Microemulsions Improve Topical Protoporphyrin IX (PpIX) Delivery for Photodynamic Therapy of Skin Cancer

Abstract We report here microemulsions (MEs) for topical delivery of protoporphyrin IX (PpIX) for Photodynamic Therapy (PDT) of skin cancers. Selected MEs consisting of Oil/Water (O/W) bicontinuous (BC) and Water/Oil (W/O) preparations were characterized as to pH, nanometric size, zeta potential, drug content, and viscosity. Sustained in vitro PpIX release was achieved from MEs 2A (O/W), 10B (BC) and 16B (W/O) through an artificial membrane for up to 24 h, characterizing MEs as drug delivery systems. None of these MEs showed permeation through the skin, demonstrating the required topical effect. After 4 h, in vitro retention of PpIX in the stratum corneum (SC) was higher from both ME 10B and control (PpIX at 60 µg/mL in PEG 300). However, in the Epidermis + Dermis ([Ep + D]), retention from ME 10B and ME 16B was ~40 times higher compared to control. Confocal Laser Scanning Microscopy (CLSM) showed higher fluorescence intensity in the SC for both control and ME 10B, whereas ME 10B fluorescence was higher in [Ep+D]. The results indicate that ME 10B is suitable for PpIX encapsulation, showing good characteristics and a localized effect for a potential delivery system for PDT-associated treatments of skin cancers.

Displasia epitelial corneal / Corneal epithelial dysplasia

Las neoplasias intraepiteliales córneo-conjuntival son lesiones premalignas, mal delimitadas, de crecimiento lento y escaso potencial de malignización. Solo el 9 por ciento progresa a carcinoma invasor de células escamosas. Posee varias formas de presentación y tiene múltiples dilataciones vasculares. La displasia epitelial corneal primaria es una forma poco frecuente de neoplasia intraepitelial córnea-epitelial. Se presenta un caso clínico confirmado por estudios anatomopatológicos. En el presente estudio se observó respuesta satisfactoria luego de un mes de tratamiento tópico con 5FU, sin efectos colaterales. El seguimiento durante tres años no ha mostrado signos de recidiva(AU)

Corneal-conjunctival intraepithelial neoplasms are premalignant, poorly demarcated, slow-growing lesions with low malignant potential. Only the 9 percent progresses to invasive squamous cell carcinoma. It appears in several forms and presents multiple vascular dilatations. Primary corneal epithelial dysplasia is a rare form of corneal-epithelial intraepithelial neoplasia. We present a clinical case, confirmed by anatomopathologic studies. In the present study we observed a satisfactory response after one month of topical treatment with 5FU, with no side effects. Follow-up for three years has shown no signs of recurrence(AU)

Cuerpos extraños intracorneales retenidos / Retained intracorneal foreign bodies

Los cuerpos extraños constituyen la segunda forma más común de traumatismos corneales. Pueden localizarse superficiales o intracorneales. Se realiza presentación de caso de paciente con cuerpos extraños intracorneales retenidos de 34 años de evolución secundario a trauma por onda expansiva, con el objetivo de mostrar los hallazgos clínicos y resultados por pruebas diagnósticas como la microscopía confocal corneal, frente de onda corneal por topografía y microscopía especular. La presencia de cuerpos extraños intracorneales en este paciente provocó respuestas en el tejido corneal en dependencia de su composición, nivel de profundidad; evidentes a pesar de tiempo de retención. La presencia de estos induce niveles altos de aberraciones corneales que dependen del grado de afectación(AU)

Foreign bodies are the second most common form of corneal trauma. They can be superficial or intracorneal. A case of a patient was presented, with retained intracorneal foreign bodies of 34 years of secondary evolution due to blast wave trauma. The objective is to show the clinical findings and results of diagnostic tests such as corneal confocal microscopy, corneal wavefront topography and specular microscopy. The presence of intracorneal foreign bodies in this patient provoked responses in the corneal tissue depending on its composition, depth level; evident in spite of retention time. The presence of intracorneal foreign bodies induces high levels of corneal aberrations depending on the degree of involvement(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.

Endotelitis por citomegalovirus y su diagnóstico mediante la microscopia confocal / Cytomegalovirus endotheliitis and its diagnosis by confocal microscopy

Objetivo: Describir las características del endotelio corneal por microscopia confocal en pacientes con lesiones basofílicas de inclusión como patognomónicas de endotelitis por citomegalovirus. Métodos: Se realizó un estudio observacional de casos clínicos en pacientes con diagnóstico de endotelitis por citomegalovirus, atendidos en la Consulta de Córnea del Instituto Cubano de Oftalmología "Ramón Pando Ferrer", entre febrero del año 2010 y junio del 2018. La muestra incluyó 39 pacientes con diagnóstico clínico de endotelitis, a quienes se les realizó microscopia confocal con el equipo Confoscan 4 (Nidek Technologies). Resultados: De los 39 pacientes, 29 fueron del sexo masculino y 10 del femenino. Todos tenían entre 41 y 60 años de edad. En el 97,4 por ciento de los casos existió el antecedente de una conjuntivitis viral y un solo paciente fue positivo de HIV (2,6 por ciento). La mejor agudeza visual corregida de 0,3 o menos se mostró en el 100 por ciento de ellos antes del tratamiento, y después de este los 39 tenían entre 0,8 y 1,0. En el 100 por ciento de los casos se observaron cuerpos de inclusión basofílicos en el endotelio corneal, que fueron apreciables mediante la microscopia confocal. Conclusiones: Existe una relación entre la presencia de cuerpos de inclusión basofílicos en el endotelio corneal y las pruebas virológicas a citomegalovirus positivas, lo que puede permitir hacer PCR a casos que ya tienen confirmación mediante microscopia confocal de las características patognomónicas del endotelio corneal(AU)

