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1.  Mitochondrial-derived reactive oxygen species (ROS) play a causal role in aging-related intervertebral disc degeneration 
Oxidative damage is a well-established driver of aging. Evidence of oxidative stress exists in aged and degenerated discs, but it is unclear how it affects disc metabolism. In this study, we first determined whether oxidative stress negatively impacts disc matrix metabolism using disc organotypic and cell cultures. Mouse disc organotypic culture grown at atmospheric oxygen (20% O2) exhibited perturbed disc matrix homeostasis, including reduced proteoglycan synthesis and enhanced expression of matrix metalloproteinases, compared to discs grown at low oxygen levels (5% O2). Human disc cells grown at 20% O2 showed increased levels of mitochondrial-derived superoxide anions and perturbed matrix homeostasis. Treatment of disc cells with the mitochondria-targeted reactive oxygen species (ROS) scavenger XJB-5-131 blunted the adverse effects caused by 20% O2. Importantly, we demonstrated that treatment of accelerated aging Ercc1−/Δmice, previously established to be a useful in vivo model to study age-related intervertebral disc degeneration (IDD), also resulted in improved disc total glycosaminoglycan content and proteoglycan synthesis. This demonstrates that mitochondrial-derived ROS contributes to age-associated IDD in Ercc1−/Δmice. Collectively, these data provide strong experimental evidence that mitochondrial-derived ROS play a causal role in driving changes linked to aging-related IDD and a potentially important role for radical scavengers in preventing IDD.
doi:10.1002/jor.22320
PMCID: PMC3668354  PMID: 23389888
Aging; oxidative stress; reactive oxygen species (ROS); intervertebral discs; radical scavenger; nitroxide; matrix proteoglycan
2.  An overview of underlying causes and animal models for the study of age-related degenerative disorders of the spine and synovial joints 
As human lifespan increases so does the incidence of age-associated degenerative joint diseases, resulting in significant negative socioeconomic consequences. Osteoarthritis (OA) and intervertebral disc degeneration (IDD) are the most common underlying causes of joint-related chronic disability and debilitating pain in the elderly. Current treatment methods are generally not effective and involve either symptomatic relief with non-steroidal anti-inflammatory drugs and physical therapy or surgery when conservative treatments fail. The limitation in treatment options is due to our incomplete knowledge of the molecular mechanism of degeneration of articular cartilage and disc tissue. Basic understanding of the age-related changes in joint tissue is thus needed to combat the adverse effects of aging on joint health. Aging is caused at least in part by time-dependent accumulation of damaged organelles and macromolecules, leading to cell death and senescence and the eventual loss of multipotent stem cells and tissue regenerative capacity. Studies over the past decades have uncovered a number of important molecular and cellular changes in joint tissues with age. However, the precise causes of damage, cellular targets of damage, and cellular responses to damage remain poorly understood. The objectives of this review are (1) to provide an overview of the current knowledge about the sources of endogenous and exogenous damaging agents and how they contribute to age-dependent degenerative joint disease, and (2) highlight animal models of accelerated aging that could potentially be useful for identifying causes of and therapies for degenerative joint diseases.
doi:10.1002/jor.22204
PMCID: PMC3628921  PMID: 23483579
Synovial joints; intervertebral disc; DNA repair; aging; oxidative damage
3.  Plasma-derived MHCII+ exosomes from tumor-bearing mice suppress tumor antigen-specific immune responses 
European journal of immunology  2012;42(7):1778-1784.
Tumor-specific immunosuppression is frequently observed in tumor-bearing hosts. Exosomes are nano-sized, endosomal-derived membrane vesicles secreted by most tumor and hematopoietic cells and have been shown to actively participate in immune regulation. We previously demonstrated that antigen-specific immunosuppressive exosomes could be isolated from the blood plasma of antigen-immunized mice. Here we demonstrate that plasma-derived exosomes isolated from mice bearing OVA-expressing tumors were able to suppress OVA-specific immune response in a mouse delayed-type hypersensitivity model. Enrichment of tumor-derived exosomes in the plasma of mice bearing subcutaneous melanoma was not detected using an exosome-tagging approach. Instead, depletion of MHC Class II+ vesicles from plasma-derived exosomes or using plasma-derived exosomes isolated from MHC Class II deficient mice resulted in significant abrogation of the suppressive effect. These results demonstrate that circulating host-derived, MHC Class II+ exosomes in tumor-bearing hosts are able to suppress the immune response specific to tumor antigens.
