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Age-related macular degeneration (AMD) and diabetic retinopathy (DR) are common retinal diseases responsible for most blindness in working-age and elderly populations. Oxidative stress and mitochondrial dysfunction play roles in these pathogenesis, and new therapies counteracting these contributors could be of great interest. Some molecules, like coenzyme Q10 (CoQ10), are considered beneficial to maintain mitochondrial homeostasis and contribute to the prevention of cellular apoptosis. We investigated the impact of adding CoQ10 (Q) to a nutritional antioxidant complex (Nutrof Total®; N) on the mitochondrial status and apoptosis in an in vitro hydrogen peroxide (H2O2)-induced oxidative stress model in human retinal pigment epithelium (RPE) cells. H2O2 significantly increased 8-OHdG levels (p < 0.05), caspase-3 (p < 0.0001) and TUNEL intensity (p < 0.01), and RANTES (p < 0.05), caspase-1 (p < 0.05), superoxide (p < 0.05), and DRP-1 (p < 0.05) levels, and also decreased IL1ß, SOD2, and CAT gene expression (p < 0.05) vs. control. Remarkably, Q showed a significant recovery in IL1ß gene expression, TUNEL, TNFα, caspase-1, and JC-1 (p < 0.05) vs. H2O2, and NQ showed a synergist effect in caspase-3 (p < 0.01), TUNEL (p < 0.0001), mtDNA, and DRP-1 (p < 0.05). Our results showed that CoQ10 supplementation is effective in restoring/preventing apoptosis and mitochondrial stress-related damage, suggesting that it could be a valid strategy in degenerative processes such as AMD or DR.
Assuntos
Apoptose , Peróxido de Hidrogênio , Estresse Oxidativo , Epitélio Pigmentado da Retina , Ubiquinona , Humanos , Ubiquinona/análogos & derivados , Ubiquinona/farmacologia , Epitélio Pigmentado da Retina/metabolismo , Epitélio Pigmentado da Retina/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Apoptose/efeitos dos fármacos , Peróxido de Hidrogênio/metabolismo , Peróxido de Hidrogênio/farmacologia , Mitocôndrias/metabolismo , Mitocôndrias/efeitos dos fármacos , Antioxidantes/farmacologia , Células Epiteliais/metabolismo , Células Epiteliais/efeitos dos fármacos , Linhagem Celular , Suplementos NutricionaisRESUMO
Alphavirus budding is driven by interactions between nucleocapsids assembled in the cytoplasm and envelope proteins present at the plasma membrane. So far, the expression of capsid and envelope proteins in infected cells has been considered an absolute requirement for alphavirus budding and propagation. In the present study, we show that Semliki Forest virus and Sindbis virus lacking the capsid gene can propagate in mammalian and insect cells. This propagation is mediated by the release of infectious microvesicles (iMVs), which are pleomorphic and have a larger size and density than wild-type virus. iMVs, which contain viral RNA inside and viral envelope proteins on their surface, are released at the plasma membrane and infect cells using the endocytic pathway in a similar way to wild-type virus. iMVs are not pathogenic in immunocompetent mice when injected intravenously, but can infect different organs like lungs and heart. Finally, we also show that alphavirus genomes without capsid can mediate the propagation of heterologous genes, making these vectors potentially interesting for gene therapy or vaccination studies. The minimalist infectious system described in this study shows that a self-replicating RNA able to express membrane proteins with binding and fusion properties is able to propagate, providing some insights into virus evolution.
