovers and defines a signaling pathway implicated in an IAV-induced cytokine storm. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Antigen-activated dendritic cells ameliorate influenza A infections

PubMed Central

Boonnak, Kobporn; Vogel, Leatrice; Orandle, Marlene; Zimmerman, Daniel; Talor, Eyal; Subbarao, Kanta

2013-01-01

Influenza A viruses cause significant morbidity and mortality worldwide. There is a need for alternative or adjunct therapies, as resistance to currently used antiviral drugs is emerging rapidly. We tested ligand epitope antigen presentation system (LEAPS) technology as a new immune-based treatment for influenza virus infection in a mouse model. Influenza-J-LEAPS peptides were synthesized by conjugating the binding ligand derived from the β2-microglobulin chain of the human MHC class I molecule (J-LEAPS) with 15 to 30 amino acid–long peptides derived from influenza virus NP, M, or HA proteins. DCs were stimulated with influenza-J-LEAPS peptides (influenza-J-LEAPS) and injected intravenously into infected mice. Antigen-specific LEAPS-stimulated DCs were effective in reducing influenza virus replication in the lungs and enhancing survival of infected animals. Additionally, they augmented influenza-specific T cell responses in the lungs and reduced the severity of disease by limiting excessive cytokine responses, which are known to contribute to morbidity and mortality following influenza virus infection. Our data demonstrate that influenza-J-LEAPS–pulsed DCs reduce virus replication in the lungs, enhance survival, and modulate the protective immune responses that eliminate the virus while preventing excessive cytokines that could injure the host. This approach shows promise as an adjunct to antiviral treatment of influenza virus infections. PMID:23934125

Model of early self-replication based on covalent complementarity for a copolymer of glycerate-3-phosphate and glycerol-3-phosphate

NASA Technical Reports Server (NTRS)

Weber, Arthur L.

1989-01-01

Glyceraldehyde-3-phosphate acts as the substrate in a model of early self-replication of a phosphodiester copolymer of glycerate-3-phosphate and glycerol-3-phosphate. This model of self-replication is based on covalent complementarity in which information transfer is mediated by a single covalent bond, in contrast to multiple weak interactions that establish complementarity in nucleic acid replication. This replication model is connected to contemporary biochemistry through its use of glyceraldehyde-3-phosphate, a central metabolite of glycolysis and photosynthesis.

Important role of interferon regulatory factor (IRF)-3 in the interferon response of mouse macrophages upon infection by Newcastle disease virus.

PubMed

Wilden, Holger; Schirrmacher, Volker; Fournier, Philippe

2011-08-01

Newcastle disease virus (NDV) is an interesting agent for activating innate immune activity in macrophages including secretion of TNF-α and IFN-α, upregulation of TRAIL and activation of NF-κB and iNOS. However, the molecular mechanism of such cellular activities remains largely unknown. Tumor selectivity of replication of NDV has been described to be linked to deviations in tumor cells of the type I interferon response. We therefore focused on the interferon response to NDV of macrophages as part of innate anti-viral and anti-tumor activity. In particular, we investigated the functional significance of the interferon regulatory factor genes (IRF)-3 and IRF-7. Deletion of the IRF-3 or IRF-7 gene was found to increase susceptibility of mouse macrophages to virus infection. Surprisingly, NDV replicated better in IRF-3 KO than in IRF-7 KO macrophages. Further analysis showed that IRF-3 KO macrophages have a lower basal and NDV-induced RIG-I expression in comparison to IRF-7 KO macrophages. This might explain why, in IRF-3 KO macrophages, the secretion of type I interferons after NDV infection is delayed, when compared to IRF-7 KO and wild-type macrophages. In addition, IRF-3 KO cells showed reduced NDV-induced levels of IRF-7. This effect could be prevented by priming the cells first by interferon-α. Further results indicated that an early production of type I interferon rather than high maximal levels at later time points are important for resistance to infection by NDV. In conclusion, these results demonstrate an important role of IRF-3 for the innate anti-viral response to NDV of mouse macrophages.

Age-associated increase in heterochromatic marks in murine and primate tissues

PubMed Central

Kreiling, Jill A.; Tamamori-Adachi, Mimi; Sexton, Alec N.; Jeyapalan, Jessie C.; Munoz-Najar, Ursula; Peterson, Abigail L.; Manivannan, Jayameenakshi; Rogers, Elizabeth S.; Pchelintsev, Nikolay A.; Adams, Peter D.; Sedivy, John M.

2011-01-01

Summary Chromatin is highly dynamic and subject to extensive remodeling under many physiological conditions. Changes in chromatin that occur during the aging process are poorly documented and understood in higher organisms, such as mammals. We developed an immunofluorescence assay to quantitatively detect, at the single cell level, changes in the nuclear content of chromatin-associated proteins. We find increased levels of the heterochromatin-associated proteins histone macro H2A (mH2A) and heterochromatin protein 1 beta (HP1β) in human fibroblasts during replicative senescence in culture, and for the first time, an age-associated increase in these heterochromatin marks in several tissues of mice and primates. Mouse lung was characterized by monophasic mH2A expression histograms at both ages, and an increase in mean staining intensity at old age. In the mouse liver we observed increased age-associated localization of mH2A to regions of pericentromeric heterochromatin. In skeletal muscle we found two populations of cells with either low or high mH2A levels. This pattern of expression was similar in mouse and baboon, and showed a clear increase in the proportion of nuclei with high mH2A levels in older animals. The frequencies of cells displaying evidence of increased heterochromatinization are too high to be readily accounted for by replicative or oncogene-induced cellular senescence, and are prominently found in terminally differentiated, post mitotic tissues that are not conventionally thought to be susceptible to senescence. Our findings distinguish specific chromatin states in individual cells of mammalian tissues, and provide a foundation to further investigate the progressive epigenetic changes that occur during aging. PMID:21176091

Age-associated increase in heterochromatic marks in murine and primate tissues.

PubMed

Kreiling, Jill A; Tamamori-Adachi, Mimi; Sexton, Alec N; Jeyapalan, Jessie C; Munoz-Najar, Ursula; Peterson, Abigail L; Manivannan, Jayameenakshi; Rogers, Elizabeth S; Pchelintsev, Nikolay A; Adams, Peter D; Sedivy, John M

2011-04-01

Chromatin is highly dynamic and subject to extensive remodeling under many physiologic conditions. Changes in chromatin that occur during the aging process are poorly documented and understood in higher organisms, such as mammals. We developed an immunofluorescence assay to quantitatively detect, at the single cell level, changes in the nuclear content of chromatin-associated proteins. We found increased levels of the heterochromatin-associated proteins histone macro H2A (mH2A) and heterochromatin protein 1 beta (HP1β) in human fibroblasts during replicative senescence in culture, and for the first time, an age-associated increase in these heterochromatin marks in several tissues of mice and primates. Mouse lung was characterized by monophasic mH2A expression histograms at both ages, and an increase in mean staining intensity at old age. In the mouse liver, we observed increased age-associated localization of mH2A to regions of pericentromeric heterochromatin. In the skeletal muscle, we found two populations of cells with either low or high mH2A levels. This pattern of expression was similar in mouse and baboon, and showed a clear increase in the proportion of nuclei with high mH2A levels in older animals. The frequencies of cells displaying evidence of increased heterochromatinization are too high to be readily accounted for by replicative or oncogene-induced cellular senescence, and are prominently found in terminally differentiated, postmitotic tissues that are not conventionally thought to be susceptible to senescence. Our findings distinguish specific chromatin states in individual cells of mammalian tissues, and provide a foundation to investigate further the progressive epigenetic changes that occur during aging. © 2010 The Authors. Aging Cell © 2010 Blackwell Publishing Ltd/Anatomical Society of Great Britain and Ireland.

