RESUMO
Macular degeneration (MD) is characterized by the progressive deterioration of the macula and represents one of the most prevalent causes of blindness worldwide. Abnormal intracellular accumulation of lipid droplets and pericellular deposits of lipid-rich material in the retinal pigment epithelium (RPE) called drusen are clinical hallmarks of different forms of MD including Doyne honeycomb retinal dystrophy (DHRD) and age-related MD (AMD). However, the appropriate molecular therapeutic target underlying these disorder phenotypes remains elusive. Here, we address this knowledge gap by comparing the proteomic profiles of induced pluripotent stem cell (iPSC)-derived RPEs (iRPE) from individuals with DHRD and their isogenic controls. Our analysis and follow-up studies elucidated the mechanism of lipid accumulation in DHRD iRPE cells. Specifically, we detected significant downregulation of carboxylesterase 1 (CES1), an enzyme that converts cholesteryl ester to free cholesterol, an indispensable process in cholesterol export. CES1 knockdown or overexpression of EFEMP1R345W, a variant of EGF-containing fibulin extracellular matrix protein 1 that is associated with DHRD and attenuated cholesterol efflux and led to lipid droplet accumulation. In iRPE cells, we also found that EFEMP1R345W has a hyper-inhibitory effect on epidermal growth factor receptor (EGFR) signaling when compared to EFEMP1WT and may suppress CES1 expression via the downregulation of transcription factor SP1. Taken together, these results highlight the homeostatic role of cholesterol efflux in iRPE cells and identify CES1 as a mediator of cholesterol efflux in MD.
Assuntos
Colesterol/metabolismo , Degeneração Macular/metabolismo , Epitélio Pigmentado da Retina/metabolismo , Adolescente , Adulto , Hidrolases de Éster Carboxílico/genética , Diferenciação Celular/genética , Citocinas/metabolismo , Receptores ErbB/metabolismo , Proteínas da Matriz Extracelular/genética , Proteínas da Matriz Extracelular/metabolismo , Humanos , Inflamação/metabolismo , Metabolismo dos Lipídeos , Degeneração Macular/patologia , Pessoa de Meia-Idade , Drusas do Disco Óptico/congênito , Drusas do Disco Óptico/metabolismo , Proteômica , Proteínas Proto-Oncogênicas c-akt/metabolismo , Epitélio Pigmentado da Retina/patologia , Transdução de Sinais , Fator de Transcrição Sp1/metabolismo , Transcrição Gênica , Resposta a Proteínas não DobradasRESUMO
Mutations in rhodopsin (RHO) are the most common causes of autosomal dominant retinitis pigmentosa (adRP), accounting for 20% to 30% of all cases worldwide. However, the high degree of genetic heterogeneity makes development of effective therapies cumbersome. To provide a universal solution to RHO-related adRP, we devised a CRISPR-based, mutation-independent gene ablation and replacement (AR) compound therapy carried by a dual AAV2/8 system. Moreover, we developed a novel hRHOC110R/hRHOWT humanized mouse model to assess the AR treatment in vivo. Results show that this humanized RHO mouse model exhibits progressive rod-cone degeneration that phenocopies hRHOC110R/hRHOWT patients. In vivo transduction of AR AAV8 dual vectors remarkably ablates endogenous RHO expression and overexpresses exogenous WT hRHO. Furthermore, the administration of AR during adulthood significantly hampers photoreceptor degeneration both histologically and functionally for at least 6 months compared with sole gene replacement or surgical trauma control. This study demonstrates the effectiveness of AR treatment of adRP in the human genomic context while revealing the feasibility of its application for other autosomal dominant disorders.
Assuntos
Degeneração Retiniana , Retinose Pigmentar , Animais , Modelos Animais de Doenças , Genes Dominantes , Terapia Genética/métodos , Humanos , Camundongos , Mutação , Degeneração Retiniana/genética , Retinose Pigmentar/genética , Retinose Pigmentar/patologia , Retinose Pigmentar/terapia , Rodopsina/genética , Rodopsina/metabolismoRESUMO
D190N, a missense mutation in rhodopsin, causes photoreceptor degeneration in patients with autosomal dominant retinitis pigmentosa (adRP). Two competing hypotheses have been developed to explain why D190N rod photoreceptors degenerate: (a) defective rhodopsin trafficking prevents proteins from correctly exiting the endoplasmic reticulum, leading to their accumulation, with deleterious effects or (b) elevated mutant rhodopsin expression and unabated signaling causes excitotoxicity. A knock-in D190N mouse model was engineered to delineate the mechanism of pathogenesis. Wild type (wt) and mutant rhodopsin appeared correctly localized in rod outer segments of D190N heterozygotes. Moreover, the rhodopsin glycosylation state in the mutants appeared similar to that in wt mice. Thus, it seems plausible that the injurious effect of the heterozygous mutation is not related to mistrafficking of the protein, but rather from constitutive rhodopsin activity and a greater propensity for chromophore isomerization even in the absence of light.
