RESUMO
The robust integrity of the retinal pigment epithelium (RPE), which contributes to the outer brain retina barrier (oBRB), is compromised in several retinal degenerative and vascular disorders, including diabetic macular edema (DME). This study evaluates the role of a new generation of histone deacetylase inhibitor (HDACi), ITF2357, in regulating outer blood-retinal barrier function and investigates the underlying mechanism of action in inhibiting TNFα-induced damage to RPE integrity. Using the immortalized RPE cell line (ARPE-19), ITF2357 was found to be non-toxic between 50 nM and 5 µM concentrations. When applied as a pre-treatment in conjunction with an inflammatory cytokine, TNFα, the HDACi was safe and effective in preventing epithelial permeability by fortifying tight junction (ZO-1, -2, -3, occludin, claudin-1, -2, -3, -5, -19) and adherens junction (E-cadherin, Nectin-1) protein expression post-TNFα stress. Mechanistically, ITF2357 depicted a late action at 24 h via attenuating IKK, IκBα, and p65 phosphorylation and ameliorated the expression of IL-1ß, IL-6, and MCP-1. Also, ITF2357 delayed IκBα synthesis and turnover. The use of Bay 11-7082 and MG132 further uncovered a possible role for ITF2357 in non-canonical NF-κB activation. Overall, this study revealed the protection effects of ITF2357 by regulating the turnover of tight and adherens junction proteins and modulating NF-κB signaling pathway in the presence of an inflammatory stressor, making it a potential therapeutic application for retinal vascular diseases such as DME with compromised outer blood-retinal barrier.
Assuntos
Retinopatia Diabética , Ácidos Hidroxâmicos , Edema Macular , Humanos , NF-kappa B/metabolismo , Retinopatia Diabética/metabolismo , Inibidor de NF-kappaB alfa/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Edema Macular/metabolismo , Transdução de Sinais , Epitélio Pigmentado da Retina/metabolismo , Barreira Hematorretiniana/metabolismo , Junções Íntimas/metabolismo , Células Epiteliais/metabolismo , Pigmentos da Retina/metabolismo , Pigmentos da Retina/farmacologia , Pigmentos da Retina/uso terapêuticoRESUMO
PURPOSE: Diabetic Retinopathy (DR) is associated with metabolic dysfunction in cells such as retinal pigmented epithelium (RPE). Small molecular weight microRNAs can simultaneously regulate multiple gene products thus having pivotal roles in disease pathogenesis. Since miR182-5p is involved in regulating glycolysis and angiogenesis, two pathologic processes of DR, we investigated its status in DR eyes and in high glucose model in vitro. METHOD: ology: Total RNA was extracted from vitreous humor of PDR (n = 48) and macular hole (n = 22) subjects followed by quantification of miR182-5p and its target genes. ARPE-19 cells, cultured in DMEM under differential glucose conditions (5 mM and 25 mM) were used for metabolic and biochemical assays. Cells were transfected with miRNA182 mimic or antagomir to evaluate the gain and loss of function effects. RESULTS: PDR patient eyes had high levels of miR182-5p levels (p < 0.05). RPE cells under high glucose stress elevated miR182-5p expression with altered glycolytic pathway drivers such as HK2, PFKP and PKM2 over extended durations. Additionally, RPE cells under high glucose conditions exhibited reduced FoxO1 and enhanced Akt activation. RPE cells transfected with miR182-5p mimic phenocopied the enhanced basal and compensatory glycolytic rates observed under high glucose conditions with increased VEGF secretion. Conversely, inhibiting miR182-5p reduced Akt activation, glycolytic pathway proteins, and VEGF while stabilizing FoxO1. CONCLUSION: Glycolysis-associated proteins downstream of the FoxO1-Akt axis were regulated by miR182-5p. Further, miR182-5p increased expression of VEGFR2 and VEGF levels, likely via inhibition of ZNF24. Thus, the FoxO1-Akt-glycolysis/VEGF pathway driving metabolic dysfunction with concurrent angiogenic signaling in PDR may be potentially targeted for treatment via miR182-5p modulation.
