To study the antitumor activity and immunoregulatory effect of Triterpenes isolated from Betula Platyphylla (TBP)Methods: Inhihition rate (IR) on tumor growth was determined by the mice with transplantable tumors (melanoma B16, Sarcoma 180, Lewislung carcinoma and Ehrlich ascites cancer), splenocyte proliferation induced by ConA was measured by H-TdR incorporation; cytotoxicity ofmacrophage was observed by 3H-TdR release; activity of TNF and NK cell was determined by dye (natural red)release. Results: IR of 0.80 g/kg and 1.20 g/kg TBP on all of above tumor strains were greater than30％ in vivo. TBPdoes not show significant effect of splenocyte Prolifera-tion and NK cell activity, but significantly promoted the activity of TNF producted by macroghage and splenocyte. TBP also increase the cyto-toxicity of macrophage. Conclusion:TBP shows potent antitumor effect in vivo. Promoting nonspecific immunoactivity is one of the antitumormechanism of TBP.

To study the effects of growth hormone on function of T lymphocytes. Methods: Tne expression plasmid PcDNA-GHcDNA was constructed,then the plasmids were transfected into Jurkat cells,on the other hand,Jurkat cells were treated with different doses ofrhGH, to investigate the effect of overexpression GH and rhGH on function of T lymphocytes. Results: Overexpression GH could increase the ex-pression of IL-2R(140％) and secretion of IL-2(160％) and IFN-γ( 198% ) in Jurkat cells after PHA activation,but these data were broughtdown by the treatment of GH antibody. rhGH could also enhancer the function of T lymphocytes after PHA activation. Conclusion: These resultssupport the idea that GH is an autocrine and paracrine factor, and can regulate the function of T lymphocytes.

To investigate peripheral blood mononuclear cells (PBMCs) infected by hepatitis C virus (HCV) and the influenceon T lymphocyte subset. Methods: Using non-isotopic in situ hybridization (NISH) and streptadivin-biotin-enzyme complex assay (SABC) todetect HCV-RNA, NS5 antigen and T lymphocyte subset. Results: 8(40.0% ) out of 20 patients with hepatitis C virus were HCV-RNA posi-tive in PBMCs, and among them 6(30.0％) were NS5 antigen positive simultaneously. NS5 antigen signals appeared diffusely within cyto-plasm, or in cell membrane, while HCV-RNA signals were mainly distributed in cytoplasm. Both the proportion of NS5 positive cell and HCV-RNA positive cells were very low (≤3％).The proportion of positive cellsane, while HCV-RNA signals are very low (≤3％). The proportionof CD4+ T cells was lower in the patients comparing with that in control group, the proportion of CD8+ T cells was higher, and the ratio ofCD4+/CD8+ were decreased remarkably (P＜0.01). Moreover the proportion of CD4+ T cells was lower in HCV-RNA-positive group com-paring with that in HCV-RNA-negative group, the proportion of CD8+ T cells was higher, and the ratio of CD4+/CD8+ was reversed (P＜0.01). Conclusion:HCV can infect PBMCs and replicate in patients with chronic hepatitis C;CD4+ T cells are readily damaged after PBMCsinfected by HCV, which causes the decrease or reversal of the ratio of CD4+/CD8+, undermines the ability of the body clearing virus and re-sults im chronicity of HCV infection.

To explore the effects of artisense IRAK-2 oligonucleotide on the prostacyclin (PGI2) synthesis in human umbilicalvein endothelial cells (HUVEC) induced by interleukin-1 (IL-1) and tumor necrosis factor (TNF).Methods:The HUVECs were transfectedwith antisense interleulkin-1 receptor associated kinase-2 oligonucleotide (IRAK-20DN) and stimulated with IL-1 and TNF. The levels of PGI2release were analyzed by competitive ELISA. Results: Pre-transfection with antisense IRAK-20DN could remarkably decrease the levels ofPGI2 synthesis induced by IL-1 in time- and dose-dependent manner, whereas it could not attenuate the one stimulated by TNF. Conclusion:The response of antisense IRAK-2 ODN to IL-1 and TNF-stimulated PGI2 release were different. IRAK-2 plays a key role in the IL-1 signalingevents leading to PGI2 release.

