Human IgG is produced with C-terminal lysines that are cleaved off in circulation. The function of this modification was unknown and generally thought not to affect antibody function. We recently ...reported that efficient C1q binding and complement-dependent cytotoxicity (CDC) requires IgG hexamerization at the cell surface. Here we demonstrate that C-terminal lysines may interfere with this process, leading to suboptimal C1q binding and CDC of cells opsonized with C-terminal lysine-containing IgG. After we removed these lysines with a carboxypeptidase, maximal complement activation was observed. Interestingly, IgG1 mutants containing either a negative C-terminal charge or multiple positive charges lost CDC almost completely; however, CDC was fully restored by mixing C-terminal mutants of opposite charge. Our data indicate a novel post-translational control mechanism of human IgG: human IgG molecules are produced in a pro-form in which charged C-termini interfere with IgG hexamer formation, C1q binding and CDC. To allow maximal complement activation, C-terminal lysine processing is required to release the antibody's full cytotoxic potential.
Abstract
Partly due to poor blood-brain barrier drug penetration the treatment options for many brain diseases are limited. To safely enhance drug delivery to the brain, glutathione PEGylated ...liposomes (G-Technology®) were developed. In this study, in rats, we compared the pharmacokinetics and organ distribution of GSH-PEG liposomes using an autoquenched fluorescent tracer after intraperitoneal administration and intravenous administration. Although the appearance of liposomes in the circulation was much slower after intraperitoneal administration, comparable maximum levels of long circulating liposomes were found between 4 and 24 h after injection. Furthermore, 24 h after injection a similar tissue distribution was found. To investigate the effect of GSH coating on brain delivery in vitro uptake studies in rat brain endothelial cells (RBE4) and an in vivo brain microdialysis study in rats were used. Significantly more fluorescent tracer was found in RBE4 cell homogenates incubated with GSH-PEG liposomes compared to non-targeted PEG liposomes (1.8-fold, p < 0.001). In the microdialysis study 4-fold higher (p < 0.001) brain levels of fluorescent tracer were found after intravenous injection of GSH-PEG liposomes compared with PEG control liposomes. The results support further investigation into the versatility of GSH-PEG liposomes for enhanced drug delivery to the brain within a tolerable therapeutic window.
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DOBA, IJS, IZUM, KILJ, NUK, PILJ, PNG, SAZU, UILJ, UKNU, UL, UM, UPUK
Identification of hyaluronan as a crystal-binding molecule at the surface of migrating and proliferating MDCK cells.
The adherence of calcium oxalate crystals to the renal tubule epithelium is ...considered a critical event in the pathophysiology of calcium nephrolithiasis. Calcium oxalate monohydrate (COM) crystals cannot adhere to the surface of a functional Madin-Darby canine kidney (MDCK) monolayer, but they bind avidly to the surface of proliferating and migrating cells.
To identify crystal-binding molecules (CBMs) at the surface of crystal-attracting cells, we applied metabolic labeling protocols in combination with differential enzymatic digestion and gel filtration, which was compared with 14CCOM crystal binding and confirmed by confocal microscopy.
The indication that hyaluronan hyaluronic acid (HA) might act as a CBM in subconfluent cultures came from studies with glycosaminoglycan (GAG)-degrading enzymes. Subsequently, metabolic-labeling studies revealed that hyaluronidase cleaved significantly more radiolabeled glycoconjugates from crystal-attracting cells than from cells without affinity for crystals. During wound repair, crystal binding could be prevented by pretreating the healing cultures with hyaluronate lyase, an enzyme that specifically hydrolyzes HA. Binding to immobilized HA provided evidence that COM crystals physically can become associated with this polysaccharide. Finally, confocal microscopy demonstrated that fluorescently labeled HA binding protein (HABP) adhered to the surface of proliferating cells in subconfluent cultures as well as to cells involved in closing a wound, but not to cells in confluent monolayers.
These results identify HA as binding molecule for COM crystals at the surface of migrating and proliferating MDCK cells.
