RESUMO
Primary open angle glaucoma is a leading cause of visual impairment and blindness which is commonly treated with drugs or laser but may require surgery. Tenon's ocular fibroblasts are involved in wound-healing after glaucoma filtration surgery and may compromise a favourable outcome of glaucoma surgery by contributing to fibrosis. To investigate changes in gene expression and key pathways contributing to the glaucomatous state we performed genome-wide RNA sequencing. Human Tenon's ocular fibroblasts were cultured from normal and glaucomatous human donors undergoing eye surgery (n = 12). mRNA was extracted and RNA-Seq performed on the Illumina platform. Differentially expressed genes were identified using a bioinformatics pipeline consisting of FastQC, STAR, FeatureCounts and edgeR. Changes in biological functions and pathways were determined using Enrichr and clustered using Cytoscape. A total of 5817 genes were differentially expressed between Tenon's ocular fibroblasts from normal versus glaucomatous eyes. Enrichment analysis showed 787 significantly different biological functions and pathways which were clustered into 176 clusters. Tenon's ocular fibroblasts from glaucomatous eyes showed signs of fibrosis with fibroblast to myofibroblast transdifferentiation and associated changes in mitochondrial fission, remodeling of the extracellular matrix, proliferation, unfolded protein response, inflammation and apoptosis which may relate to the pathogenesis of glaucoma or the detrimental effects of topical glaucoma therapies. Altered gene expression in glaucomatous Tenon's ocular fibroblasts may contribute to an unfavourable outcome of glaucoma filtration surgery. This work presents a genome-wide transcriptome of glaucomatous versus normal Tenon's ocular fibroblasts which may identify genes or pathways of therapeutic value to improve surgical outcomes.
Assuntos
Fibroblastos , Humanos , Fibroblastos/metabolismo , Fibroblastos/patologia , Análise de Sequência de RNA , Feminino , Masculino , Glaucoma/genética , Glaucoma/patologia , Glaucoma de Ângulo Aberto/genética , Glaucoma de Ângulo Aberto/patologia , Idoso , Pessoa de Meia-Idade , Cirurgia Filtrante/efeitos adversos , Fibrose/genética , Células Cultivadas , Perfilação da Expressão GênicaRESUMO
Primary open-angle glaucoma (POAG) is a progressive optic neuropathy with a complex, multifactorial aetiology. Raised intraocular pressure (IOP) is the most important clinically modifiable risk factor for POAG. All current pharmacological agents target aqueous humour dynamics to lower IOP. Newer therapeutic agents are required as some patients with POAG show a limited therapeutic response or develop ocular and systemic side effects to topical medication. Elevated IOP in POAG results from cellular and molecular changes in the trabecular meshwork driven by increased levels of transforming growth factor ß (TGFß) in the anterior segment of the eye. Understanding how TGFß affects both the structural and functional changes in the outflow pathway and IOP is required to develop new glaucoma therapies that target the molecular pathology in the trabecular meshwork. In this study, we evaluated the effects of TGF-ß1 and -ß2 treatment on miRNA expression in cultured human primary trabecular meshwork cells. Our findings are presented in terms of specific miRNAs (miRNA-centric), but given miRNAs work in networks to control cellular pathways and processes, a pathway-centric view of miRNA action is also reported. Evaluating TGFß-responsive miRNA expression in trabecular meshwork cells will further our understanding of the important pathways and changes involved in the pathogenesis of glaucoma and could lead to the development of miRNAs as new therapeutic modalities in glaucoma.
Assuntos
MicroRNAs , Malha Trabecular , Malha Trabecular/metabolismo , Malha Trabecular/efeitos dos fármacos , Malha Trabecular/patologia , Humanos , MicroRNAs/genética , MicroRNAs/metabolismo , Glaucoma de Ângulo Aberto/genética , Glaucoma de Ângulo Aberto/metabolismo , Glaucoma de Ângulo Aberto/patologia , Fator de Crescimento Transformador beta2/metabolismo , Fator de Crescimento Transformador beta2/genética , Fator de Crescimento Transformador beta/metabolismo , Fator de Crescimento Transformador beta1/metabolismo , Células Cultivadas , Regulação da Expressão Gênica/efeitos dos fármacos , Pressão Intraocular/efeitos dos fármacosRESUMO
BACKGROUND AND OBJECTIVES: All US Food and Drug Administration-approved medications for Tourette syndrome are antipsychotics, and their use is limited by the risk of weight gain, metabolic changes, and drug-induced movement disorders. Several small trials suggest that ecopipam, a first-in-class, selective dopamine 1 receptor antagonist, reduces tics with a low risk for these adverse events. This trial sought to further evaluate the efficacy, safety, and tolerability of ecopipam in children and adolescents with moderate to severe Tourette syndrome. METHODS: This was a multicenter, randomized, double-blind, placebo-controlled, phase 2b trial. Subjects aged ≥6 to <18 years with a baseline Yale Global Tic Severity Score Total Tic Score of ≥20 were randomly assigned 1:1 to ecopipam (n = 76) or placebo (n = 77). The primary endpoint was mean change over 12 weeks in the Yale Global Tic Severity Score Total Tic Score. The Clinical Global Impression of Tourette Syndrome Severity was the secondary endpoint. Safety and tolerability were evaluated at each study visit. RESULTS: Total tic scores were significantly reduced from baseline to 12 weeks in the ecopipam group compared with placebo (least squares mean differences -3.44, 95% confidence interval -6.09 to -0.79, P = .01). Improvement in Clinical Global Impression of Tourette Syndrome Severity was also greater in the ecopipam group (P = .03). More weight gain was seen in subjects assigned to placebo. No metabolic or electrocardiogram changes were identified. Headache (15.8%), insomnia (14.5%), fatigue (7.9%), and somnolence (7.9%) were the most common adverse events. CONCLUSIONS: Among children and adolescents with TS, ecopipam reduces tics to a greater extent than placebo, without observable evidence of common antipsychotic-associated side effects.
