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1.  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
2.  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
3.  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
4.  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
5.  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
6.  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
7.  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
8.  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
9.  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
10.  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
11.  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
12.  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
13.  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
14.  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
15.  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
16.  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
17.  Ligand activation of peroxisome proliferator-activated receptor β/δ (PPARβ/δ) inhibits chemically induced skin tumorigenesis 
Carcinogenesis  2008;29(12):2406-2414.
Peroxisome proliferator-activated receptor (PPAR)β/δ-null mice exhibit enhanced tumorigenesis in a two-stage chemical carcinogenesis model as compared with wild-type mice. Previous work showed that ligand activation of PPARβ/δ induces terminal differentiation and inhibits proliferation of primary keratinocytes, and this effect does not occur in the absence of PPARβ/δ expression. In the present studies, the effect of ligand activation of PPARβ/δ on skin tumorigenesis was examined using both in vivo and ex vivo skin carcinogenesis models. Inhibition of chemically induced skin tumorigenesis was observed in wild-type mice administered GW0742, and this effect was likely the result of ligand-induced terminal differentiation and inhibition of replicative DNA synthesis. These effects were not found in similarly treated PPARβ/δ-null mice. Ligand activation of PPARβ/δ also inhibited cell proliferation and induced terminal differentiation in initiated/neoplastic keratinocyte cell lines representing different stages of skin carcinogenesis. These studies suggest that topical administration of PPARβ/δ ligands may be useful as both a chemopreventive and/or a chemotherapeutic approach to inhibit skin cancer.
PMCID: PMC2722866  PMID: 18799709
18.  Arsenic Exposure In utero Exacerbates Skin Cancer Response in Adulthood with Contemporaneous Distortion of Tumor Stem Cell Dynamics 
Cancer research  2008;68(20):8278-8285.
Arsenic is a carcinogen with transplacental activity that can affect human skin stem cell population dynamics in vitro by blocking exit into differentiation pathways. Keratinocyte stem cells (KSC) are probably a key target in skin carcinogenesis. Thus, we tested the effects of fetal arsenic exposure in Tg.AC mice, a strain sensitive to skin carcinogenesis via activation of the v-Ha-ras transgene likely in KSCs. After fetal arsenic treatment, offspring received topical 12-O-tetradecanoyl phorbol-13-acetate (TPA) through adulthood. Arsenic alone had no effect, whereas TPA alone induced papillomas and squamous cell carcinomas (SCC). However, fetal arsenic treatment before TPA increased SCC multiplicity 3-fold more than TPA alone, and these SCCs were much more aggressive (invasive, etc.). Tumor v-Ha-ras levels were 3-fold higher with arsenic plus TPA than TPA alone, and v-Ha-ras was over expressed early on in arsenic-treated fetal skin. CD34, considered a marker for both KSCs and skin cancer stem cells, and Rac1, a key gene stimulating KSC self-renewal, were greatly increased in tumors produced by arsenic plus TPA exposure versus TPA alone, and both were elevated in arsenic-treated fetal skin. Greatly increased numbers of CD34-positive probable cancer stem cells and marked over expression of RAC1 protein occurred in tumors induced by arsenic plus TPA compared with TPA alone. Thus, fetal arsenic exposure, although by itself oncogenically inactive in skin, facilitated cancer response in association with distorted skin tumor stem cell signaling and population dynamics, implicating stem cells as a target of arsenic in the fetal basis of skin cancer in adulthood.
PMCID: PMC2652700  PMID: 18922899
19.  Pathogenic Roles of CD14, Galectin-3, and OX40 during Experimental Cerebral Malaria in Mice 
PLoS ONE  2009;4(8):e6793.
