Objective To prepare and screen out monoclonal antibodies against the receptor bind-ing domain (RBD) of Middle East respiratory syndrome coronavirus ( MERS-CoV) spike ( S) protein in mice. Methods The RBD of MERS-CoV S protein expressed in the insect-baculovirus system was purified and then used to immunize the female BALB/ c mice. The spleen cells collected from the mice were fused with myeloma Sp2 / 0 cells. The positive hybridoma cells were obtained by using limited dilution method. Enzyme-linked immunosorbent assay ( ELISA), Western blot assay and neutralization test based on the MERS-CoV pseudovirus were performed for further screening and identification. Results Twelve strains of hybridoma cells that produced the monoclonal antibodies against RBD of MERS-CoV S protein were screened out. All of the 12 monoclonal antibodies (McAbs) could have specific reaction with the RBD of MERS-CoV S protein as indicated by the results of ELISA. Of the 12 McAbs, two were identified as the immunoglobulin M (IgM) isotype and the rest were IgG1 isotype by using double antibodies sandwich ELISA. Four McAbs including 1F1, 2E4, 3C3 and 3E6 were identified as having neutralizing activity by the neutralization test based on MERS-CoV pseudovirus. Results of the Western blot assay showed that the four McAbs (1F1, 2E4, 3C3 and 3E6) could have specific reaction with the RBD of MERS-CoV S protein, but no cross-reac-tion with that of SARS-CoV S protein. Conclusion Twelve mouse-derived McAbs against the RBD of MERS-CoV S protein were obtained. The prepared hybridoma cells showed the characteristics of high speci-ficity and stability in antibody secretion. Four out of the 12 McAbs were proved to have neutralizing activity.

Objective To investigate the efficacy and safety of oxycodone hydrochloride controlled-release tablets combined with gabapentin in the treatment of painful diabetic neuropathy.Methods Eighty patients with painful diabetic neuropathy were divided into group A and group B with 40 cases each by random digits table.Patients of group A received simple oxycodone hydrochloride controlled-release tablets,while patients of group B received oxycodone combined with gabapentin.The intensity of pain, oxycodone dosage, quality of life and adverse effect in two groups was observed and compared.Results The pain was obviously relieved in two groups, and the effective analgesic rate at 21-28 d after treatment in group B was superior to group A[95.0% (38/40) vs.62.5% (25/40)], there was significant difference between two groups (P＜0.05).Numerical rating scale NRS score at 28 d after treatment in group B and group A was 1.2 ± 0.3 and 2.8 ± 0.5 respectively,and there was significant difference between two groups (P＜ 0.01 );the oxycodone dosage at 28 d after treatment in group B was lower than that in group A[(32.2 ± 4.3 )mg vs.(40.3±5.5 ) mg], and there was significant difference between two groups (P ＜ 0.05 ); the quality of life in group B was superior to group A, and there was significant difference between two groups (P ＜ 0.05 or ＜ 0.01 );the incidence of dizzy at 7, 14 d after treatment in group B was higher than that in group A[35.0% (14/40) vs.15.0% (6/40) and 30.0% (12/40) vs.10.0% (4/40)],and there was significant difference between two groups (P ＜ 0.05 ).Conclusion Oxycodon hydrochloride controlled-release tablets combined with gabapentin is effective and safe for the management of painful diabetic neuropathy.

