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1.  In vivo hypoxic preconditioning protects from warm liver ischemic/reperfusion injury through the adenosine A2B receptor 
Transplantation  2012;94(9):894-902.
Liver ischemia(I)/reperfusion(R) injury(I) is a known risk factor for the postoperative outcome of patients undergoing liver surgery/transplantation. Attempts to protect from organ damage require multidisciplinary strategies and are of emerging interest in view of patients with higher age and ASA-status. Ischemic preconditioning has been successfully applied to prevent from IRI during liver resections/transplantation. Since even short periods of ischemia during preconditioning inevitably lead to hypoxia and formation of anti-inflammatory/ cytoprotective acting adenosine, we reasoned that short non-ischemic hypoxia also protects against hepatic IRI.
Mice underwent hypoxic preconditioning(HPC) by breathing 10%-oxygen for 10 minutes, followed by 10 minutes of 21%-oxygen prior to left-liver-lobe-ischemia(45 min) and reperfusion(4 hrs). The interactions of hypoxia->adenosine->adenosine-receptors were tested by pharmacologic antagonism at adenosine receptor(AR) sites in wild type mice and in mice with genetic deletions at the A1-;A2A-;A2B- and A3-ARs. Hepatocellular damage, inflammation and metabolic effects were quantified by enzyme activities, cytokines, liver-myeloperoxidase(MPO), blood adenosine and tissue-adenosinemonophosphate(AMP), respectively.
Hepatoprotection by HPC was significant in wild type and A1-, A2A-and A3 AR-knock-out mice as quantified by lower ALT serum activities, cytokine levels, histological damage-scores, tissue-myeloperoxidase-concentrations and as well as preserved AMP-concentrations. Protection by HPC was blunted in mice pretreated with the A2B-AR-antagonist MRS1754 or in A2B-AR“knock-outs”.
Because liver protective effects of HPC are negated when the A2B receptor is non-functional, the "hypoxia->adenosine->A2B receptor" pathway plays a critical role in the prevention of warm ischemia reperfusion injury in vivo. Hypoxic activation of this pathway warrants use of selective A2B-AR-agonists or even intermittent hypoxia (e.g. in deceased organ donors) to protect from liver ischemia/reperfusion injury.
PMCID: PMC3491139  PMID: 23073466
hypoxia; murine liver ischemia; preconditioning; hepatoprotection
2.  Differentiation of Rodent Immune and Hematopoietic System Reactive Lesions from Neoplasias 
Toxicologic pathology  2012;40(3):425-434.
The immune and hematopoietic systems play an important role in the normal homeostasis of blood and blood cells and for immune responses to endogenous and exogenous processes and insults. In order to interpret histopathologic changes in the immune and hematopoietic systems, it is important to understand the normal anatomy and histology of the thymus, spleen, lymph nodes, bone marrow, and other tissues. The thymus, spleen, and lymph nodes can be categorized by anatomical compartments, each of which contributes to specific immune functions. Lesions may be diagnosed by interpretive or descriptive (semiquantitative) methods. The interpretation of these tissues by lesion in anatomical compartments should allow for better understanding of these reactions and more definitive pathologic findings. Proliferative lesions may be difficult to differentiate from lymphomas and leukemias. The use of immunohistochemistry, compartmental pathology, and methods for the evaluation of clonality will make interpretation easier.
PMCID: PMC3443630  PMID: 22215512
clonality; mice; hematopoietic system; immune system;  immunohistochemistry; lymph nodes; lymphoid; lymphoid hyperplasia; lymphoma; lymphoproliferative disease; rats; spleen; Thymus
Melanoma represents a significant malignancy in humans and dogs. Different from genetically engineered models, sporadic canine melanocytic neoplasms share several characteristics with human disease that could make dogs a more relevant pre-clinical model. Canine melanomas rarely arise in sun-exposed sites. Most occur in the oral cavity, with a subset having intraepithelial malignant melanocytes mimicking the in situ component of human mucosal melanoma. The spectrum of canine melanocytic neoplasia includes benign lesions with some analogy to nevi, as well as invasive primary melanoma, and widespread metastasis. Growing evidence of distinct subtypes in humans, differing in somatic and predisposing germ-line genetic alterations, cell of origin, epidemiology, relationship to ultraviolet radiation and progression from benign to malignant tumors, may also exist in dogs. Canine and human mucosal melanomas appear to harbor BRAF, NRAS and c-kit mutations uncommonly, compared to human cutaneous melanomas, although both species share AKT and MAPK signaling activation. We conclude that there is significant overlap in the clinical and histopathological features of canine and human mucosal melanomas. This represents opportunity to explore canine oral cavity melanoma as a pre-clinical model.
