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Article in English | LILACS | ID: biblio-1056856


Abstract Objective: To determine the expression of TLR4 and MMP8 in gingival crevicular fluid [GCF] in patients with periodontitis. Material and Methods: Clinical samples were collected from 23 gingival crevicular fluid of periodontal disease subjects (n = 14) and healthy periodontal subjects (n=9). Measurement of Clinical parameters of probing pocket depth (PPD), bleeding on probing (BOP), and clinical attachment loss (CAL) were included as diagnostic criteria. Pocket Depth (PD) and CAL were defined as present if the PPD was ≥ 4 mm and the CAL ≥ 1 mm. Expression of TLR4 and MMP8 in the gingival crevicular fluid of deep pockets (PD≥ 6mm), shallow pockets (PD 4-5 mm) and healthy periodontal sulcus (0-3 mm) were evaluated by quantitative real-time polymerase chain reaction (qRT-PCR). Statistical analysis to compare the pocket was using Independent t-test and Mann-Whitney test. Correlation between mRNA expression and clinical parameters was analyzed using Spearman's correlation test Results: Expression of TLR4 was higher in shallow pockets compared to the control group, but the difference was not statistically significant (p>0.05). The expression of MMP8 was higher in shallow pockets compared to the control group, but the difference was not statistically significant (p>0.05) either. There is no significant correlation between TLR4 and MMP8 with clinical periodontal parameters Conclusion: TLR4 and MMP8 mRNA expression levels should not be used as a clinical biomarker in periodontitis diagnostic tools.

Humans , Female , Adult , Middle Aged , Periodontitis/diagnosis , RNA, Messenger/immunology , Matrix Metalloproteinase 8 , Toll-Like Receptor 4 , Radiography, Dental/instrumentation , Statistics, Nonparametric , Observational Studies as Topic , Indonesia/epidemiology
Braz. j. med. biol. res ; 48(12): 1095-1100, Dec. 2015. graf
Article in English | LILACS | ID: lil-762920


In DNA vaccines, the gene of interest is cloned into a bacterial plasmid that is engineered to induce protein production for long periods in eukaryotic cells. Previous research has shown that the intramuscular immunization of BALB/c mice with a naked plasmid DNA fragment encoding the Mycobacterium leprae 65-kDa heat-shock protein (pcDNA3-Hsp65) induces protection against M. tuberculosis challenge. A key stage in the protective immune response after immunization is the generation of memory T cells. Previously, we have shown that B cells capture plasmid DNA-Hsp65 and thereby modulate the formation of CD8+ memory T cells after M. tuberculosis challenge in mice. Therefore, clarifying how B cells act as part of the protective immune response after DNA immunization is important for the development of more-effective vaccines. The aim of this study was to investigate the mechanisms by which B cells modulate memory T cells after DNA-Hsp65 immunization. C57BL/6 and BKO mice were injected three times, at 15-day intervals, with 100 µg naked pcDNA-Hsp65 per mouse. Thirty days after immunization, the percentages of effector memory T (TEM) cells (CD4+ and CD8+/CD44high/CD62Llow) and memory CD8+ T cells (CD8+/CD44high/CD62Llow/CD127+) were measured with flow cytometry. Interferon γ, interleukin 12 (IL-12), and IL-10 mRNAs were also quantified in whole spleen cells and purified B cells (CD43−) with real-time qPCR. Our data suggest that a B-cell subpopulation expressing IL-10 downregulated proinflammatory cytokine expression in the spleen, increasing the survival of CD4+ TEM cells and CD8+ TEM/CD127+ cells.

Animals , Male , Mice , B-Lymphocytes/immunology , Heat-Shock Proteins/immunology , Immunomodulation/genetics , /genetics , RNA, Messenger/immunology , T-Lymphocyte Subsets/immunology , B-Lymphocytes/metabolism , Flow Cytometry , Gene Expression/genetics , Heat-Shock Proteins/therapeutic use , Immunologic Memory/physiology , Immunophenotyping/classification , Inflammation Mediators/analysis , Interferon-gamma/analysis , /immunology , /analysis , Mice, Knockout , Real-Time Polymerase Chain Reaction , Reverse Transcriptase Polymerase Chain Reaction , RNA, Messenger/genetics , Spleen/cytology , Spleen/immunology , T-Lymphocyte Subsets/classification , Vaccines, DNA/immunology , Vaccines, DNA/therapeutic use
Braz. j. med. biol. res ; 45(12): 1183-1194, Dec. 2012. ilus, mapas, tab
Article in English | LILACS | ID: lil-659642


