David G Besselsen

Interests

Research

Rodent pathology; rodent viral disease; diagnostic test development

Teaching

Laboratory animal pathology

Courses

No activities entered.

Scholarly Contributions

Chapters

• Besselsen, D. G. (1998). Detection of Rodent Parvoviruses by PCR. In PCR in Bioanalysis(pp 31-37). Humana Press. doi:10.1385/0-89603-497-6:31
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  The rodent parvoviruses include mouse parvovirus (MPV), minute virus of mice (MVM), H-1 parvovirus (H-1), Kilham rat virus (KRV), rat parvovirus (RPV), and hamster parvovirus (HaPV). Recent reports suggest LuIII, an autonomous parvovirus of unknown host origin, may also belong to the rodent parvovirus group (,). In general, rodent parvoviruses cause subclinical infections in their host of origin, although naturally occurring clinical disease may occasionally occur secondary to KRV or HaPV infection (,). Probably of more importance to the scientific community are the potential adverse effects these agents may have on research that utilizes rodents with inapparent and undetected infections. Subclinical rodent parvovirus infections may alter the immune response or cause fetal resorption or oncosuppression (). In addition, rodent parvoviruses can cause persistent infections in cell culture and transplantable tumor lines (,), and therefore can adversely affect experiments performed in vitro.