Systematic Determination of Replication Activity Type Highlights Interconnections between Replication, Chromatin Structure and Nuclear Localization

PubMed Central

Polten, Andreas; Hezroni, Hadas; Eldar, Yonina C.; Meshorer, Eran; Yakhini, Zohar; Simon, Itamar

2012-01-01

DNA replication is a highly regulated process, with each genomic locus replicating at a distinct time of replication (ToR). Advances in ToR measurement technology enabled several genome-wide profiling studies that revealed tight associations between ToR and general genomic features and a remarkable ToR conservation in mammals. Genome wide studies further showed that at the hundreds kb-to-megabase scale the genome can be divided into constant ToR regions (CTRs) in which the replication process propagates at a faster pace due to the activation of multiple origins and temporal transition regions (TTRs) in which the replication process propagates at a slower pace. We developed a computational tool that assigns a ToR to every measured locus and determines its replication activity type (CTR versus TTR). Our algorithm, ARTO (Analysis of Replication Timing and Organization), uses signal processing methods to fit a constant piece-wise linear curve to the measured raw data. We tested our algorithm and provide performance and usability results. A Matlab implementation of ARTO is available at http://bioinfo.cs.technion.ac.il/people/zohar/ARTO/. Applying our algorithm to ToR data measured in multiple mouse and human samples allowed precise genome-wide ToR determination and replication activity type characterization. Analysis of the results highlighted the plasticity of the replication program. For example, we observed significant ToR differences in 10–25% of the genome when comparing different tissue types. Our analyses also provide evidence for activity type differences in up to 30% of the probes. Integration of the ToR data with multiple aspects of chromosome organization characteristics suggests that ToR plays a role in shaping the regional chromatin structure. Namely, repressive chromatin marks, are associated with late ToR both in TTRs and CTRs. Finally, characterization of the differences between TTRs and CTRs, with matching ToR, revealed that TTRs are associated with compact chromatin and are located significantly closer to the nuclear envelope. Supplementary material is available. Raw and processed data were deposited in Geo (GSE17236). PMID:23145042

Use of tissue-specific microRNA to control pathology of wild-type adenovirus without attenuation of its ability to kill cancer cells.

PubMed

Cawood, Ryan; Chen, Hannah H; Carroll, Fionnadh; Bazan-Peregrino, Miriam; van Rooijen, Nico; Seymour, Leonard W

2009-05-01

Replicating viruses have broad applications in biomedicine, notably in cancer virotherapy and in the design of attenuated vaccines; however, uncontrolled virus replication in vulnerable tissues can give pathology and often restricts the use of potent strains. Increased knowledge of tissue-selective microRNA expression now affords the possibility of engineering replicating viruses that are attenuated at the RNA level in sites of potential pathology, but retain wild-type replication activity at sites not expressing the relevant microRNA. To assess the usefulness of this approach for the DNA virus adenovirus, we have engineered a hepatocyte-safe wild-type adenovirus 5 (Ad5), which normally mediates significant toxicity and is potentially lethal in mice. To do this, we have included binding sites for hepatocyte-selective microRNA mir-122 within the 3' UTR of the E1A transcription cassette. Imaging versions of these viruses, produced by fusing E1A with luciferase, showed that inclusion of mir-122 binding sites caused up to 80-fold decreased hepatic expression of E1A following intravenous delivery to mice. Animals administered a ten-times lethal dose of wild-type Ad5 (5x10(10) viral particles/mouse) showed substantial hepatic genome replication and extensive liver pathology, while inclusion of 4 microRNA binding sites decreased replication 50-fold and virtually abrogated liver toxicity. This modified wild-type virus retained full activity within cancer cells and provided a potent, liver-safe oncolytic virus. In addition to providing many potent new viruses for cancer virotherapy, microRNA control of virus replication should provide a new strategy for designing safe attenuated vaccines applied across a broad range of viral diseases.

Molecular Determinants of Hepatitis B and D Virus Entry Restriction in Mouse Sodium Taurocholate Cotransporting Polypeptide

PubMed Central

Yan, Huan; Peng, Bo; He, Wenhui; Zhong, Guocai; Qi, Yonghe; Ren, Bijie; Gao, Zhenchao; Jing, Zhiyi; Song, Mei; Xu, Guangwei; Sui, Jianhua

2013-01-01

Human hepatitis B virus (HBV) and its satellite virus, hepatitis D virus (HDV), primarily infect humans, chimpanzees, or tree shrews (Tupaia belangeri). Viral infections in other species are known to be mainly restricted at the entry level since viral replication can be achieved in the cells by transfection of the viral genome. Sodium taurocholate cotransporting polypeptide (NTCP) is a functional receptor for HBV and HDV, and amino acids 157 to 165 of NTCP are critical for viral entry and likely limit viral infection of macaques. However, the molecular determinants for viral entry restriction in mouse NTCP (mNTCP) remain unclear. In this study, mNTCP was found to be unable to support either HBV or HDV infection, although it can bind to pre-S1 of HBV L protein and is functional in transporting substrate taurocholate; comprehensive swapping and point mutations of human NTCP (hNTCP) and mNTCP revealed molecular determinants restricting mNTCP for viral entry of HBV and HDV. Remarkably, when mNTCP residues 84 to 87 were substituted by human counterparts, mNTCP can effectively support viral infections. In addition, a number of cell lines, regardless of their species or tissue origin, supported HDV infection when transfected with hNTCP or mNTCP with residues 84 to 87 replaced by human counterparts, highlighting the central role of NTCP for viral infections mediated by HBV envelope proteins. These studies advance our understanding of NTCP-mediated viral entry of HBV and HDV and have important implications for developing the mouse model for their infections. PMID:23678176

Property of hepatitis B virus replication in Tupaia belangeri hepatocytes.