Assuntos
Células Fotorreceptoras Retinianas Bastonetes/metabolismo , Retinose Pigmentar/patologia , Rodopsina/genética , Sequência de Aminoácidos , Animais , Modelos Animais de Doenças , Eletrorretinografia , Técnicas de Introdução de Genes , Glicosilação , Camundongos , Camundongos Endogâmicos C57BL , Mutação de Sentido Incorreto , Estrutura Terciária de Proteína , Retina/metabolismo , Retina/patologia , Retinose Pigmentar/metabolismo , Rodopsina/química , Rodopsina/metabolismo , Alinhamento de SequênciaRESUMO
Microglia cells (MGCs) play a key role in scavenging pathogens and phagocytosing cellular debris in the central nervous system and retina. Their activation, however, contributes to the progression of multiple degenerative diseases. Given the potential damage created by MGCs, it is important to better understand their mechanism of activation. Here, we explored the role of MGCs in the context of retinitis pigmentosa (RP) by using four independent preclinical mouse models. For therapeutic modeling, tamoxifen-inducible CreER was introduced to explore changes in MGCs when RP progression halted. The phenotypes of the MGCs were observed using live optical coherence tomography, live autofluorescence, and immunohistochemistry. We found that, regardless of genetic background, MGCs were activated in neurodegenerative conditions and migrated beyond the layers where they are typically found to the inner and outer segments, where degeneration was ongoing. Genetic rescue not only halted degeneration but also deactivated MGCs, regardless of whether the intervention occurred at the early, middle, or late stage of the disease. These findings suggest that halting long-term disease progression may be more successful by downregulating MGC activity while co-administering the therapeutic intervention.
Assuntos
Microglia/patologia , Diester Fosfórico Hidrolases/genética , Retinose Pigmentar/diagnóstico por imagem , Tamoxifeno/farmacologia , Animais , Receptor 1 de Quimiocina CX3C/metabolismo , Modelos Animais de Doenças , Terapia Genética , Humanos , Camundongos , Diester Fosfórico Hidrolases/administração & dosagem , Retinose Pigmentar/genética , Retinose Pigmentar/metabolismo , Retinose Pigmentar/terapia , Tomografia de Coerência ÓpticaRESUMO
PURPOSE: To develop a universal gene therapy to overcome the genetic heterogeneity in retinitis pigmentosa (RP) resulting from mutations in rhodopsin (RHO). DESIGN: Experimental study for a combination gene therapy that uses both gene ablation and gene replacement. PARTICIPANTS: This study included 2 kinds of human RHO mutation knock-in mouse models: RhoP23H and RhoD190N. In total, 23 RhoP23H/P23H, 43 RhoP23H/+, and 31 RhoD190N/+ mice were used for analysis. METHODS: This study involved gene therapy using dual adeno-associated viruses (AAVs) that (1) destroy expression of the endogenous Rho gene in a mutation-independent manner via an improved clustered regularly interspaced short palindromic repeats-based gene deletion and (2) enable expression of wild-type protein via exogenous cDNA. MAIN OUTCOME MEASURES: Electroretinographic and histologic analysis. RESULTS: The thickness of the outer nuclear layer (ONL) after the subretinal injection of combination ablate-and-replace gene therapy was approximately 17% to 36% more than the ONL thickness resulting from gene replacement-only therapy at 3 months after AAV injection. Furthermore, electroretinography results demonstrated that the a and b waves of both RhoP23H and RhoD190N disease models were preserved more significantly using ablate-and-replace gene therapy (P < 0.001), but not by gene replacement monotherapy. CONCLUSIONS: As a proof of concept, our results suggest that the ablate-and-replace strategy can ameliorate disease progression as measured by photoreceptor structure and function for both of the human mutation knock-in models. These results demonstrate the potency of the ablate-and-replace strategy to treat RP caused by different Rho mutations. Furthermore, because ablate-and-replace treatment is mutation independent, this strategy may be used to treat a wide array of dominant diseases in ophthalmology and other fields. Clinical trials using ablate-and-replace gene therapy would allow researchers to determine if this strategy provides any benefits for patients with diseases of interest.
