Biomedical subjects

H J Croes

Publications and source records attributed to H J Croes.

12 recordsLinked to original sources

PubMed2001

Microscopic localization of PEG-liposomes in a rat model of focal infection.

In the present study the microscopic localization of polyethylene glycol (PEG) liposomes in infected tissues was studied with both light microscopy (LM) and transmission electron microscopy (TEM) in rats with focal intramuscular Staphylococcus aureus infection. PEG-liposomes containing colloidal gold were prepared and injected intravenously in rats with focal S. aureus infection and tissues were dissected at 24 h post injection. Sections were cut and liposomes were visualized for microscopic evaluation using silver enhancement. Uptake of PEG-liposomes was visualized by both scintigraphy and LM in the abscess, liver and spleen. In the infected area, the liposomes were mainly found in the vicinity of blood vessels. TEM showed that the liposomes were localized in the macrophages and to a lesser extent in endothelial cells in the infectious tissue. In the liver, the liposomes appeared mainly localized in Kupffer cells. In the spleen, uptake was only seen in cells of the red pulp and in cells around the central arteries. Our microscopic observations indicate that uptake and retention of PEG-liposomes in the infectious focus is a result of enhanced extravasation due to increased vascular permeability and subsequent phagocytosis of PEG-liposomes by macrophages in the infected tissue.

Abscess↗

PubMed2000

The small GTPase Rab6B, a novel Rab6 subfamily member, is cell-type specifically expressed and localised to the Golgi apparatus.

Members of the Rab subfamily of small GTPases play an important role in the regulation of intracellular transport routes. Rab6A has been shown to be a regulator of membrane traffic from the Golgi apparatus towards the endoplasmic reticulum (ER). Here, we report on the identification of a Rab6 isoform, termed Rab6B. The corresponding full-length cDNA was isolated from a Caco-2 cell library. The deduced amino acid sequence showed 91% identity with the Rab6A protein and revealed that sequence divergence is dispersed over a large region of the COOH-terminal domain. Rab6B is encoded by an independent gene which is located on chromosome 3 region q21-q23. In contrast to Rab6A whose expression is ubiquitous, northern blot analysis, immunohistochemistry, and immunofluorescence demonstrated that Rab6B is expressed in a tissue and cell-type specific manner. Rab6B is predominantly expressed in brain and the neuroblastoma cell line SK-N-SH. In brain, Rab6B was found to be specifically expressed in microglia, pericytes and Purkinje cells. Endogenous Rab6B localises to the Golgi apparatus and to ERGIC-53-positive vesicles. Comparable studies between Rab6A and Rab6B revealed distinct biochemical and cellular properties. Rab6B displayed lower GTP-binding activities and in overexpression studies, the protein is distributed over Golgi and ER membranes, whereas Rab6A is more restricted to the Golgi apparatus. Since the GTP-bound form of Rab6B (Rab6B Q72L) does interact with all known Rab6A effectors, including Rabkinesin-6, the results suggest a cell-type specific role for Rab6B in retrograde membrane traffic at the level of the Golgi complex.

Animals↗

PubMed1999

Contact guidance of rat fibroblasts on various implant materials.

Providing a substrate surface with micrometer-sized parallel grooves influences the behavior of cells growing on such substrates in vitro. Cells elongate in the direction of the groove and migrate guided by the grooves. It has been suggested that cellular alignment on microgrooves is predominantly dependent on groove dimensions and that surface chemical variation of the substrate material has little effect. Therefore we seeded primary rat dermal fibroblasts (RDF) on smooth and microgrooved (groove width 1-10 microm, depth 0.5 microm) polystyrene (PS), poly-L-lactic acid (PLA), silicone (SIL), and titanium (Ti) substrates. The production process was found to be more accurate for PS and PLA than for SIL and Ti substrates. A proliferation study, scanning electron microscopy, confocal laser scanning microscopy, and transmission electron microscopy revealed differences between RDF behavior on the materials. Our conclusions are (1) the accuracy of microtexture production by casting depends greatly on the material used; (2) even if no sharp discontinuities are present, microtextures still are potent tools for inducing contact guidance; and (3) besides surface texture, surface chemistry has a definitive influence on cell morphology.

Animals↗

PubMed1998

Microgrooved subcutaneous implants in the goat.

