The expression vector of SmIg scFv fragment was constructed in patient with B cell chronic lymphocyte leukemia (B-CLL) and expressed in E. coli to obtain scFv fragment, and the effect of the protein on the proliferation of stimulated peripheral blood mononuclear cells (PBMC) was investigated in vitro. Two pairs of primers were designed, and variable region genes of light chain and heavy chain were amplified by PCR respectively from the pGEM-T vectors previously constructed in our laboratory which containing light chain gene or Fd fragment of heavy chain gene. The PCR product was digested, purified and inserted into pHEN2 vector to construct the soluble expression vector pHEN2-scFv. After the induction by IPTG, the scFv protein was identified by SDS-PAGE electrophoresis and purified by Ni-NTA-Chromatography. MTT was used to determine the effect of purified protein on the proliferation of stimulated PBMC in vitro. Plasmid PCR and restriction enzyme digestion of pHEN2-scFv revealed the pHEN2-scFv vector was constructed successfully. Id-scFv protein was expressed in positive clone after induced by IPTG. SDS-PAGE analysis showed that the relative molecular weight of fusion protein was about 30 kD (1 kD= 0.9921 ku), which was consistent with the theoretically predicted value. Proliferation of PBMC could be induced by purified Id-scFv. It was suggested that the expression vector of SmIg scFv fragment was constructed successfully, and scFv protein was expressed and secreted from E. coli, which could induce proliferation of PBMC. This may lay an experimental foundation for further research of Id-HSP complex vaccine for B-CLL.

P-450-dependent epoxygenase pathway of arachidonic acid and the products of epoxyeicosatrienoic acids (EETs) have been demonstrated to be involved in angiogenesis and tumor progression. This study examined the expression of EETs and the role of the pathway in the angiogenesis of multiple myeloma (MM). MM cell lines of U266 and RPMI8226 were cultured, and the EETs levels (11, 12-EET and 14, 15-EET) in the supernatant were determined by ELISA. Human umbilical vein endothelial cells (HUVECs) were cultured and used for analysis of the angiogenesis activity of the two MM cell lines, which was examined both in vitro and in vivo by employing MTT, chemotaxis, tube formation and matrigel plug assays. 11, 12-EET and 14, 15-EET were found in the supernatant of the cultured MM cells. The levels of the two EETs in the supernatant of U266 cells were significantly higher than those in the RPMI8226 cell supernatant (P<0.05), and the levels paralleled the respective angiogenesis activity of the two different MM cell lines. 17-octadecynoic acid (17-ODYA), as a specific inhibitor of P450 enzyme, suppressed HUVECs proliferation and tube formation induced by MM cells. Furthermore, 17-ODYA decreased the EET levels in the supernatant of MM cells. These results suggest that EETs may play an important role in the angiogenesis of MM, and the inhibitor 17-ODYA suppresses this effect.

Objective To investigate the effect of a novel lymphotoxin with selectively binding to p55 tumor necrosis factor receptor (p55TNFR) on myocardial ischemia-reperfusion injury in rats in order to explore the mechanism.Methods A total of 40 SD rats were randomly (random number) assigned into four groups (n =10 in each),namely sham operation group (group A),I/R group (group B),wild type rhLTα treatment group (group C),and p55TNFR selective rhLTα (rhLTα-Q107E) treatment group (group D).After I/R model rats were established,various therapeutic agents or saline were given by continuous intravenous infusion for 24 h via a micropump.After 24 hours of treatment,serum myocardial zymogram,such as aspartate aminotransferase (AST),lactate dehydrogenase (LDH) and creatine kinase (CK),as well as superoxide dismutase (SOD),malondialdehyde (MDA) and glutathione peroxidase (GSH-Px) activities were determined.Myocardial infarction size (MIS) was measured by nitro blue tetrazolium chloride (NBT) staining.Results Compared to sham operation group,MIS,AST,LDH,CK,MDA were increased,while the activities of SOD and GSH-Px were decreased.However,all the effects were significantly reversed by treatment with rhLTα-Q107E (P ＜ 0.05) but not rhLTα (P ＞ 0.05).Conclusions The rhLTα-Q107E plays a role in the protection against myocardial ischemia-reperfusion injury in rats by the mechanism of scavenging oxygen free radicals and increasing the activity of endogenous antioxidant system.

