Serotyping of Toxoplasma gondii Infection Using Peptide Membrane Arrays.

The intracellular parasite Toxoplasma gondii can cause chronic infections in most warm-blooded animals, including humans. In the USA, strains belonging to four different Toxoplasma clonal lineages (types 1, 2, 3, and 12) are commonly isolated, whereas strains not belonging to these lineages are predominant in other continents such as South America. Strain type plays a pivotal role in determining the severity of Toxoplasma infection. Therefore, it is epidemiologically relevant to develop a non-invasive and inexpensive method for determining the strain type in Toxoplasma infections and to correlate the genotype with disease outcome. Serological typing is based on the fact that many host antibodies are raised against immunodominant parasite proteins that are highly polymorphic between strains. However, current serological assays can only reliably distinguish type 2 from non-type 2 infections. To improve these assays, mouse, rabbit, and human infection serum were reacted against 950 peptides from 62 different polymorphic Toxoplasma proteins by using cellulose membrane peptide arrays. This allowed us to identify the most antigenic peptides and to pinpoint the most relevant polymorphisms that determine strain specificity. Our results confirm the utility of previously described peptides and identify novel peptides that improve and increase the specificity of the assay. In addition, a large number of novel proteins showed potential to be used for Toxoplasma diagnosis. Among these, peptides derived from several rhoptry, dense granule, and surface proteins represented promising candidates that may be used in future experiments to improve Toxoplasma serotyping. Moreover, a redesigned version of the published GRA7 typing peptide performed better and specifically distinguished type 3 from non-type 3 infections in sera from mice, rabbits, and humans

Administration of a peptide inhibitor of alpha4-integrin inhibits the development of experimental autoimmune uveitis

Recruitment of lymphocytes into the retina and to the vitreous during the development of experimental autoimmune uveitis (EAU) is governed by factors such as the state of activation of inflammatory cells and the repertoire of adhesion molecules expressed by the local vascular endothelia. alpha4 Integrins and their receptors play an important role during homing of cells to the inflammatory site. In the present study, the effect of alpha4-integrin inhibitor on the development of EAU was investigated.Fil: Martín, Andrea P.. Universidade de Sao Paulo; BrasilFil: Vieira de Moraes, Luciana. Universidade de Sao Paulo; BrasilFil: Tadokoro, Carlos E.. Universidade de Sao Paulo; BrasilFil: Commodaro, Alessandra G.. Universidade de Sao Paulo; BrasilFil: Urrets Zavalia, Enrique. Universidad Catolica de Córdoba. Facultad de Medicina. Clinica Universitaria Reina Fabiola; ArgentinaFil: Rabinovich, Gabriel Adrián. Universidad de Buenos Aires. Facultad de Medicina. Hospital de Clínicas General San Martín; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; ArgentinaFil: Urrets Zavalía, Julio Alberto. Universidad Catolica de Córdoba. Facultad de Medicina. Clinica Universitaria Reina Fabiola; ArgentinaFil: Rizzo, Luiz V.. Universidade de Sao Paulo; Brasil. Fundação Zerbini; BrasilFil: Serra, Horacio Marcelo. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas; Argentin

The role of cytokines in the regulation of ocular autoimmune inflammation

The eye is a unique place for the development of an immune response. Beyond the usual mechanisms of immune restraint, the eye evolved with its exclusive mechanisms such as anterior chamber associated immune deviation. Therefore, immune-mediated inflammation in the eye does not develop at the same pace as in other sites of the body. Here we will address such peculiarities as they regard to ocular autoimmunity, using the experimental autoimmune uveitis as a model to understand the participation of cytokines in the process of aggression against the eye, as well as their immunoregulatory role. (c) 2007 Published by Elsevier B.V.Univ São Paulo, Dept Immunol, Biomed Sci Inst, BR-05508900 São Paulo, BrazilFundacao Univ Fed Rio Grande, Rio Grande do Sul, BrazilUniversidade Federal de São Paulo, Dept Ophthalmol, BR-04023062 São Paulo, BrazilUniv São Paulo, Sch Med, Div Clin Immunol & Allergy, Lab Med Invest, BR-01051 São Paulo, BrazilHeart Inst InCor & Fdn Zerbini, São Paulo, BrazilInst Invest Immunol III, São Paulo, BrazilUniversidade Federal de São Paulo, Dept Ophthalmol, BR-04023062 São Paulo, BrazilWeb of Scienc

Elevated Toxoplasma gondii Infection Rates for Retinas from Eye Banks, Southern Brazil

We found significantly higher incidence of Toxoplasma gondii DNA in eye bank specimens from Joinville in southern Brazil (13/15, 87%) than in São Paulo (3/42, 7%; p = 2.1 × 10E–8). PCR DNA sequence analysis was more sensitive at locus NTS2 than at locus B1; a high frequency of mixed co-infections was detected

Serotyping of Toxoplasma gondii Infection Using Peptide Membrane Arrays.