We investigated the behavior of microgrooved implants in soft tissue using polystyrene implantable disks, either smooth or microgrooved (1-10 microm) on both sides. The implants were placed subcutaneously in a goat for 1, 4, or 12 weeks. Light and transmission electron microscopy showed that fibrous capsule formation around the implants was fairly uniform. After 1 week the implants were covered with a fibrous capsule about 80 microm thick. The collagen matrix was loose, and many inflammatory cells were present. After 4 weeks the matrix was more dense and contained many newly formed blood vessels. At the implant surface a layer of inflammatory cells about 10 microm thick had accumulated. Finally, after 12 weeks the matrix had densified. One cellular layer of inflammatory cells was present at the implant surface. We carried out histomorphometric measurements of capsule thickness, inflammatory layer thickness, and the number of blood vessels. Capsule thickness appeared not to decrease with time. Further, these measurements showed that there were no differences in tissue reaction between smooth and microgrooved implants. On the basis of our observations, we suggest that 1 microm deep and 1-10 microm wide microgrooves do not influence tissue response around polystyrene implants in soft tissue.

Animals↗

PubMed1998

Scanning electron microscopic, transmission electron microscopic, and confocal laser scanning microscopic observation of fibroblasts cultured on microgrooved surfaces of bulk titanium substrata.

During this study, microtechnology and plasma etching were used to produce gratings 1.0 (TiD01), 2.0 (TiD02), 5.0 (TiD05), and 10.0 microns wide (TiD10) into commercially pure titanium wafers. After incubation of rat dermal fibroblast (RDFs) on these surfaces for 3 days, the cells were observed with scanning electron (SEM), transmission electron (TEM), and confocal laser scanning microscopy (CLSM). Results showed that the RDFs as a whole and their stress fibers oriented strictly parallel to the surface pattern on the TiD01 and TiD02 surfaces. On the TiD05 and TiD10 surfaces, this orientation was not observed. In addition, TEM and CLSM demonstrated that the focal adhesion points (FAP) were located mainly on the surface pattern ridges. TEM revealed that FAP were wrapped occasionally around the edges of the ridges. Only the RDFs on both the TiD05 and TiD10 surfaces protruded into the grooves and possessed FAP on the walls of the grooves. Attachment to the groove floor was observed only on the TiD10 textures. Comparison of these results with earlier observations on microtextured silicone rubber substrata suggests that material-specific properties do not influence the orientational effect of the surface texture on the observed RDF cellular behavior. The proliferation rate of the RDFs, however, seems to be much higher on titanium than on silicone rubber substrata.

Animals↗

PubMed1998

Growth behavior of fibroblasts on microgrooved polystyrene.

We investigated the contact guidance phenomenon of rat dermal fibroblasts (RDF) on microgrooved polystyrene substrates. Grooves were 1 microm deep, and between 1 and 10 microm wide. Light microscopy and digital image analysis (DIA) showed that RDF were oriented on all microgrooved substrates. Scanning electron microscopy showed that RDF cultured on 1 or 2 microm wide grooves were positioned on top of the ridges. On the wider 5 and 10 microm grooves, the cells were able to descend into the grooves. In confocal laser scanning microscopy, focal adhesions were lying in the same direction as the actin filament where they attached to. DIA confirmed an orientational behavior of focal adhesions and actin filaments on microgrooves. There were no differences in the measured orientation between the different grooves. Besides, no obvious preference was found for focal adhesions to lie along edges of the surface ridges. Transmission electron microscopy showed that focal adhesions were able to bend along the edges of ridges. On the basis of our observations, we suggest that the breakdown and formation of fibrous cellular components, especially in the filopodium, is influenced by the microgrooves. The microgrooves create a pattern of mechanical stress, which influences cell spreading and cause the cell to be aligned with surface microgrooves.

Animals↗

PubMed1997

Polarised expression of human intestinal N-benzoyl-L-tyrosyl-p-aminobenzoic acid hydrolase (human meprin) alpha and beta subunits in Madin-Darby canine kidney cells.