PMCID: PMC4066658  PMID: 24128326
melanoma; animal model; comparative study; clinical trial design; image analysis; digital telepathology; signal transduction
4.  Proceedings of the 2013 National Toxicology Program Satellite Symposium 
Toxicologic pathology  2013;42(1):12-44.
The 2013 annual National Toxicology Program (NTP) Satellite Symposium, entitled “Pathology Potpourri” was held in Portland, Oregon in advance of the Society of Toxicologic Pathology's 32nd annual meeting. The goal of the NTP Symposium is to present current diagnostic pathology or nomenclature issues to the toxicologic pathology community. This article presents summaries of the speakers' presentations, including diagnostic or nomenclature issues that were presented, along with select images that were used for audience voting and discussion. Some lesions and topics covered during the symposium included a caudal tail vertebra duplication in mice; nephroblastematosis in rats; ectopic C cell tumor in a hamster; granular cell aggregates/tumor in the uterus of a hamster; Pneumocystis carinii in the lung of a rat; iatrogenic chronic inflammation in the lungs of control rats; hepatoblastoma arising within an adenoma in a mouse; humoral hypercalcemia of benignancy in a transgenic mouse; acetaminophen induced hepatoxicity in rats; electron microscopy images of iatrogenic intraerythrocytic inclusions in transgenic mice; questionable hepatocellular degeneration/cell death/artifact in rats; atypical endometrial hyperplasia in rats; malignant mixed Müllerian tumors/carcinosarcomas in rats; differential diagnoses of proliferative lesions the intestine of rodents; and finally obstructive nephropathy caused by melamine poisoning in a rat.
PMCID: PMC3992853  PMID: 24334674
NTP Satellite Symposium; duplicate vertebra; nephroblastematosis; granular cell aggregates; Pneumocystis carinii; bronchioloalveolar hyperplasia; hepatoblastoma; intraerythrocytic inclusions; hepatocellular apoptosis; atypical endometrial hyperplasia; malignant mixed Müllerian tumor; gastrointestinal diverticulum; obstructive nephropathy
5.  Oropharyngeal Aspiration of Burkholderia mallei and Burkholderia pseudomallei in BALB/c Mice 
PLoS ONE  2014;9(12):e115066.
Burkholderia mallei and Burkholderia pseudomallei are potentially lethal pathogens categorized as biothreat agents due, in part, to their ability to be disseminated via aerosol. There are no protective vaccines against these pathogens and treatment options are limited and cumbersome. Since disease severity is greatest when these agents are inhaled, efforts to develop pre- or post-exposure prophylaxis focus largely on inhalation models of infection. Here, we demonstrate a non-invasive and technically simple method for affecting the inhalational challenge of BALB/c mice with B. pseudomallei and B. mallei. In this model, two investigators utilized common laboratory tools such as forceps and a micropipette to conduct and characterize an effective and reproducible inhalational challenge of BALB/c mice with B. mallei and B. pseudomallei. Challenge by oropharyngeal aspiration resulted in acute disease. Additionally, 50% endpoints for B. pseudomallei K96243 and B. mallei ATCC 23344 were nearly identical to published aerosol challenge methods. Furthermore, the pathogens disseminated to all major organs typically targeted by these agents where they proliferated. The pro-inflammatory cytokine production in the proximal and peripheral fluids demonstrated a rapid and robust immune response comparable to previously described murine and human studies. These observations demonstrate that OA is a viable alternative to aerosol exposure.
PMCID: PMC4263729  PMID: 25503969
6.  Secretoglobin Superfamily Protein SCGB3A2 Deficiency Potentiates Ovalbumin-Induced Allergic Pulmonary Inflammation 
Mediators of Inflammation  2014;2014:216465.
Secretoglobin (SCGB) 3A2, a cytokine-like secretory protein of small molecular weight, which may play a role in lung inflammation, is predominantly expressed in airway epithelial cells. In order to understand the physiological role of SCGB3A2, Scgb3a2−/− mice were generated and characterized. Scgb3a2−/− mice did not exhibit any overt phenotypes. In ovalbumin- (OVA-) induced airway allergy inflammation model, Scgb3a2−/− mice in mixed background showed a decreased OVA-induced airway inflammation, while six times C57BL/6NCr backcrossed congenic Scgb3a2−/− mice showed a slight exacerbation of OVA-induced airway inflammation as compared to wild-type littermates. These results indicate that the loss of SCGB3A2 function was influenced by a modifier gene(s) in mixed genetic background and suggest that SCGB3A2 has anti-inflammatory property. The results further suggest the possible use of recombinant human SCGB3A2 as an anti-inflammatory agent.
PMCID: PMC4163287  PMID: 25242865
7.  Transposon mutagenesis identifies genes driving hepatocellular carcinoma in a chronic hepatitis B mouse model 
Nature genetics  2013;46(1):24-32.
