A ceratite fúngica é uma doença grave que pode levar à perda da visão. O tratamento consiste na aplicação de antifúngicos, no entanto a administração tópica não tem se mostrado efetiva devido aos mecanismos de defesa do olho que levam à baixa retenção da formulação no local de aplicação e à baixa permeação do fármaco através da córnea. Sendo assim, formulações mais adequadas e sistemas de liberação vêm sendo estudados na tentativa de melhorar a biodisponibilidade local de antifúngicos. No presente trabalho foram obtidos e caracterizados dois sistemas para a liberação ocular do fluconazol (FLU), um antifúngico de atividade reconhecida: um gel termorreversível in situ e micropartículas poliméricas. A permeação passiva e iontoforética do fármaco através da córnea foi estudada in vitro a partir dos sistemas desenvolvidos. O gel termorreversível in situ contendo 16% de poloxamer e 1,0% de quitosana apresentou temperatura de geleificação adequada, propriedades mucoadesivas, melhores parâmetros mecânicos (dureza, compressibilidade e adesividade) que ambos os polímeros separadamente e mostrou-se superior que micropartículas poliméricas, com fluxo de permeação passiva cerca de duas vezes maior. A maior quantidade de fármaco retido na córnea foi obtida após aplicação da iontoforese em solução aquosa do fármaco. Ainda assim, o fluxo iontoforético do FLU não foi significativamente diferente do fluxo passivo a partir do gel termorreversível. O desempenho in vivo desta formulação foi então avaliado em modelo animal. Estudos de permeação, utilizando a técnica de microdiálise para amostragem da câmara anterior, confirmaram a maior biodisponibilidade do fármaco. Por fim, exames de cintilografia em humanos confirmaram maior tempo de retenção na superfície ocular. Portanto, o gel termorreversível in situ obtido e caracterizado no presente trabalho e a iontoforese, representam sistemas promissores para a liberação ocular tópica do FLU. O primeiro possui características promotoras de permeação, prolongado tempo de retenção e facilidade de obtenção e administração, e o segundo é capaz de promover maior retenção do fármaco na córnea. / Fungal keratitis is a serious disease that can lead to loss of sight. The treatment consists in antifungal application; however topical administration has not shown to be effective due to defense mechanisms of the eye, which leads to low retention at the application site and low permeation of the drug trough the cornea. In this way, more suitable formulations and delivery systems have been studied in an attempt to increase local availability of antifungal agents. At the present work it was obtained and characterized two systems for the passive and iontophoretic ocular delivery of fluconazole (FLU), a well known antifungal agent: an in situ forming gel and polymeric microparticles. Passive and iontophoretic drug permeation across the cornea was studied in vitro from the developed systems. The in situ forming gel containing 16% of poloxamer and 1.0% of chitosan presented more adequate gelation temperature, mucoadhesive properties, improved mechanical parameters (strength, compressibility and adhesiveness) than both polymers separately and showed to be superior to the polymeric microparticles, with passive permeation flux almost twice higher. The greatest amount of drug retained in the cornea was achieved after iontophoresis application using an aqueous solution of the drug. Even though, the iontophoretic drug flux was not significantly different from the passive drug flux using the in situ forming gel. The in vivo performance of this formulation was then evaluated in an animal model. Permeation studies, using the microdialysis technique for the anterior chamber sampling, confirmed the increased drug availability. At the end, scintigraphy exams in humans confirmed the greater retention time at the ocular surface. Therefore, the in situ forming gel obtained and characterized at the present work and iontophoresis, represent promising systems for the topical ocular delivery of FLU. The first possess permeation enhancement properties, prolonged retention time and facility of obtention and administration, and the second is capable of promoting higher drug retention in the cornea.

A reprodução fiel da íris do olho remanescente na confecção da prótese ocular é fator fundamental no resultado estético e na dissimulação da perda para os pacientes afetados. Este trabalho apresenta uma nova técnica de confecção da íris protética, substituindo a pintura tradicional da mesma, pela imagem digitalizada da íris remanescente, obtida através de fotografia impressa em papel fotográfico. Decidiu-se avaliar a estabilidade das cores, comparando-se a técnica de pintura com a obtida através da fotografia digital, sob a ação de dois fatores: ação térmica do ciclo de polimerização da resina acrílica na confecção da prótese e a incidência da luz solar, principal agente de fotodegradação que compromete a longevidade das próteses oculares, através de teste de envelhecimento acelerado. Foram assim, confeccionados corpos de prova simulando a íris protética, divididos em dois grupos: pintura e fotografia, nas cores azul, amarelo, preto, marrom e verde. Foi realizada leitura colorimétrica inicial e após submetê-los às condições térmicas de polimerização. Posteriormente os corpos de prova foram testados durante três semanas através de ensaio de envelhecimento acelerado artificialmente com radiação ultravioleta A, com leituras colorimétricas semanais. A análise estatística sugere que, após a polimerização, as cores amarelo e marrom apresentaram diferenças significativas entre os grupos, sendo que, a cor amarelo na amostra pintura apresentou uma degradação um pouco acima do nível clinicamente aceitável. Após o ensaio de envelhecimento, não houve diferença significativa entre as amostras pintura e fotografia para as cores verde e amarelo, com alteração um pouco acima do nível clinicamente aceitável. Para as cores marrom e preto ocorreram diferenças significantes, com variações em níveis clinicamente aceitáveis; para a cor azul em níveis elevados, inviáveis clinicamente tanto na fotografia como na pintura. / The accurate iris reproduction in the making of ocular prosthesis for the remaining eye is a fundamental factor for the loss dissimulation aesthetic result in affected patients. This paper presents a new technique for the making of prosthetic iris that consists in substituting the traditional painting of the prosthesis for a digitalized image of the remaining iris, obtained through a picture printed in a photographic paper. It was decided to evaluate the stability of the colors by comparing the painting technique with the digital picture, for this two factors were considered: thermal conditions of the acrylic resin polymerization cycle in the prosthesis making and incidence of the sun light, which is the main photo degradation agent that affect the longevity of the ocular prosthesis, through the accelerated aging test. Then proof bodies simulating the prosthetic iris were made and divided into two groups: painted (in the colors blue, yellow, black, brown and green) and picture. An initial colorimetric scanning was made before and after submitting the bodies to the thermal conditions of the polymerization process. The proof bodies were then tested artificially for three weeks using the accelerated aging with A-ultraviolet radiation technique and proceeding to colorimetric scans weekly. Statistical analysis suggests that after the temperature action in the polymerization cycle, the yellow and brown color presented significative alteration between both groups and the yellow color in the painted presented degradation slightly above the acceptable clinical level. After the aging test, there was no significant difference between the samples in both groups for the green and yellow colors, however, for the brown and black colors significant differences were observed, with variations in clinically acceptable levels, and for blue color in high levels, clinically impracticable, in both groups.

