Detection of Toxoplasma gondii DNA in peripheral blood and aqueous humor of patients with Toxoplasmic active focal necrotizing retinochoroiditis using real-time PCR / Detecção de DNA do Toxoplasma gondii no sangue periférico e humor aquoso por PCR em tempo real

ABSTRACT Purpose: To evaluate the ability of real-time quantitative PCR (qPCR) for detectingToxoplasma gondii DNA in the peripheral blood and aqueous humor of patients with toxoplasmic active focal necrotizing retinochoroiditis. Methods: Fifty-five patients with infectious uveitis seen from 2009 to 2013 at the Department of Ophthalmology and Visual Sciences of the Federal University of São Paulo were enrolled in this study. Forty-three patients had toxoplasmic active focal necrotizing retinochoroiditis, and the remaining 12 had non-toxoplasmic infectious uveitis and served as controls. qPCR analysis forT. gondii DNA was performed on the patients' peripheral blood and aqueous humor samples. Results: The qPCR was positive for T. gondii DNA in 37.21% (16/43) of the aqueous humor samples and 2.33% (1/43) of the peripheral blood samples; further, 16.27% (7/43) of the patients had positive results in both their blood and aqueous humor samples. Conclusion: qPCR was able to detect T. gondii DNA in patients with toxoplasmic active focal necrotizing retinochoroiditis in the blood as well as the aqueous humor and can help with the diagnosis of the disease.

RESUMO Objetivo: Analisar o uso do PCR em tempo real (qPCR) na detecção do DNA do T. gondii no sangue periférico e no humor aquoso de pacientes com lesões de retinocoroidite focal, ativa por toxoplasmose. Métodos: Cinquenta e cinco pacientes com uveite infecciosa foram incluídos neste estudo. Os pacientes foram atendidos entre 2009 a 2013, no Departamento de Oftalmologia e Ciências Visuais da Universidade Federal de São Paulo. Quarenta e três pacientes tiveram o diagnóstico de lesões de retinocoroidite focal, ativa por toxoplasmose e, os outros 12 tiveram o diagnóstico de uveíte infecciosa não toxoplásmica e, por isso foram usados como grupo controle. A técnica de qPCR foi utilizada na detecção de DNA do T. gondii em amostras de sangue periférico e humor aquoso. Resultados: O qPCR foi positivo para o DNA do T. gondii em 37,21% (16/43) das amostras de humor aquoso, 2,33% (1/43) nas amostras de sangue periférico e, 16,27% (7/43) em ambas amostras simultaneamente. Conclusão: O qPCR foi capaz de detectar o DNA do T. gondii em pacientes com lesões de retinocoroidite focal, ativa por Toxoplasmose, no sangue bem como, no humor aquoso, podendo ajudar no diagnostico.

The imbalance between Treg and Th17 cells caused by FTY720 treatment in skin allograft rejection

OBJECTIVES: FTY720 modulates CD4+T cells by the augmentation of regulatory T cell activity, secretion of suppressive cytokines and suppression of IL-17 secretion by Th17 cells. To further understand the process of graft rejection/acceptance, we evaluated skin allograft survival and associated events after FTY720 treatment. METHODS: F1 mice (C57BL/6xBALB/c) and C57BL/6 mice were used as donors for and recipients of skin transplantation, respectively. The recipients were transplanted and either not treated or treated with FTY720 by gavage for 21 days to evaluate the allograft survival. In another set of experiments, the immunological evaluation was performed five days post-transplantation. The spleens, axillary lymph nodes and skin allografts of the recipient mice were harvested for phenotyping (flow cytometry), gene expression (real-time PCR) and cytokine (Bio-Plex) analysis. RESULTS: The FTY720 treatment significantly increased skin allograft survival, reduced the number of cells in the lymph nodes and decreased the percentage of Tregs at this site in the C57BL/6 recipients. Moreover, the treatment reduced the number of graft-infiltrating cells and the percentage of CD4+ graft-infiltrating cells. The cytokine analysis (splenocytes) showed decreased levels of IL-10, IL-6 and IL-17 in the FTY720-treated mice. We also observed a decrease in the IL-10, IL-6 and IL-23 mRNA levels, as well as an increase in the IL-27 mRNA levels, in the splenocytes of the treated group. The FTY720-treated mice exhibited increased mRNA levels of IL-10, IL-27 and IL-23 in the skin graft. CONCLUSIONS: Our results demonstrated prolonged but not indefinite skin allograft survival by FTY720 treatment. This finding indicates that the drug did not prevent the imbalance between Tr1 and Th17 cells in the graft that led to rejection.