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GEOZS, IJS, NUK, OILJ, SBJE, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
The retention of crystals in the kidney is considered to be a crucial step in the development of a renal stone. This study demonstrates the time-dependent alterations in the extent of calcium oxalate ...(CaOx) monohydrate (COM) crystal binding to Madin-Darby canine kidney (MDCK) cells during their growth to confluence and during the healing of wounds made in confluent monolayers. As determined by radiolabeled COM crystal binding studies and confirmed by confocal-scanning laser microscopy, relatively large amounts of crystals (10.4 +/- 0.4 micrograms/cm2) bound to subconfluent cultures that still exhibited a low transepithelial electrical resistance (TER < 400 omega.cm2). The development of junctional integrity, indicated by a high resistance (TER > 1,500 omega.cm2), was followed by a decrease of the crystal binding capacity to almost undetectable low levels (0.13 +/- 0.03 microgram/cm2). Epithelial injury resulted in increased crystal adherence. The highest level of crystal binding was observed 2 days postinjury when the wounds were already morphologically closed but TER was still low. Confocal images showed that during the repair process, crystals selectively adhered to migrating cells at the wound border and to stacked cells at sites were the wounds were closed. After the barrier integrity was restored, crystal binding decreased again to the same low levels as in undamaged controls. These results indicate that, whereas functional MDCK monolayers are largely protected against COM crystal adherence, epithelial injury and the subsequent process of wound healing lead to increased crystal binding.
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The blood–brain barrier (BBB) represents a major obstacle for the delivery and development of drugs curing brain pathologies. However, this biological barrier presents numerous ...endogenous specialized transport systems that can be exploited by engineered nanoparticles to enable drug delivery to the brain. In particular, conjugation of glutathione (GSH) onto PEGylated liposomes (G-Technology®) showed to safely enhance delivery of encapsulated drugs to the brain. Yet, understanding of the mechanism of action remains limited and full mechanistic understanding will aid in the further optimization of the technology. In order to elucidate the mechanism of brain targeting by GSH-PEG liposomes, we here demonstrate that the in vivo delivery of liposomal ribavirin is increased in brain extracellular fluid according to the extent of GSH conjugation onto the liposomes. In vitro, using the hCMEC/D3 human cerebral microvascular endothelial (CMEC) cell line, as well as primary bovine and porcine CMEC (and in contrast to non-brain derived endothelial and epithelial cells), we show that liposomal uptake occurs through the process of endocytosis and that the brain-specific uptake is also glutathione conjugation-dependent. Interestingly, the uptake mechanism is an active process that is temperature-, time- and dose-dependent. Finally, early endocytosis events rely on cytoskeleton remodeling, as well as dynamin- and clathrin-dependent endocytosis pathways. Overall, our data demonstrate that the glutathione-dependent uptake mechanism of the G-Technology involves a specific endocytosis pathway indicative of a receptor-mediated mechanism, and supports the benefit of this drug delivery technology for the treatment of devastating brain diseases.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK, ZRSKP
Renal stone formation requires the persistent retention of crystals in the kidney. Calcium oxalate monohydrate (COM) crystal binding to Madin Darby canine kidney strain I (MDCK-I), a cell line that ...resembles the epithelium in the renal distal tubule/collecting duct, is developmentally regulated, while LLC-PK1 cells (American Type Tissue Collection), which are widely used as a model of the renal proximal tubule, bind crystals irrespective of their stage of epithelial development. Whereas to our knowledge the binding molecules for COM at the surface of LLC-PK1 cells are still unknown, crystals adhere to the hyaluronan (HA) rich pericellular matrix transiently expressed by mobile MDCK-I cells. In the current study we investigated whether crystal binding to either cell type is influenced by urinary substances, including glycoprotein inhibitors of crystallization
We studied crystal binding to MDCK-I cells during wound repair, to confluent LLC-PK1 cells and to HA immobilized on a solid surface using
14C COM pretreated or not pretreated with urine from healthy male volunteers. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot analysis were performed to assess whether the crystals became coated with urine derived proteins
Western blot analysis demonstrated that pretreated COM crystals were covered with protein inhibitors of crystallization. However, this protein coat had no significant effect on the level of crystal binding to either cell type. In contrast, the adherence of urine treated crystals to immobilized HA was significantly reduced
The adherence of crystals to pericellular matrixes may encompass more than their simple fixation to the polysaccharide HA. Calcium oxalate crystal retention is not prevented by coating crystals with urinary constituents such as glycoproteins and, therefore, may predominantly depend on the surface properties of the renal tubular epithelium.
Sialic acid and crystal binding Verkoelen, Carl F.; van der Boom, Burt G.; Kok, Dirk J. ...
Kidney international,
03/2000, Volume:
57, Issue:
3
Journal Article, Conference Proceeding
Peer reviewed
Open access
Sialic acid and crystal binding.
We studied the role of cell surface sialic acid in the adherence of calcium oxalate monohydrate (COM) crystals to Madin-Darby canine kidney (MDCK) cells.