The most common risk factor for developing hepatocellular carcinoma (HCC) is chronic infection with hepatitis B virus (HBV). To better understand the evolutionary forces driving HCC we performed a near saturating transposon mutagenesis screen in a mouse HBV model of HCC. This screen identified 21 candidate early stage drivers, and a bewildering number (2860) of candidate later stage drivers, that were enriched for genes mutated, deregulated, or that function in signaling pathways important for human HCC, with a striking 1199 genes linked to cellular metabolic processes. Our study provides a comprehensive overview of the genetic landscape of HCC.
PMCID: PMC4131144  PMID: 24316982
8.  Animal models of human prostate cancer: The Consensus Report of the New York Meeting of the Mouse Models of Human Cancers Consortium Prostate Pathology Committee 
Cancer research  2013;73(9):2718-2736.
Animal models, particularly mouse models, play a central role in the study of the etiology, prevention and treatment of human prostate cancer (PCa). While tissue culture models are extremely useful in understanding the biology of PCa, they cannot recapitulate the complex cellular interactions within the tumor microenvironment that play a key role in cancer initiation and progression. The NCI Mouse Models of Human Cancers Consortium convened a group of human and veterinary pathologists to review the current animal models of PCa and make recommendations regarding the pathological analysis of these models. Over 40 different models with 439 samples were reviewed including genetically engineered mouse models, xenograft, rat and canine models. Numerous relevant models have been developed over the last 15 years and each approach has strengths and weaknesses. Analysis of multiple genetically engineered models has shown that reactive stroma formation is present in all the models developing invasive carcinomas. In addition, numerous models with multiple genetic alterations display aggressive phenotypes characterized by sarcomatoid carcinomas and metastases, which is presumably a histological manifestation of epithelial-mesenchymal transition. The significant progress in development of improved models of PCa has already accelerated our understanding the complex biology of PCa and promises to enhance development of new approaches to prevention, detection and treatment of this common malignancy.
PMCID: PMC3644021  PMID: 23610450
prostate cancer; animal models; transgenic; pathology; genetically engineered mice; xenograft
9.  Mice Carrying a Hypomorphic Evi1 Allele Are Embryonic Viable but Exhibit Severe Congenital Heart Defects 
PLoS ONE  2014;9(2):e89397.
The ecotropic viral integration site 1 (Evi1) oncogenic transcription factor is one of a number of alternative transcripts encoded by the Mds1 and Evi1 complex locus (Mecom). Overexpression of Evi1 has been observed in a number of myeloid disorders and is associated with poor patient survival. It is also amplified and/or overexpressed in many epithelial cancers including nasopharyngeal carcinoma, ovarian carcinoma, ependymomas, and lung and colorectal cancers. Two murine knockout models have also demonstrated Evi1's critical role in the maintenance of hematopoietic stem cell renewal with its absence resulting in the death of mutant embryos due to hematopoietic failure. Here we characterize a novel mouse model (designated Evi1fl3) in which Evi1 exon 3, which carries the ATG start, is flanked by loxP sites. Unexpectedly, we found that germline deletion of exon3 produces a hypomorphic allele due to the use of an alternative ATG start site located in exon 4, resulting in a minor Evi1 N-terminal truncation and a block in expression of the Mds1-Evi1 fusion transcript. Evi1δex3/δex3 mutant embryos showed only a mild non-lethal hematopoietic phenotype and bone marrow failure was only observed in adult Vav-iCre/+, Evi1fl3/fl3 mice in which exon 3 was specifically deleted in the hematopoietic system. Evi1δex3/δex3 knockout pups are born in normal numbers but die during the perinatal period from congenital heart defects. Database searches identified 143 genes with similar mutant heart phenotypes as those observed in Evi1δex3/δex3 mutant pups. Interestingly, 42 of these congenital heart defect genes contain known Evi1-binding sites, and expression of 18 of these genes are also effected by Evi1 siRNA knockdown. These results show a potential functional involvement of Evi1 target genes in heart development and indicate that Evi1 is part of a transcriptional program that regulates cardiac development in addition to the development of blood.
PMCID: PMC3937339  PMID: 24586749
10.  Where’s the mouse info? 
Veterinary pathology  2009;46(6):1241-1244.
PMCID: PMC3804057  PMID: 19605899
databases; genetically engineered mice; phenotyping; record keeping; mouse pathology; mouse pathology informatics
11.  Selenium and Selenoprotein Deficiencies Induce Widespread Pyogranuloma Formation in Mice, while High Levels of Dietary Selenium Decrease Liver Tumor Size Driven by TGFα 
PLoS ONE  2013;8(2):e57389.
