David G Besselsen

Interests

Research

Rodent pathology; rodent viral disease; diagnostic test development

Teaching

Laboratory animal pathology

Courses

No activities entered.

Scholarly Contributions

Journals/Publications

• 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
• 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.
• 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.
• 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.
• 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.
• 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.
• 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.
• 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.
• 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.
• 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.
• 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.
• 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.
• 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.
• 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.
• 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.
• 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.

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.).