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Chronic inflammation disrupts the colonic epithelial layer in patients afflicted by ulcerative colitis (UC). The use of inhibitors of glycogen synthase kinase three beta (GSK3β) has proven efficacious to mitigate disease symptoms in rodent models of UC by reducing the pro-inflammatory response. Less is known about whether these inhibitors promote colonic regeneration by stimulating proliferation of colonic epithelial cells. We investigated whether delivery of the GSK3β inhibitor, lithium chloride (LiCl), during the recovery period from acute DSS-induced colitis in mice promoted colonic regeneration and ameliorated disease symptoms. We also tested whether the c-MYC transcription factor (MYC) was involved in this response. Acute colitis was induced by administration of 2.5 % dextran sodium sulfate (DSS) to wild-type C57BL/6 mice for 5 days. During the recovery period, mice received a daily intraperitoneal (IP) injection of LiCl or 1X PBS as a control. Mice were weighed, colon lengths measured, disease activity index (DAI) scores were assessed, and histological analyses were performed on colonic sections. We analyzed transcripts and proteins in purified preparations of the colonic epithelium. We delivered the MYC inhibitor 10058-F4 via IP injection to assess the role of MYC in colonic regeneration. Lithium treatments promoted recovery from acute DSS-induced damage by increasing expression of Myc transcripts, MYC proteins, and expression of a subset of Wnt/MYC target genes in the colonic epithelium. Inhibiting MYC function with 10058-F4 blunted the lithium response. By inducing Myc expression in the colonic epithelium, lithium promotes colonic regeneration after DSS-induced colitis. Therefore, the use of lithium may be of therapeutic value to manage individuals afflicted by UC.

Endocardiosis or myxomatous degeneration of the cardiac valves is a well-described age-related change in humans and dogs. Lesions consist of polypoid nodular proliferations of loose extracellular matrix and valvular interstitial cells, most commonly affecting the mitral valve. This entity has not been previously described in fish. Herein we report the appearance, location, and occurrence of valvular and mural endocardiosis in a retrospective survey of aging laboratory zebrafish. Endocardiosis was present in 59 of 777 fish (7.59%), most commonly affecting the sinoatrial (34 fish; 57.6%) and atrioventricular (33 fish; 55.9%) valves. Lesions were more common in fish raised in recirculating water systems and fed commercial diets (52/230 fish; 22.6%) versus flow-through systems with fish fed semi-purified diets (4/234; 1.71%). Lesions were overrepresented in fish heterozygous for a mutant smoothened allele (34/61 fish, 55.7% vs 17/168, 10.1% wild type). There was no association between endocardiosis and intestinal carcinoids. Valvular endocardiosis is a significant age- and husbandry-related background finding in zebrafish and should be considered in the design and interpretation of research studies.

The primary objective of this study was to evaluate a novel electrocardiogram (ECG)-synchronized pulsatile extracorporeal life support (ECLS) system for adult partial mechanical circulatory support for adequate quality of pulsatility and enhanced hemodynamic energy generation in an in vivo animal model. The secondary aim was to assess end-organ protection during nonpulsatile versus synchronized pulsatile flow mode. Ten adult swine were randomly divided into a nonpulsatile group (NP, n = 5) and pulsatile group (P, n = 5), and placed on ECLS for 24 h using an i-cor system consisting of an i-cor diagonal pump, an iLA membrane ventilator, an 18 Fr femoral arterial cannula and a 23/25 Fr femoral venous cannula. Trials were conducted at a flow rate of 2.5 L/min using nonpulsatile or pulsatile mode (with assist ratio 1:1). Real-time pressure and flow data were recorded using a custom-based data acquisition system. To the best of our knowledge, the oxygenator and circuit pressure drops were the lowest for any available system in both groups. The ECG-synchronized i-cor ECLS system was able to trigger pulsatile flow in the porcine model. After 24-h ECLS, energy equivalent pressure, surplus hemodynamic energy, and total hemodynamic energy at preoxygenator and prearterial cannula sites were significantly higher in the P group than those in the NP group (P 0.05). The novel i-cor system performed well in the nonpulsatile and ECG-synchronized pulsatile mode in an adult animal ECLS model. The iLA membrane oxygenator had an extremely lower transmembrane pressure gradient and excellent gas exchange capability. Our findings suggest that ECG-triggered pulsatile ECLS provides superior end-organ protection with improved renal function and systemic vascular tone.

Overactivation of the renin-angiotensin (Ang) system, and in particular Ang II, contributes to obesity-induced insulin resistance via multiple mechanisms. Obesity is also associated with deficiency of Ang-(1-7), a beneficial hormone that opposes Ang II actions. In this study, we tested the hypothesis that Ang-(1-7) could prevent development of diet-induced obesity in mice. To test this hypothesis, adult male C57BL/6J mice received 12-week Ang-(1-7) (400 ng/kg/min; n=7) or saline (n=6) infusion via subcutaneous osmotic mini-pumps. Mice were placed on 60% high fat diet (HFD) immediately following mini-pump implantation. Body composition, energy balance, and insulin sensitivity were measured during the last week of treatment. Ang-(1-7) attenuated HFD-induced weight gain (39.7±1.3 vs. 43.9±0.7 g saline; p=0.023) and adiposity (32±1 vs. 29±1% saline; p=0.050). This weight-reducing effect was due to enhanced average energy expenditure [0.55±0.02 vs. 0.42±0.06 kcal/hour saline, p=0.038], with no changes in locomotor activity or food intake. Ang-(1-7) attenuated HFD-induced hyperinsulinemia (1.4±0.4 vs. 5.3±1.6-ng/mL saline; p=0.032) and improved the ability of intraperitoneal insulin to decrease blood glucose levels (20% reduction in area under the curve vs. saline; p=0.015), consistent with insulin-sensitizing effects. Given effects of Ang-(1-7) on energy expenditure, we performed blinded histological and semi-quantitative real-time PCR analysis of brown and white adipose tissue. Ang-(1-7) reduced HFD-induced lipid droplet accumulation in brown adipose, but did not improve thermogenesis markers (e.g. uncoupling protein-1). Ang-(1-7) improved inflammation in visceral white adipose (mean number crown-like structures: 3±1 vs. 8±2 saline; p=0.046). Ang-(1-7) reduced adipocyte size and increased gene expression of the browning marker PRDM16 (1.7±0.2 vs. 1.1±0.1; p=0.024) in subcutaneous white adipose. These data suggest that Ang-(1-7) attenuates diet-induced obesity in mice by enhancing energy expenditure, in part by browning of white adipose tissue. These overall findings provide rationale for targeting Ang-(1-7) to prevent development of obesity and related pathology such as insulin resistance and adipose inflammation.

Mouse papillomavirus has shown broad tissue tropism in nude mice. Previous studies have tested cutaneous infections in different immunocompromised and immunocompetent mouse strains. In the current study, we examined mucosal infection in several immunocompetent and immunocompromised mouse strains. Viral DNA was monitored periodically by Q-PCR of lavage samples. Immunohistochemistry and in situ hybridization were used to determine viral capsid protein and viral DNA respectively. All athymic nude mouse strains showed active infections at both cutaneous and mucosal sites. Interestingly, NOD/SCID mice, which have a deficiency in T, B, and NK cells, showed minimal disease at cutaneous sites but developed persistent infection at the mucosal sites including those of the anogenital region and the oral cavity. Three strains of immunocompetent mice supported mucosal infections. Infections of the lower genital tract in heterozygous (immunocompetent) mice of the NU/J strain progressed to high grade dysplasia and to carcinoma in situ. Anti-MmuPV1 neutralizing antibodies were detected in the sera of all immunocompetent animals. Our findings demonstrate that the mucosae may be the preferred sites for this virus in mice. The mouse model is expected to be a valuable model for the study of mucosal papillomavirus disease, progression, and host immune control.