An in-depth knowledge of the host molecules and biological pathways that contribute towards the pathogenesis of cerebral malaria would help guide the development of novel prognostics and therapeutics. Genome-wide transcriptional profiling of the brain tissue during experimental cerebral malaria (ECM ) caused by Plasmodium berghei ANKA parasites in mice, a well established surrogate of human cerebral malaria, has been useful in predicting the functional classes of genes involved and pathways altered during the course of disease. To further understand the contribution of individual genes to the pathogenesis of ECM, we examined the biological relevance of three molecules – CD14, galectin-3, and OX40 that were previously shown to be overexpressed during ECM. We find that CD14 plays a predominant role in the induction of ECM and regulation of parasite density; deletion of the CD14 gene not only prevented the onset of disease in a majority of susceptible mice (only 21% of CD14-deficient compared to 80% of wildtype mice developed ECM, p<0.0004) but also had an ameliorating effect on parasitemia (a 2 fold reduction during the cerebral phase). Furthermore, deletion of the galectin-3 gene in susceptible C57BL/6 mice resulted in partial protection from ECM (47% of galectin-3-deficient versus 93% of wildtype mice developed ECM, p<0.0073). Subsequent adherence assays suggest that galectin-3 induced pathogenesis of ECM is not mediated by the recognition and binding of galectin-3 to P. berghei ANKA parasites. A previous study of ECM has demonstrated that brain infiltrating T cells are strongly activated and are CD44+CD62L− differentiated memory T cells [1]. We find that OX40, a marker of both T cell activation and memory, is selectively upregulated in the brain during ECM and its distribution among CD4+ and CD8+ T cells accumulated in the brain vasculature is approximately equal.
PMCID: PMC2728507  PMID: 19710907
20.  Deletion of Nonstructural Proteins NS1 and NS2 from Pneumonia Virus of Mice Attenuates Viral Replication and Reduces Pulmonary Cytokine Expression and Disease▿  
Journal of Virology  2008;83(4):1969-1980.
Pneumonia virus of mice (PVM) strain 15 causes fatal pneumonia in mice and provides a convenient model for human respiratory syncytial virus pathogenesis and immunobiology. We prepared PVM mutants lacking the genes for nonstructural proteins NS1 and/or NS2. In Vero cells, which lack type I interferon (IFN), deletion of these proteins had no effect on the efficiency of virus growth. In IFN-competent mouse embryo fibroblasts, wild-type (wt) PVM and the ΔNS1 virus grew efficiently and strongly inhibited the IFN response, whereas virus lacking NS2 was highly attenuated and induced high levels of IFN and IFN-inducible genes. In BALB/c mice, intranasal infection with wt PVM caused overt disease that began on day 6 and was lethal by day 9 postinoculation. In comparison, ΔNS1 induced transient, reduced disease, and ΔNS2 and ΔNS12 caused no disease. Thus, NS1 and NS2 are virulence factors, with NS2 being a major antagonist of the type I IFN system. The pulmonary titers of wt PVM and ΔNS1 were high on day 3 and increased further by day 6; in addition, expression of IFN and representative proinflammatory cytokines/chemokines and T lymphocyte-related cytokines was undetectable on day 3 but increased dramatically by day 6 coincident with the onset of disease. The titers of ΔNS2 and ΔNS12 were somewhat lower on day 3 and decreased further by day 6; in addition, these viruses induced a more circumscribed set of cytokines/chemokines (IFN, interleukin-6 [IL-6], and CXCL10) that were detected on day 3 and had largely subsided by day 6. Lung immunohistology revealed abundant PVM-positive pneumocytes and bronchial and bronchiolar epithelial cells in wt PVM- and ΔNS1-infected mice on day 6 compared to few PVM-positive foci with ΔNS2 and ΔNS12. These results indicate that severe PVM disease is associated with high, poorly controlled virus replication driving the expression of high levels of pulmonary IFN and a broad array of cytokines/chemokines. In contrast, in the absence of NS2, there was an early, transient innate response involving moderate levels of IFN, IL-6, and CXCL10 that restricted virus replication and prevented disease.
