Insulinoma-associated protein-2 (IA-2) is a transmembrane glycoprotein belonging to the tyrosine phosphatase-like protein family as well as an important autoantigen in the diagnosis of type 1 diabetes. IA-2 products have been marketed in Europe and the United States. At present, commercially available IA-2 antigens are either the recombinant IA-2ic domain or the IA-2 naturally extracted from bovine islets. However, the recombinant IA-2 antigen displays weak positive in clinic practice, which often results in occasional detection failures, thus cannot completely replace the naturally extracted IA-2 antigen. In this study, an HEK293 expression system was used to explore the production of recombinant IA-2. An IA-2 transmembrane fragment (IA-2 TMF) located at amino acid position 449-979, also known as the natural membrane protein form of IA-2, was produced in HEK293 through transfection, and both the expression conditions and dissolution conditions of the membrane protein were also optimized. The purified membrane protein yield was 0.78 mg/L cell culture. Subsequently, the antigen activity of IA-2 TMF was compared with RSR rhIA-2 through enzyme linked immunosorbent assay. The serum of 77 type 1 diabetes patients and 32 healthy volunteers were detected. Receiver operating characteristic curve (ROC) curve was used to characterize the sensitivity and specificity of the test results. The results showed that the sensitivity of IA-2 TMF was 71.4% (55/77), while the sensitivity of RSR rhIA-2 was 63.6% (49/77), and the specificity of both antigens were all 100%. There was no significant difference in specificity between the two antigens, but the sensitivity of IA-2 TMF was appreciably better than that of the imported gold standard RSR rhIA-2 antigen. In conclusion, the recombinant IA-2 TMF produced in HEK293 cells can be used as a raw material to develop in vitro diagnostic reagents for type 1 diabetes.
Humans ; Animals ; Cattle ; HEK293 Cells ; Insulinoma ; Diabetes Mellitus, Type 1/genetics* ; Recombinant Proteins ; Membrane Proteins ; Pancreatic Neoplasms

Objective To explore the clinical features,auxiliary examinations,therapies and prognoses of patients with antibodies against contactin-associated protein-like 2 (CASPR2).Methods The clinical data of 11 anti-CASPR2 encephalitis patients who were admited to the People's Hospital of Zhengzhou University from March 2015 to April 2018 were retrospectively analyzed.Results The age of these 11 cases was (35.6± 19.4) years (ranged 20-74 years),and eight cases were females.There were seven cases with limbic encephalitis which included six cases of epilepsy,four cases of memory impairment,two cases of mental and behavioral abnormalities.Four cases had peripheral nerve hyperexcitability.Four cases had neuropathic pain.There were six cases with autonomic dysfunction including five cases of constipation,three cases of tachycardia,two cases of hyperhidrosis,two cases of urinary disorder.Seven cases had sleep disorder.Four cases had weight loss.Two cases showed cerebellar symptoms and two cases had hyponatremia.Magnetic resonance imaging scan of the brain showed abnormal signal in two cases,mainly involved medial temporal lobe and the hippocampus.Six cases underwent 18F-fluorodeoxyglucose positron emission tomography-computed tomography (PET-CT) examination,and three cases showed abnormalities,including two with temporal hypermetabolism and one with cortical hypermetabolism.Chest enhanced CT and PET-CT showed thymoma in one case.All cases received immunotherapy,and after treatment their symptoms were improved.Long-term follow-up was performed in nine cases,and three cases relapsed.Conclusions The major clinical manifestations of anti-CASPR2 encephalitis were limbic encephalitis,peripheral nerve hyperexcitability,neuropathic pain,autonomic dysfunction,insomnia and so on.Immunotherapy was effective and some patients may have recurrence.