Objective To investigate the effects of ARHI transfection on the chemokines and receptors related gene expression profile of PANC1 cells.Methods Plasmids expressing ARHI and empty plasmid were transfected into PANC1 cells, and the stably expressed cell lines were established by using G418.mRNA expression of chemokines and receptors related genes was detected by PCR Array.Real-time PCR was used to detect mRNA expression of the genes related vascular growth.Results In cells transfected with ARHI gene, the expression levels of mRNA of 36 genes were down-regulated, and 9 were up-regulated.Among the genes related to tumor metastasis and invasion CXCL12 and CXCR4 were significantly down-regulated (<-6 folds), and MMP-2 was slightly down-regulated (<-2 folds).Among the genes related to tumor angiogenesis, pro-angiogenesis genes including CCL2, CXCL1, CXCL2, CXCL8, CXCL12 and CXCR4 were significantly down-regulated, and pro-angiogenesis genes including CXCL3, CCR1 and CCR2 was slightly down-regulated.Anti-angiogenesis genes including CXCL9, CXCL10, CXCL11 and CXCR3 were significantly up-regulated (>6 folds).Among the genes related to the localization of distant organ infiltration and latency, CXCL12, CXCR4 and CCR7 were significantly down-regulated,and CXCR5 was slightly down-regulated.Among the gene with tumor immunity,CXCL8,CXCR1 and CCR7 were significantly down-regulated.Gene expression of CXCL1,CXCL8,CXCR4 and CXCR3 detected by Real-time PCR were consistent with PCR array.Conclusions ARHI gene inhibits the expression of chemokines and receptors related to tumor metastasis,angiogenesis and tumor immunity.

Objective To investigate the development strategy of novel T cell based vaccine against HCV infection.Methods BALB/c mice were primed with pSCK-based DNA vaccine and boosted with type 5 adenoviral vector-based vaccine, which expressed the structural proteins ( Core, E1 and E2) de-rived from a Chinese HCV patient (genotype 1b, Hebei strain).Enzyme linked immunospot assay (ELIS-POT) and intracellular cytokine staining ( ICS) were used to analyze the elicited antigen-specific immune re-sponses and the efficacy of cross-protection.Results Immunization of mice with the prime-boost vaccination strategy elicited stronger T cell immune responses against multiple HCV antigens than using the DNA vac-cines alone, especially the IFN-γ-secreting T cell responses against E1 protein as indicated by ELISPOT as-say.ICS data indicated that the prime-boost regimen elicited more TNF-α-producing CD4+and IFN-γ-produ-cing CD8+T cells against E1 protein and high levels of IFN-γ-producing CD4+and CD8+T cells against E2 protein in comparison with immunization with DNA vaccines.Moreover, the prime-boost vaccination was ca-pable of eliciting effective cross-protection in a surrogate challenge model based on a recombinant heterolo-gous HCV (JFH1, 2a) vaccinia virus.Conclusion The prime-boost vaccination using DNA and rAd5-based vaccine expressing HCV structural antigens induced significant cellular immune response and cross-protection in mice, suggesting the possibility of using it as a promising T cell based vaccine against HCV in-fection.

