Murine Norovirus: An Intercurrent Variable in a Mouse Model of Bacteria-Induced Inflammatory Bowel Disease

Murine norovirus (MNV) has recently been recognized as a widely prevalent viral pathogen in mouse colonies and causes disease and mortality in mice with impaired innate immunity. We tested the hypothesis that MNV infection would alter disease course and immune responses in mice with inflammatory bowel disease (IBD). FVB.129P2-Abcb1a^tm1Bor N7 (Mdr1a−/−) mice develop spontaneous IBD that is accelerated by infection with Helicobacter bilis. As compared with controls, Mdr1a−/− mice coinfected with MNV4 and H. bilis showed greater weight loss and IBD scores indicative of severe colitis, demonstrating that MNV4 can modulate the progression of IBD. Compared with controls, mice inoculated with MNV4 alone had altered levels of serum biomarkers, and flow cytometric analysis of immune cells from MNV4-infected mice showed changes in both dendritic cell (CD11c⁺) and other nonT cell (CD4⁻ CD8⁻) populations. Dendritic cells isolated from MNV4-infected mice induced higher IFNγ production by polyclonal T cells in vitro at 2 d after infection but not at later time points, indicating that MNV4 infection enhances antigen presentation by dendritic cells early after acute infection. These findings indicate that acute infection with MNV4 is immunomodulatory and alters disease progression in a mouse model of IBD.Abbreviations: DC, dendritic cell; IBD, inflammatory bowel disease; IP, IFNγ–inducible protein; MCP, macrophage chemotactic protein; MLN, mesenteric lymph node; MNV, murine norovirus; TNF, tumor necrosis factorThe genus Norovirus of the family Caliciviridae contains a large number of single-stranded, positive-sense RNA viruses that infect vertebrates, and strains have been identified in humans, cattle, swine, and (most recently) mice.^19,29,34 Murine noroviruses (MNV) are recently recognized pathogens that can cause lethal infection in immunocompromised mice that lack innate immunity.¹⁹ However, MNV did not cause clinical disease in wild-type mice or many other strains of immunodeficient mice, including those lacking the recombination-activating gene (Rag−/−) and inducible nitric oxide synthase deficient mice.^19,35,37 MNV was reported recently to be widespread in laboratory mice and may persist in immunocompetent animals, depending on the strain of MNV used.^15,16,25 Studies in Rag−/− mice and B-cell–deficient strains showed that the acquired immune system plays an important role in the clearance of MNV.^6,19,37 MNV has tropism for dendritic cells (DCs),³⁶ which are important in the presentation of antigens to T cells in draining lymph nodes and in the pathogenesis of inflammatory bowel disease (IBD). Therefore, MNV is a potential confounder for in vivo immunology studies, including murine models of IBD.Idiopathic IBD, which encompasses both ulcerative colitis and Crohn disease, is a widely studied disorder that affects approximately 1.4 million people in the United States.²⁰ Although the precise cause of human IBD has not been elucidated, studies with mouse models have demonstrated that abnormal host responses of the innate and adaptive immune systems to intestinal microbiota are important in the pathogenesis of IBD.^28,38 DCs are the sentinels of the intestinal mucosal barrier and have a pivotal role in the initiation of IBD in response to microbial ligands.³⁹ Alterations in DC responses could lead to persistence of bacterial infection, aberrant activation of the acquired immune system, and (ultimately) tissue damage.³⁸Viral stimulation of DCs leads to activation of adaptive immune responses,¹⁷ including effector T cells, and as demonstrated with murine coronavirus (mouse hepatitis virus), intercurrent viral infections in mice can alter the phenotype of mouse models of human disease.¹⁰ Additional evidence suggests that intercurrent viral infection may enhance disease in human IBD patients.^12,18 Whether infection with MNV alters DC function and, therefore, influences the progression of IBD in mouse models is unclear.Many mouse models of intestinal inflammation develop IBD that is driven by bacterial flora.^9,28 Helicobacter spp. have been shown to drive this process in several mouse models including IL10-deficient, SMAD3-deficient, severe combined immunodeficiency and T-cell–deficient mice.^4,5,13,23 FVB.129P2-Abcb1a^tm1Bor (Mdr1a−/−) mice develop spontaneous IBD that is accelerated by infection with Helicobacter bilis.^21,22 In this report, we tested the hypothesis that infection with MNV can modulate IBD in this mouse model of bacterial-induced disease. We demonstrate that intercurrent MNV4 infection accelerates the progression of bacterial-induced IBD in the Mdr1a−/− mouse and alters the immune responses in this mouse model of IBD.     

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 IL1β, 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.Abbreviations: BMDM, bone marrow-derived macrophages; IBD, inflammatory bowel disease; MLN, mesenteric lymph node; MNV, murine norovirusInflammatory bowel disease (IBD), which includes both ulcerative colitis and Crohn disease, is a chronic and relapsing inflammatory disorder of the gastrointestinal tract. In addition, patients with IBD may be at increased risk of developing colorectal cancer.^15,46 Although the exact mechanisms of disease are still not understood fully, the pathogenesis of disease is likely multifactorial, with components of the innate and adaptive immune systems, host genetics, and environmental factors (for example, the commensal gut microflora) all playing a role.^4,37,55Animal models of IBD have been used to advance our knowledge and understanding of IBD pathogenesis and treatment.^{16,20,37,38,52} One such model that has been widely used to elucidate the mechanisms of IBD is the interleukin10–deficient (Il10^−/−) mouse.^{3,5,6,20,21,29,33,57} The antiinflammatory cytokine IL10 modulates both innate and adaptive immune responses.⁴¹ Produced mainly by dendritic cells, monocytes, macrophages, and T regulatory cells, IL10 exerts its immunomodulatory effects by various mechanisms including decreasing secretion of proinflammatory cytokines (for example, interferon γ, IL1, IL2, IL6, IL12 and TNFα) and downregulating important components of innate immune responses and T-cell activation (for example, MHC class II, costimulatory molecules, and nitric oxide production) in antigen presenting cells.^14,41 As a consequence, Il10^−/− mice, which lack the suppressive effects of IL10, develop IBD in response to their commensal gut microflora or to certain microbial triggers such as Helicobacter infections.^{5,6,11,21,29,52,57}Antigen-presenting cells such as macrophages and dendritic cells play key roles in the inflammatory responses in IBD.^32,47,50 In 2003, a newly discovered murine norovirus (MNV) in laboratory mice was shown to infect macrophages and dendritic cells.^27,53 Subsequent studies indicated widespread MNV infection in laboratory mice used for biomedical research, with a serologic prevalence as high as 32%.^25,43 Members of the genus Norovirus are regarded as gastrointestinal pathogens in humans and animals, eliciting both innate and adaptive immune responses.¹⁹ Therefore, in light of the cellular (macrophages and dendritic cells) and tissue (gastrointestinal) tropisms of MNV as well as the high prevalence of MNV infection in laboratory mice, we hypothesized that MNV infection could be a potential confounder in mouse models of inflammatory diseases including IBD. In support of this idea, our laboratory recently reported that MNV infection in Mdr1a^−/− mice (FVB.129P2-Abcb1a^tm1Bor) accelerated weight loss and exacerbated IBD progression initiated by H. bilis infection.³¹ This effect potentially was mediated in part through modulating dendritic cell and cytokine responses. In addition, others have reported gastrointestinal abnormalities as a result of MNV infection in some strains of mice,^7,26,36 whereas others have described the importance of both innate and adaptive immune responses during MNV infection.^{8,9,10,28,34,36,48} Collectively, these data indicate that MNV could alter inflammatory responses in laboratory mice.Here we extended our studies of MNV beyond Mdr1a^−/− mice to Il10^−/− mice, another common animal model of IBD, to further examine the potential effect of MNV on IBD research. Disease was initiated in Il10^−/− mice with H. bilis, and we determined whether coinfection with MNV altered disease development, incidence, and severity and the production of cytokines. We demonstrated that although MNV stimulates a Th1 skewing of cytokines in Il10^−/− bone marrow-derived macrophages (BMDM) in vitro, MNV does not alter the development, incidence, or severity of disease in vivo. Therefore, although MNV may not affect disease in Il10^−/− mouse models, the virus may influence in vitro cytokine phenotypes and thus complicate interpretation of such data. To our knowledge, this report is the first to describe the evaluation of MNV infection in the Helicobacter-induced Il10^−/− mouse model of IBD.     

