Henk K Koerten | Universiteit Leiden (original) (raw)
Papers by Henk K Koerten
British Journal of Dermatology, 2000
Melanocytic dendrites consist of a central core of microtubules and a subcortical actin network. ... more Melanocytic dendrites consist of a central core of microtubules and a subcortical actin network. Several studies provide arguments supporting the hypothesis that actin-based and microtubule-based motor proteins co-operate in melanosome transport towards the dendrite tips. Melanosomes can move bidirectionally along microtubules in vitro, and in murine melanocytes, they move towards the cell periphery and back again. Microtubules have a fast-growing plus end and a slow-growing minus end. Microtubule-associated motor proteins move unidirectionally either towards the plus or towards the minus end. However, it is not known which motor protein is responsible for minus end-directed movement of melanosomes. We aimed to investigate the in vitro expression of the minus end-directed motor protein cytoplasmic dynein in normal human epidermal melanocytes, keratinocytes and dermal fibroblasts. Reverse transcription-polymerase chain reaction and Northern blot analysis were used. In addition, an attempt to obtain insight into the subcellular localization of cytoplasmic dynein, immunofluorescence studies and immunogold electron microscopic studies were performed. The three different forms of cytoplasmic dynein heavy chain were expressed in all studied skin cells. Immunofluorescence staining showed similar punctate distributions for dynein heavy chain 1 and dynein heavy chain 2 in melanocytes, with accentuation in the perinuclear area and dendrite tips. Double labelling with a melanosome marker showed apparent co-localization of both dynein heavy chains 1 and 2 with melanosomes in the perinuclear area and dendrite tips. For the dynein intermediate chain of 74 kDa, again a punctate staining pattern was seen with intense centrosomal staining. A close association of dynein intermediate chain 74 and alpha-tubulin with the melanosome surface was detected using immunogold electron microscopy. The colocalization of different subunits of the cytoplasmic dynein complex with melanosomes is consistent with the hypothesis that this motor protein supports minus end-directed melanosome movement along microtubules.
Skin Pharmacology and Physiology, 2001
One of the prerequisites for the use of human skin equivalents for scientific and screening purpo... more One of the prerequisites for the use of human skin equivalents for scientific and screening purposes is that their barrier function is similar to that of native skin. Using human epidermis reconstructed on de-epidermized dermis we demonstrated that the formation of the stratum corneum (SC) barrier in vitro proceeds similarly as in vivo as judged from the extensive production of lamellar bodies, their complete extrusion at the stratum granulosum/SC interface, and the formation of multiple broad lamellar structures in the intercorneocyte space. The presence of wellordered lipid lamellar phases was confirmed by small-angle X-ray diffraction. Although the long periodicity lamellar phase was present in both the native and the reconstructed epidermis, the short periodicity lamellar phase was present only in native tissue. In addition, the SC lipids predominantly formed the hexagonal sublattice. Analysis of lipid composition revealed that all SC lipids are synthesized in vitro. Differences in SC lipid organization in reconstructed epidermis may be ascribed to the differences in fatty acid content and profile indicating that further improvement in culture conditions is required for generation of in vitro reconstructed epidermis with stratum barrier properties of the native tissue.
Dermatology, 1994
Tightly curled 'frizzy' hair is a pathognomonic sign of giant axonal degeneration... more Tightly curled 'frizzy' hair is a pathognomonic sign of giant axonal degeneration (GAD). The present study compares the morphological structure of the scalp hair of a GAD patient with that of her parents and first-degree relatives with the aid of scanning electron microscopy. The comparison included plaited hair of two age-matched controls, in order to exclude mechanical plaiting artifacts. Trichorrhexis nodosa and fringing of the cut ends were exclusively found in the patient's hair. Longitudinal grooving was also frequently seen in hair of normal persons. Assay of carbon (C), sulfur (S) and nitrogen (N) contents of the patient's hair was normal, but the S:N ratio was significantly reduced, as compared with her relatives. However, comparison with a control group of non-related healthy volunteers showed no difference.
Pharmaceutical research, 2001
To study at the ultrastructural level which part of the skin is associated with percutaneous iodi... more To study at the ultrastructural level which part of the skin is associated with percutaneous iodide transport by passive diffusion and iontophoresis. Following passive diffusion or iontophoresis of iodide, the morphology and the ion distribution of the skin was preserved by rapid freezing. The skin was kept frozen until and during examination by transmission electron microscopy (TEM) and X-ray microanalysis (XRMA). The intrinsic electron absorbing characteristics of cryopreserved skin allow direct TEM examination without additional staining. XRMA can be used to obtain in a relatively nondestructive way in situ information on ion distributions across the skin. After passive diffusion, iodide was mainly found in the stratum corneum (SC), whereas there was little iodide in the viable epidermis. Iontophoresis up to 300 microA/cm2 did not significantly affect this distribution. With iontophoresis at 1,000 microA/cm2, the amount of iodide increased dramatically and was equally distributed...
Cellular Signalling, 1994
Although electropermeabilization has become an important tool for studying the signal requirement... more Although electropermeabilization has become an important tool for studying the signal requirements of exocytosis, relatively little is known about the morphological changes accompanying this response in electropermeabilized cells. In this study, we determined that electropermeabilization of human neutrophils by itself caused only minor changes in the morphology as determined by transmission electron microscopy. The structure of the plasma membrane did not show detectable changes, whereas the cytoplasm was more electron lucent as compared to intact cells. Activation of intact neutrophils with formyl-methionyl-leucyl-phenylalanine (FMLP), in the presence of cytochalasin-B, caused the development of invaginations of the plasma membrane. In contrast, activation of electropermeabilized cells with 1 microM Ca2+ and/or 50 microM GTP-gamma-S caused the development of vacuoles that did not seem to be in contact (or had previously been in contact) with the extracellular environment. However, fusion of azurophilic and specific granules with these vacuoles clearly had taken place. The response characteristics of this fusion induced by Ca2+ and GTP-gamma-S were quite similar to those of the direct fusion of granules with the plasma membrane. We conclude that in electropermeabilized human neutrophils, two processes involving granule fusion can be distinguished. First, a direct fusion of granules with the plasma membrane. Secondly, the fusion of granules leading to the formation of vacuoles, not in contact with the extracellular space.
