Comparison of techniques for corneal epithelium cell culture for the collection of conditioned medium.

PURPOSES: To determine the best protocol in obtaining the higher yield of conditioned culture medium to be used for the bone marrow mesenchymal stem cell differentiation into corneal epithelial cells, five techniques for the primary culture of human corneal epithelial cells were evaluated. METHODS: The studied culture techniques of corneal epithelial cells were: explants in culture flasks with and without hydrophilic surface treatment, on amniotic membrane, with enzymatic digestion, and by corneal scraping. The conditioned culture medium collected from these cultures was used to differentiate human bone marrow mesenchymal stem cells into corneal epithelial cells, which were characterized using flow cytometry with pan-cytokeratin and the corneal-specific markers, cytokeratin 3 and cytokeratin 12. RESULTS: The culture technique using flasks with hydrophilic surface treatment resulted in the highest yield of conditioned culture medium. Flasks without surface treatment resulted to a very low success rate. Enzymatic digestion and corneal scraping showed contamination with corneal fibroblasts. The culture on amniotic membranes only allowed the collection of culture medium during the 1st cell confluence. The effectiveness of cell differentiation was confirmed by cytometry analysis using the collected conditioned culture medium, as demonstrated by the expressions of cytokeratin 3 (95.3%), cytokeratin 12 (93.4%), and pan-cytokeratin (95.3%). CONCLUSION: The culture of corneal epithelial cell explants in flasks with hydrophilic surface treatment is the best technique for collecting a higher yield of conditioned culture medium to be used to differentiate mesenchymal stem cells.

Trachoma: an underdiagnosed disease revealed by a survey carried out at Jaú, São Paulo.

BACKGROUND: Trachoma is a notifiable disease in the state of São Paulo- Brazil. However, in Jaú, a municipality located in this state, in the last 10 years there are no records of cases. This study purpose is to assess if there are cases of inflammatory trachoma in schoolchildren aged 1 to 9 years in the municipality of Jaú as well if it is possible to detect clusters areas of the disease to establish elimination programs. METHODS: An epidemiological study was performed in 2018, involving a stratified random sample of schoolchildren aged 1- to 9-year-old, from public day care centers and elementary schools in the municipality of Jaú. A trachoma screening following the criteria of the World Health Organization (WHO) and the distribution of cases was assessed using geoprocessing. RESULTS: Four thousand-six hundred-nineteen children from 44 elementary schools were examined, and 126 children with active trachoma were detected, with an adjusted prevalence rate of 2.65%. The prevalence was higher (3.01%) in children aged 6- to 9-year-old compared to children aged 1-to 5-year-old (2.42%). There were clusters with a higher concentration of positive cases of the disease in three schools located in the neighborhoods with lower socioeconomic conditions. CONCLUSION: Inflammatory trachoma still present in children aged 1- to 9-year-old in the city of Jaú. The positive cases were located mainly in areas with low socioeconomic conditions. Health promotion with active search and periodical treatment must be planned to fight this important blinding cause, that persists in our municipality.

Comparison of techniques for corneal epithelium cell culture for the collection of conditioned medium / Comparação das técnicas de cultivo de células epiteliais da córnea para a coleta de meio condicionado

ABSTRACT Purposes: To determine the best protocol in obtaining the higher yield of conditioned culture medium to be used for the bone marrow mesenchymal stem cell differentiation into corneal epithelial cells, five techniques for the primary culture of human corneal epithelial cells were evaluated. Methods: The studied culture techniques of corneal epithelial cells were: explants in culture flasks with and without hydrophilic surface treatment, on amniotic membrane, with enzymatic digestion, and by corneal scraping. The conditioned culture medium collected from these cultures was used to differentiate human bone marrow mesenchymal stem cells into corneal epithelial cells, which were characterized using flow cytometry with pan-cytokeratin and the corneal-specific markers, cytokeratin 3 and cytokeratin 12. Results: The culture technique using flasks with hydrophilic surface treatment resulted in the highest yield of conditioned culture medium. Flasks without surface treatment resulted to a very low success rate. Enzymatic digestion and corneal scraping showed contamination with corneal fibroblasts. The culture on amniotic membranes only allowed the collection of culture medium during the 1st cell confluence. The effectiveness of cell differentiation was confirmed by cytometry analysis using the collected conditioned culture medium, as demonstrated by the expressions of cytokeratin 3 (95.3%), cytokeratin 12 (93.4%), and pan-cytokeratin (95.3%). Conclusion: The culture of corneal epithelial cell explants in flasks with hydrophilic surface treatment is the best technique for collecting a higher yield of conditioned culture medium to be used to differentiate mesenchymal stem cells.

