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2.
Exp Eye Res ; 205: 108527, 2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-33667466

RESUMO

The purpose of this study was to evaluate the expression of the SARS-CoV-2 receptors ACE2 and TMPRSS2 in an immortalized human conjunctival epithelial cell line and in healthy human conjunctiva excised during ocular surgery, using Western blot, confocal microscopy and immunohistochemistry. The Western blot showed that ACE2 and TMPRSS2 proteins were expressed in human immortalized conjunctival cells, and this was confirmed by confocal microscopy images, that demonstrated a marked cellular expression of the viral receptors and their co-localization on the cell membranes. Healthy conjunctival samples from 11 adult patients were excised during retinal detachment surgery. We found the expression of ACE2 and TMPRSS2 in all the conjunctival surgical specimens analyzed and their co-localization in the superficial conjunctival epithelium. The ACE2 Western blot levels and immunofluorescence staining for ACE2 were variable among specimens. These results suggest the susceptibility of the conjunctival epithelium to SARS-CoV-2 infection, even though with a possible interindividual variability.


Assuntos
/genética , Túnica Conjuntiva/metabolismo , Células Epiteliais/metabolismo , Regulação da Expressão Gênica , Peptidil Dipeptidase A/genética , Serina Endopeptidases/genética , /metabolismo , Células Epiteliais/patologia , Humanos , Imuno-Histoquímica , Peptidil Dipeptidase A/biossíntese , RNA/genética , RNA/metabolismo , Serina Endopeptidases/biossíntese
3.
Int J Mol Sci ; 22(4)2021 Feb 18.
Artigo em Inglês | MEDLINE | ID: mdl-33670592

RESUMO

In this study, we investigated the effects of blue light exposure on nucleotide-binding oligomerization domain 2 (NOD2) expression on the mouse ocular surface and evaluated the role of NOD2 activation in light-induced cell death. Mice were divided into wild-type (WT), NOD2-knock out (KO), WT + blue light (WT + BL), and NOD2-KO + blue light (NOD2-KO + BL) groups, and the mice in the WT+BL and NOD2-KO + BL groups were exposed to blue light for 10 days. After 10 days of blue light exposure, increased reactive oxygen species and malondialdehyde were observed in the WT + BL and NOD2-KO + BL groups, and the WT + BL group showed a higher expression of NOD2 and autophagy related 16 like 1. Although both WT+BL and NOD2-KO + BL groups showed an increase in the expression of light chain 3-II, NOD2-KO + BL mice had a significantly lower p62 expression than WT + BL mice. In addition, NOD2-KO+BL mice had significantly lower corneal epithelial damage and apoptosis than WT + BL mice. In conclusion, blue light exposure can induce impaired autophagy by activation of NOD2 on the ocular surface. In addition, the reactive oxygen species (ROS)-NOD2-autophagy related 16 like 1 (ATG16L) signaling pathway may be involved in the blue-light-induced autophagy responses, resulting in corneal epithelial apoptosis.


Assuntos
Autofagia/efeitos da radiação , Epitélio Anterior/efeitos da radiação , Luz , Proteína Adaptadora de Sinalização NOD2/metabolismo , Animais , Apoptose/genética , Apoptose/efeitos da radiação , Autofagia/genética , Proteínas Relacionadas à Autofagia/metabolismo , Western Blotting , Túnica Conjuntiva/metabolismo , Túnica Conjuntiva/efeitos da radiação , Epitélio Anterior/metabolismo , Feminino , Malondialdeído/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteína Adaptadora de Sinalização NOD2/genética , Espécies Reativas de Oxigênio/metabolismo
5.
J Med Virol ; 93(5): 3122-3132, 2021 May.
Artigo em Inglês | MEDLINE | ID: mdl-33570199

