Early occurrence of primary angle-closure glaucoma in a patient with retinitis pigmentosa and CRB1 gene variations / Manifestação precoce de glaucoma de ângulo fechado em paciente com retinopatia por mutação no gene CRB1

ABSTRACT We describe the case of a 15-year-old girl with decreased visual acuity associated with elevated intraocular pressure in both eyes and angle closure on gonioscopy. She also presented attenuation of retinal vessels and optic disc pallor with large excavation in the left eye. Ultrasound biomicroscopy revealed an anteriorly positioned ciliary body and absence of ciliary sulcus, confirming the plateau iris configuration. Spectral-domain optical coherence tomography revealed a bilateral cystoid macular edema. Genetic screening revealed heterozygous variants of the Crumbs homolog 1 (CRB1) gene (c.2843G>A and c.2506C>A). The patient underwent trabeculectomy for intraocular pressure control and topical treatment for macular edema. This case highlights the importance of performing gonioscopy and evaluating intraocular pressure in patients with a shallow anterior chamber despite young age. In addition, it also shows the importance of genetic screening, when available, in elucidating the diagnosis and providing patients and their families' information on the patient's prognosis and possible therapeutic options.

RESUMO Nós descrevemos um caso de uma paciente de 15 anos com queda de acuidade visual e aumento da pressão intraocular em ambos os olhos, juntamente com fechamento angular no exame de gonioscopia. Na fundoscopia a paciente apresentava atenuação dos vasos retinianos, palidez de disco e aumento de escavação em olho esquerdo. Ao exame da biomicroscopia ultrassônica, foi evidenciado corpo ciliar anteriorizado e ausência de sulco ciliar em ambos os olhos, relevando presença de íris em plateau. Ao exame de tomografia de coerência óptica, visualizamos presença de edema macular cistoide bilateral. O screening genético revelou heterozigose no gene CRB1 (c.2843G>A and c.2506C>A), confirmando o diagnóstico de retinose pigmentar. Este caso reforça a importância do exame de gonioscopia e da avaliação da pressão intraocular em pacientes em câmara rasa, mesmo em pacientes jovens. Além disso, mostra a importância do screening genético como ferramenta útil para elucidação diagnóstica.

Retinosquisis ligada al cromosoma X: presentación de un caso clínico y análisis del diagnóstico genético / X-linked Retinoschisis: a case report and genetic diagnosis analysis / Retinosquise ligada ao X: a presentação de um caso clínico e análise do diagnóstico genético

La Retinosquisis ligada al X, que se presenta fundamentalmente en varones, es una enfermedad genética caracterizada por agudeza visual reducida debido a degeneración macular. Su prevalencia es de 1/5000 varones en todo el mundo. Se manifiesta desde la primera década de la vida con pérdida de la visión que progresa hasta la adolescencia y se mantiene estable hasta la 4ta década de la vida, momento en que presenta un declive importante. El fondo de ojo suele mostrar esquisis. Las mujeres portadoras rara vez presentan síntomas. El gen involucrado es RS1, codifica para Retinosquina, proteína que participa en la integridad estructural y funcional de la retina. El mismo presenta diferentes mutaciones que generan pérdida de función de la proteína. La sospecha diagnóstica se basa en la clínica y los antecedentes familiares, y se apoya en la paraclínica confirmándose en la mayoría de los casos mediante secuenciación del gen. El tratamiento consiste en control periódico oftalmológico y cirugía de las complicaciones. Presentamos el caso de un niño de 2 años con episodios reiterados de desprendimiento de retina, con antecedentes familiares de Retinosquisis por línea materna en individuos de sexo masculino. Estos fueron estudiados demostrándose que son portadores de la variante probablemente patogénica c.466A>C (Arg156Gly) en el gen RS1 la cual había sido reportada previamente en una familia de origen chino. Se demostró que nuestro paciente presenta la mutación familiar en hemicigosis, por lo que esta es la segunda familia en que se confirma la segregación de esta variante con Retinosquisis.

X-linked Retinoschisis is a genetic disease characterized by reduced visual acuity mainly in men due to juvenile macular degeneration. Its prevalence is 1/5000 men worldwide. It manifests from the first decade of life with loss of vision that progresses to adolescence and then remains stable until the 4th decade of life, when it may present a significant decline. The fundus exam usually shows schism. Carrier women rarely have symptoms. The gene involved is RS1 (Xp22.13), which encodes for Retinoschisin, a protein that participates in the structural and functional integrity of the retina. In affected cases, mutations that generate loss of protein function were demonstrated. The diagnosis is based on the clinical and family history, and is supported by ophthalmology evaluation; in most cases it can be confirmed by sequencing of the gene. The treatment consists of periodic ophthalmological control and surgery of the complications. We describe the case of a 2 year old boy with repeated episodes of retinal detachment and who has a family history of Retinoschisis by maternal line in male individuals. These were studied, and it was shown that they are carriers of the probably pathogenic variant c.466A> C (Arg156Gly) in the RS1 gene, which had been reported previously in a family of Chinese origin. It was shown that our patient presents the family mutation in hemizygous state, so this is the second family in which the segregation of this variant with Retinoschisis is confirmed.

