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2.
PLoS One ; 16(8): e0256240, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34407118

RESUMO

BACKGROUND: Microbial keratitis (MK) is the most common non-surgical ophthalmic emergency, and can rapidly progress, causing irreversible sight-loss. This study explored whether the COVID-19 (C19) national lockdown impacted upon the clinical presentation and outcomes of MK at a UK tertiary-care centre. METHODS: Medical records were retrospectively reviewed for all patients with presumed MK requiring corneal scrapes, presenting between 23rd March and 30th June in 2020 (Y2020), and the equivalent time windows in 2017, 2018 and 2019 (pre-C19). RESULTS: In total, 181 and 49 patients presented during the pre-C19 and Y2020 periods, respectively. In Y2020, concurrent ocular trauma (16.3% vs. 5.5%, p = 0.030) and immunosuppression use (12.2% vs 1.7%, p = 0.004) were more prevalent. Despite proportionately fewer ward admissions during the pandemic (8.2% vs 32.6%, p<0.001), no differences were observed in baseline demographics; presenting visual acuity (VA; median 0.6 vs 0.6 LogMAR, p = 0.785); ulcer area (4.0 vs 3.0mm2, p = 0.520); or final VA (0.30 vs 0.30 LogMAR, p = 0.990). Whilst the overall rates of culture positivity were similar in Y2020 and pre-C19 (49.0% vs. 54.7%, p = 0.520), there were differences in the cultures isolated, with a lower rate of poly-microbial cultures in Y2020 (8.3% vs. 31.3%, p = 0.022). CONCLUSIONS: Patient characteristics, MK severity and final visual outcomes did not appear to be affected in the first UK lockdown, despite fewer patients being admitted for care. Concurrent trauma and systemic immunosuppression use were greater than in previous years. The difference in spectra of isolated organisms may relate to behavioural changes, such as increased hand hygiene.


Assuntos
COVID-19/epidemiologia , Ceratectomia/métodos , Ceratite/epidemiologia , Ceratite/cirurgia , Adulto , Idoso , Feminino , Humanos , Imunossupressores/uso terapêutico , Ceratite/microbiologia , Masculino , Pessoa de Meia-Idade , Pandemias , Admissão do Paciente/estatística & dados numéricos , Estudos Retrospectivos , Índice de Gravidade de Doença , Centros de Atenção Terciária , Reino Unido/epidemiologia
3.
Int J Mol Sci ; 22(16)2021 Aug 20.
Artigo em Inglês | MEDLINE | ID: mdl-34445684

RESUMO

The shape and transparency of the cornea are essential for clear vision. However, its location at the ocular surface renders the cornea vulnerable to pathogenic microorganisms in the external environment. Pseudomonas aeruginosa and Staphylococcus aureus are two such microorganisms and are responsible for most cases of bacterial keratitis. The development of antimicrobial agents has allowed the successful treatment of bacterial keratitis if the infection is diagnosed promptly. However, no effective medical treatment is available after progression to corneal ulcer, which is characterized by excessive degradation of collagen in the corneal stroma and can lead to corneal perforation and corneal blindness. This collagen degradation is mediated by both infecting bacteria and corneal fibroblasts themselves, with a urokinase-type plasminogen activator (uPA)-plasmin-matrix metalloproteinase (MMP) cascade playing a central role in collagen destruction by the host cells. Bacterial factors stimulate the production by corneal fibroblasts of both uPA and pro-MMPs, released uPA mediates the conversion of plasminogen in the extracellular environment to plasmin, and plasmin mediates the conversion of secreted pro-MMPs to the active form of these enzymes, which then degrade stromal collagen. Bacterial factors also stimulate expression by corneal fibroblasts of the chemokine interleukin-8 and the adhesion molecule ICAM-1, both of which contribute to recruitment and activation of polymorphonuclear neutrophils, and these cells then further stimulate corneal fibroblasts via the secretion of interleukin-1. At this stage of the disease, bacteria are no longer necessary for collagen degradation. In this review, we discuss the pivotal role of corneal fibroblasts in corneal ulcer associated with infection by P. aeruginosa or S. aureus as well as the development of potential new modes of treatment for this condition.


Assuntos
Úlcera da Córnea/metabolismo , Fibroblastos/metabolismo , Ceratite/microbiologia , Animais , Colágeno/metabolismo , Córnea/metabolismo , Córnea/fisiologia , Substância Própria/metabolismo , Úlcera da Córnea/etiologia , Úlcera da Córnea/microbiologia , Infecções Oculares Bacterianas/microbiologia , Infecções Oculares Bacterianas/fisiopatologia , Fibrinolisina/metabolismo , Humanos , Metaloproteinases da Matriz/metabolismo , Plasminogênio/metabolismo , Ativadores de Plasminogênio/metabolismo , Pseudomonas aeruginosa/metabolismo , Pseudomonas aeruginosa/patogenicidade , Staphylococcus aureus/metabolismo , Staphylococcus aureus/patogenicidade , Ativador de Plasminogênio Tipo Uroquinase/metabolismo
4.
Invest Ophthalmol Vis Sci ; 62(9): 7, 2021 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-34232259