At present, the experimental technique to produce human red blood cells in vitro is complicated, and in order to optimize the induction steps, human pluripotent stem cells were differentiated into red blood cells through two induction steps. First, human pluripotent stem cells (including Rh negative A type umbilical cord mesenchymal stem cells (hUCMSCRh-A) and human iPS cells (hiPS)) were differentiated into CD31+ and CD34+ cells in BVF medium. PCR and flow cytometry were used to exam the expression of CD31 and CD34. We found that hUCMSCRh-A derived CD31+ and CD34+ cells were 5.3% and 22.7%, respectively; hiPS derived CD31+ and CD34+ cells were 31.2% and 8.2%, respectively. For the second induction step, the obtained CD31+ and CD34+ cells were differentiated into mature erythrocytes for 36 days under the addition of various growth factors. Through Giemsa staining, we found that the obtained mature erythrocytes were similar in morphology and size to normal human erythrocytes, and some obtained erythrocytes were enucleated. Globin expression was detected by real time RT-PCR, and the expression of β-globin was more than 20%. The obtained erythrocytes are collected into the centrifuge tube, and then erythrocytes were naturally settled and showed the red color. Our findings provide a novel and effective method for the quantity generation of human red blood cells in vitro.

Objective The relationship between toll like receptor 4 (TLR4) the single nucleotide polymor-phisms of gene (SNP) and ischemic stroke reperfusion injury was evaluated by meta-analysis ,which was de-signed to provide evidence-based medicine for the prevention of ischemic stroke reperfusion injury . Methods In Medline ,PubMed ,EMBASE ,Cochrane ,CBM ,Chinese Journal Net ,academic conference materials and dissertations ,we searched for comprehensive information on the relationship between TLR4 gene SNP (rs10759932 ,rs11536891 ,rs11536879) in ischemic stroke Cohort study and case-control literature to determine whether the gene SNP (rs10759932 ,rs11536891 ,rs11536879) was associated with ischemic stroke reperfusion injury by genotype comparison .The heterogeneity test was performed by Stata11 .0 .The heterogeneity test was used to calculate the OR value .The heterogeneity between different studies was analyzed quantitatively . The fixed effect model was used and the percentage I2 was calculated .Results Meta analysis showed that 1943 cases of ischemia-reperfusion injury and 5043 cases of control group were analyzed ,TLR4 gene SNP (rs10759932 ,rs11536891 ,rs11536879) was associated with the risk of ischemic stroke reperfusion injury ,the dominant fixed effect models were (OR=1 .653 ,95％ CI:1 .416 -1 .930 ;OR=1 .653 ,95％ CI:1 .416 -1 .930 ;OR=1 .653 ,95％ CI:1 .416-1 .930 ;);a co-dominant fixed effect model (OR=1 .525 ,95％ CI:1 .350 -1 .723 ;OR= 1 .653 ,95％ CI:1 .416 -1 .930 ;OR= 1 .653 ,95％ CI:1 .416 -1 .930) .Conclusion TLR4 gene SNP (rs10759932 ,rs11536891 ,rs11536879) was associated with the occurrence of ischemic stroke reperfusion inju-ry by Meta-analysis .

BACKGROUND:Percutaneous vertebroplasty is a minimal y invasive treatment for spinal metastasis tumor, but the mechanism of the injected polymethylmethacrylate (PMMA) bone cement in the treatment of tumor is not ful y understood. OBJECTIVE:To explore the cytotoxicity of PMMA bone cement and its monomers on tumor cel s. METHODS:PMMA extracts in the wire drawing and curing periods and different mass concentrations of monomer dilutions were co-cultured with human lung cancer cel s spc-A1. The cel morphology was observed using inverted microscope, the absorbance (A) values were detected by cel counting kit-8 assay, the relative growth rate was calculated, and the toxicity of PMAA bone cement and its monomers was evaluated at 1 and 3 days of culture, respectively. RESULTS AND CONCLUSION:At 1 day of culture, the absorbance values in the 1 and 10 g/L groups were significantly lower than that in the negative control group (P<0.01). The absorbance values in the 1 g/L, 10 g/L, 100 mg/L and wire drawing extract groups were significantly lower than those in the negative control group at 3 days of culture (P<0.05). The relative growth rate in the 1 and 10 g/L groups was 26%-29%, and the level of toxicity was grade 4 after 1-day culture;at 3 days, the relative growth rate was decreased to 12%-16%, and the level of toxicity was grades 4-5. After 3-day culture, the level of toxicity reached to grade 2 in the 100 mg/L and wire drawing extract groups. These results indicate that PMMA bone cement in wiredrawing period and its monomers exert toxic effects on tumor cels.