Journals/Publications

• Laubitz, D., Besselsen, D. G., Kiela, P. R., Ghishan, F. K., Chen, H., Clutter, C., Midura-Kiela, M. T., & Gurney, M. A. (2022). Decreased NHE3 expression in colon cancer is associated with DNA damage, increased inflammation and tumor growth. Scientific Reports. doi:10.1038/s41598-022-19091-x
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  Abstract Dysregulation of intra- and extracellular pH in cancer contributes to extracellular matrix remodeling, favors cell migration, proliferation, and metastasis. Although the primary attention has been focused on the role of the ubiquitous Na + /H + exchanger isoform NHE1, the role of NHE3, the predominant apical isoform in colonic surface epithelium in the pathogenesis of colon cancer has not been investigated. Here, we show that NHE3 mRNA expression is significantly reduced in colorectal cancer patients and that low NHE3 expression is associated with poorer survival. Deletion of NHE3 in Apc Min mice evaluated at 15 weeks of age (significant mortality was observed beyond this time) led to lower body weights, increased mucosal inflammation, increased colonic tumor numbers, evidence of enhanced DNA damage in tumor surface epithelium, and to significant alteration in the gut microbiota. In the absence of the inflammatory and microbial pressors, ca. 70% knockdown of NHE3 expression in SK-CO15 cells led to reduced intracellular pH, elevated apical pH, dramatic differences in their transcriptomic profile, increased susceptibility to DNA damage, increased proliferation, decreased apoptosis and reduced adhesion to extracellular matrix proteins. Our findings suggest that loss of NHE3 in the surface epithelium of colonic tumors has profound consequences for cancer progression and behavior.
• Pryor, B. M., Besselsen, D. G., Chen, Y., Luo, Q., Liu, Y., Cunningham, A., Pereira, R. S., & Daines, M. O. (2021). Novel Mouse Models of Fungal Asthma. Frontiers in Cellular and Infection Microbiology. doi:10.3389/fcimb.2021.683194
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  Alternaria alternata is a ubiquitous fungus and a major allergen associated with the development of asthma. Inhalation of intact spores is the primary cause of human exposure to fungal allergen. However, allergen-rich cultured fungal filtrates are oftentimes used in the current models of fungal sensitization that do not fully reflect real-life exposures. Thus, establishing novel spore exposure models is imperative. In this study, we established novel fungal exposure models of both adult and neonate to live spores. We examined pathophysiological changes in the spore models as compared to the non-exposure controls and also to the conventional filtrate models. While both Alternaria filtrate- and spore-exposed adult BALB/c mice developed elevated airway hyperresponsiveness (AHR), filtrates induced a greater IgE mediated response and higher broncholavage eosinophils than spores. In contrast, the mice exposed to Alternaria spores had higher numbers of neutrophils. Both exposures induced comparable levels of lung tissue inflammation and mucous cell metaplasia (MCM). In the neonatal model, exposure to Alternaria spores resulted in a significant increase of AHR in both adult and neonatal mice. Increased levels of IgE in both neonatal and adult mice exposed to spores was associated with increased eosinophilia in the treatment groups. Adult demonstrated increased numbers of lymphocytes that was paralleled by increased IgG1 production. Both adults and neonates demonstrated similarly increased eosinophilia, IgE, tissue inflammation and MCM.
• Smithey, M. J., Uhrlaub, J. L., Smithey, M. J., Nikolich-zugich, J., Jergovic, M., Coplen, C. P., Cheng, S., & Besselsen, D. G. (2021). Infection-induced type I interferons critically modulate the homeostasis and function of CD8+ naïve T cells.. Nature communications, 12(1), 5303. doi:10.1038/s41467-021-25645-w
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  Naïve T (Tn) cells require two homeostatic signals for long-term survival: tonic T cell receptor:self-peptide-MHC contact and IL-7 stimulation. However, how microbial exposure impacts Tn homeostasis is still unclear. Here we show that infections can lead to the expansion of a subpopulation of long-lived, Ly6C+ CD8+ Tn cells with accelerated effector function. Mechanistically, mono-infection with West Nile virus transiently, and polymicrobial exposure persistently, enhances Ly6C expression selectively on CD5hiCD8+ cells, which in the case of polyinfection translates into a numerical CD8+ Tn cell increase in the lymph nodes. This conversion and expansion of Ly6C+ Tn cells depends on IFN-I, which upregulates MHC class I expression and enhances tonic TCR signaling in differentiating Tn cells. Moreover, for Ly6C+CD8+ Tn cells, IFN-I-mediated signals optimize their homing to secondary sites, extend their lifespan, and enhance their effector differentiation and antibacterial function, particularly for low-affinity clones. Our results thus uncover significant regulation of Tn homeostasis and function via infection-driven IFN-I, with potential implications for immunotherapy.
• Bartlett, M. J., Flores, A. J., Ye, T., Smidt, S. I., Dollish, H. K., Stancati, J. A., Farrell, D. C., Parent, K. L., Besselsen, D. G., Doyle, K., Heien, M. L., Cowen, S. L., Steece-Collier, K., Sherman, S. J., & Falk, T. (2020). Preclinical evidence in support of repurposing sub-anesthetic ketamine as a treatment for L-DOPA-induced dyskinesia.. Experimental Neurology, 333, 113413. doi:https://doi.org/10.1016/j.expneurol.2020.113413
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  Parkinson's disease (PD) is the second most common neurodegenerative disease. Pharmacotherapy with L-DOPA remains the gold-standard therapy for PD, but is often limited by the development of the common side effect of L-DOPA-induced dyskinesia (LID), which can become debilitating. The only effective treatment for disabling dyskinesia is surgical therapy (neuromodulation or lesioning), therefore effective pharmacological treatment of LID is a critical unmet need. Here, we show that sub-anesthetic doses of ketamine attenuate the development of LID in a rodent model, while also having acute anti-parkinsonian activity. The long-term anti-dyskinetic effect is mediated by brain-derived neurotrophic factor-release in the striatum, followed by activation of ERK1/2 and mTOR pathway signaling. This ultimately leads to morphological changes in dendritic spines on striatal medium spiny neurons that correlate with the behavioral effects, specifically a reduction in the density of mushroom spines, a dendritic spine phenotype that shows a high correlation with LID. These molecular and cellular changes match those occurring in hippocampus and cortex after effective sub-anesthetic ketamine treatment in preclinical models of depression, and point to common mechanisms underlying the therapeutic efficacy of ketamine for these two disorders. These preclinical mechanistic studies complement current ongoing clinical testing of sub-anesthetic ketamine for the treatment of LID by our group, and provide further evidence in support of repurposing ketamine to treat individuals with PD. Given its clinically proven therapeutic benefit for both treatment-resistant depression and several pain states, very common co-morbidities in PD, sub-anesthetic ketamine could provide multiple therapeutic benefits for PD in the future.
• Falk, T., Dollish, H. K., Ye, T., Steece-collier, K., Stancati, J. A., Smidt, S. I., Sherman, S. J., Parent, K. L., Heien, M. L., Flores, A. J., Farrell, D. C., Falk, T., Doyle, K. P., Dollish, H. K., Cowen, S. L., Besselsen, D. G., Bartlett, M. J., Sherman, S. J., Falk, T., , Ye, T., et al. (2020). Preclinical evidence in support of repurposing sub-anesthetic ketamine as a treatment for L-DOPA-induced dyskinesia.. Experimental neurology, 333, 113413. doi:10.1016/j.expneurol.2020.113413
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  Parkinson's disease (PD) is the second most common neurodegenerative disease. Pharmacotherapy with L-DOPA remains the gold-standard therapy for PD, but is often limited by the development of the common side effect of L-DOPA-induced dyskinesia (LID), which can become debilitating. The only effective treatment for disabling dyskinesia is surgical therapy (neuromodulation or lesioning), therefore effective pharmacological treatment of LID is a critical unmet need. Here, we show that sub-anesthetic doses of ketamine attenuate the development of LID in a rodent model, while also having acute anti-parkinsonian activity. The long-term anti-dyskinetic effect is mediated by brain-derived neurotrophic factor-release in the striatum, followed by activation of ERK1/2 and mTOR pathway signaling. This ultimately leads to morphological changes in dendritic spines on striatal medium spiny neurons that correlate with the behavioral effects, specifically a reduction in the density of mushroom spines, a dendritic spine phenotype that shows a high correlation with LID. These molecular and cellular changes match those occurring in hippocampus and cortex after effective sub-anesthetic ketamine treatment in preclinical models of depression, and point to common mechanisms underlying the therapeutic efficacy of ketamine for these two disorders. These preclinical mechanistic studies complement current ongoing clinical testing of sub-anesthetic ketamine for the treatment of LID by our group, and provide further evidence in support of repurposing ketamine to treat individuals with PD. Given its clinically proven therapeutic benefit for both treatment-resistant depression and several pain states, very common co-morbidities in PD, sub-anesthetic ketamine could provide multiple therapeutic benefits for PD in the future.
• Lybarger, L., Wong, R., Wilson, J. M., Setty, P., Paz, V. F., Midura-kiela, M. T., Lybarger, L., Laubitz, D., Kiela, P. R., Jamwal, D. R., Harrison, C. A., Gurney, M. A., Ghishan, F. K., Cox, C. M., Bhattacharya, D., & Besselsen, D. G. (2020). Intestinal Epithelial Expression of MHCII Determines Severity of Chemical, T-Cell-Induced, and Infectious Colitis in Mice.. Gastroenterology, 159(4), 1342-1356.e6. doi:10.1053/j.gastro.2020.06.049
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  Intestinal epithelial cells (IECs) provide a barrier that separates the mucosal immune system from the luminal microbiota. IECs constitutively express low levels of major histocompatibility complex (MHC) class II proteins, which are upregulated upon exposure to interferon gamma. We investigated the effects of deleting MHCII proteins specifically in mice with infectious, dextran sodium sulfate (DSS)-, and T-cell-induced colitis..We disrupted the histocompatibility 2, class II antigen A, beta 1 gene (H2-Ab1) in IECs of C57BL/6 mice (I-AbΔIEC) or Rag1-/- mice (Rag1-/-I-AbΔIEC); we used I-AbWT mice as controls. Colitis was induced by administration of DSS, transfer of CD4+CD45RBhi T cells, or infection with Citrobacter rodentium. Colon tissues were collected and analyzed by histology, immunofluorescence, xMAP, and reverse-transcription polymerase chain reaction and organoids were generated. Microbiota (total and immunoglobulin [Ig]A-coated) in intestinal samples were analyzed by16S amplicon profiling. IgA+CD138+ plasma cells from Peyer's patches and lamina propria were analyzed by flow cytometry and IgA repertoire was determined by next-generation sequencing..Mice with IEC-specific loss of MHCII (I-AbΔIEC mice) developed less severe DSS- or T-cell transfer-induced colitis than control mice. Intestinal tissues from I-AbΔIEC mice had a lower proportion of IgA-coated bacteria compared with control mice, and a reduced luminal concentration of secretory IgA (SIgA) following infection with C rodentium. There was no significant difference in the mucosal IgA repertoire of I-AbΔIEC vs control mice, but opsonization of cultured C rodentium by SIgA isolated from I-AbΔIEC mice was 50% lower than that of SIgA from mAbWT mice. Fifty percent of I-AbΔIEC mice died after infection with C rodentium, compared with none of the control mice. We observed a transient but significant expansion of the pathogen in the feces of I-AbΔIEC mice compared with I-AbWT mice..In mice with DSS or T-cell-induced colitis, loss of MHCII from IECs reduces but does not eliminate mucosal inflammation. However, in mice with C rodentium-induced colitis, loss of MHCII reduces bacterial clearance by decreasing binding of IgA to commensal and pathogenic bacteria.
• Paz, V. R., Midura-kiela, M. T., Marati, R. V., Kiela, P. R., Jamwal, D. R., Harrison, C. A., Ghishan, F. K., & Besselsen, D. G. (2020). Total CD3 T Cells Are Necessary and Sufficient to Induce Colitis in Immunodeficient Mice With Dendritic Cell-Specific Deletion of TGFbR2: A Novel IBD Model to Study CD4 and CD8 T-Cell Interaction.. Inflammatory bowel diseases, 26(2), 229-241. doi:10.1093/ibd/izz191
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  Inflammatory bowel disease (IBD) is a multifactorial disorder, with the innate and adaptive immune cells contributing to disease initiation and progression. However, the intricate cross-talk between immune cell lineages remains incompletely understood. The role of CD8+ T cells in IBD pathogenesis has been understudied, largely due to the lack of appropriate models..We previously reported spontaneous colitis in mice with impaired TGFβ signaling due to dendritic cell-specific knockout of TGFbR2 (TGFβR2ΔDC). Here, we demonstrate that crossing TGFβR2ΔDC mice with a Rag1-/- background eliminates all symptoms of colitis and that adoptive transfer of unfractionated CD3+ splenocytes is sufficient to induce progressive colitis in Rag1-/-TGFβR2ΔDC mice..Both CD4+ and CD8+ T cells are required for the induction of colitis accompanied by activation of both T-cell lineages and DCs, increased expression of mucosal IFNγ, TNFα, IL6, IL1β, and IL12, and decreased frequencies of CD4+FoxP3+ regulatory T cells. Development of colitis required CD40L expression in CD4+ T cells, and the disease was partially ameliorated by IFNγ neutralization..This novel model provides an important tool for studying IBD pathogenesis, in particular the complex interactions among innate and adaptive immune cells in a controlled fashion, and represents a valuable tool for preclinical evaluation of novel therapeutics.
• Midura-kiela, M. T., Laubitz, D., Kiela, P. R., Jamwal, D. R., Harrison, C. A., Ghishan, F. K., & Besselsen, D. G. (2019). Sexual Dimorphism in the Response to Broad-spectrum Antibiotics During T Cell-mediated Colitis.. Journal of Crohn's & colitis, 13(1), 115-126. doi:10.1093/ecco-jcc/jjy144
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  Broad-spectrum antibiotics [Abx], including combination therapy with ciprofloxacin and metronidazole, are often prescribed during the treatment of inflammatory bowel disease [IBD] to alleviate symptoms, but with varying success. In this pilot study, we studied the effects of Abx on the course of experimental colitis, with a particular focus on sex as a determinant of the microbial and inflammatory responses..The effects of Abx were tested on colonic inflammation and microbiome in male and female Rag-/- mice, using adoptive transfer of naïve T cells to induce colitis in a short-term [2-week] and long-term [9-week] study..We observed disparities between the sexes in both the response to adoptive T cell transfer and the effects of Abx. At baseline without Abx, female mice displayed a trend toward a more severe colitis than males. In both the short- and the long-term experiments, gut microbiota of some female mice exposed to Abx showed weak, delayed, or negligible shifts. Caecum weight was significantly lower in Abx-treated females. Abx exposure favoured a quick and persistent rise in Enterococcaceae exclusively in females. Males had higher relative abundance of Lactobacillaceae following Abx exposure relative to females. Abx-treated females trended toward higher colitis scores than Abx-treated males, and towards higher levels of IL-17A, NOS2, and IL-22..Although preliminary, our results suggest a differential response to both inflammation and Abx between male and female mice, The findings may be relevant to current practice and also as the basis for further studies on the differential gender effects during long-term antibiotic exposure in IBD.
• Putnam, P. T., Zimmerman, P. E., Putnam, P. T., Gothard, K. M., Doane, C. J., & Besselsen, D. G. (2019). Silicon Foreign Body in the Cerebrum of a Rhesus Macaque.. Comparative Medicine, 68.
• Abril, E. R., Hsu, C. H., Rice, P. F., Nagle, R. B., Jones, M. S., Ignatenko, N. A., Hsu, C. H., Ehrichs, K. G., Chen, H., Besselsen, D. G., Barton, J. K., & Abril, E. R. (2018). Does Mutated K-RAS Oncogene Attenuate the Effect of Sulindac in Colon Cancer Chemoprevention?. Cancer prevention research (Philadelphia, Pa.), 11(1), 16-26. doi:10.1158/1940-6207.capr-17-0230
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  The NSAID sulindac has been successfully used alone or in combination with other agents to suppress colon tumorigenesis in patients with genetic predisposition and also showed its efficacy in prevention of sporadic colon adenomas. At the same time, some experimental and clinical reports suggest that a mutant K-RAS oncogene may negate sulindac antitumor efficacy. To directly assess sulindac activity at suppressing premalignant lesions carrying K-RAS mutation, we utilized a novel mouse model with an inducible colon-specific expression of the mutant K-ras oncogene (K-rasG12D ). Tumor development and treatment effects were monitored by minimally invasive endoscopic Optical coherence tomography. Expression of the mutant K-ras allele accelerated azoxymethane (AOM)-induced colon carcinogenesis in C57BL/6 mice, a strain otherwise resistant to this carcinogen. Sulindac completely prevented AOM-induced tumor formation in K-ras wild-type (K-ras wt) animals. In K-rasG12D -mutant mice, a 38% reduction in tumor number, an 83% reduction in tumor volume (P ≤ 0.01) and an increase in the number of adenoma-free mice (P = 0.04) were observed. The partial response of K-RasG12D animals to sulindac treatment was evident by the decrease in mucosal thickness (P < 0.01) and delay in progression of the precancerous aberrant crypt foci to adenomas. Molecular analyses showed significant induction in cyclooxygenase 2 (COX-2), cleaved caspase-3 (CC3), and Ki-67 expression by AOM, but not sulindac treatment, in all genotypes. Our data underscore the importance of screening for K-RAS mutations in individuals with colon polyps to provide more personalized interventions targeting mutant K-RAS signaling pathways. Cancer Prev Res; 11(1); 16-26. ©2017 AACR.
• Besselsen, D. G., Patil, K., Doane, C. J., Hoffman, E. A., Stokes, J., & Katsanis, E. (2018). Supernumerary Incisors in CB6F1 Mice Conditioned with Chemotherapy and Total Body Irradiation before Bone Marrow Transplantation. Comparative Medicine, 68(5), 349-352. doi:10.30802/aalas-cm-18-000043
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  Multiple adult female CB6F1 mice presented with supernumerary incisors after preconditioning with chemotherapy and total body irradiation for bone marrow transplantation (BMT). Mice received nonmyeloablative total body irradiation (3 Gy) and either cyclophosphamide or bendamustine, followed by BMT and posttransplantation cyclophosphamide or bendamustine. Here we describe the clinical presentation, μCT findings, and histopathologic evaluation of the affected mice. These analyses confirmed the gross diagnosis and revealed details of the abnormal tooth morphology. We surmise that the combination of total body irradiation and chemotherapy resulted in the abnormal formation of supernumerary incisors. Supernumerary teeth should be considered as a potential confounding factor in tracking weight loss after BMT. These conditions can be managed to allow animals to reach their intended scientific endpoint.
• Besselsen, D. G., Patil, K., Stokes, J., Hoffman, E., & Doane, C. J. (2017). Supernumary Incisors Associated with Administration of Chemotherapeutic Agents in CB6F1 Mice. Journal of the American Association for Laboratory Animal Science.
• Besselsen, D. G., Patil, K., Stokes, J., Hoffman, E., & Doane, C. J. (2018). Supernumerary Incisors in Bone Marrow Transplanted CB6F1 Mice Conditioned with Chemotherapy and Total Body Irradiation. Comparative Medicine, 68(5), 349-352.
• Doane, C. J., Zimmerman, P. E., Putnam, P. T., Gothard, K. M., & Besselsen, D. G. (2018). Silicon foreign body in the cerebrum of a rhesus macaque (Macaca mulatta). Comparative Medicine, 68(2), 1-5.
• Kiela, P. R., Jobin, C., Ghishan, F. K., Jamwal, D. R., Besselsen, D. G., Patil, K., Midura-Kiela, M. T., Ohland, C. L., Lubitz, D., & Harrison, C. A. (2017). Microbial Dysbiosis Associated with Impaired Intestinal Na+/H+ Exchange Accelerates and Exacerbates Colitis in Gnotobiotic Mice. Mucosal Immunology.
• Kiela, P. R., Jobin, C., Ghishan, F. K., Jamwal, D. R., Besselsen, D. G., Patil, K., Midura-Kiela, M. T., Ohland, C. L., Lubitz, D., & Harrison, C. A. (2018). Microbial dysbiosis associated with impaired intestinal Na+/H+ exchange accelerates and exacerbates colitis in ex-germ free mice.. Mucosal Immunology, 11(5), 1329-1341. doi:10.1038/s41385-018-0035-2
• Wagner, A. M., Brownlee, R. D., Besselsen, D. G., Becker, M. D., & Ardeshir, A. (2018). A field strain of minute virus of mice (MVMm) exhibits age- and strain-specific pathogenesis.. The Journal of general virology, 99(4), 558-566. doi:10.1099/jgv.0.001044
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  The influence of mouse strain, immune competence and age on the pathogenesis of a field strain of minute virus of mice (MVMm) was examined in BALB/c, C3H, C57BL/6 and SCID mice experimentally infected as neonates, weanlings and adults. Sera, bodily excretions and tissues were harvested at 7, 14, 28 and 56 days after inoculation and evaluated by serology, quantitative PCR and histopathology. Seroconversion to recombinant viral capsid protein 2 was consistently observed in all immunocompetent strains of mice, regardless of the age at which they were inoculated, while seroconversion to the viral nonstructural protein 1 was only consistently detected in neonate inoculates. Viral DNA was detected by quantitative PCR in multiple tissues of immunocompetent mice at each time point after inoculation, with the highest levels being observed in neonate inoculates at 7 days after inoculation. In contrast, viral DNA levels in tissues and bodily excretions increased consistently over time in immunodeficient SCID mice, regardless of the age at which they were inoculated, with mortality being observed in neonatal inoculates between 28 and 56 days after inoculation. Overall, productive infection was observed more frequently in immunocompetent mice inoculated as neonates as compared to those inoculated as weanlings or adults, and immunodeficient SCID mice developed persistent, progressive infection, with mortality being observed in mice inoculated as neonates. Importantly, the clinical syndrome observed in experimentally infected SCID neonatal mice recapitulates the clinical presentation reported for the naturally infected immunodeficient NOD µ-chain knockout mice from which MVMm was initially isolated.
• Ball, C. L., Daniel, S. G., Besselsen, D. G., Hurwitz, B. L., & Doetschman, T. C. (2017). Functional changes in the gut microbiome contribute to Transforming Growth Factor β-deficient colon cancer. mSystems, 2(5), 1-17.
• Doane, C. J., Johnson, P., & Besselsen, D. G. (2017). Well-Differentiated Liposarcoma in a Bonnet Macaque (Macaca radiata).. Comparative Medicine, 67(2), 1-4.
• Hurwitz, B. L., Doetschman, T., Besselsen, D. G., Daniel, S. G., & Ball, C. L. (2017). Functional Changes in the Gut Microbiome Contribute to Transforming Growth Factor β-Deficient Colon Cancer. mSystems, 2(5). doi:10.1128/msystems.00065-17
• Buntain, B., Dial, S., Besselsen, D. G., & Burgess, S. (2016). In Defense of Funding New US Veterinary Schools. Journal of the American Veterinary Medical Association, 248(9), 989-90.
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  Letter to the editor relating merits of new UA school of veterinary medicine.
• Laubitz, D., Harrison, C. A., Midura-Kiela, M. T., Ramalingam, R., Larmonier, C. B., Chase, J. H., Caporaso, J. G., Besselsen, D. G., Ghishan, F. K., & Kiela, P. R. (2016). Reduced Epithelial Na+/H+ Exchange Drives Gut Microbial Dysbiosis and Promotes Inflammatory Response in T Cell-Mediated Murine Colitis. PloS one, 11(4), e0152044.
