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1.  Treatment of Diabetes and Long-Term Survival After Insulin and Glucokinase Gene Therapy 
Diabetes  2013;62(5):1718-1729.
Diabetes is associated with severe secondary complications, largely caused by poor glycemic control. Treatment with exogenous insulin fails to prevent these complications completely, leading to significant morbidity and mortality. We previously demonstrated that it is possible to generate a “glucose sensor” in skeletal muscle through coexpression of glucokinase and insulin, increasing glucose uptake and correcting hyperglycemia in diabetic mice. Here, we demonstrate long-term efficacy of this approach in a large animal model of diabetes. A one-time intramuscular administration of adeno-associated viral vectors of serotype 1 encoding for glucokinase and insulin in diabetic dogs resulted in normalization of fasting glycemia, accelerated disposal of glucose after oral challenge, and no episodes of hypoglycemia during exercise for >4 years after gene transfer. This was associated with recovery of body weight, reduced glycosylated plasma proteins levels, and long-term survival without secondary complications. Conversely, exogenous insulin or gene transfer for insulin or glucokinase alone failed to achieve complete correction of diabetes, indicating that the synergistic action of insulin and glucokinase is needed for full therapeutic effect. This study provides the first proof-of-concept in a large animal model for a gene transfer approach to treat diabetes.
doi:10.2337/db12-1113
PMCID: PMC3636629  PMID: 23378612
2.  Nonviral-Mediated Hepatic Expression of IGF-I Increases Treg Levels and Suppresses Autoimmune Diabetes in Mice 
Diabetes  2013;62(2):551-560.
In type 1 diabetes, loss of tolerance to β-cell antigens results in T-cell–dependent autoimmune destruction of β cells. The abrogation of autoreactive T-cell responses is a prerequisite to achieve long-lasting correction of the disease. The liver has unique immunomodulatory properties and hepatic gene transfer results in tolerance induction and suppression of autoimmune diseases, in part by regulatory T-cell (Treg) activation. Hence, the liver could be manipulated to treat or prevent diabetes onset through expression of key genes. IGF-I may be an immunomodulatory candidate because it prevents autoimmune diabetes when expressed in β cells or subcutaneously injected. Here, we demonstrate that transient, plasmid-derived IGF-I expression in mouse liver suppressed autoimmune diabetes progression. Suppression was associated with decreased islet inflammation and β-cell apoptosis, increased β-cell replication, and normalized β-cell mass. Permanent protection depended on exogenous IGF-I expression in liver nonparenchymal cells and was associated with increased percentage of intrapancreatic Tregs. Importantly, Treg depletion completely abolished IGF-I-mediated protection confirming the therapeutic potential of these cells in autoimmune diabetes. This study demonstrates that a nonviral gene therapy combining the immunological properties of the liver and IGF-I could be beneficial in the treatment of the disease.
doi:10.2337/db11-1776
PMCID: PMC3554392  PMID: 23099863
3.  Macrophage Plasticity and the Role of Inflammation in Skeletal Muscle Repair 
Mediators of Inflammation  2013;2013:491497.
Effective repair of damaged tissues and organs requires the coordinated action of several cell types, including infiltrating inflammatory cells and resident cells. Recent findings have uncovered a central role for macrophages in the repair of skeletal muscle after acute damage. If damage persists, as in skeletal muscle pathologies such as Duchenne muscular dystrophy (DMD), macrophage infiltration perpetuates and leads to progressive fibrosis, thus exacerbating disease severity. Here we discuss how dynamic changes in macrophage populations and activation states in the damaged muscle tissue contribute to its efficient regeneration. We describe how ordered changes in macrophage polarization, from M1 to M2 subtypes, can differently affect muscle stem cell (satellite cell) functions. Finally, we also highlight some of the new mechanisms underlying macrophage plasticity and briefly discuss the emerging implications of lymphocytes and other inflammatory cell types in normal versus pathological muscle repair.
doi:10.1155/2013/491497
PMCID: PMC3572642  PMID: 23509419
4.  Vestigial-like-2b (VITO-1b) and Tead-3a (Tef-5a) expression in zebrafish skeletal muscle, brain and notochord 
Gene expression patterns : GEP  2007;7(8):827-836.
The vestigial gene has been shown to control skeletal muscle formation in Drosophila and the related Vestigial-like 2 (Vgl-2) protein plays a similar role in mice. Vgl-family proteins are thought to regulate tissue-specific gene expression by binding to members of the broadly expressed Scalloped/Tef/TEAD transcription factor family. Zebrafish have at least four Vgl genes, including two Vgl-2s, and at least three TEAD genes, including two Tead3s. We describe the cloning and expression of one member from each family in the zebrafish. A novel gene, vgl-2b, with closest homology to mouse and human vgl-2, is expressed transiently in nascent notochord and in muscle fibres as they undergo terminal differentiation during somitogenesis. Muscle cells also express a TEAD-3 homologue, a possible partner of Vgl-2b, during myoblast differentiation and early fibre assembly. Tead3a is also expressed in rhombomeres, eye and epiphysis regions.
doi:10.1016/j.modgep.2007.08.001
PMCID: PMC3360971  PMID: 17916448
muscle; adaxial; zebrafish; vestigial-like; transcription enhancer factor; TEAD domain
5.  Aberrant repair and fibrosis development in skeletal muscle 
Skeletal Muscle  2011;1:21.