doi:10.1002/eji.201141978
PMCID: PMC3471134  PMID: 22585706
4.  DNA damage drives accelerated bone aging via an NF-κB-dependent mechanism 
Advanced age is one of the most important risk factors for osteoporosis. Accumulation of oxidative DNA damage has been proposed to contribute to age-related deregulation of osteoblastic and osteoclastic cells. ERCC1 (Excision Repair Cross Complementary group 1)-XPF (Xeroderma Pigmentosum Group F) is an evolutionarily conserved structure-specific endonuclease that is required for multiple DNA repair pathways. Inherited mutations affecting expression of ERCC1-XPF cause a severe progeroid syndrome in humans, including early onset of osteopenia and osteoporosis, or anomalies in skeletal development. Herein, we used progeroid ERCC1-XPF deficient mice, including Ercc1-null (Ercc1−/−) and hypomorphic (Ercc1−/Δ) mice, to investigate the mechanism by which DNA damage leads to accelerated bone aging. Compared to their wild-type littermates, both Ercc1−/− and Ercc1−/Δ mice display severe, progressive osteoporosis caused by reduced bone formation and enhanced osteoclastogenesis. ERCC1 deficiency leads to atrophy of osteoblastic progenitors in the bone marrow stromal cell (BMSC) population. There is increased cellular senescence of BMSCs and osteoblastic cells, as characterized by reduced proliferation, accumulation of DNA damage and a senescence-associated secretory phenotype (SASP). This leads to enhanced secretion of inflammatory cytokines known to drive osteoclastogenesis, such as IL-6, TNFα, and RANKL and thereby induces an inflammatory bone microenvironment favoring osteoclastogenesis. Furthermore, we found that the transcription factor NF-κB is activated in osteoblastic and osteoclastic cells of the Ercc1 mutant mice. Importantly, we demonstrated that haploinsufficiency of the p65 NF-κB subunit partially rescued the osteoporosis phenotype of Ercc1−/Δ mice. Finally, pharmacological inhibition of the NF-κB signaling via an IKK inhibitor reversed cellular senescence and SASP in Ercc1−/Δ BMSCs. These results demonstrate that DNA damage drives osteoporosis through an NF-κB-dependent mechanism. Therefore, the NF-κB pathway represents a novel therapeutic target to treat aging-related bone disease.
doi:10.1002/jbmr.1851
PMCID: PMC3662975  PMID: 23281008
osteoporosis; osteoblasts; osteoclasts; bone; nucleotide excision repair; progeria; aging; NF-κB transcription factor; ERCC1-XPF endonuclease
5.  AAV8-Mediated Gene Transfer of Interleukin-4 to Endogenous β-Cells Prevents the Onset of Diabetes in NOD Mice 
We previously demonstrated that intra-peritoneal delivery of adeno-associated virus serotype 8 (AAV8) stably transduces the pancreas, including the β-cells in the endogenous islets. We also demonstrated the ability to deliver and express genes specifically in β-cells for at least 6 months using a murine insulin promoter (mIP) in a double-stranded, self-complementary AAV vector (dsAAV8-mIP). Here we evaluated the effects of dsAAV8-mIP mediated delivery of interleukin 4 (mIL-4) to endogenous β-cells in NOD mice. In 4 week old NOD mice, the extent of gene transfer and expression in endogenous β-cells following i.p. delivery of dsAAV8-mIP-eGFP was comparable to normal BALB/c mice. Furthermore, following i.p. delivery of dsAAV8-mIP-IL4, expression of mIL-4 was detected in islets isolated and cultured from the treated mice. AAV8-mIP mediated gene expression of mIL-4 to endogenous β-cells of 4 and 8 week old NOD mice prevented the onset of hyperglycemia in NOD mice and reduced the severity of insulitis. Moreover, expression of mIL-4 also maintained the level of CD4+CD25+FoxP3+ cells and adoptive co-transfer of splenocytes from diabetes-free IL-4 vector recipients and splenocytes from wild type diabetic NOD mice prevented the onset diabetes. Taken together, these results demonstrate that local expression of mIL-4 in islets prevents islet destruction and blocks autoimmunity, in part, through regulation of T cell function. These results also demonstrate the utility of using dsAAV8-mIP gene transfer to endogenous NOD β-cells to examine the role of specific gene products in preventing or exacerbating the onset of type 1 diabetes.
doi:10.1038/mt.2008.116
PMCID: PMC3560428  PMID: 18560422
6.  The Anti-tumor Effects of Adenoviral-Mediated, Intra-tumoral delivery Interleukin 23 require Endogenous IL-12 
Cancer gene therapy  2011;19(2):135-143.
IL-23 is a member of the IL-12 family of heterodimeric cytokines, comprised of p19 and p40 subunits, which exhibits immunostimulatory properties similar to IL-12. We have demonstrated previously that adenoviral-mediated, intra-tumoral delivery of IL-23 (Ad.IL-23) was able to induce systemic anti-tumor immunity. Here we demonstrate that Ad.IL-23 requires endogenous IL-12 for conferring an anti-tumor effect after adenoviral-mediated intra-tumoral delivery. In contrast, Ad.IL-12 does not require IL-23 for its anti-tumor effects although endogenous IL-23 appears important for induction of systemic anti-tumor immunity by IL-12. However, despite the requirement for endogenous IL-12, co-delivery of IL-23 and IL-12 does not provide even an additive local or systemic anti-tumor effect, regardless of the dose. We further demonstrate that although the use of a single chain IL-23 (scIL-23) results in higher level of expression and a more pronounced IL-23-mediated anti-tumor effect, there is still no synergy with IL-12. These results demonstrate that whereas significant anti-tumor effects are achieved by intratumoral injection of adenovirus expressing either scIL-23 or IL-12 alone and that IL-23 requires endogenous IL-12 for maximum anti-tumor benefit, the combined use of these cytokines provides no additive or synergistic effect.
