AIM: To explore the significance of measuring urinary complement C5b-9 complex in various types of immune complex (IC) nephritis models of rats. METHODS: The four models of rats, namely, passive Heymann nephritis (PHN), anti-thymocyte serum nephritis(ATSN), anti-glomerular basement membrane nephritis (AGBMN) and chronic serum disease nephritis (CSDN) were reproduced. Then, the contents of complement C5b-9 complex in plasma and urine of the rats were detected with sandwich ELISA. And the deposits of C5b-9 complex in glomeruli of the rats were examined by ABC immunohistochemistry staining. RESULTS: The contents of rat plasma C5b-9 were elevated and deposits of C5b-9 in glomeruli could be detected in the four model rats. But the increased urinary excretion of C5b-9 was observed only in PHN rats. Moreover, the time of urinary C5b-9 complex excretion was earlier than that of urinary protein in the rats with PHN. CONCLUSION: Urinary C5b-9 complex excretion could be taken as one of several sensitive immunologic parameters in diagnosing of PHN and in distinguishing PHN from other type of nephritis.

Objective To determine the effect of apoptosis in different host cells induced by L. in- terrogans and the associated intracellular signaling pathway. Methods L. interrogans serogroup Icterohaem- orrhagiae serovar icterohaemorrhagiae strain Lai infected cell models in mouse mono-macrophage like cell J774A. 1, human EVC304 cells and A549 cells were established, respectively. Flow cytometry with fluores- cein labeling of FITC-Annexin V/PI was performed to examine the apoptosis or necrosis of the infected cells. Fluorometry as well as Western blot assay was applied to measure the activity of caspase-3, -8, -9 and the expression levels of apoptotic associated protein FADD in the infected cells , respectively . Results 36.70%-63.70% of the L. interrogans strain Lai infected J774A. 1 cells displayed obvious early apoptosis during the infection for 1-6 h, and then altered to later apoptosis / necrosis (53.68%) as the major injury pattern when infected for 12 h. 78.52% of the L. interrogans strain Lai infected A549 cells only showed later apoptosis / necrosis. However, no obvious apoptosis and / or necrosis could be found in the L. interrogans strain Lai infected EVC304 cells. The maximal activities of caspase-3 and -8 in the infected J774A. 1 cells were (1453.41±36.07) and (1402.15± 59.09) FU, respectively, which were the 16.38-fold and 29.99- fold of those the non-infected cells. The caspase-9 activity of the infected J774A. 1 cells slightly increased [(89.42±5.08) FU ], which significantly lower than those of caspase-3 and -8 (P <0.001). The FADD expression level of the infected J774A. 1 cells was gradually increased in an infection time-dependent man- ner. Conclusion There is a distinct diversity of apoptosis in different cells induced by L. interrogans, and FADD→caspase-8→caspase-3 is the major signaling pathway to mediate L. interrogans infection associated cell apoptosis.

AIM: To investigate the effects of FK506 on anti-glomerular basement membrane(GBM) nephritis in rats. METHODS: Anti-GBM nephritis model was elaborated by rabbit anti-rat GBM antibody injection in SD rats in this study. The rats were divided into three groups: FK506 treated group(0 5 mg?kg -1 ?d -1 , sc), untreated nephritis control group and normal control group. FK506 was administered daily six hours after injection of anti-GBM IgG. All the rats were observed urinary protein at the 4th day, the 14th day and the 21st day. At the same time, the kidney specimens were collected, and T cell transforming function was also monitored. RESULTS: Rats injected with rabbit anti-GBM Ab developed heavy proteinuria by 4 days, and serum creatinine and serum urea appeared which kept on the rising. Glmerular hypercellularity, crescents, and protein casts were observed in nephritic rats. By electron microscopy, the thickening of GBM and loss of foot processes were seen. T cell transforming function was higher than normal. But, all pathological changes obviously turned for the better in FK506 treated group. CONCLUSION: FK506 could inhibit the progression of rat anti-GBM nephritis.

AIM: To explore the localization and semi-quantification o f the glo merular complement C5b-9 complexes and synthesis of some inflammatory mediators or cytokines such as nitric oxide (NO) and tumor necrosis factor ?(TNF?) in the ra ts with anti-thymocyte serum nephritis(ATSN). METHODS: The animal mo del of rat AT SN was reproduced by a single intravenous injection of anti-thymocyte serum (ATS ). Then, the deposits of glomerular C5b-9 complexes were localized and quantifie d by immunohistochemical staining and microscopic image scanning separately. And the glomerular mesangial cells (MC) surrounded by C5b-9 complexes were counted under microscope. In addition, the expression of glomerular MC inducible NO synt hase(iNOS) mRNA and excretion of urinary NO metabolite ( NO - 2/NO - 3 ) and TNF ? in the rats with ATSN were detected. RESULTS: The MC in t h e rats with ATSN emerged necrosis followed by a rapid proliferation. In the early time of MC injury, the C5b-9 complexes were mainly seen in glomerular mesangium and MC sur fac e. But with the progression of ATSN, the MC enclosed by C5b-9 appeared gr adual decrease. Moreover, the expression of MC iNOS mRNA in early stage of ATSN obviously increased and the excretion of urinary NO - 2/NO - 3 and TNF ? also significantly increased. However, the changes of parameters mentioned abov e in ATSN proliferative stage (after 7 days) alleviated gradually. CONCLUSION: The second ary lysis of MC has relation to the deposition of C5b-9 complexes and synthesis and release of NO and TNF ? in rats with ATSN.

