Preparation of an ophthalmic formulation of TPGS-modified insulin-loaded liposomes and its in vitro corneal permeation and pharmacokinetics in rabbit eyes / 南方医科大学学报

OBJECTIVE@#To prepare vitamin E polyethylene glycol 1000 succinate (TPGS)-modified insulin-loaded liposomes (T-LPs/INS) and evaluate its safety, corneal permeability, ocular surface retention and pharmacokinetics in rabbit eyes.@*METHODS@#The safety of the preparation was investigated in human corneal endothelial cells (HCECs) using CCK8 assay and live/dead cell staining. In the ocular surface retention study, 6 rabbits were randomized into 2 equal groups for application of fluorescein sodium dilution or T-LPs/INS labeled with fluorescein in both eyes, which were photographed under cobalt blue light at different time points. In the cornea penetration test, another 6 rabbits divided into 2 groups for application of Nile red diluent or T-LPs/INS labeled with Nile red in both eyes, after which the corneas were harvested for microscopic observation. In the pharmacokinetic study, 2 groups of rabbits (n=24) were treated with eye drops of T-LPs/INS or insulin, and the aqueous humor and cornea were collected at different time points for measurement of insulin concentrations using enzyme linked immunosorbent assay. DAS2 software was used to analyze the pharmacokinetic parameters.@*RESULTS@#The prepared T-LPs/INS showed good safety in cultured HCECs. Corneal permeability assay and fluorescence tracer ocular surface retention assay demonstrated a significantly higher corneal permeability of T-LPs/INS with a prolonged drug residence in the cornea. In the pharmacokinetic study, insulin concentrations in the cornea at 6, 15, 45, 60, and 120 min (P < 0.01) and in the aqueous humor at 15, 45, 60, and 120 min after dosing were significantly higher in T-LPs/INS group. The changes in insulin concentrations in the cornea and aqueous humor were consistent with a two-compartment model in T-LPs/INS group and with the one-compartment model in the insulin group.@*CONCLUSION@#The prepared T-LPs/INS shows an improved corneal permeability, ocular surface retention capacity and eye tissue concentration of insulin in rabbits.

Application of lectins for detection of glycomic differences in the epididymal duct in water buffalo (Bubalus bubalis) and dromedary camel (Camelus dromedarius) / Aplicación de lectinas para la detección de diferencias glucómicas en el conducto epididimario en búfalos de agua (Bubalus bubalis) y dromedarios (Camelus dromedarius)

SUMMARY: The present study was conducted to detect the differences in glycohistochemical features in the epididymal duct of the dromedary camel and the water buffalo. Epididymal sections (caput, corpus and cauda) from both species were stained with fluorescein isothiocyanate (FITC) conjugated lectins. Binding sites for five lectins (DBA, GSA-1, HPA, PNA and WGA) have been found in both species. The binding sites of different lectins showed significant variations in the pattern of distribution in both a species. This included both species-specific and region-specific order. Additionally, only three (GSA-1, PNA and WGA) out the five lectins studied exhibited binding sites in all epididymal regions in both species. The other two lectins (DBA and HPA) followed the same order recorded for the other three (GSA-1, PNA and WGA) in buffalo, but failed to show any binding sites in cauda epididymis in camel. In conclusion, the variable regional and species-specific distribution features of lectins revealed that both species have diverse glycomic characteristics that may be related to their different reproductive patterns. However, the glycome-associated functional capacities remain obscured and need further profound investigations.

RESUMEN: El presente estudio se realizó para detectar las diferencias en las características glicohistoquímicas del conducto epididimal del dromedario y el búfalo de agua. Las secciones del epidídimo (cabeza, cuerpo y cola) de ambas especies se tiñeron con lectinas conjugadas con isotiocianato de fluoresceína (FITC). Se encontraron sitios de unión para cinco lectinas (DBA, GSA-1, HPA, PNA y WGA) en ambas especies. Los sitios de unión de diferentes lectinas mostraron variaciones significativas en el patrón de distribución en ambas especies. Esto incluía tanto el orden específico de la especie como el específico de la región. Además, solo tres (GSA-1, PNA y WGA) de las cinco lectinas estudiadas exhibieron sitios de unión en todas las regiones del epidídimo en ambas especies. Las otras dos lectinas (DBA y HPA) siguieron el mismo orden registrado para las tres restantes (GSA-1, PNA y WGA) en búfalos, pero no mostraron ningún sitio de union en la cola del epidídimo en camellos. En conclusión, las características de distribución regionales y específicas de especies variables de las lectinas revelaron que ambas especies tienen características glucómicas diversas que pueden estar relacionadas con sus diferentes patrones reproductivos. Sin embargo, las capacidades funcionales asociadas con el glicoma permanecen desconocidas y requieren mayor investigación.

