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1.
Arthritis Res Ther ; 18: 166, 2016 07 13.
Artigo em Inglês | MEDLINE | ID: mdl-27412524

RESUMO

BACKGROUND: Joint surface injury, a known risk factor for osteoarthritis, triggers synovial hyperplasia, which involves proliferation of mesenchymal stromal/stem cells (MSCs). Whether these proliferative MSCs are resident synovial cells or move into the tissue from elsewhere is not known. The aim of this study was to determine the contribution of bone marrow-derived cells to synovial hyperplasia following joint surface injury. METHODS: Lethally irradiated mice were transplanted with green fluorescent protein (GFP)-labelled bone marrow, and MSC chimerism was determined by the colony-forming unit fibroblast (CFU-F) assay and phenotypic analysis. To label host slow-cycling cells prior to bone marrow transplant, mice received iododeoxyuridine for 3 weeks. Mice then were subjected to GFP(+) bone marrow transplant, underwent joint surface injury and received chlorodeoxyuridine (CldU) for 7 days to label cells proliferating after injury. GFP- and nucleoside-labelled cells in normal and injured knee joint synovium were quantified in situ by immunofluorescence staining of paraffin-embedded tissue sections. The phenotype of GFP-labelled cells was determined by co-staining for the haematopoietic marker CD16/CD32 and the MSC/fibroblast marker platelet-derived growth factor receptor α (Pdgfrα). RESULTS: CFU-F assay and phenotypic analysis demonstrated successful bone marrow mesenchymal lineage chimerism in mice that underwent transplants. Bone marrow reconstitution preceded the detection of GFP-labelled cells in synovium. The percentage of GFP(+) cells in synovium increased significantly in response to injury, while the proportion of GFP(+) cells that were labelled with the proliferation marker CldU did not increase, suggesting that the expansion of the GFP(+) cell population in synovium was due mainly to bone marrow cell infiltration. In contrast, proliferation of host slow-cycling cells was significantly increased in the hyperplastic synovium. In both control and injured knee joints, the majority of marrow-derived GFP(+) cells in the synovium were haematopoietic (CD16/32(+)), while a minority of cells expressed the pan-fibroblast/MSC marker Pdgfrα. CONCLUSIONS: Our findings indicate that synovial hyperplasia following joint surface injury involves proliferation of resident slow-cycling cells, with a contribution from infiltrating bone marrow-derived cells. Understanding the process of synovial hyperplasia may reveal ways to restore homeostasis in injured joints and prevent secondary osteoarthritis.


Assuntos
Artrite Experimental/patologia , Articulação do Joelho/patologia , Células-Tronco Mesenquimais , Osteoartrite/patologia , Membrana Sinovial/patologia , Animais , Células da Medula Óssea , Transplante de Medula Óssea , Citometria de Fluxo , Imunofluorescência , Hiperplasia/patologia , Traumatismos do Joelho/complicações , Camundongos , Microscopia Confocal
2.
Mol Ther ; 21(10): 1938-49, 2013 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-23748415

RESUMO

Mucopolysaccharidosis type IIIA (MPSIIIA) is a lysosomal storage disorder caused by mutations in N-sulfoglucosamine sulfohydrolase (SGSH), resulting in heparan sulfate (HS) accumulation and progressive neurodegeneration. There are no treatments. We previously demonstrated improved neuropathology in MPSIIIA mice using lentiviral vectors (LVs) overexpressing SGSH in wild-type (WT) hematopoietic stem cell (HSC) transplants (HSCTs), achieved via donor monocyte/microglial engraftment in the brain. However, neurological disease was not corrected using LVs in autologous MPSIIIA HSCTs. To improve brain expression via monocyte/microglial specificity, LVs expressing enhanced green fluorescent protein (eGFP) under ubiquitous phosphoglycerate kinase (PGK) or myeloid-specific promoters were compared in transplanted HSCs. LV-CD11b-GFP gave significantly higher monocyte/B-cell eGFP expression than LV-PGK-GFP or LV-CD18-GFP after 6 months. Subsequently, autologous MPSIIIA HSCs were transduced with either LV-PGK-coSGSH or LV-CD11b-coSGSH vectors expressing codon-optimized SGSH and transplanted into MPSIIIA mice. Eight months after HSCT, LV-PGK-coSGSH vectors produced bone marrow SGSH (576% normal activity) similar to LV-CD11b-coSGSH (473%), but LV-CD11b-coSGSH had significantly higher brain expression (11 versus 7%), demonstrating improved brain specificity. LV-CD11b-coSGSH normalized MPSIIIA behavior, brain HS, GM2 ganglioside, and neuroinflammation to WT levels, whereas LV-PGK-coSGSH partly corrected neuropathology but not behavior. We demonstrate compelling evidence of neurological disease correction using autologous myeloid driven lentiviral-HSC gene therapy in MPSIIIA mice.


