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Objective To analyze the effects of Leptospira interrogans ( L. interrogans) infection on the activation of NLRP3 in THP-1 and J774A. 1 cells and to further understand the mechanism of inflam-mation caused by L. interrogans in different hosts. Methods Human mononuclear macrophage cell line (THP-1) and murine mononuclear macrophage cell line (J774A. 1) were infected with L. interrogans strain 56601. The expression of NLRP3 at mRNA and protein levels were measured by using real-time RT-PCR and flow cytometry analysis, respectively. The NLRP3-mediated secretion of IL-1β, IL-18 and IL-33 was detec-ted by ELISA combined with the NLRP3 inhibitory test. Results Compared with the normal cells, the ex-pression of NLRP3 at mRNA level in L. interrogans-infected THP-1 cells was respectively increased by 4. 05, 0. 34, 0. 33, 0. 06 and 1. 66 times at the time points of 1 h, 2 h, 4 h, 12 h and 24 h after infection ( P0. 05). Results of the inhibitory test showed that the up-regulation of IL-1β , IL-18 and IL-33 in THP-1 and J774A. 1 cells were effectively inhibited by the specific inhibitor of NLRP3. Conclusion NL-RP3 inflammasome was activated and involved in the production of specific inflammatory cytokines IL-1βand IL-18 in both THP-1 and J774A. 1 cells after L. interrogans infection, but the inflammatory cytokines induced by L. interrogans infection varied in different cells. L. interrogans induced earlier and higher level of IL-1βand IL-18 production in human macrophages than in murine macrophages.
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Objective To investigate the effects of Leptospira interrogans ( L. interrogans) infec-tion on the activities of NADPH oxidase ( nicotinamide adenine dinucleotide phosphate-oxidase) and the lev-els of reactive oxygen species (ROS) in THP-1 and J774A. 1 cells and to understand the bactericidal mecha-nisms of macrophages in different hosts against L. interrogans. Methods Human mononuclear macrophage cell line (THP-1 cells) and murine mononuclear macrophage cell line (J774A. 1 cells) were infected with L. interrogans strain 56601. The activities of NADPH oxidase and the levels of superoxide ion ( O-2 ) were measured with spectrophotography. Changes in the levels of ROS were detected with immunofluorescence as-say. Results Compared with the normal cells, the activities of NADPH oxidase in L. interrogans-infected J774A. 1 cells changed from 0. 619 0 μmol · min-1 · mg-1 to 0. 305 5 μmol · min-1 · mg-1 , 6. 141 5μmol·min-1 ·mg-1 , 1. 487 1μmol·min-1 ·mg-1 and 0. 964 6μmol·min-1 ·mg-1 after 2, 4, 12 and 24 hours of infection, respectively (P<0. 05), while the activities of NADPH oxidase in L. interrogans-infected THP-1 cells were up-regulated from 0. 723 5μmol·min-1 ·mg-1 to 0. 884 2μmol·min-1 ·mg-1 , 1. 897 1μmol·min-1 ·mg-1 , 1. 125 4 μmol·min-1 ·mg-1 and 0. 562 7 μmol·min-1 ·mg-1 , respectively ( P<0. 05). The levels of O-2 in L. interrogans-infected J774A. 1 cells at the time points of 2 h, 4 h, 12 h and 24 h after infection increased from 0. 189 0μmol/L to 0. 236 3μmol/L, 0. 297 7μmol/L, 0. 324 0μmol/L and 0. 305 7 μmol/L, respectively (P<0. 05), while the levels of O-2 in L. interrogans-infected THP-1 cells rose from 0. 123 7 μmol/L to 0. 149 3 μmol/ L, 0. 249 0 μmol/ L, 0. 270 0 μmol/ L and 0. 272 7μmol/L, respectively (P<0. 05). The fluorescence intensity of ROS in THP-1 and J774A. 1 cells increased gradually after infection with L. interrogans for 2 h and decreased after reaching the peak at 24 h. Conclu-sion Both the activities of NADPH oxidase and the levels of O-2 in J774A. 1 and THP-1 cells were signifi-cantly upregulated after infected with L. interrogans, especially in J774A. 1 cells. The results of this study provided references for further elucidating the bactericidal mechanisms of macrophages in different hosts against L. interrogans.