Objective To evaluate the relationship of preoperative neutrophil-lymphocyte ratio (NLR) with clinicopathological features and prognosis of colorectal cancer in middle-aged and elderly patients.Methods A retrospective analysis was performed in 212 patients with colorectal cancer in the First Affiliated Hospital of Nanjing Medical University from January 2011 to June 2013.All patients were divided into middle-aged group (46-65 year old,n=130) and old-aged group (66-89 year old,n=82),The optimal cut-off point of NLR was identified by the area under receiver operating characteristic curve,while NLR ＞ 3.13 and NLR≤3.13 were classified as high and low NLR group.The clinicopathological features and prognosis between the two groups were compared.Results There was no difference in gender,tumor growth site,depth of invasion,tumor embolus,lymphatic metastasis,distant metastasis,TNM stage between low and high NLR group (allP＞ 0.05).However,the difference between high NLR group and low NLR group in old-aged group with diabetes mellitus was statistically significant (P＜0.05).The 1-,2-,and 3-year survival rate of the overall 212 patients were 96.2％ (204/212),87.7％ (186/212) and 74.5％ (158/212) In middle-aged group,the 1-,2-,and 3-year survival rates were 98.8％ (85/86),90.7％ (78/86) and 84.9％ (73/86) respectively in low NLR group,but 95.5％ (42/44),84.1％ (37/44) and 72.7％ (32/44) respectively in high NLR group,(allP＜0.05).In old-aged group,the 1-,2-,and 3-year survival rates were 95.7％ (44/46),89.1％ (41/46) and 73.9％ (34/46) respectively in low NLR group,but 91.7％ (33/ 36),83.3％ (30/36) and 52.8％ (19/36) respectively in high NLR group (all P＜0.05).Cox regression showed that TNM stage and NLR were independent risk factors for the prognosis of the middle-aged and elderly patients with colorectal cancer (P＜0.05 or P＜0.01).Conclusions Preoperative NLR ＞ 3.13 suggest that the prognosis is poor in middle-aged and elderly patients with colorectal cancer.

Objective To investigate the effects of traditional Chinese medicine colquhounia root tablet on the expression of tight junction protein claudin-2 and ZO-1 in bronchial epithelium tissues of rats with acute lung injury (ALI), and to study the mechanism of protective effect of colquhounia root tablet on ALI. Methods Twenty-four healthy male Sprague-Dawley (SD) rats were randomly divided into control group, ALI group and colquhounia root tablet pretreatment group, with 8 rats in each group. The model of ALI was reproduced by intravenous injection of oleic acid 0.04 mL/kg, and the rats in cont rol group were given the same amount of normal saline (NS) instead. The rats in colquhounia root tablet pretreatment group were intragastric administrated with colquhounia root tablet of 600 mg·kg-1·d-1 (2 mL) for 10 days before model reproduction, and the rats in control group and ALI group were given the same amount of NS. At 4 hours after model reproduction, the blood was drawn from abdominal aorta, and bronchoalveolar lavage fluid (BALF) was collected for determination of protein content in plasma and BALF, and the lung permeability index (LPI) was calculated. The rats were sacrificed to collect lung tissues for determination of lung wet/dry weight ratio (W/D), the changes in pathology of lung tissue were observed after hematoxylin and eosin (HE) staining with light microscope, and lung injury score (LIS) was evaluated. The immunohistochemic al staining was used to detect the expression and localization of claudin-2 and ZO-1 in bronchial epithelium tissues. The protein expressions of claudin-2 and ZO-1 in bronchial epithelium tissues were determined by Western Blot. Results Compared with control group, the lung injury in ALI group was more obvious including cellular edema and structural disorder of intercellular connection by optical microscope, and LIS, W/D ratio, and LPI were significantly increased (LIS: 3.81±0.42 vs. 0.40±0.08, W/D: 7.68±0.64 vs. 4.44±0.39, LPI: 0.89±0.15 vs. 0.38±0.05, all P < 0.01). Claudin-2 and ZO-1 were mainly expressed in the bronchial epithelium cell, and the expression degrees were significantly weakened in ALI group as compared with control group. It was shown by Western Blot results that compared with control group, the protein expressions of claudin-2 and ZO-1 were significantly down-regulated in ALI group [claudin-2 protein (gray value): 0.43±0.31 vs. 2.16±1.33, ZO-1 protein (gray value): 1.25±0.41 vs. 2.82±0.76, both P < 0.01]. Compared with ALI group, colquhounia root pretreatment could effectively diminish the degree of ALI (LIS: 1.22±0.39 vs. 3.81±0.42, W/D: 4.62±0.84 vs. 7.68±0.64, LPI: 0.46±0.07 vs. 0.89±0.15, all P < 0.01), and the protein expressions of claudin-2 and ZO-1 were significantly up-regulated [claudin-2 protein (gray value): 2.98±0.91 vs. 0.43±0.31, ZO-1 protein (gray value): 2.35±0.51 vs. 1.25±0.41, both P < 0.01]. Conclusion Administration of colquhounia root table could attenuate lung injury induced by oleic acid with improving epithelial barrier function via up-regulate the expression claudin-2 and ZO-1, which play a protective effect on the lung of rats with ALI.

