Animal Models Of Human Disease Research Articles

Rd9 Is a Naturally Occurring Mouse Model of a Common Form of Retinitis Pigmentosa Caused by Mutations in RPGR-ORF15

Animal models of human disease are an invaluable component of studies aimed at understanding disease pathogenesis and therapeutic possibilities. Mutations in the gene encoding retinitis pigmentosa GTPase regulator (RPGR) are the most common cause of X-linked retinitis pigmentosa (XLRP) and are estimated to cause 20% of all retinal dystrophy cases. A majority of RPGR mutations are present in ORF15, the purine-rich terminal exon of the predominant splice-variant expressed in retina. Here we describe the genetic and phenotypic characterization of the retinal degeneration 9 (Rd9) strain of mice, a naturally occurring animal model of XLRP. Rd9 mice were found to carry a 32-base-pair duplication within ORF15 that causes a shift in the reading frame that introduces a premature-stop codon. Rpgr ORF15 transcripts, but not protein, were detected in retinas from Rd9/Y male mice that exhibited retinal pathology, including pigment loss and slowly progressing decrease in outer nuclear layer thickness. The levels of rhodopsin and transducin in rod outer segments were also decreased, and M-cone opsin appeared mislocalized within cone photoreceptors. In addition, electroretinogram (ERG) a- and b-wave amplitudes of both Rd9/Y male and Rd9/Rd9 female mice showed moderate gradual reduction that continued to 24 months of age. The presence of multiple retinal features that correlate with findings in individuals with XLRP identifies Rd9 as a valuable model for use in gaining insight into ORF15-associated disease progression and pathogenesis, as well as accelerating the development and testing of therapeutic strategies for this common form of retinal dystrophy.

Microvesicle-associated AAV Vector as a Novel Gene Delivery System

Adeno-associated virus (AAV) vectors have shown remarkable efficiency for gene delivery to cultured cells and in animal models of human disease. However, limitations to AAV vectored gene transfer exist after intravenous transfer, including off-target gene delivery (e.g., liver) and low transduction of target tissue. Here, we show that during production, a fraction of AAV vectors are associated with microvesicles/exosomes, termed vexosomes (vector-exosomes). AAV capsids associated with the surface and in the interior of microvesicles were visualized using electron microscopy. In cultured cells, vexosomes outperformed conventionally purified AAV vectors in transduction efficiency. We found that purified vexosomes were more resistant to a neutralizing anti-AAV antibody compared to conventionally purified AAV. Finally, we show that vexosomes bound to magnetic beads can be attracted to a magnetized area in cultured cells. Vexosomes represent a unique entity which offers a promising strategy to improve gene delivery.

Cytokine-mediated β-cell damage in PARP-1-deficient islets

Poly(ADP)-ribose polymerase (PARP) is an abundant nuclear protein that is activated by DNA damage; once active, it modifies nuclear proteins through attachment of poly(ADP)-ribose units derived from β-nicotinamide adenine dinucleotide (NAD(+)). In mice, the deletion of PARP-1 attenuates tissue injury in a number of animal models of human disease, including streptozotocin-induced diabetes. Also, inflammatory cell signaling and inflammatory gene expression are attenuated in macrophages isolated from endotoxin-treated PARP-1-deficient mice. In this study, the effects of PARP-1 deletion on cytokine-mediated β-cell damage and macrophage activation were evaluated. There are no defects in inflammatory mediator signaling or inflammatory gene expression in macrophages and islets isolated from PARP-1-deficient mice. While PARP-1 deficiency protects islets against cytokine-induced islet cell death as measured by biochemical assays of membrane polarization, the genetic absence of PARP-1 does not effect cytokine-induced inhibition of insulin secretion or cytokine-induced DNA damage in islets. While PARP-1 deficiency appears to provide protection from cell death, it fails to provide protection against the inhibitory actions of cytokines on insulin secretion or the damaging actions on islet DNA integrity.

