Potential of Personalized Dendritic Cell-Based Immunohybridoma Vaccines to Treat Prostate Cancer.

Prostate cancer (PCa) is the most commonly diagnosed cancer and the second most common cause of death due to cancer. About 30% of patients with PCa who have been castrated develop a castration-resistant form of the disease (CRPC), which is incurable. In the last decade, new treatments that control the disease have emerged, slowing progression and spread and prolonging survival while maintaining the quality of life. These include immunotherapies; however, we do not yet know the optimal combination and sequence of these therapies with the standard ones. All therapies are not always suitable for every patient due to co-morbidities or adverse effects of therapies or both, so there is an urgent need for further work on new therapeutic options. Advances in cancer immunotherapy with an immune checkpoint inhibition mechanism (e.g., ipilimumab, an anti-CTLA-4 inhibitor) have not shown a survival benefit in patients with CRPC. Other immunological approaches have also not given clear results, which has indirectly prevented breakthrough for this type of therapeutic strategy into clinical use. Currently, the only approved form of immunotherapy for patients with CRPC is a cell-based medicine, but it is only available to patients in some parts of the world. Based on what was gained from recently completed clinical research on immunotherapy with dendritic cell-based immunohybridomas, the aHyC dendritic cell vaccine for patients with CRPC, we highlight the current status and possible alternatives that should be considered in the future.

Dendritic cell-based vaccine prolongs survival and time to next therapy independently of the vaccine cell number.

In 2009, new EU legislation regulating advanced therapy medicinal products (ATMPs), consisting of gene therapy, tissue engineering and cell-based medicines, was introduced. Although less than 20 ATMPs were authorized since that time, the awarding of the Nobel Prize for Physiology or Medicine in 2018 revived interest in developing new cancer immunotherapies involving significant manipulation of the patient's own immune cells, including lymphocytes and dendritic cells. The lymphocytes are mainly thought to directly affect tumour cells, dendritic cells are involved in indirect mechanisms by antigen presentation to other leukocytes orchestrating the immune response. It is the latter cells that are the focus of this brief review. Based on the recent results of our study treating patients with castration-resistant prostate cancer (CRPC) with an immunohybridoma cell construct (termed aHyC), produced by electrofusion of autologous tumour and dendritic cells, we compare their effectiveness with a matched documented control group of patients. The results revealed that cancer-specific survival and the time to next in-line therapy (TTNT) were both significantly prolonged versus controls. When patients were observed for longer periods since the time of diagnosis of CRPC, 20% of patients had not yet progressed to the next in-line therapy even though the time under observation was ~ 80 months. Interestingly, analysis of survival of patients revealed that the effectiveness of treatment was independent of the number of cells in the vaccine used for treatment. It is concluded that autologous dendritic cell-based immunotherapy is a new possibility to treat not only CRPC but also other solid tumours.

Inhibiting glycolysis rescues memory impairment in an intellectual disability Gdi1-null mouse.

OBJECTIVES: GDI1 gene encodes for αGDI, a protein controlling the cycling of small GTPases, reputed to orchestrate vesicle trafficking. Mutations in human GDI1 are responsible for intellectual disability (ID). In mice with ablated Gdi1, a model of ID, impaired working and associative short-term memory was recorded. This cognitive phenotype worsens if the deletion of αGDI expression is restricted to neurons. However, whether astrocytes, key homeostasis providing neuroglial cells, supporting neurons via aerobic glycolysis, contribute to this cognitive impairment is unclear. METHODS: We carried out proteomic analysis and monitored [18F]-fluoro-2-deoxy-d-glucose uptake into brain slices of Gdi1 knockout and wild type control mice. d-Glucose utilization at single astrocyte level was measured by the Förster Resonance Energy Transfer (FRET)-based measurements of cytosolic cyclic AMP, d-glucose and L-lactate, evoked by agonists selective for noradrenaline and L-lactate receptors. To test the role of astrocyte-resident processes in disease phenotype, we generated an inducible Gdi1 knockout mouse carrying the Gdi1 deletion only in adult astrocytes and conducted behavioural tests. RESULTS: Proteomic analysis revealed significant changes in astrocyte-resident glycolytic enzymes. Imaging [18F]-fluoro-2-deoxy-d-glucose revealed an increased d-glucose uptake in Gdi1 knockout tissue versus wild type control mice, consistent with the facilitated d-glucose uptake determined by FRET measurements. In mice with Gdi1 deletion restricted to astrocytes, a selective and significant impairment in working memory was recorded, which was rescued by inhibiting glycolysis by 2-deoxy-d-glucose injection. CONCLUSIONS: These results reveal a new astrocyte-based mechanism in neurodevelopmental disorders and open a novel therapeutic opportunity of targeting aerobic glycolysis, advocating a change in clinical practice.