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.Abbreviations: ABCA1, ATP-binding cassette A1; BMDM, bone-marrow–derived macrophage; iNOS, inducible nitric oxide synthase; MNV, murine norovirusChronic viral infection, such as occurs with HIV and hepatitis C virus, has been associated with an increased risk for atherosclerosis,^2,19,46,48 although the mechanisms by which this occurs are not clearly defined. Our laboratory has been studying the effects of murine norovirus (MNV), which chronically infects immunocompetent mice, on murine models of inflammatory diseases, including atherosclerosis. MNV is a single-stranded RNA intestinal virus that belongs to the family Caliciviridae and has shown tropism toward antigen-presenting cells such as dendritic cells and macrophages.⁵⁴ Whereas human norovirus is a major cause of nonbacterial acute gastroenteritis,⁵² MNV does not cause clinical disease in immunocompetent mice.⁵⁵ However, the high prevalence of MNV in biomedical research facilities throughout the world,^42,55 combined with its tropism for antigen-presenting cells, has prompted concern regarding potential effects on disease phenotypes in murine models of human diseases. Therefore, we previously examined 2 diseases, obesity and atherosclerosis, where macrophages have critical roles.^41,42 We found that MNV infection did not influence glucose metabolism and weight gain,⁴¹ but it significantly increased the size and macrophage content of aortic sinus lesions in Ldlr^−/− mice fed an atherogenic diet.⁴² These findings suggest that MNV might be a potential tool to determine how viral infection alters the risk of atherosclerosis.Many factors influence the progression of atherosclerosis. Accordingly, we examined whether the timing of MNV infection relative to the stage of atherosclerosis progression influenced disease phenotype and evaluated potential mechanisms by which MNV could affect the disease process. To this end, we modeled the infection of macrophages by using in vitro cultures of bone-marrow–derived macrophages (BMDM).     

Anatomic,Hematologic, and Biochemical Features of C57BL/6NCrl Mice Maintained on Chronic Oral Corticosterone

Metabolic syndrome is a condition that typically includes central obesity, insulin resistance, glucose intolerance, dyslipidemia, and hypertension. Disruption of the hypothalamic–pituitary–adrenal axis, a regulator of corticosterone secretion, occurs in some cases of metabolic syndrome and obesity, and Cushing hypercortisolemia is associated with obesity and metabolic disorders. We therefore assessed anatomic and clinical pathology in C57BL/6NCrl mice to evaluate the effects of chronic corticosterone in the drinking water at doses of 25, 50, and 100 μg/mL for 25 d. Treated mice developed obesity, glucose intolerance, electrolyte aberrations, and dyslipidemia that were dose-dependent and most severe in the 100-μg/mL treatment group. To evaluate return to normal function, additional C57BL/6NCrl mice received corticosterone-free water for 2 wk after the 25-d treatment period. According to results of gross examination, mice appeared to recover within days of exogenous corticosterone withdrawal; however, adrenal gland vacuolation and protein, lipid, and electrolyte abnormalities persisted. Together, these findings support chronic corticosterone exposure through the drinking water as a potentially useful, noninvasive method to induce some features of metabolic syndrome.Obesity and associated metabolic dysfunctions are an increasing public health concern in modern Western society. In humans, obesity and metabolic syndrome heighten the risk of developing debilitating and costly illness including diabetes, cardiovascular disease, stroke, and some forms of cancer.^2,20 Mounting evidence indicates that stress and associated hormones such as cortisol (corticosterone in rodents) contribute to the development of metabolic syndrome. Furthermore, regional glucocorticoid metabolism in adipocytes is proposed to be involved in the pathogenesis of metabolic syndrome.^{6,16,17,27,56} Cushing syndrome, iatrogenic hypercortisolemia, and metabolic syndrome share clinical and physiologic similarities, including central obesity, insulin resistance, glucose intolerance, dyslipidemia, and hypertension.^{1,2,31,35,41,46} How glucocorticoids contribute to the development of these problems remains unclear.Numerous clinical and experimental studies have linked stress, diet, and lifestyle choices to changes in risk factors associated with the development of metabolic disorders.^{1,3,7,10,21,33,36,42,55} How corticosterone influences this risk remains unclear. Although corticosterone has beneficial short-term effects, long-term corticosterone exposure can result in damage to the physiologic systems it protects acutely.²⁷ Disruption of this physiologic signal occurs in numerous disparate disorders, ranging from depression to Cushing syndrome.^16,22,36,54 Therefore, understanding the effects of chronic high corticosterone on metabolism and physiology is of key importance.To clarify how chronic treatment with corticosterone alters the physiology of an organism, we treated adrenally intact adult male mice with corticosterone in drinking water for 4 wk. Furthermore, we examined the return of physiology 2 wk after withdrawal of chronic corticosterone administration. We used this approach as a rapid (3- to 4-wk), noninvasive method of altering plasma corticosterone levels that enabled us to retain some integrity in the diurnal rhythm present in normal animals.We previously characterized the gross metabolic consequences of exogenous noninvasive corticosterone delivery in the drinking water.^20,28 In those studies, we found that high doses of corticosterone (100 μg/mL) resulted in rapid and dramatic hyperphagia; weight gain; increased adiposity; elevated plasma corticosterone, leptin, insulin, and triglyceride levels; and decreased homecage locomotion.²⁰ Moreover, several studies have shown that a lower dose of corticosterone (25 μg/mL) resulted in an intermediate phenotype in some of these measures but had no effect on others.^{12,14,20,23,28,38,42,47} As such, the high corticosterone dose results in a phenotype that satisfies most of the criteria for metabolic syndrome as defined by the National Heart, Lung, and Blood Institute and the American Heart Association.¹⁵ However, little information is available on the resulting histologic, hematologic, and serum chemical profiles associated with this treatment. We sought to more fully characterize this model to support selection of the model that most accurately reflects the human disease conditions under study. In-depth characterization of the model also provides more precise measurements of response to therapies intended to ameliorate the effects of the treatment.The current study provides a detailed examination of the physiologic effect of 3 dosages of corticosterone—low (25 μg/mL), intermediate (50 μg/mL), and high (100 μg/mL) doses—in drinking water. The goal was to extend the previous findings that established this regimen as a model of metabolic syndrome by exploring the detailed physiologic changes associated with this model and to assess whether and how treated mice recover after withdrawal of the corticosterone treatment. We propose that the physiologic changes observed in the mice treated with high-dose corticosterone approximate changes observed in human patients with metabolic syndrome and that these mice potentially serve as a model for hypercortisolemia and associated obesity. In addition, we hypothesized that 2 wk of recovery from corticosterone treatment would not completely resolve cellular and clinical pathologies characterized during treatment, given the numerous changes in physiology.     

