O tratamento da ceratite por Acanthamoeba é longo, inespecífico e de baixa adesão do paciente. Assim, a busca por novas estratégias de tratamento é necessária. Extratos não aquosos de algumas espécies de Pterocaulon exibem efeito antimicótico e antiparasitário, sendo esta atividade correntemente relacionada à presença de cumarinas. Neste contexto, o presente estudo teve por objetivo o desenvolvimento de nanoemulsões contendo extrato hexânico de Pterocaulon balansae Chodat (rico em cumarinas) visando à obtenção de um produto de uso ocular com atividade amebicida. Em uma primeira etapa, o extrato hexânico foi caracterizado por cromatografia líquida de alta eficiência com detector de arranjo de diodos, detectando-se a presença de quatro cumarinas majoritárias, entre elas, a 5-metoxi-6,7-metileno dioxicumarina (5MMDC), selecionada como marcador químico do extrato vegetal. O extrato foi na sequência incorporado em nanoemulsões constituídas de um núcleo de triglicerídeos de cadeia média estabilizado por lecitina de gema de ovo, preparadas por emulsificação espontânea. Tal procedimento conduziu à obtenção de nanoemulsões monodispersas com diâmetro médio de gotícula de aproximadamente 200-300 nm, independente da quantidade de extrato adicionado à formulação (1,0 a 5,0 mg/mL). A atividade amebicida das formulções contra Acanthamoeba castellanii foi dependente da dose e do tempo de incubação, sendo 24 horas e a concentração de 1,25 mg/mL de extrato considerada como ótima (~5% de viabilidade), com efeito similar ao controle clorexidina. Enfim, os estudos de citotoxicidade in vitro demonstraram que as células de epitélio de córnea humana (HCE) não foram afetadas com a incubação com as nanoemulsões através do ensaio de MTT. Esses resultados sugerem o potencial do extrato hexânico, rico em cumarinas de Pterocaulon balansae, associado a nanoemulsões como uma nova estratégia para o tratamento da ceratite ocular causada por Acanthamoeba. / Treatment for keratitis caused by Acanthamoeba is unspecific, long term and with low patient compliance, being the search of new treatment strategies a need. Non-aqueous extracts of some species of Pterocaulon exhibit antimycotic and anti-parasitic activity, usually attributed to the presence of coumarins. In this scenario, the aim of this work is the development of nanoemulsion to associate the coumarin-rich n-hexane extract of Pterocaulon balansae. Previously, the extract was prepared and characterized by high performance liquid chromatography with photodiode array detector. The presence of 4 major coumarins was detected, where 5-methoxy-6,7-methylene dioxycoumarin (5MMDC) was selected as chemical marker. This extract was associated to nanoemulsions composed of egg lecithin and medium chain triglycerides, prepared by spontaneous emulsification. The physicochemical characterization showed the formation of monodisperse nanoemulsions with 200-300 nm diameter, regardless the amount of extract incorporated (1.0-5.0 mg/mL). The amoebicidal activity of the formulations against Acanthamoeba castellanii was both dose-dependent and incubation time-dependent, being 24h of incubation and concentration of 1.25 mg/mL of the extract the optimal (~5% viability), with effect similar to chlorexidine control. Finally, in vitro cytotoxicity studies showed that human corneal epithelial cells were unaffected after incubation with nanoemulsions by MTT assay. These results suggest a potential of the coumarin-rich n-hexane extracts of Pterocaulon balansae associated to nanoemulsion as a new strategy for the treatment of ocular keratitis caused by Acanthamoeba.

