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Cutaneous T cell lymphomas (CTCLs) represent a heterogeneous group of T cell lymphomas that primarily affect the skin. The most frequent forms of CTCL are mycosis fungoides and Sézary syndrome. Both are characterized by frequent recurrence, developing chronic conditions and high mortality with a lack of a curative treatment. In this study, we evaluated the effect of short-chain, cell-permeable C6 Ceramide (C6Cer) on CTCL cell lines and keratinocytes. C6Cer significantly reduced cell viability of CTCL cell lines and induced cell death via apoptosis and necrosis. In contrast, primary human keratinocytes and HaCaT keratinocytes were less affected by C6Cer. Both keratinocyte cell lines showed higher expressions of ceramide catabolizing enzymes and HaCaT keratinocytes were able to metabolize C6Cer faster and more efficiently than CTCL cell lines, which might explain the observed protective effects. Along with other existing skin-directed therapies, C6Cer could be a novel well-tolerated drug for the topical treatment of CTCL.
Background Bacterial DNA containing motifs of unmethylated CpG dinucleotides (CpG-ODN) initiate an innate immune response mediated by the pattern recognition receptor Toll-like receptor 9 (TLR9). This leads in particular to the expression of proinflammatory mediators such as tumor necrosis factor (TNF-alpha) and interleukin-1beta (IL-1beta). TLR9 is expressed in human and murine pulmonary tissue and induction of proinflammatory mediators has been linked to the development of acute lung injury. Therefore, the hypothesis was tested whether CpG-ODN administration induces an inflammatory response in the lung via TLR9 in vivo. Methods Wild-type (WT) and TLR9-deficient (TLR9-D) mice received CpG-ODN intraperitoneally (1668-Thioat, 1 nmol/g BW) and were observed for up to 6 hrs. Lung tissue and plasma samples were taken and various inflammatory markers were measured. Results In WT mice, CpG-ODN induced a strong activation of pulmonary NFKB as well as a significant increase in pulmonary TNF-alpha and IL-1beta mRNA/protein. In addition, cytokine serum levels were significantly elevated in WT mice. Increased pulmonary content of lung myeloperoxidase (MPO) was documented in WT mice following application of CpG-ODN. Bronchoalveolar lavage (BAL) revealed that CpG-ODN stimulation significantly increased total cell number as well as neutrophil count in WT animals. In contrast, the CpG-ODN-induced inflammatory response was abolished in TLR9-D mice. Conclusion This study suggests that bacterial CpG-ODN causes lung inflammation via TLR9.
Introduction: Sepsis remains associated with a high mortality rate. Endotoxin has been shown to influence viscoelastic coagulation parameters, thus suggesting a link between endotoxin levels and the altered coagulation phenotype in septic patients. This study evaluated the effects of systemic polyspecific IgM-enriched immunoglobulin (IgM-IVIg) (Pentaglobin® [Biotest, Dreieich, Germany]) on endotoxin activity (EA), inflammatory markers, viscoelastic and conventional coagulation parameters.
Methods: Patients with severe sepsis were identified by daily screening in a tertiary, academic, surgical ICU. After the inclusion of 15 patients, the application of IgM-IVIg (5 mg/kg/d over three days) was integrated into the unit’s standard operation procedure (SOP) to treat patients with severe sepsis, thereby generating “control” and “IgM-IVIg” groups. EA assays, thrombelastometry (ROTEM®) and impedance aggregometry (Multiplate®) were performed on whole blood. Furthermore, routine laboratory parameters were determined according to unit’s standards.
Results: Data from 26 patients were included. On day 1, EA was significantly decreased in the IgM-IVIg group following 6 and 12 hours of treatment (0.51 ±0.06 vs. 0.26 ±0.07, p<0.05 and 0.51 ±0.06 vs. 0.25 ±0.04, p<0.05) and differed significantly compared with the control group following 6 hours of treatment (0.26 ±0.07 vs. 0.43 ±0.07, p<0.05). The platelet count was significantly higher in the IgM-IVIg group following four days of IgM-IVIg treatment (200/nl ±43 vs. 87/nl ±20, p<0.05). The fibrinogen concentration was significantly lower in the control group on day 2 (311 mg/dl ±37 vs. 475 mg/dl ±47 (p = 0.015)) and day 4 (307 mg/dl ±35 vs. 420 mg/dl ±16 (p = 0.017)). No differences in thrombelastometric or aggregometric measurements, or inflammatory markers (interleukin-6 (IL-6), leukocyte, lipopolysaccharide binding protein (LBP)) were observed.
