GD2 Expression in Medulloblastoma and Neuroblastoma for Personalized Immunotherapy: A Matter of Subtype.

Neuroblastoma (NBL) and medulloblastoma (MB) are aggressive pediatric cancers which can benefit from therapies targeting gangliosides. Therefore, we compared the ganglioside profile of 9 MB and 14 NBL samples by thin layer chromatography and mass spectrometry. NBL had the highest expression of GD2 (median 0.54 nmol GD2/mg protein), and also expressed complex gangliosides. GD2-low samples expressed GD1a and were more differentiated. MB mainly expressed GD2 (median 0.032 nmol GD2/mg protein) or GM3. Four sonic hedgehog-activated (SHH) as well as one group 4 and one group 3 MBs were GD2-positive. Two group 3 MB samples were GD2-negative but GM3-positive. N-glycolyl neuraminic acid-containing GM3 was neither detected in NBL nor MB by mass spectrometry. Furthermore, a GD2-phenotype predicting two-gene signature (ST8SIA1 and B4GALNT1) was applied to RNA-Seq datasets, including 86 MBs and validated by qRT-PCR. The signature values were decreased in group 3 and wingless-activated (WNT) compared to SHH and group 4 MBs. These results suggest that while NBL is GD2-positive, only some MB patients can benefit from a GD2-directed therapy. The expression of genes involved in the ganglioside synthesis may allow the identification of GD2-positive MBs. Finally, the ganglioside profile may reflect the differentiation status in NBL and could help to define MB subtypes.

A Clathrin light chain A reporter mouse for in vivo imaging of endocytosis.

Clathrin-mediated endocytosis (CME) is one of the best studied cellular uptake pathways and its contributions to nutrient uptake, receptor signaling, and maintenance of the lipid membrane homeostasis have been already elucidated. Today, we still have a lack of understanding how the different components of this pathway cooperate dynamically in vivo. Therefore, we generated a reporter mouse model for CME by fusing eGFP endogenously in frame to clathrin light chain a (Clta) to track endocytosis in living mice. The fusion protein is expressed in all tissues, but in a cell specific manner, and can be visualized using fluorescence microscopy. Recruitment to nanobeads recorded by TIRF microscopy validated the functionality of the Clta-eGFP reporter. With this reporter model we were able to track the dynamics of Alexa594-BSA uptake in kidneys of anesthetized mice using intravital 2-photon microscopy. This reporter mouse model is not only a suitable and powerful tool to track CME in vivo in genetic or disease mouse models it can also help to shed light into the differential roles of the two clathrin light chain isoforms in health and disease.

Blockade of Glycosphingolipid Synthesis Inhibits Cell Cycle and Spheroid Growth of Colon Cancer Cells In Vitro and Experimental Colon Cancer Incidence In Vivo.

Colorectal cancer (CRC) is one of the most frequently diagnosed cancers in humans. At early stages CRC is treated by surgery and at advanced stages combined with chemotherapy. We examined here the potential effect of glucosylceramide synthase (GCS)-inhibition on CRC biology. GCS is the rate-limiting enzyme in the glycosphingolipid (GSL)-biosynthesis pathway and overexpressed in many human tumors. We suppressed GSL-biosynthesis using the GCS inhibitor Genz-123346 (Genz), NB-DNJ (Miglustat) or by genetic targeting of the GCS-encoding gene UDP-glucose-ceramide-glucosyltransferase- (UGCG). GCS-inhibition or GSL-depletion led to a marked arrest of the cell cycle in Lovo cells. UGCG silencing strongly also inhibited tumor spheroid growth in Lovo cells and moderately in HCT116 cells. MS/MS analysis demonstrated markedly elevated levels of sphingomyelin (SM) and phosphatidylcholine (PC) that occurred in a Genz-concentration dependent manner. Ultrastructural analysis of Genz-treated cells indicated multi-lamellar lipid storage in vesicular compartments. In mice, Genz lowered the incidence of experimentally induced colorectal tumors and in particular the growth of colorectal adenomas. These results highlight the potential for GCS-based inhibition in the treatment of CRC.

The prognostic value of galactosylceramide-sulfotransferase (Gal3ST1) in human renal cell carcinoma.