Objective: Describe the characteristics of the corneal endothelium by confocal microscopy in patients with inclusion basophilic lesions as pathognomonic signs of cytomegalovirus endotheliitis. Methods: An observational study was conducted of clinical cases of patients diagnosed with cytomegalovirus endotheliitis attending the Cornea Service at Ramón Pando Ferrer Cuban Institute of Ophthalmology from February 2010 to June 2018. The study sample was 39 patients with a clinical diagnosis of endotheliitis who underwent confocal microscopy with a Confoscan 4 device (Nidek Technologies). Results: Of the 39 patients examined, 29 were male and 10 were female. All were aged 41-60 years. 97.4 percent had a history of viral conjunctivitis and only one was HIV positive (2.6 percent). Best corrected visual acuity was 0.3 or less in 100 percent before treatment, and 0.8 to 1.0 after treatment. Inclusion basophilic bodies visible by confocal microscopy were observed in the corneal endothelium of all patients. Conclusions: A relationship exists between the presence of inclusion basophilic bodies in the corneal endothelium and virological tests positive for cytomegalovirus, making it possible to perform PCR testing in cases with confocal microscopy confirmation of the pathognomonic characteristics of the corneal endothelium(AU)

Imagenología diferencial por microscopia confocal in vivo entre la epiteliopatía por acanthamoeba y la queratitis por herpes simple / In vivo confocal microscopy imaging for the differential diagnosis of Acanthamoeba epitheliopathy and herpes simplex keratitis

La queratitis por Acanthamoeba es una infección corneal de baja incidencia, condicionada por varios factores, pero con manifestación clínica variada y sintomatología típica. En su fase epitelial debe ser diferenciada de otras queratitis, específicamente de la queratitis por herpes simple, por la similitud entre ambas en cuanto a las características de la lesión corneal. La microscopia confocal in vivo constituye una alternativa diagnóstica. Es una biopsia fotográfica en cuyas imágenes podemos describir los quistes y trofozoítos de Acanthamoeba desde etapas iniciales, que nos ayudan a diferenciarla de otros tipos de queratitis e iniciar el tratamiento precoz. Se realizó una búsqueda de artículos publicados, con el objetivo de mostrar las imágenes por microscopia confocal de la fase epitelial de la infección corneal por Acanthamoeba y herpes simple. Se utilizó la plataforma Infomed, específicamente la Biblioteca Virtual de Salud(AU)

Acanthamoeba keratitis is a low-incidence corneal infection caused by several factors and characterized by a variety of clinical manifestations and typical symptoms. In its epithelial phase, it should be differentiated from other keratitis, particularly from herpes simplex keratitis, due to the similar characteristics of the corneal lesion. In vivo confocal microscopy is a diagnostic alternative consisting in a photographic biopsy showing images of Acanthamoeba cysts and trophozoites since their initial stages, thus allowing differentiation from other types of keratitis and the initiation of early treatment. A search was conducted of published papers with the purpose of showing confocal microscopy images of the epithelial phase of Acanthamoeba and herpes simplex corneal infection. Use was made of the platform Infomed, specifically the Virtual Health Library(AU)

Evidence of autophagic vesicles in a patient with Lisch corneal dystrophy / Evidência de vesículas autofágicas em paciente com distrofia corneana de Lisch

ABSTRACT Lisch corneal dystrophy is a rare corneal disease characterized by the distinctive feature of highly vacuolated cells. Although this feature is important, the nature of these vacuoles within corneal cells remains unknown. Here, we sought to analyze corneal cells from a patient diagnosed with Lisch dystrophy to characterize the vacuoles within these cells. Analyses using histopathology examination, confocal microscopy, and transmission electron microscopy were all consistent with previous descriptions of Lisch cells. Importantly, the vacuoles within these cells appeared to be autophagosomes and autolysosomes, and could be stained with an anti-microtubule-associated protein 1A/1B-light chain 3 (LC3) antibody. Taken together, these findings indicate that the vacuoles we observed within superficial corneal cells of a patient with Lisch corneal dystrophy constituted autophagosomes and autolysosomes; this finding has not been previously reported and suggests a need for further analyses to define the role of autophagy in this ocular disease.

RESUMO A distrofia corneana de Lisch é uma doença rara, caracterizada principalmente pela presença de células altamente vacuoladas. Embora esta característica seja importante, a natureza desses vacúolos dentro das células da córnea permanece des conhecida. Aqui, procuramos analisar as células da córnea de um paciente diagnosticado com distrofia de Lisch para caracte rizar os vacúolos dentro dessas células. Análises utilizando exame histopatológico, microscopia confocal e microscopia eletrônica de transmissão foram todas consistentes com descrições previas de células de Lisch. Importante, os vacúolos dentro dessas células pareciam ser autofagossomos e autolisossomos, e po deriam ser corados com um anticorpo proteico 1A/1B-cadeia leve 3 (LC3) da proteína anti-microtúbulo associado a microtúbulos. Em conjunto, esses achados indicam que os vacúolos observados nas células superficiais da córnea de um paciente com distrofia corneana de Lisch constituíram autofagossomos e autolisossomos. Esse achado não foi relatado anteriormente e sugere a necessidade de mais análises para definir o papel da autofagia nessa doença ocular.