Made available in DSpace on 2014-06-11T19:30:11Z (GMT). No. of bitstreams: 0 Previous issue date: 2011-07-20Bitstream added on 2014-06-13T18:40:04Z : No. of bitstreams: 1 perches_cs_me_botfmvz_parcial.pdf: 57363 bytes, checksum: 7194bc6e69ce3bcd9c7d95014e54cd86 (MD5) Bitstreams deleted on 2015-06-25T13:01:27Z: perches_cs_me_botfmvz_parcial.pdf,. Added 1 bitstream(s) on 2015-06-25T13:03:36Z : No. of bitstreams: 1 000692053_20160720.pdf: 57206 bytes, checksum: d761ff391f9b209a38f7349c5ee3c1f1 (MD5) Bitstreams deleted on 2016-07-25T13:17:34Z: 000692053_20160720.pdf,. Added 1 bitstream(s) on 2016-07-25T13:18:43Z : No. of bitstreams: 1 000692053.pdf: 7043915 bytes, checksum: fa994284baa896bbc842afa959d5378b (MD5) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / O objetivo do presente estudo foi avaliar e comparar por meio de exames oftalmológicos, histopatológicos e imuno-histoquímica para PCNA, o processo de reparação corneal de úlceras superficiais induzidas em coelhos, frente à utilização de colírios de óleo essencial de Citrus lemon, produto até então não utilizado em oftalmologia. Foram utilizadas 50 fêmeas da espécie leporina, todas submetidas à indução da úlcera superficial, constituindo-se 5 grupos experimentais de 10 animais cada. Em três grupos foram instilados colírios à base de óleo essencial de Citrus lemon, em diferentes concentrações, sendo 1,5% (GL1,5), 3% (GL3) e 5% (GL5). O grupo controle (GC) recebeu substituto da lágrima, e o grupo Tween 80 8% (GT) foi tratado com o diluente utilizado na produção dos colírios de citrus. Todos os protocolos foram realizados quatro vezes ao dia. Os grupos foram divididos, aleatoriamente, em dois subgrupos, de acordo com o período final de avaliação, sendo M1, coelhos avaliados após 24 horas, e M5, após 5 dias. Não houve diferença entre os tratamentos utilizados quanto aos sinais clínicos secreção ocular, hiperemia conjuntival, quemose e opacidade corneal. O grupo tratado com colírio de óleo essencial de Citrus lemon na concentração 1,5%, no período final de avaliação, apresentou maior sensibilidade ocular em relação ao GC, além de aumento da celularidade corneal, representada principalmente por células inflamatórias. Nas comparações entre os momentos iniciais e finais, os grupos tratados com substituto da lágrima, Tween 80 8% e colírio à base de óleo essencial de Citrus lemon 5% promoveram aumento na espessura epitelial na periferia da córnea e maior percentual de proliferação celular, na avaliação final. Quanto à úlcera de córnea, houve redução significativa da extensão total para leve... / The purpose of this study was to evaluate and compare through ophthalmic exams, histopathology and immunohistochemistry for PCNA, the repair process in superficial corneal ulcers induced in rabbits using eyedrops of Citrus lemon essential oil, a product not used in ophthalmology yet. Fifty female rabbits were submitted to induction of the corneal ulcer and then divided into 5 experimental groups of 10 animals each. Were instilled into three groups eyedrops of Citrus lemon essential oil, in different concentrations, being 1.5% (GL1, 5), 3% (GL3) and 5% (GL5). The control group (CG) received a tear substitute, and the Tween 80 8% group (GT) was treated with the diluent used in the production of eyedrops of citrus. All protocols were performed four times a day. The groups were divided randomly into two subgroups, according to the final period of evaluation, in the M1 rabbits were evaluated after 24 hours, and in the M5 after 5 days. There was no difference between treatments for clinical signs: ocular discharge, conjunctival hyperemia, chemosis and corneal opacity. In the final period of evaluation, the group treated with eyedrops of Citrus lemon essential oil in the concentration 1.5% showed a higher ocular sensitivity compared to the GC, and increased of corneal cellularity, represented mainly by inflammatory cells. In the comparison between the initial and final moments, the groups treated with tear substitute, Tween 80 8% and eyedrops of Citrus lemon essential oil 5% led to an increase in epithelial thickness at the periphery of the cornea and a higher percentage of cell proliferation, in the final evaluation. In the clinical evaluation of corneal ulcer, there was significant reduction in total length for mild or absent at the time of final evaluation in all groups; the exception was GL 3%, that showed a lower... (Complete abstract click electronic access below)