A retinosquise ligada ao X, que ocorre principalmente em homens, é uma doença genética caracterizada pela redução da acuidade visual devido à degeneração macular. Sua prevalência é de 1/5000 homens em todo o mundo. Manifesta-se desde a primeira década de vida com perda da visão que progride até a adolescência e permanece estável até a 4ª década de vida, época em que apresenta declínio significativo. O fundo geralmente mostra esquise. Portadoras do sexo feminino raramente apresentam sintomas. O gene envolvido é o RS1, que codifica a Retinosquina, proteína que participa da integridade estrutural e funcional da retina. Apresenta diferentes mutações que geram perda de função da proteína. A suspeita diagnóstica baseia-se na história clínica e familiar, e na paraclínica, sendo confirmada na maioria dos casos pelo sequenciamento gênico. O tratamento consiste em acompanhamento oftalmológico periódico e cirurgia para complicações. Apresentamos o caso de um menino de 2 anos com episódios repetidos de descolamento de retina, com história familiar de retinosquise materna no sexo masculino. Estes foram estudados mostrando que são portadores da variante provavelmente patogênica c.466A> C (Arg156Gly) no gene RS1, que havia sido relatado anteriormente em uma família de origem chinesa. Foi demonstrado que nosso paciente apresenta a mutação familiar em hemizigose, sendo esta a segunda família em que se confirma a segregação desta variante com Retinosquise.

Association of serum levels of secreted frizzled-related protein 5 and Wnt member 5a with glomerular filtration rate in patients with type 2 diabetes mellitus and chronic renal disease: a cross-sectional study

ABSTRACT BACKGROUND: Diabetic nephropathy is a common complication of chronic kidney disease (CKD). ­Inflammation in the kidneys is crucial for promoting development and progression of this complication. Wnt member 5a (Wnt5a) and secreted frizzled-related protein 5 (Sfrp5) are proinflammatory proteins associated with insulin resistance and chronic low-grade adipose tissue inflammation. OBJECTIVE: To determine the correlation between serum Sfrp5 and Wnt5a concentrations and glomerular filtration rate in patients with type 2 diabetes mellitus and CKD. DESIGN AND SETTING: Cross-sectional, comparative and observational study in the Department of Endocrinology, Civil Hospital, Culiacán, Sinaloa, Mexico. METHODS: Eighty individuals with chronic kidney disease were recruited. Their serum Sfrp5 and Wnt5a concentrations were quantified using the enzyme-linked immunosorbent assay (ELISA) test. The statistical analysis consisted of the Mann-Whitney U test for independent samples and Spearman correlation, with statistical significance of P < 0.05. RESULTS: Serum Sfrp5 concentration continually increased through the stages of CKD progression, whereas serum Wnt5a concentration presented its highest levels in stage 3 CKD. Negative correlations between estimated glomerular filtration rate (eGFR) and serum concentrations of Sfrp5 (r = -0434, P = 0.001) and Wnt5a (r = -0481, P = 0.001) were found. CONCLUSIONS: There were negative correlations between serum Sfrp5 and Wnt5a concentrations and eGFR at each stage of CKD, with higher levels in female patients. This phenomenon suggests that Sfrp5 and Wnt5a might be involved in development and evolution towards end-stage renal disease.

Insulin resistance is associated with Sfrp5 in obstructive sleep apnea / Resistência à insulina está associada à Sfrp5 na apneia obstrutiva do sono

Abstract Introduction: Obstructive sleep apnea, a common disease, is usually complicated by insulin resistance and type 2 diabetes mellitus. Adipokine is considered to play an important role in the development of insulin resistance and type 2 diabetes mellitus in obstructive sleep apnea. Objective: To assess whether secreted frizzled-related protein 5, a new adipokine, is involved in untreated obstructive sleep apnea patients. Methods: Seventy-six subjects with obstructive sleep apnea and thirty-three control subjects without obstructive sleep apnea were recruited and matched in terms of body mass index and age. The fasting secreted frizzled-related protein 5 plasma concentration was tested using ELISA. In addition, the correlation between secreted frizzled-related protein 5 and the homeostasis model assessment of insulin resistance was obtained. Multiple linear regression analysis models with stepwise selection were performed to determine the independent associations between various factors and secreted frizzled-related protein 5. Results: Plasma secreted frizzled-related protein 5 levels were significantly lower in the obstructive sleep apnea group than in the control group (obstructive sleep apnea group: 28.44 ± 13.25 ng/L; control group: 34.16 ± 13.51 ng/L; p = 0.023). In addition, secreted frizzled-related protein 5 was negatively correlated with homeostasis model assessment of insulin resistance but positively correlated with the mean and lowest oxygen saturation with or without adjusting for age, gender, body mass index, neck circumference, waist circumference and waist-to-hip ratio. The multiple linear regression analysis showed there was an independent negative association between secreted frizzled-related protein 5 and homeostasis model assessment of insulin resistance. Conclusion: Secreted frizzled-related protein 5 was involved in obstructive sleep apnea and the decrease in secreted frizzled-related protein 5 was directly proportional to the severity of obstructive sleep apnea. There was an independent negative correlation between homeostasis model assessment of insulin resistance and secreted frizzled-related protein 5 in the obstructive sleep apnea group. Secreted frizzled-related protein 5 might be a therapeutic target for insulin resistance in obstructive sleep apnea.