To identify the expression of the molecule recognized by Pf18-3 mAb (Pf18-3 molecule) on various cells. Meth-ods: The expression of pr18-3 molecule was assayed by flow cytometry and confocal laser scanning microscope . Result: The molecule recog-nized by Pf18-3 mAb expressed on TSC and other stromal cells. Whereas, fresh thymocytes were Pf18-3 negative. Interestingly, the expressionof Pf18-3 molecule was gradually up-regulated on thymocytes after activation by ConA. This molecule mainly expressed on CD4+ CD8+ andCD4+ CD8- cells. Under confocal laser scanning microscope, the staining of fluorescence showed as ring around the cell, it changed grsduallystronger and thicker with activation. Conclusion: This study indicated that the Pf18-3 molecule was co-expressive molecule of MTSC and acti-vated tlymocytes,it was concemed closely about the activation of CD4+ C D8+ and CD4+ CD8- cells.

Objective To investigate the effects of NOD2 on inflammatory responses of macrophages to Staphylococcus aureus. Methods Real-time RT-PCR detected NOD2 gene expression of macrophages infected by S. aureus. Synthesis of siRNA against NOD2 and interfere with macrophages, observed the effects of NOD2 gene silencing to phagocytosis of 5. aureus, cytokine secretion, activation of nuclear transcription factors, cell apoptosis of the macrophages infected by S. aureus using F.I .IS A, flow cytometry etc. Results S. aureus infection of macrophages can cause increased expression of intracellular NOD2. NOD2 gene silencing of macrophage lead to the decreased ability of phagocytosis with S. aureus, the lower levels of cytokines secretion, deficiencies of NF-κB activation. S. aureus can cause macrophage apoptosis, with the apoptosis rate increased with time. Conclusion The intracellular pattern recognition receptor NOD2 play a key role in pathogen recognition, signal transduction, activation of nuclear transcription factors in the process of macrophages infected by S. aureus.

Objective To determine the effects of Che A and CheY proteins of Campylobacter jejuni regulating the bacterial chemotaxis in vitro and colonization in vivo. Methods By using pET42a plasmid and E. coli BL21DE3 as expression vector and expression strain, respectively, prokaryotic expression systems of cheA and cheY genes of C. jejuni strain NCTC11168 was constructed. Rabbits were immunized with the target recombinant expression proteins, rCheA and rCheY, to prepare the antisera. rCheA-IgG and rCheY-IgG in the antisera were separated using DEAE-52 ion exchange column. pBluescript- II -SK was applied to construct suicide plasmid which used to generate cheA gene knock-out mutant (cheA-). A chemotaxis model in vitro of C. jejuni based on DOC-HAP, the chemotactic ability of cheA' mutant as well as the effect of rCheA-IgG and closantel inhibiting the bacterial chemotaxis were demonstrated. The phosphorylation levels of CheA and CheY after DOC treatment were examined by using either rCheA-IgG or CheY-IgG capture method. The difference of colonization ability between cheA- mutant and wild-type of C. jejuni in mice were checked and then compared. Results The constructed prokaryotic expression systems could efficiently express rCheA and rCheY. The data from PCR and sequencing confirmed the cheA gene knock out from cheA- mutant chromosome. cheA- mutant lost its chemotactic ability towards DOC. Both the rCheA-IgG and closantel could inhibit the chemotaxis of wild-type of C. jejuni (P < 0.05 ). When treatment of DOC, the phosphorylation levels of CheA and CheY in wild-type of C. jejuni rapidly decreased (P < 0. 05 ). The colonization ability in murine jejunum of cheA- mutant was also lower than that of the wild-type ( P<0.05 ) . Conclusion Chemotaxis-associated two-component signaling system (Che-TCSS) of C. jejuni are composed of CheA and CheY, and the two proteins are activated by dephosphorylation. CheA in the Che-TCSS play a critical role in chemotaxis in vitro and colonization in vivo of C. jejuni, and this protein can be used as a target for developing novel anti- C. jejuni drugs.

Objective To understand the epidemic characteristics of an outbreak of panresistance Klebsiella pneumoniae occurred between 2006 and 2009 in a university hospital of Shanghai, China. Methods A total of 57 panresistance K. pneumoniae isolates were collected from August 2006 to December 2009.Antibiotic susceptibility of the isolates were determined by Kirby-Bauer disc diffusion method and microbroth dilution (MBD). ESBLs-producing initial screen test and phenotypic confirmatory test and carbapenemase-producing modified Hodge test ( MHT) were performed to detect the resistance phenotype of the isolates. Be-ta-lactamases were studied by IEF, PCR and the product sequencing. While conjugation assay were conducted to understand the transferability of these genes. The genetic relationship between isolates was established by ERIC-PCR and multilocus sequence typing (MLST). Except for the antibiotics recommended by CLSI guideline in the routine test, the other antibiotics were added to find out the effective drugs to treat the infection. Results All 57 isolates were highly resistant to all examined antibiotics. All isolates produced ESBLs and carbapenemase. IEF revealed that each isolate produced four beta-lactamases. All isolates carried blaKPC-2,blaCTX-M-14,blaSHV12,blaTEM-1,qnrB and aac(6') - I b-cr. Forty-four of the 57 (77.2% ) isolates were successful to transfer their resistance genes to E. coli recipient J53 by conjugation assay. By RAPD, all 57 isolates were grouped into two genotypes that were further identified as members of MUST types 423 and 11.Sequence types 423(ST423) only occurred before May 2008 and ST11 occurred (52 isolates) after May 2008. Most of isolates of the outbreak were ST11 (91. 2% ). A part of isolates were susceptive to added antibiotics. Conclusion The outbreak of panresistance K.pneumoniae was caused by those isolates which carried multiple resistant genes. There is a different ability of dissemination between different ST types K. pneumoniae isolate. It was necessary to add the antibiotics to find out the effective drugs to treat the infection.