Assuntos
Alphavirus/patogenicidade , Capsídeo/metabolismo , Membrana Celular/virologia , Micropartículas Derivadas de Células/virologia , Alphavirus/genética , Animais , Fusão Celular , Linhagem Celular , Micropartículas Derivadas de Células/metabolismo , Micropartículas Derivadas de Células/ultraestrutura , Feminino , Genoma Viral , Proteínas de Fluorescência Verde/metabolismo , Humanos , Camundongos Endogâmicos C57BL , Testes de Neutralização , RNA Viral/metabolismo , Vírus da Floresta de Semliki/patogenicidade , Transfecção , Proteínas do Envelope Viral/metabolismo , Proteínas Virais/metabolismoRESUMO
Semliki Forest virus vectors expressing IL-12 (SFV-IL-12) were shown to induce potent antitumor responses against s.c. MC38 colon adenocarcinomas in immunocompetent mice. However, when MC38 tumors were implanted in liver, where colon tumors usually metastasize, SFV-IL-12 efficacy was significantly reduced. We reasoned that characterization of immune responses against intrahepatic tumors in responder and nonresponder animals could provide useful information for designing more potent antitumor strategies. Remarkably, SFV-IL-12 induced a high percentage of circulating tumor-specific CD8 T cells in all treated animals. Depletion studies showed that these cells were essential for SFV-IL-12 antitumor activity. However, in comparison with nonresponders, tumor-specific cells from responder mice acquired an effector-like phenotype significantly earlier, were recruited more efficiently to the liver, and, importantly, persisted for a longer period of time. All treated mice had high levels of functional specific CD8 T cells at 8 d posttreatment reflected by both in vivo killing and IFN-γ-production assays, but responder animals showed a more avid and persistent IFN-γ response. Interestingly, differences in immune responses between responders and nonresponders seemed to correlate with the immune status of the animals before treatment and were not due to the treatment itself. Mice that rejected tumors were protected against tumor rechallenge, indicating that sustained memory responses are required for an efficacious therapy. Interestingly, tumor-specific CD8 T cells of responder animals showed upregulation of IL-15Rα expression compared with nonresponders. These results suggest that SFV-IL-12 therapy could benefit from the use of strategies that could either upregulate IL-15Rα expression or activate this receptor.
Assuntos
Interleucina-12/biossíntese , Neoplasias Hepáticas Experimentais/imunologia , Neoplasias Hepáticas Experimentais/prevenção & controle , Vírus da Floresta de Semliki/imunologia , Vírus da Floresta de Semliki/metabolismo , Adenocarcinoma/imunologia , Adenocarcinoma/prevenção & controle , Adenocarcinoma/virologia , Infecções por Alphavirus/imunologia , Infecções por Alphavirus/prevenção & controle , Infecções por Alphavirus/virologia , Animais , Linfócitos T CD8-Positivos/imunologia , Linhagem Celular , Células Cultivadas , Neoplasias Colorretais/imunologia , Neoplasias Colorretais/prevenção & controle , Neoplasias Colorretais/virologia , Cricetinae , Feminino , Vetores Genéticos/administração & dosagem , Vetores Genéticos/imunologia , Vetores Genéticos/metabolismo , Interleucina-12/genética , Neoplasias Hepáticas Experimentais/virologia , Camundongos , Camundongos Endogâmicos C57BLRESUMO
We report a new method to generate high-expressing mammalian cell lines in a quick and efficient way. For that purpose, we developed a master cell line (MCL) containing an inducible alphavirus vector expressing GFP integrated into the genome. In the MCL, recombinant RNA levels increased >4,600-fold after induction, due to a doxycycline-dependent RNA amplification loop. The MCL maintained inducibility and expression during 50 passages, being more efficient for protein expression than a conventional cell line. To generate new cell lines, mutant LoxP sites were inserted into the MCL, allowing transgene and selection gene exchange by Cre-directed recombination, leading to quick generation of inducible cell lines expressing proteins of therapeutic interest, like human cardiotrophin-1 and oncostatin-M at several mg/l/24 h. These proteins contained posttranslational modifications, showed bioactivity, and were efficiently purified. Remarkably, this system allowed production of toxic proteins, like oncostatin-M, since cells able to express it could be grown to the desired amount before induction. These cell lines were easily adapted to growth in suspension, making this methodology very attractive for therapeutic protein production.
