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1.
Molecules ; 25(16)2020 Aug 06.
Artículo en Inglés | MEDLINE | ID: mdl-32781588

RESUMEN

Bone regeneration for replacing and repairing damaged and defective bones in the human body has attracted much attention over the last decade. In this research, highly porous polyetheretherketone (PEEK)/hydroxyapatite (HA) bionanocomposite scaffolds reinforced with carbon fiber (CF) and carbon nanotubes (CNTs) were fabricated, and their structural, mechanical, and biological properties were studied in detail. Salt porogen (200-500 µm size) leaching methods were adapted to produce porous PEEK structures with controlled pore size and distribution, facilitating greater cellular infiltration and biological integration of PEEK composites within patient tissue. In biological tests, nanocomposites proved to be non-toxic and have very good cell viability. In addition, bone marrow cell growth was observed, and PEEK/HA biocomposites with carbon particles showed increased cell attachment over the neat PEEK/HA composites. In cell viability tests, bionanocomposites with 0.5 wt% CNTs established good attachment of cells on disks compared to neat PEEK/HA biocomposites. A similar performance was seen in culture tests of bone marrow cells (osteoblasts and osteoclasts). The 0.5 wt% CF for osteoblasts and 1 wt% CNTs for osteoclasts showed higher cell attachment. The addition of carbon-based nanomaterials into PEEK/HA has been identified as an effective approach to improve cell attachment as well as mechanical and biological properties. With confirmed cell attachment and sustained viability and proliferation of the fabricated PEEK/HA/CNTs, CF bionanocomposites were confirmed to possess excellent biocompatibility and will have potential uses in bone scaffolding and other biomedical applications.


Asunto(s)
Durapatita/química , Cetonas/química , Cetonas/farmacología , Nanocompuestos/química , Nanopartículas/química , Nanotubos de Carbono/química , Polietilenglicoles/química , Polietilenglicoles/farmacología , Benzofenonas , Adhesión Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Fenómenos Mecánicos , Osteoclastos/citología , Osteoclastos/efectos de los fármacos , Polímeros , Porosidad
2.
Clin Exp Immunol ; 179(2): 210-9, 2015 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-25228326

RESUMEN

Summary Recombinant human binding immunoglobulin protein (BiP) has previously demonstrated anti-inflammatory properties in multiple models of inflammatory arthritis. We investigated whether these immunoregulatory properties could be exploited using gene therapy techniques. A single intraperitoneal injection of lentiviral vector containing the murine BiP (Lenti-mBiP) or green fluorescent protein (Lenti-GFP) transgene was administered in low- or high-dose studies during early arthritis. Disease activity was assessed by visual scoring, histology, serum cytokine and antibody production measured by cell enzyme-linked immunosorbent assay (ELISA) and ELISA, respectively. Lentiviral vector treatment caused significant induction of interferon (IFN)-γ responses regardless of the transgene; however, further specific effects were directly attributable to the BiP transgene. In both studies Lenti-mBiP suppressed clinical arthritis significantly. Histological examination showed that low-dose Lenti-mBiP suppressed inflammatory cell infiltration, cartilage destruction and significantly reduced pathogenic anti-type II collagen (CII) antibodies. Lenti-mBiP treatment caused significant up-regulation of soluble cytotoxic T lymphocyte antigen-4 (sCTLA-4) serum levels and down-regulation of interleukin (IL)-17A production in response to CII cell restimulation. In-vitro studies confirmed that Lenti-mBiP spleen cells could significantly suppress the release of IL-17A from CII primed responder cells following CII restimulation in vitro, and this suppression was associated with increased IL-10 production. Neutralization of CTLA-4 in further co-culture experiments demonstrated inverse regulation of IL-17A production. In conclusion, these data demonstrate proof of principle for the therapeutic potential of systemic lentiviral vector delivery of the BiP transgene leading to immunoregulation of arthritis by induction of soluble CTLA-4 and suppression of IL-17A production.


Asunto(s)
Artritis Experimental/prevención & control , Terapia Genética , Vectores Genéticos , Proteínas de Choque Térmico/inmunología , Lentivirus , Transducción Genética , Animales , Artritis Experimental/genética , Artritis Experimental/inmunología , Artritis Experimental/patología , Antígeno CTLA-4/genética , Antígeno CTLA-4/inmunología , Progresión de la Enfermedad , Chaperón BiP del Retículo Endoplásmico , Proteínas de Choque Térmico/genética , Humanos , Interferón gamma/genética , Interferón gamma/inmunología , Interleucina-10/genética , Interleucina-10/inmunología , Interleucina-17/genética , Interleucina-17/inmunología , Ratones , Transgenes/inmunología
3.
Gene Ther ; 20(2): 128-35, 2013 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-22318091