Wound Repair and Regeneration, 1999
A fully differentiated epithelium mimicking the features of native epidermis was obtained in vitr... more A fully differentiated epithelium mimicking the features of native epidermis was obtained in vitro by culturing human or porcine epidermal keratinocytes on polyester filter substrate at the air-liquid interface. In addition, after 2 weeks of culture, hemidesmosome-like structures were formed along the basal area of the plasma membrane of the basal cells at the cell-filter interface. When grafted onto full-thickness skin wounds in pigs, the take of cell sheets detached from the filter with dispase was significantly higher (about 70%) in comparison to mechanically detached keratinocytes (about 15%). With dispase-treated keratinocytes alone, basement membrane formation took place within 7 days postgrafting as judged from the presence of a lamina lucida and positive staining for type IV collagen. Also, numerous hemidesmosomes and anchoring fibrils were observed at the basal cell-"neodermis" interface. The fully differentiated epidermis, generated by culturing keratinocytes at the air-liquid interface and detached from the substrate by dispase-treatment, is less fragile and easier to handle than epidermal autografts obtained by conventional culturing methods. Detachment by a short dispase-treatment appeared in our hands the only method for successful and complete epithelial regeneration in full-thickness wounds.
Pigment Cell Research, 2000
The two isoforms of P150 Glued and P50 are expressed in all Melanocytic dendrites consist of a ce... more The two isoforms of P150 Glued and P50 are expressed in all Melanocytic dendrites consist of a central core of microtubules (MT) and a subcortical actin network. In previous studied skin cells. Immunofluorescence staining shows punctate distributions for P150 Glued and P50 in melanocytes. reports we showed the presence of MT-associated motor P150 Glued shows a clear centrosomal staining and accentua-proteins kinesin and cytoplasmic dynein on the melanosomal tion in the dendrite tips. P50 is also accentuated in the surface, forming a link with MT (Vancoillie et al. J Invest Dermatol 2000;114:421-429; Vancoillie et al. Br J Derma-perinuclear area and dendrite tips. Immunofluorescence doutol 2000;143:258-306)
Journal of Investigative Dermatology, 1998
Calcium plays an important role in the regulation of cellular differentiation and desquamation of... more Calcium plays an important role in the regulation of cellular differentiation and desquamation of epidermal keratinocytes. In this study, we examined the calcium distribution in reconstructed epidermis in an attempt to understand the physiology of keratinocyte differentiation and desquamation in vitro. Ion capture cytochemistry (the potassium oxalate-pyroantimonate method) was employed to localize ionic calcium in reconstructed epidermis generated under three different culture conditions (in serum-containing medium, serum-free medium, and serum-free medium supplemented with retinoic acid), allowing a comparison of the physiology of incompletely and well-differentiated keratinocytes. The reconstructed epidermis generated in serum-containing medium showed features of incomplete differentiation, and compared with the native skin, a high calcium content within incompletely differentiated cells in the stratum corneum. Use of serum-free medium containing vitamin and lipid supplements led to a marked improvement of the stratum corneum ultrastructure and penetration pathway across the stratum corneum, indicating improved barrier formation of the reconstructed epidermis. In parallel, the calcium distribution pattern was normalized showing the highest levels of calcium in the stratum granulosum and low levels in the inner stratum corneum. Addition of retinoic acid to the serum-free medium resulted in an altered keratinocyte differentiation and re-appearance of large quantities of calcium precipitates in the stratum corneum. Proton probe X-ray microanalysis was applied to investigate the calcium distribution quantitatively in native and reconstructed epidermis generated in serum-free medium, and verified the calcium distribution demonstrated by the precipitation technique. Regardless of the presence or absence of calcium in the stratum corneum, all examined culture systems exhibited insufficient desquamation, which correlates with the finding that stratum corneum chymotryptic enzyme was present predominantly as an inactive precursor. This study demonstrates that improvement of the stratum corneum barrier properties in vitro is concurrent with the normalization of the epidermal calcium gradient, whereas deregulation of terminal differentiation correlates with an accumulation of calcium ions within incompletely differentiated corneocytes.
Journal of Investigative Dermatology, 1998
Mutations of the gene encoding myosin V can produce a dilute or silvery hair color and various ne... more Mutations of the gene encoding myosin V can produce a dilute or silvery hair color and various neurologic defects in mice and patients with Griscelli syndrome, leading to speculations that the myosin V motor protein plays a critical role in transporting melanosomes within melanocytes and neurosecretory vesicles within neurons. Therefore, we investigated the in vitro expression of myosin V in cultured normal human melanocytes, keratinocytes, and dermal fibroblasts using reverse transcriptase-polymerase chain reaction and northern blot analysis. Subcellular distribution of myosin V and proximity to actin bundles and melanosomes were determined by double indirect immunofluorescence labeling and immunogold electron microscopy. In all studied cells V ery little is known about the molecular mechanisms underlying dendrite extension and melanosome transport in human melanocytes. It is expected that cytoskeletal proteins such as actin and microtubule components with their respective motor proteins play a role in these processes. Indeed, Lacour et al (1992) reported that actin microfilaments play a role in dendrite formation and microtubules play a role in dendrite maintenance in cultured human melanocytes.
