Normal platelet activation profile in patients with peripheral arterial disease on aspirin.

BACKGROUND Peripheral arterial disease (PAD) is a progressive vascular disease associated with a high risk of cardiovascular morbidity and death. Antithrombotic prevention is usually applied by prescribing the antiplatelet agent aspirin. However, in patients with PAD aspirin fails to provide protection against myocardial infarction and death, only reducing the risk of ischemic stroke. Platelets may play a role in disease development, but this has not been tested by proper mechanistic studies. In the present study, we performed a systematic evaluation of platelet reactivity in whole blood from patients with PAD using two high-throughput assays, i.e. multi-agonist testing of platelet activation by flow cytometry and multi-parameter testing of thrombus formation on spotted microarrays. METHODS Blood was obtained from 40 patients (38 on aspirin) with PAD in majority class IIa/IIb and from 40 age-matched control subjects. Whole-blood flow cytometry and multiparameter thrombus formation under high-shear flow conditions were determined using recently developed and validated assays. RESULTS Flow cytometry of whole blood samples from aspirin-treated patients demonstrated unchanged high platelet responsiveness towards ADP, slightly elevated responsiveness after glycoprotein VI stimulation, and decreased responsiveness after PAR1 thrombin receptor stimulation, compared to the control subjects. Most parameters of thrombus formation under flow were similarly high for the patient and control groups. However, in vitro aspirin treatment caused a marked reduction in thrombus formation, especially on collagen surfaces. When compared per subject, markers of ADP- and collagen-induced integrin activation (flow cytometry) strongly correlated with parameters of collagen-dependent thrombus formation under flow, indicative of a common, subject-dependent regulation of both processes. CONCLUSION Despite of the use of aspirin, most platelet activation properties were in the normal range in whole-blood from class II PAD patients. These data underline the need for more effective antithrombotic pharmacoprotection in PAD.

DAPK loss in colon cancer tumor buds: implications for migration capacity of disseminating tumor cells.

Defining new therapeutic strategies to overcome therapy resistance due to tumor heterogeneity in colon cancer is challenging. One option is to explore the molecular profile of aggressive disseminating tumor cells. The cytoskeleton-associated Death-associated protein kinase (DAPK) is involved in the cross talk between tumor and immune cells at the invasion front of colorectal cancer. Here dedifferentiated tumor cells histologically defined as tumor budding are associated with a high risk of metastasis and poor prognosis. Analyzing samples from 144 colorectal cancer patients we investigated immunhistochemical DAPK expression in different tumor regions such as center, invasion front, and buds. Functional consequences for tumor aggressiveness were studied in a panel of colon tumor cell lines using different migration, wound healing, and invasion assays. DAPK levels were experimentally modified by siRNA transfection and overexpression as well as inhibitor treatments. We found that DAPK expression was reduced towards the invasion front and was nearly absent in tumor buds. Applying the ECIS system with HCT116 and HCT116 stable lentiviral DAPK knock down cells (HCTshDAPK) we identified an important role for DAPK in decreasing the migratory capacity whereas proliferation was not affected. Furthermore, the migration pattern differed with HCTshDAPK cells showing a cluster-like migration of tumor cell groups. DAPK inhibitor treatment revealed that the migration rate was independent of DAPK's catalytic activity. Modulation of DAPK expression level in SW480 and DLD1 colorectal cancer cells significantly influenced wound closure rate. DAPK seems to be a major player that influences the migratory capability of disseminating tumor cells and possibly affects the dynamic interface between pro- and anti-survival factors at the invasion front of colorectal cancer. This interesting and new finding requires further evaluation.

Human lung-derived mesenchymal stem cell-conditioned medium exerts in vitro antitumor effects in malignant pleural mesothelioma cell lines.

BACKGROUND The soluble factors secreted by mesenchymal stem cells are thought to either support or inhibit tumor growth. Herein, we investigated whether the human lung-derived mesenchymal stem cell-conditioned medium (hlMSC-CM) exerts antitumor activity in malignant pleural mesothelioma cell lines H28, H2052 and Meso4. METHODS hlMSC-CM was collected from the human lung-derived mesenchymal stem cells. Inhibition of tumor cell growth was based on the reduction of cell viability and inhibition of cell proliferation using the XTT and BrdU assays, respectively. Elimination of tumor spheroids was assessed by the anchorage-independent sphere formation assay. The cytokine profile of hlMSC-CM was determined by a chemiluminescence-based cytokine array. RESULTS Our data showed that hlMSC-CM contains a broad range of soluble factors which include: cytokines, chemokines, hormones, growth and angiogenic factors, matrix metalloproteinases, metalloproteinase inhibitors and cell-cell mediator proteins. The 48- and 72-hour hlMSC-CM treatments of H28, H2052 and Meso4 cell lines elicited significant decreases in cell viability and inhibited cell proliferation. The 72-hour hlMSC-CM incubation of H28 cells completely eliminated the drug-resistant sphere-forming cells, which is more potent than twice the half maximal inhibitory concentration of cisplatin. CONCLUSIONS Our findings indicate that the cell-free hlMSC-CM confers in vitro antitumor activities via soluble factors in the tested mesothelioma cells and, hence, may serve as a therapeutic tool to augment the current treatment strategies in malignant pleural mesothelioma.

Potent and broad-spectrum antimicrobial activity of CXCL14 suggests an immediate role in skin infections

The skin is constantly exposed to commensal microflora and pathogenic microbes. The stratum corneum of the outermost skin layer employs distinct tools such as harsh growth conditions and numerous antimicrobial peptides (AMPs) to discriminate between beneficial cutaneous microflora and harmful bacteria. How the skin deals with microbes that have gained access to the live part of the skin as a result of microinjuries is ill defined. In this study, we report that the chemokine CXCL14 is a broad-spectrum AMP with killing activity for cutaneous gram-positive bacteria and Candida albicans as well as the gram-negative enterobacterium Escherichia coli. Based on two separate bacteria-killing assays, CXCL14 compares favorably with other tested AMPs, including human beta-defensin and the chemokine CCL20. Increased salt concentrations and skin-typical pH conditions did not abrogate its AMP function. This novel AMP is highly abundant in the epidermis and dermis of healthy human skin but is down-modulated under conditions of inflammation and disease. We propose that CXCL14 fights bacteria at the earliest stage of infection, well before the establishment of inflammation, and thus fulfills a unique role in antimicrobial immunity.