Immunogenicity of quadrivalent influenza virus subunit vaccine combined with RFH01 adjuvant in mice / 中华微生物学和免疫学杂志

Objective:To evaluate the immunogenicity of a quadrivalent subunit vaccine combined with RFH01 adjuvant in a mouse model.Methods:Identification tests were performed on four monovalent influenza virus subunit vaccine stock solutions according to the methods described in Part 3 of the Chinese Pharmacopoeia 2020 Edition. In the study of the quadrivalent subunit vaccine combined with RFH01 adjuvant, 460 female BALB/c mice (6-8 weeks old) were randomly divided into 46 groups including experimental groups, vaccine control group, negative control group and blank group with 10 mice in each group. In the study of the quadrivalent subunit vaccine in old and young mice, 80 female 10-month-old and 80 female 10-week-old BALB/c mice were randomly divided into 16 groups ( n=10) including monovalent influenza virus vaccine group, quadrivalent subunit vaccine group, quadrivalent subunit vaccine+ RFH01 adjuvant group, chicken embryo quadrivalent split vaccine control group and PBS group. All mice were immunized by intramuscular injection. At 21 d after the primary immunization, a booster immunization was conducted using the same strategy. Blood samples were collected at 21 d and 42 d after the primary immunization for serum separation. Haemagglutination inhibition (HI) test was performed to detect the antibody levels in mouse serum samples. Results:After the booster immunization, the positive conversion rates in all vaccine+ RFH01 adjuvant groups reached 100%, and the geometric mean titers (GMTs) of serum antibodies were significantly higher than those of the vaccine groups without RFH01 adjuvant. There were significant differences in serum antibody titers between the monovalent/quadrivalent subunit vaccine groups with and without RFH01 adjuvant. After the booster immunization, the titers of serum antibodies against H1N1, H3N2, B/Victoria and B/Yamagata in the 10-week-old mice were significantly higher than those in the 10-month-old mice.Conclusions:The monovalent and quadrivalent influenza virus vaccines in combination with RFH01 adjuvant could elicit higher antibody titers in young (6-10 weeks old) and old (10 months old) mice, showing good immunogenicity.

Purification of H5N1 influenza virus by different chromatography media / 中华微生物学和免疫学杂志

Objective:To purify H5N1 influenza virus concentrate prepared by MDCK cells with a new mixed-mode chromatography medium Capto Core700 and the traditional medium Sepharose 4FF, and to compare the separation and purification efficacy of the two media.Methods:Capto Core700 and Sepharose 4FF were used to purify inactivated H5N1 influenza virus concentrate. The morphology of virus particles in different samples was then observed under a transmission electron microscope. Single radial immunodiffusion (SRID), Folin-Phenol (Lowry) method, double-antibody sandwich ELISA and qPCR were used to detect hemagglutinin, total protein, host cell protein (HCP) and host cell DNA (HCD) before and after purification. The recovery rate of virus antigen and the removal rate of impurities were calculated. The immunogenicity of the viruses purified with different media was analyzed using animal experiments. Difference in the purification efficacy of the two chromatography media was analyzed by t-test. Results:H5N1 influenza viruses purified by Capto Core700 or Sepharose 4FF showed the typical influenza virus morphology under transmission electron microscope. There was no significant difference in the recovery rate of hemagglutinin between the two chromatography media ( P>0.05), but compared with Sepharose 4FF, Capto Core700 had a higher removal rate of impurities (total protein, HCP, HCD) and the difference was statistically significant ( P<0.05). Animal experiments showed that the viruses purified by the two chromatography media had good immunogenicity. Conclusions:Compared with Sepharose 4FF chromatography medium, Capto Core700 could more effectively remove process-related impurities such as HCP, HCD and total protein without affecting the recovery rate of viral antigen. This study provided reference for the development of purification technology in the production of H5N1 influenza virus vaccine in MDCK cells.

