Objective:To investigate the immume mechanism and protective effect of DNA vaccine pcD flaB against pathogenic leptospira infection. Methods: DNA vaccine pcD flaB was constructed by inserting flaB into the eucaryotic expressing plasmid pcDNA3. After guinea pigs were infected with leptospira, the protective rate was observed and specific anti leptospira antibody IgG were tested by ELISA. TNF activity was tested by cell proliferation. Results:The protective rate against leptospira infection was 100%. The specific antibody IgG generated peaked at the 6th week. Activity of TNF released by macrophage of guinea pigs given DNA vaccine was higher than that not given vaccine. Conclusion:DNA vaccine pcD flaB can protect guinea pigs from leptospira challenge infection by inducing humoral immune response and increasing TNF activity.

Objective To express a recombinant protein TFPR1 ( the functional region of the snake venom proteins from Trimeresurus flavoviridis) in Pichia pastoris expression system. Methods The target gene was codon-optimized and synthesized according to the sequence of the conserved structural do-main of triflin and then cloned into the Pichia pastoris expression vector pPICZαA to construct the recombi-nant expression plasmid pPICZαA-TFPR1. The recombinant plasmid pPICZαA-TFPR1 was electroporated into the yeast strain X33. The transformed strains carrying expression plasmid were screened out with Zeocin and then induced by methanol to express the recombinant protein TFPR1. ELISA was performed for the screening of positive clones. SDS-PAGE and Western blot were used for further identification of the ex-pressed products. Results The recombinant plasmid pPICZαA-TFPR1 was successfully constructed. The recombinant protein TFPR1 was expressed in a secreted form at a molecular weight of 16×103. Conclusion The recombinant protein TFPR1 was successfully expressed in Pichia pastoris expression system, which laid a foundation for further researches on its biological function and application as an adjuvant.

Objective To obtain Chinese hamster ovary (CHO) cell lines that stably express a targeting complement inhibitor CR2-CD59.Methods The recombinant plasmid PEE14.1-CR2-CD59 was constru-cted by cloning the DNA fragment CR2-CD59 into plasmid PEE14.1,and the obtained plasmid was transfected into CHO cells by FuGENE 6.The clones with stable high expression of target fragment were selected by methionine sulfoximine (MSX),the expression of CR2-CD59 was analyzed by ELISA,SDS-PAGE and Western blotting analysis.Results Several stable expression clones were obtained,and CR2-CD59 was highly expressed in the secret form in CHO cells.SDS-PAGE analysis showed that the molecular weight of the recombined protein CR2-CD59 was consistent with the predicted one.ELISA and Western blotting results revealed that the CR2-CD59 could react with both anti-human CR2 and anti-human CD59 polyclonal antibodies.Compared with serum-containing medium,the protein was highly expressed in serum-free medium (P

Toll-like receptor 4 (TLR4), a type Ⅰ transmembrane protein, has been extensively studied in the Toll-like receptor family at present. TLR4 ligands include lipopolysaccharides ( LPS) present in the outer membrane of gram-negative bacteria and monophosphoryl lipid A ( MPLA) which is a derivative of LPS. TLR4 agonists, alone, as a major component of compound adjuvants or in combination with other TLRs agonists, have been widely used as adjuvants in various vaccines and demonstrated great potential in vaccine development. This review addressed the discovery, application, features and prospect of novel vac-cine adjuvants based on TLR4 agonists, aiming to provide reference for rational use of adjuvants and further development.

Objective To analyze and compare the pathological changes of lung tissue in mice infected with the novel H7N9 influenza virus and 2009 pandemic H1N1 influenza virus, respectively, and to preliminarily study the mecha-nisms of acute lung injury induced by those virus infection .Methods SPF 6-week old BALB/c mice ( body weight 18-20 g, male∶female=1∶1) (n=3 in each subgroup) were intranasally infected with H7N9 virus and H1N1 virus, respec-tively.The behavior and survival time of mice after virus infection were observed and the survival rates were analyzed .The heart, liver, spleen, lung, kidney, intestines, and brain were collected at indicated time points for histopathological exami-nation using H&E staining .The distribution of virus antigen was detected by immunohistochemistry .The neutrophil infiltra-tion was also observed .The correlation of lung injury with virus replication and host immune responses was analyzed .Re-sults The lung and spleen injury of mice infected with H 7N9 virus was slighter and their survival rate (100%) was high-er than those of mice infected with H1N1 virus.The damages of the lung and spleen in H1N1virus-infected mice were more severe than that in H7N9 virus-infected mice, and all the 10 mice in this group died within 9 days after virus inoculation . The distributions of both the virus antigens were mainly in the bronchial epithelial cells , a few stromal cells and alveolar ep-ithelial cells .The levels of virus replication in the two groups were not significantly different .There were more intense neu-trophil infiltration in the lung and inflammatory response in the H 1N1 virus-infected mice than those in the H7N9 virus-in-fected mice .Conclusions There are some differences of the pathological characteristics and extent of lung injury in the mice infected with H7N9 virus and H1N1 virus, respectively.The virus replication is a precipitating factor but not the deci-sive factor of the lung injury , and there is a close relationship between the host immune responses and acute lung injury .