Microcirugía mínimamente invasiva para aneurisma de la arteria comunicante anterior: abordaje supraorbitario lateral más angiografía con fluoresceína intraoperatoria / Minimally invasive micro surgery for aneurysm of the anterior communicating artery: supra-orbitary lateral approach with intraoperative Fluorescein Angiography

RESUMEN Los aneurismas de la arteria comunicante anterior son complejos y requieren de tratamiento endovascular o microquirúrgico según sus características. El uso de enfoques mínimamente invasivos en microcirugía continúa evolucionando. Presentamos el caso de un paciente de 34 años con un aneurisma cerebral no roto de AcoA de cuello ancho, con escala de Glasgow de 15 puntos. Se realizó un abordaje supraorbitario lateral más "clipaje" total del aneurisma usando angio fluoresceína intraoperatoria. Se logró el objetivo con seguridad y eficacia; al mismo tiempo se optimizó el tiempo quirúrgico y hospitalario, maximizando la comodidad del paciente, estética y reanudación a sus actividades tempranas. (AU)

SUMMARY Anterior communicating artery aneurisms (ACAA) are complex and require endovascular or microsurgery approaches. The use of minimally invasive microsurgery techniques is evolving. We present the case of a 34-yearold patient with a broad neck, non-broken, ACAA with a Glasgow coma scale of 15 points. A lateral supra-orbitary approach with total clipping of the ACAA using intra-operatory fluorescein angiography was performed. The objective was accomplished successfully and safe, optimizing both intra-operative and hospitalization times, thus,maximizing patient comfort, esthetic and prompt return to normal duties. (AU)

A Selective Fluorescent Probe for the Determination and Imaging of Fluoride in Living Cells

Abstract Fluoride anions are indispensable trace elements required for sustaining life. To investigate the homeostasis and action of fluoride in the body, a new highly sensitive and selective fluorescence detection method was designed for fluoride in aqueous solutions. A fluorescent probe for fluoride (FP-F) was synthesized for imaging F- in living cells. The design strategy for the probe was based on the specific reaction between fluoride and silica to mediate deprotection of this probe to fluorescein. Upon treatment with F-, FP-F, a closed and weakly fluorescent lactone, was transformed into an open and strongly fluorescent product. Under the optimum conditions, the detection limit for fluoride was 0.526 nM. FP-F could detect micromolar changes in F- concentrations in living cells by confocal microscopy.

Comparison of Detection Rate of Root Canal Orifices of Maxillary First Molar Using Various Techniques: An in-vivo Study

ABSTRACT Objective: To compare the detection rate of root canal orifices of maxillary first molar by various techniques in the Indian population. Material and Methods: A total of 50 maxillary 1st molar cases were selected and sequentially divided into four groups: Group I: Naked eye; Group II: Surgical loupe; Group III: Surgical operating microscope; and Group IV: Fluorescein sodium dye. After access opening, the number of root canal orifices was detected in all cases with these methods. Results: By naked eye and surgical loupe, a total of 171 root canal orifices were detected, by a surgical operating microscope, 176, and by fluorescein sodium dye, 177 root canal orifices were detected. The detection rate of root canal orifices is as follows: Group I (96.61%) = Group II (96.61%) < Group III (99.44%) < Group IV (100%) and detection rate of MB-2 canal orifices Group I (40%) = Group II (40%) < Group III (50%) < Group IV (52%). No significant difference in the number of canal orifices detected could be seen for any of the comparisons. No significant difference was observed between the naked eye and surgical loupe techniques. Although the surgical operating microscope detected more root canal orifices, it did not have a significantly higher detection than the other two techniques. Conclusion: No significant difference was seen among various methods. However, the use of a surgical operating microscope and fluorescein sodium dye increased the detection rate of root canal orifices.