RESUMO

Purpose: To investigate the role of elastase in corneal epithelial barrier dysfunction caused by the exoproteins secreted by Pseudomonas aeruginosa. Methods: Exoproteins obtained from Pseudomonas aeruginosa culture supernatant were analyzed by shotgun proteomics approach. In vitro multilayered rabbit corneal epithelial barrier model prepared by air-liquid interface technique (CECs-ALI) were treated with 2 µg/ml exoproteins and/or 8 mM elastase inhibitor. Then the epithelial barrier function was evaluated by transepithelial electrical resistance (TEER) assay and tight junction proteins immunofluorescence. Cell viability and the apoptosis rate were examined by CCK8 assay and flow cytometry. TNF-α, IL-6, IL-8, and IL-1ß levels were measured by ELISA. Mice cornea treated with exoproteins and/or elastase inhibitor were evaluated in vivo and in vitro. Results: Elastase (24.2%) is one of the major components of exoproteins. After 2 µg/ml exoproteins were applied to CECs-ALI for two hours, TEER decreased from 323.2 ± 2.7 to 104 ± 6.8 Ω/cm2 (P < 0.001). The immunofluorescence results showed a distinct separation in tight junction and significant degradation of ZO-1 and occludin (P < 0.05). Elastase inhibitor (8 mM) alleviated the decrease in TEER value (234 ± 6.8 Ω cm2) induced by exoproteins. Inhibition of elastase decreased the apoptosis rate of CECs treated with exoproteins from 30.2 ± 3.8% to 7.26 ± 1.3% and the levels of inflammatory factors (P < 0.05). Mice corneal epithelium defect could be induced by exoproteins and protected by elastase inhibitor. Conclusions: Elastase plays a critical role in corneal epithelial barrier dysfunction caused by Pseudomonas aeruginosa exoproteins via damaging tight junctions. The inhibition of elastase could protect the corneal epithelial barrier via reducing virulence and inflammation.


Assuntos
Epitélio Corneano/microbiologia , Infecções Oculares Bacterianas/enzimologia , Ceratite/enzimologia , Ocludina/metabolismo , Elastase Pancreática/metabolismo , Infecções por Pseudomonas/enzimologia , Pseudomonas aeruginosa/isolamento & purificação , Proteínas de Junções Íntimas/metabolismo , Animais , Células Cultivadas , Modelos Animais de Doenças , Epitélio Corneano/enzimologia , Epitélio Corneano/patologia , Infecções Oculares Bacterianas/microbiologia , Infecções Oculares Bacterianas/patologia , Ceratite/microbiologia , Ceratite/patologia , Infecções por Pseudomonas/microbiologia , Infecções por Pseudomonas/patologia , Coelhos
5.
Invest Ophthalmol Vis Sci ; 62(9): 12, 2021 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-34236384

RESUMO

Purpose: To determine the role of scavenger receptor expressed by endothelial cell-1 (SREC-Ⅰ) in vitro and in a mouse model of Aspergillus fumigatus keratitis. Methods: SREC-Ⅰ mRNA and protein expression were tested in both normal and A fumigatus stimulated human corneal epithelial cells (HCECs). Immunofluorescence was used to detect SREC-Ⅰ expression in human corneas with or without A fumigatus infection. HCECs were incubated with SREC-Ⅰ small interfering RNA, then the mRNA levels of LOX-1, IL-1ß, and TNF-α were detected after A fumigatus stimulation. A mouse fungal keratitis (FK) model was established and SREC-Ⅰ mRNA and protein expression were detected by RT-PCR, Western blot and immunofluorescence. The severity of FK was evaluated by clinical score. CLCX1, LOX-1, IL-1ß, and TNF-α mRNA expression levels were tested before and after anti-SREC-Ⅰ treatment. Results: SREC-Ⅰ expressed in normal and A fumigatus treated HCECs and human corneal epithelium. In vitro experiment showed that SREC-Ⅰ mRNA and protein levels were significantly increased after A fumigatus stimulation. SREC-Ⅰ small interfering RNA treatment inhibited the expressions of LOX-1, IL-1ß, and TNF-α in HCECs. The expressions of CLCX1, LOX-1, IL-1ß, and TNF-α were elevated in mice with A fumigatus keratitis, which could be decreased by SREC-Ⅰ-neutralizing antibody treatment. Conclusions: SREC-Ⅰ is a key mediator in inflammatory response induced by A fumigatus keratitis. SREC-Ⅰ blockade could be a potential therapeutic approach for FK.


Assuntos
Aspergilose/genética , Aspergillus fumigatus/isolamento & purificação , Infecções Oculares Fúngicas/genética , Regulação da Expressão Gênica/genética , Imunidade Inata/genética , Ceratite/genética , RNA Mensageiro/genética , Receptores Depuradores Classe F/genética , Adulto , Aspergilose/imunologia , Aspergilose/microbiologia , Western Blotting , Células Cultivadas , Infecções Oculares Fúngicas/imunologia , Infecções Oculares Fúngicas/microbiologia , Feminino , Humanos , Ceratite/imunologia , Ceratite/microbiologia , Masculino , Pessoa de Meia-Idade , Receptores Depuradores Classe F/biossíntese
6.
AAPS PharmSciTech ; 22(5): 195, 2021 Jun 28.
Artigo em Inglês | MEDLINE | ID: mdl-34184117

RESUMO

Microbial keratitis (MK) is a vision-threatening disease and the fourth leading cause of blindness worldwide. In this work, we aim to develop moxifloxacin (MXN)-loaded chitosan-based cationic mucoadhesive polyelectrolyte nanocapsules (PENs) for the effective treatment of MK. PENs were formulated by polyelectrolyte complex coacervation method and characterized for their particle size, surface charge, morphology, mucoadhesive property, in-vitro and ex-vivo release, ocular tolerance, and antimicrobial efficacy studies. The pharmacodynamic study was conducted on rabbit eye model of induced keratitis and it is compared with marketed formulation (MF). Developed PENs showed the size range from 230.7 ± 0.64 to 249.0 ± 0.49 nm and positive surface charge, spherical shape along with appropriate physico-chemical parameters. Both in-vitro and ex-vivo examination concludes that PENs having more efficiency in sustained release of MXN compared to MF. Ocular irritation studies demonstrated that no corneal damage or ocular irritation. The in-vivo study proved that the anti-bacterial efficacy of PENs was improved when compared with MF. These results suggested that PENs are a feasible choice for MK therapy because of their ability to enhance ocular retention of loaded MXN through interaction with the corneal surface of the mucous membrane.