PubMed

Sanada, Takahiro; Tsukiyama-Kohara, Kyoko; Yamamoto, Naoki; Ezzikouri, Sayeh; Benjelloun, Soumaya; Murakami, Shuko; Tanaka, Yasuhito; Tateno, Chise; Kohara, Michinori

2016-01-08

The northern treeshrew (Tupaia belangeri) has been reported to be an effective candidate for animal infection model with hepatitis B virus (HBV). The objective of our study was to analyze the growth characteristics of HBV in tupaia hepatocytes and the host response to HBV infection. We established primary tupaia hepatocytes (3-6-week old tupaia) and infected them with HBV genotypes A, B and C, and all the genotypes proliferated as well as those in human primary hepatocytes (>10(5) copies/ml in culture supernatant). We next generated a chimeric mouse with tupaia liver by transplantation of tupaia primary hepatocytes to urokinase-type plasminogen activator cDNA (cDNA-uPA)/severe combined immunodeficient (SCID) mice and the replacement ratio with tupaia hepatocytes was found to be more than 95%. Infection of chimeric mice with HBV (genotypes B, C, and D) resulted in HBV-DNA level of 10(4)-10(6) copies/ml after 8 weeks of infection, which were almost similar to that in humanized chimeric mouse. In contrast, serum HBV level in adult tupaia (1-year-old tupaia) was quite low (<10(3) copies/ml). Understanding the differences in the response to HBV infection in primary tupaia hepatocytes, chimeric mouse, and adult tupaia will contribute to elucidating the mechanism of persistent HBV infection and viral eradication. Thus, T. belangeri was found to be efficient for studying the host response to HBV infection, thereby providing novel insight into the pathogenesis of HBV. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

Pathogenesis of persistent hyperplastic primary vitreous in mice lacking the arf tumor suppressor gene.

PubMed

Martin, Amy C; Thornton, J Derek; Liu, Jiewiu; Wang, XiaoFei; Zuo, Jian; Jablonski, Monica M; Chaum, Edward; Zindy, Frederique; Skapek, Stephen X

2004-10-01

Persistent hyperplastic primary vitreous (PHPV) is an idiopathic developmental eye disease associated with failed involution of the hyaloid vasculature. The present work addressed the pathogenesis of PHPV in a mouse model that replicates many aspects of the human disease. Ophthalmoscopic and histologic analyses documented pathologic processes in eyes of mice lacking the Arf gene compared with Ink4a-deficient and wild-type control animals. Immunohistochemical staining, in situ hybridization, and RT-PCR demonstrated the expression of relevant gene products. Arf gene expression was determined by in situ hybridization using wholemounts of wild-type mouse eyes and by immunofluorescence staining for green fluorescent protein (GFP) in Arf(+/GFP) heterozygous knock-in mouse eyes. Abnormalities in Arf(-/-) mice mimicked those found in patients with severe PHPV. The mice had microphthalmia; fibrovascular, retrolental tissue containing retinal pigment epithelial cells and remnants of the hyaloid vascular system; posterior lens capsule destruction with lens degeneration and opacity; and severe retinal dysplasia and detachment. Eyes of mice lacking the overlapping Ink4a gene were normal. Arf was selectively expressed in perivascular cells within the vitreous of the postnatal eye. Cells composing the retrolental mass in Arf(-/-) mice expressed the Arf promoter. The remnant hyaloid vessels expressed Flk-1. Its ligand, vascular endothelial growth factor (Vegf), was expressed in the retrolental tissue and the adjacent dysplastic neuroretina. Arf(-/-) mice have features that accurately mimic severe PHPV. In the HVS, Arf expression in perivascular cells may block their accumulation or repress Vegf expression to promote HVS involution and prevent PHPV.

Pathogenesis of Persistent Hyperplastic Primary Vitreous in Mice Lacking the Arf Tumor Suppressor Gene

PubMed Central

Martin, Amy C.; Thornton, J. Derek; Liu, Jiewiu; Wang, XiaoFei; Zuo, Jian; Jablonski, Monica M.; Chaum, Edward; Zindy, Frederique; Skapek, Stephen X.

2006-01-01

Purpose Persistent hyperplastic primary vitreous (PHPV) is an idiopathic developmental eye disease associated with failed involution of the hyaloid vasculature. The present work addressed the pathogenesis of PHPV in a mouse model that replicates many aspects of the human disease. Methods Ophthalmoscopic and histologic analyses documented pathologic processes in eyes of mice lacking the Arf gene compared with Ink4a-deficient and wild-type control animals. Immunohistochemical staining, in situ hybridization, and RT-PCR demonstrated the expression of relevant gene products. Arf gene expression was determined by in situ hybridization using wholemounts of wild-type mouse eyes and by immunofluorescence staining for green fluores-cent protein (GFP) in Arf+/GFP heterozygous knock-in mouse eyes. Results Abnormalities in Arf−/− mice mimicked those found in patients with severe PHPV. The mice had microphthalmia; fibrovascular, retrolental tissue containing retinal pigment epithelial cells and remnants of the hyaloid vascular system; posterior lens capsule destruction with lens degeneration and opacity; and severe retinal dysplasia and detachment. Eyes of mice lacking the overlapping Ink4a gene were normal. Arf was selectively expressed in perivascular cells within the vitreous of the postnatal eye. Cells composing the retrolental mass in Arf−/− mice expressed the Arf promoter. The remnant hyaloid vessels expressed Flk-1. Its ligand, vascular endothelial growth factor (Vegf), was expressed in the retrolental tissue and the adjacent dysplastic neuroretina. Conclusions Arf−/− mice have features that accurately mimic severe PHPV. In the HVS, Arf expression in perivascular cells may block their accumulation or repress Vegf expression to promote HVS involution and prevent PHPV. PMID:15452040

Screening for the protective effect target of deproteinized extract of calf blood and its mechanisms in mice with CCl4-induced acute liver injury.

PubMed

Xu, Guangyu; Han, Xiao; Yuan, Guangxin; An, Liping; Du, Peige

2017-01-01

Liver injury is a common pathological basis of various liver diseases, and long-term liver injury is often an important initiation factor leading to liver fibrosis and even liver cirrhosis and hepatocellular carcinoma (HCC). It has been reported that deproteinized extract of calf blood (DECB) can inhibit the replication of hepatitis B virus and confers a protective effect on the liver after traumatic liver injury. However, few studies on the regulatory factors and mechanisms of DECB have been reported. In this current study, an acute mouse liver injury model was established with carbon tetrachloride (CCl4). The differentially expressed genes and related cell signal transduction pathways were screened using mRNA expression microarray. STEM software V1.3.6 was used for clustering gene functions, and the DAVID and KEGG databases were applied for the analysis. A total of 1355 differentially expressed genes were selected, among which nine were validated by RT-qPCR. The results showed that the Fas, IL1b, Pik3r1, Pik3r5, Traf2, Traf2, Csf2rb2, Map3k14, Pik3cd and Ppp3cc genes were involved in the regulation of DECB in an acute mouse liver injury model. Targets of the protective effects of DECB and its related mechanisms were found in mice with acute liver injury induced by carbon tetrachloride, which may provide an important theoretical basis for further DECB research.

