Tumor Infiltrating T Lymphocytes and Apoptosis in Colorectal Cancer.

Dysfunction of the immune system in colorectal cancer (CRC) can be due to a number of reasons including apoptosis of tumour infiltrating lymphocytes (TILs). The aims of this study was to investigate TILs in colorectal cancer and characterize apoptosis of TILs using terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay for detecting DNA fragments. We used monoclonal antibodies (mAbs) to T lymphocytes to detect TILs and double immunohistochemistry to assess apoptosis. T lymphocytes were detected in the immune infiltrate in CRC. TUNEL staining disclosed a high level of cell death among TILs. Apoptosis of T lymphocytes showed significant correlation with Dukes' stage (P = 0.02), lymphatic metastasis (P = 0.03), vascular metastasis (P = 0.01), lymph node metastasis (P = 0.02) and age of patient (P = 0.01). In conclusion, CRC may elude immunological surveillance by inducing apoptosis of TILs.

Apoptosis of T-cell subsets in colorectal cancer in vivo.

Dysfunction of the immune system in colorectal cancer (CRC) can be due to a number of reasons including apoptosis of tumour infiltrating lymphocytes (TILs). The aims of this study were to characterize, phenotypically, the apoptosis of TILs in CRC, and define the association of these findings with prognostic indicators. We used double immunohistochemistry to assess the apoptosis of T-cell subsets. Monoclonal antibodies to T lymphocytes, T helper cells, cytotoxic T cells (CTLs), natural killer cells (NK), CD45 and CD45RO were used. Antibodies against cleaved caspase-3 as a marker of apoptosis were used. Apoptosis of T-cell subsets was detected in the immune infiltrate in CRC. Apoptosis of T lymphocytes showed significant correlation with lymphatic metastasis (P = 0.01), Dukes' stage (P = 0.019). Apoptotic T helper cells showed significant correlation with metastasis (P = 0.04), lymphatic metastasis (P = 0.02), death (P = 0.04) and recurrence (P = 0.04). For apoptosis of CTLs, there was a significant correlation with histological classification (P = 0.02), lymphatic metastasis (P = 0.04), vascular metastasis (P = 0.03) and lymph node metastasis (P = 0.04). A significant association was found between the apoptosis of NK cells and the histological classification (P = 0.04). A significant association was found between the apoptosis of cd45RO cells and the histological classification (P = 0.04). In conclusion, apoptosis of lymphocytes provides theoretical foundation for metastasis and counterattack of colon cancer.

Selective inhibition of the human tie-1 promoter with triplex-forming oligonucleotides targeted to Ets binding sites.

The Tie receptors (Tie-1 and Tie-2/Tek) are essential for angiogenesis and vascular remodeling/integrity. Tie receptors are up-regulated in tumor-associated endothelium, and their inhibition disrupts angiogenesis and can prevent tumor growth as a consequence. To investigate the potential of anti-gene approaches to inhibit tie gene expression for anti-angiogenic therapy, we have examined triple-helical (triplex) DNA formation at 2 tandem Ets transcription factor binding motifs (designated E-1 and E-2) in the human tie-1 promoter. Various tie-1 promoter deletion/mutation luciferase reporter constructs were generated and transfected into endothelial cells to examine the relative activities of E-1 and E-2. The binding of antiparallel and parallel (control) purine motif oligonucleotides (21-22 bp) targeted to E-1 and E-2 was assessed by plasmid DNA fragment binding and electrophoretic mobility shift assays. Triplex-forming oligonucleotides were incubated with tie-1 reporter constructs and transfected into endothelial cells to determine their activity. The Ets binding motifs in the E-1 sequence were essential for human tie-1 promoter activity in endothelial cells, whereas the deletion of E-2 had no effect. Antiparallel purine motif oligonucleotides targeted at E-1 or E-2 selectively formed strong triplex DNA (K(d) approximately 10(-7) M) at 37 degrees C. Transfection of tie-1 reporter constructs with triplex DNA at E-1, but not E-2, specifically inhibited tie-1 promoter activity by up to 75% compared with control oligonucleotides in endothelial cells. As similar multiple Ets binding sites are important for the regulation of several endothelial-restricted genes, this approach may have broad therapeutic potential for cancer and other pathologies involving endothelial proliferation/dysfunction.