Resumo Introdução: A apneia obstrutiva do sono, uma doença comum, é geralmente complicada com resistência à insulina e diabetes melito tipo 2. Acredita-se que a adipocina possa ter um papel importante no desenvolvimento de resistência à insulina e diabetes melito tipo 2 na apneia obstrutiva do sono. Objetivo: Avaliar se a proteína secretada relacionada ao receptor frizzled-5, uma nova adipocina, está envolvida em pacientes com apneia obstrutiva do sono não tratada. Método: Foram recrutados 76 indivíduos com apneia obstrutiva do sono e 33 indivíduos controle sem apneia obstrutiva do sono e pareados em relação a índice de massa corporal e idade. A concentração plasmática de proteína secretada relacionada ao receptor frizzled-5 foi testada em jejum com o teste Elisa. Além disso, obteve-se correlação entre a proteína secretada relacionada ao receptor frizzled-5 e o modelo de avaliação da homeostase de resistência à insulina. Modelos de análise de regressão linear múltipla com seleção stepwise foram feitos para determinar as associações independentes entre vários fatores e a proteína secretada relacionada ao receptor frizzled-5. Resultados: Os níveis plasmáticos de proteína secretada relacionada ao receptor frizzled-5 foram significativamente menores no grupo com apneia obstrutiva do sono do que no grupo controle (grupo com apneia obstrutiva do sono: 28,44 ± 13,25 ng/L; grupo controle: 34,16 ± 13,51 ng/L; p = 0,023). Além disso, a proteína secretada relacionada ao receptor frizzled-5 foi correlacionada negativamente com o modelo de avaliação da homeostase de resistência à insulina, mas se correlacionou positivamente com a média e a saturação mínima de oxigênio com ou sem ajuste para idade, gênero, índice de massa corporal, circunferência do pescoço, circunferência da cintura e relação cintura-quadril. A análise de regressão linear múltipla mostrou que houve uma associação negativa independente entre a proteína secretada relacionada ao receptor frizzled-5 e o modelo de avaliação da homeostase de resistência à insulina. Conclusões: A proteína secretada relacionada ao receptor frizzled-5 esteve envolvida na apneia obstrutiva do sono e sua diminuição foi diretamente proporcional à gravidade da apneia obstrutiva do sono. Houve uma correlação negativa independente entre o modelo de avaliação da homeostase de resistência à insulina e a proteína secretada relacionada ao receptor frizzled-5 no grupo da apneia obstrutiva do sono. A proteína secretada relacionada ao receptor frizzled-5 pode ser um alvo terapêutico para a resistência à insulina na apneia obstrutiva do sono.

Intravitreal neurodegenerative and inflammatory mediators in proliferative diabetic retinopathy / Mediadores intravítreos da neurodegeneração e inflamação na retinopatia diabética proliferativa

ABSTRACT Purpose: To compare the intravitreal concentrations of cellular mediators involved in neurodegeneration, inflammation, and angiogenesis in patients with proliferative diabetic retinopathy and other vitreoretinal diseases. Methods: A multiplex bead immunoassay was used to measure vitreous levels of pigment epithelium-derived factor, serum amyloid P, C-reactive protein, complement C4, alpha-1 antitrypsin, vascular endothelial growth factor, platelet-derived growth factor-AA, platelet-derived growth factor-BB, interleukin-6, interleukin-8, interleukin-10, tumor necrosis factor alpha and beta in patients undergoing 23-gauge vitrectomy for proliferative diabetic retinopathy and other diagnoses (control group). Results: We evaluated 55 patients, of whom 24 had proliferative diabetic retinopathy and 31 had other diagnoses including vitreous hemorrhage, retinal detachment, macular hole, and epiretinal membrane. Patients with proliferative diabetic retinopathy demonstrated increased levels of serum amyloid P (85.49 vs. 31.38 ng/mL); C-reactive protein (59.89 vs. 41.75 ng/mL), vascular endothelial growth factor (2,330.11 vs. 554.25 pg/mL; p<0.001), platelet-derived growth factor A (127.32 vs. 39.11 pg/mL), platelet-derived growth factor B (29.37 vs. 7.12 pg/mL), interleukin-6 (69.37 vs. 33.58 pg/mL), interleukin-8 (175.25 vs. 59.71 pg/mL), and interleukin-10 (3.70 vs. 1.88 pg/mL); all p<0.004 when compared with the control group. Levels of pigment epithelium-derived factor (30.06 vs. 27.48 ng/mL; p=0.295), complement C4 (570.78 vs. 366.24 ng/mL; p=0.069), and alpha-1-antitrypsin (359.27 vs. 522.44 ng/mL; p=0.264) were not significantly different between the groups. Intravitreal levels of tumor necrosis factor-alpha and tumor necrosis factor-beta were undetectable. Serum Amyloid P, C-reactive protein, platelet-derived growth factor A, platelet-derived growth factor B, interleukin-6, and interleukin-8 were correlated positively with vascular endothelial growth factor. Conclusions: Cellular mediators involved in neurodegeneration and inflammation demonstrated increased levels in the vitreous humor of patients with proliferative diabetic retinopathy and may be part of the pathogenesis of diabetic retinopathy.

RESUMO Objetivo: Comparar as concentrações intravítreas de mediadores celulares envolvidos na neurodegeneração, inflamação e angiogênese em pacientes com retinopatia diabética proliferativa e outras doenças vítreo-retinianas. Métodos: Um ensaio imunomagnético foi utilizado para medir os níveis vítreos do fator derivado do epitélio pigmentar, amilóide P sérico, proteína-C-reativa, complemento C4, e alfa-1-antitripsina, fator de crescimento do endotélio vascular, fator de crescimento derivado das plaquetas AA, fator de crescimento derivado das plaquetas BB, interleucina-6, interleucina-8, interleucina-10, fator de necrose tumoral alfa e beta em pacientes submetidos à vitrectomia 23-gauge para retinopatia diabética proliferativa ou outros diagnósticos (grupo controle). Resultados: Foram avaliados 55 pacientes, dos quais 24 tinham retinopatia diabética proliferativa e 31 tinham outros diagnósticos, incluindo hemorragia vítrea, descolamento de retina, buraco macular e membrana epirretiniana. Pacientes com retinopatia diabética proliferativa demonstraram níveis aumentados de amilóide P sérico (85,49 vs 31,38 ng/mL), proteína-C-reativa (59,89 vs 41,75 ng/mL), fator de crescimento do endotélio vascular (2.330,11 vs 554,25 pg/mL, p<0.001), fator de crescimento derivado das plaquetas-A: (127,32 vs 39,11 pg/mL), fator de crescimento derivado das plaquetas-B (29,37 vs 7,12 pg/mL), interleucina-6 (69,37 vs 33,58 pg/mL), interleucina-8 (175,25 vs 59,71 pg/mL) e interleucina-10 (3,70 vs 1,88 pg/mL), todos com p<0,004 quando comparados ao grupo controle. Níveis de fator derivado do epitélio pigmentar (30,06 vs 27,48 ng/mL; p=0,295), complemento C4 (570,78 vs 366,24 ng/mL; p=0,069), alfa-1 antitripsina (359,27 vs 522,44 ng/mL; p=0,264) não foram significativamente diferente entre os grupos. Níveis intravítreos de fator de necrose tumoral alfa e fator de necrose tumoral beta foram indetectáveis. O amilóide P sérico, a proteína C-reativa, o fator de crescimento derivado das plaquetas A e B, a interleucina-6 e a interleucina-8 correlacionaram-se positivamente com o fator de crescimento do endotélio vascular. Conclusões: Os medidores celulares envolvidos na neurodegeneração e inflamação demonstraram níveis aumentados no humor vítreo de pacientes com retinopatia diabética proliferativa e podem ser parte da patogênese da retinopatia diabética.