The repair process of damaged tissue involves the coordinated activities of several cell types in response to local and systemic signals. Following acute tissue injury, infiltrating inflammatory cells and resident stem cells orchestrate their activities to restore tissue homeostasis. However, during chronic tissue damage, such as in muscular dystrophies, the inflammatory-cell infiltration and fibroblast activation persists, while the reparative capacity of stem cells (satellite cells) is attenuated. Abnormal dystrophic muscle repair and its end stage, fibrosis, represent the final common pathway of virtually all chronic neurodegenerative muscular diseases. As our understanding of the pathogenesis of muscle fibrosis has progressed, it has become evident that the muscle provides a useful model for the regulation of tissue repair by the local microenvironment, showing interplay among muscle-specific stem cells, inflammatory cells, fibroblasts and extracellular matrix components of the mammalian wound-healing response. This article reviews the emerging findings of the mechanisms that underlie normal versus aberrant muscle-tissue repair.
doi:10.1186/2044-5040-1-21
PMCID: PMC3156644  PMID: 21798099
6.  Movies of cellular and sub-cellular motion by digital holographic microscopy 
Background
Many biological specimens, such as living cells and their intracellular components, often exhibit very little amplitude contrast, making it difficult for conventional bright field microscopes to distinguish them from their surroundings. To overcome this problem phase contrast techniques such as Zernike, Normarsky and dark-field microscopies have been developed to improve specimen visibility without chemically or physically altering them by the process of staining. These techniques have proven to be invaluable tools for studying living cells and furthering scientific understanding of fundamental cellular processes such as mitosis. However a drawback of these techniques is that direct quantitative phase imaging is not possible. Quantitative phase imaging is important because it enables determination of either the refractive index or optical thickness variations from the measured optical path length with sub-wavelength accuracy.
Digital holography is an emergent phase contrast technique that offers an excellent approach in obtaining both qualitative and quantitative phase information from the hologram. A CCD camera is used to record a hologram onto a computer and numerical methods are subsequently applied to reconstruct the hologram to enable direct access to both phase and amplitude information. Another attractive feature of digital holography is the ability to focus on multiple focal planes from a single hologram, emulating the focusing control of a conventional microscope.
Methods
A modified Mach-Zender off-axis setup in transmission is used to record and reconstruct a number of holographic amplitude and phase images of cellular and sub-cellular features.
Results
Both cellular and sub-cellular features are imaged with sub-micron, diffraction-limited resolution. Movies of holographic amplitude and phase images of living microbes and cells are created from a series of holograms and reconstructed with numerically adjustable focus, so that the moving object can be accurately tracked with a reconstruction rate of 300ms for each hologram. The holographic movies show paramecium swimming among other microbes as well as displaying some of their intracellular processes. A time lapse movie is also shown for fibroblast cells in the process of migration.
Conclusion
Digital holography and movies of digital holography are seen to be useful new tools for visualization of dynamic processes in biological microscopy. Phase imaging digital holography is a promising technique in terms of the lack of coherent noise and the precision with which the optical thickness of a sample can be profiled, which can lead to images with an axial resolution of a few nanometres.
doi:10.1186/1475-925X-5-21
PMCID: PMC1448199  PMID: 16556319
7.  Analyses of the differentiation potential of satellite cells from myoD-/-, mdx, and PMP22 C22 mice 
Background
Sporadic and sometimes contradictory studies have indicated changes in satellite cell behaviour associated with the progressive nature of human Duchenne muscular dystrophy (DMD). Satellite cell proliferation and number are reportedly altered in DMD and the mdx mouse model. We recently found that satellite cells in MSVski transgenic mice, a muscle hypertrophy model showing progressive muscle degeneration, display a severe ageing-related differentiation defect in vitro. We tested the hypothesis that similar changes contribute to the gradual loss of muscle function with age in mdx and PMP22 mice, a model of human motor and sensory neuropathy type 1A (HMSN1A).
Methods
Single extensor digitorum longus muscle fibres were cultured from mdx and PMP22 mice and age- and genetic background-matched controls. Mice at several ages were compared with regard to the differentiation of satellite cells, assayed as the proportion of desmin-expressing cells that accumulated sarcomeric myosin heavy chain.
Results
Satellite cells of 2 month, 6 month, and 12 month old mdx mice were capable of differentiating to a similar extent to age-matched wild type control animals in an in vitro proliferation/differentiation model. Strikingly, differentiation efficiency in individual 6 month and 12 month old mdx animals varies to a much higher extent than in age-matched controls, younger mdx animals, or PMP22 mice. In contrast, differentiation of myoblasts from all myoD null mice assayed was severely impaired in this assay system. The defect in satellite cell differentiation that occurs in some mdx animals arises from a delay in differentiation that is not overcome by IGF-1 treatment at any phase of cultivation.
Conclusion
Overall, a defect in satellite cell differentiation above that arising through normal ageing does not occur in mdx or PMP22 mouse models of human disease. Nonetheless, the impaired differentiation of satellite cells from some mdx animals suggests that additional factors, environmental or epigenetic, may lead to deteriorating muscle repair through poor differentiation of satellite cells in genetically predisposed individuals.
doi:10.1186/1471-2474-6-15
PMCID: PMC1079863  PMID: 15762989

Results 1-7 (7)