Modulatory effects of heparin and short-length oligosaccharides of heparin on the metastasis and growth of LMD MDA-MB 231 breast cancer cells in vivo

Expression of the chemokine receptor CXCR4 allows breast cancer cells to migrate towards specific metastatic target sites which constitutively express CXCL12. In this study, we determined whether this interaction could be disrupted using short-chain length heparin oligosaccharides. Radioligand competition binding assays were performed using a range of heparin oligosaccharides to compete with polymeric heparin or heparan sulphate binding to I125 CXCL12. Heparin dodecasaccharides were found to be the minimal chain length required to efficiently bind CXCL12 (71% inhibition; P<0.001). These oligosaccharides also significantly inhibited CXCL12-induced migration of CXCR4-expressing LMD MDA-MB 231 breast cancer cells. In addition, heparin dodecasaccharides were found to have less anticoagulant activity than either a smaller quantity of polymeric heparin or a similar amount of the low molecular weight heparin pharmaceutical product, Tinzaparin. When given subcutaneously in a SCID mouse model of human breast cancer, heparin dodecasaccharides had no effect on the number of lung metastases, but did however inhibit (P<0.05) tumour growth (lesion area) compared to control groups. In contrast, polymeric heparin significantly inhibited both the number (P<0.001) and area of metastases, suggesting a differing mechanism for the action of polymeric and heparin-derived oligosaccharides in the inhibition of tumour growth and metastases

Monocyte chemoattractant proteins in the pathogenesis of systemic sclerosis

Activation of the immune system and increased synthesis of extracellular matrix proteins by fibroblasts are hallmarks in the pathogenesis of SSc. The molecular mechanisms underlying the infiltration of inflammatory cells into the skin and the subsequent activation of fibroblasts are still largely unknown. Chemokines are a family of small molecules that are classified according to the position of the NH(2)-terminal cysteine motif. Recent data indicate that chemokines and in particular two members of the subfamily of monocyte chemoattractant proteins, MCP-1 (CCL-2) and MCP-3 (CCL-7), might be involved in the pathogenesis of SSc. MCP-1 and -3 are overexpressed by SSc fibroblasts and in skin lesions from SSc patients compared to healthy controls. MCP-1 and -3 are chemotactic for inflammatory cells and stimulate their migration into the skin. In addition to their pro-inflammatory effects, MCP-1 and -3 contribute to tissue fibrosis by activating the synthesis of extracellular matrix proteins in SSc fibroblasts. Therapeutic strategies targeting MCP-1 have revealed promising results in several animal models of SSc. Antagonists against the receptor CCR2 are currently tested in clinical trials of a variety of diseases and also represent interesting candidates for target-directed therapy in SSc

A novel CXCL10-based GPI-anchored fusion protein as adjuvant in NK-based tumor therapy

BACKGROUND: Cellular therapy is a promising therapeutic strategy for malignant diseases. The efficacy of this therapy can be limited by poor infiltration of the tumor by immune effector cells. In particular, NK cell infiltration is often reduced relative to T cells. A novel class of fusion proteins was designed to enhance the recruitment of specific leukocyte subsets based on their expression of a given chemokine receptor. The proteins are composed of an N-terminal chemokine head, the mucin domain taken from the membrane-anchored chemokine CX3CL1, and a C-terminal glycosylphosphatidylinositol (GPI) membrane anchor replacing the normal transmembrane domain allowing integration of the proteins into cell membranes when injected into a solid tumor. The mucin domain in conjunction with the chemokine head acts to specifically recruit leukocytes expressing the corresponding chemokine receptor. METHODOLOGY/PRINCIPAL FINDINGS: A fusion protein comprising a CXCL10 chemokine head (CXCL10-mucin-GPI) was used for proof of concept for this approach and expressed constitutively in Chinese Hamster Ovary cells. FPLC was used to purify proteins. The recombinant proteins efficiently integrated into cell membranes in a process dependent upon the GPI anchor and were able to activate the CXCR3 receptor on lymphocytes. Endothelial cells incubated with CXCL10-mucin-GPI efficiently recruited NK cells in vitro under conditions of physiologic flow, which was shown to be dependent on the presence of the mucin domain. Experiments conducted in vivo using established tumors in mice suggested a positive effect of CXCL10-mucin-GPI on the recruitment of NK cells. CONCLUSIONS: The results suggest enhanced recruitment of NK cells by CXCL10-mucin-GPI. This class of fusion proteins represents a novel adjuvant in cellular immunotherapy. The underlying concept of a chemokine head fused to the mucin domain and a GPI anchor signal sequence may be expanded into a broader family of reagents that will allow targeted recruitment of cells in various settings