Preparation and immunogenicity evaluation of recombinant influenza hemagglutinin trimer vaccine / 中华微生物学和免疫学杂志

Objective:To prepare a recombinant hemagglutinin trimer (HA-Tri) vaccine against influenza viruses and to study its immunogenicity in a mouse model.Methods:A stable CHO cell line that could express HA-Tri was constructed. Western blot, single radial immunodiffusion, protein particle size detection and N-glycosylation site analysis were performed for qualitative and quantitative analysis of the recombinant protein. According to the different treatment conditions such as dosage and adjuvant, BALB/c mice were divided into 11 groups and subjected to consistent immunization procedures. Serum neutralizing antibody titers were measured on 56 d after the first immunization to evaluate the immunogenicity of HA-Tri.Results:The constructed CHO cells could secret and express HA-Tri proteins. The HA-Tri proteins were biologically active and capable of forming precipitation rings in the single radial immunodiffusion. The particle size of HA-Tri was approximately 18.79 nm and 10 N-glycosylation sites were detected, including high mannose, complex glycoforms and heterozygous glycoforms. After prime-boost immunization, there was no statistically significant difference in the titers of neutralizing antibodies induced in mice by 3.75 μg of HA-Tri in combination with RFH01 adjuvant and 15 μg of monovalent vaccine stock solution ( P=0.431 2, U=36). Serum antibody titers in the HA-Tri+ RFH01 groups were higher than those in the corresponding HA-Tri groups without RFH01 adjuvant, and the highest titer was induced in the 15 μg HA-Tri+ RFH01 group, which was 1 280. Conclusions:The recombinant HA-Tri protein was successfully prepared. HA-Tri in combination with RFH01 adjuvant could induce humoral immune responses against influenza viruses in BALB/c mice, which would provide reference for the development of influenza virus recombinant subunit vaccines.

Integrins mediate the migration of HepG2 cells induced by IL-8 / 军事医学

[ Abstract] Objective To investigate the effect of IL-8 on the viability and migration of HepG2 cells and the potential effect of integrins (αsubunits ) on the migration of HepG2 cells.Methods HepG2 cells were stimulated by IL-8 at different concentrations ranging from 0 to 125 ng/ml in vitro.MTT assay was preformed to detect the viability of HepG2 cells.Scratch wound migration assay was used to explore the effect of IL-8 on the migration of HepG2 cells at the time points of 0, 4, 8, 12 and 24 h,respectively.Transwell assay was conducted to detect the vertical migration of HepG2 cells after stimulation with IL-8 for 24 h.The F-actin of HepG2 cells was observed by immunofluorescence analysis after treatment with IL-8 at different concentrations.The expression of αsubunits of integrins in HepG2 cells treated with IL-8 for 8h was detected using Western boltting assays.Results Compared with the control group, HepG2 cells treated with IL-8 showed improved proliferation activities, but there was no significant difference between the groups of HepG2 cells treated with IL-8 at different concentrations.The cell scratch healing assays and Transwell analysis both indicated that IL-8 promoted the migration of HepG2 cells in a dose-dependent manner.Meanwhile, the cytoskeleton of HepG2 cells was rearranged and the number of filopodium-like protrusions (FLPs) was increased rapidly after treatment with IL-8.Western blotting analysis showed that IL-8 up-regulated the expression of αsubunit of integrins and there was a concentration difference betweenαsubunits.Conclusion IL-8 promotes the migration of HepG2 cells by up-regulating the expression ofαsubunits, and different αsubunits may play different roles.