Depletion of white adipocyte progenitors induces beige adipocyte differentiation and suppresses obesity development

Overgrowth of white adipose tissue (WAT) in obesity occurs as a result of adipocyte hypertrophy and hyperplasia. Expansion and renewal of adipocytes relies on proliferation and differentiation of white adipocyte progenitors (WAP); however, the requirement of WAP for obesity development has not been proven. Here, we investigate whether depletion of WAP can be used to prevent WAT expansion. We test this approach by using a hunter-killer peptide designed to induce apoptosis selectively in WAP. We show that targeted WAP cytoablation results in a long-term WAT growth suppression despite increased caloric intake in a mouse diet-induced obesity model. Our data indicate that WAP depletion results in a compensatory population of adipose tissue with beige adipocytes. Consistent with reported thermogenic capacity of beige adipose tissue, WAP-depleted mice display increased energy expenditure. We conclude that targeting of white adipocyte progenitors could be developed as a strategy to sustained modulation of WAT metabolic activity.Obesity, a medical condition predisposing to diabetes, cardiovascular diseases, cancer, and complicating other life-threatening diseases, is becoming an increasingly important social problem.^{1, 2, 3} Development of pharmacological approaches to reduction of body fat has remained a daunting task.⁴ Approved obesity treatments typically produce only moderate and temporary effects.^2,5 White adipocytes are the differentiated cells of white adipose tissue (WAT) that store triglycerides in lipid droplets.^6,7 In contrast, adipocytes of brown adipose tissue (BAT) dissipate excess energy through adaptive thermogenesis. Under certain conditions, white adipocytes can become partially replaced with brown-like ‘beige'' (‘brite'') adipocytes that simulate the thermogenic function of BAT adipocytes.^7,8 Obesity develops in the context of positive energy balance as a result of hypertrophy and hyperplasia of white adipocytes.⁹Expansion and renewal of the white adipocyte pool in WAT continues in adulthood.^10,11 This process is believed to rely on proliferation and self-renewal of mesenchymal precursor cells¹² that we term white adipocyte progenitors (WAPs). WAPs reside within the population of adipose stromal cells (ASCs)¹³ and are functionally similar to bone marrow mesenchymal stem cells (MSCs).^{14, 15, 16} ASCs can be isolated from the stromal/vascular fraction (SVF) of WAT based on negativity for hematopoietic (CD45) and endothelial (CD31) markers.^17,18 ASCs support vascularization as mural/adventitial cells secreting angiogenic factors^5,19 and, unlike bone marrow MSCs, express CD34.^19,20 WAPs have been identified within the ASC population based on expression of mesenchymal markers, such as platelet-derived growth factor receptor-β (PDGFRβ, aka CD140b) and pericyte markers.^17,18 Recently, a distinct ASC progenitor population capable of differentiating into both white and brown adipocytes has been identified in WAT based on PDGFRα (CD140a) expression and lack of PDGFRβ expression.^21,22 The physiological relevance of the two precursor populations residing in WAT has not been explored.We have previously established an approach to isolate peptide ligands binding to receptors selectively expressed on the surface of cell populations of interest.^{23, 24, 25, 26, 27} Such cell-targeted peptides can be used for targeted delivery of experimental therapeutic agents in vivo. A number of ‘hunter-killer'' peptides²⁸ composed of a cell-homing domain binding to a surface marker and of KLAKLAK₂ (sequence KLAKLAKKLAKLAK), a moiety inducing apoptosis upon receptor-mediated internalization, has been described by our group.^26,29 Such bimodal peptides have been used for depletion of malignant cells and organ-specific endothelial cells in preclinical animal models.^26,30,31 Recently, we isolated a cyclic peptide WAT7 (amino acid sequence CSWKYWFGEC) based on its specific binding to ASCs.²⁰ We identified Δ-decorin (ΔDCN), a proteolytic cleavage fragment of decorin, as the WAT7 receptor specifically expressed on the surface of CD34+PDGFRβ+CD31-CD45- WAPs and absent on MSCs in other organs.²⁰Here, we investigated whether WAPs are required for obesity development in adulthood. By designing a new hunter-killer peptide that directs KLAKLAK₂ to WAPs through WAT7/ΔDCN interaction, we depleted WAP in the mouse diet-induced obesity model. We demonstrate that WAP depletion suppresses WAT growth. We show that, in response to WAP deficiency, WAT becomes populated with beige adipocytes. Consistent with the reported thermogenic function of beige adipocytes,^32,33 the observed WAT remodeling is associated with increased energy expenditure. We identify a population of PDGFRα-positive, PDGFRβ-negative ASCs reported recently²² as a population surviving WAP depletion and responsible for WAT browning.     

Effects of Helicobacter Infection on Research: The Case for Eradication of Helicobacter from Rodent Research Colonies

Infection of mouse colonies with Helicobacter spp. has become an increasing concern for the research community. Although Helicobacter infection may cause clinical disease, investigators may be unaware that their laboratory mice are infected because the pathology of Helicobacter species is host-dependent and may not be recognized clinically. The effects of Helicobacter infections are not limited to the gastrointestinal system and can affect reproduction, the development of cancers in gastrointestinal organs and remote organs such as the breast, responses to vaccines, and other areas of research. The data we present in this review show clearly that unintentional Helicobacter infection has the potential to significantly interfere with the reliability of research studies based on murine models. Therefore, frequent screening of rodent research colonies for Helicobacter spp. and the eradication of these pathogens should be key goals of the research community.The reliability of an experiment that uses an in vivo model system depends on understanding and controlling all variables that can influence the experimental outcome. Infections of mouse colonies are important to the scientific community because they can introduce such harmful variables. Therefore, the ultimate goal of laboratory animal facilities is to maintain disease-free animals, to eliminate those unwanted variables.Numerous pathogenic microbes can interfere with animal research (reviewed in reference 57), and colonization of mouse colonies with members of the family Helicobacteriaceae is an increasing concern for the research community. Naturally acquired Helicobacter infections have been reported in all commonly used laboratory rodent species.^{3,10,36,44,45,49,82,124} A study of mice derived from 34 commercial and academic institutions in Canada, Europe, Asia, Australia, and the United States showed that 88% of these institutions had mouse colonies infected with 1 or more Helicobacter spp.¹⁰⁹ Approximately 59% of these mice were infected with Helicobacter hepaticus ; however monoinfections with other species also were encountered. In another study, at least 1 of 5 Helicobacter spp. was detected in 88% of the 40 mouse strains tested.⁴Surveys such as these have established that a broad range of Helicobacter spp. may be present in mouse research colonies. Several of those Helicobacter species cause disease in laboratory mice. H. hepaticus first was identified as a pathogen when it was discovered to be the cause of chronic hepatitis and hepatocellular carcinoma in mice,^26,31,116 either alone or in combination with other Helicobacter spp.⁷⁸ In addition, H. typhlonius causes intestinal inflammation in mice with immunodeficiency or defects in immune regulation;^28,37 H. muridarum has been associated with gastritis,⁸⁶ and H. bilis has been associated with hepatitis^35,38 and colitis.^60,61 Although, H. rodentium appears to be relatively nonpathogenic in wild-type and SCID mice,⁷⁸ combined infection with H. rodentium and H. typhlonius results in a high incidence of inflammation-associated neoplasia in IL10^−/− mice.^9,46 Further, it is becoming increasingly clear that the effects of Helicobacter infections are not limited to the gastrointestinal system. Helicobacter infections have been documented to directly or indirectly affect responses as diverse as reproduction, development of breast cancer, and altered immune responses to vaccines.^65,95,99 In addition to effects on rodents, Helicobacter spp. can infect other laboratory animals^{2,5,27,29,33,36,107} and can colonize different anatomic regions of the gastrointestinal system.³⁵ This review focuses on the potential effect of these organisms on in vivo experiments and biomedical research. The results summarized here emphasize the importance of knowledge of colony infection status and prevention of unintentional infections to achieve the goal of providing a consistent and reliable environment for research studies.     