Comparison of two techniques for assessing pancreatic islet viability: flow cytometry and fluorescein diacetate/propidium iodine staining

Introduction: The success of islet transplantation for patients with unstable type 1 diabetes mellitus depends, in part, on the number of isolated islets and their quality, which is assessed by functional and viability tests. The test currently employed to evaluate islet viability, used by the Collaborative Islet Transplant Registry to release products for transplantation, is fluorescein diacetate/propidium iodide (FDA/PI) staining. However, the efficacy of this method relies on researcher experience; in this context, a quantitative method may be useful. The aim of this study was to compare islet viability as assessed by flow cytometry and the FDA/PI assay. Methods: Viability was analyzed in islets isolated from 10 male Wistar rats. Upon FDA/PI staining, 50 islets from each animal were analyzed under fluorescence microscopy by two well-trained researchers. For flow cytometry, islets were dispersed and 100 000 single cells were incubated with the 7-amino-actinomycin D (7AAD) fluorophore (dyes necrotic and late apoptotic cells) and the Annexin V-APC antibody (marks early apoptotic cells). Results: A moderate correlation was found between techniques (r = 0.6; p = 0.047). The mean islet viability measured by flow cytometry was higher than that estimated using FDA/PI staining (95.5 ± 1.4% vs 89.5 ± 5.0%; p = 0.002). Conclusions: Although flow cytometry is more expensive and time-consuming than FDA/PI staining, it is a quantitative technique with greater reproducibility that is less subject to inter-observer variability than FDA/PI. Therefore, flow cytometry appears to be the technique of choice when aiming for a more precise determination of islet viability. (AU)

Particulate Matter 2.5 Causes Deficiency in Barrier Integrity in Human Nasal Epithelial Cells

PURPOSE: The effect of air pollution-related particulate matter (PM) on epithelial barrier function and tight junction (TJ) expression in human nasal mucosa has not been studied to date. This study therefore aimed to assess the direct impact of PM with an aerodynamic diameter less than 2.5 μm (PM2.5) on the barrier function and TJ molecular expression of human nasal epithelial cells. METHODS: Air-liquid interface cultures were established with epithelial cells derived from noninflammatory nasal mucosal tissue collected from patients undergoing paranasal sinus surgery. Confluent cultures were exposed to 50 or 100 µg/mL PM2.5 for up to 72 hours, and assessed for 1) epithelial barrier integrity as measured by transepithelial resistance (TER) and permeability of fluorescein isothiocyanate (FITC) 4 kDa; 2) expression of TJs using real-time quantitative polymerase chain reaction and immunofluorescence staining, and 3) proinflammatory cytokines by luminometric bead array or enzyme-linked immunosorbent assay. RESULTS: Compared to control medium, 50 and/or 100 µg/mL PM2.5-treatment 1) significantly decreased TER and increased FITC permeability, which could not be restored by budesonide pretreatment; 2) significantly decreased the expression of claudin-1 messenger RNA, claudin-1, occludin and ZO-1 protein; and 3) significantly increased production of the cytokines interleukin-8, TIMP metallopeptidase inhibitor 1 and thymic stromal lymphopoietin. CONCLUSIONS: Exposure to PM2.5 may lead to loss of barrier function in human nasal epithelium through decreased expression of TJ proteins and increased release of proinflammatory cytokines. These results suggest an important mechanism of susceptibility to rhinitis and rhinosinusitis in highly PM2.5-polluted areas.

Effect of 3% Diquafosol Tetrasodium on Tear Film Stability after Laser-assisted in situ Keratomileusis

PURPOSE: To evaluate the efficacy of 3% diquafosol tetrasodium (DQ) after laser-assisted in-situ keratomileusis (LASIK). METHODS: This prospective randomized study included 150 eyes in 75 patients who were scheduled for LASIK. The patients in the 3% diquafosol tetrasodium (DQ) group (37 patients, 74 eyes) were instructed to apply one drop of DQ, six times daily post-op, while the patients in the 0.3% sodium hyaluronate (HA) group (38 patients, 76 eyes) were instructed to apply one drop of HA, six times daily post-op. A Schirmer test, tear film break-up time (BUT), corneal and conjunctival fluorescein staining score (FLSS), and ocular surface disease index (OSDI) were evaluated pre-op and at 1, 4, and 12 weeks post-op while the tear osmolarity was evaluated pre-op and at 4 and 12 weeks post-op. RESULTS: There was no significant difference between the two groups regarding Schirmer test results or tear osmolarity and conjunctival FLSS. The BUT was significantly higher in the DQ group at 1 week and 12 weeks post-op. The corneal FLSS was significantly lower in the DQ group at 1 week, 4 weeks and 12 weeks post-op. The OSDI was significantly lower in the DQ group at 1 week post-op. CONCLUSIONS: Use of 3% diquafosol tetrasodium after surgery improved ocular dryness and increased the tear film stability.

