The aim of this study was to develop a semi-quantitative immunochromatographic method for rapid detection of Newcastle disease virus (NDV) antibodies by expressing HN protein in rice endosperm bioreactor. The recombinant plasmid pUC57-HN was digested by MlyⅠ and XhoⅠ to retrieve the HN gene, while the intermediate vector pMP3 containing promoter, signal peptide and terminator was digested by NaeⅠ and XhoⅠ. The HN gene and the linearized pMP3 were purified and ligated to form a recombinant plasmid pMP3-HN1. Subsequently, pMP3-HN1 and plant vector pCAMBIA1300 were digested by EcoRⅠ and Hind Ⅲ, and the HN1 gene was cloned into pCAMBIA1300. The recombinant plasmid pCAMBIA1300-HN1 was introduced into Agrobacterium tumefaciens EHA105 by electrotransformation, and the pCAMBIA1300-HN1 was transferred into rice callus by agrobacterium-mediated method. After dark culture, callus screening, differentiation, rooting and transplanting, transgenic rice seeds were obtained 4 months later. PCR identified that the HN gene has been inserted into the rice genome. SDS-PAGE and Western blotting indicated that the HN protein was successfully expressed in the positive rice endosperm. The purity of the HN protein was more than 90% by SP cation exchange chromatography and gel filtration chromatography. According to the national standards for the diagnostic techniques of Newcastle disease HI test (HI≥4log₂, positive antibody reaction), a colloidal gold labeled purified HN protein was used to prepare a semi-quantitative test strip by double-antibody sandwich method for rapid detection of NDV antibody. The results showed that the test strip did not cross-react with positive sera against other viruses, and the sensitivity of the test strip reached 1:102 400 for standard positive sera of Newcastle disease. Testing of a total of 308 clinical sera showed that the compliance rate of the test strip with HI test was 97.08%, and the Kappa value was 0.942. In conclusion, high purity recombinant HN protein was obtained from rice endosperm, and a simple, rapid, highly sensitive and highly specific semi-quantitative immunochromatographic strip was developed. The test strip could be used for immune evaluation of the Newcastle disease vaccine.
Animals ; Antibodies, Viral ; Chickens ; HN Protein/metabolism* ; Newcastle Disease/prevention & control* ; Newcastle disease virus/metabolism* ; Oryza/genetics*

Objective:To explore the value of MR elastography (MRE) and shear wave elastography (SWE) for staging liver fibrosis in a rabbit model.Methods:From March to November 2020, 200 healthy New Zealand white rabbits were randomly divided into control group ( n=40) and liver fibrosis group ( n=160) by random number table method. The volume ratio of CCl 4 and olive oil was 1∶1 to prepare 50% CCl 4 oil solution, and the experimental rabbits in the liver fibrosis group were subcutaneously injected with 50% CCl 4 olive oil solution. It was injected once a week at the dose of 0.1 ml/kg in the first to third weeks, once a week at the dose of 0.2 ml/kg in the 4th to 6th weeks. The dose of 0.1 ml/kg was injected twice a week from week 7 to 16. The control group were subcutaneously injected with an equal dose of normal saline. At the end of the 4th, 8th, 12th, and 16th week, 40 and 10 animals in the liver fibrosis group and the control group were randomly selected by random number table method for MRE and SWE, respectively, to obtain the liver elastic stiffness (LS), which were recorded as LS MRE and LS SWE. After the examination, the experimental rabbits were sacrificed and liver tissue of rabbits were taken for histopathological Scheuer staging, and they were divided into F0-F4 groups. One-way ANOVA was used to evaluate the differences of LS MRE and LS SWE in different stages of liver fibrosis. Spearman correlation was used to analyze the correlation between LS and pathological stages. The receiver operating characteristic curve was used to evaluate the efficacy of LS MRE and LS SWE in diagnosing liver fibrosis staging, and the area under the curve (AUC) was compared using the Z test. Results:Totally 162 rabbits were included, which covered F0 ( n=38), F1 ( n=33), F2 ( n=35), F3 ( n=31) and F4 ( n=25). Significant differences of LS MRE and LS SWE values were found among different stages of liver fibrosis ( F=295.29, 102.40, both P<0.001). LS MRE, LS SWE were both positively correlated with liver fibrosis stage ( r=0.93, 0.81, both P<0.001). The AUC of LS MRE for diagnosing liver fibrosis stages ≥F1, ≥F2, ≥F3, and ≥F4 were 0.955, 0.967, 0.996, and 0.980, respectively; the AUC of LS SWE were 0.856, 0.880, 0.974, and 0.953, respectively. The AUC of liver fibrosis stage ≥ F1, ≥ F2 for LS MRE value were greater than LS SWE value ( Z=2.93, 3.29, P=0.003, 0.001), and the AUC of ≥F3, ≥F4 had no significant differences ( Z=1.58, 1.68, P=0.115, 0.093). Conclusion:Both MRE and SWE can accurately predict the stage of liver fibrosis in experimental rabbits, and MRE is better than SWE in diagnosing early liver fibrosis.