Gene targeting of VEGF-A in thymus epithelium disrupts thymus blood vessel architecture

The thymus harbors an organ-typical dense network of branching and anastomosing blood vessels. To address the molecular basis for morphogenesis of this thymus-specific vascular pattern, we have inactivated a key vascular growth factor, VEGF-A, in thymus epithelial cells (TECs). Both Vegf-A alleles were deleted in TECs by a complementation strategy termed nude mouse [mutated in the transcription factor Foxn1 (forkhead box N1)] blastocyst complementation. Injection of Foxn1(+/+) ES cells into Foxn1(nu/nu) blastocysts reconstituted a functional thymus. By dissecting thymus stromal cell subsets, we have defined, in addition to medullary TECs (mTECs) and cortical TECs (cTECs), another prominent stromal cell subset designated cortical mesenchymal cells (cMes). In chimeric thymi, mTECs and cTECs but not cMes were exclusively ES cell-derived. According to this distinct origin, the Vegf-A gene was deleted in mTECs and cTECs, whereas cMes still expressed Vegf-A. This genetic mosaic was associated with hypovascularization and disruption of the organ-typical network of vascular arcades. Thus, vascular growth factor production by TECs is required for normal thymus vascular architecture. These experiments provide insights into Foxn1-dependent and Foxn1-independent stromal cell development and demonstrate the value of this chimeric approach to analyzing gene function in thymus epithelium

A role for the endothelial glycosaminoglycan hyaluronan in neutrophil recruitment by endothelial cells cultured for prolonged periods

Glycosaminoglycans (GAGs) presented on the surface of endothelial cells (ECs) are believed to influence leukocyte recruitment during inflammation, but their roles remain uncertain. Here we report an in vitro model of prolonged culture of human EC in which the contributions of heparan sulphate (HS) and hyaluronan (HA) to the process of neutrophil recruitment could be studied. Previously, we reported that increasing EC culture duration (up to 20 days) enhanced neutrophil recruitment in response to low dose (1 U/ml) but not high dose (100 U/ml) of tumour necrosis factor-α (TNF). Here we found that HS and HA were present at much higher levels on the surface of day 20 cultures than day 3 cultures. Neutrophil recruitment on both day 3 and day 20 ECs was mediated through CXCR chemokine receptors and interleukin-8 (IL-8). In addition, mRNA levels for TNF receptors, signalling pathway constituents, adhesion receptors, and chemokines involved in neutrophil recruitment were similar for day 3 and day 20 ECs. To test whether the enhanced neutrophil recruitment on day 20 EC was mediated by GAGs, they were removed enzymatically. Removal of HA (but not HS) inhibited neutrophil recruitment, as did antibody blockade of CD44, a counter-receptor for HA on neutrophils. Supernatants from hyaluronidase-treated day 20 ECs were more potent in activating neutrophils than supernatants from untreated EC. Thus, HA has a role in neutrophil recruitment that is revealed in long-term cultures where it increases potency of response to sub-optimal levels of TNF. This effect appears to occur through a dual mechanism involving chemokine presentation and interaction with CD44

Altered chemokine expression in the spinal cord and brain contributes to differential interleukin-1 beta-induced neutrophil recruitment

The pattern of neutrophil recruitment that accompanies inflammation in the CNS depends on the site of injury and the stage of development. The adult brain parenchyma is refractory to neutrophil recruitment and associated damage as compared to the spinal cord or juvenile brain. Using quantitative Taqman RT–PCR and enzyme-liked immunosorbent assay (ELISA), we compared mRNA and protein expression of the rat neutrophil chemoattractant chemokines (CINC) in spinal cord and brain of adult and juvenile rats to identify possible association with the observed differences in neutrophil recruitment. Interleukin-1? (IL-1?) injection resulted in up-regulated chemokine expression in both brain and spinal cord. CINC-3 mRNA was elevated above CINC-1 and CINC-2?, with expression levels for each higher in spinal cord than in brain. By ELISA, IL-1? induced greater CINC-1 and CINC-2? expression compared to CINC-3, with higher protein levels in spinal cord than in brain. In the juvenile brain, significantly higher levels of CINC-2? protein were observed in response to IL-1? injection than in the adult brain following an equivalent challenge. Correspondingly, neutrophil recruitment was observed in the juvenile brain and adult spinal cord, but not in the adult brain. No expression of CINC-2? mRNA was detected. Thus differential chemokine induction may contribute to variations in neutrophil recruitment in during development and between the different CNS compartments