OBJETIVO: avaliar e correlacionar parâmetros tomográficos de segmento anterior e parâmetros biomecânicos de córnea entre si, com a idade e o gênero de pacientes com olhos normais. MÉTODOS: um estudo clínico de série de casos com intervenção diagnóstica foi realizado, envolvendo um olho selecionado aleatoriamente de 235 pacientes (235 olhos). Os pacientes foram submetidos a exame oftalmológico completo, incluindo avaliação tomográfica (Pentacam) e biomecânica (ORA). Parâmetros avaliados pelo Pentacam: K1, K2, K Front Max , Astig, ISV, IVA, KI, CKI, IHA, IHD, BAD D, BAD Df, BAD Db, BAD Dp, BAD Dt, BAD Dy, ART Max, ART Avg, ART Min, Enh BFS Front 8mm, Enh BFS Back 8mm, Ele F BFS 8mm Apex, Ele F BFS 8mm Thinnest, Ele F BFS 8mm Max 4mm zone, Ele B BFS 8mm Apex, Ele B BFS 8mm Thinnest, Ele B BFS 8mm Max 4mm zone, Ele F BFTE 8mm Apex, Ele F BFTE 8mm Thinnest, Ele F BFTE 8mm 4 mm zone, Ele B BFTE 8mm Apex, Ele B BFTE 8mm Thinnest, Ele B BFTE 8mm Max 4mm zone, RPI Max, RPI Avg, RPI Min, Diff RPI Max Compl, Diff RPI Min Compl, Pachy Min, Pachy Apex, Pachy Pupil, Rel Pachy Min, Asph Q Front 30º, Asph Q Back 30º, Q Asymmetr Frontal Hor 30º, Q Asymmetr Frontal Vert 30º, AC Depth, AC Volume, Ch Angle, Volume, PNS, Densid Avg %. Parâmetros avaliados pelo ORA: IOPg, IOPcc, CH, CRF, KC Score, KC Normal, KC Suspect, KC Mild, KC Moderate, KC Severe, WS, aindex, bindex, p1area, p2area, aspect1, aspect2, uslope1, uslope2, dslope1, dslope2, w1, w2, h1, h2, dive1, dive2 , path1, path2, mslew1, mslew2, slew1, slew2, aplhf, p1area1, p2area1, aspect11, aspect21, uslope11, uslope21, dslope11, dslope21, w11, w21, h11, h21, path11, path21. RESULTADOS: K1, K2, K Max Front, BAD Df, Ele B BFS 8mm Thinnest, Ele B BFTE 8mm Thinnest, CRF e CH apresentaram valores mais altos no sexo feminino, enquanto BAD Db, Enh BFS Front 8mm, AC Depth, AC Volume, Ch Angle, p1area, w2, h1, p1area1, w21 e h11 apresentam valores mais altos no sexo masculino. CKI, BAD Db, Ele F BFS 8mm Apex, Ele F BFS 8mm Thinnest, Ele F BFTE 8mm Apex, Ele F BFTE 8mm Thinnest, Asph Q Back 30º, AC Depth, Ch Angle mostraram forte correlação negativa com a idade. Ele B BFS 8mm Apex, Ele B BFS 8mm Thinnest, Ele B BFTE 8mm Apex, Ele B BFTE 8mm Thinnest mostraram forte correlação positiva com a idade. As correlações mais altas de CH e CRF ocorreram com BAD Dt, Pachy Min, Pachy Apex e Pachy Pupil, ou seja, CH e CRF apresentaram associação positiva com a espessura corneana central. As correlações mais altas da IOPg ocorreram com BAD Dt, ART Max, ART Avg, Pachy Min, Pachy Apex e Pachy Pupil mostrando ser afetada pela espessura corneana. A IOPcc mostrou correlações mais baixas do que as obtidas com a IOPg, sendo esta menos afetada pela espessura corneana. CONCLUSÃO: Os parâmetros tomográficos de segmento anterior (Pentacam) e biomecânicos (ORA) mostraram diversas associações estatisticamente significantes entre si com a idade e com o gênero de pacientes com olhos normais / PURPOSE: to evaluate and to correlate anterior segment tomography parameters and corneal biomechanical parameters between each other, age and gender in patients with healthy eyes. METHODS: a clinical study of case series design with diagnostic intervention was conducted, involving one eye randomly selected from 235 patients (235 eyes). Patients underwent complete ophthalmological examination, including tomographic (Pentacam) and biomechanical (ORA) evaluation. Pentacam parameters assesssed: K1, K2, K Front Max , Astig, ISV, IVA, KI, CKI, IHA, IHD, BAD D, BAD Df, BAD Db, BAD Dp, BAD Dt, BAD Dy, ART Max, ART Avg, ART Min, Enh BFS Front 8mm, Enh BFS Back 8mm, Ele F BFS 8mm Apex, Ele F BFS 8mm Thinnest, Ele F BFS 8mm Max 4mm zone, Ele B BFS 8mm Apex, Ele B BFS 8mm Thinnest, Ele B BFS 8mm Max 4mm zone, Ele F BFTE 8mm Apex, Ele F BFTE 8mm Thinnest, Ele F BFTE 8mm 4 mm zone, Ele B BFTE 8mm Apex, Ele B BFTE 8mm Thinnest, Ele B BFTE 8mm Max 4mm zone, RPI Max, RPI Avg, RPI Min, Diff RPI Max Compl, Diff RPI Min Compl, Pachy Min, Pachy Apex, Pachy Pupil, Rel Pachy Min, Asph Q Front 30º, Asph Q Back 30º, Q Asymmetr Frontal Hor 30º, Q Asymmetr Frontal Vert 30º, AC Depth, AC Volume, Ch Angle, Volume, PNS, Densid Avg %. ORA Parameters assessed: IOPg, IOPcc, CH, CRF, KC Score, KC Normal, KC Suspect, KC Mild, KC Moderate, KC