Objective To investigate the long-term oncological outcomes of laparoscopic radical hysterectomy (LRH) plus lymph node dissection (LND) and abdominal radical hysterectomy (ARH) plus LND for patients with stage Ⅰ a2-Ⅱ a2 cervical cancer.Methods A retrospective review of stage Ⅰ a2-Ⅱ a2 cervical cancer patients who underwent LRH + LND (n=372) and ARH + LND (n=434) at the First Affiliated Hospital of Sun Yat-sen University from Jan.2005 to Aug.2013 was performed.Individual patient matching was performed by the risk factors for recurrence [tumor size,lymph vascular space invasion (LVSI),depth of cervical stromal invasion,lymph node metastasis,parametrialinvolvement,and resection margin involvement] between two groups.After matched,a total of 203 patient pairs (LRH-ARH) were enrolled.The survival data,surgery data,intraoperative and postoperative complications were compared between the two groups.To assess the prognosis factors,the univariate and multivariate Cox's proportional hazards modelanalysis were conducted.Stratified analysis was performed based on the independent prognosis factors to investigate the survival data between the two surgery groups.Results (1) Surgery data:The operating time [(239±44) vs (270±42) minutes],estimated blood loss [(210± 129) vs (428±320) ml],the duration of bowel motility return [(2.0±0.8) vs (3.0± 1.6) days] and hospital stay [(11 ±6) vs (13±6) days] in the LRH group were significantly shorter than those in ARH group (all P＜0.01).(2) Intraoperative and postoperative complications:The intraoperative complications rate was similar betweentwo groups [6.4％(13/203) vs 6.9％(14/203),P=1.000].The rate of postoperative complications (excluded bladder dysfunction) in the LRH group were significantly lower than those in the ARH group [9.4％ (19/203) vs 20.2％ (41/203),P=0.002].While there was no significant difference in the rates of bladder dysfunction between two groups [36.5％ (74/203) vs 37.4％ (76/203),P=0.910].(3) Recurrence and survival data:There was no significant difference in the recurrence rates between the LRH group and ARH groups [7.9％ (16/203) vs 9.4％ (19/203),P=0.850].There were similar 5-year recurrence-free survival (RFS;92.1％ vs 91.1％,P=0.790) and 5-year overall survival (OS;93.7％ vs 96.1％,P=0.900).(4) Prognosis factor:In univariate analysis,the results showed that tumor size,International Federation of Gynecology and Obstetrics (FIGO) stage,adjuvant therapy,LVSI,stromal invasion,parametrium invasion,pelvic lymph node metastasis,and para-aortic lymph node metastasis were significantly associated with poor prognosis (all P＜0.01).However,age,body mass index (BMI),surgery type,histological type,grade were not significantly associated with poor prognosis (all P＞0.05).The multivariate analysis results,showed that tumor size,pelvic lymph node metastasis,and para-aortic lymph node metastasis were significantly associated with poor prognosis (all P＜0.01).Stratified analysis showed that,even in patients with tumor size ＞4 cm,pelvic lymph node metastasis positive,and para-aortic lymph node metastasis positive in all subgroups,there were not significant difference for the estimated 5-year RFS and 5-year OS between LRH and ARH group (all P＞0.05).Conclusion For patients with stage Ⅰ a2-Ⅱ a2 cervical cancer,LRH plus lymph node dissection is an oncologically safe and surgical feasible alternative to ARH.

Objective To evaluate the influence of blood transfusion or pregnancy on blood cross-matching results.Methods 1 000 specimens with blood transfusion or pregnancy history were collected and performed the blood group typing,blood cross matching,irregular antibody screening and identification by the micro-gel method with the specimens of first time transfuse blood and without pregnancy history as the control.The inconsistency rate of positive negative blood grouping,unqualified rate of blood cross matching and the positive rate of irregular antibody were compared between the two groups by the t-test.The causes influen-cing the blood cross-matching results were analyzed.Results The inconsistency rate of blood cross-matching in the blood transfu-sion or pregnancy group was significantly higher than that in the control group(P <0.05);the inconsistency rate of positive nega-tive blood grouping was similar in the two groups(P >0.05);the positive rate of irregular antibody screening in the blood transfu-sion or pregnancy group was also significantly higher than that in the control group(P <0.05),indicating that the main causes in-fluencing blood cross matching was the existence of irregular antibodies in the patients in vivo.Conclusion Blood transfusion or pregnancy may stimulate the body to produce the irregular antibodies.Therefore the irregular antibody screening should be per-formed before blood transfusion and blood without corresponding antigen will be selected for transfusion according to the antibody identification results for ensuring safe and effective blood transfusion.

