Ammonia for Determination of Cage-change Frequency in Antelope Ground Squirrels (Ammospermophilus leucurus).

Due to a lack of evidence-based standards for cage-change intervals for antelope ground squirrels (AGS, Ammospermophilus leucurus), we evaluated cage ammonia accumulation in our colony of adult, wild-caught AGS and identified factors that influenced ammonia levels. Intracage ammonia was measured daily in singly housed AGS in static caging that contained a running wheel and 1/2, 3/4, 1, or 2 quart (qt) of corncob bedding. Cages were changed when ammonia levels reached greater than 50 ppm, our upper acceptable limit for ammonia based on mouse studies of ammonia aversion and toxicity. We also measured average daily water consumption over 2 wk to examine any correlation between water use and ammonia accumulation. We hypothesized that the desert-dwelling AGS would not reach intracage ammonia levels of greater than 50 ppm in a 2-wk interval at any bedding volume. Our data showed that intracage ammonia was highly variable among individuals and was significantly associated with water consumption and bedding volumes. Seventeen percent of AGS on 1/2 qt of bedding and 18% on 3/4 qt of bedding reached greater than 50 ppm ammonia before 7 d. All AGS on 1 and 2 qt of bedding remained below 50 ppm ammonia for 1 wk. Even when maintained on 2 qt of bedding, not all AGS remained below 50 ppm ammonia for 2 wk. Therefore, we concluded that the most appropriate option was weekly cage change for singly housed AGS on 1 qt of bedding in static caging.

ALDH1A Inhibition Suppresses Colitis and Alters α4ß7 Integrin Expression on Activated T Cells in Mdr1a-/- Mice.

There are limited pharmacological treatment options for inflammatory bowel disease (IBD), and some of these options are expensive and administered by injection or infusion. Thus, new cheaper and easier (oral) treatment options are needed. ALDH1A enzymes produce retinoic acid that can affect intestinal diseases such as IBD by regulating immune cells in the gut. We previously demonstrated that an orally deliverable ALDH1A inhibitor, WIN 18,466, can suppress colitis in an acute mouse model of IBD. Here, we tested the efficacy of ALDH1A inhibition in a chronic mouse model of IBD. Mdr1a-/- mice were treated with a diet containing WIN 18,446 starting 1 week prior to inducing colitis by H. bilis inoculation. Treatment was continued until the study end point and colitis was monitored based on clinical symptoms and confirmed by histological analysis. Immune cell phenotypes in colon-draining lymph nodes (cMLN) were analyzed. WIN 18,446 treatment reduced clinical symptoms and improved histopathologic colitis scores. This was associated with decreased expression of the gut homing integrin, α4ß7, on T cells in cMLN; increased expression of CD103, a protein associated with tissue-resident memory T cells; and changes in dendritic cells, plasmacytoid dendritic cells and B cells in inhibitor-treated mice. ALDH1A inhibition broadly influences immune cells during colitis and is a potential new target for IBD treatment. Future studies will be needed to determine the efficacy of ALDH1A inhibition on active colitis and to evaluate its relative efficacy in comparison to approved drugs.

Analysis of Salmonella Typhi Pathogenesis in a Humanized Mouse Model.

Efforts to understand molecular mechanisms of pathogenesis of the human-restricted pathogen Salmonella enterica serovar Typhi, the causative agent of typhoid fever, have been hampered by the lack of a tractable small animal model. This obstacle has been surmounted by a humanized mouse model in which genetically modified mice are engrafted with purified CD34+ stem cells from human umbilical cord blood, designated CD34+ Hu-NSG (formerly hu-SRC-SCID) mice. We have shown that these mice develop a lethal systemic infection with S. Typhi that is dependent on the presence of engrafted human hematopoietic cells. Immunological and pathological features of human typhoid are recapitulated in this model, which has been successfully employed for the identification of bacterial genetic determinants of S. Typhi virulence. Here we describe the methods used to infect CD34+ Hu-NSG mice with S. Typhi in humanized mice and to construct and analyze a transposon-directed insertion site sequencing S. Typhi library, and provide general considerations for the use of humanized mice for the study of a human-restricted pathogen.

Investigation of an ALDH1A1-specific inhibitor for suppression of weight gain in a diet-induced mouse model of obesity.

