Lifelong low-phylloquinone intake is associated with cognitive impairments in old rats.

In a previous report, we showed vitamin K to preferentially accumulate in brain regions rich in white matter and to positively correlate with certain sphingolipids. In rodents, pharmacological vitamin K deficiency has resulted in behavioral perturbations. To gain insight on the role of vitamin K status on brain function, we investigated learning abilities (Morris water maze), motor activity (open field), and anxiety (elevated plus maze) in distinct groups of 6-, 12-, and 20-mo-old female Sprague-Dawley rats that had been fed diets containing low (L; ~80 µg/kg diet), adequate (A; ~500 µg/kg diet), or high (H; ~2000 µg/kg diet) levels of phylloquinone (µg/kg diet; n = 9-12/diet) since weaning. In 20-mo-old rats, sphingolipids (cerebroside, sulfatide, sphingomyelin, ceramide, and gangliosides), phylloquinone, and menaquinone-4 were also assessed in cerebellum, midbrain, pons medulla, striatum, and hippocampus. Lifetime consumption of a low-vitamin K diet resulted in cognitive deficits in the 20-mo-old rats, with those in the L group having longer latencies than those in the H group (P < 0.05); this was associated with higher concentrations of ceramides in the hippocampus (P < 0.05) and lower gangliosides in the pons medulla and midbrain (P < 0.05). The low-vitamin K diet did not affect cognition at 6 and 12 mo of age, nor did it affect motor activity or anxiety at any age. Although much remains to be elucidated about the mechanism of action of vitamin K in cognition, this report points to vitamin K as an important nutritional factor contributing to cognitive health during aging.

The Linoleic Acid Content of the Stratum Corneum of Ichthyotic Golden Retriever Dogs Is Reduced as Compared to Healthy Dogs and a Significant Part Is Oxidized in Both Free and Esterified Forms.

Golden Retrievers may suffer from Pnpl1-related inherited ichthyosis. Our study shows that in the stratum corneum (SC) of ichthyotic dogs, linoleic acid (LA) is also present in the form of 9-keto-octadecadienoic acid (9-KODE) instead of the acylacid form as in normal dogs. The fatty acids purified from SC strips (LA, acylacids) were characterized by liquid chromatography-tandem mass spectrometry (LC-MS) and atmospheric pressure chemical ionization (APCI). Electrospray ionization (ESI) and MS2(MS/MS Tandem mass spectrum/spectra)/M3 (MS/MS/MS Tandem mass spectrum/spectra) fragmentation indicated the positions of the double bonds in 9-KODE. We showed that ichthyotic dogs have a threefold lower LA content in the form of acylacids. The MS2 fragmentation of acyl acids showed in some peaks the presenceof an ion at the m/z 279, instead of an ion at m/z 293 which is characteristic of LA. The detected variant was identified upon MS3 fragmentation as 9-keto-octadecadienoic acid (9-KODE), and the level of this keto-derivative was increased in ichthyotic dogs. We showed by the APCI that such keto forms of LA are produced from hydroperoxy-octadecadienoic acids (HpODE) upon dehydration. In conclusion, the free form of 9-KODE was detected in ichthyotic SC up to fivefold as compared to unaffected dogs, and analyses by HPLC (High performance liquid chromatography) and ESI-MS (Electrospray Ionization-Mass Spectrometry) indicated its production via dehydration of native 9-HpODE.

Human Endogenous Retrovirus Type W Envelope from Multiple Sclerosis Demyelinating Lesions Shows Unique Solubility and Antigenic Characteristics.

In multiple sclerosis (MS), human endogenous retrovirus W family (HERV-W) envelope protein, pHERV-W ENV, limits remyelination and induces microglia-mediated neurodegeneration. To better understand its role, we examined the soluble pHERV-W antigen from MS brain lesions detected by specific antibodies. Physico-chemical and antigenic characteristics confirmed differences between pHERV-W ENV and syncytin-1. pHERV-W ENV monomers and trimers remained associated with membranes, while hexamers self-assembled from monomers into a soluble macrostructure involving sulfatides in MS brain. Extracellular hexamers are stabilized by internal hydrophobic bonds and external hydrophilic moieties. HERV-W studies in MS also suggest that this diffusible antigen may correspond to a previously described high-molecular-weight neurotoxic factor secreted by MS B-cells and thus represents a major agonist in MS pathogenesis. Adapted methods are now needed to identify encoding HERV provirus(es) in affected cells DNA. The properties and origin of MS brain pHERV-W ENV soluble antigen will allow a better understanding of the role of HERVs in MS pathogenesis. The present results anyhow pave the way to an accurate detection of the different forms of pHERV-W ENV antigen with appropriate conditions that remained unseen until now.