Assuntos
Alphavirus/genética , Linhagem Celular/metabolismo , Clonagem Molecular/métodos , Vetores Genéticos/genética , Proteínas de Fluorescência Verde/genética , Transgenes , Animais , Técnicas de Cultura de Células , Linhagem Celular/citologia , Linhagem Celular/virologia , Cricetinae , Citocinas/genética , DNA/genética , Genoma , Células Hep G2 , Humanos , Oncostatina M/genética , RNA/genética , Proteínas Recombinantes/genéticaRESUMO
Intratumoral injection of Semliki Forest virus encoding interleukin-12 (SFV-IL-12) combines acute expression of IL-12 and stressful apoptosis of infected malignant cells. Agonist antibodies directed to costimulatory receptor CD137 (4-1BB) strongly amplify pre-existing cellular immune responses toward weak tumor antigens. In this study, we provide evidence for powerful synergistic effects of a combined strategy consisting of intratumoral injection of SFV-IL-12 and systemic delivery of agonist anti-CD137 monoclonal antibodies (mAbs), which was substantiated against poorly immunogenic B16 melanomas (B16-OVA and B16.F10) and TC-1 lung carcinomas. Effector CD8(ß)(+) T cells were sufficient to mediate complete tumor eradications. Accordingly, there was an intensely synergistic in vivo enhancement of cytotoxic T lymphocytes (CTL)-mediated immunity against the tumor antigens OVA and tyrosine-related protein-2 (TRP-2). This train of phenomena led to long-lasting tumor-specific immunity against rechallenge, attained transient control of the progression of concomitant tumor lesions that were not directly treated with SFV-IL-12 and caused autoimmune vitiligo. Importantly, we found that SFV-IL-12 intratumoral injection induces bright expression of CD137 on most tumor-infiltrating CD8(+) T lymphocytes, thereby providing more abundant targets for the action of the agonist antibody. This efficacious combinatorial immunotherapy strategy offers feasibility for clinical translation since anti-CD137 mAbs are already undergoing clinical trials and development of clinical-grade SFV-IL-12 vectors is in progress.
Assuntos
Anticorpos Monoclonais/imunologia , Carcinoma/terapia , Interleucina-12/imunologia , Neoplasias Pulmonares/terapia , Melanoma Experimental/terapia , Vírus da Floresta de Semliki/imunologia , Neoplasias Cutâneas/terapia , Animais , Anticorpos Monoclonais/administração & dosagem , Apoptose/efeitos dos fármacos , Linfócitos T CD8-Positivos/efeitos dos fármacos , Linfócitos T CD8-Positivos/imunologia , Carcinoma/imunologia , Carcinoma/mortalidade , Linhagem Celular Tumoral , Cricetinae , Expressão Gênica/efeitos dos fármacos , Imunidade Celular/efeitos dos fármacos , Memória Imunológica/efeitos dos fármacos , Imunoterapia/métodos , Injeções Intralesionais , Injeções Intravenosas , Interleucina-12/administração & dosagem , Interleucina-12/genética , Oxirredutases Intramoleculares/genética , Oxirredutases Intramoleculares/imunologia , Neoplasias Pulmonares/imunologia , Neoplasias Pulmonares/mortalidade , Melanoma Experimental/imunologia , Melanoma Experimental/mortalidade , Camundongos , Vírus da Floresta de Semliki/genética , Neoplasias Cutâneas/imunologia , Neoplasias Cutâneas/mortalidade , Taxa de Sobrevida , Linfócitos T Citotóxicos/efeitos dos fármacos , Linfócitos T Citotóxicos/imunologia , Membro 9 da Superfamília de Receptores de Fatores de Necrose Tumoral/agonistas , Membro 9 da Superfamília de Receptores de Fatores de Necrose Tumoral/imunologiaRESUMO
Age-related macular degeneration (AMD) is a leading cause of severe vision loss in older individuals in developed countries. Despite advances in our understanding of AMD, its pathophysiology remains poorly understood. Matrix metalloproteinases (MMPs) have been proposed to play a role in AMD development. In this study, we aimed to characterize MMP-13 in AMD. We used retinal pigment epithelial cells, a murine model of laser-induced choroidal neovascularization, and plasma samples from patients with neovascular AMD to conduct our study. Our results show that MMP13 expression significantly increased under oxidative stress conditions in cultured retinal pigment epithelial cells. In the murine model, MMP13 was overexpressed in both retinal pigment epithelial cells and endothelial cells during choroidal neovascularization. Additionally, the total MMP13 levels in the plasma of patients with neovascular AMD were significantly lower than those in the control group. This suggests a reduced diffusion from the tissues or release from circulating cells in the bloodstream, given that the number and function of monocytes have been reported to be deficient in patients with AMD. Although more studies are needed to elucidate the role of MMP13 in AMD, it could be a promising therapeutic target for treating AMD.