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Objective To investigate the expression of inducible nitric oxide synthase ( iNOS) and the levels of nitric oxide (NO) in THP-1 and J774A. 1 cells during Leptospira interrogans (L. interrogans) infection for a better understanding of the mechanism of macrophages involved defense against L. interrogans strains in different hosts. Methods The human mononuclear macrophages (THP-1) and the murine mono-nuclear macrophages (J774A. 1) were infected with L. interrogans strain 56601. The expression of iNOS at mRNA and protein levels were determined by using real-time RT-PCR and flow cytometry analysis. The lev-els of NO were detected with Griess test. Results The expression of iNOS at mRNA level in J774A. 1 and THP-1 cells infected with L. interrogans strains for 2, 4, 12 and 24 hours were respectively 1. 37, 2. 82, 25. 76, 27. 47 times and 1. 59, 3. 98, 3. 89, 8. 81 times than that of cells without infection (P<0. 05). The expression rates of iNOS protein in J774A. 1 cells were increased from 34. 16% to 85. 85%, 93. 82%, 91. 77% and 93. 65% along with the increased time of infection time (P<0. 05). The expression rates of iNOS protein in THP-1 cells were up-regulated from 22. 08% to 72. 64%, 81. 33%, 80. 03% and 65. 72%after 2, 4, 12 and 24 hours of infection (P<0. 05), respectively. Results of the Griess test indicated that the levels of NO in J774A. 1 and THP-1 cells were respectively increased from 0. 1588 μmol/L to 0. 2208μmol/L, 0. 2668μmol/L, 0. 3808μmol/L, 0. 3828μmol/L and from 0. 0988μmol/L to 0. 2848μmol/L,0. 3228 μmol/L, 0. 2608μmol/L and 0. 3308μmol/L after infection with L. interrogans strains for 2, 4, 12 and 24 hours (P<0. 05). Conclusion The expression of iNOS and the levels of NO in J774A. 1 and THP-1 cells were significantly increased during L. interrogans infection. This study might help to explain the bactericidal mechanism of macrophages derived from different hosts against L. interrogans infection.
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The complete gene sequences of eight capripoxvirus strains in GenBank were aligned and analyzed with DNAStar software. We selected a size of 64 bp gene fragment that was located in gp064 region of goat pox virus (GPV) genome, and designed a pair of primers and a TaqMan-MGB probe against the gene fragment with Primer Express 2.0 software. Then, the fluorescence quantitative PCR (FQ-PCR) assay was developed and the standard curve of different dilution series was described. We extracted the DNA samples from clinical skin pox, scab and GPV infected materials of artificial challenge animals. The FQ-PCR assay has been performed for all kinds of DNA samples. The results showed that the FQ-PCR assay was sensitive, specific, stable and could be used for clinical diagnosis. This method provided an important tool for rapid diagnosis of goat pox clinically, and for study GPV pathogenesis in the course of disease occurrence, development and convalescence.
Subject(s)
Animals , Base Sequence , Capripoxvirus , Genetics , Goats , Molecular Sequence Data , Polymerase Chain Reaction , Methods , Poxviridae Infections , Diagnosis , Virology , Sensitivity and SpecificityABSTRACT
Frogs were caught from 4 towns in Huaxi of Guiyang and dissected.The collected spargana were used to infect young dogs for species identification.Results showed that the wild frogs were identified as Rana nigromaculata, and the infection rate was 16%(131/818) with an average intensity infection of 3.44 per frog, The tapeworm obtained from an infected dog was specified as Spirometra mansoni.