Immunosuppressive networks in the tumour environment and their effect in dendritic cells.

The failure of the immune system to provide protection against tumour cells is an important immunological problem. It is now evident that inadequate function of the host immune system is one of the main mechanisms by which tumours escape from immune control, as well as an important factor that limits the success of cancer immunotherapy. In recent years, it has become increasingly clear that defects in dendritic cells have a crucial role in non-responsiveness to tumours. This article focuses on the functional consequences and recently described mechanisms of the dendritic-cell defects in cancer.

Sulfogalactosylceramides in motor and psycho-cognitive adult metachromatic leukodystrophy: relations between clinical, biochemical analysis and molecular aspects.

Metachromatic leukodystrophy (MLD) is a human autosomal recessive lysosomal neurodegenerative disorder that results from the accumulation of sulfatides in the central and peripheral nervous system. It is due to the enzyme deficiency of the sulfatide sulfatase i.e. arylsulfatase A (ASA). During adolescence and/or adulthood, there are 2 clinical presentations. It may be that of a degenerative disease of the central nervous system with mainly spastic manifestations or a spino-cerebellar ataxia, or that of a psychosis. As several lines of evidence indicate that the psychotic form of MLD could be a model of psychosis, we decided to do a pluridisciplinary study on 11 psycho-cognitive cases involving mental and psychiatric testing, in comparison with 5 adult motor cases, a biochemical study with enzyme assays and quantitative mass spectrometry of urinary sulfatides, so as to determine whether there were biochemical particularities related to the psychotic forms. For quantitative mass spectrometry (MS), a non physiological sulfatide with C17:0 fatty acid was synthesized. The major sulfatide isoforms were present in the 2 clinical forms with the following fatty acids and sphingoid bases: C22:1/d18:1, and /or C22:0/d18:2 (m/z 862.5), C22:0 (OH)/d18:1 (m/z 878,5), C24:0/d18:1 and / or C24:0/C23:1(OH)/d18:2 (m/z 890,3), C24:0 (OH)/d18:1(m/z 906.5). We had shown previously that there were different ASA mutations in the psychiatric adult form (heterozygous I179S) versus the adult motor form (homozygous P426L). We show here that there were no relations with the level of ASA and with the mass spectrometric study of the sulfatide isoforms which were identical in the 2 clinical forms.

A new approach for drug discovery from glycobiology and phage-displayed peptide library technology.

Peptides which mimic functional activities of glycosphingolipids were prepared by a technology of phage-displayed peptide library using monoclonal antibodies against glycosphingolipids. These peptides were named glyco-replica peptides. Peptides prepared with anti-GD1alpha antibody by this technology were found to contain WHW as common motif, and they showed suppressive activity not only on adhesion between hepatic sinusoidal endothelial cells and lymphosarcoma RAW117-H10 cells, but also on metastasis of the tumor cell to the liver and lung. The WHW motif seems to be important to mimic the functional activity of the ganglioside GD1alpha. Next, we prepared GD3-replica peptides using a monoclonal antibody against GD3 (4F6). A peptide, GD3-P4 with highest affinity to 4F6 was used to immunize mice to examine if the mice show their immune response to raise antibodies against GD3. We confirmed the immune response and succeeded in the production of a monoclonal antibody (3D2) against GD3. The monoclonal antibody 3D2 showed specific binding to GD3 on a thin-layer chromatography plate and also melanoma tissues. Interestingly, the amino acid sequence of the CDR regions of light and heavy chains showed high similarity with those of the original GD3 monoclonal antibody (4F6) used for the preparation of GD3-replica peptide. The technology of the phage-displayed peptide library was applied to in vivo bio-panning study using an angiogenesis experimental model. The obtained peptides were found to show strong binding property to the neo-vasculature system and to be quite useful to carry an anti-tumor drug to the tumor tissue. Based on these experimental results, we discuss about some applications of this method to drug discovery.