Studies were ...performed with undifferentiated (crystal-binding) cells in subconfluent cultures and maturated (noncrystal-binding) cells in confluent cultures. Lectins were used to study the emergence and abundance of oligosaccharides at the cell surface during epithelial development. The effect of neuraminidase treatment on crystal binding was studied with 14CCOM crystals, and the enzyme-induced release of cell surface-associated sialic acid molecules was monitored by labeling the cells metabolically with 3Hglucosamine
Binding studies with lectins derived from Maackia Amurensis II (MALII) and Sambucus Nigra (SNA) demonstrated that the cells expressed terminal sialic acids attached to penultimate galactose through α2,3 and α2,6 bonds at different stages of epithelial development. Neuraminidase treatment strongly reduced the affinity of the cell surface for COM crystals in subconfluent cultures. Nevertheless, neuraminidase cleaved more sialic acids from cells in confluent cultures than from those in subconfluent cultures. Peanut agglutinin (PNA), which binds only to sialylated terminal galactose units, adhered to developing but not to maturated cells, unless the latter were pretreated with neuraminidase. Both results indicate that the surface of maturated MDCK cells is more heavily sialylated than that of undifferentiated cells. Free sialic acid molecules showed little or no affinity for COM crystals and did not affect the adherence of the crystals to undifferentiated cells.
There are at least two models that may explain these results. First, sialic acids are presented at the surface of immature cells in an orientation that specifically matches crystal surface characteristics favoring crystal–cell interactions. Second, sialic acid molecules are not directly associated with the crystals, but may be involved in the exposure of another crystal binding molecule at the cell surface.
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GEOZS, IJS, NUK, OILJ, SBJE, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Cell cultures and nephrolithiasis Verkoelen, C F; van der Boom, B G; Schröder, F H ...
World journal of urology,
08/1997, Volume:
15, Issue:
4
Journal Article
Peer reviewed
While the physical chemistry of stone formation has been intensively studied during the last decade, it has become clear that the pathophysiology of renal stone disease cannot be explained by ...crystallization processes only. In recent years, evidence has emerged that the cells lining the renal tubules can have an active role in creating the conditions under which stones may develop. Since it is difficult to study these mechanisms in vivo, cultured renal tubular cells have become increasingly popular for the study of physiological and cell biological processes that are possibly linked to stone disease. In this paper, we discuss the possible contribution of cellular processes such as transepithelial oxalate transport and crystal--cell interaction to the formation of renal stones. Experimental studies that have been performed with cultured renal cells to elucidate the mechanisms involved in these processes will be summarized.
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EMUNI, FIS, FZAB, GEOZS, GIS, IJS, IMTLJ, KILJ, KISLJ, MFDPS, NLZOH, NUK, OBVAL, OILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, UKNU, UL, UM, UPUK, VKSCE, ZAGLJ
Cell type-specific acquired protection from crystal adherence by renal tubule cells in culture.
Adherence of crystals to the surface of renal tubule epithelial cells is considered an important step ...in the development of nephrolithiasis. Previously, we demonstrated that functional monolayers formed by the renal tubule cell line, Madin-Darby canine kidney (MDCK), acquire protection against the adherence of calcium oxalate monohydrate crystals. We now examined whether this property is cell type specific. The susceptibility of the cells to crystal binding was further studied under different culture conditions.
Cell-type specificity and the influence of the growth substrate was tested by comparing calcium oxalate monohydrate crystal binding to LLC-PK1 cells and to two MDCK strains cultured on either permeable or impermeable supports. These cell lines are representative for the renal proximal tubule (LLC-PK1) and distal tubule/collecting duct (MDCK) segments of the nephron, in which crystals are expected to be absent and present, respectively.
Whereas relatively large amounts of crystals adhered to subconfluent MDCK cultures, the level of crystal binding to confluent monolayers was reduced for both MDCK strains. On permeable supports, MDCK cells not only obtained a higher level of morphological differentiation, but also acquired a higher degree of protection than on impermeable surfaces. Crystals avidly adhered to LLC-PK1 cells, irrespective of their developmental stage or growth substrate used.
These results show that the prevention of crystal binding is cell type specific and expressed only by differentiated MDCK cells. The anti-adherence properties acquired by MDCK cells may mirror a specific functional characteristic of its in situ equivalent, the renal distal tubule/collecting ducts.
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GEOZS, IJS, NUK, OILJ, SBJE, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
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