Assuntos
Diabetes Mellitus , Retinopatia Diabética , Hiperglicemia , MicroRNAs , Humanos , Retinopatia Diabética/metabolismo , Glucose/farmacologia , MicroRNAs/genética , MicroRNAs/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
Different types of refractive surgeries often exhibit differences in wound healing responses. The current study investigated post-operative tear protein profiles in subjects who underwent LASIK and SMILE to elucidate global changes to the proteomic profile during the period the patient cornea undergoes healing. In this study, 10 patients underwent LASIK and SMILE surgery with a contralateral paired eye design. Tear samples were collected using Schirmer's strips preoperatively, at 1 month, 3 months and 6 months postoperatively. Quantitative ITRAQ labeled proteomics was performed and the tear protein ratios were normalized to pre-operative protein levels for each subject. Whole proteomics identified 1345 proteins in tears from LASIK and 1584 proteins in SMILE across time points. About 67 proteins were common in LASIK and SMILE tears across all the time points. Wound healing responses were differentially regulated between two refractive surgeries (SMILE and LASIK). The proteins Ceruloplasmin, Clusterin, Serotransferrin were upregulated at 1 month and 3 months and downregulated at 6 months post operatively in LASIK surgery where as in SMILE these were downregulated. Galectin 3 binding protein showed upregulation at 1 month and the levels decreased at 3 months and 6 months postop in LASIK tears whereas the levels increased at 3 months and 6 months post-op in SMILE tears. The levels of proteins that protect from oxidative stress were higher in SMILE as compared to LASIK postoperatively. The extracellular matrix proteins showed an increase in expression at 6 months in SMILE tears and was stabilized at 6 months in LASIK tears post operatively. Different refractive surgeries induce distinct wound healing responses as identified in tears. This study has implications in targeting key proteins for improving the clinical outcome postrefractive surgery.
Assuntos
Proteínas do Olho , Ceratomileuse Assistida por Excimer Laser In Situ , Miopia , Proteômica , Lágrimas , Cicatrização , Humanos , Lágrimas/metabolismo , Ceratomileuse Assistida por Excimer Laser In Situ/métodos , Cicatrização/fisiologia , Proteômica/métodos , Feminino , Masculino , Adulto , Proteínas do Olho/metabolismo , Miopia/cirurgia , Miopia/metabolismo , Cirurgia da Córnea a Laser/métodos , Adulto Jovem , Lasers de Excimer/uso terapêutico , Período Pós-Operatório , Substância Própria/metabolismo , Substância Própria/cirurgiaRESUMO
The emergence of the novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic poses a never before seen challenge to human health and the economy. Considering its clinical impact, with no streamlined therapeutic strategies in sight, it is crucial to understand the infection process of SARS-CoV-2. Our limited knowledge of the mechanisms underlying SARS-CoV-2 infection impedes the development of alternative therapeutics to address the pandemic. This aspect can be addressed by modeling SARS-CoV-2 infection in the human context to facilitate drug screening and discovery. Human induced pluripotent stem cell (iPSC)-derived lung epithelial cells and organoids recapitulating the features and functionality of the alveolar cell types can serve as an in vitro human model and screening platform for SARS-CoV-2. Recent studies suggest an immune system asynchrony leading to compromised function and a decreased proportion of specific immune cell types in coronavirus disease 2019 (COVID-19) patients. Replenishing these specific immune cells may serve as useful treatment modality against SARS-CoV-2 infection. Here the authors review protocols for deriving lung epithelial cells, alveolar organoids and specific immune cell types, such as T lymphocytes and natural killer cells, from iPSCs with the aim to aid investigators in making relevant in vitro models of SARS-CoV-2 along with the possibility derive immune cell types to treat COVID-19.
Assuntos
COVID-19 , Células-Tronco Pluripotentes Induzidas , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Organoides/metabolismo , Estudos Prospectivos , SARS-CoV-2RESUMO
Infection mediated ocular surface stress responses are activated as early defense mechanisms in response to host cell damage. Integrated stress responses initiate the host response to different types of infections and modulate the transcription of key genes and translation of proteins. The crosstalk between host and pathogen results in profound alterations in cellular and molecular homeostasis triggering specific stress responses in the infected tissues. The amplitude and variations of such responses are partly responsible for the disease severity and clinical sequelae. Understanding the etiology and pathogenesis of ocular infections is important for early diagnosis and effective treatment. This review considers the molecular status of infection mediated ocular surface stress responses which may shed light on the importance of the host stress-signaling pathways. In this review, we collated literature on the molecular studies of all ocular surface infections and summarize the results from such studies systematically. Identification of important mediators involved in the crosstalk between the stress response and activation of diverse signaling molecules in host ocular surface infection may provide novel molecular targets for maintaining the cellular homeostasis during infection. These targets can be then explored and validated for diagnostic and therapeutic purposes.