Severe, WS, aindex, bindex, p1area, p2area, aspect1, aspect2, uslope1, uslope2, dslope1, dslope2, w1, w2, h1, h2, dive1, dive2 , path1, path2, mslew1, mslew 2, slew1, slew2, aplhf, p1area1, p2area1, aspect11, aspect21, uslope11, uslope21, dslope11, dslope21, w11, w21, h11, h21, path11, path21. RESULTS: K1, K2, K Max Front, BAD Df, Ele B BFS 8mm Thinnest, Ele B BFTE 8mm Thinnest, CRF and CH showed higher values in females and BAD Db, Enh BFS Front 8mm, AC Depth, AC Volume, Ch Angle, p1area, w2, h1, p1area1, w21 and h11 showed higher values in males. CKI, BAD Db, Ele F BFS 8mm Apex, Ele F BFS 8mm Thinnest, Ele F BFTE 8mm Apex, Ele F BFTE 8mm Thinnest, Asph Q Back 30º, AC Depth, Ch Angle showed strong negative correlation with age. Ele B BFS 8mm Apex, Ele B BFS 8mm Thinnest, Ele B BFTE 8mm Apex, Ele B BFTE 8mm Thinnest showed strong positive correlation with age. The highest correlations of CH and CRF occurred with BAD Dt, Pachy Min, Pachy Apex and Pachy Pupil, i.e. CH and CRF presented positive association with central corneal thickness. The highest correlations of IOPg occurred with BAD Dt, ART Max, ART Avg, Pachy Min, Pachy Apex and Pachy Pupil, showing that it is affected by the thickness of the cornea. IOPcc showed lower correlations than those obtained with IOPg, showing that it is less affected by the thickness of the cornea. CONCLUSION: Anterior segment tomographic parameters (Pentacam) and biomechanical parameters (ORA) showed several statistically significant associations between each other, age and gender in patients with healthy eyes

Um dos fatores imprescindíveis no resultado estético satisfatório do tratamento reabilitador protético ocular é a reprodução fiel da íris do olho remanescente na íris protética. É a dissimulação da perda para os pacientes afetados. Este trabalho apresenta duas novas técnicas de confecção da íris protética, utilizando imagem digitalizada da íris remanescente, obtida por meio de imagem fotográfica impressa em etiqueta autocolante e em adesivo. Em função da fotodegradação que compromete a longevidade das próteses oculares, decidiu-se avaliar a estabilidade das cores, comparando-se ambas as técnicas, através de teste de envelhecimento acelerado. Para os ensaios foram confeccionados corpos de prova simulando a íris protética, divididos em dois grupos: adesivo e etiqueta autocolante, nas cores verde, azul, marrom e preto. A variação do grau de degradação foi avaliada por leitura colorimétrica inicial e, em três intervalos semanais durante o envelhecimento artificial com radiação ultravioleta. A análise estatística sugere que quando as cores foram avaliadas em função do período de ensaio de envelhecimento artificial, somente na cor azul no grupo adesivo houve diferença estatisticamente significante. Quando avaliadas em função do grupo e período de ensaio, os testes estatísticos indicam que não houve diferença entre as cores entre adesivo. Nenhuma das cores entre os grupos apresentou uma degradação de cor acima do nível clinicamente aceitável. / One of the essential factors in satisfactory final result of ocular prosthetic rehabilitation treatment is a faithful reproduction of the iris of the eye remaining in prosthetic iris. It concealment loss for affected patients. This paper presents two new techniques to manufacture prosthetic iris, using the remaining iris scanned image, obtained by photographic image printed sticker and adhesive. Depending on the photo degradation that compromises the longevity of artificial eyes, decided to evaluate the stability of colors, comparing both techniques, through accelerated aging test. For the tests were prepared specimens simulating prosthetic iris, divided into two groups: adhesive sticker and, in green, blue, brown and black. The variation of the degree of degradation was evaluated by colorimetric reading before and after and three weekly intervals during aging with artificial ultraviolet radiation. Statistical analysis suggests that when the colors were evaluated according to the period of artificial aging test, only in blue color in the adhesive group difference was statistically significant. When evaluated as a function of group and trial period, the statistical tests indicate no difference between the colors of adhesive. None of the colors between the groups showed color degradation above the clinically acceptable level.