Objective: To determine the impact of inflammatory reaction levels and the culprit plaque characteristics on preprocedural Thrombolysis in Myocardial Infarction (TIMI) flow grade in patients with ST-segment elevation myocardial infarction (STEMI) undergoing primary percutaneous coronary intervention (PCI). Methods: The is a retrospective study. A total of 1 268 STEMI patients who underwent pre-intervention optical coherence tomography (OCT) examination of culprit lesion during emergency PCI were divided into 2 groups by preprocedural TIMI flow grade (TIMI 0-1 group (n =964, 76.0%) and TIMI 2-3 group (n =304, 24.0%)). Baseline clinical data of the 2 groups were collected; blood samples were collected for the detection of inflammatory markers such as high sensitivity C-reactive protein (hsCRP), myocardial injury marker, blood lipid, etc.; echocardiography was used to determine left ventricular ejection fraction; coronary angiography and OCT were performed to define the lesion length, diameter stenosis degree of the infarct-related arteries, presence or absence of complex lesions, culprit lesion type, area stenosis degree and vulnerability of culprit plaques. Multivariable logistic regression analysis was performed to identify independent correlation factors. The receiver operating characteristic (ROC) curve of continuous independent correlation factors was analyzed, and the best cut-off value of TIMI 0-1 was respectively determined according to the maximum value of Youden index. Results: The mean age of 1 268 STEMI patients were (57.6±11.4) years old and 923 cases were males (72.8%). Compared with TIMI 2-3 group, the patients in TIMI 0-1 group were older and had higher N-terminal-pro-B-type natriuretic peptide level, lower cardiac troponin I (cTnI) level, lower left ventricular ejection fraction, and higher hsCRP level (5.16(2.06, 11.78) mg/L vs. 3.73(1.51, 10.46) mg/L). Moreover, the hsCRP level of patients in TIMI 0-1 group was higher in the plaque rupture subgroup (all P<0.05). Coronary angiography results showed that compared with TIMI 2-3 group, the proportion of right coronary artery (RCA) as the infarct-related artery was higher, the angiographical lesion length was longer, minimal lumen diameter was smaller, and diameter stenosis was larger in TIMI 0-1 group (all P<0.05). The prevalence of plaque rupture was higher (75.8% vs. 61.2%) in TIMI 0-1 group. Plaque vulnerability was significantly higher in TIMI 0-1 group than that in TIMI 2-3 group with larger mean lipid arc (241.27°±46.78° vs. 228.30°±46.32°), more thin-cap fibroatheroma (TCFA, 72.4% vs. 57.9%), more frequent appearance of macrophage accumulation (84.4% vs. 70.7%) and cholesterol crystals (39.1% vs. 25.7%). Minimal flow area was smaller [1.3(1.1-1.7)mm² vs. 1.4(1.1-1.9)mm², all P<0.05] and flow area stenosis was higher (78.2%±10.6% vs. 76.3%±12.3%) in TIMI 0-1 group. Multivariable analysis showed that mean lipid arc>255.55°, cholesterol crystals, angiographical lesion length>16.14 mm, and hsCRP>3.29 mg/L were the independent correlation factors of reduced preprocedural TIMI flow grade in STEMI patients. Conclusions: Plaque vulnerability and inflammation are closely related to reduced preprocedural TIMI flow grade in STEMI patients.
Aged ; Coronary Angiography ; Humans ; Inflammation ; Male ; Middle Aged ; Myocardial Infarction/diagnostic imaging* ; Percutaneous Coronary Intervention ; Plaque, Atherosclerotic/diagnostic imaging* ; Retrospective Studies ; ST Elevation Myocardial Infarction/surgery* ; Stroke Volume ; Thrombolytic Therapy ; Ventricular Function, Left

