Circulating cell death products predict clinical outcome of colorectal cancer patients.

BackgroundTumor cell death generates products that can be measured in the circulation of cancer patients. CK18-Asp396 (M30 antigen) is a caspase-degraded product of cytokeratin 18 (CK18), produced by apoptotic epithelial cells, and is elevated in breast and lung cancer patients.MethodsWe determined the CK18-Asp396 and total CK18 levels in plasma of 49 colorectal cancer patients, before and after surgical resection of the tumor, by ELISA. Correlations with patient and tumor characteristics were determined by Kruskal-Wallis H and Mann-Whitney U tests. Disease-free survival was determined using Kaplan-Meier methodology with Log Rank tests, and univariate and multivariate Cox proportional hazard analysis.ResultsPlasma CK18-Asp396 and total CK18 levels in colorectal cancer patients were related to disease stage and tumor diameter, and were predictive of disease-free survival, independent of disease-stage, with hazard ratios (HR) of patients with high levels (> median) compared to those with low levels (< or = median) of 3.58 (95% CI: 1.17-11.02) and 3.58 (95% CI: 0.97-7.71), respectively. The CK18-Asp396/CK18 ratio, which decreased with tumor progression, was also predictive of disease-free survival, with a low ratio (< or = median) associated with worse disease-free survival: HR 2.78 (95% CI: 1.06-7.19). Remarkably, the plasma CK18-Asp396 and total CK18 levels after surgical removal of the tumor were also predictive of disease-free survival, with patients with high levels having a HR of 3.78 (95% CI: 0.77-18.50) and 4.12 (95% CI: 0.84-20.34), respectively, indicating that these parameters can be used also to monitor patients after surgery.ConclusionCK18-Asp396 and total CK18 levels in the circulation of colorectal cancer patients are predictive of tumor progression and prognosis and might be helpful for treatment selection and monitoring of these patients

Identification of a luteinizing hormone-selective determinant in the exodomain of a follicle-stimulating hormone receptor

Mammalian glycoprotein hormone receptors (GpHRs) display a stringent selectivity for their cognate hormones. In contrast, the follicle-stimulating hormone receptor of the African catfish (cfFSHR) is promiscuously activated by catfish luteinizing hormone (cfLH). Glycoprotein hormones bind to the concave site of the cusp-shaped N-terminal GpHR exodomain, which is formed by 9-10 parallel beta-strands. Hence, hormone selectivity of each GpHR for its cognate ligand is defined by amino acid sequence divergence in these beta-strands between different GpHRs. To identify the molecular determinants that allow promiscuous activation of the cfFSHR by cfLH, beta-strands were systematically exchanged between the cfFSHR and the human FSHR. Both gain-of-function and loss-of-function mutational approaches revealed that beta-strand 2 of the cfFSHR contains determinants that contribute to the receptor's responsiveness to cfLH

Applicability of different cell line-derived dendritic cell-like cells in autophagy research

Background and aims: Immortalized cell lines have been long used as substitute for ex vivo murine and human material, but exhibit features that are not found in healthy tissue. True human dendritic cells (DC) cannot be cultured or passaged as opposed to immortalized cell lines. Research in the fields of immunogenic responses and immunotolerance in DCs has increased over the last decade. Autophagy has gained interest in these fields as well, and has been researched extensively in many other cell types as well. Here we have studied the applicability of cell line-derived dendritic cell-like cells of six myeloid cell lines aimed at research focussed on autophagy. Methods: Six myeloid leukaemia cell lines were differentiated towards cell line-derived dendritic cell-like cells (cd-DC) using GM-CSF, IL-4, Ionomycine and PMA: HL60, KG1, MM6, MV-4-11, THP1 and U937. Autophagy was modulated using Rapamycin, Bafilomycin A1 and 3MA. Cell lines were genotyped for autophagy-related SNPs using RFLP. Marker expression was determined with FACS analysis and cytokine profiles were determined using Human Cytometric Bead Assay. Antigen uptake was assessed using Fluoresbrite microspheres. Results and discussion: All researched cell lines harboured SNPs in the autophagy pathways. MM6 and THP1 derived cd-DCs resembled monocyte-derived DCs (moDC) most closely in marker expression, cytokine profiles and autophagy response. The HL60 and U937 cell lines proved least suitable for autophagy-related dendritic cell research. Conclusion: The genetic background of cell lines should be taken into account upon studying (the effects of) autophagy in any cell line. Although none of the studied cell lines recapitulate the full spectrum of DC characteristics, MM6 and THP1 derived cd-DCs are most suitable for autophagy-related research in dendritic cells

Intestinal inflammation in a murine model of autism spectrum disorders

Autism spectrum disorder (ASD) is a cluster of neurodevelopmental disorders characterized by impairments in communication, social interest and stereotypical behaviour. Dysfunction of the intestinal tract is reported in patients with ASD and implicated in the development and severity of ASD symptoms. However, more research is required to investigate the association of intestinal problems with ASD and the potential underlying mechanisms. The purpose of this study was to investigate comorbid symptoms of intestinal inflammation in a murine model of ASD induced by prenatal exposure to valproic acid (VPA). Pregnant BALB/c females were treated subcutaneously with 600. mg/kg VPA or phosphate buffered saline on gestational day 11. Offspring were housed with their mother until weaning on postnatal day 21 (P21). All pups were exposed to a social behaviour test on P28. Inflammatory correlates and activity of the serotonergic system were measured in brain and intestinal tissue. Here we demonstrate, in addition to reduced social behaviour and increased expression of neuroinflammatory markers in the brain, that VPA in utero- exposed male offspring showed epithelial cell loss and neutrophil infiltration in the intestinal tract. Furthermore, reduced levels of serotonin were not only observed the prefrontal cortex and amygdala of VPA in utero- exposed males, but also in the small intestine. Overall, we demonstrate that gender-specific inflammatory conditions are present in the small intestines of VPA in utero- exposed mice and are accompanied by a disturbed serotonergic system in the brain as well as in the intestinal tract. © 2013 Elsevier Inc

New perspective on dextran sodium sulfate colitis: antigen-specific T cell development during intestinal inflammation.

CD4+ T cell responses against oral antigens can develop in inflammatory bowel disease (IBD) patients, which may modulate disease. Dextran sodium sulfate (DSS) colitis is commonly used to study IBD, however, it is not considered the best model in which to study T cell involvement in intestinal disease. Our aim was to determine if antigen-specific T cells could be induced during DSS colitis and if they could be detected after disease resolution. To induce antigen-specific T cells, the tracking antigen, ovalbumin (OVA), was administered orally during colitis initiation. Disease severity was monitored, and the antigen-reactivity of CD4+ T cells examined using CD69 expression. While OVA-directed, CD4+ Foxp3+ regulatory T cells could be detected in the spleens of both OVA-treated control and DSS mice, OVA-reactive, CD4+ Foxp3-T cells were only found in the OVA and DSS-treated mice. These results indicate that during DSS colitis T cells develop that are specific against oral antigens, and they are found systemically after colitis resolution. This gives added depth and utility to the DSS model as well as a way to track T cells that are primed against luminal antigens