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Biomedical subjects

W Westerhof

Publications and source records attributed to W Westerhof.

At least 19 recordsLinked to original sources
PubMed1996

Presence of T cells and macrophages in inflammatory vitiligo skin parallels melanocyte disappearance.

Evidence for the involvement of cellular immunity in the etiopathogenesis of the hypopigmentary disorder vitiligo is provided by rare cases of inflammatory vitiligo. Nonlesional, perilesional, and lesional skin biopsies from three inflammatory vitiligo patients were immunohistochemically analyzed. The composition of inflammatory infiltrates present in perilesional skin was analyzed by antibodies to T cells (CD2, CD3, CD4, and CD8), Langerhans cells (CD1a), and macrophages (CD36 and CD68). The presence of activation markers on inflammatory cells was evaluated by analysis of HLA-DR, interleukin-2 receptor, and HECA452 expression. The presence or absence of melanocytes was determined by the antibody NKI-beteb. Moreover, the abundance of matrix molecule tenascin was semi-quantified using T2H5. Results indicate that within perilesional skin, epidermis-infiltrating T cells exhibit an increased CD8/CD4 ratio and increased cutaneous lymphocyte antigen and interleukin-2 receptor expression. These cells are frequently juxtapositionally apposed to remaining melanocytes. In perilesional dermis, CD68+OKM5- macrophages were more numerous than in lesional or nonlesional skin. Keratinocytes as well as melanocytes consistently express major histocompatibility complex class II antigens along stretches of basal and suprabasal layers in perilesional epidermis. Moreover, inflammation is accompanied by increased tenascin content. Although these observations do not permit differentiation between the immune infiltrates being a result as opposed to the cause of the disease process, results presented in this study are very suggestive of involvement of local immune reactivity in melanocyte destruction.

Adult↗
PubMed1995

Adherence, proliferation and collagen turnover by human fibroblasts seeded into different types of collagen sponges.

We describe an in vitro model that we have used to evaluate dermal substitutes and to obtain data on cell proliferation, the rate of degradation of the dermal equivalent, contractibility and de novo synthesis of collagen. We tested three classes of collagenous materials: (1) reconstituted non-crosslinked collagen, (2) reconstituted collagen that was chemically crosslinked with either glutaraldehyde, aluminium alginate or acetate, and (3) native collagen fibres, with or without other extracellular matrix molecules (elastin hydrolysate, hyaluronic acid or fibronectin). The non-crosslinked reconstituted collagen was degraded rapidly by human fibroblasts. The chemically crosslinked materials proved to be cytotoxic. Native collagen fibres were stable. In the absence of ascorbic acid, the addition of elastin hydrolysate to this type of matrix reduced the rate of collagen degradation. Both elastin hydrolysate and fibronectin partially prevented fibroblast-mediated contraction. Hyaluronic acid was only slightly effective in reducing the collagen degradation rate and more fibroblast-mediated contraction of the material was found than for the native collagen fibres with elastin hydrolysate and fibronectin. In the presence of ascorbate, collagen synthesis was enhanced in the native collagen matrix without additions and in the material containing elastin hydrolysate, but not in the material with hyaluronic acid. These results are indicative of the suitability of tissue substitutes for in vivo application.

Ascorbic Acid↗
PubMed1995

Repigmentation in vitiligo vulgaris by autologous minigrafting: results in nineteen patients.

BACKGROUND: Minigrafting is a successful therapy for localized vitiligo but has never been reported for vitiligo vulgaris. OBJECTIVE: Our purpose was to evaluate the efficacy of minigrafting in vitiligo vulgaris. METHODS: In 59 patients with stable vitiligo vulgaris, a minigraft test was done by implanting two minigrafts in the lesion to be grafted. Patients were selected for grafting when spread of pigment was observed within 3 months. The rate of repigmentation was evaluated by digital image analysis. RESULTS: Twenty-three patients (36 lesions), of 24 with a positive minigraft test, were grafted. The results of 19 patients were analyzed, showing 80% to 99% repigmentation in 14 lesions, 50% to 80% repigmentation in 10 lesions, and zero to 50% repigmentation in 12 lesions. Time of observation varied from 3 to 12 months after grafting. Best results were observed after 9 to 12 months. In all patients with a positive Koebner phenomenon depigmentation of the minigrafts developed. CONCLUSION: Autologous minigrafting is an effective therapy for stable vitiligo vulgaris in a selected group of patients.