RESUMEN

This study investigated the efficacy of a combination gene therapy to repress interleukin-1 (IL-1) and receptor activator of nuclear factor NF-kappa B ligand (RANKL) for the treatment of particulate debris-induced aseptic loosening, and tried to explore the molecular mechanism of the exogenous gene modifications on osteoclastogenesis. RAW cells activated by titanium particles were transduced with DFG-IL-1Ra (retroviral vector encoding IL-1 receptor antagonist) and AAV-OPG (adeno-associated viral vectors-osteoprotegerin) individually or in combination for 4 weeks. Pro-inflammatory cytokines in culture media were determined by enzyme-linked immunosorbent assay, and gene expressions of RANK, IL-1ß, c-Fos, TRAF6, JNK1 and CPK were examined using real-time PCR. An established knee-implant-failure mouse model was employed to evaluate the efficacy of the in vivo double-gene therapy. The surgical implantation of a titanium alloy pin into the proximal tibia was followed by monthly challenge with titanium debris. Peri-implant gene transfers of IL-1Ra and OPG (respectively or in combination) were given 3 weeks after surgery. The combination of OPG and IL-1Ra gene transfer exhibited strong synergetic effects in blockage of inflammation and osteoclastogenesis at 8 weeks after gene modification. The combination therapy reversed peri-implant bone resorption and restored implant stability when compared with either single gene transduction. Real-time PCR data indicated that the action of IL-1Ra gene therapy may be mediated via the JNK1 pathway, while the reduction of osteoclastogenesis by OPG gene modification may be regulated by c-Fos expression. In addition, both gene modifications resulted in significant diminishment of TRAF6 expression.


Asunto(s)
Terapia Genética , Inflamación/terapia , Interleucina-1beta/antagonistas & inhibidores , Falla de Prótesis , Ligando RANK/antagonistas & inhibidores , Animales , Regeneración Ósea/genética , Resorción Ósea/terapia , Diferenciación Celular , Línea Celular Tumoral , Implantes Experimentales , Proteína Antagonista del Receptor de Interleucina 1/genética , Prótesis de la Rodilla , Ratones , Ratones Endogámicos BALB C , Osteoclastos/metabolismo , Osteoclastos/patología , Osteoprotegerina/genética , Titanio/administración & dosificación
4.
Gene Ther ; 17(10): 1262-9, 2010 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-20428210

RESUMEN

Exogenous osteoprotegerin (OPG) gene modification appears a therapeutic strategy for osteolytic aseptic loosening. The feasibility and efficacy of a cell-based OPG gene delivery approach were investigated using a murine model of knee prosthesis failure. A titanium pin was implanted into mouse proximal tibia to mimic a weight-bearing knee arthroplasty, followed by titanium particles challenge to induce periprosthetic osteolysis. Mouse fibroblast-like synoviocytes were transduced in vitro with either AAV-OPG or AAV-LacZ before transfused into the osteolytic prosthetic joint 3 weeks post surgery. Successful transgene expression at the local site was confirmed 4 weeks later after killing. Biomechanical pullout test indicated a significant restoration of implant stability after the cell-based OPG gene therapy. Histology revealed that inflammatory pseudo-membranes existed ubiquitously at bone-implant interface in control groups, whereas only observed sporadically in OPG gene-modified groups. Tartrate-resistant acid phosphatase+osteoclasts and tumor necrosis factor α, interleukin-1ß, CD68+ expressing cells were significantly reduced in periprosthetic tissues of OPG gene-modified mice. No transgene dissemination or tumorigenesis was detected in remote organs and tissues. Data suggest that cell-based ex vivo OPG gene therapy was comparable in efficacy with in vivo local gene transfer technique to deliver functional therapeutic OPG activities, effectively halted the debris-induced osteolysis and regained the implant stability in this model.


Asunto(s)
Osteólisis/terapia , Osteoprotegerina/genética , Falla de Prótesis , Fosfatasa Ácida/genética , Fosfatasa Ácida/metabolismo , Animales , Antígenos CD/metabolismo , Antígenos de Diferenciación Mielomonocítica/metabolismo , Terapia Genética/métodos , Vectores Genéticos/genética , Isoenzimas/genética , Isoenzimas/metabolismo , Prótesis de la Rodilla , Ratones , Ratones Endogámicos BALB C , Osteólisis/patología , Fosfatasa Ácida Tartratorresistente , Soporte de Peso
5.
Ann Rheum Dis ; 64(11): 1550-6, 2005 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-15860512

RESUMEN

BACKGROUND: Collagen induced arthritis (CIA) is an animal model of rheumatoid arthritis (RA) amenable to immunotherapy directed against tumour necrosis factor alpha (TNFalpha). OBJECTIVE: To evaluate whether local TNF receptor (TNF-R) gene therapy in DBA/1 mice exerts an influence beyond anti-inflammatory effects. Two measures of CIA pathogenesis were investigated-namely, immunity to collagen II (CII) 245-270 peptide (the major immunodominant epitope within bovine CII) and the preferential activation of T cell Vbeta8.2 variable region receptors in arthritic DBA/1 mice. METHODS: DBA/1 mice received single periarticular injections of media or retroviral vectors containing LacZ or human TNF-R into affected arthritic paws at disease onset. Disease severity was monitored, immune responses towards the immunodominant bovine CII 245-270 and subdominant CII 334-360 peptide epitopes were assessed by ELISA, and T cell Vbeta usage was analysed by real time polymerase chain reaction for the LacZ transduced, TNF-R, and viral-free media treated control animals. The therapeutic influence of TNF-R gene transduction was compared with other groups at different times after treatment. RESULTS: Reduced disease severity was seen 15-35 days after treatment, with a concomitant increase in immunity towards the subdominant CII 334-360 peptide epitope rather than the immunodominant CII 245-270 peptide in TNF-R treated animals. Early in the disease, TNF-R treated animals demonstrated a reduction of bias towards the otherwise predominant Vbeta8.2 T cell subset. CONCLUSIONS: TNF-R gene therapy influences cellular immunity in CIA, leading to overall disease amelioration, thus suggesting that TNF inhibition may have therapeutic potential beyond the control of inflammation in RA.