Paricalcitol attenuates indoxyl sulfate-induced apoptosis through the inhibition of MAPK, Akt, and NF-κB activation in HK-2 cells

BACKGROUND/AIMS: Indoxyl sulfate (IS) is a uremic toxin and an important causative factor in the progression of chronic kidney disease. Recently, paricalcitol (19-nor-1,25-dihydroxyvitamin D2) was shown to exhibit protective effects in kidney injury. Here, we investigated the effects of paricalcitol treatment on IS-induced renal tubular injury. METHODS: The fluorescent dye 2ʹ,7ʹ-dichlorofluorescein diacetate was used to measure intracellular reactive oxygen species (ROS) following IS administration in human renal proximal tubular epithelial (HK-2) cells. The effects of IS on cell viability were determined using MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assays and levels of apoptosis-related proteins (Bcl-2-associated protein X [Bax] and B-cell lymphoma 2 [Bcl-2]), nuclear factor-κB (NF-κB) p65, and phosphorylation of mitogen-activated protein kinase (MAPK) and protein kinase B (Akt) were determined by semiquantitative immunoblotting. The promoter activity of NF-κB was measured by luciferase assays and apoptosis was determined by f low cytometry of cells stained with f luorescein isothiocyanate-conjugated Annexin V protein. RESULTS: IS treatment increased ROS production, decreased cell viability and induced apoptosis in HK-2 cells. IS treatment increased the expression of apoptosis-related protein Bax, decreased Bcl-2 expression, and activated phosphorylation of MAPK, NF-κB p65, and Akt. In contrast, paricalcitol treatment decreased Bax expression, increased Bcl-2 expression, and inhibited phosphorylation of MAPK, NF-κB p65, and Akt in HK-2 cells. NF-κB promoter activity was increased following IS, administration and was counteracted by pretreatment with paricalcitol. Additionally, flow cytometry analysis revealed that IS-induced apoptosis was attenuated by paricalcitol treatment, which resulted in decreased numbers of fluorescein isothiocyanate-conjugated Annexin V positive cells. CONCLUSIONS: Treatment with paricalcitol inhibited IS-induced apoptosis by regulating MAPK, NF-κB, and Akt signaling pathway in HK-2 cells.

Contact Lens Induced Limbal Stem Cell Deficiency: Clinical Features in Korean Patients

PURPOSE: To describe the clinical features of Korean patients with contact lens-induced limbal stem cell deficiency (CL-LSCD).METHODS: Medical records of 22 patients who were diagnosed with CL-LSCD between 2014 and 2019 were reviewed retrospectively. Outcome measures included demographics, clinical presentation, treatment, clinical course, and pattern of contact lens (CL) wear.RESULTS: Forty-two eyes of 22 patients were found to have typical changes associated with CL-LSCD. Twenty (91%) patients were women and mean age was 36 ± 12 years. All patients had myopia with mean spherical equivalent of −7.52 ± 3.2 diopter. Twenty (91%) patients had bilateral disease and the location of limbal involvement was diffuse in 20 eyes (47.6%) and partial in 22 eyes (52.4%, superior in 20 eyes and inferior in 2 eyes). Fourteen (63.6%) patients complained of decreased visual acuity. Average period of CL wear was 14 ± 9 years. Four patients used cosmetic colored CLs and four patients had a history of overnight CL wear. All 12 patients who completed follow-up (28 ± 42 weeks) showed improvement in visual acuity and ocular surface condition after cessation of CL wear and medical treatment. Of them, five (42%) patients showed full recovery while seven (58%) showed partial recovery.CONCLUSIONS: If a patient with a history of CL wear for an extended period of time presents with decreased visual acuity, practitioners should perform detailed examinations with suspicion of CL-LSCD, including fluorescein staining. CL-LSCD is usually reversible and close follow-up with conservative treatment is recommended as the initial treatment option.

A Removed Chestnut Thorn after Corneal Incision

PURPOSE: To report the surgical technique to remove a chestnut thorn through a corneal incision. CASE SUMMARY: A 54-year-old female visited our clinic complaining of a sudden foreign body sensation and conjunctival injection in her left eye after picking chestnuts 4 days prior to her visit. Visual acuity of both eyes was 1.0 and the intraocular pressures were within normal limits. Slit lamp examination revealed that a chestnut thorn had deeply penetrated the left corneal stroma and a small number of inflammatory cells were observed in the anterior chamber. There was no corneal defect stained with fluorescein and the Seidel test was negative. A corneal foreign body comprised of a chestnut thorn and its remnants was diagnosed and emergency surgery was performed. A partial corneal incision was made along the foreign body and the exposed foreign body was easily and completely removed. The patient was treated with topical antibiotics after surgery and no complication was observed during a follow-up period of 3 months. CONCLUSIONS: In the case of a corneal foreign body comprised of a chestnut thorn, the foreign body with its remnants were easily removed by performing a partial corneal incision.