RESUMO

OBJECTIVES: To perform a comprehensive clinic, laboratory, and instrumental evaluation of children affected by coronavirus disease (COVID-19). METHODS: Children with a positive result of nasopharyngeal swab for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) underwent laboratory tests, anal and conjunctival swab, electrocardiography, lung, abdomen, and cardiac ultrasound. Twenty-four-hour ambulatory blood pressure monitoring was performed if abnormal basal blood pressure. Patients were followed-up for 6 months. RESULTS: Three hundred and sixteen children were evaluated; 15 were finally included. Confirmed family member SARS-CoV-2 infection was present in all. Twenty-seven percent were asymptomatic. Anal and conjunctival swabs tests resulted negative in all. Patients with lower body mass index (BMI) presented significantly higher viral loads. Main laboratory abnormalities were: lactate dehydrogenase increasing (73%), low vitamin D levels (87%), hematuria (33%), proteinuria (26%), renal hyperfiltration (33%), and hypofiltration (13%). Two of the patients with hyperfiltration exhibited high blood pressure levels at diagnosis, and persistence of prehypertension at 6-month follow-up. No abnormalities were seen at ultrasound, excepting for one patient who exhibited B-lines at lung sonography. Immunoglobulin G seroconversion was observed in all at 1-month. CONCLUSIONS: Our study confirm that intra-family transmission is important. The significant higher viral loads recorded among patients with lower BMI, together with low vitamin D levels, support the impact of nutritional status on immune system. Renal involvement is frequent even among children with mild COVID-19, therefore prompt evaluation and identification of patients with reduced renal function reserve would allow a better stratification and management of patients. Seroconversion occurs also in asymptomatic children, with no differences in antibodies titer according to age, sex and clinical manifestations.


Assuntos
/diagnóstico , /patologia , Adolescente , Canal Anal/virologia , Índice de Massa Corporal , Criança , Pré-Escolar , Túnica Conjuntiva/virologia , Busca de Comunicante , Família , Feminino , Humanos , Masculino , Nasofaringe/virologia , Estudos Prospectivos , Carga Viral
6.
Exp Eye Res ; 205: 108501, 2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-33600811

RESUMO

The angiotensin-converting enzyme 2 (ACE2) receptor has been proved for SARS-CoV-2 cell entry after auxiliary cellular protease priming by transmembrane protease serine 2 (TMPRSS2), but the co-effect of this molecular mechanism was unknown. Here, single-cell sequencing was performed with human conjunctiva and the results have shown that ACE2 and TMPRSS2 were highly co-expressed in the goblet cells with genes involved in immunity process. This identification of conjunctival cell types which are permissive to virus entry would help to understand the process by which SARS-CoV-2 infection was established. These finding might be suggestive for COVID-19 control and protection.


Assuntos
/genética , Túnica Conjuntiva/metabolismo , Regulação da Expressão Gênica , Células Caliciformes/metabolismo , Peptidil Dipeptidase A/genética , Serina Endopeptidases/genética , /metabolismo , Túnica Conjuntiva/patologia , Células Caliciformes/patologia , Humanos , Peptidil Dipeptidase A/biossíntese , RNA/genética , Serina Endopeptidases/biossíntese
7.
Arerugi ; 70(1): 26-32, 2021.
Artigo em Japonês | MEDLINE | ID: mdl-33597343

RESUMO

BACKGROUND/PURPOSE: In a method evaluating conjunctival hyperemia using rabbits, it is common to visually grade the degree of vasodilation. However, this method is limited in evaluating consecutive value and in reproducibility. We quantified the degree of conjunctival hyperemia in rabbits as the area ratio of blood vessels by image analysis, and compared the vascular area percentage calculated by image analysis with the hyperemia score. METHODS: The conjunctiva was photographed before and after the instillation of 0.1% arachidonic acid using a digital medical scope VersaCam® (Nidek Co., Ltd.). Next, the area of the conjunctival blood vessels occupying the area of interest was calculated using hyperemia analysis software. The hyperemia score was visually graded for the degree of conjunctiva vasodilation. Furthermore, the hyperemia score and the vascular area ratio were compared. RESULTS: Fifteen minutes after the instillation of arachidonic acid, the area ratio of the blood vessels in the conjunctiva increased significantly and gradually decreased over time. This trend correlated with the hyperemia score. CONCLUSION: We found that the degree of conjunctival hyperemia in rabbits can be evaluated numerically and quantitatively. This method is considered to be useful for evaluating conjunctival hyperemia in allergic conjunctival diseases.