Decreased VEGF-A and sustained PEDF expression in a human retinal pigment epithelium cell line cultured under hypothermia

BACKGROUND: Previous reports have described a decrease in retinal temperature and clinical improvement of wet age-related macular degeneration (AMD) after vitrectomy. We hypothesized that the retinal temperature decrease after vitrectomy plays a part in the suppression of wet AMD development. To test this hypothesis, we evaluated the temperature dependence of the expression of vascular endothelial growth factor-A (VEGF-A) and in vitro angiogen-esis in retinal pigment epithelium (RPE). RESULTS: We cultured ARPE-19 cells at 37, 35, 33 and 31°C and measured the expression of VEGF-A, VEGF-A splicing variants, and pigment epithelium-derived factor (PEDF). We performed an in vitro tube formation assay. The dehydrogenase activity was also evaluated at each temperature. Expression of VEGF-A significantly decreased with decreased temperature while PEDF expression did not. VEGF165 expression and in vitro angiogenesis also were temperature dependent. The dehydrogenase activity significantly decreased as the culture temperature decreased. CONCLUSIONS: RPE cultured under hypothermia that decreased cellular metabolism also had decreased VEGF-A and sustained PEDF expression, creating an anti-angiogenic environment. This mechanism may be associated with a beneficial effect after vitrectomy in patients with wet AMD.

The expression of melanopsin and clock genes in Xenopus laevis melanophores and their modulation by melatonin

Vertebrates have a central clock and also several peripheral clocks. Light responses might result from the integration of light signals by these clocks. The dermal melanophores of Xenopus laevis have a photoreceptor molecule denominated melanopsin (OPN4x). The mechanisms of the circadian clock involve positive and negative feedback. We hypothesize that these dermal melanophores also present peripheral clock characteristics. Using quantitative PCR, we analyzed the pattern of temporal expression of Opn4x and the clock genes Per1, Per2, Bmal1, and Clock in these cells, subjected to a 14-h light:10-h dark (14L:10D) regime or constant darkness (DD). Also, in view of the physiological role of melatonin in the dermal melanophores of X. laevis, we determined whether melatonin modulates the expression of these clock genes. These genes show a time-dependent expression pattern when these cells are exposed to 14L:10D, which differs from the pattern observed under DD. Cells kept in DD for 5 days exhibited overall increased mRNA expression for Opn4x and Clock, and a lower expression for Per1, Per2, and Bmal1. When the cells were kept in DD for 5 days and treated with melatonin for 1 h, 24 h before extraction, the mRNA levels tended to decrease for Opn4x and Clock, did not change for Bmal1, and increased for Per1 and Per2 at different Zeitgeber times (ZT). Although these data are limited to one-day data collection, and therefore preliminary, we suggest that the dermal melanophores of X. laevis might have some characteristics of a peripheral clock, and that melatonin modulates, to a certain extent, melanopsin and clock gene expression.

Experimental models of autoimmune inflammatory ocular diseases / Modelos experimentais para o estudo de doenças inflamatórias oculares autoimunes

Ocular inflammation is one of the leading causes of blindness and loss of vision. Human uveitis is a complex and heterogeneous group of diseases characterized by inflammation of intraocular tissues. The eye may be the only organ involved, or uveitis may be part of a systemic disease. A significant number of cases are of unknown etiology and are labeled idiopathic. Animal models have been developed to the study of the physiopathogenesis of autoimmune uveitis due to the difficulty in obtaining human eye inflamed tissues for experiments. Most of those models are induced by injection of specific photoreceptors proteins (e.g., S-antigen, interphotoreceptor retinoid-binding protein, rhodopsin, recoverin, phosducin). Non-retinal antigens, including melanin-associated proteins and myelin basic protein, are also good inducers of uveitis in animals. Understanding the basic mechanisms and pathogenesis of autoimmune ocular diseases are essential for the development of new treatment approaches and therapeutic agents. The present review describes the main experimental models of autoimmune ocular inflammatory diseases.

A inflamação ocular é uma das principais causas de perda visual e cegueira. As uveítes constituem um grupo complexo e heterogêneo de doenças caracterizadas por inflamação dos tecidos intraoculares. O olho pode ser o único órgão envolvido ou a uveíte pode ser parte de uma doença sistêmica. A etiologia é desconhecida em um número significativo de casos, que são considerados idiopáticos. Modelos animais têm sido desenvolvidos para estudar a fisiopatogênese da uveíte autoimune devido às dificuldades na obtenção de tecidos de olhos humanos inflamados para experimentos. Na maioria desses modelos, que simulam as uveítes autoimunes em humanos, a uveíte é induzida com proteínas específicas de fotorreceptores (antígeno-S, proteína ligadora de retinoide do interfotoreceptor, rodopsina, recoverina e fosducina). Antígenos não retinianos, como proteínas associadas à melanina e proteína básica de mielina, são também bons indutores de uveíte em animais. Entender os mecanismos básicos e a patogênese dessas doenças oculares é essencial para o desenvolvimento de novas formas de tratamento das uveítes autoimunes e de novos agentes terapêuticos. Nesta revisão serão abordados os principais modelos experimentais utilizados para o estudo de doenças inflamatórias oculares autoimunes.