RESUMO
Age-related macular degeneration (AMD) is currently the main cause of severe visual loss among older adults in developed countries. The pathophysiology has not been clarified, but oxidative stress is believed to play a major role. Matrix metalloproteinases (MMP) may play a prominent role in several steps of the pathophysiology of AMD, especially in its neovascular form; therefore, there is of great interest in understanding their role in choroidal neovascularisation. This study aimed to elucidate the role of MMP10 in the development of choroidal neovascularisation (CNV). We have demonstrated that MMP10 was expressed by retinal pigment epithelium cells and endothelial cells of the neovascular membrane, in cell culture, mouse and human retina. MMP10 expression and activity increased under oxidative stress conditions in ARPE-19 cells. MMP10-/- mice developed smaller laser-induced areas of CNV. Furthermore, to exclude a systemic MMP10 imbalance in these patients, plasma MMP10 concentrations were assessed in an age- and sex-matched sample of 52 control patients and 52 patients with neovascular AMD and no significant differences were found between the groups, demonstrating that MMP10 induction is a local phenomenon. Our findings suggest that MMP10 participates in the development of choroidal neovascularisation and promotes MMP10 as a possible new therapeutic target.
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Myopia is the most common refractive error worldwide. This cannot be explained by genetic factors alone, therefore, environmental factors may play an important role. Hence, the main objective of this study was to analyse whether outdoor exposure could exert a protective effect against the development of myopia in a cohort of young adults and to investigate ultraviolet autofluorescence (CUVAF), as a biomarker of time spent outdoors. A cross-sectional observational study was carried out using two cohorts. A total of 208 participants were recruited, 156 medical students and 52 environmental science students. The data showed that 66.66% of the medical students were myopic, while 50% of the environmental science students were myopic (p = 0.021). Environmental science students spent significantly more hours per week doing outdoor activities than medical students (p < 0.0001), but there was no significant difference with respect to near work activities between them. In both cohorts, the degree of myopia was inversely associated with CUVAF, and a statistically significant positive correlation was observed between spherical equivalent and CUVAF (Pearson's r = 0.248). In conclusion, outdoor activities could reduce the onset and progression of myopia not only in children, but also in young adults. In addition, CUVAF represents an objective, non-invasive biomarker of outdoor exposure that is inversely associated with myopia.
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The main aim of this study was to compare refraction measurements with and without cycloplegia from two refractors devices, (TRK-2P autorefractometer (TRK-2P) and wavefront-based refraction Visionix 130 (VX130)) in children and adolescents. This descriptive observational study included 20 myopic eyes and 40 hyperopic eyes measured in two different Spanish hospitals. Cycloplegia was carried out by three drops of cyclopentolate hydrochloride 1% (Colircusí cycloplegic, Alcon Healthcare S.A., Barcelona). The mean age of the myopia group was 12.40 ± 3.48 years; for the hyperopia group, the mean age was 7.37 ± 2.47 years. In the myopia group, autorefraction and wavefront-based refraction did not show clinically significant differences in any components between with and without cycloplegia. The hyperopia group showed statistical and clinically significant differences in sphere and SE components between relaxed and non-relaxed states of accommodation, although the cylindrical components were not clinically different. In this study, we considered a value of ≥0.50D as a clinically significant difference in refraction. Therefore, both devices were capable of obtaining accurate refractions without cyclopegia in myopia children, although they did not avoid instrument myopia and accommodation involved in hyperopia children. Moreover, both refractometers could be useful for astigmatism monitoring in children without the need for cycloplegic drops.
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A promising therapy for patients with B-cell lymphoma is based on vaccination with idiotype monoclonal antibodies (mAbs). Since idiotypes are different in each tumor, a personalized vaccine has to be produced for each patient. Expression of immunoglobulins with appropriate post-translational modifications for human use often requires the use of stable mammalian cells that can be scaled-up to reach the desired level of production. We have used a noncytopathic self-amplifying RNA vector derived from Semliki Forest virus (ncSFV) to generate BHK cell lines expressing murine follicular lymphoma-derived idiotype A20 mAb. ncSFV/BHK cell lines expressed approximately 2 mg/L/24 h of A20 mAb with proper quaternary structure and a glycosylation pattern similar to that of A20 mAb produced by hybridoma cells. A20 mAb purified from the supernatant of a ncSFV cell line, or from the hybridoma, was conjugated to keyhole limpet hemocyanin and used to immunize Balb/c mice by administration of four weekly doses of 25 µg of mAb. Both idiotype mAbs were able to induce a similar antitumor protection and longer survival compared to non-immunized mice. These results indicate that the ncSFV RNA vector could represent a quick and efficient system to produce patient-specific idiotypes with potential application as lymphoma vaccines.