Different mechanisms are involved in apoptosis induced by melanoma gangliosides on human monocyte-derived dendritic cells.

Tumor escape is linked to multiple mechanisms, notably the liberation, by tumor cells, of soluble factors that inhibit the function of dendritic cells (DC). We have shown that melanoma gangliosides impair DC differentiation and induce their apoptosis. The present study was aimed to give insight into the mechanisms involved. DC apoptosis was independent of the catabolism of gangliosides since lactosylceramide did not induce cell death. Apoptosis induced by GM3 and GD3 gangliosides was not blocked by inhibitors of de novo ceramide biosynthesis, whereas the acid sphingomyelinase inhibitor desipramine only prevented apoptosis induced by GM3. Furthermore, our results suggest that DC apoptosis was triggered via caspase activation, and it was ROS dependent with GD3 ganglioside, suggesting that GM3 and GD3 induced apoptosis through different mechanisms.

Glycosylation-related gene expression profiling in the brain and spleen of scrapie-affected mouse.

A central event in the formation of infectious prions is the conformational change of a host-encoded glycoprotein, PrP(C), into a pathogenic isoform, PrP(Sc). The molecular requirements for efficient PrP conversion remain unknown. Altered glycosylation has been linked to various pathologies and the N-glycans harbored by two prion protein isoforms are different. In order to search for glycosylation-related genes that could mark prion infection, we used a glycosylation-dedicated microarray that allowed the simultaneous analysis of the expression of 165 glycosylation-related genes encoding proteins of the glycosyltransferase, glycosidase, lectin, and sulfotransferase families to compare the gene expression profiles of normal and scrapie-infected mouse brain and spleen. Eight genes were found upregulated in "scrapie brain" at the final state of the disease. In the spleen, five genes presented a modified expression. Three genes were also upregulated in the spleen of infected mice, and two (Pigq and St3gal5) downregulated. All changes were confirmed by qPCR and biochemical analyses applied to Pigq and St3gal5 proteins.

Detection of GD3 ganglioside in primary melanomas depends on histopathologic procedures used for tumor preservation.

Gangliosides, cell surface glycosphingolipids, are implicated in diverse biologic functions potentially important for tumor growth. Because expression of the GD3 ganglioside may have an impact on the melanoma malignancy, and therefore on the patient prognosis, we evaluated the feasibility of a retrospective immunohistochemical study of GD3 in paraffin embedded biopsies of primary melanomas. Immunoperoxidase staining of frozen and deparaffinized sections of melanoma lesions with two anti-GD3 antibodies was compared using Dako biotin-streptavidin detection kit. Residual ganglioside content was evaluated in the tissues submitted to routine histopathologic procedures using HPLC. A strong and reproducible staining was obtained with both antibodies on frozen sections of all 17 melanoma samples. However, only KM641 antibody could detect GD3 on deparaffinized sections. Biochemical quantification revealed that the Bouin fixative resulted in degradation of GD3. Additionally, most of GD3 was eluted from the tissue samples during dehydration and re-hydration steps. A subgroup of tumors particularly rich in GD3 could be detected on deparaffinized sections after standard formaldehyde fixation. Clinical evolution of such melanomas can now be compared to the group with low GD3 expression. However, any Bouin-fixed, paraffin-embedded biopsies should be excluded from such a retrospective study.

Linoleate-enriched diet increases both linoleic acid esterified to omega hydroxy very long chain fatty acids and free ceramides of canine stratum corneum without effect on protein-bound ceramides and skin barrier function.