Assuntos
Antipsicóticos , Tiques , Síndrome de Tourette , Adolescente , Criança , Humanos , Síndrome de Tourette/tratamento farmacológico , Síndrome de Tourette/induzido quimicamente , Síndrome de Tourette/complicações , Tiques/induzido quimicamente , Tiques/complicações , Tiques/tratamento farmacológico , Resultado do Tratamento , Antipsicóticos/efeitos adversos , Método Duplo-Cego , Aumento de Peso , Índice de Gravidade de DoençaRESUMO
Objective: In a phase 2 study, pimavanserin demonstrated efficacy as adjunctive treatment for major depressive disorder (MDD). Subsequently, two phase 3 studies (NCT03968159 in the US; NCT03999918 in Europe) were initiated to examine the efficacy and safety of adjunctive pimavanserin in subjects with MDD and inadequate response to antidepressant treatment. Studies were combined with a prespecified statistical analysis plan owing to recruitment challenges related to the COVID-19 pandemic. Experimental design: The randomized, double-blind studies enrolled 298 patients with MDD and inadequate response to current antidepressants. Patients were randomly assigned 1:1 to pimavanserin or placebo added to current antidepressant for 6 weeks. Primary endpoint was change from baseline to week 5 in the Hamilton Rating Scale for Depression, 17-item version (HAM-D-17). Principal observations: There was no effect of pimavanserin in change from baseline to week 5 in the HAM-D-17 (pimavanserin [n = 138]: least-squares mean [LSM] [standard error {SE}], -9.0 [0.58]; placebo [n = 135]: -8.1 [0.58]; mixed-effects model for repeated measures LSM [SE] difference, -0.9 [0.82], P = 0.2956). Nominal improvement with pimavanserin was observed on 2 secondary endpoints: Clinical Global Impressions-Severity scale, Karolinska Sleepiness Scale. Treatment-emergent adverse events occurred in 58.1% of pimavanserin-treated and 54.7% of placebo-treated patients. Conclusions: Adjunctive pimavanserin did not significantly improve depressive symptoms, although pimavanserin was well tolerated.
Assuntos
COVID-19 , Transtorno Depressivo Maior , Humanos , Transtorno Depressivo Maior/tratamento farmacológico , Método Duplo-Cego , Pandemias , Antidepressivos/efeitos adversos , Resultado do TratamentoRESUMO
Pseudoexfoliation glaucoma (XFG) is an aggressive form of secondary open angle glaucoma, characterised by the production of exfoliation material and is estimated to affect 30 million people worldwide. Activation of the TGF-ß pathway by TGF-ß1 has been implicated in the pathogenesis of pseudoexfoliation glaucoma. To further investigate the role of TGF-ß1 in glaucomatous changes in the trabecular meshwork (TM), we used RNA-Seq to determine TGF-ß1 induced changes in the transcriptome of normal human trabecular meshwork (HTM) cells. The main purpose of this study was to perform a hypothesis-independent RNA sequencing analysis to investigate genome-wide alterations in the transcriptome of normal HTMs stimulated with TGF-ß1 and investigate possible pathophysiological mechanisms driving XFG. Our results identified multiple differentially expressed genes including several genes known to be present in exfoliation material. Significantly altered pathways, biological processes and molecular functions included extracellular matrix remodelling, Hippo and Wnt pathways, the unfolded protein response, oxidative stress, and the antioxidant system. This cellular model of pseudoexfoliation glaucoma can provide insight into disease pathogenesis and support the development of novel therapeutic interventions.