BACKGROUND: Retinoic acid (RA) controls diverse physiological functions including weight regulation and energy metabolism. It has been reported that mice lacking ALDH1A1, one of the aldehyde dehydrogenases (ALDH) that synthesize RA, are healthy and resistant to weight gain, raising the possibility that inhibiting this enzyme might treat obesity. We previously demonstrated that treatment with a pan-ALDH1A enzyme inhibitor, WIN18446, suppressed weight gain in mice fed a high-fat diet (HFD), but caused increased hepatic lipidosis and reversible male infertility. METHODS: A series of piperazine compounds that inhibited ALDH1A1 were identified and their inhibitory activity was characterized in vitro using purified recombinant enzymes and cell-based assay systems. One potent compound, FSI-TN42 (N42) was examined for its oral bioavailability and pharmacodynamic effects. In addition, its effect on weight gain was investigated by daily oral administration to C57BL/6 male mice receiving a HFD, and compared with mice receiving WIN18446 or vehicle alone (n = 6/group, 200 mg compound/kg body weight) for 5 weeks. Body weights were measured weekly, and a glucose tolerance test was performed after 4 weeks of treatment. Tissues were collected to determine changes in adipose weight, hepatic lipidosis, retinoid metabolism, and expression of genes associated with RA and lipid metabolism. RESULTS: N42 irreversibly binds and inhibits ALDH1A1 in vitro with a low nM IC50 and 800-fold specificity for ALDH1A1 compared to ALDH1A2. Daily oral administration of N42 significantly suppressed weight gain (P < 0.05) and reduced visceral adiposity (p < 0.05) in mice fed a HFD without the hepatic lipidosis observed with WIN18446 treatment. CONCLUSIONS: We developed a potent and specific inhibitor of ALDH1A1 that suppressed weight gain in mice fed a HFD. These findings demonstrate that inhibition of ALDH1A1 is a feasible target for drug development to treat and/or prevent obesity.

Evaluation of Nutritional Gel Supplementation in C57BL/6J Mice Infected with Mouse-Adapted Influenza A/PR/8/34 Virus.

Mice are a common animal model for the study of influenza virus A (IAV). IAV infection causes weight loss due to anorexia and dehydration, which can result in early removal of mice from a study when they reach a humane endpoint. To reduce the number of mice prematurely removed from an experiment, we assessed nutritional gel (NG) supplementation as a support strategy for mice infected with mouse-adapted Influenza A/Puerto Rico/8/34 (A/PR/8/34; H1N1) virus. We hypothesized that, compared with the standard of care (SOC), supplementation with NG would reduce weight loss and increase survival in mice infected with IAV without impacting the initial immune response to infection. To assess the effects of NG, male and female C57BL/6J mice were infected with IAV at low, intermediate, or high doses. When compared with SOC, mice given NG showed a significant decrease in the maximal percent weight loss at all viral doses in males and at the intermediate dose for females. Mice supplemented with NG had no deaths for either sex at the intermediate dose and a significant increase in survival in males at the high viral dose. Supplementation with NG did not alter the viral titer or the pulmonary recruitment of immune cells as measured by cell counts and flow cytometry of cells recovered in bronchoalveolar lavage (BAL) fluid in either sex. However, mice given NG had a significant reduction in IL6 and TNFα in BAL fluid and no significant differences in CCL2, IL4, IL10, CXCL1, CXCL2, and VEGF. The results of this study show that as compared with infected SOC mice, infected mice supplemented with NG have reduced weight loss and increased survival, with males showing a greater benefit. These results suggest that NG should be considered as a support strategy and indicate that sex is an important biologic variable in mice infected with IAV.

Protective Effects of ALDH1A Enzyme Inhibition on Helicobacter-Induced Colitis in Smad3-/- Mice are Associated with Altered α4ß7 Integrin Expression on Activated T Cells.

Many inflammatory bowel disease (IBD) patients require surgical intervention due to limited pharmacological treatment options. Antibodies targeting α4ß7, a gut-homing integrin, are one of the most promising IBD treatments. As retinoic acid (RA) regulates expression of gut-homing proteins including α4ß7 integrin, we tested if ALDH1A enzymes in the RA synthesis pathway could be targeted for IBD treatment using a potent inhibitor, WIN 18,446. Age- and sex-matched Smad3-/- mice were fed a diet with and without WIN 18,446 for 3 weeks before triggering inflammation with Helicobacter bilis infection. Colitis was evaluated by histopathology one week following the IBD trigger, and T cell subsets were evaluated before and after the IBD trigger. WIN 18,446 treatment significantly reduced IBD severity in Smad3-/- mice and reduced expression of α4ß7 integrin on multiple activated CD4+ T cell subsets. This change was associated with increased ratios of induced regulatory T cells to Th17 cells during the inflammatory response in the draining lymph nodes. These studies indicate that RA reduction via ALDH1A enzyme inhibition is a potential new target for IBD treatment. Further studies are needed to examine its effects on other types of immune cells, to evaluate the efficacy window for this target, and to determine its efficacy in other animal models of IBD.

