Objective To evaluate the role of recombinant human soluble Tim-3 (hTim-3-Fc) in regulating immune response.Methods Soluble hTim-3 was incubated with human macrophage cell line U 937, human T cell line Jurkat and normal human PBMC before cytokines secreted by or expressed in different immune cells were analyzed using ELISA , RT-PCR and Western-blotting, respectively.Results Soluble hTim-3 significantly promoted the activation of different immune cells.Our data showed that IL-8 secretion by U937 cells, IL-2 secretion by Jurkat cells , IL-2 and IFN-γsecretion by human PBMCs were all significantly increased .In addition , soluble hTim-3 significantly increased the IFN-α2 and IFN-β1 mRNA expression in U937, Jurkat and PBMCs and increased the phosphorylation of stat-1 in Jurkat and U937 cells.Conclusion Recombinant soluble hTim-3 can significantly promote the activation of immune cells in vitro, which shows its therapeutic potential .

Objective To assess the in vitro antimite activity of artemether against Demodex folliculorum, and to provide evidence for the use of artemether in the treatment of skin diseases caused by Demodex folliculorum infection. Methods Artemether was diluted to different concentrations(20, 10, 5 and 2.5 g/L)with peanut oil. The pH values of working solutions of artemether and peanut oil were measured. Demodex folliculorum mites were divided into several groups(32 mites in each group)to be treated with artemether(20, 10, 5 and 2.5 g/L, artemether groups) or peanut oil(control group). Results There were significant differences in the time required for killing of Demodex folliculorum among the 20?, 10?, 5?and 2.5?g/L artemether groups and control group(Median[P25-P75]:3.00[2.00-3.88]vs. 6.00[4.13- 7.25]vs. 13.00[11.63- 14.50]vs. 17.00[15.25- 20.75]vs. 34.00[23.50- 39.50]hours, H=133.954, P<0.001). Additionally, the time required for killing of Demodex folliculorum was significantly shorter in these artemether groups than in the control group(all P<0.001), and was gradually shortened with the increase of artemether concentrations, but was similar between the 10? and 20?g/L artemether groups(P > 0.05). Moreover, the pH values of working solutions of artemether and peanut oil ranged between 7.0 and 7.1, and were close to neutral. Conclusion Artemether at 20, 10, 5 and 2.5 g/L can kill Demodex folliculorum in vitro, so artemether may serve as an alternative drug for the treatment of Demodex folliculorum infection.

OBJECTIVE To investigate the effect of bevacizumab ,an anti-human vascular endothelial growth factor monoclonal antibody,on pulmonary dissemination of colorectal cancer. METHODS A metastatic colorectal cancer mouse model was established. Mice were randomly divided into two groups(n=8). The mice in experimental group were administered ip with bevacizumab at the dosage of 5 mg · kg-1,and those in control group were given isotype IgG at the same dosage. The antibodies were administered on 2 d before initiation of model establishment and 2 d after that,then once every 5 d for 4 weeks,for a total of 7 times. Liver and lung metastases were determined by histopathological examination. The chemokine receptor C-X-C receptor 4(CXCR4)and its ligand C-X-C ligand 12(CXCL12)mRNA expression in the lung were detected by quantitative RT-PCR. Human colon cancer cells HCT116 were treated with bevacizumab(5 mg·L-1)for 24 h. The expression levels of vascular endothelial growth factor receptor 1(VEGFR1)and CXCR4/7 protein as well as CXCR3/4/7 mRNA were examined by Western blotting and quantitative RT-PCR respectively. RESULTS The number of mice(2/8) with liver metastases was reduced,while the number of mice(8/8) with lung metastases increased in experimental group compared with isotype IgG-treated group(6/8 and 2/8 respectively,P<0.05). The mRNA expression level of CXCR4 and CXCL12 in lung tissue was significantly up-regulated in bevacizumab-treated group com?pared with control group(P<0.05). The mRNA and protein expression level of CXCR4 and CXCR7 was dramatically increased in HCT116 cells treated with bevacizumab(P<0.05). CONCLUSION Bevacizumab can potentially promote lung metastases of colorectal cancer,which may be related to up-regulation of CXCR4 and CXCL12 expression.