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  Inflammatory bowel diseases (IBD) are associated with functional inhibition of epithelial Na+/H+ exchange. In mice, a selective disruption of NHE3 (Slc9a3), a major apical Na+/H+ exchanger, also promotes IBD-like symptoms and gut microbial dysbiosis. We hypothesized that disruption of Na+/H+ exchange is necessary for the development of dysbiosis, which promotes an exacerbated mucosal inflammatory response. Therefore, we performed a temporal analysis of gut microbiota composition, and mucosal immune response to adoptive T cell transfer was evaluated in Rag2-/- and NHE3-/-/Rag2-/- (DKO) mice with and without broad-spectrum antibiotics. Microbiome (16S profiling), colonic histology, T cell and neutrophil infiltration, mucosal inflammatory tone, and epithelial permeability were analyzed. In adoptive T cell transfer colitis model, Slc9a3 status was the most significant determinant of gut microbial community. In DKO mice, NHE3-deficiency and dysbiosis were associated with dramatically accelerated and exacerbated disease, with rapid body weight loss, increased mucosal T cell and neutrophil influx, increased mucosal cytokine expression, increased permeability, and expansion of CD25-FoxP3+ Tregs; this enhanced susceptibility was alleviated by oral broad-spectrum antibiotics. Based on these results and our previous work, we postulate that epithelial electrolyte homeostasis is an important modulator in the progression of colitis, acting through remodeling of the gut microbial community.
• McFadden, R. T., Larmonier, C. B., Shehab, K. W., Midura-Kiela, M., Ramalingam, R., Harrison, C. A., Besselsen, D. G., Chase, J. H., Caporaso, J. G., Jobin, C., Ghishan, F. K., & Kiela, P. R. (2015). The Role of Curcumin in Modulating Colonic Microbiota During Colitis and Colon Cancer Prevention. Inflammatory bowel diseases, 21(11), 2483-94.
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  Intestinal microbiota influences the progression of colitis-associated colorectal cancer. With diet being a key determinant of the gut microbial ecology, dietary interventions are an attractive avenue for the prevention of colitis-associated colorectal cancer. Curcumin is the most active constituent of the ground rhizome of the Curcuma longa plant, which has been demonstrated to have anti-inflammatory, antioxidative, and antiproliferative properties.
• Ramalingam, R., Midura-kiela, M. T., Mcfadden, R. T., Larmonier, C. B., Kiela, P. R., Harrison, C. A., Ghishan, F. K., Chase, J., Caporaso, G., & Besselsen, D. G. (2014). AGA Abstracts275 The Role of Curcumin in Modulating Colonic Microbiota During Colitis and Colon Cancer Prevention. Gastroenterology, 146(5), S-66. doi:10.1016/s0016-5085(14)60234-1
• Towne, J. W., Wagner, A. M., Griffin, K. J., Buntzman, A. S., Frelinger, J. A., & Besselsen, D. G. (2014). Elimination of Pasteurella pneumotropica from a mouse barrier facility by using a modified enrofloxacin treatment regimen. Journal of the American Association for Laboratory Animal Science : JAALAS, 53(5), 517-22.
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  Multiple NOD. Cg-Prkdc(scid)Il2rg(tm1Wjl)Tg(HLA-A2.1)Enge/Sz (NSG/A2) transgenic mice maintained in a mouse barrier facility were submitted for necropsy to determine the cause of facial alopecia, tachypnea, dyspnea, and sudden death. Pneumonia and soft-tissue abscesses were observed, and Pasteurella pneumotropica biotype Jawetz was consistently isolated from the upper respiratory tract, lung, and abscesses. Epidemiologic investigation within the facility revealed presence of this pathogen in mice generated or rederived by the intramural Genetically Engineered Mouse Model (GEMM) Core but not in mice procured from several approved commercial vendors. Epidemiologic data suggested the infection originated from female or vasectomized male ND4 mice obtained from a commercial vendor and then comingled by the GEMM Core to induce pseudopregnancy in female mice for embryo implantation. Enrofloxacin delivered in drinking water (85 mg/kg body weight daily) for 14 d was sufficient to clear bacterial infection in normal, breeding, and immune-deficient mice without the need to change the antibiotic water source. This modified treatment regimen was administered to 2400 cages of mice to eradicate Pasteurella pneumotropica from the facility. Follow-up PCR testing for P. pneumotropica biotype Jawetz remained uniformly negative at 2, 6, 12, and 52 wk after treatment in multiple strains of mice that were originally infected. Together, these data indicate that enrofloxacin can eradicate P. pneumotropica from infected mice in a less labor-intensive approach that does not require breeding cessation and that is easily adaptable to the standard biweekly cage change schedule for individually ventilated cages.
• Watson, J. M., Marion, S. L., Rice, P. F., Bentley, D. L., Besselsen, D. G., Utzinger, U., Hoyer, P. B., & Barton, J. K. (2014). In vivo time-serial multi-modality optical imaging in a mouse model of ovarian tumorigenesis. Cancer Biology and Therapy, 15(1), 42-60.
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  Abstract: Identification of the early microscopic changes associated with ovarian cancer may lead to development of a diagnostic test for high-risk women. In this study we use optical coherence tomography (OCT) and multiphoton microscopy (MPM) (collecting both two photon excited fluorescence [TPEF] and second harmonic generation [SH G]) to image mouse ovaries in vivo at multiple time points. We demonstrate the feasibility of imaging mouse ovaries in vivo during a longterm survival study and identify microscopic changes associated with early tumor development. These changes include alterations in tissue microstructure, as seen by OCT, alterations in cellular fluorescence and morphology, as seen by TPEF, and remodeling of collagen structure, as seen by SH G. These results suggest that a combined OCT-MPM system may be useful for early detection of ovarian cancer. © 2014 Landes Bioscience.
• Besselsen DVM, PhD, D. G. (2013). High vitamin D3 diet administered during active colitis negatively affects bone metabolism in an adoptive T cell transfer model. American Journal of Physiology- Gastrointestinal and Liver Physiology, 305(1), G35-46.
• Besselsen, D. G., Laubitz, D., Larmonier, C. B., Hill, F. M., Shehab, K. W., Lipinski, L., Midura-Kiela, M. T., McFadden, R. T., Ramalingam, R., Hassan, K. A., Golebiewski, M., Ghishan, F. K., & Kiela, P. R. (2013). Reduced colonic microbial diversity is associated with colitis in NHE3-deficient mice. American Journal of Physiology-Gastrointestinal and Liver Physiology, 305(10), G667-G677. doi:10.1152/ajpgi.00189.2013
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  Chronic inflammation and enteric infections are frequently associated with epithelial Na + /H + exchange (NHE) inhibition. Alterations in electrolyte transport and in mucosal pH associated with inflammation may represent a key mechanism leading to changes in the intestinal microbial composition. NHE3 expression is essential for the maintenance of the epithelial barrier function. NHE3 −/− mice develop spontaneous distal chronic colitis and are highly susceptible to dextran sulfate (DSS)-induced mucosal injury. Spontaneous colitis is reduced with broad-spectrum antibiotics treatment, thus highlighting the importance of the microbiota composition in NHE3 deficiency-mediated colitis. We herein characterized the colonic microbiome of wild-type (WT) and NHE3 −/− mice housed in a conventional environment using 454 pyrosequencing. We demonstrated a significant decrease in the phylogenetic diversity of the luminal and mucosal microbiota of conventional NHE3 −/− mice compared with WT. Rederivation of NHE3 −/− mice from conventional to a barrier facility eliminated the signs of colitis and decreased DSS susceptibility. Reintroduction of the conventional microflora into WT and NHE3 −/− mice from the barrier facility resulted in the restoration of the symptoms initially described in the conventional environment. Interestingly, qPCR analysis of the microbiota composition in mice kept in the barrier facility compared with reconventionalized mice showed a significant reduction of Clostridia classes IV and XIVa. Therefore, the gut microbiome plays a prominent role in the pathogenesis of colitis in NHE3 −/− mice, and, reciprocally, NHE3 also plays a critical role in shaping the gut microbiota. NHE3 deficiency may be a critical contributor to dysbiosis observed in patients with inflammatory bowel disease.
• Hoyer, P. B., Watson, J. M., Utzinger, U., Rice, P. F., Marion, S. L., Hoyer, P. B., Besselsen, D. G., Bentley, D. L., & Barton, J. K. (2013). In vivo three-dimensional optical coherence tomography and multiphoton microscopy in a mouse model of ovarian neoplasia. Proceedings of SPIE, 8577. doi:10.1117/12.1000273
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  ABSTRACT Our goal is to use optical coherence tomography (OCT) and multiphoton microscopy (MPM) to detect early tumor development in a mouse model of ovarian neoplasia. We hope to use information regarding early tumor development to create a diagnostic test for high-risk patients. In this study we collect in vivo images using OCT, second harmonic generation and two-photon excited fluorescence from non-vinylcyclohexene diepoxide (VCD)-dosed and VCD-dosed mice. VCD causes follicular apoptosis (simulating menopaus e) and leads to tumor development. Using OCT and MPM we visualized the ovarian microstructure and were able to see differences between non-VCD-dosed and VCD-dosed animals. This leads us to believe that OCT and MPM may be useful for detecting changes due to early tumor development. Keywords: imaging, fluorescence microscopy, second harmonic generation, two-photon excited fluorescence, ovarian cancer, post-menopausal mouse model, vinylcyclohexene diepoxide 1. INTRODUCTION In the United States there are more than 21,000 new cases of ovarian cancer di agnosed each year. Less than 20% of ovarian cancers are diagnosed at a localized stage and once cancer has spread from th e ovary 5-year survival rate is low. However, when found at the localized st age, the 5-year survival rate is 93%
• Larmonier, C. B., Laubitz, D., Hill, F. M., Shehab, K. W., Lipinski, L., Midura-Kiela, M. T., McFadden, R. T., Ramalingam, R., Hassan, K. A., Golebiewski, M., Besselsen, D. G., Ghishan, F. K., & Kiela, P. R. (2013). Reduced colonic microbial diversity is associated with colitis in NHE3-deficient mice. American Journal of Physiology. Gastrointestinal and liver physiology, 305(10).
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  Chronic inflammation and enteric infections are frequently associated with epithelial Na(+)/H(+) exchange (NHE) inhibition. Alterations in electrolyte transport and in mucosal pH associated with inflammation may represent a key mechanism leading to changes in the intestinal microbial composition. NHE3 expression is essential for the maintenance of the epithelial barrier function. NHE3(-/-) mice develop spontaneous distal chronic colitis and are highly susceptible to dextran sulfate (DSS)-induced mucosal injury. Spontaneous colitis is reduced with broad-spectrum antibiotics treatment, thus highlighting the importance of the microbiota composition in NHE3 deficiency-mediated colitis. We herein characterized the colonic microbiome of wild-type (WT) and NHE3(-/-) mice housed in a conventional environment using 454 pyrosequencing. We demonstrated a significant decrease in the phylogenetic diversity of the luminal and mucosal microbiota of conventional NHE3(-/-) mice compared with WT. Rederivation of NHE3(-/-) mice from conventional to a barrier facility eliminated the signs of colitis and decreased DSS susceptibility. Reintroduction of the conventional microflora into WT and NHE3(-/-) mice from the barrier facility resulted in the restoration of the symptoms initially described in the conventional environment. Interestingly, qPCR analysis of the microbiota composition in mice kept in the barrier facility compared with reconventionalized mice showed a significant reduction of Clostridia classes IV and XIVa. Therefore, the gut microbiome plays a prominent role in the pathogenesis of colitis in NHE3(-/-) mice, and, reciprocally, NHE3 also plays a critical role in shaping the gut microbiota. NHE3 deficiency may be a critical contributor to dysbiosis observed in patients with inflammatory bowel disease.
• Larmonier, C. B., McFadden, R. T., Hill, F. M., Schreiner, R., Ramalingam, R., Besselsen, D. G., Ghishan, F. K., & Kiela, P. R. (2013). High vitamin D3 diet administered during active colitis negatively affects bone metabolism in an adoptive T cell transfer model. American journal of physiology. Gastrointestinal and liver physiology, 305(1), G35-46.
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  Decreased bone mineral density (BMD) represents an extraintestinal complication of inflammatory bowel disease (IBD). Vitamin D₃ has been considered a viable adjunctive therapy in IBD. However, vitamin D₃ plays a pleiotropic role in bone modeling and regulates the bone formation-resorption balance, depending on the physiological environment, and supplementation during active IBD may have unintended consequences. We evaluated the effects of vitamin D₃ supplementation during the active phase of disease on colonic inflammation, BMD, and bone metabolism in an adoptive IL-10-/- CD4⁺ T cell transfer model of chronic colitis. High-dose vitamin D₃ supplementation for 12 days during established disease had negligible effects on mucosal inflammation. Plasma vitamin D₃ metabolites correlated with diet, but not disease, status. Colitis significantly reduced BMD. High-dose vitamin D₃ supplementation did not affect cortical bone but led to a further deterioration of trabecular bone morphology. In mice fed a high vitamin D₃ diet, colitis more severely impacted bone formation markers (osteocalcin and bone alkaline phosphatase) and increased bone resorption markers, ratio of receptor activator of NF-κB ligand to osteoprotegrin transcript, plasma osteoprotegrin level, and the osteoclast activation marker tartrate-resistant acid phosphatase (ACp5). Bone vitamin D receptor expression was increased in mice with chronic colitis, especially in the high vitamin D₃ group. Our data suggest that vitamin D₃, at a dose that does not improve inflammation, has no beneficial effects on bone metabolism and density during active colitis or may adversely affect BMD and bone turnover. These observations should be taken into consideration in the planning of further clinical studies with high-dose vitamin D₃ supplementation in patients with active IBD.
• Shehab, K. W., Woodford, R. T., Shehab, K. W., Midura-kiela, M. T., Laubitz, D., Larmonier, C. B., Kiela, P. R., Hill, F. M., Golebiewski, M., Ghishan, F. K., & Besselsen, D. G. (2013). 739 Alteration of the Gut Microbiome in NHE3-Deficient Mice. Gastroenterology, 144(5), S-133. doi:10.1016/s0016-5085(13)60479-5
• Watson, J. M., Marion, S. L., Rice, P. F., Bentley, D. L., Besselsen, D. G., Utzinger, U., Hoyer, P. B., & Barton, J. K. (2013). In vivo time-serial multi-modality optical imaging in a mouse model of ovarian tumorigenesis. Cancer Biology & Therapy, 15(1).
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  Identification of the early microscopic changes associated with ovarian cancer may lead to development of a diagnostic test for high-risk women. In this study we use optical coherence tomography (OCT) and multiphoton microscopy (MPM) (collecting both two photon excited fluorescence [TPEF] and second harmonic generation [SHG]) to image mouse ovaries in vivo at multiple time points. We demonstrate the feasibility of imaging mouse ovaries in vivo during a long-term survival study and identify microscopic changes associated with early tumor development. These changes include alterations in tissue microstructure, as seen by OCT, alterations in cellular fluorescence and morphology, as seen by TPEF, and remodeling of collagen structure, as seen by SHG. These results suggest that a combined OCT-MPM system may be useful for early detection of ovarian cancer.
• Woodford, R. T., Schreiner, R., Ramalingam, R., Larmonier, C. B., Kiela, P. R., Hill, F. M., Ghishan, F. K., & Besselsen, D. G. (2013). Su1135 High Vitamin D Diet Leads to a Paradoxical Decrease of Bone Mineral Density in Adoptive T-Cell Transfer Colitis. Gastroenterology, 144(5), S-408. doi:10.1016/s0016-5085(13)61502-4
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  Background: Inflammatory Bowel Disease is not only characterized by an aberrant immune response against commensal intestinal bacterial flora and mucosal damage but can also affect bone metabolism and bone mineral density (BMD) with osteopenia and osteoporosis as the two major extra-intestinal symptoms of IBD. Low BMD along with 25(OH) vitamin D3 deficiency have been reported in both Ulcerative colitis and Crohn's Disease and is associated with an increased risk of fracture. In IBD patients, inflammation-associated pro-inflammatory mediators, poor nutritional status and malabsortion affecting vitamin D3 and Ca2+ homeostasis, have been postulated to be the cause of altered bone morphology and metabolism. Moreover, immunomodulatory role of vitamin D3 has been reported in both innate and adaptive immune system. Therefore, vitamin D3 supplementation has been considered as a viable adjunctive therapeutic strategy in IBD. Vitamin D3 plays a pleiotropic role in bone modeling and regulates the balance of bone formation and bone resorption depending of the physiological environment. Moreover, a significant fraction of adult and pediatric IBD patients with low BMD have elevated levels of circulating 1,25(OH)2 D3. We hypothesized that vitamin D3 supplementation in active IBD may lead to a paradoxical loss in BMD. Methods: We evaluated the effect of vitamin D3 supplementation in adoptive IL-10-/-CD4+ T cell transfer model of chronic colitis. In this model, we evaluated the effect of vitamin D3 supplementation (switch from human equivalent dose of 730 IU to 3,648 IU daily) on established colitis and on BMD (μCT), bone metabolism and turnover. Results: 12-day highdose vitamin D3 supplementation during established disease had negligible effects onmucosal inflammation. Plasma level of vitamin D3 metabolites correlated with diet, but not with the disease status. Colitis reduced BMD. High vitamin D3 supplementation did not affect cortical bone structure but led to a further deterioration of trabecular bone morphology. High D3 diet negatively affected bone formation markers; osteocalcin and bone alkaline phosphatase, and increased bone resorption markers; RANKL/OPG transcript ratio, plasma OPG level and osteoclast activation marker ACp5. Consistently, expression of bone vitamin D receptor (VDR) was increased in mice with chronic colitis, especially in mice fed high vitamin D3 diet. Conclusion: Our data collectively suggests that vitamin D3 in a dose which does not improve inflammation, adversely affects BMD and bone turnover. The data provided by this study should be taken into consideration in the planning of further clinical studies with high vitamin D3 supplementation in IBD patients with active disease.
• Hahn, T., Bradley-Dunlop, D. J., Hurley, L. H., Von-Hoff, D., Gately, S., Mary, D. L., Lu, H., Penichet, M. L., Besselsen, D. G., Cole, B. B., Meeuwsen, T., Walker, E., & Akporiaye, E. T. (2011). The vitamin E analog, alpha-tocopheryloxyacetic acid enhances the anti-tumor activity of trastuzumab against HER2/neu-expressing breast cancer. BMC cancer, 11.
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  HER2/neu is an oncogene that facilitates neoplastic transformation due to its ability to transduce growth signals in a ligand-independent manner, is over-expressed in 20-30% of human breast cancers correlating with aggressive disease and has been successfully targeted with trastuzumab (Herceptin®). Because trastuzumab alone achieves only a 15-30% response rate, it is now commonly combined with conventional chemotherapeutic drugs. While the combination of trastuzumab plus chemotherapy has greatly improved response rates and increased survival, these conventional chemotherapy drugs are frequently associated with gastrointestinal and cardiac toxicity, bone marrow and immune suppression. These drawbacks necessitate the development of new, less toxic drugs that can be combined with trastuzumab. Recently, we reported that orally administered alpha-tocopheryloxyacetic acid (α-TEA), a novel ether derivative of alpha-tocopherol, dramatically suppressed primary tumor growth and reduced the incidence of lung metastases both in a transplanted and a spontaneous mouse model of breast cancer without discernable toxicity.
• Ignatenko, N. A., Gerner, E. W., & Besselsen, D. G. (2011). Defining the role of polyamines in colon carcinogenesis using mouse models. Journal of carcinogenesis, 10.
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  Genetics and diet are both considered important risk determinants for colorectal cancer, a leading cause of death in the US and worldwide. Genetically engineered mouse (GEM) models have made a significant contribution to the characterization of colorectal cancer risk factors. Reliable, reproducible, and clinically relevant animal models help in the identification of the molecular events associated with disease progression and in the development of effictive treatment strategies. This review is focused on the use of mouse models for studying the role of polyamines in colon carcinogenesis. We describe how the available mouse models of colon cancer such as the multiple intestinal neoplasia (Min) mice and knockout genetic models facilitate understanding of the role of polyamines in colon carcinogenesis and help in the development of a rational strategy for colon cancer chemoprevention.
• Wright, L. E., Frye, J. B., Lukefahr, A. L., Marion, S. L., Hoyer, P. B., Besselsen, D. G., & Funk, J. L. (2011). 4-Vinylcyclohexene diepoxide (VCD) inhibits mammary epithelial differentiation and induces fibroadenoma formation in female Sprague Dawley rats. Reproductive toxicology (Elmsford, N.Y.), 32(1).
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  4-Vinylcyclohexene diepoxide (VCD), an occupational chemical that targets ovarian follicles and accelerates ovarian failure in rodents, was used to test the effect of early-onset reproductive senescence on mammary fibroadenoma formation. One-month female Sprague Dawley rats were dosed with VCD (80 mg/kg or 160 mg/kg) and monitored for 22 months for persistent estrus and tumor development. Only high-dose VCD treatment accelerated the onset of persistent estrus relative to controls. However, both doses of VCD accelerated mammary tumor onset by 5 months, increasing incidence to 84% (vs. 38% in controls). Tumor development was independent of time in persistent estrus, 17 β-estradiol, androstenedione and prolactin. Delay in VCD administration until after completion of mammary epithelial differentiation (3 months) did not alter tumor formation despite acceleration of ovarian senescence. VCD administration to 1-month rats acutely decreased mammary alveolar bud number and expression of β-casein, suggesting that VCD's tumorigenic effect requires exposure during mammary epithelial differentiation.
• Zink, M. C., Valli, V. E., Tolwani, R. J., Sundberg, J. P., Sellers, R. S., Schwahn, D. J., Schoeb, T. R., Perle, K. L., Nikitin, A. Y., Meyerholz, D. K., Liu, C., Lairmore, M. D., Griffey, S. M., Foreman, O., Eaton, K. A., Couto, S. S., Cardiff, R. D., Brayton, C., Boyd, K. L., , Bolon, B., et al. (2011). Advancing translational research.. Science (New York, N.Y.), 331(6024), 1516-7. doi:10.1126/science.331.6024.1516-b