Aim To observe the effect of hemodialysis on pharmacokinetics of sparfloxacin in thepatients contracting chronic renal failure. Methods Sparfloxacin concentrations inserum and urine of hemodialysis and non-dialysis patients were measured with a highperformance liquid chromatography method after administration a single oral dose of 200mg sparfloxacin. The pharmacokinetic parameters were computed with the programPKBP-N1.Results The main pharmacokinetic parameters in hemodialysis group wereT1/2(ka) - (1. 25 ?0. 57) h, T1/2(?) = (11. 88?4. 13) h, Tpeak = (4. 18 ? 0. 78) h,Cmax = (0.80 ? 0. 17) mg? L-1 and AUC0-= (6. 90 ? 3. 25) mg?h?L-1, while innon-dialysis group were T1/ 2(ka) = (1. 12 ? 0. 42) h, T1/ 2(?) = (15. 93 ? 5. 20) h, Tpeak =(3. 88 ? 0. 75) h, Cmax = (0. 69 ? 0. 37) mg?L -1, AUC0-= (10.05 ? 4. 13) mg?h?L-l. The original sparfloxacin discharge rats in urine within 24 h were (8. 98 ? 3. 92) % and(10. 58 ? 5. 64) % separately. T1/2(?) and AUC in hemodialysis group were markedly lowerthan in non-dialysis group (P

Pilonidal disease is a common clinical condition which is not sufficiently recognized with regard to the clinical manifestation and treatment by colorectal surgeons in China, resulting in high misdiagnosis rate and recurrent rate. With reference to the Practice Parameters for the Management of Pilonidal Disease published in 2013 by The American Society of Colon and Rectal Surgeons, we discuss the management of pilonidal disease in four aspects, including etiology, diagnoses, nonoperative and operative management.
Colonic Diseases ; surgery ; Humans ; Rectal Diseases ; surgery ; United States

Adult ; Burns, Inhalation ; diagnostic imaging ; Explosions ; Follow-Up Studies ; Humans ; Male ; Middle Aged ; Radiography

To investigate the effect of Tripterygium wilfordii polycoride (TWP) on LPS-induced macrophage inflammatory response, particularly the inhibitory effect on inflammatory factors TNF-α and IL-1β and the regulatory effect on inflammation via TLR4/NF-κB. The MTT method was adopted to test the effects of tested drugs, TWP, dexamethasone (DXM) and azathioprine (AZA) on cell growth to define the appropriate concentration. LPS was used to induce the inflammatory reaction in mouse RAW264. 7 cell lines. The Elisa kit was adopted to test the release level of TNF-α and IL-1β. The Western blotting was applied to test the protein expressions of TNF-α and IL-1β. The RT-PCR was adopted to test the expressions of TLR4 and NF-κB. According to the results, TWP could inhibit the release of macrophage inflammatory factors TNF-α and IL-1β in a dose dependent manner. All of TWP groups showed a weaker efficacy than that of the DXM group. But the TWP high dose group revealed a better effect on TNF-α and equal effect on IL-1β compared with the AZA group. TWP show an equal or better effect in down-regulating TLR4 and NF-κB p65 expressions in a dose dependent manner than DXM and AZA. In conclusion, TWP could inhibit TLR4 and NF-κB p65, which may be related to the down-regulation of TLR4 and NF-κB p65 receptor expressions.
Animals ; Anti-Inflammatory Agents ; pharmacology ; Cell Proliferation ; drug effects ; Down-Regulation ; drug effects ; Drugs, Chinese Herbal ; pharmacology ; Humans ; Inflammation ; drug therapy ; genetics ; immunology ; physiopathology ; Interleukin-1beta ; genetics ; immunology ; Macrophages ; drug effects ; immunology ; Mice ; NF-kappa B ; genetics ; immunology ; RAW 264.7 Cells ; Toll-Like Receptor 4 ; genetics ; immunology ; Transcription Factor RelA ; genetics ; immunology ; Tripterygium ; chemistry

Adolescent ; Child ; Collagen Type I ; metabolism ; Extracellular Matrix ; metabolism ; Female ; Fibrillin-1 ; Fibrillins ; Heart Defects, Congenital ; metabolism ; pathology ; Humans ; Male ; Microfilament Proteins ; metabolism