Abstract SY24-01: Use of imaging to guide the development of immune cell therapies

Abstract Advances in in vivo imaging have enabled the study of cellular and molecular biology in living animals models of human disease. When integrated with thorough studies in cell culture and directed ex vivo analyses, these tools can reveal the nuances of cellular mechanisms and the subtlety of therapeutic responses. This is leading to the identification and interrogation of molecular targets at a level that was not previously possible, and is refining and accelerating the study of novel therapeutic strategies directed at these targets. The impact of molecular imaging has been most dramatic in preclinical studies of animal models and is moving into clinical studies. With these tools we have a unique opportunity to ask, and answer, hard questions about disease processes and novel therapies in the context of living tissues. By preserving the tissue structure and cellular function in these studies we can gain more information that is more relevant to the disease processes. The advances in stem cell biology and transgenic sciences are creating animal models that are more relevant to human disease and response to therapy, and imaging is accelerating the study of these more predictive models. Molecular imaging is leading to new insights, and enabling new approaches in the emerging fields of stem cell therapy, nanotechnology and regenerative medicine. This is leading to dramatic changes in the way we screen and develop new drugs. It is likely that these visible animal models of human biology and disease comprise one of the most important contributions of molecular imaging to human health as they serve to accelerate and refine the analyses of mammalian biology and offer a rapid readout for the development of new therapies. Using imaging we have developed a combination of two well-developed biotherapies that together maximize delivery, and improve efficacy in preclinical models of ovarian cancer. The dual biotherapy is comprised of immunotherapy with cytokine induced killer (CIK) cells, and oncolytic virotherapy with an attenuated vaccinia virus. These two therapies have a proven safety record with decades of clinical evaluation of each individual biotherapy, but alone the therapies lack efficacy. The limitations of each therapy are well understood and explain the lack of efficacy. In considering these limitations we proposed and tested the combination therapy and demonstrated a complementarity that overcomes the limitations of each individual therapy and creates a therapeutic synergy that is highly efficacious in preclinical studies. Transporting cytotoxic agents to tumor targets has been the goal of cell-mediated delivery, however, immune cells can also i) produce their own tumoricidal effect, ii) conceal a payload from an immune response, iii) amplify a selective agent at the target site and iv) facilitate an antitumor immune response. Integrating the biology of a cellular delivery vehicle with that of the therapeutic payload, vaccinia virus, leads to enhanced antitumor effects. Both of the agents display broad tumor-targeting potential and possess unique tumor killing mechanisms, together the therapies are able to recognize and destroy a far greater number and diversity of malignant cells within the heterogeneous tumor than either agent alone. Effective cancer therapy will require recognition and elimination of the root of the disease, the cancer stem cell, and this combination has this potential. The tumor-selectivity of oncolytic viruses is due to modifications that take advantage of the unique biology of the cancer cell, and similar modifications for integration into the delivery vehicle increases the safety and improves therapeutic outcome. In this dose escalation trail the cell numbers will be fixed and the dose of virus increased, and we will use imaging methods as rapid measures of treatment response to asses efficacy and guide future development. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr SY24-01. doi:1538-7445.AM2012-SY24-01

UniqueO-Methoxyethyl Ribose-DNA Chimeric Oligonucleotide Induces an Atypical Melanoma Differentiation-Associated Gene 5-Dependent Induction of Type I Interferon Response

Antisense oligonucleotides (ASO) containing 2'-O-methoxyethyl ribose (2'-MOE) modifications have been shown to possess both excellent pharmacokinetic properties and robust pharmacological activity in several animal models of human disease. 2'-MOE ASOs are generally well tolerated, displaying minimal to mild proinflammatory effect at doses far exceeding therapeutic doses. Although the vast majority of 2'-MOE ASOs are safe and well tolerated, a small subset of ASOs inducing acute inflammation in mice has been identified. The mechanism for these findings is not clear at this point, but the effects are clearly sequence-specific. One of those ASOs, ISIS 147420, causes a severe inflammatory response atypical of this class of oligonucleotides characterized by induction in interferon-β (IFN-β) at 48 h followed by acute transaminitis and extensive hepatocyte apoptosis and necrosis at 72 h. A large number of interferon-stimulated genes were significantly up-regulated in liver as early as 24 h. We speculated that a specific sequence motif might cause ISIS 147420 to be mistaken for viral RNA or DNA, thus triggering an acute innate immune response. ISIS 147420 toxicity was independent of Toll-like receptors, because there was no decrease in IFN-β in Toll/interleukin-1 receptor-domain-containing adapter-inducing IFN-β or Myd88-deficient mice. The involvement of cytosolic retinoic acid-inducible gene (RIG)-I-like pattern recognition receptors was also investigated. Pretreatment of mice with melanoma differentiation-associated gene 5 (MDA5) and IFN-β promoter stimulator-1 ASOs, but not RIG-I or laboratory of genetics and physiology 2 (LGP2) ASOs, prevented the increase in IFN-β and alanine aminotransferase induced by ISIS 147420. These results revealed a novel mechanism of oligonucleotide-mediated toxicity requiring both MDA5 and IPS-1 and resulting in the activation of the innate immune response.