Objective:There are limited data describing the clinical characteristics and prognosis of culture negative pyogenic spondylitis.The aim of this study was to investigate the treatment,prognosis and clinical characteristics of culture negative pyogenic spondylitis.Methods: A retrospective study reviewed 74 patients who were diagnosed with spondylitis in Peking University First Hospital from January 2010 to December 2015.A total of 27 patients suffered from pyogenic spondylitis.According to the pa-thogenic culture results,the patients were divided into two groups: culture negative group and culture positive group.The clinical characteristics and treatment outcomes between the two groups were compared.Results: The elder were more vulnerable to pyogenic spondylitis,and of the 27 patients,12 patients were female and 15 male.All patients had no history of administration of antibiotics prior to obtaining culture samples.A causative germ was identified in 14/27 patients (51.9％) with Staphylococcus aureus being the most common pathogen.There was no significant difference between the two groups in the patient's age,gender,visual analogue score (VAS),predisposing factor,clinical symptom,sign and spinal segment (P>0.05).Erythrocyte sedimentation rate (ESR) (P=0.056) and C-reactive protein (CRP) (P=0.040) of culture negative group were lower in contrast to culture positive group.The incidence of vertebral abscess in culture negative group was higher than in culture positive group (P=0.046).After treatment,ESR dropped almost equally in both groups,and CRP dropped faster in the culture positive group (P=0.192).At last,there was no significant difference between the two groups in hospital stay,pain relief,open debridement operation rate,and recurrence rate of infection.Conclusion: ESR and CRP of the culture negative patient were lower than those of the culture positive patient,and the incidence rate of paravertebral abscess was higher than that of the culture positive patient.After administration of antibiotics,there was no significant difference between the two groups in duration of antibiotics,open debridement operation rate and recurrence rate of infection.So,culture negative may not necessarily be a negative prognostic factor for pyogenic spondylitis.However,we should watch out for the drug resistant bacteria or double infection,due to the long term use of wide-spectrum antibiotic in culture negative patients.

OBJECTIVETo investigate a cerebral autosomal dominant arteriopathy with the subcortical infarcts and leukoencephalopathy (CADASIL) case with clinical manifestations of baldness, lumbago and Parkinson's symptoms.

METHODSClinical and imaging data of the patient were analyzed. The patient and his family members were also subjected to genetic testing.

RESULTSThe symptoms of the patient included recurrent stroke, dementia, and mood disturbance, in addition with lumbago, baldness and Parkinson's symptoms but no migraine. Cranial MRI of the patient showed bilateral symmetric leukoencephalopathy and multiple small subcortical lacunar infarcts. A point mutation in exon 11 of the NOTCH3 gene (R558C) was discovered in the proband and four asymptomatic relatives.

CONCLUSIONCADASIL is characterized by recurrent subcortical ischemic stroke, dementia, pseudobulbar palsy, and mood disturbance. Baldness, lumbago and Parkinson's symptoms may also be seen in such patients.

Alopecia ; etiology ; CADASIL ; complications ; diagnostic imaging ; genetics ; Humans ; Low Back Pain ; etiology ; Magnetic Resonance Imaging ; Male ; Middle Aged ; Mutation ; Parkinsonian Disorders ; etiology ; Receptor, Notch3 ; genetics

Objective To understand the changing characteristics of drug concentration in the serum and cerebrospinal fluid(CSF) after intravenous (IV) drip of norvancomycin in patients after neurosurgery procedure.Methods Patients with surgical cavity/ventricular drainages after neurosurgery procedure in a hospital in 2014 were selected, and they were divided into 2 groups according to the administration modes (12 in each group), conventional administration group: 0.8 g norvancomycin IV drip for 60 minutes, repeated every 12 hours;continuous administration group, 0.8 g norvancomycin, IV drip for 60 minutes, followed by 0.4 g of IV drip for 11 hours, then 0.4 g for 12 hours, serum and CSF specimens were collected at different time points after administration, concentration of norvancomycin was determined.Results Serum norvancomycin concentration reached a peak of (55.52±26.04) and (59.22±41.88) mg/L in conventional administration group and continuous administration group respectively, 24-hour serum concentration were (8.21±6.04) and (9.11±5.09)mg/L respectively;CSF norvancomycin concentration reached a peak of (16.31±11.15) and (8.82±8.91)mg/L in conventional administration group and continuous administration group respectively, 24-hour CSF concentration were (6.12±2.34)and (5.71±4.72)mg/L respectively;CSF penetration rate of conventional administration group was calculated by ratio of area under curve (AUCCSF/AUCserum), at 0-12 and 12-24 h hour were 63.3% and 59.0% respectively;in continuous administration group were 25.4% and 47.4% respectively.According to 95% of the minimum inhibitory concentration (MIC90) 2 mg/L of target bacteria methicillin-resistant Staphylococcus aureus (MRSA), AUC0-24/MIC90 in conventional administration group and continuous administration group were 192 and 184 respectively.Conclusion For patients who receives early use of standard dose of norvancomycin after neurosurgery procedure, CSF drug concentration after convention and continuous administration of norvancomycin can both reach MIC90 against target bacteria.