Assuntos
Doenças da Túnica Conjuntiva , Hiperemia , Túnica Conjuntiva , Humanos , Reprodutibilidade dos Testes , Software
8.
Zhonghua Yan Ke Za Zhi ; 57(2): 139-141, 2021 Feb 11.
Artigo em Chinês | MEDLINE | ID: mdl-33541056

RESUMO

A 34-year-old female presented to the doctor with chief complaint of redness in both eyes for 1 week. The slit lamp examination showed slight eyelid swelling, water-like discharge in the conjunctival sac of the lower eyelid, conjunctival follicles, gray-white pseudomembrane and small lamellar subconjunctival hemorrhage on the lower palpebral conjunctiva. Physical examination showed two erythematous macules(size 5 mm×6 mm) on the left palm. One erythematous macule(size 4 mm×5 mm) was seen on the right palm. Multiple erosions were located in the skin of upper and lower lips, oral cavity and the gingival mucosa. A diagnosis of hand, foot and mouth disease was made following by dermatological consultation. After topical antiviral and steroids treatment, the disease quickly relieved.(Chin J Ophthalmol, 2021, 57: 139-141).


Assuntos
Doenças da Túnica Conjuntiva , Conjuntivite , Doença de Mão, Pé e Boca , Adulto , Túnica Conjuntiva , Doenças da Túnica Conjuntiva/diagnóstico , Conjuntivite/diagnóstico , Pálpebras , Feminino , Doença de Mão, Pé e Boca/diagnóstico , Humanos
9.
Lancet Digit Health ; 3(2): e88-e97, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-33509389

RESUMO

BACKGROUND: Ocular changes are traditionally associated with only a few hepatobiliary diseases. These changes are non-specific and have a low detection rate, limiting their potential use as clinically independent diagnostic features. Therefore, we aimed to engineer deep learning models to establish associations between ocular features and major hepatobiliary diseases and to advance automated screening and identification of hepatobiliary diseases from ocular images. METHODS: We did a multicentre, prospective study to develop models using slit-lamp or retinal fundus images from participants in three hepatobiliary departments and two medical examination centres. Included participants were older than 18 years and had complete clinical information; participants diagnosed with acute hepatobiliary diseases were excluded. We trained seven slit-lamp models and seven fundus models (with or without hepatobiliary disease [screening model] or one specific disease type within six categories [identifying model]) using a development dataset, and we tested the models with an external test dataset. Additionally, we did a visual explanation and occlusion test. Model performances were evaluated using the area under the receiver operating characteristic curve (AUROC), sensitivity, specificity, and F1* score. FINDINGS: Between Dec 16, 2018, and July 31, 2019, we collected data from 1252 participants (from the Department of Hepatobiliary Surgery of the Third Affiliated Hospital of Sun Yat-sen University, the Department of Infectious Diseases of the Affiliated Huadu Hospital of Southern Medical University, and the Nantian Medical Centre of Aikang Health Care [Guangzhou, China]) for the development dataset; between Aug 14, 2019, and Jan 31, 2020, we collected data from 537 participants (from the Department of Infectious Diseases of the Third Affiliated Hospital of Sun Yat-sen University and the Huanshidong Medical Centre of Aikang Health Care [Guangzhou, China]) for the test dataset. The AUROC for screening for hepatobiliary diseases of the slit-lamp model was 0·74 (95% CI 0·71-0·76), whereas that of the fundus model was 0·68 (0·65-0·71). For the identification of hepatobiliary diseases, the AUROCs were 0·93 (0·91-0·94; slit-lamp) and 0·84 (0·81-0·86; fundus) for liver cancer, 0·90 (0·88-0·91; slit-lamp) and 0·83 (0·81-0·86; fundus) for liver cirrhosis, and ranged 0·58-0·69 (0·55-0·71; slit-lamp) and 0·62-0·70 (0·58-0·73; fundus) for other hepatobiliary diseases, including chronic viral hepatitis, non-alcoholic fatty liver disease, cholelithiasis, and hepatic cyst. In addition to the conjunctiva and sclera, our deep learning model revealed that the structures of the iris and fundus also contributed to the classification. INTERPRETATION: Our study established qualitative associations between ocular features and major hepatobiliary diseases, providing a non-invasive, convenient, and complementary method for hepatobiliary disease screening and identification, which could be applied as an opportunistic screening tool. FUNDING: Science and Technology Planning Projects of Guangdong Province; National Key R&D Program of China; Guangzhou Key Laboratory Project; National Natural Science Foundation of China.