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Glaucoma de Ângulo Aberto , Glaucoma , Humanos , Malha Trabecular/metabolismo , Fator de Crescimento Transformador beta1/genética , Fator de Crescimento Transformador beta1/metabolismo , Glaucoma de Ângulo Aberto/genética , Glaucoma de Ângulo Aberto/metabolismo , RNA/metabolismo , Glaucoma/genética , Glaucoma/metabolismo , Análise de Sequência de RNARESUMO
TWEAK (tumor necrosis factor-like weak inducer of apoptosis) is a member of the TNF superfamily that controls a multitude of cellular events including proliferation, migration, differentiation, apoptosis, angiogenesis, and inflammation. TWEAK control of these events is via an expanding list of intracellular signalling pathways which include NF-κB, ERK/MAPK, Notch, EGFR and AP-1. Two receptors have been identified for TWEAK - Fn14, which targets the membrane bound form of TWEAK, and CD163, which scavenges the soluble form of TWEAK. TWEAK appears to elicit specific events based on the receptor to which it binds, tissue type in which it is expressed, specific extrinsic conditions, and the presence of other cytokines. TWEAK signalling is protective in healthy tissues, but in chronic inflammatory states become detrimental to the tissue. Consistent data show a role for the TWEAK/FN14/CD163 axis in metabolic disease, chronic autoimmune diseases, and acute ischaemic stroke. Low circulating concentrations of soluble TWEAK are predictive of poor cardiovascular outcomes in those with and without diabetes. This review details the current understanding of the TWEAK/Fn14/CD163 axis as one of the chief regulators of immune signalling and its cell-specific role in metabolic disease development and progression.
Assuntos
Isquemia Encefálica , Doenças Metabólicas , Acidente Vascular Cerebral , Antígenos CD , Antígenos de Diferenciação Mielomonocítica , Citocina TWEAK , Humanos , Inflamação/metabolismo , Receptores de Superfície Celular , Receptores do Fator de Necrose Tumoral/metabolismo , Receptor de TWEAK , Fatores de Necrose Tumoral/metabolismoRESUMO
TNFAIP3 encodes a zinc finger protein called A20, which has potent anti-inflammatory and anti-apoptotic properties. A20 promotes beta-cell survival and protects against islet graft rejection in experimental models. The current study sought to investigate the mechanisms underlying the protective role of A20 in the pancreatic beta-cell. Two islet cell types were used for experiments: the insulin-secreting BRIN-BD11 cell line and human islet cells. A20 was silenced using siRNA against TNFAIP3, and knockdown was confirmed by qPCR and immunostaining of cells. Cell viability, cytotoxicity and apoptosis were assessed using the ApotoxGlo assay. Glucose-stimulated insulin secretion and production of inflammatory cytokines (TNFa, IL1b and IFNg) were measured by ELISA. Expression of beta-cell regulatory genes (Abcc8, Kcnj11, Kcnq1, Gck, Scl2a2) and transcription factors (Hnf1a, Pdx1, Nkx6.1, Ngn3) was determined by qPCR. A20 deficiency increased apoptosis, impaired glucose-induced insulin secretion, and reduced expression of beta-cell regulatory genes and transcription factors. Addition of recombinant A20 normalized gene expression profiles. TNFa, IL1b and IFNg were elevated in A20 deficient cells and found to independently elicit changes in gene expression. Analysis of PCR array data suggests that A20 action in the beta cell is largely, although not exclusively, driven by the P65 subunit of NF-kB. The current report demonstrates a role for A20 in controlling beta-cell integrity and survival, which likely results from the regulation of inflammatory signalling. Of particular note is the impact that A20 deficiency has on the expression of transcription factors regulating the maturation and normal function of beta cells.
Assuntos
Regulação da Expressão Gênica , Genes Reguladores , Células Secretoras de Insulina/metabolismo , Proteína 3 Induzida por Fator de Necrose Tumoral alfa/genética , Proteína 3 Induzida por Fator de Necrose Tumoral alfa/metabolismo , Animais , Apoptose/genética , Linhagem Celular , Sobrevivência Celular/genética , Citocinas/metabolismo , Perfilação da Expressão Gênica , Regulação da Expressão Gênica/efeitos dos fármacos , Inativação Gênica , Glucose/metabolismo , Humanos , Mediadores da Inflamação/metabolismo , Insulina/metabolismo , Ilhotas Pancreáticas/metabolismo , NF-kappa B/metabolismo , RNA Interferente Pequeno/genética , Ratos , Transdução de Sinais , Fatores de Transcrição/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Fator de Necrose Tumoral alfa/farmacologia , UbiquitinaçãoRESUMO
Glaucoma is a group of optic neuropathies characterised by the degeneration of retinal ganglion cells, resulting in damage to the optic nerve head (ONH) and loss of vision in one or both eyes. Increased intraocular pressure (IOP) is one of the major aetiological risk factors in glaucoma, and is currently the only modifiable risk factor. However, 30-40% of glaucoma patients do not present with elevated IOP and still proceed to lose vision. The pathophysiology of glaucoma is therefore not completely understood, and there is a need for the development of IOP-independent neuroprotective therapies to preserve vision. Neuroinflammation has been shown to play a key role in glaucoma and, specifically, the NLRP3 inflammasome, a key driver of inflammation, has recently been implicated. The NLRP3 inflammasome is expressed in the eye and its activation is reported in pre-clinical studies of glaucoma. Activation of the NLRP3 inflammasome results in IL-1ß processing. This pro inflammatory cytokine is elevated in the blood of glaucoma patients and is believed to drive neurotoxic inflammation, resulting in axon degeneration and the death of retinal ganglion cells (RGCs). This review discusses glaucoma as an inflammatory disease and evaluates targeting the NLRP3 inflammasome as a therapeutic strategy. A hypothetical mechanism for the action of the NLRP3 inflammasome in glaucoma is presented.