Endpoints for Mouse Abdominal Tumor Models: Refinement of Current Criteria

Accurate, rapid, and noninvasive health assessments are required to establish more appropriate endpoints in mouse cancer models where tumor size is not easily measured. We evaluated potential endpoints in mice with experimentally induced peritoneal lymphoma, an abdominal tumor model, by comparing body weight, body condition, and behavior with those of a control group of mice not developing lymphoma. Our hypothesis was that body weight would increase or plateau, whereas body condition and behavioral scores would decrease, as disease progressed. Results indicated that body weight did not differ significantly between the control and experimental groups, but the experimental group experienced significant decreases in both body condition and behavioral scores. Our results support the use of body condition and behavioral scoring as adjunctive assessment methods for mice involved in abdominal lymphoma tumor studies in which health may decline despite an increase or plateau in body weight.Abbreviations: BCS, body condition scoreCurrently many approaches are used to monitor morbidity in experimental mouse studies, including assessment of body weight, activity, hydration, and hair coat appearance, with assessment of body weight predominating.^{1,11,15-17,19,21,23,25} However, in cancer research, these commonly used endpoints are not always effective tools for health assessment. The assessment of activity or coat appearance is subjective. Dehydration and weight loss can be difficult to ascertain accurately because an increase in tumor mass can mask the loss of overall body weight that is associated with dehydration, loss of normal fat deposits, and muscle wasting. In mice with subcutaneous tumors, tumor size, tumor ulceration, and the animal''s ability to ambulate can be measured objectively and used to evaluate health. ⁸ However, in mice with internal tumors, these parameters may be difficult to evaluate, and body weight and overall appearance may be the only parameters that can be assessed clearly. Therefore, additional evaluation methods that are noninvasive, reliable, and easily performed would be useful.Body condition scoring (BCS) is a routinely used technique in veterinary medicine for assessing health and nutritional status. In ruminants, pigs, and horses, BCS has been used to assess health in disease and reproductive states.^{3,4,9,14,18,22} In addition, BCS techniques have been used to monitor dogs and cats with neoplasia and heart disease^2,20 and to evaluate diet choice and volume when treating obesity.^12,13 Furthermore, BCS techniques have been developed for application to laboratory species and have the potential to improve animal welfare in research.⁵,⁶,⁷,⁸,¹⁰,²⁴Body condition scoring has been adapted for rodents. In rats, BCS and body weight have been used adjunctively to evaluate diabetes models.¹¹ Although BCS techniques for mice have been used to accurately assess the health of P- and E-selectin double-deficient mice,^7,24 this technique has not been applied to or evaluated in other mouse models.⁶,²⁴ In addition, although BCS is an effective evaluation method, it alone does not give a complete picture of animal health. In this study, we used body weight, BCS, appearance, and behavioral assessments to evaluate morbidity in a mouse model of peritoneal lymphoma. Our hypothesis was that body weight would plateau or increase as the tumors increased in size, but body condition score would decrease and, therefore, more accurately reflect the true health status of the mouse. We also hypothesized that a change in appearance and behavior would accompany the decrease in BCS. A total score combining these evaluations was developed to assess overall health status in mice, helping investigators and animal care staff to reevaluate study endpoints for abdominal tumor growth. A further hypothesis was that the total score would provide more sensitivity than BCS, appearance, or behavior alone in assessing health status. To reduce the overall number of animals used for this study, we collaborated with an investigator performing abdominal lymphoma research at our institution and evaluated animals from ongoing studies of different anticancer vaccine therapies.     

Adiponectin receptor-mediated signaling ameliorates cerebral cell damage and regulates the neurogenesis of neural stem cells at high glucose concentrations: an in vivo and in vitro study

In the central nervous system (CNS), hyperglycemia leads to neuronal damage and cognitive decline. Recent research has focused on revealing alterations in the brain in hyperglycemia and finding therapeutic solutions for alleviating the hyperglycemia-induced cognitive dysfunction. Adiponectin is a protein hormone with a major regulatory role in diabetes and obesity; however, its role in the CNS has not been studied yet. Although the presence of adiponectin receptors has been reported in the CNS, adiponectin receptor-mediated signaling in the CNS has not been investigated. In the present study, we investigated adiponectin receptor (AdipoR)-mediated signaling in vivo using a high-fat diet and in vitro using neural stem cells (NSCs). We showed that AdipoR1 protects cell damage and synaptic dysfunction in the mouse brain in hyperglycemia. At high glucose concentrations in vitro, AdipoR1 regulated the survival of NSCs through the p53/p21 pathway and the proliferation- and differentiation-related factors of NSCs via tailless (TLX). Hence, we suggest that further investigations are necessary to understand the cerebral AdipoR1-mediated signaling in hyperglycemic conditions, because the modulation of AdipoR1 might alleviate hyperglycemia-induced neuropathogenesis.Adiponectin secreted by the adipose tissue^{1, 2} exists in either a full-length or globular form.^{3, 4, 5, 6} Adiponectin can cross the blood–brain barrier, and various forms of adiponectin are found in the cerebrospinal fluid.^{7, 8, 9, 10, 11} Adiponectin exerts its effect by binding to the adiponectin receptor 1 (AdipoR1) and adiponectin receptor 2 (AdipoR2)^{12, 13} that have different affinities for the various circulating adiponectins.^{12, 14, 15, 16, 17} Several studies reported that both receptor subtypes are expressed in the central nervous system (CNS).^{7, 12, 18} As adiponectin modulates insulin sensitivity and inflammation,¹⁹ its deficiency induces insulin resistance and glucose intolerance in animals fed a high-fat diet (HFD).^{19, 20, 21} In addition, adiponectin can ameliorate the glucose homeostasis and increase insulin sensitivity.^{22, 23, 24} Adiponectin, which is the most well-known adipokine, acts mainly as an anti-inflammatory regulator,^{25, 26} and is associated with the onset of neurological disorders.²⁷ In addition, a recent study reported that adiponectin promotes the proliferation of hippocampal neural stem cells (NSCs).²⁸ Considering that adiponectin acts by binding to the adiponectin receptors, investigation of the adiponectin receptor-mediated signaling in the brain is crucial to understand the cerebral effects of adiponectin and the underlying cellular mechanisms.The prevalence of type II diabetes mellitus (DM2) and Alzheimer''s disease increases with aging.²⁹ According to a cross-sectional study, in people with DM2, the risk of dementia is 2.5 times higher than that in the normal population.^{30, 31} A study performed between 1980 and 2002 suggested that an elevated blood glucose level is associated with a greater risk for dementia in elderly patients with DM2.³² In addition, according to a 9-year-long longitudinal cohort study, the risk of developing Alzheimer''s disease was 65% higher in people with diabetes than in control subjects.³³ A community-based cohort study also reported that higher plasma glucose concentrations are associated with an increased risk for dementia, because the higher glucose level has detrimental effects on the brain.³¹ High blood glucose level causes mitochondria-dependent apoptosis,^{34, 35, 36} and aggravates diverse neurological functions.^{37, 38} Inflammation and oxidative stress, which are commonly observed in people with diabetes, inhibit neurogenesis.^{39, 40, 41} Similarly, neurogenesis is decreased in mice and rats with genetically induced type I diabetes.^{42, 43} In addition, diabetic rodents have a decreased proliferation rate of neural progenitors.^{43, 44} Furthermore, several studies suggested that an HFD leads to neuroinflammation, the impairment of synaptic plasticity, and cognitive decline.^{45, 46}Here, we investigated whether AdipoR1-mediated signaling is associated with cell death in the brain of mice on a HFD, and whether high glucose level modifies the proliferation and differentiation capacity of NSCs in vitro. Our study provides novel findings about the role of AdipoR1-mediated signaling in hyperglycemia-induced neuropathogenesis.     