doi:10.1038/cgt.2011.78
PMCID: PMC3508719  PMID: 22158522
Interleukin 23; Interleukin 12; adenovirus; cancer; gene therapy
7.  Exosomes from IDO+ DC are therapeutic in CIA and DTH disease models 
Arthritis and rheumatism  2009;60(2):380-389.
Objective
We have demonstrated previously that dendritic cells (DC), modified with immunosuppressive cytokines, and exosomes derived from the DC can suppress the onset of murine CIA and reduce the severity of established arthritis. Indoleamine 2,3-dioxygenase (IDO) is a tryptophan degrading enzyme important for immune regulation and tolerance maintenance. DC expressing functional IDO can inhibit T cells by either depleting them of essential tryptophan and/or by producing toxic metabolites, as well as by generating regulatory T cells. In this study, we examined the immunosuppressive effects of bone marrow derived DC, genetically modified to express IDO, and IDO+-DC-derived exosomes.
Methods
Bone marrow derived DC were adenovirally transduced with IDO or CTLA4-Ig (an inducer of IDO), and the resulting DC and exosomes were tested for their immunosuppressive ability in the collagen-induced arthritis and delayed type hypersensitivity murine models.
Results
We demonstrate that both DC and exosomes derived from DC overexpressing IDO are anti-inflammatory in collagen-induced arthritis and delayed type hypersensitivity murine models. The suppressive effects were partially dependent on B7 costimulatory molecules. In addition, gene transfer of CTLA4-Ig to DC resulted in induction of IDO in the DC and exosomes able to reduce inflammation in an IDO-dependent manner.
Conclusion
These results demonstrate that both IDO expressing DC and DC-derived exosomes are immunosuppressive and anti-inflammatory, and are able to reverse established arthritis. Therefore, exosomes from IDO+ DC may represent a novel therapy for rheumatoid arthritis.
doi:10.1002/art.24229
PMCID: PMC3491653  PMID: 19180475
Dendritic cells; Exosomes; IDO; Arthritis; Inflammatory disease
8.  Pharmacologic IKK/NF-κB inhibition causes antigen presenting cells to undergo TNFα dependent ROS-mediated programmed cell death 
Scientific Reports  2014;4:3631.
Monocyte-derived antigen presenting cells (APC) are central mediators of the innate and adaptive immune response in inflammatory diseases. As such, APC are appropriate targets for therapeutic intervention to ameliorate certain diseases. APC differentiation, activation and functions are regulated by the NF-κB family of transcription factors. Herein, we examined the effect of NF-κB inhibition, via suppression of the IκB Kinase (IKK) complex, on APC function. Murine bone marrow-derived macrophages and dendritic cells (DC), as well as macrophage and DC lines, underwent rapid programmed cell death (PCD) after treatment with several IKK/NF-κB inhibitors through a TNFα-dependent mechanism. PCD was induced proximally by reactive oxygen species (ROS) formation, which causes a loss of mitochondrial membrane potential and activation of a caspase signaling cascade. NF-κB-inhibition-induced PCD of APC may be a key mechanism through which therapeutic targeting of NF-κB reduces inflammatory pathologies.
doi:10.1038/srep03631
PMCID: PMC3887388  PMID: 24406986
9.  Genotoxic stress accelerates age-associated degenerative changes in intervertebral discs 
Intervertebral disc degeneration (IDD) is the leading cause of debilitating spinal disorders such as chronic lower back pain. Aging is the greatest risk factor for IDD. Previously, we demonstrated IDD in a murine model of a progeroid syndrome caused by reduced expression of a key DNA repair enzyme. This led us to hypothesize that DNA damage promotes IDD. To test our hypothesis, we chronically exposed adult wild-type (Wt) and DNA repair-deficient Ercc1−/Δ mice to the cancer therapeutic agent mechlorethamine (MEC) or ionization radiation (IR) to induce DNA damage and measured the impact on disc structure. Proteoglycan, a major structural matrix constituent of the disc, was reduced 3-5x in the discs of MEC- and IR-exposed animals compared to untreated controls. Expression of the protease ADAMTS4 and aggrecan proteolytic fragments were significantly increased. Additionally, new PG synthesis was reduced 2-3x in MEC- and IR-treated discs compared to untreated controls. Both cellular senescence and apoptosis were increased in discs of treated animals. The effects were more severe in the DNA repair-deficient Ercc1−/Δ mice than in Wt littermates. Local irradiation of the vertebra in Wt mice elicited a similar reduction in PG. These data demonstrate that genotoxic stress drives degenerative changes associated with IDD.
doi:10.1016/j.mad.2012.11.002
PMCID: PMC3558562  PMID: 23262094
Intervertebral disc; aging; DNA damage; genotoxic stress; matrix proteoglycan
10.  NF-κB inhibition delays DNA damage–induced senescence and aging in mice 
The Journal of Clinical Investigation  2012;122(7):2601-2612.