Development of a Pancreatic Cancer Specific Binding Peptide Using Phage Display / 대한소화기병학회잡지

BACKGROUND/AIMS: Pancreatic cancer has a very poor prognosis, and early diagnosis is a way to increase the survival rate of patients. The purpose of this study was to develop pancreatic cancer-specific peptides for imaging studies. METHODS: Three pancreatic cancer cell lines, MIA PaCa-2, UACC-462, and BxPC-3, and a control cell line, CCD841, were used. Biopannings were performed on MIA PaCa-2 using a phage display library. After this, the peptides were synthesized and labeled with fluorescein isothiocyanate (FITC). Immunocytochemistry (ICC), enzyme-linked immunosorbent assay (ELISA), and fluorescence-activated cell sorter (FACS) were performed to examine the specific binding. To examine its therapeutic applications, a photosensitizer, chlorin e6 (Ce6), was conjugated on the peptide and photodynamic therapy was performed. Cell survival was investigated using a [3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide] assay. RESULTS: After three biopannings, the phages were amplified from 1.4×104 to 3.2×105 plaque-forming units. The most strongly binding phage was selected from the ELISA and ICC results. FITC-labeled peptide, M5, in the three pancreatic cancer cell lines showed significantly higher immunofluorescence in the ICC experiments than that of CCD841. The higher binding ability to MIA PaCa-2 cells was confirmed from FACS analysis, which showed a right shift compared to CCD841. M5 bound to Ce6 showed a significantly lower cell survival rate than that of Ce6 alone in photodynamic therapy, which was observed consistently as a change in the tumor size and fluorescence intensity in MIA PaCa-2 cell-implanted animal models. CONCLUSIONS: This study showed that the noble peptide, M5, binds specifically to the pancreatic cancer cell line, MIA PaCa-2. The M5 peptide has potential use in future optical diagnostic and therapeutic purposes.

Development of a Pancreatic Cancer Specific Binding Peptide Using Phage Display / 대한소화기학회지

BACKGROUND/AIMS: Pancreatic cancer has a very poor prognosis, and early diagnosis is a way to increase the survival rate of patients. The purpose of this study was to develop pancreatic cancer-specific peptides for imaging studies.METHODS: Three pancreatic cancer cell lines, MIA PaCa-2, UACC-462, and BxPC-3, and a control cell line, CCD841, were used. Biopannings were performed on MIA PaCa-2 using a phage display library. After this, the peptides were synthesized and labeled with fluorescein isothiocyanate (FITC). Immunocytochemistry (ICC), enzyme-linked immunosorbent assay (ELISA), and fluorescence-activated cell sorter (FACS) were performed to examine the specific binding. To examine its therapeutic applications, a photosensitizer, chlorin e6 (Ce6), was conjugated on the peptide and photodynamic therapy was performed. Cell survival was investigated using a [3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide] assay.RESULTS: After three biopannings, the phages were amplified from 1.4×104 to 3.2×105 plaque-forming units. The most strongly binding phage was selected from the ELISA and ICC results. FITC-labeled peptide, M5, in the three pancreatic cancer cell lines showed significantly higher immunofluorescence in the ICC experiments than that of CCD841. The higher binding ability to MIA PaCa-2 cells was confirmed from FACS analysis, which showed a right shift compared to CCD841. M5 bound to Ce6 showed a significantly lower cell survival rate than that of Ce6 alone in photodynamic therapy, which was observed consistently as a change in the tumor size and fluorescence intensity in MIA PaCa-2 cell-implanted animal models.CONCLUSIONS: This study showed that the noble peptide, M5, binds specifically to the pancreatic cancer cell line, MIA PaCa-2. The M5 peptide has potential use in future optical diagnostic and therapeutic purposes.