Real-time PCR in infectious uveitis as an alternative diagnosis / PCR em tempo real em uveites infecciosas como um diagnóstico alternativo

PURPOSE: Uveitis is a major visual impairment disease affecting parts or the entire uveal tract and occasionally the sclera, the cornea or the optic nerve. The disease is a major cause of ocular morbidity and blindness in immunocompetent and immunocompromised patients. In this work we analyzed the sensitivity and specificity of real-time PCR to detect the etiological agent from blood, plasma, vitreous and aqueous humor and compared with the diagnostic hypothesis. METHODS: Twenty-seven patients (13 male) were studied and Real-time PCR method was used for the detection of herpes simplex virus 1 (HSV-1), herpes simplex virus 2 (HSV-2), varicella zoster virus (VZV), cytomegalovirus (CMV), Mycobacterium tuberculosis (TB) and Toxoplasma gondii (Toxo) in the aqueous humor as well as in the vitreous, blood and plasma. RESULTS: Our results showed the presence of Toxo, CMV, VZV or HSV-2 in 19.2 percent of aqueous humor samples, and in 30 percent of vitreous humor samples. In plasma and blood samples, only CMV was detected (11.1 percent and 3.7 percent, respectively). CONCLUSION: Real-time PCR was able to detect and to confirm diagnostic hypothesis in uveitis. Our data also confirms that vitreous humor is the best source for molecular diagnosis of infectious uveitis but indicates aqueous humor samples that are easier to obtain may also be appropriate to be tested by Real-time PCR.

OBJETIVO: Uveíte é a maior causa de doença ocular que afeta o trato uveal, e ocasionalmente a esclera, cornea e o nervo óptico. Esta doença é a maior causa de morbidade ocular e cegueira em pacientes imunocompetentes e imunossuprimidos. Neste trabalho nós analisamos a sensiblidade e especificidade do PCR em tempo real para detectar agentes etiológicos no sangue, plasma, humor vítreo e aquoso, e comparamos com a hipótese diagnóstica. MÉTODOS: Vinte e sete pacientes (13 homens) foram estudados e o método de PCR em tempo real foi usado para detectar o vírus da herpes simples 1 (HSV-1), vírus da herpes simples 2 (HSV-2), vírus varicella zoster (VZV), citomegalovírus (CMV), Mycobacterium tuberculosis (TB) e Toxoplama gondii (Toxo) no humor aquoso e vítreo, além do sangue e plasma. RESULTADOS: Nossos resultados mostraram a presença de Toxo, CMV, VZV ou HSV-2 em 19,2 por cento das amostras de humor aquoso, e em 30 por cento das amostras de humor vítreo. Nas amostras de plasma e sangue somente CMV foi detectado (11,1 por cento e 3,7 por cento, respectivamente). CONCLUSÃO: PCR em tempo real foi capaz de detectar e confirmar a hipótese diagnóstica em uveíte. Nossos dados confirmam que o humor vítreo é a melhor fonte para diagnóstico molecular de uveíte infecciosa, porém o humor aquoso também foi uma fonte importante de detecção, além de ser mais fácil de se obter.

Autoimmune uveitis: study of treatment therapies / Uveíte autoimune: estudo de terapias para tratamento

Experimental autoimmune uveitis is an organ-specific T-cell mediated autoimmune disease characterized by inflammation and consequent destruction of the neural retina and adjacent tissues. Inflammation in experimental autoimmune uveitis may be induced in rodents by immunization with retinal antigens, such as interphotoreceptor retinoid-binding protein. We present a review of experimental studies that correlate primary immunobiological functions with this chronic disease and the possible use of molecules for the treatment of autoimmune uveitis.

A uveíte autoimune experimental é uma doença autoimune mediada por células T, órgão-específica e caracterizada por inflamação e subsequente destruição da retina neural e tecidos adjacentes. A inflamação na uveíte autoimune experimental pode ser induzida em roedores pela imunização com antígenos retinianos, tais como a proteína interfotorreceptora ligante de retinoide. Apresentamos aqui uma revisão de estudos experimentais que correlacionam as principais funções imunobiológicas com esta doença crônica e o possível uso de moléculas para o tratamento da uveíte autoimune.