Henk van Veen

Candidacidal effects of two antimicrobial peptides: histatin 5 causes small membrane defects, but LL-37 causes massive disruption of the cell membrane

The effects of antimicrobial peptides on artificial membranes have been well-documented; however, reports on the ultrastructural effects on the membranes of micro-organisms are relatively scarce. We compared the effects of histatin 5 and LL-37, two antimicrobial peptides present in human saliva, on the functional and morphological properties of the Candida albicans cell membrane. Fluorescence microscopy and immunogold transmission electron microscopy revealed that LL-37 remained associated with the cell wall and cell membrane, whereas histatin 5 transmigrated over the membrane and accumulated intracellularly. Freeze-fracture electron microscopy revealed that LL-37 severely affected the membrane morphology, resulting in the disintegration of the membrane bilayer into discrete vesicles, and an instantaneous efflux of small molecules such as ATP as well as larger molecules such as proteins with molecular masses up to 40 kDa. The effects of histatin 5 on the membrane morphology were less pronounced, but still resulted in the efflux of nucleotides. As the morphological defects induced by histatin 5 are much smaller than those induced by LL-37, but the efflux of nucleotides is similar at comparable candidacidal concentrations, we suggest that the loss of nucleotides plays an important role in the killing process.

The value of enzyme leakage for the prediction of necrosis in liver ischemia.

SummaryFollowing the clamping of the afferent vessels of the left lateral and median lobes in rat liver, a considerable part of these lobes show signs of necrosis 24 h after 90 min of ischemia, wheras no necrotic areas can be detected after 30 min interruption of the blood flow. The purpose of this study was to examine the value of an analysis of the leakage of enzymes from the liver parenchyma in the early phase after restoration of the blood flow after ichemia for a prediction of the occurrence of necrosis. Leakage of the enzymes GPT, GOT and LDH can be detected in the blood plasma with a maximum activity between 1 and 5 h both following 30 and 90 min of ischemia; a considerable difference in clearance is observed, however, in the period afterwards, the normal situation being reached after 24 h with the 30-min ischemic period, but not following the 90-min period. With use of an enzyme histochemical reaction, in situ a depletion of LDH-activity in the hepatocytes could be detected within a short period of time after 30 min temporary ischemia and a restoration during the following period of 24 h; the decrease in LDH-activity persisted during 24 h with a 90-min period of ischemia. Electronmicroscopically cytoplasmic blebs arosen from hepatocytes are observed in the lumen of sinusoids immediately after 30 min of ischemia, whereas after 90 min of ischemia actual leakage of cytoplasmic material takes place through the damaged surface of the hepatocytes. Enzyme leakage probably takes place via these both types of shedding of cytoplasm. It is concluded that the enzyme leakage as such cannot be used as a discriminating test between reversible and irreversible damage of the liver parenchyma.

Skin-Depigmenting Agent Monobenzone Induces Potent T-Cell Autoimmunity toward Pigmented Cells by Tyrosinase Haptenation and Melanosome Autophagy

In this study, we report the previously unknown mechanism of inducing robust anti-melanoma immunity by the vitiligo-inducing compound monobenzone. We show monobenzone to increase melanocyte and melanoma cell immunogenicity by forming quinone-haptens to the tyrosinase protein and by inducing the release of tyrosinase- and melanoma antigen recognized by T cells-1 (MART-1)-containing CD63+ exosomes following melanosome oxidative stress induction. Monobenzone further augments the processing and shedding of melanocyte-differentiation antigens by inducing melanosome autophagy and enhanced tyrosinase ubiquitination, ultimately activating dendritic cells, which induced cytotoxic human melanoma-reactive T cells. These T cells effectively eradicate melanoma in vivo, as we have reported previously. Monobenzone thereby represents a promising and readily applicable compound for immunotherapy in melanoma patients.