The accumulation of cellular damage, including DNA damage, is thought to contribute to aging-related degenerative changes, but how damage drives aging is unknown. XFE progeroid syndrome is a disease of accelerated aging caused by a defect in DNA repair. NF-κB, a transcription factor activated by cellular damage and stress, has increased activity with aging and aging-related chronic diseases. To determine whether NF-κB drives aging in response to the accumulation of spontaneous, endogenous DNA damage, we measured the activation of NF-κB in WT and progeroid model mice. As both WT and progeroid mice aged, NF-κB was activated stochastically in a variety of cell types. Genetic depletion of one allele of the p65 subunit of NF-κB or treatment with a pharmacological inhibitor of the NF-κB–activating kinase, IKK, delayed the age-related symptoms and pathologies of progeroid mice. Additionally, inhibition of NF-κB reduced oxidative DNA damage and stress and delayed cellular senescence. These results indicate that the mechanism by which DNA damage drives aging is due in part to NF-κB activation. IKK/NF-κB inhibitors are sufficient to attenuate this damage and could provide clinical benefit for degenerative changes associated with accelerated aging disorders and normal aging.
doi:10.1172/JCI45785
PMCID: PMC3386805  PMID: 22706308
11.  Immunosuppressive Exosomes: A New Approach for Treating Arthritis 
Rheumatoid arthritis (RA) is a chronic autoimmune disease and one of the leading causes of disability in the USA. Although certain biological therapies, including protein and antibodies targeting inflammatory factors such as the tumor necrosis factor, are effective in reducing symptoms of RA, these treatments do not reverse disease. Also, although novel gene therapy approaches have shown promise in preclinical and clinical studies to treat RA, it is still unclear whether gene therapy can be readily and safely applied to treat the large number of RA patients. Recently, nanosized, endocytic-derived membrane vesicles “exosomes” were demonstrated to function in cell-to-cell communication and to possess potent immunoregulatory properties. In particular, immunosuppressive DC-derived exosomes and blood plasma- or serum-derived exosomes have shown potent therapeutic effects in animal models of inflammatory and autoimmune disease including RA. This paper discusses the current knowledge on the production, efficacy, mechanism of action, and potential therapeutic use of immunosuppressive exosomes for arthritis therapy.
doi:10.1155/2012/573528
PMCID: PMC3324137  PMID: 22548070
12.  ISSLS PRIZE WINNER: INHIBITION OF NF-κB ACTIVITY AMELIORATES AGE-ASSOCIATED DISC DEGENERATION IN A MOUSE MODEL OF ACCELERATED AGING 
Spine  2012;37(21):1819-1825.
Study Design
NF-κB activity was pharmacologically and genetically blocked in an accelerated aging mouse model to mitigate age-related disc degenerative changes.
Objective
To study the mediatory role of NF-κB signaling pathway in age-dependent intervertebral disc degeneration.
Summary of Background Data
Aging is a major contributor to intervertebral disc degeneration (IDD), but the molecular mechanism behind this process is poorly understood. NF-κB is a family of transcription factors which play a central role in mediating cellular response to damage, stress, and inflammation. Growing evidence implicates chronic NF-κB activation as a culprit in many aging-related diseases, but its role in aging-related IDD has not been adequately explored. We studied the effects of NF-κB inhibition on IDD using a DNA repair-deficient mouse model of accelerated aging (Ercc1-/Δ mice) previously been reported to exhibit age-related IDD.
Methods
Systemic inhibition of NF-κB activation was achieved either genetically by deletion of one allele of the NF-κB subunit p65 (Ercc1-/Δp65+/- mice) or pharmacologically by chronic intra-peritoneal administration of the Nemo Binding Domain (8K-NBD) peptide to block the formation of the upstream activator of NF-κB, IκB Inducible Kinase (IKK), in Ercc1-/Δ mice. Disc cellularity, total proteoglycan content and proteoglycan synthesis of treated mice and untreated controls were assessed.
Results
Decreased disc matrix proteoglycan content, a hallmark feature of IDD, and elevated disc NF-κB activity were observed in discs of progeroid Ercc1-/Δ mice and naturally aged wild-type compared to young WT mice. Systemic inhibition of NF-κB by the 8K-NBD peptide in Ercc1-/Δ mice increased disc proteoglycan synthesis and ameriolated loss disc cellularity and matrix proteoglycan. These results were confirmed genetically by using the p65 haploinsufficient Ercc1-/Δp65+/- mice.