A tuberculose ocular afeta 1 a 2% dos pacientes com diagnóstico de tuberculose sistêmica. O tratamento convencional consiste na administração oral dos agentes antituberculosos. Devido às barreiras oculares, o tratamento tópico requer dose elevada e repetidas administrações para atingir efeito terapêutico no olho. Assim, a toxicidade, nos tratamentos convencionais, pode ser relevante. Considerando tais limitações, o desenvolvimento de preparações que permitam a obtenção de produtos com maior eficácia e segurança é de fundamental importância. Nesse sentido, o presente trabalho teve como objetivos o desenvolvimento, a caracterização físico-química e microbiológica de nanoemulsão contendo rifampicina (NR) revestidas empregando cloreto de quitosana (NR-Quit) e sulfato de polimixina B (NR-Poli) com potencial aplicação para o tratamento da tuberculose ocular por via tópica. A NR foi preparada empregando método por homogeneização a alta pressão e apresentou diâmetro hidrodinâmico médio (DHM), índice de polidispersão (IP), potencial zeta (PZ) e pH entre 131,0 e 137,3 nm, entre 0,19 e 0,24, entre -31,0 e -35,4 mV e entre 5,10 e 5,26, respectivamente. A eficiência de encapsulação da rifampicina, determinada por espectrofotometria UV-vis, foi de 82,5 % m/v. Para obtenção de NR-Quit e NR-Poli foi empregado planejamento fatorial completo. Foi observada alteração do PZ de NR após adição de cloreto de quitosana de -35,4 para +51,3 mV. No caso da adição de sulfato de polimixina B o PZ foi alterado de -35,4 para +5,5 mV. Nesse sentido, a abordagem metodológica elucidou que a concentração do cloreto de quitosana e sulfato de polimixina B influenciou significativamente no potencial zeta da NR-Quit e da NR-Poli. Além disso, foi observada a relação linear entre a concentração dos agentes catiônicos empregados e o potencial zeta da NR-Quit e NR-Poli. Essas preparações, no estudo de estabilidade, mostraram aspecto visual, DHM, IP e PZ inalterados por período maior que 90 dias. Também, os valores de pH, viscosidade e osmolalidade foram ajustados entre 4,07 e 4,55, 1,03 e 1,08 cP, 209,7 e 213,4 mOsm/Kg, respectivamente. A atividade antimicrobiana realizada frente ao Mycobacterium tuberculosis H37Rv da NR, NR-Quit, NR-Poli e solução padrão de rifampicina, determinada pela concentração mínima inibitória (CMI), revelou CMI igual 0,125 µg/mL para todas as preparações. Tal resultado demonstrou que os processos de obtenção da nanoemulsão e do revestimento não alteraram a eficácia antimicrobiana da rifampicina. O presente trabalho permitiu o desenvolvimento de preparações inovadoras para o tratamento da tuberculose ocular, por via tópica, com potencial maior eficácia e segurança. / Ocular tuberculosis affects 1 to 2% of the patients diagnosed with systemic tuberculosis. The conventional treatment is the oral administration of the anti-tuberculosis agents. Due to eye barriers, topical treatment requires high dose and repeated administrations to achieve a therapeutic effect on the eye. Thus, toxicity is a major concern in these conventional treatments. Considering these limitations, development of preparations that enable products with higher efficacy and safety is of fundamental importance. In this sense, the present work aimed preparation, physicochemical properties evaluation and microbiological characterization of the rifampicin nanoemulsion (RN) coated using chitosan chloride (RN-Chit) and polymyxin B sulfate (RN-Poly) with potential application for topical treatment of ocular tuberculosis. The RN was prepared by high-pressure homogenization and present droplet mean size (DMS), polydispersity index (PdI), zeta potential and pH between 131.0 and 137.3 nm, between 0.19 and 0.24, between -31.0 and -35.4 mV and between 5.10 and 5.26, respectively. The encapsulation efficiency of rifampicin determined of using spectrophotometric UV-vis method was 82.5% w/v. For preparations RN-Chit and RN-Poly factorial experimental design was employed. The change in the zeta potential of RN-Chit was observed after the addition of chitosan chloride, from -35.4 to +51.3 mV. In the other case, an addition of polymyxin B sulfate changed the PZ from -35.4 to +5.5 mV. Therefore, the methodological approach elucidated that the concentration of chitosan chloride and polymyxin B sulfate significantly influenced the zeta potential of RN-Chit and RN-Poly. Furthermore, the linear relationship between the concentration of cationic agents employed and the zeta potential of RN-Chit and RN-Poly was observed. These preparations, in the stability study, showed visual appearance, DMS, PdI and PZ unchanged for a period greater than 90 days. Additionally, pH, viscosity and osmolality values were adjusted between 4.07 and 4.55, 1.03 and 1.08 cP, 209.7 and 213.4 mOsm/kg, respectively. The antimicrobial activity against Mycobacterium tuberculosis H37Rv of RN, RN-Chit, RN-Poly and the standard solution of rifampicin, determined by the minimum inhibitory concentration (MIC), showed MIC of 0.125 µg/mL for all preparations. This result demonstrated that the processes of nanoemulsion preparation and coating did not affect the antimicrobial efficacy of rifampicin. The present work allowed the development of innovative preparations for the treatment of topical ocular tuberculosis with potential high efficacy and safety.

Submitted by Fabíola Silva (fabiola.silva@famerp.br) on 2017-09-29T14:01:26Z No. of bitstreams: 1 anavitoriadascamargo_dissert.pdf: 2267502 bytes, checksum: 7b4d6187bc2712721b24d6875f2fc823 (MD5) / Made available in DSpace on 2017-09-29T14:01:27Z (GMT). No. of bitstreams: 1 anavitoriadascamargo_dissert.pdf: 2267502 bytes, checksum: 7b4d6187bc2712721b24d6875f2fc823 (MD5) Previous issue date: 2016-06-06 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / Fundação de Amparo à Pesquisa do Estado de São Paulo - FAPESP / Introduction: Toxoplasmosis, a disease resulting from Toxoplasma gondii infection, is clinically manifested through ocular, cerebral and congenital ways. This pararsito Apicomplexa is capable of infecting cells of all nucleated tissues and may remain in a latent state or cause irreversible cell damage. The HLA class II genes control the adaptive immune response humoral and influence susceptibility and the resistance to infectious and parasitic diseases. The ocular toxoplasmosis, besides being dependent on infection