O tratamento da ceratite por Acanthamoeba é longo, inespecífico e de baixa adesão do paciente. Assim, a busca por novas estratégias de tratamento é necessária. Extratos não aquosos de algumas espécies de Pterocaulon exibem efeito antimicótico e antiparasitário, sendo esta atividade correntemente relacionada à presença de cumarinas. Neste contexto, o presente estudo teve por objetivo o desenvolvimento de nanoemulsões contendo extrato hexânico de Pterocaulon balansae Chodat (rico em cumarinas) visando à obtenção de um produto de uso ocular com atividade amebicida. Em uma primeira etapa, o extrato hexânico foi caracterizado por cromatografia líquida de alta eficiência com detector de arranjo de diodos, detectando-se a presença de quatro cumarinas majoritárias, entre elas, a 5-metoxi-6,7-metileno dioxicumarina (5MMDC), selecionada como marcador químico do extrato vegetal. O extrato foi na sequência incorporado em nanoemulsões constituídas de um núcleo de triglicerídeos de cadeia média estabilizado por lecitina de gema de ovo, preparadas por emulsificação espontânea. Tal procedimento conduziu à obtenção de nanoemulsões monodispersas com diâmetro médio de gotícula de aproximadamente 200-300 nm, independente da quantidade de extrato adicionado à formulação (1,0 a 5,0 mg/mL). A atividade amebicida das formulções contra Acanthamoeba castellanii foi dependente da dose e do tempo de incubação, sendo 24 horas e a concentração de 1,25 mg/mL de extrato considerada como ótima (~5% de viabilidade), com efeito similar ao controle clorexidina. Enfim, os estudos de citotoxicidade in vitro demonstraram que as células de epitélio de córnea humana (HCE) não foram afetadas com a incubação com as nanoemulsões através do ensaio de MTT. Esses resultados sugerem o potencial do extrato hexânico, rico em cumarinas de Pterocaulon balansae, associado a nanoemulsões como uma nova estratégia para o tratamento da ceratite ocular causada por Acanthamoeba. / Treatment for keratitis caused by Acanthamoeba is unspecific, long term and with low patient compliance, being the search of new treatment strategies a need. Non-aqueous extracts of some species of Pterocaulon exhibit antimycotic and anti-parasitic activity, usually attributed to the presence of coumarins. In this scenario, the aim of this work is the development of nanoemulsion to associate the coumarin-rich n-hexane extract of Pterocaulon balansae. Previously, the extract was prepared and characterized by high performance liquid chromatography with photodiode array detector. The presence of 4 major coumarins was detected, where 5-methoxy-6,7-methylene dioxycoumarin (5MMDC) was selected as chemical marker. This extract was associated to nanoemulsions composed of egg lecithin and medium chain triglycerides, prepared by spontaneous emulsification. The physicochemical characterization showed the formation of monodisperse nanoemulsions with 200-300 nm diameter, regardless the amount of extract incorporated (1.0-5.0 mg/mL). The amoebicidal activity of the formulations against Acanthamoeba castellanii was both dose-dependent and incubation time-dependent, being 24h of incubation and concentration of 1.25 mg/mL of the extract the optimal (~5% viability), with effect similar to chlorexidine control. Finally, in vitro cytotoxicity studies showed that human corneal epithelial cells were unaffected after incubation with nanoemulsions by MTT assay. These results suggest a potential of the coumarin-rich n-hexane extracts of Pterocaulon balansae associated to nanoemulsion as a new strategy for the treatment of ocular keratitis caused by Acanthamoeba.