Renal cell carcinoma (RCC) is the deadliest primary genitourinary malignancy typically associated with asymptomatic initial presentation and poorly predictable survival. Next to established risk factors, tumor microenvironment may alter metastatic capacity and immune landscape. Due to their high concentrations, sulfoglycolipids (sulfatides) were among the first well-described antigens in RCC that are associated with worse prognosis. As sulfatide detection in routine diagnostics is not possible, we aimed to test the prognostic value of its protein counterpart, sulfatide-producing enzyme Gal3ST1. We performed retrospective long-term follow up analysis of Gal3ST1 expression as prognostic risk factor in a representative RCC patient cohort. We observed differentially regulated Gal3ST1 expression in all RCC types, being significantly more associated with clear cell RCC than to chromophobe RCC (p = 0.001). Surprisingly, in contrast to published observations from in vitro models, we could not confirm an association between Gal3ST1 expression and a malignant clinical behaviour of the RCC. In our cohort, Gal3ST1 did not significantly influence progression-free survival (Hazard Ratio (HR): 1.7 95% CI (0.6-4.9), p = 0.327). Particularly after adjusting for histology, T-stage, N-status and M-status at baseline, we observed no independent prognostic effect (HR = 1.0 95% CI (0.3-3.3), p = 0.96). The analysis of Gal3ST1 mRNA expression in a TCGA dataset supported the results of our cohort. Thus, Gal3ST1 might help to differentiate between chromophobe RCC and other frequent RCC entities but-despite previously published data from cell culture models-does not qualify as a prognostic marker for RCC. Further investigation of regulatory mechanisms of sulfatide metabolism in human RCC microenvironment is necessary to understand the role of this quantitatively prominent glycosphingolipid in RCC progression.

Glycolipid-dependent and lectin-driven transcytosis in mouse enterocytes.

Glycoproteins and glycolipids at the plasma membrane contribute to a range of functions from growth factor signaling to cell adhesion and migration. Glycoconjugates undergo endocytic trafficking. According to the glycolipid-lectin (GL-Lect) hypothesis, the construction of tubular endocytic pits is driven in a glycosphingolipid-dependent manner by sugar-binding proteins of the galectin family. Here, we provide evidence for a function of the GL-Lect mechanism in transcytosis across enterocytes in the mouse intestine. We show that galectin-3 (Gal3) and its newly identified binding partner lactotransferrin are transported in a glycosphingolipid-dependent manner from the apical to the basolateral membrane. Transcytosis of lactotransferrin is perturbed in Gal3 knockout mice and can be rescued by exogenous Gal3. Inside enterocytes, Gal3 is localized to hallmark structures of the GL-Lect mechanism, termed clathrin-independent carriers. These data pioneer the existence of GL-Lect endocytosis in vivo and strongly suggest that polarized trafficking across the intestinal barrier relies on this mechanism.

Effect of Increased Lactate Dehydrogenase A Activity and Aerobic Glycolysis on the Proinflammatory Profile of Autoimmune CD8+ T Cells in Rheumatoid Arthritis.