Assuntos
Infecções Oculares , HumanosRESUMO
The differentiation of keratocytes to fibroblasts and myofibroblasts is an essential requisite during corneal wound closure. The aim of this study is to uncover factors involved in differentiation-dependent alteration in the protein profile of human corneal stromal cells using quantitative proteomics. Human corneal fibroblasts were cultured and differentiated into keratocytes in serum-free media and myofibroblasts through treatment with TGF-ß. The protein cell lysates from the donors were tryptic and were digested and labeled using a 3-plex iTRAQ kit. The labeled peptides were subjected to LCMS analysis. Biological functional analysis revealed a set of crucial proteins involved in the differentiation of human corneal stromal cells which were found to be significantly enriched. The selected proteins were further validated by immunohistochemistry. Quantitative proteomics identified key differentially expressed proteins which are involved in cellular signaling pathways. Proteins involved in integrin signaling (Ras-RAP1b, TLN and FN) and SLIT-ROBO pathways (PFN1, CAPR1, PSMA5) as well as extracellular matrix proteins (SERPINH1, SPARC, ITGß1, CRTAP) showed enhanced expression in corneal fibroblasts and myofibroblasts compared to keratocytes, indicating their possible role in wound healing. Corneal stromal cell differentiation is associated with the activation of diverse molecular pathways critical for the repair of fibroblasts and myofibroblasts. Identified proteins such as profilin 1 and talin could play a tentative role in corneal healing and serve as a potential target to treat corneal fibrosis.
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Lesões da Córnea , Proteômica , Diferenciação Celular/fisiologia , Células Cultivadas , Córnea/metabolismo , Lesões da Córnea/metabolismo , Fibroblastos/metabolismo , Humanos , Profilinas/metabolismo , Células Estromais/metabolismo , Cicatrização/fisiologiaRESUMO
The end of 2019 saw the beginning of the coronavirus disease 2019 (COVID-19) pandemic that soared in 2020, affecting 215 countries worldwide, with no signs of abating. In an effort to contain the spread of the disease and treat the infected, researchers are racing against several odds to find an effective solution. The unavailability of timely and affordable or definitive treatment has caused significant morbidity and mortality. Acute respiratory distress syndrome (ARDS) caused by an unregulated host inflammatory response toward the viral infection, followed by multi-organ dysfunction or failure, is one of the primary causes of death in severe cases of COVID-19 infection. Currently, empirical management of respiratory and hematological manifestations along with anti-viral agents is being used to treat the infection. The quest is on for both a vaccine and a more definitive management protocol to curtail the spread. Researchers and clinicians are also exploring the possibility of using cell therapy for severe cases of COVID-19 with ARDS. Mesenchymal stromal cells are known to have immunomodulatory properties and have previously been used to treat viral infections. This review explores the potential of mesenchymal stromal cells as cell therapy for ARDS.
Assuntos
COVID-19/epidemiologia , COVID-19/cirurgia , Transplante de Células-Tronco Mesenquimais/métodos , Pandemias , Síndrome do Desconforto Respiratório/epidemiologia , Síndrome do Desconforto Respiratório/cirurgia , SARS-CoV-2 , Animais , COVID-19/virologia , Ensaios Clínicos como Assunto , Comorbidade , Humanos , Imunomodulação , Células-Tronco Mesenquimais/imunologia , Síndrome do Desconforto Respiratório/virologia , Resultado do TratamentoRESUMO
Prostaglandin analogues (PG), beta-blockers (BB) or their combination (PG+BB) are used primarily to reduce the intraocular pressure (IOP) pathologically associated with glaucoma. Since, fibrosis of the trabecular meshwork (TM) is a major aetiological factor in glaucoma, we studied the effect of these drugs on fibrosis-associated gene expression in TM of primary glaucoma patients. In the present study, TM and iris of primary open-angle (n = 32) and angle-closure (n = 37) glaucoma patients were obtained surgically during trabeculectomy and categorized based on the type of IOP-lowering medications use as PG, BB or PG+BB. mRNA expression of pro-fibrotic and anti-fibrotic genes was quantified using qPCR in these tissues. The gene expression levels of pro-fibrotic genes were significantly lower in PG+BB as compared to other groups. These observations and underlying signalling validated in vitro in human TM cells also showed reduced fibrotic gene and protein expression levels following PG+BB treatment. In conclusion, it is observed that PG+BB combination rather than their lone use renders a reduced fibrotic status in TM. This further suggests that IOP-lowering medications, in combination, would also modulate fibrosis-associated molecular changes in the TM, which may be beneficial for maintaining aqueous out-flow mechanisms over the clinical treatment duration.