Microscopia confocal de la córnea en pacientes diabéticos / Confocal microscopy of the cornea in diabetic patients

RESUMEN Objetivo: Identificar las características morfológicas del epitelio, el estroma y el endotelio corneal, así como la densidad celular de este último mediante el empleo de la microscopia confocal de la córnea en pacientes diabéticos. Métodos: Se realizó un estudio descriptivo, comparativo, en 90 ojos; 60 de ellos pertenecientes a pacientes diabéticos (30 tipo 1 y 30 tipo 2) y 30 ojos a pacientes supuestamente sanos. El estudio se realizó en el Instituto Cubano de Oftalmología "Ramón Pando Ferrer" entre enero del año 2012 y enero de 2017. Resultados: Predominó el sexo masculino con 66,7 por ciento en los pacientes con diabetes mellitus tipo 1; el sexo femenino en los pacientes con diabetes mellitus tipo 2 (60 por ciento) y aparentemente sanos (56,7 por ciento). En los pacientes con diabetes mellitus tipo 1 fueron más frecuentes las edades entre 45 y 54 años (33,3 por ciento) y entre 55 y 66 años en los pacientes con diabetes mellitus tipo 2 y aparentemente sanos con 60 y 40 por ciento respectivamente. La morfología del epitelio y el estroma corneal fue normal en el 86,7 y 87,3 por ciento respectivamente. Predominaron las alteraciones de la morfología endotelial en pacientes diabéticos tipo 1 (73,3 por ciento), así como el polimegatismo y el pleomorfismo (73,3 y 56,7 por ciento respectivamente) y la densidad celular más baja (2 222,76 células /mm2). Conclusiones: La ausencia de alteraciones morfológicas del epitelio y el estroma corneal y la presencia de polimegatismo y de pleomorfismo fueron los hallazgos más frecuentes(AU)

ABSTRACT Objective: Identify the morphological characteristics of the corneal epithelium, stroma and endothelium, as well as the cell density of the endothelium by means of confocal microscopy of the cornea in diabetic patients. Methods: A descriptive comparative study was conducted of 90 eyes: 60 from diabetic patients (30 type 1 and 30 type 2) and 30 from supposedly healthy patients, at Ramón Pando Ferrer Cuban Institute of Ophthalmology from January 2012 to January 2017. Results: A predominance was found of the male sex (66.7 percent) among patients with diabetes mellitus type 1 and of the female sex among patients with diabetes mellitus type 2 (60 percent) and seemingly healthy patients (56.7 percent). The most common age ranges were 45-54 years for patients with diabetes mellitus type 1 (33.3 percent) and 55-66 years for patients with diabetes mellitus type 2 (60 percent) and seemingly healthy patients (40 percent). Morphology of the corneal epithelium and stroma was normal in 86.7 percent and 87.3 percent, respectively. In type 1 diabetic patients there was a predominance of endothelial morphological alterations (73.3 percent), polymegethism and pleomorphism (73.3 percent and 56.7 percent, respectively) and the lowest cell density (2 222.76 cells /mm2). Conclusions: Absence of morphological alterations of the corneal epithelium and stroma, as well as the presence of polymegethism and pleomorphism were the most common findings(AU)

Influence of alumina air-abrasion for highly translucent partially stabilized zirconia on flexural strength, surface properties, and bond strength of resin cement

Abstract Objective This study aims to evaluate the influence of different air-abrasion pressures and subsequent heat treatment on the flexural strength, surface roughness, and crystallographic phases of highly translucent partially stabilized zirconia (Y-PSZ), and on the tensile bond strength of resin cement to Y-PSZ. Methodology Fully sintered zirconia specimens were ground with SiC paper (control) and/or air-abraded with 50 µm particles of alumina at 0.1, 0.15, 0.2, or 0.3 MPa or left as-sintered. After air-abrasion at 0.2 MPa (0.2AB), additional specimens were then heated to 1500°C, and held for one hour at this temperature (0.2AB+HT1h). Flexural strength and surface roughness were evaluated. Crystalline phase identification was also carried out using X-ray diffraction. Bonded zirconia specimens with self-adhesive resin cement were stored in distilled water at 37°C for 24 h, either with or without aging (thermal cycling 4-60°C/20000). Results were analyzed statistically by ANOVA and Tukey-Kramer tests. Results The flexural strength decreased with the increase in air-abrasion pressure, while in contrast, the surface roughness increased. The lowest flexural strength and the highest roughness value were found for the 0.2AB and 0.3AB groups, respectively. All groups contained cubic-, tetragonal ( t )-, and rhombohedral ( r )-ZrO2 phases with the exception of the as-sintered group. Upon increasing the air-abrasion pressure, the relative amount of the r -ZrO2 phase increased, with a significant amount of r -ZrO2 phase being detected for the 0.2AB and 0.3AB groups. The 0.2AB+HT1h group exhibited a similar flexural strength and t -ZrO2 phase content as the as-sintered group. However, the 0.2AB group showed a significantly higher tensile bond strength (p<0.05) than the 0.2AB+HT1h group before and after aging. Conclusion Micromechanical retention by alumina air-abrasion at 0.2 MPa, in combination with chemical bonding of a resin to highly translucent Y-PSZ using a MDP-containing resin cement may enable durable bonding.

Caracterización de la membrana basal, los plexos nerviosos y el grosor corneal mediante microscopia confocal en pacientes diabéticos / Characterization of basal membrane, nervous plexus and corneal thickness by confocal microscopy in diabetic patients

RESUMEN Objetivo: Identificar las características morfológicas y morfométricas de las capas de la córnea mediante microscopia confocal en pacientes diabéticos. Métodos: Se realizó un estudio descriptivo, comparativo, en 90 ojos, 60 de ellos pertenecientes a pacientes diabéticos (30 tipo 1 y 30 tipo 2) y 30 a pacientes supuestamente sanos. El estudio se realizó en el Instituto Cubano de Oftalmología "Ramón Pando Ferrer" entre enero del año 2012 y enero de 2017. Resultados: El espesor del epitelio, el estroma y el endotelio corneal fue mayor en los ojos de diabéticos tipo 1 con una media de 54,6; 506,7 y 26,7 micras respectivamente. La membrana basal epitelial se observó en el 20 por ciento de los ojos de pacientes con diabetes mellitus tipo 1, y en el 10 por ciento de los diabéticos tipo 2. El grupo de diabéticos tipo 1 mostró uno y dos plexos nerviosos por campo de microscopia confocal para el 33,3 por ciento cada uno. En los diabéticos tipo 2, predominó un plexo nervioso (40 por ciento) y en pacientes sanos predominaron 4 plexos nerviosos (66,7 por ciento). Ambos grupos de pacientes diabéticos presentaron plexos verticales tortuosos (40 por ciento y 53,3 por ciento respectivamente) y plexos nerviosos oblicuos en el grupo de pacientes supuestamente sanos (80 por ciento). Conclusiones: El estudio de la córnea por microscopia confocal en pacientes diabéticos evidencia mayor espesor corneal total y por capas, membrana basal visible, disminución del plexo nervioso sub-basal con disposición vertical y tortuosidad de las fibras(AU)