Adolescent↗
PubMed1995

Reduced wound contraction and scar formation in punch biopsy wounds. Native collagen dermal substitutes. A clinical study.

In full-thickness skin wounds dermal regeneration usually fails, resulting in scar formation and wound contraction. We studied dermal regeneration by implantation of collagenous matrices in a human punch biopsy wound model. Matrices were made of native bovine collagen I fibres, and either hyaluronic acid, fibronectin, or elastin was added. Matrices were placed in 6-mm punch biopsy holes in seven patients (biopsies were used for the grafting of leg ulcers), and covered with a protective semi-permeable polyether urethane membrane. Histology, wound contraction and dermal architecture were studied. Dermal architecture was evaluated using a recently developed laser scatter technique. All collagen matrices showed a tendency to reduce wound contraction, compared with control wounds; elastin- and fibronectin-treated matrices showed significantly less contraction than control wounds. Only the addition of elastin had a clear beneficial effect on dermal architecture; collagen bundles were more randomly organized, compared with control wounds, and wounds treated with collagen matrices coated with fibronectin or hyaluronic acid, or without coating. We conclude that the punch biopsy wound model provides important information on dermal regeneration in humans. Native collagen matrices with elastin contributed to dermal regeneration and reduced wound contraction, in contrast with matrices coated with fibronectin or hyaluronic acid, or without coating. Future clinical studies of large-area, full-thickness wounds will be required to establish their clinical relevance for leg ulcer and burn treatment.

Aged↗
PubMed1995

A new psoralen-containing gel for topical PUVA therapy: development, and treatment results in patients with palmoplantar and plaque-type psoriasis, and hyperkeratotic eczema.

Topical photochemotherapy with psoralen and its derivatives 4,5',8-trimethylpsoralen (TMP) and 8-methoxypsoralen (8-MOP), with UVA irradiation, was evaluated with regard to minimum phototoxic dose, concentration, timing of UVA irradiation and systemic and local side-effects, in healthy volunteers. Psoralen (0.005%) in aqueous gel was found to be superior to TMP and 8-MOP in aqueous gel. No hyperpigmentation was seen after topical PUVA treatment with psoralen in aqueous gel. Patients with plaque-type psoriasis (n = 7), palmoplantar psoriasis (n = 7) and hyperkeratotic eczema (n = 2) were treated. Topical PUVA therapy was effective in most psoriasis patients, without the occurrence of local or systemic side-effects. Moreover, hyperkeratotic eczema patients who did not respond to conventional therapy showed partial remission. These results indicate that topical PUVA therapy with psoralen in aqueous gel is a useful therapeutic modality for treatment of psoriasis patients, and patients with recalcitrant dermatoses such as palmoplantar psoriasis and hyperkeratotic eczema.

Eczema↗
PubMed1995

Some aspects of melanin formation of melanocytes cultured on collagen-coated microcarrier beads.

Melanocytes cultured on collagen-coated Cytodex 3 microcarrier Sephadex beads caused remarkable pigmentation of the beads during the period of culture when optimal density was reached. Electron microscopy of melanocytes on the microcarriers revealed that the cells and their dendrites invaginate into the microcarrier surface layer. Removal of the cells by trypsinization showed that some pigment granules were left on the carrier surface and within the cavities present on the microcarrier surface. In order to investigate whether the pigmentation of the microcarriers could be a result of indole intermediates of melanogenesis present in the culture medium, extracts were studied by gas chromatography/mass spectrometry for the presence of these compounds. Two compounds (5,6-dihydroxyindole-2-carboxylic acid and 6-hydroxy-5-methoxyindole-2-carboxylic acid) so far have been identified in the medium extracts. Results indicate that microcarrier culture of melanocytes can serve as an interesting model for electron microscopy studies of melanocytes with regard to pigmentation and cell attachment.

Cell Adhesion↗
PubMed1995

Stimulation of cultured melanocytes in medium containing a serum substitute: Ultroser-G.