Assuntos
Terapia Genética/métodos , Retinose Pigmentar/genética , Animais , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Modelos Animais de Doenças , Eletrorretinografia , Vetores Genéticos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Retinose Pigmentar/diagnóstico , Retinose Pigmentar/terapiaRESUMO
A single amino acid mutation near the active site of the CAPN5 protease was linked to the inherited blinding disorder, autosomal dominant neovascular inflammatory vitreoretinopathy (ADNIV, OMIM #193235). In homology modeling with other calpains, this R243L CAPN5 mutation was situated in a mobile loop that gates substrate access to the calcium-regulated active site. In in vitro activity assays, the mutation increased calpain protease activity and made it far more active at low concentrations of calcium. To test whether the disease allele could yield an animal model of ADNIV, we created transgenic mice expressing human (h) CAPN5(R243L) only in the retina. The resulting hCAPN5(R243L) transgenic mice developed a phenotype consistent with human uveitis and ADNIV, at the clinical, histological and molecular levels. The fundus of hCAPN5(R243L) mice showed enhanced autofluorescence (AF) and pigment changes indicative of reactive retinal pigment epithelial cells and photoreceptor degeneration. Electroretinography showed mutant mouse eyes had a selective loss of the b-wave indicating an inner-retina signaling defect. Histological analysis of mutant mouse eyes showed protein extravasation from dilated vessels into the anterior chamber and vitreous, vitreous inflammation, vitreous and retinal fibrosis and retinal degeneration. Analysis of gene expression changes in the hCAPN5(R243L) mouse retina showed upregulation of several markers, including members of the Toll-like receptor pathway, chemokines and cytokines, indicative of both an innate and adaptive immune response. Since many forms of uveitis share phenotypic characteristics of ADNIV, this mouse offers a model with therapeutic testing utility for ADNIV and uveitis patients.
Assuntos
Calpaína , Doenças Genéticas Inatas , Uveíte , Animais , Calpaína/genética , Calpaína/metabolismo , Linhagem Celular , Doenças Genéticas Inatas/genética , Doenças Genéticas Inatas/metabolismo , Doenças Genéticas Inatas/patologia , Humanos , Inflamação , Camundongos , Camundongos Transgênicos , Uveíte/genética , Uveíte/metabolismo , Uveíte/patologiaRESUMO
Massive parallel sequencing enables identification of numerous genetic variants in mutant organisms, but determining pathogenicity of any one mutation can be daunting. The most commonly studied preclinical model of retinitis pigmentosa called the "rodless" (rd1) mouse is homozygous for two mutations: a nonsense point mutation (Y347X) and an intronic insertion of a leukemia virus (Xmv-28). Distinguishing which mutation causes retinal degeneration is still under debate nearly a century after the discovery of this model organism. Here, we performed gene editing using the CRISPR/Cas9 system and demonstrated that the Y347X mutation is the causative variant of disease. Genome editing in the first generation produced animals that were mosaic for the corrected allele but still showed neurofunction preservation despite low repair frequencies. Furthermore, second-generation CRISPR-repaired mice showed an even more robust rescue and amelioration of the disease. This predicts excellent outcomes for gene editing in diseased human tissue, as Pde6b, the mutated gene in rd1 mice, has an orthologous intron-exon relationship comparable with the human PDE6B gene. Not only do these findings resolve the debate surrounding the source of neurodegeneration in the rd1 model, but they also provide the first example of homology-directed recombination-mediated gene correction in the visual system.