Assuntos
Algoritmos , Simulação por Computador , Aprendizado Profundo , Doenças do Sistema Digestório/diagnóstico , Olho , Programas de Rastreamento/métodos , Modelos Biológicos , Adulto , Área Sob a Curva , China , Túnica Conjuntiva/diagnóstico por imagem , Doenças do Sistema Digestório/complicações , Olho/diagnóstico por imagem , Fundo de Olho , Humanos , Iris/diagnóstico por imagem , Fígado , Pessoa de Meia-Idade , Fotografação/métodos , Estudos Prospectivos , Curva ROC , Esclera/diagnóstico por imagem , Microscopia com Lâmpada de Fenda/métodos
10.
Life Sci ; 270: 119088, 2021 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-33482188

RESUMO

This study aimed to determine whether MG-132 as a proteasome inhibitor can effectively hinder pterygium progression, and to screen out potential regulators involved in MG-132 mediated process. Human pterygium fibroblasts (HPFs) were derived from pterygium tissues from 5 patients. Cell proliferation was examined by MTT, cell cycle and apoptosis were detected by flow cytometry. The overgrowth pterygium tissues were characterized by H&E staining and IHC compared with normal tissues. Differential mRNA expression with MG-132 treatment was determined by RNA sequencing and analyzed by GO and KEGG pathways. The expression levels of Nrf2, MCPIP1, CDKN1B and XBP1, four genes closely associated with pterygium, were detected by RT-qPCR and western blotting. MG-132 dose-dependently inhibited the growth of HPFs, induced G2/M phase arrest of cell cycle at a certain dose, and also caused cell apoptosis, with the levels of cleaved caspase3, cleaved PARP, Bax and p21 increased. Ki-67 and Bcl-2 were highly expressed while Bax was decreased in pterygium tissues. Total 7199 differentially expressed genes (DEGs) were identified, including HSPA family most significantly increased, and AL590428.1, AL122125.1 and lincRNAs such as FGF14-AS2 decreased. The up-regulated DEGs were mainly enriched in RNA degradation pathway, while down-regulated DEGs were related to the regulation of cell cycle. The expressions of Nrf2 and MCPIP1 were significantly increased, while XBP1 and CDKN1B were decreased. In conclusion, MG-132 inhibited the proliferation and induced apoptosis of HPFs in vitro with 7199 DEGs participated in, which may provide a useful reference for the exploitation of MG-132 in treating pterygium.


Assuntos
Leupeptinas/farmacologia , Pterígio/genética , Pterígio/metabolismo , Adulto , Apoptose/efeitos dos fármacos , Ciclo Celular/efeitos dos fármacos , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , China , Túnica Conjuntiva/metabolismo , Feminino , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Humanos , Leupeptinas/metabolismo , Masculino , Pessoa de Meia-Idade , Cultura Primária de Células , Inibidores de Proteassoma/farmacologia , Pterígio/tratamento farmacológico
12.
Rev Med Suisse ; 17(723): 206-208, 2021 Jan 27.
Artigo em Francês | MEDLINE | ID: mdl-33507662

