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ABSTRACT: Mouse parvoviruses (MPVs) are small, single-stranded, 5 Kb DNA viruses that can be subclinical and endemic in many laboratory mouse colonies. MPVs have more distinctive deleterious effects on immune-compromised or genetically-engineered mice than immuno-competent mice. At the University of Louisville (U of L), there was a sudden increase of MPV sero-positivity for MPV infections in mouse colonies between January 2006 and February 2007, resulting in strategic changes aimed at controlling MPV spread throughout the animal facility. To investigate these MPVs, VP2 genes of seven MPVs were cloned and sequenced from eight documented incidences by PCR technology. The mutations in these VP2 genes were compared to those found at the Genbank database (NCBI; http://www.ncbi.nlm.nih.gov) and an intra-institutional phylogenetic tree for MPV infections at U of L was constructed. We discovered that the seven MPV isolates were different from those in Genbank and were not identical to each other. These MPVs were designated MPV-UL1 to 7; none of them were minute virus of mice parvoviruses (MVMs). Four isolates could be classified as MPV1, one was classified as MPV2, and two were defined as novel types with less than 96% and 94% homology with existing MPV types. Considering that all seven isolates had mutations in their VP2 genes and no mutations were observed in VP2 genes of MPV during a four-month time period, we concluded that all seven MPVs isolated at U of L between 2006 and 2007 probably originated from different sources. Serological survey for MPV infections verified that each MPV outbreak was controlled without further contamination within the institution.
Experimental and Molecular Pathology 03/2013; · 2.42 Impact Factor
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ABSTRACT: A number of single gene mutations in laboratory mice produce hair follicle defects resulting in deformed hair shafts. The radiation-induced (SB/LeJ-Foxq1(sa) ) satin mutant mice have a satin-like sheen to their hair and dilute colouration. This sheen is due to failure of the hair shafts to develop normal medullas, while the pigment dilution is due to the unrelated beige (lysosomal trafficking regulator, Lyst(bg) ) mutation. A new allelic mutation, Foxq1(sa-J) , arose spontaneously on the albino (tyrosinase, Tyr(c) ) MRL/MpJ-Fas(lpr) background. The Foxq1(sa-J) allele has a C to T transition at position 490. By contrast, the Foxq1(sa) mutant allele was confirmed to be a 67 base pair deletion followed by two base changes (GA to AT). Morphologic changes were similar to those seen in Hoxc13 transgenic and targeted mutant mice. This new allelic mutation provides yet another tool to investigate formation of the interior structures of hair shafts.
Experimental Dermatology 03/2013; 22(3):234-236. · 3.54 Impact Factor
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ABSTRACT: Peyer's patches (PP) are an important component in the immune response against intestinal pathogens. Two independent, spontaneous mutations in the mouse Sharpin gene (Sharpin(cpdm) and Sharpin(cpdm-Dem)) result in the absence of PP and disrupted splenic white pulp in adult mice, although a full complement of lymph nodes is present. Here we report that rudimentary PP begin to develop in Sharpin(cpdm) mice during embryogenesis, but lack the organizational patterns that are typical of this tissue. In the present study, small intestines examined at weekly intervals from birth to maturity showed spontaneous regression of PP in mutant mice with concurrent infiltration of granulocytes. At 5 to 6 weeks of age, only indistinct remnants of granulocytic accumulations remain. Transplantation of normal bone marrow into Sharpin(cpdm) mice at 7 days of age did not prevent regression of PP in bone marrow chimeras examined at 7 to 8 weeks of age. These findings indicate that SHARPIN expression is required for the normal development and maintenance, but not initiation, of PP.
