Effect of expressing of anti-PD-1 antibody in mouse mammary gland on spleen T cells in transgenic mice / 生物工程学报

This study aims to investigate the effect of anti-PD-1 antibody expressed in mouse mammary gland on the surface antigen protein of spleen T cells, cytokine expression, spleen CD4+ T cell proliferation and proliferation related pathways of transgenic mice at the cellular level. Transgenic mice expressing anti-human PD-1 antibody at 8 weeks of age without pregnancy and 18 weeks of age with lactation were divided into two groups, with transgenic negative mice in each group as the control. Spleen lymphocytes were extracted and the changes of spleen lymphocytes were detected. Compared with transgenic negative mice, the proportion of effector T cells of spleen T cells in the immune system of transgenic mice with anti-PD-1 antibody expressed in breast increased, the proportion of Treg cells decreased, and the IFN-γ, IL-17 and IL-2 expressed in CD4+ T cells increased in varying degrees. Moreover, IL-4, IL-10 and TGF-β in CD4+ T cells did not change, nor did some cell surface protein molecules related to T cell stimulate. There was no significant difference in T cell proliferation between transgenic positive and transgenic negative mice. In transgenic positive mice, the expression of phosphorylated proteins in PI3K/Akt/mTOR and RAS/MEK/ERK pathways were partially up-regulated, but the whole pathway was not completely up-regulated. Therefore, it is feasible to use transgenic mice as host to express monoclonal antibodies related to immune system such as anti-PD-1 antibody.

Enzyme-linked immunosorbent assays for quantification of MMMAE-conjugated ADCs and total antibodies in cynomolgus monkey sera / 药物分析学报

Antibody-drug conjugates(ADCs)are commonly heterogeneous and require extensive assessment of exposure-efficacy and exposure-safety relationships in preclinical and clinical studies.In this study,we report the generation of a monoclonal antibody against monomethyl auristatin E(MMAE)and the development,validation,and application of sensitive and high-throughput enzyme-linked immunosor-bent assays(ELISA)to measure the concentrations of MMAE-conjugated ADCs and total antibodies(tAb,antibodies in ADC plus unconjugated antibodies)in cynomolgus monkey sera.These assays were suc-cessfully applied to in vitro plasma stability and pharmacokinetic(PK)studies of SMADC001,an MMAE-conjugated ADC against trophoblast cell surface antigen 2(TROP-2).The plasma stability of SMADC001 was better than that of similar ADCs coupled with PEG4-Val-Cit,Lys(m-dPEG24)-Cit,and Val-Cit linkers.The developed ELISA methods for the calibration standards of ADC and tAb revealed a correlation be-tween serum concentrations and the OD450 values,with R2 at 1.000,and the dynamic range was 0.3-35.0 ng/mL and 0.2-22.0 ng/mL,respectively;the intra-and inter-assay accuracy bias％ranged from-12.2％to-5.2％,precision ranged from-12.4％to-1.4％,and the relative standard deviation(RSD)was less than 6.6％and 8.7％,respectively.The total error was less than 20.4％.The development and validation steps of these two assays met the acceptance criteria for all addressed validation parameters,which suggested that these can be applied to quantify MMAE-conjugated ADCs,as well as in PK studies.Furthermore,these assays can be easily adopted for development of other similar immunoassays.

Thy-1 promotes EMTprocess of livercancercells by regulating Notch1 pathway / 中国肿瘤生物治疗杂志