RESUMO

The most frequent ocular manifestation of the SARS-CoV-2 is a conjunctivitis. It is found in 1-3% of patients and has unusually a benign course. In those patients but also in patients without ocular involvement, the virus can be detected by PCR in conjunctival swabs or in the tears. Whereas the presence of the virus in the conjunctiva is proven, its transmission through the conjunctiva is still discussed. Despite the poor level of evidence, the use of protective eyewear is recommended. Ocular thromboembolic events have been described in Covid-19. They can be found in the context of the Covid-related coagulopathy. A multidisciplinary approach should be provided in these cases. In the ICU, severe ocular complications might be indirectly related to Covid-19 in ventilated patients.


Assuntos
Túnica Conjuntiva , Humanos , Lágrimas
13.
Gene ; 775: 145447, 2021 Apr 05.
Artigo em Inglês | MEDLINE | ID: mdl-33482278

RESUMO

Limbal stem cells (LSCs) reside in the basal layer of limbal epithelial cells (LECs). They are crucial for maintenance of corneal epithelium homeostasis and corneal wound healing. Their stemness is determined by their gene expression pattern. Despite of several positive identifiers have been reported, the unique biomarker for LSCs still remain elusive. Differentially expressed genes (DEGs) between stem cells and differentiated cells affect the fate of stem cells via specific signaling pathway. In order to understand the DEGs in the LSCs, RNA-seq was firstly conducted using a mouse model. A total of 1907 up-regulated DEGs and 395 down-regulated DEGs were identified in the limbus (L) compared to central cornea (CC) and conjunctiva (Cj). Reliability of the expression of genes from RNA-seq analysis was evaluated by quantitative real-time polymerase chain reaction (qRT-PCR) and immunofluorescence staining. The expression pattern of putative biomarkers was considered to be age-related. In up-regulated DEGs GO analysis, 570 gene ontology (GO) terms were significantly enriched. Five groups of genes related with biological processes from these significantly enriched GO terms comprised ionic transport, regulation of tissue development, muscle contraction, visual perception, and cell adhesion, which were clustered as a weighted similar network. Whereas, in down-regulated DEGs GO analysis, 61 GO terms were significantly enriched and only one group of ATP biosynthesis and metabolic process were clustered. Furthermore, we identified 55 signaling pathways by the Kyoto Encyclopedia of Genes and Genomes (KEGG) database based on up-regulated genes and 14 KEGG pathways based on down-regulated genes. In this study, we provide a landscape of the expression of putative LSCs biomarkers and stemness-related signaling pathways in a mouse model. Our findings could aid in the identification of LSC niche factors that may be related to the stemness of the LSCs.


Assuntos
Epitélio Anterior/química , Perfilação da Expressão Gênica/veterinária , Redes Reguladoras de Genes , Animais , Células Cultivadas , Túnica Conjuntiva/química , Regulação da Expressão Gênica , Sequenciamento de Nucleotídeos em Larga Escala , Camundongos , Mapas de Interação de Proteínas , Análise de Sequência de RNA , Células-Tronco/química
14.
Nat Commun ; 12(1): 259, 2021 01 11.
Artigo em Inglês | MEDLINE | ID: mdl-33431815

RESUMO

Although identified as the key environmental driver of common cutaneous melanoma, the role of ultraviolet radiation (UVR)-induced DNA damage in mucosal melanoma is poorly defined. We analyze 10 mucosal melanomas of conjunctival origin by whole genome sequencing and our data shows a predominance of UVR-associated single base substitution signature 7 (SBS7) in the majority of the samples. Our data shows mucosal melanomas with SBS7 dominance have similar genomic patterns to cutaneous melanomas and therefore this subset should not be excluded from treatments currently used for common cutaneous melanoma.