Changes in dietary selenium and selenoprotein status may influence both anti- and pro-cancer pathways, making the outcome of interventions different from one study to another. To characterize such outcomes in a defined setting, we undertook a controlled hepatocarcinogenesis study involving varying levels of dietary selenium and altered selenoprotein status using mice carrying a mutant (A37G) selenocysteine tRNA transgene (TrsptG37) and/or a cancer driver TGFα transgene. The use of TrsptG37 altered selenoprotein expression in a selenoprotein and tissue specific manner and, at sufficient dietary selenium levels, separate the effect of diet and selenoprotein status. Mice were maintained on diets deficient in selenium (0.02 ppm selenium) or supplemented with 0.1, 0.4 or 2.25 ppm selenium or 30 ppm triphenylselenonium chloride (TPSC), a non-metabolized selenium compound. TrsptG37 transgenic and TGFα/TrsptG37 bi-transgenic mice subjected to selenium-deficient or TPSC diets developed a neurological phenotype associated with early morbidity and mortality prior to hepatocarcinoma development. Pathology analyses revealed widespread disseminated pyogranulomatous inflammation. Pyogranulomas occurred in liver, lungs, heart, spleen, small and large intestine, and mesenteric lymph nodes in these transgenic and bi-transgenic mice. The incidence of liver tumors was significantly increased in mice carrying the TGFα transgene, while dietary selenium and selenoprotein status did not affect tumor number and multiplicity. However, adenoma and carcinoma size and area were smaller in TGFα transgenic mice that were fed 0.4 and 2.25 versus 0.1 ppm of selenium. Thus, selenium and selenoprotein deficiencies led to widespread pyogranuloma formation, while high selenium levels inhibited the size of TGFα–induced liver tumors.
PMCID: PMC3583866  PMID: 23460847
12.  Proceedings of the 2011 National Toxicology Program Satellite Symposium 
Toxicologic pathology  2011;40(2):321-344.
The 2011 annual National Toxicology Program (NTP) Satellite Symposium, entitled “Pathology Potpourri,” was held in Denver, Colorado in advance of the Society of Toxicologic Pathology’s 30th Annual Meeting. The goal of the NTP Symposium is to present current diagnostic pathology or nomenclature issues to the toxicologic pathology community. This article presents summaries of the speakers’ presentations, including diagnostic or nomenclature issues that were presented, along with select images that were used for audience voting or discussion. Some lesions and topics covered during the symposium include: proliferative lesions from various fish species including ameloblastoma, gas gland hyperplasia, nodular regenerative hepatocellular hyperplasia, and malignant granulosa cell tumor; spontaneous cystic hyperplasia in the stomach of CD1 mice and histiocytic aggregates in the duodenal villous tips of treated mice; an olfactory neuroblastoma in a cynomolgus monkey; various rodent skin lesions, including follicular parakeratotic hyperkeratosis, adnexal degeneration, and epithelial intracytoplasmic accumulations; oligodendroglioma and microgliomas in rats; a diagnostically challenging microcytic, hypochromic, responsive anemia in rats; a review of microcytes and microcytosis; nasal lesions associated with green tea extract and Ginkgo biloba in rats; corneal dystrophy in Dutch belted rabbits; valvulopathy in rats; and lymphoproliferative disease in a cynomolgus monkey.
PMCID: PMC3490626  PMID: 22089839
NTP Satellite Symposium; ameloblastoma; gas gland hyperplasia; stomach cystic hyperplasia; sodium dichromate dihydrate; olfactory neuroblastoma; cynomolgus monkey; adnexal degeneration; parakeratotic hyperkeratosis; oligodendroglioma; microglioma; microcytic hypochromic anemia; microcytosis; spherocytosis; poikilocytosis; green tea; Ginkgo biloba; corneal dystrophy; Dutch belted rabbit valvulitis; valvulopathy; post-transplant lymphoproliferative disease
13.  Deficiency of CCAAT/enhancer binding protein family DNA binding prevents malignant conversion of adenoma to carcinoma in NNK-induced lung carcinogenesis in the mouse 
Molecular Cancer  2012;11:90.
The CCAAT/enhancer binding proteins (C/EBPs) play important roles in carcinogenesis of many tumors including the lung. Since multiple C/EBPs are expressed in lung, the combinatorial expression of these C/EBPs on lung carcinogenesis is not known.
A transgenic mouse line expressing a dominant negative A-C/EBP under the promoter of lung epithelial Clara cell secretory protein (CCSP) gene in doxycycline dependent fashion was subjected to 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK)-induced lung carcinogenesis bioassay in the presence and absence of doxycycline, and the effect of abolition of DNA binding activities of C/EBPs on lung carcinogenesis was examined.
A-C/EBP expression was found not to interfere with tumor development; however, it suppressed the malignant conversion of adenoma to carcinoma during NNK-induced lung carcinogenesis. The results suggested that Ki67 may be used as a marker for lung carcinomas in mouse.
The DNA binding of C/EBP family members can be used as a potential molecular target for lung cancer therapy.