Omentin protects against LPS-induced ARDS through suppressing pulmonary inflammation and promoting endothelial barrier via an Akt/eNOS-dependent mechanism

Acute respiratory distress syndrome (ARDS) is characterized by increased pulmonary inflammation and endothelial barrier permeability. Omentin has been shown to benefit obesity-related systemic vascular diseases; however, its effects on ARDS are unknown. In the present study, the level of circulating omentin in patients with ARDS was assessed to appraise its clinical significance in ARDS. Mice were subjected to systemic administration of adenoviral vector expressing omentin (Ad-omentin) and one-shot treatment of recombinant human omentin (rh-omentin) to examine omentin''s effects on lipopolysaccharide (LPS)-induced ARDS. Pulmonary endothelial cells (ECs) were treated with rh-omentin to further investigate its underlying mechanism. We found that a decreased level of circulating omentin negatively correlated with white blood cells and procalcitonin in patients with ARDS. Ad-omentin protected against LPS-induced ARDS by alleviating the pulmonary inflammatory response and endothelial barrier injury in mice, accompanied by Akt/eNOS pathway activation. Treatment of pulmonary ECs with rh-omentin attenuated inflammatory response and restored adherens junctions (AJs), and cytoskeleton organization promoted endothelial barrier after LPS insult. Moreover, the omentin-mediated enhancement of EC survival and differentiation was blocked by the Akt/eNOS pathway inactivation. Therapeutic rh-omentin treatment also effectively protected against LPS-induced ARDS via the Akt/eNOS pathway. Collectively, these data indicated that omentin protects against LPS-induced ARDS by suppressing inflammation and promoting the pulmonary endothelial barrier, at least partially, through an Akt/eNOS-dependent mechanism. Therapeutic strategies aiming to restore omentin levels may be valuable for the prevention or treatment of ARDS.Acute respiratory distress syndrome (ARDS) is a devastating condition with a 30–60% mortality rate.^{1, 2} Although the pathogenesis of ARDS is complex, the inflammatory response and endothelial barrier disruption play important roles in the development of ARDS.^{3, 4, 5} Therefore, in addition to conventional anti-inflammatory treatments, therapeutic strategies aim to restore pulmonary endothelial barrier integrity and function through regulating inter-endothelial AJs and the endothelial cytoskeleton to minimize protein leakage and leukocyte infiltration under ARDS conditions.^{6, 7}Obesity, especially visceral obesity, has clearly been shown to impair systemic vasculature and to lead to the initiation and progression of vascular disorders.^{8, 9, 10} Although different from the well-documented impacts of obesity on cardiovascular disease, the relationships between obesity and ARDS have not been well elucidated. Clinical and experimental data focused on pertinent physiological changes in obesity indicate that the obesity may alter ARDS pathogenesis by ‘priming'' the pulmonary endothelial barrier for insult and amplifying the early inflammatory response, thus lowering the threshold to initiate ARDS.^{11, 12} Contrary to conventional dogma, adipose tissue is now appreciated as an important endocrine tissue that secretes various bioactive molecules called adipokines, which contribute to the progression of diverse vascular diseases, including hypertension, cardiovascular disease and atherosclerosis.^{13, 14, 15, 16} Although ARDS is not a classified pulmonary vascular disease, it is a severe inflammatory lung condition with widespread pulmonary endothelial breakdown. Clinical evidence has indicated that the obesity might be an emerging risk factor for ARDS and that circulating adipokines levels are associated with the initiation and progression of ARDS.^{11, 12, 17, 18} Moreover, experimental studies have suggested that some anti-inflammatory adipokines, such as adiponectin and apelin, exert beneficial actions on ARDS.^{19, 20, 21}Omentin is an anti-inflammatory adipokine that is abundant in human visceral fat tissue.^{22, 23} Paradoxically, higher circulating omentin-1 levels are present in lean and healthy individuals compared with the obese and diabetic patients. Moreover, as a novel biomarker of endothelial dysfunction, reduced circulating omentin levels are related to the pathological mechanism of obesity-linked vascular disorders, including type 2 diabetes, atherosclerosis, hypertension and cardiovascular disease.^{24, 25, 26, 27, 28} Furthermore, experimental studies have found that omentin stimulates vasodilation in isolated blood vessels and suppresses cytokine-stimulated inflammation in endothelial cells (ECs).^{29, 30, 31} Thus, these data suggest that omentin may protect against obesity-related vascular complications through its anti-inflammatory and vascular-protective properties; however, little is known regarding its role in lung tissue. It was reported that decreased circulating omentin-1 levels could be regarded as an independent predictive marker for the obstructive sleep apnea syndrome and that omentin protects against pulmonary arterial hypertension through inhibiting vascular structure remodeling and abnormal contractile reactivity.^{32, 33, 34} However, to our knowledge, no study has assessed the impact of omentin on ARDS.Akt-related signaling pathways function as an endogenous negative feedback mechanism in response to the injurious stimulus. Our prior studies have demonstrated that Akt-related signaling contributes to protection against ARDS.^{35, 36} Moreover, omentin has been reported to exert anti-inflammatory, pro-survival and pro-angiogenic functions in various cells via an Akt-dependent mechanism.^{30, 31, 37, 38, 39, 40, 41, 42}Collectively, given that ARDS is ultimately an obesity-related disorder of vascular function and that omentin is a favorable pleiotropic adipokine capable of anti-inflammatory, pro-angiogenic and anti-apoptotic abilities; omentin may exert beneficial effects on ARDS. In the present study, we first aimed to appraise the clinical significance of omentin in ARDS and then specifically evaluated its impact on inflammation and the endothelial barrier. Furthermore, we mechanistically investigated the role of Akt-related signaling pathways in these effects induced by omentin in vivo and in vitro.     

Electrocardiographic Characterization of Cardiac Hypertrophy in Mice that Overexpress the ErbB2 Receptor Tyrosine Kinase

Electrocardiography is an important method for evaluation and risk stratification of patients with cardiac hypertrophy. We hypothesized that the recently developed transgenic mouse model of cardiac hypertrophy (ErbB2^tg) will display distinct ECG features, enabling WT (wild type) mice to be distinguished from transgenic mice without using conventional PCR genotyping. We evaluated more than 2000 mice and developed specific criteria for genotype determination by using cageside ECG, during which unanesthetized mice were manually restrained for less than 1 min. Compared with those from WT counterparts, the ECG recordings of ErbB2^tg mice were characterized by higher P- and R-wave amplitudes, broader QRS complexes, inverted T waves, and ST interval depression. Pearson''s correlation matrix analysis of combined WT and ErbB2^tg data revealed significant correlation between heart weight and the ECG parameters of QT interval (corrected for heart rate), QRS interval, ST height, R amplitude, P amplitude, and PR interval. In addition, the left ventricular posterior wall thickness as determined by echocardiography correlated with ECG-determined ST height, R amplitude, QRS interval; echocardiographic left ventricular mass correlated with ECG-determined ST height and PR interval. In summary, we have determined phenotypic ECG criteria to differentiate ErbB2^tg from WT genotypes in 98.8% of mice. This inexpensive and time-efficient ECG-based phenotypic method might be applied to differentiate between genotypes in other rodent models of cardiac hypertrophy. Furthermore, with appropriate modifications, this method might be translated for use in other species.Abbreviations: HCM, hypertrophic cardiomyopathy; LV, left ventricle; QT_c, QT interval corrected for heart rateElectrocardiography is an important method used in human patients for evaluation of cardiac hypertrophy, for example, hypertrophic cardiomyopathy (HCM).^30,35 Although echocardiography is considered to be the ‘gold standard’ in HCM diagnostics, ECG evaluation may provide additional information needed for diagnosis.³⁰ In some patients with HCM, ECG changes precede echocardiographic changes;^30,36 therefore the 2 modalities are often used together to screen family members of patients with HCM. Similarly, there are reports of athletes who suddenly die during training, in whom HCM was confirmed at autopsy and in whom ECG abnormalities were recorded in the absence of overt clinical signs.²³ In contrast, although developed before echocardiography, ECG is often underutilized in the characterization of mouse models of cardiac disease.The first reports on mouse ECG were published in the 1950s^15,40 and were followed by the rapid development of rodent ECG methods, recording, and analysis.¹⁶ Several approaches support the recording and analysis of mouse ECG, including 12-lead ECG,^7,51 open-chest models,^7,51 telemetry using radiofrequency transmitters,¹⁶ and recording in anesthetized mice.^7,51 All of these methods provide information on cardiac electrophysiology, yet each has its specific advantages and limitations.⁵¹ Further use of ECG in mouse models of cardiac disease could improve our understanding of the electrophysiologic remodeling in these diseases.Several mouse models of HCM (due to genetic modifications of genes related to human HCM^18,21,50 or to lysosomal storage disease-related cardiomyopathies^2,4,6,46) were developed to study these hypertrophic conditions and the resulting electrical disturbances in the myocardium. However, despite carrying the same genetic alterations that cause human disease, many mouse models do not have phenotypic ECG changes or even hypertrophy.^18,21,50 Therefore the development of a small animal model of cardiac hypertrophy with ECG features similar to those in human patients is of particular interest. We recently developed a mouse model with cardiac hypertrophy and pathologic features compatible with HCM⁴⁷ and hypothesized that various electrocardiographic features could enable us to distinguish between wildtype (WT) mice and transgenic littermates after weaning. In the current study, we established an ECG method that identifies the hypertrophic phenotype and thus assists in determining the genotype of mice. This ECG method thus reduced laboratory costs and the time necessary to isolate and analyze DNA for genotyping.     