The AAV vector toolkit: poised at the clinical crossroads.

The discovery of naturally occurring adeno-associated virus (AAV) isolates in different animal species and the generation of engineered AAV strains using molecular genetics tools have yielded a versatile AAV vector toolkit. Promising results in preclinical animal models of human disease spurred the much awaited transition toward clinical application, and early successes in phase I/II clinical trials for a broad spectrum of genetic diseases have recently been reported. As the gene therapy community forges ahead with cautious optimism, both preclinical and clinical studies using first generation AAV vectors have highlighted potential challenges. These include cross-species variation in vector tissue tropism and gene transfer efficiency, pre-existing humoral immunity to AAV capsids and vector dose-dependent toxicity in patients. A battery of second generation AAV vectors, engineered through rational and combinatorial approaches to address the aforementioned concerns, are now available. This review will provide an overview of preclinical studies with the ever-expanding AAV vector portfolio in large animal models and an update on new lead AAV vector candidates poised for clinical translation.

Sulphide quinone reductase contributes to hydrogen sulphide metabolism in murine peripheral tissues but not in the CNS

Hydrogen sulphide (H(2) S) is gaining acceptance as a gaseous signal molecule. However, mechanisms regarding signal termination are not understood. We used stigmatellin and antimycin A, inhibitors of sulphide quinone reductase (SQR), to test the hypothesis that the catabolism of H(2) S involves SQR. H(2) S production and consumption were determined in living and intact mouse brain, liver and colonic muscularis externa using gas chromatography and HPLC. Expressions of SQR, ethylmalonic encephalopathy 1 (Ethe1) and thiosulphate transferase (TST; rhodanese) were determined by RT-PCR and immunohistochemistry. In the colonic muscularis externa, H(2) (35) S was catabolized to [(35) S]-thiosulphate and [(35) S]-sulphate, and stigmatellin reduced both the consumption of H(2) (35) S and formation of [(35) S]-thiosulphate. Stigmatellin also enhanced H(2) S release by the colonic muscularis externa. In the brain, catabolism of H(2) (35) S to [(35) S]-thiosulphate and [(35) S]-sulphate, which was stigmatellin-insensitive, partially accounted for H(2) (35) S consumption, while the remainder was captured as unidentified (35) S that was probably bound to proteins. Levels of mRNA encoding SQR were higher in the colonic muscularis externa and the liver than in the brain. These data support the concept that termination of endogenous H(2) S signalling in the colonic muscularis externa occurs via catabolism to thiosulphate and sulphate partially via a mechanism involving SQR. In the brain, it appears that H(2) S signal termination occurs partially through protein sequestration and partially through catabolism not involving SQR. As H(2) S has beneficial effects in animal models of human disease, we suggest that selective inhibition of SQR is an attractive target for pharmaceutical development.

The Therapeutic Potential of Sigma (σ) Receptors for the Treatment of Central Nervous System Diseases: Evaluation of the Evidence

Since their proposal in 1976, sigma (σ) receptors have been increasingly implicated in the pathophysiology of virtually all major central nervous system (CNS) disorders, including anxiety, depression, schizophrenia, and drug addiction. Due to their involvement in motor function and higher cognitive function,σ receptors have also been implicated in movement disorders (such as Parkinson's disease) and memory deficits (including Alzheimer's disease). In most cases the precise mechanism(s) linking σ receptors to CNS disease are unknown or yet to be fully elucidated. However, many σ ligands have shown promise in pharmacological studies and animal models of the aforementioned diseases, and some have entered clinical trials. This review will assess the validity of receptors as a target for various CNS diseases based on evidence from animal models of human diseases, preclinical studies in humans, and full clinical trials.