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  The National Institutes of Health (NIH) proposal to launch a National Center for Advancing Translational Sciences (NCATS) by dismembering the National Center for Research Resources (NCRR) (“Collins sparks furor with proposed NIH reshuffling,” J. Kaiser, News and Analysis, 28 January, p. [386][1]) is more likely to hinder than bolster translational research. Developing new treatments requires understanding of disease mechanisms. Such studies depend on superior facilities, resources, and training, which for years have been effectively supported by the NCRR Division of Comparative Medicine (DCM) ([ 1 ][2]). The original NCATS proposal sought to divide core NCRR functions, including those in DCM, between NCATS and a hodgepodge Interim Infrastructure Unit ([ 2 ][3]). The updated proposal preserves major DCM roles within an Infrastructure Entity ([ 2 ][3]). This revision clearly concedes that reducing disruption to existing NCRR resources is a surer way for NIH to sustain its historical vigor in mechanistic research while also boosting its translational science attainments than an impulsive shift to chemical screening and preclinical testing by an untried NCATS. Furthermore, the utility of the NCATS compared with the proven value of NCRR has not been adequately considered by the scientific community. To date, the NCATS concept and original “Straw Model” have received hundreds of comments, many of which are critical ([ 3 ][4]–[ 7 ][5]); deliberations over the revised NCATS proposal have barely begun ([ 8 ][6]). Hurried implementation of NCATS over such widespread objections will immediately call into question the credibility of the new Center. As veteran comparative biologists, we believe that the best way to rapidly advance NIH translational science efforts will be to build rather than break the successful, integrated program within the NCRR. Any other choice should be recognized for what it is: good politics, but bad policy. 1. [↵][7]National Center for Research Resources Program Overview ([www.ncrr.nih.gov/about\_us/program\_overview/index.asp][8]). 2. [↵][9]Feedback NIH, Proposed National Center for Advancing Translational Sciences ( ). 3. [↵][10]Feedback NIH, “Archived NCATS comments, 12/9/10-1/13/11” ( ). 4. Feedback NIH, “Comments to NCRR task force straw model” ( ). 5. 1. J. Mervis , “Collins's plan to reshuffle NIH draws more flak,” ScienceInsider (28 January 2011). 6. 1. M. Wadman , “Collins defends decision to dismantle NIH center,” The Great Beyond, 21 January 2011; [http://blogs.nature.com/news/thegreatbeyond/2011/01/collins\_defends\_decision\_to\_di.html][11]. 7. [↵][12]1. M. Wadman , “NIH revamp rushes ahead,” Nature News, 1 March 2011; [www.nature.com/news/2011/110301/full/471015a.html][13]. 8. [↵][14]Feedback NIH, “Comments to NCRR Task Force Recommendations” ( ). [1]: /lookup/doi/10.1126/science.331.6016.386 [2]: #ref-1 [3]: #ref-2 [4]: #ref-3 [5]: #ref-7 [6]: #ref-8 [7]: #xref-ref-1-1 "View reference 1 in text" [8]: http://www.ncrr.nih.gov/about_us/program_overview/index.asp [9]: #xref-ref-2-1 "View reference 2 in text" [10]: #xref-ref-3-1 "View reference 3 in text" [11]: http://blogs.nature.com/news/thegreatbeyond/2011/01/collins_defends_decision_to_di.html [12]: #xref-ref-7-1 "View reference 7 in text" [13]: http://www.nature.com/news/2011/110301/full/471015a.html [14]: #xref-ref-8-1 "View reference 8 in text"
• Besselsen, D., Christie, R. D., Marcus, E. C., Wagner, A. M., & Besselsen, D. G. (2010). Experimental infection of mice with hamster parvovirus: evidence for interspecies transmission of mouse parvovirus 3. Comparative medicine, 60(2).
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  Hamster parvovirus (HaPV) was isolated 2 decades ago from hamsters with clinical signs similar to those induced in hamsters experimentally infected with other rodent parvoviruses. Genetically, HaPV is most closely related to mouse parvovirus (MPV), which induces subclinical infection in mice. A novel MPV strain, MPV3, was detected recently in naturally infected mice, and genomic sequence analysis indicates that MPV3 is almost identical to HaPV. The goal of the present studies was to examine the infectivity of HaPV in mice. Neonatal and weanling mice of several mouse strains were inoculated with HaPV. Tissues, excretions, and sera were harvested at 1, 2, 4, and 8 wk after inoculation and evaluated by quantitative PCR and serologic assays specific for HaPV. Quantitative PCR detected viral DNA quantities that greatly exceeded the quantity of virus in inocula in multiple tissues of infected mice. Seroconversion to both nonstructural and structural viral proteins was detected in most immunocompetent mice 2 or more weeks after inoculation with HaPV. In neonatal SCID mice, viral transcripts were detected in lymphoid tissues by RT-PCR and viral DNA was detected in feces by quantitative PCR at 8 wk after inoculation. No clinical signs, gross, or histologic lesions were observed. These findings are similar to those observed in mice infected with MPV. These data support the hypothesis that HaPV and MPV3 are likely variants of the same viral species, for which the mouse is the natural rodent host with rare interspecies transmission to the hamster.
• Cray, C., Besselsen, D. G., Hart, J. L., Yoon, D., Rodriguez, M., Zaias, J., & Altman, N. H. (2010). Quantitation of acute phase proteins and protein electrophoresis in monitoring the acute inflammatory process in experimentally and naturally infected mice. Comparative medicine, 60(4).
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  Serologic screening for infectious disease in sentinel mice from rodent colonies is expensive and labor-intensive, often involving multiple assays for several different infectious agents. Previously, we established normal reference ranges for the protein fractions of several laboratory strains of mice by using a commercially available agarose system of protein electrophoresis. In the current study, we address protein fractionation and quantitation of acute phase proteins (APP) in mice experimentally infected with Sendai virus or mouse parvovirus. We further investigate this methodology by using samples from sentinel mice from colonies with endemic infection. All study groups showed significant increases in gamma globulins. Various other protein fractions showed mild variable changes; significant differences were not detected for individual APP. These results contrast the significant changes observed in APP and protein electrophoresis by using the standard methods of inducing inflammatory responses through injection of complete Freund adjuvant or LPS. These present data suggest that although quantitation of individual APP may not be helpful, gamma globulin levels may reflect infection in laboratory mice and provide a possible adjunct to traditional screening methods.
• Flowers, M., Schroeder, J. A., Borowsky, A. D., Besselsen, D. G., Thomson, C. A., Pandey, R., & Thompson, P. A. (2010). Pilot study on the effects of dietary conjugated linoleic acid on tumorigenesis and gene expression in PyMT transgenic mice. Carcinogenesis, 31(9).
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  Conjugated linoleic acid (CLA) is a class of commercially available fatty acids that have been associated with anticancer properties in rodent models of chemical carcinogenesis. We conducted a pilot study to examine the antitumor effect of dietary CLA in a polyoma virus-middle T antigen (PyMT) mouse model of invasive breast cancer. Virgin 4-week-old PyMT mice were administered a mixed-isomer CLA diet (1% wt/wt) or control AIN-93G diet for 4 weeks (N = 6 and 5, respectively) and tumor burden was assessed at 8 weeks of age. Thoracic mammary glands were prepared as whole mounts with other glands being formalin fixed and paraffin embedded for histology and immunohistochemistry (IHC). Total RNA was prepared for microarray and real-time reverse transcription-polymerase chain reaction analysis. Western blots were performed for protein expression analysis. Tumor incidence was significantly increased in CLA-treated animals compared with controls (P = 0.009) and occurred with extensive lobular-alveolar expansion and loss of mammary adipose tissue. More than 100 genes were downregulated > or = 2-fold in the CLA-treated group compared with controls, including adipose-specific markers, as wells as cytoskeletal and adhesion-related genes. This was supported by dramatic decreases in the epithelial adherens E-cadherin and beta-catenin as demonstrated by IHC. Taken together, these results suggest that dietary CLA affects the mammary stromal environment, leading to tumor progression and cellular expansion in the PyMT mouse model. Further studies of the potential for cancer promotion are needed, especially because mixed-isomer CLA formulations are sold commercially as a nutritional supplement.
• Mast, T. G., Gard, C., Doetschman, T., Chen, D., Besselsen, D. G., Ball, C., Azhar, M., & Aronow, B. J. (2010). Abstract 1957: Loss of TGFβ1 alters host-microbial interactions, predisposing the colonic epithelium to inflammation. Cancer Research, 70, 1957-1957. doi:10.1158/1538-7445.am10-1957
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  Introduction: The TGFB pathway is mutated in up to 30% of human colon cancers. Genetically Engineered Mouse Models (GEMs) with deficient TGFβ signaling model several characteristics of IBD associated human colon cancers. Introduction of Helicobacter sp. into the Tgfb1 −/− Rag2 −/− mouse model is necessary for the development of inflammatory lesions which progress to adenoma and carcinoma. The exact role of TGFβ1 and bacterial-associated inflammation has yet to be elucidated and offers a potential target for the prevention of colon cancer. Methods: To determine the function of TGFβ1 on colonic bacterial composition we used GEM models, Tgfb1 −/− Rag2 −/− and Tgfb1 +/+ Rag2 −/− . The cecal microflora of 10 Tgfb1 −/− Rag2 −/− and 10 Tgfb1 +/+ Rag2 −/− mice were isolated and serially diluted onto brucella (BRU), bacteroides bile esculin (BBE), and laked kanomycin-vancomycin (LKV) media under anaerobic conditions. Colony forming units (CFUs) were enumerated. Individual colony types were then streaked onto trypitcase soy agar with 5% sheep blood and grown anaerobically and aerobically. The bacteria were then gram-stained and biotyped utilizing a Dade Berhing MicroScan instrument. To further examine the bacterial composition we designed primers specific to the 16s rRNA subunit of different Bacteroides species, and using the Roche LightCycler preformed quantitative Real Time-PCR (qRT-PCR) on fecal DNA. Results: The Dade Berhing Instrument showed that Tgfb1 −/− Rag2 −/− mice had a 4 fold increase in bacterial load and a 28 fold increase in Bacteroides species when compared with Tgfb1 +/+ Rag2 −/− mice. The qRT-PCR results showed an increase in Bacteroides fragilis and Bacteroides distasonis and a significant decrease in Bacteroides thetaiotaomicron in the Tgfb1 −/− Rag2 −/− mice. These data suggest that loss of TGFβ1 alters the colonic microflora. Previous studies illustrated the importance of bacterial nutrient sources on bacterial composition. To examine if TGFβ1 is altering nutrient availability in the colon a previous micro-array was analyzed for candidate genes associated with glycoprotein metabolism. This showed changes in fucose metabolizing enzymes with the loss of TGFβ1. Conclusion: These findings suggest that TGFβ1 plays a role in bacterial load maintenance, possibly by altering available nutrient sources and when disrupted, can cause abnormalities in pathobionts (commensal bacterial with pathogenic potential) which could then lead to increased inflammation. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1957.
• Thurston, R. D., Larmonier, C. B., Majewski, P. M., Ramalingam, R., Midura-Kiela, M., Laubitz, D., Vandewalle, A., Besselsen, D. G., Mühlbauer, M., Jobin, C., Kiela, P. R., & Ghishan, F. K. (2010). Tumor necrosis factor and interferon-gamma down-regulate Klotho in mice with colitis. Gastroenterology, 138(4).
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  Klotho (KL) is an anti-inflammatory protein that protects the endothelium from nitric oxide (NO)-induced dysfunction, reduces the expression of endothelial adhesion molecules, and potentially regulates T-cell functions. KL deficiency leads to premature senescence and impaired Ca2+/Pi homeostasis, which can lead to inflammatory bowel disease (IBD)-associated osteopenia/osteoporosis. We investigated the changes in renal expression of Kl as a consequence of colitis.
• Beilke, L. D., Aleksunes, L. M., Holland, R. D., Besselsen, D. G., Beger, R. D., Klaassen, C. D., & Cherrington, N. J. (2009). Constitutive androstane receptor-mediated changes in bile acid composition contributes to hepatoprotection from lithocholic acid-induced liver injury in mice. Drug metabolism and disposition: the biological fate of chemicals, 37(5), 1035-45.
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  Pharmacological activation of the constitutive androstane receptor (CAR) protects the liver during cholestasis. The current study evaluates how activation of CAR influences genes involved in bile acid biosynthesis as a mechanism of hepatoprotection during bile acid-induced liver injury. CAR activators phenobarbital (PB) and 1,4-bis[2-(3,5-dichloropyridyloxy)]benzene (TCPOBOP) or corn oil (CO) were administered to C57BL/6 wild-type (WT) and CAR knockout (CAR-null) mice before and during induction of intrahepatic cholestasis using the secondary bile acid, lithocholic acid (LCA). In LCA-treated WT and all the CAR-null groups (excluding controls), histology revealed severe multifocal necrosis. This pathology was absent in WT mice pretreated with PB and TCPOBOP, indicating CAR-dependent hepatoprotection. Decreases in total hepatic bile acids and hepatic monohydroxy, dihydroxy, and trihydroxy bile acids in PB- and TCPOBOP-pretreated WT mice correlated with hepatoprotection. In comparison, concentrations of monohydroxylated and dihydroxylated bile acids were increased in all the treated CAR-null mice compared with CO controls. Along with several other enzymes (Cyp7b1, Cyp27a1, Cyp39a1), Cyp8b1 expression was increased in hepatoprotected mice, which could be suggestive of a shift in the bile acid biosynthesis pathway toward the formation of less toxic bile acids. In CAR-null mice, these changes in gene expression were not different among treatment groups. These results suggest CAR mediates a shift in bile acid biosynthesis toward the formation of less toxic bile acids, as well as a decrease in hepatic bile acid concentrations. We propose that these combined CAR-mediated effects may contribute to the hepatoprotection observed during LCA-induced liver injury.
• Beilke, L. D., Aleksunes, L. M., Olson, E. R., Besselsen, D. G., Klaassen, C. D., Dvorak, K., & Cherrington, N. J. (2009). Decreased apoptosis during CAR-mediated hepatoprotection against lithocholic acid-induced liver injury in mice. Toxicology letters, 188(1), 38-44.
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  Myeloid cell leukemia-1 (Mcl-1) is an anti-apoptotic protein that is regulated by the constitutive androstane receptor (CAR). Activation of CAR can protect the liver against bile acid-induced toxicity and it may have a role in cell death via apoptosis by altering expression of Bcl-2 family proteins such as myeloid cell leukemia-1 (Mcl-1). Our aim was to determine if activation of CAR reduces hepatocellular apoptosis during cholestasis as a mechanism of hepatoprotection. CAR(+/+) (WT) and CAR(-/-) (CAR-null) mice were pre-treated with compounds known to activate CAR prior to induction of intrahepatic cholestasis using the secondary bile acid lithocholic acid (LCA). Pre-treatment with the CAR activators phenobarbital (PB) and TCPOBOP (TC), as well as the non-CAR activator pregnenolone 16alpha-carbontrile (PCN), protected against LCA-induced liver injury in WT mice, whereas liver injury was more extensive without CAR (CAR-null). Unexpectedly, expression of anti-apoptotic Mcl-1 and Bcl-x(L) was not increased in hepatoprotected mice. Compared to unprotected groups, apoptosis was decreased in hepatoprotected mice as evidenced by the absence of cleaved caspase 3 (cCasp3). In contrast to the cytoplasmic localization in the injured livers (LCA and oltipraz), Mcl-1 protein was localized in the nucleus of hepatoprotected livers to potentially promote cell survival. This study demonstrates that although apoptosis is reduced in hepatoprotected mice pre-treated with CAR and non-CAR activators; hepatoprotection is not directly a result of CAR-induced Mcl-1 expression.
• Fisher, C. D., Lickteig, A. J., Augustine, L. M., Oude Elferink, R. P., Besselsen, D. G., Erickson, R. P., & Cherrington, N. J. (2009). Experimental non-alcoholic fatty liver disease results in decreased hepatic uptake transporter expression and function in rats. European journal of pharmacology, 613(1-3), 119-27.
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  Non-alcoholic fatty liver disease (NAFLD) encompasses a spectrum of diagnoses ranging from simple fatty liver (SFL), to non-alcoholic steatohepatitis (NASH). This study aimed to determine the effect of moderate and severe NAFLD on hepatic transporter expression and function in vivo. Rats were fed a high-fat diet (SFL model) or a methionine-choline-deficient diet (NASH model) for eight weeks. Hepatic uptake transporter function was determined by bromosulfophthalein (BSP) disposition. Transporter expression was determined by branched DNA signal amplification assay and western blotting; inflammation was identified by immunostaining of liver slices for interleukin 1 beta (IL-1beta). MC- rats showed significant retention of BSP in the plasma when compared to control rats. Hepatic NTCP, OATP1a1, 1a4, 1b2 and 2b1; and OAT 2 and 3 mRNA levels were significantly decreased in high-fat and MC- diet rats when compared to control. Protein expression of OATP1a1 was significantly decreased in high-fat animals, while OATP1a1 and OATP1b2 expressions were significantly lower in MC- rats when compared to control. Liver tissue from high-fat and MC- rats stained positive for IL-1beta, a pro-inflammatory cytokine known to decrease expression of NTCP, OATP and OAT transporters, suggesting a plausible mechanism for the observed transporter alterations. These data suggest that different stages of NAFLD result in altered hepatic uptake transporter expression that can lead to a functional impairment of xenobiotic uptake from the blood. Furthermore, NAFLD may alter the plasma retention time of clinically relevant drugs that are reliant on these transporters and may increase the potential drug toxicity.
• Rausch, M. P., Hahn, T., Ramanathapuram, L., Bradley-Dunlop, D., Mahadevan, D., Mercado-Pimentel, M. E., Runyan, R. B., Besselsen, D. G., Zhang, X., Cheung, H., Lee, W., Ling, L. E., & Akporiaye, E. T. (2009). An orally active small molecule TGF-beta receptor I antagonist inhibits the growth of metastatic murine breast cancer. Anticancer research, 29(6), 2099-109.
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  Transforming growth factor beta (TGF-beta) plays a complex role in breast carcinogenesis. Initially functioning as a tumor suppressor, this cytokine later contributes to the progression of malignant cells by enhancing their invasive and metastatic potential as well as suppressing antitumor immunity. The purpose of this study was to investigate the efficacy of SM16, a novel small molecule ALK5 kinase inhibitor, to treat a highly metastatic, TGF-beta-producing murine mammary carcinoma (4T1).
• Udovich, J. A., Besselsen, D. G., & Gmitro, A. F. (2009). Assessment of acridine orange and SYTO 16 for in vivo imaging of the peritoneal tissues in mice. Journal of microscopy, 234(2), 124-9.
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  The effect of peritoneal injection of acridine orange and SYTO 16 in mice was investigated. Images of peritoneal tissues stained with these dyes and obtained through a confocal micro-endoscope are presented. Seventy-five Balb/c mice were split into five groups and given peritoneal injections of dye or saline. The proportions of negative outcomes in each group were compared using confidence intervals and the Fisher's exact statistical test. A statistically significant increase in adverse events due to dye injection was not observed. These data provide an initial investigation into the safety of acridine orange and SYTO 16 for in vivo imaging.
• Beilke, L. D., Besselsen, D. G., Cheng, Q., Kulkarni, S., Slitt, A. L., & Cherrington, N. J. (2008). Minimal role of hepatic transporters in the hepatoprotection against LCA-induced intrahepatic cholestasis. Toxicological sciences : an official journal of the Society of Toxicology, 102(1), 196-204.
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  The multidrug resistance-associated proteins (Mrps) are a family of adenosine triphosphate-dependent transporters that facilitate the movement of various compounds, including bile acids, out of hepatocytes. The current study was conducted to determine whether induction of these transporters alters bile acid disposition as a means of hepatoprotection during bile acid-induced cholestasis. Lithocholic acid (LCA) was used to induce intrahepatic cholestasis. C57BL/6 mice were pretreated with corn oil (CO) or known transporter inducers, phenobarbital (PB), oltipraz (OPZ), or TCPOBOP (TC) for 3 days prior to cotreatment with LCA and inducer for 4 days. Histopathology revealed that PB and TC pretreatments provide a protective effect from LCA-induced toxicity, whereas OPZ pretreatment did not. Both PB/LCA and TC/LCA cotreatment groups also had significantly lower alanine aminotransferase values than the LCA-only group. In TC/LCA cotreated mice compared with LCA only, messenger RNA (mRNA) expression of uptake transporters Ntcp and Oatp4 was significantly increased, as were sinusoidal efflux transporters Mrp3 and Mrp4. Although in PB/LCA cotreated mice, the only significant change compared with LCA-only treatment was an increase in uptake transporter Oatp4. Oatp1 was reduced in all groups compared with CO controls. No significant changes in mRNA expression were observed in Oatp2, Bsep, Mrp2, Bcrp, Mrp1, Mrp5, or Mrp6. Mrp4 protein expression was induced in the OPZ/LCA and TC/LCA cotreated groups, whereas Mrp3 protein levels remained unchanged between groups. Protein expression of Mrp1 and Mrp5 was increased in the unprotected LCA-only and OPZ/LCA mice. Thus, transporter expression did not correlate with histologic hepatoprotection, however, there was a correlation between hepatoprotection and significantly reduced total liver bile acids in the PB/LCA and TC/LCA cotreated mice compared with LCA only. In conclusion, changes in transporter expression did not correlate with hepatoprotection, and therefore, transport may not play a critical role in the observed hepatoprotection from LCA-induced cholestasis in the C57BL/6 mouse.
• Besselsen, D. G., Franklin, C. L., Livingston, R. S., & Riley, L. K. (2008). Lurking in the shadows: emerging rodent infectious diseases. ILAR journal / National Research Council, Institute of Laboratory Animal Resources, 49(3), 277-90.
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  Rodent parvoviruses, Helicobacter spp., murine norovirus, and several other previously unknown infectious agents have emerged in laboratory rodents relatively recently. These agents have been discovered serendipitously or through active investigation of atypical serology results, cell culture contamination, unexpected histopathology, or previously unrecognized clinical disease syndromes. The potential research impact of these agents is not fully known. Infected rodents have demonstrated immunomodulation, tumor suppression, clinical disease (particularly in immunodeficient rodents), and histopathology. Perturbations of organismal and cellular physiology also likely occur. These agents posed unique challenges to laboratory animal resource programs once discovered; it was necessary to develop specific diagnostic assays and an understanding of their epidemiology and transmission routes before attempting eradication, and then evaluate eradication methods for efficacy. Even then management approaches varied significantly, from apathy to total exclusion, and such inconsistency has hindered the sharing and transfer of rodents among institutions, particularly for genetically modified rodent models that may not be readily available. As additional infectious agents are discovered in laboratory rodents in coming years, much of what researchers have learned from experiences with the recently identified pathogens will be applicable. This article provides an overview of the discovery, detection, and research impact of infectious agents recently identified in laboratory rodents. We also discuss emerging syndromes for which there is a suspected infectious etiology, and the unique challenges of managing newly emerging infectious agents.