RESUMO Objetivos: Foram estudadas cinco técnicas de cultivo primário de células epiteliais de córnea humana para se determinar o melhor protocolo para a obtenção do maior rendimento de meio de cultivo condicionado para ser utilizado na diferenciação de células tronco mesenquimais para células epiteliais de córnea. Métodos: As técnicas de cultivo estudadas foram: explantes em frascos de cultivo com e sem tratamento hidrofílico de superfície, sobre membrana amniótica, com digestão enzimática e por raspado de córnea. O meio de cultivo condicionado foi coletado e as células tronco mesenquimais induzidas a se diferenciarem em células epiteliais da córnea utilizando o meio de cultivo condicionado. As células foram caracterizadas por citometria de fluxo com pan-citoqueratina e com os marcadores específicos da córnea, citoqueratina 3 e citoqueratina 12. Resultados: A técnica utilizando frascos com o tratamento de superfície apresentou o maior rendimento de meio de cultivo condicionado. Os frascos sem tratamento de superfície levaram a uma taxa de sucesso muito baixa. A digestão enzimática e a raspagem da córnea mostraram contaminação das culturas com fibroblastos de córnea. A cultura sobre membranas amnióticas só permitiu a coleta do meio de cultivo condicionado durante a 1ª confluência celular. A análise de citometria de fluxo confirmou o sucesso da diferenciação celular utilizando o meio de cultivo condicionado coletado, demonstrada pela expressão de citoqueratina 3 (95,3%), citoqueratina 12 (93,4%) e pan-citoqueratina (95,3%). Conclusão: O cultivo de explantes de células tronco mesenquimais em frascos com tratamento hidrofílico de superfície é a melhor técnica para a obtenção de um alto rendimento de meio de cultivo condicionado.

Laceração de canalículo lacrimal em centro de referência do centro-oeste paulista / Lacrimal canaliculus laceration in a reference center in the Center-West of the state of São Paulo

RESUMO Objetivo: Descrever as características clínico-epidemiológicas, técnicas cirúrgicas e resultado do tratamento das lacerações canaliculares operadas em nosso serviço. Métodos: Estudo retrospectivo, realizado de janeiro de 2012 a junho de 2020, considerando-se as lesões de canalículo lacrimal operadas em um serviço de referência. Dados demográficos, características das lesões, detalhes das cirurgias e resultado do tratamento foram obtidos de prontuários eletrônicos e analisados estatisticamente. Resultados: Foram incluídos 26 portadores de lesões canaliculares, com idade entre 2 e 71 anos, sendo 73,1% homens. A lesão acometia o canalículo superior em 53,9%; 80,8% pacientes procuraram pelo serviço nas primeiras 24 horas, e 46,2% tiveram a cirurgia realizada entre 24 e 72 horas após o traumatismo. Todos os pacientes tiveram intubação mono ou bicanalicular, e o tempo transcorrido entre a cirurgia e a retirada do silicone, variou de zero a 183 dias. Após a cirurgia, 21 pacientes (80,8%; p<0,05) não apresentaram complicações, 2 (7,7%) evoluíram com obstrução canalicular, 2 (7,7%) com granuloma e 1 (3,8%) com ectrópio de ponto lacrimal. Conclusão: As lesões de canalículo encontradas em nossa prática são mais comuns em crianças ou homens jovens, acometem mais o canalículo superior e as nossas condutas levam a sucesso no tratamento na maior parte dos casos. As grandes controvérsias no assunto persistem, como o tipo e o tempo de permanência do tubo de silicone na via lacrimal. Somente estudos com grandes amostras podem consolidar esses conceitos.

