The effect of surgically induced ischaemia on gene expression in a colorectal cancer xenograft model

Delays in tissue fixation following tumour vascular clamping and extirpation may adversely affect subsequent protein and mRNA analysis. This study investigated the effect of surgically induced ischaemia in a xenograft model of a colorectal cancer on the expression of a range of prognostic, predictive, and hypoxic markers, with a particular emphasis on thymidylate synthase. Vascular occlusion of human tumour xenografts by D-shaped metal clamps permitted defined periods of tumour ischaemia. Alterations in protein expression were measured by immunohistochemistry and spectral imaging, and changes in mRNA were measured by reverse transcriptase–polymerase chain reaction. Thymidylate synthase expression decreased following vascular occlusion, and this correlated with cyclin A expression. A similar reduction in dihydropyrimidine dehydrogenase was also seen. There were significant changes in the expression of several hypoxic markers, with carbonic anhydrase-9 showing the greatest response. Gene transcriptional levels were also noted to change following tumour clamping. In this xenograft model, surgically induced tumour ischaemia considerably altered the gene expression profiles of several prognostic and hypoxic markers, suggesting that the degree of tumour ischaemia should be minimised prior to tissue fixation

Purified protein derivative of tuberculin upregulates the expression of vascular endothelial growth factor in T lymphocytes in vitro

The purpose of this study was to investigate the cellular source and significance of vascular endothelial growth factor (VEGF) which, as reported previously, is elevated in the sera of pulmonary tuberculous patients. We obtained peripheral blood mononuclear cells (PBMCs) from 28 patients with active pulmonary tuberculosis, from 11 healthy controls who were positive for purified protein derivative of tuberculin (PPD), and from eight healthy individuals who were negative for PPD. We incubated the PBMCs with PPD in the presence or absence of major histocompatibility (MHC) class I or class II antibody in vitro, and measured the VEGF levels of culture supernatants. We also analysed the source of cells that secrete VEGF by using flow cytometry with intracellular staining. The T lymphocytes of active tuberculous patients secreted a higher level of VEGF than those of healthy controls. This production of VEGF was inhibited by adding MHC class II antibody. The addition of MHC class I antibody, however, did not inhibit. We propose that CD4(+) T lymphocytes are almost certainly the cells that produce VEGF in response to PPD. VEGF production might be associated with an antigen-specific immune reaction via CD4(+) T lymphocytes in tuberculosis