An objective tool to measure the effect of botulinum toxin in blepharospasm and hemifacial spasm.

BACKGROUND AND PURPOSE: It is challenging to assess patients with blepharospasm (BSP) and hemifacial spasm (HFS) as these patients exhibit a wide range of amplitudes of eyelid movements. In order to quantify these movements, a mathematical algorithm, i.e. Fast Fourier Transform, can be employed to convert the signal from the time domain to the frequency domain. The result of this quantification represents the energy generated during the eyelid movements. In order to objectively assess the therapeutic effects of botulinum toxin (BoNT) in these patients, we evaluated the energy generated by the upper eyelid during spontaneous eyelid movements before and after treatment. METHODS: A total of 39 patients with BSP and HFS were evaluated before and 30 days after receiving onabotulinum toxin A injections. A high-speed camera and micro light-emitting diodes were used to register the spontaneous eyelid movements. The result of the quantification obtained using Fast Fourier Transform permitted assessment of the activity associated with the eyelid movements. RESULTS: We studied 78 eyelids. The total energy generated during spontaneous eyelid movements was significantly reduced after treatment in the patients with BSP (P = 0.0018) and on the affected side in the patients with HFS (P = 0.0058). CONCLUSIONS: The assessment of the energy generated by the upper eyelid during spontaneous eyelid movements enabled us to measure the therapeutic effects of BoNT in patients with these conditions. The use of this system could enable customized and fine adjustments to BoNT doses based on each patient's needs.

[Ocular manifestations of Zika virus: What we do and do not know]. / Manifestations oculaires du virus Zika : où en sommes-nous ?

Zika virus (ZIKV) disease outbreak, which was declared by the end of 2015 in Brazil, has become the largest one to date. Being reported in the Americas and in certain islands of the Pacific, it has the potential to spread worldwide. Although ZIKV infections are mostly self-limiting and/or asymptomatic in the healthy adult, they are responsible for devastating congenital neurologic malformations ZIKV (mainly microcephaly) when contracted during the first months of pregnancy. Ocular manifestations during the acute adult infection include conjunctivitis and more rarely ocular inflammation. Congenital infection is associated with chorioretinal atrophy pigment mottling of the retina retinal vasculature abnormalities and optic nerve atrophy. Therefore, complete ophthalmological evaluation is recommended for suspected congenital infections.

Cigarette toxicity triggers Leber's hereditary optic neuropathy by affecting mtDNA copy number, oxidative phosphorylation and ROS detoxification pathways.

Leber's hereditary optic neuropathy (LHON), the most frequent mitochondrial disease, is associated with mitochondrial DNA (mtDNA) point mutations affecting Complex I subunits, usually homoplasmic. This blinding disorder is characterized by incomplete penetrance, possibly related to several genetic modifying factors. We recently reported that increased mitochondrial biogenesis in unaffected mutation carriers is a compensatory mechanism, which reduces penetrance. Also, environmental factors such as cigarette smoking have been implicated as disease triggers. To investigate this issue further, we first assessed the relationship between cigarette smoke and mtDNA copy number in blood cells from large cohorts of LHON families, finding that smoking was significantly associated with the lowest mtDNA content in affected individuals. To unwrap the mechanism of tobacco toxicity in LHON, we exposed fibroblasts from affected individuals, unaffected mutation carriers and controls to cigarette smoke condensate (CSC). CSC decreased mtDNA copy number in all cells; moreover, it caused significant reduction of ATP level only in mutated cells including carriers. This implies that the bioenergetic compensation in carriers is hampered by exposure to smoke derivatives. We also observed that in untreated cells the level of carbonylated proteins was highest in affected individuals, whereas the level of several detoxifying enzymes was highest in carriers. Thus, carriers are particularly successful in reactive oxygen species (ROS) scavenging capacity. After CSC exposure, the amount of detoxifying enzymes increased in all cells, but carbonylated proteins increased only in LHON mutant cells, mostly from affected individuals. All considered, it appears that exposure to smoke derivatives has a more deleterious effect in affected individuals, whereas carriers are the most efficient in mitigating ROS rather than recovering bioenergetics. Therefore, the identification of genetic modifiers that modulate LHON penetrance must take into account also the exposure to environmental triggers such as tobacco smoke.