Assuntos
Glaucoma/metabolismo , Glaucoma/terapia , Inflamassomos/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Células Ganglionares da Retina/metabolismo , Animais , Anti-Inflamatórios/química , Axônios , Humanos , Inflamação , Interleucina-1beta/metabolismo , Camundongos , Neuroproteção , Espécies Reativas de Oxigênio , Receptores de Reconhecimento de Padrão , Fatores de RiscoRESUMO
Autosomal dominantly inherited genetic disorders such as corneal dystrophies are amenable to allele-specific gene silencing with small interfering RNA (siRNA). siRNA delivered to the cornea by injection, although effective, is not suitable for a frequent long-term treatment regimen, whereas topical delivery of siRNA to the cornea is hampered by the eye surface's protective mechanisms. Herein we describe an attractive and innovative alternative for topical application using cell-penetrating peptide derivatives capable of complexing siRNA non-covalently and delivering them into the cornea. Through a rational design approach, we modified derivatives of a cell-penetrating peptide, peptide for ocular delivery (POD), already proved to diffuse into the corneal layers. These POD derivatives were able to form siRNA-peptide complexes (polyplexes) of size and ζ-potential similar to those reported able to undergo cellular internalization. Successful cytoplasmic release and gene silencing in vitro was obtained when an endosomal disruptor, chloroquine, was added. A palmitoylated-POD, displaying the best delivery properties, was covalently functionalized with trifluoromethylquinoline, an analog of chloroquine. This modified POD, named trifluoromethylquinoline-palmitoyl-POD (QN-Palm-POD), when complexed with siRNA and topically applied to the eye in vivo, resulted in up to 30% knockdown of luciferase reporter gene expression in the corneal epithelium. The methods developed within represent a valid standardized approach that is ideal for screening of a range of delivery formulations.
RESUMO
Pterygium is a pathological proliferative condition of the ocular surface, characterised by formation of a highly vascularised, fibrous tissue arising from the limbus that invades the central cornea leading to visual disturbance and, if untreated, blindness. Whilst chronic ultraviolet (UV) light exposure plays a major role in its pathogenesis, higher susceptibility to pterygium is observed in some families, suggesting a genetic component. In this study, a Northern Irish family affected by pterygium but reporting little direct exposure to UV was identified carrying a missense variant in CRIM1 NM_016441.2: c.1235 Aâ¯>â¯C (H412P) through whole-exome sequencing and subsequent analysis. CRIM1 is expressed in the developing eye, adult cornea and conjunctiva, having a role in cell differentiation and migration but also in angiogenesis, all processes involved in pterygium formation. We demonstrate elevated CRIM1 expression in pterygium tissue from additional individual Northern Irish patients compared to unaffected conjunctival controls. UV irradiation of HCE-S cells resulted in an increase in ERK phosphorylation and CRIM1 expression, the latter further elevated by the addition of the MEK1/2 inhibitor, U0126. Conversely, siRNA knockdown of CRIM1 led to decreased UV-induced ERK phosphorylation and increased BCL2 expression. Transient expression of the mutant H412P CRIM1 in corneal epithelial HCE-S cells showed that, unlike wild-type CRIM1, it was unable to reduce the cell proliferation, increased ERK phosphorylation and apoptosis induced through a decrease of BCL2 expression levels. We propose here a series of intracellular events where CRIM1 regulation of the ERK pathway prevents UV-induced cell proliferation and may play an important role in the in the pathogenesis of pterygium.
Assuntos
Epitélio Corneano/efeitos da radiação , Regulação da Expressão Gênica/fisiologia , Proteínas de Membrana/genética , Mutação de Sentido Incorreto , Pterígio/genética , Raios Ultravioleta , Adulto , Western Blotting , Receptores de Proteínas Morfogenéticas Ósseas , Células Cultivadas , Epitélio Corneano/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Feminino , Humanos , Imuno-Histoquímica , Marcação In Situ das Extremidades Cortadas , Masculino , Proteínas de Membrana/metabolismo , Pessoa de Meia-Idade , Mutagênese Sítio-Dirigida , Linhagem , Fosforilação , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Pterígio/etiologia , Pterígio/metabolismo , RNA Interferente Pequeno/genética , Reação em Cadeia da Polimerase em Tempo Real , Sequenciamento Completo do GenomaRESUMO
The objective of this study was to investigate the efficacy and safety of adjunctive lanicemine (NMDA channel blocker) in the treatment of major depressive disorder (MDD) over 12 weeks. This phase IIb, randomized, parallel-arm, double-blind, placebo-controlled study was conducted at 49 centers in four countries between December 2011 and August 2013 in 302 patients aged 18-70 years, meeting criteria for single episode or recurrent MDD and with a history of inadequate treatment response. Patients were required to be taking an allowed antidepressant for at least four weeks prior to screening. Patients were randomized equally to receive 15 double-blind intravenous infusions of adjunctive lanicemine 50 mg, lanicemine 100 mg, or saline over a 12-week course, in addition to ongoing antidepressant. The primary efficacy end point was change in Montgomery-Åsberg Depression Rating Scale (MADRS) total score from baseline to week 6. Secondary efficacy outcome variables included change in MADRS score from baseline to week 12, response and remission rates, and changes in Clinical Global Impression scale, Quick Inventory of Depressive Symptomology Self-Report score, and Sheehan Disability Scale score. Of 302 randomized patients, 240 (79.5%) completed treatment. Although lanicemine was generally well tolerated, neither dose was superior to placebo in reducing depressive symptoms on the primary end point or any secondary measures. There was no significant difference between lanicemine and placebo treatment on any outcome measures related to MDD. Post hoc analyses were performed to explore the possible effects of trial design and patient characteristics in accounting for the contrasting results with a previously reported trial.
