Paneth Cell-Derived Lysozyme Defines the Composition of Mucolytic Microbiota and the Inflammatory Tone of the Intestine.

Paneth cells are the primary source of C-type lysozyme, a ß-1,4-N-acetylmuramoylhydrolase that enzymatically processes bacterial cell walls. Paneth cells are normally present in human cecum and ascending colon, but are rarely found in descending colon and rectum; Paneth cell metaplasia in this region and aberrant lysozyme production are hallmarks of inflammatory bowel disease (IBD) pathology. Here, we examined the impact of aberrant lysozyme production in colonic inflammation. Targeted disruption of Paneth cell lysozyme (Lyz1) protected mice from experimental colitis. Lyz1-deficiency diminished intestinal immune responses to bacterial molecular patterns and resulted in the expansion of lysozyme-sensitive mucolytic bacteria, including Ruminococcus gnavus, a Crohn's disease-associated pathobiont. Ectopic lysozyme production in colonic epithelium suppressed lysozyme-sensitive bacteria and exacerbated colitis. Transfer of R. gnavus into Lyz1-/- hosts elicited a type 2 immune response, causing epithelial reprograming and enhanced anti-colitogenic capacity. In contrast, in lysozyme-intact hosts, processed R. gnavus drove pro-inflammatory responses. Thus, Paneth cell lysozyme balances intestinal anti- and pro-inflammatory responses, with implications for IBD.

Ammonia Accumulation as a Proxy to Determine 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 50ppm, 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 50ppm 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 1/2qt of bedding and 18% on 3/4qt of bedding reached greater than 50ppm ammonia before 7 d. All AGS on 1 and 2qt of bedding remained below 50ppm ammonia for 1 wk. Even when maintained on 2qt of bedding, not all AGS remained below 50ppm ammonia for 2 wk. Therefore, we concluded that the most appropriate option was weekly cage change for singly housed AGS on 1qt of bedding in static caging.

Pharmacokinetics and Efficacy of a Long-lasting, Highly Concentrated Buprenorphine Solution in Mice.

Buprenorphine is a commonly used opioid for mitigating pain in laboratory mice after surgical procedures; however, the dosing interval necessary for standard buprenorphine may require treatment every 4 to 6 h to maintain an adequate plane of analgesia. An alternative formulation that provides prolonged plasma concentration with long-lasting effects would be beneficial in achieving steady-state analgesia. We evaluated a long-lasting and highly concentrated formulation of buprenorphine (Bup-LHC) in mice. Pharmacokinetic analysis was performed to assess plasma concentrations in male C57BL/6J (B6) and female CD1 mice after subcutaneous injection of 0.9 mg/kg. The Bup-LHC formulation provided plasma drug levels that exceeded the therapeutic level for at least 12 h in male B6 mice and was below therapeutic levels by 8 h in female CD1 mice. An experimental laparotomy model was used to assess analgesic efficacy. Female CD1 mice were treated with either Bup-LHC (0.9 mg/kg) or saline at 1 h before undergoing an ovariectomy via a ventral laparotomy. At 3, 6, 12, 24, and 48 h after surgery, pain was assessed based on the following behaviors: orbital tightness, grooming, wound licking, rearing, arched posture, ataxia, piloerection, nest building, and general activity. At 3 and 6 h after surgery, Bup-LHC-treated mice had significantly less wound licking and orbital tightness and considerably higher activity levels than did saline-treated mice. At 12 h, wound licking, orbital tightness and activity in Bup-LHC-treated mice were no longer significantly different from those of saline-treated mice. The results of this study suggest that Bup-LHC at 0.9 mg/kg provides sufficient plasma concentrations for analgesia in mice for 6 to 12 h after administration, as demonstrated behaviorally for at least 6 h after surgery.

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.

Supernumerary Incisors in CB6F1 Mice Conditioned with Chemotherapy and Total Body Irradiation before Bone Marrow Transplantation.

Multiple adult female CB6F1 mice presented with supernumerary incisors after preconditioning with chemotherapy and total body irradiation for bone marrow transplantation (BMT). Mice received nonmyeloablative total body irradiation (3 Gy) and either cyclophosphamide or bendamustine, followed by BMT and posttransplantation cyclophosphamide or bendamustine. Here we describe the clinical presentation, µCT findings, and histopathologic evaluation of the affected mice. These analyses confirmed the gross diagnosis and revealed details of the abnormal tooth morphology. We surmise that the combination of total body irradiation and chemotherapy resulted in the abnormal formation of supernumerary incisors. Supernumerary teeth should be considered as a potential confounding factor in tracking weight loss after BMT. These conditions can be managed to allow animals to reach their intended scientific endpoint.

Microbial dysbiosis associated with impaired intestinal Na+/H+ exchange accelerates and exacerbates colitis in ex-germ free mice.

Intestinal epithelial Na+/H+ exchange facilitated by the apical NHE3 (Slc9a3) is a highly regulated process inhibited by intestinal pathogens and in inflammatory bowel diseases. NHE3-/- mice develop spontaneous, bacterially mediated colitis, and IBD-like dysbiosis. Disruption of epithelial Na+/H+ exchange in IBD may thus represent a host response contributing to the altered gut microbial ecology, and may play a pivotal role in modulating the severity of inflammation in a microbiome-dependent manner. To test whether microbiome fostered in an NHE3-deficient environment is able to drive mucosal immune responses affecting the onset or severity of colitis, we performed a series of cohousing experiments and fecal microbiome transplants into germ-free Rag-deficient or IL-10-/- mice. We determined that in the settings where the microbiome of NHE3-deficient mice was stably engrafted in the recipient host, it was able accelerate the onset and amplify severity of experimental colitis. NHE3-deficiency was characterized by the reduction in pH-sensitive butyrate-producing Firmicutes families Lachnospiraceae and Ruminococcaceae (Clostridia clusters IV and XIVa), with an expansion of inflammation-associated Bacteroidaceae. We conclude that the microbiome fostered by impaired epithelial Na+/H+ exchange enhances the onset and severity of colitis through disruption of the gut microbial ecology.

A Dynamic Anesthesia System for Long-Term Imaging in Adult Zebrafish.

Long-term in vivo imaging in adult zebrafish (i.e., 1-24 h) has been limited by the fact that regimens for long-term anesthesia in embryos and larvae are ineffective in adults. Here, we examined the potential for dynamic administration of benzocaine to enable long-term anesthesia in adult zebrafish. We developed a computer-controlled perfusion system comprised of programmable peristaltic pumps that enabled automatic exchange between anesthetic and system water. Continuous administration of benzocaine in adult zebrafish resulted in a mean time to respiratory arrest of 5.0 h and 8-h survival of 14.3%. We measured characteristic sedation and recovery times in response to benzocaine, and used them to devise an intermittent dosing regimen consisting of 14.5 min of benzocaine followed by 5.5 min of system water. Intermittent benzocaine administration in adult zebrafish resulted in a mean time to respiratory arrest of 7.6 h and 8-h survival of 71.4%. Finally, we performed a single 24-h trial and found that intermittent dosing maintained anesthesia in an adult zebrafish over the entire 24-h period. In summary, our studies demonstrate the potential for dynamic administration of benzocaine to enable prolonged anesthesia in adult zebrafish, expanding the potential for imaging in adult physiologies that unfold over 1-24 h.

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.