NAAG, NMDA Receptor and Psychosis

At central synapses, glutamate is the main excitatory neurotransmitter. Once released from presynaptic terminals, glutamate activates a number of different glutamatergic receptors one of which is the ligand gated ionophore glutamatergic subtype N-methyl-D-aspartate receptors (NMDARs). NMDARs play a crucial role in controlling various determinants of synaptic function. N-acetylaspartylglutamate (NAAG) is the most prevalent peptide transmitter in the mammalian central nervous system. NAAG is released upon neuronal depolarization by a calcium-dependent process from glutamatergic and GABAergic neurons. It is cleaved by a specific peptidase located on astrocytes, glutamate carboxypeptidase type II (GCP-II), to N-acetylaspartate (NAA) and glutamate. Current evidence supports the hypothesis that NAAG is an endogenous agonist at G protein coupled mGluR3 receptors and an antagonist at NMDAR. In several disorders and animal models of human diseases, the levels of NAAG and the activity of GCP-II are altered in ways that are consistent with NAAG's role in regulation of glutamatergic neurotransmission. Several lines of evidence suggest that a dysfunction in glutamatergic via the NMDAR might be involved in schizophrenia. This hypothesis has evolved from findings that NMDAR antagonists such as phencyclidine (PCP or "angel dust"), produces a syndrome in normal individuals that closely resembles schizophrenia and exacerbates psychotic symptoms in patients with chronic schizophrenia. Recent postmortem, metabolic and genetic studies have provided evidence that hypofunction of discrete populations of NMDAR can contribute to the symptoms of schizophrenia, at least in some patients. The review outlines the role of endogenous NAAG at NMDAR neurotransmission and its putative role in the pathophysiology of schizophrenia.

Thermodynamic stability, unfolding kinetics, and aggregation of the N‐terminal actin‐binding domains of utrophin and dystrophin

Muscular dystrophy (MD) is the most common genetic lethal disorder in children. Mutations in dystrophin trigger the most common form of MD, Duchenne, and its allelic variant Becker MD. Utrophin is the closest homologue and has been shown to compensate for the loss of dystrophin in human disease animal models. However, the structural and functional similarities and differences between utrophin and dystrophin are less understood. Both proteins interact with actin through their N-terminal actin-binding domain (N-ABD). In this study, we examined the thermodynamic stability and aggregation of utrophin N-ABD and compared with that of dystrophin. Our results show that utrophin N-ABD has spectroscopic properties similar to dystrophin N-ABD. However, utrophin N-ABD has decreased denaturant and thermal stability, unfolds faster, and is correspondingly more susceptible to proteolysis, which might account for its decreased in vivo half-life compared to dystrophin. In addition, utrophin N-ABD aggregates to a lesser extent compared with dystrophin N-ABD, contrary to the general behavior of proteins in which decreased stability enhances protein aggregation. Despite these differences in stability and aggregation, both proteins exhibit deleterious effects of mutations. When utrophin N-ABD mutations analogous in position to the dystrophin disease-causing mutations were generated, they behaved similarly to dystrophin mutants in terms of decreased stability and the formation of cross-β aggregates, indicating a possible role for utrophin mutations in disease mechanisms.

Animal models for human disease – reflections from an animal researcher’s perspective

Neuronal ceroid lipofuscinoses (NCL) are a group of lethal inherited neurodegenerative disorders in humans and many animal species. My research on sheep and cattle with NCL raises critical questions on a range of ethical issues, specifically the claim that sheep and cattle are useful models for the disease in humans and other related moral problems. My reflections on moral status of animals and validity of animal models are outlined in this paper.

Formulation of the Total Western Diet (TWD) as a Basal Diet for Rodent Cancer Studies

Rodent cancer studies typically use defined diets with nutrient profiles optimized for rodent health. However, a defined rodent diet that represents typical American nutrition in all aspects, including calorie sources and macro- and micronutrient composition, is not yet available. Thus, a nutrient density approach was used to formulate the new Total Western Diet (TWD) based on NHANES data for macro- and micronutrient intakes. The TWD has fewer calories from protein and carbohydrate sources and twice that from fat as compared to the AIN-93 diet. The new diet contains more saturated and monounsaturated fats, less polyunsaturated fat, fewer complex carbohydrates, and twice the level of simple sugars. The TWD includes less calcium, copper, folate, thiamin, and vitamins B6, B12, D, and E, but much more sodium. This newly devised diet that better represents typical American nutrition will be highly useful for studies employing animal models of human disease, including cancer.