Assuntos
Antidepressivos/farmacologia , Transtorno Depressivo Maior/tratamento farmacológico , Transtorno Depressivo Resistente a Tratamento/tratamento farmacológico , Avaliação de Resultados em Cuidados de Saúde , Fenetilaminas/farmacologia , Piridinas/farmacologia , Adulto , Antidepressivos/administração & dosagem , Método Duplo-Cego , Quimioterapia Combinada , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Fenetilaminas/administração & dosagem , Piridinas/administração & dosagemRESUMO
Meesmann epithelial corneal dystrophy (MECD) is a rare autosomal dominant disorder caused by dominant-negative mutations within the KRT3 or KRT12 genes, which encode the cytoskeletal protein keratins K3 and K12, respectively. To investigate the pathomechanism of this disease, we generated and phenotypically characterized a novel knock-in humanized mouse model carrying the severe, MECD-associated, K12-Leu132Pro mutation. Although no overt changes in corneal opacity were detected by slit-lamp examination, the corneas of homozygous mutant mice exhibited histological and ultrastructural epithelial cell fragility phenotypes. An altered keratin expression profile was observed in the cornea of mutant mice, confirmed by western blot, RNA-seq and quantitative real-time polymerase chain reaction. Mass spectrometry (MS) and immunohistochemistry demonstrated a similarly altered keratin profile in corneal tissue from a K12-Leu132Pro MECD patient. The K12-Leu132Pro mutation results in cytoplasmic keratin aggregates. RNA-seq analysis revealed increased chaperone gene expression, and apoptotic unfolded protein response (UPR) markers, CHOP and Caspase 12, were also increased in the MECD mice. Corneal epithelial cell apoptosis was increased 17-fold in the mutant cornea, compared with the wild-type (P < 0.001). This elevation of UPR marker expression was also observed in the human MECD cornea. This is the first reporting of a mouse model for MECD that recapitulates the human disease and is a valuable resource in understanding the pathomechanism of the disease. Although the most severe phenotype is observed in the homozygous mice, this model will still provide a test-bed for therapies not only for corneal dystrophies but also for other keratinopathies caused by similar mutations.
Assuntos
Distrofia Corneana Epitelial Juvenil de Meesmann/genética , Queratina-12/genética , Queratina-3/genética , Mutação de Sentido Incorreto , Adulto , Animais , Apoptose/genética , Modelos Animais de Doenças , Éxons , Feminino , Heterozigoto , Humanos , Camundongos , Camundongos Transgênicos , Mutação , Linhagem , Resposta a Proteínas não DobradasRESUMO
PURPOSE: Transforming growth factor beta-induced (TGFBI)-related dystrophies constitute the most common heritable forms of corneal dystrophy worldwide. However, other than the underlying genotypes of these conditions, a limited knowledge exists of the exact pathomechanisms of these disorders. This study expands on our previous research investigating dystrophic stromal aggregates, with the aim of better elucidating the pathomechanism of two conditions arising from the most common TGFBI mutations: granular corneal dystrophy type 1 (GCD1; R555W) and lattice corneal dystrophy type 1 (LCD1; R124C). METHODS: Patient corneas with GCD1 and LCD1 were stained with hematoxylin and eosin and Congo red to visualize stromal nonamyloid and amyloid deposits, respectively. Laser capture microdissection was used to isolate aggregates and extracted protein was analyzed by mass spectrometry. Proteins were identified and their approximate abundances were determined. Spectra of TGFBIp peptides were also recorded and quantified. RESULTS: In total, three proteins were found within GCD1 aggregates that were absent in the healthy control corneal tissue. In comparison, an additional 18 and 24 proteins within stromal LCD1 and Bowman's LCD1 deposits, respectively, were identified. Variances surrounding the endogenous cleavage sites of TGFBIp were also noted. An increase in the number of residues experiencing cleavage was observed in both GCD1 aggregates and LCD1 deposits. CONCLUSIONS: The study reveals previously unknown differences between the protein composition of GCD1 and LCD1 aggregates, and confirms the presence of the HtrA1 protease in LCD1-amyloid aggregates. In addition, we find mutation-specific differences in the processing of mutant TGFBIp species, which may contribute to the variable phenotypes noted in TGFBI-related dystrophies.