New influenza A viruses that emerge frequently elicit composite inflammatory responses to both infection and structural damage of alveolar-capillary barrier cells that hinders regeneration of respiratory function. The host factors that relinquish restoration of lung health to enduring lung injury are insufficiently understood. Here, we investigated the role of endophilin B2 (B2) in susceptibility to severe influenza infection. WT and B2-deficient mice were infected with H1N1 PR8 by intranasal administration and course of influenza pneumonia, inflammatory, and tissue responses were monitored over time. Disruption of B2 enhanced recovery from severe influenza infection as indicated by swift body weight recovery and significantly better survival of endophilin B2-deficient mice compared to WT mice. Compared to WT mice, the B2-deficient lungs exhibited induction of genes that express surfactant proteins, ABCA3, GM-CSF, podoplanin, and caveolin mRNA after 7 days, temporal induction of CCAAT/enhancer binding protein CEBPα, β, and δ mRNAs 3–14 days after infection, and differences in alveolar extracellular matrix integrity and respiratory mechanics. Flow cytometry and gene expression studies demonstrated robust recovery of alveolar macrophages and recruitment of CD4+ lymphocytes in B2-deficient lungs. Targeting of endophilin B2 alleviates adverse effects of IAV infection on respiratory and immune cells enabling restoration of alveolar homeostasis.

Intensely eosinophilic and glassy intracytoplasmic inclusions were present in the neurons of the peripheral autonomic ganglia, Meissner's and Auerbach's plexus, and spinal ganglia in 20 aged white-nosed coatis ( Nasua narica, 7-19 years old) and in 4 of 7 brown-nosed coatis ( Nasua nasua, 2-21 years old) from multiple zoological institutions. Inclusions were single to numerous, sometimes distorting the cell. Pheochromocytomas were present in 5 of 16 white-nosed and 2 of 6 brown-nosed coatis, although no inclusions were present in the adrenal glands. Histochemically, immunohistochemically, and ultrastructurally, these inclusions were consistent with dense neurosecretory granules. Although similar inclusions have been reported sporadically in the adrenal medulla of humans and several other mammalian species as both incidental and pathologic findings, ganglionic inclusions reported herein appear to be unique and related to age in these species.

Mer tyrosine kinase (Mer) signaling maintains immune tolerance by clearing apoptotic cells (ACs) and inducing immunoregulatory signals. We previously showed that Mer-deficient mice (Mer−/−) have increased germinal center (GC) responses, T cell activation, and AC accumulation within GCs. Accumulated ACs in GCs can undergo necrosis and release self-ligands, which may influence the outcome of a GC response and selection. In this study, we generated Mer−/− mice with a global MyD88, TLR7, or TLR9 deficiency and cell type–specific MyD88 deficiency to study the functional correlation between Mer and TLRs in the development of GC responses and autoimmunity. We found that GC B cell–intrinsic sensing of self-RNA, but not self-DNA, released from dead cells accumulated in GCs drives enhanced GC responses in Mer−/− mice. Although self-ligands directly affect GC B cell responses, the loss of Mer in dendritic cells promotes enhanced T cell activation and proinflammatory cytokine production. To study the impact of Mer deficiency on the development of autoimmunity, we generated autoimmune-prone B6.Sle1b mice deficient in Mer (Sle1b.Mer−/−). We observed accelerated autoimmunity development even under conditions where Sle1b.Mer−/− mice did not exhibit increased AC accumulation in GCs compared with B6.Sle1b mice, indicating that Mer immunoregulatory signaling in APCs regulates B cell selection and autoimmunity. We further found significant expansion, retention, and class-switching of autoreactive B cells in GCs under conditions where ACs accumulated in GCs of Sle1b.Mer−/− mice. Altogether, both the phagocytic and immunomodulatory functions of Mer regulate GC responses to prevent the development of autoimmunity.

OBJECTIVEIntracranial aneurysms are vascular abnormalities associated with neurological morbidity and mortality due to risk of rupture. In addition, many aneurysm treatments have associated risk profiles that can preclude the prophylactic treatment of asymptomatic lesions. Gamma Knife radiosurgery (GKRS) is a standard treatment for trigeminal neuralgia, tumors, and arteriovenous malformations. Aneurysms associated with arteriovenous malformations have been noted to resolve after treatment of the malformation. The aim of this study was to determine the efficacy of GKRS treatment in a saccular aneurysm animal model.METHODSAneurysms were surgically produced using an elastase-induced aneurysm model in the right common carotid artery of 10 New Zealand white rabbits. Following initial observation for 4 years, each rabbit aneurysm was treated with a conformal GKRS isodose of 25 Gy to the 50% margin. Longitudinal MRI studies obtained over 2 years and terminal measures obtained at multiple time points were used to track aneurysm size and shape index modifications.RESULTSAneurysms did not rupture or involute during the observation period. Whole aneurysm and blood volume averages decreased with a linear trend, at rates of 1.7% and 1.6% per month, respectively, over 24 months. Aneurysm wall percent volume increased linearly at a rate of 0.3% per month, indicating a relative thickening of the aneurysm wall during occlusion. Nonsphericity of the average volume, aspect ratio, and isoperimetric ratio of whole aneurysm volume all remained constant. Histopathological samples demonstrated progressive reduction in aneurysm size and wall thickening, with subintimal fibrosis. Consistent shape indices demonstrate stable aneurysm patency and maintenance of minimal rupture risk following treatment.CONCLUSIONSThe data indicate that GKRS targeted to saccular aneurysms is associated with histopathological changes and linear reduction of aneurysm size over time. The results suggest that GKRS may be a viable, minimally invasive treatment option for intracranial aneurysm obliteration.

Adoptive T cell transfer (ACT) has achieved clinical success in treating established cancer, particularly in combination with lymphodepleting regimens. Our group previously demonstrated that ACT following whole-body irradiation (WBI) promotes high-level T cell accumulation, regression of established brain tumors, and long-term protection from tumor recurrence in a mouse model of SV40 T antigen-induced choroid plexus tumors. Here we asked whether an approach that can promote strong donor T-cell responses in the absence of WBI might also produce this dramatic and durable tumor elimination following ACT. Agonist anti-CD40 antibody can enhance antigen-specific CD8(+) T-cell responses and has shown clinical efficacy as a monotherapy in the setting of cancer. We show that anti-CD40 conditioning promotes rapid accumulation of tumor-specific donor CD8(+) T cells in the brain and regression of autochthonous T antigen-induced choroid plexus tumors, similar to WBI. Despite a significant increase in the lifespan, tumors eventually recurred in anti-CD40-conditioned mice coincident with loss of T-cell persistence from both the brain and lymphoid organs. Depletion of CD8(+) T cells from the peripheral lymphoid organs of WBI-conditioned recipients failed to promote tumor recurrence, but donor cells persisted in the brains long-term in CD8-depleted mice. These results demonstrate that anti-CD40 conditioning effectively enhances ACT-mediated acute elimination of autochthonous tumors, but suggest that mechanisms associated with WBI conditioning, such as the induction of long-lived T cells, may be critical for protection from tumor recurrence.