N-Benzoyl-L-tyrosyl-p-aminobenzoic acid hydrolase (PPH, human meprin), is a peptidase found in the microvillus membrane of human small intestinal epithelial cells. PPH belongs to the astacin family of zinc-metalloendopeptidases and is a protein complex composed of two glycosylated subunits, alpha and beta. The present report describes the cloning of the complete beta subunit and the remaining N2-terminal end of the alpha subunit for analysis of their primary structures in addition to the examination of their biogenesis in transfected cell cultures. The complete open reading frame of the PPH beta cDNA translates into 700 amino acid residues compared with 746 residues for the PPH alpha cDNA. The primary structure of beta and alpha subunits are 44% identical and 61% similar. As predicted from their primary structure, the two subunits of PPH have identical modular structures; starting at the N2-terminus both contain a signal peptide, a propeptide, a protease domain containing the astacin signature, a meprin A5 protein tyrosine phospatase mu (MAM) and a meprin and TRAF homology domain (MATH) domain, an epidermal growth factor(EGF)-like domain, a putative transmembrane anchor domain and a short cytosolic tail. Pulse/chase labelling and immuno-Gold electronmicroscopy of recombinant PPH beta and alpha subunits expressed in transfected Madin-Darby canine kidney (MDCK) cells show that post-translational processing and transport of the two subunits are very different. When expressed alone, the beta subunit acquired complex glycan residues, readily formed homodimers and was transported to the plasma membrane. Small amounts of PPH beta were found in the culture medium. In contrast, the cell-bound alpha subunit, when expressed alone, remained primarily in the high-mannose form, was aggregated and not expressed at the cell surface. However, the bulk of mostly endo-beta-N-acetylglucosaminidase H-resistant alpha subunit was found in the filtered culture medium. The proteolytic event that leads to the formation of this soluble transport-competent form occurs in the endoplasmic reticulum (ER). Coexpression of the alpha subunit with the beta subunit allowed the localisation of the alpha subunit to the plasma membrane. These studies indicate that assembly of the two subunits of PPH is required for the localisation of the alpha subunit to the plasma membrane. In contrast to rodent meprin, both PPH subunits are apically secreted from MDCK cells.

Amino Acid Sequence↗

PubMed1997

The nucleoporin CAN/Nup214 binds to both the cytoplasmic and the nucleoplasmic sides of the nuclear pore complex in overexpressing cells.

CAN/Nup214, an essential component of the vertebrate nuclear pore complex (NPC), is required for proper cell cycle progression and nucleocytoplasmic transport. It is a member of the FG-repeat-containing family of nucleoporins and has been localized to the cytoplasmic face of the NPC. Indirect immunofluorescence studies with specific antibodies have shown that moderate overexpression of human CAN in HeLa cells causes an increase in CAN/Nup214 levels at the nuclear envelope. Here, we demonstrate that in such HeLa cells, CAN/Nup214 does not localize exclusively to the cytoplasmic side of the NPC. Cryosections, stained with CAN-specific antibodies and examined by electron microscopy, showed that about one-third of the gold-labeled NPCs were decorated at the cytoplasmic face and the remaining two-thirds at the nucleoplasmic face. These data indicate that both the cytoplasmic fibrils and the nuclear basket of the vertebrate NPC contain specific binding sites for either CAN/Nup214 or for its interacting proteins, Nup88 and hCRM1. Thus, it is conceivable that CAN/Nup214 functions in nucleocytoplasmic transport at both faces of the NPC.

Cell Polarity↗

PubMed1996

In vivo ANA is a fixation artifact: nucleosome-complexed antinucleosome autoantibodies bind to the cell surface and are internalized.

It has been suggested that binding of anti-double-standed DNA antibodies to cell surfaces, followed by internalization and nuclear binding (so called in vivo ANA) is of pathophysiological significance for tissue damage in systemic lupus erythematosus. We have shown before that pathogenic antinuclear antibodies complexed to nucleosomal antigens can bind to heparan sulfate in the glomerular basement membrane in vivo. Because nucleosomes are also reported to bind to the cell surface, we hypothesized that in vivo ANA is a property of antinuclear antibodies bound to nucleosomal antigens. Therefore, we studied three antinucleosome monoclonal antibodies (mAb) that exhibit in vivo ANA as seen by immunofluorescence in mice inoculated intraperitoneally with the hybridoma producing the mAb. The same mAb complexed to nucleosomal antigens after intravenous injection into mice induced in vivo ANA, in contrast to purified noncomplexed mAb. To study this in more detail, we incubated complexed mAb with various cell lines and found binding to the cell surface and subsequent internalization into cytoplasmic vesicles. However, no binding to the nucleus was observed by immunoelectron microscopy (IEM) and confocal laser microscopy. Noncomplexed mAb did not bind to the cell surface. Next, from mice bearing the hybridomas producing the mAb intraperitoneally, a small part of the kidney was snap frozen in liquid N2, fixed with acetone, and studied in immunofluorescence, whereas the remaining part of the kidney was fixed in vivo by renal perfusion with a mixture of 0.01 M sodium periodate, 0.075 M lysine HCl, 0.0375 M Na2HPO4, and 2% paraformaldehyde (PLP) and studied in both immunofluorescence and IEM. In the acetone-fixed kidney sections obtained without in vivo fixation we again observed in vivo ANA. However, after in vivo PLP perfusion fixation, no nuclear binding was found. In IEM, localization in cytoplasmic vesicles was seen. In conclusion, antinucleosome antibodies complexed to nucleosomal antigens can bind to the cell surface and are transported into the cytoplasm, but do not bind to the nucleus. The reported nuclear localization of antinuclear antibodies is caused by a fixation artifact.