Asunto(s)
Artritis Experimental/terapia , Terapia Genética/métodos , Receptores del Factor de Necrosis Tumoral/genética , Animales , Artritis Experimental/inmunología , Colágeno Tipo II/inmunología , Femenino , Expresión Génica , Vectores Genéticos , Inmunidad Celular , Epítopos Inmunodominantes/inmunología , Interferón gamma/biosíntesis , Ratones , Ratones Endogámicos DBA , Fragmentos de Péptidos/metabolismo , Receptores de Antígenos de Linfocitos T alfa-beta/metabolismo , Retroviridae/genética , Índice de Severidad de la Enfermedad , Transducción Genética , Transgenes/genética
6.
Scand J Rheumatol ; 33(5): 349-57, 2004.
Artículo en Inglés | MEDLINE | ID: mdl-15513686

RESUMEN

OBJECTIVE: To develop a mouse model of bone resorption to quantitatively evaluate wear-debris induced osteolysis. METHODS: Air pouches were established on the back of BALB/c mice, followed by the surgical introduction of a section of femur or calvaria from a syngeneic mouse donor. One group of bone-implanted pouches was stimulated with ultra-high molecular weight polyethylene (UHMWPE) debris, and the remaining bone-implanted pouches received saline alone as controls. The tissues were harvested at 2, 7, and 14 days after bone implantation for molecular and histological analyses. RESULTS: Marked inflammatory responses (thicker membrane and increased cellular infiltration) were observed in UHMWPE-stimulated pouches, compared with the saline control. Intensive tartrate-resistant acid phosphatase (TRAP) staining was identified in the UHMWPE-stimulated pouches, especially at the attachment site of inflammatory tissue with implanted bone, where active osteolysis occurred. Image analysis showed that the bone collagen loss was closely related to the amount of UHMWPE within the tissue, and was most prevalent at the contact site of bone with inflammatory tissue. UHMWPE stimulation also significantly increased the release of free calcium into the pouch fluids. CONCLUSION: This model demonstrates a sensitive, rapid, and reproducible method for studying wear-debris induced osteolysis seen in patients with aseptic loosening.


Asunto(s)
Dispositivos de Fijación Ortopédica/efectos adversos , Osteólisis/etiología , Falla de Prótesis , Animales , Resorción Ósea/etiología , Modelos Animales de Enfermedad , Femenino , Ratones , Ratones Endogámicos BALB C
7.
Clin Exp Rheumatol ; 22(5): 617-20, 2004.
Artículo en Inglés | MEDLINE | ID: mdl-15485016

RESUMEN

OBJECTIVE: The tight skin (Tsk-1) mouse has been proposed as a model for systemic sclerosis on the basis of increased accumulation of collagen and glycosaminoglycans in the skin, and by the presence of serum autoantibodies. The genetic basis of the mutation has been identified as a genomic duplication within the fibrillin-1 (Fbn-1) gene that results in a larger than normal Fbn-1 transcript, but the mechanism that leads to dermal fibrosis is unclear Fibrillin molecules associate into a polymer that is coated with elastin molecules to form elastic fibers. To further evaluate the Tsk-1 mouse model of scleroderma, we have studied elastic fibers in the skin of these mice. METHODS: Skin sections obtained from C57BL/6-TSK+ (Tsk-1) and C57BL6-pa/+ (control) mice were stained with Masson's trichrome for evaluation of collagen and Gomori's aldehyde fuchsin stain for elastic tissue. Computer assisted image analysis was performed to quantify differences in histologic sections. RESULTS: Tsk-1 mice had a highly significant increase in the percentage of elastic fibers (19.6%) in the dermis compared to control mice (7.9%) [p < 0.001]. This correlates with the findings in the skin of systemic sclerosis patients where increased elastic fibers have been observed. In addition, an increased level of dermal collagen staining was also observed in the Tsk-1 dermis (82.9%) compared with the level in normal sections (73.7%) [p < 0.01]. CONCLUSION: These data support the use of the Tsk-1 mouse as a model for the connective tissue abnormalities of human scleroderma.


Asunto(s)
Dermis/metabolismo , Tejido Elástico/metabolismo , Esclerodermia Sistémica/metabolismo , Animales , Colágeno/biosíntesis , Dermis/química , Ratones , Ratones Endogámicos C57BL , Modelos Animales
8.
Gene Ther ; 11(4): 402-7, 2004 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-14724679

RESUMEN

Although total joint replacement surgery is one of the most successful clinical procedures performed today, bone loss around knee and hip implants (osteolysis), resulting in aseptic loosening of the prosthesis, remains a major problem for many patients. Over the last decade much has been learned about this process, which is caused by wear debris particles that simulate a local inflammatory response and osteoclastic bone resorption. Aseptic loosening cannot be prevented or treated by existing nonsurgical methods. Gene transfer, however, offers novel possibilities. Here, we review the current state of the field and the experimental gene therapy approaches that have been investigated toward a solution to aseptic loosening of prosthetic implants.