Few studies have investigated the influence of increased amounts of dietary linoleic acid on the epidermal lipid biochemistry and TEWL in healthy subject. The influence of dietary linoleic acid on canine stratum corneum (SC) lipids was studied by feeding two groups of five dogs differential amounts of linoleic acid (LA) for three months. SC was harvested by tape stripping and lipids were analyzed by thin-layer chromatography and mass spectrometry. The dogs that were fed the higher concentration of LA showed high increases in the contents of both linoleic acid and free ceramides in the SC, whereas the protein-bound ceramide content was unchanged. Acylacids that represent the esterified form of linoleic acid in omega hydroxy very long chain fatty acids (ω-OH VLCFA) accounted for most of the elevation of LA, whereas the concentration of the free form was not significantly changed. Corroborating the absence of change in the protein-bound ceramides content of healthy dogs SC, TEWL was nearly unaffected by the linoleic acid-enriched diet.

An ancestral retroviral protein identified as a therapeutic target in type-1 diabetes.

Human endogenous retroviruses (HERVs), remnants of ancestral viral genomic insertions, are known to represent 8% of the human genome and are associated with several pathologies. In particular, the envelope protein of HERV-W family (HERV-W-Env) has been involved in multiple sclerosis pathogenesis. Investigations to detect HERV-W-Env in a few other autoimmune diseases were negative, except in type-1 diabetes (T1D). In patients suffering from T1D, HERV-W-Env protein was detected in 70% of sera, and its corresponding RNA was detected in 57% of peripheral blood mononuclear cells. While studies on human Langerhans islets evidenced the inhibition of insulin secretion by HERV-W-Env, this endogenous protein was found to be expressed by acinar cells in 75% of human T1D pancreata. An extensive immunohistological analysis further revealed a significant correlation between HERV-W-Env expression and macrophage infiltrates in the exocrine part of human pancreata. Such findings were corroborated by in vivo studies on transgenic mice expressing HERV-W-env gene, which displayed hyperglycemia and decreased levels of insulin, along with immune cell infiltrates in their pancreas. Altogether, these results strongly suggest an involvement of HERV-W-Env in T1D pathogenesis. They also provide potentially novel therapeutic perspectives, since unveiling a pathogenic target in T1D.

GD3-replica peptides selected from a phage peptide library induce a GD3 ganglioside antibody response.

GD3-replica peptides were obtained from a phage peptide library and an anti-GD3 monoclonal antibody (Mab) (4F6), and anti-GD3 Mabs were generated by immunizing a peptide GD3P4. A Mab, 3D2 was found to recognize GD3 by immunohistochemical approaches. Amino acid analysis of heavy and light chain variable regions of 4F6 and 3D2 showed that the respective chains had the same length, and only a few different amino acid substitutions were found. The present data indicate that the immunogenic GD3P4 is processed in a certain size and exposed on the antigen-presenting cells with a molecular shape quite similar to that of the GD3 epitope in 4F6.

Subcellular compartmentalization of ceramide metabolism: MAM (mitochondria-associated membrane) and/or mitochondria?

Recent studies by our group and others have disclosed the presence of ceramides in mitochondria, and the activities of ceramide synthase and reverse ceramidase in mitochondria have also been reported. Since a possible contamination with the ER (endoplasmic reticulum)-related compartment MAM (mitochondria-associated membrane) could not be ruled out in previous studies, we have re-investigated the presence of the enzymes of ceramide metabolism in mitochondria and MAM highly purified from rat liver. In the present paper, we show that purified mitochondria as well as MAM are indeed able to generate ceramide in vitro through both ceramide synthase or reverse ceramidase, whereas the latter enzyme activity is barely detectable in microsomes. Moreover, ceramide synthase activities were recovered in outer mitochondrial membranes as well as in inner mitochondrial membranes. Using radiolabelled sphingosine as a substrate, mitochondria could generate ceramide and phytoceramide. However, the in vitro sensitivity of ceramide synthase toward FB1 (fumonisin B1) in mitochondria as well as in MAM was found to depend upon the sphingoid base: whereas dihydrosphingosine N-acyltransferase was inhibited by FB1 in a concentration-dependent manner, FB1 actually activated the ceramide synthase when using sphingosine as a substrate. Acylation of sphingosine 1-phosphate and dihydrosphingosine 1-phosphate, generating ceramide 1-phosphate, was also shown with both subcellular fractions. Moreover, the same difference in sensitivity towards FB1 for the ceramide synthase activities was seen between the two phosphorylated sphingoid bases, raising the possibility that distinct base-specific enzymes may be involved as ceramide synthases. Collectively, these results demonstrate the involvement of mitochondria in the metabolism of ceramides through different pathways, thereby supporting the hypothesis that topology of ceramide formation could determine its function.

