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

Zinc transporter 8 (ZnT8) is an important candidate antigen for type Ⅰ diabetes. The autoantibody detection kit based on ZnT8 can be used to help diagnose type Ⅰ diabetes, and the related products have been launched in Europe and the United States. Since the recombinant production system of active ZnT8 has not been established in China, this key raw material is heavily dependent on imports. We used Saccharomyces cerevisiae to carry out the recombinant expression of ZnT8. First, multiple antigenic forms of ZnT8 were designed as C-terminal haploid (C), C-terminal diploid (C-C), and N-terminal and C-terminal concatemers (N-C). The proteins were expressed, purified and tested for antigenicity by bridging-type ELISA. The serum of 13 patients with type Ⅰ diabetes and the serum of 16 healthy volunteers were detected. C, N-C, and C-C proteins had similar detection rates, which were 53.8% (7/13), 61.5% (8/13) and 53.8% (7/13). The specificity of the three groups was 100% (16/16). The detection value on positive samples P3, P4, and P8 increased by more than 90%, indicating better serum antibody recognition ability. Finally, N-C protein was selected for further serum sample testing, and the test results were characterized by receiver operating characteristic (ROC) curve for sensitivity and specificity. Compared with imported gold standard antigen, the sensitivity was 76.9% (10/13) and the specificity was 87.5% (14/16). There was no significant difference in the sensitivity of the method, but the specificity needed to be improved. In conclusion, the ZnT8 N-terminal and C-terminal concatemer protein developed based on S. cerevisiae expression system is expected to be a key alternative raw material in the development of in vitro diagnostic reagents for type Ⅰ diabetes.
Antigens ; Autoantibodies ; Diabetes Mellitus, Type 1/diagnosis* ; Enzyme-Linked Immunosorbent Assay ; Humans ; Saccharomyces cerevisiae/genetics* ; Zinc Transporter 8/genetics*

Objective:To analyze the clinical phenotype, imaging characteristics and genetic characteristics of a family of early-onset dementia caused by a new mutation in the triggerring receptor expressing on myeloid cells 2 gene (TREM2).Methods:Clinical data were collected from a patient with early-onset dementia. Then whole exome sequencing was performed for the proband, followed by Sanger sequencing for the family members.Results:The clinical manifestations of the proband (a 49-year-old female) was personality changes, mental and behavioral abnormalities, memory loss, ataxia, and seizures. Whole-exon sequencing revealed a novel homozygous mutation in exon 2 of TREM2, namely c.154C>T (p.R52C) heterozygosity in four family members, and one patient with similar clinical manifestations was deceased. The proband′s brain magnetic resonance imaging showed bilateral frontotemporal atrophy, bilateral white matter hyperintensity, thin corpus callosum. No bone cysts of the hands and feet were found by digital radiographic imaging.Conclusions:A homozygous mutation in TREM2 gene was detected in a patient with frontotemporal dementia-like dementia, epilepsy, but without bone cysts. This mutation is probably pathogenic. This research highlights the importance of TREM2 gene mutation screening in early-onset dementia, especially in those with atypical presentations.

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.

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 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.

Objective To analyze the clinical characteristics,imaging manifestations and prognoses of anti-GABAB receptor antibodies encephalitis.Methods The clinical manifestations,laboratory findings and radiological data of 13 patients with anti-GABAB receptor encephalitis,admitted to our hospital from September 2015 to March 2017,were retrospectively analyzed.Modified Rankin scale (mRs) was performed to evaluate the prognoses (good prognosis:mRs scores ＜ 2;poor prognosis mRs scores≥3).Results These 13 patients had an average age of 58 years (ranged 49-76 years) with a male to female ratio of 12:1.The major clinical features,including epileptic seizure,were found in 12 patients,psychiatric symptoms in 11 patients,cognitive disorder in 7 patients,and disturbance of consciousness in 4 patients.Brain MR imaging showed abnormal signal in 5 patients:4 were located in the hippocampus and amygdaloid,and one in the pons and left temporal lobe.Five patients showed abnormalities in PET-CT,including 4 with temporal hypermetabolism and 1 with cortical hypometabolism.Chest CT showed lung occupying lesions in 4 patients,of which 2 patients were diagnosed as having small cell lung cancer (SCLC) by pathological examination.Ten patients received immunomodulatory therapy,and three were with supportive care.After the average of 8 months of follow-up,7 patients had good prognosis,5 patients had poor prognosis and one patient lost of follow up.Conclusions Anti-GABAB receptor encephalitis frequently occurs in elderly male subjects and the main characteristic includes prominent refractory epilepsy and shows neurological improvement on immunotherapy.It can accompany by SCLC and have a relatively poor prognosis.