Asunto(s)
Terapia Genética/métodos , Prótesis Articulares , Falla de Prótesis , Animales , Modelos Animales de Enfermedad , Humanos , Osteólisis
9.
Gene Ther ; 11(5): 483-91, 2004 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-14724688

RESUMEN

The current study evaluated the protective effects of anti-inflammatory cytokine gene transfer on osteolysis provoked by orthopedic biomaterial particles using a murine model of inflammatory bone loss. A section of bone was surgically implanted into an air pouch established on a syngeneic recipient mouse. Inflammation was provoked by introduction of ultra-high-molecular-weight polyethylene (UHMWPE) particles into the pouch, and retroviruses encoding for interleukin-1 receptor antagonist (hIL-1Ra), viral interleukin-10 (vIL-10), or LacZ genes were injected. Pouch fluid and tissue were harvested 7 days later for histological and molecular analyses. The results indicated that IL-1Ra or vIL-10 gene transfer significantly inhibited IL-1beta and tumor necrosis factor (TNF) expression at both mRNA and protein levels. There were significantly lower mRNA expressions of calcitonin receptor and cathepsin K in RNA isolated from hIL-1Ra- or vIL-10-transduced pouches than LacZ-transduced and virus-free controls. Both anti-inflammatory cytokine gene transfers significantly reduced the mRNA expression of M-CSF (70-90%) and RANK (>65%) in comparison with LacZ- and virus-free controls. Histological examination showed that hIL-1Ra or vIL-10 gene transfer dramatically abolished UHMWPE-induced inflammatory cellular infiltration and bone pit erosion compared to LacZ-transduced and virus-free controls. Histochemical staining revealed significantly fewer osteoclast-like cells in samples treated with IL-1Ra or vIL-10 gene transfer. In addition, bone collagen content was markedly preserved in the groups with anti-inflammatory cytokine gene transfers compared with the other two groups. Overall, retrovirus-mediated hIL-1Ra or vIL-10 gene transfer effectively protected against UHMWPE-particle-induced bone resorption, probably due to the inhibition of IL-1/TNF-induced M-CSF production and the consequent osteoclast recruitment and maturation.


Asunto(s)
Terapia Genética/métodos , Interleucina-10/genética , Osteólisis/prevención & control , Sialoglicoproteínas/genética , Animales , Modelos Animales de Enfermedad , Regulación de la Expresión Génica/genética , Vectores Genéticos/genética , Proteína Antagonista del Receptor de Interleucina 1 , Interleucina-1/metabolismo , Ratones , Ratones Endogámicos BALB C , Osteoclastos/fisiología , Osteólisis/etiología , Osteólisis/patología , ARN Mensajero/genética , Retroviridae/genética , Transducción Genética , Factor de Necrosis Tumoral alfa/metabolismo
10.
Ann Rheum Dis ; 62(10): 983-90, 2003 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-12972478

RESUMEN

BACKGROUND: Transgenic deficiency in interferon gamma (IFNgamma) or IFNgamma receptor makes resistant strains of mice bearing H-2(b) or H-2(d) susceptible to collagen induced arthritis (CIA). OBJECTIVE: To determine whether the escape from regulation of disease susceptibility at the major histocompatibility complex level involves a new use of autoimmune T cells expressing T cell receptor (TCR) Vbeta that vary from the cell populations previously identified within arthritic joints. METHODS: Arthritis was induced by a standard protocol with type II bovine collagen (CII) in complete Freund's adjuvant. Clinical features, histopathology, immunological responses, and TCR profile in arthritic joints in IFNgamma knockout C57BL/6 (B6.IFNgamma KO) mice (H-2(b)) were compared directly with those in DBA/1 mice (H-2(q)). RESULTS: 60-80% of B6.IFNgamma KO mice developed a progressive arthritis with a similar clinical course to classical CIA in DBA/1 mice. The affected joints in B6.IFNgamma KO mice had an erosive form of arthritis with similar features to joint disease in DBA/1 mice. B6.IFNgamma KO mice produced significantly higher levels of IgG2b and IgG1 autoantibodies to murine CII and showed increased proliferative response to CII compared with B6 mice. Comparable levels of interleukin 1beta and tumour necrosis factor alpha expression were detected in arthritic joints from beta6.IFNgamma KO and DBA/1 mice. B6.IFNgammaKO mice used predominantly TCR Vbeta6 and Vbeta8 in arthritic joints. This TCR Vbeta profile is similar to that found in DBA/1 mice with CIA. CONCLUSIONS: C57BL/6 mice deficient in IFNgamma production can develop arthritis that resembles classical CIA. These data suggest that IFNgamma is a key factor mediating susceptibility to CIA.


Asunto(s)
Artritis Experimental/inmunología , Interferón gamma/deficiencia , Articulaciones/inmunología , Receptores de Antígenos de Linfocitos T alfa-beta , Receptores de Antígenos de Linfocitos T alfa-beta/inmunología , Animales , Artritis Experimental/patología , Distribución de Chi-Cuadrado , Colágeno , Susceptibilidad a Enfermedades , Femenino , Interleucina-1/análisis , Articulaciones/patología , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos DBA , Ratones Noqueados , Modelos Animales , ARN Mensajero/análisis , Receptores de Antígenos de Linfocitos T alfa-beta/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factor de Necrosis Tumoral alfa/análisis
11.
Ann Rheum Dis ; 62(8): 707-14, 2003 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-12860724