PMCID: PMC3548712  PMID: 23234329
C/EBPs; Lung chemical carcinogenesis bioassay; Dominant negative; A-C/EBP; Transgenic mouse; 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone; NNK
14.  Training Pathologists in Mouse Pathology 
Veterinary Pathology  2010;49(2):393-397.
Expertise in the pathology of mice has expanded from traditional regulatory and drug safety screening (toxicologic pathology), primarily performed by veterinary pathologists to the highly specialized area of mouse research pathobiology performed by veterinary and medical pathologists encompassing phenotyping of mutant mice and analysis of research experiments exploiting inbred mouse strains and genetically engineered lines. With increasing use of genetically modified mice in research, mouse pathobiology and, by extension, expert mouse research-oriented pathologists have become integral to the success of basic and translational biomedical research. Training for today’s research-oriented mouse pathologist must go beyond knowledge of anatomic features of mice and strain-specific background diseases to the specialized genetic nomenclature, husbandry, and genetics, including the methodology of genetic engineering and complex trait analysis. While training can be accomplished through “apprenticeships” in formal programs, these are often heavily service-related and do not provide the necessary comprehensive training. Specialty courses and short term mentoring with expert specialists are opportunities that, when combined with active practice and publication, will lead to acquisition of the skills required for cutting-edge mouse-based experimental science.
PMCID: PMC3329931  PMID: 20817889
15.  Secretoglobin 3A2/uteroglobin-related protein 1 is a novel marker for pulmonary carcinoma in mice and humans 
Secretoglobin (SCGB) 3A2, also called uteroglobin-related protein (UGRP) 1, is a downstream target for a homeodomain transcription factor NKX2-1, which is critical for the development of lung, thyroid and ventral forebrain. Both SCGB3A2 and NKX2-1 are expressed in airway epithelial cells and the latter also in alveolar Type II cells. NKX2-1 has been used clinically for diagnosis of human pulmonary tumors. Recently, the expression of SCGB3A2 was reported in human carcinomas, suggesting the use of this protein as a tumor marker. In this study, twenty eight lung tumors from aging B6;129 mice and nine lung adenocarcinomas from CC10TAg transgenic mice that express SV40 large T antigen under the mouse Scgb1a1 (CC10) gene promoter, were subjected to histopathological and immunohistochemical analyses for the expression of NKX2-1 and SCGB3A2. NKX2-1 was expressed in all types of tumors albeit more focally in carcinomas. In contrast, SCGB3A2 normally expressed in Clara cells, was negative in Type II cell hyperplasias and adenomas. However, it was expressed in alveolar Type II cell carcinomas and Clara cell adenocarcinomas. In these carcinomas, SCGB3A2 expression was observed in the portion of the tumor where NKX2-1 expression was reduced or almost abolished. As a comparison, the expression of SCGB3A2 and NKX2-1 from twenty-three human non-small cell lung carcinoma specimens was also examined. The results demonstrate that SCGB3A2 is a useful marker for diagnosis of pulmonary tumors both in mice and humans.
PMCID: PMC2950211  PMID: 20466451
SCGB3A2; UGRP1; NKX2-1; TTF1; NSCLC; aging B6; 129 mice; CC10TAg transgenic mice; pulmonary carcinoma; carcinoma marker; histopathological and immunohistochemical analysis
16.  Carcinogenic Effects of “Whole-Life” Exposure to Inorganic Arsenic in CD1 Mice 
Toxicological Sciences  2010;119(1):73-83.
In a previously developed mouse model, arsenic exposure in utero induces tumors at multiple sites in the offspring as adults, often duplicating human targets. However, human environmental inorganic arsenic exposure occurs during the entire life span, not just part of gestation. Thus, “whole-life” inorganic arsenic carcinogenesis in mice was studied. CD1 mice were exposed to 0, 6, 12, or 24 ppm arsenic in the drinking water 2 weeks prior to breeding, during pregnancy, lactation, and after weaning through adulthood. Tumors were assessed in offspring until 2 years of age. Arsenic induced dose-related increases in lung adenocarcinoma (both sexes), hepatocellular carcinoma (both sexes), gallbladder tumors (males), and uterine carcinomas. Arsenic induced dose-related increases in ovarian tumors (including carcinomas) starting with the lowest dose. Adrenal tumors increased at all doses (both sexes). Arsenic-induced lung and liver cancers were highly enriched for cancer stem cells, consistent with prior work with skin cancers stimulated by prenatal arsenic. Reproductive tract tumors overexpressed cyclooxygenase-2 and estrogen receptor-α. Arsenic target sites were remarkably similar to prior transplacental studies, although tumors from whole-life exposure were generally more aggressive and frequent. This may indicate that arsenic-induced events in utero dictate target site in some tissues, whereas other exposure periods of arsenic enhance incidence or progression, though other factors could be at play, like cumulative dose. Whole-life arsenic exposure induced tumors at dramatically lower external doses than in utero arsenic only while more realistically duplicating human exposure.