Assuntos
Distrofias Hereditárias da Córnea/genética , Substância Própria/metabolismo , DNA/genética , Mutação , Fator de Crescimento Transformador beta/genética , Idoso , Idoso de 80 Anos ou mais , Amiloide/metabolismo , Distrofias Hereditárias da Córnea/metabolismo , Distrofias Hereditárias da Córnea/patologia , Substância Própria/patologia , Análise Mutacional de DNA , Feminino , Genótipo , Humanos , Microdissecção e Captura a Laser , Masculino , Pessoa de Meia-Idade , Linhagem , Proteômica/métodos , Fator de Crescimento Transformador beta/químicaRESUMO
PURPOSE: The aim of this study is to further assess our previously reported keratin 12 (K12)-Leu132Pro specific siRNA in silencing the mutant allele in Meesmann's Epithelial Corneal Dystrophy (MECD) in experimental systems more akin to the in vivo situation through simultaneous expression of both wild-type and mutant alleles. METHODS: Using KRT12 exogenous expression constructs transfected into cells, mutant allele specific knockdown was quantified using pyrosequencing and infrared Western blot analysis, while the silencing mechanism was assessed by a modified rapid amplification of cDNA ends (5'RACE) method. Corneal limbal biopsies taken from patients suffering from MECD were used to establish cultures of MECD corneal limbal epithelial stem cells and the ability of the siRNA to silence the endogenous mutant KRT12 allele was assessed by a combination of pyrosequencing, qPCR, ELISA, and quantitative-fluorescent immunohistochemistry (Q-FIHC). RESULTS: The siRNA displayed a potent and specific knockdown of K12-Leu132Pro at both the mRNA and protein levels with exogenous expression constructs. Analysis by the 5'RACE method confirmed siRNA-mediated cleavage. In the MECD cells, an allele-specific knockdown of 63% of the endogenous mutant allele was observed without effect on wild-type allele expression. CONCLUSIONS: Combined with an effective delivery vehicle this siRNA approach represents a viable treatment option for prevention of the MECD pathology observed in K12-Leu132Pro heterozygous individuals.
Assuntos
Distrofia Corneana Epitelial Juvenil de Meesmann/genética , DNA/genética , Queratina-12/genética , Limbo da Córnea/patologia , Mutação de Sentido Incorreto , Alelos , Proliferação de Células , Células Cultivadas , Distrofia Corneana Epitelial Juvenil de Meesmann/metabolismo , Distrofia Corneana Epitelial Juvenil de Meesmann/patologia , Ensaio de Imunoadsorção Enzimática , Éxons , Heterozigoto , Humanos , Imuno-Histoquímica , Queratina-12/metabolismo , Limbo da Córnea/metabolismo , Linhagem , RNA Interferente Pequeno/genética , Reação em Cadeia da Polimerase em Tempo RealRESUMO
OBJECTIVE: The purpose of this study was to evaluate the efficacy and safety of duloxetine flexible dose in children (7-11 years) and adolescents (12-17 years) with major depressive disorder (MDD). METHODS: Patients (n=337) in this 36 week study (10 week acute and 26 week extension treatment) received duloxetine (60-120 mg once daily [QD], n=117), fluoxetine (20-40 mg QD, n=117), or placebo (n=103). Measures included: Children's Depression Rating Scale-Revised (CDRS-R), treatment-emergent adverse events (TEAEs), and Columbia-Suicide Severity Rating Scale (C-SSRS). RESULTS: Neither active drug (duloxetine or fluoxetine) separated significantly (p<0.05) from placebo on mean change from baseline to end-point (10 weeks) on the CDRS-R total score. There were no significant differences between the duloxetine or fluoxetine groups compared with placebo on serious AEs (SAEs), total TEAEs, or discontinuation for AE during acute treatment. There were no completed suicides or deaths, and no clinically significant electrocardiogram (ECG) abnormalities observed during the study. One fluoxetine and one duloxetine patient experienced alanine aminotransferase (ALT) three or more times the upper limit of normal, which resolved during the study. A total of 8 (7.1%) duloxetine patients, 7 (6.8%) placebo patients, and 9 (8.0%) fluoxetine patients had worsening of suicidal ideation from baseline during acute treatment. Of the patients with suicidal ideation at baseline, 15/19 (79%) duloxetine, 19/19 (100%) placebo, and 16/19 (84%) fluoxetine had improvement in suicidal ideation at end-point during acute treatment. One duloxetine and two fluoxetine patients had treatment-emergent suicidal behavior during the 36 week study. CONCLUSION: Trial results were inconclusive, as neither the investigational drug (duloxetine) nor the active control (fluoxetine) separated from placebo on the CDRS-R at 10 weeks. No new duloxetine safety signals were identified relative to those seen in adults. Clinical Trial Registry Number: NCT00849901.