ABSTRACT Objective: To identify the morphological and morphometric characteristics of the corneal layers by confocal microscopy in diabetic patients. Methods: A descriptive and comparative study was carried out in 90 eyes, 60 of which belonged to diabetic patients (30 type 1 and 30 type 2) and 30 to supposedly healthy patients. The study was conducted at Ramón Pando Ferrer Cuban Institute of Ophthalmology, between January 2012 and January 2017. Results: Epithelial thickness, stroma and corneal endothelium was greater in the eyes of type 1 diabetics with a mean of 54.6, 506.7 and 26.7 microns, respectively. The epithelial basement membrane was observed in 20 percent of the eyes of patients with type 1 diabetes mellitus, and in 10 percent of type 2 diabetics. The group of type 1 diabetics showed one and two nerve plexuses per confocal microscopy field for 33.3 percent each. In type 2 diabetics, one nerve plexus (40 percent) predominated, while in healthy patients, four nerve plexuses (66.7 percent) predominated. Both groups of diabetic patients presented tortuous vertical plexuses (40 percent and 53.3 percent, respectively) and oblique nerve plexuses in the group of supposedly healthy patients (80 percent). Conclusions: The study of the cornea by confocal microscopy in diabetic patients showed greater total and layered corneal thicknesses, visible basal membrane, decrease in the sub-basal nerve plexus with vertical arrangement, and tortuous fiber(AU)

Concordancia entre los diferentes conteos celulares según los parámetros morfológicos y morfométricos del endotelio corneal / Concordance between different cell counts according to morphologic and morphometric parameters of the corneal endothelium

RESUMEN Objetivo: Estimar los valores morfológicos y morfométricos del endotelio corneal según la cantidad de células y evaluar la concordancia interobservadores para los diferentes parámetros, considerados según los diferentes conteos celulares en adultos sin alteraciones corneales. Métodos: Se realizó una investigación observacional, descriptiva y transversal de serie de casos en el Servicio de Cirugía Refractiva del Instituto Cubano de Oftalmología "Ramón Pando Ferrer" en dos años de estudio. Después de aplicar los criterios de exclusión, la muestra quedó conformada por 200 ojos de 100 pacientes adultos sin alteraciones corneales. Se realizó microscopia endotelial de no contacto SP-3000P, para identificar los valores morfológicos (hexagonalidad y coeficiente de variabilidad) y morfométricos (densidad celular), así como el promedio del tamaño celular corneal según cantidad de células evaluadas. Resultados: Según la cantidad de células endoteliales evaluadas, no existieron diferencias significativas de las variables morfológicas y morfométricas (p> 0,05) en ambos ojos. La concordancia entre los diferentes conteos celulares según los valores de los coeficientes de correlación intraclase fueron todos altos. La concordancia interobservadores (excepto para la hexagonalidad) y los coeficientes de correlación intraclase fueron altos. Conclusiones: Los valores morfológicos y morfométricos del endotelio corneal según cantidad de células evaluadas, son similares en todos los conteos celulares. Se demuestra una buena concordancia entre los diferentes conteos celulares estudiados para los diferentes parámetros estimados(AU)

ABSTRACT Objective: To estimate the morphological and morphometric values of the corneal endothelium according to the number of cells and evaluate the interobserver concordance for the different parameters, estimated according to the different cell counts in adults without corneal alterations. Methods: An observational, descriptive and cross-sectional case series research was carried out in the Refractive Surgery Service of Ramón Pando Ferrer Cuban Institute of Ophthalmology during two years of study. After applying the exclusion criteria, the sample was made up of 200 eyes of 100 adult patients without corneal alterations. Non-contact endothelial microscopy SP-3000P was performed to identify morphological values (hexagonality and coefficient of variability) and morphometric values (cell density), as well as the average corneal cell size according to the number of cells evaluated. Results: According to the amount of endothelial cells evaluated, there were no significant differences between morphological and morphometric variables (p>0.05) in both eyes. The agreement between the different cell counts according to the values of the interclass correlation coefficients (ICC) were all high. The interobserver concordance and ICCs were also high, except for hexagonality. Conclusions: The morphological and morphometric values of the corneal endothelium, according to the number of cells evaluated, are similar in all cell counts. Good concordance between the different cell counts studied for the different estimated parameters is demonstrated(AU)

Melanoacanthoma: a potential pitfall of reflectance confocal microscopy

Abstract Melanoacanthoma is a rare variant of seborrheic keratosis, which is notable for dark pigmentation and fast radial growth, making it difficult to distinguish from melanoma. Histologically, it is characterized by proliferation of keratinocytes and dendritic melanocytes. The authors report a scalp lesion, fast growing, suspected by dermoscopy and confocal microscopy examination, with dendritic cells distributed throughout the lesion. Based on these findings, it was not possible to classify this lesion as clearly benign, so it was excised. Histopathologic evaluation and immunostain were consistent with melanoacanthoma.

Microscopia confocal no auxílio diagnóstico de Distrofia Corneana de Schnyder / In vivo confocal microscopy as a diagnostic tool in Schnyder Corneal Dystrophy's case

Resumo Neste relato, descrevemos um caso de Distrofia corneana de Schnyder que apresentou o desfecho de seu diagnóstico baseado em achados característicos na microscopia confocal, ferramenta que se aponta em destaque no universo oftalmológico.

