The lysosomal degradation of ceramide is catalyzed by acid ceramidase and requires sphingolipid activator proteins (SAP) as cofactors in vivo. The aim of this study was to investigate how ceramide is hydrolyzed by acid ceramidase at the water-membrane interface in the presence of sphingolipid activator proteins in a liposomal assay system. The degradation of membrane-bound ceramide was significantly increased both in the absence and presence of SAP-D when anionic lysosomal phospholipids such as bis (monoacylglycero)

Ceramides which arise in part from the breakdown of sphingomyelin comprise a class of antiproliferative lipids and have been implicated in the regulation of programmed cell death better known as apoptosis. In the present study, two new synthetic ceramide analogues, N‐thioacetylsphingosine and FS‐5, were used in Molt 4 cells to induce cell death. Besides their cytotoxic effects at concentrations ≥14 μM the data obtained clearly show that both analogues induced apoptosis at concentrations below this critical concentration as assessed by trypan blue exclusion and cleavage of the death substrate poly‐(ADP‐ribose) polymerase (PARP). Additional experiments in bcl‐2‐transfected Molt 4 cells revealed that the apoptotic but not the lytic effects of the analogues were antagonized by the apoptosis inhibitor Bcl‐2. Furthermore, neither N‐thio‐acetylsphingosine nor FS‐5 induced PARP cleavage in bcl‐2‐transfected Molt 4 cells indicating that the induction of apoptotic cell death by cell permeable ceramides is not due to unspecific disturbance of the cell membrane.

Prognosis of patients with advanced extrahepatic cholangiocarcinoma (eCCA) is poor. The current standard first-line treatment is systemic chemotherapy (CT) with gemcitabine and a platinum derivate. Additionally, endobiliary radiofrequency ablation (eRFA) can be applied to treat biliary obstructions. This study aimed to evaluate the additional benefit of scheduled regular eRFA in a real-life patient cohort with advanced extrahepatic cholangiocarcinoma under standard systemic CT. All patients with irresectable eCCA treated at University Hospital Bonn between 2010 and 2020 were eligible for inclusion. Patients were stratified according to treatment: standard CT (n = 26) vs. combination of eRFA with standard CT (n = 40). Overall survival (OS), progression free survival (PFS), feasibility and toxicity were retrospectively analyzed using univariate and multivariate approaches. Combined eRFA and CT resulted in significantly longer median OS (17.3 vs. 8.6 months, p = 0.004) and PFS (12.9 vs. 5.7 months, p = 0.045) compared to the CT only group. While groups did not differ regarding age, sex, tumor stage and chemotherapy treatment regimen, mean MELD was even higher (10.1 vs. 6.7, p = 0.015) in the eRFA + CT group. The survival benefit of concomitant eRFA was more evident in the subgroup with locally advanced tumors. Severe hematological toxicities (CTCAE grades 3 – 5) did not differ significantly between the groups. However, therapy-related cholangitis occurred more often in the combined treatment group (p = 0.031). Combination of eRFA and systemic CT was feasible, well-tolerated and could significantly prolong survival compared to standard CT alone. Thus, eRFA should be considered during therapeutic decision making in advanced eCCA.

We have studied the incorporation of [ 14 C]serine and of [ 3 H]sphingosine into sphingomyelin in the presence or absence of brefeldin A (BFA) in three different cell types. Administration of BFA (1 W Wg/ml) to fibroblasts for 24 h increased the incorporation of label into sphingomyelin 1.5^3 fold compared with untreated controls. In contrast, BFA strongly decreased sphingomyelin biosynthesis (4^5 fold) in cerebellar neurons as well as in neuroblastoma cells. The effect of BFA on glycosphingolipid formation, however, was similar in all three cell types studied: an increased labeling of the precursor glycolipids GlcCer, LacCer, GM3 and GD3 was paralleled by a decreased formation of complex gangliosides, GM1, GD1a, GT1b and GQ1b. Our data therefore suggest that in neuronal cells sphingomyelin synthesis, like the formation of complex gangliosides, is localized primarily distal to the BFA block, in a postGolgi compartment, most probably the trans-Golgi network, whereas in fibroblasts sphingomyelin biosynthesis is mainly localized prior to the BFA block, in the Golgi apparatus, as has been shown for LacCer, GlcCer, GM3 and GD3 synthases. ß

Introduction Gastrointestinal (GI) malignancies, such as cholangiocarcinoma, pancreatic carcinoma, and metastatic colorectal carcinoma, have a poor prognosis and effective therapeutic approaches are still challenging. Checkpoint inhibition with PD-1 or PDL-1 antibodies revealed promising results in different tumor entities; however, only few patients with GI tumors can potentially benefit from PD1/PDL1 inhibiting immunotherapy. Further immunotherapeutic strategies for GI malignancies are urgently needed. The aim of this study was to demonstrate that in vitro activation of the immune checkpoint CD40/CD40L can improve DC action towards bile duct, pancreas, and colorectal carcinoma. Methods Human DC were isolated from buffy coats from healthy donors, pulsed with tumor lysates and then transduced with adenoviruses encoding human CD40L (Ad-hCD40L). Using transwell assays, the effects of (m)CD40L on DC immunoactivation compared to (s)CD40L were analyzed. Surface marker and cytokine/chemokine expression were measured by flow cytometry, ELISA and cytokine arrays. Capacity of Ad-hCD40L-transduced DC to induce tumor-specific effector cells was tested using MTT proliferation assay and cytotoxicity assays. Apoptosis induction on tumor cells after culturing with supernatants of Ad-hCD40L-transduced DC was analyzed by flow cytometry. Results Ad-hCD40L transduction induced a high expression of (s)CD40L and (m)CD40L on DC and seemed to induce a strong cellular CD40/CD40L interaction among DC, leading to the formation of cell aggregates. Due to the CD40/CD40L interaction, a significant upregulation of DC maturation markers and a Th1-shift on cytokines/chemokines in the supernatant of DC were achieved. Interestingly, a pure Th1-shift was only achieved, when a cellular CD40/CD40L interaction among DC took place. (s)CD40L induced almost no upregulation of maturation markers and rather resulted in a Th2-cytokine expression, such as IL-10. Correspondingly, (m)CD40L-expressing DC led to significant proliferation and stimulation of tumor-specific effector cells with increased cytotoxicity towards pancreatic, bile duct and colorectal tumor cells. Supernatants of Ad-hCD40L-transduced DC could also induce apoptosis in the different tumor cells in vitro. Conclusion Stimulation of the immune checkpoint CD40L/CD40 by endogenous expression of (m)CD40L provokes a cellular interaction, which increases the immunomodulatory capacity of DC. A Th1 cytokine/chemokine expression is induced, leading to a significant proliferation and enabling cytotoxicity of effector cells towards human bile duct, pancreatic and colorectal tumor cells. The present data point to the promising approach for DC-based immunotherapy of gastrointestinal malignances by activating the CD40/CD40L immune checkpoint.