Assuntos
Melanoma/genética , Membrana Mucosa/patologia , Mutação/genética , Neoplasias Cutâneas/genética , Raios Ultravioleta , Adulto , Idoso , Idoso de 80 Anos ou mais , Túnica Conjuntiva/patologia , Dano ao DNA , Feminino , Genoma Humano , Humanos , Masculino , Pessoa de Meia-Idade
15.
Methods Mol Biol ; 2223: 133-149, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33226592

RESUMO

Mouse models of allergic conjunctivitis mimic various aspects of human allergic conjunctivitis. They are useful as acute models of allergic conjunctivitis to study immunological aspects of this condition. In this chapter, we will describe ragweed-pollen-induced experimental allergic conjunctivitis (mostly driven by adaptive immunity), and papain-soaked contact lens-induced experimental allergic conjunctivitis (mostly driven by innate immunity). Giemsa staining of histological sections is used for quantification of the number of infiltrating eosinophils, which is useful to evaluate the severity of the allergic inflammation. Immunohistochemical staining and quantitative PCR are used to clarify spatiotemporal expression of proinflammatory molecules in the conjunctival tissue. Flow cytometric analysis of conjunctival tissue is used for the detection of innate lymphoid cell type 2 (ILC2) in the ocular surface tissues.


Assuntos
Ambrosia/imunologia , Túnica Conjuntiva/efeitos dos fármacos , Conjuntivite Alérgica/imunologia , Modelos Animais de Doenças , Linfócitos/efeitos dos fármacos , Papaína/administração & dosagem , Imunidade Adaptativa/efeitos dos fármacos , Adjuvantes Imunológicos/administração & dosagem , Alérgenos/administração & dosagem , Hidróxido de Alumínio/administração & dosagem , Ambrosia/química , Animais , Biomarcadores/metabolismo , Túnica Conjuntiva/imunologia , Túnica Conjuntiva/patologia , Conjuntivite Alérgica/induzido quimicamente , Conjuntivite Alérgica/genética , Conjuntivite Alérgica/patologia , Eosinófilos/efeitos dos fármacos , Eosinófilos/imunologia , Eosinófilos/patologia , Feminino , Citometria de Fluxo/métodos , Expressão Gênica , Imunidade Inata/efeitos dos fármacos , Imunoglobulina E/genética , Imunoglobulina E/imunologia , Interleucinas/genética , Interleucinas/imunologia , Linfócitos/imunologia , Linfócitos/patologia , Camundongos , Camundongos Endogâmicos C57BL , Pólen/efeitos adversos , Pólen/imunologia , Reação em Cadeia da Polimerase em Tempo Real/métodos
16.
Exp Eye Res ; 203: 108402, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-33326809

RESUMO

PURPOSE: To develop and characterize a new type of plasma rich in growth factors (PRGF) membrane for patients in which immune system is involved in the disease etiology. METHODS: Blood from three healthy donors was collected to obtain the different fibrin membranes by PRGF technology. PRGF obtained volumes were activated and divided into two groups: PRGF membrane (mPRGF) obtained after incubation at 37 °C for 30 min (control); and is-mPRGF: mPRGF obtained after incubation for 30 min at 56 °C. The concentration of several growth factors, proteins, immunoglobulin E and the complement activity was determined in the different mPRGF. The proliferative potential of heat-inactivated mPRGF were assayed on keratocytes (HK) and conjunctival fibroblasts (HConF). In addition, morphological and physical features of the inactivated mPRGF were evaluated in contrast to the control mPRGF. RESULTS: Heat-inactivation of the mPRGF preserves the content of most of the growth factors involved in the ocular wound healing while reducing drastically the content of IgE and the complement activity. The heat-inactivated mPRGF conserve the morphological and physical characteristics of the fibrin meshwork in comparison with the control mPRGF. Furthermore, no significant differences were found in the biological activity of the control mPRGF regarding the heat-inactivated mPRGF (is-mPRGF) in any of both ocular cell types evaluated. CONCLUSIONS: The heat-inactivation of the PRGF membranes (is-mPRGF) reduces drastically the content of IgE and complement activity while preserving the content of most of the proteins and morphogens involved in ocular wound healing. Furthermore, the morphological and physical features of the immunosafe mPRGF were also preserved after heat-inactivation.