Yes / In the study of microbial keratitis, in vivo animal models often require a large number of animals, and in vitro monolayer cell culture does not maintain the three-dimensional structure of the tissues or cell-to-cell communication of in vivo models. Here, we propose reproducible ex vivo models of single- and dual-infection keratitis as an alternative to in vivo and in vitro models. / Wellcome Trust

Yes / Microbial keratitis occurs from the infection of the cornea by fungi and or bacteria. It remains one of the most common global causes of irreversible blindness accounting for 3.5% (36 million) of blind people as of 2015. This paper looks at the use of a bacteria binding polymer designed to bind Staphylococcus aureus and remove it from the corneal surface. Mechanical unbinding measurements were used to probe the interactions of a thermo-active bacteria-binding polymer, highly-branched poly(N-isopropyl acrylamide), functionalised with modified vancomycin end groups (HB-PNIPAM-Van) to bacteria placed on rabbit corneal surfaces studied ex-vivo. This was conducted during sequential temperature phase transitions of HB-PNIPAM-Van-S. aureus below, above and below the lower critical solution temperature (LCST) in 3 stages, in-vitro, using a novel micro-bead force spectroscopy (MBFS) approach via atomic force microscopy (AFM). The effect of temperature on the functionality of HB-PNIPAM-Van-S. aureus showed that the polymer-bacteria complex reduced the work done in removing bacterial aggregates at T > LCST (p < 0.05), exhibiting reversibility at T < LCST (p < 0.05). At T < LCST, the breaking force, number of unbinding events, percentage fitted segments in the short and long range, and the percentage of unbinding events occurring in the long range (> 2.5 µm) increased (p < 0.05). Furthermore, the LCST phase transition temperature showed 100 × more unbinding events in the long-range z-length (> 2.5 µm) compared to S. aureus aggregates only. Here, we present the first study using AFM to assess the reversible mechanical impact of a thermo-active polymer-binding bacteria on a natural corneal surface. / This work was funded by the Medical Research Council (MRC-DBT 'UKICAT-minimising antibiotic use', MR/N501888).

Acanthamoeba culbertsoni (A. culbertsoni) is an amphizoic amoeba that is the causative agent of Granulomatous Amoebic Encephalitis (GAE), an often fatal central nervous system infection that is seen most frequently in severely immunocompromised patients and is characterized by hemorrhagic and necrotic lesions of the brain as well as varying degrees of granuloma formation. A.culbertsoni isolates have also been identified in a few cases of Amoebic Keratitis, a painful, sight-threatening corneal infection that disproportionately affects contact lens users irrespective of immune status. Common features of both infections include amoebic interaction with host extracellular matrix (ECM) components as requisites for both attachment to, and subsequent invasion of, host tissues to facilitate disease establishment. Previous studies have demonstrated that pathogenic species of Acanthamoeba , such as A.culbertsoni, bind to the ECM proteins Laminin-1 and Collagen I to a greater extent than non-pathogenic species. It has also been documented in the literature that secreted Acanthamoeba proteases have the ability to degrade components of the extracellular matrix. The role of amoebic proteases in mediating the attachment and invasion processes is not entirely understood. Initial experiments conducted in the present study revealed secretion of approximately 150 and 55-kDa serine proteases during attachment as well as invasion of the ECM by A. culbertsoni. However, inhibition of these serine proteases using phenylmethylsulfonyl fluoride (PMSF) did not diminish the ability of amoebae to attach or invade. It was demonstrated that secretion of the observed proteases occurred in a constitutive rather than substrate-induced manner and that amoebae secrete these proteases under a number of different conditions. Additionally, a 140-kDa membrane-associated serine protease was identified which may prove to play a role in focal proteolytic degradation. Collectively, our results suggest that attachment to extracellular matrix components is mediated through non-protease-dependent mechanisms. We also suggest that ECM invasion by A.culbertsoni is predominately a mechanical process that may be supplemented or enhanced by focal proteolytic degradation of extracellular matrix components by membrane-associated proteases.