PMCID: PMC2643780  PMID: 19052095
21.  Th1, Th2 and Th17 Effector T Cell-Induced Autoimmune Gastritis Differs in Pathological Pattern and in Susceptibility to Suppression by Regulatory T Cells 
Th cells can be subdivided into IFNγ-secreting Th1, IL-4/IL-5 secreting Th2, and IL-17 secreting Th17 cells. We have evaluated the capacity of fully differentiated Th1, Th2, and Th17 cells derived from a mouse bearing a transgenic TCR specific for the gastric parietal cell antigen, H/K ATPase, to induce autoimmune gastritis after transfer to immunodeficient recipients. We have also determined the susceptibility of the disease induced by each of the effector T cell types to suppression by polyclonal regulatory T cells (Treg) in vivo. Each type of effector cell induced autoimmune gastritis with distinct histological patterns. Th17 cells induced the most destructive disease with cellular infiltrates composed primarily of eosinophils accompanied by high levels of serum IgE. Polyclonal Treg could suppress the capacity of Th1 cells, moderately suppress Th2 cells, but could only suppress Th17 induced disease at early time points. The major effect of the Treg was to inhibit the expansion of the effector T cells. However, effector cells isolated from protected animals were not anergic and were fully competent to proliferate and produce effector cytokines ex vivo. The strong inhibitory effect of polyclonal Treg on the capacity of some types of differentiated effector cells to induce disease provides an experimental basis for the clinical use of polyclonal Treg in the treatment of autoimmune disease in man.
PMCID: PMC2561289  PMID: 18641328
Rodent; Th1/Th2 cells; autoimmunity; tolerance/suppression/anergy
22.  A paramyxovirus-vectored intranasal vaccine against Ebola virus is immunogenic in vector-immune animals 
Virology  2008;377(2):255-264.
Ebola virus (EBOV) causes outbreaks of a highly lethal hemorrhagic fever in humans. The virus can be transmitted by direct contact as well as by aerosol and is considered a potential bioweapon. Because direct immunization of the respiratory tract should be particularly effective against infection of mucosal surfaces, we previously developed an intranasal vaccine based on replication-competent human parainfluenza virus type 3 (HPIV3) expressing EBOV glycoprotein GP (HPIV3/EboGP) and showed that it is immunogenic and protective against a high dose parenteral EBOV challenge. However, because the adult human population has considerable immunity to HPIV3, which is a common human pathogen, replication and immunogenicity of the vaccine in this population might be greatly restricted. Indeed, in the present study, replication of the vaccine in the respiratory tract of HPIV3-immune guinea pigs was found to be restricted to undetectable levels. This restriction appeared to be based on both neutralizing antibodies and cellular or other components of the immunity to HPIV3. Surprisingly, even though replication of HPIV3/EboGP was highly restricted in HPIV3-immune animals, it induced a high level of EBOV-specific antibodies that nearly equaled that obtained in HPIV3-naïve animals. We also show that the previously demonstrated presence of functional GP in the vector particle was not associated with increased replication in the respiratory tract nor with spread beyond the respiratory tract of HPIV3-naive guinea pigs, indicating that expression and functional incorporation of the attachment/penetration glycoprotein of this systemic virus did not mediate a change in tissue tropism.
PMCID: PMC2519172  PMID: 18570964
Ebola; virus; vaccine; immunogenicity; respiratory; immunization; antibody
23.  Control of TLR7 expression is essential to restrict autoimmunity and dendritic cell expansion 
Immunity  2007;27(5):801-810.
Nucleic acid-binding innate immune receptors such as TLR7 and TLR9 have been implicated in the development of some autoimmune pathologies. The Y chromosome-linked genomic modifier Yaa, which correlates with a duplication of TLR7 and 16 other genes, exacerbates lupus-like syndromes in several mouse strains. Here we demonstrate that duplication of the TLR7 gene is the sole requirement for this accelerated autoimmunity, as reduction of TLR7 gene dosage abolishes the Yaa phenotype. Further, we describe new transgenic lines that overexpress TLR7 alone, and find that at levels beyond a 2-fold increase, spontaneous autoimmunity develops. While a modest increase in TLR7 gene dosage promotes autoreactive lymphocytes with RNA specificities and myeloid cell expansion, a substantial increase in TLR7 expression causes fatal acute inflammatory pathology and profound dendritic cell dysregulation. These results underscore the importance of tightly regulating expression of TLR7 to prevent spontaneous triggering of harmful autoreactive and inflammatory responses.