The genetic and molecular basis of congenital cataract / Base genética e molecular da catarata congênita

Congenital cataracts are one of the most treatable causes of visual impairment and blindness during infancy, with an estimated prevalence of 1 to 6 cases per 10,000 live births. Approximately fifty percent of all congenital cataract cases may have a genetic cause. All three types of Mendelian inheritance have been reported for cataract; however, autosomal dominant transmission seems to be the most frequent. The transparency and high refractive index of the lens are achieved by the precise architecture of the fiber cells and the homeostasis of the lens proteins in terms of their concentration, stability, and supramolecular organization. Research on hereditary congenital cataract led to the identification of several classes of candidate genes that encode proteins such crystallins, lens specific connexins, aquaporine, cytoskeletal structural proteins, and developmental regulators. The purpose of this study was to review the literature on the recent advances made in understanding the molecular genetic basis of congenital cataracts.

A catarata congênita é uma das principais causas tratáveis de cegueira na infância, com prevalência estimada em 1 a 6 casos por 10.000 nascidos vivos, sendo a causa hereditária responsável por até metade dos casos. Dentre os padrões de herança já descritos para a catarata, a transmissão autossômica dominante é a mais frequente. A transparência e o alto índice refrativo do cristalino são resultados da disposição regular das fibras lenticulares e do equilíbrio homeostático; além da estabilidade e da organização supramolecular das proteínas do cristalino. Pesquisas sobre catarata congênita hereditária têm levado à identificação de várias classes de genes responsáveis pela codificação das proteínas do cristalino, tais como: cristalinas, conexinas, aquaporinas, proteínas do citoesqueleto e reguladores do desenvolvimento. O objetivo deste estudo foi a revisão da literatura sobre os recentes avanços na compreensão da base genética e molecular da catarata congênita.

Alternative PCR protocol using a single primer set for assessing DNA quality in several tissues from a large variety of mammalian species living in areas endemic for leishmaniasis

The aim of this work was to establish a modified pre-diagnostic polymerase chain reaction (PCR) protocol using a single primer set that enables successful amplification of a highly conserved mammalian sequence in order to determine overall sample DNA quality for multiple mammalian species that inhabit areas endemic for leishmaniasis. The gene encoding interphotoreceptor retinoid-binding protein (IRBP), but not other conserved genes, was efficiently amplified in DNA samples from tail skin, ear skin, bone marrow, liver and spleen from all of the species tested. In tissue samples that were PCR-positive for Leishmania, we found that DNA from 100 percent, 55 percent and 22 percent of the samples tested resulted in a positive PCR reaction for the IRBP, beta-actin and beta-globin genes, respectively. Nucleotide sequencing of an IRBP amplicon resolved any questions regarding the taxonomical classification of a rodent, which was previously based simply on the morphological features of the animal. Therefore, PCR amplification and analysis of the IRBP amplicon are suitable for pre-diagnostically assessing DNA quality and identifying mammalian species living in areas endemic to leishmaniasis and other diseases.

Absence of mutations in Pax6 gene in three cases of Morning Glory syndrome associated with isolated growth hormone deficiency / A ausência de mutações no gene PAX6 em três casos de síndrome de Morning Glory associada à deficiência isolada de hormônio do crescimento

Morning glory syndrome (MGS) is a congenital optic disc dysplasia often associated with craniofacial anomalies, especially basal encephalocele and hypopituitarism. Clinical signs are varied and often occult. The PAX6 gene is involved in ocular morphogenesis and is expressed in numerous ocular tissues during development especially in the developing central nervous system. The aim of the present study is to evaluate PAX6 in MGS associated with isolated growth hormone deficiency. Three pre-pubertal males (A, B and C) with MGS and short stature due to growth hormone deficiency, treated with recombinant human growth hormone with limited response, were reported. Two of them had basal encephalocele. Coding and non-coding sequences corresponding of PAX6 different transcripts were analyzed by direct sequencing. Nucleotide variations causing putative aminoacid change were not observed. Patient A presented the new IVS2+9G>A transition, whereas patients A and C were heterozygous for known single nucleotide polymorphisms (SNP) within the intron 4. In addition, two SNP heterozygoses were observed for patient C in both intron 9 and 13. Sequencing also revealed several nucleotide variations in patient B. Two heterozygoses for known polymorphisms were identified along with a novel C>A nucleotide change in intron 4. This patient also presented a low number on the TG repeat in intron 9 and a new IVS11+33A>T transversion. Gene regulation and transcription of PAX6 are complex processes; there are two major protein isoforms, PAX6(-5a) and PAX6(+5a), and nine transcripts described. Furthermore, extra transcription regulatory elements have been postulated within PAX6 introns. Considering that neither population distributions on PAX6 polymorphism nor their linkeages with diseases have been reported, a functional effect due to alterations described here cannot be discarded.