ABSTRACT Objective: To describe the clinical and epidemiological characteristics, surgical techniques, and results of the canalicular laceration treatment at our service. Methods: A retrospective study was carried out from January 2012 to June 2020, considering canalicular injuries operated at a reference center. Demographic data, lesion characteristics, surgical details, and treatment outcomes were obtained from electronic medical records and were statistically analyzed. Results: Twenty-six cases of people with canalicular lesions aged between 2 to 71 years old were included, of whom 73.1% were men. The superior canaliculus was affected in 53.9%; 80.8% of patients searched for care within the first 24 hours, and 46.2% had the surgery performed between 24-72 hours after trauma. All patients had mono or bicanalicular intubation and the time elapsed between surgery and silicone removal ranged from 0 to 183 days. After surgery, 21 patients (80.8%, p<0.05) did not present any complications, two (7.7%) evolved with canalicular obstruction, two (7.7%) with granuloma, and one (3.8 %) with lacrimal puncta ectropion. Conclusion: In our practice, canalicular injuries are more common in children or young men, affecting mainly the superior canaliculus, and treatment success using our approach can be achieved in most of the cases. However, great controversies remain on the subject, such as type of intubation and when to remove the silicone tube from the lacrimal pathway. Larger series are required to consolidate controversial concepts.

Distiquíase congênita: uma afecção de difícil tratamento / Congenital distichiasis: a condition difficult to be treated

RESUMO A distiquíase congênita é uma condição rara, de origem controversa, com opções terapêuticas cujos resultados nem sempre são favoráveis. Este é o relato de caso de uma criança do sexo masculino, de 7 anos, portadora de distiquíase congênita afetando as quatro pálpebras, com permanência de alguns folículos pilosos distiquiáticos mesmo tratados cirurgicamente pela técnica da divisão interlamelar, com aplicação seletiva de crioterapia e de eletrólise. O tratamento da distiquíase congênita envolveu a necessidade de associar técnicas, a fim de se obterem resultados mais efetivos.

ABSTRACT Congenital distichiasis is a rare condition, of controversial origin, with therapeutic options not always having favorable results. This is a case report of a seven-year-old male child with congenital distichiasis affecting all eyelids, with the presence of some distichiasis hair follicles despite surgical treatment using the interlamellar division technique, selective application of cryotherapy and electrolysis. The treatment of congenital distichiasis involves the need to combine techniques to achieve more effective results.

Management of external ocular prosthesis by ocularists: results of an online survey conducted in Brazil and Spain.

PURPOSE: To analyse the ocularist's perspective on the management of the anophthalmic socket and external ocular prosthesis (EOP). METHODS: Ocularists from two countries were invited to participate in an online questionnaire. Data were collected on demographics, anophthalmic socket and EOP management (manufacturing, use, cleaning), complications, follow-up visits and multidisciplinary care. The frequency and proportions of the responses were statistically analysed. RESULTS: The questionnaire was addressed to 20 Brazilian and 17 Spanish ocularists, obtaining a response rate of 65% and 64.7%, respectively. 62.5% of respondents were men. The most common cause of anophthalmia in Brazil (69.2%) and Spain (36.4%) is an eye disease (chi square: p = 0.188). Polymethylmethacrylate (PMMA) is the most commonly used material in EOP manufacture (chi square: p = 0.448), and 70.8% reported using customized EOPs (chi square: p = 0.069). Deposits are frequently observed in both countries (chi square: p = 0.157). Changing the prosthesis is recommended after 5 to 10 years by Brazilian ocularists, and after less than 5 years of use by Spanish ocularists (81.8%) (chi square: p = 0.041). Annual follow-up is recommended by Spanish ocularists (45.5%), while semestral (38.5%) and case-dependent (38.5%) follow-up is recommended by Brazilian ocularists (chi square: p = 0.267). Daily cleaning is advocated by 61.5% of Brazilian ocularists and once a month by 45.5% of Spanish ocularists (chi square: p = 0.098), with 75% of ocularists from both countries not recommending EOP removal at night (Fisher´s exact test: p = 0.166). Good communication between ocularists and ophthalmologists was reported by 87.5% of our responders (chi square: p = 0.642). CONCLUSION: Although there are no unified protocols on the management of EOPs, Brazilian and Spanish ocularists follow similar guidelines. Differences between countries were the patients´ referral and the prosthesis´ useful life.

