Jan-Eric Månsson

Dynamic association of human insulin receptor with lipid rafts in cells lacking caveolae

Cholesterol‐sphingolipid rich plasma membrane domains, known as rafts, have emerged as important regulators of signal transduction. The adipocyte insulin receptor (IR) is localized to and signals via caveolae that are formed by polymerization of caveolins. Caveolin binds to IR and stimulates signalling. We report that, in liver‐derived cells lacking caveolae, autophosphorylation of the endogenous IR is dependent on raft lipids, being compromised by acute cyclodextrin‐mediated cholesterol depletion or by antibody clustering of glycosphingolipids. Moreover, we provide evidence that IR becomes recruited to detergent‐resistant domains upon ligand binding and that clustering of GM2 ganglioside inhibits IR signalling apparently by excluding the ligand‐bound IR from these domains. Our results indicate that, in cells derived from liver, an important insulin target tissue, caveolae are not required for insulin signalling. Rather, the dynamic recruitment of the ligand‐bound IR into rafts may serve to regulate interactions in the initiation of the IR signalling cascade.

Mucopolysaccharidoses in the Scandinavian countries: incidence and prevalence.

Aim: The aim of this study was to estimate the incidence and prevalence of mucopolysaccharidoses (MPS disorders) in Scandinavia.

Bone marrow stem cell-based gene transfer in a mouse model for metachromatic leukodystrophy: effects on visceral and nervous system disease manifestations

Arylsulfatase A (ASA) knockout mice represent an animal model for the lysosomal storage disease metachromatic leukodystrophy (MLD). Stem cell gene therapy with bone marrow overexpressing the human ASA cDNA from a retroviral vector resulted in the expression of high enzyme levels in various tissues. Treatment partially reduces sulfatide storage in livers exceeding 18 ng ASA/mg tissue, while complete reduction was observed in livers exceeding 50 ng ASA/mg tissue. This corresponds to about 80% and 200% of normal enzyme activity. Similar values seem to apply for kidney. A partial correction of the lipid metabolism was detectable in the brain where the galactoerebroside/sulfatide ratio, which is diminished in ASA-deficient mice, increased upon treatment. This partial correction was accompanied by amelioration of neuropathology; axonal cross-sectional areas, which are reduced in deficient mice, were significantly increased in the saphenic and sciatic nerve but not in the optic nerve. Behavioral tests suggest some improvement of neuromotor abilities. The gene transfer did not delay the degeneration occurring in the acoustic ganglion of ASA-deficient animals. The limited success of the therapy appears to be due to the requirement of unexpected high levels of ASA for correction of the metabolic defect.

Analysis of phosphatidylcholine and sphingomyelin molecular species from brain extracts using capillary liquid chromatography electrospray ionization mass spectrometry

One feature of complex lipids is that many subtypes of these molecules exist as a diverse mixture in a biological sample. Qualitative and quantitative analysis of these closely related molecules require sensitive and specific analytical methods to detect intact phospholipids (PL) and sphingomyelin (SM) species and to differentiate between them. Conventional analytical methods require laborious procedures including separation by column, argentation thin-layer chromatography or liquid chromatography (LC) after pre- or post-column derivatization. In the present work, a method based on reversed phase capillary LC coupled on-line to electrospray ionization mass spectrometry (LC/ESI/MS) has been developed to gather tools for lipidomic studies, i.e. the profiling of complex mixtures of lipids in small amounts of various cells and tissues. The LC/MS system used consisted of an LC pump in an isocratic elution, a reversed phase capillary column and a single quadrupole mass spectrometer operated in the positive ion mode. A successful separation of phosphatidylcholine (PC) and SM molecular species was obtained with a minimum detectable quantity (MDQ) in the low fmol range injected on column. The method was applied to human brain extracts. Furthermore, the extraction efficiencies of the traditional Folch method and pressurized fluid extraction (PFE) were compared using the human brain. It was found that the intensity of the PC and SM molecular species extracted by PFE is two times that of Folch.

Human serum for culture of articular chondrocytes.

In the field of cell and tissue engineering, culture expansion of human cells in monolayer plays an important part. Traditionally, cell cultures have been supplemented with serum to support attachment and proliferation, but serum is a potential source of foreign protein contamination and viral protein transmission. In this study, we evaluated the use of human serum for experimental human articular chondrocyte expansion and to develop a method for preparation of large volumes of high-quality human serum from healthy blood donors. Human autologous serum contained high levels of epidermal-derived growth factor and platelet-derived growth factor-AB and supported proliferation up to 7 times higher than FCS in primary chondrocyte cultures. By letting the coagulation take place in a commercially available transfusion bag overnight, up to 250 ml of growth factor-rich human serum could be obtained from one donor. The allogenic human serum supported high proliferation rate without loosing expression of cartilage-specific genes. The expanded chondrocytes were able to redifferentiate and form cartilage matrix in comparable amounts to autologous serums. In conclusion, the transfusion bags allow preparation of large volumes of growth factor-rich human serum with the capacity to support in vitro cell expansion. The data further indicate that by controlling the coagulation process there are possibilities of optimizing the release of growth factors for other emerging cell therapies.