OBJECTIVE: CD8+ T cells contribute to rheumatoid arthritis (RA) by releasing proinflammatory and cytolytic mediators, even in a challenging hypoxic and nutrient-poor microenvironment such as the synovial membrane. This study was undertaken to explore the mechanisms through which CD8+ T cells meet their metabolic demands in the blood and synovial membrane of patients with RA. METHODS: Purified blood CD8+ T cells from patients with RA, patients with psoriatic arthritis (PsA), and patients with spondyloarthritis (SpA), as well as healthy control subjects, and CD8+ T cells from RA synovial membrane were stimulated in medium containing 13 C-labeled metabolic substrates in the presence or absence of metabolic inhibitors, under conditions of normoxia or hypoxia. The production of metabolic intermediates was quantified by 1 H-nuclear magnetic resonance. The expression of metabolic enzymes, transcription factors, and immune effector molecules was assessed at both the messenger RNA (mRNA) and protein levels. CD8+ T cell functional studies were performed. RESULTS: RA blood CD8+ T cells met their metabolic demands through aerobic glycolysis, production of uniformly 13 C-enriched lactate in the RA blood (2.6 to 3.7-fold higher than in patients with SpA, patients with PsA, and healthy controls; P < 0.01), and induction of glutaminolysis. Overexpression of Warburg effect-linked enzymes in all RA CD8+ T cell subsets maintained this metabolic profile, conferring to the cells the capacity to proliferate under hypoxia and low-glucose conditions. In all RA CD8+ T cell subsets, lactate dehydrogenase A (LDHA) was overexpressed at the mRNA level (P < 0.03 versus controls; n = 6 per group) and protein level (P < 0.05 versus controls; n = 17 RA patients, n = 9 controls). In RA blood, inhibition of LDHA with FX11 led to reductions in lipogenesis, migration and proliferation of CD8+ T cells, and CD8+ T cell effector functions, while production of reactive oxygen species was increased by 1.5-fold (P < 0.03 versus controls). Following inhibition of LDHA with FX11, RA CD8+ T cells lost their capacity to induce healthy B cells to develop a proinflammatory phenotype. Similar metabolic alterations were observed in RA CD8+ T cells from the synovial membrane. CONCLUSION: Remodeling glucose and glutamine metabolism in RA CD8+ T cells by targeting LDHA activity can reduce the deleterious inflammatory and cytolytic contributions of these cells to the development of autoimmunity.

Endogenous levels of 1-O-acylceramides increase upon acidic ceramidase deficiency and decrease due to loss of Dgat1 in a tissue-dependent manner.

Except for epidermis and liver, little is known about endogenous expression of 1-O-acylceramides (1-OACs) in mammalian tissue. Therefore, we screened several organs (brain, lung, liver, spleen, lymph nodes, heart, kidney, thymus, small intestine, and colon) from mice for the presence of 1-OACs by LC-MS2. In most organs, low levels of about 0.25-1.3 pmol 1-OACs/mg wet weight were recorded. Higher levels were detected in liver, small and large intestines, with about 4-13 pmol 1-OACs/mg wet weight. 1-OACs were esterified mainly with palmitic, stearic, or oleic acids. Esterification with saturated very long-chain fatty acids, as in epidermis, was not observed. Western-type diet induced 3-fold increased 1-OAC levels in mice livers while ceramides were unaltered. In a mouse model of Farber disease with a decrease of acid ceramidase activity, we observed a strong, up to 50-fold increase of 1-OACs in lung, thymus, and spleen. In contrast, 1-OAC levels were reduced 0.54-fold in liver. Only in lung 1-OAC levels correlated to changes in ceramide levels - indicating tissue-specific mechanisms of regulation. Glucosylceramide synthase deficiency in liver did not cause changes in 1-OAC or ceramide levels, whereas increased ceramide levels in glucosylceramide synthase-deficient small intestine caused an increase in 1-OAC levels. Deficiency of Dgat1 in mice resulted in a reduction of 1-OACs to 30% in colon, but not in small intestine and liver, going along with constant free ceramides levels. From these data, we conclude that Dgat1 as well as lysosomal lipid metabolism contribute in vivo to homeostatic 1-OAC levels in an organ-specific manner.

Gangliosides modulate insulin secretion by pancreatic beta cells under glucose stress.

In pancreatic beta cells, the entry of glucose and downstream signaling for insulin release is regulated by the glucose transporter 2 (Glut2) in rodents. Dysfunction of the insulin-signaling cascade may lead to diabetes mellitus. Gangliosides, sialic acid-containing glycosphingolipids (GSLs), have been reported to modulate the function of several membrane proteins.Murine islets express predominantly sialylated GSLs, particularly the simple gangliosides GM3 and GD3 having a potential modulatory role in Glut2 activity. Conditional, tamoxifen-inducible gene targeting in pancreatic islets has now shown that mice lacking the glucosylceramide synthase (Ugcg), which represents the rate-limiting enzyme in GSL biosynthesis, displayed impaired glucose uptake and showed reduced insulin secretion. Consequently, mice with pancreatic GSL deficiency had higher blood glucose levels than respective controls after intraperitoneal glucose application. High-fat diet feeding enhanced this effect. GSL-deficient islets did not show apoptosis or ER stress and displayed a normal ultrastructure. Their insulin content, size and number were similar as in control islets. Isolated beta cells from GM3 synthase null mice unable to synthesize GM3 and GD3 also showed lower glucose uptake than respective control cells, corroborating the results obtained from the cell-specific model. We conclude that in particular the negatively charged gangliosides GM3 and GD3 of beta cells positively influence Glut2 function to adequately respond to high glucose loads.