Diagnosis and treatment of autoimmune pancreatitis / 中华消化外科杂志

Objective To summarize the clinical characteristics,diagnosis and treatment of autoimmune pancreatitis(AIP).Methods The retrospective descriptive study was adopted.The clinical data of 43 patients with AIP who were admitted to the First Affiliated Hospital of China Medical University between January 2010 and December 2014 were collected.All patients underwent laboratory tests and imaging examinations.For the patients who had the definite diagnosis of AIP,corticosteroid therapy was adopted,and for the patients who had the suspicion of pancreatic cancer,surgical operation was carried out.Observed indexes included clinical manifestations,laboratory findings,imaging findings,treatment,pathological findings and follow-up status.The follow-up of imaging examinations and laboratory tests via outpatient and inpatient examinations was performed on the patients until January 2016.Measurement data with skewed distribution were presented as average (range).Results (1) Clinical manifestations:of the 43 patients,33 demonstrated obstructive jaundice,19 had loss of weight,18 had upper abdominal pain and 5 had acute mild pancreatitis (the same patient with a variety of symptoms).Extrapancreatic organ involvement was detected in 23 of the 43 patients,including focal nephritis in 15 patients,sicca syndrome in 7 patients,pulmonary involvement in 5 patients,autoimmune thyroiditis in 5patients,hilar IgG4-associated cholangitis in 3 patients,retroperitoneal fibrosis in 3 patients and ulcerative colitis in 1 patient (the same patient with a variety of diseases).(2) Results of laboratory tests:17 patients underwent serum IgG4 detection and increased expression of IgG4 was found in 15 patients.(3) Imaging findings:all the 43patients underwent enhanced computed tomography (CT) scan of pancreas.Diffused enlargement of pancreas with appearance of sausage was observed in 34 patients and localized pancreatic head enlargement was observed in 9patients.Twenty patients had peripancreatic vascular involvement including stenosis or occlusion of splenic vein,stricture at the confluence of superior mesenteric vein,portal vein and splenic vein resulting in regional portal hypertension which led to gastric venous varices and splenomegaly.Three patients showed that superior mesenteric artery was surrounded by tissue masses with massive ascites.Five patients had pancreatic pseudocyst with splenic vein involvement and peripancreatic effusion.The abdominal CT examination of 15 patients with renal involvement showed renal cortical wedge or nodular lesions.Pulmonary inflammatory pseudotumor and interstitial pneumonia appeared in 5 patients,CT of whom showed solid pulmonary nodules or diffuse ground-glass opacities and patchy shadows in pulmonary interstitium.Thirty-two patients underwent magnetic resonance cholangiopancreatography and 28 patients underwent endoscopic retrograde cholangiopancreatography.The results showed the focal and diffuse or segmental stenosis of the main pancreatic duct were detected in 28 patients (diffuse stenosis in 23patients,local stenosis in 5 patients),stenosis of the intrapancreatic common bile duct in 32 patients,stenosis of hilar bile duct in 3 patients.Nine patients underwent endoscopic ultrasonography examination.The results showed 3 patients had hypoechoic mass in the head of pancreas and 6 patients had diffuse or heterogeneous hypoechoic of the pancreatic parenchyma.(4) Treatment status:of 43 patients,30 patients received initial steroid treatment.After steroid treatment,the clinical symptoms of 27 patients gradually improved and laboratory and imaging findings were both improved.Eleven patients didn 't receive initial steroid treatment,of whom 2 patients with obstructive jaundice underwent endoscopic biliary stent implantation,7 patients underwent surgical biliary drainage and 2 received neither endoscopic treatment nor surgical treatment.Two patients underwent pancreatoduodenectomy due to suspicion of pancreatic adenocarcinoma and were diagnosed as AIP based on postoperative pathological results and treated with steroid after operation.(5) Pathological examination:of the 43 patients,22 underwent pathological examination.Results of pathological examination of superficial lymph nodes showed lymph follicles hyperplasia,and immunohistochemistry staining showed massive IgG4 positive plasmocytes in 5 patients,supporting the diagnosis.Results of fine needle aspiration cytology in 15 patients showed no cytological evidence were detected in 14 patients and epithelial cells of malignant tumors in 1 patient.Results of pathological examination of surgical specimens showed dense infiltration of lymphocyte and immunohistochemistry staining showed massive IgG4 positive plasmocytes.(6) Follow-up status:of 43 patients,41 were followed up for an average time of 28 months (range,12-71 months).Of the 30 patients who received steroid treatment,27 had complete response and 3 had partial response.Of the 27 with complete response,4 patients relapsed during followup and were cured by continuous steroid treatment and extending treatment course,3 patients did not relapse,1 patient relapsed again,and long-term low dose steroid maintenance therapy was needed.The 3 patients with partial response were treated with mycophenolate mofetil for 6,4,4 months and imaging abmormalities disappeaved absolutely.Eleven patients received no steroid for the initial treatment,in whom 2 patients were lost to follow-up,1 patient with common bile duct jejunum anastomosis died in 1 year after surgery.In the other 8 patients,the imaging abnormalities were spontaneous remission or clinical and imaging manifestations were improved after steroid treatment and no recurrence was found.Two patients who underwent pancreatoduodenectomy due to suspicion of pancreatic adenocarcinoma received steroid treatment after operation and no recurrence was observed.Conclusion Clinicians should combine clinical manifestations,serological examination,imaging findings and pathological examination to make definite diagnosis of AIP and make differential diagnosis of pancreatic cancer to avoid unnecessary surgical treatment by misdiagnosis.

