Objective To prepare humanized monoclonal antibodies(Mabs)targeting Acinetobacter baumannii(Ab)based on single memory B cell sequencing technology,construct the immune repertoire of the core protein of Ab,hemolysin-coregulated protein(Hcp),and express its Mabs with binding activity.Methods E.coli BL21 harboring the recombinant plasmid pGEX-6p-1-Hcp was constructed.Hcp protein was obtained using protein expression and affinity chromatography.Female SPF BALB/c mice(6～8 weeks old,weighing 18～20 g)were immunized intramuscularly with antigen Hcp to generate specific memory B cells.Single antigen-specific memory B cells were sorted using flow cytometry.The immune repertoire of Hcp was constructed using single-cell sequencing technology,and bioinformatics analysis was performed on the sequencing results.Mabs were obtained using antibody humanization techniques.The in vitro binding activity of the antibodies was detected by ELISA.Results The target protein Hcp with a purity>95%was obtained after expression and purification.The immune repertoire of Hcp was successfully constructed,and the results of BCR clonotype identification and analysis,CDR3 region characteristic analysis,and V-J gene pairing characteristic analysis were achieved.Antibody humanization got 7 Mabs,that is,IgG1-1,IgG1-2,IgG2-1,IgG2-2,IgG3-1,IgG4-1 and IgG4-2.ELISA results showed IgG1-1,IgG3-1,IgG4-1,and IgG4-2 had an antibody binding titer of 1∶1 280,IgG2-2 of 1∶10 240,IgG2-1 of 1∶5 120,and IgG1-2 of 1∶160.Conclusion Single-cell sequencing technology enables rapid,accurate,and efficient construction of an Hcp protein immune repertoire containing extensive antibody information.Utilizing this immune repertoire allows for the expression of Mabs with binding activity.

Objective:To explore the influencing factors of continuous renal replacement therapy(CRRT)after heart transplantation(HT).Methods:For this retrospective cohort study, the relevant clinical data were retrospectively reviewed for 145 recipients undergoing HT at No.7 Municipal People's Hospital from April 2018 to December 2022.They were assigned into two groups of non-CRRT(n=124)and CRRT(n=21). And t, χ2or rank-sum test was utilized for comparing baseline data, intraoperative and postoperative general conditions of two groups.Variables with P<0.05 in univariate analysis and significant indicators in previous studies were included in multivariate logistic regression analysis to analyze the influencing factors of CRRT post-HT.Receiver operating characteristic curve(ROC)was utilized for selecting the optimal predictive cut-off value. Results:Among them, 66 cases(45.52%)developed AKI and 21(14.48%)required CRRT.Through univariate analysis, preoperative estimated glomerular filtration rate(eGFR), erythrocyte count, platelet, hemoglobin, total bilirubin, intraoperative volume of blood loss, volume of blood transfusion, urine volume, operative duration, cardiopulmonary bypass time, postoperative mechanical ventilation time, ICU stay and postoperative acute kidney injury were compared.The inter-group differences were statistically significant( P<0.05). Further multivariate logistic regression analysis revealed that preoperative hemoglobin level( OR=0.869, 95% CI: 0.770-0.980, P=0.022), preoperative platelet count( OR=0.959, 95% CI: 0.925-0.993, P=0.019), intraoperative volume of hemorrhage( OR=1.004, 95% CI: 1.000-1.009, P=0.049), intraoperative urine volume( OR=0.997, 95% CI: 0.993-1.000, P=0.035), operative duration( OR=1.022, 95% CI: 1.000-1.044, P=0.047)and mechanical ventilation time( OR=1.036, 95% CI: 1.005-1.069, P=0.024)were the independent influencing factors of CRRT post-HT.ROC curve results indicated that area under curve(AUC)of operative duration, mechanical ventilation time and intraoperative volume of hemorrhage were 0.745(95% CI: 0.636-0.855), 0.835(95% CI: 0.735-0.934)and 0.669(95% CI: 0.506-0.830)with a sensitivity of 0.714, 0.857, 0.571 and a specificity of 0.710, 0.685, 0.895.And the cut-off values were 283.5 min, 25.46 h and 825 ml respectively. Conclusions:Hemoglobin level, preoperative platelet count, intraoperative volume of hemorrhage, urine volume, operative duration, mechanical ventilation time and intraoperative urine volume are independent influencing factors of CRRT post-HT.Operative duration >283 min, mechanical ventilation time >25.46 h and intraoperative volume of hemorrhage >825 ml have some predictive values for CRRT post-HT.