Studies in a Murine Model Confirm the Safety of Griffithsin and Advocate Its Further Development as a Microbicide Targeting HIV-1 and Other Enveloped Viruses.

PubMed

Kouokam, Joseph Calvin; Lasnik, Amanda B; Palmer, Kenneth E

2016-11-17

Griffithsin (GRFT), a lectin from Griffithsia species, inhibits human immunodeficiency virus-1 (HIV-1) replication at sub-nanomolar concentrations, with limited cellular toxicity. However, in vivo safety of GRFT is not fully understood, especially following parenteral administration. We first assessed GRFT's effects in vitro, on mouse peripheral blood mononuclear cell (mPBMC) viability, mitogenicity, and activation using flow-cytometry, as well as cytokine secretion through enzyme-linked immunosorbent assay (ELISA). Toxicological properties of GRFT were determined after a single subcutaneous administration of 50 mg/kg or 14 daily doses of 10 mg/kg in BALB/c mice. In the context of microbicide development, toxicity of GRFT at 2 mg/kg was determined after subcutaneous, intravaginal, and intraperitoneal administrations, respectively. Interestingly, GRFT caused no significant cell death, mitogenicity, activation, or cytokine release in mPBMCs, validating the usefulness of a mouse model. An excellent safety profile for GRFT was obtained in vivo: no overt changes were observed in animal fitness, blood chemistry or CBC parameters. Following GRFT treatment, reversible splenomegaly was observed with activation of certain spleen B and T cells. However, spleen tissues were not pathologically altered by GRFT (either with a single high dose or chronic doses). Finally, no detectable toxicity was found after mucosal or systemic treatment with 2 mg/kg GRFT, which should be further developed as a microbicide for HIV prevention.

Leukocyte populations and cytokine expression in the mammary gland in a mouse model of Streptococcus agalactiae mastitis.

PubMed

Trigo, Gabriela; Dinis, Márcia; França, Angela; Bonifácio Andrade, Elva; Gil da Costa, Rui M; Ferreira, Paula; Tavares, Delfina

2009-07-01

Streptococcus agalactiae is a contagious, mastitis-causing pathogen that is highly adapted to survive in the bovine mammary gland. This study used a BALB/c mouse model of Streptococcus agalactiae mastitis to evaluate leukocyte populations in regional lymph nodes and cytokine expression in the mammary gland involved in the immune response against Streptococcus agalactiae. It was found that the bacteria replicated efficiently in the mammary gland, peaking after 24 h and increasing by 100-fold. Dissemination of bacteria to systemic organs was observed 6 h after infection. At the same time, a massive infiltration of polymorphonuclear cells and an increase in the inflammatory cytokines interleukin (IL)-1beta, IL-6 and tumour necrosis factor-alpha were detected in mammary glands, indicating an early inflammatory response. A decrease in the levels of inflammatory cytokines in mammary glands was observed 72 h after infection, accompanied by an increase in the levels of IL-12 and IL-10, which were related to a gradual decrease in bacterial load. An increase in the number of macrophages and B220(+) lymphocytes and similar increases in both CD4(+) and CD8(+) T cells in regional lymph nodes were observed, being most pronounced 5 days after infection. Moreover, increased levels of anti-Streptococcus agalactiae antibodies in the mammary gland were observed 10 days after infection. Overall, these data suggest that the host exhibits both innate and acquired immune responses in response to Streptococcus agalactiae mastitis.

Why is alpha-actinin-3 deficiency so common in the general population? The evolution of athletic performance.

PubMed

North, Kathryn

2008-08-01

'We can now explain how this common genetic variation influences athletic performance as well as why it has become so common in the general population. There is a fascinating link between factors that influence survival in ancient humans and the factors that contribute to athletic abilities in modern man.' The human ACTN3 gene encodes the protein alpha-actinin-3, a component of the contractile apparatus in fast skeletal muscle fibers. In 1999, we identified a common polymorphism in ACTN3 (R577X) that results in absence of alpha-actinin-3 in more than one billion people worldwide, despite the ACTN3 gene being highly conserved during human evolution. In 2003, we demonstrated that ACTN3 genotype influences elite athletic performance, and the association between ACTN3 genotype and skeletal muscle performance has since been replicated in athletes and non-athlete cohorts. We have also studied the evolution of the R577X allele during human evolution and demonstrated that the null (X) allele has undergone strong, recent positive selection in Europeans and Asian populations. We have developed an Actn3 knockout mouse model that replicates alpha-actinin-3 deficiency in humans and has already provided insight into the role of alpha-actinin-3 in the regulation of skeletal muscle metabolism, fibre size, muscle mass and contractile properties. In particular, mouse muscle lacking alpha-actinin-3 uses energy more efficiently, with the fast fibers displaying metabolic and contractile properties of slow oxidative fibers. While this favors endurance activities, the trade off is that the muscle cannot generate the rapid contractions needed to excel in sprinting. We propose that the shift towards more efficient aerobic muscle metabolism associated with alpha-actinin-3 deficiency also underlies the adaptive benefit of the 577X allele. Our future studies will focus on the effect of ACTN3 genotype on response to exercise and ageing, and the onset and severity of muscle disease phenotype.

Systemic Delivery of Oncolytic Adenoviruses Targeting Transforming Growth Factor-β Inhibits Established Bone Metastasis in a Prostate Cancer Mouse Model

PubMed Central

Hu, Zebin; Gupta, Janhavi; Zhang, Zhenwei; Gerseny, Helen; Berg, Arthur; Chen, Yun Ju; Zhang, Zhiling; Du, Hongyan; Brendler, Charles B.; Xiao, Xianghui; Pienta, Kenneth J.; Guise, Theresa; Lee, Chung; Stern, Paula H.; Stock, Stuart

2012-01-01

Abstract We have examined whether Ad.sTβRFc and TAd.sTβRFc, two oncolytic viruses expressing soluble transforming growth factor-β receptor II fused with human Fc (sTGFβRIIFc), can be developed to treat bone metastasis of prostate cancer. Incubation of PC-3 and DU-145 prostate tumor cells with Ad.sTβRFc and TAd.sTβRFc produced sTGFβRIIFc and viral replication; sTGFβRIIFc caused inhibition of TGF-β-mediated SMAD2 and SMAD3 phosphorylation. Ad(E1-).sTβRFc, an E1– adenovirus, produced sTGFβRIIFc but failed to replicate in tumor cells. To examine the antitumor response of adenoviral vectors, PC-3-luc cells were injected into the left heart ventricle of nude mice. On day 9, mice were subjected to whole-body bioluminescence imaging (BLI). Mice bearing hind-limb tumors were administered viral vectors via the tail vein on days 10, 13, and 17 (2.5×1010 viral particles per injection per mouse, each injection in a 0.1-ml volume), and subjected to BLI and X-ray radiography weekly until day 53. Ad.sTβRFc, TAd.sTβRFc, and Ad(E1-).sTβRFc caused significant inhibition of tumor growth; however, Ad.sTβRFc was the most effective among all the vectors. Only Ad.sTβRFc and TAd.sTβRFc inhibited tumor-induced hypercalcemia. Histomorphometric and synchrotron micro-computed tomographic analysis of isolated bones indicated that Ad.sTβRFc induced significant reduction in tumor burden, osteoclast number, and trabecular and cortical bone destruction. These studies suggest that Ad.sTβRFc and TAd.sTβRFc can be developed as potential new therapies for prostate cancer bone metastasis. PMID:22551458

