Cloning of foot-and-mouth disease virus integrin receptor beta1 subunit and antibody production to its ligand-binding domain / 生物工程学报

We produced beta1 gene which is about 2400 bp by reverse transcription polymerase chain reaction (RT-PCR) from bovine trachea, reclaimed and purified, then cloned the amplified fragment to pGEM-T easy vector, confirmed by sequencing. The immune-dominant epitope of beta1 gene was chosen by computer analysis and then syncretized ligand-binding domain from 346 bp to 843 bp of ecytoplasm with six histidine, expressed LBD protein massly in E. coli BL21 (DE3), and identified by SDS-PAGE. The fusion protein was purified with Ni-NTA affinity chromatography and immunized New Zealand rabbits preparing of its polyclonal antibody, the specific antibody titer was above 1:12,800 detected by indirect ELISA, the result of Western blot showed that this antibody could be recognized by LBD fusion protein.

Expression of VLA1, VLA2 and VLA3 integrin molecules in uterine endometrium of infertile women with unexplained aetiology in Ahwaz-Iran

Recent attention has focused on the expression of integrin molecules within the endometrium, and their relation to infertility. The present prospective study was undertaken to determine whether the endometrium of women with unexplained infertility differs in the expression of very late activation antigens [VLA] from the endometrium of normal fertile women. Thirty samples of endometrial biopsies from hysterectomies with non-endometrial pathology and 28 endometrial samples by uterine curetting from infertile women in secretary phase at implantation time were collected, stained with three monoclonal antibodies against a1 integrin subunits including VLA-1 to VLA-3 by immunohistochemical technique and then assessed semi-quantitatively by microscope. Chi-Square test was used to compare the expression of VLA antigens on epithelial cells, stromal cells, lymphocytes and vessels within endometrial tissues between two groups. The results showed that most VLA integrins were present in fertile and infertile endometrium tissues. There were similarities and differences in the expression of VLA molecules in different compartments. VLA-2, VLA-3 expression on endometrial compartments showed an unaltered pattern of staining during the putative window of implantation in either fertile or infertile women with no significant differences [Pvalue> 0.5]. VLA-1 expression on endometrial compartments changed in fertile and unexplained infertile women, the differences were related to the presence or lack of the molecules on epithelial and stromal cells respectively. Differences may explain causes of unexplained infertility, and suggests that certain integrins may participate in the cascade of molecular events leading to successful implantation and early placental development which requires more investigations

Modulation of alpha1beta1 integrin mediated adhesion of hepatocytes to collagen IV and laminin by divalent cations.

Cell matrix interactions play a critical role in hepatic development and regeneration after acute injury. These interactions are mediated by transmembrane receptors belonging mainly to the integrin family. We have tried to assess the role of divalent cations in mediating attachment of hepatocytes to matrix proteins like collagen IV (Col IV) and laminin (Ln). The three cations examined viz. Ca2+, Mg2+ and Mn2+ showed attachment promoting activity. Since alpha1beta1 integrin is a common receptor for col IV and LN in liver, the effect of cations in its binding to these matrix proteins was studied. Although cations in general enhanced the binding, different cations exhibited differential effect in promoting the binding for different ligands. Mg2+ ions were more effective in promoting the binding of alpha1beta1 integrin to col IV but Ca2+ proved to be more effective one for Ln. Kinetic analysis of binding in dot blot assays using different concentrations of cations showed that while Mg2+ was active at low concentrations Ca2+ and Mn2+ promoted the binding more at higher concentrations. Absence of competitive effect in binding studies showed that they bind at different sites on the receptor. Differential effects of cations in promoting the binding of alpha1beta1 integrin to Col IV and Ln suggest that changes in level of diffusible cations can modulate affinity of the common receptor alpha1beta1 integrin to its ligands and can influence adhesion of hepatic cells to different matrix proteins during hepatic development and regeneration.

Distribution of Adhesion Molecules in Rabiit Cornea with Aspergillus fumigatus Keratitis

In order to help define the possible role of adhesion molecules in corneal inflammation, we investigated the distribution of adhesion molecules in normal and Aspergillus fumigatus keratitis-induced corneas of rabbits in process if time. Each 4 corneas were resected at 3, 12, 24, and 72 hours after intracorneal injection with A. fumigatus. Normal corneas (4 eyes) were used as a control. Monoclonal antibodies to beta 1 subunit of VLAs, alpha 1 subunit of VLA-1, LFA-1, ICAM-1,and ELAM-1 were used for immunohistochemical staining of 20 corneas.The results were as follows: In normal cornea, beta 1 subunit of VLAs was expressed on all parts of the cornea, and ICAM-1 was expressed on corneal stroma and endothelium. Vascular endothelium showed expression of beta 1 subunit of VLAs and ICAM-1 after 12 hours of intracorneal injection, and alpha1 subunit of VLA-1 and ELAM-1 at 72 hours after injection. In inflamed cornea, beta 1 subunit of VLAs was expressed strongly at 72 hours after injection. Alpha1 subunit of VLA-1 was detected on corneal stroma after 12 hours of injection, and on corneal endothelium at 72hours after injection. Expression of beta 2 subunit of LFA-1 was weak on corneal stroma after 3 hours injection, and on corneal endothelium at 72 hours after injection. ICAM-1 expression was detected weakly on corneal epithelium, and increased on corneal stroma and endothelium at 72 hours after injection. ELAM-1 was expressed weakly on corneal stroma after 3 hours of injection, and on corneal endothelium at 72 hours after injection.In this study, it was found that the invasion of A. fumigatusinto the cornea causes localized inflammatory reaction that results in activation of corneal cells (keratocytes, corneal endothelial cells and epithelial cells), and subsequent expression of adhesion molecules in the cornea. Expression of adhesion molecules facilitates the inflammatory cells to be migrated into the cornea with inflammmation.