• Besselsen, D. G., Myers, E. L., Franklin, C. L., Korte, S. W., Wagner, A. M., Henderson, K. S., & Weigler, B. J. (2008). Transmission probabilities of mouse parvovirus 1 to sentinel mice chronically exposed to serial dilutions of contaminated bedding. Comparative medicine, 58(2), 140-4.
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  Intermittent serodetection of mouse parvovirus (MPV) infections in animal facilities occurs frequently when soiled bedding sentinel mouse monitoring systems are used. We evaluated induction of seroconversion in naïve single-caged weanling ICR mice (n = 10 per group) maintained on 5-fold serially diluted contaminated bedding obtained from SCID mice persistently shedding MPV1e. Soiled bedding from the infected SCID mice was collected, diluted, and redistributed weekly to cages housing ICR mice to represent chronic exposure to MPV at varying prevalence in a research colony. Sera was collected every other week for 12 wk and evaluated for reactivity to MPV nonstructural and capsid antigens by multiplex fluorescent immunoassay. Mice were euthanized after seroconversion, and DNA extracted from lymph node and spleen was evaluated by quantitative PCR. Cumulative incidence of MPV infection for each of the 7 soiled bedding dilution groups (range, 1:5 to 1:78125 [v/v]) was 100%, 100%, 90%, 20%, 70%, 60%, and 20%, respectively. Most seropositive mice (78%) converted within the first 2 to 3 wk of soiled bedding exposure, correlating to viral exposure when mice were 4 to 7 wk of age. Viral DNA was detected in lymphoid tissues collected from all mice that were seropositive to VP2 capsid antigen, whereas viral DNA was not detected in lymphoid tissue of seronegative mice. These data indicate seroconversion occurs consistently in young mice exposed to high doses of virus equivalent to fecal MPV loads observed in acutely infected mice, whereas seroconversion is inconsistent in mice chronically exposed to lower doses of virus.
• Besselsen, D. G., Romero-Aleshire, M. J., Munger, S. J., Marcus, E. C., Henderson, K. S., & Wagner, A. M. (2008). Embryo transfer rederivation of C.B-17/Icr-Prkdc(scid) mice experimentally infected with mouse parvovirus 1. Comparative medicine, 58(4), 353-9.
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  We determined whether embryos derived from C.B-17/Icr-Prkdc(scid) (SCID) mice infected with mouse parvovirus (MPV) 1b and mated to MPV-naive B6C3F1 mice would transmit virus to naive recipient female mice and rederived progeny. Viral DNA was detected by quantitative PCR (qPCR) in lymphoid tissues, gonad, sperm, and feces of all MPV1b-inoculated SCID mice. Viral DNA was detected in 1 of 16 aliquots of embryos from infected male SCID mice and in 12 of 18 aliquots of embryos from infected female SCID mice. All recipient female mice implanted with embryos from infected SCID male mice and their progeny were negative by serology and qPCR. In contrast, 3 of 5 recipient female mice implanted with embryos from infected SCID female mice and 14 of 15 progeny mice from these recipients were seropositive by multiplex fluorescent immunoassay (MFI) for MPV capsid antigen (rVP2). All of these mice were negative by MFI for parvovirus nonstructural protein antigen (rNS1) and by qPCR, with the exception of 1 recipient female mouse that displayed weak rNS1 seroreactivity and low levels of MPV DNA in lymphoid tissues. Seroreactivity to rVP2 declined over time in all progeny mice from infected SCID female mice until all were seronegative by 20 wk of age, consistent with maternal antibody transfer. Given that the high levels of MPV contamination detected in our experimentally infected SCID mice are unlikely in naturally infected immunocompetent mice, these data indicate that embryo transfer rederivation is effective for the eradication of MPV from infected colonies.
• Ignatenko, N. A., Besselsen, D. G., Stringer, D. E., Blohm-Mangone, K. A., Cui, H., & Gerner, E. W. (2008). Combination chemoprevention of intestinal carcinogenesis in a murine model of familial adenomatous polyposis. Nutrition and cancer, 60 Suppl 1, 30-5.
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  Familial adenomatous polyposis (FAP) is an autosomal dominantly inherited syndrome in humans. The Apc(Min/+) mouse, which expresses a mutant homolog of the adenomatous polyposis coli gene, is a model of FAP in humans. Treatment with the nonsteroidal anti-inflammatory drugs (NSAIDS) sulindac or celecoxib can suppress polyp development in FAP patients, but responses are generally transient and incomplete. Combination chemoprevention with the ornithine decarboxylase inhibitor difluoromethylornithine (DFMO) and either celecoxib or sulindac was evaluated in the Apc(Min/+) mouse. Combinations of DFMO and either NSAID reduced intestinal tumor number by more than 80% (P < 0.0001) compared to untreated controls. In addition to the dramatic reduction in tumor number, the combination of DFMO and sulindac reduced the development of high-grade intestinal adenomas compared to sulindac alone (P = 0.003). The fraction of high-grade intestinal adenomas remaining after treatment was similar for the combination of DFMO and celecoxib and celecoxib alone. Only combinations of DFMO plus sulindac reduced total intestinal polyamine contents compared to untreated mice. These data support the rationale for treatment of FAP patients postcolectomy with DFMO combined with either celecoxib or sulindac but indicate that sulindac may be more effective than celecoxib in reducing intestinal polyamine contents and the incidence of high-grade intestinal adenomas when combined with DFMO.
• Akporiaye, E. T., Bradley-Dunlop, D., Gendler, S. J., Mukherjee, P., Madsen, C. S., Hahn, T., Besselsen, D. G., Dial, S. M., Cui, H., & Trevor, K. (2007). Characterization of the MUC1.Tg/MIN transgenic mouse as a model for studying antigen-specific immunotherapy of adenomas. Vaccine, 25(39-40), 6965-6974.
• Akporiaye, E. T., Bradley-Dunlop, D., Gendler, S. J., Mukherjee, P., Madsen, C. S., Hahn, T., Besselsen, D. G., Dial, S. M., Cui, H., & Trevor, K. (2007). Characterization of the MUC1.Tg/MIN transgenic mouse as a model for studying antigen-specific immunotherapy of adenomas. Vaccine, 25(39-40), 6965-74.
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  A bigenic MUC1.Tg/MIN mouse model was developed by crossing Apc/(MIN/+) (MIN) mice with human MUC1 transgenic mice to evaluate MUC1 antigen-specific immunotherapy of intestinal adenomas. The MUC1.Tg/MIN mice developed adenomas at a rate comparable to that of MIN mice and had similar levels of serum MUC1 antigen. A MUC1-based vaccine consisting of MHC class I-restricted MUC1 peptides, a MHC class II-restricted pan-helper peptide, unmethylated CpG oligodeoxynucleotide and GM-CSF caused flattening of adenomas and significantly reduced the number of large adenomas. Immunization was successful in generating a MUC1-directed immune response evidenced by increased MUC1 peptide-specific anti-tumor cytotoxicity and IFN-gamma secretion by lymphocytes.
• Besselsen, D. G., Becker, M. D., Henderson, K. S., Wagner, A. M., Banu, L. A., & Shek, W. R. (2007). Temporal transmission studies of mouse parvovirus 1 in BALB/c and C.B-17/Icr-Prkdc(scid) mice. Comparative medicine, 57(1), 66-73.
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  Fecal shedding and transmission of mouse parvovirus 1 (MPV) to naive sentinels, breeding mates, and progeny were assessed. Neonatal SCID and BALB/c mice inoculated with MPV were evaluated over 24 wk; several mice from each strain were mated once during this period. Fecal MPV loads for each cage were determined weekly by quantitative polymerase chain reaction (PCR) analysis, and all mice were evaluated by quantitative PCR analysis of lymphoid tissues and seroconversion to MPV antigens in immunocompetent mice. Results indicated persistently high fecal shedding of MPV in SCID mice throughout the evaluation period sufficient to allow transmission to sentinels, naive breeding partners, and the progeny of infected male mice and naive partners. Lymphoid tissue viral loads in the progeny of infected female SCID mice were high at weaning but low at 6 wk of age. Infected BALB/c mice shed high levels of MPV in feces for 3 wk postinoculation, with seroconversion only in sentinels exposed during the first 2 wk postinoculation. Thereafter the feces of infected BALB/c mice and the lymphoid tissues of sentinels, naive breeding partners, and progeny intermittently contained extremely low levels of MPV DNA. Although pregnancy and lactation did not increase viral shedding in BALB/c mice, MPV exposure levels were sufficient to induce productive infection in some BALB/c progeny. These data indicate that the adaptive immune response suppresses, but does not eliminate, MPV shedding; this suppression is sufficient to inhibit infection of weanling and adult mice but allows productive infection of some progeny.
• Dvorak, K., Gerner, E. W., Payne, C. M., Padilla-torres, J. L., Ignatenko, N. A., Holubec, H., Gerner, E. W., Garewal, H. S., Dvorakova, B., Dvorak, K., Blohm-mangone, K. A., Besselsen, D. G., Bernstein, H., & Bernstein, C. (2007). Deoxycholate-induced colitis is markedly attenuated in Nos2 knockout mice in association with modulation of gene expression profiles.. Digestive diseases and sciences, 52(3), 628-42. doi:10.1007/s10620-006-9608-0
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  Nos2 knockout mice were compared to wild-type mice for susceptibility to colitis in response to a diet supplemented with deoxycholate, a bile acid increased in the colon of individuals on a high-fat diet. Wild-type mice fed a fat-related diet, supplemented with 0.2% DOC, develop colonic inflammation associated with increases in nitrosative stress, proliferation, oxidative DNA/RNA damage, and angiogenesis, as well as altered expression of numerous genes. However, Nos2 knockout mice fed a diet supplemented with deoxycholate were resistant to these alterations. In particular, 35 genes were identified whose expression was significantly altered at the mRNA level in deoxycholate-fed Nos2(+/+) mice but not in deoxycholate-fed Nos2(-/-) mice. Some of these alterations in NOS2-dependent gene expression correspond to those reported in human inflammatory bowel disease. Overall, our results indicate that NOS2 expression is necessary for the development of deoxycholate-induced colitis in mice, a unique dietary-related model of colitis.
• Gerner, E. W., Ziogas, A., Zell, J. A., Yerushalmi, H. F., Ignatenko, N. A., Gerner, E. W., Besselsen, D. G., & Anton-culver, H. (2007). Risk and risk reduction involving arginine intake and meat consumption in colorectal tumorigenesis and survival.. International journal of cancer, 120(3), 459-68. doi:10.1002/ijc.22311
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  Elevated polyamine and nitric oxide levels (both derived from arginine) promote tumorigenesis, whereas non-steroidal anti-inflammatory drugs (NSAIDs) inhibit colorectal cancer (CRC) incidence in experimental and epidemiologic studies. We investigated dietary arginine-induced intestinal tumorigenesis and NSAID-inhibitory effects in Apc(Min/+) mice differentially expressing nitric oxide synthase-2 (Nos2). We also studied effects of estimated arginine exposures through meat consumption on tumor characteristics and survival in human CRC cases. Dietary arginine increased high-grade colon adenoma incidence in Apc(Min/+)Nos2(+/+) mice, but not in Nos2 knockout mice. Additionally, celecoxib suppressed intestinal steady state ornithine decarboxylase RNA levels (p < 0.001), induced steady state spermidine/spermine N(1)-acetyltransferase RNA levels (p = 0.002), decreased putrescine levels (p = 0.04) and decreased tumor number in the small intestines of Apc(Min/+)Nos2(+/+) mice (p = 0.0003). Five hundred and eleven cases from our NCI-supported CRC gene-environment study were analyzed based on self-reported meat (as a surrogate for arginine) consumption. Familial CRC cases (n = 144) in the highest meat consumption quartile (Q4) had no statistically significant differences in tumor grade compared to cases in Q1-Q3 (p = 0.32); however, they were observed to have decreased overall survival (OS) (10-year OS = 42% vs. 65%; p = 0.017), and increased risk of death in an adjusted analysis (hazards ratio [HR] = 2.24; p = 0.007). No differences in tumor grade, OS or adjusted HR were observed for sporadic CRC cases (n = 367) based on meat consumption. Our results suggest important roles for arginine and meat consumption in CRC pathogenesis, and have implications for CRC prevention.
• Hariri, L. P., Qiu, Z., Tumlinson, A. R., Besselsen, D. G., Gerner, E. W., Ignatenko, N. A., Povazay, B., Hermann, B., Sattmann, H., McNally, J., Unterhuber, A., Drexler, W., & Barton, J. K. (2007). Serial endoscopy in azoxymethane treated mice using ultra-high resolution optical coherence tomography. Cancer biology & therapy, 6(11), 1753-62.
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  Optical coherence tomography (OCT) is a minimally invasive, depth-resolved imaging tool that can be implemented in a small diameter endoscope for imaging mouse models of colorectal cancer (CRC). In this study, we utilized ultrahigh resolution (UHR) OCT to serially image the lower colon of azoxymethane (AOM) treated A/J mouse models of CRC in order to monitor the progression of neoplastic transformations and determine if OCT is capable of identifying early disease.
• Hariri, L. P., Tumlinson, A. R., Wade, N. H., Besselsen, D. G., Utzinger, U., Gerner, E. W., & Barton, J. K. (2007). Ex vivo optical coherence tomography and laser-induced fluorescence spectroscopy imaging of murine gastrointestinal tract. Comparative medicine, 57(2), 175-85.
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  Optical coherence tomography (OCT) and laser-induced fluorescence (LIF) spectroscopy each have clinical potential in identifying human gastrointestinal (GI) pathologies, yet their diagnostic capability in mouse models is unknown. In this study, we combined the 2 modalities to survey the GI tract of a variety of mouse strains and ages and to sample dysplasias and inflammatory bowel disease (IBD) of the intestines. Segments (length, 2.5 cm) of duodenum and lower colon and the entire esophagus were imaged ex-vivo with combined OCT and LIE We evaluated 30 normal mice (A/J and 10- and 21-wk-old and retired breeder C57BL/6J) and 10 mice each of 2 strains modeling colon cancer and IBD (Apc(Min) and IL2-deficient mice, respectively). Histology was used to classify tissue regions as normal, Peyer patch, dysplasia, adenoma, or IBD. Features in corresponding OCT images were analyzed. Spectra from each category were averaged and compared via Student t tests. OCT provided structural information that led to identification of the imaging characteristics of healthy mouse GI. With histology as the 'gold standard,' we developed preliminary image criteria for early disease in the form of adenomas, dysplasias, and IBD. LIF characterized the endogenous fluorescence of mouse GI tract, with spectral features corresponding to collagen, NADH, and hemoglobin. In the IBD sample, LIF emission spectra displayed potentially diagnostic peaks at 635 and 670 nm, which we attributed to increased porphyrin production by bacteria associated with IBD. OCT and LIF appear to be useful and complementary modalities for ex vivo imaging of mouse GI tissues.
• Lickteig, A. J., Fisher, C. D., Augustine, L. M., Aleksunes, L. M., Besselsen, D. G., Slitt, A. L., Manautou, J. E., & Cherrington, N. J. (2007). Efflux transporter expression and acetaminophen metabolite excretion are altered in rodent models of nonalcoholic fatty liver disease. Drug metabolism and disposition: the biological fate of chemicals, 35(10), 1970-8.
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  Efflux transporters are responsible for the excretion of numerous xenobiotics and endobiotics and thus play an essential role in proper liver and kidney function. Nonalcoholic fatty liver diseases (NAFLDs) comprise a spectrum of disorders that range from simple fatty liver (SFL) to nonalcoholic steatohepatitis (NASH). Although the precise events leading to NAFLD are unclear, even less is known about the effects on efflux transporter expression and drug disposition. The purpose of this study was to determine the effect of NAFLD on efflux transporter expression in rat liver as well as on acetaminophen (APAP) metabolite excretion. To simulate SFL and NASH, rats were fed either a high-fat (HF) or a methionine- and choline-deficient (MCD) diet for 8 weeks. In the livers of MCD rats, there were striking increases in both mRNA and protein levels of multidrug resistance-associated protein (Mrp) 3, Mrp4, and breast cancer resistance protein, as well as increased Mrp2 protein. After administration of a nontoxic dose of APAP, biliary concentrations of APAP-sulfate, APAP-glucuronide (APAP-GLUC), and APAP-glutathione were reduced in MCD rats. The effects of the HF diet on both transporter expression and APAP disposition were by comparison far less dramatic than the MCD diet-induced alterations. Whereas APAP-sulfate levels were also decreased in MCD rat plasma, the levels of the Mrp3 substrate APAP-GLUC were elevated. Urinary elimination of APAP metabolites was identical between groups, except for APAP-GLUC, the concentration of which was 80% higher in MCD rats. These studies correlate increased hepatic Mrp3 protein in the MCD model of NASH with increased urinary elimination of APAP-GLUC. Furthermore, the proportional shift in elimination of APAP metabolites from bile to urine indicates that MCD-induced alterations in efflux transporter expression can affect the route of drug elimination.
• Dial, S. M., Walker, E. B., Ramanathapuram, L. V., Kobie, J. J., Hahn, T., Fulton, A. M., Dial, S. M., Besselsen, D. G., Alvarez, I., & Akporiaye, E. T. (2006). Short-term dietary administration of celecoxib enhances the efficacy of tumor lysate-pulsed dendritic cell vaccines in treating murine breast cancer.. International journal of cancer, 118(9), 2220-31. doi:10.1002/ijc.21616
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  Cyclooxygenase-2 (COX-2) is a rate-limiting enzyme in the synthesis of prostaglandins. It is over-expressed in multiple cancers and has been associated with diminished tumor immunity. Dendritic cells (DCs) are considered candidates for cancer immunotherapy due to their ability to process and present antigens to T cells and stimulate immune responses. However, DC-based vaccines have exhibited minimal effectiveness against established tumors. In this study, we evaluated the effect of short-term administration of the selective COX-2 inhibitor celecoxib on the efficacy of DC-based vaccines in preventing and treating established 4T1 murine mammary tumors. We show that dietary celecoxib alone significantly suppresses the growth of primary tumors and the incidence of lung metastases in the prophylactic setting but is less effective against pre-established tumors. However, we demonstrate that celecoxib administered after primary tumor establishment synergizes with tumor lysate-pulsed DC and the adjuvant, GM-CSF, to improve the antitumor immune response by suppressing primary tumor growth and markedly reducing the occurrence of lung metastases. This triple combination therapy elicits a tumor-specific immune response evidenced by elevated IFN-gamma and IL-4 secretion by CD4+ T cells and results in increased infiltration of CD4+ and CD8+ T cells to the tumor site. In addition, dietary celecoxib inhibits angiogenesis evidenced by decreased vascular proliferation within the tumor and serum vascular endothelial growth factor levels. These studies suggest that short-term celecoxib therapy in combination with DC vaccines may be safely used for treating metastatic breast cancer.
• Dvorak, K., Gerner, E. W., Ramsey, L., Payne, C. M., Padilla-torres, J. L., Ignatenko, N. A., Holubec, H., Gerner, E. W., Garewal, H. S., Dvorak, K., Dall'agnol, M., Cui, H., Blohm-mangone, K. A., Besselsen, D. G., Bernstein, H., & Bernstein, C. (2006). Unique dietary-related mouse model of colitis.. Inflammatory bowel diseases, 12(4), 278-93. doi:10.1097/01.mib.0000209789.14114.63
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  A high-fat diet is a risk factor for the development of inflammatory bowel disease (IBD) in humans. Deoxycholate (DOC) is increased in the colonic contents in response to a high-fat diet. Thus, an elevated level of DOC in the colonic lumen may play a role in the natural course of development of IBD..Wild-type B6.129 mice were fed an AIN-93G diet, either supplemented with 0.2% DOC or unsupplemented and sacrificed at 1 week, 1 month, 3 months, 4 months, and 8 months. Colon samples were assessed by histopathological, immunohistochemical, and cDNA microarray analyses..Mice fed the DOC-supplemented diet developed focal areas of colonic inflammation associated with increases in angiogenesis, nitrosative stress, DNA/RNA damage, and proliferation. Genes that play a central role in inflammation and angiogenesis and other related processes such as epithelial barrier function, oxidative stress, apoptosis, cell proliferation/cell cycle/DNA repair, membrane transport, and the ubiquitin-proteasome pathway showed altered expression in the DOC-fed mice compared with the control mice. Changes in expression of individual genes (increases or reductions) correlated over time. These changes were greatest 1 month after the start of DOC feeding..The results suggest that exposure of the colonic mucosa to DOC may be a key etiologic factor in IBD. The DOC-fed mouse model may reflect the natural course of development of colitis/IBD in humans, and thus may be useful for determining new preventive strategies and lifestyle changes in affected individuals.
• Gerner, E. W., Padilla-torres, J. L., Nagle, R. B., Ignatenko, N. A., Holubec, H., Guillen-r, J. M., Gerner, E. W., Blohm-mangone, K. A., Besselsen, D. G., & Alboranc, I. M. (2006). Role of c-Myc in intestinal tumorigenesis of the ApcMin/+ mouse.. Cancer biology & therapy, 5(12), 1658-64. doi:10.4161/cbt.5.12.3376
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  The c-MYC oncogene plays an important role in tumorigenesis and is commonly highly expressed in gastrointestinal cancers. In colon cells, c-MYC is regulated by the adenomatous polyposis coli (Apc) tumor suppressor gene. Multiple intestinal neoplasia (ApcMin/+ or Min) mice are heterozygous for a truncating Apc mutation and serve as a model of familial adenomatous polyposis (FAP) disease. To study the role of c-Myc in the mutant Apc-mediated colon tumorigenesis, we have developed a transgenic mouse with the conditional deletion of the floxed c-Myc alleles in the intestinal crypts of ApcMin/+ mice (ApcMin/+; c-Mycfl/fl). The floxed c-Myc deletion was initiated via a Cre recombinase controlled by the intestine-specific transcriptional regulatory elements of the liver fatty acid-binding protein gene (Fabpl4xat-132). Fabpl4xat-132-mediated Cre expression and recombination resulted in a two-fold decrease in c-MYC protein expression with no effect on intestinal tract morphology. Small intestinal tumorigenesis was significantly suppressed throughout the small intestinal tract of ApcMin/+; c-Mycfl/fl mice compared to c-Myc wild type littermates. In ApcMin/+; c-Mycfl/fl mice, the intestinal apoptosis was higher in the areas of the small intestine with the decreased c-Myc protein expression (P=0.0016, compared to their littermates with the wild type c-Myc). Thus, conditional inactivation of c-Myc, mediated by Fabpl4xat-132-driven Cre-recombinase, suppresses Apc-dependent intestinal tumorigenesis in adult ApcMin/+ mice, without apparent effect on normal intestinal mucosa.
• Gerner, E. W., Utzinger, U., Tumlinson, A. R., Hariri, L. P., Gerner, E. W., Besselsen, D. G., & Barton, J. K. (2006). Endoscopic optical coherence tomography and laser-induced fluorescence spectroscopy in a murine colon cancer model.. Lasers in surgery and medicine, 38(4), 305-13. doi:10.1002/lsm.20305
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  The diagnostic feasibility of optical coherence tomography (OCT) and laser-induced fluorescence (LIF) have been evaluated for human colorectal cancer. This study applies these technologies to a murine model of colorectal adenoma..The lower colon of 10 Apc(Min) and two C57BL/6J mice was surveyed over five 4-week intervals using a prototype 2.0 mm diameter OCT-LIF endoscope-based system. Four categories were histologically classified: control C57BL/6J, adenomatous, non-diseased regions of adenomatous, and non-diseased Apc(Min). OCT images were compared to histology. Spectra from the four categories were compared via the Student's t-test..Three Apc(Min) and two control mice completed the study. One adenoma was histologically identified; OCT visualized mucosal thickening/abnormal mass development over the imaging timepoints. LIF spectral comparisons revealed decreased 405 nm intensity and the presence of a peak at 680 nm in the adenomatous Apc(Min)..These preliminary data indicate endoscopic OCT-LIF has the potential to identify colorectal adenomas in murine models.
