Dan Brudzewsky

Molecular profiling from liquid biopsy, in particular cell-free DNA (cfDNA), represents an attractive alternative to tissue biopsies for the detection of actionable targets and tumor monitoring. In addition to PCR-based assays, Next Generation Sequencing (NGS)-based cfDNA assays are now commercially available and are being increasingly adopted in clinical practice. However, the validity of these products as well as the clinical utility of cfDNA in the management of patients with cancer has yet to be proven. Within framework of the Innovative Medicines Initiative (IMI) program CANCER-ID we evaluated the use of commercially available reference materials designed for ctDNA testing and cfDNA derived from Diagnostic Leukaphereses (DLA) for inter- and intra-assay as well as intra- and inter-laboratory comparisons. In three experimental setups, a broad range of assays including ddPCR, MassARRAY and various NGS-based assays were tested. We demonstrate that both reference materials with predetermined VAFs and DLA samples are extremely useful for the performance assessment of mutation analysis platforms. Moreover, our data indicate a substantial variability of NGS assays with respect to sensitivity and specificity highlighting the importance of extensive validation of the test performance before offering these tests in clinical routine practice.

Ascorbate is an important antioxidant, which also displays important functions in neuronal tissues, including the retina. The retina is responsible for the initial steps of visual processing, which is further refined in cerebral high-order centers. The retina is also a prototypical model for studying physiologic aspects of cells that comprise the nervous system. Of major importance also is the cellular messenger nitric oxide (NO). Previous studies have demonstrated the significance of NO for both survival and proliferation of cultured embryonic retinal cells. Cultured retinal cells express a high-affinity ascorbate transporter, and the release of ascorbate is delicately regulated by ionotropic glutamate receptors. Therefore, we proposed whether there is interplay between the ascorbate transport system and NO signaling pathway in retinal cells. Here we show compelling evidence that ascorbate uptake is tightly controlled by NO and its downstream signaling pathway in culture. NO also modulates the expression of SVCT-2, an effect mediated by cGMP and PKG. Kinetic studies suggest that NO increases the transport capacity for ascorbate, but not the affinity of SVCT-2 for its substrate. Interestingly, NO utilizes the NF-κB pathway, in a PKG-dependent manner, to modulate both SVCT-2 expression and ascorbate uptake. These results demonstrate that NO exerts a fine-tuned control of the availability of ascorbate to cultured retinal cells and strongly reinforces ascorbate as an important bioactive molecule in neuronal tissues.

Neuropeptide Y (NPY) is expressed in mammalian retina but the location and potential modulatory effects of NPY receptor activation remain largely unknown. Retinal ganglion cell (RGC) death is a hallmark of several retinal degenerative diseases, particularly glaucoma. Using purified RGCs and ex vivo rat retinal preparations, we have measured RGC intracellular free calcium concentration ([Ca2+]i) and RGC spiking activity, respectively. We found that NPY attenuated the increase in the [Ca2+]i triggered by glutamate mainly via Y1 receptor activation. Moreover, (Leu31, Pro34)-NPY, a Y1/Y5 receptor agonist, increased the initial burst response of OFF-type RGCs, although no effect was observed on RGC spontaneous spiking activity. The Y1 receptor activation was also able to directly modulate RGC responses by attenuating the NMDA-induced increase in RGC spiking activity. These results suggest that Y1 receptor activation, at the level of inner or outer plexiform layers, leads to modulation of RGC receptive field properties. Using in vitro cultures of rat retinal explants exposed to NMDA, we found that NPY pretreatment prevented NMDA-induced cell death. However, in an animal model of retinal ischemia-reperfusion injury, pretreatment with NPY or (Leu31, Pro34)-NPY was not able to prevent apoptosis or rescue RGCs. In conclusion, we found modulatory effects of NPY application that for the first time were detected at the level of RGCs. However, further studies are needed to evaluate whether NPY neuroprotective actions detected in retinal explants can be translated into animal models of retinal degenerative diseases.

Chemokines are small proteins involved in the direction of migration of immune cells both during normal homeostasis and inflammation. Chemokines have been implicated in the pathology of many different inflammatory disorders and are therefore appealing therapeutic targets. Using a chemokine/chemokine receptor-specific gene expression profiling system of 67 genes, the authors have determined the expression profile of chemokine and chemokine receptor genes in the rectum of colitic mice and in mice that have been protected fromcolitis by CD4CD25 regulatory T cells. In mice protected from colitis, the authors found down regulation of the mRNA expression of the inflammatory chemokine receptors CCR1 and CXCR3 and their ligands CXCL9, CXCL10, CCL5, and CCL7. Also the transcripts for CCR9, CCL25, CCL17, and CXCL1 are found down regulated in protected compared with colitic animals. In addition, the authors' results suggest that CCL20 is used by CCR6 regulatory T cells in the complex process of controlling colitis because transcripts for this chemokine were expressed to a higher level in protected animals. The chemokine pathways identified in the present study may be of importance for the development of new targets for anti-inflammatory treatment strategies in human inflammatory bowel disease.

Inflammatory bowel disease (IBD) is a multifactorial disorder with an unknown aetiology. The aim of this study is to employ a murine model of IBD to identify pathways and genes, which may play a key role in the pathogenesis of IBD and could be important for discovery of new disease markers in human disease. Here, we have investigated severe combined immunodeficient (SCID) mice, which upon adoptive transfer with concanavalin A-activated CD4(+) T cells develop inflammation of the colon with predominance in rectum. Mice with increasing level of inflammation was studied. RNA from rectum of transplanted and non-transplanted SCID mice was investigated by a genome-wide gene expression analysis using the Affymetrix mouse expression array 430A (MOE430A) including 22,626 probe sets. A significant change in gene expression (P = 0.00001) is observed in 152 of the genes between the non-transplanted control mice and colitis mice, and among these genes there is an overrepresentation of genes involved in inflammatory processes. Some of the most significant genes showing higher expression encode S100A proteins and chemokines involved in trafficking of leucocytes in inflammatory areas. Classification by gene clustering based on the genes with the significantly altered gene expression corresponds to two different levels of inflammation as established by the histological scoring of the inflamed rectum. These data demonstrate that this SCID T-cell transfer model is a useful animal model for human IBD and can be used for suggesting candidate genes involved in the pathogenesis and for identifying new molecular markers of chronic inflammation in human IBD.