Abstract Schnyder's corneal dystrophy (SCD) is a rare corneal condition characterized by cholesterol and phospholipids deposition in the stroma and Bowman's layer. We present a case report of a patient who had a progressive corneal stromal haze in both eyes since he was 15 years old. Etiological diagnosis of SCD was well established by In Vivo Confocal Microscopy (IVCM).

The effect of panretinal photocoagulation on confocal laser scanning ophthalmoscopy and stereo photographic parameters of optic disk topography in patients with diabetic retinopathy / Efeito da panfotocoagulação retiniana na oftalmoscopia confocal de varredura a laser e parâmetros da estereofotografia do disco óptico em pacientes com retinopatia diabética

ABSTRACT Purpose: To determine the effect of panretinal photocoagulation on optic disk topographic parameters in non-glaucomatous patients with proliferative diabetic retinopathy. Methods: This was a prospective, single-center, observational study. Thirty-eight eyes of 26 patients with diabetes underwent panretinal photocoagulation for proliferative diabetic retinopathy. Stereoscopic disk photographs and optic nerve head parameters were evaluated using the Zeiss fundus camera and the confocal scanning laser ophthalmoscope (Heidelberg Retinal Tomograph), respectively, at baseline and 12 months after the completion of panretinal photocoagulation. Results: Thirty-eight eyes of 26 patients (15 female) with a mean age of 53.7 (range 26-74) years were recruited. No significant difference was found between the stereo photography determined mean horizontal and vertical cup-to-disk ratio before and after panretinal photocoagulation treatment (p=0.461 and 0.839, respectively). The global values of the optic nerve head parameters analyzed with the HRT3 showed no significant change from baseline to 12 months, including the disk area, cup area, rim area, cup volume, rim volume, cup-to-disk area ratio, linear cup-to-disk ratio, mean cup depth, maximum cup depth, cup shape measure, height variation contour, mean retinal nerve fiber layer thickness, and cross-sectional area. Conclusion: Our results suggest that panretinal photocoagulation does not cause morphological optic disk changes in patients with diabetic proliferative retinopathy after 1 year of follow-up.

RESUMO Objetivo: Determinar o efeito da panfotocoagulação retiniana nos parâmetros topográficos do disco óptico em pacientes não glaucomatosos com retinopatia diabética proliferativa. Métodos: Este é um estudo observacional pros­pectivo e unicêntrico. Trinta e oito olhos de 26 pacientes dia­béticos foram submetidos à panfotocoagulação retiniana para retinopatia diabética proliferativa. As estereofotografias e os parâmetros do disco óptico foram avaliados usando o retinógrafo Visucam da Zeiss e o oftalmoscópio confocal de varredura a laser (Heidelberg Retinal Tomograph), respectivamente, no início e 12 meses após a conclusão da panfotocoagulação. Resultados: Trinta e oito olhos de 26 pacientes (15 mulheres) com média de idade de 53,7 anos (intervalo de 26-74) foram recrutados. Nenhuma diferença significativa foi encontrada entre a média horizontal e vertical para relação escavação/disco óptico determinadas pelas estereo­fotografias antes e após o tratamento com panfotocoagulação retiniana (p=0,461 e 0,839, respectivamente). Os valores globais dos parâmetros do disco óptico analisados com a tomografia de varredura a laser não mostraram nenhuma mudança significativa entre o início até os 12 meses, incluindo disk area, cup area, rim area, cup volume, rim volume, C/D area ratio, linear C/D ratio, mean cup depth, maximum cup depth, cup shape measure, height variation contour, mean retinal nerve fiber layer thickness e cross-sectional area. Conclusão: Nossos resultados sugerem que a panfotocoagulação retiniana não causa alterações morfológicas no disco óptico em pacientes com retinopatia diabética proliferativa após um ano de seguimento.

In-vivo assessment of a case of cutaneous sarcoidosis using reflectance confocal microscopy

Abstract: Reflectance confocal microscopy (RCM) is a noninvasive imaging technique that allows visualization of the epidermis and papillary dermis with cellular-level resolution. Granulomatous reactions such as sarcoidosis could be assessed using RCM. The identification of bright beaded-like structures that could correspond to reticulin fibers overlying granulomas, in association with dermoscopy, may be a very useful approach in the diagnosis of sarcoidosis and for the differentiation of this granulomatous entity with superficial cutaneous metastasis.

In vivo confocal microstructural analysis of corneas presenting Kayser-Fleischer rings in patients with Wilson's disease / Análise microestrutural confocal in vivo de córneas apresentando anel de Kayser-Fleischer em pacientes com doença de Wilson

ABSTRACT Purpose: To evaluate microstructural differences between corneas with and without Kayser-Fleischer rings in age-matched subjects with Wilson's disease with neurological symptoms, using confocal laser scanning microscopy. Methods: The study included 12 subjects with Wilson's disease with neurological symptoms. Twelve corneas presented clinically with classic Kayser-Fleischer rings, visible on slit lamp examination; the other 12 served as controls. The subjects underwent a comprehensive clinical examination. Microstructural analysis using confocal laser scanning microscopy evaluated increased corneal thickness, decreased number of cells, increased debris or specific deposits, and unusual microstructures. Results: Clinically, the subjects with Kayser-Fleischer rings had similar corneal findings and normal intraocular pressure; two had typical sunflower cataracts and decreased visual acuity. The control eyes all presented normal visual acuity, intraocular pressure, and corneal appearance. The microstructural analysis demonstrated similar findings in all the affected corneas. Compared with the control corneas, there were fewer keratocytes in the anterior stroma (17.380 vs. 22.380/mm3). Round, "hollow" dark areas were observed between the keratocytes; these were universal and similar in appearance in all affected corneas and all cornea layers. In the peripheral posterior stroma, there were dust-like, bright, granular deposits that tended to increase in number and density toward Descemet's membrane, masking the peripheral endothelium. The control corneas presented a normal microstructure apart from dust-like granular deposits in the periphery. Conclusions: In vivo confocal microscopy is a useful tool for evaluating the corneal microstructure when a Kayser-Fleischer ring is clinically present. The ring consists of granular, bright particles that increase in density toward Descemet's membrane, and is associated with a decreased number of keratocytes and peculiar dark, round areas in all stromal layers, probably a sign of corneal damage. When the ring is not visible in subjects with Wilson's disease, changes to the corneal microstructure are insignificant.