Objective:To analyze the genomic epidemiological subtyping of carbapenem resistant Klebsiella pneumoniae (CRKP) isolated from a Third-class A hospital in Zhengzhou. Methods:From December 4, 2019 to January 10, 2020, 67 strains of CRKP were isolated from the samples submitted by the clinical departments of a Third-class A teaching hospital in Zhengzhou for microbiological testing. Multi-locus sequence typing (MLST) and carbapenem resistance genes were identified by whole genome sequencing and sequence analysis. Based on the whole genome SNP, the phylogenetic tree was constructed, and 67 CRKP strains were divided into clonal groups. The isolation ward and date of each clone group were analyzed.Results:Sixty-seven CRKP strains were classified into four MLST types (STs), of which 64 were ST11. There were 62 ST11 strains carrying blaKPC-2 gene. Based on genome-wide SNP phylogenetic tree, 64 ST11 strains were divided into four clone groups, two of which were dominant clone groups, including 33 and 27 strains respectively; the other two clone groups only contained 2 strains respectively. There was no aggregation of the dominant clones in the isolation department and date. Conclusion:Multiple clonal groups of ST11 strain carrying blaKPC-2 gene are differentiated during spreading, and they can spread in parallel and independently in the same hospital.

Objective:To analyze the genomic epidemiological subtyping of carbapenem resistant Klebsiella pneumoniae (CRKP) isolated from a Third-class A hospital in Zhengzhou. Methods:From December 4, 2019 to January 10, 2020, 67 strains of CRKP were isolated from the samples submitted by the clinical departments of a Third-class A teaching hospital in Zhengzhou for microbiological testing. Multi-locus sequence typing (MLST) and carbapenem resistance genes were identified by whole genome sequencing and sequence analysis. Based on the whole genome SNP, the phylogenetic tree was constructed, and 67 CRKP strains were divided into clonal groups. The isolation ward and date of each clone group were analyzed.Results:Sixty-seven CRKP strains were classified into four MLST types (STs), of which 64 were ST11. There were 62 ST11 strains carrying blaKPC-2 gene. Based on genome-wide SNP phylogenetic tree, 64 ST11 strains were divided into four clone groups, two of which were dominant clone groups, including 33 and 27 strains respectively; the other two clone groups only contained 2 strains respectively. There was no aggregation of the dominant clones in the isolation department and date. Conclusion:Multiple clonal groups of ST11 strain carrying blaKPC-2 gene are differentiated during spreading, and they can spread in parallel and independently in the same hospital.

Objective:To reveal the differentially expressed genes and long non-coding RNAs (lncRNAs) by sequencing the transcriptome of bovine alveolar macrophages (BAM) infected with Bacillus Calmette-Guérin (BCG) and analyzing their bioinformations, and to provide theoretical foundation for the research on immune regulation mechanism of anti-infection of Mycobacteriumtuberculosisof macrophages.Methods:The BAM were collected by pulmonary lavage and centrifugation and cultured and divided into infected group and uninfected group.After infection for 12hin infected group, the expression profiles of mRNA and lncRNA in infected group and uninfected group were detected by RNA-Seq, and bioinformatics analysis was carried out.Results:compared with uninfected group, there were 119differentially expressed lncRNAs and 1111differentially expressed mRNA in infected group (P＜0.05) .Gene Ontology functional enrichment analysis showed that the most significant enrichment was immune response (GO:0006955, P＜0.05) , including 125genes, in which 63 were up-regulated and 62 were downregulated, and the expressions of proinflammatory factors interleukin-1 (IL-6) , interleukin-7 (IL-7) , and interleukin-23A (IL-23A) were up-regulated.Cis target gene prediction and KEGG pathway analysis showed that the differentially expressed lncRNAs were involved in the regulation of transforming growth factor-β (TGF-β) signaling pathway and ATP-binding cassette transporter (ABC transporter) signaling pathway (P＜0.05) .Conclusion:The host cells are stimulated to produce a strong immune response after the BAM are infected by BCG and results in the changes of lncRNA and mRNA expression profiles.