Collagen morphology in human skin and scar tissue: no adaptations in response to mechanical loading at joints

Dermal collagen displays a random-like structure that has a major role in strength and function of the human integument. It is hypothesised that collagen bundles align in a parallel fashion in the direction of mechanical tension during scarring, which may explain the problematic scar formation that occurs specifically at joints. Scar tissue and normal skin were biopsied from joints and control areas and evaluated by the Fourier analysis. Collagen orientation was represented by an index ranging from 0 (perfectly random) to 1 (perfectly parallel). Collagen bundle packing signifies the average distance between the centres of collagen bundles. No differences were shown in collagen morphology of scar tissue and normal skin between joints and control areas. Normal skin had a significantly lower collagen orientation index than scar tissue (0.26 versus 0.44, P<0.001). The bundle packing of scar tissue differed significantly from normal skin (18.1 microm versus 23.7 microm, P<0.001). Collagen appeared less parallel orientated in deep dermis compared to superficial dermis especially for normal skin (0.27 versus 0.33, P=0.06). Normal skin had a less parallel organisation in sections that were cut parallel compared to those that were cut perpendicular to the epidermis (0.24 versus 0.30, P=0.02). Collagen orientation of scar tissue is more parallel compared to normal skin. Morphology differs with respect to superficial and deep dermal layers and parallel and perpendicular planes, but appears not to respond to mechanical tension.

Heparin blocks transfer of extracellular vesicles between donor and recipient cells

Extracellular vesicles (EVs) have been implicated in tumorigenesis. Biomolecules which can block EV binding and uptake into recipient cells may be of therapeutic value as well as enhance understanding of EV biology. Here, we show that heparin interacts with uptake of tumor-derived as well as non-tumor-derived EVs into recipient cells. Incubation of glioma cell-derived EVs with heparin resulted in micron-sized structures observed by transmission electron microscopy, with EVs clearly visible within these structures. Inclusion of heparin greatly diminished transfer of labeled EVs from donor to recipient tumor cells. We also show a direct interaction between heparin and EVs using confocal microscopy. We found that the block in EV uptake was at the level of cell binding and not internalization. Finally, incubation of glioma-derived EVs containing EGFRvIII mRNA with heparin reduced transfer of this message to recipient cells. The effect of heparin on EVs uptake may provide a unique tool to study EV function. It may also foster research of heparin or its derivatives as a therapeutic for disease in which EVs play a role.

Mimicking proteasomal release of polyglutamine peptides initiates aggregation and toxicity

Several neurodegenerative disorders, including Huntingtons disease, are caused by expansion of the polyglutamine (polyQ) tract over 40 glutamines in the disease-related protein. Fragments of these proteins containing the expanded polyQ tract are thought to initiate aggregation and represent the toxic species. Although it is not clear how these toxic fragments are generated, in vitro data suggest that proteasomes are unable to digest polyQ tracts. To examine whether the resulting polyQ peptides could initiate aggregation in living cells, we mimicked proteasomal release of monomeric polyQ peptides. These peptides lack the commonly used starting methionine residue or any additional tag. Only expanded polyQ peptides seem to be peptidase resistant, and their accumulation initiated the aggregation process. As observed in polyQ disorders, these aggregates subsequently sequestered proteasomes, ubiquitin and polyQ proteins, and recruited Hsp70. The generated expanded polyQ peptides were toxic to neuronal cells. Our approach mimics proteasomal release of pure polyQ peptides in living cells, and represents a valuable tool to screen for proteins and compounds that affect aggregation and toxicity.

The DNAJB6 and DNAJB8 protein chaperones prevent intracellular aggregation of polyglutamine peptides

Background: Intracellular aggregation of polyglutamine (polyQ) proteins can be prevented by the chaperones DNAJB6 and DNAJB8. Results: DNAJB6 and DNAJB8 prevent the aggregation of pure polyQ peptides. Conclusion: The polyQ tract is sufficient for DNAJB6 and DNAJB8 to prevent aggregation. Significance: By interacting with polyQ fragments, DNAJB6 and DNAJB8 reduce polyQ protein aggregation and may be potential therapeutic targets in polyQ disorders. Fragments of proteins containing an expanded polyglutamine (polyQ) tract are thought to initiate aggregation and toxicity in at least nine neurodegenerative diseases, including Huntingtons disease. Because proteasomes appear unable to digest the polyQ tract, which can initiate intracellular protein aggregation, preventing polyQ peptide aggregation by chaperones should greatly improve polyQ clearance and prevent aggregate formation. Here we expressed polyQ peptides in cells and show that their intracellular aggregation is prevented by DNAJB6 and DNAJB8, members of the DNAJ (Hsp40) chaperone family. In contrast, HSPA/Hsp70 and DNAJB1, also members of the DNAJ chaperone family, did not prevent peptide-initiated aggregation. Intriguingly, DNAJB6 and DNAJB8 also affected the soluble levels of polyQ peptides, indicating that DNAJB6 and DNAJB8 inhibit polyQ peptide aggregation directly. Together with recent data showing that purified DNAJB6 can suppress fibrillation of polyQ peptides far more efficiently than polyQ expanded protein fragments in vitro, we conclude that the mechanism of DNAJB6 and DNAJB8 is suppression of polyQ protein aggregation by directly binding the polyQ tract.