The intracellular parasite Toxoplasma gondii can cause chronic infections in most warm-blooded animals, including humans. In the USA, strains belonging to four different Toxoplasma clonal lineages (types 1, 2, 3, and 12) are commonly isolated, whereas strains not belonging to these lineages are predominant in other continents such as South America. Strain type plays a pivotal role in determining the severity of Toxoplasma infection. Therefore, it is epidemiologically relevant to develop a non-invasive and inexpensive method for determining the strain type in Toxoplasma infections and to correlate the genotype with disease outcome. Serological typing is based on the fact that many host antibodies are raised against immunodominant parasite proteins that are highly polymorphic between strains. However, current serological assays can only reliably distinguish type 2 from non-type 2 infections. To improve these assays, mouse, rabbit, and human infection serum were reacted against 950 peptides from 62 different polymorphic Toxoplasma proteins by using cellulose membrane peptide arrays. This allowed us to identify the most antigenic peptides and to pinpoint the most relevant polymorphisms that determine strain specificity. Our results confirm the utility of previously described peptides and identify novel peptides that improve and increase the specificity of the assay. In addition, a large number of novel proteins showed potential to be used for Toxoplasma diagnosis. Among these, peptides derived from several rhoptry, dense granule, and surface proteins represented promising candidates that may be used in future experiments to improve Toxoplasma serotyping. Moreover, a redesigned version of the published GRA7 typing peptide performed better and specifically distinguished type 3 from non-type 3 infections in sera from mice, rabbits, and humans

Oral tolerance reduces Th17 cells as well as the overall inflammation in the central nervous system of EAE mice

Multiple sclerosis (MS) is an autoimmune disease characterized by inflammatory immune response directed against myelin antigens of the central nervous system. in its murine model, EAE, Th17 cells play an important role in disease pathogenesis. These cells can induce blood-brain barrier disruption and CNS immune cells activation, due to the capacity to secrete high levels of IL-17 and IL-22 in an IL-6 + TGF-beta dependent manner. Thus, using the oral tolerance model, by which 200 mu g of MOG 35-55 is given orally to C57BL/6 mice prior to immunization, we showed that the percentage of Th17 cells as well as IL-17 secretion is reduced both in the periphery and also in the CNS of orally tolerated animals. Altogether, our data corroborates with the pathogenic role of IL-17 and IFN-gamma in EAE, as its reduction after oral tolerance, leads to an overall reduction of pro-inflammatory cytokines, such as IL-1 alpha, IL-6, IL-9, IL-12p70 and the chemokines MIP-1 beta, RANTES, Eotaxin and KC in the CNS. It is noteworthy that this was associated to an increase in IL-10 levels. Thus, our data clearly show that disease suppression after oral tolerance induction, correlates with reduction in target organ inflammation, that may be caused by a reduced Th1/Th17 response. Crown Copyright (c) 2010 Published by Elsevier B.V. All rights reserved.Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Univ São Paulo, Inst Biomed Sci, Clin Immunol Lab, BR-05508900 São Paulo, BrazilHarvard Univ, Brigham & Womens Hosp, Sch Med, Ctr Neurol Dis, Boston, MA 02115 USAUniversidade Federal de São Paulo, UNIFESP, Dept Microbiol Immunol & Parasitol, BR-04023062 São Paulo, BrazilAlbert Einstein Jewish Inst Educ & Res, BR-056519 São Paulo, BrazilUniversidade Federal de São Paulo, UNIFESP, Dept Microbiol Immunol & Parasitol, BR-04023062 São Paulo, BrazilCAPES: 4499-05-0CAPES: PNPD 0188085FAPESP: 2009/13109-5Web of Scienc

Galectin-1 suppresses autoimmune retinal disease by promoting concomitant Th2- and T regulatory-mediated anti-inflammatory responses

Intraocular inflammatory diseases are a common cause of severe visual impairment and blindness. In this study, we investigated the immunoregulatory role of galectin-1 (Gal-1), an endogenous lectin found at sites of T cell activation and immune privilege, in experimental autoimmune uveitis (EAU), a Th1-mediated model of retinal disease. Treatment with rGal-1 either early or late during the course of interphotoreceptor retinoid-binding protein-induced EAU was sufficient to suppress ocular pathology, inhibit leukocyte infiltration, and counteract pathogenic Th1 cells. Administration of rGal-1 at the early or late phases of EAU ameliorated disease by skewing the uveitogenic response toward nonpathogenic Th2 or T regulatory-mediated anti-inflammatory responses. Consistently, adoptive transfer of CD4(+) regulatory T cells obtained from rGal-1-treated mice prevented the development of active EAU in syngeneic recipients. In addition, increased levels of apoptosis were detected in lymph nodes from mice treated with rGal-1 during the efferent phase of the disease. Our results underscore the ability of Gal-1 to counteract Th1-mediated responses through different, but potentially overlapping anti-inflammatory mechanisms and suggest a possible therapeutic use of this protein for the treatment of human uveitic diseases of autoimmune etiology.Fil: Toscano, Marta Alicia. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; Argentina. Universidad de Buenos Aires. Facultad de Medicina. Hospital de Clínicas General San Martín; ArgentinaFil: Commodaro, Alessandra G.. Universidade de Sao Paulo; BrasilFil: Ilarregui, Juan Martin. Universidad de Buenos Aires. Facultad de Medicina. Hospital de Clínicas General San Martín; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; ArgentinaFil: Bianco, German Ariel. Universidad de Buenos Aires. Facultad de Medicina. Hospital de Clínicas General San Martín; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; ArgentinaFil: Liberman, Ana Clara. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Serra, Horacio M.. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas; ArgentinaFil: Jun Hirabayashi. National Institute of Advanced Industrial Science and Technology; JapónFil: Rizzo, Luiz V.. Universidade de Sao Paulo; BrasilFil: Rabinovich, Gabriel Adrián. Universidad de Buenos Aires. Facultad de Medicina. Hospital de Clínicas General San Martín; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; Argentin