with T. gondii as well as the variability of the infecting strain, is influenced by host genetic factors. Aim: To test the hypothesis that the HLA class II genes (HLA-DRB1 and HLA-DQB1) are associated with ocular toxoplasmosis. Materials and Methods: Samples of 249 patients undergoing ophthalmologic evaluation and positive serology to T. gondii were analyzed. According to the clinical conditions, two distinct groups were composed: one formed by patients with ocular toxoplasmosis (n=123); and another group with patients without the disease ocular form (n=126). The patients with ocular toxoplasmosis were subdivided into two groups, according to the type of ocular manifestation: primary (n=93 samples) or recurrent (n=30). Genotyping of Class II HLA alleles were performed by the polymerase chain reaction technique with specific oligonucleotide sequence (PCR-SSO; One Lambda®). Results: The average ages of the group of patients with ocular toxoplasmosis was less (40.9±19.9) than the average age of patients without ocular toxoplasmosis (57.6±17.2) (p<0.0001).The alleles HLA-DRB1*03 (OR=1,94; IC 95% 1.09-3.45; p=0.031; pc=0.404) and HLA-DQB1*02 (OR=1.52; IC 95% 1.03-2.24; p=0.039; pc=0.197) showed a more allelic frequency in patients without ocular toxoplasmosis when compared to the group with ocular toxoplasmosis. The HLA-DRB1*14 alelle was more frequent in the subgroup of the recurrent manifestation, when compared to the group without ocular toxoplasmosis (OR=0.32; IC 95% 0.12-0.83; p=0.032; pc=0.417) and to the primary manifestation subgroup (OR=0.25; IC95% 0.08-0.73; p=0.017; pc=0.223). The HLA-DRB1*03_DQB1*02 haplotype was not associated with the lower risk of ocular toxoplasmosis (OR=1.86; IC 95%: 1.03-3.36; p=0.052). Conclusions: The obtained results suggest that the Class II HLA genes (DRB1 and DQB1) are not associated with the ocular toxoplasmosis development; and that none HLA DRB1_DQB1 haplotype influences the ocular toxoplasmosis development in the study population. / Introdução: A toxoplasmose, uma doença resultante da infecção por Toxoplasma gondii, manifesta-se clinicamente nas formas ocular, cerebral e congênita. Este parasito Apicomplexa infecta células nucleadas de todos os tecidos e pode permanecer em estado latente ou provocar danos celulares irreversíveis. Os genes HLA de classe II controlam a resposta imune adaptativa humoral e influenciam a suscetibilidade e a resistência às doenças infecciosas e parasitárias. A toxoplasmose ocular, além de ser dependente da infecção por T. gondii e da variabilidade das cepas infectantes, é fortemente influenciada por fatores genéticos do hospedeiro. Objetivo: Testar a hipótese de que os genes HLA de classe II (HLA-DRB1 e HLA-DQB1) estão associados à toxoplasmose ocular. Materiais e Métodos: Foram analisadas amostras de DNA de 249 indivíduos submetidos à avaliação oftalmológica e com sorologia reagente para T. gondii. De acordo como quadro clínico, dois grupos distintos foram compostos: um formado por pacientes com toxoplasmose ocular (n=123) e outro grupo, por pacientes sem a forma ocular da doença (n=126). Os pacientes com toxoplasmose ocular foram subdivididos em dois grupos de acordo com o tipo de manifestação ocular: primária (n=93) ou recorrente (n=30). A genotipagem dos alelos HLA de classe II foi realizada pela técnica reação da cadeia de polimerase com sequência de oligonucleotídeos específicos (PCR-SSO; One Lambda®). Resultados: A média de idade dos pacientes com toxoplasmose ocular foi menor (40.9±19.9) que a daqueles sem toxoplasmose ocular (57.6±17.2) (p<0.0001). Os alelos HLA-DRB1*03 (OR=1,94; IC 95% 1.09-3.45; p=0.031; pc=0.404) e HLA-DQB1*02 (OR=1.52; IC 95% 1.03-2.24; p=0.039; pc=0.197) apresentaram maior frequência alélica no grupo sem toxoplasmose ocular em comparação ao grupo com toxoplasmose ocular. O alelo HLA-DRB1*14 foi mais frequente no subgrupo com a manifestação recorrente em comparação ao grupo sem toxoplasmose ocular (OR=0.32; IC 95% 0.12-0.83; p=0.032; pc=0.417) e com o subgrupo manifestação primária (OR=0.25; IC95% 0.08-0.73; p=0.017; pc=0.223). O haplótipo HLA-DRB1*03_DQB1*02 não se mostrou associado ao menor risco de toxoplasmose ocular (OR=1.86; IC 95%: 1.03-3.36; p=0.052). Conclusões: Os resultados obtidos sugerem que os genes HLA de classe II (DRB1 e DQB1) não estão associados com o desenvolvimento da toxoplasmose ocular e que nenhum haplótipo HLA DRB1_DQB1 influencia o desenvolvimento desta doença na população analisada.

Submitted by Suzana Dias (suzana.dias@famerp.br) on 2018-11-09T18:03:41Z No. of bitstreams: 1 ChristianeAyo_tese.pdf: 8833199 bytes, checksum: e3a470634a8f41b17966d4b370464f0c (MD5) / Made available in DSpace on 2018-11-09T18:03:41Z (GMT). No. of bitstreams: 1 ChristianeAyo_tese.pdf: 8833199 bytes, checksum: e3a470634a8f41b17966d4b370464f0c (MD5) Previous issue date: 2017-06-30 / Fundação de Amparo à Pesquisa do Estado de São Paulo - FAPESP / Ocular toxoplasmosis, characterized by an intraocular inflammation, is the most common clinical manifestation of toxoplasmosis, the infectious disease caused by the protozoan Toxoplasma gondii. The lesions can affect the macula and other layers of the retina and the choroid, resulting in retinochoroiditis with different degrees of ocular involvement. Chagas disease is resulting from infection by the protozoan Trypanosoma cruzi. After 20 years of infection, about 30% develop chronic Chagas heart disease, which is clinically manifested by malignant ventricular arrhythmia, thromboembolism, sudden cardiac death, and chronic heart failure. Ten per cent of Chagas patients present the digestive form of the disease, characterized mainly by dilatations of the esophagus and/or colon, due to the denervation process. The progression of the infection, as well as the development of the different clinical forms and different degrees of severity may be related to genetic characteristics of the pathogen and the host. Among the factors related to the host, the immunological response is of special interest with genetic markers playing an important modulating role in this context as they may contribute to the pathogenesis or resistance in the clinical course of these infections. Objective: The present study aimed to verify the hypothesis that KIR genes, their HLA ligands and MICA gene polymorphisms are associated with ocular toxoplasmosis (OT) and