@# Objective: To explore the role of Thy-1 cell surface antigen（Thy-1）in promoting epithelial-mesenchymal transition (EMT) in liver cancer HepG2 and MHCC-97 cells by regulating Notch1 pathway. Methods: MHCC-97 cells with high metastatic characteristics and HepG2 cells with low metastatic characteristics were selected as subjects. WB was used to detect the expression levels of Thy-1 and Notch1 in cells. MHCC-97 and HepG2 cells were transfected with lentivirus to construct cells with high and low expression of Thy-1 protein. Cells were treated with Notch1 agonist rhNF-κB (1 gsu/ml) and Notch1 inhibitor MW167 (100 μmol/L) for 24 h respectively. Transwell assay was used to detect the effect of Thy-1 expression on cell invasion; qPCR was used to detect the effect on Notch1 mRNA expression; WB was used to detect the effect on intracellular EMT-related protein expression. Results: The expression levels of Thy-1 and Notch1 in MHCC-97 cells were higher than those in HepG2 cells (P<0.05). Thy-1 overexpressing HepG2 cells and Thy-1 low expressing MHCC-97 cells were successfully constructed. Compared with HepG2 cells, the invasion ability of Thy-1 overexpressing HepG2 cells was significantly enhanced (183.23±55.34 vs 475.78±80.37, P<0.05), vimentin expression was significantly increased (P<0.05), epithelial cadherin protein expression was significantly decreased (P<0.05), and the expression level of Notch1 mRNAwas significantly increased (P<0.05). Compared with MHCC-97 cells, the invasion ability of Thy-1 silenced MHCC97 cells was significantly decreased (543.56±77.94 vs 237.44±62.18, P<0.05), the expression of vimentin was significantly decreased (P<0.05), epithelial cadherin protein expression was significantly increased (P<0.05), and Notch1 mRNA expression level was significantly decreased (P<0.05). Treatment of liver cancer cells with Notch1 activators or inhibitors can reverse the changes caused by Thy-1 silencing or overexpression. Conclusion: Thy-1 can affect the EMT process of HepG2 and MHCC-97 cells by regulating the expression of Notch1.

Value of combined detection of serum IL-8 ,TNF-α,KL-6 and SP-D in assisted diagnosis of idiopathic pulmonary fibrosis / 国际检验医学杂志

Objective To study the clinical value of combined detection of serum interleukin(IL)-8 ,tumor necrosis factor(TNF)-α,alvedar cell surface antigen Ⅱ(KL-6) and surface protein D(SP-D) in the diagnosis of idiopathic pulmonary fibrosis(IPF) .Meth-ods Seventy three patients with IPF were selected as the research subjects ,other 73 patients with bacterial pneumonia were taken as the bacterial pneumonia group .The levels of serum IL-8 ,TNF-α,KL-6 and SP-D were detected by enzyme-linked immunosorbent assay (ELISA) .The serum levels of IL-8 ,TNF-α,KL-6 and SP-D were compared between the IPF group and bacterial pneumonia group .The sensitivity and specificity of IPF detection were compared between the 4-index combined detection and single item de-tection .Results The levels of IL-8 ,TNF-α,KL-6 and SP-D in the IPF group were significantly higher than those in the bacterial pneumonia group (P<0 .05) .The positive rate of single detection of four indexes in the IPF group was significantly higher than that in the bacterial pneumonia group (P<0 .05) .The sensitivity and specificity of the 4-index combined detection for diagnosing IPF were 90 .4% and 93 .2% respectively ,which were significantly higher than the those of single index detection (P<0 .05) .Con-clusion The combined detection of IL-8 ,TNF-α,KL-6 and SP-D has better sensitivity and specificity in IPF diagnosis compared with single detection of IL-8 ,TNF-α,KL-6 and SP-D .

Evaluation of SAA and CD64 in early diagnosis of neonatal septicemia / 临床儿科杂志

10.3969/j.issn.1000-3606.2013.06.008

Characterization of a Novel Gene in the Extended MHC Region of Mouse, NG29/Cd320, a Homolog of the Human CD320

BACKGROUND: The MHC region of the chromosome contains a lot of genes involved in immune responses. Here we have investigated the mouse NG29/Cd320 gene in the centrometrically extended MHC region of chromosome 17. METHODS: We cloned the NG29 gene by RT-PCR and confirmed the tissue distribution of its gene expression by northern blot hybridization. We generated the NG29 gene expression constructs and polyclonal antibody against the NG29 protein to perform the immunofluorescence, immunoprecipitation and flow cytometric analysis. RESULTS: The murine NG29 gene and its human homologue, the CD320/8D6 gene, were similar in the gene structure and tissue expression patterns. We cloned the NG29 gene and confirmed its expression in plasma membrane and intracellular compartments by transfecting its expresssion constructs into HEK 293T cells. The immunoprecipitation studies with rabbit polyclonal antibody raised against the NG29-NusA fusion protein indicated that NG29 protein was a glycoprotein of about 45 kDa size. A flow cytometric analysis also showed the NG29 expression on the surface of Raw 264.7 macrophage cell line. CONCLUSION: These findings suggested that NG29 gene in mouse extended MHC class II region was the orthologue of human CD320 gene even though human CD320/8D6 gene was located in non-MHC region, chromosome 19p13.