Effect of Chronic Vitamin D Deficiency on the Development and Severity of DSS-Induced Colon Cancer in Smad3-/- Mice.

Both human epidemiologic data and animal studies suggest that low serum vitamin D increases the risk of inflammatory bowel disease (IBD) and consequently IBD-associated colorectal cancer. We tested the hypothesis that vitamin D deficiency increases the risk for colitis-associated colon cancer (CAC) by using an established CAC mouse model, 129-Smad3tm1Par/J (Smad3-/-) mice, which have defective transforming growth factor ß-signaling and develop colitis and CAC after the administration of dextran sodium sulfate (DSS). After determining a dietary regimen that induced chronic vitamin D deficiency in Smad3-/- mice, we assessed the effects of vitamin D deficiency on CAC. Decreasing dietary vitamin D from 1 IU/g diet (control diet) to 0.2 IU /g diet did not decrease serum 25-hydroxyvitamin D (25(OH)D) levels in Smad3-/- mice. A diet devoid of vitamin D (< 0.02 IU/g diet [no added vitamin D]; vitamin D-null) significantly decreased serum 25(OH)D levels in mice after 2 wk (null compared with control diet: 8.4 mg/mL compared with 12.2 ng/mL) and further decreased serum levels to below the detection limit after 9 wk but did not affect weight gain, serum calcium levels, bone histology, or bone mineral density. In light of these results, Smad3-/- mice were fed a vitamin D-null diet and given DSS to induce colitis. Unexpectedly, DSS-treated Smad3-/- mice fed a vitamin D-null diet had improved survival, decreased colon tumor incidence (8% compared with 36%), and reduced the incidence and severity of colonic dysplasia compared with mice fed the control diet. These effects correlated with increased epithelial cell proliferation and repair early in the disease, perhaps reducing the likelihood of developing chronic colitis and progression to cancer. Our results indicate that vitamin D deficiency is beneficial in some cases of CAC, possibly by promoting intestinal healing.

Lack of Effect of Murine Norovirus Infection on the CD4+ CD45RBhigh T-cell Adoptive Transfer Mouse Model of Inflammatory Bowel Disease.

Murine norovirus (MNV) infection is highly prevalent in laboratory mice. Although MNV infection does not typically induce clinical disease in most laboratory mice, infection may nonetheless affect mouse models of disease by altering immune responses. We previously reported that MNV altered the bacterial-induced mouse model of inflammatory bowel disease (IBD) using Helicobacter-infected Mdr1a-/- mice. Therefore, we hypothesized that MNV infection would exacerbate another mouse model of IBD, the T-cell adoptive transfer (AT) model. In this model, Helicobacter infection is used to accelerate the progression of IBD induced by AT of naïve CD4+CD45RBhigh T cells into B6.129S7- Rag1tm1Mom/J (Rag1-/-) mice. We evaluated the effects of MNV infection in both Helicobacter-accelerated as well as Helicobacter-free AT models. In our studies, Helicobacter-infected Rag1-/- mice that received CD4+CD45RBhigh T cells through AT rapidly developed weight loss and typhlocolitis; MNV infection had no effect on disease severity or rate of progression. In the absence of Helicobacter infection, progression of IBD caused by AT of CD4+CD45RBhigh T cells was slower and typhlocolitis was less severe; this inflammation likewise was unaltered by MNV infection. These results indicate that MNV infection does not alter IBD progression and severity in the CD4+CD45RBhigh T-cell AT model in Rag1-/- mice.

Validation studies for germ-free Smad3-/- mice as a bio-assay to test the causative role of fecal microbiomes in IBD.