Induction of COX-2-PGE2 synthesis by activation of the MAPK/ERK pathway contributes to neuronal death triggered by TDP-43-depleted microglia

Neuroinflammation is a striking hallmark of amyotrophic lateral sclerosis (ALS) and other neurodegenerative disorders. Previous studies have shown the contribution of glial cells such as astrocytes in TDP-43-linked ALS. However, the role of microglia in TDP-43-mediated motor neuron degeneration remains poorly understood. In this study, we show that depletion of TDP-43 in microglia, but not in astrocytes, strikingly upregulates cyclooxygenase-2 (COX-2) expression and prostaglandin E2 (PGE2) production through the activation of MAPK/ERK signaling and initiates neurotoxicity. Moreover, we find that administration of celecoxib, a specific COX-2 inhibitor, greatly diminishes the neurotoxicity triggered by TDP-43-depleted microglia. Taken together, our results reveal a previously unrecognized non-cell-autonomous mechanism in TDP-43-mediated neurodegeneration, identifying COX-2-PGE2 as the molecular events of microglia- but not astrocyte-initiated neurotoxicity and identifying celecoxib as a novel potential therapy for TDP-43-linked ALS and possibly other types of ALS.Amyotrophic lateral sclerosis (ALS) is an adult-onset neurodegenerative disease characterized by the degeneration of motor neurons in the brain and spinal cord.¹ Most cases of ALS are sporadic, but 10% are familial. Familial ALS cases are associated with mutations in genes such as Cu/Zn superoxide dismutase 1 (SOD1), TAR DNA-binding protein 43 (TARDBP) and, most recently discovered, C9orf72. Currently, most available information obtained from ALS research is based on the study of SOD1, but new studies focusing on TARDBP and C9orf72 have come to the forefront of ALS research.^{1, 2} The discovery of the central role of the protein TDP-43, encoded by TARDBP, in ALS was a breakthrough in ALS research.^{3, 4, 5} Although pathogenic mutations of TDP-43 are genetically rare, abnormal TDP-43 function is thought to be associated with the majority of ALS cases.¹ TDP-43 was identified as a key component of the ubiquitin-positive inclusions in most ALS patients and also in other neurodegenerative diseases such as frontotemporal lobar degeneration,^{6, 7} Alzheimer''s disease (AD)^{8, 9} and Parkinson''s disease (PD).^{10, 11} TDP-43 is a multifunctional RNA binding protein, and loss-of-function of TDP-43 has been increasingly recognized as a key contributor in TDP-43-mediated pathogenesis.^{5, 12, 13, 14}Neuroinflammation, a striking and common hallmark involved in many neurodegenerative diseases, including ALS, is characterized by extensive activation of glial cells including microglia, astrocytes and oligodendrocytes.^{15, 16} Although numerous studies have focused on the intrinsic properties of motor neurons in ALS, a large amount of evidence showed that glial cells, such as astrocytes and microglia, could have critical roles in SOD1-mediated motor neuron degeneration and ALS progression,^{17, 18, 19, 20, 21, 22} indicating the importance of non-cell-autonomous toxicity in SOD1-mediated ALS pathogenesis.Very interestingly, a vital insight of neuroinflammation research in ALS was generated by the evidence that both the mRNA and protein levels of the pro-inflammatory enzyme cyclooxygenase-2 (COX-2) are upregulated in both transgenic mouse models and in human postmortem brain and spinal cord.^{23, 24, 25, 26, 27, 28, 29} The role of COX-2 neurotoxicity in ALS and other neurodegenerative disorders has been well explored.^{30, 31, 32} One of the key downstream products of COX-2, prostaglandin E2 (PGE2), can directly mediate COX-2 neurotoxicity both in vitro and in vivo.^{33, 34, 35, 36, 37} The levels of COX-2 expression and PGE2 production are controlled by multiple cell signaling pathways, including the mitogen-activated protein kinase (MAPK)/ERK pathway,^{38, 39, 40} and they have been found to be increased in neurodegenerative diseases including AD, PD and ALS.^{25, 28, 32, 41, 42, 43, 44, 45, 46} Importantly, COX-2 inhibitors such as celecoxib exhibited significant neuroprotective effects and prolonged survival or delayed disease onset in a SOD1-ALS transgenic mouse model through the downregulation of PGE2 release.²⁸Most recent studies have tried to elucidate the role of glial cells in neurotoxicity using TDP-43-ALS models, which are considered to be helpful for better understanding the disease mechanisms.^{47, 48, 49, 50, 51} Although the contribution of glial cells to TDP-43-mediated motor neuron degeneration is now well supported, this model does not fully suggest an astrocyte-based non-cell autonomous mechanism. For example, recent studies have shown that TDP-43-mutant astrocytes do not affect the survival of motor neurons,^{50, 51} indicating a previously unrecognized non-cell autonomous TDP-43 proteinopathy that associates with cell types other than astrocytes.Given that the role of glial cell types other than astrocytes in TDP-43-mediated neuroinflammation is still not fully understood, we aim to compare the contribution of microglia and astrocytes to neurotoxicity in a TDP-43 loss-of-function model. Here, we show that TDP-43 has a dominant role in promoting COX-2-PGE2 production through the MAPK/ERK pathway in primary cultured microglia, but not in primary cultured astrocytes. Our study suggests that overproduction of PGE2 in microglia is a novel molecular mechanism underlying neurotoxicity in TDP-43-linked ALS. Moreover, our data identify celecoxib as a new potential effective treatment of TDP-43-linked ALS and possibly other types of ALS.     

New-Onset Diabetes Mellitus After Transplantation in a Cynomolgus Macaque (Macaca fasicularis)

A 5.5-y-old intact male cynomolgus macaque (Macaca fasicularis) presented with inappetence and weight loss 57 d after heterotopic heart and thymus transplantation while receiving an immunosuppressant regimen consisting of tacrolimus, mycophenolate mofetil, and methylprednisolone to prevent graft rejection. A serum chemistry panel, a glycated hemoglobin test, and urinalysis performed at presentation revealed elevated blood glucose and glycated hemoglobin (HbA1c) levels (727 mg/dL and 10.1%, respectively), glucosuria, and ketonuria. Diabetes mellitus was diagnosed, and insulin therapy was initiated immediately. The macaque was weaned off the immunosuppressive therapy as his clinical condition improved and stabilized. Approximately 74 d after discontinuation of the immunosuppressants, the blood glucose normalized, and the insulin therapy was stopped. The animal''s blood glucose and HbA1c values have remained within normal limits since this time. We suspect that our macaque experienced new-onset diabetes mellitus after transplantation, a condition that is commonly observed in human transplant patients but not well described in NHP. To our knowledge, this report represents the first documented case of new-onset diabetes mellitus after transplantation in a cynomolgus macaque.Abbreviations: NODAT, new-onset diabetes mellitus after transplantationNew-onset diabetes mellitus after transplantation (NODAT, formerly known as posttransplantation diabetes mellitus) is an important consequence of solid-organ transplantation in humans.^{7-10,15,17,19,21,25-28,31,33,34,37,38,42} A variety of risk factors have been identified including increased age, sex (male prevalence), elevated pretransplant fasting plasma glucose levels, and immunosuppressive therapy.^{7-10,15,17,19,21,25-28,31,33,34,37,38,42} The relationship between calcineurin inhibitors, such as tacrolimus and cyclosporin, and the development of NODAT is widely recognized in human medicine.^{7-10,15,17,19,21,25-28,31,33,34,37,38,42} Cynomolgus macaques (Macaca fasicularis) are a commonly used NHP model in organ transplantation research. Cases of natural and induced diabetes of cynomolgus monkeys have been described in the literature;^14,43,45 however, NODAT in a macaque model of solid-organ transplantation has not been reported previously to our knowledge.     