Objective:To determine whether chewing gum during the postoperative period facilitates the recovery of bowel function in patients after radical cystectomy with ileum urinary diversion.Methods:In the study,60 patients who underwent radical cystectomy followed by ileum urinary diversions during Nov.2014 and Nov.2015 in Department of Urology of Peking University First Hospital were randomized into three groups:gum chewing group,placebo group treated with the abdomen physical therapy machine and control group treated with ordinary method.Time to flatus,time to bowel movement,incidence of postoperative distension of the abdomen and abdominal pain,and gut related complications (such as ileus,intestinal fistula,and volrulus)of all the patients were recorded and analysed.Results:In gum chewing group,the median time to flatus was 57 hours (49 -72 hours),and the median time to bowel movement was 95 hours (88 -109 hours),which were significantly shortened compared with the other two groups of patients (82 hours,109 hours in placebo group and 81 hours,108 hours in control group, respectively).No significant difference of the median time to flatus and to bowel movement was observed between placebo group and control group.There were no significant differences in the incidence of post-operative distension of the abdomen and abdominal pain,and gut related complications among the three groups.Conclusion:Chewing gum had stimulatory effect on bowel function recovery after cystectomy fol-lowed by ileum urinary diversion.Chewing gum was safe and simple,and could be routinely used for postoperative treatment after cystectomy and ileum urinary diversion.

Objective To assess the safety,efficacy and reliability of IUPU(Abbreviation of Institute of Urology,Peking University)technique in setting up the retroperitoneal cavity for retroperitoneoscopy. Methods From February 1996 to March 2006,more than 1100 retroperitoneoscopic procedures were performed with the IUPU technique in setting up the retroperitoneal cavity,First,at the cross point of the line 2 cm above the iliac crest and the longitudirml line close to the anterior auxiliary line,a 1 cm skin incision was made and the Veress needle was penetrated into the retroperitoneal space(RPS)with a 0°-30° angle to the perpendicular line.The CO2 gas was pumped into the RPS till the pressure increases to 14mmHg and the first port was inserted into the RPS.The laparoscope was then inserted into the RPS through the first port and kept swinging right and left with its tip and trunk tO set up the retroperitoneal cavity.Other 2 ports were put into the RPS at cross points of the sub-costal line and anterior and posterior auxiliary lines under monitor observation.Then other appliances are introduced into the RPS to expand the cavity.More than 1100 procedures had been done with the IUPU technique,including 54 cases of simple nephrectomy (loss of function due to tuberculosis,hydronephrosis and atrophy), 188 radical nephrectomy, 154 ureteronephrectomy, 344 adrenalectomy, 302 renal cyst decortications, 35 partial nephrectomy, 37 pyeloplasty. Results The average time for the IUPU technique was (5.4±2.8)min (range 4. 5 to 14. 5 min) to set up the RPS. Complications included converting to open surgery due to bleeding in 8 cases(8/1114,0. 72%) when the first port was inserted into the RPS and entered into the peritoneal cavity for the first port penetration in 32 cases (32/1114,2. 87%),although the retroperitoneal cavity could be set up successfully by adjusting the laparoscope into the RPS.There was no injury to other viscera. Conclusions The IUPU technique is safe, efficient and reliable in setting up the RPS and no other special instrument is needed. It can be finished within 5 min on skilled hands and is valuable as a routine method to set up the retroperitoneal cavity.