Discuss the electromyography and imaging characteristics of Hirayama disease，and improve clinicians’ awareness of the disease. Methods A retrospective analysis of clinical data of five cases of Hirayama disease patients in our hospital，analyze its clinical manifestations，EMG and imaging features. Results 5 patients were male，age 18 to 28 years old，（3 cases） unilateral，bilateral involvement in two cases of asymmetry. Five patients showed the hand and forearm muscle weakness with atrophy，forearm ramp-like changes，with cold paralysis. Motor nerve conduction delay mainly ulnar nerve terminal during latent，median nerve，ulnar nerve compound muscle action potential （the CMAP） amplitude decreased，no motor nerve conduction block；sensory nerve conduction were normal. Needle electrode EMG neurogenic damage，abnormal muscle mainly in the C7-T1 segment dominated area. MRI in the neutral position of the cervical spine in 5 patients showed that the physiological curvature was straightened or the spinal cord was slightly thinned；2 cases showed LOA phenomenon in the cross section；5 cases showed different degrees of compression of the cervical spinal cord forward and the epidural space signal shadow in the flexion position，enhanced scanning Abnormal enhancement in the epidural space can be seen. Conclusion According to the clinical manifestations of Hirayama disease combined with its characteristic electromyography and MRI features，early diagnosis of Hirayama disease can be made.

Objective:To explore the relationship between new biomarkers in donor blood and delayed graft function after kidney transplantation and evaluate the clinical value of new biomarkers in the diagnosis of DGF (delayed graft function).Methods:For recipients of donor kidney transplantation from August 2016 to December 2017, blood samples were collected from operations of donor organ resection within 12 hours of the day. Enzyme-linked immunosorbent assay (ELISA) was employed for detecting neutrophil gelatinase-associated lipocalin (NGAL), L-type fatty acid binding protein (L-FABP), kidney injury molecule-1 (KIM-1) and interleukin-18(IL-18). They were divided into DGF and EGF (early graft function) groups according to the diagnosis of DGF. The inter-group differences of four new biomarkers were calculated. Receiver operating characteristic curve (ROC curve) was plotted for finding the best positive cutoff value and the sensitivity and specificity of new donor blood marker for diagnosing delayed graft function were calculated.Results:Among them, 8 had postoperative DGF and 62 had none. The overall incidence of DGF was 11.43%. The serum concentration of NGAL was 521.01±132.84 ng/ml in DGF group versus (299.99±100.03) ng/ml in EGF group ( P<0.001). The ROC curve was plotted. With a NGAL concentration >425.15 ng/ml, the sensitivity and specificity for diagnosing DGF were 87.5% and 90.3% respectively. The area under the curve (AUC) was 0.891. The serum concentration of IL-18 was (14.10±12.36) ng/ml in DGF group and (4.61±1.83) ng/ml in EGF group ( P=0.047). With a IL-18 concentration of >5.345 ng/ml, the sensitivity and specificity for diagnosing DGF were 100% and 64.5% respectively. The AUC was 0.914. No significant inter-group difference existed in serum L-FABP/KIM-1. The sensitivity of donor creatinine in the diagnosis of DGF was 62.5%, specificity 75.8% and AUC 0.692. Conclusions:With an excellent level of sensitivity and specificity, an elevated concentration of NGAL/IL-18 in donor blood is superior to traditional creatinine in the diagnosis of DGF after renal transplantation.