RESUMO Objetivo: Avaliar, ao nível microestrutural, através de microscopia confocal in vivo a lazer, 12 córneas com anel de Kayser-Fleischer visível ao exame da lâmpada de fenda e compará-las com 12 córneas clinicamente normais de indivíduos com idades correspondentes aos pacientes com doença de Wilson e sintomas neurológicos. Métodos: O estudo incluiu 12 indivíduos com doença de Wilson e sintomas neurológicos (24 córneas). Doze córneas apresentavam clinicamente o anel clássico de Kayser-Fleischer e as outras 12 serviram como controle. Todos os pacientes foram submetidos a um exame clínico abrangente e a uma análise microestrutural subsequente utilizando microscopia confocal in vivo de varredura a laser. Os principais resultados observados foram: aumento da espessura da córnea, diminuição do número de células, aumento de resíduos/depósitos específicos e microestrutura atípica. Resultados: Clinicamente, todos os indivíduos com anel de Kayser-Fleischer (12 olhos) apresentaram achados similares da córnea e pressão intraocular normal. Dois indivíduos também apresentaram uma catarata de girassol típica e diminuição da acuidade visual. Todos os olhos do grupo controle apresentaram acuidade visual, pressão intraocular e aparência corneana normais. A microscopia confocal in vivo com varredura a laser revelou achados semelhantes em todas as córneas afetadas. O número de ceratócitos no estroma anterior era menor, 17.380/mm3 (22.380/mm3 no grupo controle), e entre eles foram identificadas áreas escuras arredondadas "vazias". Essas zonas escuras eram generalizadas e similares em todas as córneas examinadas e em todas as camadas da córnea. No estroma posterior periférico, havia presença de depósitos granulares brilhantes e com aparência de pó que tendiam a aumentar em número e densidade no sentido da membrana de Descemet, mascarando o endotélio periférico. As córneas controle apresentaram estrutura normal, com exceção de depósitos granulares com aparência de pó na periferia. Conclusões: A microscopia confocal in vivo é uma ferramenta útil para a avaliação da microestrutura da córnea quando o anel de Kayser-Fleischer está clinicamente presente. O anel é constituído de partículas granulares brilhantes com densidade aumentada no sentido da membrana de Descemet. Sua presença está associada com uma diminuição do número de ceratócitos e com áreas circulares escuras "peculiares" em todas as camadas estromais, que representam, provavelmente, um sinal de dano da córnea. Quando o anel não está clinicamente visível, a estrutura da córnea in vivo encontra-se insignificantemente alterada.

A avaliação dos nevos comuns, nevos atípicos e melanomas iniciais (lesões melanocíticas duvidosas) através da microscopia confocal in vivo / The evaluation of common nevi, atypical nevi and early melanomas (doubtful melanocytic lesions) by in vivo confocal microscopy

Introdução: O diagnóstico clínico, dermatoscópico e histopatológico dos nevos atípicos é um desafio, uma vez que podem apresentar padrões regulares, observados em nevos melanocíticos comuns, ou padrões irregulares e assimétricos, que se assemelham aos padrões encontrados em melanomas e assim persiste a necessidade de novas técnicas de imagem não invasivas, como microscopia confocal in vivo, para se melhorar a acurácia diagnóstica dessas lesões. Objetivos: Descrever as características. encontradas no exame de microscopia confocal in vivo nas lesões melanocíticas, correlacioná-las com seus diagnósticos histopatológicos,elaborar e validar um algoritmo com as características observadas para diferenciar as lesões melanocíticas entre si. Material e Método: Trata-se de um estudo observacional realizado no Departamento de Oncologia Cutânea do A.C.Camargo Cancer Center. Foram incluídas 110 lesões suspeitas pela análise clínica e dermatoscópica de nevo atípico. Todas as lesões foram analisadas com exame de microscopia confocal e posterior exame histopatológico. Os dados obtidos foram compilados para análise estatística, a fim de estabelecer um algoritmo. Foi realizada a regressão logística simples e regressão logística múltipla em três grupos: 1) comparação do nevo melanocítico comum versus nevo melanocítico atípico; 2) comparação do nevo melanocítico atípico versus melanoma e 3) comparação do nevo melanocítico comum e atípico versus melanoma. Com os dados obtidos na regressão logística múltipla e identificação dos critérios estatisticamente. significantes, foi criado um algoritmo e um nomograma para ajudar a diferenciar as lesões melanocíticas, com base nos critérios da microscopia confocal in vivo. Para validar esse algoritmo, 30 novos casos foram analisados por três examinadores experientes. Resultados: A análise através da regressão logística simples e múltipla mostrou que no grupo 3 é possível diferenciar estatisticamente os nevos comuns e atípicos dos melanomas com base na presença de três critérios: células redondas na. JDE (p = 0,048), hotspot periférico na JDE (p = 0,032) e "sheet of cells" (p = 0,04). Essas mesmas três características foram usadas como critérios na elaboração de um algoritmo através de um nomograma cuja finalidade é indicar a probabilidade de uma dada lesão ser um melanoma. A validação desse algoritmo foi realizada em 30 novos casos por três examinadores independentes, os quais apresentaram resultados semelhantes com alta sensibilidade e especificidade: 100% sensibilidade, 83,3% especificidade e acurácia de 86,67%. Esses valores foram obtidos através da curva ROC. onde o ponto de corte de 158 pontos confere uma probabilidade estimada de 70% para o diagnóstico do melanoma. Conclusão: Ao correlacionar as características encontradas no exame microscopia confocal in vivo de cada lesão com seu diagnóstico histopatológico final, foi observado que essas características (células redondas na JDE, hotspot na JDE de localização periférica e sheet of cells) apresentavam prevalências distintas e estatisticamente significantes entre as diferentes lesões melanocíticas, que foram usadas e validadas como critérios em um algoritmo para diferenciar nevos comuns e atípicos dos melanomas