Development of a Glycoprotein D-Expressing Dominant-Negative and Replication-Defective Herpes Simplex Virus 2 (HSV-2) Recombinant Viral Vaccine against HSV-2 Infection in Mice ▿

PubMed Central

Akhrameyeva, Natalie V.; Zhang, Pengwei; Sugiyama, Nao; Behar, Samuel M.; Yao, Feng

2011-01-01

Using the T-REx (Invitrogen, California) gene switch technology and a dominant-negative mutant polypeptide of herpes simplex virus 1 (HSV-1)-origin binding protein UL9, we previously constructed a glycoprotein D-expressing replication-defective and dominant-negative HSV-1 recombinant viral vaccine, CJ9-gD, for protection against HSV infection and disease. It was demonstrated that CJ9-gD is avirulent following intracerebral inoculation in mice, cannot establish detectable latent infection following different routes of infection, and offers highly effective protective immunity against primary HSV-1 and HSV-2 infection and disease in mouse and guinea pig models of HSV infections. Given these favorable safety and immunological profiles of CJ9-gD, aiming to maximize levels of HSV-2 glycoprotein D (gD2) expression, we have constructed an ICP0 null mutant-based dominant-negative and replication-defective HSV-2 recombinant, CJ2-gD2, that contains 2 copies of the gD2 gene driven by the tetracycline operator (tetO)-bearing HSV-1 major immediate-early ICP4 promoter. CJ2-gD2 expresses gD2 as efficiently as wild-type HSV-2 infection and can lead to a 150-fold reduction in wild-type HSV-2 viral replication in cells coinfected with CJ2-gD2 and wild-type HSV-2 at the same multiplicity of infection. CJ2-gD2 is avirulent following intracerebral injection and cannot establish a detectable latent infection following subcutaneous (s.c.) immunization. CJ2-gD2 is a more effective vaccine than HSV-1 CJ9-gD and a non-gD2-expressing dominant-negative and replication-defective HSV-2 recombinant in protection against wild-type HSV-2 genital disease. Using recall response, we showed that immunization with CJ2-gD2 elicited strong HSV-2-specific memory CD4+ and CD8+ T-cell responses. Collectively, given the demonstrated preclinical immunogenicity and its unique safety profiles, CJ2-gD2 represents a new class of HSV-2 replication-defective recombinant viral vaccines in protection against HSV-2 genital infection and disease. PMID:21389121

Synthesis and structure-activity relationships of varied ether linker analogues of the antitubercular drug (6S)-2-nitro-6-{[4-(trifluoromethoxy)benzyl]oxy}-6,7-dihydro-5h-imidazo[2,1-b][1,3]oxazine (PA-824).

PubMed

Thompson, Andrew M; Sutherland, Hamish S; Palmer, Brian D; Kmentova, Iveta; Blaser, Adrian; Franzblau, Scott G; Wan, Baojie; Wang, Yuehong; Ma, Zhenkun; Denny, William A

2011-10-13

New analogues of antitubercular drug PA-824 were synthesized, featuring alternative side chain ether linkers of varying size and flexibility, seeking drug candidates with enhanced metabolic stability and high efficacy. Both α-methyl substitution and removal of the benzylic methylene were broadly tolerated in vitro, with a biaryl example of the latter class exhibiting an 8-fold better efficacy than the parent drug in a mouse model of acute Mycobacterium tuberculosis infection and negligible fragmentation to an alcohol metabolite in liver microsomes. Extended linkers (notably propenyloxy, propynyloxy, and pentynyloxy) provided greater potencies against replicating M. tb (monoaryl analogues), with propynyl ethers being most effective under anaerobic (nonreplicating) conditions (mono/biaryl analogues). For benzyloxybenzyl and biaryl derivatives, aerobic activity was maximal with the original (OCH(2)) linker. One propynyloxy-linked compound displayed an 89-fold higher efficacy than the parent drug in the acute model, and it was slightly superior to antitubercular drug OPC-67683 in a chronic infection model.

Control of temporal activation of hepatitis C virus-induced interferon response by domain 2 of nonstructural protein 5A.

PubMed

Hiet, Marie-Sophie; Bauhofer, Oliver; Zayas, Margarita; Roth, Hanna; Tanaka, Yasuhito; Schirmacher, Peter; Willemsen, Joschka; Grünvogel, Oliver; Bender, Silke; Binder, Marco; Lohmann, Volker; Lotteau, Vincent; Ruggieri, Alessia; Bartenschlager, Ralf

2015-10-01

Hepatitis C virus (HCV) nonstructural protein 5A (NS5A) is a multifunctional protein playing a crucial role in diverse steps of the viral replication cycle and perturbing multiple host cell pathways. We showed previously that removal of a region in domain 2 (D2) of NS5A (mutant NS5A(D2Δ)) is dispensable for viral replication in hepatoma cell lines. By using a mouse model and immune-competent cell systems, we studied the role of D2 in controlling the innate immune response. In vivo replication competence of NS5A(D2Δ) was studied in transgenic mice with human liver xenografts. Results were validated using primary human hepatocytes (PHHs) and mechanistic analyses were conducted in engineered Huh7 hepatoma cells with reconstituted innate signaling pathways. Although the deletion in NS5A removed most of the interferon (IFN) sensitivity determining-region, mutant NS5A(D2Δ) was as sensitive as the wild type to IFN-α and IFN-λ in vitro, but severely attenuated in vivo. This attenuation could be recapitulated in PHHs and was linked to higher activation of the IFN response, concomitant with reduced viral replication and virus production. Importantly, immune-reconstituted Huh7-derived cell lines revealed a sequential activation of the IFN-response via RIG-I (retinoic acid-inducible gene I) and MDA5 (Myeloma differentiation associated factor 5), respectively, that was significantly higher in the case of the mutant lacking most of NS5A D2. Our study reveals an important role of NS5A D2 for suppression of the IFN response that is activated by HCV via RIG-I and MDA5 in a sequential manner. Copyright © 2015 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.