Assuntos
Desenvolvimento de Medicamentos/métodos , Ceratite/tratamento farmacológico , Moxifloxacina/síntese química , Nanocápsulas/química , Polieletrólitos/síntese química , Animais , Antibacterianos/administração & dosagem , Antibacterianos/síntese química , Antibacterianos/farmacocinética , Embrião de Galinha , Córnea/efeitos dos fármacos , Córnea/metabolismo , Córnea/microbiologia , Cabras , Ceratite/metabolismo , Ceratite/microbiologia , Moxifloxacina/administração & dosagem , Moxifloxacina/farmacocinética , Nanocápsulas/administração & dosagem , Polieletrólitos/administração & dosagem , Polieletrólitos/farmacocinética , Coelhos
7.
Invest Ophthalmol Vis Sci ; 62(6): 10, 2021 05 03.
Artigo em Inglês | MEDLINE | ID: mdl-33970198

RESUMO

Purpose: Interleukin (IL)-36 cytokines have been shown to play either beneficial or detrimental roles in the infection of mucosal tissues in a pathogen-dependent manner, but their involvement in fungal keratitis remains elusive. We herein investigated their expression and function in mediating corneal innate immunity against Candida albicans infection. Methods: Gene expression in mouse corneas with or without C. albicans infection was determined by regular RT- and real-time (q)-PCR, Western blot analysis, ELISA or proteome profile assay. The severity of C. albicans keratitis was assessed using clinical scoring, bacterial counting, and myeloperoxidase (MPO) activity as an indicator of neutrophil infiltration. IL36R knockout mice and IL-33-specific siRNA were used to assess the involvement IL-33 signaling in C. albicans-infected corneas. B6 CD11c-DTR mice and clodronate liposomes were used to define the involvement of dendritic cells (DCs) and macrophages in IL-36R signaling and C. albicans keratitis, respectively. Results: IL-36γ were up-regulated in C57BL6 mouse corneas in response to C. albicans infection. IL-36 receptor-deficient mice display increased severity of keratitis, with a higher fungal load, MPO, and IL-1ß levels, and lower soluble sIL-1Ra and calprotectin levels. Exogenous IL-36γ prevented fungal keratitis pathogenesis with lower fungal load and MPO activity, higher expression of sIL-1Ra and calprotectin, and lower expression of IL-1ß, at mRNA or protein levels. Protein array analysis revealed that the expression of IL-33 and REG3G were related to IL-36/IL36R signaling, and siRNA downregulation of IL-33 increased the severity of C. albicans keratitis. Depletion of dendritic cells or macrophages resulted in severe C. albicans keratitis and yet exhibited minimal effects on exogenous IL-36γ-induced protection against C. albicans infection in B6 mouse corneas. Conclusions: IL-36/IL36R signaling plays a protective role in fungal keratitis by promoting AMP expression and by suppressing fungal infection-induced expression of proinflammatory cytokines in a dendritic cell- and macrophage-independent manner.


Assuntos
Úlcera da Córnea/prevenção & controle , Infecções Oculares Fúngicas/prevenção & controle , Imunidade Inata/fisiologia , Interleucina-1/fisiologia , Ceratite/prevenção & controle , Receptores de Interleucina-1/fisiologia , Transdução de Sinais/fisiologia , Animais , Western Blotting , Candida albicans , Úlcera da Córnea/imunologia , Úlcera da Córnea/microbiologia , Células Dendríticas/imunologia , Ensaio de Imunoadsorção Enzimática , Infecções Oculares Fúngicas/imunologia , Infecções Oculares Fúngicas/microbiologia , Regulação da Expressão Gênica/fisiologia , Ceratite/imunologia , Ceratite/microbiologia , Macrófagos/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Reação em Cadeia da Polimerase em Tempo Real
8.
Invest Ophthalmol Vis Sci ; 62(6): 26, 2021 05 03.
Artigo em Inglês | MEDLINE | ID: mdl-34038512

RESUMO

Purpose: To investigate the antifungal and anti-inflammatory effects of baicalein on Aspergillus fumigatus (A. fumigatus) keratitis and the underlying mechanisms. Methods: The noncytotoxic antifungal concentration of baicalein was determined using CCK8, cell scratch assay, minimum inhibitory concentration, biofilm formation, scanning electron microscopy, propidium iodide uptake test and adherence assay in vitro and Draize test in vivo. In fungal keratitis (FK) mouse models, clinical score and plate count were used to evaluate FK severity, and myeloperoxidase assay and immunofluorescence staining were performed to examine neutrophil infiltration and activity. Real-time PCR, ELISA, and Western blot were performed to explore the anti-inflammatory activity of baicalein and the underlying mechanisms in vivo and in vitro. Results: Baicalein at 0.25 mM (noncytotoxic) significantly inhibited A. fumigatus growth, biofilm formation, and adhesion in vitro. In A. fumigatus keratitis mice, baicalein mitigated FK severity, reduced fungal load, and inhibited neutrophil infiltration and activity. Baicalein not only suppressed mRNA and protein levels of proinflammatory factors IL-1ß, IL-6, and TNF-α, but also inhibited the expression of thymic stromal lymphopoietin (TSLP) and TSLP receptor (TSLPR) in vivo and in vitro. In HCECs, mRNA and protein levels of IL-1ß, IL-6, and TNF-α were significantly lower in the TSLP siRNA-treated group, while higher in the rTSLP-treated group than in the corresponding control. Baicalein treatment significantly inhibited rTSLP induced the expression of IL-1ß, IL-6, and TNF-α. Conclusions: Baicalein plays a protective role in mouse A. fumigatus keratitis by inhibiting fungal growth, biofilm formation, and adhesion, and suppressing inflammatory response via downregulation of the TSLP/TSLPR pathway.