Introduction: Clinical, dermatoscopic and histopathological diagnosis of atypical nevi is a challenge, because they may present regular patterns, observed in common melanocytic nevi, or irregular and asymmetric patterns, which resemble the patterns found in melanomas and thus still existing the need for development of new non-invasive imaging techniques such as in vivo microscopy confocal, in order to improve the diagnostic accuracy of these lesions. Objectives: Describe the characteristics found at in vivo confocal microscopic examination of melanocytic lesions and correlate them with their histopathological diagnoses. Also, elaborate and validate an algorithm with the characteristics observed to differentiate this lesions. Material and Method: This observational study was conducted at the Cutaneous Oncology Department of the A.C.Camargo Cancer Center. 110 suspicious lesions were included as atypical nevi after clinical and dermaoscopy examination. All lesions were later analyzed with confocal microscopy and histopathological examination. All data obtained were compiled for statistical analysis in order to establish an algorithm to aid the diagnosis of atypical nevi and other melanocytic lesions. Comparisons by simple and multiple logistic regression were performed in three groups: 1) common melanocytic nevi versus atypical melanocytic nevi; 2) atypical melanocytic nevi versus melanoma; and 3) common and atypical melanocytic nevi versus melanoma. With data obtained from multiple logistic regression and identification of the statistically significant criteria, an algorithm and a nomogram were created to help differentiate the melanocytic lesions based on in vivo confocal microscopy findings. To validate this algorithm, 30 new cases were analyzed by three experienced examiners. Results: Differentiation of common and atypical nevi from melanoma was statistically possible based on the presence of three criteria: round cells at the DEJ (p = 0.048), peripheral hotspot at the DEJ (p = 0.032) and sheet of cells (p = 0.04). These same three features were used as criteria to elaborate an algorithm and a nomogram capable to indicate the probability of a given lesion being a melanoma. The validation of this algorithm was performed by three independent examiners, who presented similar results with high sensitivity and specificity: 100% sensitivity, 83.3% specificity and 86.67% accuracy. These values were obtained through the ROC curve where the cut-off point of 158 points is related to 70% estimated probability for melanoma diagnosis. Conclusion: The correlation of in vivo confocal microscopy features found in each lesion with its final histopathological diagnosis revealed that round cells at DEJ, hotspot peripheral location at DEJ and sheet of cells had statistically significant different prevalence between different melanocytic lesions, and these features were used and validated as criteria in an algorithm to differentiate common and atypical nevi from melanomas

Chitosan and carboxymethylated derivative nanoparticles as delivery systems for biological products: preparation, characterization, stability and in vitro/in vivo evaluation / Nanopartículas de quitosana e derivado carboximetilado como sistemas de fornecimento (delivery) de produtos biológicos: preparo, caracterização, estabilidade e avaliação in vitro/in vivo

Chitosan is a biocompatible and biodegradable mucoadhesive polymer with unique advantages, such as the distinct trait of opening the junctions to allow paracellular transport of antigen and good tolerability. However, the poor solubility of chitosan in neutral or alkalinized media has restricted its applications in the pharmaceutical field. Chitosan can be easily carboxymethylated to improve its solubility in aqueous media, while its biodegradability and biocompatibility are preserved. Apart from this, carboxymethyl chitosan (CMCS) can be easily processed into nanoparticles which highlight its suitability and extensive usage for preparing different drug delivery formulations. The present study deals with the development and characterization of a delivery system based on CMCS nanoparticles using ovalbumin as model protein. We demonstrated that ovalbumin loaded nanoparticles were successfully synthetized using calcium chloride as a cross-linker by ionic gelation. The nanoparticles exhibited an average size of approximately 169 nm and presented a pseudo-spherical shape. The nanoparticles size increased according to the addition of CaCl2 due to the strong electrostatic attraction. During storage the nanoparticles size increased was attributed to swelling and aggregation. The loading efficiency of ovalbumin was found to be 17%. Confocal microscopy clearly showed the association between ovalbumin and CMCS chains into nanoparticles. Therefore, we suggest these nanoparticles can be considered as an attractive and promising carrier candidate for proteins and antigens. The major challenge that limits the use of such carriers is their instability in an aqueous medium. Thus, the next step of this work was to determine the robustness of several formulations using distinct freeze-drying protocols. This study demonstrated that mannitol in concentration of 10% (w/v) is well suited to preserve ovalbumin loaded CMCS nanocapsules from aggregation during lyophilization and subsequent reconstitution. Importantly, the results showed that an annealing step has a huge impact on porosity of freeze-dried cake by nearly complete crystallization of mannitol, once the crystalline matrix prevents the partial collapse and the formation of larger pores observed without annealing. Therefore, the usual observation that annealing increases the pore size due to growth of ice crystal size does not always apply, at least when crystallization of solute is involved. Since all characterizations and stability studies had been performed, the main purpose of this study was to develop a stable antigen delivery system for oral immunization using CMCS and inactivated rabies virus (RV) as the antigen. RV loaded nanoparticles was found to enhance both systemic (IgG) and local (IgA) immune responses against RV after oral delivery in mice. The effective doses 50% were 50-times higher than the negative controls, indicating that the immune response started only after the third boosting dose. Furthermore, enough neutralizing antibodies was produced to be protected against the harmful effects of the rabies virus. It is therefore concluded, that the CMCS nanoparticles formulated in this study, are suitable for oral vaccine delivery, and can be suggested as a promising delivery system for a diverse range of antigens as well as a gene/protein delivery system, especially for those positively charged. Since several approaches show that effective intervention in airway allergic inflammation can be achieved with allergen-activated interleukin-10-secreting cells, the final part of this work was dedicated to assessing whether IL-10 loaded chitosan nanoparticles (IL10-CSNPs) could be used as a possible inhalable therapeutic tool for preventing exacerbations in asthmatic patients. As positive controls, we also assess whether interleukin 17A and interleukin 9 have the ability to stimulate human airway smooth muscle (HASM) cell contractility using magnetic twisting cytometry (MTC). Significant decreased baseline cell stiffness was observed in HASM cells pre-treated with IL-10, but not with IL10-CSNPs, whereas treatment with IL-17A significantly enhanced baseline cell stiffening. Our findings reveal a previously unknown mechanism underlying immunotherapy for prevention and treatment of asthma