the different clinical forms of Chagas disease. Patients and Methods: This study included 297 patients with toxoplasmosis, 148 clinically diagnosed with OT and 149 without OT. Moreover, 267 patients with Chagas disease were enrolled: 78 clinically diagnosed with the digestive form of the disease, 107 with the cardiac form and 82 with the mixed form. The ELISA technique was used to confirm infection by T. gondii and T. cruzi. Polymorphisms of the KIR and MICA genes were identified by PCR-SSOP and nested PCR. Continuous variables were compared using the unpaired t test and the Chi-square test with Yates correction or the Fisher's exact test were used compare the other results. Results: In relation to the toxoplasmosis, MICA genotypes and alleles did not differ between patients with and without OT, or between patients with the primary or recurrent manifestations of the disease. KIR3DS1 gene was positively associated with the development of OT. KIR activating genes along with their HLA ligands (KIR3DS1+/Bw4-80Ile+, KIR2DS1+/C2+ and KIR3DS1+/Bw4-80Ile+) were associated with susceptibility to OT and both its primary and recurrent clinical manifestations. The inhibitory pairs - KIR2DL3/2DL3-C1/C1 and KIR2DL3/2DL3-C1 - were associated with resistance to OT and its clinical manifestations, whereas the combination KIR3DS1-/KIR3DL1+/Bw4-80Ile+ was a protection factor for the development of OT and, in particular, against recurrent manifestations. As for Chagas disease, The MICA-129met allele was associated with the development of left ventricular systolic dysfunction (LVSD) in patients with chronic chagasic cardiomyopathy, while the MICA-129val allele was associated with a protection of developing LVSD. In particular, the MICA-129 met/met homozygous haplotype was associated with the development of severe LVSD and the MICA-129 val/val homozygous genotype protected against this condition. It was also possible to demonstrate that the haplotype MICA*008~HLA-C*06 and the combination between KIR genes and their HLA ligands - KIR2DS2-/KIR2DL2-/KIR2DL3+/C1+ - were associated with susceptibility for the digestive clinical form of Chagas disease. Conclusions: Our results demonstrate that KIR genes may influence both OT and the clinical digestive form of Chagas disease, whereas the MICA gene may influence the clinical forms of Chagas disease, but not the development of OT. / A toxoplasmose ocular, caracterizada por uma inflamação intraocular, é a manifestação clínica mais comum da toxoplasmose, doença infecciosa causada pelo protozoário Toxoplasma gondii. As lesões podem afetar a mácula, as diversas camadas da retina e a coróide resultando em retinocoroidite com diferentes graus de comprometimento ocular. A doença de Chagas é resultante da infecção pelo protozoário Trypanosoma cruzi. Após 20 anos de infecção, cerca de 30% dos pacientes desenvolvem a cardiopatia chagásica crônica, que se manifesta clinicamente por arritimia ventricular malígna, tromboembolismo, morte súbita cardíaca e insuficiência cardíaca crônica. Dez por cento dos pacientes apresentam a forma digestiva, caracterizada principalmente por dilatações do esôfago e/ou cólon devido ao processo de denervação. A progressão de uma infecção, assim como o desenvolvimento de diferentes formas clínicas e diferentes graus de gravidade, podem estar relacionadas com as características genéticas do patógeno e do hospedeiro. Dentre os fatores relacionados ao hospedeiro, a resposta imunológica desperta um interesse especial e os marcadores genéticos exercem importante papel modulador neste contexto, pois podem contribuir para patogênese ou resistência do curso clínico dessas infecções. Objetivo: O presente estudo teve como objetivo verificar a hipótese de que os genes KIR, seus ligantes HLA e o polimorfismo do gene MICA estão associados à toxoplasmose ocular (TO) e às diferentes formas clínicas da doença de Chagas. Casuística e métodos: Foram incluídos no estudo 297 pacientes com toxoplasmose, 148 clinicamente classificados com TO e 149 sem TO. Também participaram deste estudo 267 pacientes com doença de Chagas, 78 clinicamente classificados com a forma digestiva da doença, 107 com a forma cardíaca, 82 com a forma mista. O teste de ELISA foi realizado para confirmar as infecções por T. gondii e T. cruzi. Os polimorfismos destes genes foram identificados por PCR-SSOP e PCR nested. As variáveis contínuas foram comparadas utilizando o teste t não pareado. Para comparação dos demais resultados foram realizados o Teste Qui-quadrado com correção de Yates ou o Teste Exato de Fisher. Resultados: Em relação à toxoplasmose, alelos e genótipos MICA não diferiram entre os pacientes com e sem TO, nem entre os pacientes com a manifestação primária ou recorrente da doença. O gene KIR3DS1 foi associado positivamente com o desenvolvimento da TO. Genes KIR ativadores juntamente com os seus ligantes HLA (KIR3DS1+/Bw4-80Ile+ e KIR2DS1+/C2+ + KIR3DS1+/Bw4-80Ile+) foram associados com suscetibilidade à TO e às suas manifestações clínicas primária e recorrente. Os pares inibidores - KIR2DL3/2DL3-C1/C1 e KIR2DL3/2DL3-C1 – foram associados com a resistência à TO e suas manifestações clínicas, enquanto que a combinação KIR3DS1-/KIR3DL1+/Bw4-80Ile+ foi associada como fator de proteção para o desenvolvimento da TO e, em particular, para a manifestação recorrente. Quanto à doença de Chagas, o alelo MICA-129met foi associado como fator de risco para o desenvolvimento da disfunção sistólica ventricular esquerda (DSVE) em pacientes com cardiopatia chagásica crônica, enquanto que o alelo MICA-129val foi associado com a proteção ao desenvolvimento da DSVE. Em especial o haplótipo homozigoto MICA-129 met/met foi associado com o desenvolvimento da DSVE grave e o genótipo homozigoto MICA-129 val/val foi associado com a proteção desta condição. Também foi possível demonstrar que o haplótipo MICA*008~HLA-C*06 e a combinação entre KIR e seus ligantes HLA - KIR2DS2-/KIR2DL2-/KIR2DL3+/C1+ - foram associados como fatores de suscetibilidade à forma clínica digestiva da doença de Chagas. Conclusões: Nossos resultados demonstram que os genes KIR podem exercer influência tanto na toxoplasmose ocular quanto na forma clínica digestiva da doença de Chagas, enquanto que MICA pode exercer influência nas formas clínicas da doença de Chagas, mas não no desenvolvimento da toxoplasmose ocular.