Assuntos
Alphavirus/genética , Anticorpos Monoclonais/administração & dosagem , Vacinas Anticâncer/administração & dosagem , Vetores Genéticos/administração & dosagem , Idiótipos de Imunoglobulinas/imunologia , Linfoma de Células B/terapia , Vacinação/métodos , Animais , Anticorpos Monoclonais/imunologia , Apoptose , Vacinas Anticâncer/genética , Vacinas Anticâncer/imunologia , Proliferação de Células , Feminino , Vetores Genéticos/genética , Humanos , Linfoma de Células B/imunologia , Linfoma de Células B/patologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Age-related macular degeneration (AMD) is a multifactorial disease of the retina featured by dysfunction of retinal pigmented epithelial (RPE) and loss of photoreceptor cells under oxidative stress and inflammatory conditions. Vitamin D and antioxidants have beneficial effects against retinal degenerative diseases, such as AMD. We investigated the impact of associating vitamin D (ND) with a nutritional antioxidant complex (Nutrof Total®; N) on oxidative stress and inflammation-like induced conditions by H2O2 and LPS, respectively, in human retinal epithelial (ARPE-19) and human retinal endothelial (HREC) cells. Application of either N or ND treatments to H2O2-induced media in ARPE-19 cells counteracted late apoptosis, attenuated oxidative DNA damage, and increased cell proliferation. Significant reduction in the expression levels of MCP1, IL-8, and IL6 cytokines was observed following application of either N or ND treatments under LPS-induced conditions in ARPE-19 cells and in MCP-1 and IL12p70 cytokine levels in HREC cells. ND and not N revealed significant downregulation of IFNγ in ARPE-19 cells, and of IL-6 and IL-18 in HREC cells. In conclusion, adding vitamin D to Nutrof Total® protects in a synergistic way against oxidative and inflammatory stress-induced conditions in retinal epithelial and endothelial cells.
Assuntos
Anti-Inflamatórios/farmacologia , Células Endoteliais/patologia , Degeneração Macular/patologia , Epitélio Pigmentado da Retina/patologia , Vitamina D/farmacologia , 8-Hidroxi-2'-Desoxiguanosina/metabolismo , Antioxidantes/farmacologia , Apoptose/efeitos dos fármacos , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Citocinas/metabolismo , Células Endoteliais/efeitos dos fármacos , Humanos , Oxirredução/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Epitélio Pigmentado da Retina/efeitos dos fármacosRESUMO
Direct cardiac reprogramming has emerged as an interesting approach for the treatment and regeneration of damaged hearts through the direct conversion of fibroblasts into cardiomyocytes or cardiovascular progenitors. However, in studies with human cells, the lack of reporter fibroblasts has hindered the screening of factors and consequently, the development of robust direct cardiac reprogramming protocols.In this study, we have generated functional human NKX2.5GFP reporter cardiac fibroblasts. We first established a new NKX2.5GFP reporter human induced pluripotent stem cell (hiPSC) line using a CRISPR-Cas9-based knock-in approach in order to preserve function which could alter the biology of the cells. The reporter was found to faithfully track NKX2.5 expressing cells in differentiated NKX2.5GFP hiPSC and the potential of NKX2.5-GFP + cells to give rise to the expected cardiac lineages, including functional ventricular- and atrial-like cardiomyocytes, was demonstrated. Then NKX2.5GFP cardiac fibroblasts were obtained through directed differentiation, and these showed typical fibroblast-like morphology, a specific marker expression profile and, more importantly, functionality similar to patient-derived cardiac fibroblasts. The advantage of using this approach is that it offers an unlimited supply of cellular models for research in cardiac reprogramming, and since NKX2.5 is expressed not only in cardiomyocytes but also in cardiovascular precursors, the detection of both induced cell types would be possible. These reporter lines will be useful tools for human direct cardiac reprogramming research and progress in this field.