• Gerner, E. W., Utzinger, U., Tumlinson, A. R., Mcnally, J. B., Kirkpatrick, N. D., Hariri, L. P., Gerner, E. W., Besselsen, D. G., & Barton, J. K. (2006). Task-based imaging of colon cancer in the Apc(Min/+) mouse model.. Applied optics, 45(13), 3049-62. doi:10.1364/ao.45.003049
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  Optical coherence tomography (OCT), laser-induced fluorescence (LIF), and laser-scanning confocal microscopy (LSCM) were used for the task of multimodal study of healthy and adenomatous mouse colon. The results from each modality were compared with histology, which served as the gold standard. The Apc(Min/+) genetic mouse model of colon cancer was compared with wild-type mice. In addition, a special diet was used for the task of studying the origins of a 680 nm autofluorescent signal that was previously observed in colon. The study found close agreement among each of the modalities and with histology. All four modalities were capable of identifying diseased tissue accurately. The OCT and LSCM images provided complementary structural information about the tissue, while the autofluorescence signal measured by LIF and LSCM provided biochemical information. OCT and LIF were performed in vivo and nondestructively, while the LSCM and histology required extraction of the tissue. The magnitude of the 680 nm signal correlates with chlorophyll content in the mouse diet, suggesting that the autofluorescent compound is a dietary metabolite.
• Gerner, E. W., Yerushalmi, H. F., Stringer, D. E., Payne, C. M., Padilla-torres, J. L., Ignatenko, N. A., Holubec, H., Gerner, E. W., Cui, H., Blohm-mangone, K. A., & Besselsen, D. G. (2006). The role of NO synthases in arginine-dependent small intestinal and colonic carcinogenesis.. Molecular carcinogenesis, 45(2), 93-105. doi:10.1002/mc.20168
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  Arginine is catabolized by NOS2 and other nitric oxide synthases to form nitric oxide. We evaluated the roles of dietary arginine and Nos2 in Apc-dependent intestinal tumorigenesis in Min mice with and without a functional Nos2 gene. NOS2 protein was expressed only in intestinal tissues of Apc(Min/+) Nos2+/+ mice. NOS3 expression was higher in intestinal tissues of mice lacking Nos2, mainly in the small intestine. When diet was supplemented with arginine (0.2% and 2% in drinking water), lack of Nos2 results in decreased tumorigenesis in both small intestine and colon. In Nos2 knockout mice, supplemental arginine (up to 2%) caused a decrease in small intestinal tumor number and size. The arginine-dependent decrease was associated with an increase in nitrotyrosine formation and apoptosis in the region of intestinal stem cells. Mice expressing Nos2 did not show these changes. These mice did, however, show an arginine-dependent increase in colon tumor number and incidence, while no effect on apoptosis was seen. These changes were associated with increased nitrotyrosine formation in epithelial cells. Mice lacking Nos2 did not show changes in tumorigenesis or nitrotyrosine formation, while demonstrating an arginine-dependent increase in apoptosis. These data suggest that Nos2 and dietary arginine have significant effects on intestinal and colonic tumorigenesis in Min mice. In both tissues, loss of Nos2 is associated with decreased tumorigenesis when mice are supplemented with dietary arginine. In the small intestine, Nos2 prevents the arginine-induced decrease in tumor number and size, which is associated with NOS3 expression and increased apoptosis. In the colon, Nos2 is required for the arginine-induced increase in tumor number and incidence.
• Gerner, E. W., Yerushalmi, H. F., Stringer, D. E., Payne, C. M., Padilla-torres, J. L., Ignatenko, N. A., Holubec, H., Guillen, J. M., Gerner, E. W., Blohm-mangone, K. A., & Besselsen, D. G. (2006). Role of polyamines in arginine-dependent colon carcinogenesis in Apc(Min) (/+) mice.. Molecular carcinogenesis, 45(10), 764-73. doi:10.1002/mc.20246
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  We evaluated the role of polyamines in arginine-dependent intestinal tumorigenesis in Apc(Min) (/+) mice. Arginine is a substrate for ornithine synthesis and thus can influence polyamine production. Supplementing the diet with arginine increased intestinal and colonic polyamine levels and colonic carcinogenesis. Inhibiting polyamine synthesis with D,L-alpha-diflouromethylornithine (DFMO) decreased small intestinal and colonic polyamine pools. In mice provided basal diet, but not when supplemented with arginine, DFMO decreased small intestinal tumor number and burden, and increased intestinal apoptosis. In mice provided supplemental arginine in the diet, DFMO induced late apoptosis and decreased tumorigenesis in the colon. DFMO slightly reduced tumor incidence, number, and size while significantly decreasing tumor burden and grade. These changes in colon tumorigenesis did not occur in mice not provided supplemental arginine. Our study indicates that polyamines play unique roles in intestinal and colonic carcinogenesis in Apc(Min) (/+) mice. Inhibition of polyamine synthesis suppresses the arginine-dependent risk of colon tumorigenesis, resulting in apoptosis induction and decreased tumorigenesis, in this murine model.
• Ignatenko, N. A., Besselsen, D. G., Roy, U. K., Stringer, D. E., Blohm-Mangone, K. A., Padilla-Torres, J. L., Guillen-R, J. M., & Gerner, E. W. (2006). Dietary putrescine reduces the intestinal anticarcinogenic activity of sulindac in a murine model of familial adenomatous polyposis. Nutrition and cancer, 56(2), 172-81.
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  The nonsteroidal antiinflammatory drug sulindac displays chemopreventive activity in patients with familial adenomatous polyposis (FAP). Sulindac metabolites induce apoptosis in colon tumor cells, in part, by a polyamine-dependent mechanism that can be suppressed with exogenous putrescine. To determine the relevance of this mechanism in animals, we treated Apc(Min/+) mice, a model of human FAP, with sulindac alone or in combination with dietary putrescine. Sulindac increased steady-state RNA levels and enzymatic activity of the polyamine catabolic enzyme spermidine/spermine N(1)-acetyltransferase and intestinal levels of monoacetylspermidine, spermidine, and spermine in the small intestine of mice. Sulindac also decreased the activity of the biosynthetic enzyme ornithine decarboxylase but not adenosylmethionine decarboxylase (AMD). Dietary putrescine increased intestinal putrescine contents, whereas the combination of dietary putrescine and sulindac yielded the highest levels of intestinal putrescine and correlated with a statistically significant reduction in AMD enzyme activity. Dietary putrescine did not statistically significantly increase tumorigenesis, although it significantly increased the grade of adenoma dysplasia (P < 0.05). The effectiveness of sulindac to suppress intestinal carcinogenesis was partially abrogated by dietary putrescine. These data suggest that sulindac exerts at least some of its anticarcinogenic effects in mice via a polyamine-dependent mechanism. Because high concentrations of putrescine can be found in certain dietary components, it may be advantageous to restrict dietary putrescine consumption in patients undergoing treatment with sulindac.
• Wagner, A. M., Romero, M. J., Livingston, R. S., Henderson, K. S., & Besselsen, D. G. (2006). Identification of novel murine parvovirus strains by epidemiological analysis of naturally infected mice.. The Journal of general virology, 87(Pt 6), 1543-1556. doi:10.1099/vir.0.81547-0
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  Random-source DNA samples obtained from naturally infected laboratory mice (n=381) were evaluated by PCR and RFLP analysis to determine the prevalence of murine parvovirus strains circulating in contemporary laboratory mouse colonies. Mouse parvovirus (MPV) was detected in 77% of samples, Minute virus of mice (MVM) was detected in 16% of samples and both MVM and MPV were detected in 7% of samples. MVMm, a strain recently isolated from clinically ill NOD-mu chain knockout mice, was detected in 91% of MVM-positive samples, with the Cutter strain of MVM (MVMc) detected in the remaining samples. The prototypic and immunosuppressive strains of MVM were not detected in any of the samples. MPV-1 was detected in 78% of the MPV-positive samples and two newly identified murine parvoviruses, tentatively named MPV-2 and MPV-3, were detected in 21 and 1% of the samples, respectively. The DNA sequence encompassing coding regions of the viral genome and the predicted protein sequences for MVMm, MPV-2 and MPV-3 were determined and compared with those of other rodent parvovirus strains and LuIII parvovirus. The genomic organization for the newly identified viral strains was similar to that of other rodent parvoviruses, and nucleotide sequence identities indicated that MVMm was most similar to MVMc (96.1%), MPV-3 was most similar to hamster parvovirus (HaPV) (98.1%) and MPV-2 was most similar to MPV-1 (95.3%). The genetic similarity of MPV-3 and HaPV suggests that HaPV epizootics in hamsters may result from cross-species transmission, with mice as the natural rodent host for this virus.
• Besselsen, D., Loganbill, J. K., Wagner, A. M., & Besselsen, D. G. (2005). Detection of Mycoplasma pulmonis by fluorogenic nuclease polymerase chain reaction analysis. Comparative medicine, 55(5).
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  Mycoplasma pulmonis induces persistent infections in laboratory mice and rats and can contaminate biological materials. We developed a fluorogenic nuclease polymerase chain reaction (fnPCR) assay to detect M. pulmonis specifically. Primer and probe sequences for the assay were targeted to 16S rRNA sequences specific to M. pulmonis. The assay consistently detected the equivalent of fewer than 10 copies of template DNA. When evaluated against a panel of 24 species of bacteria, the M. pulmonis assay detected only M. pulmonis isolates. Evaluation of 10-fold serial dilutions of cultured M. pulmonis showed that the M. pulmonis fnPCR assay and culture on Dutch agar had comparable sensitivity in detecting viable M. pulmonis organisms, whereas the mouse antibody production test displayed positive serologic results at dilutions higher than those in which viable organisms could be detected. Finally, the M. pulmonis fnPCR assay was able to detect M. pulmonis DNA in nasopharyngeal wash fluid and trachea, lung, and uterus tissue collected from mice naturally infected with M. pulmonis but did not detect the organism in similar samples collected from uninfected, negative control mice. The M. pulmonis fnPCR assay provides a high-throughput, PCR-based method to detect M. pulmonis in infected rodents and contaminated biological materials.
• Gerner, E. W., Padilla-tores, J. L., Nagle, R. B., Ignatenko, N. A., Holubec, H., Gerner, E. W., Dexter, E. I., Blohm-mangone, K. A., & Besselsen, D. G. (2005). Role of c-MYC in normal intestinal development and tumorigenesis in the ApcMin/+ mouse. Cancer Research, 65, 464-464.
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  Proc Amer Assoc Cancer Res, Volume 46, 2005 1982 The c-Myc oncogene plays an important role in tumorigenesis and is commonly activated in gastrointestinal cancers. In human colon tumor cells, c-Myc is regulated by the adenomatous polyposis coli (APC) tumor suppressor gene, which is mutated or deleted in humans with familial adenomatous polyposis (FAP). The ApcMin/+ (Min) mice are a model of FAP and are heterozygous for a truncating Apc mutation. We have developed a mouse model for studying the role of c-myc in the mutant APC-mediated colon tumorigenesis. Mice harboring a transgene containing the Cre recombinase under the control of transcriptional regulatory elements from a fatty acid-binding protein gene ( Fabp 4×at132) were crossed with mice homozygous for the c-mycflox allele. Progeny were then interbred to initiate the deletion of loxP flanked c-myc sequences in rapidly self-renewing mouse small intestinal and colonic epithelium. Cre expression and recombination in the transgenic c-myc null mouse ( Fabp 4×at 132 Cre transgenic mice x c-myc null) resulted in mosaic loss of the expression of the c-myc protein in ileum, cecum and colon tissues, in which the Fabp4×at 132 promoter is active. Conditional c-myc null mice exhibited underdeveloped intestinal crypts and villous atrophy. The results of recombination were evaluated microscopically using methylene-blue stained whole-mount preparations and were confirmed by morphology in H&E stained sections and by immunohistochemical analysis using c-MYC antibody. We crossed the Fabp4×at 132Cre c-myc null mouse to ApcMin/+ and then to their progeny - to create the ApcMin/+ mouse with intestinal specific knockout of the c-myc oncogene ( ApcMin/+ c-myc null). The compound ApcMin/+ c-myc null mouse was used to evaluate the effect of colon-specific c-myc knockout on the ApcMin/+ phenotype. We observed a relative reduction in the tumor number in the small intestine in ApcMin/+ c-myc conditional knockout mice compared to control ApcMin/+ mice. These data demonstrate that c-Myc is essential for normal development of intestinal epithelia and impacts either directly or indirectly Apc-dependent intestinal tumorigenesis in mice.
• Gerner, E. W., Utzinger, U., Tumlinson, A. R., Mcnally, J. B., Kirkpatrick, N. D., Hariri, L. P., Gerner, E. W., Besselsen, D. G., & Barton, J. K. (2005). Imaging of Colon Cancer in a Mouse Model (ApcMin). Frontiers in Optics. doi:10.1364/fio.2005.fmc3
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  Healthy and adenomatous mouse colon were studied using optical coherence tomography (OCT), laser induced fluorescence (LIF) and laser scanning confocal microscopy (LSCM). All three modalities were accurately able to identify diseased tissue.
• Gerner, E. W., Wade, N. H., Utzinger, U., Tumlinson, A. R., Hariri, L. P., Gerner, E. W., Besselsen, D. G., & Barton, J. K. (2005). Ex vivo optical coherence tomography and laser induced fluorescence spectroscopy imaging of murine gastrointestinal tract. Biomedical optics, 5692, 295-306. doi:10.1117/12.588768
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  Optical Coherence Tomography (OCT) and Laser Induced Fluorescence Spectroscopy (LIF) have separately been found to have clinical potential in identifying human gastrointestinal (GI) pathologies, yet their diagnostic capability in mouse models of human disease is unknown. We combine the two modalities to survey the GI tract of a variety of mouse strains and sample dysplasias and inflammatory bowel disease (IBD) of the small and large intestine. Segments of duodenum and lower colon 2.5 cm in length and the entire esophagus from 10 mice each of two colon cancer models (ApcMin and AOM treated A/J) and two IBD models (Il-2 and Il-10) and 5 mice each of their respective controls were excised. OCT images and LIF spectra were obtained simultaneously from each tissue sample within 1 hour of extraction. Histology was used to classify tissue regions as normal, Peyer’s patch, dysplasia, adenoma, or IBD. Features in corresponding regions of OCT images were analyzed. Spectra from each of these categories were averaged and compared via the student's t-test. Features in OCT images correlated to histology in both normal and diseased tissue samples. In the diseased samples, OCT was able to identify early stages of mild colitis and dysplasia. In the sample of IBD, the LIF spectra displayed unique peaks at 635nm and 670nm, which were attributed to increased porphyrin production in the proliferating bacteria of the disease. These peaks have the potential to act as a diagnostic for IBD. OCT and LIF appear to be useful and complementary modalities for imaging mouse models.
• Timmermann, B. N., Solyom, A. M., Midura, A. J., Kuscuoglu, N., Kiela, P. R., Jolad, S. D., Ghishan, F. K., & Besselsen, D. G. (2005). Effects of Boswellia serrata in mouse models of chemically induced colitis.. American journal of physiology. Gastrointestinal and liver physiology, 288(4), G798-808. doi:10.1152/ajpgi.00433.2004
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  Extracts from Boswellia serrata have been reported to have anti-inflammatory activity, primarily via boswellic acid-mediated inhibition of leukotriene synthesis. In three small clinical trials, boswellia was shown to improve symptoms of ulcerative colitis and Crohn's disease, and because of its alleged safety, boswellia was considered superior over mesalazine in terms of a benefit-risk evaluation. The goal of this study was to evaluate the effectiveness of boswellia extracts in controlled settings of dextran sulfate- or trinitrobenzene sulfonic acid-induced colitis in mice. Our results suggest that boswellia is ineffective in ameliorating colitis in these models. Moreover, individual boswellic acids were demonstrated to increase the basal and IL-1beta-stimulated NF-kappaB activity in intestinal epithelial cells in vitro as well as reverse proliferative effects of IL-1beta. We also observed hepatotoxic effect of boswellia with pronounced hepatomegaly and steatosis. Hepatotoxity and increased lipid accumulation in response to boswellia were further confirmed in vitro in HepG2 cells with fluorescent Nile red binding/resazurin reduction assay and by confocal microscopy. Microarray analyses of hepatic gene expression demonstrated dysregulation of a number of genes, including a large group of lipid metabolism-related genes, and detoxifying enzymes, a response consistent with that to hepatotoxic xenobiotics. In summary, boswellia does not ameliorate symptoms of colitis in chemically induced murine models and, in higher doses, may become hepatotoxic. Potential implications of prolonged and uncontrolled intake of boswellia as an herbal supplement in inflammatory bowel disease and other inflammatory conditions should be considered in future clinical trials with this botanical.
• Besselsen, D., Wagner, A. M., Loganbill, J. K., & Besselsen, D. G. (2004). Detection of lactate dehydrogenase-elevating virus by use of a fluorogenic nuclease reverse transcriptase-polymerase chain reaction assay. Comparative medicine, 54(3).
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  Lactate dehydrogenase-elevating virus (LDEV) induces persistent infections in laboratory mice, alters in vivo physiology, and is a common contaminant of biological materials such as transplantable tumor cell lines. The fluorogenic nuclease reverse transcriptase polymerase chain reaction (fnRT-PCR) assay combines RT-PCR analysis with an internal fluorogenic hybridization probe, thereby eliminating post-PCR processing and potentially enhancing specificity. An fnRT-PCR assay specific for LDEV was therefore developed by targeting primer and probe sequences to a unique region of the LDEV nucleocapsid (VP1) gene. Using the LDEV fnRT-PCR assay, we detected only LDEV and did not detect other RNA viruses that are capable of naturally infecting rodents. Using this assay, we detected as little as 10 fg of LDEV RNA; the assay was 10-fold less sensitive when directly compared with the mouse bioassay (measurement of serum LD after inoculation), without the problematic false-positive serum LD enzyme elevations associated with the mouse bioassay. Using the fnRT-PCR assay, we also were able to detect viral RNA in numerous tissues and in feces collected from experimentally inoculated C3H/HeN mice, but we did not detect any viral RNA in similar samples collected from age- and strain-matched mock-infected mice. Finally, using the fnRT-PCR assay, we were able to detect LDEV RNA in biological samples that had previously been determined to be contaminated with LDEV by use of the mouse bioassay and an RT-PCR assay at another laboratory. In conclusion, the LDEV fnRT-PCR assay is a potentially high-throughput diagnostic assay for detection of LDEV in mice and contaminated biological materials.
• Gerner, E. W., Zhang, H., Yerushalmi, H. F., Yamauchi, P., Payne, C. M., Padilla-torres, J. L., Ignatenko, N. A., Holubec, H., Gerner, E. W., Blohm-mangone, K. A., & Besselsen, D. G. (2004). Gene-diet interactions in arginine-dependent intestinal carcinogenesis. Cancer Research, 64, 900-900.
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  3904 L-arginine, an essential amino acid, is converted by arginase to ornithine, the substrate for polyamine synthesis. Polyamines (putrescine, spermidine and spermine) are small ubiquitous molecules involved in various processes and required for cell proliferation. L-arginine is also metabolized by nitric oxide synthases (NOSs) to form nitric oxide (NO) that can influence signal transduction and DNA damage. NOSs are found as constitutively expressed isoforms (NOS1/NOS3) or the inducible isoform (NOS2). In this study, we evaluated NOS2 and dietary L-arginine effects on intestinal tumorgenesis in Min (multiple intestinal neoplasia) mice. Min mice were crossed with NOS2 knockout mice or wild-type NOS2 mice. NOS2 protein was expressed in small intestine, but not the colon, of Min X NOS2 (+/+) mice. The protein was not detected in either intestinal tissue of Min X NOS2 (−/−) mice. When animals were fed an arginine-restricted diet, loss of NOS2 alleles was associated with a small (50% or less) increase in small intestinal tumor number and colonic tumor incidence, compared to Min X NOS2 (+/+) mice. Supplemental arginine (up to 2% in the drinking water) slightly decreased small intestinal tumor number, in a concentration-dependent manner, in Min X NOS2 (−/−) mice. In mice expressing NOS2, intestinal tumor number was minimally affected by dietary arginine. In these mice, dietary arginine increased small intestinal arginine and ornithine contents, whereas in Min X NOS2 (−/−) mice no changes were observed. Colon tumor incidence showed an increase with dietary arginine, independent of NOS2 expression. This increase can result from dietary-arginine dependent increase in ornithine and putrescine contents. These data suggest that NOS2 expression and dietary arginine have only minimal effects on small intestinal carcinogenesis in the Min mouse. However, dietary arginine increases the incidence of colonic tumors, resulting from lack of expression of the adenomatous polyposis coli (APC) gene, by a NOS2-independent mechanism that may involve polyamines.
• Besselsen, D., Gerner, E. W., Ignatenko, N. A., & Besselsen, D. G. (2003). Preclinical models for chemoprevention of colon cancer. Recent results in cancer research. Fortschritte der Krebsforschung. Progrès dans les recherches sur le cancer, 163.
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  Colon cancer is the second leading cause of cancer incidence and death in the USA in 2002. Specific genetic defects have been identified which cause hereditary colon cancers in humans. In addition, a number of intestinal luminal risk factors for colon cancer have been described. This information has been exploited to develop experimental cell and rodent models which recapitulate features of human colon cancer. In this chapter, we will discuss the strengths and limitations of these models to further our understanding of basic mechanisms of colon carcinogenesis and to develop strategies for colon cancer chemoprevention.
• Besselsen, D., Uchiyama, A., & Besselsen, D. G. (2003). Detection of Reovirus type 3 by use of fluorogenic nuclease reverse transcriptase polymerase chain reaction. Laboratory animals, 37(4).