Assuntos
Antifúngicos/uso terapêutico , Aspergilose/tratamento farmacológico , Citocinas/antagonistas & inibidores , Infecções Oculares Fúngicas/tratamento farmacológico , Flavanonas/uso terapêutico , Inflamação/tratamento farmacológico , Ceratite/tratamento farmacológico , Animais , Aspergilose/metabolismo , Aspergilose/microbiologia , Aspergillus fumigatus/efeitos dos fármacos , Aspergillus fumigatus/isolamento & purificação , Aspergillus fumigatus/ultraestrutura , Western Blotting , Células Cultivadas , Citocinas/genética , Citocinas/metabolismo , Modelos Animais de Doenças , Ensaio de Imunoadsorção Enzimática , Epitélio Corneano/efeitos dos fármacos , Epitélio Corneano/metabolismo , Infecções Oculares Fúngicas/metabolismo , Infecções Oculares Fúngicas/microbiologia , Feminino , Ceratite/metabolismo , Ceratite/microbiologia , Camundongos , Camundongos Endogâmicos C57BL , Testes de Sensibilidade Microbiana , Microscopia Eletrônica de Varredura , Reação em Cadeia da Polimerase em Tempo Real
9.
Invest Ophthalmol Vis Sci ; 62(6): 28, 2021 05 03.
Artigo em Inglês | MEDLINE | ID: mdl-34043748

RESUMO

Purpose: C-type lectin-like receptor-1 (CLEC-1) is a member of the Dectin-1 cluster of pattern recognition receptors (PRRs). It is involved in host immunity, has immunoregulatory function, and supports allograft tolerance. Our study aimed to describe the role of CLEC-1 in response to fungal keratitis, in situ, in vivo, and in vitro. Methods: Quantitative polymerase chain reaction (qRT-PCR) and immunofluorescence were used to detect the expression of CLEC-1 in corneas of patients with Aspergillus fumigatus (A. fumigatus) keratitis. In vitro and in vivo experiments were designed in THP-1 macrophages and C57BL/6 mouse models, respectively. The expression of CLEC-1 in corneas of mice model was determined by qRT-PCR, Western blot, and immunofluorescence. CLEC-1 overexpression in mouse corneas was achieved by intrastromal injection of adeno-associated virus (AAV) vectors. Disease response was evaluated by slit-lamp photography, clinical score, and colony forming unit (CFU). Bioluminescence imaging system image acquisition, myeloperoxidase (MPO) assays, immunofluorescence staining, qRT-PCR, and Western blot were used to investigate the role of CLEC-1. To further define the role of CLEC-1, we used lentivirus vectors to overexpress CLEC-1 or/and Dectin-1 in THP-1 macrophages. Results: The expression of CLEC-1 was increased in corneas of patients with A. fumigatus keratitis. In corneas of mice from the A. fumigatus keratitis model, the expression of CLEC-1 was decreased in the acute inflammatory stage and increased during convalescence. Following Natamycin treatment, CLEC-1 was upregulated in A. fumigatus keratitis mice. Compared with normal C57BL/6 mice, overexpression of CLEC-1 converted the characteristic susceptible response to resistance, as demonstrated by slit-lamp photography and clinical score. In vivo studies revealed decreased MPO levels and neutrophils recruitment and higher fungal load after the upregulation of CLEC-1. Compared with control corneas, CLEC-1 overexpression impaired corneal pro-inflammatory cytokine IL-1ß production. Conclusions: These findings demonstrate that CLEC-1 may act as a negative regulator of Dectin-1 induced host inflammatory response via suppressing neutrophils recruitment and production of pro-inflammatory cytokine IL-1ß production in response to A. fumigatus keratitis.


Assuntos
Aspergilose/metabolismo , Infecções Oculares Fúngicas/metabolismo , Regulação da Expressão Gênica/fisiologia , Ceratite/metabolismo , Lectinas Tipo C/genética , Lectinas Tipo C/fisiologia , Proteínas de Membrana/fisiologia , Animais , Aspergilose/imunologia , Aspergilose/microbiologia , Aspergillus fumigatus , Western Blotting , Citocinas/metabolismo , Dependovirus/genética , Modelos Animais de Doenças , Infecções Oculares Fúngicas/imunologia , Infecções Oculares Fúngicas/microbiologia , Feminino , Vetores Genéticos , Humanos , Ceratite/imunologia , Ceratite/microbiologia , Macrófagos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Microscopia de Fluorescência , Infiltração de Neutrófilos , Peroxidase/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Receptores Mitogênicos/fisiologia , Microscopia com Lâmpada de Fenda
10.
Infect Immun ; 89(8): e0011121, 2021 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-33820815

RESUMO

In this study, we tested the hypothesis that the conserved bacterial IgaA-family protein, GumB, mediates microbial pathogenesis associated with Serratia marcescens ocular infections through regulation of the Rcs stress response system. The role of the Rcs system and bacterial stress response systems for microbial keratitis is not known, and the role of IgaA proteins in mammalian pathogenesis models has only been tested with partial-function allele variants of Salmonella. Here, we observed that an Rcs-activated gumB mutant had a >50-fold reduction in proliferation compared to the wild type within rabbit corneas at 48 h and demonstrated a notable reduction in inflammation based on inflammatory signs, including the absence of hypopyons, and proinflammatory markers measured at the RNA and protein levels. The gumB mutant phenotypes could be complemented by wild-type gumB on a plasmid. We observed that bacteria with an inactivated Rcs stress response system induced high levels of ocular inflammation and restored corneal virulence to the gumB mutant. The high virulence of the ΔrcsB mutant was dependent upon the ShlA cytolysin transporter ShlB. Similar results were found for testing the cytotoxic effects of wild-type and mutant bacteria on a human corneal epithelial cell line in vitro. Together, these data indicate that GumB regulates virulence factor production through the Rcs system, and this overall stress response system is a key mediator of a bacterium's ability to induce vision-threatening keratitis.