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Diabetic retinopathy is a vision-threatening microvascular complication of diabetes and is one of the leading causes of blindness. Oxidative stress and inflammation play a major role in its pathogenesis, and new therapies counteracting these contributors could be of great interest. In the current study, we investigated the role of vitamin D against oxidative stress and inflammation in human retinal pigment epithelium (RPE) and human retinal endothelial cell lines. We demonstrate that vitamin D effectively counteracts the oxidative stress induced by hydrogen peroxide (H2O2). In addition, the increased levels of proinflammatory proteins such as Interleukin (IL)-6, IL-8, Monocyte chemoattractant protein (MCP)-1, Interferon (IFN)-γ, and tumor necrosis factor (TNF)-α triggered by lipopolysaccharide (LPS) exposure were significantly decreased by vitamin D addition. Interestingly, the increased IL-18 only decreased by vitamin D addition in endothelial cells but not in RPE cells, suggesting a main antiangiogenic role under inflammatory conditions. Moreover, H2O2 and LPS induced the alteration and morphological damage of tight junctions in adult retinal pigment epithelium (ARPE-19) cells that were restored under oxidative and inflammatory conditions by the addition of vitamin D to the media. In conclusion, our data suggest that vitamin D could protect the retina by enhancing antioxidant defense and through exhibiting anti-inflammatory properties.
RESUMO
High myopia and the subsequent degenerative changes of the retina, choroid, and sclera, known as myopic maculopathy (MM), are a serious visual problem in many Asian countries, and are beginning to be so in the south of Europe, especially in the Mediterranean. It is therefore necessary to carry out genetic and environmental studies to determine the possible causes of this disease. This study aims to verify if the genetic factors that have been most related to Asian populations are also associated in two Spanish cohorts. Eight SNPs from six genes (PAX6, SCO2, CCDC102B, BLID, chromosome 15q14, and COL8A1) along with demographic, ophthalmic and environmental factors were analysed in two cohorts from a total of 365 highly myopic subjects and 177 control subjects. The genetic analysis showed that COL8A1 SNP rs13095226 was associated with the development of choroidal neovascularization (CNV) and also seems to play an important role in the increase of axial length. The SNP rs634990 of chromosome 15q14 also showed a significant association with MM, although this was lost after the Bonferroni correction. Additional demographic and environmental factors, namely age, sex, smoking status, and pregnancy history, were also found to be associated with MM and CNV in this population.
Assuntos
Meio Ambiente , Degeneração Macular/epidemiologia , Degeneração Macular/genética , Miopia/complicações , Adulto , Idoso , Alelos , Olho/metabolismo , Feminino , Genótipo , Humanos , Degeneração Macular/complicações , Masculino , Pessoa de Meia-Idade , Espanha/epidemiologiaRESUMO
Transforming growth factor-beta (TGF-beta) is a cytokine with potent immunosuppressive effects and is overexpressed in many tumors. Therefore, development of molecules able to inhibit TGF-beta is of paramount importance to improve the efficacy of antitumor immunotherapy. TGF-beta inhibitor peptides P144 and P17 were combined with the administration of adjuvant molecules poly(I:C) and agonistic anti-CD40 antibodies, and their effect on the growth of E.G7-OVA established tumors and on antitumor immune response was evaluated. Tumor rejection efficacy of a single administration of adjuvants was enhanced from 15 to 70 % when combined with repeated injections of TGF-beta inhibitor peptides. Simultaneous administration of adjuvants and TGF-beta inhibitor peptides was required for maximal therapeutic efficacy. Although tumor cells produced TGF-beta, it was found that the beneficial effect of peptide administration was mainly due to the inhibition of TGF-beta produced by regulatory CD4(+)CD25(+) T cells rather than by tumor cells. The enhanced antitumor effect was accompanied by a higher activity of dendritic cells, natural killer cells and tumor antigen-specific T cells, as well as by a decrease in the number of myeloid-derived suppressor cells. In conclusion, administration of peptide inhibitors of TGF-beta in therapeutic vaccination enhances the efficacy of immunotherapy by increasing antitumor immune responses. These peptide inhibitors may have important applications for current immunotherapeutic strategies.