Paravascular channels, cisterns, and the subarachnoid space in the rat brain: A single compartment with preferential pathways:

Recent evidence suggests an extensive exchange of fluid and solutes between the subarachnoid space and the brain interstitium, involving preferential pathways along blood vessels. We studied the anatomical relations between brain vasculature, cerebrospinal fluid compartments, and paravascular spaces in male Wistar rats. A fluorescent tracer was infused into the cisterna magna, without affecting intracranial pressure. Tracer distribution was analyzed using a 3D imaging cryomicrotome, confocal microscopy, and correlative light and electron microscopy. We found a strong 3D colocalization of tracer with major arteries and veins in the subarachnoid space and large cisterns, attributed to relatively large subarachnoid space volumes around the vessels. Confocal imaging confirmed this colocalization and also revealed novel cisternal connections between the subarachnoid space and ventricles. Unlike the vessels in the subarachnoid space, penetrating arteries but not veins were surrounded by tracer. Correlative light and electron microscopy images indicated that this paravascular space was located outside of the endothelial layer in capillaries and just outside of the smooth muscle cells in arteries. In conclusion, the cerebrospinal fluid compartment, consisting of the subarachnoid space, cisterns, ventricles, and para-arteriolar spaces, forms a continuous and extensive network that surrounds and penetrates the rat brain, in which mixing may facilitate exchange between interstitial fluid and cerebrospinal fluid.

A scanning electron microscopic study of biliary stent materials

BACKGROUND Clogging of biliary stents remains an important problem. In vitro studies have shown less sludge formation in Teflon stents. Recently, clinical studies with Teflon stents have produced contradictory results. The aim of this study was to investigate whether the surface properties of the endoprostheses could explain the variation observed in clinical studies. METHODS A total of 9 different types of unused 10F endoprostheses were examined by scanning electron microscopy (SEM): polyethylene Amsterdam-type, polyurethane Amsterdam-type, Teflon Amsterdam-type, Teflon Tannenbaum-type and a Tannenbaum-type stent with a thin stainless steel mesh between inner and outer layers. RESULTS All polyethylene stents had a relief with tiny lumps. All Teflon stents had multiple shallow pits and ridges along the entire longitudinal axis. Both Tannenbaum-type stents also had multiple particles protruding into the stent lumen with adjacent holes in the wall of the stent. The polyurethane stent had an extremely smooth surface. CONCLUSION SEM of Teflon made stents showed a markedly irregular inner surface, which may explain the controversial results of clinical studies. Our results indicate that the inner surface of a new stent should first be evaluated by SEM before clinical trials are initiated.

Ultrastructural changes in the rat liver during Euro-Collins storage, compared with hypothermic in vitro ischemia.

SummaryThe ultrastructural alterations in liver tissue induced by in vitro ischemia at 4° C under conditions commonly used for transplantation (Euro-Collins perfused and stored liver tissue) have been compared with changes due to hypothermic in vitro ischemia in non-perfused liver. It was found that the process of cell deterioration in nonperfused liver occurred very slowly; signs of irreversible damage appeared in sinusoidal lining cells before hepatocytes (after 24 and 96 h, respectively). Liver perfused with, and stored in Euro-Collins solution showed acceleration of the ischemical damage in both types of cell (irreversible damage to sinusoidal lining cells after 12 h and to hepatocytes after 52 h), compared with non-perfused liver.These findings indicate that the safe period for storage of rat liver in Euro-Collins before damage to the microcirculatory system is less than 12 h. It might also be questioned whether Euro-Collins treatment is the optimal procedure for tissue preservation before liver transplantation.