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  Reovirus type 3 (Reo-3) can infect numerous rodent species and induces the clinical syndrome 'oily skin disease' in neonatal mice, and is a common contaminant of biological materials. The reverse transcriptase polymerase chain reaction (RT-PCR) assay has proven useful for the detection of Reo-3 in rodents and contaminated biological materials. Fluorogenic nuclease reverse transcriptase polymerase chain reaction assays (fnRT-PCR) combine RT-PCR with an internal fluorogenic hybridization probe, thereby potentially enhancing specificity and eliminating post-PCR processing. Therefore, an fnRT-PCR assay specific for Reo-3 was developed by targeting primer and probe sequences to a unique region of the Reo-3 M3 gene. The fnRT-PCR detected both strains of Reo-3 (Dearing and Abney), but did not detect Reovirus types 1 or 2, other viruses in the family Reoviridae, or other RNA viruses that naturally infect rodents. The fnRT-PCR detected less than 1 fg of target template and detected viral RNA in tissues obtained from mice experimentally infected with Reo-3. The assay also displayed comparable sensitivity when compared to the mouse antibody production test commonly used to detect viral contamination of biological materials. In conclusion, this fnRT-PCR assay offers a potentially high-throughput diagnostic assay for detecting Reo-3 RNA in infected mice and contaminated biological materials.
• Besselsen, D., Wagner, A. M., Loganbill, J. K., & Besselsen, D. G. (2003). Detection of sendai virus and pneumonia virus of mice by use of fluorogenic nuclease reverse transcriptase polymerase chain reaction analysis. Comparative medicine, 53(2).
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  Sendai virus may induce acute respiratory tract disease in laboratory mice and is a common contaminant of biological materials. Pneumonia virus of mice (PVM) also infects the respiratory tract and, like Sendai virus, may induce a persistent wasting disease syndrome in immunodeficient mice. Reverse transcriptase-polymerase chain reaction (RT-PCR) assays have proven useful for detection of Sendai virus and PVM immunodeficient animals and contaminated biomaterials. Fluorogenic nuclease RT-PCR assays (fnRT-PCR) combine RT-PCR with an internal fluorogenic hybridization probe, thereby potentially enhancing specificity and eliminating post-PCR processing. Therefore, fnRT-PCR assays specific for Sendai virus and PVM were developed by targeting primer andprobe sequences to unique regions of the Sendai virus nucleocapsid (NP) gene and the PVM attachment (G) gene, respectively. The Sendai virus and PVM fnRT-PCR assays detected only Sendai virusand PVM , respectively. Neither assay detected other viruses of the family Paramyxoviridae or other RNA viruses that naturally infect rodents. The fnRT-PCR assays detected as little as 10 fg of Sendai virus RNA and one picogram of PVM RNA, respectively, andthe Sendai virus fnRT-PCR assay had comparable sensitivity when directly compared with the mouse antibody production test. The fnRT-PCR assays were also able to detect viral RNA in respiratory tract tissues and cage swipe specimens collected from experimentally inoculated C.B-17 severe combined immunodeficient mice, but did not detect viral RNA in age- and strain-matched mock-infected mice. In conclusion, these fnRT-PCR assays offer potentially high-throughput diagnostic assays to detect Sendai virus and PVM in immunodeficient mice, and to detect Sendai virus in contaminated biological materials.
• Doetschman, T., Yang, K., Washington, K., Ward, J. M., Russell, R. L., Pretlow, T. P., Pitot, H. C., Itzkowitz, S. H., Halberg, R. B., Groden, J., Godfrey, V. L., Dove, W. F., Doetschman, T., Coffey, R. J., Boivin, G. P., & Besselsen, D. G. (2003). Pathology of mouse models of intestinal cancer: consensus report and recommendations.. Gastroenterology, 124(3), 762-77. doi:10.1053/gast.2003.50094
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  GREGORY P. BOIVIN,* KAY WASHINGTON,‡ KAN YANG,§ JERROLD M. WARD, THERESA P. PRETLOW,¶ ROBERT RUSSELL,# DAVID G. BESSELSEN,** VIRGINIA L. GODFREY,‡‡ TOM DOETSCHMAN,§§ WILLIAM F. DOVE, HENRY C. PITOT, RICHARD B. HALBERG, STEVEN H. ITZKOWITZ,## JOANNA GRODEN,§§ and ROBERT J. COFFEY¶¶ *Department of Pathology and Laboratory Medicine, University of Cincinnati and Cincinnati Veterans Affairs Medical Center, Cincinnati, Ohio; ‡Department of Pathology, Vanderbilt University Medical Center, Nashville, Tennessee; §Strang Cancer Research Laboratory at Rockefeller University, New York, New York; National Cancer Institute, Frederick, Maryland; ¶Institute of Pathology, Case Western Reserve University, Cleveland, Ohio; #Lombardi Cancer Center, Georgetown University, Washington, DC; **University Animal Care, University of Arizona, Tucson, Arizona; ‡‡Division of Laboratory Animal Medicine, University of North Carolina, Chapel Hill, North Carolina; §§Department of Molecular Genetics, Biochemistry, and Microbiology, University of Cincinnati College of Medicine, Cincinnati, Ohio; McArdle Laboratory for Cancer Research, University of Wisconsin, Madison, Wisconsin; ¶¶Medicine and Cell and Developmental Biology, Vanderbilt University Medical Center and Nashville Veterans Association, Nashville, Tennessee; and ##Division of Gastroenterology, Mount Sinai School of Medicine, New York, New York
• Besselsen, D., Drazenovich, N. L., Franklin, C. L., Livingston, R. S., & Besselsen, D. G. (2002). Detection of rodent Helicobacter spp. by use of fluorogenic nuclease polymerase chain reaction assays. Comparative medicine, 52(4).
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  Polymerase chain reaction (PCR) analysis is the standard method for detection of Helicobacter spp. infections in laboratory rodents, with H. hepaticus, H. bilis, and H. typhlonius considered primary pathogens. Fluorogenic nuclease PCR assays that detect all known rodent Helicobacter spp., or that specifically detect H. hepaticus, H. bilis, or H. typhlonius were developed to eliminate post-PCR processing, enhance specificity, and provide quantitative data on starting template concentration. Each fluorogenic PCR assay detected a minimum of 10 copies of target template, had comparable or greater sensitivity when compared directly with corollary gel detection PCR assays, and detected only targeted species when numerous Helicobacter spp. and other enteric bacteria were analyzed. Fluorogenic nuclease PCR analysis of fecal DNA samples obtained from numerous laboratory mice sources detected all samples with positive results by use of Helicobacter spp., H. hepaticus, H. bilis, and/or H. typhlonius gel detection PCR analysis, except for one sample that had positive results by H. typhlonius gel detection PCR but negative results by H. typhlonius fluorogenic nuclease PCR analysis. Among fecal DNA samples that were Helicobacter spp. negative by use of all gel detection PCR assays, the fluorogenic nuclease PCR assays detected target template in only one sample that was positive by use of the Helicobacter spp. and the H. bilis fluorogenic nuclease PCR assays. In conclusion, fluorogenic nuclease PCR assays provide sensitive, specific, and high-throughput diagnostic assays for detection of Helicobacter spp., H. hepaticus, H. bilis, and H. typhlonius in laboratory rodents, and the quantitative data generated by these assays make them potentially useful for bacterial load determination.
• Besselsen, D., Johnson, P. D., & Besselsen, D. G. (2002). Practical aspects of experimental design in animal research. ILAR journal / National Research Council, Institute of Laboratory Animal Resources, 43(4).
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  A brief overview is presented of the key steps involved in designing a research animal experiment, with reference to resources that specifically address each topic of discussion in more detail. After an idea for a research project is conceived, a thorough review of the literature and consultation with experts in that field are pursued to refine the problem statement and to assimilate background information that is necessary for the experimental design phase. A null and an alternate hypothesis that address the problem statement are then formulated, and only then is the specific design of the experiment developed. Likely the most critical step in designing animal experiments is the identification of the most appropriate animal model to address the experimental question being asked. Other practical considerations include defining the necessary control groups, randomly assigning animals to control/treatment groups, determining the number of animals needed per group, evaluating the logistics of the actual performance of the animal experiments, and identifying the most appropriate statistical analyses and potential collaborators experienced in the area of study. All of these factors are critical to designing an experiment that will generate scientifically valid and reproducible data, which should be considered the ultimate goal of any scientific investigation.
• Gerner, E. W., Ignatenko, N. A., Gerner, E. W., & Besselsen, D. G. (2002). Session 4 S9. Genetically altered rodents and human tumor cells as preclinical models for prevention of intestinal cancers. European Journal of Cancer, 38, S12. doi:10.1016/s0959-8049(02)80617-7
• Steffen, E. K., Riley, L. K., Livingston, R. S., Franklin, C. L., Besselsen, D. G., & Besch-williford, C. L. (2002). Serodiagnosis of mice minute virus and mouse parvovirus infections in mice by enzyme-linked immunosorbent assay with baculovirus-expressed recombinant VP2 proteins.. Clinical and diagnostic laboratory immunology, 9(5), 1025-31. doi:10.1128/cdli.9.5.1025-1031.2002
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  Mice minute virus (MMV) and mouse parvovirus (MPV) type 1 are the two parvoviruses known to naturally infect laboratory mice and are among the most prevalent infectious agents found in contemporary laboratory mouse colonies. Serologic assays are commonly used to diagnose MMV and MPV infections in laboratory mice; however, highly accurate, high-throughput serologic assays for the detection of MMV- and MPV-infected mice are needed. To this end, the major capsid viral protein (VP2) genes of MMV and MPV were cloned and MMV recombinant VP2 (rVP2) and MPV rVP2 proteins were expressed by using a baculovirus system. MMV rVP2 and MPV rVP2 spontaneously formed virus-like particles that were morphologically similar to empty parvovirus capsids. These proteins were used as antigens in enzyme-linked immunosorbent assays (ELISAs) to detect anti-MMV or anti-MPV antibodies in the sera of infected mice. Sera from mice experimentally infected with MMV (n = 43) or MPV (n = 35) and sera from uninfected mice (n = 30) were used to evaluate the ELISAs. The MMV ELISA was 100% sensitive and 100% specific in detecting MMV-infected mice, and the MPV ELISA was 100% sensitive and 98.6% specific in detecting MPV-infected mice. Both assays outperformed a parvovirus ELISA that uses a recombinant nonstructural protein (NS1) of MMV as antigen. The MMV rVP2 and MPV rVP2 proteins provide a ready source of easily produced antigen, and the ELISAs developed provide highly accurate, high-throughput assays for the serodiagnosis of MMV and MPV infections in laboratory mice.
• Watson, R. R., Watson, R. R., Sepulveda, R. T., Jiang, S., & Besselsen, D. G. (2002). Alcohol consumption during murine acquired immunodeficiency syndrome accentuates heart pathology due to coxsackievirus.. Alcohol and alcoholism (Oxford, Oxfordshire), 37(2), 157-63. doi:10.1093/alcalc/37.2.157
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  Alcohol, especially after prolonged and excessive consumption, results in marked alteration of host immunity and increased susceptibility to infection. To determine whether ethanol consumption exacerbates coxsackievirus B3 cardiomyopathy during murine acquired immunodeficiency syndrome (AIDS), female C57BL/6 mice were infected with LP-BM5 retrovirus and administered 40% ethanol both in water and in solid agar-based form. Cardiac histopathology was semi-quantitatively assessed for lesion severity and induced production of splenocyte: interleukin (IL)-2, IL-4, IL-6, tumour necrosis factor-alpha and interferon-gamma were determined. Ethanol consumption during murine retrovirus infection increased coxsackievirus-induced myocarditis in 85% of the animals and also exacerbated the lesion severity. Mice infected with retrovirus and co-infected with coxsackievirus showed significant heart lesions. Retrovirus infection suppressed Th1 responses, causing cytokine dysregulation and immunosuppression, which facilitated coxsackievirus-induced myocarditis. Our data suggest that ethanol consumption heightens the cytokine imbalance to favour a Th2 response by enhancing Th2 and/or by suppressing Th1 function. In conclusion, murine AIDS facilitated severe cardiotoxicity during coxsackievirus infection, while non-retrovirus-infected mice were resistant. These effects were accentuated by ethanol consumption.
• Williams, C. S., Payne, C. M., Meza, Y. G., Mcwilliam, D. L., Mccuskey, R. S., Holubec, H., Halpern, M. D., Dvorak, B., Dominguez, J. A., & Besselsen, D. G. (2002). Up-regulation of IL-18 and IL-12 in the ileum of neonatal rats with necrotizing enterocolitis.. Pediatric research, 51(6), 733-9. doi:10.1203/00006450-200206000-00012
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  Necrotizing enterocolitis (NEC) is a common and devastating gastrointestinal disease of premature infants. Because the proinflammatory cytokines IL-18, IL-12, and interferon (IFN)-gamma have been implicated in other diseases of the small intestine, we hypothesized that these cytokines would play an important role in NEC pathogenesis. NEC was induced in newborn rats via enteral feeding with rat milk substitute and asphyxia and cold stress (RMS). Dam-fed, asphyxia- and cold-stressed littermates were used as controls (DF). After 96 h, the distal ileum was removed from all animals and processed to determine expression and localization of IL-18, IL-12, and IFN-gamma using real-time reverse transcriptase PCR and immunohistology. IL-18 and IL-12 mRNA from the RMS group were increased (p < or = 0.05) compared with DF controls, and there was a correlation between increasing IL-18 and IL-12 mRNA levels and progression of tissue damage (r = 0.629 and 0.588, respectively; p < or = 0.05). Immunohistology revealed IL-18 in the cytoplasm of villi and crypt enterocytes and IL-12-positive monocytes/macrophages were increased with disease progression (r = 0.503, p < or = 0.05). No differences in the number of IFN-gamma-positive cells were observed between groups. These data demonstrate up-regulation of IL-18 and IL-12 in experimental NEC and a correlation between production of these proinflammatory cytokines and progression of tissue damage.
• Besselsen, D., Redig, A. J., & Besselsen, D. G. (2001). Detection of rodent parvoviruses by use of fluorogenic nuclease polymerase chain reaction assays. Comparative medicine, 51(4).
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  Polymerase chain reaction (PCR) assays have proven useful for detection of rodent parvoviruses in animals and contaminated biological materials. Fluorogenic nuclease PCR assays combine PCR with an internal fluorogenic hybridization probe, eliminating post-PCR processing and potentially enhancing specificity. Consequently, three fluorogenic nuclease PCR assays were developed, one that detects all rodent parvoviruses, one that specifically detects minute virus of mice (MVM), and one that specifically detects mouse parvovirus 1 (MPV) and hamster parvovirus (HaPV). When rodent parvoviruses and other rodent DNA viruses were evaluated, the rodent parvovirus assay detected only rodent parvovirus isolates, whereas the MVM and MPV/HaPV assays detected only the MVM or MPV/ HaPV isolates, respectively. Each assay detected the equivalent of 10 or fewer copies of target template, and all fluorogenic nuclease PCR assays exceeded the sensitivities associated with previously reported PCR assays and mouse antibody production testing. In addition, each fluorogenic nuclease PCR assay detected the targeted parvovirus DNA in tissues obtained from mice experimentally infected with MVM or MPV. Results of these studies indicate that fluorogenic nuclease PCR assays provide a potentially high-throughput, PCR-based method to detect rodent parvoviruses in infected mice and contaminated biological materials.
• Wu, R. S., Mcearchern, J. A., Mack, V. D., Kobie, J. J., Fong, T. C., Besselsen, D. G., & Akporiaye, E. T. (2001). Comparative analysis of IFN-gamma B7.1 and antisense TGF-beta gene transfer on the tumorigenicity of a poorly immunogenic metastatic mammary carcinoma.. Cancer immunology, immunotherapy : CII, 50(5), 229-40. doi:10.1007/s002620100197
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  Cancer progression is attributed in part to immune evasion strategies that include lack of co-stimulation, down-regulation of cell surface MHC molecules, and secretion of immunosuppressive factors, such as transforming growth factor-beta (TGF-beta). Gene therapy has been employed to counter these mechanisms of immune evasion by transference of B7.1, IFN-gamma or antisense TGF-beta genes into tumor cells, resulting in cell surface expression of B7.1, upregulation of MHC class I and class II molecules, or elimination of tumor-derived TGF-beta, respectively. Although each of these transgenes has been shown to alter tumorigenicity in murine models, a direct comparison of their efficacy has not been performed. In this study, we have employed a very aggressive, poorly immunogenic and highly metastatic mammary model, 4T1, to compare the efficacy of B7.1, IFN-gamma and antisense TGF-beta gene transfer in stimulating an anti-tumor response. We demonstrate that both IFN-gamma and antisense TGF-beta gene expression significantly reduced the tumorigenicity of these cells compared to mock transduced cells, with IFN-gamma having a greater effect. In contrast, B7.1 gene transfer did not affect the tumorigenicity of 4T1 cells. The anti-tumor response directed against antisense TGF-beta-expressing 4T1 tumors was mediated by CD4+ and CD8+ T cells. However, CD8+ T cells, and not CD4+ T cells, appeared to mediate the anti-tumor response against IFN-gamma-expressing tumors. Treatment of tumor-bearing animals with IFN-gamma or antisense TGF-beta gene-modified tumor cell vaccines reduced the number of clonogenic metastases to the lungs and liver compared to treatment with mock-transduced cells. Finally, in a residual disease model in which the primary tumor was excised and mice were vaccinated with irradiated tumor cells, treatment of mice with vaccinations consisting of 4T1 cells expressing both antisense TGF-beta and IFN-gamma genes was the most effective in prolonging survival.
• Wu, R. S., Seftor, E. A., Meade-tollin, L. C., Mcearchern, J. A., Mack, V. D., Kobie, J. J., Katsanis, E., Hendrix, M. J., Dumont, N., Besselsen, D. G., Arteaga, C. L., & Akporiaye, E. T. (2001). Invasion and metastasis of a mammary tumor involves TGF-beta signaling.. International journal of cancer, 91(1), 76-82. doi:10.1002/1097-0215(20010101)91:1<76::aid-ijc1012>3.0.co;2-8
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  Several studies have correlated escape from TGF-beta-mediated cell cycle arrest with the tumorigenic phenotype. Most often, this escape from growth control has been linked to dysfunctional TGF-beta receptors or defects in the TGF-beta-mediated SMAD signaling pathway. In this report, we found that highly metastatic 4T1 mammary carcinoma cells express functional TGF-beta receptors capable of initiating SMAD-mediated transcription, yet are not growth inhibited by TGF-beta1. We further observed that TGF-beta directly contributes to the metastatic behavior of this cell line. Exposure to TGF-beta caused 4T1 cells to undergo morphological changes associated with the metastatic phenotype and invade more readily through collagen coated matrices. Furthermore, expression of a dominant negative truncated type II receptor diminished TGF-beta signaling and significantly restricted the ability of 4T1 cells to establish distant metastases. Our results suggest that regardless of 4T1 resistance to TGF-beta-mediated growth inhibition, TGF-beta signaling is required for tumor invasion and metastases formation.
• Besselsen, D. G., Gerner, E. W., Rodriguez, J. G., Mangone, K. B., Valle, K., Powell, M., & Ignatenko, N. A. (2000). Chemoprevention of colon carcinogenesis by DFMO and sulindac in the Min mouse. Proceedings of the American Association for Cancer Research Annual Meeting.
• Ramberg, F. B., Besselsen, D. G., Maré, C. J., & Mead, D. G. (2000). Transmission of Vesicular Stomatitis Virus from Infected to Noninfected Black Flies Co-Feeding on Nonviremic Deer Mice. Science, 287(5452), 485-487. doi:10.1126/science.287.5452.485
• Riley, L. K., Kendall, L. V., Dg, B., & Besselsen, D. G. (2000). Fluorogenic 5' nuclease PCR (real time PCR).. Contemporary topics in laboratory animal science, 39(5), 41.
• Wagner, A. M., Loganbill, J. K., & Besselsen, D. G. (2000). Effect of mouse strain and age on detection of mouse parvovirus 1 by use of serologic testing and polymerase chain reaction analysis.. Comparative medicine, 50(5), 498-502.
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  Detection of mouse parvovirus 1 (MPV) depends on use of serologic and polymerase chain reaction (PCR) assays. These assays were evaluated for their ability to detect virus-specific antibodies or viral DNA in multiple strains and ages of mice inoculated with MPV..Twelve-week-old ICR, BALB/c, C3H, C57BL/6, and DBA/2 mice and four- and eight-week-old ICR mice were inoculated with MPV. Serum was harvested four weeks after inoculation and analyzed by use of recombinant non structural protein 1 (rNS1) enzyme-linked immunosorbent assay (ELISA), minute virus of mice (MVM) ELISA, and MPV indirect fluorescent antibody (IFA), MVM IFA, and MPV hemagglutination inhibition (HAI) assays. Select tissues were harvested and analyzed by use of an MPV-specific PCR assay..The number of mice in each group with detectable MPV-specific antibodies or MPV DNA varied with mouse strain, mouse age when inoculated, and viral dose. Seroconversion in mice inoculated at 12 weeks of age was detected almost exclusively by use of the MPV IFA and MPV HAI assays, whereas seroconversion in almost all mice inoculated at 4 and 8 weeks of age was detected by use of all immunoassays except the MVM ELISA. Viral DNA was detected by use of PCR analysis in all strains and ages of mice except DBA/2 mice..Mouse strain and age have important roles in seroconversion to nonstructural and structural MPV antigens and persistence of viral DNA in mouse tissues. Therefore, diagnostic serologic testing and PCR analysis should be considered within the context of mouse strain and age at the time of MPV exposure, especially when sentinel mice are used for surveillance.
• Mcearchern, J. A., Besselsen, D. G., & Akporiaye, E. T. (1999). Interferon gamma and antisense transforming growth factor beta transgenes synergize to enhance the immunogenicity of a murine mammary carcinoma.. Cancer immunology, immunotherapy : CII, 48(2-3), 63-70. doi:10.1007/s002620050549
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  Transforming growth factor beta (TGFbeta) is an immunosuppressive cytokine that contributes to the immunological escape of tumor cells. In a previous study we demonstrated that inhibition of TGFbeta production by EMT6 murine mammary tumor cells expressing an antisense TGF-beta transgene reduces their tumorigenicity. On the basis of this observation we hypothesized that down-regulation of TGFbeta production coupled with interferon gamma (IFNgamma) stimulation would induce an immune response superior to that generated by either strategy alone. In this study, EMT6 tumor cells expressing antisense TGFbeta were transduced with the murine IFNgamma gene. Tumor cells expressing either or both transgenes grew more slowly than mock-transduced tumors. Dual-transgene-expressing tumor cells were more immunogenic than tumor cells expressing either transgene alone. Studies in mice depleted of T cell subsets indicated that CD8+ T cells are the primary effectors of the antitumor activity observed. These results suggest that down-regulation of immunosuppression combined with cytokine-mediated immune augmentation is a useful strategy to improve antitumor immunity.
• Wagner, J. E., Sv, G., Rr, H., Rl, K., Riley, L. K., Purdy, G. A., Pintel, D. J., Knowles, R. L., Je, W., Hook, R. R., Gibson, S. V., Ga, P., Franklin, C. L., Besselsen, D. G., & Besch-williford, C. L. (1999). Natural and experimentally induced infection of Syrian hamsters with a newly recognized parvovirus.. Laboratory animal science, 49(3), 308-12.
• Besselsen, D. G., Riley, L. K., Rr, H., Franklin, C. L., Besch-Williford, C., Purdy, G. A., & Pintel, D. J. (1996).