Assuntos
Antagonistas Adrenérgicos beta/uso terapêutico , Regulação da Expressão Gênica , Glaucoma/tratamento farmacológico , Glaucoma/genética , Prostaglandinas/agonistas , Malha Trabecular/metabolismo , Antagonistas Adrenérgicos beta/farmacologia , Estudos de Coortes , Quimioterapia Combinada , Feminino , Fibrose , Perfilação da Expressão Gênica , Regulação da Expressão Gênica/efeitos dos fármacos , Glaucoma/fisiopatologia , Humanos , Pressão Intraocular/efeitos dos fármacos , Masculino , Pessoa de Meia-Idade , Prostaglandinas Sintéticas/farmacologia , Prostaglandinas Sintéticas/uso terapêutico , Malha Trabecular/efeitos dos fármacos , Malha Trabecular/patologiaRESUMO
We studied the early protein profile in the ocular tissue extracted after LASIK and SMILE surgery. SMILE and LASIK was performed in contralateral eyes and stromal tissue samples were collected from 10 eyes of 5 donors. The stromal tissue samples were analyzed using label free quantification approach and ITRAQ labelling approach in LC-MS/MS. Combined functional analysis revealed many differentially expressed proteins which were involved in important biological processes. About 117 unique differentially expressed proteins were identified using two different proteomic approaches. Collagens, proteoglycans, corneal crystallins were enriched and showed differential expression in SMILE and LASIK as compared to the non-surgical control. Apart from these, 14-3-3 class of proteins, Lysozyme (LYZ), Macrophage Migratory Inhibitory Factor protein (MIF), Pigment Epithelial Derived Factor (PEDF) were differentially expressed when compared between LASIK and SMILE. Peroxiredoxin 1 (PRDX1) expression was found to be reduced in LASIK as compared to SMILE. The expression of Lysozyme C and Macrophage Migratory Inhibitory Factor inflammatory response was found to be less in SMILE as compared to LASIK. Western blot validation of specific markers such as Collagen IV (COL4), Keratocan (KERA), Lumican (LUM), Aldehyde dehydrogenase 3 A1 (ALDH3A1), Lysozyme C (LYZC) confirmed the differences in the protein levels observed in SMILE and LASIK operated tissues as compared to non-surgical controls. In conclusion, this study revealed the early molecular changes occurring in the cornea resulting from these two surgical procedures which may have implications on managing post-operative complications.
Assuntos
Substância Própria/cirurgia , Proteínas do Olho/metabolismo , Ceratomileuse Assistida por Excimer Laser In Situ/métodos , Proteoma/metabolismo , Idoso , Idoso de 80 Anos ou mais , Western Blotting , Cromatografia Líquida , Colágeno/metabolismo , Substância Própria/metabolismo , Cirurgia da Córnea a Laser , Cristalinas/metabolismo , Humanos , Masculino , Metaboloma , Pessoa de Meia-Idade , Estudos Prospectivos , Proteoglicanas/metabolismo , Espectrometria de Massas em TandemRESUMO
BACKGROUND: Medical big data analytics has revolutionized the human healthcare system by introducing processes that facilitate rationale clinical decision making, predictive or prognostic modelling of the disease progression and management, disease surveillance, overall impact on public health and research. Although, the electronic medical records (EMR) system is the digital storehouse of rich medical data of a large patient cohort collected over many years, the data lack sufficient structure to be of clinical value for applying deep learning methods and advanced analytics to improve disease management at an individual patient level or for the discipline in general. Ophthatome™ captures data contained in retrospective electronic medical records between September 2012 and January 2018 to facilitate translational vision research through a knowledgebase of ophthalmic diseases. METHODS: The electronic medical records data from Narayana Nethralaya ophthalmic hospital recorded in the MS-SQL database was mapped and programmatically transferred to MySQL. The captured data was manually curated to preserve data integrity and accuracy. The data was stored in MySQL database management system for ease of visualization, advanced search functions and other knowledgebase applications. RESULTS: Ophthatome™ is a comprehensive and accurate knowledgebase of ophthalmic diseases containing curated clinical, treatment and imaging data of 581,466 ophthalmic subjects from the Indian population, recorded between September 2012 and January 2018. Ophthatome™ provides filters and Boolean searches with operators and modifiers that allow selection of specific cohorts covering 524 distinct ophthalmic disease types and 1800 disease sub-types across 35 different anatomical regions of the eye. The availability of longitudinal data for about 300,000 subjects provides additional opportunity to perform clinical research on disease progression and management including drug responses and management outcomes. The knowledgebase captures ophthalmic diseases in a genetically diverse population providing opportunity to study genetic and environmental factors contributing to or influencing ophthalmic diseases. CONCLUSION: Ophthatome™ will accelerate clinical, genomic, pharmacogenomic and advanced translational research in ophthalmology and vision sciences.