Enhanced cellular uptake and long-term retention of chitosan-modified iron-oxide nanoparticles for MRI-based cell tracking

Tracking cells after therapeutic transplantation is imperative for evaluation of implanted cell fate and function. In this study, ultrasmall superparamagnetic iron oxide nanoparticles (USPIO NPs) were surface functionalized with water-soluble chitosan, a cationic polysaccharide that mediates enhanced endocytic uptake, endosomal escape into the cytosol, and subsequent long-term retention of nanoparticles. NP surface and chitosan were independently fluorescently labeled. Our NPs enable NP trafficking studies and determination of fate beyond uptake by fluorescence microscopy as well as tracking of labeled cells as localized regions of hypointensity in T2*-weighted magnetic resonance imaging (MRI) images. Adult rat neural stem cells (NSCs) were labeled with NPs, and assessment of NSC proliferation rates and differentiation potential revealed no significant differences between labeled and unlabeled NSCs. Significantly enhanced uptake of chitosan NPs in comparison to native NPs was confirmed by transmission electron microscopy, nuclear magnetic resonance (NMR) spectroscopy and in vitro cellular MRI at 11.7 Tesla. While only negligible fractions of native NPs enter cells, chitosan NPs appear within membranous vesicles within 2 hours of exposure. Additionally, chitosan-functionalized NPs escaped from membrane-bound vesicles within days, circumventing NP endo-lysosomal trafficking and exocytosis and hence enabling long-term tracking of labeled cells. Finally, our labeling strategy does not contain any NSC-specific reagents. To demonstrate general applicability across a variety of primary and immortalized cell types, embryonic mouse NSCs, mouse embryonic stem cells, HEK 293 kidney cells, and HeLa cervical cancer cells were additionally exposed to chitosan-USPIO NPs and exhibited similarly efficient loading as verified by NMR relaxometry. Our efficient and versatile labeling technology can support cell tracking with close to single cell resolution by MRI in vitro, for example, in complex tissue models not optically accessible by confocal or multi-photon fluorescence microscopy, and potentially in vivo, for example, in animal models of human disease or injury.

Human Stromal (Mesenchymal) Stem Cells: Basic Biology and Current Clinical Use for Tissue Regeneration

Human stromal (mesenchymal) stem cells (hMSC) represent a group of non-hematopoietic stem cells present in the bone marrow stroma and the stroma of other organs including subcutaneous adipose tissue, placenta, and muscles. They exhibit the characteristics of somatic stem cells of self-renewal and multi-lineage differentiation into mesoderm-type of cells, e.g., to osteoblasts, adipocytes, chondrocytes and possibly other cell types including hepatocytes and astrocytes. Due to their ease of culture and multipotentiality, hMSC are increasingly employed as a source for cells suitable for a number of clinical applications, e.g., non-healing bone fractures and defects and also non-skeletal degenerative diseases like heart failure. Currently, the numbers of clinical trials that employ MSC are increasing. However, several biological and biotechnological challenges need to be overcome to benefit from the full potential of hMSC. In this current review, we present some of the most important and recent advances in understanding of the biology of hMSC and their current and potential use in therapy.