Conclusion
These findings demonstrate that the IKK/NF-κB signaling pathway is a key mediator of age-dependent IDD and represents a therapeutic target for mitigating disc degenerative diseases associated with aging.
doi:10.1097/BRS.0b013e31824ee8f7
PMCID: PMC3395770  PMID: 22343279
NF-kB;aging; proteoglycan; disc degeneration; DNA damage repair; ERCC1-deficient mice
13.  In Vivo Expression of HGF/NK1 and GLP-1 From dsAAV Vectors Enhances Pancreatic β-Cell Proliferation and Improves Pathology in the db/db Mouse Model of Diabetes 
Diabetes  2010;59(12):3108-3116.
OBJECTIVE
The purpose of the current study was to determine whether double-stranded adeno-associated virus (dsAAV)-mediated in vivo expression of β-cell growth factors, glucagon-like peptide-1 (GLP-1) and the NK1 fragment of hepatocyte growth factor (HGF/NK1) in β-cells, improves pathology in the db/db mouse model of type 2 diabetes.
RESEARCH DESIGN AND METHODS
The glucoregulatory actions of GLP-1 and full-length HGF are well characterized. Here, we test the ability of HGF/NK1 to induce proliferation of exogenous islets and MIN6 β-cells. In addition, we target both GLP-1 and HGF/NK1 to endogenous β-cells using dsAAV vectors containing the mouse insulin-II promoter. We compare the abilities of these gene products to induce islet proliferation in vitro and in vivo and characterize their abilities to regulate diabetes after AAV-mediated delivery to endogenous islets of db/db mice.
RESULTS
Recombinant HGF/NK1 induces proliferation of isolated islets, and dsAAV-mediated expression of both GLP-1 and HGF/NK1 induces significant β-cell proliferation in vivo. Furthermore, both GLP-1 and HGF/NK1 expressed from dsAAV vectors enhance β-cell mass and insulin secretion in vivo and significantly delay the onset of hyperglycemia in db/db mice.
CONCLUSIONS
A single treatment with dsAAV vectors expressing GLP-1 or HGF/NK1 enhances islet growth and significantly improves pathology in a mouse model of type 2 diabetes. This represents the first example of a successful use of HGF/NK1 for diabetes therapy, providing support for direct AAV-mediated in vivo delivery of β-cell growth factors as a novel therapeutic strategy for the treatment of type 2 diabetes.
doi:10.2337/db09-1886
PMCID: PMC2992772  PMID: 20841608
14.  The Roles of Tumor-Derived Exosomes in Cancer Pathogenesis 
Exosomes are endosome-derived, 30–100 nm small membrane vesicles released by most cell types including tumor cells. They are enriched in a selective repertoire of proteins and nucleic acids from parental cells and are thought to be actively involved in conferring intercellular signals. Tumor-derived exosomes have been viewed as a source of tumor antigens that can be used to induce antitumor immune responses. However, tumor-derived exosomes also have been found to possess immunosuppressive properties and are able to facilitate tumor growth, metastasis, and the development of drug resistance. These different effects of tumor-derived exosomes contribute to the pathogenesis of cancer. This review will discuss the roles of tumor-derived exosomes in cancer pathogenesis, therapy, and diagnostics.
doi:10.1155/2011/842849
PMCID: PMC3235485  PMID: 22190973
15.  Isolation of Muscle-Derived Stem/Progenitor Cells based on adhesion characteristics to collagen-coated surfaces 
Summary
Our lab developed and optimized a method, known as the modified pre-plate technique, to isolate stem/progenitor cells from skeletal muscle. This method separates different populations of myogenic cells based on their propensity to adhere to collagen I-coated surface. Based on their surface markers and stem-like properties, including self-renewal, multi-lineage differentiation, and ability to promote tissue regeneration, the last cell fraction or slowest to adhere to the collagen-coated surface (pre-plate 6; pp6) appear to be early, quiescent progenitor cells termed muscle-derived stem/progenitor cells (MDSPCs). The cell fractions preceding pp6 (pp1–5) are likely populations of more committed (differentiated) cells, including fibroblast- and myoblastlike cells. This technique may be used to isolate MDSPCs from skeletal muscle of humans or mice regardless of age, sex or disease state, although the yield of MDSPCs varies with age and health. MDSPCs can be used for regeneration of a variety of tissues including bone, articular cartilage, skeletal and cardiac muscle and nerve. MDSPCs are currently being tested in clinical trials for treatment of urinary incontinence and myocardial infarction. MDSPCs from young mice have also been demonstrated to extend lifespan and healthspan in mouse models of accelerated aging through an apparent paracrine/endocrine mechanism. Here we detail methods for isolation and characterization of MDSPCs.
doi:10.1007/978-1-62703-317-6_5
PMCID: PMC3731996  PMID: 23400434
Pre-plate technique; muscle-derived stem/progenitor cells; muscle; adult stem cells; regenerative medicine
16.  The oxidative DNA lesions 8,5′-cyclopurines accumulate with aging in a tissue-specific manner 
Aging Cell  2012;11(4):714-716.