A simple and rapid immunochromatographic test strip incorporating a colloidal gold-labeled recombinant Nsp7 antigen probe was successfully developed for the detection of anti-porcine reproductive and respiratory syndrome virus (PRRSV) antibodies in swine. Recombinant Nsp7 protein of PRRSV labeled with colloidal gold was dispensed on a conjugate pad for use as the detector. Staphylococcal protein A and purified porcine anti-Nsp7 antibodies were blotted on a nitrocellulose membrane to form test and control lines, respectively. A comparison of the strip with standard diagnostic tests, enzyme-linked immunosorbent assays and immunoperoxidase monolayer assay, was also performed. The immunochromatographic test strip was shown to be of high specificity and sensitivity. Furthermore, the strip assay is rapid and easy to perform with no requirement for professional-level skills or equipment. It is suggested that the immunochromatographic test strip can be used to quickly and accurately detect PRRSV antibody and to be suitable for diagnostic purposes in the field.
Antibodies* ; Collodion ; Colloids ; Diagnostic Tests, Routine ; Enzyme-Linked Immunosorbent Assay ; Gold Colloid ; Immunochromatography ; Membranes ; Porcine Reproductive and Respiratory Syndrome* ; Porcine respiratory and reproductive syndrome virus* ; Sensitivity and Specificity ; Staphylococcal Protein A ; Swine

Objective To summarize the clinical experience of the application of bortezomib desensitization regime prior to secondary renal transplantation in a highly-sensitized recipient. Methods At 13, 10 and 6 d prior to secondary renal transplantation, one patient positive for donor specific antibody (DSA) was subcutaneously administered with bortezomib at a dose of 1.3 mg/m2 combined with a low dose of immunoglobulin. Postoperatively, immunosuppressive regime of tacrolimus (FK506), mycophenolat sodium and methylprednisolone was adopted. The serum creatinine (Scr), blood urea nitrogen (BUN) levels, FK506 concentration, DSA titre, C3d binding DSA (C3d-DSA) titre, pathological biopsy of the renal graft and adverse reactions were observed. Results During 12-month follow-up after administration of bortezomib, the Scr level was declined and maintained at 130 μmol/L, and the BUN level was remained at 3.9 mmol/L. The DSA level was significantly decreased and the C3d-DSA was negative. At postoperative 4 and 9 months, pathological biopsy of the renal graft revealed that the patient was positive for C4d, prompting the chronic active antibody mediated rejection (AMR). The patient presented with grade Ⅲ peripheral neuropathy. Conclusions Application of preoperative bortezomib desensitization regime can effectively down-regulate the DSA level in the recipient and avert the incidence of acute rejection in highly-sensitized patients undergoing secondary renal transplantation. Comprehensive treatment using bortezomib is recommended for preoperative desensitization in the highly-sensitized transplant recipients.