Differences in cytosolic glucose dynamics in astrocytes and adipocytes measured by FRET-based nanosensors.

The cellular response to fluctuations in blood glucose levels consists of integrative regulation of cell glucose uptake and glucose utilization in the cytosol, resulting in altered levels of glucose in the cytosol. Cytosolic glucose is difficult to be measured in the intact tissue, however recently methods have become available that allow measurements of glucose in single living cells with fluorescence resonance energy transfer (FRET) based protein sensors. By studying the dynamics of cytosolic glucose levels in different experimental settings, we can gain insights into the properties of plasma membrane permeability to glucose and glucose utilization in the cytosol, and how these processes are modulated by different environmental conditions, agents and enzymes. In this review, we compare the cytosolic regulation of glucose in adipocytes and astrocytes - two important regulators of energy balance and glucose homeostasis in whole body and brain, respectively, with particular emphasis on the data obtained with FRET based protein sensors as well as other biochemical and molecular approaches.

Enhancement of Astroglial Aerobic Glycolysis by Extracellular Lactate-Mediated Increase in cAMP.

Besides being a neuronal fuel, L-lactate is also a signal in the brain. Whether extracellular L-lactate affects brain metabolism, in particular astrocytes, abundant neuroglial cells, which produce L-lactate in aerobic glycolysis, is unclear. Recent studies suggested that astrocytes express low levels of the L-lactate GPR81 receptor (EC50 ≈ 5 mM) that is in fat cells part of an autocrine loop, in which the Gi-protein mediates reduction of cytosolic cyclic adenosine monophosphate (cAMP). To study whether a similar signaling loop is present in astrocytes, affecting aerobic glycolysis, we measured the cytosolic levels of cAMP, D-glucose and L-lactate in single astrocytes using fluorescence resonance energy transfer (FRET)-based nanosensors. In contrast to the situation in fat cells, stimulation by extracellular L-lactate and the selective GPR81 agonists, 3-chloro-5-hydroxybenzoic acid (3Cl-5OH-BA) or 4-methyl-N-(5-(2-(4-methylpiperazin-1-yl)-2-oxoethyl)-4-(2-thienyl)-1,3-thiazol-2-yl)cyclohexanecarboxamide (Compound 2), like adrenergic stimulation, elevated intracellular cAMP and L-lactate in astrocytes, which was reduced by the inhibition of adenylate cyclase. Surprisingly, 3Cl-5OH-BA and Compound 2 increased cytosolic cAMP also in GPR81-knock out astrocytes, indicating that the effect is GPR81-independent and mediated by a novel, yet unidentified, excitatory L-lactate receptor-like mechanism in astrocytes that enhances aerobic glycolysis and L-lactate production via a positive feedback mechanism.

The uptake, retention and clearance of drug-loaded dendrimer nanoparticles in astrocytes - electrophysiological quantification.