RESUMEN

BACKGROUND: Therapeutic strategies to block tumour necrosis factor alpha (TNFalpha) activity in experimental autoimmune arthritis models and rheumatoid arthritis (RA) have proved highly successful, and provide sustained beneficial effects. OBJECTIVE: To examine whether TNFalpha inhibition has immunological activity beyond the reduction of inflammation in collagen induced arthritis (CIA), an established experimental model of RA. METHODS: Arthritic DBA/1 mice received single periarticular injections of retroviral constructs encoding human TNF receptor (TNF-R) into the affected arthritic paw, at the onset of arthritis. Severity of arthritis, antibodies to collagen type II (CII), and extent of pathological joint damage of arthritic paws were compared between TNF-R and media treated (control) animals 3, 7, 14, 21, and 49 days after disease onset. RESULTS: Severity of CIA was significantly decreased in TNF-R treated animals compared with controls, 14-34 days after disease onset. Joint destruction was reduced in TNF-R injected joints and in the uninjected contralateral and ipsilateral paws of TNF-R treated animals. Seven days after disease onset, TNF-R treated mice had lower levels of inflammatory Th1 driven IgG2a antibodies to CII (p<0.05) than controls. This altered the anticollagen IgG2a:IgG1 ratio towards Th2 driven IgG1. CONCLUSIONS: Local TNF-R gene therapy in CIA appears to have systemic effects on the anti-CII antibodies. The overall influence of TNF-R gene therapy is that it inhibits the progression of CIA mainly by suppressing the inflammatory Th1 response rather than by stimulating a Th2 response. Therefore, periarticular TNF-R gene therapy may have excellent therapeutic potential in RA.


Asunto(s)
Artritis Experimental/terapia , Autoanticuerpos/biosíntesis , Terapia Genética/métodos , Terapia de Inmunosupresión/métodos , Receptores del Factor de Necrosis Tumoral/genética , Animales , Artritis Experimental/inmunología , Artritis Experimental/patología , Artritis Reumatoide/inmunología , Artritis Reumatoide/patología , Artritis Reumatoide/terapia , Colágeno Tipo II/inmunología , Femenino , Vectores Genéticos , Inmunoglobulina G/biosíntesis , Ratones , Ratones Endogámicos DBA , Receptores del Factor de Necrosis Tumoral/inmunología , Retroviridae/genética , Células TH1/inmunología , Factor de Necrosis Tumoral alfa/antagonistas & inhibidores , Factor de Necrosis Tumoral alfa/metabolismo
12.
Inflamm Res ; 51(7): 342-50, 2002 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-12146725

RESUMEN

OBJECTIVE: This study examined anti- inflammatory gene therapy to ameliorate tissue responses to ultra high molecular weight polyethylene (UHMWPE) particles in the murine air pouch. METHODS: Retroviruses encoding human interleukin- 1 receptor antagonist (IL-1Ra), viral interleukin-10 (vIL-10), or LacZ (reporter) genes were injected into murine air pouches stimulated by UHMWPE particles. Pouch membranes and fluids were harvested at 1, 3 and 7 days post gene-transduction, and assayed for markers of inflammation using histological, molecular, and immunological techniques. RESULTS: Real time RT-PCR and ELISA showed a strong production of IL-1beta in pouch tissue and lavage fluid induced by particle stimulation, accompanied by a lower expression of IL-6, TNF-alpha and IL-4. Transduction of IL-1Ra or vIL-10 genes resulted in a significant reduction of IL-1beta both at the mRNA and at the protein level. The gene therapy also resulted in diminution of IL-6 and TNF-alpha expression. In addition, significant elevation of TGF-beta expression was observed in IL-1Ra transduced pouches. Histological analysis revealed that the membranes of pouches transduced with vIL-10 or IL-1Ra were significantly less inflamed than the membranes of non-viral and LacZ-transduced pouches, with less cellular proliferation and lowered monocyte/macrophage influx. CONCLUSIONS: IL-1Ra or vIL-10 gene transduction was effective in ameliorating local inflammation by reducing the IL-1 production and subsequent cellular events elicited in response to UHMWPE particles in this model. These findings suggest that IL-1 directed gene therapy might be excellent therapeutic candidates to prevent or retard the inflammatory response to wear debris that contributes to the pathology of aseptic loosening.


Asunto(s)
Terapia Genética/métodos , Inflamación/prevención & control , Interleucina-10/biosíntesis , Interleucina-10/genética , Retroviridae/genética , Sialoglicoproteínas/biosíntesis , Sialoglicoproteínas/genética , Aire , Animales , Anticuerpos Monoclonales , Recuento de Células , Citocinas/biosíntesis , Citocinas/genética , Ensayo de Inmunoadsorción Enzimática , Regulación de la Expresión Génica/genética , Vectores Genéticos/genética , Inflamación/patología , Proteína Antagonista del Receptor de Interleucina 1 , Ratones , Ratones Endogámicos BALB C , Peso Molecular , Polietileno , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa
13.
J Immunol ; 166(3): 1492-8, 2001 Feb 01.
Artículo en Inglés | MEDLINE | ID: mdl-11160188

RESUMEN

Rheumatoid arthritis (RA) is the most common, crippling human autoimmune disease. Using Western blotting and tandem mass spectroscopy, we have identified the endoplasmic reticulum chaperone BiP, a 78-kDa glucose-regulated protein, as a possible autoantigen. It preferentially stimulated increased proliferation of synovial T cells from patients with RA but not from patients with other arthritides. Mice with established collagen- or pristane-induced arthritis developed IgG Abs to BiP. Although BiP injected in CFA failed to induce arthritis in several strains of rats and mice, including HLA-DR4(+/-)- and HLA-DR1(+/+)-transgenic animals, it completely inhibited the development of arthritis when given i.v. 1 wk before the injection of type II collagen arthritis. Preimmunization with BiP suppressed the development of adjuvant arthritis in Lewis rats in a similar manner. This is the first report of a mammalian chaperone that is an autoantigen in human RA and in experimental arthritis and that can also prevent the induction of experimental arthritis. These findings may stimulate the development of new immunotherapies for the treatment of RA.