Journal of Investigative Dermatology, 2000
Microtubuli play an important role in the organization of organelles and membrane traffic. They a... more Microtubuli play an important role in the organization of organelles and membrane traffic. They are present in melanocytic dendrites through which melanosomes are transported towards keratinocytes. Besides the actin-based motility systems, microtubuli-associated motor proteins also play a critical role in melanosome movement, as has recently been confirmed in mouse melanocytes. We investigated the in vitro expression of two forms of human conventional kinesin and its receptor kinectin in normal human epidermal melanocytes, keratinocytes, and dermal fibroblasts by reverse transcription polymerase chain reaction and northern blot analysis. In an attempt to gain insight into the subcellular distribution of kinesin and kinectin in melanocytes, double immunofluorescent staining and immunogold electron microscopy were performed. In all studied skin cells ubiquitous and neuronal kinesin are expressed, as well as the kinectin receptor. Immunofluorescent staining shows distinct but partially overlapping distributions for kinesin heavy chain and melanosomes, suggesting that kinesin is associated with some but not all of the melanosomes. Similar observations for kinectin indicate that this receptor can colocalize with melanosomes, which was confirmed by immunoelectron microscopy. The latter technique allowed us to demonstrate a close association between kinesin heavy chain, microtubuli, and melanosomes. The combined data from reverse transcription polymerase chain reaction, northern blot analysis, double immunofluorescent staining, and immunogold electron microscopy suggest that kinesins and kinectin have an important role in microtubuli-based melanosome transport in human melanocytes.
Journal of Biomedical Materials Research, 1997
To analyze the bone-bonding property of hydroxyapatite ceramics (HA), composites of rat marrow ce... more To analyze the bone-bonding property of hydroxyapatite ceramics (HA), composites of rat marrow cells and porous HA were implanted subcutaneously and harvested at 3 to 4 weeks postimplantation. De novo bone formation was observed primarily on the HA surface without fibrous tissue interposition. The HA/tissue interface was analyzed by the observations of thin undecalcified histological sections and fractured surfaces of the implants. The observations were done with a light microscope and a scanning electron microscope (SEM) connected to an energy dispersive spectrometer. The interfacial analyses showed the appearance of osteoblastic cells on the HA surface and that the cells had initiated partially mineralized bone (osteoid) formation directly onto the surface. The osteoid matured into fully mineralized bone, resulting in firm bone bonding to the HA surface. Characterization of osteoblastic cells on the surface was done by determining levels of protein and gene expression of bone Gla protein (BGP, a.k.a. Osteocalcin), i.e., immunohistochemistry and in situ hybridization, respectively. The existence of BGP and mRNA in the cytoplasmic area of the cells confirmed that active osteoblast apposition fabricated primary bone on the HA surface. All of these results indicate the importance of the HA surface in supporting osteoblastic differentiation of marrow stromal stem cells, which leads to firm bone bonding.
Developmental Dynamics, 2006
During cardiovascular development, fluid shear stress patterns change dramatically due to extensi... more During cardiovascular development, fluid shear stress patterns change dramatically due to extensive remodeling. This biomechanical force has been shown to drive gene expression in endothelial cells and, consequently, is considered to play a role in cardiovascular development. The mechanism by which endothelial cells sense shear stress is still unidentified. In this study, we postulate that primary cilia function as fluid shear stress sensors of endothelial cells. Such a function already has been attributed to primary cilia on epithelial cells of the adult kidney and of Hensen's node in the embryo where they transduce mechanical signals into an intracellular Ca2+ signaling response. Recently, primary cilia were observed on human umbilical vein endothelial cells. These primary cilia disassembled when subjected to high shear stress levels. Whereas endocardial-endothelial cells have been reported to be more shear responsive than endothelial cells, cilia are not detected, thus far, on endocardial cells. In the present study, we use field emission scanning electron microscopy to show shear stress-related regional differences in cell protrusions within the cardiovasculature of the developing chicken. Furthermore, we identify one of these cell protrusions as a monocilium with monoclonal antibodies against acetylated and detyrosinated alpha-tubulin. The distribution pattern of the monocilia was compared to the chicken embryonic expression pattern of the high shear stress marker Krüppel-like factor-2. We demonstrate the presence of monocilia on endocardial-endothelial cells in areas of low shear stress and postulate that they are immotile primary cilia, which function as fluid shear stress sensors.
Cell and Tissue Research, 1996
A fully differentiated epidermis generated in vitro by culturing normal human keratinocytes at th... more A fully differentiated epidermis generated in vitro by culturing normal human keratinocytes at the air-liquid interface shares many similarities with native tissue. However, in contrast to native epidermis, its stratum corneum consists of a larger number of compactly packed corneocyte layers, indicating that the processes involved in corneocyte desquamation are disturbed. Although the tri-lamellar appearance of desmosomes in viable cell layers of both types of epidermis is similar, abnormalities in the transformation of desmosomes into the corneosomal plug in the stratum corneum of reconstructed epidermis have been observed. In the native epidermis, desmosomes are transformed into corneosomes at the stratum granulosum/stratum corneum interface. This process is retarded in vitro, since desmosomal structures with preserved lamellar appearance are present in the lower parts of the stratum corneum. Moreover, the corneosome frequency in reconstructed epidermis is significantly higher than in native human skin. A comparison of reconstructed epidermis with hyperproliferative epidermis, such as UV-irradiated epidermis, psoriatic epidermis and recently healed burn-wounds treated with cultured epidermal autografts, has revealed that only the structure of the stratum corneum derived from psoriatic skin is similar to that of reconstructed epidermis. The stratum corneum organization in the UV-treated epidermis and in recently healed burn-wound is, however, close to that seen in native skin.