While the association between microbiomes and inflammatory bowel disease (IBD) is well known, establishing causal relationships between the two is difficult in humans. Germ-free (GF) mice genetically susceptible to IBD can address this question. Smad3-/- mice with defective transforming growth factor ß signaling are a model of IBD and colon cancer. They develop IBD upon colonization with Helicobacter under specific pathogen-free conditions, suggesting a role of the microbiome in IBD in this model. Thus, we rederived Smad3-/- mice GF to determine the potential of using these mice for testing the causative role of microbiomes in IBD. We found that fecal microbiomes from mice with IBD cause more severe gut inflammation in GF Smad3-/- and wild type mice compared to microbiomes from healthy mice and that Helicobacter induces gut inflammation within the context of other microbiomes but not by itself. Unexpectedly, GF Smad3+/+ and Smad3+/- mice given IBD microbiomes develop IBD despite their lack of disease in SPF conditions upon Helicobacter infection. This was not unique to the background strain of our Smad3 model (129); both wild type C57BL/6 and 129 strains developed IBD upon fecal transfer. However, wild type Swiss Webster stock was not susceptible, indicating that the genetic background of recipient mice influences the severity of IBD following fecal transfer. Our data suggest that the microbiome is an independent risk factor contributing to IBD development, and careful characterization of new GF models is needed to understand potential sources of confounding factors influencing microbiome studies in these mice.

Obstructive Lymphangitis Precedes Colitis in Murine Norovirus-Infected Stat1-Deficient Mice.

Murine norovirus (MNV) is an RNA virus that can prove lethal in mice with impaired innate immunity. We found that MNV-4 infection of Stat1-/- mice was not lethal, but produced a 100% penetrant, previously undescribed lymphatic phenotype characterized by chronic-active lymphangitis with hepatitis, splenitis, and chronic cecal and colonic inflammation. Lesion pathogenesis progressed from early ileal enteritis and regional dilated lymphatics to lymphangitis, granulomatous changes in the liver and spleen, and, ultimately, typhlocolitis. Lesion development was neither affected by antibiotics nor reproduced by infection with another enteric RNA virus, rotavirus. MNV-4 infection in Stat1-/- mice decreased expression of vascular endothelial growth factor (Vegf) receptor 3, Vegf-c, and Vegf-d and increased interferon (Ifn)-γ, tumor necrosis factor-α, and inducible nitric oxide synthase. However, anti-IFN-γ and anti-tumor necrosis factor-α antibody treatment did not attenuate the histologic lesions. Studies in Ifnαßγr-/- mice suggested that canonical signaling via interferon receptors did not cause MNV-4-induced disease. Infected Stat1-/- mice had increased STAT3 phosphorylation and expressed many STAT3-regulated genes, consistent with our findings of increased myeloid cell subsets and serum granulocyte colony-stimulating factor, which are also associated with increased STAT3 activity. In conclusion, in Stat1-/- mice, MNV-4 induces lymphatic lesions similar to those seen in Crohn disease as well as hepatitis, splenitis, and typhlocolitis. MNV-4-infected Stat1-/- mice may be a useful model to study mechanistic associations between viral infections, lymphatic dysfunction, and intestinal inflammation in a genetically susceptible host.

Murine norovirus inhibits B cell development in the bone marrow of STAT1-deficient mice.

Noroviruses are a leading cause of gastroenteritis in humans and it was recently revealed that noroviruses can infect B cells. We demonstrate that murine norovirus (MNV) infection can significantly impair B cell development in the bone marrow in a signal transducer and activator of transcription 1 (STAT1) dependent, but interferon signaling independent manner. We also show that MNV replication is more pronounced in the absence of STAT1 in ex vivo cultured B cells. Interestingly, using bone marrow transplantation studies, we found that impaired B cell development requires Stat1-/- hematopoietic cells and Stat1-/- stromal cells, and that the presence of wild-type hematopoietic or stromal cells was sufficient to restore normal development of Stat1-/- B cells. These results suggest that B cells normally restrain norovirus replication in a cell autonomous manner, and that wild-type STAT1 is required to protect B cell development during infection.

Pharmacological inhibition of ALDH1A enzymes suppresses weight gain in a mouse model of diet-induced obesity.