Objective:To study the method of measuring iodine content in urine with automatic iodine element analyzer.Methods:The iodine content in urine was measured by automatic iodine element analyzer DAT-50S, and the applicability of the method was tested, including the standard curve and detection limit, precision test, standard substance determination test, standard addition recovery test, and method comparison test.Results:Within the range of iodine content 0 - 800 μg/L, the correlation coefficient of the standard curve was - 0.999 7, and the detection limit was 6.2 μg/L. Precision: the relative standard deviation ( RSD) values of three urine samples with low, medium and high iodine contents within the range of the standard curve were 1.6%, 1.1% and 0.7%, respectively ( n = 8). Accuracy: the measured values of two urine iodine standard substances measured by the automatic iodine element analyzer were all within the reference value range; the average recovery rate of three urine samples with low, medium and high iodine contents within the range of the standard curve was 99.6%, and the recovery rate ranged from 97.6% to 101.2%. Method comparison test: there was no statistically significant difference between the results of 20 urine samples determined by arsenic-cerium catalytic spectrophotometry (WS/T 107.1-2016) and the automatic iodine element analyzer ( t = 0.984, P > 0.05). Conclusion:The precision and accuracy of urinary iodine determination using the automatic iodine element analyzer are high, the operation is simple, and it is suitable for popularization and application in grassroots disease control institutions.

Objective To compare the adjuvant activity of oil-in-water emulsion MF59 and aluminum hydroxide on immune responses to HIV-1 multi-epitope protein MEP1 in BALB/c mice. Methods HIV-1 multi-epitope protein MEP1 with MF59 or aluminum was prepared to intramuscularly vaccinate female BALB/c mice for three times. Serum and splenocytes were isolated from the mice 10 d after the first and last vaccination. Specific anti-MEP1 antibodies and the subclasses in serum were detected by ELISA. The num-bers of splenic lymphocytes secreting IFN-γ and IL-4 were measured by ELISPOT. Differences in humoral and cellular immune responses induced by the two different adjuvants were compared. Results After a sin-gle-dose immunization, aluminum adjuvant promoted early humoral and cellular immune responses to MEP1 compared with MF59. After the three-dose immunization, both aluminum adjuvant and MF59 promoted MEP1 to induce Th2-biased humoral immune response, while MF59 also enhanced a balanced Th1/Th2 cel-lular immune response. Conclusions Aluminum adjuvant was a more suitable adjuvant than MF59 for HIV-1 multi-epitope protein MEP1.

