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Carmen W van den Berg

Publications and source records attributed to Carmen W van den Berg.

16 recordsLinked to original sources
PubMed2007

Tetracycline protects against dermonecrosis induced by Loxosceles spider venom.

Envenomation by spiders belonging to the Loxosceles genus (brown spider) often results in local dermonecrotic lesions. We have previously shown that Loxosceles sphingomyelinase D (SMase D), the venom component responsible for all the pathological effects, induced the expression of matrix metalloproteinases (MMPs) in rabbits and in human keratinocytic cells. We also showed that the SMase D-induced apoptosis and MMP expression of keratinocytes was inhibited by tetracyclines. We have further investigated the ability of tetracyclines to inhibit or prevent the dermonecrotic lesion induced by Loxosceles venom in vivo and in vitro models. Primary cultures of rabbit fibroblasts incubated with increasing concentrations of venom or SMase D showed a decrease in cell viability, which was prevented by tetracyclines. In vivo experiments showed that topical treatments with tetracycline of rabbits, inoculated with crude Loxosceles intermedia venom or recombinant SMase D, significantly reduced the progression of the dermonecrotic lesion. Furthermore, tetracyclines also reduced the expression of MMP-2 and prevented the induction of MMP-9. Our results suggest that tetracycline may be an effective therapeutic agent for the treatment of cutaneous loxoscelism.

Animals↗
PubMed2006

Structural and functional comparison of native pentameric, denatured monomeric and biotinylated C-reactive protein.

There are many controversies surrounding the biological activities of native C-reactive protein (nCRP) and its various modified forms such as monomerized and biotinylated CRP (mCRP and bCRP). No simple methods have been described to distinguish among these forms. By adapting established electrophoresis methods, we have developed a useful quality control method with which we have investigated the structural and functional characteristics of these forms of CRP. Under all electrophoresis conditions, biotinylation altered the electrophoretic mobility of CRP. nCRP was sensitive to sodium dodecyl sulphate (SDS)-induced monomerization, and only mCRP was susceptible to digestion by trypsin or neutrophil-derived serine proteases. bCRP and mCRP but not nCRP bound to cells, suggesting that chemical modification by biotin and denaturation had altered the structural integrity of CRP. Neither nCRP nor mCRP had the ability to induce secretion of chemokines, nor did they increase intracellular adhesion molecule 1 (ICAM-1) expression in endothelial cells.

Biotin↗
PubMed2006

Characterisation of the complement susceptibility of the rat aortic smooth muscle cell line A7r5.

Complement (C) activation is thought to contribute to the initiation and progression of atherosclerosis. Proliferation of smooth muscle cells plays an important role in atherosclerotic plaque formation. Our aim was to investigate the suitability of the rat aortic smooth muscle cell line A7r5 as an in vitro model to study C-induced events in smooth muscle cells. A7r5 cells abundantly expressed membrane bound C-regulators (CReg) Crry and CD59 as assessed by flow-cytometry, but no DAF or MCP was detected. Using RT-PCR in addition to Crry and CD59, also mRNA for rat DAF but not for MCP was detected. Flow-cytometry of cells removed by EDTA instead of trypsin demonstrated that A7r5 did express cell surface DAF. Upon prolonged culturing under either logarithmic growing conditions or under conditions where cells were kept over-confluent, two different sub cell lines were obtained, one which had lost the expression of CD59, while the other showed increased expression of DAF and Crry. The change in expression of these CReg resulted in a change in C-susceptibility. Incubation of the A7r5 cells with human serum induced membrane attack complex dependent proliferation. Transfection with human CD59 efficiently protected the cells from C-mediated killing and C-induced cell proliferation. Our results show that A7r5 cells can be used as an in vitro model for C-induced events, but care has to be taken to use the cells at an early stage of passaging as they readily change their phenotype.

Animals↗
PubMed2006

Role of matrix metalloproteinases in HaCaT keratinocytes apoptosis induced by loxosceles venom sphingomyelinase D.