In the last several years, the use of dendritic cells has been studied as a therapeutic strategy against tumors. Dendritic cells can be pulsed with peptides or full-length protein, or they can be transfected with DNA or RNA. However, comparative studies suggest that transfecting dendritic cells with messenger RNA (mRNA) is superior to other antigen-loading techniques in generating immunocompetent dendritic cells. In the present study, we evaluated a new therapeutic strategy to fight tuberculosis using dendritic cells and macrophages transfected with Hsp65 mRNA. First, we demonstrated that antigen-presenting cells transfected with Hsp65 mRNA exhibit a higher level of expression of co-stimulatory molecules, suggesting that Hsp65 mRNA has immunostimulatory properties. We also demonstrated that spleen cells obtained from animals immunized with mock and Hsp65 mRNA-transfected dendritic cells were able to generate a mixed Th1/Th2 response with production not only of IFN-γ but also of IL-5 and IL-10. In contrast, cells recovered from mice immunized with Hsp65 mRNA-transfected macrophages were able to produce only IL-5. When mice were infected with Mycobacterium tuberculosis and treated with antigen-presenting cells transfected with Hsp65 mRNA (therapeutic immunization), we did not detect any decrease in the lung bacterial load or any preservation of the lung parenchyma, indicating the inability of transfected cells to confer curative effects against tuberculosis. In spite of the lack of therapeutic efficacy, this study reports for the first time the use of antigen-presenting cells transfected with mRNA in experimental tuberculosis.

Animals , Male , Mice , Antigen-Presenting Cells/immunology , Bacterial Proteins/administration & dosage , /administration & dosage , Mycobacterium tuberculosis/immunology , RNA, Messenger/immunology , Tuberculosis Vaccines/administration & dosage , Tuberculosis/immunology , Bacterial Proteins/adverse effects , Bacterial Proteins/immunology , /adverse effects , /immunology , Mice, Inbred BALB C , RNA, Messenger/adverse effects , Spleen/immunology , Transfection , Tuberculosis Vaccines/adverse effects , Tuberculosis Vaccines/immunology , Tuberculosis/prevention & control
Salvador; s.n; 1993. 64 p. ilus.
Thesis in Portuguese | LILACS | ID: lil-175779


O gene completo codificando a enzima triose fosfato isomerase (TPI) do Schistosoma mansoni foi caracterizado a partir do estudo de dois ciclones exibindo áreas de superposiçäo, isolados, de uma biblioteca genômica em fago de lambda. Estes clones genômicos foram caracterizados através de mapas de restriçäo, sequenciamento do DNA envolvendo a regiäo a montante do gene ("5' flanking region"), exons, limites dos entrons e o sítio de poli-adenilaçäo. O gene da TPI do Schistosoma mansoni é codificado por 6 exons ocupando uma regiäo de aproximadamente 12 Kb. Os cinco introns estäo situados em posiçöes análogas aquelas para os genes da TPI de mamíferos, porém um dos 6 introns da TPI de mamíferos está ausente no S. mansoni. Nós näo encontramos evidências da participaçäo de "spliced leader" na expressäo do gene do TPI. O gene é precedido de pelo menos 4 cópias de sequências repetitivas de 2.5 Kb dispostas de forma linear. Apesar do gene de TPI de S. mansoni com 12 Kb, ser muito maior que um gene típico da TPI de um mamífero, o qual tem em média 3-4 Kb, ele apresenta o primeiro intron com apenas 42 bp. O sítio de iniciaçäo de transiçäo do S. mansoni é heterogêneo. A análise pela técnica de "Southern blot" sugere que o gene da TPI de S. mansoni se expressa a partir de uma única cópia do gene

Mice , Rabbits , DNA, Recombinant/immunology , Exons , Hybridomas/immunology , In Vitro Techniques , Introns , Polymerase Chain Reaction , RNA, Messenger/immunology , Schistosoma mansoni , Triose-Phosphate Isomerase , Antibodies, Monoclonal , Antigens , Biomphalaria/immunology , Cloning, Molecular , Genomic Library , Immunologic Tests , Mice/immunology , Molecular Biology