Summary
Accumulation of DNA damage is implicated in aging. This is supported by the fact that inherited defects in DNA repair can cause accelerated aging of tissues. However, clear-cut evidence for DNA damage accumulation in old age is lacking. Numerous studies report measurement of DNA damage in nuclear and mitochondrial DNA from tissues of young and old organisms, with variable outcomes. Variability results from genetic differences between specimens or the instability of some DNA lesions. To control these variables and test the hypothesis that elderly organisms have more oxidative DNA damage than young organisms, we measured 8,5′-cyclopurine-2′-deoxynucleosides (cPu), which are relatively stable, in tissues of young and old wild-type and congenic progeroid mice. We found that cPu accumulate spontaneously in the nuclear DNA of wild-type mice with age and to a greater extent in DNA repair-deficient progeroid mice, with a similar tissue-specific pattern (liver>kidney>brain). These data, generated under conditions where genetic and environmental variables are controlled, provide strong evidence that DNA repair mechanisms are inadequate to clear endogenous lesions over the lifespan of mammals. The similar, although exaggerated, results obtained from progeroid, DNA repair-deficient mice and old normal mice support the conclusion that DNA damage accumulates with, and likely contributes to aging.
doi:10.1111/j.1474-9726.2012.00828.x
PMCID: PMC3399950  PMID: 22530741
ageing; progeria; DNA damage; nucleotide excision repair; oxidative DNA lesion
17.  LIM DOMAIN PROTEIN-3 (LMP3) COOPERATES WITH BMP7 TO PROMOTE TISSUE REGENERATION BY LIGAMENT PROGENITOR CELLS 
Gene therapy  2012;20(1):1-6.
Gene transfer of key regulators of osteogenesis for mesenchymal stem cells (MSCs) represents a promising strategy to regenerate bone. It has been reported that LMP3, a transcription variant of LIM domain mineralization protein (LMP) lacking LIM domains, can induce osteogenesis in vitro and in vivo. Since little is known about the effects of LMP3 gene therapy on periodontal ligament (PDL) cell osteogenic differentiation, this study sought to explore whether gene delivery of LMP3 can promote PDL cell mineralization and bone formation. Our results showed that adenoviral mediated gene transfer of LMP3 (AdLMP3) significantly upregulated ALP, BSP, and BMP2 gene expression and increased in vitro matrix mineralization in human PDL. Although AdLMP3 gene delivery to PDL cells did not induce ectopic bone formation in vivo, we found that AdLMP3 augments new bone formation, which co-delivered with AdBMP7 gene transfer. Our study provides evidence that there is a synergistic effect between LMP3 and BMP-7 in vivo, suggesting that LMP3 delivery may be used to augment BMP-mediated osteogenesis. LMP3 and BMP-7 combinatory gene therapy may also have specific applications for oral and periodontal regenerative medicine.
doi:10.1038/gt.2011.203
PMCID: PMC3330200  PMID: 22241179
gene therapy; LIM domain mineralization protein; bone morphogenetic protein; osteogenesis; regenerative medicine
18.  B7-1/2, but not PD-L1/2 molecules, are required on IL-10-treated tolerogenic DC and DC-derived exosomes for in vivo function 
European journal of immunology  2009;39(11):3084-3090.
SUMMARY
Costimulatory molecules such as B7-1/2 and PD-L1/2 play an important role in the function of APC. The regulation of the surface levels of costimulatory molecules is one mechanism by which APC maintain the balance between tolerance and immunity. We examined the contributions of B7-1/2 and PD-L1/2 to the function of IL-10-treated, immunosuppressive DC as well as therapeutic exosomes derived from these DC. IL-10 treatment of DC significantly downregulated surface expression of MHC II, B7-1, B7-2, and decreased levels of MHC I and PD-L2. IL-10 treatment of DC resulted in a modified co-stimulatory profile of DC-secreted exosomes with a reduction in B7-1, PD-L1 and PD-L2. We further demonstrate that absence of B7-1 or B7-2 on donor DC results in a loss of ability of IL-10 treated DC and their exosomes to suppress the delayed-type hypersensitivity (DTH) response, whereas IL-10 treated DC deficient in PD-L1/2 as well as their secreted exosomes retained the ability to suppress DTH responses. We conclude that B7-1 and B7-2, but not PD-L1 and PD-L2, on IL-10 treated DC and DC-derived exosomes play a critical role in immunosuppressive functions of both DC and exosomes.
doi:10.1002/eji.200939407
PMCID: PMC3075967  PMID: 19757438
Dendritic cells; Exosomes; B7-1/2; PD-L1/2; Delayed-type hypersensitivity
19.  Synovial fibroblasts spread rheumatoid arthritis to unaffected joints 
Nature medicine  2009;15(12):1414-1420.