Envenomation by the spider Loxosceles (brown spider) can result in dermonecrosis and severe ulceration. We have previously shown that Loxosceles sphingomyelinase D (SMaseD), the enzyme responsible for these pathological effects, induced expression of matrix metalloproteinase-9 (MMP-9), which is possibly one of the main factors involved in the pathogenesis of the cutaneous loxoscelism. The aim of this study was to further investigate the molecular mechanisms triggered by Loxosceles SMaseD involved in the initiation of the dermonecrotic lesion, using HaCaT cultures, a human keratinocyte cell line, as an in vitro model for cutaneous loxoscelism. We show here that SMaseD from Loxosceles spider venom induces apoptosis in human keratinocytes, which is associated with an increased expression of metalloproteinase-2 and -9, and that the use of metalloproteinase inhibitors, such as tetracycline, may prevent cell death and potentially may prevent tissue destruction after envenomation.

Annexin A5↗
PubMed2005

C-reactive protein-induced in vitro endothelial cell activation is an artefact caused by azide and lipopolysaccharide.

OBJECTIVE: C-reactive protein (CRP) has been proposed to be an independent risk factor for cardiovascular disease. In vitro studies investigating the mechanism behind this have used purified commercial CRP (cCRP) and endothelial cells. We investigated the role of contaminants in cCRP preparations. METHODS AND RESULTS: Human umbilical vein endothelial cells and the human endothelial cell line EA.hy926 were incubated with Escherichia coli-derived cCRP, in-house-generated azide-free recombinant, and ascites-purified CRP, azide, or lipopolysaccharide (LPS) equivalent to the concentration present in cCRP preparations. Cells were investigated for change in cell proliferation, morphology, apoptosis, and expression of endothelial NO synthase and intercellular adhesion molecule-1. Cell supernatants were assessed for monocyte chemoattractant protein-1 (MCP-1), interleukin-8, von Willebrand factor secretion, and pH change. Only cCRP was able to induce all activation events analyzed; however, this ability was lost on extensive dialysis, suggesting that low molecular weight contaminants were responsible for these events. Indeed, the effects of cCRP were mirrored by azide or LPS. CONCLUSIONS: We investigated a wide range of effects on endothelial cells ascribed to CRP; however, azide and LPS, but never CRP itself, were responsible for the cell activation events. We conclude that CRP, per se, does not activate endothelial cells.

Artifacts↗
PubMed2005

Conformational changes of Loxosceles venom sphingomyelinases monitored by circular dichroism.

Envenomation by arachnids of the genus Loxosceles can induce a variety of biological effects, including dermonecrosis and hemolysis. We have previously identified in L. intermedia venom two highly homologous proteins with sphingomyelinase activity, termed P1 and P2, responsible for all these pathological events, and also an inactive isoform P3. The toxins P1 and P2 displayed 85% identity with each other at the amino acid level and showed a 57% identity with SMase I, an active toxin from L. laeta venom. Circular dichroism was used to determine and compare the solution structure of the active and inactive isoforms. Effects of pH and temperature change on the CD spectra of the toxins were investigated and correlated with the biological activities. This study sheds new light on the structure-function relationship of homologous proteins with distinct biological properties and represents the first report on the structure-function relationship of Loxosceles sphingomyelinases D.

Animals↗
PubMed2005

Analysis of the toxic potential of venom from Loxosceles adelaida, a Brazilian brown spider from karstic areas.

Loxosceles adelaida spiders (Araneae, Sicariidae) are found near and inside the caves in the Parque Estadual Turistico do Alto Ribeira (PETAR), Sao Paulo, Brazil, which are visited by thousands of tourists every year. Several Loxosceles species are a public health problem in many regions of the world, by causing severe dermonecrosis and/or complement dependent haemolysis upon envenomation. The aim of this study was to characterize the biochemical and biological properties of L. adelaida venom and evaluate the toxic potential of envenomation by this non-synanthropic Loxosceles species. The biological activities of the L. adelaida venom was compared to that of Loxosceles gaucho, a synanthropic species of medical importance in Brazil. L. adelaida venom showed a similar potential to induce haemolysis, dermonecrosis and lethality as L. gaucho venom. L. adelaida crude venom was purified, yielding a 31 kDa component endowed with haemolytic and dermonecrotic activities. In conclusion, we show here that the troglophile Loxosceles species, L. adelaida, commonly found in the complex of caves from PETAR, is potentially able to cause envenomation with the same gravity of those produced by synanthropic species.