British Journal of Dermatology, 2000
Melanocytic dendrites consist of a central core of microtubules and a subcortical actin network. ... more Melanocytic dendrites consist of a central core of microtubules and a subcortical actin network. Several studies provide arguments supporting the hypothesis that actin-based and microtubule-based motor proteins co-operate in melanosome transport towards the dendrite tips. Melanosomes can move bidirectionally along microtubules in vitro, and in murine melanocytes, they move towards the cell periphery and back again. Microtubules have a fast-growing plus end and a slow-growing minus end. Microtubule-associated motor proteins move unidirectionally either towards the plus or towards the minus end. However, it is not known which motor protein is responsible for minus end-directed movement of melanosomes. We aimed to investigate the in vitro expression of the minus end-directed motor protein cytoplasmic dynein in normal human epidermal melanocytes, keratinocytes and dermal fibroblasts. Reverse transcription-polymerase chain reaction and Northern blot analysis were used. In addition, an attempt to obtain insight into the subcellular localization of cytoplasmic dynein, immunofluorescence studies and immunogold electron microscopic studies were performed. The three different forms of cytoplasmic dynein heavy chain were expressed in all studied skin cells. Immunofluorescence staining showed similar punctate distributions for dynein heavy chain 1 and dynein heavy chain 2 in melanocytes, with accentuation in the perinuclear area and dendrite tips. Double labelling with a melanosome marker showed apparent co-localization of both dynein heavy chains 1 and 2 with melanosomes in the perinuclear area and dendrite tips. For the dynein intermediate chain of 74 kDa, again a punctate staining pattern was seen with intense centrosomal staining. A close association of dynein intermediate chain 74 and alpha-tubulin with the melanosome surface was detected using immunogold electron microscopy. The colocalization of different subunits of the cytoplasmic dynein complex with melanosomes is consistent with the hypothesis that this motor protein supports minus end-directed melanosome movement along microtubules.
The American journal of pathology, 1990
This report describes the cell biology of the development of asbestos bodies after a single intra... more This report describes the cell biology of the development of asbestos bodies after a single intraperitoneal injection of a suspension of crocidolite asbestos fibers into the mouse peritoneal cavity. The majority of the infected fibers were found in aggregates of peritoneal macrophages, exudate cells, and fibrous tissue. These aggregates developed into granulomas containing not only numerous asbestos fibers, but also cells of various types, including macrophages, multinucleated giant cells, fibroblasts, plasma cells, granulocytes, and mast cells. Cytoplasmic ferritin was abundantly present in macrophages and giant cells. In addition, iron-rich inclusion bodies were detected. The results of this study show that asbestos body formation can occur outside the pleural cavity. Asbestos body formation occurred in the granulomas after periods of 1 month and longer. On the basis of morphologic criteria, various types of asbestos body were distinguished. X-ray microanalysis showed that variati...
The Anatomical Record, 2004
The beta-geo (LacZ) reporter gene encodes for -galactosidase (-gal) in all cells of the ROSA26 ... more The beta-geo (LacZ) reporter gene encodes for -galactosidase (-gal) in all cells of the ROSA26 mouse during embryonic development. As such, -gal activity constitutes an excellent marker for in situ labeling of expressing cells. However, the intracellular distribution of -gal differs between cells, and changes during embryonic development. Therefore, we studied LacZ-encoded -gal using light and electron microscopy in the heart, lung, liver, and small intestine on days 13 and 16 of gestation, and the kidney on day 16 of gestation in ROSA26 mice. The Bluo-gal method was carried out under standardized conditions, including fixation, washing, and incubation procedures. Intracellular -gal staining is encountered in a combination of membranous compartments, including the nuclear envelope, the endoplasmic reticulum, and the plasma membrane. Its exact localization depends on the cell type and is regulated during development. Therefore, one must take the compartmental transition of intracellular -gal staining into consideration when interpreting results obtained from experiments using ROSA26 mice.
Photochemical & Photobiological Sciences, 2002
The application of a novel model for sunscreen photoimmunoprotection studies was assessed using a... more The application of a novel model for sunscreen photoimmunoprotection studies was assessed using a systemic infection of rats with herpes simplex virus type 1 (HSV-1). Rats were irradiated daily with 1 minimal erythemal/oedematous dose of UVB for 7 consecutive days on their shaved backs with or without application of a broad-spectrum sunscreen (containing TiO2) with a sun protection factor of 10. Subsequently, rats were infected intranasally with HSV. UV exposure prior to HSV infection induced increased severity and incidence of clinical signs of disease, suppression of cellular immune responses as assessed by delayed type hypersensitivity and increased viral load in the brain. The sunscreen provided protection against all these UV-induced effects. We conclude that this novel model is a promising way of testing the immunoprotective qualities of sunscreens, based on the response to a common infectious agent of human subjects.
Journal of Virology, 2004
Equine arteritis virus (EAV) is an enveloped, positive-stranded RNA virus belonging to the family... more Equine arteritis virus (EAV) is an enveloped, positive-stranded RNA virus belonging to the family Arteriviridae of the order Nidovirales. EAV particles contain seven structural proteins: the nucleocapsid protein N, the unglycosylated envelope proteins M and E, and the N-glycosylated membrane proteins GP 2b (previously named G S ), GP 3 , GP 4 , and GP 5 (previously named G L ). Proteins N, M, and GP 5 are major virion components, E occurs in virus particles in intermediate amounts, and GP 4 , GP 3 , and GP 2b are minor structural proteins. The M and GP 5 proteins occur in virus particles as disulfide-linked heterodimers while the GP 4 , GP 3 , and GP 2b proteins are incorporated into virions as a heterotrimeric complex. Here, we studied the effect on virus assembly of inactivating the structural protein genes one by one in the context of a (full-length) EAV cDNA clone. It appeared that the three major structural proteins are essential for particle formation, while the other four virion proteins are dispensable. When one of the GP 2b , GP 3 , or GP 4 proteins was missing, the incorporation of the remaining two minor envelope glycoproteins was completely blocked while that of the E protein was greatly reduced. The absence of E entirely prevented the incorporation of the GP 2b , GP 3 , and GP 4 proteins into viral particles. EAV particles lacking GP 2b , GP 3 , GP 4 , and E did not markedly differ from wild-type virions in buoyant density, major structural protein composition, electron microscopic appearance, and genomic RNA content. On the basis of these results, we propose a model for the EAV particle in which the GP 2b /GP 3 /GP 4 heterotrimers are positioned, in association with a defined number of E molecules, above the vertices of the putatively icosahedral nucleocapsid.