Assuntos
Membrana Celular/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Fibrina Rica em Plaquetas/metabolismo , Tecnologia Biomédica , Doadores de Sangue , Células Cultivadas , Via Clássica do Complemento/fisiologia , Túnica Conjuntiva/citologia , Ceratócitos da Córnea/metabolismo , Ensaio de Imunoadsorção Enzimática , Fator de Crescimento Epidérmico/metabolismo , Fator 2 de Crescimento de Fibroblastos/metabolismo , Fibroblastos/metabolismo , Técnica Indireta de Fluorescência para Anticorpo , Humanos , Imunoglobulina E/imunologia , Microscopia Eletrônica de Varredura , Fator de Crescimento Derivado de Plaquetas/metabolismo , Fator de Crescimento Transformador beta1/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo
17.
Exp Eye Res ; 203: 108421, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-33359326

RESUMO

Glaucoma, the second leading cause of blindness worldwide, is characterized by aberrant elevations of intraocular pressure (IOP), which can damage the optic nerve. IOP reduction is the only effective therapy for prevention of visual impairment and blindness in both hypertensive and normotensive individuals, and in some cases, trabeculectomy is a major surgical procedure that can lower IOP in patients with glaucoma. No matter how surgical technique and postoperative care advances, excessive scarring and tissue fibrosis could result from increased human conjunctival fibroblast (HCF) proliferation and extracellular matrix (ECM) deposition of the subconjunctival tissue and scleral flaps would persist after trabeculectomy. And these issues are major impediments to IOP reduction and filtering of bleb formations, so the modulation of the factors which can induce fibrosis could used as a novel strategy to control scarring after trabeculectomy. In this study, we examined the effects of mammalian target of rapamycin (mTOR) inhibitors (rapamycin or Torin1) on the fibrotic response induced by transforming growth factor-beta 1 (TGF-ß1) in cultured human conjunctival fibroblast (HCF) cells. The study also examined the effects of mTOR inhibitor on fibrosis after trabeculectomy in rabbit eyes. In in vitro studies, we stimulated HCFs with TGF-ß1, and confirmed that the expression levels of fibronectin, collagen type I alpha 1 chain (COL1A1), and α-smooth muscle actin (SMA) were significantly upregulated in HCFs with TGF-ß1, by means of quantitative real-time polymerase chain reaction and immunocytochemistry. And those TGF-ß1-induced changes were significantly attenuated with mTOR inhibitors, rapamycin or Torin1. Additionally the migration rate of HCFs was examined under conditions of TGF-ß1 induction, TGF-ß1-induced changes were significantly attenuated with mTOR inhibitors. A rabbit model of trabeculectomy was examined in vivo, and the effects of topical mTOR inhibitor were also examined, and found that topical treatment with mTOR inhibitor significantly suppressed collagen deposition in rabbit eyes after trabeculectomy. These results have demonstrated that mTOR inhibitors may provide a novel treatment modality for reducing the fibrotic response in HCFs and improving bleb scarring after filtration surgery.


Assuntos
Túnica Conjuntiva/patologia , Naftiridinas/farmacologia , Sirolimo/farmacologia , Serina-Treonina Quinases TOR/antagonistas & inibidores , Trabeculectomia , Actinas/genética , Actinas/metabolismo , Animais , Western Blotting , Células Cultivadas , Colágeno Tipo I/genética , Colágeno Tipo I/metabolismo , Túnica Conjuntiva/metabolismo , Fibroblastos/metabolismo , Fibroblastos/patologia , Fibronectinas/genética , Fibronectinas/metabolismo , Fibrose , Humanos , Imuno-Histoquímica , Masculino , RNA Mensageiro/genética , Coelhos , Reação em Cadeia da Polimerase em Tempo Real , Fator de Crescimento Transformador beta1/farmacologia
18.
Exp Eye Res ; 203: 108418, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-33359511