Nanoparticle-based drug delivery systems may impose risks to patients due to potential toxicity associated with a lack of clearance from cells or prolonged carrier-cell retention. This work evaluates vesicular cell uptake, retention and the possible transfer of endocytosed methylprednisolone-loaded carboxymethylchitosan/poly(amidoamine) dendrimer nanoparticles (NPs) into secretory vesicles of rat cultured astrocytes. The cells were incubated with NPs and unitary vesicle fusions/fissions with the plasma membrane were monitored employing high-resolution membrane capacitance measurements. In the NP-treated cells the frequency of unitary exocytotic events was significantly increased. The presence of NPs also induces an increase in the size of exocytotic vesicles interacting with the plasma membrane, which exhibit transient fusion with prolonged fusion pore dwell-time. Live-cell confocal imaging revealed that once NPs internalize into endocytotic compartments they remain in the cell for 7 days, although a significant proportion of these merge with secretory vesicles destined for exocytosis. Co-localization studies show the route of clearance of NPs from cells via the exocytotic pathway. These findings bring new insight into the understanding of the intracellular trafficking and biological interactions of drug-loaded dendrimer NPs targeting astrocytes.

Systemic Hypoxia Increases the Expression of DPP4 in Preadipocytes of Healthy Human Participants.

Dipeptidyl peptidase 4 (DPP4) is a transmembrane glycoprotein involved in protein degradation. Due to its action on incretins, which increase insulin secretion, DPP4 is considered a therapeutic target for type 2 diabetes. Here we have studied the role of single and combined effects of hypoxia and inactivity on the expression of DPP4 in human adipose tissue of 12 adult normal-weight males. Fat biopsies were obtained at baseline and after each of three experimental campaigns. The results revealed that in isolated human preadipocytes the expression of DPP4 was significantly increased by exposure of participants to hypoxia. Physical inactivity per se had no apparent effect on the DPP4 expression. It is concluded that DPP4 may be a marker to monitor indirectly tissue hypoxia, as occurs in obese subjects.

Hypoxia Alters the Expression of Dipeptidyl Peptidase 4 and Induces Developmental Remodeling of Human Preadipocytes.

Dipeptidyl peptidase 4 (DPP4), a transmembrane protein, has been identified in human adipose tissue and is considered to be associated with obesity-related type 2 diabetes. Since adipose tissue is relatively hypoxic in obese participants, we investigated the expression of DPP4 in human preadipocytes (hPA) and adipocytes in hypoxia, during differentiation and upon insulin stimulation. The results show that DPP4 is abundantly expressed in hPA but very sparsely in adipocytes. During differentiation in vitro, the expression of DPP4 in hPA is reduced on the addition of differentiation medium, indicating that this protein can be hPA marker. Long term hypoxia altered the expression of DPP4 in hPA. In in vitro hypoxic conditions the protease activity of shed DPP4 is reduced; however, in the presence of insulin, the increase in DPP4 expression is potentiated by hypoxia.

Insulin and Insulin-like Growth Factor 1 (IGF-1) Modulate Cytoplasmic Glucose and Glycogen Levels but Not Glucose Transport across the Membrane in Astrocytes.

Astrocytes contain glycogen, an energy buffer, which can bridge local short term energy requirements in the brain. Glycogen levels reflect a dynamic equilibrium between glycogen synthesis and glycogenolysis. Many factors that include hormones and neuropeptides, such as insulin and insulin-like growth factor 1 (IGF-1) likely modulate glycogen stores in astrocytes, but detailed mechanisms at the cellular level are sparse. We used a glucose nanosensor based on Förster resonance energy transfer to monitor cytosolic glucose concentration with high temporal resolution and a cytochemical approach to determine glycogen stores in single cells. The results show that after glucose depletion, glycogen stores are replenished. Insulin and IGF-1 boost the process of glycogen formation. Although astrocytes appear to express glucose transporter GLUT4, glucose entry across the astrocyte plasma membrane is not affected by insulin. Stimulation of cells with insulin and IGF-1 decreased cytosolic glucose concentration, likely because of elevated glucose utilization for glycogen synthesis.

Insulin induces an increase in cytosolic glucose levels in 3T3-L1 cells with inhibited glycogen synthase activation.