Renal globotriaosylceramide facilitates tubular albumin absorption and its inhibition protects against acute kidney injury.

To elucidate the physiologic function of renal globotriaosylceramide (Gb3/CD77), which up-to-date has been associated exclusively with Shiga toxin binding, we have analyzed renal function in Gb3-deficient mice. Gb3 synthase KO (Gb3S-/-) mice displayed an increased renal albumin and low molecular weight protein excretion compared to WT. Gb3 localized at the brush border and within vesicular structures in WT proximal tubules and has now been shown to be closely associated with the receptor complex megalin/cubilin and with albumin uptake. In two clinically relevant mouse models of acute kidney injury caused by myoglobin as seen in rhabdomyolysis and the aminoglycoside gentamicin, Gb3S-/- mice showed a preserved renal function and morphology, compared to WT. Pharmacologic inhibition of glucosylceramide-based glycosphingolipids, including Gb3, in WT mice corroborated the results of genetically Gb3-deficient mice. In conclusion, our data significantly advance the current knowledge on the physiologic and pathophysiologic role of Gb3 in proximal tubules, showing an involvement in the reabsorption of filtered albumin, myoglobin and the aminoglycoside gentamicin.

Glucosylceramide Synthase Is Involved in Development of Invariant Natural Killer T Cells.

Invariant natural killer T (iNKT) cells represent a unique population of CD1d-restricted T lymphocytes expressing an invariant T cell receptor encoded by Vα14-Jα18 and Vα24-Jα18 gene segments in mice and humans, respectively. Recognition of CD1d-loaded endogenous lipid antigen(s) on CD4/CD8-double positive (DP) thymocytes is essential for the development of iNKT cells. The lipid repertoire of DP thymocytes and the identity of the decisive endogenous lipid ligands have not yet been fully elucidated. Glycosphingolipids (GSL) were implicated to serve as endogenous ligands. However, further in vivo investigations were hampered by early embryonal lethality of mice deficient for the key GSL-synthesizing enzyme glucosylceramide (GlcCer) synthase [GlcCer synthase (GCS), EC 2.4.1.80]. We have now analyzed the GSL composition of DP thymocytes and shown that GlcCer represented the sole neutral GSL and the acidic fraction was composed of gangliosides. Furthermore, we report on a mouse model that by combination of Vav-promoter-driven iCre and floxed GCS alleles (Vav Cre GCS f/f ) enabled an efficient depletion of GCS-derived GSL very early in the T cell development, reaching a reduction by 99.6% in DP thymocytes. Although the general T cell population remained unaffected by this depletion, iNKT cells were reduced by approximately 50% in thymus, spleen, and liver and showed a reduced proliferation and an increased apoptosis rate. The Vß-chains repertoire and development of iNKT cells remained unaltered. The GSL-depletion neither interfered with expression of CD1d, SLAM, and Ly108 molecules nor impeded the antigen presentation on DP thymocytes. These results indicate that GlcCer-derived GSL, in particular GlcCer, contribute to the homeostatic development of iNKT cells.

Diastereomer-specific quantification of bioactive hexosylceramides from bacteria and mammals.

