ENU mutagenesis in the mouse: application to human genetic disease.

Genetic approaches in model organisms provide a powerful means by which to examine the biological basis of human diseases as well as the physiological processes that are affected by them. Although not without its drawbacks, the mouse has become the mammalian species of choice in studying the molecular basis of disease. Targeted mutagenesis approaches in the mouse have led to dramatic increases in our understanding of human disease processes. As a complement to these gene-driven studies, three developments have led to the reassessment of a phenotype-driven approach in the mouse--the accumulation of information that has emerged from human and mouse genome sequencing projects, the use of high-efficiency point mutagens such as N-ethyl-N-nitrosourea (ENU) and the application of systematic hierarchical screening protocols for the mouse. In this paper, progress with existing phenotypic screening programmes is discussed and opportunities for the development of new mouse disease models are presented.     

A functional analysis of mouse models of cardiac disease through metabolic profiling

Since the completion of the human and mouse genomes, the focus in mammalian biology has been on assessing gene function. Tools are needed for assessing the phenotypes of the many mouse models that are now being generated, where genes have been "knocked out," "knocked in," or mutated, so that gene expression can be understood in its biological context. Metabolic profiling of cardiac tissue through high resolution NMR spectroscopy in conjunction with multivariate statistics has been used to classify mouse models of cardiac disease. The data sets included metabolic profiles from mouse models of Duchenne muscular dystrophy, two models of cardiac arrhythmia, and one of cardiac hypertrophy. The metabolic profiles demonstrate that the strain background is an important component of the global metabolic phenotype of a mouse, providing insight into how a given gene deletion may result in very different responses in diverse populations. Despite these differences associated with strain, multivariate statistics were capable of separating each mouse model from its control strain, demonstrating that metabolic profiles could be generated for each disease. Thus, this approach is a rapid method of phenotyping mouse models of disease.     

Mouse models for the study of Crohn's disease

Crohn's Disease (CD) is a chronic inflammatory bowel disease (IBD) that can affect any portion of the gastrointestinal tract and can cause significant morbidity. A variety of animal models of both acute and chronic intestinal inflammation have been developed to investigate disease pathogenesis and novel treatment modalities. These include chemically induced, genetically manipulated and immune-mediated models of gut inflammation, each of which possesses similarities to human IBD and offers unique advantages for studying specific aspects of disease pathogenesis. However, the majority of these models are characterized by colitis and, unlike human CD, do not involve the small intestine. More recently, murine models of chronic ileal inflammation have been characterized that spontaneously develop and closely resemble human CD with regard to disease location, histologic features and clinical response to therapy. Two mouse models of experimental ileitis will be discussed in this review: the TNF DeltaARE and SAMP1/YitFc strains. Studies using these new models might provide important insight into the pathogenesis of human CD and test the efficacy of potential therapies to treat this devastating disease.     

Preface<Emphasis Type="Bold">:</Emphasis> Perspectives on progress in ornitholimnology

An introduction to papers presented in this volume is given describing the topics of interest dealt with in this special issue in the context of past and recent articles published on aquatic birds and limnology. Lack of holistic approach is observed in this review, while it is observed that most studies deal with community ecology, environmental factors regulating bird population distribution through space and time, and their management. However, the holistic approach taking into account interactions between different trophic levels and human regulations, and further studies relating ornitholimnology to global changes are not well enough represented in the literature.     

From bedside to bench to clinic trials: identifying new treatments for severe asthma

Asthmatics with a severe form of the disease are frequently refractory to standard medications such as inhaled corticosteroids, underlining the need for new treatments to prevent the occurrence of potentially life-threatening episodes. A major obstacle in the development of new treatments for severe asthma is the heterogeneous pathogenesis of the disease, which involves multiple mechanisms and cell types. Furthermore, new therapies might need to be targeted to subgroups of patients whose disease pathogenesis is mediated by a specific pathway. One approach to solving the challenge of developing new treatments for severe asthma is to use experimental mouse models of asthma to address clinically relevant questions regarding disease pathogenesis. The mechanistic insights gained from mouse studies can be translated back to the clinic as potential treatment approaches that require evaluation in clinical trials to validate their effectiveness and safety in human subjects. Here, we will review how mouse models have advanced our understanding of severe asthma pathogenesis. Mouse studies have helped us to uncover the underlying inflammatory mechanisms (mediated by multiple immune cell types that produce Th1, Th2 or Th17 cytokines) and non-inflammatory pathways, in addition to shedding light on asthma that is associated with obesity or steroid unresponsiveness. We propose that the strategy of using mouse models to address clinically relevant questions remains an attractive and productive research approach for identifying mechanistic pathways that can be developed into novel treatments for severe asthma.     