Assuntos
Proteínas de Bactérias/genética , Ceratite/microbiologia , Infecções por Serratia/microbiologia , Serratia marcescens/fisiologia , Estresse Fisiológico , Animais , Proteínas de Bactérias/metabolismo , Regulação Bacteriana da Expressão Gênica , Interações Hospedeiro-Patógeno , Mutação , Coelhos , Estresse Fisiológico/genética , Virulência , Fatores de Virulência/genética
11.
Invest Ophthalmol Vis Sci ; 62(4): 16, 2021 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-33851975

RESUMO

Purpose: To explore the role of IL-36α in corneas infected by Aspergillus fumigatus. Methods: The experimental group was comprised of 15 corneas with fungal keratitis, and 15 healthy donor corneas were included in the control group. IL-36α was detected in normal and infected corneas of humans and C57BL/6 mice. Mice corneas were infected with A. fumigatus with or without pretreatment of recombinant mouse (rm) IL-36α and IL-36α neutralizing antibody (Ab). Primary macrophages were stimulated with 75% ethanol-killed A. fumigatus with or without pretreatment of rmIL-36α. The severity of the disease was documented by clinical score and photographs with a slit lamp. PCR, western blot, and immunostaining were used to determine the expression of IL-36α, IL-1ß, IL-6, and TNF-α. Polymorphonuclear neutrophilic leukocyte infiltration was assessed by myeloperoxidase (MPO) assay and flow cytometry. Macrophage infiltration was tested by immunofluorescent staining and flow cytometry. Results: IL-36α mRNA and protein were significantly elevated in human and mice corneas after infection. The rmIL-36α treatment of C57BL/6 mice increased clinical score, MPO levels, macrophage infiltration, and expression of the proinflammatory cytokines IL-1ß, IL-6, and TNF-α compared with the infected controls, which showed a decrease due to IL-36α Ab treatment. In primary macrophages, IL-36α expression was also significantly increased by A. fumigatus. The rmIL-36α treatment upregulated IL-1ß, IL-6, and phosphorylated nuclear factor (NF)-κB expression, which was significantly inhibited by rmIL-36Ra. Conclusions: IL-36α act as a proinflammatory cytokine in A. fumigatus keratitis by promoting the infiltration of neutrophils and macrophages and increasing the secretion of IL-1ß, IL-6, and TNF-α, in addition to regulating expression of phosphorylated NF-κB.


Assuntos
Aspergilose/tratamento farmacológico , Aspergillus fumigatus/isolamento & purificação , Regulação da Expressão Gênica , Interleucina-1/genética , Interleucina-1/farmacologia , Ceratite/tratamento farmacológico , NF-kappa B/genética , Animais , Aspergilose/metabolismo , Modelos Animais de Doenças , Epitélio Corneano/metabolismo , Epitélio Corneano/microbiologia , Epitélio Corneano/patologia , Infecções Oculares Fúngicas/microbiologia , Feminino , Humanos , Ceratite/metabolismo , Ceratite/microbiologia , Macrófagos/metabolismo , Camundongos Endogâmicos C57BL , NF-kappa B/biossíntese , Neutrófilos/metabolismo , Ratos , Transdução de Sinais/efeitos dos fármacos
12.
Molecules ; 26(9)2021 Apr 28.
Artigo em Inglês | MEDLINE | ID: mdl-33925130

RESUMO

In this research, polyvinyl-alcohol (PVA)/gelatin (GEL)/propolis (Ps) biocompatible nanofiber patches were fabricated via electrospinning technique. The controlled release of Propolis, surface wettability behaviors, antimicrobial activities against the S. aureus and P. aeruginosa, and biocompatibility properties with the mesenchymal stem cells (MSCs) were investigated in detail. By adding 0.5, 1, and 3 wt.% GEL into the 13 wt.% PVA, the morphological and mechanical results suggested that 13 wt.% PVA/0.5 wt.% GEL patch can be an ideal matrix for 3 and 5 wt.% propolis addition. Morphological results revealed that the diameters of the electrospun nanofiber patches were increased with GEL (from 290 nm to 400 nm) and Ps addition and crosslinking process cause the formation of thicker nanofibers. The tensile strength and elongation at break enhancement were also determined for 13 wt.% PVA/0.5 wt.% GEL/3 wt.% Ps patch. Propolis was released quickly in the first hour and arrived at a plateau. Cell culture and contact angle results confirmed that the 3 wt.% addition of propolis reinforced mesenchymal stem cell proliferation and wettability properties of the patches. The antimicrobial activity demonstrated that propolis loaded patches had antibacterial activity against the S. aureus, but for P. aeruginosa, more studies should be performed.


Assuntos
Anti-Infecciosos/administração & dosagem , Materiais Biocompatíveis , Ceratite/tratamento farmacológico , Ceratite/microbiologia , Nanofibras , Própole/administração & dosagem , Materiais Biocompatíveis/química , Portadores de Fármacos/química , Liberação Controlada de Fármacos , Testes de Sensibilidade Microbiana , Nanofibras/química , Nanofibras/ultraestrutura , Álcool de Polivinil/química , Própole/química , Pseudomonas aeruginosa/efeitos dos fármacos , Espectroscopia de Infravermelho com Transformada de Fourier , Staphylococcus aureus/efeitos dos fármacos , Propriedades de Superfície
13.
Int J Biol Macromol ; 180: 365-374, 2021 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-33676980

RESUMO

Post-operative endophthalmitis (POE) is one of the most dreadful complications after intraocular surgery. For cataract surgery patients, both commercially available topical 0.5% levofloxacin and 1% prednisolone acetate (PA) ophthalmic solution require at least 3 to 4 times application daily. In this study, we develop a dual drug delivery system composed of the thermosensitive chitosan/gelatin-based hydrogel containing PA and levofloxacin-loaded nanoparticles (LNPs). LNPs with negative surface charge show the monodisperse (polydispersity index ~0.045), nanosize (~154.7 nm) and sphere-like structure. The optimal concentration of LNPs and PA to corneal epithelial cells was 5 µg/mL and 50 µg/mL, respectively. The developed dual drug delivery system (PAgel-LNPs) could gel at 34 °C within 63 s. The osmolarity of PAgel-LNPs was 301.2 ± 1.5 mOsm/L. PAgel-LNPs showed a sustained-release profile for 7 days. Post-treatment of PAgel-LNPs in TNF-α-damaged corneal epithelial cells could decrease the inflammation (inflammatory genes (TNF-α, IL-6, MMP-3 andMMP-9) and IL-6 production) and cell death. In ex-vivo rabbit model of S. aureus keratitis, the anti-inflammation and anti-bacterial property have been demonstrated. These results suggest that thermosensitive PAgel-LNPs may have the potential to use for the prevention of POE.


