Correlação entre o diagnóstico clínico e análise do PCR de amostras do soro, aquoso e vítreo em pacientes com doença inflamatória ocular

PURPOSE: To study the applicability (sensitivity, specificity) of polymerase chain reaction (PCR) tests in the detection of cytomegalovirus (CMV), herpes virus (HSV) and varicella zoster (VZV), Epstein-Barr virus (EBV), Mycobacterium sp and Toxoplasma gondii in the diagnosis of patients with or without AIDS, with presumably infectious uveitis, using serum, aqueous humor and vitreous humor samples. METHODS: Twenty individuals with uveitis of presumed infectious origin were evaluated. Sixteen of them had AIDS, four were immunocompetent individuals. We also evaluated 4 normal controls who underwent vitrectomy surgery. Clinical evaluation of the patients was performed together by three clinicians. PCR evaluations of the serum, aqueous, and vitreous humor were performed in a masked fashion by the laboratory staff. RESULTS: Twelve patients had a clinical diagnosis of CMV retinitis. Of these 6 (50%) had a positive PCR for CMV in the vitreous, three (25%) had a positive PCR for CMV in the serum, and none were positive in the aqueous. Five patients had a clinical diagnosis of acute retinal necrosis (ARN). Three (60%) of these had positive PCR for HSV/VZV in the vitreous. One of these patients had a positive PCR reaction for both EBV and HSV/VZV in the vitreous samples. One patient with cutaneous herpes zoster had a positive PCR reaction for HSV/VZV in the serum. Four patients had a presumed diagnosis of ocular toxoplasmosis, one patient (25%) had a positive PCR for Toxoplasma gondii in the serum, 3 (75%) had positive results in the aqueous, and 2 (50%) had positive results in the vitreous. One patient with presumed ocular tuberculosis had a positive PCR reaction both in the serum and in the vitreous samples. Finally, none of the four control individuals revealed any positive PCR reaction. CONCLUSION: PCR is an auxiliary diagnostic procedure that should be evaluated together with ophthalmological aspects of the patient.OBJETIVOS: Avaliar a aplicabilidade (especificidade, sensibilidade) do teste da reação da cadeia de polimerase (PCR) na detecção de citomegalovírus (CMV), herpes vírus e varicela zoster (HSV, VZV), Epstein-Barr vírus (EBV), Mycobacterium sp e Toxoplasma gondii no diagnóstico de pacientes com ou sem AIDS, com uveíte infecciosa presumível, utilizando amostras de humor aquoso, humor vítreo e soro. MÉTODOS: Vinte pacientes com uveíte infecciosa presumível foram estudados. Dezesseis destes apresentavam AIDS e quatro eram imunocompetentes. Foram utilizados quatro pacientes como grupo controle que se submeteram a vitrectomia. A avaliação clínica foi feita conjuntamente com três oftalmologistas. O exame do PCR do soro, aquoso e vítreo foi feito sem o conhecimento da hipótese diagnóstica pela equipe do laboratório. RESULTADOS: Doze pacientes tinham o diagnóstico clínico de retinite por CMV. Deste subgrupo 6 (50%) eram PCR positivo para CMV no vítreo, 3 (25%) eram PCR positivos para CMV no soro e nenhum destes foi positivo no aquoso. Cinco pacientes tinham o diagnóstico clínico de necrose aguda de retina (ARN). Três (60%) destes eram PCR positivos para HSV/VZV no vítreo. Um destes pacientes era PCR positivo tanto para EBV e HSV/VZV na amostra do vítreo. Um destes pacientes com herpes zoster cutâneo era PCR positivo para HSV/VZV no soro. Quatro pacientes tinham o diagnóstico de toxoplasmose ocular presumida, um paciente (25%) era PCR positivo para Toxoplasma gondii no soro, 3 (75%) eram positivos no aquoso e 2 (50%) eram positivo no vítreo. Um paciente com tuberculose ocular presumível era PCR positivo tanto no soro quanto no vítreo. Nenhum dos pacientes do grupo controle era PCR positivo em qualquer amostra. CONCLUSÃO: O exame do PCR é procedimento diagnóstico auxiliar que deve ser utilizado conjuntamente com os aspectos clínicos.Universidade Federal de São Paulo (UNIFESP) Setor de Uveite e AIDSUNIFESP Departamento de OftalmologiaUniversidade de São Paulo Departamento de ImunologiaUNIFESP, Setor de Uveite e AIDSUNIFESP, Depto. de OftalmologiaSciEL