Active rheumatoid arthritis is characterized by originating from few but affecting subsequently the majority of joints. Thus far, the pathways of the progression of the disease are largely unknown. As rheumatoid arthritis synovial fibroblasts (RASFs) are key players in joint destruction and migrate in vitro, the current study evaluated the potential of RASFs to spread the disease in vivo. To simulate the primary joint of origin, healthy human cartilage was co-implanted subcutaneously into SCID mice together with RASFs. At the contralateral flank, healthy cartilage was implanted without cells. RASFs showed an active movement to the naïve cartilage via the vasculature independent of the site of application of RASFs into the SCID mouse, leading to a strong destruction of the target cartilage. These findings support the hypothesis that the characteristic clinical phenomenon of destructive arthritis spreading between joints is mediated, at least in part, by the transmigration of activated RASFs.
doi:10.1038/nm.2050
PMCID: PMC3678354  PMID: 19898488
20.  Regenerating the injured kidney with human umbilical cord mesenchymal stem cell-derived exosomes 
Transplantation of adult stem cells is being used to facilitate repair or regeneration of damaged or diseased tissues. However, in many cases, the therapeutic effects of the injected stem cells are mediated by factors secreted by stem cells and not by differentiation of the transplanted stem cells. Recent reports have identified a class of microvesicles, termed exosomes, released by stem cells that are able to confer therapeutic effects on injured renal and cardiac tissue. In this issue of Stem Cell Research & Therapy, Zhou and colleagues demonstrate the ability of exosomes derived from human umbilical cord mesenchymal stem cells (hucMSCs), but not non-stem cell-derived exosomes, to improve acute kidney injury induced by cisplatin in rats. The authors demonstrate that hucMSC exosomes can reduce cisplatin-mediated renal oxidative stress and apoptosis in vivo and increase renal epithelial cell proliferation in culture. These results suggest that stem cell-derived exosomes, which are easy to isolate and safer to use than the parental stem cells, could have significant clinical utility.
doi:10.1186/scrt187
PMCID: PMC3706756  PMID: 23680102
21.  Adenoviral Mediated, Intra-Tumor Gene Transfer of Interleukin 23 Induces a Therapeutic Anti-tumor Response 
Cancer gene therapy  2009;16(10):776-785.
IL-23 is a member of the IL-12 family of heterodimeric cytokines, comprised of p19 and p40 subunits, which exhibits immunostimulatory properties similar to IL-12. IL-23 has been shown to possess potent anti-tumor activities in several establishment models of cancer and a few therapeutic models, but the efficacy of local, adenoviral-mediated expression of IL-23 in established tumors has yet to be investigated. Here we have examined the anti-tumor activity of adenovirally-delivered IL-23 in a day 7 MCA205 murine fibrosarcoma tumor model. Three intratumoral injections of adenovirus expressing IL-23 (Ad.IL-23) significantly increased animal survival and resulted in complete rejection of 40 percent of tumors, with subsequent generation of protective immunity and MCA205-specific cytotoxic T-lymphocytes (CTLs). Additionally, we have shown that the anti-tumor activity of IL-23 is independent of IL-17, perforin and Fas ligand, but dependent on IFN-γ, CD4 and CD8 positive T-cells. These results demonstrate that direct intratumoral injection of adenovirus expressing IL-23 results in enhanced survival, tumor eradication and generation of protective immunity by generation of a Th1-type immune response.
doi:10.1038/cgt.2009.27
PMCID: PMC2745505  PMID: 19390568
Interleukin 23; adenovirus; cancer; gene therapy
22.  Gene therapy of the rheumatic diseases: 1998 to 2008 
During the decade since the launch of Arthritis Research, the application of gene therapy to the rheumatic diseases has experienced the same vicissitudes as the field of gene therapy as a whole. There have been conceptual and technological advances and an increase in the number of clinical trials. However, funding has been unreliable and a small number of high-profile deaths in human trials, including one in an arthritis gene therapy trial, have provided ammunition to skeptics. Nevertheless, steady progress has been made in a number of applications, including rheumatoid arthritis and osteoarthritis, Sjögren syndrome, and lupus. Clinical trials in rheumatoid arthritis have progressed to phase II and have provided the first glimpses of possible efficacy. Two phase I protocols for osteoarthritis are under way. Proof of principle has been demonstrated in animal models of Sjögren syndrome and lupus. For certain indications, the major technological barriers to the development of genetic therapies seem to have been largely overcome. The translational research necessary to turn these advances into effective genetic medicines requires sustained funding and continuity of effort.
doi:10.1186/ar2563
PMCID: PMC2688220  PMID: 19232068
23.  Targeting of XJB-5-131 to mitochondria suppresses oxidative DNA damage and motor decline in a mouse model of Huntington’s disease 
Cell reports  2012;2(5):1137-1142.
