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 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.

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 .

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 evaluate the feasibility and strategy of the lesser omentum approach for laparoscopic pancreatic enucleation. Methods: Between June 2011 and October 2013, 6 laparoscopic pancreatic enucleations were performed by lesser omentum approach.The average age was 42 years, including 1 male and 5 female.The preoperation diagnosis of 6 cases were pancreatic islet cell tumors determined by abdominal CT/MRI, ultrasound and digital subtraction angiography.The tumors of 3 cases located in pancreatic neck, 2 tumors located in neck and body of pancreas, and 1 tumor located in pancreatic body.Their biggest tumor diameter were 0.8-2.5 cm. Results: Among the 6 cases, all laparoscopic pancreatic enucleations were successfully performed.None of the patients were converted to open operation.Eestimated blood loss was (26.7±18.6)ml, operating time was (82.5±19.4)minutes, and postoperative length of stay was (5.17±1.17)days.Additionally, postoperative complication included grade A pancreatic fistula in 1 case.After 36-64 months followed-up, there was no tumor recurrence and clinical symptom disappeared. Conclusion For the islet cell tumors located in pancreatic neck and body, the lesser omentumapproach may contribute to good surgical view and operative space, which can make pancreatectomy safer and easier for clinical application.

Objective To analyze the relationship between hyperhomocystinemia (HHcy) and occurrence of Alzheimer disease (AD) .Methods A total of 100 cases of elderly patients with dementia were collected ,from April 2010 to May 2013 ,and divided in‐to AD group and vascular dementia (VD) group according to patients′condition .Other 50 cases of healthy elderly individuals were collected in the control group .Levels of homocysteine (Hcy) were detected ,and the relationship between level of Hcy and occur‐rence of AD was analysed .Results Levels of Hcy in the AD group and VD group were higher than that in the control group ,and were decreased after treatment ,there were statistically significant differences(P<0 .05) .After treatment ,the scores of mini‐mental state examination(MMSE) in the AD group and VD group both were increased ,and the score of activity of daily living scale(ADL) was decreased in the AD group ,there were statistically significant differences (P<0 .05) .The level of Hcy in mild AD patients was lower than that in severe AD patients ,the difference was statistically significant (P<0 .05) .The AD odds ratio(OR) was 4 .7 ,and 95% confidence interval(CI) was 1 .76 -7 .09 .The level of Hcy in patients with AD was significantly negatively correlated with score of MMSE ,the coefficient value(r) was -0 .32 ,-0 .40 and -0 .27 in mild ,moderate and severe AD(P<0 .05) .Conclusion HHcy is an independent risk factor for the onset of AD ,so attention should be paid on high Hcy level ,in order to prevent AD .

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

Objective To explore rapamycin's inhibitory effect on proliferation of H_(22) hepatic cancer in mice. Methods In vitro study: H_(22) hepatic cancer cell lines were cultured with rapamycin, CsA, FK506, and proliferation was determined through MTT. The influences of different agents on the H_(22) hepatic cancer cell cycle were observed by flow cytometry. The vascular endothelial growth factor (VEGF) concentration of the supernatant fluid of the cultured H_(22) hepatic cancer cell was detected by ELISA. In vivo study: C57BL/6 to Balb/c mice allogenic skin transplant was established, and the H_(22) hepatic cancer cell was implanted under skin. Rapamycin, CsA, FK506 and 5-FU were fed to the mice, respectively. The effect of different immunosuppressors on the survival of skin graft was observed while the proliferation of the transplant tumor was investigated. VEGF concentration of treated mice serum was examined by ELISA. The microvessel density of the transplanted tumor was observed through immunohistochemistry staining of CD34. Results The proliferation of the H_(22) hepatic cancer cells was inhibited by rapamycin at the concentration different dose of rapamycin, the VEGF concentration of the supernatant fluid decreased significantly (P<0.05). The number of S phase cells decreased significantly compared to that of other agents (P<0.05). When rapamycin, the lengthened survival time of the skin grafts was similar to that in CsA and FK506 groups. But the tumor volume was smaller than that in CsA and FK506 groups (P<0.05). Compared to that in the control group, the VEGF concentration of mice serum decreased in rapamycin group (P<0.05), and the microvessel density of the transplant tumor was reduced greatly (P<0.05). Conclusion Rapamycin, as an immunosuppressor, significantly resists immunologic rejection and inhibits the proliferation of H_(22) hepatic cancer, thus having its advantage in treating malignant hepatic cancer with liver transplantation.

Objective To report one case with congenital ichthyosiform eruption, neurosensory deafness and vascularizing keratitis. Methods The overall clinical and laboratory examinations were conducted to confirm the diagnosis of keratitis, ichthyosis and deafness (KID) syndrome. Results The case presented with the typical hypotrichosis features of the eye lashes and eyebrows, alopecia of the scalp, and ophtalmological lesions. The keratotic plaques over the face, nose, ears, and the extremities were characterstic, and the skin of the trunk was leather-like, dry and hyperkeratotic. Dysplasia of cerebellum, and cystic enlargement of the fourth ventricle of cerebrum, and Dandy Walker syndrome were observed on MRI scanning. Treatment with oral acitretin for 3 weeks cleared the hyperkeratotic ichthyotic lesions on her posterior scalp and also improved other lesions on the extremities and the trunk. Conclusion Acitretin seems to be promising in the treatment of keratotic skin lesions in KID syndrome.