Evaluation of CXCL9 and CXCL10 as circulating biomarkers of human cardiac allograft rejection

BACKGROUND: Cardiac allograft rejection remains a significant clinical problem in the early phase after heart transplantation and requires frequent surveillance with endomyocardial biopsy. However, this is an invasive procedure, which is unpleasant for the patient and carries a certain risk. Therefore, a sensitive non-invasive biomarker of acute rejection would be desirable. METHODS: Endomyocardial tissue samples and serum were obtained in connection with clinical biopsies from twenty consecutive heart transplant patients followed for six months. A rejection episode was observed in 14 patients (11 men and 3 women) and biopsies obtained before, during and after the episode were identified. Endomyocardial RNA, from three patients, matching these three points in time were analysed with DNA microarray. Genes showing up-regulation during rejection followed by normalization after the rejection episode were evaluated further with real-time RT-PCR. Finally, ELISA was performed to investigate whether change in gene-regulation during graft rejection was reflected in altered concentrations of the encoded protein in serum. RESULTS: Three potential cardiac allograft rejection biomarker genes, chemokine (C-X-C motif) ligand 9 (CXCL9), chemokine (C-X-C motif) ligand 10 (CXCL10) and Natriuretic peptide precursor A (NPPA), from the DNA microarray analysis were selected for further evaluation. CXCL9 was significantly upregulated during rejection (p < 0.05) and CXCL10 displayed a similar pattern without reaching statistical significance. Serum levels of CXCL9 and CXCL10 were measured by ELISA in samples from 10 patients before, during and after cardiac rejection. There were no changes in CXCL9 and CXCL10 serum concentrations during cardiac rejection. Both chemokines displayed large individual variations in the selected samples, but the serum levels between the two chemokines correlated (p < 0.001). CONCLUSION: We conclude, that despite a distinct up-regulation of CXCL9 mRNA in human hearts during cardiac allograft rejection, this was not reflected in the serum levels of the encoded protein. Thus, in contrast to previous suggestions, serum CXCL9 does not appear to be a promising serum biomarker for cardiac allograft rejection

Exosomes Communicate Protective Messages during Oxidative Stress; Possible Role of Exosomal Shuttle RNA

BACKGROUND: Exosomes are small extracellular nanovesicles of endocytic origin that mediate different signals between cells, by surface interactions and by shuttling functional RNA from one cell to another. Exosomes are released by many cells including mast cells, dendritic cells, macrophages, epithelial cells and tumour cells. Exosomes differ compared to their donor cells, not only in size, but also in their RNA, protein and lipid composition. METHODOLOGY/PRINCIPAL FINDINGS: In this study, we show that exosomes, released by mouse mast cells exposed to oxidative stress, differ in their mRNA content. Also, we show that these exosomes can influence the response of other cells to oxidative stress by providing recipient cells with a resistance against oxidative stress, observed as an attenuated loss of cell viability. Furthermore, Affymetrix microarray analysis revealed that the exosomal mRNA content not only differs between exosomes and donor cells, but also between exosomes derived from cells grown under different conditions; oxidative stress and normal conditions. Finally, we also show that exposure to UV-light affects the biological functions associated with exosomes released under oxidative stress. CONCLUSIONS/SIGNIFICANCE: These results argue that the exosomal shuttle of RNA is involved in cell-to-cell communication, by influencing the response of recipient cells to an external stress stimulus