A Study on the Cell Surface Antigens of Bladder Carcinoma by Means of Monoclonal Antibodies / 대한비뇨기과학회지

This study was carried out to investigate the localization and change of the cell surface antigens, such as URO-9(Om5) and URO-10 (T43), in transitional cell carcinomas of bladder. The subjects used were normal bladder mucosae of 10 autopsy cases of fetus and 10 biopsy cases of child or adult patients with disease of other organs as controls, and neoplastic tissue of 15 cases of patient with transitional cell in carcinoma of bladder. Avidin-Biotin complex(ABC) kits(Cambridge Research Lab.) and URO-9 and URO-10 monoclonal antibodies were used and the data observed were analyzed. The results obtained were summarized as follows: 1. URO-9 expression was none of 10 fetal bladder mucosae and 3 or 10 normal bladder mucosae and URO-10 expression was none showed. 2. Of 15 cases with transitional cell carcinoma of bladder, URO-9 expression was 5, of which 1 was Ta grade II, 1 T1 grade II and the rest T2 grade III. URO-10 expression was 10, of which I was T1 grade III, 3 T2 grade III, 3 T3 grade III and 3 T4 grade III, of 15 cases of transitional cell carcinoma of bladder. As above results, the transitional cell carcinoma of bladder in low grade and low stage have tendency to express URO-9 monoclonal antibody but not URO-10 monoclonal antibody, whereas the transitional cell carcinoma of bladder in high grade and high stage were inclined to express URO-10 monoclonal antibody with variable expression of URO-9 monoclonal antibody.

Cell Surface Antigen in Bladder Tumors: An Approach Using Morning Urine of Bladder Washing Specimen / 대한비뇨기과학회지

The major blood group antigens A, B or O (H) are present on normal bladder, epithelium. Several recent studies have suggested that the loss of cell surface antigens may be a precursor of subsequent invasion of the bladder by tumor. Because the specific and cell adherence test demonstrates the presence or absence of these antigens the test may have an important prognostic and screening value. We have examined cells in the morning urines or bladder washing specimens for 7 cages of bladder tumors and 5 cases of normal controls for specific red cell adherence. Even if we have studied insufficient cases, our results suggest that a correlation exists between absence of the cell surface antigen and histopathologic dedifferentiation. And seen is a progressive logs of cell surface antigen as malignant potential increase as measured by clinical staging.

The Cell Surface Antigen A,B,O(H) as An Indicator of Malignant Potential in Bladder Carcinoma: A Preliminary Report / 대한비뇨기과학회지

Currently, the cell surface antigen A,B,O(H) is thought to be an important indicator of malignant potential in bladder carcinoma. Herein, we performed SRCA test in 54 bladder carcinoma for detection of such an isoantigen, comparing the SRCA result to its tumor grade and stage. Also, various significances including the clinical application of SRCA test for the management of the bladder carcinoma were discussed. The results were as follows: 1. Of 54 patients, 34 patients were low stage(0-A) and low grade(1-2). 2. There is a significant correlation between tumor grade and SRCA test: Of 38 patients with low grade. 19 patients were SRCA positive, but of 16 patients with high grade. all were SRCA negative. 3. There is a significant correlation between tumor stage and SRCA test: Of 36 patients with low stage, 18 patients were SRCA positive, but of 18 patients with high stage(above B1), only one patient was SRCA positive. 4. There is a high possibility of false-negative results in detecting O(H) isoantigen: Of 36 patients with low stage, 6 patients were blood group 0 who were all SRCA negative. but 30 patients with other blood groups showed variable SRCA results. 5. There is a considerable correlation between tumor recurrence and SRCA result: Of 20 patients who were followed more than one year after initial TUR, 8 patients were SRCA positive, of these 4 patients were recurred, but 9 patients of 12 patients with SRCA negative were recurred.