PLoS ONE 01/2013; 8(2):e55224. · 4.09 Impact Factor
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ABSTRACT: C57BL/6 mice develop dermatitis and scarring alopecia resembling human cicatricial alopecias (CAs), particularly the central centrifugal CA (CCCA) type. To evaluate the role of retinoids in CA, the expression of retinoid metabolism components were examined in these mice with mild, moderate, or severe CA compared with hair cycle-matched mice with no disease. Two feeding studies were conducted with dams fed either NIH 31 diet (study 1) or AIN93G diet (study 2). Adult mice were fed AIN93M diet with 4 (recommended), 28, or 56 IU vitamin A g(-1) diet. Feeding the AIN93M diet to adults increased CA frequency over NIH 31 fed mice. Increased follicular dystrophy was seen in study 1 and increased dermal scars in study 2 in mice fed the 28 IU diet. These results indicate that retinoid metabolism is altered in CA in C57BL/6J mice that require precise levels of dietary vitamin A. Human patients with CCCA, pseudopelade (end-stage scarring), and controls with no alopecia were also studied. Many retinoid metabolism proteins were increased in mild CCCA, but were undetectable in pseudopelade. Studies to determine whether these dietary alterations in retinoid metabolism seen in C57BL/6J mice are also involved in different types of human CA are needed.Journal of Investigative Dermatology advance online publication, 25 October 2012; doi:10.1038/jid.2012.393.
Journal of Investigative Dermatology 10/2012; · 6.31 Impact Factor
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ABSTRACT: Lung cancer is the most common cause of cancer-related deaths in both men and women, and effective preventatives are rare due to the difficulty of early detection. Specific gene expression signatures have been identified in individuals that already developed lung cancer. To identify if gene expression differences could be detected in individuals before the onset of the disease, we obtained lung tissues for microarray analysis from young, healthy mice of 9 inbred strains with known differences in their susceptibility to spontaneous pulmonary adenomas when aged. We found that the most common differentially expressed genes among all possible 36 strain comparisons showed significant associations with cancer- and inflammation-related processes. Significant expression differences between susceptible and resistant strains were detected for Aldh3a1, Cxcr1 and 7, Dpt, and Nptx1-genes with known cancer-related functions, and Cd209, Cxcr1 and 7, and Plag2g1b-genes with known inflammatory-related functions. Whereas Aldh3a1, Cd209, Dpt, and Pla2g1b had increased expression, Cxcr1 and 7, and Nptx1 had decreased expression in strains susceptible to pulmonary adenomas. Thus, our study shows that expression differences between susceptible and resistant strains can be detected in young and healthy mice without manifestation of pulmonary adenomas and, thus, may provide an opportunity of early detection. Finally, the identified genes have previously been reported for human non-small cell lung cancer suggesting that molecular pathways may be shared between these two cancer types.
Experimental and Molecular Pathology 09/2012; · 2.42 Impact Factor
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Journal of Investigative Dermatology 09/2012; · 6.31 Impact Factor
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F Jason Duncan,
Kathleen A Silva,
Charles J Johnson,
Benjamin L King,
Jin P Szatkiewicz,
Sonya P Kamdar,
David E Ong,
Joseph L Napoli,
Jinshan Wang,
Lloyd E King,
David A Whiting,
Kevin J McElwee, John P Sundberg,
Helen B Everts
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ABSTRACT: Alopecia areata (AA) is an autoimmune disease that attacks anagen hair follicles. Gene array in graft-induced C3H/HeJ mice revealed that genes involved in retinoic acid (RA) synthesis were increased, whereas RA degradation genes were decreased in AA compared with sham controls. This was confirmed by immunohistochemistry in biopsies from patients with AA and both mouse and rat AA models. RA levels were also increased in C3H/HeJ mice with AA. C3H/HeJ mice were fed a purified diet containing one of the four levels of dietary vitamin A or an unpurified diet 2 weeks before grafting and disease progression followed. High vitamin A accelerated AA, whereas mice that were not fed vitamin A had more severe disease by the end of the study. More hair follicles were in anagen in mice fed high vitamin A. Both the number and localization of granzyme B-positive cells were altered by vitamin A. IFNγ was also the lowest and IL13 highest in mice fed high vitamin A. Other cytokines were reduced and chemokines increased as the disease progressed, but no additional effects of vitamin A were seen. Combined, these results suggest that vitamin A regulates both the hair cycle and immune response to alter the progression of AA.Journal of Investigative Dermatology advance online publication, 27 September 2012; doi:10.1038/jid.2012.344.