A síndrome de Morning Glory (SMG) é uma displasia óptica congênita associada a anomalias craniofaciais, principalmente encefalocele basal e hipopituitarismo. Os sinais clínicos são variados e muitas vezes ocultos. O gene PAX6 está envolvido na morfogênese ocular e se expressa em vários tecidos oculares durante o desenvolvimento do sistema nervoso central. O objetivo deste estudo foi avaliar o gene PAX6 na SMG associada com deficiência isolada de hormônio de crescimento. Foram relatados três pacientes pré-púberes (A, B e C) com SMG e baixa estatura por deficiência de hormônio de crescimento tratados com hormônio de crescimento recombinante humano. As seqüências codificadoras e não-codificadoras correspondentes ao PAX6 em diferentes transcritos foram analisadas por seqüenciamento direto. Variações nucleotídeas com trocas pontuais de aminoácidos não foram encontradas. O paciente A apresentou uma transição nova IVS2+9G>A, enquanto os pacientes A e C foram heterozigotos para um polimorfismo (SNP) no íntron 4. Ainda, dois SNPs em heterozigose nos íntrons 9 e 13 foram observados no paciente C. O seqüenciamento também mostrou várias variações nucleotídeas no paciente B. Dois SNPs conhecidos com a alteração nucleotídea nova C>A no íntron 4 foram observados em heterozigose. Este paciente também apresentou um baixo número de repetições TG no íntron 9 e uma nova transversão IVS11+33A>T. A regulação e a transcrição do gene PAX6 são um processo complexo; existem 2 isoformas principais da proteína, PAX6(-5a) e PAX6(+5a) e 9 transcritos descritos. Considerando que nem a distribuição de SNPs no PAX6 e nem as suas ligações com as doenças foram relatadas, um defeito funcional devido às alterações descritas não pode ser descartado.

Immunohistochemical characterization of the chick marginal retina

The retina is a highly differentiated tissue with a complex layered structure that has been extensively characterized. However, most of the previous studies focused on the histology of the central retina while little is known about the cellular composition, organization and function of the marginal retina. Recent research has identified a subpopulation of multipotential progenitor cells in the marginal regions of the retina, closest to the ciliary body ("ciliary marginal zone"). These cells are capable of differentiation in response to an appropriate stimulus. Thus, it is possible that the structure and composition of the marginal retina are distinct from those of the central retina to accommodate the potential addition of newly formed neurons. To characterize the cellular profile of the chick marginal retina, we labeled it immunohistochemically for markers whose staining pattern is well established in the central retina: calbindin, calretinin, protein kinase C, and choline acetyltransferase. Calbindin was present at very low levels in the marginal retina putative photoreceptor layer. Calretinin-positive horizontal cells were also sparse close to the ciliary marginal zone. The bipolar cells in the marginal outer plexiform layer were positive for anti-protein kinase C antibodies, but the density of labeling was also decreased in relation to the central retina. In contrast, the marginal starburst cholinergic amacrine cell pattern was very similar to the central retina. From these data we conclude that the structure of the marginal retina is significantly different from that of the central retina. In particular, the expression of late retina markers in the marginal retina decreased in comparison to the central retina.

Concentração de proteínas no humor aquoso de pacientes com glaucoma primário de ângulo aberto em tratamento clínico / Aqueous humor protein concentration in patients with primary open-angle glaucoma under clinical treatment

OBJETIVOS: Comparar a concentração total de proteínas no humor aquoso entre pacientes com glaucoma primário de ângulo aberto e sem glaucoma. MÉTODOS: Foram coletadas amostras de humor aquoso de 22 pacientes com glaucoma primário de ângulo aberto (grupo GPAA) no momento da trabeculectomia. Na coleta, 0,1 mL de humor aquoso foi aspirado da câmara anterior através de uma agulha de calibre 26, no início do procedimento cirúrgico. Coleta semelhante foi realizada em 22 pacientes sem glaucoma no início da cirurgia de catarata (grupo controle). A amostra de humor aquoso foi armazenada a -20°C após a coleta. A concentração total de proteínas no humor aquoso foi determinada por meio de um teste colorimétrico. RESULTADOS: A média geométrica da concentração total de proteínas no humor aquoso foi de 32 mg/dL (amplitude: 8-137 mg/dL) no grupo glaucoma primário de ângulo aberto e de 16 mg/dL (amplitude: 2-85 mg/dL) no grupo controle. A razão da concentração total de proteínas no humor aquoso entre estes dois grupos foi de 2,0 (intervalo de confiança de 95 por cento: 1,3 a 3,2; p=0,003). CONCLUSÕES: A concentração total de proteínas no humor aquoso de pacientes com glaucoma primário de ângulo aberto foi aproximadamente duas vezes maior quando comparada aos pacientes sem glaucoma.

PURPOSE: To compare total protein concentration in the aqueous humor of primary open-angle glaucoma and non-glaucomatous patients. METHODS: Aqueous humor samples were obtained from 22 patients just before trabeculectomy for clinically uncontrolled primary open angle glaucoma (POAG group). Aqueous humor (0.1 mL) was aspirated by inserting a 26-gauge needle into the anterior chamber. The same procedure was performed in 22 non-glaucomatous patients just before cataract surgery (control group). Immediately after collection, the aqueous humor was stored at -20°C. Aqueous humor total protein concentration was determined using a colorimetric assay. RESULTS: The geometric mean of total protein concentration of the aqueous humor samples was 32 mg/dL (range: 8-137 mg/dL) in the primary open angle glaucoma group and 16 mg/dL (range: 2-85 mg/dL) in the control group. The ratio of the protein concentration between the two groups was 2.0 (95 percent confidence interval: 1.3 to 3.2; p=0.003). CONCLUSIONS: The total protein concentration in primary open-angle glaucoma aqueous humor was approximately two times higher than that in non-glaucomatous subjects.