Interrelationship of Primary Virus Replication, Level of Latency, and Time to Reactivation in the Trigeminal Ganglia of Latently Infected Mice

PubMed Central

Matundan, Harry H.; Mott, Kevin R.; Allen, Sariah J.; Wang, Shaohui; Bresee, Catherine J.; Ghiasi, Yasamin N.; Town, Terrence

2016-01-01

ABSTRACT We sought to determine the possibility of an interrelationship between primary virus replication in the eye, the level of viral DNA in the trigeminal ganglia (TG) during latency, and the amount of virus reactivation following ocular herpes simplex virus type 1 (HSV-1) infection. Mice were infected with virulent (McKrae) or avirulent (KOS and RE) strains of HSV-1, and virus titers in the eyes and TG during primary infection, level of viral gB DNA in TG on day 28 postinfection (p.i.), and virus reactivation on day 28 p.i. as measured by explant reactivation were calculated. Our results suggest that the avirulent strains of HSV-1, even after corneal scarification, had lower virus titers in the eye, had less latency in the TG, and took a longer time to reactivate than virulent strains of HSV-1. The time to explant reactivation of avirulent strains of HSV-1 was similar to that of the virulent LAT(−) McKrae-derived mutant. The viral dose with the McKrae strain of HSV-1 affected the level of viral DNA and time to explant reactivation. Overall, our results suggest that there is no absolute correlation between primary virus titer in the eye and TG and the level of viral DNA in latent TG and time to reactivation. IMPORTANCE Very little is known regarding the interrelationship between primary virus replication in the eye, the level of latency in TG, and the time to reactivate in the mouse model. This study was designed to answer these questions. Our results point to the absence of any correlation between the level of primary virus replication and the level of viral DNA during latency, and neither was an indicator of how rapidly the virus reactivated following explant TG-induced reactivation. PMID:27512072

An innate defense peptide BPIFA1/SPLUNC1 restricts influenza A virus infection.

PubMed

Akram, K M; Moyo, N A; Leeming, G H; Bingle, L; Jasim, S; Hussain, S; Schorlemmer, A; Kipar, A; Digard, P; Tripp, R A; Shohet, R V; Bingle, C D; Stewart, J P

2018-01-01

The airway epithelium secretes proteins that function in innate defense against infection. Bactericidal/permeability-increasing fold-containing family member A1 (BPIFA1) is secreted into airways and has a protective role during bacterial infections, but it is not known whether it also has an antiviral role. To determine a role in host defense against influenza A virus (IAV) infection and to find the underlying defense mechanism, we developed transgenic mouse models that are deficient in BPIFA1 and used these, in combination with in vitro three-dimensional mouse tracheal epithelial cell (mTEC) cultures, to investigate its antiviral properties. We show that BPIFA1 has a significant role in mucosal defense against IAV infection. BPIFA1 secretion was highly modulated after IAV infection. Mice deficient in BPIFA1 lost more weight after infection, supported a higher viral load and virus reached the peripheral lung earlier, indicative of a defect in the control of infection. Further analysis using mTEC cultures showed that BPIFA1-deficient cells bound more virus particles, displayed increased nuclear import of IAV ribonucleoprotein complexes, and supported higher levels of viral replication. Our results identify a critical role of BPIFA1 in the initial phase of infection by inhibiting the binding and entry of IAV into airway epithelial cells.

Decreased VIP and VPAC2 receptor expression in the biological clock of the R6/2 Huntington's disease mouse.

PubMed

Fahrenkrug, Jan; Popovic, Natalija; Georg, Birgitte; Brundin, Patrik; Hannibal, Jens

2007-01-01

Huntington's disease (HD) is a fatal genetic neurodegenerative disorder caused by a CAG triplet repeat expansion in the gene encoding the protein huntingtin. The most studied model of HD, the R6/2 transgenic mouse, replicates many features of the disease. In addition to motor, cognitive, and endocrine dysfunctions, these mice exhibit a progressive disruption of circadian rhythms. This is accompanied by an altered expression of the circadian clock genes in the suprachiasmatic nucleus/nuclei (SCN), the principal circadian pacemaker in the brain. The neuropeptide vasoactive intestinal polypeptide (VIP) and its receptor VPAC2 are highly expressed in the SCN, and VIPergic signaling plays an essential role in maintenance of ongoing circadian rhythmicity. We found a marked reduction in both VIP mRNA and VPAC2 receptor mRNA, quantified by RT-PCR, as well as a decrease in VIP immunostaining in the SCN of R6/2 mice. These changes were coupled to a disruption of circadian rhythm. We observed no loss of neurons in the SCN and therefore suggest that the changes in VIP and VPAC2 receptor are due to their decreased expression. In conclusion, we propose that impaired VIPergic signaling is an additional candidate mechanism for disruption of circadian rhythms in R6/2 mice.

RNAi screening uncovers Dhx9 as a modifier of ABT-737 resistance in an Eμ-myc/Bcl-2 mouse model

PubMed Central

Mills, John R.; Malina, Abba; Lee, Teresa; Di Paola, Domenic; Larsson, Ola; Miething, Cornelius; Grosse, Frank; Tang, Hengli; Zannis-Hadjopoulos, Maria; Lowe, Scott W.

2013-01-01

ABT-737 is a promising chemotherapeutic agent that promotes apoptosis by acting as a selective BH3 mimetic to neutralize Bcl-2–like family members. One shortcoming with its use is that Mcl-1, a member of the Bcl-2 family, is poorly inhibited by ABT-737 and thus is a major cause of resistance. We performed a short hairpin RNA (shRNA)-based drop-out screen to identify novel genes and pathways that could reverse resistance to ABT-737 treatment in Eµ-myc/Bcl-2 lymphoma cells engineered to rely on endogenous Mcl-1 for survival. Several drug-sensitive shRNAs were identified that were selectively depleted in the presence of ABT-737. Of these, 2 independent shRNAs targeting the RNA/DNA helicase Dhx9 were found to sensitize lymphomas to ABT-737 to an extent comparable to control Mcl-1 shRNAs. Although Dhx9 suppression sensitized both mouse and human cells to ABT-737 treatment, it did so without altering MCL-1 levels. Rather, loss of Dhx9 appeared to activate a p53-dependent apoptotic program, through aggravation of replicative stress, which was found to be both necessary and sufficient for the ABT-737–shDhx9 synthetic lethal relationship. PMID:23440244

Animal models in burn research.

PubMed

Abdullahi, A; Amini-Nik, S; Jeschke, M G

2014-09-01

Burn injury is a severe form of trauma affecting more than 2 million people in North America each year. Burn trauma is not a single pathophysiological event but a devastating injury that causes structural and functional deficits in numerous organ systems. Due to its complexity and the involvement of multiple organs, in vitro experiments cannot capture this complexity nor address the pathophysiology. In the past two decades, a number of burn animal models have been developed to replicate the various aspects of burn injury, to elucidate the pathophysiology, and to explore potential treatment interventions. Understanding the advantages and limitations of these animal models is essential for the design and development of treatments that are clinically relevant to humans. This review aims to highlight the common animal models of burn injury in order to provide investigators with a better understanding of the benefits and limitations of these models for translational applications. While many animal models of burn exist, we limit our discussion to the skin healing of mouse, rat, and pig. Additionally, we briefly explain hypermetabolic characteristics of burn injury and the animal model utilized to study this phenomena. Finally, we discuss the economic costs associated with each of these models in order to guide decisions of choosing the appropriate animal model for burn research.