Journal of Investigative Dermatology 09/2012; · 6.31 Impact Factor
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ABSTRACT: MusPV, a novel papillomavirus (PV) that naturally infects laboratory mice, was isolated and characterized from a colony of NMRI-Foxn1(nu)/Foxn1(nu) (nude) mice in India. Because MusPV may have been missed during routine pathogen screening of mice in colonies worldwide, a variety of detection methods are described to detect MusPV. The clinical and histologic lesions of productive MusPV infections fit PV-associated features, including papillomas, koilocytes within the stratum granulosum of the hyperplastic/acanthotic papillomatous epithelium, and the presence of intranuclear virus particles in koilocytotic cells is visualized by electron microscopy. Antiserum against disrupted PV virions, isolated from another species (canine), identified conserved viral antigens in productively infected cells by immunohistochemistry. A rolling circle technique was used to amplify viral circular DNAs followed by endonuclease restriction enzyme digestion to determine the correct size of PV DNA. Consensus PV degenerative primers, My09/11, commonly used to detect many different types of PVs by polymerase chain reaction (PCR), particularly mucosotropic HPVs and also identified MusPV and all rodent PVs tested. Since there was one nucleotide mismatch between the My09/11 primer set and MusPV template, a new primer set, MusPV-My09/11, was designed to specifically detect MusPV in latent infections and spontaneous MusPV-induced papillomas. Southern blot analysis verified the presence of full size PV DNA in infected tissues. Virus-like particles (VLPs), generated from MusPV L1 genes, provided a substrate for serological testing of naturally and experimentally infected mice. In summary, a series of diagnostic assays were developed and validated to detect MusPV infection in skin tumors and serological response in laboratory mice.
Experimental and Molecular Pathology 07/2012; · 2.42 Impact Factor
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ABSTRACT: Mice with spontaneous mutations in the Sharpin gene develop chronic proliferative dermatitis that is characterized by eosinophilic inflammation of the skin and other organs with increased expression of type 2 cytokines and dysregulated development of lymphoid tissues. The mutant mice share phenotypic features with human hypereosinophilic syndromes. The biological function of SHARPIN and how its absence leads to such a complex inflammatory phenotype in mice are poorly understood. However, recent studies identified SHARPIN as a novel modulator of immune and inflammatory responses. The emerging mechanistic model suggests that SHARPIN functions as an important adaptor component of the linear ubiquitin chain assembly complex that modulates activation of NF-κB signalling pathway, thereby regulating cell survival and apoptosis, cytokine production and development of lymphoid tissues. In this review, we will summarize the current understanding of the ubiquitin-dependent regulatory mechanisms involved in NF-κB signalling, and incorporate the recently obtained molecular insights of SHARPIN into this pathway. Recent studies identified SHARPIN as an inhibitor of β1-integrin activation and signalling, and this may be another mechanism by which SHARPIN regulates inflammation. Furthermore, the disrupted lymphoid organogenesis in SHARPIN-deficient mice suggests that SHARPIN-mediated NF-κB regulation is important for de novo development of lymphoid tissues.
Journal of Cellular and Molecular Medicine 03/2012; 16(10):2271-9. · 4.13 Impact Factor
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Toxicologic Pathology 03/2012; 40(5):823-5. · 1.91 Impact Factor
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ABSTRACT: Epidermolysis bullosa (EB) describes a spectrum of rare, incurable, inherited mechanobullous disorders unified by the fact that they are caused by structural defects in the basement membrane zone which disrupt adhesion between the epidermis and dermis. Mouse models provide valuable tools to define the molecular basis of these diseases and to test novel therapeutic approaches. There is need for rapid, quantitative tests that measure the integrity of dermal-epidermal adhesions in such models. To address this need, we describe a novel quantitative method to determine the mechanical strength of the adhesion between tail skin epidermis and dermis. We show that this test reliably measures the force required to cause dermal-epidermal separation in tails of mice that are genetically predisposed to a form of non-Herlitz Junctional EB which develops as the result of a hypomorphic mutation in the laminin gamma 2 gene (Lamc2(jeb) ). This simple, quantitative method of directly measuring the tensile strength of dermal-epidermal adhesion provides a novel dimension to the pathophysiological screening, evaluation, and therapeutic treatment of mice that may develop progressive forms of EB and potentially other disorders that compromise cutaneous integrity.
