Objective To detect the difference of cytokines and antibodies productions by immunologic system from mice vaccinated with recombinant P6 protein with different immunization routes and adiuvants.Methods 6 weeks female BALB/c mice were vaccinated with meombinant protein P6 combined with A1(OH)3,CpG ODN or the mix of them through intramuscular and intranasal.The mice were boosted twice with the same dose by the same route.Serum and respiratory tract specimen were collected to detect rP6 specific antibodies by ELISA.Results In the 6th week after immunization.The higher titer of serum rP6 specific IgG antibody were detected from the two groups vaccinated with A1(OH)3+CpG ODN+rP6 by intramuscular and vaccinated with CpG ODN+rP6 by intranasal(F=41.259.P=0.000).The ELISPOT experiment showed that,Inoculation with the CpG ODN+rP6 either by intranasal or intramuscular immunization could induce a large number of antigen-specific T lymphocyte activation.In addition.The mice vaccinated with the CpG ODN+rP6 through intranasal can be detected high titer rP6 specific mucosal IgA antibody(F=70.966.P=0.000).Conclusion Inoculation with the CoG ODN+rP6 by jntranasal immunization not only can induce high-titer serum IgG antibody,but also can induce high-titer mucosal IsA antibody and CD4+Th1.CD8+T lymphoeyte activation.

Objective To clone and express recombinant outer membrane protein P6, determine its optimal expression conditions and to investigate the immunoprotective effects of the P6 protein on mice. Methods The P6 gene of nontypeable Haemophilus influertzae(NTHi) was amplified by PCR from the NTHi genome and cloned into expression vector pET-32a (+) to generate the pET-32a-P6 recombinants. They were confirmed by nuclease digestion and sequence analysis. The verified recombinant was transformed into E. coli BL21 (DE3). Its optimal expression conditions were determined such as engineering strains, the concentration of IPTG, inducing temperature, inducing time, different medium etc. The recombinant protein was purified by Q Sepharose~(TM) XL ion exchange and gel filtration chromatography. The protein was analyzed by SDS-PAGE, Western blot and sequencing. BALB/c mice were immunized with recombinant protein be-fore challenged by NTHi through intraperitoneal injection. Then the mortality rate of different group was com-pared. Results The recombinant P6 of NTHi was successfully constructed and expressed in E. coli at a rel-atively high level. The purity was up to 95% after purification. The relative molecular mass of the protein is 14 145. 848. The recombinant protein was confirmed to show specific reaction on the antiserum through Western blot. The animal experiments showed the mortality rates of immunization groups were significantly lower than that of the control group (P < 0.05). Conclusion The successful expression of the recombinant P6 will be very helpful for the further study on development of vaccine, its purified, immunological activity and antibody preparation.

This article reviews and summarizes the research data about vertebrobasilar dolichoectasia in recent years from the aspects of epidemiology,pathogeny,pathology,diagnosis,clinical manifestations,as well as its mechanism and treatment.

Objective To construct a stable infectious clone of S191 virus strain used for the pro-duction of live attenuated measles vaccine. Methods Full length cDNA of S191 strain and gene fragments encoding nucleocapsid(N),phosphoprotein(P)and RNA polymerase(L)were synthesis and respectively cloned into the vector pVAX1. The 293T cells were respectively transfected with the recombinant expression plasmids and co-cultured with Vero cells. The supernatants of cell culture were collected for identifying res-cued viruses. The indirect immunofluorescence assay was performed for virus identification. The rescued viruses at different passages in Vero cells and the sequences of the rescued viruses were analyzed. Results Restriction enzyme digestion and sequence analysis showed that the recombinant expression plasmids contai-ning the full length cDNA with an artificially engineered mutation at nucleotide 2101(C-A)and gene frag-ments encoding N,P and L proteins of S191 strain were constructed successfully. The N and P proteins were detected in Vero cells with immunofluorescence assay. A cytopathogenic effect on Vero cells was induced by rescued viruses. Conclusion The stable infectious clones of S191 virus used for the production of live at-tenuated measles vaccine were rescued successfully. An approach by using reverse genetics technique for S191 strain study was established which could be used for the development of new chimeric vaccines against measles virus.