Objective:To observe the effect of transdermal fentanyl combined with gabapentin for the treatment of malignant neu-ropathic pain (MNP). Methods:A total of 60 patients with MNP were randomly divided into two groups. A total of 30 cases in the con-trol group received transdermal fentanyl according to the dosages of opioid medicine that patients used. Such dosages were gradually in-creased until the pain relief visual analogue scale (VAS) fell below 3 or until the times of breakthrough pain became less than 3. For the combined group, gabapentin was co-administered with transdermal fentanyl, similar to the control group. Initially, 100 mg of gabapen-tin was administered thrice a day. This dosage was gradually increased until pain relief. However, gabapentin dosages were kept below 2,400 mg a day. VAS, quality of life (QOL), degree of pain relief, dosages of fentanyl and morphine, and side effects were evaluated be-fore treatment and at one, two, three, and four weeks after the treatment. Results:Both groups exhibited lower VAS after treatment (P<0.05), but the difference was observed to be more significant in the combined group than that in the control group (P<0.05). Both groups exhibited improved QOL (P<0.05), which was observed to be more significant in the combined group than in the control group (P<0.05). The effective rate was 96.7%in the combined group and 83.3%in the control group. The dosage of opioid medicine and the side effects in the combined group were less than those in the control group. Conclusion:Transdermal fentanyl combined with gabapen-tin is effective for the treatment of malignant neuropathic pain.

ObjectiveTo investigate the effect of individual donation-nucleic acid amplification test (ID-NAT) and minipool of 16 donations-NAT (P16-NAT) on the results of NAT of blood donors.Methods From February 2009 to June 2009,samples randomly collected from voluntary blood donors in Beijing were tested individually or in pooling of 16 donations by the PROCLEIX ULTRIO assay.For ID-NAT reactive samples with HBsAg,anti-HCV,or anti-HIV serologically unqualified,ID-NAT repeat reactive samples with serologically qualified,and P16-NAT reactive and followed resolution ID-NAT reactive samples,were performed for further discriminatory assays for HIV-1,samples and followed resolution ID-NAT reactive samples,were performed for further discriminatory assays for HBV,HCV and HIV-1discriminatory reagents.Samples which were HBV NAT + alone with serologically qualified were further quantified and confirmed of HBV DNA by Roche HBV quantitative PCR,analyzed by HBV serology and were diluted to simulate if they could be detected in P16-NAT.Results ( 1 ) Among 7613 samples tested by ID-NAT,26 were NAT positive,i.e.the ID-NAT positive rate was 0.34％ ( 26/7613 ). ( 2 ) Among 1004 P16 samples from 16 064 blood donations,27 were NAT positive,i.e.the P16-NAT positive rate was 0.17％ (27/16 064).(3)In serological qualified donations,ID-NAT yield rate (1 in 826,9/7438 ) was much higher than P16-NAT ( 1 in 7875,2/15 750) (x2 =11.880,P ＜ 0.05 ).All these 9 ID-NAT positive and 2 P16-NAT positive donations were discriminated as HBV NAT positive.There were no HCV NAT yield or HIV NAT yield samples. (4) Dilution assay showed only 2 of the 9 (22.22％ ) ID-NAT HBV yields were detected by P16-NAT.(5)Eight ID-NAT and 2 P16-NAT positive samples were quantified for HBV DNA and confirmed as HBV NAT yield,although the virus loads were very low:2 samples had HBV viral loads of 15 IU/ml and 472 IU/ml,6 samples ＜ 12 IU/ml,and 2 could not be detected in the original samples while had ＜ 12 IU/ml and 14.3 IU/ml in the 10 times concentrated samples.(6)Among 11 HBV NAT yield cases,3 (27.3％ ) were possible HBV window-period donors with all HBV seromarkers negative,the other 8 (72.7％ ) had occult HBV infections with anti-HBc or anti-HBe positive,however anti-HBc IgM negative.(7) The rate of initial P16-NAT reactive pools needed to be further tested by ID-NAT was 2.49％(25/1004).Initial P16-NAT reactive pools which caused by serologically qualified donations was 0.20％(2/1004).ConclusionsHBV NAT yield cases are detected at a higher frequency with ID-NAT than P16-NAT.In order to avoid samples with low viral loads would be undetected,NAT assay with high sensitivity should be selected and tested in minimized minipool donations or even with individual donation.