PMCID: PMC2706502  PMID: 17997333
24.  TGFβ-induced FoxP3+ Regulatory T Cells Rescue Scurfy Mice 
European journal of immunology  2008;38(7):1814-1821.
Scurfy mice have a deletion in the forkhead domain of Foxp3, fail to develop thymic-derived Foxp3+ regulatory T cells (nTreg), and develop a fatal lymphoproliferative syndrome with multi-organ inflammation. Transfer of thymic-derived Foxp3+ nTreg into neonatal Scurfy mice prevents the development of disease. Stimulation of conventional CD4+Foxp3− via the TCR in the presence of TGFβ and IL-2 induces the expression of Foxp3 and an anergic/suppressive phenotype. To determine whether the TGFβ-induced Treg (iTR) were capable of suppressing disease in the Scurfy mouse, we reconstituted newborn Scurfy mice with polyclonal iTR. iTR-treated Scurfy mice do not show any signs of disease and have drastically reduced cell numbers in peripheral lymph nodes and spleen in comparison to untreated Scurfy controls. The iTR retained their expression of FoxP3 in vivo for 21 days, migrated into the skin, and prevented the development of inflammation in skin, liver and lung. Thus, TGFβ-differentiated Foxp3+ Treg appear to possess all of the functional properties of thymic-derived nTreg and represent a potent population for the cellular immunotherapy of autoimmune and inflammatory diseases.
PMCID: PMC2574868  PMID: 18546144
TGFβ; regulatory T cells; Scurfy mice; autoimmune disease
25.  The Secreted Form of Respiratory Syncytial Virus G Glycoprotein Helps the Virus Evade Antibody-Mediated Restriction of Replication by Acting as an Antigen Decoy and through Effects on Fc Receptor-Bearing Leukocytes▿  
Journal of Virology  2008;82(24):12191-12204.
Respiratory syncytial virus (RSV) readily infects and reinfects during infancy and throughout life, despite maternal antibodies and immunity from prior infection and without the need for significant antigenic change. RSV has two neutralization antigens, the F and G virion glycoproteins. G is expressed in both membrane-bound (mG) and secreted (sG) forms. We investigated whether sG might act as a decoy for neutralizing antibodies by comparing the in vitro neutralization of wild-type (wt) RSV versus recombinant mG RSV expressing only mG. wt RSV indeed was less susceptible than mG RSV to monovalent G-specific and polyvalent RSV-specific antibodies, whereas susceptibility to F-specific antibodies was equivalent. This difference disappeared when the virus preparations were purified to remove sG. Thus, sG appears to function as a neutralization decoy. We evaluated this effect in vivo in mice by comparing the effects of passively transferred antibodies on the pulmonary replication of wt RSV versus mG RSV. Again, wt RSV was less sensitive than mG RSV to G-specific and RSV-specific antibodies; however, a similar difference was also observed with F-specific antibodies. This confirmed that sG helps wt RSV evade the antibody-dependent restriction of replication but indicated that in mice, it is not acting primarily as a decoy for G-specific antibodies, perhaps because sG is produced in insufficient quantities in this poorly permissive animal. Rather, we found that the greater sensitivity of mG versus wt RSV to the antiviral effect of passively transferred RSV antibodies required the presence of inflammatory cells in the lung and was Fcγ receptor dependent. Thus, sG helps RSV escape the antibody-dependent restriction of replication via effects as an antigen decoy and as a modulator of leukocytes bearing Fcγ receptors.
PMCID: PMC2593351  PMID: 18842713

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