Experimental Dermatology 03/2012; 21(6):453-5. · 3.54 Impact Factor
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Journal of Investigative Dermatology 02/2012; 132(6):1736-8. · 6.31 Impact Factor
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ABSTRACT: Spontaneous mutations of the Sharpin (SHANK-associated RH domain-interacting protein, other aliases: Rbckl1, Sipl1) gene in mice result in systemic inflammation that is characterized by chronic proliferative dermatitis and dysregulated secretion of T helper1 (Th1) and Th2 cytokines. The cellular and molecular mechanisms underlying this inflammatory phenotype remain elusive. Dendritic cells may contribute to the initiation and progression of the phenotype of SHARPIN-deficient mice because of their pivotal role in innate and adaptive immunity. Here we show by flow cytometry that SHARPIN- deficiency did not alter the distribution of different DC subtypes in the spleen. In response to TOLL-like receptor (TLR) agonists LPS and poly I:C, cultured bone marrow-derived dendritic cells (BMDC) from WT and mutant mice exhibited similar increases in expression of co-stimulatory molecules CD40, CD80, and CD86. However, stimulated SHARPIN-deficient BMDC had reduced transcription and secretion of pro-inflammatory mediators IL6, IL12P70, GMCSF, and nitric oxide. Mutant BMDC had defective activation of NF-κB signaling, whereas the MAPK1/3 (ERK1/2) and MAPK11/12/13/14 (p38 MAP kinase isoforms) and TBK1 signaling pathways were intact. A mixed lymphocyte reaction showed that mutant BMDC only induced a weak Th1 immune response but stimulated increased Th2 cytokine production from allogeneic naïve CD4(+) T cells. In conclusion, loss of Sharpin in mice significantly affects the immune function of DC and this may partially account for the systemic inflammation and Th2-biased immune response.
PLoS ONE 01/2012; 7(2):e31809. · 4.09 Impact Factor
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ABSTRACT: Mutant laboratory mice with distinctive hair phenotypes are useful for identifying genes responsible for hair diseases. The work presented here demonstrates that shotgun proteomic profiling can distinguish hair shafts from different inbred mouse strains. For this purpose, analyzing the total hair shaft provided better discrimination than analyzing the isolated solubilized and particulate (cross-linked) fractions. Over 100 proteins exhibited significant differences among the 11 strains and 5 mutant stocks across the wide spectrum of strains surveyed. Effects on the profile of single gene mutations causing hair shaft defects were profound. Since the hair shaft provides a discrete sampling of the species proteome, with constituents serving important functions in epidermal appendages and throughout the body, this work provides a foundation for non-invasive diagnosis of genetic diseases of hair and perhaps other tissues.
PLoS ONE 01/2012; 7(12):e51956. · 4.09 Impact Factor
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ABSTRACT: Three methods are currently available to reconstitute hair follicles in mice. Direct comparisons have yet to be made to determine which method is most efficient. In this study, mouse epithelial cells (MECs) and mouse dermal cells (MDCs) were grafted onto the dorsal skin of nude mice using the chamber, flap or patch assays. Comparisons were made based on gross, scanning electron microscopic and histological observations. MDCs alone induced hair follicle reconstitution with the production of hairs yielding false-positive results caused by contamination by hair follicle remnants. Neither primary MECs nor cultured MDCs alone formed hair follicles but did result in hair follicle formation when mixed together. Frozen MECs or MDCs resulted in decreased hair follicle-inductive activity but could still regenerate hairs. The hair patch assay was the quickest model (20±3 days) to determine whether cell mixtures would reconstitute hair follicles that produce hairs; however, the hair follicles were randomly orientated and often associated with foreign body granulomas. The flap assay took the longest time (29±2 days) to produce follicles and hairs to develop with a clinically natural appearance, but an epidermal sheet was needed. The chamber assay was the most labour-intensive and cell number-dependent procedure but follicles developed in a dense, clinically normal manner.