Asunto(s)
Artritis Experimental/inmunología , Artritis Experimental/prevención & control , Artritis Reumatoide/inmunología , Autoantígenos/inmunología , Proteínas Portadoras/administración & dosificación , Proteínas Portadoras/inmunología , Proteínas de Choque Térmico , Chaperonas Moleculares/administración & dosificación , Chaperonas Moleculares/inmunología , Adulto , Animales , Artritis Experimental/etiología , Artritis Reumatoide/patología , Autoanticuerpos/biosíntesis , Autoanticuerpos/sangre , Autoantígenos/sangre , Autoantígenos/aislamiento & purificación , Retículo Endoplásmico/inmunología , Chaperón BiP del Retículo Endoplásmico , Femenino , Humanos , Esquemas de Inmunización , Inyecciones Intradérmicas , Inyecciones Intravenosas , Activación de Linfocitos , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Endogámicos DBA , Ratones Transgénicos , Persona de Mediana Edad , Ratas , Ratas Endogámicas Lew , Ratas Wistar , Membrana Sinovial/inmunología , Membrana Sinovial/patología , Linfocitos T/inmunología , Linfocitos T/patología , Células Tumorales Cultivadas
14.
Gene Ther ; 8(23): 1770-6, 2001 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-11803396

RESUMEN

Various cytokines and cytokine antagonists hold promise as new therapeutic agents for osteoporosis, but their application is hindered by delivery problems. Gene transfer offers an attractive technology with which to obviate these restrictions. Its utility was evaluated in an animal model of osteoporosis. Disease was induced by surgical ovariectomy and monitored by measuring bone weight after 12 days, and by histomorphometry after 5 weeks. Genes were transferred to the mice by intramedullary injection of adenoviral vectors. LacZ and luciferase marker genes were used to identify the bone marrow cells transduced by this procedure, and to track the possible spread of transgenes to other organs. The effect on bone loss of transferring a cDNA encoding the human interleukin-1 receptor antagonist (IL-1Ra) was then evaluated. The intramedullary injection of adenoviral vectors transduced lining osteoblasts, osteocytes and cells within the bone marrow. Luciferase activity persisted within the injected femora and adjacent musculature for at least 3 weeks, and in the draining lymph nodes for 2 weeks. Transient, low level expression was present in the liver, but no luciferase was detected at any time in the lung or spleen. Intramedullary introduction of the IL-1Ra gene resulted in circulation of the corresponding protein at concentrations that peaked on day 3, and returned to baseline by day 12. Transfer of the IL-1Ra gene strongly reduced the early loss of bone mass occurring in response to ovariectomy. Furthermore, it completely inhibited the loss of matrix detected by histomorphometry at 5 weeks. The protective effect of this gene was not restricted to bones receiving intramedullary injection of the vector, but occurred in all bones that were evaluated. This proof of concept encourages further development of gene therapy approaches to the treatment of osteoporosis.


Asunto(s)
Terapia Genética/métodos , Osteoporosis Posmenopáusica/prevención & control , Adenoviridae/genética , Animales , ADN Complementario/genética , Modelos Animales de Enfermedad , Femenino , Fémur/patología , Expresión Génica , Técnicas de Transferencia de Gen , Vectores Genéticos , Humanos , Húmero/patología , Proteína Antagonista del Receptor de Interleucina 1 , Ratones , Ratones Endogámicos BALB C , Osteoporosis Posmenopáusica/patología , Ovariectomía , Sialoglicoproteínas/genética
15.
Inflammation ; 25(6): 361-72, 2001 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-11831439

RESUMEN

Retroviral vectors encoding the human IL-1 antagonist (IL-1Ra) gene and the human tumor necrosis factor soluble receptor (sTNF-R) gene were investigated using an in vivo model of the inflammatory response to orthopedic wear debris. Air pouches established in BALB/c mice were injected with polymethylmethacrylate (PMMA) particles to provoke an inflammatory reaction, and infected with retroviral vectors expressing IL-1Ra, sTNF-R or a LacZ marker gene. Pouch membranes and fluids were harvested after 48 or 72 hours for analyses. Positive PCR reactions for Neo genes were observed specifically in DNA extracted from the membrane of retroviral-infected pouches. ELISA assays revealed the presence of human IL-1 Ra in pouch fluid from DFG-IRAP-Neo transduced mice, but not control animals. Histological evaluation indicated that the IL-1Ra gene transfer was associated with markedly decreased inflammation in the model, with resolution of the edematous phase of the reaction, decreased pouch fluid accumulation, and lowered macrophage influx. The data suggest that the air pouch model represents a useful tool to evaluate gene therapy, and demonstrate that IL-1Ra gene therapy may be an appropriate therapeutic approach to inflammation.