Mammals synthesize, cell-type specifically, the diastereomeric hexosylceramides, ß-galactosylceramide (GalCer) and ß-glucosylceramide (GlcCer), which are involved in several diseases, such as sphingolipidosis, diabetes, chronic kidney diseases, or cancer. In contrast, Bacteroides fragilis, a member of the human gut microbiome, and the marine sponge, Agelas mauritianus, produce α-GalCer, one of the most potent stimulators for invariant natural killer T cells. To dissect the contribution of these individual stereoisomers to pathologies, we established a novel hydrophilic interaction chromatography-based LC-MS2 method and separated (R > 1.5) corresponding diastereomers from each other, independent of their lipid anchors. Testing various bacterial and mammalian samples, we could separate, identify (including the lipid anchor composition), and quantify endogenous ß-GlcCer, ß-GalCer, and α-GalCer isomers without additional derivatization steps. Thereby, we show a selective decrease of ß-GlcCers versus ß-GalCers in cell-specific models of GlcCer synthase-deficiency and an increase of specific ß-GlcCers due to loss of ß-glucoceramidase 2 activity. Vice versa, ß-GalCer increased specifically when cerebroside sulfotransferase (Gal3st1) was deleted. We further confirm ß-GalCer as substrate of globotriaosylceramide synthase for galabiaosylceramide synthesis and identify additional members of the human gut microbiome to contain immunogenic α-GalCers. Finally, this method is shown to separate corresponding hexosylsphingosine standards, promoting its applicability in further investigations.

Inhibition of hepatocellular carcinoma growth by blockade of glycosphingolipid synthesis.

Hepatocellular carcinoma (HCC) is one of the most frequent cancers. In vitro studies suggest that growth and response to therapy of human carcinomas may depend on glycosphingolipid (GSL) expression. Glucosylceramide synthase (GCS), encoded by the gene Ugcg, is the basic enzyme required for the synthesis of GSLs. Gene array analysis implied that Ugcg is significantly overexpressed in human HCC as compared to non-tumorous liver tissue. Therefore we have investigated whether tumor - genesis and - growth is altered in the absence of GSLs. An endogenous liver cancer model has been initiated by application of diethylnitrosamine in mice lacking Ugcg specifically in hepatocytes. We have now shown that hepatocellular tumor initiation and growth in mice is significantly inhibited by hepatic GSL deficiency in vivo. Neither the expression of cell cycle proteins, such as cyclins and pathways such as the MAP-kinase/Erk pathway nor the mTOR/Akt pathway as well as the number of liver infiltrating macrophages and T cells were essentially changed in tumors lacking GSLs. Significantly elevated bi-nucleation of atypical hepatocytes, a feature for impaired cytokinesis, was detected in tumors of mice lacking liver-specific GSLs. A reduction of proliferation and restricted growth of tumor microspheres due to delayed, GSL-dependent cytokinesis, analogous to the histopathologic phenotype in vivo could be demonstrated in vitro. GSL synthesis inhibition may thus constitute a potential therapeutic target for hepatocellular carcinoma.

Male meiotic cytokinesis requires ceramide synthase 3-dependent sphingolipids with unique membrane anchors.

Somatic cell cytokinesis was shown to involve the insertion of sphingolipids (SLs) to midbodies prior to abscission. Spermatogenic midbodies transform into stable intercellular bridges (ICBs) connecting clonal daughter cells in a syncytium. This process requires specialized SL structures. (1) Using high resolution-mass spectrometric imaging, we show in situ a biphasic pattern of SL synthesis with testis-specific anchors. This pattern correlates with and depends on ceramide synthase 3 (CerS3) localization in both, pachytene spermatocytes until completion of meiosis and elongating spermatids. (2) Blocking the pathways to germ cell-specific ceramides (CerS3-KO) and further to glycosphingolipids (glucosylceramide synthase-KO) in mice highlights the need for special SLs for spermatid ICB stability. In contrast to somatic mitosis these SLs require ultra-long polyunsaturated anchors with unique physico-chemical properties, which can only be provided by CerS3. Loss of these anchors causes enhanced apoptosis during meiosis, formation of multinuclear giant cells and spermatogenic arrest. Hence, testis-specific SLs, which we also link to CerS3 in human testis, are quintessential for male fertility.

Zeb1 affects epithelial cell adhesion by diverting glycosphingolipid metabolism.

This study proposes that the transcription factor Zeb1 modulates epithelial cell adhesion by diverting glycosphingolipid metabolism. Zeb1 promotes expression of a-series glycosphingolipids via regulating expression of GM3 synthase (St3gal5), which mechanistically involves Zeb1 binding to the St3gal5 promoter as well as suppressing microRNA-mediated repression of St3gal5. Functionally, the repression of St3gal5 suffices to elevate intercellular adhesion and expression of distinct junction-associated proteins, reminiscent of knockdown of Zeb1. Conversely, overexpressing St3gal5 sensitizes cells towards TGF-ß1-induced disruption of cell-cell interaction and partially antagonizes elevation of intercellular adhesion imposed by Zeb1 knockdown. These results highlight a direct connection of glycosphingolipid metabolism and epithelial cell adhesion via Zeb1.