Assuntos
Imunoterapia , Melanoma Experimental/terapia , Fragmentos de Peptídeos/farmacologia , Fator de Crescimento Transformador beta/antagonistas & inibidores , Adjuvantes Imunológicos/uso terapêutico , Animais , Antígenos CD40/imunologia , Células Dendríticas/imunologia , Feminino , Citometria de Fluxo , Proteínas de Homeodomínio/fisiologia , Humanos , Células Matadoras Naturais/imunologia , Melanoma Experimental/imunologia , Melanoma Experimental/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Ovalbumina/imunologia , Poli I-C/imunologia , Poli I-C/uso terapêutico , Receptores de Fatores de Crescimento Transformadores beta , Taxa de Sobrevida , Linfócitos T Citotóxicos/imunologia , Linfócitos T Reguladores/imunologia , Células Tumorais CultivadasRESUMO
Age-related macular degeneration (AMD) is a progressive retinal disorder characterized by imbalanced pro- and antiangiogenic signals. The aim of this study was to evaluate the effect of ex vivo cell-based gene therapy with stable expression of human pigment epithelium-derived factor (PEDF) release using the non-viral Sleeping Beauty (SB100X) transposon system delivered by miniplasmids free of antibiotic resistance markers (pFAR4). Retinal pigment epithelial (RPE) cells and iris pigment epithelial (IPE) cells were co-transfected with pFAR4-inverted terminal repeats (ITRs) CMV-PEDF-BGH and pFAR4-CMV-SB100X-SV40 plasmids. Laser-induced choroidal neovascularization (CNV) was performed in rats, and transfected primary cells (transfected RPE [tRPE] and transfected IPE [tIPE] cells) were injected into the subretinal space. The leakage and CNV areas, vascular endothelial growth factor (VEGF), PEDF protein expression, metalloproteinases 2 and 9 (MMP-2/9), and microglial/macrophage markers were measured. Injection with tRPE/IPE cells significantly reduced the leakage area at 7 and 14 days and the CNV area at 7 days. There was a significant increase in PEDF and the PEDF/VEGF ratio with tRPE cells and a reduction in the MMP-2 activity. Our data demonstrated that ex vivo non-viral gene therapy reduces CNV and could be an effective and safe therapeutic option for angiogenic retinal diseases.
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Pigment epithelium derived factor (PEDF) is a potent antiangiogenic, neurotrophic, and neuroprotective molecule that is the endogenous inhibitor of vascular endothelial growth factor (VEGF) in the retina. An ex vivo gene therapy approach based on transgenic overexpression of PEDF in the eye is assumed to rebalance the angiogenic-antiangiogenic milieu of the retina, resulting in growth regression of choroidal blood vessels, the hallmark of neovascular age-related macular degeneration. Here, we show that rat pigment epithelial cells can be efficiently transfected with the PEDF-expressing non-viral hyperactive Sleeping Beauty transposon system delivered in a form free of antibiotic resistance marker miniplasmids. The engineered retinal and iris pigment epithelium cells secrete high (141 ± 13 and 222 ± 14 ng) PEDF levels in 72 hr in vitro. In vivo studies showed cell survival and insert expression during at least 4 months. Transplantation of the engineered cells to the subretinal space of a rat model of choroidal neovascularization reduces almost 50% of the development of new vessels.
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We assessed the effect of single and repeated doses of bevacizumab, ranibizumab, and aflibercept on cell viability, proliferation, permeability, and apoptosis of ARPE-19 cells. MTT and BrdU assays were used to determine viability and proliferation after single or repeated doses of anti-VEGF drugs under normal and oxidative stress conditions. Caspase-3 expression after single and repeated doses of the 3 drugs was assessed using immunofluorescence. Transepithelial-electrical-resistance (TER) was measured to study the effect of anti-VEGFs on retinal pigment epithelium (RPE) permeability under normal and oxidative stress conditions. Flow cytometry was used to detect intracellular accumulation of the drugs. Finally, a wound healing assay was performed to investigate the effect of the drugs on RPE cell migration. Single and multiple doses of anti-VEGF drugs had no effect on cell viability and proliferation. The oxidative effect of H2O2 decreased cell viability and proliferation; however, no difference was observed between anti-VEGF treatments. Immunofluorescence performed after single and repeated doses of the drugs revealed some caspase-3 expression. Interestingly, anti-VEGFs restored the increased permeability induced by H2O2. The 3 drugs accumulated inside the cells and were detectable 5 days after treatment. Finally, none of the drugs affected migration. In conclusion, no measureable toxic effect was observed after single or repeated doses of VEGF antagonists under normal and oxidative stress. Intracellular accumulation of the drugs does not seem to be toxic or affect cell functions. Our study suggests that anti-VEGFs could have a preventive effect on the maintenance of the RPE barrier under oxidative stress.