Mutation analysis of gene PAX6 in human gliomas

Gliomas are the most common tumors of the central nervous system. In spite of the marked advances in the characterization of the molecular pathogenesis of gliomas, these tumors remain incurable and, in most of the cases, resistant to treatments, due to their molecular heterogeneity. Gene PAX6, which encodes a transcription factor that plays an important role in the development of the central nervous system, was recently recognized as a tumor suppressor in gliomas. The objective of the present study was to analyze the mutational status of the coding and regulating regions of PAX6 in 94 gliomas: 81 astrocytomas (11 grade I, 23 grade II, 8 grade III, and 39 grade IV glioblastomas), 5 oligodendrogliomas (3 grade II, and 2 grade III), and 8 ependymomas (5 grade II, and 3 grade III). Two regulating regions (SX250 and EIE) and the 11 coding regions (exons 4-13, plus exon 5a resulting from alternative splicing) of gene PAX6 were analyzed and no mutation was found. Therefore, we conclude that the tumor suppressor role of PAX6, reported in previous studies on gliomas, is not due to mutation in its coding and regulating regions, suggesting the involvement of epigenetic mechanisms in the silencing of PAX6 in these tumors.

The anti-IRBP IgG1 and IgG2a response does not correlate with susceptibility to experimental autoimmune uveitis

Susceptibility to experimental autoimmune uveitis (EAU) in inbred mice has been associated with a dominant Th1 response. Elevated anti-inter-photoreceptor retinoid-binding protein (anti-IRBP) IgG2a/IgG1 antibody ratios have been implicated as candidate markers to predict disease severity. In the present study, both the anti-IRBP antibody isotype and severity of EAU phenotypes were examined in 4 non-isogenic genetically selected mouse lines to determine if they can be used as general markers of disease. Mice between 8 and 12 weeks old selected for high (H III) or low (L III) antibody response and for maximum (AIR MAX) or minimum (AIR MIN) acute inflammatory reaction (AIR) were immunized with IRBP. Each experiment was performed with at least 5 mice per group. EAU was evaluated by histopathology 21 days after immunization and the minimal criterion was inflammatory cell infiltration of the ciliary body, choroid and retina. Serum IgG1- and IgG2a-specific antibodies were determined by ELISA. EAU was graded by histological examination of the enucleated eyes. The incidence of EAU was lower in AIR MIN mice whereas in the other strains approximately 40 percent of the animals developed the disease. Low responder animals did not produce anti-IRBP IgG2a antibodies or interferon-gamma. No correlation was observed between susceptibility to EAU and anti-IRBP isotype profiles. Susceptibility to EAU is related to the intrinsic capacity to mount higher inflammatory reactions and increased production of anti-IRBP IgG2a isotype is not necessarily a marker of this immunologic profile.

Correlation between genotype and phenotype in primary open angle glaucoma of Brazilian families with mutations in exon 3 of the TIGR/MYOC gene

OBJETIVO: Identificar nos representantes de famílias com glaucoma primário de ângulo aberto (GPAA) mutações no exon 3 do gene TIGR/MYOC e avaliar a expressão fenotípica associada às mutações encontradas em seus respectivos núcleos familiares. MÉTODOS: Setenta e oito pacientes (81,2%), com pelo menos um representante na família com GPAA, e dezoito pacientes (18,7%) com glaucoma esporádico tiveram o exon 3, do gene TIGR/MYOC, submetido a seqüenciamento automático para identificação de mutações. Os pacientes, com mutação não silenciosa identificadas nesta triagem inicial, tiveram os heredogramas de suas famílias construídos. Todos os seus familiares disponíveis foram submetidos a exame oftalmológico e seqüenciamento automático do exon 3, do gene TIGR/MYOC.RESULTADOS: Foram identificados quatro tipos de variações na seqüência do exon 3 do TIGR/MYOC (Cys433Arg, Pro370Pro, Lys398Arg e Tyr347Tyr) nos 96 pacientes inicialmente estudados. A mutação Lys398Arg previamente descrita como polimorfismo não segregou com a doença na família estudada. A mutação Cys433Arg foi a mais prevalente afetando 3,1% da amostra inicial (3/96). Em duas diferentes famílias (56 integrantes disponíveis para exame), 8/56 carregavam a mutação Cys433Arg e tinham GPAA, 5/56 com mutação eram hipertensos oculares e 8/56 com mutação não apresentavam manifestações da doença. Pacientes com GPAA apresentaram mediana de idade de diagnóstico de 43,25 anos, variando entre 17-58, e média de pressão intra-ocular (PIO) de 36,3±3,8 mmHg para olho direito e 37,6±9,75 mmHg para olho esquerdo. O grupo com a mutação Cys433Arg apresentou PIO significantemente mais elevada (p<0,0007) e relação escavação/disco vertical mais comprometida (p<0,023) que o grupo de pacientes sem mutação.CONCLUSÃO: A mutação no exon 3 do gene TIGR/MYOC associa-se com famílias brasileiras portadoras de GPAA de início precoce. O fenótipo desta mutação é caracterizado por variável idade de diagnóstico, causando GPAA-juvenil e GPAA do adulto, PIO bastante elevada, de difícil controle, freqüentemente levando a grave comprometimento visual.