Objective:To investigate the effect of teriparatide on residual back pain (RBP) after percutaneous kyphoplasty (PKP) for osteoporotic vertebral compression fracture (OVCF).Methods:A retrospective cohort study was used to analyze the clinical data of 90 OVCF patients sustaining RBP after PKP admitted to Second Affiliated Hospital of Kunming Medical University from September 2015 to March 2019, including 18 males and 72 females, at age of 57-85 years［(68.0±5.9) years］. Teriparatide treatment was applied regularly in 32 patients (teriparatide group) and antiosteoporosis drug was administered routinely in 58 patients (routine treatment group). Visual analogue scale (VAS) and Oswestry disability index (ODI) were compared between the two groups before operation, at 24 hours, 1 month, 3 months, 6 months and 12 months after operation. Anterior vertebral body height (ABH), middle vertebral body height (MBH), kyphosis angle (KA), maintenance rate of anterior vertebral body height (MRABH), maintenance rate of middle vertebral body height (MRMBH) and difference of kyphosis angle (DKA) were measured at 24 hours and 12 months after operation to evaluate the maintenance of vertebral height and incidence of vertebral refracture. Levels of type I collagen carboxy-terminal peptide (β-CTX) and serum N-terminal osteocalcin (N-MID) were measured before operation and at 12 months after operation to evaluate the improvement of bone metabolism. The adverse reactions of teriparatide group were observed.Results:All patients were followed up for 12-36 months［(14.3±0.6)months］. VAS and ODI were decreased gradually with time in both groups (all P<0.01). There were no significant differences in VAS between the two groups before operation and at 24 hours after operation (all P>0.05). Teriparatide group showed VAS of (4.4±0.6)points, (3.2±0.5)points, (2.0±0.5)points, (1.1±0.1)points at 1, 3, 6 and 12 months after operation, significantly lower than those in routine treatment group［(4.9±0.6)points, (4.0±0.6)points, (3.2±0.7)points, (2.7±0.1)points, respectively］(all P<0.01). Teriparatide group showed ODI of 26.5±1.3 and 20.6±1.2 at 6 months and 12 months after operation, significantly lower than those in routine treatment group (28.2±1.6, 23.6±1.6) (all P<0.01). There were no significant differences in ODI between the two groups at other time points (all P>0.05). Both groups presented significantly lowered levels of ABH and MBH at 12 months after operation as compared with those at 24 hours after operation (all P<0.01). There were no significant differences in ABH or MBH between the two groups at 24 hours after operation (all P>0.05). ABH, MBH, MRABH and MRMBH in teriparatide group were (1.9±0.2)cm, (1.7±0.2)cm, 0.91±0.02 and 0.92±0.02 at 12 months after operation, significantly higher than those in routine treatment group［(1.7±0.2)cm, (1.6±0.2)cm, 0.86±0.02 and 0.87±0.02］(all P<0.01). KA in both groups showed significant increase at 12 months after operation as compared with that at 24 hours after operation (all P<0.01). There was no significant difference in KA between the two groups at 24 hours after operation ( P>0.05). KA in teriparatide group was (7.3±0.7)° at 12 months after operation, significantly lower than (9.5±0.5)° in routine treatment group ( P<0.01). DKA in teriparatide group was (5.3±1.3)° at 12 months after operation, significantly lower than (6.6±1.4)° in routine treatment group ( P<0.01). Incidence of vertebral refracture in teriparatide group was 7% (2/32), significantly lower than 35% (15/58) in routine treatment group ( P<0.05). Level of β-CTX was not significantly different between and within the two groups before operation and at 12 months after operation (all P>0.05). There was no significant difference in N-MID between the two groups before operation ( P>0.05). After treatment for 12 months, level of N-MID in teriparatide group was significantly increased［19.5 (17.6, 20.9)pg/ml］as compared with that before operation［18.2 (14.6, 21.0)pg/ml］( P<0.01), and was significantly higher than that in routine treatment group［17.6 (15.3, 19.9)pg/ml］( P<0.01). Routine treatment group showed no significant difference in level of N-MID before operation and at 12 months after operation ( P>0.05). Two patients in teriparatide group had orthostatic hypotension after treatment. Conclusion:For OVCF patients with RBP after PKP, teriparatide can effectively alleviate pain, improve motor dysfunction, maintain the height of bone cement vertebral body, reduce incidence of vertebral refracture and enhance the activity of osteoblasts, with less adverse reactions.

Objective:To explore the risk factors of adjacent segment diseases (ASDis) after lumbar fusion, summarize the prevention strategies and provide reference for clinical treatment.Methods:All of 258 patients who underwent lumbar interbody fusion from March 2014 to March 2019 were retrospectively analyzed, including 95 males and 163 females, the age of whom was 61.8±8.4 years (range, 39-77 years). The patients were divided into ASDis group and non-ASDis group according to whether ASDis occurred at the follow-up of 24 months after operation. The patient's individual factors [gender, age, body mass index (BMI), main diagnosis, preoperative paraspinal muscle fatty degree, etc.] and surgical factors (operation type, fixed segment, fusion segment, etc.), sagittal parameters [lumbar lordosis (LL), pelvic incidence (PI), pelvic tilt (PT), sacral slope (SS), PI-LL] were recorded. After univariate analysis of potential risk factors, the factors with P<0.05 were substituted into logistic regression model for multivariate analysis to determine the risk factors of ASDis after lumbar fusion. Results:ASDis occurred in 24 patients after lumbar fusion, with an incidence of 9.3% (24/258); univariate analysis showed that age ≥ 60 years old, complicated with osteoporosis, preoperative fatty degree of paraspinal muscle (GCS grade≥3), PLIF operation, suspension fixation, total laminectomy and multi-segment fusion (≥ 3 segments) were the potential risk factors for ASDis after operation (P<0.05); Gender, education level, partner status, type of work, BMI, obesity (BMI≥24 kg/m 2) , smoking, use of bisphosphonates, concomitant lumbar spinal stenosis, lumbar lordosis angle, pelvic incidence angle, pelvic tilt angle, sacral slope angle, and PI-LL had no significant correlation with ASDis. Logistic regression analysis showed that age ≥ 60 years ( OR=5.63, 95% CI: 1.56, 20.29, P=0.008), preoperative paravertebral muscle fatty GCS ≥ 3 ( OR=4.82, 95% CI: 1.36, 17.13, P=0.015), combined with osteoporosis ( OR=14.04, 95% CI: 2.53, 77.79, P=0.002), PLIF ( OR=9.69, 95% CI: 1.91, 49.03, P=0.001), and multi-segment fixation ( OR=9.36, 95% CI: 1.77, 49.41, P=0.008) were the risk factors for ASDis after lumbar fusion; Incomplete laminectomy ( OR=0.09, 95% CI: 0.02, 0.37, P=0.001) and suspension fixation ( OR=0.16, 95% CI: 0.02, 0.94, P=0.042) were the protective factors of ASDis after lumbar fusion. Conclusion:The patients with age ≥ 60 years old, osteoporosis and preoperative paraspinal muscle fatty degree ≥ 3 grade GCS should be more careful in choosing the surgical methods, and try to choose transforaminal interbody fusion, posterolateral fusion, short segment fusion, decompression with preservation of vertebral lamina, suspension fixation and other surgical methods to reduce the incidence of postoperative ASDis.