Assuntos
Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Reparo do DNA , Mutação , Retinose Pigmentar/genética , Animais , Sistemas CRISPR-Cas , Nucleotídeo Cíclico Fosfodiesterase do Tipo 6/genética , Nucleotídeo Cíclico Fosfodiesterase do Tipo 6/metabolismo , Modelos Animais de Doenças , Eletrorretinografia , Éxons , Edição de Genes , Loci Gênicos , Recombinação Homóloga , Camundongos , Camundongos Transgênicos , Células Fotorreceptoras de Vertebrados/metabolismo , Células Fotorreceptoras de Vertebrados/patologia , RNA Guia de Cinetoplastídeos , Degeneração Retiniana/genética , Degeneração Retiniana/patologiaRESUMO
While the past decade has seen great progress in mapping loci for common diseases, studying how these risk alleles lead to pathology remains a challenge. Age-related macular degeneration (AMD) affects 9 million older Americans, and is characterized by the loss of the retinal pigment epithelium (RPE). Although the closely linked genome-wide association studies ARMS2/HTRA1 genes, located at the chromosome 10q26 locus, are strongly associated with the risk of AMD, their downstream targets are unknown. Low population frequencies of risk alleles in tissue banks make it impractical to study their function in cells derived from autopsied tissue. Moreover, autopsy eyes from end-stage AMD patients, where age-related RPE atrophy and fibrosis are already present, cannot be used to determine how abnormal ARMS2/HTRA1 expression can initiate RPE pathology. Instead, induced pluripotent stem (iPS) cell-derived RPE from patients provides us with earlier stage AMD patient-specific cells and allows us to analyze the underlying mechanisms at this critical time point. An unbiased proteome screen of A2E-aged patient-specific iPS-derived RPE cell lines identified superoxide dismutase 2 (SOD2)-mediated antioxidative defense in the genetic allele's susceptibility of AMD. The AMD-associated risk haplotype (T-in/del-A) impairs the ability of the RPE to defend against aging-related oxidative stress. SOD2 defense is impaired in RPE homozygous for the risk haplotype (T-in/del-A; T-in/del-A), while the effect was less pronounced in RPE homozygous for the protective haplotype (G-Wt-G; G-Wt-G). ARMS2/HTRA1 risk alleles decrease SOD2 defense, making RPE more susceptible to oxidative damage and thereby contributing to AMD pathogenesis.
Assuntos
Alelos , Estudo de Associação Genômica Ampla/métodos , Linhagem Celular , Predisposição Genética para Doença/genética , Genótipo , Haplótipos/genética , HumanosRESUMO
Defects in Membrane Frizzled-related Protein (MFRP) cause autosomal recessive retinitis pigmentosa (RP). MFRP codes for a retinal pigment epithelium (RPE)-specific membrane receptor of unknown function. In patient-specific induced pluripotent stem (iPS)-derived RPE cells, precise levels of MFRP, and its dicistronic partner CTRP5, are critical to the regulation of actin organization. Overexpression of CTRP5 in naïve human RPE cells phenocopied behavior of MFRP-deficient patient RPE (iPS-RPE) cells. AAV8 (Y733F) vector expressing human MFRP rescued the actin disorganization phenotype and restored apical microvilli in patient-specific iPS-RPE cell lines. As a result, AAV-treated MFRP mutant iPS-RPE recovered pigmentation and transepithelial resistance. The efficacy of AAV-mediated gene therapy was also evaluated in Mfrp(rd6)/Mfrp(rd6) mice--an established preclinical model of RP--and long-term improvement in visual function was observed in AAV-Mfrp-treated mice. This report is the first to indicate the successful use of human iPS-RPE cells as a recipient for gene therapy. The observed favorable response to gene therapy in both patient-specific cell lines, and the Mfrp(rd6)/Mfrp(rd6) preclinical model suggests that this form of degeneration caused by MFRP mutations is a potential target for interventional trials.