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Oftalmopatias , Oftalmologia , Registros Eletrônicos de Saúde , Oftalmopatias/diagnóstico , Oftalmopatias/epidemiologia , Oftalmopatias/terapia , Humanos , Bases de Conhecimento , Estudos RetrospectivosRESUMO
PURPOSE: The pathogenesis of pterygium has been linked to limbal stem cell damage, abnormal apoptosis and cellular proliferation. In this study, we investigated the epigenetic regulation through microRNA expression in the pathogenesis of pterygium. METHODS: Human full-length primary pterygia were microdissected into head and body regions. Specific microRNA and mRNA expression was assayed by TaqMan® real-time quantitative polymerase chain reaction (qPCR). Tissue localization of target microRNAs was performed by LNA-based in situ hybridization. MicroRNA-145 (miR-145) mimics were transfected to primary culture of human pterygial cells, followed by analyses of cell cycle changes, apoptosis, p53 and MDM2 expression using flow cytometry and qPCR. RESULTS: The expression of miR-145 was markedly higher in primary human pterygium than in limbus and conjunctiva. Both miR-143 and miR-145 were predominantly expressed in the basal pterygial epithelium. Oncogene MDM2 expression was abundant in pterygial epithelium and stroma, while the expression pattern was opposite to that of miR-145. Ectopic expression of miR-145 in pterygial cells induced G1 arrest, down-regulated MDM2 and elevated p53 expression. CONCLUSIONS: Our study showed that miR-145 suppressed MDM2 expression, which subsequently influenced the p53-related cell growth pattern in pterygial epithelium. The regulatory miR-145/MDM2-p53 loop can serve as a potential target for treatment of pterygium.
Assuntos
Regulação da Expressão Gênica/fisiologia , MicroRNAs/genética , Proteínas Proto-Oncogênicas c-mdm2/genética , Pterígio/genética , Apoptose , Proliferação de Células , Células Cultivadas , Epigênese Genética , Células Epiteliais/patologia , Citometria de Fluxo , Técnica Indireta de Fluorescência para Anticorpo , Humanos , Hibridização In Situ , Reação em Cadeia da Polimerase em Tempo Real , TransfecçãoRESUMO
In the present study we investigated the phosphorylation status of the 12 most important signaling cascades in glioblastomas. More than 60 tumor and control biopsies from tumor center and periphery (based on neuronavigation) were subjected to selective protein expression analysis using reverse-phase protein arrays (RPPA) incubated with antibodies against posttranslationally modified cancer pathway proteins. The ratio between phosphorylated (or modified) and non-phosphorylated protein was assessed. All samples were histopathologically validated and proteomic profiles correlated with clinical and survival data. By RPPA, we identified three distinct activation patterns within glioblastoma defined by the ratios of pCREB1/CREB1, NOTCH-ICD/NOTCH1, and pGSK3ß/GSK3ß, respectively. These subclasses demonstrated distinct overall survival patterns in a cohort of patients from a single-institution and in an analysis of publicly available data. In particular, a high pGSK3ß/GSK3ß-ratio was associated with a poor survival. Wnt-activation/GSK3ß-inhibition in U373 and U251 cell lines halted glioma cell proliferation and migration. Gene expression analysis was used as an internal quality control of baseline proteomic data. The protein expression and phosphorylation had a higher resolution, resulting in a better class-subdivision than mRNA based stratification data. Patients with different proteomic profiles from multiple biopsies showed a worse overall survival. The CREB1-, NOTCH1-, GSK3ß-phosphorylation status correlated with glioma grades. RPPA represent a fast and reliable tool to supplement morphological diagnosis with pathway-specific information in individual tumors. These data can be exploited for molecular stratification and possible combinatorial treatment planning. Further, our results may optimize current glioma grading algorithms.
Assuntos
Neoplasias Encefálicas/metabolismo , Glioblastoma/metabolismo , Transdução de Sinais , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Fosforilação , Análise Serial de Proteínas , Proteômica , Análise de SobrevidaRESUMO
PURPOSE: To investigate the tear cytokine profile in HIV patients with dry eye disease (DED) and study the association between the severity of ocular inflammatory complications and tear cytokines levels. We postulate that HIV-mediated inflammation may be the underlying pathogenic mechanism for HIV-associated DED. METHODS: The current prospective case-control study compared tear film cytokine profiles in DED patients with HIV infection (n=34) and age/gender-matched DED patients without HIV infection [controls (n=32)]. Participants were recruited from tertiary referral eye care centre and communicable disease clinics, Singapore. Ocular surface health was documented using tear film, Schirmer's test, corneal staining, and conjunctival injection measurements. Tear samples were collected using Schirmer's strips and analysed for the levels of 41 cytokines using Luminex bead assay. Logistic regression models were performed to determine correlation and significance. RESULTS: Among the 41 cytokines analysed, statistically significant differences were observed in the mean values of epithelial growth factor (EGF), growth related oncogene (GRO) and interferon gamma-induced protein 10 (IP-10). EGF and IP-10 levels were higher and GRO levels were lower in the tears of DED patients with HIV infection compared to DED patients without HIV infection. No significant association was found between varying levels of ocular surface parameters and cytokine concentrations in HIV patients with DED (p>0.05). CONCLUSIONS: EGF and IP-10 were significantly elevated and GRO levels were lower in the tear profile of HIV patients with DED compared to immunocompetent patients with DED. This study suggests a novel cytokine driven paradigm for ocular inflammatory complications of HIV infection. Additional studies in large organised cohorts can validate the results.