An intraocular dye solution based on lutein and zeaxanthin in a surrogate internal limiting membrane model: a Langmuir monolayer study.

Investigating the role of biomolecules and bioactive molecules associated with membranes is fundamental to comprehend at the molecular point-of-view biochemical and clinical processes that occur at biointerfaces. In this paper we exploit the interaction of an intraocular dye solution based on lutein and zeaxanthin in surrogate internal limiting membrane (ILM) models, consisting of dipalmitoyphosphatidylcholine (DPPC) Langmuir monolayers, pure or mixed with collagen, proteoglycan and laminin. The interactions between the film components occurring at the air-water interface were investigated with surface pressure-area isotherms and polarization modulation infrared reflection-absorption spectroscopy (PM-IRRAS). A natural dye solution based on lutein and zeaxanthin, employed to label ILM in ophthalmic surgery, was incorporated in the ILM model, and the data suggested non-rupture of the structure of the membrane, with predominance of interactions based on intermolecular forces.

The Standardization of Uveitis Nomenclature (SUN) Project. Development of a clinical evidence base utilizing informatics tools and techniques.

BACKGROUND: Given the recent increased focus on evidence-based medicine, it is critical that diseases and syndromes have accurate and complete descriptions, including standardized and widely accepted terminologies. Standardizing these descriptions and terminologies is necessary to develop tools such as computerized data entry forms and classification criteria. This need is especially true for diseases that are relatively uncommon, such as uveitis. OBJECTIVES: To develop a standardized and internationally accepted terminology for the field of uveitis. METHODS: The Standardization of Uveitis Nomenclature (SUN) Working Group (WG) is an international group of 79 uveitis experts from 18 countries and 62 clinical centers. Initial terminology was developed utilizing a "modified" green field approach, which was enhanced through web-based surveys and teleconferences via a "modified" Delphi technique. Terms were mapped provisionally into ontologic dimensions for each syndrome. The Working Group then met and utilized nominal group techniques as a formalized method of finalizing the mappings. RESULTS: Mapping of terms into dimensions to describe 28 major uveitic diseases was confirmed using nominal group techniques (achieving super-majority consensus) for each of the diseases at a meeting of the entire WG. CONCLUSIONS: The SUN WG utilized an informatics-based approach to develop a standardized and internationally accepted terminology for the uveitides.

IL-10 and TGF-beta immunoregulatory cytokines rather than natural regulatory T cells are associated with the resolution phase of Vogt-Koyanagi-Harada (VKH) syndrome.

The pro-inflammatory cytokines play a critical role in the initiation and propagation of ocular autoimmune diseases. Regulation of these cytokines is generally mediated by the immunoregulatory cytokine such as IL-10 or TGF-beta. In this study, we investigated the immunoregulatory cytokine profile and frequency of natural regulatory T cells (nTregs) in patients with Vogt-Koyanagi-Harada (VKH). We obtained the peripheral blood mononuclear cells (PBMC) from patients with VKH and healthy controls. The cytokine profile from supernatants of PBMC cultured with or without phytohaemagglutinin (PHA) was measured by ELISA, the percentage of CD4(+) Foxp3(+) and CD25(high)Foxp3(+) T regulatory cells were analysed by flow cytometry, and the transcriptional level of Foxp3 expression was analysed by real-time quantitative PCR. The immunoregulatory cytokines, TGF-beta and IL-10, increased in patients with VKH in the inactive stage of the disease. We observed no significant difference in the CD4(+) Foxp3(+) and CD25(high)Foxp3(+) T cells as well as no reduction in FOXP3 mRNA expression in the patients with VKH when compared to healthy controls. We showed in our work, an increase in IFN-gamma secretion by PBMC of patients with VKH in the active stage of the disease when compared to healthy controls and patients in the inactive stage. Our data suggest that IL-10 and TGF-beta cytokines, rather than nTregs are associated with the resolution phase of the disease and may have a more relevant role in controlling this disease.