A quitosana é um polímero mucoadesivo biocompatível e biodegradável, com vantagens únicas, tais como a característica distinta de abrir as junções que permitim o transporte paracelular de antígenos e boa tolerabilidade. No entanto, sua baixa solubilidade em meios neutros ou alcalinizados tem restringido suas aplicações no campo farmacêutico. A quitosana pode ser facilmente carboximetilada para melhorar de sua solubilidade em meios aquosos, enquanto sua biodegradabilidade e biocompatibilidade são preservadas. Além disso, a carboximetilquitosana (CMCS) pode ser facilmente processada na forma de nanopartículas, o que destaca sua adequabilidade para uso extensivo no preparo de sistemas de delivery de medicamentos. O presente estudo trata do desenvolvimento e caracterização de um sistema de delivery baseado em nanopartículas de CMCS utilizando ovalbumina como proteína modelo. Nós demonstramos que as nanopartículas carregadas com ovalbumina foram sintetizadas com sucesso utilizando cloreto de cálcio como agente de reticulação por gelificação iônica. As nanopartículas exibiram um tamanho médio de aproximadamente 169 nm e apresentaram uma forma pseudo-esférica. O tamanho das nanopartículas aumentou de acordo com a adição de CaCl2 devido à forte atração eletrostática. Durante o armazenamento, o tamanho aumentado das nanopartículas foi atribuído a incorporação de água e agregação. A eficiência de encapsulamento da ovalbumina foi de aproximadamente 17%. A microscopia confocal mostrou claramente a associação entre ovalbumina e a cadeias de CMCS nas nanopartículas. Sugerimos, portanto, que tal sistema pode ser considerado como candidato atraente e promissor para o carreamento de proteínas e antígenos. O principal desafio que limita o uso desses carreadores consiste na instabilidade em meio aquoso. Assim, o próximo passo deste trabalho foi determinar a robustez de várias formulações utilizandose diferentes protocolos de liofilização. Este estudo demonstrou que o manitol em uma concentração de 10% (p/v) é adequado para preservar da agregação as nanocápsulas de CMCS carregadas com ovalbumina durante a liofilização e subsequente reconstituição. Mais importante, os resultados mostraram que uma etapa de annealing tem um enorme impacto sobre a porosidade da amostra liofilizada devido a quase completa cristalização do manitol, uma vez que a matriz cristalina evita o colapso parcial e a formação de poros maiores observados na ausência do annealing. Portanto, a observação comum de que o annealing aumenta o tamanho doporos devido ao crescimento dos cristais de gelo nem sempre se aplica, pelo menos quando a cristalização de um soluto está envolvida. Uma vez que todas as caracterizações e estudos de estabilidade foram realizados, o principal objetivo deste estudo foi desenvolver um sistema estável de delivery de antígeno para imunização oral utilizando CMCS e vírus rábico inativado (RV) como antígeno. Verificou-se que as nanopartículas carregadas com RV aumentam as respostas imune sistêmica (IgG) e local (IgA) contra o RV após administração oral em camundongos. As doses efetivas 50% foram 50 vezes maiores que os controles negativos, indicando que a resposta imune foi iniciada apenas após a terceira dose da vacina. Além disso, foram produzidos anticorpos neutralizantes suficientes para proteção contra os efeitos nocivos do vírus rábico. Conclui-se, portanto, que as nanopartículas de CMCS formuladas neste estudo, são adequadas para o delivery oral de vacinas, e podem ser sugeridas como um sistema promissor de delivery para uma gama diversa de antígenos, bem como para o delivery de genes/proteínas, especialmente para aqueles carregados positivamente. Uma vez que diversas abordagens mostram que uma intervenção efetiva em casos de inflamação alérgica de vias aéreas pode ser conseguida por meio de células secretoras de interleucina 10 (IL-10) mediante ativação por alergenos, a parte final deste trabalho esteve dedicada a avaliação de nanopartículas de quitosana carregadas com IL-10 (IL10-CSNPs) como possível ferramenta terapêutica inalável para prevenção de exacerbações em pacientes asmáticos. Como controles positivos, avaliou-se adicionalmente se as interleucinas 17A (IL-17A) e 9 (IL-9) possuem a capacidade de estimular a contratilidade de células humanas de músculo liso de vias aéreas humanas (HASM) por meio de citometria de torção magnética (MTC). Uma diminuição significativa da rigidez celular basal foi observada em células HASM pré-tratadas com IL-10, mas não com IL10-CSNPs, enquanto que o tratamento com IL-17A aumentou significativamente a magnitude rigidez celular basal. Nossos resultados revelam um mecanismo previamente desconhecido subjacente à imunoterapia para prevenção e tratamento da asma