Anti-inflammatory effects of α-humulene and ß-caryophyllene on pterygium fibroblasts.

AIM: To investigate the anti-inflammatory effects of the sesquiterpenes α-humulene and ß-caryophyllene on pterygium fibroblasts. METHODS: Primary cultures of pterygium fibroblasts were established. Third passage pterygium fibroblasts were exposed to α-humulene and ß-caryophyllene separately and together. The cell viability was assessed by 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide (MTT) assay at 12, 24, 48, and 72h after exposure. The levels of the inflammatory cytokines interleukin (IL)-1ß, IL-6, IL-8, tumor necrosis factor (TNF)-α and IL-10 in the conditioned culture medium were assessed by enzyme-linked immunosorbent assay (ELISA) at 12, 24 and 48h after exposure. Data were statistically analyzed using Friedman repeated measures analysis of variances on ranks. RESULTS: The 25 µmol/L ß-caryophyllene induced significant decrease in the IL-6 production by pterygium fibroblasts 48h after the exposure (P=0.041). The levels of IL-1ß, IL-8, IL-10, and TNF-α were very low and had no statistically significant variations after exposure to α-humulene, ß-caryophyllene, or both compounds together. CONCLUSION: The exposure to 25 µmol/L of ß-caryophyllene significantly reduce the production of IL-6 by pterygium fibroblasts after 48h. This sesquiterpene may be a potential alternative adjuvant agent for the treatment of pterygium.

Expression of inflammatory cytokines in mesenchymal stem cells derived from proximal humerus fractures.

Background: Mesenchymal stem cells (MSCs) are an excellent treatment option for a wide variety of orthopaedic conditions. This study aimed to establish if bone marrow MSCs obtained from proximal humerus fractures can be an alternative source for obtaining primary cultures of human MSCs. Methods: Human bone marrow was obtained during osteosynthesis surgeries on closed proximal humerus fractures within 48 hours of injury. MSCs were harvested using the Ficoll gradient separation protocol and in vitro cultured until the third passage. Then, the cells were immunophenotyped by flow cytometry using stem cell specific surface markers. The cells were also induced to differentiate into osteoblasts and adipocytes for the characterization and confirmation of MSCs. The production of cytokines interleukin (IL)-1ß, IL-6, IL-8, IL-10, tumor necrosis factor α (TNF-α), and interferon γ (IFN-γ) was assessed using enzyme-linked immunosorbent assay (ELISA) in the supernatant of the cultures after 3, 5 or 7 days. Results: Immunophenotyping showed high expression of the stem cell surface markers CD73, CD90, and CD105 and negative or very low expression of CD34, CD45, CD11b, CD19, and human leukocyte antigen (HLA)-DR. The bone marrow derived MSCs were able to differentiate into osteoblasts and adipocytes. The quantification of secreted cytokines revealed that IL-8 was the most produced cytokine, followed by IL-6 and IL-10 at similar quantities and lower levels of IL-1ß. TNF-α and IFN-γ were not detected. Conclusions: Proximal humerus fractures can be an alternative source for the collection of bone marrow MSCs. The cytokine production of these cells is very similar to the production profile of fracture haematomas previously reported and may be used for improving bone repair.

In vitro effect of triamcinolone and platelet-rich plasma on cytokine levels of elbow lateral epicondylitis-derived cells.

