Objective To observe the changes in the pathological diagnosis rate of Graves'disease(GD) and Hashimoto's thyroiditis(HT)in the past 28 years and to probe its relationship to the environmental factors. Methods Statistic analysis was performed on data of thyroid disease,GD,HT and the complications of HT in Department of Pathology(1978-2005)in General Hospital of Tianjin Medical University with Run test,Cox-stuart test,ANOVA,t or t'test and Chi-square test.Results In 28 years there were 243 403 biopsies(average 8 693/ year)including 6 771 cases of thyroid disease(27.8‰),216 cases of GD(0.89‰),352 cases of HT (1.45‰).The biopsy rate of thyroid disease and GD showed a descending tendency since 1982 but an ascending tendency from 1995,furthermore,with GD it showed a descending tendency from 2001 to 2005 once more.While the biopsy rate of HT and GD+HT showed a descending tendency from 1986 and an ascending tendency after 1996,being 1-4 years later than thyroid disease and GD.The biopsy rate of GD+HT and the proportion of HT showed a descending tendency in 2002-2005 and 2003-2005 respecitvely.The average age of total and female patients with GD showed a descending tendency from 1997.The average age of GD was 13.7 years younger than HT,the patients of GDⅠ-type andⅡ-type being younger thanⅢ-type.The average age of lymphoid type(L), oxyphilie epithelium type(O),pronounced epithelium destruction type(P)in HT increased sequentially.In 28 years,there were more female patients with GD and HT,especially with HT.There existed 7.95% hyperthyroidism,6.25% nodular goiter,2.27% carcinoma in HT(more in L-type)and 2.56% hypothyroidism in HT(more in P-type).Conclusion Within 28 years,according to the pathological diagnosis rate,GD,HT,and GD+HT showed phasic tendencies related to drinking low iodine water and the implement of universal salt iodization policies in 1994 and 2000.Therefore,on the levels of genetics and environment,the factors that influence autoimmunity of thyroid gland could not exclude the variations of iodine intake,infection,affection or emotional stress.The mechanism of the changes in the pathological diagnosis rate needs further study.

Adult ; Antibodies, Monoclonal, Murine-Derived ; administration & dosage ; therapeutic use ; Female ; Hemophilia A ; drug therapy ; Humans ; Rituximab

Brain ; diagnostic imaging ; pathology ; Cerebral Palsy ; diagnosis ; pathology ; Cognition Disorders ; diagnosis ; pathology ; Early Diagnosis ; Humans ; Infant, Newborn ; Infant, Premature ; Infant, Premature, Diseases ; diagnosis ; pathology ; Language Disorders ; diagnosis ; pathology ; Leukomalacia, Periventricular ; complications ; diagnosis ; pathology ; Magnetic Resonance Imaging ; methods ; Prognosis ; Radiography ; Severity of Illness Index

Humans ; Pulmonary Embolism ; diagnosis ; therapy ; Venous Thromboembolism ; diagnosis ; therapy

@@

Objective To prepare recombinant human prothrombin-2 expressed in Pichia pastoris, and assay the enzymatic and clotting activities of prothrombin-2 activated by prothrombin activator ecarin.Methods Human prothrombin-2 gene and Echis carinatus ecarin gene were synthesized separately on the basis of the cDNA sequences published in GenBank.The gene of prothrombin-2 was cloned into the expression vector pPICZαA.The expression vector pPICZαA/prothrombin-2 was transformed into glycoengineered P.pastoris, and then prothrombin-2 engineered P.pastoris was screened.The expression products were induced by methanol, purified by two-step chromatography and identified by diges-tion by PNGase F and analysis of pepetide fingerprint.The ecarin gene was cloned into the expression vector pcDNA3.1. The expression vector pcDNA3.1/Ecarin was transformed into HEK 293T cells and the culture supernatant of HEK 293T/Ecarin was collected.The reaction product of HEK 293T/Ecarin cell culture supernatant and purified prothrombin-2 was analyzed by S-2238,which was the chromogenic substrate for thrombin.Fibrinogen was used to measure blood clotting time. Results The purified protein of P.pastoris expressed prothrombin-2 culture supernatant was 37 ×103 .The relative molecular mass(Mr) of the purified protein was reduced to 35 ×103, which was consistent with the theoretical Mr of prothrombin-2 molecular weight.The purified protein was proved to be prothrombin-2 by peptide fingerprint identification. The purified prothrombin-2 processed by HEK 293T/Ecarin culture supernatant could hydrolyze S-2238 to produce yellow pNA, and D405 of pNA increased with the volume of the processed prothrombin-2 that could promote the plasma coagulation.The blood clotting time was close to that of the thrombin kit.Conclusion Prothrombin-2 is prepared by P.pastoris and activated toα-thrombin by ecarin.This technique may replace the method of extraction of prothrombin from plasma and can be used for the treatment of war wounds or for future clinical research.