Assuntos
Citocinas/metabolismo , Síndromes do Olho Seco/metabolismo , Infecções por HIV/metabolismo , HIV-1 , Lágrimas/metabolismo , Adulto , Síndromes do Olho Seco/etiologia , Feminino , Infecções por HIV/complicações , Humanos , Masculino , Pessoa de Meia-Idade , Estudos ProspectivosRESUMO
BACKGROUND: Visual system homeobox gene (VSX1) plays a major role in the early development of craniofacial and ocular tissues including cone opsin gene in the human retina. To date, few disease-causing mutations of VSX1 have been linked to familial and sporadic keratoconus (KC) in humans. In this study, we describe the clinical features and screening for VSX1 gene in families with KC from India. METHODS: Clinical data and genomic DNA were collected from patients with clinically diagnosed KC and their family members. The study was conducted on 20 subjects of eight families from India. The coding exons of VSX1 gene were amplified using PCR and amplicons were analyzed by direct sequencing. Predictive effect of the mutations was performed using Polyphen-2, SIFT and mutation assessor algorithms. Additionally, haplotypes of VSX1 gene were constructed for affected and unaffected individuals using SNPs. RESULTS: In the coding region of VSX1, one novel missense heterozygous change (p.Leu268His) was identified in five KC patients from two unrelated families. Another family of three members had a novel heterozygous change (p.Ser251Thr). These variants co-segregated with the disease phenotype in all affected individuals but not in the unaffected family members and 105 normal controls. In silico analysis suggested that p.Leu268His could have a deleterious effect on the protein coded by VSX1, while p.Ser251Thr has a neutral effect on the functional properties of VSX1. Haplotype examination revealed common SNPs around the missense change (p.Leu268His) in two unrelated KC families. CONCLUSIONS: In this study, we add p.Leu268His, a novel missense variation in the coding region of VSX1 to the existing repertoire of VSX1 coding variations observed in Indian patients with the characteristic phenotype of KC. The variant p.Ser251Thr might be a benign polymorphism, but further biophysical studies are necessary to evaluate its molecular mechanism. The shared haplotype by two families with the same variant suggests the possibility of a founder effect, which requires further elucidation. We suggest that p.Leu268His might be involved in the pathogenesis of KC, which may help in the genetic counselling of patients and their family.
Assuntos
Proteínas do Olho/genética , Proteínas de Homeodomínio/genética , Ceratocone/genética , Mutação de Sentido Incorreto , Adolescente , Adulto , Sequência de Aminoácidos , Animais , Sequência de Bases , Biologia Computacional , Proteínas do Olho/química , Feminino , Haplótipos , Proteínas de Homeodomínio/química , Humanos , Índia , Masculino , Pessoa de Meia-Idade , Dados de Sequência Molecular , Linhagem , Adulto JovemRESUMO
PURPOSE: Keratoconus (KC) is characterized by progressive vision loss due to corneal thinning and structural abnormalities. It is hypothesized that KC is caused by deregulated collagen levels and collagen fibril-maturating enzyme lysyl oxidase (LOX). Further, it is currently not understood whether the gene expression deregulated by the corneal epithelium influences KC pathogenesis. We studied (i) the expressions of the LOX, collagen I (COL IA1), collagen IV (COL IVA1), MMP9, and IL6 genes in KC corneal epithelia, (ii) validated their expression levels in patient tissues, and (iii) correlated expression levels with KC disease severity. The primary goal of this study was to evaluate the importance of these genes in the progression of KC. METHODS: We analyzed the gene expression levels of the key proteins LOX, collagens (COL IA1 and COL IVA1), MMP9, and IL6 in debrided corneal epithelia from a large cohort of KC patients (90 eyes) and compared them to control patients (52 eyes) without KC. We measured the total LOX activity in the tears of KC patients compared to controls. We also correlated the protein expression levels of LOX and collagens by immunohistochemistry (IHC) in primary tissues from KC patients (27 eyes) undergoing keratoplasty compared to healthy donor corneas (15 eyes). RESULTS: We observed a significant reduction in LOX transcript levels in KC corneal epithelia, and LOX activity in KC tears correlated with disease severity. Collagen transcripts were also reduced in KC while MMP9 transcript levels were upregulated and correlated with disease severity. IL6 was moderately increased in KC patients. IHC demonstrated a reduction in the protein expression levels of LOX in the epithelium and collagen IV in the basement membrane of KC patients compared to healthy donor corneas. CONCLUSIONS: The data demonstrates that the structural deformity of the KC cornea may be dependent on reduced expressions of collagens and LOX, as well as on MMP9 elevated by the corneal epithelium.