Glucose is an important source of energy for mammalian cells and enters the cytosol via glucose transporters. It has been thought for a long time that glucose entering the cytosol is swiftly phosphorylated in most cell types; hence the levels of free glucose are very low, beyond the detection level. However, the introduction of new fluorescence resonance energy transfer-based glucose nanosensors has made it possible to measure intracellular glucose more accurately. Here, we used the fluorescent indicator protein (FLIPglu-600µ) to monitor cytosolic glucose dynamics in mouse 3T3-L1 cells in which glucose utilization for glycogen synthesis was inhibited. The results show that cells exhibit a low resting cytosolic glucose concentration. However, in cells with inhibited glycogen synthase activation, insulin induced a robust increase in cytosolic free glucose. The insulin-induced increase in cytosolic glucose in these cells is due to an imbalance between the glucose transported into the cytosol and the use of glucose in the cytosol. In untreated cells with sensitive glycogen synthase activation, insulin stimulation did not result in a change in the cytosolic glucose level. This is the first report of dynamic measurements of cytosolic glucose levels in cells devoid of the glycogen synthesis pathway.

Cholesterol and regulated exocytosis: a requirement for unitary exocytotic events.

Since the 1970s, much effort was been expended researching mechanisms of regulated exocytosis. Early work focused mainly on the role of proteins. Most notably the discovery of SNARE proteins in the 1980s and the zippering hypothesis brought us much closer to understanding the complex interactions in membrane fusion between vesicle and plasma membranes, a pivotal component of regulated exocytosis. However, most likely due to the predictions of the Singer-Nicholson fluid mosaic membrane model, the lipid components of the exocytotic machinery remained largely overlooked. Lipids were considered passive constituents of cellular membranes, not contributing much, if anything, to the process of exocytosis and membrane fusion. Since the 1990s, this so-called proteocentric view has been gradually giving way to the new perspective best described with the term proteolipidic. Many lipids were found to be of great importance in the regulation of exocytosis. Here we highlight the role of cholesterol. Furthermore, by using high-resolution cell-attached membrane capacitance measurements, we have monitored unitary exocytotic events in cholesterol-depleted membranes. We show that the frequency of these events is attenuated, providing evidence at the single vesicle level that cholesterol directly influences the merger of the vesicle and the plasma membranes.

Rab4 and Rab5 GTPase are required for directional mobility of endocytic vesicles in astrocytes.

Rab4 and Rab5 GTPases are key players in the regulation of endocytosis. Although their role has been studied intensively in the past, it is still unclear how they regulate vesicle mobility. In particular, in astrocytes, the most abundant glial cells in the brain, vesicles have been shown to exhibit nondirectional and directional mobility, which can be intermittent, but the underlying switching mechanisms are not known. By using quantitative imaging, we studied the dynamics of single vesicle movements in astrocytes in real time, by transfecting them with different GDP- and GTP-locked mutants of Rab4 and Rab5. Along with the localization of Rab4 and Rab5 on early and late endocytic compartments, we measured the apparent vesicle size by monitoring the area of fluorescent puncta and determined the patterns of vesicle mobility in the presence of wild-type and Rab mutants. Dominant-negative and dominant-positive mutants, Rab4 S22N, Rab5 S34N and Rab4 Q67L, Rab5 Q79L, induced an increase in the apparent vesicle size, especially Rab5 mutants. These mutants also significantly reduced vesicle mobility in terms of vesicle track length, maximal displacement, and speed. In addition, significant reductions in the fraction of vesicles exhibiting directional mobility were observed in cells expressing Rab4 S22N, Rab4 Q67L, Rab5 S34N, and Rab5 Q79L. Our data indicate that changes in the GDP-GTP switch apparently not only affect fusion events in endocytosis and recycling, as already proposed, but also affect the molecular interactions determining directional vesicle mobility, likely involving motor proteins and the cytoskeleton.

New insights into cytosolic glucose levels during differentiation of 3T3-L1 fibroblasts into adipocytes.