ObjectiveTo detect the difference of cytokines and antibodies productions by immunologic system from mice and rabbits vaccinated with the M.RCAg-1 chimeric protein,expressed in E.coli,formulation with different adjuvants,including Freund's adjuvant and three clinically acceptable adjuvants,namely,Al(OH)3,Montanide ISA720 and Montanide ISA51.MethodsSix weeks female BALB/c mice were vaccinated with recombinant protein formulated with different adjuvants through intranasal.Serum were collected to detect specific antibodies of M.RCAg-1 and individual Epitope by ELISA ; natural parasite antigen was recognized by indirect immunofluorescence assay; mouse specific T lymphocyte activation was detected by enzyme-linked immunosorbent spot test (ELISPOT) ; Affinity assay between protein and immune IgG of rabbits with the biosensor,and the growth of Plasmodiumfalciparum in vitro to evaluate by growth inhibition assay(GIA).ResultsDifferent formulation can induce different levels of antibody titers,the effection of ISA51 adjuvant was most closely with Freund's adjuvant,and can induce a higher specific antibody of 11 epitopes within proteins,can effectively stimulate cellular immune response based on the IFN-γ,to avidity Montanide ISA51 adjuvant immune antibodies and M.RCAg-1 protein affinity than the other two adjuvants;and Montanide ISA720 adjuvants and Al (OH)3 adjuvant group in mice can't induce a significant IFN-γresponse(P＞0.05).On avidity assay,the Montanide ISA51 formulation group was better than the other two adjuvants; and Montanide ISA720 and Al (OH)3 adjuvant formulation group can't induce a significant IFN-γresponse in mice(P＞0.05) ; the inhibition rates were 60% and 100% in 3D7 and Dd2 Plasmodium falciparum at a concentration of 2 mg/ml IgG by Montanide ISA51 formulated protein,and IgG of Al( OH)3 formulation could not effectively inhibit the in vitro growth of Plasmodium falciparum( 10% ),while IgG of Montanide ISA720 formulation could not inhibit growth of parasite in vitro.ConclusionBy comparing three clinically acceptable adjuvants and Freund's adjuvant in BALB/c mice and New Zealand rabbit,Montanide ISA51 adjuvants can be acceptable formulated M.RCAg-1 protein induced humoral and cellular immune responses,can be used as one of the candidate adjuvants.

ObjectiveTo explore the effects of vector fusion peptides on the conformation and immune reactivity of recombinant polyepitope antigens,M.RCAg-1.MethodsWe subcloned polyepitope artificial antigen gene,M.RCAg-1,into prokaryotic expression vectors,pDS-ex,that contain different fusion tags at the N-terminus or no any tag by different restriction enzyme cutting site.Three recombinant proteins expressed by these vectors,named P312-1,P312-2,and P312-3,were purified and purity is greater than 95％.Then BALB/c mice were vaccinated with the three proteins formulated with Freund's adjuvant through intranasal.Serum were collected to detect specific antibodies of M.RCAg-1 and individual epitope by ELISA ; natural parasite antigen was recognized by indirect immunofluorescence assay; mouse specific T lymphocyte activation was detected by enzyme-linked immunosorbent spot test (ELISPOT) ; and the growth of Plasmodium falciparum in vitro to evaluate by growth inhibition assay(GIA),and analyze secondary and tertiary structures of recombinant proteins from different expression vectors by bioinformatics and circular dichroism technique.ResultsThe P312-1,P312-2 have almost the same amino acid sequence,and the three proteins have the same immunogenicity in animal models(P＞0.05),however,the different proteins elicited various T-cell responses,the rabbit antibody induced by these proteins showed diverse efficacy in malaria parasite growth inhibition assaysin vitro ( respectively,93.9％,14.7％,54.3％ ).The significant differences of secondary and tertiary structures were shown in recombinant proteins from different expression vectors,analyzed by bioinformatics and circular dichroism technique,which demonstrated the change of protein molecule spaces conformation,and the obviously change of some epitope locations.ConclusionThese results suggest that the expressed polyepitope artificial antigens originating from the different vector fusion peptides indeed affect the protein folding and,subsequently,the epitope exposure.Thus,these proteins are able to induce both distinct humoral and cellular immune responses in animal models,and they affect the efficacy of immune inhibition against the parasite,the enhancement or suppresses.