Anaplastic Renal Carcinoma Expressing SV40 T Antigen in a Female TRAMP Mouse

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.Abbreviations: Tag, large T antigen; TRAMP, transgenic adenocarcinoma mouse prostateThe transgenic adenocarcinoma of the mouse prostate (TRAMP) strain is a genetically engineered mouse strain developed on a C57BL/6 background as a model of human prostate cancer. Human prostatic cancer is characterized by a loss of wildtype tumor protein 53 (TP53) and retinoblastoma (RB1) genes.^9,10,13 The TRAMP model was developed to mimic that mechanism by using the SV40 polyomavirus large T antigen (Tag) to act as an oncogene.¹⁰ Tag protein binds to and inactivates the tumor suppressor proteins TRP53 and RB1. In TRAMP mice, Tag expression is high and limited to the dorsolateral and ventral prostatic lobes, as well as the stromal cells of the seminal vesicles, through the use of the rat probasin promoter (Tg[rPb-SV40Tag]), and affects the secretory epithelial cells.^6,8,9,20 This gene–promoter combination results in epithelial transformation in vivo under the regulation of androgens and zinc.^9,20 Whereas TRAMP mice on an FVB background readily develop neuroendocrine tumors of the prostate, TRAMP mice with a B6 background are less likely to form neuroendocrine-type prostatic tumors and instead were shown to produce prostatic epithelial origin tumors that can be described as glandular, papillary, or cribriform.²⁰ Seminal vesicle tumors in TRAMP mice do not express Tag within the epithelial component but do express it in the mesenchymal component.²⁰Male TRAMP mice of B6 background begin expressing abnormal differentiation of prostatic epithelial cells at sexual maturity and by 10 to 12 wk of age have mild to severe prostatic hyperplasia with the formation of cribriform structures. By 24 to 30 wk of age, these mice typically develop primary prostatic adenocarcinomas.^9,10 Other abnormalities noted in male TRAMP mice include epithelial–stromal (phyllodes) tumors in the seminal vesicles involving mainly the stroma with low epithelial involvement.^20,22 Female mice are reported as being fertile, with no expression of the transgene or associated neoplasia.¹⁰ This report presents the first documented occurrence of a transgene-expressing neoplasm in a female TRAMP mouse.     

Neuritin can normalize neural deficits of Alzheimer's disease

Reductions in hippocampal neurite complexity and synaptic plasticity are believed to contribute to the progressive impairment in episodic memory and the mild cognitive decline that occur particularly in the early stages of Alzheimer''s disease (AD). Despite the functional and therapeutic importance for patients with AD, intervention to rescue or normalize dendritic elaboration and synaptic plasticity is scarcely provided. Here we show that overexpression of neuritin, an activity-dependent protein, promoted neurite outgrowth and maturation of synapses in parallel with enhanced basal synaptic transmission in cultured hippocampal neurons. Importantly, exogenous application of recombinant neuritin fully restored dendritic complexity as well as spine density in hippocampal neurons prepared from Tg2576 mice, whereas it did not affect neurite branching of neurons from their wild-type littermates. We also showed that soluble recombinant neuritin, when chronically infused into the brains of Tg2576 mice, normalized synaptic plasticity in acute hippocampal slices, leading to intact long-term potentiation. By revealing the protective actions of soluble neuritin against AD-related neural defects, we provide a potential therapeutic approach for patients with AD.Efficient neuronal communications through synapses are crucial for normal brain functions, whereas alterations in synapse numbers, dendritic spine morphology, and dendritic complexity are thought to be reflected by different forms of synaptic plasticity and are also causally associated with a variety of neurological disorders.^{1, 2, 3, 4, 5} For example, synapse loss and neurite atrophy are the major neurobiological substrates underlying memory impairment in neurodegenerative diseases such as Alzheimer''s disease (AD).^{6, 7} The increased dendritic mislocalization of hyperphosphorylated tau protein, a microtubule-associated protein enriched at axons of mature neurons,⁸ and abundance of soluble oligomeric forms of β-amyloid (Aβ) appear to cause the synaptic defects and disruption of synaptic plasticity involving the progression of AD pathology.^{6, 9, 10} The apparent decreases in neurotrophic factors observed in brains of patients with AD¹¹ have prompted several trials for administration of neurotrophic factors, such as brain-derived neurotrophic factor (BDNF), to attenuate and possibly reverse synaptic defects.^{11, 12, 13} However, the truncation or decreased expression of its cognate receptors in AD brains have limited their potential usage as AD therapeutics.^{12, 14, 15}Neuritin, also known as the candidate plasticity gene 15, was originally identified in a screening study for activity-regulated genes and was subsequently found to be one of the signaling molecules downstream to BDNF and its receptor tropomyosin-related kinase receptor type B.^{16, 17} Ensuing studies indicated that neuritin could also be induced by experimental seizure or by normal life experiences, such as sensory stimulation and exercise.^{17, 18, 19, 20, 21, 22} Located in the 6p24-p25 interval on chromosome 6,²³ the neuritin gene encodes a small, highly conserved protein containing a secretory signal sequence at the N-terminus and a consensus sequence for glycosylphosphatidylinositol (GPI) at the C-terminus.¹⁶ This GPI linkage enables neuritin to anchor at cell surfaces, and upon cleavage of GPI by phospholipase the resultant soluble neuritin is released into the extracellular space.^{16, 20, 24, 25, 26}During embryonic neural development, neuritin is mainly expressed in brain regions that undergo a rapid proliferation of neuronal progenitor pools, suggesting a protective role of neuritin for differentiated neurons.^{26, 27} Interestingly, the expression level of neuritin remains elevated after birth or even increases, especially in brain regions presumably exhibiting high neural activity and synaptic plasticity, such as the hippocampus, visual cortex, and external granular layer of the cerebellum.^{16, 19, 20, 26} In addition, neuritin promotes neuritic arbor growth and synaptic formation.^{16, 20, 24, 25, 28, 29, 30, 31} Although various studies have suggested these potent neuritogenetic activities of neuritin, the contribution of neuritin expression to or its effectiveness against neurodegenerative diseases that display neurite atrophy and synapse loss has been largely unexplored.Here we determined that neuritin expression increased neurite complexity and promoted the maturation of individual spines in cultured hippocampal neurons. Consistent with these findings, basal synaptic transmission was enhanced by transient expression of neuritin. Importantly, when exogenously applied, the soluble neuritin peptide rescued the dendrite complexity of neurons prepared from Tg2576 mice, a transgenic mouse model of AD, such that the complexity was comparable to that in wild-type (WT) mice and also normalized synaptic plasticity in the hippocampus of the Tg2576 mice. Taken together, these results suggest that neuritin, particularly a soluble form of neuritin, reverses synaptic defects manifest in Tg2576 mice and that manipulations to increase neuritin levels may be beneficial therapeutic approaches in AD.     