Experimental Dermatology 12/2011; 20(12):1011-5. · 3.54 Impact Factor
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ABSTRACT: Genetic influences that underlie spontaneous lung oncogenesis are poorly understood. The objective of this study was to determine the genetic influences on spontaneous pulmonary adenoma frequency and severity in 28 strains of mice as part of a large-scale aging study conducted at the Jackson Aging Center (http://agingmice.jax.org/). Genome-wide association studies were conducted in these strains with both low-density (132,000) and high-density (4,000,000) panel of single-nucleotide polymorphisms (SNP). Our analysis revealed that adenomas were relatively less frequent and less severe in females than males, and that loci implicated in frequency and severity were often different between male and female mice. While some of the significant loci identified mapped to genomic locations known to be responsible for carcinogen-induced cancers (e.g., Pas1), others were unique to our study. In particular, Fat4 was influential in males and Tsc22d1 was influential in females. SNPs implicated were predicted to alter amino acid sequence and change protein function. In summary, our results suggested that genetic influences that underlie pulmonary adenoma frequency are dependent on gender, and that Fat4 and Tsc22d1 are likely candidate genes to influence formation of spontaneous pulmonary adenoma in aging male and female mice, respectively.
Cancer Research 07/2011; 71(17):5779-91. · 7.86 Impact Factor
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Journal of Investigative Dermatology 07/2011; 131(11):2323-4. · 6.31 Impact Factor
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Journal of Investigative Dermatology 05/2011; 131(9):1936-8. · 6.31 Impact Factor
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ABSTRACT: The chronic proliferative dermatitis mutation (CPDM) in mice, due to Sharpin deficiency (Sharpin(cpdm)), is a multisystem disorder characterized by peripheral blood eosinophilia and eosinophil infiltration of affected tissues including the skin, bone marrow, spleen, lung, heart, and other organs. The epidermis has numerous apoptotic keratinocytes which increase with age, coalesce, form vesicles, and rupture causing ulceration.
To clarify the molecular pathways involved in the keratinocyte apoptosis caused by loss of function of SHARPIN in mice.
10-week-old Sharpin(cpdm) and wildtype mice were used for experiments. Ultrastructural changes of skin were evaluated by transmission electron microscopy. Cross points of mitochondrial pathway were analyzed by in vitro and in vivo cellular and molecular assays.
77.5% skin cells in Sharpin(cpdm) mice were functionally apoptotic and dead cells, compared to only 18.1% unhealthy skin cells in wildtype mice, indicated by annexin-V/propidium iodide FACS analysis. Mitochondria in keratinocytes were disrupted containing prominent electron dense inclusions and membrane potential depolarization, accompanied by a shift in protein expression between the anti-apoptotic BCL2 and pro-apoptotic BAX proteins. Enzymatic activities of caspases 9 and 3, but not 8, were markedly increased in Sharpin(cpdm) keratinocytes. Caspase-3 was cleaved in most cells in skin of 10-week-old mutant mice.
The present results indicated that keratinocyte apoptosis in Sharpin(cpdm) mice was regulated by an intrinsic caspase-dependent mitochondria pathway.
Journal of dermatological science 05/2011; 63(3):148-53. · 3.71 Impact Factor
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ABSTRACT: The NF-κB pathway performs pivotal roles in diverse physiological processes such as immunity, inflammation, proliferation, and apoptosis. NF-κB is kept inactive in the cytoplasm through association with inhibitors (IκB), and translocates to the nucleus to activate its target genes after the IκBs are phosphorylated and degraded. Here, we demonstrate that loss of function of SHANK-associated RH domain interacting protein (SHARPIN) leads to activation of NF-κB signaling in skin, resulting in the development of an idiopathic hypereosinophilic syndrome (IHES) with eosinophilic dermatitis in C57BL/KaLawRij-Sharpin(cpdm)/RijSunJ mice, and clonal expansion of B-1 B cells and CD3(+)CD4(-)CD8(-) T cells. Transcription profiling in skin revealed constitutive activation of classical NF-κB pathways, predominantly by overexpressed members of IL1 family. Compound-null mutants for both the IL1 receptor accessory protein (Il1rap(tm1Roml)) and SHARPIN (Sharpin(cpdm)) resulted in mice having decreased skin disease severity. Inhibition of IκBA degradation by the proteasome inhibitor bortezomib alleviated the dermatitis in Sharpin(cpdm) mice. These results indicate that absence of SHARPIN causes IHES with eosinophilic dermatitis by NF-κB activation, and bortezomib may be an effective treatment for skin problems of IHES.
Journal of Investigative Dermatology 01/2011; 131(1):141-9. · 6.31 Impact Factor