PMCID: PMC3003832  PMID: 20937726
arsenic; carcinogenesis; mice; whole-life exposure
17.  Immunogenicity and Protective Efficacy in Mice and Hamsters of a β-Propiolactone Inactivated Whole Virus SARS-CoV Vaccine 
Viral Immunology  2010;23(5):509-519.
The immunogenicity and efficacy of β-propiolactone (BPL) inactivated whole virion SARS-CoV (WI-SARS) vaccine was evaluated in BALB/c mice and golden Syrian hamsters. The vaccine preparation was tested with or without adjuvants. Adjuvant Systems AS01B and AS03A were selected and tested for their capacity to elicit high humoral and cellular immune responses to WI-SARS vaccine. We evaluated the effect of vaccine dose and each adjuvant on immunogenicity and efficacy in mice, and the effect of vaccine dose with or without the AS01B adjuvant on the immunogenicity and efficacy in hamsters. Efficacy was evaluated by challenge with wild-type virus at early and late time points (4 and 18 wk post-vaccination). A single dose of vaccine with or without adjuvant was poorly immunogenic in mice; a second dose resulted in a significant boost in antibody levels, even in the absence of adjuvant. The use of adjuvants resulted in higher antibody titers, with the AS01B-adjuvanted vaccine being slightly more immunogenic than the AS03A-adjuvanted vaccine. Two doses of WI-SARS with and without Adjuvant Systems were highly efficacious in mice. In hamsters, two doses of WI-SARS with and without AS01B were immunogenic, and two doses of 2 μg of WI-SARS with and without the adjuvant provided complete protection from early challenge. Although antibody titers had declined in all groups of vaccinated hamsters 18 wk after the second dose, the vaccinated hamsters were still partially protected from wild-type virus challenge. Vaccine with adjuvant provided better protection than non-adjuvanted WI-SARS vaccine at this later time point. Enhanced disease was not observed in the lungs or liver of hamsters following SARS-CoV challenge, regardless of the level of serum neutralizing antibodies.
PMCID: PMC2967819  PMID: 20883165
18.  Diversity of Murine Norovirus Strains Isolated from Asymptomatic Mice of Different Genetic Backgrounds within a Single U.S. Research Institute 
PLoS ONE  2011;6(6):e21435.
Antibody prevalence studies in laboratory mice indicate that murine norovirus (MNV) infections are common, but the natural history of these viruses has not been fully established. This study examined the extent of genetic diversity of murine noroviruses isolated from healthy laboratory mice housed in multiple animal facilities within a single, large research institute- the National Institute of Allergy and Infectious Diseases of the National Institutes of Health (NIAID-NIH) in Bethesda, Maryland, U.S. Ten distinct murine norovirus strains were isolated from various tissues and feces of asymptomatic wild type sentinel mice as well as asymptomatic immunodeficient (RAG 2−/−) mice. The NIH MNV isolates showed little cytopathic effect in permissive RAW264.7 cells in early passages, but all isolates examined could be adapted to efficient growth in cell culture by serial passage. The viruses, although closely related in genome sequence, were distinguishable from each other according to facility location, likely due to the introduction of new viruses into each facility from separate sources or vendors at different times. Our study indicates that the murine noroviruses are widespread in these animal facilities, despite rigorous guidelines for animal care and maintenance.
PMCID: PMC3125191  PMID: 21738664
19.  Role of NKX2-1 in N-bis(2-hydroxypropyl)-nitrosamine-induced thyroid adenoma in mice 
Carcinogenesis  2009;30(9):1614-1619.
NKX2-1 is a homeodomain transcription factor that is critical for genesis of the thyroid and transcription of the thyroid-specific genes. Nkx2-1-thyroid-conditional hypomorphic mice were previously developed in which Nkx2-1 gene expression is lost in 50% of the thyroid cells. Using this mouse line as compared with wild-type and Nkx2-1 heterozygous mice, a thyroid carcinogenesis study was carried out using the genotoxic carcinogen N-bis(2-hydroxypropyl)-nitrosamine (DHPN), followed by sulfadimethoxine (SDM) or the non-genotoxic carcinogen amitrole (3-amino-1,2,4-triazole). A significantly higher incidence of adenomas was obtained in Nkx2-1-thyroid-conditional hypomorphic mice as compared with the other two groups of mice only when they were treated with DHPN + SDM, but not amitrole. A bromodeoxyuridine incorporation study revealed that thyroids of the Nkx2-1-thyroid-conditional hypomorphic mice had >2-fold higher constitutive cell proliferation rate than the other two groups of mice, suggesting that this may be at least partially responsible for the increased incidence of adenoma in this mouse line after genotoxic carcinogen exposure. Thus, NKX2-1 may function to control the proliferation of thyroid follicular cells following damage by a genotoxic carcinogen.