Objective To analyze the clinical effect through 20 adult atrial septal defect repair cases with totally thoracoscopic and summarize the experience of totally thoracoscopic cardiac surgeries.Methods From March 2014 to August 2016, 20 adult atrial septal defect cases repaired by totally thoracoscopic were enrolled in the study. Extracorporeal circulation was established by inserting artery pump tubes into the right femoral artery and vein tubes in to the right femoral vein, cold blood cardioplegia was perfused ante gradely through the aortic root in order to protect the myocardium, then atrial septal defect repair cardiac surgeries were implemented by perforating 3 holes through the right chest wall. Analyze the operation time, aortic cross clamping time, CPB time, amount of drainage, hospital stay, postoperative complications and so on.Results The operation time was 3.5~5.0 h, and the average level was (3.8 ± 0.5) h. The blocking duration of the ascending aorta ranged from 28 to 46 min, and the average level was (29.8 ± 8.2) min. The duration of extracorporeal circulation ranged from 86 to 108 min, and the average level was (80.6 ± 11.5) min. Ventilation time ranged from 5 to 8h, and the average level was (6.0 ± 0.8) h. The amount of thoracic drainage ranged from 100 to 260 ml, and the average level was (150.0 ± 35.0) ml. Hospital stay ranged from 6 to 9 days, and the average level was (6.5 ± 1.2) days. All the operations were completed successfully. There were no in-hospital death or serious post-operative complications. UCG performed 3~5 days after the operation revealed surgical results were satisfactory. Follow up to 1~28 months were available in all cases. During the period, the heart function was conifrmed asⅠ level.Conclusion Adult atrial septal defect repair surgeries with totally thoracoscope is safe and reliable and have advantages of less injury, rapid discovery, light pain, and less postoperative drainage.

Objective To analyze the clinical presentation , the mutation of the pathogenic genes and imaging features in a Chinese Han early-onset Alzheimer's disease pedigree.Methods A pedigree of Alzheimer's disease was collected.The DNA sequence of presenilin 1 (PSEN1), presenilin 2, micro-tubule associated protein tau ,β-amyloid precursor protein gene was analyzed , the clinical presentation , results of accessory examination , neuropsychological evaluation of the proband were investigated and the point mutations of some members of the family , 50 sporadic Alzheimer's disease patients , 50 normal controls were verified.Results The proband of the family appeared as language impairment , memory loss, personality change, repeated language, visuospatial impairment, mental and behavior disorder.The gene detection showed p.L226R mutation in the condon 226 in the exon 7 of PSEN1 gene of the proband and five other family members (Ⅲ1 ,Ⅲ2 ,Ⅲ4 ,Ⅲ6 ,Ⅲ7 ).The mother of the proband had the suspicious symptoms , and the sister and the brother of the proband had the similiar symptoms with the proband , all of whom died.Fifty sporadic Alzheimer'disease patients and 50 unrelated normal subjects did not have the mutation .The computed tomographic angiography showed that the brain blood vessels were normal and 18 F-fludeoxyglucose positron emission tomography (18F-FDG-PET) showed brain atrophy and hypometabolism in frontotemporal regions, parietal regions, hippocampal areas, however, the MRI, MRA and 18F-FDG-PET of the two mutation carriers (Ⅲ6 ,Ⅲ7 ) were all normal.Conclusion We reported a novel mutation in an early-onset Alzheimer's disease family presented as language impairment in the early stage of the disease , the p.L226R mutation of PSEN1, which may be a pathogenic mutation to cause the family's dementia.