Animal Models in Burn Research

PubMed Central

Abdullahi, A.; Amini-Nik, S.; Jeschke, M.G

2014-01-01

Burn injury is a severe form of trauma affecting more than two million people in North America each year. Burn trauma is not a single pathophysiological event but a devastating injury that causes structural and functional deficits in numerous organ systems. Due to its complexity and the involvement of multiple organs, in vitro experiments cannot capture this complexity nor address the pathophysiology. In the past two decades, a number of burn animal models have been developed to replicate the various aspects of burn injury; to elucidate the pathophysiology and explore potential treatment interventions. Understanding the advantages and limitations of these animal models is essential for the design and development of treatments that are clinically relevant to humans. This review paper aims to highlight the common animal models of burn injury in order to provide investigators with a better understanding of the benefits and limitations of these models for translational applications. While many animal models of burn exist, we limit our discussion to the skin healing of mouse, rat, and pig. Additionally, we briefly explain hypermetabolic characteristics of burn injury and the animal model utilized to study this phenomena. Finally, we discuss the economic costs associated with each of these models in order to guide decisions of choosing the appropriate animal model for burn research. PMID:24714880

A Survey for Novel Imprinted Genes in the Mouse Placenta by mRNA-seq

PubMed Central

Wang, Xu; Soloway, Paul D.; Clark, Andrew G.

2011-01-01

Many questions about the regulation, functional specialization, computational prediction, and evolution of genomic imprinting would be better addressed by having an exhaustive genome-wide catalog of genes that display parent-of-origin differential expression. As a first-pass scan for novel imprinted genes, we performed mRNA-seq experiments on embryonic day 17.5 (E17.5) mouse placenta cDNA samples from reciprocal cross F1 progeny of AKR and PWD mouse strains and quantified the allele-specific expression and the degree of parent-of-origin allelic imbalance. We confirmed the imprinting status of 23 known imprinted genes in the placenta and found that 12 genes reported previously to be imprinted in other tissues are also imprinted in mouse placenta. Through a well-replicated design using an orthogonal allelic-expression technology, we verified 5 novel imprinted genes that were not previously known to be imprinted in mouse (Pde10, Phf17, Phactr2, Zfp64, and Htra3). Our data suggest that most of the strongly imprinted genes have already been identified, at least in the placenta, and that evidence supports perhaps 100 additional weakly imprinted genes. Despite previous appearance that the placenta tends to display an excess of maternally expressed imprinted genes, with the addition of our validated set of placenta-imprinted genes, this maternal bias has disappeared. PMID:21705755

System parameters for erythropoiesis control model: Comparison of normal values in human and mouse model

NASA Technical Reports Server (NTRS)

1979-01-01

The computer model for erythropoietic control was adapted to the mouse system by altering system parameters originally given for the human to those which more realistically represent the mouse. Parameter values were obtained from a variety of literature sources. Using the mouse model, the mouse was studied as a potential experimental model for spaceflight. Simulation studies of dehydration and hypoxia were performed. A comparison of system parameters for the mouse and human models is presented. Aside from the obvious differences expected in fluid volumes, blood flows and metabolic rates, larger differences were observed in the following: erythrocyte life span, erythropoietin half-life, and normal arterial pO2.

Prenatal pharmacotherapy rescues brain development in a Down's syndrome mouse model.

PubMed

Guidi, Sandra; Stagni, Fiorenza; Bianchi, Patrizia; Ciani, Elisabetta; Giacomini, Andrea; De Franceschi, Marianna; Moldrich, Randal; Kurniawan, Nyoman; Mardon, Karine; Giuliani, Alessandro; Calzà, Laura; Bartesaghi, Renata

2014-02-01

Intellectual impairment is a strongly disabling feature of Down's syndrome, a genetic disorder of high prevalence (1 in 700-1000 live births) caused by trisomy of chromosome 21. Accumulating evidence shows that widespread neurogenesis impairment is a major determinant of abnormal brain development and, hence, of intellectual disability in Down's syndrome. This defect is worsened by dendritic hypotrophy and connectivity alterations. Most of the pharmacotherapies designed to improve cognitive performance in Down's syndrome have been attempted in Down's syndrome mouse models during adult life stages. Yet, as neurogenesis is mainly a prenatal event, treatments aimed at correcting neurogenesis failure in Down's syndrome should be administered during pregnancy. Correction of neurogenesis during the very first stages of brain formation may, in turn, rescue improper brain wiring. The aim of our study was to establish whether it is possible to rescue the neurodevelopmental alterations that characterize the trisomic brain with a prenatal pharmacotherapy with fluoxetine, a drug that is able to restore post-natal hippocampal neurogenesis in the Ts65Dn mouse model of Down's syndrome. Pregnant Ts65Dn females were treated with fluoxetine from embryonic Day 10 until delivery. On post-natal Day 2 the pups received an injection of 5-bromo-2-deoxyuridine and were sacrificed after either 2 h or after 43 days (at the age of 45 days). Untreated 2-day-old Ts65Dn mice exhibited a severe neurogenesis reduction and hypocellularity throughout the forebrain (subventricular zone, subgranular zone, neocortex, striatum, thalamus and hypothalamus), midbrain (mesencephalon) and hindbrain (cerebellum and pons). In embryonically treated 2-day-old Ts65Dn mice, precursor proliferation and cellularity were fully restored throughout all brain regions. The recovery of proliferation potency and cellularity was still present in treated Ts65Dn 45-day-old mice. Moreover, embryonic treatment restored dendritic development, cortical and hippocampal synapse development and brain volume. Importantly, these effects were accompanied by recovery of behavioural performance. The cognitive deficits caused by Down's syndrome have long been considered irreversible. The current study provides novel evidence that a pharmacotherapy with fluoxetine during embryonic development is able to fully rescue the abnormal brain development and behavioural deficits that are typical of Down's syndrome. If the positive effects of fluoxetine on the brain of a mouse model are replicated in foetuses with Down's syndrome, fluoxetine, a drug usable in humans, may represent a breakthrough for the therapy of intellectual disability in Down's syndrome.

Diversity, Replication, Pathogenicity and Cell Biology of Crimean Congo Hemorrhagic Fever Virus

DTIC Science & Technology

2008-10-01

author. Mailing address: Department of Microbi - ology, University of Pennsylvania, 225 Johnson Pavilion, 3610 Hamil- ton Walk, Philadelphia, PA 19104...to 15% Tris-HCl gels (Bio-Rad, Hercules, CA), followed by Western blot analysis with mouse anti-V5 (Invitrogen) as the primary antibody and sheep anti...bovine serum. In addition, TGN46, a sheep antibody specific for a heavily glycosylated protein localized primarily in the trans-Golgi network, was

Human mammary microenvironment better regulates the biology of human breast cancer in humanized mouse model.