Immunocytochemical Study on the Change of the Activated T Cells in Peripheral Blood of the Pulmonary Tuberculosis Patients / 결핵

BACKGROUND: It has been found that Helper T cells in the peripheral blood are decreased in the cell mediated immunity in the pulmonary tuberculosis But it has not been confirmed yet that only decrease in number of cells which has phenotype in the peripheral blood is defined to decrease in cell mediated immunity. The immunocytochemical study was performed to observe the change of the percentage of T-lymphocytes with their subsets and activated T cells in the peripheral blood of pulmonary tuberculosis and to know how many T cells would be activated, relative to resting cells in the peripheral blood. METHODS: The peripheral blood obtained from twenty two patients and ten healthy controls were smeared on the gelatin coated slide glass prepared for of mononuclear cells. The double bridge technique of alkaline phosphatase-antialkaline phosphatase(APAAP) method was used. As the primary antibodies, T1(anti-human T cell), T4(anti-human helper/inducer T cells) and T8(anti-human supressor/cytotoxic T cell) antibodies and interleukin-2 receptor (for early activated T cell),very late activation antigen (for activated cytotoxic T cell), T cell lineage specific activation antigen monoclonal actibodies were used. RESULTS: 1) There were significantly decrease in the absolute number of T4(+) cells but significantly increase of T8(+) cells in the peripheral blood of pulmonary tuberculosis (p<0.05). 2) The percentage of T4(+) cells showed significantly decrease in pulmonary tuberculosis but T8 (+)cells significantly increase(p<0.05). T4(+)/T8 (+) ratio showed significantly decrease in the peripheral blood of the pulmonary tuberculosis(p <0.05) 3) There were significantly increase in the absolute number of variable stages of activated T cells in the peripheral blood of the pulmonary tuberculosis(p<0.05). 4) The percentage of IL-2R, VLA-1, TLiSA were 6.45+1.56%, 7.64+1.34*, 10.45+1.16% in order which showed significantly increase in the peripheral blood of the pulmonary tuberculosis(p <0.05). CONCLUSION: We speculate that only a few percentage of T lymphocyte is activated in cell mediated immunity in pulmonary tuberculosis.

Vla-Integrin Exprfs Ion In Articular Cartilage / 대한류마티스학회지

OBJECTIVE: The adhesion molecule that mediate cell-cell and cell-extracellular matrix adhes.ion provides very important role in growth and differentiation of cells and tissue. VLA integrin is a prototype of adhesion molecule which participate in cell-cell and cell-extracellular matrix interacton, especially for collagen, laminin and fibronectin. The biologic functions of VLA-integrin are very diverse. Cartilage is the target tissue of various arthritides including rheumatoid arthritis and osteoarthritis and the process of homeostasis in cartilage matrix may be very important in preservation of cartilage. Although VLA integrin may participate in the process of cartilage degeneration and repair mechanism, tissue.distribution and exact role of VLA integtin in cartilage was not yet clearly defined. METHODS:Immunohistochemical analysis of VLA-integrin in cryostat section of articular cartilage was conducted using monoclonal antibody and avidin-biotin complex method. Analysis was performed in 10 rheumatoid arthritis specimens, 7 osteoarthritis specimens and 1 normal control. RESULTS: 1) Normal cartilage showed strong and diffuse stain with CD29, CD51 and moderate stain of VLA-5. Staining pattern of VLA-1 and 3 was inconstant and weak in intensity. 2) The intensity of VLA expression in articular cartilage of osteoarthritis was upregulated chiefly in CD29, CD51 and slightly in VLA-5. The positive rate of VLA-1 and 3 was similar to that of normal cartilage though the intensity was increased especially at cluster of chondrocytes. 3) VLA-integrin expression of rheumatoid arthritis cartilage was similar to that of osteoarthritis cartilage. CONCLUSION: VLA integrins functioning as fibronectin receptor such as VLA-5 and alpha, beta1 were upregulated in osteoarthritis and rheumatoid arthritis. Intensity was increased at clusters of chondrocytes. It was able to presume from above findings that VLA molecule has some role in the maintenance and repair of articular cartilage. The regulation of expression by cytokines and growth factors and exact function of VLA molecule in cartilage have to be elucidated.