Clinical features and corneal optical anisotropies in a rabbit model of limbal stem cell deficiency / Fenótipos clínicos e anisotropias ópticas da córnea em coelhos com deficiência de células tronco limbais

ABSTRACT Purposes: To investigate the intra-laboratory reproducibility of clinical features and to evaluate corneal optical anisotropies in a rabbit model of limbal stem cell deficiency. Methods: Limbal injury was induced in the right eye of 23 adult New Zealand White rabbits using a highly aggressive protocol that combined 360 degrees limbal peritomy, keratolimbectomy, alkaline chemical burn, and mechanical removal of the epithelium. Clinical evaluation of the injured eyes was performed for 28 days and included corneal impression cytology. Corneas with a severe clinical outcome set typical of limbal stem cell deficiency were then collected, subjected to a histopathological examination, and examined for optical anisotropies. Corneas from healthy rabbit eyes were used as controls. Differences in optical path due to stromal collagen birefringence, as well as linear dichroism related to the expression and spatial orientation of glycosaminoglycan chains from proteoglycans, were measured from cross-sections under a quantitative polarized light microscope. Results: One eye showed signs of hypopyon and was excluded. Signs of ocular inflammation were observed in all eyes studied (n=22). Corneal impression cytology did not detect goblet cells. Twelve of the 22 corneas presented a clinical outcome set typical of limbal stem cell deficiency, which is characterized by the presence of epithelial defects, inflammatory cells, moderate-to-severe opacity, and neovascularization. Microscopic studies under polarized light revealed that relative to controls, limbal stem cell deficiency caused a 24.4% increase in corneal optical path differences. Further, corneas with limbal stem cell deficiency were less dichroic than controls. Conclusions: These results suggest that rabbit models of limbal stem cell deficiency must be rigorously screened for use in preclinical studies to ensure experimental homogeneity because protocols used to create limbal stem cell deficiency could be not associated with good intra-laboratory reproducibility of clinical features. Limbal stem cell deficiency, as induced herein, altered the optical anisotropic properties of the corneal stroma. Such alterations are indicative of changes in collagen packing and the spatial orientation of glycosaminoglycan chains from proteoglycans. Knowledge of these changes is important to potentiate strategies aimed at restoring the morphofunctional integrity of the corneal stroma affected by limbal stem cell deficiency.

RESUMO Objetivos: Investigar a reprodutibilidade intra-la­boratorial dos fenótipos clínicos e avaliar anisotropias ópticas em córneas de coelhos com deficiência de células tronco limbais. Métodos: Lesões ao limbo foram feitas no olho direito de 23 coelhos adultos da Nova Zelândia Branco, usando um protocolo altamente agressivo, que envolveu peritomia limbal em 360 graus, ceratolimbectomia, cauterização por álcali, e remoção mecânica de epitélio remanescente. Os olhos foram clinicamente avaliados por 28 dias, inclusive por citologia de impressão corneal. As córneas que manifestaram um conjunto de alterações típicas de deficiência de células tronco limbais foram coletadas e submetidas à estudos em histopatologia e em anisotropias ópticas. Córneas saudáveis foram usadas como controles. Diferenças de caminho óptico de birrefringência relacionada à organização do colágeno estromal, e dicroísmo linear relacionado à expressão e à orientação das cadeias de glicosaminoglicanos dos proteoglicanos estromais, foram quantificados por microscopia de luz polarizada. Resultados: Um olho apresentou hipópio e foi excluído do estudo. Todos os olhos estudados (n=22) apresentaram sinais de inflamação ocular. A citologia de impressão não detectou células caliciformes na superfície corneal. Doze de 22 córneas manifestaram alterações clínicas típicas de deficiência de células tronco limbais, caracterizado por defeitos epiteliais, infiltrados inflamatórios, opacidade de moderada à severa, e neovascularização. Estudos por microscopia de luz polarizada mostraram que a deficiência de células tronco limbais aumentou a diferenças de caminho óptico corneal em 24,4% (versus controles). As cór­neas com deficiência de células tronco limbais foram menos dicroicas do que as córneas controle. Conclusões: Coelhos com deficiência de células tronco limbais, para aplicações em estudos pré-clínicos, devem ser rigorosamente selecionados para assegurar homogeneidade experimental, pois há evidências de que protocolos utilizados para indução de deficiência de células tronco limbais não estão associados com boa reprodutibilidade intra-laboratorial de fenótipos clínicos. A deficiência de células tronco limbais, como induzida aqui, alterou as propriedades ópticas anisotrópicas do estroma corneal. Tais alterações são indicativas de mudanças no empacotamento de colágeno e na orientação das cadeias de glicosaminoglicanos dos proteoglicanos. Conhecimentos nessas alterações são importantes para potencializar estratégias que visam a restabelecer a integridade morfofuncional do estromal corneal acometido pela deficiência de células tronco limbais.