Objective To examine whether Tim-3 plays a protective role in paraquat poisoning induced excessive immune response and tissue damage based on the critical roles of Tim-3 controlling inflammatory response.Methods A paraquat poisoning model was established in wild type and in Tim-3 transgenic C57BL/6 mice by intraperitoneal injection of paraquat (40 mg/kg) .In addition, C57BL/6 mice with paraquat poisoning were injected with Tim-3 soluble protein( sTim-3) or control protein to see the effect of Tim-3 blocking on the progression of paraquat poisoning.Samples were collected at 6 and 24 h after paraquat injection respectively and were examined for tissue damage, cytokine expression and paraquat metabolism.Results After paraquat poisoning, there was significantly attenuated tissue damage in the lungs and kidneys and decreased TNF-α,IL-6 and IL-1 beta expression in the PBMCs or in the serum from Tim-3 transgenic mice compared to wild type mice.The serum concentration of paraquat in Tim-3 transgenic mice was also significantly decreased.However, in sTim-3 treated paraquat poisoning mice, there was significantly increased cytokine expression and tissue damage compared to control protein treated mice.The in vitro data showed that Tim-3 signaling negatively regulated macrophages mediated inflammatory response.Conclusion Tim-3 plays a critical role in maintaining the homeostasis after paraquat poisoning. Further investigation on the regulatory roles of Tim-3 in inflammation will shed new light on the pathogenesis of paraquat poisoning and provide new therapeutic strategies.

Complement system is an important component of innate immunity and has been recognized as an effective means to inhibit tumor. However,in last decades,accumulating studies showed unexpected results that the complement components and their activation products could promote development of malignancies by secreting growth factors,activating signal pathway and promoting tumor angiogenesis. Herein,the relationship between the complement system and tumor is reviewed.

Objective To develop a human Tim-3 specific monoclonal antibody and evaluate its biological activity and possible use in clinical diseases associated with dysregulated Tim-3 expression .Methods The BALB/c mice were immu-nized by conventional method, and positive clones were used to develop anti-human Tim-3 antibody, the binding and neutralization activities of which in vitro and in vivo were investigated.Results ①A monoclonal antibody (clone L3D) which could specifically bind to human Tim-3 protein in ELISA assay was obtained and the subtype of the monoclonal antibody was IgG2a .②Flow cytometry indicated that the monoclonal antibody could bind to Tim-3 expressed in human U937 cells.This antibody also showed a cross activity to mice′Tim-3.③The monoclonal antibody inhibited the apoptosis of THP1 cells induced by Gal-9, the ligand of Tim-3.④Injection of Tim-3 antibody exacerbated sepsis in mice as marked by the decreased survival rate and increased expression of pro-inflammatory cytokines .Conclusion An anti-human Tim-3 monoclonal antibody is successfully obtained.The excellent binding and neutralization activities of this antibody enable it to be widely used in clinical diseases associated with deregulated Tim-3 expression .

Objective To investigate and compare the killing effect of photodynamic therapy (PDT)induced by hematoporphyrin derivative (HpD),hematoporphyrin monomethyl ether (HMME) and photocarcinorin (PsD007) on human leukemia cells K562 in vitro.Methods Human leukemia cells were cultured with serial concentrations of photosensitizers followed by irradiation of different dosage of laser light,then MTT colorimetric assay was applied to measure the relative survival rate of PDT for the cells.Results Significant difference in the inhibitory between the PDT group and control group was observed (P＜0.05).The survival rate of PDT for the cells elevated along with the increase in the concentration of sensitizer and dose of laser light.When the photosensitizer concentration was bigger (25 μg/ml) or the energy density was bigger (7.2 J/cm2),the effect of PsD007 was better than HMME,and they were significantly better than HpD (P＜0.05).Conclusion PDT has significant killing effect on human leukemia cells K562,and its relative inhibitory rate appears to be correlated with the dose of sensitizer and laser light irritation.The effect of PDT is related to the photosensitizers.The effect of HpD-PDT is not as effective as PsD007 and HMME.On the conditions of higher energy density and larger photosensitizer concentration,the effect of PsD007-PDT is better than HMME-PDT.