British Journal of Dermatology, 2000
Melanocytic dendrites consist of a central core of microtubules and a subcortical actin network. ... more Melanocytic dendrites consist of a central core of microtubules and a subcortical actin network. Several studies provide arguments supporting the hypothesis that actin-based and microtubule-based motor proteins co-operate in melanosome transport towards the dendrite tips. Melanosomes can move bidirectionally along microtubules in vitro, and in murine melanocytes, they move towards the cell periphery and back again. Microtubules have a fast-growing plus end and a slow-growing minus end. Microtubule-associated motor proteins move unidirectionally either towards the plus or towards the minus end. However, it is not known which motor protein is responsible for minus end-directed movement of melanosomes. We aimed to investigate the in vitro expression of the minus end-directed motor protein cytoplasmic dynein in normal human epidermal melanocytes, keratinocytes and dermal fibroblasts. Reverse transcription-polymerase chain reaction and Northern blot analysis were used. In addition, an attempt to obtain insight into the subcellular localization of cytoplasmic dynein, immunofluorescence studies and immunogold electron microscopic studies were performed. The three different forms of cytoplasmic dynein heavy chain were expressed in all studied skin cells. Immunofluorescence staining showed similar punctate distributions for dynein heavy chain 1 and dynein heavy chain 2 in melanocytes, with accentuation in the perinuclear area and dendrite tips. Double labelling with a melanosome marker showed apparent co-localization of both dynein heavy chains 1 and 2 with melanosomes in the perinuclear area and dendrite tips. For the dynein intermediate chain of 74 kDa, again a punctate staining pattern was seen with intense centrosomal staining. A close association of dynein intermediate chain 74 and alpha-tubulin with the melanosome surface was detected using immunogold electron microscopy. The colocalization of different subunits of the cytoplasmic dynein complex with melanosomes is consistent with the hypothesis that this motor protein supports minus end-directed melanosome movement along microtubules.
Skin Pharmacology and Physiology, 2001
One of the prerequisites for the use of human skin equivalents for scientific and screening purpo... more One of the prerequisites for the use of human skin equivalents for scientific and screening purposes is that their barrier function is similar to that of native skin. Using human epidermis reconstructed on de-epidermized dermis we demonstrated that the formation of the stratum corneum (SC) barrier in vitro proceeds similarly as in vivo as judged from the extensive production of lamellar bodies, their complete extrusion at the stratum granulosum/SC interface, and the formation of multiple broad lamellar structures in the intercorneocyte space. The presence of wellordered lipid lamellar phases was confirmed by small-angle X-ray diffraction. Although the long periodicity lamellar phase was present in both the native and the reconstructed epidermis, the short periodicity lamellar phase was present only in native tissue. In addition, the SC lipids predominantly formed the hexagonal sublattice. Analysis of lipid composition revealed that all SC lipids are synthesized in vitro. Differences in SC lipid organization in reconstructed epidermis may be ascribed to the differences in fatty acid content and profile indicating that further improvement in culture conditions is required for generation of in vitro reconstructed epidermis with stratum barrier properties of the native tissue.
Dermatology, 1994
Tightly curled 'frizzy' hair is a pathognomonic sign of giant axonal degeneration... more Tightly curled 'frizzy' hair is a pathognomonic sign of giant axonal degeneration (GAD). The present study compares the morphological structure of the scalp hair of a GAD patient with that of her parents and first-degree relatives with the aid of scanning electron microscopy. The comparison included plaited hair of two age-matched controls, in order to exclude mechanical plaiting artifacts. Trichorrhexis nodosa and fringing of the cut ends were exclusively found in the patient's hair. Longitudinal grooving was also frequently seen in hair of normal persons. Assay of carbon (C), sulfur (S) and nitrogen (N) contents of the patient's hair was normal, but the S:N ratio was significantly reduced, as compared with her relatives. However, comparison with a control group of non-related healthy volunteers showed no difference.
Pharmaceutical research, 2001
To study at the ultrastructural level which part of the skin is associated with percutaneous iodi... more To study at the ultrastructural level which part of the skin is associated with percutaneous iodide transport by passive diffusion and iontophoresis. Following passive diffusion or iontophoresis of iodide, the morphology and the ion distribution of the skin was preserved by rapid freezing. The skin was kept frozen until and during examination by transmission electron microscopy (TEM) and X-ray microanalysis (XRMA). The intrinsic electron absorbing characteristics of cryopreserved skin allow direct TEM examination without additional staining. XRMA can be used to obtain in a relatively nondestructive way in situ information on ion distributions across the skin. After passive diffusion, iodide was mainly found in the stratum corneum (SC), whereas there was little iodide in the viable epidermis. Iontophoresis up to 300 microA/cm2 did not significantly affect this distribution. With iontophoresis at 1,000 microA/cm2, the amount of iodide increased dramatically and was equally distributed...
Cellular Signalling, 1994
Although electropermeabilization has become an important tool for studying the signal requirement... more Although electropermeabilization has become an important tool for studying the signal requirements of exocytosis, relatively little is known about the morphological changes accompanying this response in electropermeabilized cells. In this study, we determined that electropermeabilization of human neutrophils by itself caused only minor changes in the morphology as determined by transmission electron microscopy. The structure of the plasma membrane did not show detectable changes, whereas the cytoplasm was more electron lucent as compared to intact cells. Activation of intact neutrophils with formyl-methionyl-leucyl-phenylalanine (FMLP), in the presence of cytochalasin-B, caused the development of invaginations of the plasma membrane. In contrast, activation of electropermeabilized cells with 1 microM Ca2+ and/or 50 microM GTP-gamma-S caused the development of vacuoles that did not seem to be in contact (or had previously been in contact) with the extracellular environment. However, fusion of azurophilic and specific granules with these vacuoles clearly had taken place. The response characteristics of this fusion induced by Ca2+ and GTP-gamma-S were quite similar to those of the direct fusion of granules with the plasma membrane. We conclude that in electropermeabilized human neutrophils, two processes involving granule fusion can be distinguished. First, a direct fusion of granules with the plasma membrane. Secondly, the fusion of granules leading to the formation of vacuoles, not in contact with the extracellular space.