BACKGROUND: Tumor necrosis factor-α (TNF-α) is one of important cytokines to promote the maturation of dendritic cells. Blockage of TNF-α action by binding with soluble tumor necrosis factor receptor 1 (sTNFR1) may arrest dendritic cells in an immature state and induce stable, long-term tolerance. OBJECTIVE: To construct the lentiviral vectors carrying sTNFR1 gene and investigate sTNFR1 expression in immature dendritic cells. METHODS: Total RNA of human peripheral blood mononuclear cells was taken as a template. The sTNFR1 gene fragment was amplified by RT-PCR, subcloned to the lentiviral vectors pXZ208, and ligated to the enhanced green fluorescent protein (eGFP) reporter gene to establish lentiviral vector, called pXZ9-sTNFR1. DNA sequencing was performed for lentiviral vector identification. Lentivirus was prepared by transfection of 293 FT cells with pXZ9-sTNFR1. Viral titer was determined by eGFP expression. C57BL/6 mouse bone marrow-derived dendritic cells were in vitro cultured with low-dose granulocyte-macrophage colony stimulating factors and interleukin 4. On day 5 of culture, immature dendritic cells were transfected with pXZ9-sTNFR1 recombinant lentiviral supernatant, sTNFR1 transcription was detected by RT-PCR, sTNFR1 protein expression by Western blot analysis. Following sTNFR1 gene modification and lipopolysaccharide stimulation, the phenotype characteristics of dendritic cells were observed. RESULTS AND CONCLUSION: Recombinant plasmid pXZ9-sTNFR1 was successfully constructed. Twenty-four hours after 293 FT cell transfection, eGFP expression was observed and viral titer was over 10<'6> U/L. RT-PCR demonstrated that pXZ9-sTNFRl-transfected immature dendritic cells showed sTNFR1 positive expression. Western blot analysis revealed that sTNFR1 protein appeared in the immature dendritic cells and supernatant following 293 FT cell transfection. On day 5 of culture, dendritic cells expressed low level of class Ⅱ major histocompatibility complex (MHC Ⅱ), as well as CD40, CD86, CD80, molecules. However, following lipopolysaccharide stimulation, dendritic cells expressed high level of MHC Ⅱ, as well as CD40, CD80, and CD86, molecules, exhibiting the phenotype characteristics of mature dendritic cells. But after sTNFR modification, the expression level of MHC Ⅱ, as well as CD40, CD80, and CD86, molecules was not altered obviously. Lentiviral vectors carrying sTNFR1 gene and eGFP reporter gene were successfully constructed, and recombinant lentiviral plasmids with high titer were acquired. Following high efficacy of lentiviral gene transfection, immature dendritic cells stably express sTNFR1 mRNA and protein, which prevents immature dendritic cells from activation by exogenous lipopolysaccharide and maintains the immature state.

Objective To study the molecular mechanism of glutamate receptor-6 (GluR6) in the pathogenesis of epilepsy. Methods Seizure model of SD rats was induced by intraperitoneal injection of kainate (KA). Immunoprecipitation and immunoblotting were performed to examine the interactions of GluR6 and MLK3 with PSD95 at various time points after KA injection. The effect of Tat-GluR6-9c on the MLK3 phospharylation induced by kainate was observed with immunoblotting and immunohistochemistry. Results The assembly of GluR6 and MLK3 with PSD95 was induced after KA hippocampal CA3 region, and bagan to decrease one day later. Pretreatment after KA injection in CA3 region (P<0.05). Conclusion KA induces the assembly of the GluR6-PSD95-MLK3 signaling module and subsequently activates MLK3, which ultimately results in brain injury.