Objectives Exogenous administration of cholecystokinin (CCK) induces hypertrophy and hyperplasia of the pancreas with an increase in DNA content. We hypothesized that endogenous CCK is involved in the malignant progression of pancreatic intraepithelial neoplasia (PanIN) lesions and the fibrosis associated with pancreatic cancer. Methods The presence of CCK receptors in early PanIN lesions was examined by immunohistochemistry in mouse and human pancreas. Pdx1-Cre/LSL-KrasG12D transgenic mice were randomized to receive either untreated drinking water or water supplemented with a CCK receptor antagonist (proglumide, 0.1 mg/mL). Pancreas from the mice were removed and examined histologically for number and grade of PanINs after 1, 2, or 4 months of antagonist therapy. Results Both CCK-A and CCK-B receptors were identified in early stage PanINs from mouse and human pancreas. The grade of PanIN lesions was reversed, and progression to advanced lesions arrested in mice treated with proglumide compared with the controls (P = 0.004). Furthermore, pancreatic fibrosis was significantly reduced in antagonist-treated animals compared with vehicle (P Conclusions These findings demonstrate that endogenous CCK is in part responsible for the development and progression of pancreatic cancer. The use of CCK receptor antagonists may have a role in cancer prophylaxis in high-risk subjects and may reduce fibrosis in the microenvironment.

BACKGROUND Prostate cancer (PCa) is a major aging-related disease for which little progress has been made in developing preventive strategies. Over the past several years, methionine restriction (MR), the feeding of a diet low in methionine (Met), has been identified as an intervention which significantly extends lifespan and reduces the onset of chronic diseases, including cancer, in laboratory animals. We, therefore, hypothesized that MR may be an effective strategy for inhibiting PCa. METHODS Control (0.86% Met) or MR (0.12% Met) diets were fed to 5-week old TRansgenic Adenocarcinoma of the Mouse Prostate (TRAMP) mice, a well-characterized model for PCa. The mice were sacrificed at 16 weeks of age and prostate and other tissues were harvested for histological and biochemical analyses. RESULTS As previously reported, MR was associated with a decrease in body weight which was not associated with lowered food intake. MR led to significant reductions in the development of Prostatic Intraepithelial Neoplasia (PIN) lesions, specifically in the anterior and dorsal lobes of the prostate where the incidence of high-grade PIN was reduced by ∼50% (P

Obesity and dietary fat are associated with increased risk of several malignancies including pancreatic cancer. The incidence of pancreatic cancer is increased in countries that consume diets high in fat.The purpose of this study was to assess the relationship and mechanism of action between dietary fat and endogenous cholecystokinin (CCK) on pancreatic tumor growth and metastasis in an immunocompetent animal model.C57BL/6 mice were placed on regular, low-fat, or high-fat diets for 8 weeks before establishment of Panc-02 orthotopic pancreatic tumors. Mice were then treated with a CCK-A receptor antagonist, devazepide, or vehicle for an additional 2.5 weeks. Pancreas tumors were weighed and metastases counted. Blood CCK levels were measured by radioimmunoassay (RIA). Tissues were examined histologically and studied for genes associated with metastasis by RT-PCR array. Effects of the CCK antagonist on Panc-02 cells invasiveness was assessed in a Matrigel invasion assay.Mice that received the high-fat diet had larger tumors and tenfold higher serum CCK levels by RIA compared to normal diet controls (p 0.01). Pancreatic tumors in high-fat diet mice treated with the antagonist had fewer intravascular tumor emboli and metastases compared to controls. The reduction in tumor emboli correlated with decreased vascular endothelial growth factor-A (VEGF-A) expression in tumors (p 6 × 10(-9)). In vitro invasiveness of Panc-02 cells also was reduced by CCK-A receptor antagonist treatment (p = 1.33 × 10(-6)).CCK is a mediator of dietary fat-associated pancreatic cancer. CCK is also involved in the invasiveness of pancreatic tumors through a mechanism involving VEGF-A.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Monocyte/macrophage recruitment correlates strongly with the progression of renal impairment in diabetic nephropathy (DN), yet their direct role is not clear. We hypothesized that macrophages contribute to direct podocyte injury and/or an abnormal podocyte niche leading to DN. Experiments were conducted in CD11b-DTR mice treated with diphtheria toxin (DT) to deplete macrophages after streptozotocin-induced diabetes. Additional experiments were conducted in bone marrow chimeric (CD11b-DTR→ C57BL6/J) mice. Diabetes was associated with an increase in the M1-to-M2 ratio by 6 wk after the induction of diabetes. Macrophage depletion in diabetic CD11b-DTR mice significantly attenuated albuminuria, kidney macrophage recruitment, and glomerular histological changes and preserved kidney nephrin and podocin expression compared with diabetic CD11b-DTR mice treated with mutant DT. These data were confirmed in chimeric mice indicating a direct role of bone marrow-derived macrophages in DN. In vitro, podocytes grown in high-glucose media significantly increased macrophage migration compared with podocytes grown in normal glucose media. In addition, classically activated M1 macrophages, but not M2 macrophages, induced podocyte permeability. These findings provide evidence showing that macrophages directly contribute to kidney injury in DN, perhaps by altering podocyte integrity through the proinflammatory M1 subset of macrophages. Attenuating the deleterious effects of macrophages on podocytes could provide a new therapeutic approach to the treatment of DN.

Recently, we showed that pharmacological blockade or genetic deficiency of arginase-2 confers kidney protection in diabetic mouse models. Here, we tested whether the protective effect of arginase inhibition is nitric oxide synthase 3 (eNOS) dependent in diabetic nephropathy. Experiments were conducted in eNOS-knockout and their wild-type littermate mice using multiple low doses of vehicle or streptozotocin, and treated with continuous subcutaneous infusion of vehicle or the arginase inhibitor S-(2-boronoethyl)-L-cysteine by an osmotic pump. Inhibition of arginases for 6 weeks in diabetic wild-type mice significantly attenuated albuminuria, the increase in plasma creatinine and blood urea nitrogen, histopathological changes, kidney fibronectin and TNF-α expression, kidney macrophage recruitment, and oxidative stress compared with vehicle-treated diabetic wild-type mice. Arginase inhibition in diabetic eNOS-knockout mice failed to affect any of these parameters, but reduced kidney macrophage recruitment and kidney TNF-α expression compared with vehicle-treated diabetic eNOS-knockout mice. Furthermore, diabetic wild-type and eNOS-knockout mice exhibited increased kidney arginase-2 protein, arginase activity, and ornithine levels. Thus, arginase inhibition mediates renal tissue protection in diabetic nephropathy by an eNOS-dependent mechanism and has an eNOS-independent effect on kidney macrophage recruitment.

We report here a detailed time course study of the individual and combined chemopreventive effects of Tamoxifen (Tam) and a high fish oil (FO) diet on multiple histologic parameters of mammary carcinogenesis. Groups of female Sprague-Dawley rats were injected ip with 1-methyl-1-nitrosourea at 50 days of age and assigned to either a control diet (20% corn oil [CO]) or a FO-rich diet (10% FO + 10% CO) in the presence and absence of Tam in the diet (0.6 ppm). Rats were sacrificed at weeks 4 (before palpable tumors), 8 and 12 (when ∼90% of control rats had palpable tumors). Our results demonstrate a major effect of Tam in inhibiting the development of early preneoplastic lesions. FO, while having a marginal protective effect of it own, enhanced the antitumor action of Tam on all histologic parameters of carcinogenesis, although the effects of the combination were not statistically different from those of Tam alone. The combination of FO and Tam was the only intervention that induced regression of established preneoplastic lesions. We also found that in contrast to plasma, only target tissue n-3 fatty acids (FAs) levels correlated with select tissue biomarkers of carcinogenesis whose expression was altered in a manner predictive of a protective effect. Our results demonstrating the potentially superior chemopreventive efficacy of Tam and n-3FA have important translational implications. Our data also emphasize the importance of local factors in affecting target tissue levels and biologic effects of n-3FA.