Assuntos
Bevacizumab/toxicidade , Estresse Oxidativo , Ranibizumab/toxicidade , Receptores de Fatores de Crescimento do Endotélio Vascular/toxicidade , Proteínas Recombinantes de Fusão/toxicidade , Epitélio Pigmentado da Retina/efeitos dos fármacos , Fator A de Crescimento do Endotélio Vascular/antagonistas & inibidores , Bevacizumab/metabolismo , Caspase 3/metabolismo , Linhagem Celular , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Impedância Elétrica , Humanos , Espaço Intracelular/metabolismo , Ranibizumab/metabolismo , Receptores de Fatores de Crescimento do Endotélio Vascular/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Epitélio Pigmentado da Retina/citologia , Epitélio Pigmentado da Retina/metabolismoRESUMO
Choroidal neovascularization (CNV) commonly occurs in age related macular degeneration and pathological myopia patients. In this study we conducted a case-control prospective study including 431 participants. The aim of this study was to determine the potential association between 10 single nucleotide polymorphisms (SNPs) located in 4 different genetic regions (CFI, COL8A1, LIPC, and APOE), and choroidal neovascularization in age-related macular degeneration and the development of choroidal neovascularization in highly myopic eyes of a Caucasian population. Univariate and multivariate logistic regression analysis adjusted for age, sex and hypertension was performed for each allele, genotype and haplotype frequency analysis. We found that in the univariate analysis that both single-nucleotide polymorphisms in COL8A1 gene (rs13095226 and rs669676) together with age, sex and hypertension were significantly associated with myopic CNV development in Spanish patients (p<0.05). After correcting for multiple testing none of the polymorphisms studied remained significantly associated with myopic CNV (p>0.05); however, analysis of the axial length between genotypes of rs13095226 revealed an important influence of COL8A1 in the development of CNV in high myopia. Furthermore we conducted a meta-analysis of COL8A1, CFI and LIPC genes SNPs (rs669676, rs10033900 and rs10468017) and found that only rs669676 of these SNPs were associated with high myopia neovascularization.
Assuntos
Neovascularização de Coroide/genética , Degeneração Macular/genética , Miopia Degenerativa/genética , Polimorfismo de Nucleotídeo Único , Idoso , Apolipoproteínas E/genética , Corioide/irrigação sanguínea , Corioide/patologia , Neovascularização de Coroide/complicações , Neovascularização de Coroide/patologia , Colágeno Tipo VIII/genética , Fator I do Complemento/genética , Feminino , Humanos , Lipase/genética , Degeneração Macular/complicações , Degeneração Macular/patologia , Masculino , Pessoa de Meia-Idade , Miopia Degenerativa/complicações , Miopia Degenerativa/patologia , Retina/patologiaRESUMO
Peptide vaccines derived from CD8+ T-cell epitopes have shown variable efficacy in cancer patients. Thus, some peptide vaccines are capable of activating CD8+ T-cell responses, even in the absence of CD4+ T-cell epitopes or dendritic cell (DC)-activating adjuvants. However, the mechanisms underlying the clinical activity of these potent peptides are poorly understood. Using CT26 and ovalbumin-expressing B16 murine allograft tumor models, we found that the antitumor effect of helper cell-independent CD8 T-cell peptide vaccines is inhibited by the blockade of CD40 ligand (CD40L) in vivo. Furthermore, in vitro stimulation with antigenic peptides of cells derived from immunized mice induced the expression of CD40L on the surface of CD8+ T cells and fostered DC maturation, an effect that was partially inhibited by CD40L-blocking antibodies. Interestingly, CD40L blockade also inhibited CD8+ T-cell responses, even in the presence of fully mature DCs, suggesting a role for CD40L not only in promoting DC maturation but also in mediating CD8+ T-cell co-stimulation. Importantly, these potent peptides share features with bona fide CD4 epitopes, since they foster responses against less immunogenic CD8+ T-cell epitopes in a CD40L-dependent manner. The analysis of peptides used for the vaccination of cancer patients in clinical trials showed that these peptides also induce the expression of CD40L on the surface of CD8+ T cells. Taken together, these results suggest that CD40L expression induced by potent CD8+ T-cell epitopes can activate antitumor CD8+ T-cell responses, potentially amplifying the immunological responses to less immunogenic CD8+ T-cell epitopes and bypassing the requirement for CD4+ helper T cells in vaccination protocols.