Sulfatide with short fatty acid dominates in astrocytes and neurons

Glycosphingolipids are located in cell membranes and the brain is especially enriched. We speculated that the subcellular location of glycosphingolipids depends on their fatty acid chain length because their sugar residues are constant, whereas fatty acid chain length can vary within the same molecule. To test this hypothesis we analysed the glycosphingolipid sulfatide, which is highly abundant in myelin and has mostly long fatty acids. We used a negative ion electrospray tandem mass spectrometry precursor ion scan to analyse the molecular species of sulfatide in cultured astrocytes and a mouse model of the human disease metachromatic leukodystrophy. In these arylsulfatase A (ASA)‐deficient mice sulfatide accumulates intracellularly in neurons and astrocytes. Immunocytochemistry was also performed on cultured astrocytes and analysed using confocal laser scanning microscopy. Analyses of the molecular species showed that cultured astrocytes contained sulfatide with a predominance of stearic acid (C18), which was located in large intracellular vesicles throughout the cell body and along the processes. The same was seen in ASA‐deficient mice, which accumulated a higher proportion (15 mol% compared with 8 mol% in control mice) of sulfatide with stearic acid. We conclude that the major fatty acid composition of sulfatide differs between white and grey matter, with neurons and astrocytes containing mostly short‐chain fatty acids with an emphasis on stearic acid. Based on our results, we speculate that the fatty acid chain length of sulfatide might determine its intracellular (short chain) or extracellular (long chain) location and thereby its functions.

NKT Cells Stimulated by Long Fatty Acyl Chain Sulfatides Significantly Reduces the Incidence of Type 1 Diabetes in Nonobese Diabetic Mice

Sulfatide-reactive type II NKT cells have been shown to regulate autoimmunity and anti-tumor immunity. Although, two major isoforms of sulfatide, C16:0 and C24:0, are enriched in the pancreas, their relative role in autoimmune diabetes is not known. Here, we report that sulfatide/CD1d-tetramer+ cells accumulate in the draining pancreatic lymph nodes, and that treatment of NOD mice with sulfatide or C24:0 was more efficient than C16:0 in stimulating the NKT cell-mediated transfer of a delay in onset from T1D into NOD.Scid recipients. Using NOD.CD1d−/− mice, we show that this delay of T1D is CD1d-dependent. Interestingly, the latter delay or protection from T1D is associated with the enhanced secretion of IL-10 rather than IFN-g by C24:0-treated CD4+ T cells and the deviation of the islet-reactive diabetogenic T cell response. Both C16:0 and C24:0 sulfatide isoforms are unable to activate and expand type I iNKT cells. Collectively, these data suggest that C24:0 stimulated type II NKT cells may regulate protection from T1D by activating DCs to secrete IL-10 and suppress the activation and expansion of type I iNKT cells and diabetogenic T cells. Our results raise the possibility that C24:0 may be used therapeutically to delay the onset and protect from T1D in humans.

Metachromatic leukodystrophy: consequences of sulphatide accumulation

Metachromatic leukodystrophy is a lysosomal lipid storage disorder. It is caused by mutations in the gene for arylsulphatase A, an enzyme involved in the degradation of the sphingolipid 3′‐O‐sulphogalactosylceramide (sulphatide). This membrane lipid can be found in various cell types, but in particularly high concentrations in the myelin of the nervous system. Patients suffer from progressive, finally lethal, demyelination due to accumulation of sulphatide. In the nervous system, lipid storage not only affects oligodendrocytes but also neurons and, in addition, leads to astrogliosis and activation of microglia. At the cellular level, lysosomal sulphatide storage also affects the lipid composition of myelin itself and has consequences for the amount and localization of particular myelin membrane‐associated proteins. Here we review data, largely based on an arylsulphatase A knock‐out mouse model of metachromatic leukodystrophy.

Successful low-risk hematopoietic cell therapy in a mouse model of type 1 Gaucher disease.

Hematopoietic stem cell‐based gene therapy offers the possibility of permanent correction for genetic disorders of the hematopoietic system. However, optimization of present protocols is required before gene therapy can be safely applied as general treatment of genetic diseases. In this study we have used a mouse model of type 1 Gaucher disease (GD) to demonstrate the feasibility of a low‐risk conditioning regimen instead of standard radiation, which is associated with severe adverse effects. We first wanted to establish what level of engraftment and glucosylceramidase (GCase) activity is required to correct the pathology of the type 1 GD mouse. Our results demonstrate that a median wild‐type (WT) cell engraftment of 7%, corresponding to GCase activity levels above 10 nmoles/hour and mg protein, was sufficient to reverse pathology in bone marrow and spleen in the GD mouse. Moreover, we applied nonmyeloablative doses of busulfan as a pretransplant conditioning regimen and show that even WT cell engraftment in the range of 1%–10% can confer a beneficial therapeutical outcome in this disease model. Taken together, our data provide encouraging evidence for the possibility of developing safe and efficient conditioning protocols for diseases that require only a low level of normal or gene‐corrected cells for a permanent and beneficial therapeutic outcome. STEM CELLS 2009;27:744–752

Differences in cerebrospinal fluid dynamics do not affect the levels of biochemical markers in ventricular CSF from patients with aqueductal stenosis and idiopathic normal pressure hydrocephalus

To compare levels of biochemical markers in ventricular cerebrospinal fluid (vCSF) between patients with aqueductal stenosis (AS) and idiopathic normal pressure hydrocephalus (INPH) and relate these results to clinical outcome after surgery. Neurofilament light protein, tau protein, sulfatide, vasoactive intestinal peptide (VIP), neuropeptide PYY (NPY) and CSF/serum albumin ratio were measured in vCSF from 18 consecutive AS and 19 consecutive INPH patients. Clinical outcome was evaluated after surgery by standardized indices. The levels of markers were related to clinical outcome. No differences in any of the markers were found between AS and INPH patients. The concentration of sulfatide and albumin ratio correlated inversely with psychometric improvement, whilst VIP and NPY correlated inversely with improvement in alertness. The similar levels of biochemical markers in vCSF from AS and INPH patients indicate similarities in pathophysiology and turnover rate of vCSF despite differences in CSF dynamics. High albumin ratio and sulfatide concentrations in vCSF in hydrocephalus patients have negative implications for surgical outcome and might indicate concomitant cerebrovascular disorder.