Objective To develop an interleukin-4(IL-4) antagonist named M5-IgG1Fc protein constructed by genetic engineering of antibody Fc fragment-cytokine mutein fusion protein which has a long half-life time in plasma.M5-IgG1 Fc protein binds to IL-4 receptor but cannot activate downstream signalling pathway , which provides a basis for drug develop-ment for allergic diseases .Methods The synthesized interleukin-4 mutant gene ( named M5 ) was cloned into the expres-sion vector pBV220 and transformed into E.coli DH5α.Chimeric gene M5-IgG1Fc obtained by overlap extension (SOE) method was transformed into glycoengineered Pichia pastoris GJK01 through expression vector pPICZαA .Then M5-IgGFc fusion protein was obtained by protein purification after being induced by methanol in 72 hours.The anti-IL-4 biologicial ac-tivity assay of M5 and M5-IgG1 Fc was performed with CTLL-2/IL-4R cells and detected with MTT colormetry .Finally,the half-life time of M5 and M5-IgG1 Fc protein in mice was compared by detecting the remaining amount in plasma with ELISA kit.Results The M5 protein expressed in E.coli and M5-IgG1 Fc fusion protein expressed in P.pastoris GJK01 both had IL-4 antagonistic bioactivity .The EC50 of both, which inhibited 5.6 ×10 -2 nmol/ml of IL-4, were 0.31 ±0.05 and 0.77 ± 0.03 nmol/ml,respectively.The maximum of M5 in plasma at 0.5 h was 5.8 ×10 -2 nmol/ml but the remaining amount was 2.8%of the maximum at 2 h.M5 protein could not be detected after administration at 8 h because of the detection line . The maximum of M5-IgG1 Fc fusion protein was 4.7 ×10 -2 nmol/ml,while fusion protein M5-IgG1 Fc decreased to 4.3%of its maximum at 120 h and could not be detected at 168 h.Conclusion M5 protein has IL-4 antagonistic bioactivity .M5-IgG1 Fc fusion protein expressed in glycoengineered P.pastoris GJK01 has IL-4 antagonistic bioactivity and long retention time in mice,which can be potentially used for treatment of allergic diseases .

Objective:To assess the survival-predictive value of TNM and Lugano staging systems in patients with primary gastro-intestinal lymphoma (PGL). Methods:A total of 73 patients with PGL were recruited from February 2001 to August 2013. All patients were diagnosed according to the TNM and Lugano staging systems. Five-year survival rate was used as the major clinical outcome. Sur-vival curves were plotted using the Kaplan–Meier method and analyzed with the log-rank test. The prognostic value of different vari-ables for clinical outcomes was assessed using the Cox multiple regression model. Results:The median follow-up time of surviving pa-tients was 42.4 months (range:1.3-158.6 months). The estimated 5-year overall survival rate was 77.82%. When diagnosed with the TNM system, the 5-year survival rates in stagesⅠ,Ⅱ,Ⅲ, andⅣwere 100%, 90.0%, 67.4%, and 22.2%, respectively (χ2=17.7956, P=0.0005). When staged by the Lugano system, the 5-year survival rates in stagesⅠ,Ⅱ,ⅡE , andⅣwere 100%, 100%, 70.7%, and 46.2%, respectively (χ2=15.6776, P=0.0013). Cox analysis showed that the invasion depth (T) (P=0.0181) and metastasis (M) (P=0.0031) were covariates that were prognostically significant for the overall survival. Conclusion:The TNM staging system is more ac-curate than the Lugano system in predicting the 5-year survival rate of patients with PGL.

Objective To study the association between serum interleukin-33 (IL-33) level and human stromelysin-2 (ST2) level in patients with rheumatoid arthritis (RA) associated with interstitial lung disease (RA-ILD) and its correlation with lung function and other laboratory parameters.Methods Two hundred and forty-five newly diagnosed RA patients during March 2012 to March 2013 in the in-patient and out-patient clinic of the First Affiliated Hospital of Liaoning Medical College were enrolled into this study.Patients were further divided into RA group (n=187) and RA-ILD group (n=58).Sixty subjects who came to the hospital for routine health check-up was composed of the normal control group.The clinical data of the two groups and controls were collected and their serum IL-33 and ST2 concentrations were measured.The t test was used to compare the difference between the two groups.Multiple variance analysis was used to com-pare the difference between groups.Pearson's correlation analysis was applied to explore the relationship between IL-33 concentrations and related variables.Results ① This study showed that the prevalence of RA associated interstitial lung disease was 23.7％(58/245).② The concentration of IL-33 [(746±43) pg/ml] and ST2 [(3413±169)pg/ml] of the RA-ILD group was significantly higher than that of the RA group [(433±42) pg/ml,(1500±147) pg/ml] (P＜0.01).③The vital capacity (VC％),forced vital capac-ity (FVC％),maximal midexpiratary flow curve (MMF％) and carbon monoxide diffusing capacity (DLCO) of the RA-ILD group were significantly lower than those of the RA group.④ The serum level of IL-33 was negatively correlated with that of RF and ACPA (IL-33 and RF,r=0.82,P＜0.01; IL-33 and ACPA,r=0.55,P＜0.01).Serum level of IL-33 was negaitively correlated with DLCO (r=-0.80,P＜0.01).Conclusion IL-33 participates in the pathogenesis of RA; and may be involved in the pathogenesis of RA-ILD.

Objective To express and purify mammalian glycosylation modified trimeric Ebola virus trimeric glycoprotein (EBOV-GP) using novel glycoengineered Pichia pastoris.Methods The EBOV-GP and EBOV-GPΔMLDΔTM genes were cloned into the pPICZ-αA vector,electrochemically converted to glycoengineered Pichia pastoris,and compared with EBOV-GP expressed in HEK-293T cells.Glycosylation was analyzed by PNGaseF and EndoH digestion,while the target protein was purified by affinity chromatography and ion exchange chromatography.N-terminal sequencing was used to determine whether the signal peptide was correctly cleaved during protein translation and gel column analysis was used to find out whether the trimeric structure was formed.Results The results of PNGaseF showed that EBOV-GP expressed by glycoengineered Pichia pastoris and HEK-293T cells had the same relative molecular mass and N-glycosylation degree.EndoH digestion showed that the N-glycosylation modification of EBOV-GPΔMLDΔTM was in a non-high mannose form.N-terminal sequencing showed that the signal peptide of the GP protein itself was correctly excised.Gel column analysis showed that the purified protein was in a trimeric form.Conclusion An EBOV-GP is obtained with complex glycosylation modification based on Glycoengineered Pichia pastoris.