OBJECTIVETo develop a recombinant single domain antibody against hTERT, human telomerase catalytic subunit.

METHODSA previously prepared His-tagged hTERT fusion protein was used as the antigen, and the variable regions in heavy chain (VH) of immunized mice were RT-PCR amplified and cloned into the pCANTAB 5E, a phagemid vector. By transfection, the display library of mouse VH was developed. The candidate clones were selected by affinity panning, and soluble VH were obtained after expression in E. coli, HB2151. The resultant single VH antibodies were characterized on their binding potentials by western blotting.

RESULTSAn about 350 bp VH fragment was amplified from spleen cells of mice immunized by His-tagged hTERT and expressed by phage displayed as VH library. The size of the library was 8 x 10(4). After three rounds of affinity panning, 4 independent clones were chosen and consequently expressed as soluble single domain antibodies (Mr = 16 000). In Western blot analysis, the single domain antibody from 2 of 4 clones proved to react with the His-tagged hTERT fusion protein (Mr = 167 000) without dependence of His-tags and also detect the native hTERT (Mr = 127 000) extracted from the human HeLa cancer cell line. DNA sequencing showed both of the single domain antibodies were encoded by the heavy chain variable region of the mouse.

CONCLUSIONSThe single domain antibodies developed were hTERT recognizable and hTERT specific, thus providing a basis for application of recombinant single domain antibody in inhibition of telomerase activity and anticancer therapy.

Amino Acid Sequence ; Animals ; Antibodies, Monoclonal ; genetics ; immunology ; Base Sequence ; Cloning, Molecular ; Complementarity Determining Regions ; genetics ; DNA-Binding Proteins ; HeLa Cells ; Humans ; Immunoglobulin Heavy Chains ; genetics ; Immunoglobulin Variable Region ; genetics ; Mice ; Molecular Sequence Data ; Sequence Analysis, DNA ; Telomerase ; immunology

Objective:To investigate the incidence, risk factors and prognosis of acute renal injury(AKI)in elderly patients with respiratory distress syndrome(ARDS).Methods:The elderly patients with ARDS treated in the Department of Respiratory Medicine, Emergency Department and Geriatrics of the Second People's Hospital of Lianyungang from July 2016 to July 2019 were divided into AKI group and non-AKl group according to KDIGO diagnostic criteria.The clinical data and the differences were compared and analyzed between the two groups.Binary Logistic regression was used to analyze Risk factors for AKI.Kaplan-Meier cure was used to analyze the influence of different stages of AKI on the prognosis of ARDS patients.The Cox proportional hazards model was used to analysis risk factors for AKI and ARDS on elderly patients＇prognosis.Results:A total of 432 elderly patients with ARDS were enrolled in the study, in which the mean age was 74.7 ± 8.8 years, and AKI occurred in 129 cases(29.9%). Compared with non-AKI group, AKI group showed older age, and higher proportion of the incidences of hypertension, diabetes, atrial fibrillation, consciousness disturbance, mechanical ventilation and a low mean arterial pressure(all P<0.05). The incidence of AKI was increased significantly in patients with moderate to severe ARDS( P< 0.001). The levels of basal creatinine, AST and NT-proBNP were significantly higher in AKI Group than in non-AKI Group( P= 0.001, P< 0.001, P< 0.001). AKI Group patients had the more elevated inflammatory marker level of neutrophil / lymphocyte ratio(NLR)( P= 0.003)and D-dimer( P< 0.001), and the level of high-sensitivity c-reactive protein(hsCRP)( P=0.040). AKI group showed the increased incidence of urine protein( P< 0.001), low ejection fraction( P= 0.040), and positive rate of pleural effusion( P= 0.003). Logistic Regression analysis showed the following independent risk factors for the development of ARDS-associated AKI, included hypertension( OR: 1.789, 95%, CI: 1.105-2.894, P=0.018), diabetes( OR: 1.976, 95% CI: 1.076-3.628, P=0.028), consciousness disturbance( OR: 2.531, 95% CI: 1.203-5.251, P=0.014), mechanical ventilation( OR: 3.421, 95% CI: 1.521-7.694, P=0.003), AST>40 U/L( OR: 2.495, 95% CI: 1.431-4.348, P=0.001), increased basal creatinine levels( OR: 1.014, 95% CI: 1.002-1.027, P=0.024), and NLR( OR: 1.015, 95% CI: 1.001-1.029, P=0.042). Kaplan-Meier survival curve showed that there was a significant difference in the prognosis between patients with different AKI stages( χ2=19.790, P<0.001), and there was no significant difference in the prognosis between stage 1-AKI and non-AKI( χ2=2.188, P=0.139). The risk of poor prognosis was higher in AKI(stage 2-3)group( χ2=18.268, P<0.001; χ2=6.347, P=0.012)than in patients without AKI or stage 1 AKI.Multivariate Cox Proportional Hazard Model Analysis elucidated that AKI( HR: 1.858, 95% CI: 1.207-2.861, P= 0.005)and moderate-severe ARDS( HR: 1.815, 95% CI: 1.167-2.822, P=0.008)were independent risk factors for poor prognosis of ARDS in the elderly. Conclusions:Hypertension, diabetes, disturbance of consciousness, mechanical ventilation, AST>40 U/L, elevated levels of basal creatinine and NLR are independent risk factors for ARDS-associated AKI in elderly patients with ARDS.Patients with moderate-severe ARDS and AKI(2-3 phases)have the increased risk of poor prognosis.

Objective To investigate the diagnostic value of fiberoptic ductoscopy (FDS) in pathological milky white nipple discharge.Methods The data of 1688 patients with pathological milky white nipple discharge who underwent FDS examination in Chengdu Third People's Hospital from Oct.2011 to Oct.2016 were analyzed retrospectively.Results Among the 1688 cases,the proportion of patients with milky white nipple discharge was 30％,higher than that of the bloody discharge (15％) and yellow liquid (24.5％).The detection rate of lesions in patients with milk nipple discharge was 9.3％,among whom 6.1％ was breast cancer.Conclusions FDS should be routinely performed in patients with pathological milky white nipple discharge,as an examination tool to exclude the intra ductal lesion.The disease should be paid more attention by physicians.