Bone turnover is regulated by local concentrations of cytokines such as osteoprotegerin (OPG) and receptor activator of nuclear factor kappaB ligand (RANKL). To explore the in vivo biological function of Opg and the mechanism of osteoporosis due to deficiency of Opg, Opg knockout mice have been generated through homologous recombination. Opg-/- mice exhibit a sharply decrease in bone density and strength as expected. The number of osteoclasts in Opg-/- mice significantly increases. Morphologically, osteoclasts appear more cuboidal in shape in Opg-/- mice than those of wt mice, suggesting that active osteoclastogenesis occurs in the absence of Opg. In consistent with this finding, an increase of osteoblast activity was also observed with accelerated mineral accumulation rate by histomorphometric measurement and elevated serum alkaline phosphatase activity (ALP) in Opg-/- mice. Interestingly, more than 50% of 2-month-old Opg-/- mice manifest medial calcification of aorta with comparable serum concentrations of calcium and phosphorus to wt mice. In conclusion, Opg-/- mice have a high-bone-rurnover type osteoporosis. The aortic calcification in Opg-/- mice is not due to abnormality of calcium and phosphorus metabolism. The mechanism underlying aortic calcification in Opg-/- mice needs to be further investigated.

Diamond-like carbon (DLC) films were deposited by acetylene plasma immersion ion implantation-deposition (PIII-D) on biomedical polyethylene terephthalate (PET). The capacities of Staphylococcus aureus (SA), Staphylococcus epidermidis (SE), Escherichia coli (EC), Pseudomonas aeruginosa (PA) and Candida albicans (CA) for adhesion to PETs are quantitatively determined by the plate counting and Gamma-ray counting of 125I radio labeled bacteria in vitro. The results indicate that the capacities of five types of bacteria for adhesion to PETs are all suppressed by C2H2 PIII-D (P<0.05). The surface energy components of the various substrates and bacteria are calculated based on measurements in water, formamide and diiodomethane and Lifshitz-van del Waals/acid-base approach (LW-AB). The surface free energies obtained are used to calculate the interfacial free energies of adhesion (deltaF(adh)) of five kinds of bacteria on various substrates, and the results show that it is energetically unfavorable for bacterial adhesion to the DLC films already deposited on PET by C2H2 PIII-D.
Bacterial Adhesion ; drug effects ; physiology ; Carbon ; chemistry ; Coated Materials, Biocompatible ; chemistry ; Diamond ; chemistry ; Escherichia coli ; drug effects ; Heart Valve Prosthesis ; microbiology ; Materials Testing ; Polyethylene Terephthalates ; chemistry ; Staphylococcus aureus ; drug effects ; Staphylococcus epidermidis ; drug effects

Adiponectin is an adipocyte-derived secretory protein. It was found to be associated with insulin resistance, inflammation and arteriosclerosis. To further study the biological function and expression of adiponection in vivo, adipoenctin gene knock-out and LacZ gene knock-in mouse model was constructed. Gene targeting strategy was designed to replace part of exon 2 and exon 3 of adiponectin gene with full length LacZ gene in frame with remaining upstream ATG and signal peptide sequence of exon 2. The targeting vector (Adipo-LacZ-XpPNT) was constructed and verified by restriction enzyme digestion and sequencing. CJ7 ES cells were transfected with targeting vector linearized by NotⅠ digestion, selected in the medium containing both G418 and ganciclovoir. Resistant clones were screened by PCR and further confirmed by Southern blot for correct homologous recombinants. Chimera mice were obtained by routing microinjection of homologous recombined ES cells into blastocysts. After mating, mice heterozygous and further homozygous for adiponectin knockout and LacZ gene knock-in were established. Expression of both endogenous adiponectin and exogenous LacZ gene in mouse tissues and sera were detected by RT-PCR, Northern-blot, Western blot and ELISA. The results show that adiponectin was disrupted at both mRNA and protein levels. LacZ gene is expressed exclusively in adipose tissue of mutant mice. Its expression profile is identical to endogenous adiponection. Unexpectedly, LacZ activity could not be detected in both adipose tissue and serum although LacZ protein can be detected in adipose tissue but not in serum of mutant mice. In conclusion, mice homozygous for adiponectin knockout and LacZ gene knock-in have been successfully constructed. Mutant mice display LacZ expression profile identical to endogenous adiponectin albeit neither LacZ activity nor protein can be detected in serum of mutant mice.