Análise da microbiota em tonômetros de aplanação de Goldmann / Analysis of the microbiota in Goldmann applanation tonometers

RESUMO Objetivos: Analisar a prevalência da microbiota nos tonômetros de aplanação de Goldmann nos consultórios do SUS e definir o grau de contaminação dos tonômetros e a eficácia da assepsia do cone do tonômetro de aplanação. Métodos: Estudo transversal em que foi realizado a coleta de 60 "swabs", divididos nos três tonômetros de aplanação dos ambulatórios do SUS em dois momentos distintos. No primeiro realizou-se a coleta no início dos atendimentos e no segundo momento, a coleta foi realizada ao final de todos os atendimentos. Todos "swabs" foram colhidos no meio Stuart e foi realizada a cultura em meio de bactérias. Resultados: Das 60 amostras, apenas uma apresentou crescimento de agente patogênico, a Escherichia coli. Conclusão: Independente dos vários métodos que o oftalmologista escolher para realizar a assepsia, a mesma é imprescindível para a manutenção de uma boa saúde ocular do paciente, evitando assim a transmissão e propagação de patógenos por meio do exame oftalmológico e concluímos também que o método utilizado pelo nosso serviço parece ser eficaz nesta profilaxia.

ABSTRACT Objective: Analyze the microbiota prevalence in the Goldmann applanation tonometers in the clinic of the SUS to define the contamination of the tonometers and the efficacy of asepsis of the applanation tonometer cone. Methods: A cross-sectional study was carried out to collect 60 "swabs" divided into the three aplanation tonometers of SUS clinics at two different times. In the first one, the collection will be performed at the beginning of the visits and at the second moment, the collection will be performed at the end of all the visits. All swabs will be harvested in the Stuart medium and culture was carried to sow bacteria. Results: Of the 60 samples, only one showed pathogen growth, Escherichia coli. Conclusion: Regardless of the various ways the ophthalmologist chooses to perform asepsis, it is essential for the maintenance of good patient eye health, thus avoiding the transmission and propagation of pathogens through ophthalmologic examination, and we also conclude that the method used by our patient seems to be effective in this prophylaxis.

Diagnosis and treatment of dry eye syndrome

Dry eye syndrome is a multifactorial disease of the ocular surface characterized by a loss of homeostasis of the tear film, and accompanied by ocular symptoms. Tear film instability and hyperosmolarity, ocular surface inflammation and damage, and neurosensory abnormalities play etiological roles. Symptom screening with the Five-item Dry Eye Questionnaire or Ocular Surface Disease Index can be used to identify the possibility that a patient might have DED dry eye syndrome and triggers. The results of these screening tools can lead to performing diagnostic tests of (ideally non-invasive) breakup time, osmolarity and ocular surface staining with fluorescein and lissamine green (observing the cornea, conjunctiva and eyelid margin). Meibomian gland dysfunction, lipid thickness/dynamics and assessments of the volume and severity of tear allow the subclassification of dry eye syndrome as predominantly evaporative or aqueous deficient, which informs the management of dry eye syndrome. A staged management algorithm was derived that presents a stepwise approach to implementing the various management and therapeutic options according to disease severity.

Novel Cell Permeable Peptide Based on Multi Basic Furin-dependent Cleavage Site of Respiratory Syncytial Virus Fusion Protein

Cell permeable peptide (CPP) is able to transport itself or conjugated molecules such as nucleotides, peptides, and proteins into cells. Since short peptide of human immunodeficiency virus-1 Tat has been discovered as CPP, it has been continuously studied for their ability to transport heterologous cargoes into cells. In this study, we have focused on the fusion protein of respiratory syncytial virus (RSV), which has six basic amino acids in multi basic furin-dependent cleavage site (MBFCS) required to be cationic CPP. To develop more efficient CPP, the sequence, which linked two MBFCS, was synthesized (called RS-CPP). To assess cell permeable efficiency of RS-CPP or MBFCS, the peptides was conjugated with fluorescein isothiocyanate, and cell permeable efficiency was measured by fluorescence-activated cell sorting. Cell permeability of RS-CPP or MBFCS was increased in a dose-dependent manner, but RS-CPP showed more efficient cell permeability than MBFCS in MDCK, HeLa, Vero E6, and A549 cells. To evaluate whether RS-CPP can transport its conjugated functional peptide (VIVIT) in CD8+ T cell, it was confirmed that IL-2 and β-galactosidase expression were significantly inhibited through selective block of nuclear factor activated T-cell. To investigate endocytic pathways, Cre-mediated DNA recombination (loxP-STOP-loxP-LacZ reporter system) was investigated with divergent endocytosis inhibitors in TE671 cells, and RS-CPP endocytosis is occurred via binding cell surface glycosaminoglycan and clathrin-mediated endocytosis, or macropinocytosis. These results indicated that RS-CPP could be a novel cationic CPP, and it would help understanding for delivery of biologically functional molecules based on viral basic amino acids.