Animals↗

PubMed1993

Titin aggregates associated with intermediate filaments align along stress fiber-like structures during human skeletal muscle cell differentiation.

Differentiating human skeletal muscle cell cultures were used to study the association of titin with other sarcomeric and cytoskeletal proteins during myofibrillogenesis. Several developmental stages of these cultures were double stained with antibodies to titin in combination with antibodies to alpha-actin, alpha-actinin, myosin heavy chain (MHC), nebulin, desmin, and beta-tubulin. The first indications of titin expression were found in postmitotic mononuclear myoblasts where it is located in a random, punctate fashion. At the light microscope level no evidence was found for an association of these titin spots with any of the other proteins studied, with the exception of MHC, which colocalized with titin in a small minority of the titin expressing cells. Subsequently the titin spots were found to be linked to longitudinally oriented stress fiber-like structures (SFLS), containing alpha-actinin and sarcomeric alpha-actin, but not MHC, nebulin or desmin. Upon further maturation titin antibodies seemed to stain SFLS in a rather homogeneous fashion together with MHC, alpha-actin and alpha-actinin. Thereafter a more periodic localization of titin, MHC, alpha-actin and alpha-actinin on SFLS became obvious. From these structures myofibrils developed as a result of further differentiation. Initially only short stretches with a striated titin, MHC, F-actin and alpha-actinin organization were found. Nebulin was integrated in these young myofibrils at a later developmental stage. Desmin was not found to be incorporated in these myofibrils until complete alignment of the sarcomeres in mature myotubes had occurred. At the ultrastructural level titin antibodies recognized aggregates that were associated with intermediate filaments (IF) in postmitotic mononuclear myoblasts. At a later maturational stage, prior to the development of cross-striated myofibrils, the IF-associated titin aggregates were found in close association with subsarcolemmally located SFLS. We conclude that IF and SFLS play an important role in the very early stages of in vitro human myofibrillogenesis. On the basis of our results we assume that titin aggregates are targeted to SFLS through IF. The association of titin with SFLS might be crucial for the unwinding of titin necessary for the assembly of sarcomeres and the first association of titin with other sarcomeric proteins.

Cell Differentiation↗

PubMed1989

ETN-1: a new human endometrial carcinoma cell line producing ascites and distant metastases in nude mice.

A human carcinoma cell line (ETN-1) has been established from a skin metastasis of a moderately differentiated adenocarcinoma of endometrial origin. The cell line has been so far maintained for 27 months through 55 passages, growing as a monolayer as well as in 3-dimensional clusters with a population doubling time of 72 hr. The number of chromosomes per cell varied from 39 to 107 (average number 61.0 +/- 19.8), with a modal number of 46-48. Seven clonal marker chromosomes were detected. Flow cytometric analysis revealed a population of pseudo-tetraploid cells (DNA index 2.1) next to a pseudo-diploid population (DNA index 1.1). The epithelial character of the cells was confirmed by a positive immunocytochemical reaction using monoclonal antibodies (MAbs) to different keratins, the epithelial cell markers BW 495/36 and HMFG-2, as well as by the presence of many junctional complexes. The tumour cells retained a positive reaction with the anti-ovarian carcinoma OV-TL 3, OV-TL 10 and OC 125 MAbs, although the reaction was markedly diminished in comparison with the original tumour. Tumour cells inoculated subcutaneously in nude mice produced well differentiated adenomatous tumour nodules with formation of glandular lumina and basal lamina. Tumour cells injected intraperitoneally produced malignant ascites and regional as well as distant metastases of adenomatous character.

Adenocarcinoma↗

PubMed1989

Characterization of a hormone-producing ovarian carcinoma cell line.

An ovarian carcinoma cell line (OTN 11) was produced from the ascitic fluid of a patient with a moderately to well differentiated papilliferous cystadenocarcinoma of the ovary. The cell line was characterized using electron microscopy karyotyping, immunohistochemical techniques with monoclonal antibodies against keratins as epithelial markers, and the monoclonal antibodies OV-TL 3 and OC 125 as ovarian carcinoma markers. These techniques revealed the epithelial and adenocarcinomatous nature of the cell line and the presence of ovarian carcinoma-related surface markers. The adenocarcinomatous nature of the cell line also became apparent after heterotransplantation of cell suspensions into nude mice and nude rats, in which adenomatous tumor structures were formed. These xenografts had the same ultrastructural and immunohistochemical properties as the cell line. Despite the adenocarcinomatous character of the tumor the cultured cells release estradiol into the culture medium. We may conclude that OTN 11 is an ovarian carcinoma cell line which has retained highly differentiated functions, such as the production of an ovarian hormone.

Adult↗