SUMMARY
Oxidative damage and mitochondrial dysfunction are implicated in aging and age-related neurodegenerative diseases, including Huntington’s disease (HD). Many naturally occurring antioxidants have been tested to correct for deleterious effects of reactive oxygen species, but often they lack specificity, are tissue variable, and the efficacy is marginal in human clinical trials. To increase specificity and efficacy, we have designed a synthetic antioxidant, XJB-5-131, to target mitochondria. We demonstrate in a mouse model of HD that XJB-5-131 has remarkably beneficial effects. XJB-5-131 reduces oxidative damage to mitochondrial DNA, maintains mitochondrial DNA copy number, suppresses motor decline and weight loss, enhances neuronal survival, and improves mitochondrial function. The findings poise XJB-5-131 as a promising therapeutic compound.
doi:10.1016/j.celrep.2012.10.001
PMCID: PMC3513647  PMID: 23122961
24.  Safety Assessment of Intradiscal Gene Therapy II: Effect of Dosing and Vector Choice 
Spine  2008;33(14):1509-1517.
Study Design
Clinical, biochemical, and histological analysis was performed following in vivo delivery of cDNA encoding various anabolic cytokines and marker genes to the lumbar epidural space of New Zealand white rabbits, using both adenoviral and adeno-associated viral vectors.
Objective
To mimic errant or misplaced doses of gene therapy in order to better ascertain the potential risks associated with alternative vectors and transgene products with regard to their application to problems of the intervertebral disc.
Summary of Background Data
Work done with several anabolic cytokines including bone morphogenic proteins and transforming growth factors, has demonstrated the potential of gene therapy. Recently, data has been published demonstrating that improperly dosed or delivered adenoviral-mediated gene therapy within the subarachnoid space can result in significant morbidity in rabbits. There are currently no studies examining the effect of these errors within the epidural space or using an adeno-associated viral (AAV) vector.
Methods
Using either adenoviral or adeno-associated viral vectors, complementary DNA (cDNA) encoding anabolic cytokines bone morphogenic protein-2 (BMP-2) and transforming growth factor-beta 1 (TGF-β1) and marker proteins LacZ and green fluorescent protein (GFP) were injected into the epidural space of 37 New Zealand white rabbits at the L5/6 level. Rabbits were then observed clinically for up to six weeks, after which the rabbits were sacrificed in order to perform a comprehensive biochemical and histological analysis.
Results
Following adenoviral-mediated delivery of anabolic cytokine cDNA, up to eighty percent of rabbits suffered significant clinical, biochemical, and histological morbidity. Conversely, AAV-mediated delivery of any cDNA and adenoviral-mediated delivery of marker protein cDNA resulted in no clinical, histological, or biochemical morbidity.
Conclusion
Properly dosed and directed gene therapy seems to be both safe and potentially efficacious. This study suggests that side effects of gene therapy may be due to a combination of dosing, transgene product, and vector choice, and that newer AAV vectors may reduce these side-effects and decrease the risk of this technology.
doi:10.1097/BRS.0b013e318178866c
PMCID: PMC3510668  PMID: 18520636
25.  Biologic Response of Human Intervertebral Disc Cells to Gene Therapy Cocktail 
Spine  2008;33(17):1850-1855.
Study Design
In vitro experiment using human intervertebral disc (IVD) cells and adenovirus-therapeutic gene constructs.
Objectives
To examine the biologic effect of “cocktail” therapeutic gene transfer to human IVD cells in three dimensional cultures.
Summary of Background Data
. Gene therapy is regarded as a potential option for the treatment of degenerative disc disease. Although various anabolic genes have previously been introduced for this purpose, cocktail gene transfer of anabolic genes to IVD cells has never been attempted.
Materials and Methods
Human IVDs were harvested during surgical disc procedures and cultured. We prepared recombinant adenovirus constructs bearing the TGF-β1 gene (Ad/TGF-β1), the IGF-1 gene (Ad/IGF-1), and the BMP-2 gene (Ad/BMP-2). Transgene expression was detected by luciferase assays, enzyme linked immuno-sorbent assays, and Western blot analysis. Newly synthesized proteoglycan was measured by 35S-sulfate incorporation on Sephadex G-25M in PD 10 columns. Human IVD cells were transduced by single, double, and triple combination of Ad/TGF-β1, Ad/IGF-1, Ad/BMP-2 with an MOI of 75, then cultured three-dimensionally in alginate beads.
Results
Transgene expression was detected at 18 hours after viral transduction. IVD cultures with Ad/TGF-β1, Ad/IGF-1, Ad/BMP-2 (MOI of 75) showed 2.9, 1.8, and 1.9 fold increases, respectively, in proteoglycan synthesis compared to control. Human IVD cultures with double gene combination (MOI of 75) showed 3.2 to 3.9 fold increases of proteoglycan synthesis. Lastly, Human IVD cultures with triple gene combination (TGF-β1+IGF-1+BMP-2 genes with an MOI of 75) transfer demonstrated 4.7 fold increase in proteoglycan synthesis compared control.
Conclusion
Combination or “cocktail” gene therapy offers a promising mechanism for maximizing matrix synthesis with low dose of adenoviral mixtures, circumventing systemic, local toxic effect, and immune response.
doi:10.1097/BRS.0b013e31817e1cd7
PMCID: PMC3508786  PMID: 18622355
Gene Therapy; Intervertebral Disc; Proteoglycan

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