Assuntos
Antibacterianos/administração & dosagem , Anti-Inflamatórios/administração & dosagem , Quitosana/química , Portadores de Fármacos/química , Células Epiteliais/efeitos dos fármacos , Hidrogéis/química , Ceratite/tratamento farmacológico , Levofloxacino/administração & dosagem , Nanopartículas/química , Complicações Pós-Operatórias/prevenção & controle , Prednisolona/análogos & derivados , Infecções Estafilocócicas/tratamento farmacológico , Staphylococcus aureus , Animais , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Citocinas/biossíntese , Modelos Animais de Doenças , Liberação Controlada de Fármacos , Quimioterapia Combinada/métodos , Endoftalmite/prevenção & controle , Células Epiteliais/metabolismo , Epitélio Corneano/citologia , Ceratite/microbiologia , Tamanho da Partícula , Prednisolona/administração & dosagem , Coelhos , Infecções Estafilocócicas/microbiologia , Temperatura
14.
Optom Vis Sci ; 98(3): 182-198, 2021 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-33771951

RESUMO

SIGNIFICANCE: Think Tank 2019 affirmed that the rate of infection associated with contact lenses has not changed in several decades. Also, there is a trend toward more serious infections associated with Acanthamoeba and fungi. The growing use of contact lenses in children demands our attention with surveillance and case-control studies. PURPOSE: The American Academy of Optometry (AAO) gathered researchers and key opinion leaders from around the world to discuss contact lens-associated microbial keratitis at the 2019 AAO Annual Meeting. METHODS: Experts presented within four sessions. Session 1 covered the epidemiology of microbial keratitis, pathogenesis of Pseudomonas aeruginosa, and the role of lens care systems and storage cases in corneal disease. Session 2 covered nonbacterial forms of keratitis in contact lens wearers. Session 3 covered future needs, challenges, and research questions in relation to microbial keratitis in youth and myopia control, microbiome, antimicrobial surfaces, and genetic susceptibility. Session 4 covered compliance and communication imperatives. RESULTS: The absolute rate of microbial keratitis has remained very consistent for three decades despite new technologies, and extended wear significantly increases the risk. Improved oxygen delivery afforded by silicone hydrogel lenses has not impacted the rates, and although the introduction of daily disposable lenses has minimized the risk of severe disease, there is no consistent evidence that they have altered the overall rate of microbial keratitis. Overnight orthokeratology lenses may increase the risk of microbial keratitis, especially secondary to Acanthamoeba, in children. Compliance remains a concern and a significant risk factor for disease. New insights into host microbiome and genetic susceptibility may uncover new theories. More studies such as case-control designs suited for rare diseases and registries are needed. CONCLUSIONS: The first annual AAO Think Tank acknowledged that the risk of microbial keratitis has not decreased over decades, despite innovation. Important questions and research directions remain.


Assuntos
Ceratite por Acanthamoeba/epidemiologia , Lentes de Contato/efeitos adversos , Infecções Oculares Bacterianas/epidemiologia , Infecções Oculares Fúngicas/epidemiologia , Infecções Oculares Parasitárias/epidemiologia , Ceratite/epidemiologia , Optometria/organização & administração , Academias e Institutos , Ceratite por Acanthamoeba/parasitologia , Estudos Epidemiológicos , Infecções Oculares Bacterianas/microbiologia , Infecções Oculares Fúngicas/microbiologia , Infecções Oculares Parasitárias/parasitologia , Humanos , Incidência , Ceratite/microbiologia , Fatores de Risco , Estados Unidos/epidemiologia
15.
Infect Immun ; 89(6)2021 05 17.
Artigo em Inglês | MEDLINE | ID: mdl-33753415

RESUMO

Human corneal epithelial (HCE) cells play a significant role in the innate immune response by secreting cytokines and antimicrobial peptides when they encounter fungal pathogens. But the detailed mechanism of attachment and engulfment of the fungal conidia by HCE cells is not well understood. Here, we show the phagocytosis of Aspergillus flavus conidia by RCB2280 cells and primary HCE cultures using confocal microscopy and proteomic analysis of conidium-containing phagosomes. Phalloidin staining showed actin polymerization, leading to an actin ring around engulfed conidia. Cytochalasin D inhibited the actin-mediated endocytosis of the conidia. Immunolabeling of the early endosomal markers CD71 and early endosomal antigen (EEA1) and the late endosomal markers lysosome-associated membrane protein 1 (LAMP1), Rab7, and cathepsin G showed that endosomal proteins were recruited to the site of conidia and showed maturation of the conidium-containing phagosomes. Lysotracker red DND 99 labeling showed the acidification of the phagosomes containing conidia. Phagosome-specific proteome analysis confirmed the recruitment of various phagosomal and endosomal proteins to the conidium-containing phagosomes. These results show that the ocular surface epithelium contributes actively to antifungal defense by the phagocytosis of invading fungal conidia.