Microarray analysis of gene expression in human adipocytes and adipose tissue

Obesity has reached epidemic proportions worldwide and is associated with several serious conditions such as insulin resistance, type 2 diabetes, hyperlipidemia and atherosclerosis. Adipose tissue exerts important endocrine and immune functions through the release of adipokines. Adipokines are involved in the regulation of adipose tissue metabolism and associated with alterations in insulin resistance. The aim of this thesis was to identify genes, expressed in adipose tissue and adipocytes, that may contribute to insulin resistance and metabolic diseases related to obesity. Enlarged adipocytes are associated with insulin resistance and type 2 diabetes. A technique to separate human adipocytes from an adipose tissue biopsy into populations of small and large adipocytes was developed and the expression profiles of the populations were compared. This showed that serum amyloid A (SAA) and NAD(P)H:quinone oxidoreductase 1 (NQO1) were higher expressed in large versus small adipocytes. The expression of both SAA and NQO1 correlated to adipocyte size. SAA has been implicated in inflamma-tion and insulin resistance and NQO1 is known to be involved in oxidative stress suggesting that these findings may provide novel insights into the connection between hypertrophic obesity and insulin resistance/type 2 diabetes. SAA, NQO1 and also the cell death-inducing DFFA-like effector A (CIDE-A) were predominantly expressed in human adipocytes as compared to a panel of 32 other human tissues and cell types. During diet-induced weight loss in obese subjects, adipose tissue expression of NQO1 was reduced and CIDE-A was elevated. NQO1 expression correlated to measures of adiposity, insulin and the markers of liver dysfunction, AST and ALT. These findings indicate a role for NQO1 in the metabolic complications of human obesity. CIDE-A expression was inversely associated with basal metabolic rate independently of body composition, age, and gender. These data suggest that human CIDE-A plays a role in adipose tissue energy balance. Adipokines may play a key role in the rapid development of insulin resistance during critical illness. We identified gene expression changes in human adi-pose tissue in subjects with subarachnoidal hemorrhage during intensive care. Zinc-alpha2-glycoprotein (ZAG) was the only adipokine that was increased in adipose tissue during critical illness, and this increase was accom-panied by elevated plasma ZAG levels. Plasma levels of SAA and CRP were increased and adiponectin levels decreased of during intensive care. In summary, gene expression profiling of human adipocytes and adipose tissue during different conditions suggest that SAA, NQO1, CIDE-A and ZAG may be implicated in human obesity-related metabolic disease. During intensive care, increased plasma levels of ZAG, SAA, and CRP together with decreased levels of adiponectin may be involved in the decrease in insulin sensitivity

MicroRNA regulate immunological pathways in T-cells in immune thrombocytopenia (ITP)

MicroRNA are small non-coding RNA molecules that regulate gene expression. To investigate the role of microRNA in ITP, we performed genome-wide expression analyses of mRNA and microRNA in T-cells from ITP patients and controls. We identified 1,915 regulated genes and 22 regulated microRNA that differed between ITP patients and controls. Seventeen of the 22 regulated microRNA were linked to changes in target gene expression; 57 of these target genes were associated with the immune system, e.g. T-cell activation and regulation of immunoglobulin production. CXCL13 and IL-21 were two microRNA target genes significantly increased in ITP. We could demonstrate increased plasma levels of CXCL13 and others have reported increased plasma levels of IL-21 in ITP. Thus, regulated microRNA were significantly associated with both gene and protein expression of molecules in immunological pathways. We suggest that microRNA may be important regulatory molecules involved in the loss of tolerance in ITP

MC/9 cells pre-treated with exosomes released under oxidative stress obtain a resistance to oxidative stress.

<p>Time course of viability of MC/9 cells (n = 6) (%) after exposure to oxidative stress (H₂O₂ 125 µM) when pre-treated with exosomes derived from other MC/9 cells that were either exposed (oxi exo) or not exposed (norm exo) to H₂O₂ at the same concentration. Treatment of MC/9 cells with exosomes released under oxidative stress increased viability with approximately 15-20% at different time points after the initiation of H₂O₂ exposure. ***p<0.001.</p

Induced genes in exosomes released under oxidative stress.

<p>This table shows the 20 most induced mRNA transcripts in exosomes derived from MC/9 cells exposed to oxidative stress (H₂O₂, 125 µM for 24 h, oxi exo) compared to exosomal mRNA transcripts after exposure of cells to vehicle (norm exo). A fold-change of e.g. 2 indicated that the gene is 2 fold up-regulated in the exosomes derived from cells exposed to oxidative stress.</p

Oxidative stress induced by H₂O₂ results in a dose related loss of viability.

<p>Dose response relationship between viability of cultured MC/9 cells (%) and concentration of H₂O₂ (50 µM-500 µM) for 24 h. The dose versus viability correlation coefficient was 0.86.</p