Assuntos
Células-Tronco Pluripotentes Induzidas/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Epitélio Pigmentado da Retina/citologia , Retinose Pigmentar/terapia , Animais , Linhagem Celular , Colágeno/metabolismo , Dependovirus/genética , Dependovirus/metabolismo , Modelos Animais de Doenças , Feminino , Terapia Genética , Vetores Genéticos/administração & dosagem , Humanos , Células-Tronco Pluripotentes Induzidas/virologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Pessoa de Meia-Idade , Fenótipo , Retinose Pigmentar/patologia , Adulto JovemRESUMO
BACKGROUND AND AIMS: Acute exacerbation (AE) of chronic hepatitis B virus (HBV) infection is common and negatively impacts the clinical outcome. Although upsurge of viral load always precedes or coincides with AE, the underlying immunological mechanisms remain unclear and were investigated. METHODS: We prospectively followed the serum cytokine/chemokine profiles, viral load, and alanine aminotransferase (ALT) levels in 250 patients and identified 44 consecutive patients (male: 72.7%; age: 40.4 ± 9.7 years; hepatitis B e antigen [HBeAg] positivity: 63.6%; genotype B/C: 75%/25%) who developed AE during the follow-up in a medical center. The impact of clinical characteristics (age, gender, HBeAg, ALT, HBV genotype), cytokines (tumor necrosis factor-alpha, interferon gamma, interleukin [IL]-2, IL-4, IL-6, and IL-10), and chemokines (CXCL10/interferon gamma-induced protein [IP]-10, CCL2/MCP-1, CXCL9/MIG, CCL5/RANTES, and CXCL8/IL-8) on the serum HBV DNA dynamics at different time points (baseline, peak of serum HBV DNA level, peak of serum ALT level, and after AE) were analyzed. RESULTS: Of 44 patients, serum HBV DNA level surged before the peak of serum ALT level in 23 (52.3%), and coincided with the peak of ALT in 21 (47.7%). The upsurge of serum viral load significantly correlated with the increase of IL-10 (P = 0.0037) and CXCL10/IP-10 (P = 0.0044). Upsurge of serum viral load was preceded by an increase in serum IL-4 (P < 0.05), IL-6 (P < 0.05), and IL-10 (P < 0.05). Combination of HBV genotype, IL-6 level at baseline, and ALT level at the peak of serum HBV DNA reliably predicted subsequent AE pattern (P = 0.0116). CONCLUSIONS: Enhanced Th2 activity is likely involved in the surge of HBV DNA level before hepatitis exacerbation.
Assuntos
Quimiocinas/sangue , Citocinas/sangue , DNA Viral/sangue , Vírus da Hepatite B/genética , Hepatite B Crônica , Adulto , Alanina Transaminase/sangue , Biomarcadores/sangue , Quimiocina CXCL10/sangue , Progressão da Doença , Feminino , Seguimentos , Hepatite B Crônica/sangue , Hepatite B Crônica/virologia , Humanos , Interleucina-10/sangue , Interleucina-4/sangue , Interleucina-6/sangue , Masculino , Pessoa de Meia-Idade , Carga ViralRESUMO
PURPOSE: The objective of this study was to determine whether cells from the conjunctiva could be reprogrammed into induced pluripotent stem (iPS) cells, providing an alternative source of stem cells. METHODS: We employed a doxycycline-induced reprogrammable mouse strain to generate iPS cells from conjunctiva. The identity of the stem cells was confirmed by reverse transcription polymerase chain reaction (RT-PCR) and immunofluorescence assays. Immunocytochemistry and teratoma assays are established means for scoring stem cell pluripotency. The reprogramming efficiencies of conjunctival cells and ear fibroblasts were compared. RESULTS: We confirmed the identity of the stem cells and demonstrated expression of pluripotency markers (OCT4, SOX2, NANOG, and SSEA1), as tested by RT-PCR and immunofluorescence assays. In addition, derived iPS cells differentiated successfully into embryoid bodies, and showed teratoma formation when injected into immunodeficient mice. Reprogramming conjunctival tissue is as efficient as reprogramming ear fibroblasts. Conjunctiva-iPS exhibited classic features of embryonic stem (ES) cells with respect to morphology, expression of surface antigens, and pluripotency-associated transcription factors, capacity to differentiate in vitro, and the ability to form all three germ layers in vivo. CONCLUSION: The present study demonstrated that conjunctival cells, which are readily obtained during the course of many routine conjunctival biopsies and ophthalmic procedures, can be another reliable source of iPS cells.