PMCID: PMC2736302  PMID: 19581346
20.  Plasmodium falciparum-infected erythrocytes induce Tissue Factor expression in endothelial cells and support the assembly of multimolecular coagulation complexes 
Plasmodium falciparum malaria infects 300–500 million people every year causing 1–2 million deaths annually. Evidence of a coagulation disorder, activation of endothelial cells (EC) and increase in inflammatory cytokines are often present in malaria.
We have asked whether parasitized red blood cells (pRBC) interaction with EC induces Tissue Factor expression in vitro and in vivo. The potential of phosphatidylserine-containing pRBC to support the assembly of blood coagulation complexes was also investigated.
We demonstrate that mature forms of pRBC induce functional expression of tissue factor (TF) by endothelial cells (EC) in vitro with productive assembly of the extrinsic Xnase complex and initiation of the coagulation cascade. Late stage pRBC also support the prothrombinase and intrinsic Xnase complex formation in vitro, and may function as activated platelets in the amplification phase of the blood coagulation. Notably, postmortem brain sections obtained from P. falciparum-infected children who died from Cerebral Malaria and other causes display a consistent staining for TF in the EC.
These findings place TF expression by endothelium and the amplification of the coagulation cascade by pRBC and/or activated platelets as potentially critical steps in the pathogenesis of malaria. Furthermore, it may allow investigators to test other therapeutic alternatives targeting TF or modulators of EC function in the treatment of malaria and/or its complications.
PMCID: PMC2892312  PMID: 17002660
endothelial cell; malaria; prothrombinase; platelets; Plasmodium falciparum; Tissue Factor
21.  A Single-Amino-Acid Substitution in a Polymerase Protein of an H5N1 Influenza Virus Is Associated with Systemic Infection and Impaired T-Cell Activation in Mice▿ †  
Journal of Virology  2009;83(21):11102-11115.
The transmission of H5N1 influenza viruses from birds to humans poses a significant public health threat. A substitution of glutamic acid for lysine at position 627 of the PB2 protein of H5N1 viruses has been identified as a virulence determinant. We utilized the BALB/c mouse model of H5N1 infection to examine how this substitution affects virus-host interactions and leads to systemic infection. Mice infected with H5N1 viruses containing lysine at amino acid 627 in the PB2 protein exhibited an increased severity of lesions in the lung parenchyma and the spleen, increased apoptosis in the lungs, and a decrease in oxygen saturation. Gene expression profiling revealed that T-cell receptor activation was impaired at 2 days postinfection (dpi) in the lungs of mice infected with these viruses. The inflammatory response was highly activated in the lungs of mice infected with these viruses and was sustained at 4 dpi. In the spleen, immune-related processes including NK cell cytotoxicity and antigen presentation were highly activated by 2 dpi. These differences are not attributable solely to differences in viral replication in the lungs but to an inefficient immune response early in infection as well. The timing and magnitude of the immune response to highly pathogenic influenza viruses is critical in determining the outcome of infection. The disruption of these factors by a single-amino-acid substitution in a polymerase protein of an influenza virus is associated with severe disease and correlates with the spread of the virus to extrapulmonary sites.
PMCID: PMC2772766  PMID: 19692471
22.  SARS-CoV Pathogenesis Is Regulated by a STAT1 Dependent but a Type I, II and III Interferon Receptor Independent Mechanism 
PLoS Pathogens  2010;6(4):e1000849.
Severe acute respiratory syndrome coronavirus (SARS-CoV) infection often caused severe end stage lung disease and organizing phase diffuse alveolar damage, especially in the elderly. The virus-host interactions that governed development of these acute end stage lung diseases and death are unknown. To address this question, we evaluated the role of innate immune signaling in protection from human (Urbani) and a recombinant mouse adapted SARS-CoV, designated rMA15. In contrast to most models of viral pathogenesis, infection of type I, type II or type III interferon knockout mice (129 background) with either Urbani or MA15 viruses resulted in clinical disease outcomes, including transient weight loss, denuding bronchiolitis and alveolar inflammation and recovery, identical to that seen in infection of wildtype mice. This suggests that type I, II and III interferon signaling play minor roles in regulating SARS pathogenesis in mouse models. In contrast, infection of STAT1−/− mice resulted in severe disease, high virus titer, extensive pulmonary lesions and 100% mortality by day 9 and 30 post-infection with rMA15 or Urbani viruses, respectively. Non-lethal in BALB/c mice, Urbani SARS-CoV infection in STAT1−/− mice caused disseminated infection involving the liver, spleen and other tissues after day 9. These findings demonstrated that SARS-CoV pathogenesis is regulated by a STAT1 dependent but type I, II and III interferon receptor independent, mechanism. In contrast to a well documented role in innate immunity, we propose that STAT1 also protects mice via its role as an antagonist of unrestrained cell proliferation.