Assuntos
Colágeno Tipo IV/genética , Colágeno Tipo I/genética , Epitélio Corneano/metabolismo , Ceratocone/genética , Proteína-Lisina 6-Oxidase/genética , Adulto , Estudos de Casos e Controles , Colágeno Tipo I/metabolismo , Colágeno Tipo IV/metabolismo , Epitélio Corneano/patologia , Feminino , Regulação da Expressão Gênica , Humanos , Interleucina-6/genética , Interleucina-6/metabolismo , Ceratocone/metabolismo , Ceratocone/patologia , Masculino , Metaloproteinase 9 da Matriz/genética , Metaloproteinase 9 da Matriz/metabolismo , Proteína-Lisina 6-Oxidase/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Índice de Gravidade de Doença , Lágrimas/químicaRESUMO
Inherited genetic disorders are progressive in nature and lead to organ dysfunction or death in severe cases. At present, there are no permanent treatment options for >95% of inherited disorders. Different modes of inheritance, type of gene(s) involved, and population-based variations add further complexity to finding suitable cures for approximately 400 million patients worldwide. Gene therapy is a very promising molecular technique for the treatment of rare genetic disorders. Gene therapy functions on the basis of restoration, replacement, inhibition, and, most recently, editing of gene(s) to rescue the disease phenotype. Recent reports show that increasing numbers of gene therapy clinical trials are using viral vectors (64.2%) when compared with non-viral vectors. Rapid development of efficient viral vector systems like the adeno-associated virus (AAV) and lentivirus has significantly contributed to this progress. Notably, AAV-mediated gene therapy has shown high potential for genetic disease treatment as evident from recent clinical trials for the eye (NCT00999609), blood (NCT00979238), and neuro-muscular systems (NCT02122952). Safety and efficacy are the two most critical features required for vector(s) to qualify for pre-clinical and clinical trial approval. The process of clinical-grade vector production, evaluation, and approvals for gene therapy products requires significant technological development, knowledge enhancement, and large financial investments. Additionally, trained manpower is required to meet the demands for constant technical innovation. These factors together contribute towards exorbitant prices for every dose of a gene therapy product and thus pose a challenge for the gene therapy field. The Indian subcontinent has traditionally lagged behind North America, Europe, Japan, and others in gene therapy clinical trials due to factors like inadequate industrial-scientific infrastructure, lack of accessible and organized patient databases, low financial investments, etc. However, over the last decade, increasing awareness of rare diseases, and international approvals of gene therapies such as Luxturna, Zolgensma, Hemgenix, etc., have spurred gene therapy development in India as well. In view of these advances, this article outlines gene therapy research, regulatory processes, and the launch of gene therapy clinical trials in India in the context of major developments worldwide. We briefly describe ongoing gene therapy research across Indian organizations and the nascent gene therapy product manufacturing. Further, we highlight the various initiatives from the medical and patient community to avail rehabilitation and gene therapy options. We briefly discuss the roles of regulatory agencies and guidelines for gene therapy clinical trials in India. We anticipate that this concise review will highlight the promise of gene therapy for the large population of rare disease patients in India.
Assuntos
Ensaios Clínicos como Assunto , Terapia Genética , Humanos , Terapia Genética/efeitos adversos , Vetores Genéticos/genética , Índia , Lentivirus/genéticaRESUMO
Diseases leading to retinal cell loss can cause severe visual impairment and blindness. The lack of effective therapies to address retinal cell loss and the absence of intrinsic regeneration in the human retina leads to an irreversible pathological condition. Progress in recent years in the generation of human three-dimensional retinal organoids from pluripotent stem cells makes it possible to recreate the cytoarchitecture and associated cell-cell interactions of the human retina in remarkable detail. These human three-dimensional retinal organoid systems made of distinct retinal cell types and possessing contextual physiological responses allow the study of human retina development and retinal disease pathology in a way animal model and two-dimensional cell cultures were unable to achieve. We describe the derivation of retinal organoids from human pluripotent stem cells and their application for modeling retinal disease pathologies, while outlining the opportunities and challenges for its application in academia and industry.