Assuntos
Túnica Conjuntiva/citologia , Células-Tronco Pluripotentes Induzidas/citologia , Animais , Antibacterianos/farmacologia , Biomarcadores/metabolismo , Técnicas de Cultura de Células , Técnicas de Cocultura , Túnica Conjuntiva/efeitos dos fármacos , Túnica Conjuntiva/metabolismo , Doxiciclina/farmacologia , Fibroblastos/citologia , Técnica Indireta de Fluorescência para Anticorpo , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Células-Tronco Pluripotentes Induzidas/metabolismo , Cariotipagem , Antígenos CD15/genética , Antígenos CD15/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Proteína Homeobox Nanog , Fator 3 de Transcrição de Octâmero/genética , Fator 3 de Transcrição de Octâmero/metabolismo , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Transcrição SOXB1/genética , Fatores de Transcrição SOXB1/metabolismoRESUMO
BACKGROUND: Retinitis pigmentosa (RP) is a genetically heterogeneous group of degenerative disorders causing progressive vision loss due to photoreceptor death. RP affects other retinal cells, including the retinal pigment epithelium (RPE). MicroRNAs (miRs) are implicated in RP pathogenesis, and downregulating miR-181a/b has shown therapeutic benefit in RP mouse models by improving mitochondrial function. This study investigates the expression profile of miR-181a/b in RPE cells and the neural retina during RP disease progression. We also evaluate how miR-181a/b downregulation, by knocking out miR-181a/b-1 cluster in RPE cells, confers therapeutic efficacy in an RP mouse model and explore the mechanisms underlying this process. RESULTS: Our findings reveal distinct expression profiles, with downregulated miR-181a/b in RPE cells suggesting a protective response and upregulated miR-181a/b in the neural retina indicating a role in disease progression. We found that miR-181a/b-2, encoded in a separate genomic cluster, compensates for miR-181a/b-1 ablation in RPE cells at late time points. The transient downregulation of miR-181a/b in RPE cells at post-natal week 6 (PW6) led to improved RPE morphology, retarded photoreceptor degeneration and decreased RPE aerobic glycolysis. CONCLUSIONS: Our study elucidates the underlying mechanisms associated with the therapeutic modulation of miR-181a/b, providing insights into the metabolic processes linked to its RPE-specific downregulation. Our data further highlights the impact of compensatory regulation between miR clusters with implications for the development of miR-based therapeutics.
RESUMO
Retinitis pigmentosa (RP) is one of the most common forms of hereditary neurodegeneration. It is caused by one or more of at least 3,100 mutations in over 80 genes that are primarily expressed in rod photoreceptors. In RP, the primary rod-death phase is followed by cone death, regardless of the underlying gene mutation that drove the initial rod degeneration. Dampening the oxidation of glycolytic end products in rod mitochondria enhances cone survival in divergent etiological disease models independent of the underlying rod-specific gene mutations. Therapeutic editing of the prolyl hydroxylase domain-containing protein gene (PHD2, also known as Egln1) in rod photoreceptors led to the sustained survival of both diseased rods and cones in both preclinical autosomal-recessive and dominant RP models. Adeno-associated virus-mediated CRISPR-based therapeutic reprogramming of the aerobic glycolysis node may serve as a gene-agnostic treatment for patients with various forms of RP.
Assuntos
Células Fotorreceptoras Retinianas Bastonetes , Retinose Pigmentar , Animais , Humanos , Células Fotorreceptoras Retinianas Bastonetes/metabolismo , Retinose Pigmentar/genética , Retinose Pigmentar/metabolismo , Retinose Pigmentar/terapia , Células Fotorreceptoras Retinianas Cones/metabolismo , Modelos Animais de DoençasRESUMO
Selective detection of biomarkers at low concentrations in blood is crucial for the clinical diagnosis of many diseases but remains challenging. In this work, we aimed to develop an ultrasensitive immunoassay that can detect biomarkers in serum with an attomolar limit of detection (LOD). We proposed a sandwich-type heterogeneous immunosensor in a 3 × 3 well array format by integrating a resonant waveguide grating (RWG) substrate with upconversion nanoparticles (UCNPs). UCNPs were used to label a target biomarker captured by capture antibody molecules immobilized on the surface of the RWG substrate, and the RWG substrate was used to enhance the upconversion luminescence (UCL) of UCNPs through excitation resonance. The LOD of the immunosensor was greatly reduced due to the increased UCL of UCNPs and the reduction of nonspecific adsorption of detection antibody-conjugated UCNPs on the RWG substrate surface by coating the RWG substrate surface with a carboxymethyl dextran layer. The immunosensor exhibited an extremely low LOD [0.24 fg/mL (9.1 aM)] and wide detection range (1 fg/mL to 100 pg/mL) in the detection of cardiac troponin I (cTnI). The cTnI concentrations in human serum samples collected at different times during cyclophosphamide, epirubicin, and 5-fluorouracil (CEF) chemotherapy in a breast cancer patient were measured by an immunosensor, and the results showed that the CEF chemotherapy did cause cardiotoxicity in the patient. Having a higher number of wells in such an array-based biosensor, the sensor can be developed as a high-throughput diagnostic tool for clinically important biomarkers.
