[Isolation of human umbilical vein endothelial cells and development of an angiogenesis model in fibrin matrix]

Angiogenesis plays a key role in different physiologic and pathologic processes. Evaluation of endothelial cells and finally new vessels development in-vivo is a complex and formidable task. Thus, we designed an in-vitro experimental angiogenesis model using human umbilical vein endothelial cells [HUVEC] which is highly reproducible and controllable. This model has applicability to study various parameters involved in angiogenesis and its harness. HUVEC cells were isolated from umbilical vein by enzyme treatment as the source of endothelial cells. Then, the three-dimensional model was designed using fibrin gel and cytodex beads which was covered by HUVEC cells. In this model 10-12 days after culturing HUVEC, the resulting capillary formation was observed as branching of the source endothelial cells in microscopic field. The model is controllable and highly reproducible to study various parameters involved in angiogenesis. Moreover, the model provides a reliable method to screen angiogenesis and anti-angiogenesis substances

[Isolation and purification of Ag 85 complex from mycobacterium bovis [BCG] and assessment of its cell proliferation response in whole blood]

Introduction: Tuberculosis [TB] caused by Mycobacterium tuberculosis remains a major worldwide health problem. The only TB vaccine currently available is an attenuated strain of mycobacterium bovis termed Bacillus Calmette -Guerin [BCG]. The efficacy of BCG remains controversial. Mycobacterium secretory proteins are generally considered important antigens for protection against TB. A major protein component of mycobacterial culture filtrate is Antigen 85 complex which is a promising potential vaccine candidate

Material and Methods: Antigen 85 complex was purified from Mycobacterium bovis [BCG] culture filtrate by sequential chromatography on phenyl Sepharose 4B, DEAE-Sephacel and Superdex G75. Purification was confirmed by SDS-PAGE and immunoblotting with a specific monoclonal antibody. The in vitro ability of Ag 85 complex to stimulate cell proliferation was compared with that of Purified Protein Derivative [PPD] and the polyclonal T cell mitogen PHA in a whole blood assay in which the target cells were derived from 25 healthy PPD-positive and 25 healthy PPD-negative subjects

Results: Antigen 85 complex was found to have a molecular weight of 30-32 KDa by SDS-polyacrylamide gel electrophoresis and reacted strongly by immunoblotting with the monoclonal antibody specified against Ag 85. The responses to Ag 85 and PPD were significantly higher in cells of PPD- positive than in cells of PPD-negative donors. Eighty eight percent [22/25] of the PPD- positive cells responded to Ag 85 whereas only 16% [4/25] of the PPD-negative cells responded

Conclusion: The cell proliferation response to Ag 85 complex is significantly different between cells of skin-test positive and skin- test negative subjects and may suggest an immuno-protective role for Ag 85 complex against M. tuberculosis infection