Assuntos
Antidepressivos/uso terapêutico , Transtorno Depressivo Maior/tratamento farmacológico , Fluoxetina/uso terapêutico , Tiofenos/uso terapêutico , Adolescente , Antidepressivos/administração & dosagem , Antidepressivos/efeitos adversos , Criança , Transtorno Depressivo Maior/fisiopatologia , Relação Dose-Resposta a Droga , Método Duplo-Cego , Cloridrato de Duloxetina , Feminino , Humanos , Masculino , Escalas de Graduação Psiquiátrica , Inibidores Seletivos de Recaptação de Serotonina/administração & dosagem , Inibidores Seletivos de Recaptação de Serotonina/efeitos adversos , Inibidores Seletivos de Recaptação de Serotonina/uso terapêutico , Índice de Gravidade de Doença , Ideação Suicida , Suicídio , Tiofenos/administração & dosagem , Tiofenos/efeitos adversos , Resultado do TratamentoRESUMO
PURPOSE: This study aimed to investigate the potency and specificity of short-interfering RNA (siRNA) treatment for TGFBI-Arg124Cys lattice corneal dystrophy type I (LCDI) using exogenous expression constructs in model systems and endogenous gene targeting in an ex vivo model using corneal epithelial cell cultures. METHODS: A panel of 19 TGFBI-Arg124Cys-specific siRNAs were assessed by a dual-luciferase reporter assay. Further assessment using pyrosequencing and qPCR was used to identify the lead siRNA; suppression of mutant TGFBIp expression was confirmed by Western blot and Congo red aggregation assays. An ex vivo model of LCDI was established using limbal biopsies from corneal dystrophy patients harboring the Arg124Cys mutation. Treatment efficiency of the siRNA was assessed for the inhibition of the mutant allele in the primary patient's corneal epithelial cells using pyrosequencing, quantitative PCR (qPCR), and an ELISA. RESULTS: A lead siRNA was identified, and demonstrated to be potent and specific in inhibiting the TGFBI-Arg124Cys mutant allele at the mRNA and protein levels. Besides high allele specificity, siRNA treatment achieved a 44% reduction of the endogenous Arg124Cys allele in an ex vivo model of LCDI. CONCLUSIONS: We have identified a lead siRNA specific to the TGFBI-Arg124Cys mutant allele associated with LCDI. Silencing of exogenous TGFBI was observed at mRNA and protein levels, and in an ex vivo model of LCDI with an efficient suppression of the endogenous mutant allele. This result indicates the potential of siRNA treatment as a personalized medicine approach for the management of heritable TGFBI-associated corneal dystrophies.
Assuntos
Distrofias Hereditárias da Córnea/genética , Proteínas da Matriz Extracelular/genética , Inativação Gênica , Mutação Puntual , RNA Interferente Pequeno/genética , Fator de Crescimento Transformador beta/genética , Alelos , Western Blotting , Técnicas de Cultura de Células , Ensaio de Imunoadsorção Enzimática , Humanos , Reação em Cadeia da Polimerase em Tempo Real , Análise de Sequência de DNA , TransfecçãoRESUMO
BACKGROUND/AIMS: Cross-linking of the cornea is usually carried out at a young age as a treatment to manage ectasia. The corneal limbal region contains delicate long-lived stem cells, which could potentially be deleteriously affected by Ultraviolet A (UV-A) radiation. Damage to these stem cells may not demonstrate as a clinical problem for many years subsequent to cross-linking treatment. UV-A radiation is known to have potential mutagenic effects upon mammalian DNA and can result in cancer. METHODS: Cultured corneal epithelial cells and ex vivo corneal tissue were treated with the standard clinical cross-linking protocol for UV-A irradiation. 8-hydroxydeoxyguansoine (8-OHdG) and cyclin-dependent kinase inhibitor genes (CDKN1A and CDKN2A) were assayed as markers of DNA damage using immunohistochemistry, ELISA and quantitative real time PCR. RESULTS: Staining of treated limbal tissue demonstrated the presence of 8-OHdG within p63 positive basal limbal cells. Levels of 8-OHdG and CDKN1A mRNA were found to be significantly increased in cultured corneal epithelial cells and limbal epithelial cells but no increase was demonstrated with the use of a polymethyl methylacrylate protective cover. CONCLUSIONS: This study provides evidence that oxidative nuclear DNA damage can occur through cross-linking in layers of corneal epithelial cells at the limbus and that this can be easily prevented by covering the limbus.