Assuntos
Terapia Genética/métodos , Transplante de Células-Tronco Hematopoéticas , Células-Tronco Hematopoéticas/fisiologia , Hidrolases/genética , Hidrolases/metabolismo , Microglia/fisiologia , Mucopolissacaridose III/terapia , Animais , Encéfalo/enzimologia , Antígeno CD11b/genética , Linhagem Celular , Modelos Animais de Doenças , Feminino , Vetores Genéticos , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Heparitina Sulfato/metabolismo , Humanos , Lentivirus/genética , Leucócitos/metabolismo , Lisossomos/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Microglia/enzimologia , Mucopolissacaridose III/metabolismo , Mucopolissacaridose III/patologia , Células Mieloides/enzimologia , Células Mieloides/fisiologia , Especificidade de Órgãos , Regiões Promotoras Genéticas
3.
Mol Ther ; 21(4): 868-76, 2013 04.
Artigo em Inglês | MEDLINE | ID: mdl-23423338

RESUMO

Hematopoietic stem cell gene therapy for neurological disorders relies on transmigration of donor-derived monocytes to the brain, where they can engraft as microglia and deliver therapeutic proteins. Many mouse studies use whole-body irradiation to investigate brain transmigration pathways, but chemotherapy is generally used clinically. The current evidence for transmigration to the brain after chemotherapy is conflicting. We compared hematopoietic donor cell brain engraftment after bone marrow (BM) transplants in busulfan- or irradiation-conditioned mice. Significantly more donor-derived microglial cells engrafted posttransplant in busulfan-conditioned brain compared with the irradiated, in both the short and long term. Although total Iba-1(+) microglial content was increased in irradiated brain in the short term, it was similar between groups over long-term engraftment. MCP-1, a key regulator of monocyte transmigration, showed long-term elevation in busulfan-conditioned brain, whereas irradiated brains showed long-term elevation of the proinflammatory chemokine interleukin 1α (IL-1α), with increased in situ proliferation of resident microglia, and significant increases in the relative number of amoeboid activated microglia in the brain. This has implications for the choice of conditioning regimen to promote hematopoietic cell brain engraftment and the relevance of irradiation in mouse models of transplantation.


Assuntos
Encéfalo/efeitos dos fármacos , Encéfalo/efeitos da radiação , Bussulfano/uso terapêutico , Células-Tronco Hematopoéticas/citologia , Animais , Transplante de Medula Óssea , Encéfalo/citologia , Encéfalo/metabolismo , Quimiocina CCL2/metabolismo , Interleucina-1/metabolismo , Camundongos , Condicionamento Pré-Transplante , Irradiação Corporal Total
4.
Mol Ther ; 20(8): 1610-21, 2012 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-22547151

RESUMO

Mucopolysaccharidosis IIIA (MPS IIIA or Sanfilippo disease) is a neurodegenerative disorder caused by a deficiency in the lysosomal enzyme sulfamidase (SGSH), catabolizing heparan sulfate (HS). Affected children present with severe behavioral abnormalities, sleep disturbances, and progressive neurodegeneration, leading to death in their second decade. MPS I, a similar neurodegenerative disease accumulating HS, is treated successfully with hematopoietic stem cell transplantation (HSCT) but this treatment is ineffectual for MPS IIIA. We compared HSCT in MPS IIIA mice using wild-type donor cells transduced ex vivo with lentiviral vector-expressing SGSH (LV-WT-HSCT) versus wild-type donor cell transplant (WT-HSCT) or lentiviral-SGSH transduced MPS IIIA cells (LV-IIIA-HSCT). LV-WT-HSCT results in 10% of normal brain enzyme activity, near normalization of brain HS and GM2 gangliosides, significant improvements in neuroinflammation and behavioral correction. Both WT-HSCT and LV-IIIA-HSCT mediated improvements in GM2 gangliosides and neuroinflammation but were less effective at reducing HS or in ameliorating abnormal HS sulfation and had no significant effect on behavior. This suggests that HS may have a more significant role in neuropathology than neuroinflammation or GM2 gangliosides. These data provide compelling evidence for the efficacy of gene therapy in conjunction with WT-HSCT for neurological correction of MPS IIIA where conventional transplant is ineffectual.


Assuntos
Terapia Genética/métodos , Células-Tronco Hematopoéticas/fisiologia , Mucopolissacaridoses/patologia , Mucopolissacaridoses/terapia , Animais , Feminino , Citometria de Fluxo , Células-Tronco Hematopoéticas/citologia , Imuno-Histoquímica , Camundongos
5.
Pharm Res ; 28(11): 2863-70, 2011 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-21739320

RESUMO

PURPOSE: Development of a human mitochondrial gene delivery vector is a critical step in the ability to treat diseases arising from mutations in mitochondrial DNA. Although we have previously cloned the mouse mitochondrial genome in its entirety and developed it as a mitochondrial gene therapy vector, the human mitochondrial genome has been dubbed unclonable in E. coli, due to regions of instability in the D-loop and tRNA(Thr) gene. METHODS: We tested multi- and single-copy vector systems for cloning human mitochondrial DNA in E. coli and Saccharomyces cerevisiae, including transformation-associated recombination. RESULTS: Human mitochondrial DNA is unclonable in E. coli and cannot be retained in multi- or single-copy vectors under any conditions. It was, however, possible to clone and stably maintain the entire human mitochondrial genome in yeast as long as a single-copy centromeric plasmid was used. D-loop and tRNA(Thr) were both stable and unmutated. CONCLUSIONS: This is the first report of cloning the entire human mitochondrial genome and the first step in developing a gene delivery vehicle for human mitochondrial gene therapy.


Assuntos
DNA Mitocondrial/genética , Sistemas de Liberação de Medicamentos , Terapia Genética/métodos , Vetores Genéticos , Genoma Humano , Sequência de Bases , Células Clonais , Composição de Medicamentos , Escherichia coli/genética , Genoma Mitocondrial , Humanos , Mitocôndrias/genética , Dados de Sequência Molecular , Terapia de Alvo Molecular , Plasmídeos , Recombinação Genética , Saccharomyces cerevisiae/genética , Análise de Sequência de DNA , Leveduras/genética
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