Cysteine oxidation targets peroxiredoxins 1 and 2 for exosomal release through a novel mechanism of redox-dependent secretion

Non-classical protein secretion is of major importance as a number of cytokines and inflammatory mediators are secreted via this route. Current evidence indicates that there are several mechanistically distinct methods of non-classical secretion. We have recently shown that peroxiredoxin (Prdx) 1 and Prdx2 are released by various cells upon exposure to inflammatory stimuli such as LPS or TNF-α. The released Prdx then acts to induce production of inflammatory cytokines. However, Prdx1 and 2 do not have signal peptides and therefore must be secreted by alternative mechanisms as has been postulated for the inflammatory mediators IL-1β and HMGB1. We show here that circulating Prdx1 and 2 are present exclusively as disulphide-linked homodimers. Inflammatory stimuli also induce in vitro release of Prdx1 and 2 as disulfide-linked homodimers. Mutation of cysteines Cys51 or Cys172 (but not Cys70) in Prdx2, and Cys52 or Cys173 (but not Cys71 or Cys83) in Prdx1 prevented dimer formation and this was associated with inhibition of their TNF-α-induced release. Thus, the presence and oxidation of key cysteine residues in these proteins are a prerequisite for their secretion in response to TNF-α and this release can be induced with an oxidant. In contrast, the secretion of the nuclear-associated danger signal HMGB1 is independent of cysteine oxidation, as shown by experiments with a cysteine-free HMGB1 mutant. Release of Prdx1 and 2 is not prevented by inhibitors of the classical secretory pathway; instead, both Prdx1 and 2 are released in exosomes from both HEK cells and monocytic cells. Serum Prdx1 and 2 are also associated with the exosomes. These results describe a novel pathway of protein secretion mediated by cysteine oxidation that underlines the importance of redox-dependent signalling mechanisms in inflammation

Lymphatic density and metastatic spread in human malignant melanoma

Lymphatic density and metastatic spread in human malignant melanoma. Malignant melanoma (MM), the most common cause of skin cancer deaths, metastasises to regional lymph nodes. In animal models of other cancers, lymphatic growth is associated with metastasis. To assess if lymphatic density (LD) was increased in human MM, and its association with metastasis, we measured LD inside and around archival MM samples (MM, n = 21), and compared them with normal dermis (n = 11), basal cell carcinoma (BCC, n = 6) and Merkel cell carcinoma (MCC), a skin tumour thought to metastasise through a vascular route (MCC, n = 6). Lymphatic capillary density (mm(-2)), as determined by immunohistochemical staining with the lymphatic specific marker LYVE-1, was significantly increased around MM (10.0+/-2.5 mm(-2)) compared with normal dermis (2.4+/-0.9 mm(-2)), BCC (3.0+/-0.9 mm(-2)) and MCC (2.4+/-1.4 mm(-2)) (P<0.0001). There was a small decrease in LD inside MM (1.1+/-0.7 mm(-2)) compared with normal dermis, but a highly significant decrease in BCC (0.14+/-0.13) and MCC (0.12+/-2.4) (P<0.01 Kruskal-Wallis). Astonishingly, LD discriminated between melanomas that subsequently metastasised (12.8+/-1.6 mm(-2)) and those that did not (5.4+/-1.1 mm(-2), P<0.01, Mann-Whitney). Lymphatic invasion by tumour cells was seen mainly in MM that metastasised (70% compared with 12% not metastasising, P<0.05 Fisher's Exact test). The results show that LD was increased around MMs, and that LD and tumour cell invasion of lymphatics may help to predict metastasis. To this end, a prognostic index was calculated using LD, lymphatic invasion and thickness that clearly discriminated metastatic from nonmetastatic tumours

Gene expression profiles in rat mesenteric lymph nodes upon supplementation with Conjugated Linoleic Acid during gestation and suckling