BACKGROUND: Retinoic acid (RA) is known to play a role in weight regulation. Because mice without ALDH1A1, a major RA synthesizing enzyme, are resistant to diet-induced obesity, we tested a hypothesis that pharmacological inhibition of RA synthesis can suppress weight gain in a murine model of diet-induced obesity. METHODS: C57BL/6J male mice were fed a high fat diet (HFD) for 8 weeks to induce obesity and then randomized to a HFD with or without WIN 18,446, an RA synthesis inhibitor, for an additional 9 weeks. Body weight, body composition, energy expenditure, activity, and food intake were measured. Levels of retinoids, lipids, and genes involved in the metabolism of retinoid and lipids were also determined. RESULT: s Mice treated with WIN 18,446 gained significantly less weight and had decreased adipose tissue weight, adipocyte size, and macrophage infiltration in adipose tissue. In addition, we observed higher UCP1 expression in adipose tissues and decreased expression of RA responsive genes and genes involved in fatty acid synthesis in the livers and lungs of mice treated with WIN 18,446. CONCLUSIONS: Pharmacological suppression of RA synthesis via inhibition of ALDH1A1 may be a potential target for treatment of obesity.

Effects of Murine Norovirus on Chlamydia pneumoniae-Accelerated Atherosclerosis in ApoE(-/-) Mice.

Chlamydia pneumoniae (Cpn), a common respiratory pathogen of humans, is associated with human cardiovascular disease and the acceleration of atherosclerosis in hyperlipidemic animal models. Our laboratory has demonstrated that murine norovirus (MNV), a prevalent infection of laboratory mice, can unpredictably alter atherosclerosis in hyperlipidemic Ldlr(-/-) and ApoE(-/-) mice. Given that MNV has a tropism for macrophages and may exacerbate atherogenesis, we investigated whether coinfection with MNV and Cpn might alter macrophage phenotypes in vitro and atherosclerosis in ApoE(-/-) mice. In the presence of oxidized low-density lipoprotein, coinfection of ApoE(-/-) bone marrow-derived macrophages (BMDM) with MNV and Cpn resulted in significant increases in gene expression of IL6, MCP1, iNOS, and TNFα compared with Cpn-monoinfected BMDM. On the basis of these findings, we hypothesized that concurrent MNV-Cpn infection might increase plaque lesion size in vivo. As expected, Cpn monoinfection of ApoE(-/-) mice increased mean plaque size by 62% compared with that in uninfected mice. However, MNV did not significantly alter plaque lesion size in MNV-Cpn-coinfected mice compared with Cpn-monoinfected mice. There were no differences in aortic cytokines locally at the site of plaque development or in peritoneal macrophages at 1 wk after infection in MNV-Cpn-coinfected mice compared with Cpn-monoinfected mice. MNV was not detected in the aortic tissue of MNV-infected mice at 1 or 8 wk after infection regardless of Cpn status. These data suggest that MNV infection does not appreciably alter plaque development in Cpn-accelerated atherosclerosis in ApoE(-/-) mice.

Murine Norovirus Infection Variably Alters Atherosclerosis in Mice Lacking Apolipoprotein E.

Macrophages play a key role in the development of atherosclerosis. Murine noroviruses (MNV) are highly prevalent in research mouse colonies and infect macrophages and dendritic cells. Our laboratory found that MNV4 infection in mice lacking the LDL receptor alters the development of atherosclerosis, potentially confounding research outcomes. Therefore, we investigated whether MNV4 likewise altered atherosclerosis in ApoE(-/-) mice. In the presence of oxidized LDL, MNV4 infection of ApoE(-/-) bone marrow-derived macrophages increased the gene expression of the inflammatory markers inducible nitric oxide synthase, monocyte chemoattractant protein 1, and IL6. In addition, proteins involved in cholesterol transport were altered in MNV4-infected ApoE -/- bone marrow-derived macrophages and consisted of increased CD36 and decreased ATP-binding cassette transporter A1. MNV4 infection of ApoE(-/-) mice at 12 wk of age (during the development of atherosclerosis) had a variable effect on atherosclerotic lesion size. In one study, MNV4 significantly increased atherosclerotic plaque area whereas in a second study, no effect was observed. Compared with controls, MNV4-infected mice had higher circulating Ly6C-positive monocytes, and viral RNA was detected in the aortas of some mice, suggesting potential mechanisms by which MNV4 alters disease progression. Plaque size did not differ when ApoE -/- mice were infected at 4 wk of age (early during disease development) or in ApoE -/- mice maintained on a high-fat, high-cholesterol diet. Therefore, these data show that MNV4 has the potential to exert a variable and unpredictable effect on atherosclerosis in ApoE(-/-) mice. We therefore propose that performing experiments in MNV-free mouse colonies is warranted.