A incidência de ceratite infecciosa relacionada a lentes de contato ocasionada por espécies de Acanthamoeba e Fusarium tem aumentado nos últimos anos. Esses organimos compartilham vários ambientes e casos de coinfecção foram relatados. Interações entre amebas e outros micro-organismos podem resultar em mudanças significativas para ambos, como o aumento da virulência em hospedeiros mamíferos. Neste estudo, foi avaliada a interação das cepas Neff, T4 e T4 pulmão de Acanthamoeba castellanii com Fusarium solani, bem como as possíveis implicações no desenvolvimento de ceratite. A. castellanii foi capaz de fagocitar F. solani e os conídios foram capazes de germinar dentro da ameba. A cocultura com ameba viva, bem como o sobrenadante da cultura e o lisado amebiano aumentaram significativamente o crescimento do fungo. Além disso, F. solani vivo e o seu sobrenadante de cultura aumentaram a sobrevivência da ameba, mas de forma diferente em cada cepa amebiana. A presença do fungo resultou em aumento de encistamento das cepas amebianas T4 pulmão e T4, bem como aumentou a resistência da cepa Neff à clorexidina. Curiosamente, a passagem pela ameba aumentou o efeito citopático de F. solani em células de mamíferos. Esses resultados mostram que A. castellanii e F. solani têm uma relação de protocooperação, com benefícios para ambos, e especialmente, indicam um possível efeito sobre as características de virulência de ambos os organismos. Assim a interação entre A. castellanii e F. solani pode resultar em impactos graves nos casos de ceratite. / The incidence of Acanthamoeba and Fusarium species has increased in contact lens-related infectious keratitis. They share several environments and cases of co-infection have been reported. The interaction between the amoeba and other microorganisms may result in significant changes for both, like increased virulence in mammalian hosts. In this study, we evaluated the interaction of Neff, T4 and T4 lung strains of Acanthamoeba castellanii with Fusarium solani and the implications in keratitis. A. castellanii phagocyted the fungi, and conidia were able to germinate inside the amoeba. The co-culture with live amoeba as well as the amebic culture supernatant and lysate significantly increased fungal growth. Moreover, live F. solani and the its culture supernatant enhanced the survival of amoeba, but differently in each A. castellanii strain. The presence of fungi resulted in increased amoebal encystment of T4 and T4 lung strains, end increased the resistence of Neff strain to chlorhexidine. The encystment of T4 and T4 lung strains was increased by the fungi. Interestingly, the passage by Neff amoeba increased the cytophatic effect of F. solani on mammalian cells linage. These results show that A. castellanii and F. solani have a protocooperation relationship, with benefits for both, and especially indicate a possible effect on virulence characteristics of both organisms. These data suggest that the A. castellanii-F. solani interaction may cause severe impacts in keratitis.

This thesis describes the impact of contact lens-related microbial keratitis in terms of incidence and severity. Disease outcome is defined by visual outcome, costs to the healthcare system, costs to the individual and duration of disease. A successful 12-month surveillance study was conducted of the populations of Australia and New Zealand to detect all cases of contact lens-related microbial keratitis. A random telephone survey of 32,000 households in Australia and 7,500 in New Zealand accurately determined the level of use of various contact lenses in the community. The impact of new contact lens types: silicone hydrogels and daily disposables were investigated. Increased risk persisted in overnight wear with silicone hydrogel materials. Microbial keratitis associated with silicone hydrogel materials had slightly shorter disease duration however other factors had a stronger influence on severity. Rigid gas permeable and frequent replacement soft lenses when used for daily wear constitute the lowest risk. Cost analysis was developed in a hospital case series of microbial keratitis. This analysis was applied in the surveillance study including cases managed in the private health care sector. Disease duration and associated costs are novel indices of severity for contact lens-related disease. The most dramatic effects on disease severity were seen with the type of organism involved. Keratitis attributed to environmental organisms (Gram-negative bacteria, Acanthamoeba, fungi and Nocardia species) were 10x more likely to cause loss of visual acuity, had longer duration of symptoms and incurred higher costs. Importantly, delays in receiving treatment increased disease duration and associated costs. Greater awareness of the need for specialist healthcare is indicated amongst health care providers and contact lens wearers. The hypothesis that overnight wear in silicone hydrogel lenses would not increase the risk of infection has been disproven. This information is of value to practitioners who are responsible for informing contact lens wearers about the risk of contact lens-related infections and should be weighed against the benefits of continuous wear. The identification of factors which contribute to the outcomes of disease will be used in education campaigns amongst health care providers and contact lens wearers to minimise the impact of disease.