Assuntos
Células-Tronco Pluripotentes , Doenças Retinianas , Animais , Humanos , Retina , Células-Tronco Pluripotentes/metabolismo , Organoides/metabolismo , Doenças Retinianas/tratamento farmacológico , Doenças Retinianas/metabolismo , Descoberta de Drogas , Diferenciação CelularRESUMO
Peripheral artery disease (PAD), a significant health burden worldwide, affects lower extremities due to atherosclerosis in peripheral vessels. Although the mechanisms of PAD have been well studied, the molecular milieu of the plaques localized within peripheral arteries are not well understood. Thus, to identify PAD-lesion-specific gene expression profiles precluding genetic, environmental, and dietary biases, we studied the transcriptomic profile of nine plaque tissues normalized to non-plaque tissues from the same donors. A total of 296 upregulated genes, 274 downregulated genes, and 186 non-coding RNAs were identified. STAG1, SPCC3, FOXQ1, and E2F3 were key downregulated genes, and CD93 was the top upregulated gene. Autophagosome assembly, cellular response to UV, cytoskeletal organization, TCR signaling, and phosphatase activity were the key dysregulated pathways identified. Telomerase regulation and autophagy were identified as novel interacting pathways using network analysis. The plaque tissue was predominantly composed of immune cells and dedifferentiated cell populations indicated by cell-specific marker-imputed gene expression analysis. This study identifies novel genes, non-coding RNAs, associated regulatory pathways, and the cell composition of the plaque tissue in PAD patients. The autophagy and immunoregulatory genes may drive novel mechanisms, resulting in atheroma. These novel interacting networks and genes have potential for PAD-specific therapeutic applications.
Assuntos
Autofagia , Perfilação da Expressão Gênica , Doença Arterial Periférica , Placa Aterosclerótica , Transcriptoma , Humanos , Autofagia/genética , Doença Arterial Periférica/genética , Doença Arterial Periférica/patologia , Transcriptoma/genética , Placa Aterosclerótica/genética , Placa Aterosclerótica/patologia , Masculino , Feminino , Redes Reguladoras de Genes , Pessoa de Meia-Idade , Idoso , Regulação da Expressão GênicaRESUMO
The gene therapy approach for retinal disorders has been considered largely over the last decade owing to the favorable outcomes of the US Food and Drug Administration-approved commercial gene therapy, Luxturna. Technological advances in recent years, such as next-generation sequencing, research in molecular pathogenesis of retinal disorders, and precise correlations with their clinical phenotypes, have contributed to the progress of gene therapies for various diseases worldwide, and more recently in India as well. Thus, considerable research is being conducted for the right choice of vectors, transgene engineering, and accessible and cost-effective large-scale vector production. Many retinal disease-specific clinical trials are presently being conducted, thereby necessitating the collation of such information as a ready reference for the scientific and clinical community. In this article, we present an overview of existing gene therapy research, which is derived from an extensive search across PubMed, Google Scholar, and clinicaltrials.gov sources. This contributes to prime the understanding of basic aspects of this cutting-edge technology and information regarding current clinical trials across many different conditions. This information will provide a comprehensive evaluation of therapies in existing use/research for personalized treatment approaches in retinal disorders.
Assuntos
Terapia Genética , Doenças Retinianas , Humanos , Terapia Genética/métodos , Doenças Retinianas/terapia , Doenças Retinianas/genética , Vetores GenéticosRESUMO
PURPOSE: To report a case of mumps-associated outer retinitis, diagnostic, and therapeutic challenges associated with the disease. METHOD: Retrospective observational case report. RESULTS: An 8-year-old male child on presentation had a history of mumps infection following which he developed outer retinitis. Upon evaluation, he had bilateral multifocal perivascular cerebriform retinitis. MRI revealed increased uptake of contrast by bilateral parotid gland and with serum mumps IgM and IgG antibodies being raised, a diagnosis of mumps associated outer retinitis was made. In terms of treatment post-systemic steroid therapy, hyperbaric oxygen therapy was tried as a rescue therapy in this patient. Improvement in vision was noted in the left eye more than the right eye. CONCLUSION: Hyperbaric oxygen therapy can be considered as an additional therapy to systemic steroid therapy in mumps associated retinitis. In such a situation, since there is no specific antiviral drug available for mumps infection, the most effective treatment is prevention by vaccination.