Assuntos
Aspergillus flavus/imunologia , Córnea/citologia , Endocitose , Células Epiteliais/microbiologia , Esporos Fúngicos/imunologia , Suscetibilidade a Doenças , Endossomos/metabolismo , Células Epiteliais/imunologia , Células Epiteliais/metabolismo , Humanos , Ceratite/imunologia , Ceratite/metabolismo , Ceratite/microbiologia , Fagossomos/metabolismo , Proteoma , Proteômica/métodos
16.
Invest Ophthalmol Vis Sci ; 62(3): 38, 2021 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-33783487

RESUMO

Purpose: Isorhamnetin is a natural flavonoid with both antimicrobial and anti-inflammatory properties, but its effect on fungal keratitis (FK) remains unknown. The current study aims to investigate the antifungal and anti-inflammatory effects of isorhamnetin against mouse Aspergillus fumigatus keratitis. Methods: In vitro, the lowest effective concentration of isorhamnetin was assessed by minimum inhibitory concentration and cytotoxicity tests in human corneal epithelial cells (HCECs) and RAW264.7 cells. The antifungal property was investigated by scanning electron microscopy and propidium iodide uptake test. The anti-inflammatory effect of isorhamnetin in HCECs and RAW264.7 cells was observed by quantitative real-time polymerase chain reaction (qRT-PCR). In the eyes of mice with A. fumigatus keratitis, FK severity was evaluated using clinical score, plate counting, histological staining and periodic acid Schiff staining. In vivo, the anti-inflammatory effect of isorhamnetin was examined by immunofluorescence staining, myeloperoxidase assay, Western blot, enzyme-linked immunosorbent assay, and qRT-PCR. Results: In HCECs and RAW264.7 cells, isorhamnetin significantly inhibited A. fumigatus conidia growth and hyphae viability at 80 µg/mL without affecting cell viability. In vitro, isorhamnetin altered A. fumigatus hyphal morphology and membrane integrity. In A. fumigatus keratitis mouse model, isorhamnetin treatment alleviated the severity of FK by reducing corneal fungal load and inhibiting neutrophil recruitment. In addition, the mRNA and protein expression levels of TLR-2, TLR-4, Dectin-1, IL-1ß, and tumor necrosis factor-α were significantly decreased in isorhamnetin-treated groups in vivo and in vitro. Conclusions: Isorhamnetin improves the prognosis of A. fumigatus keratitis in mice by inhibiting the growth of A. fumigatus, reducing the recruitment of neutrophils and downregulating inflammatory factors.


Assuntos
Antifúngicos/uso terapêutico , Aspergilose/tratamento farmacológico , Citocinas/antagonistas & inibidores , Infecções Oculares Fúngicas/tratamento farmacológico , Ceratite/tratamento farmacológico , Quercetina/análogos & derivados , Receptores de Reconhecimento de Padrão/antagonistas & inibidores , Animais , Aspergilose/metabolismo , Aspergilose/microbiologia , Aspergillus fumigatus/efeitos dos fármacos , Western Blotting , Citocinas/metabolismo , Ensaio de Imunoadsorção Enzimática , Epitélio Corneano/efeitos dos fármacos , Epitélio Corneano/ultraestrutura , Infecções Oculares Fúngicas/metabolismo , Infecções Oculares Fúngicas/microbiologia , Feminino , Ceratite/metabolismo , Ceratite/microbiologia , Camundongos , Camundongos Endogâmicos C57BL , Testes de Sensibilidade Microbiana , Microscopia Eletrônica de Varredura , Microscopia de Fluorescência , Quercetina/uso terapêutico , Receptores de Reconhecimento de Padrão/metabolismo
17.
Sci Rep ; 11(1): 6015, 2021 03 16.
Artigo em Inglês | MEDLINE | ID: mdl-33727638

RESUMO

Infectious keratitis is a potentially sight threatening ophthalmological emergency. Contact lens wear is a common risk factor. Diagnostic advances such as MALDI-TOF MS provides new insights into the spectrum of corneal pathogens and on microbes previously considered as commensals. Corynebacterium macginleyi was described in 1995, and in 2018, the genomic features of three isolates were reported after whole-genome sequencing. Here we describe the clinical characteristics of patients with infectious keratitis (n = 29) presumably caused by Corynebacterium macginleyi, and analyze the genomic features of C. macginleyi (n = 22) isolated from the corneal ulcers of these patients. The disease course was uneventful apart from minor interventions such as corneal cross-linking and amniotic membrane transplant. Genome sequencing and comparison revealed a highly conserved core genome of C. macginleyi. Based on the analyses of single nucleotide polymorphisms, the population could be divided into two main clades that also differed in a few clade-specific genomic islands. Patients infected with an isolate belonging to the minor clade (n = 7) presented a more severe disease. Comparisons with other corynebacterial species clearly separated C. macginleyi. C. macginleyi may be considered a corneal pathogen; genomic analysis provided insights into its population structure and disease-causing potential.


Assuntos
Infecções por Corynebacterium/genética , Corynebacterium/genética , Ceratite , Adulto , Idoso , Corynebacterium/isolamento & purificação , Feminino , Humanos , Ceratite/genética , Ceratite/microbiologia , Masculino , Pessoa de Meia-Idade , Estudos Retrospectivos , Sequenciamento Completo do Genoma
18.
Lancet Infect Dis ; 21(3): e49-e57, 2021 03.
Artigo em Inglês | MEDLINE | ID: mdl-33645500

RESUMO

Fungal keratitis is a severe corneal infection that often results in blindness and eye loss. The disease is most prevalent in tropical and subtropical climates, and infected individuals are frequently young agricultural workers of low socioeconomic status. Early diagnosis and treatment can preserve vision. Here, we discuss the fungal keratitis diagnostic literature and estimate the global burden through a complete systematic literature review from January, 1946 to July, 2019. An adapted GRADE score was used to evaluate incidence papers-116 studies provided the incidence of fungal keratitis as a proportion of microbial keratitis and 18 provided the incidence in a defined population. We calculated a minimum annual incidence estimate of 1 051 787 cases (736 251-1 367 323), with the highest rates in Asia and Africa. If all culture-negative cases are assumed to be fungal, the annual incidence would be 1 480 916 cases (1 036 641-1 925 191). In three case series, 8-11% of patients had to have the eye removed, which represents an annual loss of 84 143-115 697 eyes. As fungal keratitis probably affects over a million people annually, an inexpensive, simple diagnostic method and affordable treatment are needed in every country.