Asunto(s)
Materiales Biocompatibles/efectos adversos , Citocinas/uso terapéutico , Terapia Genética/métodos , Inflamación/terapia , Animales , Citocinas/genética , Modelos Animales de Enfermedad , Femenino , Vectores Genéticos/administración & dosificación , Vectores Genéticos/uso terapéutico , Inflamación/etiología , Inflamación/patología , Ratones , Ratones Endogámicos BALB C , Polimetil Metacrilato/efectos adversos , Receptores de Interleucina-1/genética , Receptores de Interleucina-1/uso terapéutico , Receptores del Factor de Necrosis Tumoral/genética , Receptores del Factor de Necrosis Tumoral/uso terapéutico , Retroviridae/genética , Resultado del Tratamiento
16.
Clin Orthop Relat Res ; (379 Suppl): S120-5, 2000 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-11039760

RESUMEN

Gene therapy has much to offer in the treatment of conditions in which it is necessary to increase the formation of bone. Nonunions, segmental defects, and aseptic loosening are examples of conditions where the local expression of genes that inhibit osteolysis and promote osteogenesis might be helpful. Studies in which one such possibility has been evaluated experimentally are described. These investigations used a surgically produced segmental defect in the femurs of New Zealand White rabbits as the model system. Adjacent muscle was fashioned around the defect to form a chamber into which adenoviral vectors were injected. High levels of transgene expression were found in the muscle surrounding the defect after injection of vectors carrying marker genes. Transgene expression also was seen in the cut ends of the bone and the scar tissue within the gap. No transgene expression was seen in the contralateral limb, spleen, or lung; transient, low levels of expression were found in the liver. Transgene expression declined with time, disappearing from all tissue but bone by Day 26; expression persisted in bone for at least 6 weeks. The control defects did not heal spontaneously. Injection of adenovirus carrying a human bone morphogenetic protein-2 complementary deoxyribonucleic acid led to healing of the segmental defect within 12 weeks, as judged by radiographic, histologic, and biomechanical criteria. Adenovirus carrying a human transforming growth factor-beta 1 complementary deoxyribonucleic acid showed signs of improved healing, but not to the extent seen with the bone morphogenetic protein-2 complementary deoxyribonucleic acid. This approach to therapy holds much promise as a novel means of promoting osteogenesis.


Asunto(s)
Adenoviridae , Curación de Fractura , Técnicas de Transferencia de Gen , Terapia Genética , Vectores Genéticos , Animales , Proteína Morfogenética Ósea 2 , Proteínas Morfogenéticas Óseas/genética , Expresión Génica , Osteogénesis/genética , Conejos , Factor de Crecimiento Transformador beta/genética , Transgenes
17.
Clin Exp Immunol ; 121(2): 399-405, 2000 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-10931159

RESUMEN

Unlike other agents associated with drug-induced lupus, the isoprenoid alkane pristane induces autoantibodies pathognomonic of lupus, including anti-Sm, anti-dsDNA, and anti-ribosomal P in BALB/c and SJL/J mice. The susceptibility of other strains of mice to pristane-induced lupus is unknown and is the focus of the present study. Anti-nRNP/Sm, anti-Su, and anti-ribosomal P autoantibodies were produced by most strains of mice surveyed within several months of pristane treatment, although there was marked interstrain variability in their frequencies, levels, and times of onset. In sharp contrast, the production of autoantibodies against the double-stranded RNA binding proteins NF45/NF90/p110 was restricted to B6 and B10.S mice. We conclude that pristane selectively induces lupus-specific autoantibodies in virtually any strain of mouse regardless of its genetic background. However, H-2-linked as well as non-H2 genes influenced the expression of individual autoantibody markers. The widespread susceptibility of pristane-treated mice to lupus autoantibody production and the relatively small effect of MHC are unique features of this chemically induced lupus syndrome, with potential implications for understanding the pathogenesis of autoantibodies in idiopathic human systemic lupus erythematosus.


Asunto(s)
Autoanticuerpos/biosíntesis , Enfermedades Autoinmunes/inducido químicamente , Lupus Eritematoso Sistémico/inducido químicamente , Ratones Endogámicos/genética , Terpenos/toxicidad , Animales , Anticuerpos Antinucleares/biosíntesis , Anticuerpos Antinucleares/inmunología , Autoanticuerpos/inmunología , Autoantígenos/inmunología , Enfermedades Autoinmunes/genética , Enfermedades Autoinmunes/inmunología , Femenino , Predisposición Genética a la Enfermedad , Antígenos H-2/genética , Haplotipos , Vivienda para Animales , Humanos , Inmunoglobulina G/biosíntesis , Inmunoglobulina G/inmunología , Células K562 , Lupus Eritematoso Sistémico/genética , Lupus Eritematoso Sistémico/inmunología , Ratones , Ratones Endogámicos A , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Endogámicos DBA , Ratones Endogámicos/inmunología , Fragmentos de Péptidos/inmunología , Fosfoproteínas/inmunología , Proteínas Ribosómicas/inmunología , Especificidad de la Especie
18.
Ann Rheum Dis ; 59(8): 626-30, 2000 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-10913060

RESUMEN

OBJECTIVE: To evaluate the contribution of polymorphisms in the vitamin D receptor (VDR) gene to ethnic variations in bone mass in mother and children from different ethnic origins. METHODS: VDR genotypes and bone mass in 43 African-American and white women, mean age 38.2 years, and 41 of their children were studied. All children had a whole body bone mass measurement at age 9, and 39 had follow up measurements at age 11, while all the mothers had a single measurement. DNA was extracted from peripheral blood samples, subjected to polymerase chain reactions using primers specific for the VDR gene, and the Bsm1 restriction fragment length polymorphism defined. RESULTS: There was a significant ethnic difference in the VDR genotype frequencies among the adults and the children. No African-American subjects had the genotype "BB". In contrast, there was a 25% frequency of the "BB" genotype in the white adults and 24% in the white children. After pooling the ethnic groups, the mean bone mass in the "bb" genotype was significantly higher than in the "BB" genotype among the mothers, but this was not found in the children at baseline. However, by age 11, those with the "Bb" or "bb" genotypes had a larger gain in bone mass than those with "BB". CONCLUSION: These data support the suggestion that the ethnic difference in VDR genotype frequencies, together with the association between the genotypes and bone mass, may help to explain the well known ethnic differences in bone mass. Further, our observations suggest that VDR polymorphism may have an effect on bone mass during puberty as peak bone mass is accumulated.