A heparin mimetic isolated from a marine shrimp suppresses neovascularization.

BACKGROUND: Choroidal neovascularization (CNV) is the main cause of severe visual loss in age-related macular degeneration (AMD). Heparin/heparan sulfate are known to play important roles in neovascularization due to their abilities to bind and modulate angiogenic growth factors and cytokines. Previously, we have isolated from marine shrimp a heparin-like compound with striking anti-inflammatory action and negligible anticoagulant and hemorrhagic activities. OBJECTIVES: To investigate the role of this novel heparin-like compound in angiogenic processes. METHODS AND RESULTS: The anti-angiogenic effect of this heparinoid in laser-induced CNV and in vitro models is reported. The compound binds to growth factors (FGF-2, EGF and VEGF), blocks endothelial cell proliferation and shows no cytotoxic effect. The decrease in proliferation is not related to cell death either by apoptosis or secondary necrosis. The results also showed that the heparinoid modified the 2-D network organization in capillary-like structures of endothelial cells in Matrigel and reduced the CNV area. The effect on CNV area correlates with decreases in the levels of VEGF and TGF-ß1 in the choroidal tissue. The low content of 2-O-sulfate groups in this heparinoid may explain its potent anti-angiogenic effect. CONCLUSIONS: The properties of the shrimp heparinoid, such as potent anti-angiogenic and anti-inflammatory activities but insignificant anticoagulant or hemorrhagic actions, point to this compound as a compelling drug candidate for treating neovascular AMD and other angioproliferative diseases. A mechanism for the anti-angiogenic effect of the heparinoid is proposed.

Bilateral retinochoroiditis caused by an atypical strain of Toxoplasma gondii.

BACKGROUND: A 53-year-old man presented with an acute bilateral posterior uveitis with extensive necrotising retinochoroiditis but without chorioretinal scarring. A thorough workup did not reveal any underlying disease. The possibilities of atypical ocular toxoplasmosis as well as herpetic retinal necrosis were considered and specific therapy instituted, with little improvement. The patient died within 2 months as result of an undifferentiated squamous cell carcinoma. METHODS: Histopathological examination, immunohistochemistry and multilocus polymerase chain reaction confirmed Toxoplasma gondii infection of the retina RESULTS: Macroscopic examination of enucleated globe showed extensive retinal necrosis and vitreous detachment. Histological examination of retinal tissue identified numerous round-to-elliptical toxoplasmic cysts within the retina, with retinal necrosis and minimal choroidal inflammation. Immunohistochemical analyses confirmed that the cysts were due to T gondii. DNA extracted from formalin-fixed, paraffin-embedded tissue sections was subjected to multilocus polymerase chain reaction (PCR) analysis at the following typing loci: SAG1, SAG2, SAG3, SAG4, B1, NTS2, GRA6 and GRA7. DNA sequencing of positive PCR products at the NTS2, SAG1 and GRA7 loci confirmed the presence of a non-archetypal strain of T gondii infecting the eye of the patient experiencing a severe, atypical ocular toxoplasmosis CONCLUSION: A highly divergent, non-archetypal strain of T gondii was responsible for causing a severe, atypical bilateral retinochoroiditis in a patient from Brazil.

Twenty years of acanthamoeba keratitis.

