Objective To compare the sensitivity and specificity of Leptospira interrogans using loop-mediated isothermal amplification (LAMP) and real-time PCR technology, then looking for a rapid,sensitive and specific methods for the detection of Leptospira interrogans. MethodsIn accordance with lipL41 gene from Leptospira interrogans, primers for LAMP and real-time PCR were designed and used to detect Leptospira interrogans in cultured 15 reference strains of 15 serogroups in China, then compared the sensitivity and specificity of the two methods in the detection of Leptospira interrogans. ResultsThe LAMP reaction could be completed within 30 min, and whole process of it less than 60 min. The whole real-time PCR reaction could be finished at about 60 min. Both of them had the same detection sensitivity and specificity,the lower detection limits in the reactions was approximately 100 copies and there was no false positive occurred. ConclusionBoth LAMP and real-time PCR were time-saving and had the same sensitivity and specificity. But LAMP reaction could be done under a constant temperature conditions, and need not a special expensive equipment. Therefore, as a sensitive and specific method for quantifying Leptospira interrogans,the LAMP assay was more rapidly and convenient than conventional methods.

Objective To construct the T-cells and B-cells combined epitope peptide gene based on the LipL32,OmpL1 and LipL21 protein from Leptospira interrogans and E.coli expression system,and better understanding of the immunological activity of the recombinant protein. MethodsThe immunodomaint T- and B-cells combined epitopes of LipL32,OmpL1 and LipL21 were identified and used to synthetic a new gene and then construct its prokaryotic expression system.The expression of recombinant protein was determined by SDS-PAGE; MAT was used to determine the titer of the antiserum to L. interrogans standard strains of China ; Western blot and ELISA were used to identify the immunity activity of the recombinant protein.Results The synthetic gene was effectively expressed in E.coli BL21 ( DE3 ) strain and mainly presented in dissoluble protein.Western blot result showed that the expression protein react well with the antibodies from immunized rabbit by Leptospira or recombinant protein.ELISA and MAT results showed that the multiepitope protein could cross-react with different serogroup or serovar of Leptospira.Conclusion In this study,we successfully constructed the recombinant T- and B-cells combine epitope gene of leptospires and expressed it in E.coli.The recombinant protein had a good immune activity,and could cross-reacted with antibodies from different serogroups Leptospira infected patients.

Objective To determine the modality of Leptospira interrogans invading human and murine mononuclear-macrophages and diversity of leptospiral phagocytotic vesicle formation. Methods Transmission electron microscopy was applied to observe the invasion of L. interrogans serogroup Icterohaemorrhagiae serovar Lai strain Lai into murine mononuclear-macrophage-like cell line J774A. 1 and PMA-activated human monocyte line THP-1 and the formation of leptospiral phagocytotic vesicles. By using immunofluorescence plus either laser confocal microscopy or fluorescence spectrophotometry, the changes of intracellular leptospiral numbers in J774A. 1 and PMA-activated THP-1 cells before and after block with endocytosis inhibitors monodansylcadaverin (MDC), phenylarsine oxide (PAO) and clathrin antibody were investigated. Results The leptospires in J774A. 1 cells were located in phagocytotic vesicles while the leptospires in THP-1 cells had no package with phagocytotic vesicle membrane. Both MDC and PAO presented the effect inhibiting endocytosis of L. interrogans into J774A. 1 and THP-1 cells in dose-dependent manner. The numbers of leptospires in J774A. 1 and THP-1 cells that pre-blocked with 10 μmol/L or above MDC and 1 μmol/L or above PAO were significantly less than that in the two cells untreated with MDC and PAO (P＜0.05＝. After J774A. 1 and THP-1 cells were blocked with clathrin antibody, the numbers of intracellular leptospires were also remarkbly decreased ( P<0.05 ).Conclusion Leptospira interrogans can invade into both human and murine mononuclear-macrophages through the way of clathrin-dependent endocytosis. There is an opposite diversity of leptospiral phagocytotic vesicle formations in human and murine mononuclear-macrophages, which may result in the difference of pathogenesis in human and mice after infected with L. interrogans.