[Truncated abstract] The cornea plays a major role in the refraction of light and thus the maintenance of its transparency is critical for optimal vision. Infection or trauma can initiate a host inflammatory response, which can cause edema of the collagenous stroma. This tissue edema compromises vision by disrupting the regular arrangement of the corneal stromal lamellae, whose organization is critical to its refractive properties. Until recently, it was the accepted dogma that the cornea was an immune privileged tissue owing in part to its avascular nature and paucity of resident macrophages and dendritic cells (DCs) in the central region of the cornea. However, recent studies have identified heterogenous populations of macrophages and DCs in both the corneal stroma and epithelium. Despite the recognition of the existence of these cells in the cornea, very little is known about their biological role. The overall purpose of the experiments described in this thesis is to characterise corneal macrophages and DCs in homeostatic conditions and investigate their role in the initiation of inflammatory responses to bacterial ligands that induce corneal inflammation and contribute to the severity and resolution of bacterial keratitis. Experiments described in this thesis utilized a range of transgenic, knock-out and bone marrow (BM) chimeric mice to address the immunological function and characterization of BM-derived cells in the mouse cornea. Of particular importance was the use of Cx3cr1 transgenic mice, which contain an enhanced green fluorescent protein (eGFP) encoding cassette knocked into the Cx3cr1 gene that disrupts its expression but facilitates GFP expression under the control of the Cx3cr1 promoter. ... This highlights a novel functional role for corneal BM-derived cells in the recognition and initiation of inflammatory responses to LPS. Finally, a novel observation of a potential mechanism by which DC in the cornea communicate with neighbouring DCs via fine membrane extensions was identified in both chimeric and wild-type mice. These membrane nanotubes, found exclusively on MHC class II+ cells in the corneal stroma, significantly increased in density in the central cornea under inflammatory conditions, suggesting a role for these cell protrusions in the immune response. These data represent the first ever description of nanotubes in vivo, the only previous evidence of their presence being in vitro studies. In summary, the data presented in this thesis supports a role for Cx3cr1 in the homing of DCs to the normal corneal epithelium and also suggests that Cx3cr1-deficiency may influence the ability of corneal macrophages and DCs to respond to bacteria. In addition, the thesis supports a role for resident corneal macrophages and DCs in the initiation of immune responses following challenge with LPS, which is possibly supported by a newly discovered system of membrane nanotubes. A greater understanding of the biology of the resident corneal immune cells could lead to the development of potential therapies aimed at targeting macrophages and DCs as a means of regulating potentially harmful inflammatory responses in the cornea.

Fusarium solani is a soil-borne pathogen devastating agricultural crops throughout the world. While most pathogens are host specific, the fusaria are able to infect both plants and animals. In 2004, an outbreak of Fusarium occurred in association with contact lens wear. Several species of Fusarium were involved but F. solani and F. oxysporum were most prominent. In this work, we have identified a MAP kinase (MAPK), highly similar to fmk1 in F. oxysporum, belonging to the YERK1 subfamily of extracellular regulated kinases. Directed disruption of fmk1 in F. solani AFR4 (FSSC 1) affected several aspects of fungal growth and pathogenicity. Colonies of AFR4„½fmk1 displayed pigmented colony-like formation as opposed to the cottony-white diffuse growth observed with the wild-type strain. Mutants displayed changes in morphology and conidiation patterns with AFR4„½fmk1 mutants producing increased amounts of macroconidia vs. microconidia. AFR4„½fmk1 germ tube emergence was similar to that of wild-type AFR4 and wet weight growth was equivalent but germinules were not able to sense nutrient in chemotropic assays. The disruption of fmk1 increased spore surface hydrophobicity leading to a decrease in association with commercially available hydrogel contact lenses. FMK1 did not affect unworn lens penetration in phosphate buffered saline as both wild-type and mutant strains were able to penetrate commercially available silicone hydrogel contact lenses. AFR4 displayed increased penetration of silicone lenses and this is likely due to: increased spore/lens association, and the inability of AFR4„½fmk1 macroconidia to germinate in phosphate buffered saline. Diminished invasive growth was also noted with disruption of fmk1. Wild-type AFR4 was detected throughout the eye after conidial microinjection while AFR4„½fmk1 was limited to the vitreous fluid. The reduced invasive growth seen is attributed to the loss of chemotropism. The ability to sense and respond to the environment is essential for pathogenicity. MAPK has been deemed essential for plant pathogenicity and now, with its affect on chemotropism, appears to be necessary for animal pathogenicity. FMK1 plays a conserved role in the pathogenicity of Fusarium.