A sensitive flow cytometry method for anti-GM1 antibodies detection.

High-affinity anti-GM1 antibodies are frequently described in several nervous system diseases, mainly in multifocal motor neuropathy (MMN) and some acute neuropathies. These antibodies are currently detected using enzyme-linked immunosorbent assay (ELISA) and immuno-thin-layer-chromatography (immuno-TLC) methods. We report in this article a new method based on the incorporation of exogenous GM1 in a selected cell line to detect anti-GM1 antibodies using flow cytometry (FC). This method is evaluated on 80 sera from normal blood donors (NBD) and patients suffering various nervous system diseases. It appears to be as sensitive as our method ELISA for the diagnosis of some motor neuron syndromes.

Sulfatide and GM1 ganglioside modulate the high-affinity dopamine uptake in rat striatal synaptosomes: evidence for the involvement of their ionic charges.

The present study was undertaken to examine the effects of the anionic glycolipids GM1 ganglioside and sulfatide on the high-affinity dopamine (DA) uptake in rat striatal synaptosomes. After 1h of incubation, GM1 stably bound to synaptosomes and modified the activity of the neuronal dopamine transporter (DAT). With 1.2 and 12 microM GM1, V(max) decreased by 13 and 23%, respectively, reflecting a slight reduction of the number of functional uptake sites and K(m) was lowered by 21 and 33%, thus showing an increase of the affinity. Treatment of synaptosomes with 1.2 microM of sulfatide, which possesses an anionic sulfated group, led to a similar decrease of V(max) (19%) than GM1, but to a significantly higher reduction of K(m) (35%). In fact, sulfatide associated to synaptosomes in a 3.5-fold higher extent than GM1. Conversely, when GM1 and sulfatide were replaced by GM1 alcohol and galactosylceramide, respectively, no modification of the DA uptake occurred, although these neutral glycolipids incorporated into the synaptosomes to the same extent as the related anionic compounds.Altogether, these results demonstrate the key role of negative charges linked to the oligosaccharide chains of glycolipids in the modulation of DA transport across the synaptosomal membrane.

Human endogenous retrovirus protein activates innate immunity and promotes experimental allergic encephalomyelitis in mice.

Multiple sclerosis (MS) is a complex multifactorial disease of the central nervous system (CNS) for which animal models have mainly addressed downstream immunopathology but not potential inducers of autoimmunity. In the absence of a pathogen known to cause neuroinflammation in MS, Mycobacterial lysate is commonly used in the form of complete Freund's adjuvant to induce autoimmunity to myelin proteins in Experimental Allergic Encephalomyelitis (EAE), an animal model for MS. The present study demonstrates that a protein from the human endogenous retrovirus HERV-W family (MSRV-Env) can be used instead of mycobacterial lysate to induce autoimmunity and EAE in mice injected with MOG, with typical anti-myelin response and CNS lesions normally seen in this model. MSRV-Env was shown to induce proinflammatory response in human macrophage cells through TLR4 activation pathway. The present results demonstrate a similar activation of murine dendritic cells and show the ability of MSRV-Env to trigger EAE in mice. In previous studies, MSRV-Env protein was reproducibly detected in MS brain lesions within microglia and perivascular macrophages. The present results are therefore likely to provide a model for MS, in which the upstream adjuvant triggering neuroinflammation is the one detected in MS active lesions. This model now allows pre-clinical studies with therapeutic agents targeting this endogenous retroviral protein in MS.

Production of multiple brain-like ganglioside species is dispensable for fas-induced apoptosis of lymphoid cells.