PURPOSE: We described the rate of Acanthamoeba keratitis (AK) in a referral eye center in São Paulo, Brazil, through a retrospective review of clinical and laboratorial records of patients over 2 decades. METHODS: From 1987 to 2006, a total of 581 requests for amoebic laboratory workup in cases of infectious keratitis were investigated. Statistical analyses were applied to analyze a tendency of AK cases. RESULTS: Acanthamoeba species were cultured from corneal scrapings of 185 patients, 5 of them with bilateral infection. Eighty-three percent of those patients were related with contact lens wear. CONCLUSIONS: The results suggested that patients with AK have persisted and increased over time at our ophthalmology center. Contact lenses showed to be a potential risk factor. Amoebic corneal infection can be considered as a new but well-established disease in Brazilian ophthalmology and visual sciences.

Dose-dependent in vitro inhibition of rabbit corneal matrix metalloproteinases by an extract of Pothomorphe umbellata after alkali injury.

The in vitro ability of Pothomorphe umbellata ethanolic crude extract to inhibit matrix metalloproteinase (MMP) in normal cornea and in cornea after alkali injury was demonstrated. Corneas of albino rabbits were injured with 1 N NaOH for 20 s. After 48 h the corneas were excised, homogenized and analyzed for MMP-9 (92 kDa), pro-MMP-2 (72 kDa) and MMP-2 (67 kDa) activity by gelatin zymography. The activity was also measured in untreated corneas. After electrophoresis of 20 microg protein, gels were incubated with 50, 100, or 250 microg/mL lyophilized hydroethanolic (1:1) root crude extract of P. umbellata standardized for 4-nerolidylcatechol (7.09%). The activity of the enzymes was compared with that of untreated gel. At 48 h after injury, the activity of all MMPs was increased compared with untreated eyes. When the gels were incubated with P. umbellata extract the activity of MMP-2, pro-MMP-2 and MMP-9 decreased in a dose-dependent manner. MMP-9 activity decreased by approximately 50% after incubation with 50 microg/mL and was completely abolished at 100 and 250 microg/mL of the extract. After incubation with 50 microg/mL the activity of pro-MMP-2 and MMP-2 also decreased by 50%. The activity of pro-MMP-2 was almost completely abolished after incubation with 250 microg/mL of the extract. For MMP-2 the incubation with 100 or 250 microg/mL of the extract of P. umbellata promoted a 10-fold decrease in activity. In conclusion, P. umbellata root crude extract can be useful as an alternative therapy to control MMP activity after corneal injury.

NK1.1 cells downregulate murine endotoxin-induced uveitis following intraocular administration of interleukin-12.

To evaluate the role of IFN-gamma (interferon gamma) in IL-12- (interleukin-12)-induced inhibition of the inflammatory response in the eye during endotoxin-induced uveitis (EIU). C57BL/6 wild type mice and IFN-gamma-deficient (GKO) mice were injected with 250 microg of Salmonella typhymurium endotoxin as a model for EIU. Animals were then injected intraocularly with 100 ng of rIL-12 or the equivalent volume of Phosphate-buffer saline (PBS). Histopathologic grading of disease was performed 12, 36 and 72 h after endotoxin injection. Chemokine mRNA expression in the eye was evaluated by reverse transcriptase-polymerase chain reaction. Depletion of NK1.1+ cells in vivo was performed using a PK136 antibody. Depletion of IFN-gamma was performed using the R4-6A2 antibody. C57BL/6 mice treated with rIL-12 intraocularly were protected from the development of EIU. Neutralization of IFN-gamma with a monoclonal antibody abrogated such protection. The IL-12 protective effects were lost in NK1.1-depleted mice. Intraocular IL-12 decreased the expression of keratinocyte-derived chemokines (KC) gene but had no effect on macrophage inflammatory protein (MIP-2) gene. The protective effect of IL-12 during EIU occurs through production of IFN-gamma by NK1.1+ cells. IL-12-induced higher levels of IFN-gamma are also correlated with lower expression of the chemokine KC, resulting in diminished attraction of neutrophils to the inflammatory site.

Nonspecific provocation of target organs in allergic diseases: EAACI-GA(2)LEN consensus report.