Cytosolic glucose concentration reflects the balance between glucose entry across the plasma membrane and cytosolic glucose utilization. In adipocytes, glucose utilization is considered very rapid, meaning that every glucose molecule entering the cytoplasm is quickly phosphorylated. Thus, the cytosolic free glucose concentration is considered to be negligible; however, it was never measured directly. In the present study, we monitored cytosolic glucose dynamics in 3T3-L1 fibroblasts and adipocytes by expressing a fluorescence resonance energy transfer (FRET)-based glucose nanosensor: fluorescent indicator protein FLIPglu-600µ. Specifically, we monitored cytosolic glucose responses by varying transmembrane glucose concentration gradient. The changes in cytosolic glucose concentration were detected in only 56% of 3T3-L1 fibroblasts and in 14% of 3T3-L1 adipocytes. In adipocytes, the resting cytosolic glucose concentration was reduced in comparison with the one recorded in fibroblasts. Membrane permeabilization increased cytosolic glucose concentration in adipocytes, and glycolytic inhibitor iodoacetate failed to increase cytosolic glucose concentration, indicating low adipocyte permeability for glucose at rest. We also examined the effects of insulin and adrenaline. Insulin significantly increased cytosolic glucose concentration in adipocytes by a factor of 3.6; however, we recorded no effect on delta ratio (ΔR) in fibroblasts. Adrenaline increased cytosolic glucose concentration in fibroblasts but not in adipocytes. However, in adipocytes in insulin-stimulated conditions, glucose clearance was significantly faster following adrenaline addition in comparison with controls (p < 0.001). Together, these results demonstrate that during differentiation, adipocytes develop more efficient mechanisms for maintaining low cytosolic glucose concentration, predominantly with reduced membrane permeability for glucose.

Changes in cytosolic glucose level in ATP stimulated live astrocytes.

Astrocytes which lie between brain capillaries and neuronal terminals are the primary site of glucose uptake and have a key role in coupling synaptic activity to glucose utilization in the central nervous system (CNS). We used a fluorescence resonance energy transfer (FRET) based approach to monitor cytosolic glucose in astrocytes. We determined the effect of increasing extracellular glucose concentrations on FRET ratio as a measure of increased cytosolic glucose in astrocytes. By briefly raising extracellular glucose concentration, astrocytes responded promptly by increased cytosolic glucose levels, which was manifested by decreased time-dependent FRET ratio. The FRET ratio fall-time recorded at low extracellular D-glucose concentration change (from 0 to 0.5 mM) was 53 s, whereas 17 s was recorded by raising extracellular concentration of D-glucose from 0 to 10 mM, which is likely due to facilitated d-glucose entry along the increased D-glucose gradient across the plasmalemma. The relationship between the extracellular glucose concentration and the FRET ratio change is limited to the maximal ratio change, where the D-glucose plasma membrane permeability is balanced by the cytosolic utilization. We measured the effect of extracellular ATP, an important extracellular messenger for astrocyte-to-astrocyte communication, on intracellular glucose concentration. The results show that stimulation of astrocytes with ATP (1 mM) decreases cytosolic glucose concentration with a time constant of ∼145 s. The mechanism of this change is discussed.

Rosiglitazone balances insulin-induced exo- and endocytosis in single 3T3-L1 adipocytes.

Rosiglitazone (Rosi) improves insulin sensitivity and increases the translocation of glucose transporter 4 (GLUT4) to the plasma membrane (PM). This involves the fusion of membrane-bound compartments with the plasma membrane, thus increasing the plasma membrane area. However, recent work has shown that in Rosi-pretreated 3T3-L1 adipocytes membrane area did not increase following insulin application, suggesting that the rates of exo- and endocytosis are balanced. Here we examined whether Rosi differentially affects the rates of exo- and endocytosis in 3T3-L1 adipocytes. The immunolabelling of GLUT4 revealed the 3.1-fold increase in PM-resident GLUT4 in Rosi-pretreated, insulin-stimulated cells. By monitoring cumulative exocytosis and endocytosis we found that in Rosi-pretreated cells insulin substantially stimulated the rate of exocytosis and to a similar extent also the rate of endocytosis. We conclude that Rosi-pretreatment balances insulin-stimulated exocytosis and endocytosis, which may prevent insulin-mediated adipocyte cell size increase.