Corticosteroide por via espinal é uma das alternativas no processo de tratamento da dor radicular quando o tratamento conservador falhou. Sua administração por via epidural vem sendo reavaliada devido ao risco de sequelas, provenientes da obstrução de vasos sanguíneos, associadas a outros efeitos adversos próprios dos corticosteroides. O presente estudo visou avaliar o efeito analgésico da administração de diferentes corticosteroides, exemplificados pela dexametasona e pela betametasona, administrados por via epidural. Vinte e seis pacientes portadores de dor neuropática secundária à hérnia de disco participaram do estudo. A punção sacral foi realizada com combinação de 40 mg de lidocaína, 30 µg de clonidina e 10 mg de dexametasona ou 10 mg de betametasona, diluídos para volume final 10 ml, com solução fisiológica a 0,9%, após comprovação com contraste não-iodado do perfeito posicionamento da agulha no espaço epidural. Este procedimento foi repetido uma vez por semana por 2 semanas seguidas, e após um intervalo de 3 semanas foi realizado mais duas vezes, uma vez por semana, sendo o total de 4 procedimentos por paciente. Treze pacientes iniciaram com dexametasona epidural durante as primeiras duas semanas, e prosseguiram com betametasona após período de descanso de três semanas, sendo que cada paciente atuou como seu próprio controle. Os pacientes foram avaliados em relação à analgesia, incidência de efeitos adversos e alteração das concentrações plasmáticas de íons, cortisol, adrenocorticotrofina e glicemia. Vinte e três pacientes completaram o estudo. Cada paciente foi o seu próprio controle e, no mesmo paciente, o efeito analgésico da dexametasona foi superior ao da betametasona. (p<0,05). Cortisol e adrenocorticotrofina plasmáticos reduziram no sétimo dia após realização do bloqueio (p<0,05), As demais medidas de concentrações plasmáticas de íons Na+, K+, Ca++, glicemia inicial, pós-prandial e hemoglobina glicada foram semelhantes entre os grupos, e sem alteração quando comparados com valores iniciais (p>0,05). Ambos grupos relataram insônia relativa nos primeiros dias pós-bloqueio (p<0,05). Não houve alteração de peso corporal e da pressão arterial durante o tratamento entre os fármacos avaliados (p>0,05). Entretanto, 3 pacientes que receberam dexametasona e 2 que receberam betametasona inicialmente, apresentam medida de pressão ocular aumentada (p<0,05). Como conclusões, a analgesia da dexametasona foi superior quando comparada à da betametasona, (p<0,05). Entretanto ambos grupos apresentaram aumento da pressão ocular e prejuízo do sono noturno, não relevante este último. Finalmente, uma vez que a dexametasona foi superior à betametasona em relação à analgesia e demonstrada ser mais segura em relação à formação de agregados, sugere-se a dexametasona como escolha. / Although the primarily indication of epidural corticosteroids as part of the treatment of acute neuropathic pain when the conservative treatment have failed, recently there have been concerns related to which corticosteroid would be the best indication, as there is the risk of particulate aggregation and serious adverse effects secondary to their own pharmacological properties or intra vessel aggregation and obliteration, as well their efficacy as analgesics. This actual study was designed to evaluate two different corticosteroids, i.e., dexamethasone and betamethasone, as coajuvants in epidural management of acute radicular pain. Twenty-six patients with history of neuropathic pain secondary to disc herniation acted as their own control related to the epidural administration of dexamethasone and betamethasone. Sacral puncture was performed with a combination of 40 mg of lidocaine, 30 µg of clonidine and 10 mg of dexamethasone or 10 mg of betamethasone, diluted to final volume 10 ml with 0.9% physiological solution after non-iodinated contrast of the perfect positioning of the needle in the epidural space.Thirteen patients have started with dexamethasone during the first two weekly procedures, and after 3 weeks of wash-out were submitted to two weekly sequence of sacral betamethasone. The other patients have started with epidural betamethasone, followed by dexamethasone after 3-week rest. Each patient acted as its own control. Patients were evaluated related to analgesia, blood pressure, weight gain, adverse effects and plasmatic measurements of ions, glicemia, ACTH and cortisol. Twenty-three patients completed the study. The analgesic effect was higher when dexamethasone was used in the same patient (p <0.05) Plasma cortisol and ACTH reduced on the 7th day after the block (p<0.05). The plasmatic concentrations of the ions Na+, K+, Ca++, control and post-prandial glicemia, blood pressure, weight were similar between groups and did no differ from initial control values (p>0.05). Both groups reported relative insomnia in the first post-blockade days (P <0.05). However, 3 patients that received dexamethasone and 2 that received betamethasone had ocular pressure increase (p<0.05). As conclusions, both drugs resulted in still unaware increase in ocular pressure and sleep disturbance. Because dexamethasone analgesia was superior and there were no differences regarding adverse effects between either epidural betamethasone or dexamethasone, based on the literature, it would be more appropriate the utilization of dexamethasone.