Renal sulfatides: sphingoid base-dependent localization and region-specific compensation of CerS2-dysfunction.

Mammalian kidneys are rich in sulfatides. Papillary sulfatides, especially, contribute to renal adaptation to chronic metabolic acidosis. Due to differences in their cer-amide (Cer) anchors, the structural diversity of renal sulfatides is large. However, the underling biological function of this complexity is not understood. As a compound's function and its tissue location are intimately connected, we analyzed individual renal sulfatide distributions of control and Cer synthase 2 (CerS)2-deficient mice by imaging MS (IMS) and by LC-MS(2) (in controls for the cortex, medulla, and papillae separately). To explain locally different structures, we compared our lipid data with regional mRNA levels of corresponding anabolic enzymes. The combination of IMS and in source decay-LC-MS(2) analyses revealed exclusive expression of C20-sphingosine-containing sulfatides within the renal papillae, whereas conventional C18-sphingosine-containing compounds were predominant in the medulla, and sulfatides with a C18-phytosphingosine were restricted to special cortical structures. CerS2 deletion resulted in bulk loss of sulfatides with C23/C24-acyl chains, but did not lead to decreased urinary pH, as previously observed in sulfatide-depleted kidneys. The reasons may be the almost unchanged C22-sulfatide levels and constant total renal sulfatide levels due to compensation with C16- to C20-acyl chain-containing compounds. Intriguingly, CerS2-deficient kidneys were completely depleted of phytosphingosine-containing cortical sulfatides without any compensation.

Quantitative imaging mass spectrometry of renal sulfatides: validation by classical mass spectrometric methods.

Owing to its capability of discriminating subtle mass-altering structural differences such as double bonds or elongated acyl chains, MALDI-based imaging MS (IMS) has emerged as a powerful technique for analysis of lipid distribution in tissue at moderate spatial resolution of about 50 µm. However, it is still unknown if MS(1)-signals and ion intensity images correlate with the corresponding apparent lipid concentrations. Analyzing renal sulfated glycosphingolipids, sulfatides, we validate for the first time IMS-signal identities using corresponding sulfatide-deficient kidneys. To evaluate the extent of signal quenching effects interfering with lipid quantification, we surgically dissected the three major renal regions (papillae, medulla, and cortex) and systematically compared MALDI IMS of renal sulfatides with quantitative analyses of corresponding lipid extracts by on-target MALDI TOF-MS and by ultra-performance LC-ESI-(triple-quadrupole)tandem MS. Our results demonstrate a generally strong correlation (R(2) > 0.9) between the local relative sulfatide signal intensity in MALDI IMS and absolute sulfatide quantities determined by the other two methods. However, high concentrations of sulfatides in the papillae and medulla result in an up to 4-fold signal suppression. In conclusion, our study suggests that MALDI IMS is useful for semi-quantitative dissection of relative local changes of sulfatides and possibly other lipids in tissue.

Depletion of globosides and isoglobosides fully reverts the morphologic phenotype of Fabry disease.

Fabry disease is a monogenic X-linked lysosomal storage disease caused by α-galactosidase A (αGalA) deficiency. Enzyme replacement therapy through administration of the missing αGalA is currently the only accepted therapeutic option. However, this treatment is connected to high costs, has ill-defined indication criteria and its efficacy is controversially discussed. Our aim was to explore the possibility of a novel targeted substrate reduction therapy for Fabry disease. Owing to the fact that αGalA-deficient humans and mice accumulate the same glycosphingolipids (i.e. globosides, galabiosylceramide and isoglobosides), αGalA-deficient mice were crossed with mice deficient in enzymes synthesizing these classes of glycosphingolipids (i.e. globotrihexosylceramide and isoglobotrihexosylceramide synthase, respectively). Functional heart and kidney tests were performed together with an extensive biochemical analysis of urine and serum in aged mice. Lysosomal storage was assessed by thin layer chromatography and electron microscopy. We showed that depletion of globosides was sufficient to fully abolish the storage of glycosphingolipids in heart, kidney and liver and was paralleled by a complete restoration of lysosomal morphology in these organs. In contrast, in dorsal root ganglia, a depletion of both globosides and isoglobosides was necessary to fully counteract the lysosomal storage. The deficiency in globosides and/or isoglobosides did not cause any adverse effects. We conclude that substrate reduction therapy through inhibition of the synthesis of globosides and isoglobosides represents a valuable therapeutic option for Fabry disease, all the more as globosides and isoglobosides seem to be dispensable.