Activation of an acid sphingomyelinase (aSMase) leading to a biosynthesis of GD3 disialoganglioside has been associated with Fas-induced apoptosis of lymphoid cells. The present study was undertaken to clarify the role of this enzyme in the generation of gangliosides during apoptosis triggered by Fas ligation. The issue was addressed by using aSMase-deficient and aSMase-corrected cell lines derived from Niemann-Pick disease (NPD) patients. Fas cross-linking elicited a rapid production of large amounts of complex a- and b-series species of gangliosides with a pattern and a chromatographic behavior as single bands reminiscent of brain gangliosides. The gangliosides were synthesized within the first ten minutes and completely disappeared within thirty minutes after stimulation. Noteworthy is the observation that GD3 was not the only ganglioside produced. The production of gangliosides and the onset of apoptotic hallmarks occurred similarly in both aSMase-deficient and aSMase-corrected NPD lymphoid cells, indicating that aSMase activation is not accountable for ganglioside generation. Hampering ganglioside production by inhibiting the key enzyme glucosylceramide synthase did not abrogate the apoptotic process. In addition, GM3 synthase-deficient lymphoid cells underwent Fas-induced apoptosis, suggesting that gangliosides are unlikely to play an indispensable role in transducing Fas-induced apoptosis of lymphoid cells.

Analysis of epidermal lipids in normal and atopic dogs, before and after administration of an oral omega-6/omega-3 fatty acid feed supplement. A pilot study.

Alterations of the lipid expression in the skin of human and canine atopic subjects may be one of the key factors in the disease development. We have analyzed the ultrastructure of the clinically uninvolved skin of atopic dogs and compared it with the lipid composition of their tape-stripped stratum corneum (SC). The effect of a 2 month treatment of atopic dogs by food supplementation with a mixture of essential fatty acids was evaluated on skin samples taken before and after the treatment period. Electron microscopy revealed that the non-lesional skin of atopic dogs exhibited an abnormal and largely incomplete structure of the lamellar lipids with little cohesion between the corneocyte strata. The SC of atopic dogs was characterized by a significant decrease in the lipid content when compared to the healthy controls. Following oral supplementation with the mixture of essential fatty acids, the overall lipid content of the SC markedly increased. This feature was observed both with the free and, most importantly, with the protein-bound lipids (cholesterol, fatty acids and ceramides), the latter constituting the corneocyte-bound scaffold for ordinate organisation of the extracellular lipid bi-layers. Indeed, the semi-quantitative electron microscopy study revealed that the treatment resulted in a significantly improved organization of the lamellar lipids in the lower SC, comparable to that of the healthy dogs. Our results indicate the potential interest of long-term alimentary supplementation with omega-6 and omega-3 essential fatty acids in canine atopic dermatitis.

Atopic dermatitis in dogs is associated with a high heterogeneity in the distribution of protein-bound lipids within the stratum corneum.

The stratum corneum (SC) was taken from five atopic dogs by tape stripping (12 strips) of non-lesional areas of the abdomen. The free and protein-bound lipids were extracted and analyzed by thin-layer chromatography after fractionation on aminopropyl-bonded silica gel columns. A very frequent feature was the heterogeneity in the lipid content of consecutive layers. This was even more accentuated for the covalently bound lipids, with variations from one layer to another in the concentrations of cholesterol, omega hydroxylated ceramides and omega hydroxylated long-chain fatty acids. Among the free lipids, large amounts of glucosylceramides were present in canine atopic SC although they are nearly absent from the SC of normal dogs. A heterogeneous distribution of lipids was seen in canine atopic SC. These results suggest that strikingly deep variations occur in the lipid metabolism of keratinocytes in the skin of atopic dogs. In order to gain insight into this phenomenon, further studies should be focused on the activity of enzymes involved in both biosynthetic and catabolic processes.

Analysis of free and protein-bound ceramides by tape stripping of stratum corneum from dogs.

The free and protein-bound ceramides of dog stratum corneum (SC) were analyzed by thin-layer chromatography after tape stripping of the abdomen of five dogs. The sphingoid bases were identified by gas-liquid chromatography as sphingosine, phytosphingosine, and 6-hydroxysphingosine. Electrospray ionization-ion trap mass spectrometry was used to characterize the protein-bound ceramides containing sphingosine and omega-hydroxy long-chain fatty acids. Although the molecular species were the same ones in all dogs, wide quantitative variations in the patterns of SC ceramides were observed in different breeds of dogs. The free ceramide concentration changed with the depth of SC, with a higher concentration in the deep layers, whereas the concentration of protein-bound ceramides remained constant. These results show that canine SC is close to that of humans with respect to ceramides.