Author Summary
The SARS coronavirus is a highly pathogenic respiratory virus that caused the first epidemic of the 21st century. During the epidemic ∼10% of those infected died and the elderly were particularly vulnerable. Severe cases developed acute lung injury with pulmonary fibrosis and Acute Respiratory Distress Syndrome (ARDS). Little is known about the molecular mechanisms governing its virus pathogenesis and high lethality. Using a mouse model of infection with the epidemic strain of SARS-CoV (Urbani) as well as a recombinant mouse adapted strain of SARS-CoV (rMA15), we showed that a protein normally associated with the innate immune response, STAT1, plays an important role in the development of severe end stage lung injury. However, the lack of a normal innate immune type I, type II and type III interferon response did not enhance virus pathogenesis. Our work suggests that STAT1 may play a key role in development of acute lung injury and other chronic lung pathology, most likely by affecting cell proliferation and wound repair pathways.
PMCID: PMC2851658  PMID: 20386712
23.  Spindle assembly checkpoint and p53 deficiencies cooperate for tumorigenesis in mice 
The spindle assembly checkpoint (SAC) guards against chromosomal mis-segregation during mitosis. To investigate the role of SAC in tumor development, mice heterozygously knocked-out for the mitotic arrest deficient (Mad) genes Mad1 and/or Mad2 were mated with p53+/− mice. Increased tumor frequencies were reproducibly observed in Mad2+/−p53+/− (88.2%) and Mad1+/−Mad2+/−p53+/− (95.0%) mice compared to p53+/− (66.7%) mice. Moreover, 53% of Mad2+/−p53+/− mice developed lymphomas compared to 11% of p53+/− mice. By examining chromosome content, increased loss in diploidy was seen in cells from Mad2+/−p53+/− versus p53+/− mice, correlating loss of SAC function, in a p53+/− context, with increased aneuploidy and tumorigenesis. The findings here provide evidence for a cooperative role of Mad1/Mad2 and p53 genes in preventing tumor development.
PMCID: PMC2706662  PMID: 19065665
Mad1; Mad2; p53; spindle assembly checkpoint; tumorigenesis
24.  Delivery to the lower respiratory tract is required for effective immunization with Newcastle disease virus-vectored vaccines intended for humans 
Vaccine  2009;27(10):1530-1539.
Newcastle disease virus (NDV), an avian virus, is being evaluated for the development of vectored human vaccines against emerging pathogens. Previous studies of NDV-vectored vaccines in a mouse model suggested their potency after delivery by injection or by the intranasal route. We compared the efficacy of various routes of delivery of NDV-vectored vaccines in a non-human primate model. While delivery of an NDV vectored vaccine by the combined intranasal/intratracheal route elicited protective immune responses, delivery by the subcutaneous route or the intranasal route alone elicited limited or no protective immune responses, suggesting the necessity for vaccine delivery to the lower respiratory tract. Furthermore, direct comparison of a vaccine based on an NDV mesogenic strain (NDV-BC) with a similarly designed NDV vector based on a modified lentogenic strain carrying a polybasic F cleavage site (NDV-VF) suggested that the two NDV strains were similar in immunogenicity and were equally protective.
PMCID: PMC2723768  PMID: 19168110
25.  Chimeric human parainfluenza virus bearing the Ebola virus glycoprotein as the sole surface protein is immunogenic and highly protective against Ebola virus challenge 
Virology  2008;383(2):348-361.
We generated a new live-attenuated vaccine against Ebola virus (EBOV) based on a chimeric virus HPIV3/ΔF-HN/EboGP that contains the EBOV glycoprotein (GP) as the sole transmembrane envelope protein combined with the internal proteins of human parainfluenza virus type 3 (HPIV3). Electron microscopy analysis of the virus particles showed that they have an envelope and surface spikes resembling those of EBOV and a particle size and shape resembling those of HPIV3. When HPIV3/ΔF-HN/EboGP was inoculated via apical surface of an in vitro model of human ciliated airway epithelium, the virus was released from the apical surface; when applied to basolateral surface, the virus infected basolateral cells but did not spread through the tissue. Following intranasal (IN) inoculation of guinea pigs, scattered infected cells were detected in the lungs by immunohistochemistry, but infectious HPIV3/ΔF-HN/EboGP could not be recovered from the lungs, blood, or other tissues. Despite the attenuation, the virus was highly immunogenic, and a single IN dose completely protected the animals against a highly lethal intraperitoneal challenge of guinea pig-adapted EBOV.
PMCID: PMC2649782  PMID: 19010509

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