Autoimmunity in Chagas' disease: immunomodulation of autoimmune and T. cruzi-specific immune responses

Faculdade de Medicina (FMD

Use of albumin in exfoliative cytology in patients with allergic conjunctivitis

PURPOSE: To evaluate albumin as fixative material of conjunctival scrapings for exfoliative cytology. METHODS: Conjunctival exfoliative cytology was performed through a prospective study in forty-six eyes of twenty-three patients with clinical diagnosis of allergic conjunctivitis. Three scrapings were performed: (1) in the pretreatment period, (2) after use of corticosteroids (treatment A) and (3) after use of mast cell stabilizer (treatment B); in two types of smears, with and without albumin, which were then submitted to staining by the Giemsa method. RESULTS: Of the twenty-three patients, five (21.7%) were female and eighteen (78.3%) male, with an age average of 10.8 years. Eighteen (78.3%) presented vernal conjunctivitis and five (21.7%) atopic keratoconjunctivitis. By the statistical analysis of the conjunctival cytology, the smears treated with albumin presented a larger number of intact epithelial cells in relation to the nontreated, only in the pretreatment and in the B treatment groups. The number of neutrophils was significantly greater in the B treatment group than in the smears not treated with albumin in relation to the treated ones. Regarding the number of eosinophils there was no difference between the smears treated with albumin in relation to the nontreated, while in the B treatment group there was a statistical difference between the right and left eye regarding smears without albumin. CONCLUSION: There were no statistical differences between smears treated with albumin and those not treated for most researched cells; in addition, albumin adheres mucus hindering the observation of smears, and thus is not indicated for conjunctival exfoliative cytology.OBJETIVO: Avaliar a albumina como fixador de material obtido de raspado conjuntival para citologia esfoliativa. MÉTODOS: Foi realizada citologia esfoliativa conjuntival por meio de estudo prospectivo em quarenta e seis olhos de vinte e três pacientes com conjuntivite alérgica. Foram realizadas três colheitas: (1) na fase aguda, (2) após uso de corticosteróides (tratamento A) e (3) após uso de estabilizador de membrana de mastócitos (tratamento B); em dois tipos de lâminas, com e sem albumina, sendo submetidas à coloração pelo método de Giemsa. RESULTADOS: Dos vinte e três pacientes, cinco (21,7%) eram do sexo feminino e dezoito (78,3%) do sexo masculino, com média de 10,8 anos. Dezoito (78,3%) apresentaram conjuntivite primaveril e cinco (21,7%) ceratoconjuntivite atópica. Após análise estatística da citologia conjuntival, as lâminas tratadas com albumina apresentaram maior número de células epiteliais íntegras em relação às não tratadas, apenas nos grupos pré-tratamento e no grupo do tratamento B. A contagem de neutrófilos foi significativamente maior no grupo de tratamento B nas lâminas não tratadas com albumina em relação às tratadas. Na contagem de eosinófilos não houve diferença entre as lâminas tratadas com albumina em relação às não tratadas, já no grupo de tratamento B houve diferença estatística entre olho direito e esquerdo na lâmina sem albumina. CONCLUSÃO: Não houve diferenças estatísticas entre as lâminas tratadas com albumina e as não tratadas para a maioria das células pesquisadas, além disso, a albumina adere muco dificultando a observação das lâminas, não sendo indicada na citologia esfoliativa de conjuntiva.Universidade Federal de São Paulo (UNIFESP) Departamento de OftalmologiaUniversidade de São Paulo Instituto de Ciências Biomédicas Laboratório de Imunologia ClínicaUniversidade Federal de São Paulo (UNIFESP) Departamento de Oftalmologia Laboratório de Doença Externa Ocular e CórneaUniversidade Federal de São Paulo (UNIFESP) Departamento de Oftalmologia Setor de Doença Externa Ocular e CórneaUNIFESP, Depto. de OftalmologiaUNIFESP, Depto. de Oftalmologia Laboratório de Doença Externa Ocular e CórneaUNIFESP, Depto. de Oftalmologia Setor de Doença Externa Ocular e CórneaSciEL