BACKGROUND: The pathogenesis and treatment of lateral elbow epicondylitis (LEE) are still controversial. The purpose of the current study was to evaluate the production of inflammatory cytokines by LEE-derived cells and to compare the anti-inflammatory effect of triamcinolone acetonide with platelet-rich plasma (PRP) on cytokines production in primary culture of these cells. METHODS: Third passage cells from primary cultures of LEE were assessed for the production of the cytokines IL-1ß, IL-6, IL-8, IL-10 and TNF-α by immune-enzymatic assay (ELISA), after the treatment with 1, 10 and 100 µM triamcinolone compared to no treated controls at the time points 6, 12, 18, 24, 48, 72 and 96 h, and to PRP at 48, 72 and 96 h. RESULTS: The cytokines IL-6 and IL-8 were produced in high concentrations by LEE cells. One, 10 and 100 µM triamcinolone induced significant decrease in the production of IL-6 and IL-8 at 48, 72 and 96 h, adding the time point 12 h for IL-8. Compared to controls, PRP caused a significant increase in the production of IL-6 and IL-8 and there was a significant increase in IL-10 production with the use of 100 µM triamcinolone at 48 h. The production of IL1-ß and TNF-α was very low and did not change when the cultures were treated with triamcinolone or PRP. CONCLUSION: LEE-derived cells produce IL-6 and IL-8, confirming the inflammatory nature of this condition. While triamcinolone inhibited the production of IL-6 and IL-8 by LEE cells, PRP induced an increase in these cytokines compared with controls.

Amplitude of movements with conical or spherical implants in anophthalmic socket.

PURPOSE: To evaluate the amplitude of movement in anophthalmic sockets reconstructed with conical or spherical orbital implants with and without an external ocular prosthesis (EOP), and whether the fornix depth could play a role. METHODS: Prospective observational study involving unilateral anophthalmic sockets evaluated the amplitude of movement with conical (20 subjects) or spherical (16) non-porous orbital implants, with and without an EOP, having the contralateral eye as the control group. Standardized photographs were obtained in the four gaze directions and measurements were performed using the Image J software. The upper and lower fornix depths were measured using rulers. RESULTS: Compared to the contralateral eye, the median movement amplitude without EOP was smaller with conical implants in supraduction (-0.88 mm, p=0.008), abduction (-2.26 mm, p<0.001) and adduction (-0.91 mm, p=0.008). Spherical implants had reduced movement only in abduction (-2.63 mm, p<0.001). Conical and spherical implants had similar amplitudes of movement in all versions, and were always smaller compared to the control. The median movement amplitude with the EOP was -3.05 mm (p=0.001) than without the EOP in abduction and -2.07 mm (p=0.020) in adduction, regardless of implant format. The fornix depth did not affect the orbital implants or EOP movement amplitude's median. CONCLUSION: Conical and spherical implants provide similar amplitude of movement and fornix depth did not have an influence on it. The amplitude of movement was significantly limited compared to the contralateral eye and was even more reduced if the EOP was in place with conical or spherical implant formats.

Reasons for no-show to referrals at a university eye clinic after eye examination via a mobile ophthalmic unit in a Brazilian region / Razão de não comparecimento à clínica oftalmológica da universidade após triagem em uma unidade móvel oftalmológica em uma região brasileira

ABSTRACT Purpose: This study aimed to identify patient's reason for no-show at a university eye clinic after ophthalmic examination via a mobile ophthalmic unit, which provides comprehensive ophthalmic care to underserved communities in a region of Brazil. Methods: In 2017/2018, this prospective observational study searched for no-shows at referrals to a university eye clinic after an outreach program screening via a mobile ophthalmic unit in 10 municipalities in the central-western region of São Paulo, Brazil. A total of 1,928 patients underwent a comprehensive eye examination at no cost, and 37.1% of them needed referral to a university eye clinic for specialized examinations or surgeries. We used the following two main tools: (1) comparative analysis between patients who attended the referral and those who did not; (2) active search using a questionnaire to assess reasons for no-show. Results: Attendance to referrals was not influenced by age, gender, distance from the university hospital, number of ophthalmologists in the municipality, average family income, and visual acuity. The main cause for referrals was cataract (350 cases). No-show was most common among glaucoma/suspected glaucoma (54.1%) cases, followed by strabismus (45%) and anterior segment disease (33.6%) cases. Many patients who did not attend the referral sought another service. Conclusion: Patient's issues and lack of knowledge regarding their ophthalmic condition are the main reasons for no-show at referrals for free ophthalmic care. Thus, educational campaigns are needed to achieve consistently high attendance to prevent avoidable blindness.