Pigment epithelium-derived factor (PEDF) is a multifunctional protein with antiangiogenic, antioxidative, and anti-inflammatory properties. PEDF is involved in the pathogenesis of diabetic retinopathy, but its direct role in the kidneys remains unclear. We hypothesize that a PEDF fragment (P78-PEDF) confers kidney protection in diabetic nephropathy (DN). The localization of the full-length PEDF protein were determined in DBA mice following multiple low doses of streptozotocin. Using immunohistochemistry, PEDF was localized in the kidney vasculature, interstitial space, glomeruli, tubules, and renal medulla. Kidney PEDF protein and mRNA expression were significantly reduced in diabetic mice. Continuous infusion of P78-PEDF for 6 wk resulted in protection from diabetic neuropathy as indicated by reduced albuminuria and blood urea nitrogen, increased nephrin expression, decreased kidney macrophage recruitment and inflammatory cytokines, and reduced histological changes compared with vehicle-treated diabetic mice. In vitro, P78-PEDF blocked the increase in podocyte permeability to albumin and disruption of the actin cytoskeleton induced by puromycin aminonucleoside treatment. These findings highlight the importance of P78-PEDF peptide as a potential therapeutic modality in early phase diabetic renal injury.

Papillomavirus disease poses a special challenge to people with compromised immune systems. Appropriate models to study infections in these individuals are lacking. We report here the development of a model that will help to address these deficiencies. The MmuPV1 genome was synthesized and used successfully to produce virus from DNA infections in immunocompromised mice. In these early studies, we have demonstrated both primary and secondary infections, expanded tissue tropism, and extensive dysplasia.

We previously reported that dibenzo[a,l]pyrene (DB[a,l]P), the most potent known environmental carcinogen among polycyclic aromatic hydrocarbons (PAH) congeners, is carcinogenic in the oral tissues of mice. We have now developed a new mouse model which employs the oral application of the fjord region diol epoxide, (±)-anti−11,12-dihydroxy-13,14-epoxy-11,12,13,14-tetrahydrodibenzo[a,l]pyrene (DB[a,l]PDE), a metabolite of the tobacco smoke constituent DB[a,l]P, and we show its specific induction of oral squamous cell carcinoma (OSCC) in both tongue and other oral tissues. Groups of B6C3F1 mice (20/group) received 6 or 3 nmol of (±)-anti-DB[a,l]PDE administered into the oral cavity; 3 times per week for 38 weeks. Additional groups received the vehicle alone or were left untreated. Mice were sacrificed 42 weeks after the first carcinogen administration. The high dose induced 74 and 100% OSCC in the tongue and other oral tissues, respectively; the corresponding values at the lower dose were 45 and 89%. Using immunohistochemistry, we showed that DB[a,l]PDE resulted in overexpression of p53 and COX-2 proteins in malignant tissues when compared to normal oral tissues and tongues. Consistent with the carcinogenicity, we demonstrated powerful mutagenicity in cII gene in B6C3F1 (Big Blue) mouse tongue. The mutational profile in lacI reporter gene is similar to those detected in human head and neck cancer, and p53 mutations were observed in mouse oral tumor tissues. Taken together, we conclude that the formation of diol epoxides plays a major role among the mechanisms by which DB[a,l]P exerts its oral mutagenicity and tumorigenicity.

Spontaneous age-related lesions of laboratory rabbits are not well documented in the contemporary scientific literature. A retrospective study of diagnostic necropsies of 36 rabbits >2 years of age found a number of common lung lesions. Fibromuscular intimal hyperplasia affected medium and to a lesser extent large pulmonary arteries and was present to a variable extent in all 36 rabbits >2 years of age. The lesions were characterized by fragmentation and/or reduplication of the internal elastic lamina (IEL), proliferation of smoothelin+/alpha-smooth muscle actin (α-SMA)+/vimentin− smooth muscle cells and fewer smoothelin−/α-SMA+/vimentin+ myofibroblasts, and intimal deposition of collagen without thrombosis, embolism, or evidence of pulmonary hypertension. Pulmonary emphysema, present in 30/36 rabbits, was characterized by the loss of alveolar septa; most affected rabbits did not have clinical signs of respiratory disease. In 8/13 rabbits of the inbred EIII/JC audiogenic strain, we identified a unique syndrome of granulomatous pneumonia containing hyaline brown to gray, globular to ring-like acellular material that was Alcian blue and periodic acid-Schiff positive. The material was immunoreactive for surfactant protein-A and had the ultrastructural appearance of multilamellar vesicles, suggesting a genetic defect in surfactant metabolism. Additionally, we found small benign primary lung tumors (fibropapillomas, 5 rabbits) not previously described. Other findings included heterotopic bone (5 rabbits), subacute to chronic suppurative bronchopneumonia, pyogranulomatous pneumonia with plant material, and pulmonary artifacts from barbiturate euthanasia solution.

Dietary methionine restriction (MR) improves healthspan in part by reducing adiposity and by increasing insulin sensitivity in rodent models. The purpose of this study was to determine whether MR inhibits tumor progression in breast cancer xenograft model and breast cancer cell lines. Athymic nude mice were injected with MCF10AT1 cells in Matrigel® and fed a diet containing either 0.86 % methionine (control fed, CF), or 0.12 % methionine (MR) for 12 weeks. Plasma amino acid concentrations were measured by UPLC, and proliferation and apoptosis were examined using RT-PCR, immunohistochemistry, and Cell Titer 96® Aqueous One Solution Cell Proliferation assay. Mice on the MR diet had reduced body weight and decreased adiposity. They also had smaller tumors when compared to the mice bearing tumors on the CF diet. Plasma concentrations of the sulfur amino acids (methionine, cysteine, and taurine) were reduced, whereas ornithine, serine, and glutamate acid were increased in mice on the MR diet. MR mice exhibited decreased proliferation and increased apoptosis in cells that comprise the mammary glands and tumors of mice. Elevated expression of P21 occurred in both MCF10AT1-derived tumor tissue and endogenously in mammary gland tissue of MR mice. Breast cancer cell lines MCF10A and MDA-MB-231 grown in methionine-restricted cysteine-depleted media for 24 h also up-regulated P21 and P27 gene expression, and MDA-MB-231 cells had decreased proliferation. MR hinders cancer progression by increasing cell cycle inhibitors that halt cell cycle progression. The application of MR in a clinical setting may provide a delay in the progression of cancer, which would provide more time for conventional cancer therapies to be effective.

Inbred mice are a unique model system for studying aging because of the genetic homogeneity within inbred strains, the short life span of mice relative to humans, and the rich array of analytic tools that are available. A large-scale aging study was conducted on 28 inbred strains representing great genetic diversity to determine, via histopathology, the type and diversity of spontaneous diseases that aging mice develop. A total of 20 885 different diagnoses were made, with an average of 12 diagnoses per mouse in the study. Eighteen inbred strains have had their genomes sequenced, and many others have been partially sequenced to provide large repositories of data on genetic variation among the strains. This vast amount of genomic information can be utilized in genome-wide association studies to find candidate genes that are involved in the pathogenesis of spontaneous diseases. As an illustration, this article presents a genome-wide association study of the genetic associations of age-related intestinal amyloidosis, which implicated 3 candidate genes: translocating chain-associated membrane protein 1 ( Tram1); splicing factor 3b, subunit 5 ( Sf3b5); and syntaxin 11 ( Stx11). Representative photomicrographs are available on the Mouse Tumor Biology Database and Pathbase to serve as a reference when evaluating inbred mice used in other genetic or experimental studies to rule out strain background lesions. Many of the age-related mouse diseases are similar, if not identical, to human diseases; therefore, the genetic discoveries have direct translational benefit.