Effects of murine norovirus on atherosclerosis in ldlr(-/-) mice depends on the timing of infection.

We previously reported that murine norovirus (MNV), a virus prevalent in United States research institutions, increased atherosclerotic lesion size in Ldlr(-/-) mice when the mice were infected 8 wk after feeding an atherogenic diet. To determine whether the timing of MNV infection relative to atherosclerosis development altered the disease phenotype and to examine potential mechanisms by which MNV influences the disease process, we fed Ldlr(-/-) mice an atherogenic diet for 16 wk. Three days after initiating the atherogenic diet, half of the mice received MNV4 and the other half vehicle only (clarified cell-culture lysate; controls). Both groups of mice developed large aortic sinus lesions (control compared with MNV4: 133 ± 8 × 10³ µm² compared with 140 ± 7 × 10³ µm²) that were not significantly different in size. Because the timing of MNV infection relative to atherosclerosis development and hypercholesterolemia differed between our previous and the current studies, we examined whether hypercholesterolemia altered MNV4-induced changes in bone-marrow-derived macrophages. MNV4 infection increased the potential of macrophages to take up and store cholesterol by increasing CD36 expression while suppressing the ABCA1 transporter. Thus, the effects of MNV4 infection on atherosclerotic lesion size appear to be dependent on the timing of the infection: MNV4 infection promotes only established lesions. This effect may be due to MNV4's ability to increase cholesterol uptake and decrease efflux by regulating CD36 and ABCA1 protein expression.

Hepatic Lipidosis in a Research Colony of Big Brown Bats (Eptesicus fuscus).

During a nearby construction project, a sudden decrease in food intake and guano production occurred in an outdoor colony of big brown bats (Eptesicus fuscus), and one animal was found dead. Investigation revealed that the project was generating a large amount of noise and vibration, which disturbed the bats' feeding. Consequently the bats were moved into an indoor enclosure away from the construction noises, and the colony resumed eating. Over the next 3 wk, additional animals presented with clinical signs of lethargy, weight loss, ecchymoses, and icterus and were necropsied. Gross necropsy of the affected bats revealed large, pale yellow to tan, friable livers with rounded edges that floated when placed in 10% neutral-buffered formalin. Some bats had ecchymoses on the webbing and skin and gross perirenal hemorrhage. Histologic examination showed hepatic and renal tubular lipidosis. The clinical and pathologic signs of hemorrhage and icterus were suggestive of hepatic failure. Hepatic lipidosis was attributed to stress and inappetence associated with environmental perturbations. Once the environmental stressor was removed, the colony morbidity and mortality decreased. However, 2 y later, a series of new environmental stressors triggered additional deaths associated with hepatic lipidosis. Over a 9-y period, 21 cases of hepatic lipidosis were diagnosed in this bat colony.

Infection with murine norovirus 4 does not alter Helicobacter-induced inflammatory bowel disease in Il10(-/-) mice.

Infection of laboratory mice with murine noroviruses (MNV) is widely prevalent. MNV alters various mouse models of disease, including the Helicobacter bilis-induced mouse model of inflammatory bowel disease (IBD) in Mdr1a(--) mice. To further characterize the effect of MNV on IBD, we used mice deficient in the immunoregulatory cytokine IL10 (Il10(-/-) mice). In vitro infection of Il10(-/-) bone marrow-derived macrophages (BMDM) with MNV4 cocultured with H. bilis antigens increased the gene expression of the proinflammatory cytokines IL1ß, IL6, and TNFα as compared with that of BMDM cultured with H. bilis antigens only. Therefore, to test the hypothesis that MNV4 infection increases inflammation and alters disease phenotype in H. bilis-infected Il10(-/-) mice, we compared the amount and extent of inflammation in Il10(-/-) mice coinfected with H. bilis and MNV4 with those of mice singly infected with H. bilis. IBD scores, incidence of IBD, or frequency of severe IBD did not differ between mice coinfected with H. bilis and MNV4 and those singly infected with H. bilis. Mice infected with MNV4 only had no appreciable IBD, comparable to uninfected mice. Our findings suggest that, unlike in Mdr1a(-/-) mice, the presence of MNV4 in Il10(-/-) mouse colonies is unlikely to affect the IBD phenotype in a Helicobacter-induced model. However, because MNV4 altered cytokine expression in vitro, our results highlight the importance of determining the potential influence of MNV on mouse models of inflammatory disease, given that MNV has a tropism for macrophages and dendritic cells and that infection is widely prevalent.