RESUMO Objetivo: Avaliar as razões para não comparecimento à clínica oftalmológica da universidade após triagem oftalmológica realizada usando uma unidade móvel oftalmológica que fornece exame oftalmológico para comunidades não assistidas em uma região do Brasil. Métodos: Foi realizado um estudo observacional prospectivo no ano de 2017/2018 para avaliar as razões que fizeram com que os indivíduos triados usando uma unidade móvel oftalmológica e referenciados para a clínica oftalmológica da universidade não comparecessem à consulta agendada. A triagem foi feita em 10 municípios da região centro-oeste do estado de São Paulo, Brasil. Todos os 1.928 participantes fizeram o exame oftalmológico sem custo e 37,1% deles necessitaram de encaminhamento para a clínica oftalmológica da universidade para exames especializados ou cirurgias. O estudo usou duas ferramentas: (1) análise comparativa entre os dados dos indivíduos encaminhados que compareceram ao agendamento com os que não compareceram; (2) busca ativa dos indivíduos que não compareceram à consulta agendada, aplicando-se um questionário para avaliar os motivos para o não comparecimento. Resultados: Fatores como idade, sexo, distância entre a cidade de origem e o hospital universitário, número de oftalmologistas na cidade de procedência, renda familiar média e acuidade visual não influenciaram no comparecimento ao encaminhamento. Catarata foi a maior causa para o encaminhamento (350 casos). O não comparecimento foi maior nos portadores de glaucoma/glaucoma suspeitos (54,1%), estrabismo (45%) e afecções do segmento anterior (33,6%). Muitos indivíduos que não compareceram ao serviço de referência procuraram por outro local para o atendimento oftalmológico. Conclusão: O não comparecimento para tratamento oftalmológico sem custo depende de fatores relacionados ao paciente ou à falta de conhecimento das próprias condições oftalmológicas. Campanhas educativas nas comunidades assistidas devem ser feitas para alcançar maior comparecimento às consultas e melhor prevenir a cegueira evitável.

Reasons for no-show to referrals at a university eye clinic after eye examination via a mobile ophthalmic unit in a Brazilian region.

PURPOSE: This study aimed to identify patient's reason for no-show at a university eye clinic after ophthalmic examination via a mobile ophthalmic unit, which provides comprehensive ophthalmic care to underserved communities in a region of Brazil. METHODS: In 2017/2018, this prospective observational study searched for no-shows at referrals to a university eye clinic after an outreach program screening via a mobile ophthalmic unit in 10 municipalities in the central-western region of São Paulo, Brazil. A total of 1,928 patients underwent a comprehensive eye examination at no cost, and 37.1% of them needed referral to a university eye clinic for specialized examinations or surgeries. We used the following two main tools: (1) comparative analysis between patients who attended the referral and those who did not; (2) active search using a questionnaire to assess reasons for no-show. RESULTS: Attendance to referrals was not influenced by age, gender, distance from the university hospital, number of ophthalmologists in the municipality, average family income, and visual acuity. The main cause for referrals was cataract (350 cases). No-show was most common among glaucoma/suspected glaucoma (54.1%) cases, followed by strabismus (45%) and anterior segment disease (33.6%) cases. Many patients who did not attend the referral sought another service. CONCLUSION: Patient's issues and lack of knowledge regarding their ophthalmic condition are the main reasons for no-show at referrals for free ophthalmic care. Thus, educational campaigns are needed to achieve consistently high attendance to prevent avoidable blindness.