Recent research data on post-stroke seizures and epilepsy are reviewed and summarized.They are reviewed from the aspects of concept, risk factors, epidemiology, pathogenesis, clinical manifestations, diagnosis, as well as prevention and treatment.

Objective: The mitotic count of gastrointestinal stromal tumors (GIST) is closely associated with the risk of planting and metastasis. The purpose of this study was to develop a predictive model for the mitotic index of local primary GIST, based on deep learning algorithm. Materials and Methods: Abdominal contrast-enhanced CT images of 148 pathologically confirmed GIST cases were retrospectively collected for the development of a deep learning classification algorithm. The areas of GIST masses on the CT images were retrospectively labelled by an experienced radiologist. The postoperative pathological mitotic count was considered as the gold standard (high mitotic count, > 5/50 high-power fields [HPFs]; low mitotic count, ≤ 5/50 HPFs). A binary classification model was trained on the basis of the VGG16 convolutional neural network, using the CT images with the training set (n = 108), validation set (n = 20), and the test set (n = 20). The sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) were calculated at both, the image level and the patient level. The receiver operating characteristic curves were generated on the basis of the model prediction results and the area under curves (AUCs) were calculated. The risk categories of the tumors were predicted according to the Armed Forces Institute of Pathology criteria. Results: At the image level, the classification prediction results of the mitotic counts in the test cohort were as follows:sensitivity 85.7% (95% confidence interval [CI]: 0.834–0.877), specificity 67.5% (95% CI: 0.636–0.712), PPV 82.1% (95% CI: 0.797–0.843), NPV 73.0% (95% CI: 0.691–0.766), and AUC 0.771 (95% CI: 0.750–0.791). At the patient level, the classification prediction results in the test cohort were as follows: sensitivity 90.0% (95% CI: 0.541–0.995), specificity 70.0% (95% CI: 0.354–0.919), PPV 75.0% (95% CI: 0.428–0.933), NPV 87.5% (95% CI: 0.467–0.993), and AUC 0.800 (95% CI: 0.563–0.943). Conclusion We developed and preliminarily verified the GIST mitotic count binary prediction model, based on the VGG convolutional neural network. The model displayed a good predictive performance.

Objective:To compare the efficacy and safety of robot-assisted laparoscopic and laparoscopic partial nephrectomy (RAPN and LPN) for patients with completely endophytic renal tumor.Methods:A total of 73 patients with completely endophytic renal tumor receiving RAPN (n=29) or LPN (n=44) in our center between January 2015 and June 2021 were retrospectively collected. There were 21 males and 8 females in RAPN group. The average age was 48.6±13.7 years old, average tumor size was 2.9±0.9 cm with 13 left tumors and 16 right tumors, average R. E.N.A.L. score was 9.2±1.0, and average preoperative eGFR was 82.6±10.7 ml/(min·1.73 m 2). There were 27 males and 17 females in LPN group. The average age was 50.1±12.3 years old, average tumor size was 2.9±0.9 cm with 24 left tumors and 20 right tumors, average R. E.N.A.L. score was 9.1±1.3, and average preoperative eGFR was 81.7±9.6 ml/(min·1.73 m 2). There was no significant difference in above variables between two groups. The operative time, warm ischemia time, blood loss, postoperative complication, postoperative hospital stay and postoperative 3 months renal function of two groups were compared. Results:All 73 patients successfully underwent RAPN or LPN and no patient converted to radical nephrectomy or open surgery. There was no significant difference in operation time [140(80, 160) min vs. 150 (90, 180) min, P=0.264], intraoperative estimated blood loss[150 (100, 200)vs. 180 (120, 200) ml, P=0.576]and postoperative hospital stay (7.0±2.7 vs. 7.4±2.1 days, P=0.480) between two groups. Compared with LPN group, RAPN group had obvious less warm ischemia time (23.1±3.3 vs. 27.6±4.7 min, P<0.001). No obvious complication occurred in RAPN group and one case with postoperative hemorrhage occurred in LPN group. No positive margin occurred in either group. There was no difference in renal function 3 months after operation between the two groups [73.2±6.3 vs.70.5±7.6ml/(min·1.73 m 2), P=0.117]. The median follow-up period was 22.6 months with no tumor recurrence or metastasis. Conclusions:For experienced surgeons, both RAPN and LPN are safe and feasible for patients with completely endophytic renal tumor. Compared with LPN, RAPN has advantages of perioperative curative effect, which could reduce the operating difficulty and shorten the warm ischemia time.