Nanomedicine: current status and future prospects.

Applications of nanotechnology for treatment, diagnosis, monitoring, and control of biological systems has recently been referred to as "nanomedicine" by the National Institutes of Health. Research into the rational delivery and targeting of pharmaceutical, therapeutic, and diagnostic agents is at the forefront of projects in nanomedicine. These involve the identification of precise targets (cells and receptors) related to specific clinical conditions and choice of the appropriate nanocarriers to achieve the required responses while minimizing the side effects. Mononuclear phagocytes, dendritic cells, endothelial cells, and cancers (tumor cells, as well as tumor neovasculature) are key targets. Today, nanotechnology and nanoscience approaches to particle design and formulation are beginning to expand the market for many drugs and are forming the basis for a highly profitable niche within the industry, but some predicted benefits are hyped. This article will highlight rational approaches in design and surface engineering of nanoscale vehicles and entities for site-specific drug delivery and medical imaging after parenteral administration. Potential pitfalls or side effects associated with nanoparticles are also discussed.

Identification of an HLA-A*0201 cytotoxic T lymphocyte epitope specific to the endothelial antigen Tie-2.

Tie-2 stabilises pericyte-endothelial interactions during angiogenesis and is highly expressed on endothelium during several diseases, including arthritis, age-related macular degeneration and cancer. A vaccine that targets endothelium overexpressing Tie-2 may result in vessel damage and stimulate an inflammatory cascade resulting in disease regression. We have identified a region unique to Tie-2 (amino acids 1-196) that is homologous in humans and mice. Using computer algorithms, several HLA-A*0201 epitopes that are identical in mice and humans were predicted within this region; however, binding assays showed that the majority of these epitopes were of low affinity. Modification of the anchor residues of 4 epitopes enhanced HLA binding. These epitopes were incorporated by site-directed mutagenesis into a Tie-2 DNA construct. Immunisation of HLA*0201 transgenic mice with one of the modified Tie-2 constructs stimulated CTLs that recognised both wild-type and modified peptide-pulsed target cells. In contrast, no CTLs were generated in mice immunised with wild-type Tie-2 construct, demonstrating that the modified epitope was necessary in the generation of CTLs. Moreover, CTLs from mice immunised with the modified construct killed HLA-A*0201 endothelial cells overexpressing Tie-2. Our study demonstrates that it is possible to break tolerance to the endothelial antigen Tie-2, suggesting that it may be feasible to design a vaccine to activate CTLs to kill endothelial cells overexpressing Tie-2.

Colorectal cancer cells induce lymphocyte apoptosis by an endothelial monocyte-activating polypeptide-II-dependent mechanism.

Endothelial monocyte-activating polypeptide-II (EMAP-II) was first isolated from cell growth medium conditioned by tumor cells, and is closely related or identical with the p43 component of the mammalian multisynthase complex. In its secreted form, EMAP-II has multiple cytokine-like activities in vitro, inducing procoagulant activity on the surface of endothelial cells, increasing expression of E- and P-selectins and TNF-R1, and directing migration of monocytes and neutrophils. EMAP-II has also been shown to induce apoptosis in endothelial cells, leading to the suggestion that it is a proinflammatory polypeptide with antiangiogenic activity. The role of secreted EMAP-II in tumors remains poorly understood, and we hypothesized that EMAP-II may play a role in immune evasion by tumor cells. We investigated its effects on lymphocytes, using recombinant protein, or colorectal cancer cell lines, as a source of native EMAP-II. Recombinant EMAP-II inhibits DNA synthesis and cell division, and induces apoptosis in mitogen-activated lymphocytes in PBMC preparations, and in Jurkat T cells. Native EMAP-II, released by or expressed on the surface of colorectal carcinoma cells, also induces activation of caspase 8 and apoptosis of PBLs and Jurkat cells, which are partially blocked by addition of Abs against EMAP-II. Thus, activated lymphocytes, along with proliferating endothelial cells, are targets for the cytotoxic activity of EMAP-II. Membrane-bound and soluble EMAP-II appear to play multiple roles in the tumor microenvironment, one of which is to assist in immune evasion.