Bacterial-mucosal interactions in inflammatory bowel disease: an alliance gone bad

The complex interaction of genetic, microbial, and environmental factors may result in continuous activation of the mucosal immune system leading to inflammatory bowel disease (IBD). Most present treatments for IBD involve altering or suppressing the aberrant immune response; however, the role of the intestinal microbiota in the pathophysiology of IBD is becoming more evident. The epithelial layer is essential for the proper functioning of the gastrointestinal tract, and its increased permeability to the luminal antigens may lead to the inflammatory processes and mucosal damage observed in IBD. Factors affecting the efficacy of the epithelial barrier include presence of pathogenic bacteria (e.g., Helicobacter spp.), presence of probiotic bacteria, availability of selected nutrients, and others. Defective function of the mucosal barrier might facilitate the contact of bacterial antigens and adjuvants with innate and adaptive immune cells to generate prolonged inflammatory responses. This review will briefly describe the complex structure of the epithelial barrier in the context of bacterial-mucosal interactions observed in human IBD and mouse models of colitis.     

An antibiotic-responsive mouse model of fulminant ulcerative colitis

Background

The constellation of human inflammatory bowel disease (IBD) includes ulcerative colitis and Crohn''s disease, which both display a wide spectrum in the severity of pathology. One theory is that multiple genetic hits to the host immune system may contribute to the susceptibility and severity of IBD. However, experimental proof of this concept is still lacking. Several genetic mouse models that each recapitulate some aspects of human IBD have utilized a single gene defect to induce colitis. However, none have produced pathology clearly distinguishable as either ulcerative colitis or Crohn''s disease, in part because none of them reproduce the most severe forms of disease that are observed in human patients. This lack of severe IBD models has posed a challenge for research into pathogenic mechanisms and development of new treatments. We hypothesized that multiple genetic hits to the regulatory machinery that normally inhibits immune activation in the intestine would generate more severe, reproducible pathology that would mimic either ulcerative colitis or Crohn''s disease.

Methods and Findings

We generated a novel mouse line (dnKO) that possessed defects in both TGFβRII and IL-10R2 signaling. These mice rapidly and reproducibly developed a disease resembling fulminant human ulcerative colitis that was quite distinct from the much longer and more variable course of pathology observed previously in mice possessing only single defects. Pathogenesis was driven by uncontrolled production of proinflammatory cytokines resulting in large part from T cell activation. The disease process could be significantly ameliorated by administration of antibodies against IFNγ and TNFα and was completely inhibited by a combination of broad-spectrum antibiotics.

Conclusions

Here, we develop to our knowledge the first mouse model of fulminant ulcerative colitis by combining multiple genetic hits in immune regulation and demonstrate that the resulting disease is sensitive to both anticytokine therapy and broad-spectrum antibiotics. These findings indicated the IL-10 and TGFβ pathways synergize to inhibit microbially induced production of proinflammatory cytokines, including IFNγ and TNFα, which are known to play a role in the pathogenesis of human ulcerative colitis. Our findings also provide evidence that broad-spectrum antibiotics may have an application in the treatment of patients with ulcerative colitis. This model system will be useful in the future to explore the microbial factors that induce immune activation and characterize how these interactions produce disease.     

Treatment with IL-27 attenuates experimental colitis through the suppression of the development of IL-17-producing T helper cells

Inflammatory bowel disease (IBD) represents a group of chronic inflammatory diseases characterized by inflammation and relapsing gastrointestinal disorders. Recent studies have shown that Th17 cells, which are well known as key mediators of chronic inflammation, have a pivotal role in onset and development of IBD in humans and mice, alike. In recent years, it has been reported that IL-27, which is an IL-12-related heterodimeric cytokine consisting of EBI3 and p28 subunits, act directly on naive T cells to suppress the differentiation of Th17 cells. However, effects of exogenous IL-27 on the IBD are not well elucidated. To clarify the suppressive effect of IL-27 treatment on IBD, we applied the flexible linking method to EBI3 and p28 subunits and generated a single-chain human IL-27 (scIL-27). scIL-27 inhibited xenogenic mouse Th17 cell differentiation in vitro, indicating that scIL-27 also acts in mouse immune systems. In a 2,4,6-trinitrobenzene sulfonic acid (TNBS)-induced mouse acute colitis model, subcutaneous scIL-27 treatment significantly improved the colon length, extent of necrosis, and ulceration and thickened epithelium and several pathological scores in a dose-dependent manner. scIL-27 clearly suppressed several inflammatory cytokines, including IL-17, in inflamed colon, except for anti-inflammatory cytokine IL-10. The mesenteric lymph node cells from scIL-27-treated mice also exhibited a reduced inflammatory response and, furthermore, a lower population of Th17 cells than those of PBS-treated mice. Finally, we showed the therapeutic efficacy of scIL-27 on TNBS-induced colitis even after active colitis was established. These results suggest new possible therapeutic approaches for IBD, including disorders such as Crohn's disease and ulcerative colitis.     