PubMed

Zheng, Ming-Jie; Wang, Jue; Xu, Lu; Zha, Xiao-Ming; Zhao, Yi; Ling, Li-Jun; Wang, Shui

2015-02-01

During the past decades, many efforts have been made in mimicking the clinical progress of human cancer in mouse models. Previously, we developed a human breast tissue-derived (HB) mouse model. Theoretically, it may mimic the interactions between "species-specific" mammary microenvironment of human origin and human breast cancer cells. However, detailed evidences are absent. The present study (in vivo, cellular, and molecular experiments) was designed to explore the regulatory role of human mammary microenvironment in the progress of human breast cancer cells. Subcutaneous (SUB), mammary fat pad (MFP), and HB mouse models were developed for in vivo comparisons. Then, the orthotopic tumor masses from three different mouse models were collected for primary culture. Finally, the biology of primary cultured human breast cancer cells was compared by cellular and molecular experiments. Results of in vivo mouse models indicated that human breast cancer cells grew better in human mammary microenvironment. Cellular and molecular experiments confirmed that primary cultured human breast cancer cells from HB mouse model showed a better proliferative and anti-apoptotic biology than those from SUB to MFP mouse models. Meanwhile, primary cultured human breast cancer cells from HB mouse model also obtained the migratory and invasive biology for "species-specific" tissue metastasis to human tissues. Comprehensive analyses suggest that "species-specific" mammary microenvironment of human origin better regulates the biology of human breast cancer cells in our humanized mouse model of breast cancer, which is more consistent with the clinical progress of human breast cancer.

Spatial aspects of prebiotic replicator coexistence and community stability in a surface-bound RNA world model

PubMed Central

2013-01-01

Background The coexistence of macromolecular replicators and thus the stability of presumed prebiotic replicator communities have been shown to critically depend on spatially constrained catalytic cooperation among RNA-like modular replicators. The necessary spatial constraints might have been supplied by mineral surfaces initially, preceding the more effective compartmentalization in membrane vesicles which must have been a later development of chemical evolution. Results Using our surface-bound RNA world model – the Metabolic Replicator Model (MRM) platform – we show that the mobilities on the mineral substrate surface of both the macromolecular replicators and the small molecules of metabolites they produce catalytically are the key factors determining the stable persistence of an evolvable metabolic replicator community. Conclusion The effects of replicator mobility and metabolite diffusion on different aspects of replicator coexistence in MRM are determined, including the maximum attainable size of the metabolic replicator system and its resistance to the invasion of parasitic replicators. We suggest a chemically plausible hypothetical scenario for the evolution of the first protocell starting from the surface-bound MRM system. PMID:24053177

Differential Plasma Glycoproteome of p19 Skin Cancer Mouse Model Using the Corra Label-Free LC-MS Proteomics Platform.

PubMed

Letarte, Simon; Brusniak, Mi-Youn; Campbell, David; Eddes, James; Kemp, Christopher J; Lau, Hollis; Mueller, Lukas; Schmidt, Alexander; Shannon, Paul; Kelly-Spratt, Karen S; Vitek, Olga; Zhang, Hui; Aebersold, Ruedi; Watts, Julian D

2008-12-01

A proof-of-concept demonstration of the use of label-free quantitative glycoproteomics for biomarker discovery workflow is presented here, using a mouse model for skin cancer as an example. Blood plasma was collected from 10 control mice, and 10 mice having a mutation in the p19(ARF) gene, conferring them high propensity to develop skin cancer after carcinogen exposure. We enriched for N-glycosylated plasma proteins, ultimately generating deglycosylated forms of the modified tryptic peptides for liquid chromatography mass spectrometry (LC-MS) analyses. LC-MS runs for each sample were then performed with a view to identifying proteins that were differentially abundant between the two mouse populations. We then used a recently developed computational framework, Corra, to perform peak picking and alignment, and to compute the statistical significance of any observed changes in individual peptide abundances. Once determined, the most discriminating peptide features were then fragmented and identified by tandem mass spectrometry with the use of inclusion lists. We next assessed the identified proteins to see if there were sets of proteins indicative of specific biological processes that correlate with the presence of disease, and specifically cancer, according to their functional annotations. As expected for such sick animals, many of the proteins identified were related to host immune response. However, a significant number of proteins also directly associated with processes linked to cancer development, including proteins related to the cell cycle, localisation, trasport, and cell death. Additional analysis of the same samples in profiling mode, and in triplicate, confirmed that replicate MS analysis of the same plasma sample generated less variation than that observed between plasma samples from different individuals, demonstrating that the reproducibility of the LC-MS platform was sufficient for this application. These results thus show that an LC-MS-based workflow can be a useful tool for the generation of candidate proteins of interest as part of a disease biomarker discovery effort.

Inoculation of α-synuclein preformed fibrils into the mouse gastrointestinal tract induces Lewy body-like aggregates in the brainstem via the vagus nerve.

PubMed

Uemura, Norihito; Yagi, Hisashi; Uemura, Maiko T; Hatanaka, Yusuke; Yamakado, Hodaka; Takahashi, Ryosuke

2018-05-11

Intraneuronal α-synuclein (α-Syn) aggregates known as Lewy bodies (LBs) and the loss of dopaminergic neurons in the substantia nigra pars compacta (SNpc) are the pathological hallmarks of Parkinson's disease (PD). Braak's hypothesis based on autopsy studies suggests that Lewy pathology initially occurs in the enteric nervous system (ENS) and then travels retrogradely to the dorsal motor nucleus of the vagus nerve (dmX), proceeding from there in a caudo-rostral direction. Recent evidence that α-Syn aggregates propagate between interconnected neurons supports this hypothesis. However, there is no direct evidence demonstrating this transmission from the ENS to the dmX and then to the SNpc. We inoculated α-Syn preformed fibrils (PFFs) or phosphate-buffered saline (PBS) into the mouse gastric wall and analyzed the progression of the pathology. The mice inoculated with α-Syn PFFs, but not with PBS, developed phosphorylated α-Syn (p-α-Syn)-positive LB-like aggregates in the dmX at 45 days postinoculation. This aggregate formation was completely abolished when vagotomy was performed prior to inoculation of α-Syn PFFs, suggesting that the aggregates in the dmX were retrogradely induced via the vagus nerve. Unexpectedly, the number of neurons containing p-α-Syn-positive aggregates in the dmX decreased over time, and no further caudo-rostral propagation beyond the dmX was observed up to 12 months postinoculation. P-α-Syn-positive aggregates were also present in the myenteric plexus at 12 months postinoculation. However, unlike in patients with PD, there was no cell-type specificity in neurons containing those aggregates in this model. These results indicate that α-Syn PFF inoculation into the mouse gastrointestinal tract can induce α-Syn pathology resembling that of very early PD, but other factors are apparently required if further progression of PD pathology is to be replicated in this animal model.