Repeated bolus intravenous (IV) administration of large doses of beta-lactams and aminoglycosides has previously been associated with the development of eosinophilic and occlusive arterial lesions limited to the lungs in calves. Reviewing 13 years worth of records from left ventricular assist device implantation studies, morphologically identical segmental arterial lesions were present in 32 of the 56 calves receiving IV antibiotics, affecting lungs (6/50), kidneys (12/56), or lungs and kidneys (14/50). In 16 of these calves, renal arterial lesions spatially colocalized with renal cortical infarctions. Lesions were noted in additional abdominal organs in 4 of the 50 calves and were exclusively present in the liver in a single calf. Similar arterial lesions were also noted in the lungs (3/4), kidneys (1/4), liver (1/4), and spleen (1/4) of unimplanted calves receiving similar IV antibiotic regimens for bacterial infections. Lesions were observed with therapeutic IV doses of cephalosporins with or without aminoglycosides over shorter intervals than previously implicated. Lesions were significantly associated with increased peripheral eosinophil counts and mildly elevated, not reduced, arterial pulse pressures. This report documents the features of an idiosyncratic drug reaction with features strongly suggestive of an acute type-I hypersensitivity in this species.

Neoplastic growth is associated with increased polyamine biosynthetic activity and content. Tumor promoter treatment induces the rate-limiting enzymes in polyamine biosynthesis, ornithine decarboxylase (ODC), and S-adenosylmethionine decarboxylase (AdoMetDC), and targeted ODC overexpression is sufficient for tumor promotion in initiated mouse skin. We generated a mouse model with doxycycline (Dox)-regulated AdoMetDC expression to determine the impact of this second rate-limiting enzyme on epithelial carcinogenesis. TetO–AdoMetDC (TAMD) transgenic founders were crossed with transgenic mice (K5-tTA) that express the tetracycline-regulated transcriptional activator within basal keratinocytes of the skin. Transgene expression in TAMD/K5-tTA mice was restricted to keratin 5 (K5) target tissues and silenced upon Dox treatment. AdoMetDC activity and its product, decarboxylated AdoMet, both increased approximately 8-fold in the skin. This enabled a redistribution of the polyamines that led to reduced putrescine, increased spermine, and an elevated spermine:spermidine ratio. Given the positive association between polyamine biosynthetic capacity and neoplastic growth, it was somewhat surprising to find that TAMD/K5-tTA mice developed significantly fewer tumors than controls in response to 7,12-dimethylbenz[a]anthracene/12-O-tetradecanoylphorbol-13-acetate chemical carcinogenesis. Importantly, tumor counts in TAMD/K5-tTA mice rebounded to nearly equal the levels in the control group upon Dox-mediated transgene silencing at a late stage of tumor promotion, which indicates that latent viable initiated cells remain in AdoMetDC-expressing skin. These results underscore the complexity of polyamine modulation of tumor development and emphasize the critical role of putrescine in tumor promotion. AdoMetDC-expressing mice will enable more refined spatial and temporal manipulation of polyamine biosynthesis during tumorigenesis and in other models of human disease.

Evaluation of thrombogenicity is a critical component in the preclinical testing and development of blood pumps. Left ventricular assist devices (LVADs), because of their device routing, can produce thromboembolic showers to the kidney resulting in renal cortical ischemia or infarctions. Although postmortem evaluation of renal pathology can confirm ischemic events and infarctions, there are no validated and highly sensitive real-time measures of renal ischemia in the preclinical models. In this article, we report the evaluation of urinary biomarkers of ischemic tubular damage in a lamb preclinical LVAD model. We found that urinary excretion of glutathione-S-transferase-π, heat shock protein 1B, and hepatitis A virus cellular receptor 1 homologue precursor (HAVCR1/kidney injury molecule 1) were upregulated in toxic ischemic renal injury as well as in the immediate postoperative period in an LVAD-implanted lamb. These markers were consistent with both gross and histologic pathology, and proved far more sensitive for renal injury than serum blood urea nitrogen or creatinine concentrations.

Seventy-four 9-week old female C57BL/6J mice housed in a conventional facility were manipulated to induce experimental autoimmune encephalomyelitis, among which 26 developed clinical signs including lethargy, absence of defecation, and abdominal distension. By gross necropsy examination, there was distension of the cecum and colon with fecal impaction. By histologic examination, there was severe ulcerative and proliferative typhlocolitis. Fecal ELISA confirmed the presence of toxins A and B of Clostridium difficile. Alteration in immune status of the immunocompetent mice, due to stress caused by experimental manipulation or autoimmune disease, may have led to intestinal dysbiosis, followed by opportunistic infections resulting in C. difficile–associated disease. This report brings to light the occurrence of the disease in immunocompetent laboratory mice during experimental manipulations associated with alteration in immune status, and it discusses potential hazards associated with conventional housing within a hospital-associated research institute.

OBJECTIVE To determine 1) whether renal arginase activity or expression is increased in diabetes and 2) whether arginase plays a role in development of diabetic nephropathy (DN). RESEARCH DESIGN AND METHODS The impact of arginase activity and expression on renal damage was evaluated in spontaneously diabetic Ins2Akita mice and in streptozotocin (STZ)-induced diabetic Dilute Brown Agouti (DBA) and arginase-2–deficient mice (Arg2−/−). RESULTS Pharmacological blockade or genetic deficiency of arginase-2 conferred kidney protection in Ins2Akita mice or STZ-induced diabetic renal injury. Blocking arginases using S-(2-boronoethyl)-l-cysteine for 9 weeks in Ins2Akita mice or 6 weeks in STZ-induced diabetic DBA mice significantly attenuated albuminuria, the increase in blood urea nitrogen, histopathological changes, and kidney macrophage recruitment compared with vehicle-treated Ins2Akita mice. Furthermore, kidney arginase-2 expression increased in Ins2Akita mice compared with control. In contrast, arginase-1 expression was undetectable in kidneys under normal or diabetes conditions. Arg2−/− mice mimicked arginase blockade by reducing albuminuria after 6 and 18 weeks of STZ-induced diabetes. In wild-type mice, kidney arginase activity increased significantly after 6 and 18 weeks of STZ-induced diabetes but remained very low in STZ-diabetic Arg2−/− mice. The increase in kidney arginase activity was associated with a reduction in renal medullary blood flow in wild-type mice after 6 weeks of STZ-induced diabetes, an effect significantly attenuated in diabetic Arg2−/− mice. CONCLUSIONS These findings indicate that arginase-2 plays a major role in induction of diabetic renal injury and that blocking arginase-2 activity or expression could be a novel therapeutic approach for treatment of DN.

The vascular form of Ehlers-Danlos syndrome (vEDS), a rare disease with grave complications resulting from rupture of major arteries, is caused by mutations of collagen type III [α1 chain of collagen type III (COL3A1)]. The only, recently proven, preventive strategy consists of the reduction of arterial wall stress by β-adrenergic blockers. The heterozygous (HT) Col3a1 knockout mouse has reduced expression of collagen III and recapitulates features of a mild presentation of the disease. The objective of this study was to determine whether changing the balance between synthesis and degradation of collagen by chronic treatment with doxycycline, a nonspecific matrix metalloproteinase (MMP) inhibitor, could prevent the development of vascular pathology in HT mice. After 3 months of treatment with doxycycline or placebo, 9-month-old HT or wild-type (WT) mice were subjected to surgical stressing of the aorta. A 3-fold increase in stress-induced aortic lesions found in untreated HT mice 1 week after intervention (cumulative score 4.5 ± 0.87 versus 1.3 ± 0.34 in WT, p < 0.001) was fully prevented in the doxycycline-treated group (1.1 ± 0.56, p < 0.001). Untreated HT mice showed increased MMP-9 activity in the carotid artery and decreased collagen content in the aorta; however, in doxycycline-treated animals there was normalization to the levels observed in WT mice. Doxycycline treatment inhibits the activity of tissue MMP and attenuates the decrease in the collagen content in aortas of mice haploinsufficient for collagen III, as well as prevents the development of stress-induced vessel pathology. The results suggest that doxycycline merits clinical testing as a treatment for vEDS.