Efficient Derivation of Multipotent Neural Stem/Progenitor Cells from Non-Human Primate Embryonic Stem Cells

The common marmoset (Callithrix jacchus) is a small New World primate that has been used as a non-human primate model for various biomedical studies. We previously demonstrated that transplantation of neural stem/progenitor cells (NS/PCs) derived from mouse and human embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) promote functional locomotor recovery of mouse spinal cord injury models. However, for the clinical application of such a therapeutic approach, we need to evaluate the efficacy and safety of pluripotent stem cell-derived NS/PCs not only by xenotransplantation, but also allotransplantation using non-human primate models to assess immunological rejection and tumorigenicity. In the present study, we established a culture method to efficiently derive NS/PCs as neurospheres from common marmoset ESCs. Marmoset ESC-derived neurospheres could be passaged repeatedly and showed sequential generation of neurons and astrocytes, similar to that of mouse ESC-derived NS/PCs, and gave rise to functional neurons as indicated by calcium imaging. Although marmoset ESC-derived NS/PCs could not differentiate into oligodendrocytes under default culture conditions, these cells could abundantly generate oligodendrocytes by incorporating additional signals that recapitulate in vivo neural development. Moreover, principal component analysis of microarray data demonstrated that marmoset ESC-derived NS/PCs acquired similar gene expression profiles to those of fetal brain-derived NS/PCs by repeated passaging. Therefore, marmoset ESC-derived NS/PCs may be useful not only for accurate evaluation by allotransplantation of NS/PCs into non-human primate models, but are also applicable to analysis of iPSCs established from transgenic disease model marmosets.     

Evaluation of molecular model-based discovery of ecto-5′-nucleotidase inhibitors on the basis of X-ray structures

The enzyme ecto-5′-nucleotidase (e5NT, CD73), a metallophosphoesterase, is a critical component of adenosine metabolism and signaling and implicated in different disease states. Therefore, attempts have been made to discover inhibitors of e5NT. For example, a virtual screening study using a molecular model of the enzyme has led to the identification of a new series of sulfonamide-containing e5NT inhibitors. The recent availability of several X-ray structures of human e5NT in complex with inhibitors has made it possible to re-evaluate this model building and virtual screening effort. We have assessed the quality of the model in detail and analyzed the question why it was possible to identify a new series of inhibitors on the basis of model-based docking calculations. The model utilized for virtual screening was found to be topologically correct and approach experimental accuracy in the active site region. Two key features within the active site were identified as major determinants for the successfully identification of inhibitors. Taken together, the results rationalize the computer-aided discovery of sulfonamide inhibitors of e5NT and provide further support for the use of carefully built protein models for virtual screening.     

Overexpression of Ste20-related proline/alanine-rich kinase exacerbates experimental colitis in mice

Inflammatory bowel disease, mainly Crohn's disease and ulcerative colitis, are characterized by epithelial barrier disruption and altered immune regulation. Colonic Ste20-like proline/alanine-rich kinase (SPAK) plays a role in intestinal inflammation, but its underlying mechanisms need to be defined. Both SPAK-transfected Caco2-BBE cells and villin-SPAK transgenic (TG) FVB/6 mice exhibited loss of intestinal barrier function. Further studies demonstrated that SPAK significantly increased paracellular intestinal permeability to FITC-dextran. In vivo studies using the mouse models of colitis induced by dextran sulfate sodium (DSS) and trinitrobenzene sulfonic acid showed that TG FVB/6 mice were more susceptible to DSS and trinitrobenzene sulfonic acid treatment than wild-type FVB/6 mice, as demonstrated by clinical and histological characteristics and enzymatic activities. Consistent with this notion, we found that SPAK increased intestinal epithelial permeability, which likely facilitated the production of inflammatory cytokines in vitro and in vivo, aggravated bacterial translocation in TG mice under DSS treatment, and consequently established a context favorable for the triggering of intestinal inflammation cascades. In conclusion, overexpression of SPAK inhibits maintenance of intestinal mucosal innate immune homeostasis, which makes regulation of SPAK important to attenuate pathological responses in inflammatory bowel disease.     