Conclusion: Treatment with IgM-enriched immunoglobulin attenuates the EA levels in patients with severe sepsis and might have an effect on septic thrombocytopenia and fibrinogen depletion. Viscoelastic, aggregometric or inflammatory parameters were not influenced.
Hepatocellular carcinoma (HCC) shows a remarkable heterogeneity and is recognized as a chemoresistant tumor with dismal prognosis. In previous studies, we observed significant alterations in the serum sphingolipids of patients with HCC. This study aimed to investigate the in vitro effects of sorafenib, which is the most widely used systemic HCC medication, on the sphingolipid pathway as well as the effects of inhibiting the sphingolipid pathway in HCC. Huh7.5 and HepG2 cells were stimulated with sorafenib, and inhibitors of the sphingolipid pathway and cell proliferation, viability, and concentrations of bioactive metabolites were assessed. We observed a significant downregulation of cell proliferation and viability and a simultaneous upregulation of dihydroceramides upon sorafenib stimulation. Interestingly, fumonisin B1 (FB1) and the general sphingosine kinase inhibitor SKI II were able to inhibit cell proliferation more prominently in HepG2 and Huh7.5 cells, whereas there were no consistent effects on the formation of dihydroceramides, thus implying an involvement of distinct metabolic pathways. In conclusion, our study demonstrates a significant downregulation of HCC proliferation upon sorafenib, FB1, and SKI II treatment, whereas it seems they exert antiproliferative effects independently from sphingolipids. Certainly, further data would be required to elucidate the potential of FB1 and SKI II as putative novel therapeutic targets in HCC.
Hepatitis C virus (HCV) substantially affects lipid metabolism, and remodeling of sphingolipids appears to be essential for HCV persistence in vitro. The aim of the current study is the evaluation of serum sphingolipid variations during acute HCV infection. We enrolled prospectively 60 consecutive patients with acute HCV infection, most of them already infected with human immunodeficiency virus (HIV), and serum was collected at the time of diagnosis and longitudinally over a six-month period until initiation of antiviral therapy or confirmed spontaneous clearance. Quantification of serum sphingolipids was performed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Spontaneous clearance was observed in 11 out of 60 patients (18.3%), a sustained viral response (SVR) in 43 out of 45 patients (95.5%) receiving an antiviral treatment after follow-up, whereas persistence of HCV occurred in six out of 60 patients (10%). C24-ceramide (C24-Cer)-levels increased at follow-up in patients with spontaneous HCV eradication (p < 0.01), as compared to baseline. Sphingosine and sphinganine values were significantly upregulated in patients unable to clear HCV over time compared to patients with spontaneous clearance of HCV infection on follow-up (p = 0.013 and 0.006, respectively). In summary, the persistence of HCV after acute infection induces a downregulation of C24Cer and a simultaneous elevation of serum sphingosine and sphinganine concentrations.
Background: Sphingolipids constitute bioactive molecules with functional implications in liver homeostasis. Particularly, ablation of very long chain ceramides in a knockout mouse model has been shown to cause a severe hepatopathy.
Methods: We aimed to evaluate the serum sphingolipid profile of 244 patients with cirrhosis prospectively followed for a median period of 228±217 days via mass spectrometry.
Results: We thereby observed a significant decrease of long and very long chain ceramides, particularly of C24ceramide, in patients with increasing severity of cirrhosis (p<0.001). Additionally, hydropic decompensation, defined by clinical presentation of ascites formation, was significantly correlated to low C24ceramide levels (p<0.001) while a significant association to hepatic decompensation and poor overall survival was observed for low serum concentrations of C24ceramide (p<0.001) as well. Multivariate analysis further identified low serum C24ceramide to be independently associated to overall survival (standard beta = -0.001, p = 0.022).