RecQ DNA helicases resolve Rad-51-mediated recombination and suppress aberrant homologous recombination. RecQ gene loss is associated with cancer susceptibility and increased mitotic recombination. We have developed an in vivo assay based on a zebrafish pigment mutant for suppression of RecQ activity, and demonstrate that zebrafish RecQ genes have conserved function in suppressing mitotic recombination.

Noninvasive and practical techniques to longitudinally track viral infection are sought after in clinical practice. We report a proof-of-principle study to monitor the viral DNA copy number using a newly established mouse papillomavirus (MmuPV1) mucosal infection model. We hypothesized that viral presence could be identified and quantified by collecting lavage samples from cervicovaginal, anal and oral sites. Nude mice infected at these sites with infectious MmuPV1 were tracked for up to 23 weeks starting at 6 weeks post-infection. Viral DNA copy number was determined by SYBR Green Q-PCR analysis. In addition, we tracked viral DNA load through three complete oestrous cycles to pinpoint whether there was a correlation between the DNA load and the four stages of the oestrous cycle. Our results showed that high viral DNA copy number was reproducibly detected from both anal and cervicovaginal lavage samples. The infection and disease progression were further confirmed by histology, cytology, in situ hybridization, immunohistochemistry and transmission electron microscopy. Interestingly, the viral copy number fluctuated over the oestrous cycle, with the highest level at the oestrus stage, implying that multiple sampling might be necessary to provide a reliable diagnosis. Virus DNA was detected in oral lavage samples at a later time after infection. Lower viral DNA load was found in oral samples when compared with those in anal and vaginal tracts. To our knowledge, our study is the first in vivo study to sequentially monitor papillomavirus infection from mucosal anal, oral and vaginal tracts in a preclinical model.

Sex differences in the incidence of respiratory diseases have been reported. Women are more susceptible to inflammatory lung disease induced by air pollution and show worse adverse pulmonary health outcomes than men. However, the mechanisms underlying these differences remain unknown. In the present study, we hypothesized that sex differences in the expression of lung inflammatory mediators affect sex-specific immune responses to environmental toxicants. We focused on the effects of ground-level ozone, a major air pollutant, in the expression and regulation of lung immunity genes. We exposed adult male and female mice to 2 ppm of ozone or filtered air (control) for 3 h. We compared mRNA levels of 84 inflammatory genes in lungs harvested 4 h postexposure using a PCR array. We also evaluated changes in lung histology and bronchoalveolar lavage fluid cell counts and protein content at 24 and 72 h postexposure. Our results revealed sex differences in lung inflammation triggered by ozone exposure and in the expression of genes involved in acute phase and inflammatory responses. Major sex differences were found in the expression of neutrophil-attracting chemokines ( Ccl20, Cxcl5, and Cxcl2), the proinflammatory cytokine interleukin-6, and oxidative stress-related enzymes ( Ptgs2, Nos2). In addition, the phosphorylation of STAT3, known to mediate IL-6-related immune responses, was significantly higher in ozone-exposed mice. Together, our observations suggest that a differential regulation of the lung immune response could be implicated in the observed increased susceptibility to adverse health effects from ozone observed in women vs. men.

The primary objective of this study was to evaluate a novel electrocardiogram (ECG)-synchronized pulsatile extracorporeal life support (ECLS) system for adult partial mechanical circulatory support for adequate quality of pulsatility and enhanced hemodynamic energy generation in an in vivo animal model. The secondary aim was to assess end-organ protection during nonpulsatile versus synchronized pulsatile flow mode. Ten adult swine were randomly divided into a nonpulsatile group (NP, n = 5) and pulsatile group (P, n = 5), and placed on ECLS for 24 h using an i-cor system consisting of an i-cor diagonal pump, an iLA membrane ventilator, an 18 Fr femoral arterial cannula and a 23/25 Fr femoral venous cannula. Trials were conducted at a flow rate of 2.5 L/min using nonpulsatile or pulsatile mode (with assist ratio 1:1). Real-time pressure and flow data were recorded using a custom-based data acquisition system. To the best of our knowledge, the oxygenator and circuit pressure drops were the lowest for any available system in both groups. The ECG-synchronized i-cor ECLS system was able to trigger pulsatile flow in the porcine model. After 24-h ECLS, energy equivalent pressure, surplus hemodynamic energy, and total hemodynamic energy at preoxygenator and prearterial cannula sites were significantly higher in the P group than those in the NP group (P 0.05). Urine output was higher in P versus NP (3379 ± 443 mL vs. NP, 2598 ± 1012 mL), and the P group seemed to require less inotropic support, but both did not reach statistical significances (P 0.05). The novel i-cor system performed well in the nonpulsatile and ECG-synchronized pulsatile mode in an adult animal ECLS model. The iLA membrane oxygenator had an extremely lower transmembrane pressure gradient and excellent gas exchange capability. Our findings suggest that ECG-triggered pulsatile ECLS provides superior end-organ protection with improved renal function and systemic vascular tone.

The ceramide nanoliposome (CNL) has shown promise in being able to treat a variety of primary tumors. However, its potential for treating metastatic cancer remains unknown. In this study, we demonstrate that CNL increases anoikis while preventing cancer cell extravasation under both static and physiological fluid flow conditions. Mechanistically, CNL limits metastases by decreasing CD44 protein levels in human breast and pancreatic cancer cells via lysosomal degradation of CD44, independent of palmitoylation or proteasome targeting. siRNA down-regulation of CD44 mimics CNL-induced anoikis and diminished extravasation of cancer cells. Taken together, our data indicate that ceramide limits CD44-dependent cancer cell migration, suggesting that CNL could be used to prevent and treat solid tumor metastasis.

Our recent publication showed that a small bioactive pigment epithelium derived factor (PEDF) peptide (P78-PEDF) prevents the development of diabetic nephropathy (DN). However, its effects on the progression of established DN were not clear. Therefore, the purpose of this study was to determine the effect of P78-PEDF in the progression of DN and to compare the effects of P78-PEDF and an ACE inhibitor (ACEi), a standard of care in DN. Experiments were conducted in Ins2(Akita) mice treated with P78-PEDF or captopril starting at 6 wks of age for 12 wks (early treatment) or starting at 12 wks of age for 6 wks (late treatment). We first established the optimal dose of the P78-PEDF peptide to ameliorate DN in Ins2(Akita) mouse for a 6 wk study period and found that the peptide was effective at 0.1- 0.5 µg/g/day. We next showed that early or late treatment with P78-PEDF resulted in protection from DN as indicated by reduced albuminuria, kidney macrophage recruitment, histological changes, inflammatory cytokines and fibrotic markers (kidney TNF-α, fibronectin, VEGFA and EGFR), and restored nephrin expression compared with vehicle-treated Ins2(Akita) mice. Interestingly, only early but not late treatment with captopril was as effective as P78-PEDF in reducing most DN complications, despite its lack of effect on nephrin, VEGFA and EGFR expression. These findings highlight the importance of P78-PEDF peptide as a potential therapeutic modality in both the development and progression of diabetic renal injury.