A clinical drug library screen identifies astemizole as an antimalarial agent

The high cost and protracted time line of new drug discovery are major roadblocks to creating therapies for neglected diseases. To accelerate drug discovery we created a library of 2,687 existing drugs and screened for inhibitors of the human malaria parasite Plasmodium falciparum. The antihistamine astemizole and its principal human metabolite are promising new inhibitors of chloroquine-sensitive and multidrug-resistant parasites, and they show efficacy in two mouse models of malaria.     

Discovery genetics: serendipity in basic research

The role of serendipity in science has no better example than the discovery of spontaneous mutations that leads to new mouse models for research. The approach of finding phenotypes and then carrying out genetic analysis is called forward genetics. Serendipity is a key component of discovering and developing mice with spontaneous mutations into animal models of human disease. In this article, the role of serendipity in discovering and developing mouse models is described within a program at The Jackson Laboratory that capitalizes on serendipitous discoveries in large breeding colonies. Also described is how any scientists working with mice can take advantage of serendipitous discoveries as a research strategy to develop new models. Spontaneous mutations cannot be planned but happen in all research mouse colonies and are discovered as unexpected phenotypes. The alert scientist or technician can rationally exploit such chance observations to create new research opportunities.     

Suppression of colitis in mice by Cl-amidine: a novel peptidylarginine deiminase inhibitor

Inflammatory bowel diseases (IBDs), mainly Crohn's disease and ulcerative colitis, are dynamic, chronic inflammatory conditions that are associated with an increased colon cancer risk. Inflammatory cell apoptosis is a key mechanism for regulating IBD. Peptidylarginine deiminases (PADs) catalyze the posttranslational conversion of peptidylarginine to peptidylcitrulline in a calcium-dependent, irreversible reaction and mediate the effects of proinflammatory cytokines. Because PAD levels are elevated in mouse and human colitis, we hypothesized that a novel small-molecule inhibitor of the PADs, i.e., chloramidine (Cl-amidine), could suppress colitis in a dextran sulfate sodium mouse model. Results are consistent with this hypothesis, as demonstrated by the finding that Cl-amidine treatment, both prophylactic and after the onset of disease, reduced the clinical signs and symptoms of colitis, without any indication of toxic side effects. Interestingly, Cl-amidine drives apoptosis of inflammatory cells in vitro and in vivo, providing a mechanism by which Cl-amidine suppresses colitis. In total, these data help validate the PADs as therapeutic targets for the treatment of IBD and further suggest Cl-amidine as a candidate therapy for this disease.     

IL‐35 recombinant protein reverses inflammatory bowel disease and psoriasis through regulation of inflammatory cytokines and immune cells

Interleukin‐35 (IL‐35), a member of the IL‐12 family, functions as a new anti‐inflammatory factor involved in arthritis, psoriasis, inflammatory bowel disease (IBD) and other immune diseases. Although IL‐35 can significantly prevent the development of inflammation in many diseases, there have been no early studies accounting for the role of IL‐35 recombinant protein in IBD and psoriasis. In this study, we assessed the therapeutic potential of IL‐35 recombinant protein in three well‐known mouse models: the dextransulfate sodium (DSS)‐induced colitis mouse model, the keratin14 (K14)‐vascular endothelial growth factor A (VEGF‐A)‐transgenic (Tg) psoriasis mouse model and the imiquimod (IMQ)‐induced psoriasis mouse model. Our results indicated that IL‐35 recombinant protein can slow down the pathologic process in DSS‐induced acute colitis mouse model by decreasing the infiltrations of macrophages, CD4⁺T and CD8⁺T cells and by promoting the infiltration of Treg cells. Further analysis demonstrated that IL‐35 recombinant protein may regulate inflammation through promoting the secretion of IL‐10 and inhibiting the expression of pro‐inflammatory cytokines such as IL‐6, TNF‐α and IL‐17 in acute colitis model. In addition, lower dose of IL‐35 recombinant protein could achieve long‐term treatment effects as TNF‐α monoclonal antibody did in the psoriasis mouse. In summary, the remarkable therapeutic effects of IL‐35 recombinant protein in acute colitis and psoriasis mouse models indicated that IL‐35 recombinant protein had a variety of anti‐inflammatory effects and was expected to become an effective candidate drug for the treatment of inflammatory diseases.     