Conclusions: In our current analysis serum levels of very long chain ceramides show a significant reciprocal correlation to disease severity and hepatic decompensation and are independently associated with overall survival in patients with cirrhosis. Serum sphingolipid metabolites and particularly C24ceramide may constitute novel molecular targets of disease severity, hepatic decompensation and overall prognosis in cirrhosis and should be further evaluated in basic research studies.
Ceramides induce important intracellular signaling pathways, modulating proliferation, migration, apoptosis, and inflammation. However, the relevance of the ceramide metabolism in the reconvalescence phase after stroke is unclear. Besides its well-known property as a selective serotonin reuptake inhibitor, fluoxetine has been reported to inhibit the acid sphingomyelinase (ASM), a key regulator of ceramide levels which derives ceramide from sphingomyelin. Furthermore, fluoxetine has shown therapeutic potential in a randomized controlled rehabilitation trial in stroke patients. Our aim was to investigate and modulate ceramide concentrations in the peri-infarct cortex, whose morphological and functional properties correlate with long-term functional outcome in stroke. We show that certain ceramide species are modulated after experimental stroke and that these changes do not result from alterations of ASM activity, but rather from nontranscriptional induction of the ceramide de novo pathway. Unexpectedly, although reducing lesion size, fluoxetine did not improve functional outcome in our model and had no significant influence on ASM activity or the concentration of ceramides. The ceramide metabolism could emerge as a potential therapeutic target in the reconvalescence phase after stroke, as its accumulation in the peri-infarct cortex potentially influences membrane functions as well as signaling events in the tissue essential for neurological recovery.
Introduction: Systemic inflammation (e.g. following surgery) involves Toll-like receptor (TLR) signaling and leads to an endocrine stress response. This study aims to investigate a possible influence of TLR2 and TLR4 single nucleotide polymorphisms (SNPs) on perioperative adrenocorticotropic hormone (ACTH) and cortisol regulation in serum of cardiac surgical patients. To investigate the link to systemic inflammation in this context, we additionally measured 10 different cytokines in the serum. Methods: 338 patients admitted for elective cardiac surgery were included in this prospective observational clinical cohort study. Genomic DNA of patients was screened for TLR2 and TLR4 SNPs. Serum concentrations of ACTH, cortisol, interferon (IFN)-, interleukin (IL)-1, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, tumor necrosis factor (TNF)- and granulocyte macro-phage-colony stimulating factor (GM-CSF) were determined before surgery, immediately post surgery and on the first postoperative day. Results: 13 patients were identified as TLR2 SNP carrier, 51 as TLR4 SNP carrier and 274 pa-tients as non-carrier. Basal levels of ACTH, cortisol and cytokines did not differ between groups. In all three groups a significant, transient perioperative rise of cortisol could be ob-served. However, only in the non-carrier group this was accompanied by a significant ACTH rise, TLR4 SNP carriers had significant lower ACTH levels compared to non-carriers ((mean[95% confidence intervals]) non-carriers: 201.9[187.7 to 216.1]pg/ml; TLR4 SNP car-riers: 149.9[118.4 to 181.5]pg/ml; TLR2 SNP carriers: 176.4[110.5 to 242.3]pg/ml). Compared to non-carriers, TLR4 SNP carriers showed significant lower serum IL-8, IL-10 and GM-CSF peaks ((mean[95% confidence intervals]): IL-8: non-carriers: 42.6[36.7 to 48.5]pg/ml, TLR4 SNP carriers: 23.7[10.7 to 36.8]pg/ml; IL-10: non-carriers: 83.8[70.3 to 97.4]pg/ml, TLR4 SNP carriers: 54.2[24.1 to 84.2]pg/ml; GM-CSF: non-carriers: 33.0[27.8 to 38.3]pg/ml, TLR4 SNP carriers: 20.2[8.6 to 31.8]pg/ml). No significant changes over time or between the groups were found for the other cytokines. Conclusions: Regulation of the immunoendocrine stress response during systemic inflamma-tion is influenced by the presence of a TLR4 SNP. Cardiac surgical patients carrying this ge-notype showed decreased serum concentrations of ACTH, IL-8, IL-10 and GM-CSF. This finding might have impact on interpreting previous and designing future trials on diagnosing and modulating immunoendocrine dysregulation (e.g. adrenal insufficiency) during systemic inflammation and sepsis.