Analysis of confocal images using variable-width line profiles.

A line profile of fluorescent intensities in confocal images is frequently examined. We have developed the computer software tool to analyse the profiles of intensities of fluorescent probes in confocal images. The software averages neighbouring pixels, adjacent to the central line, without reducing the spatial resolution of the image. As an experimental model, we have used the skeletal muscle fibre isolated from the rat skeletal muscle extensor digitorum brevis. As a marker of myofibrils' structure, we have used phalloidin-rhodamine staining and the anti-TIM antibody to label mitochondria. We also tested the distribution of the protein kinase B/Akt. Since signalling is ordered in modules and large protein complexes appear to direct signalling to organelles and regulate specific physiological functions, a software tool to analyse such complexes in fluorescent confocal images is required. The software displays the image, and the user defines the line for analysis. The image is rotated by the angle of the line. The line profile is calculated by averaging one dimension of the cropped rotated image matrix. The spatial resolution in averaged line profile is not decreased compared with single-pixel line profile, which was confirmed by the discrete Fourier transform computed with a fast Fourier transform algorithm. We conclude that the custom software tool presented here is a useful tool to analyse line profiles of fluorescence intensities in confocal images.

Rosiglitazone modulates insulin-induced plasma membrane area changes in single 3T3-L1 adipocytes.

In this study we hypothesized that rosiglitazone, an antidiabetic high-affinity agonist for the peroxisome proliferator-activated receptor gamma, affects the plasma membrane (PM) turnover in single 3T3-L1 adipocytes. To study the PM turnover, the patch-clamp electrophysiological method was used to measure changes in membrane capacitance (Cm), a parameter linearly related to the PM area. Microscopy results show that the presence of rosiglitazone in the differentiating medium significantly increased the differentiation of 3T3-L1 adipocytes in cell culture, based on oil red O-stained area (11.4 +/- 1.2%) vs. controls (3.1 +/- 0.5%). Moreover, rosiglitazone treatment significantly reduced the size of single 3T3-L1 adipocytes; their average radius of 21.1 +/- 1.1 microm in controls was reduced to 17.5 +/- 0.5 microm in rosiglitazone-treated cells. Consistent with this, insulin application increased the rate of Cm increase to 2.34 +/- 0.10%/min, which was significantly different from controls (0.12 +/- 0.08%/min). However, pretreatment of cells with rosiglitazone prior to the treatment with insulin resulted in an attenuated rate of Cm increase. These data support the involvement of insulin in the modulation of membrane area and show that treatment by rosiglitazone reduced the insulin-mediated membrane area increase in 3T3-L1 adipocytes.

Prolactin secretion sites contain syntaxin-1 and differ from ganglioside monosialic acid rafts in rat lactotrophs.

In neuroendocrine cells, discharge of hormones follows the fusion of exocytotic vesicles with the plasma membrane at confined sites; however, the molecular nature of these distinct sites remains poorly understood. We studied intact pituitary lactotrophs and plasma membrane lawns by confocal microscopy in conjunction with antibodies against rat prolactin (rPRL), soluble N-ethylmaleimide-sensitive factor-attachment protein receptor (SNARE) proteins (syntaxin-1 and synaptobrevin-2,) and fluorescent cholera toxin subunit B (CT-B), a marker of ganglioside monosialic acid (GM1) lipid rafts, to examine 1) whether rPRL vesicles discharge cargo at GM1 rafts, 2) whether discharging rPRL vesicles interact with SNAREs, and 3) to examine the overlap of GM1 rafts, rPRL, and syntaxin-1 sites in plasma membrane lawns. In intact cells, immunofluorescently labeled rPRL poorly colocalized (<6%) with CT-B. In conditions favoring endocytotic trafficking, vesicle SNARE synaptobrevin-2 modestly colocalized (35%) with CT-B, whereas it highly colocalized (58%) with retrieved rPRL. Although partial mixing between rPRL and CT-B intracellular trafficking pathways is likely, our results indicated that rPRL discharge involves interactions with plasma membrane SNAREs, but not with GM1 rafts. In support of this, the plasma membrane SNARE syntaxin-1 poorly colocalized with CT-B (<5%), whereas it highly colocalized (75%) with rPRL in inside-out plasma membrane lawns. Spontaneous and stimulated rPRL discharge in live lactotrophs is thus associated with plasma membrane sites enriched with SNARE proteins, however, spatially confined to plasma membrane areas other than GM1 rafts.