ObjectiveTo explore the significance of colonoscopy examination in healthy subjects.MethodsA total of 537 health examination cases who underwent colonoscopy from January 2010 to June 2011 in Peking Union Medical College Hospital (PUMCH) health examination center were enrolled,in which 529 cases underwent total colonoscopy.Detection of colorectal lesion,polyp types and pathology were recorded and the correlation between detection rate of polyps,lesion sites and ages as well as gender were analyzed.ResultsThere were 315 male and 214 female,with a mean age of 48.3 ± 9.3 years.A total of 158 colorectal polyps were detected in 104 cases ( 19.7％ ),including 84 ( 15.9％ ) adenomas,17 (3.2％ ) advanced polyps and 1 (0.2％ ) rectal cancer.Other abnormalities were hemorrhoids in 61 cases ( 11.5％ )and inflammation in 22 (4.2％ ).Colorectal polyps were more frequent in men (P ＜0.001 ).Adults older than 50 had higher rates of colorectal polyps (P ＜0.001 ) and advanced polyps ( P ＜0.05).The proportion of patients with proximal polyps increased with age,whereas that of patients with distal polyps decreased with age ( P ＜0.05).ConclusionColonoscopy is an effective screening modality for colorectal lesions and the importance of colonoscopy on health examination population to prevent colorectal cancer should be stressed.

Objective Inflammation is a defensive reaction of body , but excessive inflammatory response can lead to physi-cal injury.The aim of this study was to explore the effects of luteolin on the secretion of inflammatory cytokines from lipopolysaccharide (LPS) and interferon-g(IFN-γ) activated RAW264.7 cells. Methods RAW264.7 cells were divided into 5 groups: control group (without any medicine), M1 group (polarized M1 cells activated by final concentration of 10 ng/mL LPS+20 ng/mL IFN-γ), M1+5L group (simultaneous activation of LPS and IFN-γplus final concentration of 5μmol/L luteolin), M1+10L group(simultaneous activa-tion of LPS and IFN-γplus 10μmol/L luteolin), M1+20L group(simultaneous activation of LPS and IFN-γplus 20μmol/L luteolin). The cell morphological transformation was observed by laser confocal microscope ;the mRNA levels of iNOS , IL-1βand IL-6 were test-ed by real-time quantitative PCR respectively;the secretion levels of TNF-αand IL-6 in culture supernatant were detected by ELISA;the changes of p-STAT3 (ser727) protein pathways were examined by western blot. Results Cellular morphology of activated RAW 264.7 cells changed obviously .Compared with the control group , the mRNA levels of iNOS, IL-1βand IL-6 decreased significantly in the other 4 groups(P<0.05).The iNOS level in M1+20L group significantly de-creased compared with M1 group[(29.52±3.07) vs (98.91±10.65), P<0.01].As to IL-1βlevel, it decreased significantly in M1+10L group(78.38±8.65) and M1+20L group(41.59±6.80) compared with M1 group(110.69±4.12)(P<0.05).While the IL-6 levels decreased significantly in M1+5L group(177.51±19.28), M1+10L group (106.14±5.63), M1+20L group(27.15±1.26), compared with M1 group(394.10±33.47)(P<0.05).LPS+IFN-γcould induce in-creased p-STAT3 (ser727) expression in M1 phenotype of RAW264.7 cells which was proved by its significant increase in M 1 group, M1+5L group and M1+10L group compared with control group (P<0.05).In comparison to M1 group, p-STAT3-ser expression in M1 phenotype downregulated in M1+5L group, M1+10L group, M1+20L group(P<0.05), along with dose-dependent characteristic.Com-pared with control group, the levels of IL-6 and TNF-αincreased significantly in M1 group, M1+5L group and M1+10L group.Com-pared with M1 group, the levels of IL-6 and TNF-αdecreased significantly in M1+5L group, M1+10L group and M1+20L group(P<0.05) , in which IL-6 showed concentration independence and TNF-αshowed no concentration independence . Conclusion Luteolin inhibits the secretion of pro-inflammatory cytokines through the down-regulation of p-STAT3 so as to exert anti-inflammatory effects .