Topical use of amitriptyline and linoleic acid to restore ceramide rheostat in atopic dermatitis lesions - a case report.

Rebuilding, stabilizing and maintaining the dermal lipid barrier is an encouraging disease management concept (relief and care) in the treatment and prevention of atopic dermatitis. Prevention and topical treatment, however, lack a simple, safe, effective and modular approach. For decades, the mainstay of topical therapy of atopic dermatitis has been corticosteroids, with innovations being rare. Our case report demonstrates the struggle of a patient with little relief of itchy dermal lesions and the recurrence of skin lesions following current therapeutic guidelines which proved to be ineffective. Therefore we decided to try an advanced C16-ceramide pathomechanism derived topical therapeutic measure since it offers hope of re-establishing skin and alleviating suffering. Amitriptyline in combination with linoleic acid offers a chance to release from dry and itchy skin, mild to moderate atopic dermatitis lesions without known serious adverse effects of topical corticosteroids, while preventing recurrence.

Molecular characterization of EZH2 mutant patients with myelodysplastic/myeloproliferative neoplasms.

Mutations in the epigenetic regulator gene EZH2 are frequently observed in patients with myelodysplastic/myeloproliferative neoplasms (MDS/MPN; 10-13%) and are associated with a poor outcome. To gain more insight into EZH2 pathology, we sought to genetically characterize a cohort of 41 EZH2-mutated MDS/MPN patients using targeted deep next-generation sequencing (NGS), colony-forming progenitor assays and transcriptome analysis. Stable short hairpin RNA (shRNA)-mediated downregulation of EZH2 was performed in MDS-derived F-36P, MOLM-13 and OCI-M2 cells to study EZH2-specific changes. Targeted NGS revealed a complex pattern of mutations with a total of 190 individual mutations. EZH2 mutations frequently co-occur with TET2 (58%), RUNX1 (40%) and ASXL1 (34%) mutations. Colony assays indicated EZH2 mutations to be mostly early events in leukemogenesis and showed a complex mutational hierarchy. Gene expression data revealed a number of differently expressed genes between EZH2 wild-type and mutant patients including known EZH2 targets. Comparison of patient transcriptome to EZH2-downregulated cell line data revealed several genes as novel EZH2 targets, showing opposite as well as unidirectional regulation between cell lines and patients. Some genes, such as CXXC5, ETS1 and VAV3 have previously been implied to have a role in leukemogenesis. Their precise role in MDS/MPN needs to be further investigated.

Transcriptomic and proteomic patterns of systemic inflammation in on-pump and off-pump coronary artery bypass grafting.

BACKGROUND: Coronary artery bypass grafting (CABG) using cardiopulmonary bypass (CPB) provides controlled operative conditions but induces a whole-body inflammatory response capable of initiating devastating morbidity and mortality. Although technically more demanding, deliberate avoidance of CPB in off-pump surgery attenuates the physiological insult associated with CABG. METHODS AND RESULTS: To systematically assess the molecular mechanisms underlying the better-preserved remote organ function, we studied gene expression patterns in leukocytes and plasma proteomic response to on-pump and off-pump CABG. Proteomic analysis confirmed (tumor necrosis factor-alpha, interleukin [IL]-6, IL-10) and expanded (eg, interferon [IFN]-gamma, granulocyte colony-stimulating factor [G-CSF], monocyte chemotactic protein-1, macrophage inflammatory protein-1beta) the mediators released on CPB, whereas blood leukocyte transcriptomics suggested that circulating leukocytes are not primarily responsible for this response. Interestingly, release of some cytokines (eg, IL-6, IFN-gamma, G-CSF) was observed on off-pump surgery to a similar extent but with delayed kinetics. A total of 45 of 4868 transcripts were identified to be significantly altered as a result of initiation of CPB. Systematic analysis of transcriptional activation by CPB revealed primarily genes involved in inflammation-related cell-cell communication (such as L-selectin or intercellular adhesion molecule-2) and signaling (such as IL-1, IL-8, or IL-18 receptors and toll-like receptors 4, 5, and 6), thus confirming a "primed" phenotype of circulating peripheral blood mononuclear cells. CONCLUSIONS: Gene array and multiplex protein analysis, only in concert, can illuminate the molecular mechanisms responsible for systemic sequelae of CPB and indicate that circulating leukocytes overexpress adhesion and signaling factors after contact with CPB, which potentially facilitates their trapping, eg, in the lungs and may promote a subsequent tissue-associated inflammatory response.

Stereospecific induction of apoptosis in tumor cells via endogenous C16-ceramide and distinct transcripts.

Concentration and distribution of individual endogenous ceramide species is crucial for apoptosis induction in response to various stimuli. Exogenous ceramide analogs induce apoptosis and can in turn modify the composition/concentrations of endogenous ceramide species and associated signaling. In this study, we show here that the elevation of endogenous C16-ceramide levels is a common feature of several known apoptosis-inducing triggers like mmLDL, TNF-alpha, H2O2 and exogenous C6-ceramide. Vice versa apoptosis requires elevation of endogenous C16-ceramide levels in cells. Enantiomers of a synthetic ceramide analog HPL-1RS36N have been developed as probes and vary in their capacity to inducing apoptosis in macrophages and HT-29 cells. Apoptosis induction by the two synthetic ceramide analogs HPL-39N and HPL-1R36N correlates with generation of cellular C16-ceramide concentration. In contrast to the S-enantiomer HPL-1S36N, the R-enantiomer HPL-1R36N shows significant effects on the expression of distinct genes known to be involved in cell cycle, cell growth and cell death (CXCL10, CCL5 and TNF-alpha), similarly on apoptosis induction. Enantioselective effects on transcription induced by metabolically stable synthetic probes provide clues on molecular mechanisms of ceramide-induced signaling, as well as leads for future anti-cancer agents.

Ceramide induces aSMase expression: implications for oxLDL-induced apoptosis.

Sphingomyelinase (SMase) stimulation and subsequent ceramide generation are suggested to be involved in signal transduction of stress-induced apoptosis. We now show that apoptosis of human macrophages (MPhi) and fibroblasts initiated by oxidized low density lipoproteins (minimally modified LDL, mmLDL) is associated with an increase in acid SMase (aSMase, E.C. 3.1.4.12) expression and ceramide concentration. Application of a novel, potent, and specific inhibitor of aSMase expression (NB6) diminished the effects of mmLDL and C6-ceramide treatment by inhibiting transcription via Sp1 and AP-2. Moreover, apoptosis was abolished after mmLDL and C6-ceramide treatment of hereditary aSMase-deficient fibroblasts (from Niemann-Pick patients). We suggest that in mmLDL-initiated apoptosis 1) enhanced ceramide generation via aSMase appears to be required as well as 2) a positive feedback control of aSMase expression by the increase in intracellular ceramide concentration.