Animal Models of Human Pathology 2012

In this special issue we gather together 23 articles (15 reviews, 7 research articles, and 1 methodology report) with diverse information presently available on animal models of human disease. The use of animal for studying human diseases, from the pathogenesis to the clinical aspects of diagnosis and therapy, has always been a very useful tool and indeed, has contributed enormously to our understanding of the mechanisms of diseases and to the development of numerous drugs. The 15 comprehensive reviews critically inform the reader of the latest developments in the animal models of diseases such as Parkinson (Blesa et al.), glaucoma (Bouhenni et al.), dermatophytoses (Shimamura et al.), colitis (Perse and Cerar), HCV infections (Carcamo and Nguyen), Glomerulonephritis (Urushihara et al.), and posterior circulation stroke (Lekic and Ani). Moreover, Underbayev et al. provide a clear and concise review on the applications of induced pluripotent stem cells (iPS) for human disease mouse modeling, focusing on the use of microRNA manipulation in the generation and differentiation of iPS. Goldsmith and Jobin propose the use of zebrafish model to study human disease. Moro and colleagues review the significance of mouse models in the study of human cancer progression with particular emphasis on the possible application of patient-derived xenograft in the evaluation of chemoresistance, while Mendes-Braz et al. review the mechanisms of hepatic ischemia-reperfusion injury in animal models, and its translation to the clinical practice. Greco et al. describe the basic principles of high resolution imaging ultrasound equipment and the most important applications in preclinical studies, Li et al. examine different MRI sequences to explore a murine model of intracerebral hemorrhage, whereas Gargiulo et al. provide a review of the use of PET/CT for examining mouse models of myocardial ischemia and Sergi et al. propose an important role of hepatocellular to cholangiocytic metaplasia (as expressed by switch in keratin expression) in the causation of liver graft dysfunction (including cholestasis) in case of ischemia/reperfusion injury, based on personal observations and evidence from the literature. Di Domenico et al. present an exhaustive analysis of the role played by the vascular endothelial growth factor and some cytokines in periodontal ligament remodeling and bone resorption at the pressure side of orthodontic tooth movement. The research articles deal with the molecular mechanisms involved in the pathogenesis of a particular disease. Vitale et al. report synaptic differences between wild-type and FDDki mice, essentially in the expression levels of selected proteins, in a model of familial dementia; Venancio et al. demonstrate that sleep deprivation alters rat ventral prostate morphology; Neri et al. analyze the effect of colic vein ligature in rats with loperamide-induced constipation, and Allan et al. evaluate the ability of an antimicrobial peripherally inserted central catheter (PICC) to reduce microbial migration and colonization compared to an uncoated PICC. Dunn and Pinkert provide evidence supporting allotopic expression as a tool for mtDNA disease research with implications in development of DNA-based therapeutics, while Mingchang et al. propose a pilot study to examine the use of T1, T2, and T2* weighted images for evaluating hematoma size and extent of edema in mouse brain at high field. In further contributions animal models have been used to implement therapeutic approaches. It is the case of the studies (a research article and a methodology report) by Pereira et al. which develop an animal model to study deep second-degree thermal burning and characterize in vivo hydrogel formulation for burn healing. In conclusion, as it is shown in the collection of contributions assembled in this special issue, we believe that animal models of human pathologies, because of the tremendous bulk of information that they can supply to scientific community, particularly genetic and alternative models, might lead us into a new era of disease research and drug discovery.

Zebrafish-a new animal models of anterior segment embryonic development and diseases basic research

In recent years, zebrafish has become ideal animal models of human disease with its unique characteristics, such as small body, fecundity, fast development and growth, embryo transparency, and so on. Furthermore, the structure and gene of zebrafish eye are highly conservative with human eye, which make ophthalmologists to pay close attention to zebrafish. This review focus on the studies and applications on zebrafish embryonic development of anterior segment, including the morphogenesis of cornea, lens and anterior chamber, and diseases of anterior segment (corneal diseases, cataract, glaucoma).

Usefulness of clinical magnetic resonance scanners for imaging experimental changes in laboratory rodents' central nervous system

Abstract Introduction Magnetic resonance imaging (MRI) is a noninvasive technique applied in medical diagnosis and for studying animal models of human diseases. MRI offers longitudinal in vivo studies without the need to sacrifice animals, thus making data easier to compare. The number of required animals can be limited. Aim The aim of this article was to present the potential role of clinical MRI scanners in the management of central nervous system visualization and injury in rodents on the basis of the current literature. Materials and methods Clinical small bore scanners with field strength from 0.1 T to 3 T are used for imaging the nervous system of rodents in vivo . Results and discussion The employment of clinical scanners equipped with dedicated human coils, for small objects imaging, results in the reduction of image quality. It is caused by a small signal-to-noise ratio (SNR). The way to increase the SNR is to use clinical scanners for imaging particular parts of the human body, e.g. head, or dedicated coils for imaging small parts of the human body, e.g., thumb or wrist, or to use dedicated small animal coils to image multiple animals in the larger bore of the clinical scanner at the same time. For some neurobiological experiments clinical scanners seem to be sufficient. Although clinical MRI scanners are widespread, not many laboratories use them for small animal research. Conclusions Clinical scanners with surface coils dedicated to small human organs, or with dedicated small animal coils, are useful for imaging experimental changes in the central nervous system of laboratory rodents.