Animals↗
PubMed2005

Loxosceles sphingomyelinase induces complement-dependent dermonecrosis, neutrophil infiltration, and endogenous gelatinase expression.

Envenomation by the spider Loxosceles can result in dermonecrosis and severe ulceration. Our aim was to investigate the role of the complement system and of the endogenous metalloproteinases in the initiation of the pathology of dermonecrosis. Histological analysis of skin of rabbits injected with Loxosceles intermedia venom and purified or recombinant sphingomyelinases showed a large influx of neutrophils, concomitant with dissociation of the collagenous fibers in the dermis. Decomplementation, using cobra venom factor, largely prevented the influx of neutrophils, while influx of neutrophils was also reduced in genetically C6-deficient rabbits, suggesting roles for both C5a and the membrane attack complex in the induction of dermonecrosis. However, C-depletion and C6 deficiency did not prevent the haemorrhage and the collagen injury. Zymography analysis of skin extracts showed the induction of expression of the endogenous gelatinase MMP-9 in the skin of envenomated animals. Rabbit neutrophils contained high levels of MMP-9, expression of which was further increased after incubation with venom, suggesting that these cells may be a source of the MMP-9 found in the skin of envenomated animals. Furthermore, skin fibroblasts also secreted MMP-9 and MMP-2 upon incubation with venom, suggesting that locally produced MMPs can also contribute to proteolytic tissue destruction.

Animals↗
PubMed2005

Porcine complement regulators protect aortic smooth muscle cells poorly against human complement-induced lysis and proliferation: consequences for xenotransplantation.

BACKGROUND: Accelerated atherosclerosis after transplantation has been observed and is characterized by smooth muscle cell proliferation in the graft. Porcine cells are frequently used in models of atherosclerosis and porcine organs are considered for use in transplantation. Complement (C) activation is known to play a major role in rejection of xenografts and is also considered to play a role in the development of atherosclerosis. The aim of this study was to investigate the expression and function of membrane bound regulators of complement (CReg) on porcine aortic smooth muscle cells (PASMC). METHODS: The PASMC were assessed for expression of CReg and susceptibility to lysis by human C by flow-cytometry. The effect of various cytokines on CReg expression and C-susceptibility was investigated. The ability of human C to induce cell proliferation was assessed using the Alamar blue assay. RESULTS: The PASMC only express the CReg membrane cofactor protein (MCP) and CD59 on their cell surface. MCP expression was increased by interleukin (IL)-4. In contrast to porcine aortic endothelial cells (PAEC), PASMC were found to be surprisingly sensitive to C-mediated lysis, mainly due to a low level of expression of CD59. Human C-induced proliferation of PASMC, which was dependent on complete membrane attack complex (MAC) formation. CONCLUSIONS: Endogenously expressed CReg on PASMC poorly protect these cells to human C. Human C can induce proliferation of PASMC. In order to prevent accelerated atherosclerosis in porcine xenografts, increased levels of CReg not only have to be obtained on the endothelial cells but also on the smooth muscle cells.

Animals↗
PubMed2004

C-reactive protein-induced in vitro vasorelaxation is an artefact caused by the presence of sodium azide in commercial preparations.

OBJECTIVE: Although C-reactive protein (CRP) is increasingly recognized as an independent risk factor for acute myocardial events, recent evidence suggests that it can directly induce vasorelaxation. This study aimed to investigate the mechanism of this CRP-induced response. METHODS AND RESULTS: Isometric tension recordings were used to measure endothelium-dependent and endothelium-independent vascular smooth muscle relaxation in isolated rabbit aortic rings. CRP generated in-house by genetic engineering and expressed in Chinese hamster ovary cells, CRP purified from ascites, and CRP obtained from commercial sources were assessed for vasorelaxing properties. Only the commercial CRP preparation induced vasorelaxation; more than half maximal relaxation was observed at 0.025 microg/mL and maximum relaxation attained at 0.25 microg/mL. Commercial CRP contains high levels of sodium azide, a well-known vasorelaxant. Removal of this agent by dialysis abolished the vasodilatory effect of commercial CRP. Sodium azide alone at concentrations equivalent to that present in the commercial CRP produced a near-identical relaxation pattern to the undialyzed commercial product. CONCLUSIONS: CRP has no vasorelaxant properties per se, and the reported vasorelaxant ability of CRP is an artifact caused by sodium azide present in commercial preparations of this agent.