Introduction: It has been proposed that individual genetic variation contributes to the course of severe infections and sepsis. Recent studies of single nucleotide polymorphisms (SNPs) within the endotoxin receptor and its signaling system showed an association with the risk of disease development. This study aims to examine the response associated with genetic variations of TLR4, the receptor for bacterial LPS, and a central intracellular signal transducer (TIRAP/Mal) on cytokine release and for susceptibility and course of severe hospital acquired infections in distinct patient populations. Methods: Three intensive care units in tertiary care university hospitals in Greece and Germany participated. 375 and 415 postoperative patients and 159 patients with ventilator associated pneumonia (VAP) were included. TLR4 and TIRAP/Mal polymorphisms in 375 general surgical patients were associated with risk of infection, clinical course and outcome. In two prospective studies, 415 patients following cardiac surgery and 159 patients with newly diagnosed VAP predominantly caused by Gram-negative bacteria were studied for cytokine levels in-vivo and after ex-vivo monocyte stimulation and clinical course. Results: Patients simultaneously carrying polymorphisms in TIRAP/Mal and TLR4 and patients homozygous for the TIRAP/Mal SNP had a significantly higher risk of severe infections after surgery (odds ratio (OR) 5.5; confidence interval (CI): 1.34 - 22.64; P = 0.02 and OR: 7.3; CI: 1.89 - 28.50; P < 0.01 respectively). Additionally we found significantly lower circulating cytokine levels in double-mutant individuals with ventilator associated pneumonia and reduced cytokine production in an ex-vivo monocyte stimulation assay, but this difference was not apparent in TIRAP/Mal-homozygous patients. In cardiac surgery patients without infection, the cytokine release profiles were not changed when comparing different genotypes. Conclusions: Carriers of mutations in sequential components of the TLR signaling system may have an increased risk for severe infections. Patients with this genotype showed a decrease in cytokine release when infected which was not apparent in patients with sterile inflammation following cardiac surgery.
Sphingolipids are characterized by a broad range of bioactive properties. Particularly, the development of insulin resistance, a major pathophysiological hallmark of Type 2 Diabetes mellitus (T2D), has been linked to ceramide signaling. Since vitamin D supplementation may slow down T2D progression by improving glucose concentrations and insulin sensitivity, we investigated whether vitamin D supplementation impacts on plasma sphingolipid levels in T2D patients. Thus, plasma samples of 59 patients with non-insulin-requiring T2D from a placebo-controlled, randomized, and double-blind study were retrospectively analyzed. Once per week, patients received either 20 drops of Vigantol oil, corresponding to a daily dose of 1904 IU/d vitamin D (verum: n = 31), or a placebo oil consisting of medium chain triglycerides (placebo: n = 28). Blood samples were taken from all of the participants at three different time points: 1) at the beginning of the study (baseline), 2) after 6 months supplementation, and 3) after an additional 6 months of follow-up. Plasma sphingolipids were measured by high-performance liquid chromatography tandem mass spectrometry. At baseline and 6 months follow-up, no significant differences in plasma sphingolipid species were detected between the placebo and verum groups. After 6 months, vitamin D supplementation significantly enhanced plasma C18dihydroceramide (dhCer; N-stearoyl-sphinganine (d18:0/18:0)) and C18ceramide (Cer; N-stearoyl-sphingosine (d18:1/18:0)) levels were observed in the verum group compared to the placebo group. This was accompanied by significantly higher 25-hydroxyvitamin D3 (25(OH)D3) blood levels in patients receiving vitamin D compared to the placebo group. Taken together, vitamin D supplementation induced changes of the C18 chain-length-specific dhCer and Cer plasma levels in patients with T2D. The regulation of sphingolipid signaling by vitamin D may thus unravel a novel mechanism by which vitamin D can influence glucose utilization and insulin action. Whether this acts favorably or unfavorably for the progression of T2D needs to be clarified.