It is widely accepted that nonspecific tissue reactivity is a distinct pathophysiological hallmark of allergic diseases, influenced by genetic and environmental factors different from those involved in causing sensitization and allergen response of target organs. This consensus document aims at reviewing procedures currently used for nonspecific provocation of the bronchi, nose and eye and for measuring their responsiveness to nonspecific stimuli.

TGFBI gene mutations in Brazilian patients with corneal dystrophy.

PURPOSE: To investigate the transforming growth factor beta-induced gene (TGFBI) mutations in Brazilian patients with corneal dystrophy and to evaluate the phenotype-genotype correlation in these patients. METHODS: A total of 11 unrelated families were studied. The diagnosis of corneal dystrophy was based on clinical and histopathological findings. Genomic DNA was extracted from peripheral blood leucocytes, and exons 4 and 12 of the TGFBIgene were amplified by polymerase chain reaction followed by direct sequencing on both strands. RESULTS: Five different mutations in the TGFBIgene were found in the probands. We identified the following mutations: lattice corneal dystrophy--R124C and A546T; Reis-Bücklers corneal dystrophy--R555Q and R124L; granular corneal dystrophy--R555W and Avellino dystrophy--R555W. In three of the 11 studied families there was no mutation in exons 4 and 12. CONCLUSIONS: This is the first report of mutations in the TGFBIgene in a series of Brazilian patients with corneal dystrophy. The findings indicate that TGFBIgene screening should be considered in the diagnosis of corneal dystrophy.

Colour vision defects in asymptomatic carriers of the Leber's hereditary optic neuropathy (LHON) mtDNA 11778 mutation from a large Brazilian LHON pedigree: a case-control study.

AIMS: To determine if asymptomatic carriers from a previously identified large pedigree of the Leber's hereditary optic neuropathy (LHON) 11778 mtDNA mutation have colour vision deficits. METHODS: As part of a comprehensive analysis of over 200 members of a large Brazilian LHON pedigree spanning seven generations, colour vision tests were obtained from 91 members. Colour vision was tested one eye at a time using the Farnsworth-Munsell 100 (FM-100) hue colour vision test. The test was administered under uniform conditions, taking into account: ambient light levels, daylight colour temperature of 6700 kelvin, and neutral uniform background. Tests were scored using the FM-100 MS-Excel computer scoring program. Defects were determined and categorised as tritan, deutan, or protan. Categorisation of each dyschromatopsia was based on review of demonstrated axis computer generated plots and age adjusted error scores which coincided with Verriest 95% confidence intervals. Only the axis with the greatest magnitude error score was used to classify the defect. 55 of the 91 test subjects were LHON mtDNA 11778 J haplotype mutation carriers, proved by mtDNA analysis. The remaining 36 subjects were age matched non-blood relatives (off pedigree), who served as controls. RESULTS: 27 of 55 carriers (49.10%) were shown to have colour vision defects in one or both eyes. 13 of the 27 (48%) abnormal tests in the carrier group were tritan defects and the remaining 14 (52%) were deutan defects. Nine of the 27 (33%) abnormals in the carrier group were identified as having bilateral defects. Six of these were deutan, and the remaining three were tritan dyschromatopsias. Only six of the 36 (16.66%) age matched controls were found to have any type of dyschromatopsia. Five (83.3%) of these were deutan defects. The remaining one was a tritan defect. The difference between the two groups using a chi(2) test with one degree of freedom was statistically significant with a p value less that 0.001. CONCLUSIONS: Until now, LHON has always been characterised by a sudden, devastating vision loss. Asymptomatic carriers, those without vision loss, were considered unaffected by the disease. It now appears that asymptomatic carriers of the LHON mutation are affected by colour vision defects and may manifest other subtle, yet chronic, changes.

Adiponectin receptors gene expression and insulin sensitivity in non-diabetic Mexican Americans with or without a family history of Type 2 diabetes.

