Objective To describe the phenotype of NK cells in Bama miniature pigs, and establish an efficient activation and culture method for porcine cytokine-induced killer ( CIK) cells in vitro. Methods The porcine peripheral blood mononuclear cells ( PBMCs) were isolated by Percoll gradient centrifugation, and the phenotype of NK cells was test-ed by detecting the CD2 + /CD8 + /CD3 - cell compartment. To establish an efficient activation and culture method for por-cine CIK cells, we optimized the culture conditions to improve the CIK activation efficiency. Results Using the optimized induction culture conditions, the ratio of CIK ( CD2 + /CD8 + /CD3 -) cells was up to 43. 63% at the fifth day, approxi-mately 5. 59 times increased compared with the initially separated PBMCs. Cell proliferation experiments showed that three obvious fluorescence peaks were observed on the fifth day. The results indicated that the induced CIK cells underwent three times cell division, in theory, about increased 8-fold compared with the initial separation of PBMCs. Furthermore, the qRT-PCR result of the surface markers of porcine NK cells also showed a similar variation tendency as the flow cytometry results. Conclusions Our findings demonstrate the successful establishment of an efficient activation and culture method for porcine CIK cells in vitro.

Objective To develop a real-time RT-PCR assay ( rRT-PCR) for efficient detection of duck hepatitis virus type 1 ( DHV-I) .Method According to the different gene sequences of DHV-I from different provinces download from NCBI and to find the conserved sequences.One pair of the specific primers and one TaqMan probe were designed. Then reaction parameters were optimized to develop a real-time RT-PCR assay ( rRT-PCR) .Results This developed rRT-PCR assay could detect 20 template copies of RNA, and its sensitivity was higher than that of the conventional RT-PCR. This rRT-PCR assay was found to be specific and able to detect DHV-I, and no positive results were observed when nucleic acid from Muscovy duck parvovirus, goose parvovims, Newcastle disease and avian influenza virus, egg drop syndrome virus, reticuloendotheliosis virus, duck Tembusu virus, poultry intestinal arc virus were used as rRT-PCR templates.The results of this developed rRT-PCR assay used for 100 duck clinical samples showed a positive rate of 92%, indicating that DHV exists in duck group of Jiangsu province in China.Conclusion This rRT-PCR assay can be used as a rapid tool for detection of DHV-I.

Objective:To investigate the clinical efficacy of neoadjuvant chemo-hormonal therapy(NCHT)followed by radical prostatectomy(RP) plus extended pelvic lymphadenectomy for very-high-risk locally advanced prostate cancer.Methods:The data of 327 cases of very-high-risk locally advanced prostate cancer treated in Renji Hospital Affiliated to Shanghai Jiaotong University School of Medicine, The Second Hospital of Tianjin Medical University, and The Third Affiliated Hospital of Sun Yat-sen University from December 2014 to July 2019 were retrospectively analyzed. Patients were divided into two groups according to treatment regimens: the RP group (direct RP + extended pelvic lymphadenectomy 4-6 weeks after the biopsy of prostate) and the NCHT group (4-6 cycles of NCHT prior to RP). There were 171 cases in RP group and 156 cases in NCHT group, respectively. In the RP group, the median age was 67 (ranging 44-83)years. The median PSA at diagnosis was 27.24 (ranging 4.55-207.00) ng/ml. Patients’numbers of clinical T 2, T 3a, T 3b, T 4 stage were 13, 85, 57, 16, respectively, and clinical N 1, N 0 stage were 33 and 138, respectively. Patients’numbers of ISUP grade groups of 1, 2, 3, 4, 5 were 5, 35, 41, 51, 39, respectively. In the NCHT group, The median age was 67 years, ranging 46-78 years. The median PSA at diagnosis was 72.09(ranging 4.08-722.95)ng/ml. Patients’ numbers of clinical T 2, T 3a, T 3b, T 4 stage were 11, 47, 58, 40, respectively, and clinical N 1, N 0stage were 76 and 80, respectively. Patients’numbers of ISUP grade groups of 1, 2, 3, 4, 5 were 1, 11, 33, 43, 68, respectively. At baseline, the NCHT group showed higher PSA, higher ISUP grade, and more advanced clinical stage at diagnosis( P<0.05). The PSA, pathological down-staging rate, and positive surgical margin rate as well as the biochemical recurrence free survival(bRFS)were compared between the two groups. Results:After radical prostatectomy, compared with the RP group, the NCHT group had a higher proportion of patients achieving PSA<0.2 ng/ml at 6-week postoperative follow-up ( P<0.001), a higher pathologic tumor stage down-staging rate ( P<0.001), a higher ISUP down-grading rate ( P<0.001), and a lower positive surgical margins rate ( P<0.001). In addition, 10.9% of the NCHT group achieved pT 0 or minimal residual disease in postoperative pathology exams. Eighty-three patients (48.5%) in the RP group and 125 patients (80.1%) in the NCHT group achieved undetectable PSA after surgery and entered further analysis for bRFS, which showed NCHT group had significantly longer bRFS (19.46 months vs. 6.35 months). NCHT significantly reduced the risk for biochemical recurrence in locally advanced prostate cancer patients( HR=0.278, 95% CI 0.198-0.390, P<0.001). Such a reduce in risk for biochemical recurrence was seen in all subgroups( P<0.001). Conclusions:NCHT might improve surgical outcomes as well as bRFS in very-high-risk locally advanced prostate cancer patients.

Objective:To study the risk factors and treatment of portal vein thrombosis (PVT) in patients after liver transplantation.Methods:The clinical data of 290 recipients who underwent liver transplantation at the Department of Hepatology, the Fifth Medical Center of PLA General Hospital from July 2015 to April 2019 were retrospectively analyzed. There were 245 males and 45 females, with a median age of 51(44, 56) years old. The liver transplantation recipients were divided into two groups according to whether PVT occurred or not after operation: the PVT group ( n=16) and the non-PVT group ( n=274). Gender, age and other clinical data of the recipients were compared between the two groups. Outpatient and inpatient follow-up were performed. The risk factors of postoperative PVT were analysed in the liver transplantation recipients. Results:The median follow-up of these 290 liver transplant recipients was 59(42, 73) months, and 16 patients were confirmed to have PVT after operation, with an incidence of 5.5%(16/290). Multivariate logistic regression analysis showed that preoperative PVT ( OR=12.773, 95% CI: 3.887-41.973) was an independent risk factor for PVT after liver transplantation. For the 16 patients with postoperative PVT, 10 were treated with portal vein intervention, and the remaining 6 patients were treated with oral aspirin or rivaroxaban anticoagulation due to mild symptoms. The 3-year survival rate of the PVT group was 93.8% (15/16), while that of the non-PVT group was 90.1% (247/274). There was no significant difference in the 3-year survival rates between the two groups (χ 2<0.01, P=0.969). Conclusions:Preoperative PVT in recipients was an independent risk factor for PVT after liver transplantation. For patients with postoperative PVT, appropriate treatment resulted in good results without affecting the long-term prognosis of these patients.