Objective To evaluate the safety and efficacy of the anatomical retroperitoneoscopic nephrectomy(RSN)and standardize the procedure of RSN. Methods The retrospective analysis was performed on 405 consecutive patients underwent anatomical RSN in Our institute from January 2002 to June 2008.There were 232 male and 173 female patients with the average age of(57.2±14.2)years,among whom there were 228 renal cell carcinoma patients accepted RSU,96 and 49 renal pelvic carcinoma and ureteral carcinoma cases accepted retroperitoneoscopic ureteronephrectomy (RSUN) and 32 cases accepted simple RSN due to loss of renal function caused by benign renal discsses.The tadical RSN was performed by dissecting outside Gerota's fascia and in the latent cavities between this fascia and lateral conal fascia in the dorsal side and between this fascia and prerenal fusion fascia in the ventral side,whereas the simple RSN was done inside Gerota's fascia by making direct incision on it and dissecting between this fascia and perirenal adipose tissue.Kidneys and perirenal adipose tissue were completely removed by dissection along several avascular planes around the kidney under the amplified view of laparoscopy. The software SPSS 12.0 was used for the statistical analysis of all data. Results The mean operative time was (132±48)min for radical and simple RSN and (245 ± 62)min for radical RSUN, which included the time for position change and second skin preparation. The medium estimated blood loss was 100 ml(10-2500 ml) and the average drainage volume was 150 ml (0-1152 ml) postoperatively. 15 cases (3. 70%) required blood transfusion with the median volume of 400ml (400-1650 ml). Four cases (0. 99%) were converted to open surgery due to severe adhesion (2 cases), difficult exposure of renal helium (1 case) and severe bleeding (1 case).The mean drainage time was (3. 9±1.8)d, the mean time to first oral intake was (2.7±1.2)d and the mean postoperative hospital stay was (8.6±3. 8)d. Conclusion The anatomical RSN is safe and effective and should be the standard surgical procedure for laparoscopic nephrectomy.

Objective To observe prospectively and systematically the effect and safety of rhendostati injection (endostar) combined with FOLFOX as a second line chemotherapy for advanced/metastatic colorectal cancer. Methods 23 patients with histological confirmed advanced/metastatic colorectal cancer after first line chemotherapy failure were observed. The dosage of 15 mg/time of endostar solved in 500ml normal saline was slowly intravenously dropped 4 h from day 1 to day 14. Oxaliplatin 85 mg/m~2 iv 2-3 h dl, d15. CF 200 mg/m~2 iv 2 h followed by 5-Fu 400 mg iv bolus and 5-Fu 600 mg/m~2 iv 22 h dl-2, d15-16 were given, every 4 weeks as one cycle. Efficacy was evaluated after 2 cycles according to RECIST criteria. Results 23 cases had been completed totally 56 cycles. Among 23 cases, 8 cases were PR, 12 cases SD, and 3 cases PD. The objective response rate (RR) was 34.8 % (8/23), and the disease control rate (DCR) was 87.0 % (20/23). The median time to progression was 7 months. The 1-year survival rate were 50.0 %. The 2-year survival rate was 40.0 %. The occurrence rate of G3/4 toxicities was low, including neutropenia(21.7 %), anemia(4.3 %), thrombocytopenia (13.0 %). Those toxicities were mainly related with the chemotherapy agents. Meanwhile transient electrocardiogram changes mild ST-T of changes occurred in 3 cases. 2 cases were mild hypertension and were symptomatically controlled. Conclusion There are better efficacies of endostar combined with FOLFOX chemotherapy for advanced/metastatic colorectal cancer, and it is low toxic and tolerable. It is worth of further clinical observation. More experiences need to be accumulated.

Objective To summarize the experience in open surgery for huge adrenal tumors in order to improve its safety and efficiency of this complicated surgical procedure. Methods Fortyfour consecutive patients with huge adrenal tumors underwent open surgery with mean long tumor diameter of 13 cm (9-34 cm), and autologous blood transfusion was prepared in routine. It was analyzed retrospectively for clinical data, perioperative complications and the effective and safety results of this procedure. Results The incision was oblique in lumbar region in 5 cases, subcostal unilaterally in 32 cases and abdomino-thoracic joint in 7 cases. There were 27 malignant tumors (61.4%) in 44 cases, 3 with hepatic invasion, 6 with thrombi extending into inferior vena cava, among which 2 needed translocation of artificial blood vessels and 3 needed cardio-pulmonary bypass. The mean blood loss was 1309 ml (100-3000 ml) in 41 cases(93.2%)and the autologous blood transfusion was used in 20 case (45.5%). There were 1 diaphragmatic injury, 1 pleura injury, 3 hemorrhage in large amount more than 15 000 ml and 2 peritoneal cavity infection.There were no perioperative deaths and 42 tumors (95.5%) were curatively resected. Conclusions Open surgery for huge adrenal tumors is a complicated surgical technique with high risk and large amount of blood loss. The key points to success are proper selection of incision, routine autologous blood transfusion, perfect surgical skills and good cooperation between different specialties.