Fluorescein Angiographic Abnormalities in the Contralateral Eye with Normal Fundus in Children with Unilateral Coats' Disease

PURPOSE: We report the detection of peripheral retinal vascular abnormalities in the fellow eye with normal fundus in children with unilateral Coats' disease. METHODS: The clinical records of patients diagnosed with Coats' disease were retrospectively reviewed. We recorded the subjects' characteristics and obtained fundus photography and fluorescein angiography (FA) images. The main outcome measure was peripheral vascular abnormalities in the contralateral eye with normal fundus in children with unilateral Coats' disease, observed with FA. RESULTS: Out of 47 patients with Coats' disease, two (4.3%) were diagnosed with clinically bilateral Coats' disease. Of the 45 patients with presumed unilateral Coats' disease, four (8.9%) had bilateral abnormal peripheral vasculature in FA. The mean age of these four patients was 6.4 ± 5.4 years (range, 1 to 14 years), and three patients were male (75%). All four had peripheral retinal nonperfusion, and two (50%) received laser photocoagulation due to peripheral leakage with telangiectatic vessels. CONCLUSIONS: Coats' disease may more often be a bilateral disease with asymmetry than previously thought. Patients with Coats' disease should undergo careful examination of the fellow eye with FA in order to detect and treat vascular abnormalities that are not visible clinically.

The Clinical Efficacy of Silicone Stents for Endoscopic Dacryocystorhinostomy: A Meta-Analysis

We evaluated the effect of silicone stent use during endoscopic dacryocystorhinostomy on postoperative morbidities in comparison with versus without a silicone stent. Two authors independently searched six databases (PubMed, Embase, Scopus, the Web of Science, the Cochrane library, and Google Scholar) from inception of article collection to July 2017. The analysis included prospective randomized studies that compared intraoperative silicone stent insertion (silicone group) with no application of a silicone stent (control group), in which the outcomes of interest were success rate (lacrimal passage patent check with syringing, symptom relief, or endoscopic confirmation of fluorescein dye from the opening of Hasner's valve) and morbidities (e.g., postoperative bleeding, rhinostomy closure, granulation tissue, synechia, and eyelid problems) after certain follow-up periods (over 10 weeks). Nine studies involving a total of 587 participants were included. Functional success rates tended to be higher in the silicone group than in the control, but there was no statistically significant difference in success rates (odds ratio, 1.45; 95% confidence interval, 0.77 to 2.73). According to the surgical type such as mucosal removal and mucosal flap surgery, the results from types didn't demonstrate any significant effect, but the mucosal flap technique seemed to be more beneficial. Regarding postoperative morbidities, although the outcomes of the groups did not present any statistically significant difference, eyelid problems and postoperative bleeding tended to occur more frequently in the silicone group, but rhinostomy closure tended to occur more frequently in the control group. Success and morbidity rates showed no difference between the silicone stent group and control group in the meta-analysis. However, additional analyses revealed that the success rate of endonasal dacryocystorhinostomy using silicone intubation with mucosal flap has shown an improving trend, and morbidities such as granulation and synechia showed decreasing trends compared with the group without silicone intubation.

Fluorescent test accurately predicts leak of ischemic colon anastomosis in rats

Abstract Purpose: To investigate if fluorescein fluorescent test can predict dehiscence in a model of ischemic colonic anastomosis in rats. Methods: This experimental controlled trial randomly assigned 55 rats to four groups. Anastomoses were performed in non-ischemic colon segments (control group) and in ischemic colon segments measuring 1, 2 or 3 cm long (groups 1, 2 and 3, respectively). Fluorescein was injected and the tissues were examined under ultraviolet light. Seven days later, a second-look surgery was performed to check for the presence or absence of anastomosis dehiscence. Results: Twenty-four rats presented anastomotic dehiscence during the second-look surgery. Reticular and nonfluorescent patterns were significantly associated with the occurrence of anastomotic dehiscence. Fluorescein fluorescence had a sensitivity of 95.8%, specificity of 89.2%, positive predictive value of 88.4%, negative predictive value of 96.2%, and accuracy of 92.3% to predict anastomotic dehiscence. Conclusion: Fluorescein fluorescent test can accurately predict leak in a model of ischemic colonic anastomosis in rats.