Increased dietary vitamin D suppresses MAPK signaling, colitis, and colon cancer.

Epidemiologic studies associate low serum vitamin D levels with an increased risk of colon cancer and inflammatory diseases such as inflammatory bowel disease (IBD). 129-Smad3(tm1Par)/J (Smad3(-/-)) mice are a model of bacteria-driven colitis and colon cancer when infected with Helicobacter bilis (H. bilis). Thus, we used this mouse model to determine whether increased dietary vitamin D would reduce inflammation and colon cancer. Smad3(-/-) mice were fed purified diet with either maintenance (1 IU vitamin D/g diet; maintenance) or increased concentrations of vitamin D (5 IU vitamin D/g diet; high vitamin D). One week after diet initiation, mice were inoculated with broth or H. bilis and were necropsied at several time points postinoculation to assess inflammation, dysplasia, and neoplasia incidence. At 16 weeks postinfection, 11% of mice fed high vitamin D diet had cancer compared with 41% of mice fed maintenance diet (P = 0.0121). Evaluation at an early time point (1 week postinfection) showed that animals fed high vitamin D had decreased MAPK (p-P38 and p-JNK) activation in lamina propria leukocytes as well as decreased NFκB activation in colonic epithelial cells. Reduction in MAPK and NFκB activation correlated with decreased IBD scores (2.7 vs. 15.5; P < 0.0001) as well as decreased inflammatory cell infiltrates and reduced expression of proinflammatory cytokines in cecal tissue. These findings suggest that increased dietary vitamin D is beneficial in preventing inflammation-associated colon cancer through suppression of inflammatory responses during initiation of neoplasia or early-stage carcinogenesis.

Inhibition of retinoic acid biosynthesis by the bisdichloroacetyldiamine WIN 18,446 markedly suppresses spermatogenesis and alters retinoid metabolism in mice.

Knowledge of the regulation of testicular retinoic acid synthesis is crucial for understanding its role in spermatogenesis. Bisdichloroacetyldiamines strongly inhibit spermatogenesis. We reported previously that one of these compounds, WIN 18,446, potently inhibited spermatogenesis in rabbits by inhibiting retinoic acid synthesis. To understand how WIN 18,446 inhibits retinoic acid synthesis, we characterized its effects on human retinal dehydrogenase ALDH1A2 in vitro as well as its effects on retinoid metabolism in vivo using mice. WIN 18,446 strongly and irreversibly inhibited ALDH1A2 in vitro. In vivo, WIN 18,446 treatment completely abolished spermatogenesis after 4 weeks of treatment and modestly reduced adiposity in mice fed a chow diet. Effects of WIN 18,446 on retinoid concentrations were tissue-dependent. Although lung and liver retinyl ester concentrations were lower in WIN 18,446-treated animals, adipose retinyl ester levels were increased following the treatment. Interestingly, animals treated with WIN 18,446 had significantly higher circulating retinol concentrations compared with control mice. The effect on spermatogenesis by WIN 18,446 was not prevented by simultaneous treatment with retinoic acid, whereas effects on other tissues were partially or completely reversed. Cessation of WIN 18,446 treatment for 4 weeks reversed most retinoid-related phenotypes except for inhibition of spermatogenesis. Our data suggest that WIN 18,446 may be a useful model of systemic acquired retinoic acid deficiency. Given the effects observed in our study, inhibition of retinoic acid biosynthesis may have relevance for the treatment of obesity and in the development of novel male contraceptives.

Institutional animal care and use committee considerations for animal models of peripheral neuropathy.

Peripheral neuropathy and neuropathic pain are debilitating, life-altering conditions that affect a significant proportion of the human population. Animal models, used to study basic disease mechanisms and treatment modalities, are diverse and provide many challenges for institutional animal care and use committee (IACUC) review and postapproval monitoring. Items to consider include regulatory and ethical imperatives in animal models that may be designed to study pain, the basic mechanism of neurodegeneration, and different disease processes for which neuropathic pain is a side effect. Neuropathic pain can be difficult to detect or quantify in many models, and pain management is often unsuccessful in both humans and animals, inspiring the need for more research. Design of humane endpoints requires clear communication of potential adverse outcomes and solutions. Communication with the IACUC, researchers, and veterinary staff is also key for successful postapproval monitoring of these challenging models.