Melanocyte cultures were established and maintained routinely in Ham's F-10 medium containing 12-O-tetradecanoyl-phorbol-13-acetate (TPA), isobutylmethylxanthine (IBMX), cholera toxin (CT) and fetal calf serum (FCS). Three serum substitutes (Ultroser-G, Nutridoma-Hu and Nutricyte-H) were tested in order to obtain a medium without FCS having a more constant composition. Melanocyte proliferation was examined in long-term culture experiments by in situ cell counts at different periods of time. Only with Ultroser-G (1-2%) was the proliferation of melanocytes maintained without both FCS and CT, whereas the addition of the other two serum substitutes resulted in stabilization of melanocyte densities in the cultures up to 28 days. In the medium containing 1% Ultroser-G and IBMX without TPA minimal or no increases in melanocyte density were found. Addition of basic fibroblast growth factor (bFGF, 1 ng/ml) to the medium without TPA resulted in a partial restimulation of growth in different experiments. In this system with 1% Ultroser-G and 1 ng/ml bFGF, IBMX could also be replaced by other factors (dbcAMP, LTC4 and a purified form of alpha-melanocyte stimulating hormone). The culture medium with 1% Ultroser-G containing TPA and IBMX is now used for routine melanocyte culture. In this medium TPA/IBMX can easily be replaced by bFGF/dbcAMP with optimal growth stimulation. The combination bFGF/alpha-MSH and other more physiological stimulators offers an alternative to study responses of melanocytes in culture with respect to proliferation, metabolism, and phenotype.

1-Methyl-3-isobutylxanthine↗
PubMed1995

Stromal cells from subcutaneous adipose tissue seeded in a native collagen/elastin dermal substitute reduce wound contraction in full thickness skin defects.

BACKGROUND: Dermal substitutes seeded with cultured fibroblasts have been developed to improve dermal regeneration in full thickness wounds. Because of cell cultivation, 3 weeks are required before patients can be treated with these autologous adipose tissue. This substitute is easily fabricated within hours, which allows immediate treatment of full thickness defects. EXPERIMENTAL DESIGN: Porcine full thickness wounds were substituted with native collagen/alpha-elastin hydrolysate matrices. One group of matrices was left unseeded as negative control. The second was seeded with cultured dermal fibroblasts as positive control. The third was seeded with a stromal-vascular-fraction of adipose tissue, and the fourth was seeded with a stromal fraction with few vascular fragments (SF). All substitutes were covered with split skin mesh grafts and were protected against dehydration and infection with a microporous polyether urethane membrane. For 8 weeks, weekly biopsies were taken, myofibroblasts and fibroblasts were counted, thickness of the granulation tissue band was measured, and wound contraction and histology were evaluated. RESULTS: Negative control and stromal-vascular-fraction substitutes were invaded by high numbers of myofibroblasts and fibroblasts. They did not reduce wound contraction, and scar tissue was formed. SF substitutes reduced the accumulation of myofibroblasts and fibroblasts and prevented the formation of granulation tissue. As a result, dermal regeneration improved, and wound contraction was less than by the other substitutes. CONCLUSIONS: Adipose tissue cell isolates included vascular fragments containing endothelial cells. Seeded in dermal substitutes, these vascular fragments induced hypergranulation tissue formation and caused wound contraction. SF substitutes contained few endothelial cells. As a result, the contraction arresting effect of the seeded stromal cell fraction was effective. Our concept of a cellular dermal substitute seeded with stromal cells from adipose tissue is feasible and allows immediate treatment of full thickness skin defects.

Adipose Tissue↗
PubMed1994

Catechol-O-methyltransferase as a target for melanoma destruction?

Catechols may interfere in melanogenesis by causing increased levels of toxic quinones. Several catechols and known inhibitors of the enzyme catechol-O-methyltransferase (COMT) were therefore tested for their toxicity towards a pigmented melanoma cell line, UCLA-SO-(M14). The inhibition of thymidine incorporation as a result of exposure to the compounds was measured. All agents were compared to 4-hydroxyanisole (4HA), a depigmenting agent extensively studied as an antimelanoma drug. The compounds were also tested on the epithelial cell line, CNCM-I-(221) in the presence and absence of tyrosinase. All the compounds were more effective than 4HA towards the M14-cells at either 10(-4) M or 10(-5) M. The toxicity of 4HA towards the 221-cells was shown to be completely dependent on the presence of tyrosinase. Effects of the test agents on the 221-cells were also observed in the absence of tyrosinase. Although some of them were shown to be good substrates for tyrosinase only small changes in toxicity were observed as a result of the presence of the enzyme in comparison with 4HA. No direct correlation of the toxicity of the agents and COMT inhibition was observed. The possible mode of action of the compounds through inhibition of COMT and interference in melanogenesis is discussed together with other possibilities and factors involved.

Anisoles↗
PubMed1994

Catechol-O-methyltransferase in vitiligo.