Thesis (M.Sc. (Optometry)) --University of Limpopo (Turfloop Campus), 2010 / Human Immunodeficiency Virus (HIV) and the associated Acquired Immunodeficiency Syndrome (AIDS) are major public health concerns worldwide and present socioeconomic burden to many governments. To combat this scourge, a deep knowledge about this pandemic is required as one of the preventive methods. Also, the knowledge can serve as a tool to help those who are infected. HIV/AIDS is common among adolescents; therefore, one of the aims of this study is to establish the knowledge of basics of HIV/AIDS among high school learners in the Capricorn district of the Limpopo Province, South Africa.HIV/AIDS also affects the eye; identification of the ocular problem can sometimes help with the diagnosis of HIV/AIDS the condition. Therefore may have both sight and life-saving implications. The school curriculum and HIV/ AIDS awareness campaigns in South Africa do not include basic knowledge of the effects of HIV/AIDS on the eye; therefore an additional aim of this study was to establish the basic knowledge of the effects of HIV/AIDS on the eye among the participants, so that an informed recommendation can be made for the inclusion of the eye component in the awareness campaign and school curriculum. Methodology Following relevant research and ethics approval of the proposal, relevant permissions were obtained before the study commenced. A quantitative survey design was used for the study. Stratified random sampling method was used to select high schools and students that took part in the research. The participants were learners from 18 selected high schools in the Capricorn district of the Limpopo Province. A questionnaire containing demographic data, information on general and ocular effects of HIV/AIDS was used to collect information from the learners, following a pilot study involving 20 high school learners. Data was analyzed with Statistical Packages for Social Sciences (SPSS) computer program version 16. Descriptive statistics (range, mean, standard deviations and frequencies) was used to describe the findings, and analysis of covariance (ANCOVA) model was used to model the scores on each of the dependent variables i.e. general knowledge of HIV/AIDS and basic knowledge of the effects of HIV/AIDS on the eye. Results The participants were 2659 black South African learners in grades 10 to 12 in the high schools. Eighty eight percent were from rural high schools and (12%) were from a semi-urban high school. Their ages ranged from 14 to 28 years with a mean age of 17.82 and SD of ±1.766 years. The participants included (46.4%) males,(53.1%) females, and (0.5%) learners did not indicate their gender.The meaning of the acronym HIV was known by 63.6% of the participants, and these included 65.8% of the females and 61.5% of males. A lesser proportion (61.5%) knew what the acronym AIDS stands for. They included 70.9% of the females and 60.8% of the males. Knowledge about the different modes of HIV transmission varied from 87.0% (transmission through unprotected sex) to 62.5% (transmission from mother-to–child during pregnancy). Regarding the misconception on the modes of HIV/AIDS transmission, a majority (81.5%) of the learners knew that HIV/AIDS could not be transmitted from one person to another through hugging a person infected with HIV; this percentage includes 82.6% of the females and 80.3% of the males.Only two respondents (0.1%) out of the total population knew the meaning of window period in HIV and they were both females. Only about a third (33.2%) knew the difference between HIV positive and negative results. Majority (85.9%) knew that it is important for one to know his or her HIV status. However, only 5.6% knew the reason why people should know their HIV status. Many (60.1%) knew that the manifestation of HIV symptoms can take many years to develop on an HIV infected person, and 54.9% knew that immune system suppression is the first effect of HIV on the body. Knowledge of the effects of HIV on the body during the seroconversion phase (acute phase), varies from 29.9% (HIV can cause continuous nausea) to 73.0% (HIV can cause tiredness). On the facilities that can be used to test for HIV status, knowledge of the participants varied from 93.4% (knew that HIV test can be done at a hospital) to 34.2% (HIV test can be done at the offices of family general medical practitioner). Only a few (27.8%) knew that HIV/ AIDS can affect the eye. Many (65.5%) of the participants knew that an ophthalmologist or an optometrist should be consulted for ocular problems that are related to HIV/AIDS. Knowledge on the effects of HIV on the eye ranges from 20.6% (HIV can cause an itching and burning sensation in the eye) to 58.6% (HIV can cause severe headaches). Forty six percent knew that HIV can cause blindness. Less than half of the participants (46.9%) knew that exchanging contact lenses with the infected person could not transmit HIV from an HIV positive person. Generally the performance was poor on the ocular manifestation of HIV/AIDS. Young learners were more knowledgeable than the older ones, for both general knowledge of HIV/AIDS and knowledge of the effect of HIV/AIDS on the eye (p < 0.05). Gender of the participants was not statistically significant for both components of the survey (p > 0.05). The pattern of performance among the grades across the schools was not consistent for both components of the survey. Also ages of the learners and their grades did not correspond in terms of performance in this study. Conclusion and recommendations General knowledge of HIV/AIDS was fairly good, but the basic knowledge of the effects of HIV/AIDS on the eye was poor. Therefore, to improve the knowledge about HIV/AIDS of the target population people, there is a need for more awareness campaigns in the rural areas of South Africa. Also, it is recommended that basic knowledge of the effects of HIV/AIDS on the eye should be included in the awareness campaigns (e.g. TV, newspapers, radio, magazines, fliers and HIV/AIDS workshops) and should also be integrated into HIV education syllabus as early as primary level. Educators teaching life orientation and life skills should have regular continuing educational programmes to increase their knowledge on the subject of HIV/AIDS and its effects on organs of the body such as the eye. / University of Limpopo

The primary aim of this research is to test the therapeutic potential of certain new generation corticosteroid drugs in order to develop safe and effective treatment for eye diseases that result in oedema, or swelling. The rising incidence of diabetes and the ageing population of developed countries mean that the prevalence of uveitis, diabetic retinopathy and age related macular degeneration will rise. Often, oedema is one of the reasons for vision loss. Corticosteroids are often used to reduce inflammation. Inflammation is one of several sources of oedema. Glucocorticoids, a class of corticosteroids that have anti-inflammatory properties, are thus used to treat ocular oedema. There is an unmet need to support clinical experience of the efficacy of steroids for ocular inflammation and oedema with more substantial scientific evidence. None of the drugs under investigation, with the exceptions of dexamethasone and triamcinolone, have been used for any ocular therapeutic purpose before. This thesis investigates “repurposing” fludrocortisone to the ophthalmic area. 11-Desoxycorticosterone (11D) and Deoxycorticosterone (DCS), other potentially valuable mineralocorticoids, remain completely untested. Lastly, Kenacort ®, or triamcinolone acetonide (TCA), is only used off-label by ophthalmologists. Methods: In the first study, corticosteroids, and especially mineralocorticoids, were investigated for their treatment efficacy in experimental uveitis, or intraocular inflammation (using a model known as endotoxin induced uveitis). In the second study, endothelial cells from choroidal blood vessels in the back of the eye were used in vitro to study whether corticosteroids reduce paracellular (between cells) permeability. Lastly, since endophthalmitis due to frequent injections is a side effect of corticosteroid use, the pharmacokinetics of different size formulations of corticosteroids were studied in an effort to find a formula that would have a prolonged dwell time within the eye.