Assuntos
Técnicas Biossensoriais , Nanopartículas , Humanos , Troponina I , Imunoensaio/métodos , Nanopartículas/química , Epirubicina , BiomarcadoresRESUMO
Ferulic acid (FA) is an antioxidant and photoprotective agent used in biomedical and cosmetic formulations to prevent skin cancer and senescence. Although FA exhibits numerous health benefits, physicochemical instability leading to decomposition hinders its efficacy. To minimize inherent decomposition, a FA-containing biodegradable polymer was prepared via solution polymerization to chemically incorporate FA into a poly(anhydride-ester). The polymer was characterized using nuclear magnetic resonance and infrared spectroscopies. The molecular weight and thermal properties were also determined. In vitro studies demonstrated that the polymer was hydrolytically degradable, thus providing controlled release of the chemically incorporated bioactive with no detectable decomposition. The polymer degradation products were found to exhibit antioxidant and antibacterial activity comparable to that of free FA, and in vitro cell viability studies demonstrated that the polymer is noncytotoxic toward fibroblasts. This renders the polymer a potential candidate for use as a controlled release system for skin care formulations.
Assuntos
Anidridos/química , Antioxidantes/química , Materiais Biocompatíveis/síntese química , Ácidos Cumáricos/análise , Ésteres/química , Poliésteres/síntese química , Anidridos/farmacologia , Animais , Antibacterianos/química , Antibacterianos/farmacologia , Antioxidantes/farmacologia , Materiais Biocompatíveis/farmacologia , Sobrevivência Celular/efeitos dos fármacos , Ácidos Cumáricos/química , Ácidos Cumáricos/farmacologia , Escherichia coli/efeitos dos fármacos , Ésteres/farmacologia , Células L , Espectroscopia de Ressonância Magnética , Camundongos , Peso Molecular , Poliésteres/farmacologia , Soluções/químicaRESUMO
Cytokine and antiangiogenic gene therapies have proved effective in implanted hepatocellular carcinoma (HCC) models in which small tumor burdens were established in small rodents. These models, however, may not reflect human HCCs, which are frequently detected at a stage when tumors are large and multifocal. In addition, HCC in patients is often associated with viral hepatitis. To investigate the effectiveness of a mixture type of gene therapy strategy on large tumor burdens, we used the woodchuck model in which woodchuck hepatitis virus-induced HCCs are large and multifocal, simulating the conditions in humans. Adenoviruses encoding antiangiogenic factors (pigment epithelium-derived factor and endostatin) or cytokines (GM-CSF and IL-12) were delivered via the hepatic artery separately or in combination into woodchuck livers bearing HCCs. Our results showed that the mixture type of strategy, which contained two cytokines and two antiangiogenic factors, had better antitumor effects on large tumors as compared with monotherapy either with antiangiogenic or cytokine genes. The immunotherapy recruited significant levels of CD3(+) T cells that infiltrated the tumors, whereas the antiangiogenesis-based therapy significantly reduced tumor vasculature. The mixture type of gene therapy achieved both effects. In addition, it induced high levels of natural killer cells and apoptotic cells and reduced the levels of immunosuppressive effectors in the tumor regions. Hence, antiangiogenic therapy may provide the advantage of reducing immune tolerance in large tumors, making them more vulnerable to the immune reactions. Our study implies that in the future, the combination therapy may prove effective for the treatment of patients with advanced HCC.