Background Diet plays a role on the development of the immune system, and polyunsaturated fatty acids can modulate the expression of a variety of genes. Human milk contains conjugated linoleic acid (CLA), a fatty acid that seems to contribute to immune development. Indeed, recent studies carried out in our group in suckling animals have shown that the immune function is enhanced after feeding them with an 80:20 isomer mix composed of c9,t11 and t10,c12 CLA. However, little work has been done on the effects of CLA on gene expression, and even less regarding immune system development in early life. Results The expression profile of mesenteric lymph nodes from animals supplemented with CLA during gestation and suckling through dam's milk (Group A) or by oral gavage (Group B), supplemented just during suckling (Group C) and control animals (Group D) was determined with the aid of the specific GeneChip® Rat Genome 230 2.0 (Affymettrix). Bioinformatics analyses were performed using the GeneSpring GX software package v10.0.2 and lead to the identification of 89 genes differentially expressed in all three dietary approaches. Generation of a biological association network evidenced several genes, such as connective tissue growth factor (Ctgf), tissue inhibitor of metalloproteinase 1 (Timp1), galanin (Gal), synaptotagmin 1 (Syt1), growth factor receptor bound protein 2 (Grb2), actin gamma 2 (Actg2) and smooth muscle alpha actin (Acta2), as highly interconnected nodes of the resulting network. Gene underexpression was confirmed by Real-Time RT-PCR. Conclusions Ctgf, Timp1, Gal and Syt1, among others, are genes modulated by CLA supplementation that may have a role on mucosal immune responses in early life

Gene expression profiles in rat mesenteric lymph nodes upon supplementation with Conjugated Linoleic Acid during gestation and suckling

Background Diet plays a role on the development of the immune system, and polyunsaturated fatty acids can modulate the expression of a variety of genes. Human milk contains conjugated linoleic acid (CLA), a fatty acid that seems to contribute to immune development. Indeed, recent studies carried out in our group in suckling animals have shown that the immune function is enhanced after feeding them with an 80:20 isomer mix composed of c9,t11 and t10,c12 CLA. However, little work has been done on the effects of CLA on gene expression, and even less regarding immune system development in early life. Results The expression profile of mesenteric lymph nodes from animals supplemented with CLA during gestation and suckling through dam's milk (Group A) or by oral gavage (Group B), supplemented just during suckling (Group C) and control animals (Group D) was determined with the aid of the specific GeneChip® Rat Genome 230 2.0 (Affymettrix). Bioinformatics analyses were performed using the GeneSpring GX software package v10.0.2 and lead to the identification of 89 genes differentially expressed in all three dietary approaches. Generation of a biological association network evidenced several genes, such as connective tissue growth factor (Ctgf), tissue inhibitor of metalloproteinase 1 (Timp1), galanin (Gal), synaptotagmin 1 (Syt1), growth factor receptor bound protein 2 (Grb2), actin gamma 2 (Actg2) and smooth muscle alpha actin (Acta2), as highly interconnected nodes of the resulting network. Gene underexpression was confirmed by Real-Time RT-PCR. Conclusions Ctgf, Timp1, Gal and Syt1, among others, are genes modulated by CLA supplementation that may have a role on mucosal immune responses in early life

Generation and characterization of novel stromal specific antibodies

Rheumatoid synovial fibroblasts were used as an immunogen to produce monoclonal antibodies selected for their reactivity with stromal cell antigens. Mice were immunised with low passage whole cell preparations and the subsequent hybridomas were screened by immunohistochemistry on rheumatoid synovium and tonsil sections. The aim was to identify those antibodies that recognised antigens that were restricted to stromal cells and were not expressed on CD45 positive leucocytes. A significant number of antibodies detected antigen that identified endothelial cells. These antibodies were further characterised to determine whether the vessels identified by these antibodies were vascular or lymphatic. From five fusions clones were identified with predominant reactivity with: 1) fibroblasts and endothelial cells; or 2) broad stromal elements (fibroblast, endothelium, epithelium, follicular dendritic cells). A fibroblast-specific antibody that did not also identify vessels was not generated. Examples of each reactivity pattern are discussed