Asunto(s)
Población Negra/genética , Densidad Ósea/genética , Polimorfismo de Longitud del Fragmento de Restricción , Receptores de Calcitriol/genética , Población Blanca/genética , Absorciometría de Fotón , Adulto , Niño , ADN/análisis , Femenino , Genotipo , Humanos , Masculino , Reacción en Cadena de la Polimerasa/métodos
19.
Gene Ther ; 7(9): 734-9, 2000 May.
Artículo en Inglés | MEDLINE | ID: mdl-10822299

RESUMEN

This study evaluated the ability of gene transfer to enhance bone healing. Segmental defects were created surgically in the femora of New Zealand white rabbits. First generation adenoviruses were used as vectors to introduce into the defects genes encoding either human bone morphogenetic protein-2 (BMP-2) or, as a negative control, firefly luciferase. Representative specimens were evaluated histologically after 8 weeks. Healing of the defects was monitored radiographically for 12 weeks, after which time the repair tissue was evaluated biomechanically. By radiological criteria, animals receiving the BMP-2 gene had healed their osseous lesions after 7 weeks, whereas those receiving the luciferase gene had not. Histologic examination of representative rabbits at 8 weeks confirmed ossification across the entire defect in response to the BMP-2 gene, whereas the control defect was predominantly fibrotic and sparsely ossified. At the end of the 12-week experiment, the control femora still showed no radiological signs of stable healing. The difference in radiologically defined healing between the experimental and control groups was statistically significant (P < 0. 002). Biomechanical testing of the femora at 12 weeks demonstrated statistically significant increases in the mean bending strength (P < 0.005) and bending stiffness (P < 0.05) of the animals treated with the BMP-2 gene. Direct, local adenoviral delivery of an osteogenic gene thus led to the healing of an osseous lesion that otherwise would not do so. These promising data encourage the further development of genetic approaches to enhancing bone healing. Gene Therapy (2000) 7, 734-739.


Asunto(s)
Adenoviridae/genética , Proteínas Morfogenéticas Óseas/genética , Fracturas del Fémur/terapia , Terapia Genética/métodos , Transfección/métodos , Factor de Crecimiento Transformador beta , Cicatrización de Heridas , Animales , Fenómenos Biomecánicos , Proteína Morfogenética Ósea 2 , Fracturas del Fémur/diagnóstico por imagen , Fracturas del Fémur/patología , Fémur/diagnóstico por imagen , Fémur/patología , Conejos , Radiografía
20.
J Rheumatol ; 26(10): 2215-21, 1999 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-10529143

RESUMEN

OBJECTIVE: The use of silicone breast implants has been implicated in the development of autoimmune connective tissue diseases including systemic lupus erythematosus (SLE). We examined the influence of implanted silicones in MRL lpr/lpr and MRL +/+ mice, to determine whether silicone increases autoimmunity and exacerbates experimental lupus. METHODS: Mice were implanted with either silicone gel or silicone oil (polydimethylsiloxane; PDMS), while saline injected mice were used as controls. Proteinuria levels, palpation of lymphadenopathy, serum autoantibodies, circulating cytokines, and weight change were monitored for 18 weeks, when terminal glomerulonephritis was evaluated by histopathological techniques. Proteins were extracted from the surface of recovered implants, and the composition and immune reactive status of the silicone-binding proteins (SBP) were investigated. RESULTS: No adverse influence of silicone gel or silicone oil on the clinical aspects of lupus was observed. However, anti-DNA antibodies were significantly increased in MRL mice implanted with silicone gel compared to the control animals, and rheumatoid factor titers were modestly increased in implanted MRL lpr/lpr mice. Serum cytokine levels were influenced by silicone implantation in MRL lpr/lpr mice (but not MRL +/+ mice), with interleukin 1 (IL-1) levels increased in gel implanted animals and IL-2 levels elevated in PDMS (silicone oil) implanted mice. Different SBP were detected on implants recovered from MRL lpr/lpr mice compared with MRL +/+ mice, and Western blotting revealed the presence of strong autoantibodies to SBP in sera from MRL lpr/lpr mice, but not MRL +/+ mice. CONCLUSION: These findings suggest that silicone implantation may influence immunological responses during murine lupus, including the provocation or exacerbation of autoantibodies. However, these immune modifications did not appear to influence the clinical variables of this experimental lupus model.


Asunto(s)
Lupus Vulgar/etiología , Prótesis e Implantes/efectos adversos , Geles de Silicona/efectos adversos , Aceites de Silicona/efectos adversos , Animales , Anticuerpos Antinucleares/sangre , Autoanticuerpos/sangre , Autoinmunidad , Implantes de Mama/efectos adversos , Citocinas/sangre , Lupus Vulgar/sangre , Lupus Vulgar/inmunología , Ratones , Ratones Endogámicos MRL lpr , Factor Reumatoide/sangre
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