Objective To determine the change of expression level of Leptospira interrogans sph2 gene, and hemolytic and cell apoptosis-inducing activities of sphingomyelinase hemolysin Sph2. Methods Entire sph2 gene fragment was amplified by PCR from genomic DNA of L. Interrogans serovar serogroup Icterohaemorrhagiae serovar Lai strain Lai, and sequenced after T-A cloning. Subsequently, a prokaryotic expression system of sph2 gene was constructed. The expression of target recombinant Sph2( rSph2 ) was examined by SDS-PAGE and the expressed rSph2 was extracted by Ni-NTA affinity chromatogaphy. The hemolytic activity of rSph2 was measured by hemolytic test in sheep blood agar plate and spectrophotometry-based hemoglobin measurement, and the apoptosis-inducing activity of rSph2 to murine mononuclear-macrophagelike cell line(J774A. 1) and hepatic cell line(IAR20) was determined by flow cytometry. A real-time fluorescence quantitative RT-PCR was applied to detect the change of sph2 mRNA levels before and after L. Interrogans strain Lai infecting J774A. 1 and IAR20 cells. Results The cloned sph2 gene had 100% sequence identity to the corresponding gene in GenBank. The constructed prokaryotic expression system was able to efficiently express rSph2. The rSph2 could lyse sheep erythrocytes in concentration-dependent pattern. 10μg/ml rSph2 could induce the apoptosis of J774A. 1 cells and IAR20 cells, and the peak apoptotic rates were 23.96% and 32.92%, respectively. The mRNA level of sph2 gene was significantly elevated within 0.5-2 h of L. Interrogans strain Lai infecting either J774A. 1 or IAR20 cells, and then the mRNA level was quickly descended. Conclusion The sph2 gene of L. Interrogans strain Lai has a transient expression when the microbe contacts host cells. rSph2 possesses activities of sheep erythrocyte lysis and inducing macrophage and hepatocyte apoptosis, indicating Sph2 as an important virulence factor during pathogenic process of Leptospira.

Adult ; Drug Resistance, Microbial ; Female ; Humans ; Male ; Middle Aged ; Pneumonia ; complications ; microbiology ; Silicotuberculosis ; complications ; microbiology

Objective:To confirm the Sigma N transcription factor activity of a gene product encoded by LA2404 gene of Leptospira interrogans ( L. interrogans) and to identify the target genes of Sigma N signaling system. Methods:L. interrogans LA2404 gene and its regulated target genes were predicted using bioinformatic analysis according to the promoter sequence signature in Sigma N-regulated genes. A LA2404 gene-knockout (ΔLA2404) strain of L. interrogans was constructed based on homologous sequence recombinant of suicide plasmid. Real-time fluorescent quantitative RT-PCR (qRT-PCR) was used to detect the changes in the expression of target genes at mRNA level in the ΔLA2404 mutant. A prokaryotic expression system for LA2404 gene was established and the target recombinant protein rSigma N was extracted by Ni-NTA affinity chromatography. Gel electrophoresis mobility shift assay (EMSA) was used to screen out the target genes regulated by rSigma N. Results:Pathogenic L. interrogans serogroup Icterohaemorrhagiae serovar Lai strain Lai carried one Sigma N gene and 22 Sigma N promoter sequence-containing target genes. Qualitative examination of the ΔLA2404 mutant by microscopy revealed no defect in motility and appearance. Expression of LA1188, LA2306, LA3426, LA1968, LA1313, LA3806 and LA0773 genes at mRNA level in the ΔLA2404 mutant was significantly down-regulated ( P<0.05), but no significant changes in the expression of other target genes at mRNA level were detected. EMSA results confirmed that rSigma N could bind to the promotor sequences of the target genes mentioned above. Conclusions:Sigma N transcription factor was encoded by LA2404 gene. LA1188, LA2306, LA3426, LA1968, LA1313, LA3806 and LA0773 genes contained Sigma N promoter sequence and the expression of them was regulated by Sigma N signaling system.