Wound Repair and Regeneration, 1999
A fully differentiated epithelium mimicking the features of native epidermis was obtained in vitr... more A fully differentiated epithelium mimicking the features of native epidermis was obtained in vitro by culturing human or porcine epidermal keratinocytes on polyester filter substrate at the air-liquid interface. In addition, after 2 weeks of culture, hemidesmosome-like structures were formed along the basal area of the plasma membrane of the basal cells at the cell-filter interface. When grafted onto full-thickness skin wounds in pigs, the take of cell sheets detached from the filter with dispase was significantly higher (about 70%) in comparison to mechanically detached keratinocytes (about 15%). With dispase-treated keratinocytes alone, basement membrane formation took place within 7 days postgrafting as judged from the presence of a lamina lucida and positive staining for type IV collagen. Also, numerous hemidesmosomes and anchoring fibrils were observed at the basal cell-"neodermis" interface. The fully differentiated epidermis, generated by culturing keratinocytes at the air-liquid interface and detached from the substrate by dispase-treatment, is less fragile and easier to handle than epidermal autografts obtained by conventional culturing methods. Detachment by a short dispase-treatment appeared in our hands the only method for successful and complete epithelial regeneration in full-thickness wounds.
Pigment Cell Research, 2000
The two isoforms of P150 Glued and P50 are expressed in all Melanocytic dendrites consist of a ce... more The two isoforms of P150 Glued and P50 are expressed in all Melanocytic dendrites consist of a central core of microtubules (MT) and a subcortical actin network. In previous studied skin cells. Immunofluorescence staining shows punctate distributions for P150 Glued and P50 in melanocytes. reports we showed the presence of MT-associated motor P150 Glued shows a clear centrosomal staining and accentua-proteins kinesin and cytoplasmic dynein on the melanosomal tion in the dendrite tips. P50 is also accentuated in the surface, forming a link with MT (Vancoillie et al. J Invest Dermatol 2000;114:421-429; Vancoillie et al. Br J Derma-perinuclear area and dendrite tips. Immunofluorescence doutol 2000;143:258-306)
Journal of Investigative Dermatology, 1998
Calcium plays an important role in the regulation of cellular differentiation and desquamation of... more Calcium plays an important role in the regulation of cellular differentiation and desquamation of epidermal keratinocytes. In this study, we examined the calcium distribution in reconstructed epidermis in an attempt to understand the physiology of keratinocyte differentiation and desquamation in vitro. Ion capture cytochemistry (the potassium oxalate-pyroantimonate method) was employed to localize ionic calcium in reconstructed epidermis generated under three different culture conditions (in serum-containing medium, serum-free medium, and serum-free medium supplemented with retinoic acid), allowing a comparison of the physiology of incompletely and well-differentiated keratinocytes. The reconstructed epidermis generated in serum-containing medium showed features of incomplete differentiation, and compared with the native skin, a high calcium content within incompletely differentiated cells in the stratum corneum. Use of serum-free medium containing vitamin and lipid supplements led to a marked improvement of the stratum corneum ultrastructure and penetration pathway across the stratum corneum, indicating improved barrier formation of the reconstructed epidermis. In parallel, the calcium distribution pattern was normalized showing the highest levels of calcium in the stratum granulosum and low levels in the inner stratum corneum. Addition of retinoic acid to the serum-free medium resulted in an altered keratinocyte differentiation and re-appearance of large quantities of calcium precipitates in the stratum corneum. Proton probe X-ray microanalysis was applied to investigate the calcium distribution quantitatively in native and reconstructed epidermis generated in serum-free medium, and verified the calcium distribution demonstrated by the precipitation technique. Regardless of the presence or absence of calcium in the stratum corneum, all examined culture systems exhibited insufficient desquamation, which correlates with the finding that stratum corneum chymotryptic enzyme was present predominantly as an inactive precursor. This study demonstrates that improvement of the stratum corneum barrier properties in vitro is concurrent with the normalization of the epidermal calcium gradient, whereas deregulation of terminal differentiation correlates with an accumulation of calcium ions within incompletely differentiated corneocytes.
Journal of Investigative Dermatology, 1998
Mutations of the gene encoding myosin V can produce a dilute or silvery hair color and various ne... more Mutations of the gene encoding myosin V can produce a dilute or silvery hair color and various neurologic defects in mice and patients with Griscelli syndrome, leading to speculations that the myosin V motor protein plays a critical role in transporting melanosomes within melanocytes and neurosecretory vesicles within neurons. Therefore, we investigated the in vitro expression of myosin V in cultured normal human melanocytes, keratinocytes, and dermal fibroblasts using reverse transcriptase-polymerase chain reaction and northern blot analysis. Subcellular distribution of myosin V and proximity to actin bundles and melanosomes were determined by double indirect immunofluorescence labeling and immunogold electron microscopy. In all studied cells V ery little is known about the molecular mechanisms underlying dendrite extension and melanosome transport in human melanocytes. It is expected that cytoskeletal proteins such as actin and microtubule components with their respective motor proteins play a role in these processes. Indeed, Lacour et al (1992) reported that actin microfilaments play a role in dendrite formation and microtubules play a role in dendrite maintenance in cultured human melanocytes.