MicroRNAs and Induced Pluripotent Stem Cells for Human Disease Mouse Modeling

Human disease animal models are absolutely invaluable tools for our understanding of mechanisms involved in both physiological and pathological processes. By studying various genetic abnormalities in these organisms we can get a better insight into potential candidate genes responsible for human disease development. To this point a mouse represents one of the most used and convenient species for human disease modeling. Hundreds if not thousands of inbred, congenic, and transgenic mouse models have been created and are now extensively utilized in the research labs worldwide. Importantly, pluripotent stem cells play a significant role in developing new genetically engineered mice with the desired human disease-like phenotype. Induced pluripotent stem (iPS) cells which represent reprogramming of somatic cells into pluripotent stem cells represent a significant advancement in research armament. The novel application of microRNA manipulation both in the generation of iPS cells and subsequent lineage-directed differentiation is discussed. Potential applications of induced pluripotent stem cell—a relatively new type of pluripotent stem cells—for human disease modeling by employing human iPS cells derived from normal and diseased somatic cells and iPS cells derived from mouse models of human disease may lead to uncovering of disease mechanisms and novel therapies.

Immunization strategies for Parkinson's disease

Parkinson's disease (PD) is the most common neurodegenerative movement disorder. Currently, no curative treatments or treatments that interdict disease progression are available. Over the past decade, immunization strategies were developed in our laboratories to combat disease progression. These strategies were developed in laboratory and animal models of human disease. Induction of humoral immune responses can be elicited against misfolded protein aggregates. Robust cell-mediated immunity against nitrated misfolded protein(s) accelerates disease progression through effector T cell responses that facilitate neuronal death. We propose that shifting the balance between effector and regulatory T cell activity can attenuate neurotoxic inflammatory events. We now summarize our works that support immune regulation in PD with the singular goal of restoring homeostatic glial responses. New methods to optimize immunization schemes and measure their clinical efficacy are discussed.

Abstract P331: Metabolic Dynamics and Phosphometabolomic Fingerprinting Using 18 O-Assisted 31 P NMR and Mass Spectrometry

Evaluation of disease phenotypes and drug effects requires knowledge not only metabolite levels but also their turnover rates from which metabolic fluxes and status of the whole energetic system can be determined. Oxygen-18-assited 31 P NMR and mass spectrometric technique uniquely allow simultaneous measurements of phosphorus-containing metabolite levels and their turnover rates. This includes simultaneous recordings of 18 O-labeling rates reflecting ATP synthesis (γ-ATP) and ATP utilization (Pi), phosphotransfer fluxes through adenylate kinase (β-ATP/ADP), creatine kinase (CrP) and glycolytic pathways (G-6-P) as well as Krebs cycle associated mitochondrial nucleotide turnover (γ/β-GTP), substrate shuttle (G-3-P) and glycogen metabolism (G-1-P). Metabolomic and fluxomic profiling of hearts and body fluids of phosphotransfer enzyme deficient transgenic animals using 18 O-assisted GC/MS, 1 H and 18 O-assisted 31 P NMR indicate selective metabolic perturbations and adaptations in the whole energetic system. Adenylate kinase and creatine kinase deficiencies were associated with altered metabolomic profiles, increased mitochondrial capacities and redistribution of phosphotransfer flux through glycolytic and guanine nucleotide systems. These phosphometabolomic alterations were more expressed when animals were subjected to treadmill exercise, mitochondria-targeting drugs such as metformin and high-fat diet. Metabolite turnover rates are correlated with heart performance unveiling robustness and dynamics rearrangements in cell bioenergetics infrastructure. Glycerophosphate, G-3-P, metabolic dynamics is altered in transgenic animal models indicating defects in substrate shuttle and supply of reducing equivalents to mitochondria. This is of importance since G-3-P metabolic abnormalities and metabolic arrest are linked to human diseases such as sudden death syndrome. Thus, our study demonstrates that phosphometabolomic and fluxomic profiling is a valuable tool for metabolic phenotyping of transgenic animal models of human diseases as well as for plasma biomarker identification and monitoring of treatment efficacy and drug toxicity.