OBJECTIVE: To explore the changes of the serum immune cytokines in medical radiation workers exposure to low dose ionizing radiation. METHODS: Totally 244 medical professionals working with radiation(61 diagnosis radiology,51 nuclear medicine,74 radio therapeutics and 58 interventional radiology) from 7 hospitals of Guangdong Province were selected as study subjects by using the typical sampling method; 51 administration workers who did not expose to radiation were selected as control group. The radiation dose of these individuals was monitored by thermoluminescent measurement instrument for one year. Venous blood was collected and the levels of interferon γ(IFN-γ),interleukin 10(IL-10),transforming growth factor-β1(TGF-β1) in serum were examined by enzyme-linked immuno sorbent assay. RESULTS: The maximum annual average dose of radiation per person of the medical radiation workers was 0. 41 mSv/a. It was smaller than the occupational exposure limit(20. 00 mSv/a). The annual average dose of radiation per person in the group of nuclear medicine was significantly higher than those of diagnosis radiology,radio therapeutics and interventional radiology(P <0. 01). Among the male staffs,the expression of IL-10 in the diagnosis radiology group,radio therapeutics group and interventional radiology group was lower than that in the control group(P < 0. 05); the expression of IL-10 in radio therapeutics group was lower than those in nuclear medicine group and interventional radiology group(P < 0. 05); the ratio of IFN-γ/IL-10 in radio therapeutics group was higher than those in diagnosis radiology group,nuclear medicine group,interventional radiology group and control group(P < 0. 05). These individuals were divided into 3 different dose group(0. 03-,0. 06-and > 0. 15 m Sv/a) based on their average radiation dose. The expression of IL-10 in male staffs of these3 dose groups was lower than that of the male control group(P < 0. 05). CONCLUSION: Long-term low dose ionizing radiation may restrain the expression level of IL-10 in the male staffs.

Neuroinflammation is a common pathological feature of neurodegenerative diseases （NDs）. Microglia （MG）， a resident macrophage in the brain with a unique developmental origin， is the core driver of neuroinflammation. It can participate in the occurrence and development of NDs through different polarization states and play a key role in regulating neurogenesis and synapse shaping and maintaining homeostasis. MG can be divided into M1 pro-inflammatory phenotype and M2 anti-inflammatory phenotype according to its function. The inflammatory mediators released by the M1 phenotype can lead to nerve degeneration and myelin sheath damage， while the activation of the M2 phenotype is required to inhibit the inflammatory response and promote tissue repair. With the advantages of multi-pathway， multi-target， and bidirectional regulation， traditional Chinese medicine can regulate the polarization balance of MG and has dual effects on NDs such as Alzheimer's disease， Parkinson's disease， and multiple sclerosis. The active components of traditional Chinese medicine and its compound can inhibit the activation of MG by regulating phosphatidylinositol-3-kinases/protein kinase B（PI3K/Akt）， NOD-like receptor thermal protein domain associated protein 3（NLRP3）， signal transducer and activator of transcription factor1（STAT1）， nuclear transcription factor kappa B（NF-κB）， and other pathways， promote the polarization of M1 phenotype to M2 phenotype， reduce the expression of interleukin（IL）-6， tumor necrosis factor-α（TNF-α）， and other pro-inflammatory factors， and increase the secretion of IL-10， arginase-1（Arg-1）， and other anti-inflammatory factors. It can also reduce β-amyloid deposition and tau protein expression in Alzheimer's disease， alleviate dopaminergic neuronal damage in Parkinson's disease， and relieve demyelination， inflammatory cell infiltration， and related clinical symptoms of multiple sclerosis. The bidirectional regulation of the M1/M2 polarization balance of MG by traditional Chinese medicine is a potential strategy for the treatment of NDs. This paper focused on the targets of the regulation of MG polarization balance by traditional Chinese medicine monomer and its compound in the treatment of NDs， so as to further study and summarize the existing research results and provide ideas and basis for the future treatment of NDs.