RESUMO

Microbial keratitis is an infectious disease of the eye, in which the cornea is inflamed. Under severe conditions, keratitis can lead to significant loss of vision and enucleation of the eye. Ocular trauma is the major risk factor causing keratitis and microorganisms viz., bacteria, fungi, viruses are the causative agents. The current study characterized the conjunctival bacterial microbiomes of healthy individuals and individuals with bacterial keratitis (BK) and assessed whether ocular microbiome dysbiosis is prevalent in BK patients. Ocular bacterial microbiomes were generated from the conjunctival swabs of healthy controls (HC-SW) and conjunctival swabs (BK-SW) and corneal scrapings (BK-CR) of BK patients using V3-V4 amplicon sequencing and data analysed using QIIME and R software. The Alpha diversity indices, diversity and abundance of different phyla and genera, heat map analysis, NMDS plots and inferred functional pathway analysis clearly discriminated the bacterial microbiomes of conjunctival swabs of healthy controls from that of BK patients. Preponderance of negative interactions in the hub genera were observed in BK-CR and BK-SW compared to HC-SW. In addition, a consistent increase in the abundance of pathogenic bacteria, as inferred from published literature, was observed in the conjunctiva of BK patients compared to HC and this may be related to causing or exacerbating ocular surface inflammation. This is the first study demonstrating dysbiosis in the ocular bacterial microbiome of conjunctiva of bacterial keratitis patients compared to the eye of healthy controls. The bacterial microbiome associated with the corneal scrapings of keratitis individuals is also described for the first time.


Assuntos
Bactérias/genética , Túnica Conjuntiva/microbiologia , Úlcera da Córnea/microbiologia , Infecções Oculares Bacterianas/microbiologia , Microbiota/genética , Adulto , Idoso , Bactérias/isolamento & purificação , Técnicas Bacteriológicas , DNA Bacteriano/genética , Feminino , Voluntários Saudáveis , Humanos , Masculino , Pessoa de Meia-Idade , Reação em Cadeia da Polimerase , Análise de Sequência de DNA , Adulto Jovem
20.
Int J Mol Sci ; 21(24)2020 Dec 10.
Artigo em Inglês | MEDLINE | ID: mdl-33321932

RESUMO

How ion channels impact the response of the ocular surface to dry eye is only beginning to be explored. Here, we review recent progress and provide new experimental data clarifying the exocytosis-altering actions of ion channels in conjunctival goblet cells whose release of tear-stabilizing mucin is a key adaptive response to the pre-ocular hyperosmolarity that characterizes dry eye. Patch-clamp recordings of goblet cells located in freshly excised rat conjunctiva reveal that these mucin-releasing cells respond to sustained hyperosmolarity by sequentially activating their ATP-sensitive potassium (KATP), nonspecific cation (NSC), voltage-gated calcium (VGCC), and P2X7 channels; each of which modulates exocytosis. Based on these and other new findings, we now identify four stages in the bioelectric response of conjunctival goblet cells to extracellular hyperosmolarity. To better characterize these stages, we report that high-resolution membrane capacitance (Cm) measurements of the exocytotic activity of single goblet cells demonstrate that the replenishment of mucin-filled granules after neural-evoked exocytosis is a multi-hour process, which VGCCs markedly accelerate. Yet, we also discovered that VGCC activation is high-risk since hyperosmotic-induced goblet cell death is boosted. With dry eye treatments being far from optimal, elucidating the physiologic and pathobiologic impact of the KATP/NSC/VGCC/P2X7 pathway provides a new opportunity to identify novel therapeutic strategies.


Assuntos
Canais de Cálcio/metabolismo , Síndromes do Olho Seco/metabolismo , Exocitose , Células Caliciformes/metabolismo , Canais KATP/metabolismo , Potenciais da Membrana , Receptores Purinérgicos P2X7/metabolismo , Animais , Sobrevivência Celular , Túnica Conjuntiva/citologia , Túnica Conjuntiva/metabolismo , Células Caliciformes/fisiologia , Mucinas/metabolismo , Concentração Osmolar , Ratos , Ratos Long-Evans , Ratos Sprague-Dawley
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