Animals↗
PubMed2004

Molecular cloning, expression, function and immunoreactivities of members of a gene family of sphingomyelinases from Loxosceles venom glands.

Loxoscelism is the clinical condition produced by the venom of spiders belonging to the genus Loxosceles, which can be observed as two well-defined clinical variants: cutaneous loxoscelism and systemic or viscerocutaneous loxoscelism. We have recently identified, purified and characterised the toxins (sphingomyelinases) from Loxosceles intermedia venom that are responsible for all the local (dermonecrosis) and systemic effects (complement dependent haemolysis) induced by whole venom. In the present study, we have cloned and expressed the two functional sphingomyelinases isoforms, P1 and P2, and shown that the recombinant proteins display all the functional characteristics of whole L. intermedia venom, e.g., dermonecrotic and complement-dependent hemolytic activities and ability of hydrolyzing sphingomyelin. We have also compared the cross-reactivities of antisera raised against the toxins from different Loxosceles species and show here that the cross-reactivity is high when toxins are from the same species (P1 and P2 from L. intermedia) but low when the toxins are from different species (L. intermedia versus L. laeta). These data suggest that in order to obtain a suitable comprehensive neutralizing antiserum using the recombinant toxin as an immunogen, a mixture of the recombinant toxins from the different species has to be used. The use of anti-recombinant toxin antisera may have clinical benefits to those individuals displaying acute loxoscelic lesions.

Amino Acid Sequence↗
PubMed2002

Molecular cloning and expression of a functional dermonecrotic and haemolytic factor from Loxosceles laeta venom.

The bite of spiders of the genus Loxosceles can induce a variety of biological effects, including dermonecrosis and complement-dependent haemolysis. The aim of this study was to generate recombinant proteins from the Loxosceles spider gland to facilitate structural and functional studies in the mechanisms of loxoscelism. Using "Expressed Sequencing Tag" strategy of aleatory clones from, L. laeta venom gland cDNA library we have identified clones containing inserts coding for proteins with significant similarity with previously obtained N-terminus of sphingomyelinases from Loxosceles intermedia venom [1]. Clone H17 was expressed as a fusion protein containing a 6x His-tag at its N-terminus and yielded a 33kDa protein. The recombinant protein was endowed with all biological properties ascribed to the whole L. laeta venom and sphingomyelinases from L. intermedia, including dermonecrotic and complement-dependent haemolytic activities. Antiserum raised against the recombinant protein recognised a 32-kDa protein in crude L. laeta venom and was able to block the dermonecrotic reaction caused by whole L. laeta venom. This study demonstrates conclusively that the sphingomyelinase activity in the whole venom is responsible for the major pathological effects of Loxosceles spider envenomation.

Amino Acid Sequence↗
PubMed2002

GPI-anchored GFP signals Ca2+ but is homogeneously distributed on the cell surface.

Glycosyl-phosphatidylinositol (GPI)-anchored proteins are unique in that they penetrate only the outer leaflet of the plasma membrane but are still able to mediate intracellular signalling events following antibody-induced ligation. Detergent solubilisation studies suggest that microdomains exist at the cell surface within which are sequestered GPI-linked proteins. Here we report the construction and expression of a fluorescent GPI anchor on the surface of CHO, EL4, and U937 cells by fusing green fluorescent protein (GFP) to the GPI-attachment site of CD59. The resultant GFP-GPI has properties comparable to that of endogenously expressed GPI-anchored molecules as shown by Triton X-114 partitioning. However, sucrose gradient floatation showed that GFP-GPI was only partially resistant to detergent solubilisation. Furthermore confocal scanning laser microscopy revealed a homogeneous distribution of GFP-GPI at the cell surface, which only became clustered following cross-linking of the GPI anchor via an anti-GFP antibody. Surprisingly, GFP-GPI signalled Ca2+ change upon cross-linking demonstrating its signalling competence. Our results suggest that the GPI-anchor itself does not confer a clustered distribution to molecules but that clustering occurs following ligation with antibody, which allows the protein to become Ca2+ signalling competent.

Animals↗