To investigate the N-glycosylation characteristics of recombinant human erythropoietin (rhEPO) produced by an industrial Chinese hamster ovary (CHO) cell line that is currently used in a large scale manufacturing process, we cultured this cell strain in static mode. The produced rhEPO in the culture supernatant was analyzed using isoelectric focusing (IEF) and Ricinus communis agglutinin-I (RCA-I) lectin precipitation. The lactate dehydrogenase (LDH) and sialidase activity in the serum-free supernatant were assayed as well. The analyses revealed that this cell strain could produce rhEPO with high sialic acid content, but during prolonged culture, cell viability decreased with time whilst the activity of sialidase present in the supernatant increased. The loss in rhEPO quality was due to a decrease in terminal sialic acid on the N-glycans, caused by sialidase degradation. The methods and findings in this paper serve as basis for further investigation of industrial production process.
Animals ; CHO Cells ; metabolism ; Cell Culture Techniques ; methods ; Cricetinae ; Cricetulus ; Erythropoietin ; biosynthesis ; genetics ; metabolism ; Genetic Engineering ; Humans ; N-Acetylneuraminic Acid ; metabolism ; Neuraminidase ; metabolism ; Proteolysis ; Recombinant Proteins ; biosynthesis ; genetics ; metabolism

The extracelluar domain I-IV of target gene VEGFR2 (Vascular endothelial growth factor receptor 2) was cloned from villus of trimester abortion by RT-PCR, and linked to the expression vectors. Then, the transfection conditions were optimized in serum-free suspension culture HEK293 using GFP (Green fluorescence protein) as the report gene. The results showed that the optimal transfection efficiency and cell number were obtained when the ratio of foreign DNA: PEI = 1:2 (W/W), DNA = 1.5 g /10(6) cells and shaking speed (120 r/min) in serum free medium in the beginning 4 hours of transfection. After optimizing the transfection conditions, the expression vector was successfully constructed for transient gene expression in HEK293, COS-7, and CHO-K1. The result shows that the target protein was only detected in CHO-K1 supernatant. Because of the C-terminal 8-His tag of target protein, target protein was subsequently purified using Ni2+-IDA and 5 mg purified protein was obtained in 1.5 L supernatant of CHOK1.
Animals ; CHO Cells ; Cell Line ; Chorionic Villi ; metabolism ; Cloning, Molecular ; Cricetinae ; Cricetulus ; Culture Media, Serum-Free ; Gene Expression Regulation ; Genetic Vectors ; genetics ; Green Fluorescent Proteins ; genetics ; Humans ; RNA, Messenger ; biosynthesis ; genetics ; Recombinant Proteins ; biosynthesis ; genetics ; Solubility ; Transfection ; Vascular Endothelial Growth Factor Receptor-2 ; biosynthesis ; genetics

OBJECTIVE:To scientifically establish the variety scope of expensive drugs and to reduce the stock level to a maximum degree under the premise of supply security. METHODS: The variety scope of expensive drugs was established by modified ABC (activity-based costing) analytical method,and the management on expensive drugs was carried out by fixed-quantity system (FQS) together with fixed-interval system (FIS). RESULTS: The practice of the optimized management effectively reduced the stock quantity of expensive drugs,increased the amount of money of 75 stocked drugs to 2.473 7 million yuan from 3.935 7 million yuan;the turnover rate rose from 238.36% to 461.86%,and the turnover days were decreased from 13.03 days to 8.29 days. CONCLUSION: Computer management in combination with the modified ABC analytical method together with FQS and FIS management led to an effective and accurate inventory management of expensive drugs,which then resulted in a speedy turnover rate,decreased fund occupying and lowered drug cost.