Potential role of metabolomics in the improvement of research on traditional African medicine

The global market for herbal medicine is growing steadily. The usage of herbal medicine is particularly common in many parts of Africa; the World Health Organization estimates that approximately 80% of Africans rely on traditional African medicines (TAMs) for treating various diseases. TAMs hold promise in preventive treatment, early disease intervention and personalized medicine. However, clinical integration of TAMs is restricted due to limited information concerning their characterization. Presently, many studies on TAMs utilize a reductionist approach, making it extremely difficult to understand the holistic modifying effects that these therapeutic agents may have on biological systems. Fortunately, emerging technologies such as metabolomics platforms adopt a ⿿top-down⿿ strategy that permits a holistic evaluation of the components, metabolic pathways and biomarkers modified by TAMs, which can aid in addressing common concerns over safety and toxicity, while also ensuring that quality control standards are met. Metabolomics approaches may also be beneficial for advancing our understanding of the efficacy and mechanism of action of TAMs, and may contribute to the advancement of research and drug discovery, early diagnosis, preventive treatment and TAMs-driven personalized medicine in Africa. This review also considers the main challenges that may hinder the adoption and integration of metabolomics approaches in research on TAMs in Africa and suggests possible solutions.     

The mouse as a model for investigation of human granulocytic ehrlichiosis: current knowledge and future directions

The use of laboratory mice to investigate correlates of infectious disease, including infection kinetics, cellular alterations, cytokine profiles, and immune response in the context of an intact host has expanded exponentially in the last decade. A marked increase in the availability of transgenic mice and research tools developed specifically for the mouse parallels and enhances this research. Human granulocytic ehrlichiosis (HGE) is an emerging, zoonotic disease caused by tick-borne bacteria. The HGE agent (Anaplasma phagocytophila) is one of two recognized pathogens to cause human granulocytic ehrlichiosis (HGE). The mouse model of HGE complements in vitro tissue culture studies, limited in vivo large animal studies, and ex vivo studies of human and ruminant neutrophils, and promises new avenues to approach mechanisms of disease. In the overview reported here, we focus principally on current research into HGE pathogenesis using the mouse model. Included is a discussion of current changes in ehrlichial classification and nomenclature, a review of ehrlichial biology and ecology, and highlights of clinical disease in animals and people.     

An endogenously anti-inflammatory role for methylation in mucosal inflammation identified through metabolite profiling

Tissues of the mucosa are lined by an epithelium that provides barrier and transport functions. It is now appreciated that inflammatory responses in inflammatory bowel diseases are accompanied by striking shifts in tissue metabolism. In this paper, we examined global metabolic consequences of mucosal inflammation using both in vitro and in vivo models of disease. Initial analysis of the metabolic signature elicited by inflammation in epithelial models and in colonic tissue isolated from murine colitis demonstrated that levels of specific metabolites associated with cellular methylation reactions are significantly altered by model inflammatory systems. Furthermore, expression of enzymes central to all cellular methylation, S-adenosylmethionine synthetase and S-adenosylhomocysteine hydrolase, are increased in response to inflammation. Subsequent studies showed that DNA methylation is substantially increased during inflammation and that epithelial NF-κB activity is significantly inhibited following treatment with a reversible S-adenosylhomocysteine hydrolase inhibitor, DZ2002. Finally, these studies demonstrated that inhibition of cellular methylation in a murine model of colitis results in disease exacerbation while folate supplementation to promote methylation partially ameliorates the severity of murine colitis. Taken together, these results identify a global change in methylation, which during inflammation, translates to an overall protective role in mucosal epithelia.     

Sarm1-mediated neurodegeneration within the enteric nervous system protects against local inflammation of the colon

Axonal degeneration is one of the key features of neu-rodegenerative disorders.In the canonical view,axonal degeneration destructs neural connections and pro-motes detrimental disease defects.Here,we assessed the enteric nervous system (ENS) of the mouse,non-human primate,and human by advanced 3D imaging.We observed the profound neurodegeneration of cate-cholaminergic axons in human colons with ulcerative colitis,and similarly,in mouse colons during acute dextran sulfate sodium-induced colitis.However,we unexpectedly revealed that blockage of such axonal degeneration by the Sarm1 deletion in mice exacerbated the colitis condition.In contrast,pharmacologic ablation or chemogenetic inhibition of catecholaminergic axons suppressed the colon inflammation.We further showed that the catecholaminergic neurotransmitter nore-pinephrine exerted a pro-inflammatory function by enhancing the expression of IL-17 cytokines.Together,this study demonstrated that Sarm1-mediated neurode-generation within the ENS mitigated local inflammation of the colon,uncovering a previously-unrecognized beneficial role of axonal degeneration in this disease context.