Comparative study of application effect of BD preset syringe and regular syringe in arterial blood collection in standardized nursing process / 中国实用护理杂志

ObjectiveTo explore the best practice and normalization of arterial blood collection by comparing performance of BD preset syringe with regular syringes in arterial blood collection in standardized nursing process. MethodsThe nurses were trained to use BD preset syringe and regular syringes to collect arterial blood sample.Five hundred subjects were randomly divided into the preset syringe group and the regular syringes group,250 subjects in each.Agglutination of blood samples was observed.Time spent in every step of collections was recorded for 23 subjects in each group to calculate workflow efficiency.A questionnaire was used for nurses participated in arterial blood collection for their evaluation of both types of syringes. Results The incidence of micro-clot formation in the preset syringe group was 2 cases,lower than 15 cases in the regular syringes group,the preset syringe group also showed higher workflow efficiency as well as safe and easy-to-use characters.The difference between two groups was statistically significant. ConclusionsUsing BD preset syringe in nursing process management has lower incidence of micro-clot formation,higher workflow efficiency,safe and easy-to-use character.It should be promoted in clinical practice.

Imaging features of autoimmune paacreatitis and the diagnostic value of imaging examinations / 中华胰腺病杂志

Objective To investigate the imaging characteristics of autoimmune pancreatitis (AIP)and its diagnostic value.Methods Imaging examinations and clinical data of 13 patients with AIP were reviewed retrospectively.Results All patients had enlargement of pancreas either diffusely ( n =11 ) or focally in pancreatic head ( n =2 ).The swollen pancreas was of homogeneous density on CT scan ( n =13 ),hypointense on T1-weighted images and mildly hyperintense on T2-weighted images in 4 patients.It was demonstrated as mild enhancement on artery phase of dynamic imaging and progressive enhancement on portal and delayed phase images.Capsule-like enhanced rim was presented around the pancreas in 9 patients.Stricture or obliteration of the common bile duct located in pancreatic head was found in 10 patients accompanied by intrahepatic bile duct dilation.Pancreatic duct was invisible on axial images in 11 cases.Among the 6 patients underwent MRCP,it was showed diffuse and irregular narrowing of the main pancreatic duct and stricture of common bile duct located in pancreatic head in 4 cases.ERCP showed diffuse and irregular narrowing of the main pancreatic duct in 7 cases.Extra-pancreatic lesion included veins around pancreas involvement in 8 cases;multiple low density renal lesions in 6 cases; retroperitoneal fibrosis in 2 cases; stricture of hilar duct,interstitial lung disease and ankylosing spondylitis in 1 case,respectively.Conclusions AIP shows some characteristic imaging features,and imaging examinations play an important role in the diagnosis of AIP.Recognition of extra-pancreatic lesions aids in the correct diagnosis of AIP.

Findings of bile flow artifacts on MR cholangiopancreatography / 中华放射学杂志

Objective Toexplore the findings of bile flow artifacts (BFA)on MR cholangiopancreatography(MRCP). MethodsEighty five patients with normal common bile duct (CBD)and 95 patients with dilated CBD who had MRCP examination were studied retrospectively. Findings of BFA on original thin images of MRCP were recorded. Date were tested by Chi-square test and Spearman ceefficient of rank correlation analysis. ResultsThe rate of BFA in CBD with normal group was 38. 8％ ( 33/85 ), in dilated group was 83.2％ (79/95), there was statistically significant difference between them( x2 ＝ 37. 512,P ＝ 0. 000). BFA was not observed when the diameter of CBD was no more than 4 mm. BFA was partly observed when the diameter of CBD was 4 mm ＜ ψ≤ 8 mm, single- string sign was predominant findings (52.6％ ,30/57 ). Three-string sign was predominant findings in CBD with slight-dilated group ( 85.7％,36/42). Multi-string sign was predominant findings in CBD with medium-dilated group (84. 6％, 22/26 ).There was statistically significant difference between each groups ( x2 ＝ 284. 561, P ＝ 0. 000 ).BFA had different manifestations with CBD of different diameter, there was relative between them ( r ＝ 0. 553, P ＝0. 000). ConclusionBFA can occur in CBD with normal diameter. CBD with dilated group has higher rate of BFA than CBD with normal diameter. Manifestations of BFA are relative with the diameter of CBD.