AIMS/HYPOTHESIS: The recent discovery of two adiponectin receptors (AdipoR1 and AdipoR2) will improve our understanding of the molecular mechanisms underlying the insulin-sensitising effect of adiponectin. The aim of this study was to determine for the first time whether skeletal muscle AdipoR1 and/or AdipoR2 gene expression levels are associated with insulin resistance. METHODS: Using RT-PCR and northern analysis we measured AdipoR1 and AdipoR2 gene expression in skeletal muscle from healthy Mexican Americans with normal glucose tolerance who had (n=8) or did not have (n=10) a family history of Type 2 diabetes. RESULTS: Gene expression profiling indicated that the AdipoR1 and AdipoR2 isoforms are highly expressed in human skeletal muscle, unlike in mice where AdipoR2 expression was highest in the liver, and AdipoR1 was highest in skeletal muscle. In the study subjects, the expression levels of AdipoR1 (p=0.004) and AdipoR2 (p=0.04), as well as plasma adiponectin concentration (p=0.03) were lower in people with a family history of Type 2 diabetes than in those with no family history of the disease. Importantly, the expression levels of both receptors correlated positively with insulin sensitivity (r=0.64, p=0.004 and r=0.47, p=0.048 respectively). CONCLUSIONS/INTERPRETATION: Collectively, these data indicate that both isoforms of the adiponectin receptor play a role in the insulin-sensitising effect of adiponectin.

Ocular toxoplasmosis: more than just what meets the eye.

Toxoplasma gondii is an intracellular parasite whose life cycle may include the man as an intermediate host. Close to a billion people are infected with this parasite worldwide. Ocular lesions may occur in up to 25% of those individuals infected. The infection may occur intra-uterus, through the placenta when the mother is infected during pregnancy. The parasite may also infect adults after the ingestion of contaminated food products, most notably meats or water. We have shown that although congenital and post-natal (acquired) infection results in similar ocular lesions, the immunological mechanisms behind the development of disease are different. On the other hand, contrary to published data obtained in mice, we were unable to find evidence that the T. gondii express superantigen activity for human lymphocytes. Our findings are important because they suggest that superantigen activity is not important as a pathological mechanism in human disease. Our data also suggest that, whereas the ocular lesion caused by infection after birth is the result of an excessive or dysfunctional immune response, the lesions caused by congenital infection may be due to a lack of an appropriate response to the parasite.

Anterior chamber intraocular lens for high myopia: five year results.

PURPOSE: To evaluate the effectiveness of the surgical correction of high myopia 5 years after anterior chamber intraocular lens (IOL) implantation and to analyze the lens position and related complications. SETTING: Federal University of São Paulo-Escola Paulista de Medicina, São Paulo, Brazil. METHODS: This prospective study comprised 26 eyes that were implanted with an anterior chamber IOL (model ZB5M) and had a minimum follow-up of 5 years. Before and after surgery, manifest and cycloplegic refractions were done; slitlamp examination was performed; anterior chamber depth was measured; and keratometry, ophthalmoscopy, and central corneal endothelial cell count were performed. At the last follow-up, computerized biomicroscopy of the anterior segment was also done. RESULTS: The spherical equivalent decreased from -16.5 diopters (D) +/- 4.1 (SD) preoperatively to -0.9 +/- 0.9 D postoperatively. At the last examination, 57.7% of eyes had a spherical equivalent refraction within +/-1.0 D of emmetropia. Of eyes with a preoperative best spectacle-corrected visual acuity of 20/40 or better, 73.3% had an uncorrected visual acuity of 20/40 or better postoperatively. The mean tilt caused by the IOL was 4.4 +/- 2.7 degrees (range 0.5 to 12.2 degrees) and the mean IOL decentration, 0.3 +/- 0.2 mm (range 0.02 to 0.8 mm). The mean decrease in the endothelial cell count was 1.5%, with a mean cell density of 2808 +/- 305 cells/mm2 preoperatively and 2765 +/- 242 cells/mm2 postoperatively. Pupillary ovalization was observed in 12 eyes (46.1%). CONCLUSIONS: The anterior chamber IOL safely and effectively corrected high myopia.