Trypanosoma Cruzi: Early Resistance Induced By Culture-derived Trypomastigotes

Previous observations in this laboratory showed that injection of culture-derived trypomastigotes (CT), in CBA/J mice, induced an early increased resistance that was detected 24-72 hr after antigen injection and permitted mice to survive a challenge of 105 blood trypomastigotes (BT) corresponding to 2000 LD50%. Present experiments were conducted to determine the optimal conditions for inducing this early resistance and to investigate the early morphological changes which occurred in blood and lymphoid organs of mice infected with either BT or CT. Among nine antigens tested, only living CT showed a protective effect permiting most of mice to survive 30 days after BT challenge, while control mice injected with PBS or other antigens died at 10 ± 1 days. A dose-response relationship was seen when different doses of CT were tested, higher doses of CT inducing higher survival and lower parasitemia. Injection of CT by either an im or ip route induced similar degrees of resistance but significantly different results were obtained when mice were challenged by using ip or im routes. Higher parasitemia and lower survival were always obtained when animals were challenged by the ip route. Within 72 hr, mice injected with BT presented a lymphopenia which reached a maximum at 48 hr, a depletion of thymic cortical zone, and splenomegaly with hyperplasia of the white pulp and congestion of the red pulp. No gross alterations were observed in animals infected with CT. Overall data suggest that the early resistance is a specifically induced phenomenon and that BT and CT induce different early reactions in the CBA/J lymphoid organs. 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The Influence Of Type I Diabetes Mellitus On The Expression And Activity Of Gelatinases (matrix Metalloproteinases-2 And -9) In Induced Periodontal Disease