Journal of Investigative Dermatology, 2000
Microtubuli play an important role in the organization of organelles and membrane traffic. They a... more Microtubuli play an important role in the organization of organelles and membrane traffic. They are present in melanocytic dendrites through which melanosomes are transported towards keratinocytes. Besides the actin-based motility systems, microtubuli-associated motor proteins also play a critical role in melanosome movement, as has recently been confirmed in mouse melanocytes. We investigated the in vitro expression of two forms of human conventional kinesin and its receptor kinectin in normal human epidermal melanocytes, keratinocytes, and dermal fibroblasts by reverse transcription polymerase chain reaction and northern blot analysis. In an attempt to gain insight into the subcellular distribution of kinesin and kinectin in melanocytes, double immunofluorescent staining and immunogold electron microscopy were performed. In all studied skin cells ubiquitous and neuronal kinesin are expressed, as well as the kinectin receptor. Immunofluorescent staining shows distinct but partially overlapping distributions for kinesin heavy chain and melanosomes, suggesting that kinesin is associated with some but not all of the melanosomes. Similar observations for kinectin indicate that this receptor can colocalize with melanosomes, which was confirmed by immunoelectron microscopy. The latter technique allowed us to demonstrate a close association between kinesin heavy chain, microtubuli, and melanosomes. The combined data from reverse transcription polymerase chain reaction, northern blot analysis, double immunofluorescent staining, and immunogold electron microscopy suggest that kinesins and kinectin have an important role in microtubuli-based melanosome transport in human melanocytes.
Journal of Biomedical Materials Research, 1997
To analyze the bone-bonding property of hydroxyapatite ceramics (HA), composites of rat marrow ce... more To analyze the bone-bonding property of hydroxyapatite ceramics (HA), composites of rat marrow cells and porous HA were implanted subcutaneously and harvested at 3 to 4 weeks postimplantation. De novo bone formation was observed primarily on the HA surface without fibrous tissue interposition. The HA/tissue interface was analyzed by the observations of thin undecalcified histological sections and fractured surfaces of the implants. The observations were done with a light microscope and a scanning electron microscope (SEM) connected to an energy dispersive spectrometer. The interfacial analyses showed the appearance of osteoblastic cells on the HA surface and that the cells had initiated partially mineralized bone (osteoid) formation directly onto the surface. The osteoid matured into fully mineralized bone, resulting in firm bone bonding to the HA surface. Characterization of osteoblastic cells on the surface was done by determining levels of protein and gene expression of bone Gla protein (BGP, a.k.a. Osteocalcin), i.e., immunohistochemistry and in situ hybridization, respectively. The existence of BGP and mRNA in the cytoplasmic area of the cells confirmed that active osteoblast apposition fabricated primary bone on the HA surface. All of these results indicate the importance of the HA surface in supporting osteoblastic differentiation of marrow stromal stem cells, which leads to firm bone bonding.
Developmental Dynamics, 2006
During cardiovascular development, fluid shear stress patterns change dramatically due to extensi... more During cardiovascular development, fluid shear stress patterns change dramatically due to extensive remodeling. This biomechanical force has been shown to drive gene expression in endothelial cells and, consequently, is considered to play a role in cardiovascular development. The mechanism by which endothelial cells sense shear stress is still unidentified. In this study, we postulate that primary cilia function as fluid shear stress sensors of endothelial cells. Such a function already has been attributed to primary cilia on epithelial cells of the adult kidney and of Hensen's node in the embryo where they transduce mechanical signals into an intracellular Ca2+ signaling response. Recently, primary cilia were observed on human umbilical vein endothelial cells. These primary cilia disassembled when subjected to high shear stress levels. Whereas endocardial-endothelial cells have been reported to be more shear responsive than endothelial cells, cilia are not detected, thus far, on endocardial cells. In the present study, we use field emission scanning electron microscopy to show shear stress-related regional differences in cell protrusions within the cardiovasculature of the developing chicken. Furthermore, we identify one of these cell protrusions as a monocilium with monoclonal antibodies against acetylated and detyrosinated alpha-tubulin. The distribution pattern of the monocilia was compared to the chicken embryonic expression pattern of the high shear stress marker Krüppel-like factor-2. We demonstrate the presence of monocilia on endocardial-endothelial cells in areas of low shear stress and postulate that they are immotile primary cilia, which function as fluid shear stress sensors.
Cell and Tissue Research, 1996
A fully differentiated epidermis generated in vitro by culturing normal human keratinocytes at th... more A fully differentiated epidermis generated in vitro by culturing normal human keratinocytes at the air-liquid interface shares many similarities with native tissue. However, in contrast to native epidermis, its stratum corneum consists of a larger number of compactly packed corneocyte layers, indicating that the processes involved in corneocyte desquamation are disturbed. Although the tri-lamellar appearance of desmosomes in viable cell layers of both types of epidermis is similar, abnormalities in the transformation of desmosomes into the corneosomal plug in the stratum corneum of reconstructed epidermis have been observed. In the native epidermis, desmosomes are transformed into corneosomes at the stratum granulosum/stratum corneum interface. This process is retarded in vitro, since desmosomal structures with preserved lamellar appearance are present in the lower parts of the stratum corneum. Moreover, the corneosome frequency in reconstructed epidermis is significantly higher than in native human skin. A comparison of reconstructed epidermis with hyperproliferative epidermis, such as UV-irradiated epidermis, psoriatic epidermis and recently healed burn-wounds treated with cultured epidermal autografts, has revealed that only the structure of the stratum corneum derived from psoriatic skin is similar to that of reconstructed epidermis. The stratum corneum organization in the UV-treated epidermis and in recently healed burn-wound is, however, close to that seen in native skin.