A cellular screen identifies ponatinib and pazopanib as inhibitors of necroptosis

Necroptosis is a form of regulated necrotic cell death mediated by receptor-interacting serine/threonine-protein kinase 1 (RIPK1) and RIPK3. Necroptotic cell death contributes to the pathophysiology of several disorders involving tissue damage, including myocardial infarction, stroke and ischemia-reperfusion injury. However, no inhibitors of necroptosis are currently in clinical use. Here we performed a phenotypic screen for small-molecule inhibitors of tumor necrosis factor-alpha (TNF-α)-induced necroptosis in Fas-associated protein with death domain (FADD)-deficient Jurkat cells using a representative panel of Food and Drug Administration (FDA)-approved drugs. We identified two anti-cancer agents, ponatinib and pazopanib, as submicromolar inhibitors of necroptosis. Both compounds inhibited necroptotic cell death induced by various cell death receptor ligands in human cells, while not protecting from apoptosis. Ponatinib and pazopanib abrogated phosphorylation of mixed lineage kinase domain-like protein (MLKL) upon TNF-α-induced necroptosis, indicating that both agents target a component upstream of MLKL. An unbiased chemical proteomic approach determined the cellular target spectrum of ponatinib, revealing key members of the necroptosis signaling pathway. We validated RIPK1, RIPK3 and transforming growth factor-β-activated kinase 1 (TAK1) as novel, direct targets of ponatinib by using competitive binding, cellular thermal shift and recombinant kinase assays. Ponatinib inhibited both RIPK1 and RIPK3, while pazopanib preferentially targeted RIPK1. The identification of the FDA-approved drugs ponatinib and pazopanib as cellular inhibitors of necroptosis highlights them as potentially interesting for the treatment of pathologies caused or aggravated by necroptotic cell death.Programmed cell death has a crucial role in a variety of biological processes ranging from normal tissue development to diverse pathological conditions.^{1, 2} Necroptosis is a form of regulated cell death that has been shown to occur during pathogen infection or sterile injury-induced inflammation in conditions where apoptosis signaling is compromised.^{3, 4, 5, 6} Given that many viruses have developed strategies to circumvent apoptotic cell death, necroptosis constitutes an important, pro-inflammatory back-up mechanism that limits viral spread in vivo.^{7, 8, 9} In contrast, in the context of sterile inflammation, necroptotic cell death contributes to disease pathology, outlining potential benefits of therapeutic intervention.¹⁰ Necroptosis can be initiated by death receptors of the tumor necrosis factor (TNF) superfamily,¹¹ Toll-like receptor 3 (TLR3),¹² TLR4,¹³ DNA-dependent activator of IFN-regulatory factors¹⁴ or interferon receptors.¹⁵ Downstream signaling is subsequently conveyed via RIPK1¹⁶ or TIR-domain-containing adapter-inducing interferon-β,^{8, 17} and converges on RIPK3-mediated^{13, 18, 19, 20} activation of MLKL.²¹ Phosphorylated MLKL triggers membrane rupture,^{22, 23, 24, 25, 26} releasing pro-inflammatory cellular contents to the extracellular space.²⁷ Studies using the RIPK1 inhibitor necrostatin-1 (Nec-1) ²⁸ or RIPK3-deficient mice have established a role for necroptosis in the pathophysiology of pancreatitis,¹⁹ artherosclerosis,²⁹ retinal cell death,³⁰ ischemic organ damage and ischemia-reperfusion injury in both the kidney³¹ and the heart.³² Moreover, allografts from RIPK3-deficient mice are better protected from rejection, suggesting necroptosis inhibition as a therapeutic option to improve transplant outcome.³³ Besides Nec-1, several tool compounds inhibiting different pathway members have been described,^{12, 16, 21, 34, 35} however, no inhibitors of necroptosis are available for clinical use so far.^{2, 10} In this study we screened a library of FDA approved drugs for the precise purpose of identifying already existing and generally safe chemical agents that could be used as necroptosis inhibitors. We identified the two structurally distinct kinase inhibitors pazopanib and ponatinib as potent blockers of necroptosis targeting the key enzymes RIPK1/3.     

Role of Retinoic Acid and Fibroblast Growth Factor 2 in Neural Differentiation from Cynomolgus Monkey (Macaca fascicularis) Embryonic Stem Cells

Retinoic acid is a widely used factor in both mouse and human embryonic stem cells. It suppresses differentiation to mesoderm and enhances differentiation to ectoderm. Fibroblast growth factor 2 (FGF2) is widely used to induce differentiation to neurons in mice, yet in primates, including humans, it maintains embryonic stem cells in the undifferentiated state. In this study, we established an FGF2 low-dose-dependent embryonic stem cell line from cynomolgus monkeys and then analyzed neural differentiation in cultures supplemented with retinoic acid and FGF2. When only retinoic acid was added to culture, neurons differentiated from FGF2 low-dose-dependent embryonic stem cells. When both retinoic acid and FGF2 were added, neurons and astrocytes differentiated from the same embryonic stem cell line. Thus, retinoic acid promotes the differentiation from embryonic stem cells to neuroectoderm. Although FGF2 seems to promote self-renewal in stem cells, its effects on the differentiation of stem cells are influenced by the presence or absence of supplemental retinoic acid.Abbreviations: EB, embryoid body; ES, embryonic stem; ESM, embryonic stem cell medium; FGF, fibroblast growth factor; GFAP, glial fibrillary acidic protein; LIF, leukemia inhibitory factor; MBP, myelin basic protein; RA, retinoic acid; SSEA, stage-specific embryonic antigen; TRA, tumor-related antigenPluripotent stem cells are potential sources of material for cell replacement therapy and are useful experimental tools for in vitro models of human disease and drug screening. Embryonic stem (ES) cells are capable of extensive proliferation and multilineage differentiation, and thus ES-derived cells are suitable for use in cell-replacement therapies.^18,23 Reported ES cell characteristics including tumorigenic potential, DNA methylation status, expression of imprinted genes, and chromatin structure were elucidated by using induced pluripotent stem cells.^2,11,17 Because the social expectations of regeneration medicine are growing, we must perform basic research with ES cells, which differ from induced pluripotent stem cells in terms of origin, differentiation ability, and epigenetic status.^2,8Several advances in research have been made by using mouse ES cells. Furthermore, primate ES cell lines have been established from rhesus monkeys (Macaca mulatta),²⁴ common marmosets (Callithrix jacchus),²⁵ cynomolgus monkeys (M. fascicularis),²⁰ and African green monkeys (Chlorocebus aethiops).¹⁹ Mouse and other mammalian ES cells differ markedly in their responses to the signaling pathways that support self-renewal.^8,28 Mouse ES cells require leukemia inhibitory factor (LIF)–STAT3 signaling.¹⁴ In contrast, primate ES cells do not respond to LIF. Fibroblast growth factor 2 (FGF2) appears to be the most upstream self-renewal factor in primate ES cells. FGF2 also exerts its effects through indirect mechanisms, such as the TGFβ–Activin–Nodal signaling pathway, in primate ES cells.²¹ In addition to the biologic similarities between monkeys and humans, ES cells derived from cynomolgus monkeys or human blastocysts have extensive similarities that are not apparent in mouse ES cells.^8,14,21,28 Numerous monkey ES cell lines are now available, and cynomolgus monkeys are an efficient model for developing strategies to investigate the efficacy of ES-cell–based medical treatments in humans.Several growth factors and chemical compounds, including retinoic acid (RA),^4,9,13,22,26 FGF2,^9,10,16,22 epidermal growth factor,^9,22 SB431542,^1,4,10 dorsomorphin,^10,27 sonic hedgehog,^{12,13,16,27,29} and noggin,^1,4,9,27 are essential for the differentiation and proliferation or maintenance of neural stem cells derived from primate ES cells. Of these factors, active RA signaling suppresses a mesodermal fate by inhibiting Wnt and Nodal signaling pathways during in vitro culture and leads to neuroectoderm differentiation in ES cells.^4,13,26 RA is an indispensable factor for the specialization to neural cells. FGF2 is important during nervous system development,¹² and FGF2 and RA both are believed to influence the differentiation to neural cells. The current study was done to clarify the mechanism of RA and FGF2 in the induction of differentiation along the neural lineage.We recently established a monkey ES cell line that does not need FGF2 supplementation for maintenance of the undifferentiated state. This ES cell line allowed us to study the role of differentiation to neural cells with RA and enabled us to compare ES cell differentiation in the context of supplementation with RA or FGF2 in culture. To this end, we established a novel cynomolgus monkey cell line derived from ES cells and maintained it in an undifferentiated state in the absence of FGF2 supplementation.