Assuntos
Colágeno/farmacologia , Epitélio Corneano/citologia , Ceratocone/terapia , Células-Tronco/citologia , Raios Ultravioleta/efeitos adversos , 8-Hidroxi-2'-Desoxiguanosina/análogos & derivados , Apoptose/efeitos da radiação , Linhagem Celular , DNA/genética , Dano ao DNA , Ensaio de Imunoadsorção Enzimática , Epitélio Corneano/efeitos dos fármacos , Epitélio Corneano/efeitos da radiação , Guanina/análogos & derivados , Guanina/metabolismo , Humanos , Ceratocone/metabolismo , Ceratocone/patologia , Reação em Cadeia da Polimerase em Tempo Real , Células-Tronco/efeitos dos fármacos , Células-Tronco/efeitos da radiação , Terapia Ultravioleta/efeitos adversosRESUMO
PURPOSE: To identify an allele-specific short interfering RNA (siRNA), against the common KRT12 mutation Arg135Thr in Meesmann epithelial corneal dystrophy (MECD) as a personalized approach to treatment. METHODS: siRNAs against the K12 Arg135Thr mutation were evaluated using a dual luciferase reporter gene assay and the most potent and specific siRNAs were further screened by Western blot. Off-target effects on related keratins were assessed and immunological stimulation of TLR3 was evaluated by RT-PCR. A modified 5' rapid amplification of cDNA ends method was used to confirm siRNA-mediated mutant knockdown. Allele discrimination was confirmed by quantitative infrared immunoblotting. RESULTS: The lead siRNA, with an IC(50) of thirty picomolar, showed no keratin off-target effects or activation of TLR3 in the concentration ranges tested. We confirmed siRNA-mediated knockdown by the presence of K12 mRNA fragments cleaved at the predicted site. A dual tag infrared immunoblot showed knockdown to be allele-specific, with 70% to 80% silencing of the mutant protein. CONCLUSIONS: A potent allele-specific siRNA against the K12 Arg135Thr mutation was identified. In combination with efficient eyedrop formulation delivery, this would represent a personalized medicine approach, aimed at preventing the pathology associated with MECD and other ocular surface pathologies with dominant-negative or gain-of-function pathomechanisms.
Assuntos
Distrofia Corneana Epitelial Juvenil de Meesmann/genética , DNA/genética , Inativação Gênica , Queratina-12/genética , Mutação , RNA Interferente Pequeno/genética , Alelos , Técnicas de Cultura de Células , Distrofia Corneana Epitelial Juvenil de Meesmann/metabolismo , Distrofia Corneana Epitelial Juvenil de Meesmann/patologia , Éxons , Humanos , Queratina-12/metabolismo , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Epidermolysis bullosa simplex (EBS) is an incurable, inherited skin-blistering disorder predominantly caused by dominant-negative mutations in the genes encoding keratins K5 or K14. RNA interference, particularly in the form of small interfering RNA (siRNA), offers a potential therapy route for EBS and related keratin disorders by selectively silencing the mutant allele. Here, using a systemic screening system based on a luciferase reporter gene assay, we have developed mutant-specific siRNAs for two independent EBS-causing missense mutations in the K5 gene (p.Ser181Pro and p.Asn193Lys). The specificity of the allele-specific inhibitors identified in the screen was subsequently confirmed at the protein level, where the lead inhibitors were shown to strongly knock down the expression of mutant proteins with negligible effect on wild-type K5 expression. In a cell-based model system, the lead inhibitors were able to significantly reverse the cytoskeletal aggregation phenotype. Overall, this approach shows promise for the treatment of EBS and paves the way for future clinical trials.
Assuntos
Epidermólise Bolhosa Simples/genética , Epidermólise Bolhosa Simples/terapia , Queratina-5/genética , Mutação , RNA Interferente Pequeno/metabolismo , Alelos , Animais , Dipodomys , Inativação Gênica , Proteínas de Fluorescência Verde/metabolismo , Humanos , Queratina-14/genética , Fenótipo , Interferência de RNA , TransfecçãoRESUMO
PURPOSE: To investigate the effect of tear hyperosmolarity and signs of clinical ocular surface pathology on conjunctival goblet cell population. METHODS: 111 participants were assessed using tear osmolarity (TO) measurements and a comprehensive selection of clinical ophthalmic tests. The resultant clinical database was assessed for evidence of patterns of composite increasing pathology. The total, filled, and empty goblet cell numbers were measured: total number of goblet cells as per cytokeratin 7 (CK7) immunofluorescence and number of filled goblet cells as per periodic acid Schiff's reagent (PAS) or lectin helix pomatia agglutinin (HPA). Goblet cell profile was correlated with composite clinical pathologic grades. RESULTS: No significant correlation was found between TO and goblet cell number or function (as indicated by number of filled or unfilled goblet cells). Distinct composite clinical pathologic groups 0-IV with increasing pathology were created based on the frequency of positive pathologic signs, which adhered to the Dry Eye Workshop purported mechanism. Only in group IV was there significantly increased mean tear osmolarity of 344 mOsm/L (P < 0.000) along with significantly decreased empty goblet cell number (CK7+ and HPA-) compared to filled (CK7+ and HPA+, P = 0.000). When the number of filled goblet cells (PAS+) was analyzed there was significant increase in tear osmolarity for the two most severe grades; 3 and 4. CONCLUSIONS: The goblet cell population does not appear to be affected by isolated tear hyperosmolarity. Hyperosmolarity when combined with other ocular surface pathology or inflammation alters the goblet cell population.