Objective: To investigate the clinical effect of percutaneous curved vertebroplasty in the treatment of thoracolum-bar osteoporotic vertebral compression fractures (OVCFs). Methods: All of 85 patients with single thoracolumbar vertebral OVCFs who met the admission criteria from January 2017 to July 2018 were divided into three groups according to the random dig-its table method. They were treated with percutaneous curved vertebroplasty, routine unipedicular PVP and routine bipedicular PVP respectively. There were 25 patients in the percutaneous curved vertebroplasty group, 6 males and 19 females; aged 56-80 years, with an average age of 70.6±9.7 years. Fracture vertebral body distribution: T₁₀ 2 cases, T₁₁ 4 cases, T₁₂ 3 cases, L₁ 9 cases, L₂ 3 cases, L₃ 1 case, L₄ 1 case and L₅ 2 cases. There were 32 patients in the routine unipedicular PVP group, 6 males and 26 fe-males; aged 58-75 years, with an average age of 69.5±9.3 years. Fracture vertebral body distribution: T₁₀ 2 cases, T₁₁ 4 cases, T₁₂ 5 cases, L₁ 11 cases, L₂ 6 cases, L₃ 1 case, L₄ 1 case and L₅ 2 cases. There were 28 patients in the routine bipedicular PVP group, 5 males and 23 females; aged 59-81 years, with an average age of 69.8±8.8 years. Fracture vertebral body distribution: T₁₀ 2 cases, T₁₁ 4 cases, T₁₂ 4 cases, L₁ 10 cases, L₂ 4 cases, L₃ 1 case, L₄ 1 case and L₅ 2 cases. The operation time, injected cement volume, in-traoperative blood loss were recorded and analyzed. Preoperative, postoperative 1 week and 3 months visual analogue scale scores and oswestry disability index were adopted to value the clinical improvements. Preoperative, postoperative 1 week and 3 months relative vertebral height and kyphosis correction, and the cement leakage rate were measured and analyzed. Results: There was no significant difference in the data of gender, age, VAS scores, ODI and distribution of fracture vertebrae among the three groups (P>0.05), and the baseline data was comparable. The average VAS score in the percutaneous curved vertebroplasty group was 2.3±0.5 at 1 week after surgery, that of the routine unipedicular PVP group was 2.4±0.4 and that of the routine bipe-dicular PVP group was 2.4±0.4; the average ODI in the percutaneous curved vertebroplasty group was 19.8%±3.9%, that of the routine unipedicular PVP group was 20.0%±4.1% and that of the routine bipedicular PVP group was 19.9%±3.8%; they were lower than the preoperative data, which were statistically significant (P<0.001). The average relative vertebral height in the percutaneous curved vertebroplasty group was 48.99%±9.23% at 3 months after surgery, that of the routine unipedicular PVP group was 47.11%±10.12% and that of the routine bipedicular PVP group was 46.71%±11.16%; the average kyphosis cor-rection in the percutaneous curved vertebroplasty group was 6.21%±1.94%, that of the routine unipedicular PVP group was 5.22%±2.07% and that of the routine bipedicular PVP group was 5.97%±2.09%; there was 1 cement leakage case in the per-cutaneous curved vertebroplasty group; those of the routine unipedicular PVP group were 4 cases and those of the routine bipe-dicular PVP group were 6 cases; there was no significant difference among the three groups (P>0.05). Operation time 39.10±2.00 min vs 38.70±1.70 min, injected cement volume 3.60±0.11 ml vs 3.50±0.13 ml and blood loss 5.10±0.30 ml vs 5.00±0.40 ml of the percutaneous curved vertebroplasty group and the routine unipedicular PVP group were less than those of the routine bipedicular PVP group, which were statistically significant (P<0.05). Conclution Percutaneous curved vertebroplasty could achieve satisfactory clinical outcomes for OVCFs, with advantages of less operation time, less blood loss, limited X-ray expo-sure, less injected cement volume, and more balanced augmentation for stabilization of the affected vertebrae and total verte-bral column.