BACKGROUND:Studies have shown that Schwann cells form a Bunger band in the basement tube and guide the extension of regenerating axons after peripheral nerve injury, but the exact mechanism remains to be explored.
OBJECTIVE:To explore the effect of Wal erian degeneration on biological characteristics and secretory function of Schwann cells in rats with sciatic nerve injury.
METHODS:A rat model of sciatic nerve injury was established and divided into two groups:sciatic nerve transection group and surgical control group. Schwann cells were isolated and cultured from sciatic nerve segments by one enzyme digestion. The cellmorphology was observed under light microscope and S-100 protein expression was determined by immunofluorescence staining. After subculture, the first generation of Schwann cells were chosen to draw the growth curve by the counting method within 14 days. The cellactivity was detected by MTT assay. The adhesion of Schwann cells was examined by acid phosphatase analysis and the concentration of nerve growth factor was detected by ELISA method.
RESULTS AND CONCLUSION:At 14 days after primary culture, a great number of Schwann cells were observed near the edges of nerve segments in the sciatic nerve transection group, but only smal number of Schwann cells scattered around nerve segments in the control group. Schwann cells in both groups showed S-100 positive expression. At 3 days after subculture, Schwann cells reached the logarithm proliferative phase, the cellnumber and proliferation absorbance values in both groups were increased along with time extension. Furthermore, the number of Schwann cells and absorbance value in the sciatic nerve transection group were significantly higher than those of control group (P<0.05). The adhesion ability in the sciatic nerve transection group was also significantly higher than those in the control group (P<0.05). ELISA results showed that, the concentrations of nerve growth factor in the sciatic nerve transection group were significantly higher than those in the control group at 4, 6, 8, 10, 12 and 14 days (P<0.05). After sciatic nerve injury, Wal erian degeneration can induce Schwann cells dedifferentiate into the precursors, significantly influence the biological function of Schwann cells, promote the proliferation of Schwann cells within the short term, secrete large amounts of neurotrophic factors, enhance celladhesion, and provide a suitable microenvironment for regenerated axons. In addition, it creates the necessary microenvironment for peripheral nerve regeneration.

BACKGROUND:PA6 cells are bone marrow stromal stem cells from the mouse skul , and scientists have found that PA6 cells co-cultured with some kinds of stem cells have shown neural differentiation, based on which, PA6 cells can be used for repair of nerve injury. Therefore, researchers give more and more attentions to PA6, but few studies have addressed isolation and culture methods of bone marrow stromal stem cells from the skul . OBJECTIVE:To isolate and culture bone marrow stromal stem cells from the skul of Sprague-Dawley rats and to observe cellular morpholopy in vitro and perform immunofluorescence identification. METHODS:Under sterile conditions, newborn Sprague-Dawley rat’s skul was cut into pieces. Smal skul pieces were washed using Dulbecco’s modified Eagle’s medium containing 10%fetal bovine serum. Single cellsuspension was made, and placed in culture flask. The culture medium was changed many times for cellpurification. The second passage of cells was obtained for morphology observation under an inverted microscope. cellsurface markers were detected by using immunofluorescence staining. RESULTS AND CONCLUSION:After the primary culture for 24 hours, cells exhibited adherent growth;after 3 days, cells were increased in number, and presented with irregular shapes, such as polygon, triangle, spindle and flat shape. After passage, cells were uniform in morphology, arranged in radial and bunch patterns, and exhibited strong adherent ability. Some cells grew in cluster, and proliferated faster than primary cells. Immunofluorescence staining showed that cells were positive for CD105, CD73, CD44, CD90, but negative for CD45, D34,CD14, HLA-DR. Results indicate that this method can obtain bone marrow stromal stem cells.

Objective To establish the Cookie Theft Test for Chinese norms. Methods 29 normal participants and 17 patients with stroke finished the Cookie Theft Test. The language samples were analyzed in 7 different indexes which are incorrect statement (IS), dysfluencies (DF), providing structure support (PS), repetitions (RP), content units (CU), elaborations of content units (EC) and irrelevancies (IR). Besides, the total words of language samples were counted. The scores of normal participants in 8 indexes were regarded as norms. And the scores of patients in 8 indexes were used to test the validity of the norms. Results The test-retest reliability was r(IS)=0.92, r(DF)=0.89, r(PS)= 0.98, r(RP)=0.84, r(CU)=0.96, r(EC)=0.88 and r(IR)=0.99, respectively. 12 out of 17 patients were distinguished by the norms. Conclusion The norms of Cookie Theft have acceptable reliability and validity and can be applied to clinical diagnoses and scientific researches