British Journal of Dermatology, 2000
Melanocytic dendrites consist of a central core of microtubules and a subcortical actin network. ... more Melanocytic dendrites consist of a central core of microtubules and a subcortical actin network. Several studies provide arguments supporting the hypothesis that actin-based and microtubule-based motor proteins co-operate in melanosome transport towards the dendrite tips. Melanosomes can move bidirectionally along microtubules in vitro, and in murine melanocytes, they move towards the cell periphery and back again. Microtubules have a fast-growing plus end and a slow-growing minus end. Microtubule-associated motor proteins move unidirectionally either towards the plus or towards the minus end. However, it is not known which motor protein is responsible for minus end-directed movement of melanosomes. We aimed to investigate the in vitro expression of the minus end-directed motor protein cytoplasmic dynein in normal human epidermal melanocytes, keratinocytes and dermal fibroblasts. Reverse transcription-polymerase chain reaction and Northern blot analysis were used. In addition, an attempt to obtain insight into the subcellular localization of cytoplasmic dynein, immunofluorescence studies and immunogold electron microscopic studies were performed. The three different forms of cytoplasmic dynein heavy chain were expressed in all studied skin cells. Immunofluorescence staining showed similar punctate distributions for dynein heavy chain 1 and dynein heavy chain 2 in melanocytes, with accentuation in the perinuclear area and dendrite tips. Double labelling with a melanosome marker showed apparent co-localization of both dynein heavy chains 1 and 2 with melanosomes in the perinuclear area and dendrite tips. For the dynein intermediate chain of 74 kDa, again a punctate staining pattern was seen with intense centrosomal staining. A close association of dynein intermediate chain 74 and alpha-tubulin with the melanosome surface was detected using immunogold electron microscopy. The colocalization of different subunits of the cytoplasmic dynein complex with melanosomes is consistent with the hypothesis that this motor protein supports minus end-directed melanosome movement along microtubules.
The American journal of pathology, 1990
This report describes the cell biology of the development of asbestos bodies after a single intra... more This report describes the cell biology of the development of asbestos bodies after a single intraperitoneal injection of a suspension of crocidolite asbestos fibers into the mouse peritoneal cavity. The majority of the infected fibers were found in aggregates of peritoneal macrophages, exudate cells, and fibrous tissue. These aggregates developed into granulomas containing not only numerous asbestos fibers, but also cells of various types, including macrophages, multinucleated giant cells, fibroblasts, plasma cells, granulocytes, and mast cells. Cytoplasmic ferritin was abundantly present in macrophages and giant cells. In addition, iron-rich inclusion bodies were detected. The results of this study show that asbestos body formation can occur outside the pleural cavity. Asbestos body formation occurred in the granulomas after periods of 1 month and longer. On the basis of morphologic criteria, various types of asbestos body were distinguished. X-ray microanalysis showed that variati...
The Anatomical Record, 2004
The beta-geo (LacZ) reporter gene encodes for -galactosidase (-gal) in all cells of the ROSA26 ... more The beta-geo (LacZ) reporter gene encodes for -galactosidase (-gal) in all cells of the ROSA26 mouse during embryonic development. As such, -gal activity constitutes an excellent marker for in situ labeling of expressing cells. However, the intracellular distribution of -gal differs between cells, and changes during embryonic development. Therefore, we studied LacZ-encoded -gal using light and electron microscopy in the heart, lung, liver, and small intestine on days 13 and 16 of gestation, and the kidney on day 16 of gestation in ROSA26 mice. The Bluo-gal method was carried out under standardized conditions, including fixation, washing, and incubation procedures. Intracellular -gal staining is encountered in a combination of membranous compartments, including the nuclear envelope, the endoplasmic reticulum, and the plasma membrane. Its exact localization depends on the cell type and is regulated during development. Therefore, one must take the compartmental transition of intracellular -gal staining into consideration when interpreting results obtained from experiments using ROSA26 mice.
Photochemical & Photobiological Sciences, 2002
The application of a novel model for sunscreen photoimmunoprotection studies was assessed using a... more The application of a novel model for sunscreen photoimmunoprotection studies was assessed using a systemic infection of rats with herpes simplex virus type 1 (HSV-1). Rats were irradiated daily with 1 minimal erythemal/oedematous dose of UVB for 7 consecutive days on their shaved backs with or without application of a broad-spectrum sunscreen (containing TiO2) with a sun protection factor of 10. Subsequently, rats were infected intranasally with HSV. UV exposure prior to HSV infection induced increased severity and incidence of clinical signs of disease, suppression of cellular immune responses as assessed by delayed type hypersensitivity and increased viral load in the brain. The sunscreen provided protection against all these UV-induced effects. We conclude that this novel model is a promising way of testing the immunoprotective qualities of sunscreens, based on the response to a common infectious agent of human subjects.
Journal of Virology, 2004
Equine arteritis virus (EAV) is an enveloped, positive-stranded RNA virus belonging to the family... more Equine arteritis virus (EAV) is an enveloped, positive-stranded RNA virus belonging to the family Arteriviridae of the order Nidovirales. EAV particles contain seven structural proteins: the nucleocapsid protein N, the unglycosylated envelope proteins M and E, and the N-glycosylated membrane proteins GP 2b (previously named G S ), GP 3 , GP 4 , and GP 5 (previously named G L ). Proteins N, M, and GP 5 are major virion components, E occurs in virus particles in intermediate amounts, and GP 4 , GP 3 , and GP 2b are minor structural proteins. The M and GP 5 proteins occur in virus particles as disulfide-linked heterodimers while the GP 4 , GP 3 , and GP 2b proteins are incorporated into virions as a heterotrimeric complex. Here, we studied the effect on virus assembly of inactivating the structural protein genes one by one in the context of a (full-length) EAV cDNA clone. It appeared that the three major structural proteins are essential for particle formation, while the other four virion proteins are dispensable. When one of the GP 2b , GP 3 , or GP 4 proteins was missing, the incorporation of the remaining two minor envelope glycoproteins was completely blocked while that of the E protein was greatly reduced. The absence of E entirely prevented the incorporation of the GP 2b , GP 3 , and GP 4 proteins into viral particles. EAV particles lacking GP 2b , GP 3 , GP 4 , and E did not markedly differ from wild-type virions in buoyant density, major structural protein composition, electron microscopic appearance, and genomic RNA content. On the basis of these results, we propose a model for the EAV particle in which the GP 2b /GP 3 /GP 4 heterotrimers are positioned, in association with a defined number of E molecules, above the vertices of the putatively icosahedral nucleocapsid.