Direct acute tubular damage contributes to Shigatoxin-mediated kidney failure.

The pathogenesis and therapy of Shigatoxin 2 (Stx2)-mediated kidney failure remain controversial. Our aim was to test whether, during an infection with Stx2-producing E. coli (STEC), Stx2 exerts direct effects on renal tubular epithelium and thereby possibly contributes to acute renal failure. Mice represent a suitable model because they, like humans, express the Stx2-receptor Gb3 in the tubular epithelium but, in contrast to humans, not in glomerular endothelia, and are thus free of glomerular thrombotic microangiopathy (TMA). In wild-type mice, Stx2 caused acute tubular dysfunction with consequent electrolyte disturbance, which was most likely the cause of death. Tubule-specific depletion of Gb3 protected the mice from acute renal failure. In vitro, Stx2 induced secretion of proinflammatory cytokines and apoptosis in human tubular epithelial cells, thus implicating a direct effect of Stx2 on the tubular epithelium. To correlate these results to human disease, kidney biopsies and outcome were analysed in patients with Stx2-associated kidney failure (n = 11, aged 22-44 years). The majority of kidney biopsies showed different stages of an ongoing TMA; however, no glomerular complement activation could be demonstrated. All biopsies, including those without TMA, showed severe acute tubular damage. Due to these findings, patients were treated with supportive therapy without complement-inhibiting antibodies (eculizumab) or immunoadsorption. Despite the severity of the initial disease [creatinine 6.34 (1.31-17.60) mg/dl, lactate dehydrogenase 1944 (753-2792) U/l, platelets 33 (19-124)/nl and haemoglobin 6.2 (5.2-7.8) g/dl; median (range)], all patients were discharged after 33 (range 19-43) days with no neurological symptoms and no dialysis requirement [creatinine 1.39 (range 0.84-2.86) mg/dl]. The creatinine decreased further to 0.90 (range 0.66-1.27) mg/dl after 24 months. Based on these data, one may surmise that acute tubular damage represents a separate pathophysiological mechanism, importantly contributing to Stx2-mediated acute kidney failure. Specifically in young adults, an excellent outcome can be achieved by supportive therapy only.

1-O-acylceramides are natural components of human and mouse epidermis.

The lipid-rich stratum corneum functions as a barrier against pathogens and desiccation inter alia by an unbroken meshwork of extracellular lipid lamellae. These lamellae are composed of cholesterol, fatty acids, and ceramides (Cers) in an equimolar ratio. The huge class of skin Cers consists of three groups: group I, "classical" long and very long chain Cers; group II, ultra-long chain Cers; and group III, ω-esterified ultra-long chain Cers, which are esterified either with linoleic acid or with cornified envelope proteins and are required for the water permeability barrier. Here, we describe 1-O-acylceramides as a new class of epidermal Cers in humans and mice. These Cers contain, in both the N- and 1-O-position, long to very long acyl chains. They derive from the group I of classical Cers and make up 5% of all esterified Cers. Considering their chemical structure and hydrophobicity, we presume 1-O-acylceramides to contribute to the water barrier homeostasis. Biosynthesis of 1-O-acylceramides is not dependent on lysosomal phospholipase A2. However, glucosylceramide synthase deficiency was followed by a 7-fold increase of 1-O-acylceramides, which then contributed 30% to all esterified Cers. Furthermore, loss of neutral glucosylceramidase resulted in decreased levels of a 1-O-acylceramide subgroup. Therefore, we propose 1-O-acylceramides to be synthesized at endoplasmic reticulum-related sites.