Low expression of pro-apoptotic proteins Bax, Bak and Smac indicates prolonged progression-free survival in chemotherapy-treated metastatic melanoma.

Despite the introduction of novel targeted therapies, chemotherapy still remains the primary treatment for metastatic melanoma in poorly funded healthcare environments or in case of disease relapse, with no reliable molecular markers for progression-free survival (PFS) available. As chemotherapy primarily eliminates cancer cells by apoptosis, we here evaluated if the expression of key apoptosis regulators (Bax, Bak, Bcl-2, Bcl-xL, Smac, Procaspase-9, Apaf-1, Procaspase-3 and XIAP) allows prognosticating PFS in stage III/IV melanoma patients. Following antibody validation, marker expression was determined by automated and manual scoring of immunohistochemically stained tissue microarrays (TMAs) constructed from treatment-naive metastatic melanoma biopsies. Interestingly and counter-intuitively, low expression of the pro-apoptotic proteins Bax, Bak and Smac indicated better prognosis (log-rank p < 0.0001, p = 0.0301 and p = 0.0227 for automated and p = 0.0422, p = 0.0410 and p = 0.0073 for manual scoring). These findings were independently validated in the cancer genome atlas (TCGA) metastatic melanoma cohort (TCGA-SKCM) at transcript level (log-rank p = 0.0004, p = 0.0104 and p = 0.0377). Taking expression heterogeneity between the markers in individual tumour samples into account allowed defining combinatorial Bax, Bak, Smac signatures that were associated with significantly increased PFS (p = 0.0002 and p = 0.0028 at protein and transcript level, respectively). Furthermore, combined low expression of Bax, Bak and Smac allowed predicting prolonged PFS (> 12 months) on a case-by-case basis (area under the receiver operating characteristic curve (ROC AUC) = 0.79). Taken together, our results therefore suggest that Bax, Bak and Smac jointly define a signature with potential clinical utility in chemotherapy-treated metastatic melanoma.

Systemic IL-1ß production as a consequence of corneal HSV-1 infection-contribution to the development of herpes simplex keratitis.

This study sought to identify potential therapeutic targets in herpes simplex keratitis (HSK) patients with active and inactive infection by investigating peripheral cytokine production. Peripheral blood mononuclear cells (PBMCs) and serum were prepared from healthy controls and HSK patients during active infection or following treatment (inactive infection). Serum antibody titres were determined by ELISA. Protein expression levels were analysed by Western blot. Cytokine levels were determined by multiplex ELISA. Active corneal herpes simplex virus type 1 (HSV-1) infection resulted in significantly elevated peripheral levels of IL-1ß in HSK patients compared to healthy controls, and remained significantly increased following treatment. Elevated production of IL-1ß in inactive patients was associated with significantly increased levels of IRF3 and STAT1, key proteins involved in promoting anti-viral immune responses. Our data suggest that inflammation persists beyond the period that it is clinically evident and that enhanced peripheral production of IL-1ß may have implications for HSV-1 viral clearance in active and inactive HSK patients.

The Formation of Microthrombi in Parenchymal Microvessels after Traumatic Brain Injury Is Independent of Coagulation Factor XI.

Microthrombus formation and bleeding worsen the outcome after traumatic brain injury (TBI). The aim of the current study was to characterize these processes in the brain parenchyma after experimental TBI and to determine the involvement of coagulation factor XI (FXI). C57BL/6 mice (n = 101) and FXI-deficient mice (n = 15) were subjected to controlled cortical impact (CCI). Wild-type mice received an inhibitory antibody against FXI (14E11) or control immunoglobulin G 24 h before or 30 or 120 min after CCI. Cerebral microcirculation was visualized in vivo by 2-photon microscopy 2-3 h post-trauma and histopathological outcome was assessed after 24 h. TBI induced hemorrhage and microthrombus formation in the brain parenchyma (p < 0.001). Inhibition of FXI activation or FXI deficiency did not reduce cerebral thrombogenesis, lesion volume, or hemispheric swelling. However, it also did not increase intracranial hemorrhage. Formation of microthrombosis in the brain parenchyma after TBI is independent of the intrinsic coagulation cascade since it was not reduced by inhibition of FXI. However, since targeting FXI has well-established antithrombotic effects in humans and experimental animals, inhibition of FXI could represent a reasonable strategy for the prevention of deep venous thrombosis in immobilized patients with TBI.

Prognostic and predictive biomarkers in melanoma: an update.

Malignant melanoma is one of the most aggressive cancers. Several new therapeutic strategies that focus on immuno- and/or targeted therapy have been developed, which have entered clinical trials or already been approved. This review provides an update on prognostic and predictive biomarkers in melanoma that may be used to improve the clinical management of patients. Prognostic markers include conventional histopathological characteristics, chromosomal aberrations, gene expression patterns and miRNA profiles. There is a trend towards multi-marker assays and whole-genome molecular screening methods to determine the prognosis of individual patients. Predictive biomarkers, including targeted components of signal transduction, developmental or transcriptional pathways, can be used to determine patient response towards a particular treatment or combination thereof. The rapid evolution of sequencing technologies and multi-marker screening will change the spectrum of patients who become candidates for therapeutic agents, and in addition create new ethical and regulatory challenges.

Platelet signalling networks: pathway perturbation demonstrates differential sensitivity of ADP secretion and fibrinogen binding.

Platelet signalling responses to single agonists have been identified previously. However, a model of the total platelet signalling network is still lacking. In order to gain insights into this network, we explored the effects of a range of platelet-function inhibitors in two independent assays of platelet function, namely fibrinogen binding and ADP secretion. In this study, we targeted the intracellular signalling molecules targeted intracellular signalling molecules, Syk and PI3K and targeted intracellular signalling molecules, Syk and PI3K, the prostaglandin synthesis enzyme cyclooxygenase, surface receptors for TxA(2) and ADP (P2Y1 and P2Y12) and the integrin cell adhesion molecule, αIIbß3. We demonstrate that the platelet responses of fibrinogen binding and secretion can be differentially affected by the individual inhibitors permitting the generation of a model delineating novel regulatory links in the platelet signalling network. Importantly, the model illustrates the interconnections among portions that are traditionally studied as separate modules, promoting a more integrated view of the platelet.

Electrochemical desorption of fibrinogen from gold.

The electrochemically induced desorption of Oregon green labeled fibrinogen layers from clean gold surfaces at negative potentials has been probed using capacitance, fluorescence microscopy, and atomic force microscopy. Capacitance measurements on fibrinogen layers indicate that desorption occurs at potentials more negative than -0.8 V and that complete desorption occurs when the electrode is biased at -1.2 V. Significantly, the fluorescence intensity initially increases as the dye labeled protein is electrochemically desorbed due to a decrease in quenching by the gold surface. Following this initial increase, the protein diffuses into solution and the fluorescence intensity decreases over time. More than 90% of the dye labeled fibrinogen is desorbed and diffuses out of the confocal volume in less than 2000 s when the potential is stepped to -1.2 V. AFM before and after application of the desorbing potential confirms removal of the protein. Collection of the desorbed protein in solution reveals a surface coverage of (4.0 +/- 2.3) x 10(-13) mol cm(-2) or an area of occupation of 400 +/- 140 nm(2) per molecule, which indicates that the protein is not extensively spread on the bare gold surface. Significantly, SDS-PAGE analysis indicates that the adsorption-desorption cycle dramatically effects the protein structure, with the electrochemically desorbed fibrinogen showing extensive fragmentation compared to native protein.