Assuntos
Terapia Genética/métodos , Imunoterapia/métodos , Neoplasias Hepáticas Experimentais/terapia , Doenças dos Roedores/terapia , Alanina Transaminase/sangue , Inibidores da Angiogênese/genética , Inibidores da Angiogênese/metabolismo , Animais , Aspartato Aminotransferases/sangue , Terapia Combinada , Endostatinas/genética , Endostatinas/metabolismo , Proteínas do Olho/genética , Proteínas do Olho/metabolismo , Fator Estimulador de Colônias de Granulócitos e Macrófagos/genética , Fator Estimulador de Colônias de Granulócitos e Macrófagos/metabolismo , Hepatite B/complicações , Hepatite B/virologia , Vírus da Hepatite B da Marmota/crescimento & desenvolvimento , Humanos , Neoplasias Hepáticas Experimentais/sangue , Neoplasias Hepáticas Experimentais/etiologia , Marmota , Fatores de Crescimento Neural/genética , Fatores de Crescimento Neural/metabolismo , Doenças dos Roedores/sangue , Doenças dos Roedores/etiologia , Serpinas/genética , Serpinas/metabolismo , Resultado do Tratamento , Carga Tumoral , gama-Glutamiltransferase/sangueRESUMO
Epidemiological data suggests that certain groups such as the elderly are more susceptible to listeriosis than the general population. Repeated exposure to low levels of Listeria monocytogenes may increase the probability of infection. Increased susceptibility to infection in the elderly has been attributed in part to reduced activity of T cells. We investigated the impact of consecutive daily intragastric challenge with L. monocytogenes on the development of listeriosis within an aged guinea pig population. Approximately 63% of animals became infected following oral L. monocytogenes challenge with 10(4) CFU daily for 3 days, based on recovery of the pathogen from the liver or spleen. At day 4 postchallenge, 100% of animals were infected based on recovery of the bacterium in spleen and liver, decreasing to 14% by day 6 and then steadily increasing to 83% by day 13. During the 13-day postchallenge period, in the blood, numbers of total CD3(+) T cells decreased significantly; CD8(+) T-cell population underwent two shifts; CD4(+) T-cell population decreased and then increased. The results suggest that listerial infection can occur following repeated daily exposure to low levels of L. monocytogenes and that during infection, CD3(+) T-cell immune response may be depressed, potentially increasing susceptibility to other diseases.
Assuntos
Suscetibilidade a Doenças/imunologia , Listeria monocytogenes/isolamento & purificação , Listeriose/veterinária , Animais , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/microbiologia , Contagem de Colônia Microbiana , Feminino , Cobaias , Listeria monocytogenes/crescimento & desenvolvimento , Listeriose/imunologia , Listeriose/microbiologia , Fígado/microbiologia , Contagem de Linfócitos , Baço/microbiologiaRESUMO
Background: In this research, we propose probabilistic approaches to identify pairwise patterns of species co-occurrence by using presence-absence maps only. In particular, the two-by-two contingency table constructed from a presence-absence map of two species would be sufficient to compute the test statistics and perform the statistical tests proposed in this article. Some previous studies have investigated species co-occurrence through incidence data of different survey sites. We focus on using presence-absence maps for a specific study plot instead. The proposed methods are assessed by a thorough simulation study. Methods: A Chi-squared test is used to determine whether the distributions of two species are independent. If the null hypothesis of independence is rejected, the Chi-squared method can not distinguish positive or negative association between two species. We propose six different approaches based on either the binomial or Poisson distribution to obtain p-values for testing the positive (or negative) association between two species. When we test to investigate a positive (or negative) association, if the p-value is below the predetermined level of significance, then we have enough evidence to support that the two species are positively (or negatively) associated. Results: A simulation study is conducted to demonstrate the type-I errors and the testing powers of our approaches. The probabilistic approach proposed by Veech (2013) is served as a benchmark for comparison. The results show that the type-I error of the Chi-squared test is close to the significance level when the presence rate is between 40% and 80%. For extremely low or high presence rate data, one of our approaches outperforms Veech (2013)'s in terms of the testing power and type-I error rate. The proposed methods are applied to a tree data of Barro Colorado Island in Panama and a tree data of Lansing Woods in USA. Both positive and negative associations are found among some species in these two real data.
Assuntos
Benchmarking , Decoração de Interiores e Mobiliário , Colorado , Simulação por Computador , PanamáRESUMO
Due to the clinically established safety and efficacy profile of recombinant adeno-associated viral (rAAV) vectors, they are considered the "go to" vector for retinal gene therapy. Design of a rAAV-mediated gene therapy focuses on cell tropism, high transduction efficiency, and high transgene expression levels to achieve the lowest therapeutic treatment dosage and avoid toxicity. Human retinal explants are a clinically relevant model system for exploring these aspects of rAAV-mediated gene delivery. In this chapter, we describe an ex vivo human retinal explant culture protocol to evaluate transgene expression in order to determine the selectivity and efficacy of rAAV vectors for human retinal gene therapy.