Background and Objective: Periodontal disease corresponds to a group of lesions that affect the tooth-supporting tissues present in the dental follicle. Although bacterial plaque is important, the immune response also contributes to the destruction of periodontal tissues. Diabetes mellitus is closely associated with the development, progression and severity of periodontal disease because it not only affects extracellular matrix organization but also the tissue response to inflammation. The objective of the present investigation was to study the influence of diabetes on experimental periodontal disease by evaluating the degradation of extracellular matrix through the analysis of matrix metalloproteinase (MMP)-2 and MMP-9 expression and activity, using immunofluorescence, zymography and real-time reverse transcription-polymerase chain reaction. Material and Methods: Wistar rats were divided into normal and diabetic groups and evaluated 0, 15 and 30 d after the induction of periodontal disease by ligature. Results: MMP-2 and -9 were detected in epithelial cells, in the blood vessel endothelium and in connective tissue cells. The same profile of enzymatic expression of MMP-2 and -9 was observed in normal and diabetic animals, with a peak in activity at day 15 of inflammation. However, in diabetic animals, MMP-2 gelatinolytic activity was reduced after the inflammatory stimulus, whereas that of MMP-9 was increased. MMP-2 gene expression decreased with inflammation in both normal groups and groups with diabetes. In contrast, MMP-9 expression increased in normal animals and decreased in diabetic animals after inflammation. Conclusion: The results suggest the involvement of MMP-2 and -9 in the dynamics of periodontal disease and that variation in their expression levels results in differences in tissue organization and wound healing in normal and diabetic animals. © 2007 Blackwell Munksgaard.4314854Lindhe, J., (1999) Tratado de Periodontologia Clínica e Implantologia Oral, 3rd Edn., pp. 46-67. , Rio de Janeiro: Rio de JaneiroGarlet, G.P., Avila-Campos, M.J., Milanezi, C.M., Ferreira, B.R., Silva, J.S., Actinobacillus actinomycetemcomitans-induced periodontal disease in mice: Patterns of cytokine, chemokine and chemokine receptor expression and leukocyte migration (2005) Microbes Infect, 7, pp. 738-747Potempa, J., Banbula, A., Travis, J., Role of bacterial proteinases in matrix destruction and modulation of host responses (2000) Periodontol 2000, 24, pp. 153-192Page, R.C., Schroeder, H.E., Pathogenesis of inflammatory periodontal disease. 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(2001) J Periodontol, 72, pp. 1398-1406Liu, R.K., Cao, C.F., Meng, H.X., Gao, Y., Polymorphonuclear neutrophils and their mediators in gingival tissues from generalized aggressive periodontitis (2001) J Periodontol, 72, pp. 1545-1553Ejeil, A.-L., Gaultier, F., Igondjo-Tchen, S., Are cytokines linked to collagen breakdown during periodontal disease progression? 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Campinas, SP: State University of Campinas, DissertationCarson, K.A., (1979) Cytochemical and Cytopathological Studies of Nerve, Vascular and Salivary Gland Tissues in An Hereditary Mouse Model for Diabetes Mellitus, , Chapel Hill, NC: University of North Carolina, DissertationJohnson, I.H., Effects of local irritation and dextran sulphate administration on the periodontium of the rat (1975) J Periodont Res, 10, pp. 332-345Bradford, M.M., A rapid and sensitive method of quantification of microgram quantities of protein utilizing the principle of protein-dye binding (1976) Anal Biochem, 72, pp. 248-254Ponte, E., Tabaj, D., Maglione, M., Melato, M., Diabetes mellitus and oral disease (2001) Acta Diabetol, 38, pp. 57-62Southerland, J.H., Taylor, G.W., Moss, K., Beck, J.D., Offenbacher, S., Commonality in chronic inflammation diseases: Periodontitis, diabetes, and coronary artery disease (2006) Periodontol 2000, 40, pp. 130-143Taylor, G.W., Bidirectional interrelationships between diabetes and periodontal diseases: An epidemiologic perspective (2001) Ann Periodontol, 6, pp. 99-112Holzhausen, M., Garcia, D.F., Pepato, M.T., Marcantonio Jr., E., The influence of short-term diabetes mellitus and insulin therapy on alveolar bone loss in rats (2004) J Periodont Res, 39, pp. 188-193Makela, M., Sato, T., Uitto, V., Larjava, H., Matrix metalloproteinases (MMP-2 and MMP-9) of the oral cavity: Cellular origin and relationship to periodontal status (1994) J Dent Res, 73, pp. 1397-1406Aiba, T., Akeno, N., Kawane, T., Okamoto, H., Horiuchi, N., Matrix metalloproteinases-1 and -8 and TIMP-1 mRNA levels in normal and diseased human gingivae (1996) Eur J Oral Sci, 104, pp. 562-569Garlet, G.P., Cardoso, C.R., Silva, T.A., Cytokine pattern determines the progression of experimental periodontal disease induced by Actinobacillus actinomycetemcomitans through the modulation of MMPs, RANKL, and their physiological inhibitors (2006) Oral Microbiol Immunol, 21, pp. 12-20Ingman, T., Tervahartala, T., Ding, Y., Matrix metalloproteinases and their inhibitors in gingival crevicular fluid and saliva of periodontitis patients (1996) J Clin Periodontol, 23, pp. 1127-1132Zhou, J., Windsor, J., Porphyromonas gingivalis affects host collagen degradation by affecting expression, activation, and inhibition of matrix metalloproteinases (2006) J Periodont Res, 41, pp. 47-54Saito, T., Shimazaki, Y., Kiyohaba, Y., The severity of periodontal disease is associated with the development of glucose intolerance in non-diabetics: The Hisama study (2004) J Dent Res, 83, pp. 485-490Bartold, P.M., Narayanan, A.S., Molecular and cell biology of healthy and diseased periodontal tissues (2006) Periodontol 2000, 40, pp. 29-4

Genetic Transformation Of Citrus Sinensis With Citrus Tristeza Virus (ctv) Derived Sequences And Reaction Of Transgenic Lines To Ctv Infection