Assuntos
Infecções Oculares Fúngicas/diagnóstico , Infecções Oculares Fúngicas/epidemiologia , Ceratite/diagnóstico , Ceratite/epidemiologia , Infecções Oculares Fúngicas/microbiologia , Fungos/isolamento & purificação , Saúde Global , Humanos , Incidência , Ceratite/microbiologia , Fatores de Risco
19.
Arch. Soc. Esp. Oftalmol ; 96(2): 106-109, feb. 2021. ilus
Artigo em Espanhol | IBECS | ID: ibc-200797

RESUMO

Este caso clínico describe el primer caso publicado de queratitis fúngica de la interfaz (QI) de inicio temprano después de una queratoplastia endotelial de la membrana de Descemet (DMEK) tratada satisfactoriamente con queratoplastia penetrante (QP) durante la etapa activa de la infección. Un paciente con fracaso de injerto después de una queratoplastia endotelial automatizada con pelado de Descemet (DSAEK) fue intervenido con DMEK. El cultivo y el medio de cultivo del donante dieron positivo para Candida albicans. Se confirmaron y localizaron varios infiltrados de la interface utilizando tomografía de coherencia óptica de segmento anterior. Tres días después del diagnóstico, observándose signos claros de infección intraocular, se retiró el injerto con lavado simultáneo de cámara anterior con fluconazol al 1% seguido de una QP e inyecciones corneales intraestromales de fluconazol. Se consiguió una mejor agudeza visual corregida de 20/20. Este caso resalta la importancia de analizar cada pieza y medio de cultivo de donantes a pesar de que el paciente no exhiba síntomas o signos durante el periodo posoperatorio. La QP es una opción de tratamiento viable para la queratitis de interface de inicio temprano


In this case-report we describe the first reported case of early-onset fungal interface keratitis (IK) after Descemet Membrane Endothelial Keratoplasty (DMEK) successfully treated with penetrating keratoplasty (PK) during the active stage of infection. A patient with graft failure after Descemet Stripping Automated Endothelial Keratoplasty (DSAEK) was operated on with DMEK. Donor rim culture and broth were positive for Candida albicans. Several interface infiltrates were confirmed and localized using anterior segment optical coherence tomography. Three days after diagnosis, observing clear signs of intraocular infection, the graft was removed with simultaneous washed-up of anterior chamber with fluconazole 1% followed by a PK and intrastromal corneal injections of fluconazole. A best-corrected visual acuity of 20/20 was achieved. This case highlights the importance of analysing every donor rim and broth, despite the patient doesn't show any symptoms or signs during the post-operative period. PK is a viable treatment option in early-onset interface keratitis


Assuntos
Humanos , Masculino , Idoso , Ceratoplastia Penetrante/métodos , Ceratite/microbiologia , Ceratite/cirurgia , Ceratoplastia Endotelial com Remoção da Lâmina Limitante Posterior/efeitos adversos , Infecções Oculares Fúngicas/etiologia , Candida albicans/isolamento & purificação , Infecções Oculares Fúngicas/diagnóstico , Ceratite/diagnóstico , Fatores de Tempo
20.
Drug Des Devel Ther ; 15: 617-628, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33623371

RESUMO

Purpose: Efficacy of norvancomycin (NVCM) through continuous topical ocular instillation drug delivery (CTOIDD) system for treating severe acute bacterial keratitis infection with Staphylococcus aureus was investigated. Methods: Rabbits with bacterial keratitis were treated using CTOIDD with NVCM (n=13), topical NVCM eye drops (n=11), and CTOIDD with saline (n=8). Clinical signs of keratitis in all groups were assessed consecutively for a week. Bacterial quantification of excised corneas was counted on the fourth and eighth days. Histopathologic examinations were performed to assess inflammatory cell infiltration on the eighth day. Results: All signs of bacterial keratitis were alleviated in CTOIDD with NVCM according to criteria, and the CTOIDD-NVCM group had significantly less inflammation than CTOIDD-saline (p<0.05), and eye drop-NVCM (p<0.05). Two eyes in the eye drop-NVCM group, four eyes in the CTOIDD-saline group had corneal perforation (CP), while none of the rabbits showed CP in the CTOIDD-NVCM group. Bacterial counts were significantly less in the CTOIDD with NVCM group in comparison to the eye drop-NVCM (p<0.05), and CTOIDD-saline (p<0.05) groups. Severe inflammation and marked inflammatory cell infiltration were found in histopathologic examinations in the CTOIDD-saline and eye drop-NVCM groups, while significantly less inflammation was documented in the CTOIDD-NVCM (p<0.05) group. Conclusion: CTOIDD with NVCM effectively reduced the severity and treated acute bacterial S. aureus keratitis infection in a rabbit model. The presented approach of CTOIDD with NVCM appears to be a promising therapeutic approach for severe acute bacterial keratitis.


Assuntos
Antibacterianos/farmacologia , Infecções Oculares Bacterianas/tratamento farmacológico , Ceratite/tratamento farmacológico , Soluções Oftálmicas/farmacologia , Staphylococcus aureus/efeitos dos fármacos , Vancomicina/análogos & derivados , Doença Aguda , Administração Oftálmica , Animais , Antibacterianos/administração & dosagem , Relação Dose-Resposta a Droga , Infecções Oculares Bacterianas/microbiologia , Feminino , Ceratite/microbiologia , Testes de Sensibilidade Microbiana , Estrutura Molecular , Soluções Oftálmicas/administração & dosagem , Coelhos , Relação Estrutura-Atividade , Vancomicina/administração & dosagem , Vancomicina/farmacologia
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