Objective To study the activation of TLRs/NF-κB signal pathway and production of different functional cytokines during invasive pulmonary aspergillosis( IPA) , in order to probe the pathogene-sis of IPA. Methods Mouse were randomly divided into normal, normal + inoculated with Aspergillus fumigatus( normal inoculation group), and immune suppression + inoculation with Aspergillus fumigatus (IPAmodel group) , the mouse were killed at different time points after inhaling Aspergillus fumigatus spores by nose. Removing the lung tissue in a sterile manner and making pathological section respectively, counting Aspergillus fumigatus colony, dynamiclly detecting the expression of TLR2, TLR4 mRNA, variation of NF-κB p65 protein, pro-inflammatory cytokines TNF-α, IL-1β and anti-inflammatory cytokines IL-10 levels in the lung tissue by RT-PCR and Western blot method during Aspergillus fumigatus infection in mouse. Results (1) When it's 72 h after inhaling Aspergillus fumigatus by nose, IPA model emerged severe lung tissue inflammation, and generated a large number of hyphae, meanwhile, burthen of Aspergillus fumigatus was higher than normal inoculation group at each time point. (2)Compared with the normal inoculation group, IPA group whose TLR2 mRNA was low expression at early stage of infection (24 h), and emerged high expression at late stage of infection (120 h, 144 h); and TLR4 mRNA has been at a state of low expression in the infection process; NF-κB p65 suddenly increased at early stage of infection(24 h) and then continued to decline. (3) After infected by Aspergillus fumigatus in normal mouse, proinflammatory cytokine TNF-α, IL-1β in lung exhibited high expression at the early stages of infection, and the highest expression levels appeared at 48 h or 72 h, then decreased and recovered to normal level. And the expression level of anti-inflammatory cytokine IL-10 rised at late stage of infection; The IP A mouse released a lot of anti-inflammatory cytokine IL-10 at early stage of infection, which significantly reduced at late stage, and released pro-inflammatory cyto-kines TNF-α, IL-1β at slow and low level. Conclusion The abnormal activation of TLRs/NF-β signaling pathway caused the loss of dynamic balance between pro-inflammatory cytokines and anti-inflammatory cytokines, leading to the occurrence and development of IPA.

Objective To reduce the turnaround time for laboratory diagnosis of bacteremia, the feasibility of rapid identification and susceptibility testing using samples taken directly from positive blood culture bottles was evaluated. Methods The growth of microorganisms in blood culture bottles was screened by the BACTEC 9000 blood culture system. 65 positive blood culture bottles containing gram-negative bacteria were adopted to test. Culture fluid was injected into BD SST vacutainer and centrifuged to pellet blood cells. After collecting required McFarland units, they were cultured on Phoenix 100 NMIC/ID-4(identification-gram-negative bacteria and susceptibility testing) cards using 0.25 McF and 0.5 McF methods respectively. They were also evaluated by the standard method, involving subculture tests from positive blood culture bottles. Results 63 of 65 gram-negative bacteria (96. 9% ) were correctly identified with 0. 25 McF method. 59 of 65 gram-negative bacteria(90.8% ) were correctly identified with 0.5 McF method. For antimicrobial susceptibility testing, the 0.25 McF direct method had an agreement rate more than 94% , the 0.5 McF method was more than 85.7% and direct blood sample KB method was more than 93.8% compared to the standard method. But the overall minor error rate in susceptibility testing of direct blood sample KB method is higher than other methods. Conclusion Applying 0. 25 McF and 0. 5 McF rapid identification and susceptibility test was practical. During to possessing more prominent advantages, laboratory put the 0. 25 McF direct method into practice had a timely, remarkable significance.