Transgenic Citrus sinensis (L.) Osb. plants, cvs. Valencia and Hamlin, expressing Citrus tristeza virus (CTV) derived sequences were obtained by genetic transformation. The gene constructs were pCTV-CP containing the 25 kDa major capsid protein gene (CTV-CP), pCTV-dsCP containing the same CTV-CP gene in an intron-spliced hairpin construct, and pCTV-CS containing a 559 nt conserved region of the CTV genome. The transgenic lines were identified by PCR and the transgene integration was confirmed by Southern blot. Transgene mRNA could be detected in most transgenic lines containing pCTV-CP or pCTV-CS transgene. The mRNA of pCTV-dsCP transgene was almost undetectable, with very light bands in most analyzed plants. The transgene transcription appears to be closely linked to the type of gene construct. The virus challenge assays reveals that all transgenic lines were infected. However, it was possible to identify propagated clones of transgenic plants of both cultivars studied with a low virus titer, with values similar to the noninoculated plants (negative control). These results suggested that the transgenic plants present some level of resistance to virus replication. The higher number of clones with low virus titer and where mRNA could not be detected or was presented in a very light band was found for pCTV-dsCP-derived transgenic lines. © 2011 Springer Science+Business Media B.V.1

Characterization of Citrus tristeza virus isolates from grapefruit (Citrus paradisi Macf.) accessions of Citrus Active Germplasm Bank Caracterização de isolados do vírus da tristeza dos citros de acessos de pomelos (Citrus paradisi Macf.) do Banco Ativo de Germoplasma de Citros

Citrus tristeza virus (CTV) isolates from 35 grapefruit accessions belonging to Citrus Active Germplasm Bank of the "Instituto Agronômico de Campinas" located at the "Centro APTA Citros Sylvio Moreira", Cordeirópolis, São Paulo state, Brazil, were characterized and evaluated through symptoms in the trees, biological indexing, immunological diagnosis with different monoclonal antibodies and SSCP analysis (single-strand conformation polymorphism) of the coat protein gene. Symptomatology indicated that, in general, the group of plants with smaller canopy volume and severe stem pitting differed significantly from the group that presented greater vegetative development and mild to moderate stem pitting. However, the isolates from most of the accessions induced mild reaction on Mexican lime. The serological evaluation through the DAS-ELISA using monoclonal antibodies did not reveal any association between virus titer in the plant tissue and symptoms. The reaction with different monoclonal antibodies and the distinct electrophoresis patterns obtained through SSCP showed that there is a high degree of diversity among the isolates that infect these grapefruit accessions. High complexity within the same isolate was also observed in the SSCP profiles. This finding indicates that the CTV isolates from these plants are a complex mixture of CTV haplotypes. Similar SSCP banding patterns were observed among some plants with strong stem pitting symptoms, and among some plants with weak or moderate stem pitting symptoms.<br>Isolados do vírus da tristeza dos citros (CTV) de 35 acessos de pomelos que fazem parte do Banco Ativo de Germoplasma de Citros, localizado no Centro APTA Citros Sylvio Moreira, Cordeirópolis, São Paulo, Brasil, pertencente ao Instituto Agronômico de Campinas (IAC), foram caracterizados através dos sintomas observados nas árvores, indexação biológica, diagnóstico imunológico e análise SSCP (polimorfismo de conformação de fita simples) do gene da proteína do capsídeo. O grupo de plantas que, em geral, apresentou menor volume de copa e severo sintoma de canelura diferenciou-se significativamente do grupo com maior desenvolvimento vegetativo e fraco a moderado sintoma de canelura. No entanto, a maioria dos isolados de CTV das plantas de ambos os grupos induziu fraca reação em limão galego e nenhuma relação entre títulos do vírus nos tecidos e sintomatologia foi observada na avaliação sorológica conduzida por DAS-ELISA. A reação com diferentes anticorpos monoclonais e os distintos padrões eletroforéticos obtidos por SSCP demonstraram que há uma grande diversidade entre os isolados de CTV que infectam os acessos de pomelos. Alta complexidade de bandas dentro de um mesmo isolado foi também observada nos perfis SSCP, demonstrando que cada isolado é constituído por uma mistura de diferentes haplótipos de CTV. Padrões SSCP semelhantes foram observados entre algumas plantas com fortes sintomas de caneluras e entre algumas plantas com sintomas fracos ou moderados de caneluras