Marfan syndrome is a disease resulting from a mutation of the gene coding for fibrillin-1 ([1][1]), a protein that forms fibrils in the extracellular matrix, thus depleting elastic tissue of a key building block. As a result, the ascending aorta of these patients weakens and enlarges progressively

Aortic aneurysm and dissection are manifestations of Marfan syndrome (MFS), a disorder caused by mutations in the gene that encodes fibrillin-1. Selected manifestations of MFS reflect excessive signaling by the transforming growth factor–β (TGF-β) family of cytokines. We show that aortic aneurysm in a mouse model of MFS is associated with increased TGF-β signaling and can be prevented by TGF-β antagonists such as TGF-β–neutralizing antibody or the angiotensin II type 1 receptor (AT1) blocker, losartan. AT1 antagonism also partially reversed noncardiovascular manifestations of MFS, including impaired alveolar septation. These data suggest that losartan, a drug already in clinical use for hypertension, merits investigation as a therapeutic strategy for patients with MFS and has the potential to prevent the major life-threatening manifestation of this disorder.

The gene hypermethylated in cancer 1 (HIC1) is epigenetically inactivated, but not mutated, in cancer. Here we show that cooperative loss of Hic1 with p53, but not INK4a, yields distinct tumor phenotypes in mice. Germline deletion of one allele of each gene on the opposite chromosome yields breast and ovarian carcinomas and metastatic osteosarcomas with epigenetic inactivation of the wild-type Hic1 allele. Germline deletion of the two genes on the same chromosome results in earlier appearance and increased prevalence and aggressiveness of osteosarcomas with genetic deletion of both wild-type genes. In human osteosarcomas, hypermethylation of HIC1 is frequent only in tumors with p53 mutations. Our results indicate the importance of genes altered only through epigenetic mechanisms in cancer progression in conjunction with genetically modified tumor suppressor genes.

Monocyte/macrophage recruitment correlates strongly with the progression of diabetic nephropathy. Tumor necrosis factor-α (TNF-α) is produced by monocytes/macrophages but the direct role of TNF-α and/or macrophage-derived TNF-α in the progression of diabetic nephropathy remains unclear. Here we tested whether inhibition of TNF-α confers kidney protection in diabetic nephropathy via a macrophage-derived TNF-α-dependent pathway. Compared to vehicle-treated mice, blockade of TNF-α with a murine anti-TNF-α antibody conferred kidney protection in Ins2(Akita) mice as indicated by reductions in albuminuria, plasma creatinine, histopathologic changes, kidney macrophage recruitment, and plasma inflammatory cytokine levels at 18 weeks of age. To assess the direct role of macrophage-derived TNF-α in diabetic nephropathy, we generated macrophage-specific TNF-α-deficient mice (CD11b(Cre)/TNF-α(Flox/Flox)). Conditional ablation of TNF-α in macrophages significantly reduced albuminuria, the increase in plasma creatinine and blood urea nitrogen, histopathologic changes, and kidney macrophage recruitment compared to diabetic TNF-α(Flox/Flox) control mice after 12 weeks of streptozotocin-induced diabetes. Thus, production of TNF-α by macrophages plays a major role in diabetic renal injury. Hence, blocking TNF-α could be a novel therapeutic approach for treatment of diabetic nephropathy.

A 1-yr-old female Canada lynx (Lynx canadensis) presented for sudden onset of rapidly progressive bilateral pelvic limb paralysis. The lynx was chemically immobilized to perform a physical examination but expired shortly thereafter. On postmortem radiographs, there were myriad small irregular, round-to-spherical gas densities within the skeletal muscle of the right thigh and epaxial musculature. At gross necropsy, the muscles of the right thigh, right lateral abdominal wall, and epaxial region were emphysematous and necrohemorrhagic, with subcutaneous and muscular crepitant swelling. Multiple skin puncture wounds, consistent with bites, were present over the affected tissues. Clostridium septicum was isolated in pure anaerobic culture from the musculature of the right hind limb. Histopathologic examination confirmed the diagnosis of acute, severe necrohemorrhagic and gangrenous myositis and cellulitis. Gram stains demonstrated large gram-positive bacilli with subterminal spores. This is the first known documented case of C. septicum myonecrosis in a nondomestic felid.

Our recent publication showed that pharmacological blockade of arginases confers kidney protection in diabetic nephropathy via a nitric oxide (NO) synthase (NOS)3-dependent mechanism. Arginase competes with endothelial NOS (eNOS) for the common substrate l-arginine. Lack of l-arginine results in reduced NO production and eNOS uncoupling, which lead to endothelial dysfunction. Therefore, we hypothesized that l-arginine or l-citrulline supplementation would ameliorate diabetic nephropathy. DBA mice injected with multiple low doses of vehicle or streptozotocin (50 mg/kg ip for 5 days) were provided drinking water with or without l-arginine (1.5%, 6.05 g·kg−1·day−1) or l-citrulline (1.66%, 5.73 g·kg−1·day−1) for 9 wk. Nonsupplemented diabetic mice showed significant increases in albuminuria, blood urea nitrogen, glomerular histopathological changes, kidney macrophage recruitment, kidney TNF-α and fibronectin mRNA expression, kidney arginase activity, kidney arginase-2 protein expression, and urinary oxidative stress along with a significant reduction of nephrin and eNOS protein expression and kidney nitrite + nitrate compared with normal mice after 9 wk of diabetes. Surprisingly, l-arginine or l-citrulline supplementation in diabetic mice did not affect any of these parameters despite greatly increasing kidney and plasma arginine levels. These findings demonstrate that chronic l-arginine or l-citrulline supplementation does not prevent or reduce renal injury in a model of type 1 diabetes.

Seven male Hartley guinea pigs, 3 to 18 months old, died or had to be euthanized because of nonspecific clinical signs unresponsive to supportive treatment. Gross necropsy and histopathology findings in all animals included severe soft tissue calcification affecting the myocardium, kidneys, and occasionally the liver.

Objective To investigate the occurrence of spontaneous cataracts in a breeding colony of the inbred EIII/JC strain of New Zealand White rabbits (Oryctolagus cuniculi) and the congenic strain of EIII/JC-HLA-A2.1transgenic rabbits. Procedure A retrospective study was conducted by collecting and analyzing data from clinical records for individual rabbits filed between January 2011 and October 2013. Results Thirteen cases (eight females and five males) of cataract were identified in a group of 51 EIII/JC inbred rabbits with a morbidity of 25.5%. The median age of the rabbits identified with unilateral or bilateral cataracts was 43 months in contrast to the median age of 23 months of the entire group of 51 rabbits. Additionally, seven cases (five females and two males) of cataracts were identified in a group of 21 EIII/JC-HLA-A2.1 transgenic rabbits. The EIII/JC-HLA-A2.1 transgenic rabbits showed similar morbidity (33.3%) and median age (41 months) for the development of cataracts as the EIII/JC rabbits. In both groups, none of the rabbits younger than 37 months developed cataracts while 13 (93%) of 14 EIII/JC rabbits aged 37–49 months and seven (63.6%) of 11 EIII/JC-HLA-A2.1 transgenic rabbits aged 37–43 months developed cataracts. In contrast, none of 78 outbred rabbits with a median age of 26 months (10–67 months) developed cataracts. Conclusion Results of this study indicate that the occurrence and high incidence of spontaneous cataracts in this inbred strain (EIII/JC) of rabbits were strictly age related and consistently transmitted through inbreeding.

Chordomas are uncommon neoplasms arising from notochord remnants, most commonly occurring in the axial skeleton. Extraskeletal soft tissue chordomas are rare primary tumors, and primary alimentary tract chordomas have not been reported. Herein we report 24 cases of spontaneous primary intestinal chordomas in zebrafish, as well as 9 spontaneous vertebral chordomas. Both intestinal and vertebral tumors showed invasive behavior, although more commonly in the latter. In all cases of primary intestinal chordomas, there was no axial or peripheral skeletal or other nonvisceral involvement. Although uncommon, intestinal chordomas represent a unique background lesion in aged zebrafish.