  Molecular characterization of newly recognized rodent parvoviruses

  . Journal of General Virology. doi:10.1099/0022-1317-77-5-899
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  Several autonomous rodent parvoviruses distinct from the prototypic rodent parvoviruses have been isolated. These include variants of a mouse parvovirus (MPV), a hamster isolate designated hamster parvovirus (HaPV), and a variant strain of minute virus of mice (MVM) designated MVM-Cutter or MVM(c). In this study, the DNA sequence of the coding regions of the viral genome and the predicted protein sequences for each of these new isolates were determined and compared to the immunosuppressive and prototypic strains of MVM [MVM(i) and MVM(p)], the rodent parvovirus H-1, and LuIII, an autonomous parvovirus of uncertain host origin. Sequence comparisons showed that the MPV isolates were almost identical, HaPV was very similar to MPV, and MVM(c) was most similar to MVM(i) and MVM(p). Haemagglutination inhibition assays revealed that MPV and HaPV represent two serotypes distinct from previously characterized rodent parvovirus serotypes while MVM(c) belongs to the MVM serotype. Each of the newly isolated rodent parvoviruses was shown to encapsidate a predominantly negative-sense 5 kb DNA genome and to encode two nonstructural proteins (NS1 and NS2) and two structural viral proteins (VP1 and VP2). These studies indicate that MPV and HaPV are autonomous parvoviruses distinct from previously characterized parvoviruses and MVM(c) is a variant strain of MVM distinct from MVM(i) and MVM(p).
• Besselsen, D. G., Riley, L. K., Rr, H., Franklin, C. L., Besch-Williford, C., Purdy, G. A., & Pintel, D. J. (1996). Molecular characterization of newly recognized rodent parvoviruses. Journal of General Virology. doi:10.1099/0022-1317-77-5-899
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  Several autonomous rodent parvoviruses distinct from the prototypic rodent parvoviruses have been isolated. These include variants of a mouse parvovirus (MPV), a hamster isolate designated hamster parvovirus (HaPV), and a variant strain of minute virus of mice (MVM) designated MVM-Cutter or MVM(c). In this study, the DNA sequence of the coding regions of the viral genome and the predicted protein sequences for each of these new isolates were determined and compared to the immunosuppressive and prototypic strains of MVM [MVM(i) and MVM(p)], the rodent parvovirus H-1, and LuIII, an autonomous parvovirus of uncertain host origin. Sequence comparisons showed that the MPV isolates were almost identical, HaPV was very similar to MPV, and MVM(c) was most similar to MVM(i) and MVM(p). Haemagglutination inhibition assays revealed that MPV and HaPV represent two serotypes distinct from previously characterized rodent parvovirus serotypes while MVM(c) belongs to the MVM serotype. Each of the newly isolated rodent parvoviruses was shown to encapsidate a predominantly negative-sense 5 kb DNA genome and to encode two nonstructural proteins (NS1 and NS2) and two structural viral proteins (VP1 and VP2). These studies indicate that MPV and HaPV are autonomous parvoviruses distinct from previously characterized parvoviruses and MVM(c) is a variant strain of MVM distinct from MVM(i) and MVM(p).
• Besselsen, D. G., Smith, A. M., Riley, L. K., McKisic, M. D., Ball-Goodrich, L. J., & Jacoby, R. O. (1996). Rodent parvovirus infections.. Laboratory Animal Science.
• Besselsen, D. G., Riley, L. K., Rr, H., Franklin, C. E., Pintel, D. J., & Besch-Williford, C. (1995). Detection of newly recognized rodent parvoviruses by PCR. Journal of Clinical Microbiology. doi:10.1128/jcm.33.11.2859-2863.1995
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  Several autonomous parvovirus isolates distinct from the prototypic rodent parvoviruses have recently been identified. These include variants of a mouse orphan parvovirus (MOPV) and a hamster isolate designated hamster orphan parvovirus (HOPV). In this study, a PCR primer set specific for these newly identified rodent parvoviruses was designed on the basis of DNA sequence comparisons of these isolates with other autonomous parvoviruses. The specificity of the primer set was determined by testing viral preparations of seven different parvoviruses and eight other viruses known to infect rodents. The PCR assay amplified the expected 260-bp product only in the presence of DNA from MOPV, HOPV, or LuIII a parvovirus of unknown species origin. The assay was able to detect as little as 10 pg of MOPV viral DNA or 1 pg of HOPV viral DNA, and it was able to detect MOPV in tissues from naturally infected mice and HOPV in tissues from experimentally infected hamsters. In contrast, the 260-bp product was not amplified from tissues of MOPV-negative mice or mock-infected hamsters. Our findings indicate that this PCR assay provides a rapid, specific, and sensitive method for the detection of MOPV in mice, HOPV in hamsters, and MOPV and HOPV in cell culture systems and that it may also be useful for the detection of LuIII contamination of cell culture systems.
• Besselsen, D. G., Friskey, S. A., Fish, R., Wright, J., C, B., M, S. C., & Penner, J. E. (1994). Pseudomonas sp. bacteremia associated with chronic vascular catheterization in rabbits.. Laboratory Animal Science.

Presentations

• Besselsen DVM, PhD, D. G. (2018, January). Reid Park Zoo partnership with UA College of Veterinary Medicine. Dedication of Reid Park Zoo Veterinary Hospital. Reid Park Zoo, Tucson, AZ: Reid Park Zoo.
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  Comments related to how partnership will engage and educate future veterinary students.
• Besselsen DVM, PhD, D. G. (2017, December). College of Veterinary Medicine Update. UA Community Presentation. UA Main Campus: College of Veterinary Medicine.
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  Update on UA College of Veterinary Medicine presented to veterinarians and other stakeholders in AZ.
• Besselsen DVM, PhD, D. G. (2017, January). University Animal Care Annual Update. UAC Seminar Series. UA Health Sciences, Tucson, AZ: University Animal Care.
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  Annual update on status of UAC.
• Besselsen DVM, PhD, D. G. (2017, November). College of Veterinary Medicine Update. Ag-100 Meeting. Phoenix Biomedical Campus, Phoenix, AZ: Ag-100.
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  An update of the status of the UA College of Veterinary Medicine was presented to the Ag-100, which represent the major agricultural industries and producers in the state of AZ.
• Besselsen DVM, PhD, D. G. (2016, January). UAC Annual Update. UAC Seminar Series. University of Arizona: University Animal Care.
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  Provided departmental update to UAC employees and user group.
• Besselsen DVM, PhD, D. G. (2016, October). ACLAM Exam Question Development Training. WebinarsACLAM.
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  Presented two webinars on the development of board certification exam questions to the ACLAM Exam Resource Committee membership (15 total individuals).
• Besselsen DVM, PhD, D. G. (2016, September). Going Down the Rabbit Hole: Meeting Regulatory and Accreditation Standards for UA Research and Teaching Animals. School of Animal and Comparative Biomedical Sciences Seminar Series. University of Arizona: School of Animal and Comparative Biomedical Sciences.
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  A review of laws and regulations related to animal care and use was presented in a departmental seminar series, with emphasis on current issues, citations, and animal rights activities.

Poster Presentations

• Besselsen, D. G., Diaz, M., Johnson, C., Mexas, A., & Patil, K. (2018, November). Microenvironmental Findings and Undetectable Changes in Inflammatory Cytokines Support Extended Cage-change Frequency for Rats. 69th National American Association for Laboratory Animal Science Meeting.

Others

• Bolon, B., Altrock, B., Barthold, S. W., Baumgarth, N., Besselsen, D., Boivin, G., Boyd, K. L., Brayton, C., Cardiff, R. D., Couto, S., Eaton, K. A., Foreman, O., Griffey, S. M., La Perle, K., Lairmore, M. D., Liu, C., Meyerholz, D. K., Nikitin, A. Y., Schoeb, T. R., , Schwahn, D., et al. (2011, Mar). Advancing translational research. Science (New York, N.Y.).