Catechol-O-methyltransferase (COMT) is involved in the metabolism of neurotransmitters such as epinephrine, norepinephrine and dopamine. For melanocytes, the enzyme is of particular importance in preventing the formation of toxic o-quinones during melanin synthesis. It has been suggested that COMT plays a regulatory role in melanin synthesis. Indeed, when the melanin precursor molecule DHI(2C) is methylated by COMT it is no longer available for incorporation into melanin. Auto-destruction by intermediates of melanin metabolism has been implicated in the aetiology of vitiligo. Therefore enzyme activities in vitiligo patients and in healthy controls were compared. Systemic COMT activities were measured using red blood cells (RBC) as starting material. However, as local alterations in COMT activity may be specifically involved in vitiligo, the enzyme activity was also measured in epidermal homogenates. Finally, to ascribe epidermal COMT activity to the responsible cell type(s), enzyme activity was measured in cultured vitiligo non-lesional melanocytes and melanocytes from healthy controls as well as in cultured keratinocytes from lesional skin and in purified keratinocytes from control skin. It was found that epidermal homogenates from vitiligo patients expressed higher levels of COMT activity than homogenates from healthy controls. Such differences were not found at the systemic level (i.e. in RBC) nor could they be explained by measurements on separately cultured epidermal cell types, indicating that the COMT activity was induced at the tissue level by extracellular factors.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗
PubMed1994

Organotypic culture of human skin to study melanocyte migration.

An ex vivo model system was developed to investigate melanocyte migration. Within this model system, melanocytes migrate among other epidermal cells in the epibolic outgrowth of skin explants. This process is initiated by loss of contact inhibition of epidermal cells at the rim of the explants and by locally produced chemotactic factors. Punch biopsies provided explants of reproducible diameter. Optimal culture conditions include medium consisting of Dulbecco's Minimal Essential Medium containing 10% inactivated normal human serum and placement of explants epidermal side up at the air-liquid interphase. Within 7 days, epidermal cells completely surround the explant. Approximately 3 days after the onset of keratinocyte migration, melanocytes distribute themselves within the newly formed epidermis. Throughout the 7-day culture period, melanocytes and keratinocytes show maintenance of subcellular morphology, and the dermo-epidermal junction remains intact. Melanocyte migration was quantified using immunoperoxidase staining in combination with light microscopy and computer-aided image analysis. Preliminary results using the model system to compare migration in control and nonlesional vitiligo skin indicate that no inherent migration defect is responsible for impaired repigmentation of vitiligo lesions. The organotypic culture model system allows for investigations on melanocytes within their environment of autologous epidermal and dermal components, closely resembling in vivo circumstances in human skin.

Adult↗
PubMed1994

O-methylation of L-dopa in melanin metabolism and the presence of catechol-O-methyltransferase in melanocytes.

O-Methylation of L-dopa was investigated as a possible regulatory mechanism in melanin metabolism. The methylation product of L-dopa, 3-O-methoxytyrosine was detected in extracts of cultured human melanocytes. The enzyme catechol-O-methyltransferase is responsible for this O-methylation and that of the dihydroxyindolic intermediates of melanogenesis. The enzyme is present in melanocytes in its soluble and membrane-bound isoforms. Immuno-electron microscopy suggests the presence of the membrane-bound enzyme in the endoplasmic reticulum. This localization may indicate a role of catechol-O-methyltransferase in protecting the melanocyte against reactive dihydroxyphenolic intermediates of melanogenesis leaking from the melanogenic compartments. On the other hand, the O-methylation of L-dopa may serve as a regulatory point in melanogenesis during early stage of tyrosinase processing in the endoplasmic reticulum.

1-Methyl-3-isobutylxanthine↗
PubMed1994

L-phenylalanine and UVA irradiation in the treatment of vitiligo.

In order to evaluate the efficacy of L-phenylalanine (L-Phe) in combination with UVA therapy for vitiligo an open trial (149 patients, 18 months) and a small double-blind trial (32 patients, 6 months) were conducted. Oral L-Phe loading resulted in peak plasma levels of L-Phe after 30-60 min and a slight increase in the plasma tyrosine level. Response to L-Phe plus UVA irradiation was positive, and various grades of repigmentation not exceeding 77% in the open and 60% in the blind trial were observed. An increased L-Phe dose resulted in increased L-Phe plasma levels but not in improved clinical results. The optimal L-Phe dose appears to be lower than 50 mg/kg/day. Although it is difficult to draw firm conclusions from the present investigation, we think that L-Phe may have a place in the treatment of vitiligo and its role merits further investigation.

Administration, Oral↗