Prostate cancer (PCa) is a major aging-related disease for which little progress has been made in developing preventive strategies. Over the past several years, methionine restriction (MR), the feeding of a diet low in methionine (Met), has been identified as an intervention which significantly extends lifespan and reduces the onset of chronic diseases, including cancer, in laboratory animals. We, therefore, hypothesized that MR may be an effective strategy for inhibiting PCa.Control (0.86% Met) or MR (0.12% Met) diets were fed to 5-week old TRansgenic Adenocarcinoma of the Mouse Prostate (TRAMP) mice, a well-characterized model for PCa. The mice were sacrificed at 16 weeks of age and prostate and other tissues were harvested for histological and biochemical analyses.As previously reported, MR was associated with a decrease in body weight which was not associated with lowered food intake. MR led to significant reductions in the development of Prostatic Intraepithelial Neoplasia (PIN) lesions, specifically in the anterior and dorsal lobes of the prostate where the incidence of high-grade PIN was reduced by ∼50% (P 0.02). The reduction in PIN severity was associated with 46-64% reductions in cell proliferation rates (P 0.02) and plasma IGF-1 levels (P 0.0001), which might, in part, explain the effects on carcinogenesis. Additionally, no adverse consequences of MR on immune function were observed in the TRAMP mice.Overall, these findings indicate that MR is associated with a reduction in prostate cancer development in the TRAMP model and supports the continued development of MR as a potential PCa prevention strategy.

Adoptive immunotherapy has demonstrated efficacy in a subset of clinical and preclinical studies, but the T cells used for therapy often are rendered rapidly nonfunctional in tumor-bearing hosts. Recent evidence indicates that prostate cancer can be susceptible to immunotherapy, but most studies using autochthonous tumor models demonstrate only short-lived T-cell responses in the tolerogenic prostate microenvironment. Here, we assessed the efficacy of sublethal whole-body irradiation (WBI) to enhance the magnitude and duration of adoptively transferred CD8+ T cells in the transgenic adenocarcinoma of the mouse prostate (TRAMP) model. We demonstrate that WBI promoted high-level accumulation of granzyme B (GzB, Gzmb)–expressing donor T cells both in lymphoid organs and in the prostate of TRAMP mice. Donor T cells remained responsive to vaccination in irradiated recipients, but a single round of WBI-enhanced adoptive immunotherapy failed to affect significantly the existing disease. Addition of a second round of immunotherapy promoted regression of established disease in half of the treated mice, with no progression observed. Regression was associated with long-term persistence of effector/memory phenotype CD8+ donor cells. Administration of the second round of adoptive immunotherapy led to reacquisition of GzB expression by persistent T cells from the first transfer. These results indicate that WBI conditioning amplifies tumor-specific T cells in the TRAMP prostate and lymphoid tissue, and suggest that the initial treatment alters the tolerogenic microenvironment to increase antitumor activity by a second wave of donor cells. Cancer Immunol Res; 2(8); 777–88. ©2014 AACR.

Our recent publication showed that pharmacological blockade of arginases confers kidney protection in diabetic nephropathy via a nitric oxide synthase 3 (NOS3)-dependent mechanism. Arginases compete with NOS for the common substrate L-arginine. L-arginine or L-citrulline supplementation showed beneficial effect in patients with hypertension, angina and erectile dysfunction by improving endothelial function with enhanced NO synthesis. Therefore, we hypothesized that L-arginine or L-citrulline supplementation would prevent or reduce diabetic renal complication. DBA mice injected with multiple low doses of vehicle or streptozotocin (STZ; 50 mg/kg ip for 5 days) were provided drinking water (DW) supplemented with L-arginine, L-citrulline, isonitrogenous nonessential amino acid control or no amino acids (DW control) for 9 weeks. Diabetic mice with DW control showed significant increases in urine albumin excretion (p

To the Editors, I enjoyed the recent review on left ventricular assist devices (LVADs) and kidney function by Tromp et al. [1]. I would, however, like to clarify one point related to animal studies on continuous flow (cf) versus pulsatile flow (pf) LVADs. The authors state ‘‘Animal studies report proliferation of SMCs in the afferent arteriole in the renal cortex [2, 3] and perivascular tissue [3], but could not determine whether this change in morphology affected afferent arteriolar constriction and renal function. Infiltration of inflammatory cells in the renal cortical matrix has been observed, suggesting an immunologic mechanism for SMC hypertrophy [2]. Interestingly, reduced pulsatility may induce (severe) periarteritis in the kidneys, an observation not made in control animals supported by pf-devices.’’ Ohnishi et al. do indeed report proliferation of smooth muscle cells in the renal afferent arterioles of goats implanted with cf-LVADs. Ootaki et al., on the other hand, do not report any changes to the afferent arterioles in six calves implanted with cf-LVADs. Rather, the lesions reported by Ootaki et al. were observed in medium arcuate and interlobular arteries at the corticomedullary junction. These medium artery findings were consistent with previously described renal medium arterial lesions in calves implanted with cf-LVADs as reported by Kihara et al. [4], and with pulmonary artery lesions more recently reported in calves with cf-RVADs [5]. This critical distinction on lesion location (and species) is germane because, while smooth muscle cell proliferation in the afferent arteriole is fully consistent with fine control of glomerular blood pressure, lesions in the arcuate and interlobular (and pulmonary) arteries of calves may represent something else entirely. We have recently published a large retrospective study describing lesions of the renal arcuate and radial arteries and pulmonary arteries in 56 calves, including those implanted with either pulsatile or non-pulsatile LVADs as well as unimplanted calves [6]. Lesions included both smooth muscle cell hyperplasia and inflammation. The common thread to these cases was the use of intravenous cephalosporin antibiotics, unrelated to pump design (or even presence). Characteristic pulmonary arterial lesions in calves receiving intravenous penicillin or cephalosporin antibiotics were originally described in studies by Majeski and Fitts [7], and appear to be a specific idiopathic reaction in cattle. Regrettably, neither Ootaki et al. nor Kihara et al. indicate whether the calves in their studies similarly received intravenous beta-lactam class antibiotics. It therefore remains to be seen the translatability of the arterial findings in the calf LVAD preclinical model to human patients. It should be noted that humans and sheep implanted with cf-LVADs do not appear to show any renal arterial lesions [8, 9].

Congenital malformations may occur during early embryogenesis in cases of genetic abnormalities or various environmental factors. Affected subjects most often have only one or 2 abnormalities; subjects rarely have several unrelated congenital defects. Here we describe a case of a stillborn New Zealand white rabbit with multiple complex congenital malformations, including synophthalmia, holoprosencephaly, gastroschisis, and a supernumerary hindlimb, among other anomalies. There was no historical exposure to teratogens or other known environmental causes. Although not confirmed, this case was most likely a rare spontaneous genetic event.

An 8-mo-old female transgenic adenocarcinoma of the mouse prostate (C57BL/6-Tg(TRAMP)8247Ng/J) mouse presented with abdominal distention, lethargy, and serosanguineous vaginal discharge. A large primary renal tumor with metastases to lung and liver was present at necropsy. The tumor was composed of poorly differentiated and crowded epithelial cells forming ducts, acini, and cribriform patterns, with comedonecrosis and frequent bizarre mitoses. Immunohistochemistry revealed that neoplastic cells expressed nuclear SV40 T antigen, confirming aberrant expression of the transgene. In addition, cells were positive for pancytokeratin and negative for synaptophysin and estrogen and progesterone receptors. This report details the first transgene-induced tumor in a female TRAMP mouse.