Biologic effects induced in vitro by PM10 from three different zones of Mexico City.

Exposure to urban airborne particles is associated with an increase in morbidity and mortality. There is little experimental evidence of the mechanisms involved and the role of particle composition. We assessed cytotoxicity (crystal violet assay), apoptosis [terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) or annexin V assay], DNA breakage (comet assay), and production of proinflammatory mediators [tumor necrosis factor Alpha (TNF-Alpha), interleukin 6 (IL-6), prostaglandin E2 (PGE2)] (enzyme-linked immunosorbent assay), and E-selectin (flow cytometry) in cell lines exposed to particulate matter < 10 microm in size (PM10) obtained from the northern, central, and southern zones of Mexico City. Particle concentrations ranged from 2.5 to 160 microg/cm(2). We used epithelial, endothelial, fibroblastic, and monocytic cells and assessed DNA damage in Balb-c cells, TNF-Alpha and IL-6 production in mouse monocytes, and PGE2 in rat lung fibroblasts. We determined the expression of E-selectin in human endothelial cells and evaluated the cytotoxic potential of the PM10 samples in all cell types. PM10 from all three zones of Mexico City caused cell death, DNA breakage, and apoptosis, with particles from the north and central zones being the most toxic. All of these PM10 samples induced secretion of proinflammatory molecules, and particles from the central zone were the most potent. Endothelial cells exposed to PM10 from the three zones expressed similar E-selectin levels. Mexico City PM10 induced biologic effects dependent on the zone of origin, which could be caused by differences in the mixture or size distribution within particle samples. Our data suggest that particle composition as well as particle size should be considered in assessing the adverse effects of airborne particulate pollution.

Vascular endothelial growth factor-D expression is an independent prognostic marker for survival in colorectal carcinoma.

The angiogenic factor vascular endothelial growth factor-D (VEGF-D) isa ligand for VEGF receptor-3 (VEGFR-3/Flt-4) and receptor-2 (VEGFR-2/KDR)and is implicated in the development of lymphatic vessels and promotion of lymphatic metastases. We assessed the expression of VEGF-D and VEGFR-3 in relation to microvessel density (MVD) in colorectal carcinomas (CRC), adenomas, and adjacent normal tissue by immunohistochemistry on consecutive archival sections. VEGF-D was detected in malignant and benign epithelium and in some smooth muscle of the colorectum. High-grade VEGF-D expression was observed frequently (74%) in CRC compared with adenomas (0%) and adjacent normal mucosa (22%). High-grade VEGF-D expression was not correlated with MVD, Dukes' stage (A to C), or tumor differentiation, but was associated with lymphatic involvement and patient survival. By multivariate analysis, VEGF-D expression was found to be an independent prognostic factor for both disease-free and overall survival. VEGFR-3 expression was detected in a subset of vessels, typically thin-walled and devoid of RBCs, in 89% of CRC cases examined. VEGFR-3-positive vessel densities increased progressively from normal mucosa to adenomas and carcinomas and were correlated with MVD, but not with Dukes' stage (A to C), tumor differentiation, or VEGF-D expression. VEGFR-3 expression was spatially associated with macrophage-rich inflammatory infiltrates, which were significantly more frequent among VEGFR-3-positive cases. We conclude that VEGF-D expression, but not that of its receptor VEGFR-3, is an independent prognostic indicator in CRC. VEGF-D expression may be associated with disease outcome through the promotion of lymphatic involvement/metastases.