Impacto da adiposidade nos parâmetros imunológicos

Studies evaluating immune function in obese humans and experimental animals indicate that the excess adiposity is associated with impaired in immune responses. Obesity is related to a higher rate of infections and to some types of cancer. Nutritional, metabolic and endocrine factors are implicated in the immunological changes. The adipose tissue directly produces substances with various functions related to immune system. Furthermore, some investigations suggest that certain types of weight reduction strategies can alter the immune function. Nevertheless, long-term studies should be carried out to address whether these changes positively affects the ability of these obese individuals to control infections and tumor development.Estudos acerca da função imunológica em animais experimentais e humanos obesos indicam que o excesso de adiposidade associa-se ao prejuízo da resposta imune. A obesidade está relacionada a uma taxa maior de infecções e a alguns tipos de câncer. Fatores nutricionais, metabólicos e endócrinos estão implicados nessas alterações imunológicas. O próprio tecido adiposo produz diretamente substâncias com várias funções relacionadas ao sistema imune. Além disso, algumas investigações sugerem que certas estratégias para perda de peso podem alterar a função imune. Entretanto, estudos em longo prazo são necessários para avaliar se tais alterações afetam positivamente a capacidade desses pacientes obesos de controlar infecções e desenvolver tumores

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

Early IL-10 production is essential for syngeneic graft acceptance

We performed a comparative study and evaluated cellular infiltrates and anti-inflammatory cytokine production at different time-points after syngeneic or allogeneic skin transplantation. We observed an early IL-10 production in syngeneic grafts compared with allografts. This observation prompted us to investigate the role of IL-10 in isograft acceptance. For this, we used IL-10 KO and WT mice to perform syngeneic transplantation, where IL-10 was absent in the graft or in the recipient. The majority of syngeneic grafts derived from IL-10 KO donors did not engraft or was only partially accepted, whereas IL-10 KO mice transplanted with skin from WT donors accepted the graft. We evaluated IL-10 producers in the transplanted skin and observed that epithelial cells were the major source. Taken together, our data show that production of IL-10 by donor cells, but not by the recipient, is determinant for graft acceptance and strongly suggest that production of this cytokine by keratinocytes immediately upon transplantation is necessary for isograft survival. J. Leukoc. Biol. 92: 259-264; 2012.FAPESPFAPESPCNPqCNPqFCTFC

Lack of evidence for superantigen activity of Toxoplasma gondii towards human T cells

Toxoplasma gondii is an obligatory intracellular parasite whose life cycle may include man as an intermediate host. More than 500 million people are infected with this parasite worldwide. It has been previously reported that T. gondii contains a superantigen activity. The purpose of the present study was to determine if the putative superantigen activity of T. gondii would manifest towards human T cells. Peripheral blood mononuclear cells (PBMC) from individuals with no previous contact with the parasite were evaluated for proliferation as well as specific Vß expansion after exposure to Toxoplasma antigens. Likewise, PBMC from individuals with the congenital infection were evaluated for putative Vß family deletions in their T cell repertoire. We also evaluated, over a period of one year, the PBMC proliferation pattern in response to Toxoplasma antigens in patients with recently acquired infection. Some degree of proliferation in response to T. gondii was observed in the PBMC from individuals never exposed to the parasite, accompanied by specific Vß expansion, suggesting a superantigen effect. However, we found no specific deletion of Vß (or Valpha) families in the blood of congenitally infected individuals. Furthermore, PBMC from recently infected individuals followed up over a period of one year did not present a reduction of the Vß families that were originally expanded in response to the parasite antigens. Taken together, our data suggest that T. gondii does not have a strong superantigen activity on human T cells.Universidade de São Paulo Faculdade de Medicina Instituto de Ciências BiomédicasUniversidade de São Paulo Faculdade de Medicina Divisão de OftalmologiaUniversidade de São Paulo Faculdade de Medicina Divisão de Alergia e Imunologia ClínicaUniversidade Federal de São Paulo (UNIFESP) Departamento de OftalmologiaFundação E.J. ZerbiniUNIFESP, Depto. de OftalmologiaSciEL