Lysophospholipids prevent binding of a cytolytic protein ostreolysin to cholesterol-enriched membrane domains.

Ostreolysin, a 15kDa pore-forming protein from the oyster mushroom (Pleurotus ostreatus), binds specifically to cholesterol-enriched membrane domains existing in the liquid-ordered phase, and lyses cells and lipid vesicles made of cholesterol and sphingomyelin. We have monitored binding of sub-lytic concentrations of ostreolysin to membranes of Chinese Hamster Ovary cells and rat somatotrophs, using primary anti-ostreolysin and fluorescence-labeled secondary antibodies detected by confocal microscopy. Depletion of more than 40% membrane cholesterol content by methyl-beta-cyclodextrin dramatically decreased ostreolysin binding. Immunostaining showed that ostreolysin is not co-localized with raft-binding proteins, cholera toxin B-subunit or caveolin, suggesting that natural membranes display heterogeneity of cholesterol-enriched raft-like microdomains. Impaired ostreolysin binding was also observed after treating the cells with lysophosphatidylinositol. Effects of lysophosphatidylinositol on binding of ostreolysin to immobilized large sphingomyelin/cholesterol (1/1, mol/mol) unilamellar vesicles were studied by a surface plasmon resonance technique. Injection of ostreolysin during the lysophosphatidylinositol dissociation phase showed an inverse relationship between ostreolysin binding and the quantity of lysophosphatidylinositol in the membranes of lipid vesicles. It was concluded that lysophospholipids prevent binding of ostreolysin to cell and to artificial lipid membranes resembling lipid rafts, by partitioning into the lipid bilayer and altering the properties of cholesterol-rich microdomains.

Effect of ostreolysin, an Asp-hemolysin isoform, on human chondrocytes and osteoblasts, and possible role of Asp-hemolysin in pathogenesis.

Aspergillus fumigatus, a pathogenic mould causing a wide range of diseases including aspergillosis, produces a series of toxic substances which appear to act in an additive and/or synergic way on cells. Aspergillosis generally occurs in immunocompromised hosts or is associated with organ transplantation. From the muscul skeleton system the vertebrae, ribs and orbit are the most commonly affected, while the joints are less frequent targets. The cytolytic protein Asp-hemolysin is one of the toxins produced by Aspergillus fumigatus during infection. It belongs to the aegerolysin protein family and shares 43% identity in amino acid sequence with ostreolysin, a cytolysin from the mushroom Pleurotus ostreatus. In this work, ostreolysin was used in an experimental model to study the in vitro effects of aegerolysin-like proteins on human chondrocytes and osteoblasts. Immunostaining analyses showed selective binding and clustering of the protein on chondrocyte membranes, pointing to its association with distinctive membrane microdomains. Consequently, ostreolysin can induce effective permeabilization of both chondrocytes and osteoblasts. Based on sequence similarities of ostreolysin and Asp-hemolysin, their comparable cytolytic effects towards different cells, and similar signs of intoxication in experimental animals, our results indicate that Asp-hemolysin might be considered as a possible virulence factor of Aspergillus fumigatus during the infection of bone and cartilage.