Glaucoma in Costa Rica. Initial approaches: [minireview]

Glaucoma is the second most frequent cause of irreversible blindness worldwide. Genetic factors have been implicated in the development of the disease. So far six loci (GLC1A-GLC1F) and two genes (TIGR/MYOC and OPTN) are involved in the development of juvenile (JOAG) and adult onset or chronic primary open angle glaucoma (COAG), while two loci (GLC3A,GLC3B) and one gene (CYP1B1) are known for primary congenital glaucoma (PCG). Here we summarize the results of the first genetic studies of glaucoma in Costa Rica. Nine families: 1 with JOAG, 1 with PCG and 7 with COAG were screened for mutations at the known genes. A 10 bp duplication, 1546-1555dupTCATGCCACC, at the CYP1B1 gene, causes, in homozygous state, glaucoma in the consanguineous PCG family. This mutation has been found in different countries and generates an early stop codon that termitates protein synthesis 140 amino acids earlier than the normal allele. In exon 1 of the T1GR/MYOC the innocuous Arg76Lys variant was found in two of the COAG families. In the OPTN gene two variants in the coding region (Thr34Thr, Met 98Lys) and 7 intronic changes were found in other Costa Rican glaucoma patients. One of the COAG families was chosen for a genome scan with 379 microsatellite markers and linkage analysis. LOD scores [quot ]suggestive[quot ] of linkage were obtained for several chromosomal regions. Evidence indicates that hereditary glaucoma in Costa Rica is highly heterogeneous and that further studies in the country will probably disclose some up to now unknown genes responsible for the disease.

Antioxidant status of dog aqueous humor after extracapsular lens extraction

We determined the antioxidant status of the aqueous humor after extracapsular lens extraction in 14 mongrel dogs weighing about 10 kg. The animals were examined by slit lamp biomicroscopy, applanation tonometry and indirect ophthalmoscopy. One eye was submitted to conventional extracapsular lens extraction and the other was used as control. Samples of aqueous humor were obtained by anterior chamber paracentesis before and at days 1, 2, 3, 7 and 15 after surgery. Total antioxidant status was determined as the capacity of aqueous humor to inhibit free radical generation by 2,2-azobis(2-amidopropane) chlorine. Ascorbic acid concentration was measured by HPLC with UV detection. Protein content was determined with the biuret reagent. Statistical analysis was performed by ANOVA followed by the Tukey-Kramer test. Protein concentration increased from 0.61 to 22 mg/ml 24 h after surgery. These levels were maintained and returned to normal at day 7. Total antioxidant capacity was reduced from 50 to about 30 min until day 3 and at day 7 it was equal to control. Ascorbic acid levels were reduced from 252 to about 110 æM and then returned to control values at day 15. Considering the importance of ascorbic acid concentration in aqueous humor for the maintenance of the antioxidant status of the anterior segment of the eye, the decrease of antioxidant defenses suggests that the surgical procedures promote an oxidative stress condition in the eye.

Neuropatía óptica epidémica cubana: parte II: aspectos neuro-oftalmológicos, neurológicos, nutricionales e históricos / Cuban epidemic optic neuropathy: part II: aspects neuro-ophthalmologic, neurological, ntritional and history

En el presente trabajo se definen y establecen los elementos clínicos diagnósticos de lo que se dió en llamar la neuropatia óptica epidémica cubana que afectó a más de 50.000 personas desde su inicio en 1991, hasta su control parcial en 1993, cuando adquirió rango de endemia. Por invitación del Ministerio Cubano de Salud miembros de la Organización Mundial de la Salud/Oficina Sanitaria Panamericana y del Grupo Orbis estructuraron un equipo que viajó a la Isla de Cuba en mayo de 1993. Para la definición de caso el grupo examinó a un total de 20 pacientes desde el punto de vista oftalmológico, neurológico, neuro-oftalmológico incluyendo campos visuales, estudios de función del nervio óptico, sensibilidad al contraste y evaluación fundoscópica exhaustiva. Los elementos básicos fueron los de una neuropatía óptica tóxico-nutricional tipificada por disminución subaguda de la agudeza visual central en un período de uno o dos meses, discromatopsia en el eje verde-rojo, escotoma central, disminución de la sensibilidad al contraste, seguimiento sacádico, y el hallazgo más prominente, una pérdida de las estriaciones en la capa de fibras, ópticas retinianas que conforman el haz máculo-papilar. Posteriormente, esta definición fue adoptada por el grupo de médicos cubanos lo cual trajo aparejado una disminución del número de falsos positivos, inicialemente de un 50 por ciento. Adicionalmente los pacientes presentaron polineuropatía periférica con o sin mielopatía asociada, desnutrición con pérdida de peso variable entre 10 y 20 libras y deficiencias vitamínicas. Luego de asegurarse suplentación vitamínica a toda la población, en subsecuente visita, se comprobó que el número de casos había disminuido en forma dramática y todos los afectados habían mejorado en algún grado. La epidemia cubana se caracterizó por una multiplicidad de síndromes de naturaleza nutricional-carencial que ocurren al unísono. Muestras gran similitud con otras epidemias en prisiones de guerra observadas en el trópico durante la Segunda Guerra Mundial

MIP 28 forms channels in planar lipid bilayers

Fiber cells, which constitute most the lens tissue, have large amounts of a protein named the main intrinsic protein (MIP) in their plasma membrane, MIP seems to vary among species. On SDS-PAGE, MIP from bovine lens (MIP 26) migrates faster than MIP from chicken lens (MIP 28), which runs as a 28-KDa protein. Recently a number of laboratories have shown that MIP 26 forms channels in lipid bilayers. We have isolated membrane fractions highy enriched in MIP 28 from chicken lens and incorporated channel activity into planar bilayers from these membrane fractions before and after treatment with the detergent Triton X-100. Detergent treatment does not seem to affect channel properties. We have attempted to block channel activity with polyclonal antivodies against bovine and chicken MIP but failed to detect blockade using either detergent-free or detergent-treated membranes. Single channel size in symmetric solutions of 300 m§K2SO4(3-00-400pS) agrees well with published results one allows for corrections in ionic strenght. Preliminary experiments indicate that the incorporated channels display voltage dependence. The channel activity recorded from MIP 28-enriched membrane fractions ins qualitatively similar to that described for MIP 26 membrane fractions incorporated into bilayers. In contrast to previous reports, we do not find it necessary to add the membrane fractions to both sides of the bilayer to obtain channel incorporation. This may reflect the fact that MIP does not span two bilayers