Objective:To investigate the efficacy and safety of modified early unclamping technique in robot-assisted laparoscopic partial nephrectomy (RAPN) for patients with renal tumors.Methods:A total of 32 renal tumor patients undergoing RAPN with modified early unclamping technique between January 2019 and August 2020 were retrospectively collected, including 18 males and 14 females. The average age was (48.5±11.2) years old, average BMI was (23.8±3.7) kg/m 2, average tumor size was (4.2±1.4)cm with 18 left tumors and 14 right tumors, average R. E.N.A.L. score was 7.6±0.4, and average preoperative eGFR was (84.0±18.6)ml/(min·1.73 m 2). The control group included 66 renal tumor patients undergoing RAPN with standard unclamping technique during the same period by the same surgeon, including 42 males and 24 females. The average age was (50.2±13.8) years old, average BMI was (24.0±4.5)kg/m 2, average tumor size was (4.1±1.6)cm with 35 left tumors and 31 right tumors, average R. E.N.A.L. score was 7.5±0.5, and average preoperative eGFR was (82.8±20.2) ml/(min·1.73 m 2). There was no significant difference in above variables between two groups. Modified early unclamping technique used barbed wire to continually suture 2-3 needles in a short time to close the large space at the outer after the inner suture, and then loosen the blocking clip to restore renal blood supply. The operative time, warm ischemia time, blood loss, postoperative tube removal time, postoperative hospital stay and 3 months postoperative renal function of two groups were compared. Results:All of the 98 RAPN were performed successfully and no patient was converted to radical nephrectomy or open surgery. There was no significant difference in operation time [(120.9±22.8)vs.(111.6±25.0)min, P=0.079], postoperative tube removal time [(4.0±0.6)day vs.(3.8±0.8) day, P=0.214] and postoperative hospital stay [(5.1±0.7)day vs.(5.2±0.5) day, P=0.419] between the two groups. Compared with the standard unclamping group, the modified early unclamping group had obvious less warm ischemia time [(13.5±3.6)min vs.(21.2±4.4) min, P<0.001]. There was no difference in intraoperative estimated blood loss between two groups (110 ml vs. 100 ml, P=0.480). No blood transfusion, urine leakage, postoperative hemorrhage occurred in either group. The 3 months postoperative renal function decline of modified early unclamping group was slightly less than standard unclamping group [(10.5±7.6)ml/(min·1.73m 2)vs.(13.2±6.4) ml/(min·1.73m 2)], but did not reach statistical significance ( P=0.069). The median follow-up period was 12.4 months(4-24 months) without any recurrence or metastasis. Conclusions:The modified early unclamping technique in RAPN for patients with renal tumors is safe and feasible. Compared with the standard unclamping technique, the modified early unclamping technique could shorten the warm ischemia time without increasing blood loss and complications, and might protect the postoperative renal function, which has high value in clinical practice.