Patients with diffuse uveitis and inactive toxoplasmic

Background/aims To determine if patients with inactive chorioretinitis lesions who experience chronic toxoplasmic uveitis test PCR positive for Toxoplasma in their ocular fluids.Methods Two patients undergoing long-term anti-toxoplasmic treatment developed chronic uveitis and vitritis. They underwent therapeutic and diagnostic pars plana vitrectomy. Patient specimens were tested for toxoplasmosis by real-time PCR and nested PCR. Patient specimens were also tested for the presence of Toxoplasma antibodies that recognise allelic peptide motifs to determine parasite serotype.Results Patients tested positive for Toxoplasma by real-time PCR at the B1 gene in the vitreous and aqueous humours of patient 1, but only the vitreous of patient 2. Patients were not parasitemic by real-time PCR in plasma and blood. During surgery, only old hyperpigmented toxoplasmic scars were observed; there was no sign of active retinitis. Multilocus PCR-DNA sequence genotyping at B1, NTS2 and SAG1 loci established that two different non-archetypal Toxoplasma strains had infected patients 1 and 2. A peptide-based serotyping ELISA confirmed the molecular findings.Conclusions No active lesions were observed, but both patients possessed sufficient parasite DNA in their vitreous to permit genotyping. Several hypotheses to explain the persistence of the vitritis and anterior uveitis in the absence of active retinitis are discussed.National Institute of Allergy and Infectious Diseases, National Institutes of HealthConselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Universidade Federal de São Paulo, Dept Ophthalmol, BR-04023062 São Paulo, BrazilNIAID, Parasit Dis Lab, Bethesda, MD 20892 USAHosp Albert Einstein, São Paulo, BrazilUniversidade Federal de São Paulo, Dept Ophthalmol, BR-04023062 São Paulo, BrazilCNPq: 237252/2012Web of Scienc

Intestinal microbiota regulates tryptophan metabolism following oral infection with Toxoplasma gondii

The intestinal microbiota plays an important role in modulating host immune responses. Oral Toxop/asma gondii infection can promote intestinal inflam­mation in certain mice strains. The 1DO-AhR axis may control tryptophan (Trp) me­tabolism constituting an important immune regulatory mechanism in inflammatory settings. Aims: In the present study, we investigated the role of the intestinal microbiota on Trp metabolism during oral infection with T gondii. Methods and results: Mice were treated with antibiotics for four weeks and then infected with T gondii by gavage. Histopathology and immune responses were eval­uated 8 days after infection. We found that depletion of intestinal microbiota by antibiotics contributed to resistance against T gondii infection and led to reduced expression of AhR on dendritic and Treg cells. Mice depleted of Gram-negative bac­teria presented higher levels of systemic Trp, downregulation of AhR expression and increased resistance to infection whereas depletion of Gram-positive bacteria did not affect susceptibility or expression of AhR on immune cells. Conclusion: Our findings indicate that the intestinal microbiota can control Trp avail­ability and provide a link between the AhR pathway and host-microbiota interaction in acute infection with T gondii.Fil: Santos, Liliane M.. Universidade Federal de Minas Gerais; BrasilFil: Commodaro, Alessandra G.. Universidade de Sao Paulo; BrasilFil: Vasquez, Alicia R. R.. Universidade Federal de Minas Gerais; BrasilFil: Kohlhoff, Markus. Instituto Federal de Educacao Ciencia E Tecnologia Do Sul de Minas.; BrasilFil: de Paula Guerra, Daniel A.. Universidade Federal de Minas Gerais; BrasilFil: Coimbra, Roney S.. Universidade Federal de Minas Gerais; BrasilFil: Martins Filho, Olindo A.. Universidade Federal de Minas Gerais; BrasilFil: Teixeira Carvalho, Andrea. Universidade Federal de Minas Gerais; BrasilFil: Rizzo, Luiz V.. Instituto Israelita de Pesquisa E Ensino, São Paulo; BrasilFil: Vieira, Leda Q.. Universidade Federal de Minas Gerais; BrasilFil: Serra, Horacio Marcelo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Centro de Investigaciones en Bioquímica Clínica e Inmunología; Argentina. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Departamento de Bioquímica Clínica; Argentin