Objective To investigate the effects of 14-3-3 phosphorylation (p-14-3-3) induced by C-Jun N-termi-nal kinase (JNK) on ischemic brain injury in rats. Methods Twenty rats were divided into 4 groups:sham operation group, ischemia-reperfusion group, SP600125 group and solvent control group. The rat model of cerebral ischemia was established. The p-14-3-3, the binding of 14-3-3 and Bax and the protein expression of Bax in cytoplasm and mitochondria in hippo-campal CA1 region were detected by immunoprecipitation (IP) and immunoblotting 12-hour after ischemia-reperfusion in four groups. Results Compared with the sham operation group, protein expression levels of p-14-3-3 in cytoplasm and Bax in mitochondria were significantly increased, the binding of 14-3-3 and Bax was significantly decreased in ischemia-re-perfusion group, solvent control group and SP600125 group. The protein expressions of p-14-3-3 and Bax were significantly lower in SP600125 group than those of ischemia-reperfusion group and solvent control group. The binding of 14-3-3 and Bax was significantly higher in SP600125 group than that of ischemia-reperfusion group and solvent control group (P <0.05). Conclusion 14-3-3 phosphorylation induced by JNK plays important effects on ischemic brain injury in rats.

AIM:To study the effect of human complement C 5b～9 complex on nitric oxide(NO) synthesis of glomerular mesangial cells (MC). METHODS: First, the human complement C 5b～9 complexes were isolated and glomerular MC of rats were cultured. Second, the MC were stimulated with C 5b～9 complex and changes of metabolism products of NO(NO 3 and NO 2) in MC culture supernatant at 6,24 and 48 hours after C 5b～9 stimulating were detected. Moreover, cGMP levels in cultured MC were also measured. RESULTS: NO 3/NO 2 contents from culture supernatant and cGMP levels in MC were increased parallelly after C 5b～9 complex stimulation. Further, NO synthesis was inhibited by L-NG-nitro-arginine-methylester(L-NAME). CONCLUSION: NO synthesis of rat glomerular MC was incerased by human complement C 5b～9 stimulation.

Using recombinant TpNs proteins of Treponema pallidum as antigens, ELISAs are proved to be of higher sensitivity and specificity. However, they can be further increased by using multiple TpNs antigens. According to the epitope analysis, we firstly used linking primers PCRs to obtain an artificial fusion gene segment tpE17-47 containing epitopes of both TpN17 and TpN47. Subsequently, we conducted the prokaryotic expression systems of entire tpN17 and tpN47 genes and tpE17-47 fusion gene. SDS-PAGE analysis and BioRad Gel Image Analysis System showed that the recombinant proteins rTpN17, rTpN47 and rTpE17-47 expressed stably, with 36%, 20% and 28% yields of total bacterial protein, respectively. After purified by Ni-NTA affinity chromatography, all the three recombinant proteins could be recognized by T. pallidum antibody positive sera from syphilis patients. The positive rate of rTpE17-47-ELISA for detecting serum specimens in clinically 630 cases with syphilis was 98.6%. This rate was slightly higher than that by Treponema pallidum particle agglutination (TPPA) (97.9%) (P > 0.05), but significantly higher than those by rTpN17-ELISA (83.8%), rTpN47-ELISA (83.3%) and rapid plasma reagin (RPR) (72.1%) (P < 0.01). Furthermore, both ELISAs and TPPA for detecting the serum specimens in 25 cases with SLE, 36 cases with RA and 250 healthy cases were all negative. RPR showed positive in 1 case with SLE, 2 cases with RA and 2 healthy cases. This could be a novel serological screening or diagnostic method of syphilis with advantages of quickness, convenience, safety, sensitivity and specificity.
Amino Acid Sequence ; Antigens, Bacterial ; biosynthesis ; genetics ; Base Sequence ; Enzyme-Linked Immunosorbent Assay ; Humans ; Immunodominant Epitopes ; immunology ; Molecular Sequence Data ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; immunology ; Sensitivity and Specificity ; Syphilis ; diagnosis ; microbiology ; Syphilis Serodiagnosis ; methods ; Treponema pallidum ; immunology

Objective To explore the effects of the down-regulating peroxiredoxin 2 (PRDX2) on invasion, migration and proliferation of human gastric cancer MGC803 cells by using RNA interference. Methods MGC803 cells were divided into 3 groups:blank control group,negative control group and PRDX2 siRNA group. Transwell assay was used to examine the invasive ability change of MGC803 cells after transfection. Scratch test was used to detect the change of MGC803 cells migration ability after transfection. Western blot was used to examine the expression changes of PRDX2, matrix metalloproteinase (MMP)-2 and MMP-9. The proliferation ability of MGC803 cells was assessed by using CCK-8 assay. Results The expressions of PRDX2(0.345±0.006,0.721±0.013,0.720±0.014),MMP-2(0.067±0.012, 0.391±0.015, 0.371± 0.016) and MMP-9 (0.073±0.013, 0.341±0.028, 0.346±0.024) in the PRDX2 siRNA group were lower than those in the blank control group and negative control group (all P < 0.05). The cell invasion, migration and proliferation were inhibited in MGC803 cells (all P < 0.05). Conclusion PRDX2 is overexpressed in MGC803 cells. Down-regulating the expression of PRDX2 could inhibit the invasion, migration and proliferation of MGC803 cells.