Biomedical subjects

M Detmar

Publications and source records attributed to M Detmar.

At least 19 recordsLinked to original sources

PubMed1992

Cytokine regulation of proliferation and ICAM-1 expression of human dermal microvascular endothelial cells in vitro.

The effects of recombinant human interleukin 1 alpha (IL-1 alpha), interleukin 1 beta (IL-1 beta), interleukin 6 (IL-6), granulocyte/macrophage colony-stimulating factor (GM-CSF) and tumor necrosis factor-alpha (TNF) on the cell proliferation and the expression of intercellular adhesion molecule-1 (ICAM-1) were assessed in cultured human dermal microvascular endothelial cells (HDMEC). IL-1 alpha and IL-1 beta stimulated the proliferation of HDMEC in a dose-dependent manner, whereas in control experiments using human umbilical vein endothelial cells (HUVEC), IL-1 alpha and IL-1 beta did not stimulate HUVEC growth. Also GM-CSF stimulated the proliferation of HDMEC, whereas IL-6 did not affect endothelial cell growth in vitro. Treatment with IL-1 alpha, IL-1 beta, and TNF markedly increased the expression of ICAM-1 on HDMEC in a time- and dose-dependent manner, in contrast to IL-6 and GM-CSF. By pre-embedding immunoelectron microscopy, membrane-bound expression of ICAM-1 was visualized with pronounced labeling in areas of microvillous cell protrusions. The TNF-induced expression of ICAM-1 on HDMEC was blocked by co-incubation with a neutralizing antibody against TNF, but not with neutralizing antibodies against IL-1 alpha, IL-1 beta, or IL-6. In addition, co-incubation of HDMEC with TNF and the retinoid compound acitretin, dexamethasone, or indomethacin did not abrogate the TNF-induced ICAM-1 expression. These results disclose IL-1 as a major, multifunctional endothelial cell-targeted cytokine and further confirm the concept that pro-inflammatory cytokines exert differential regulatory effects on dermal microvascular endothelial cell proliferation and expression of cell-adhesion molecules.

Adult↗

PubMed1992

Growth characteristics and differentiation of basal cell carcinoma in vitro--immunohistochemical, gel electrophoretic, and ultrastructural analysis.

Cell cultures were established from 48 solid basal cell carcinomas (BCC) and from the normal epidermis of the same patients. The growth characteristics and differentiation of BCC cells in vitro were compared with normal keratinocytes (nKC) by using immunohistochemistry, two-dimensional gel electrophoresis including immunoblots, transmission electron microscopy, and soft agar suspension culture. After isolation of the tumor tissue under a stereodissection microscope, explants were cultured on feeder layers of mitomycin-treated 3T3 cells. After 3-5 d, 73% of all explants of BCC could be successfully cultured showing spindle-shaped outgrowing cells. Compared to nKC, cultured BCC cells had a lower growth rate and showed a wider intercellular polymorphism regarding size and shape. Their labeling pattern with a wide panel of monoclonal antibodies showed significant differences from that of nKC. In particular, only weak reactions for various cytokeratins, filaggrin and vimentin depending on the BCC cell type (small, middle, large) were found. Two-dimensional gel electrophoresis revealed expression of keratins 5, 6, 14, 16, and 17 in BCC cells and of K 5, 6, 13, 14, 16, 17, and 19 in nKC. These findings were confirmed by immunoblot. On the ultrastructural level, only a few desmosomes and a lower degree of keratinization markers were detected in BCC cells; finally, when cultured in soft agar BCC cells formed colonies whereas nKC did not. Our findings indicate that cultured BCC cells may preserve in vitro some in vivo characteristics and maintain a growth and differentiation pattern that differs from cultured nKC. The culture model presented here provides further insights into the cytogenetic and histogenetic characteristics of BCC.

Aged↗

PubMed1992

Cytokine-stimulated human dermal microvascular endothelial cells produce interleukin 6--inhibition by hydrocortisone, dexamethasone, and calcitriol.

The effects of lipopolysaccharide (LPS), recombinant human tumor necrosis factor-alpha (TNF), recombinant human interleukin 1-beta (IL-1 beta), and interferon-gamma (IFN-gamma) on IL-6 production were determined by enzyme-linked immunosorbent assay (ELISA) and by Northern blot analysis in cultured human dermal microvascular endothelial cells (HDMEC). Unstimulated HDMEC did not produce significant amounts of IL-6, whereas lipopolysaccharide (LPS), TNF, and IL-1 beta were potent inducers of HDMEC-derived IL-6 production. Treatment with IFN-gamma had no effect. IL-1 beta stimulation resulted in pronounced IL-6 production after 4 h, followed by complete downregulation at the transcriptional level after 24 h. In contrast, LPS and TNF induced prolonged stimulation of IL-6 production by HDMEC as IL-6 mRNA transcripts were still detected after 24 h treatment and IL-6 protein was markedly increased at this timepoint. The effects of hydrocortisone, dexamethasone, calcitriol, acitretin, and cyclosporin A on TNF- or IL-1 beta-induced IL-6 production by HDMEC were determined by ELISA. Both hydrocortisone and dexamethasone dose-dependently inhibited the cytokine-induced IL-6 production, whereas the inhibition by calcitriol was less pronounced. In contrast, acitretin and cyclosporine A had no influence on cytokine-induced HDMEC IL-6 production. These results disclose dermal endothelial cells as a major source for the pro-inflammatory cytokine IL-6, involved in the regulation of inflammatory skin processes. As IL-6 seems to play a key role in the pathogenesis of psoriasis, the beneficial effects of corticosteroids and calcitriol in this disease may partly be explained by their ability to inhibit HDMEC-derived IL-6 production.

Acitretin↗

PubMed1992

[Granulocyte colony stimulating factor (G-CSF) in treatment of patients with HIV-associated mucocutaneous Kaposi sarcoma. Successful use in virus and drug-induced leukopenia].

Three patients with HIV-associated Kaposi sarcoma were treated with human recombinant granulocyte colony stimulating factor (G-CSF). They had all developed leucopenia during treatment with recombinant interferon-alpha-2a, in two cases combined with vincristine. In all three patients, there was an obvious rapid stimulation after s.c. injection of 300 or 150 micrograms G-CSF per day; the white blood count reached normal values within only a few days and partial transformation to leucocytosis took place. After discontinuation of G-CSF, leucocyte counts regressed rapidly to pretreatment levels. A dose of 150 micrograms of G-CSF twice to three times per week proved to be sufficient to keep the white blood cell count in the normal range allowing the treatment necessary for Kaposi sarcoma. G-CSF therapy had no serious side effects. One of the patients developed a tumour-like infiltration in his left upper jaw, which histologically simulated Burkitt's lymphoma and which regressed spontaneously after discontinuation of the G-CSF therapy. G-CSF plays an important role in the treatment of patients with HIV-associated Kaposi sarcoma and enables combined treatment with zidovudine, interferon, and cytostatic drugs.

Adult↗

PubMed1992

[Linear hyperpigmentation caused by bleomycin].

We report on an uncommon but characteristic cutaneous side-effect of bleomycin. A 52-year-old woman being treated for carcinoma of the cervix developed linear hyperpigmentation in wheals in the lumbosacral region, the lateral thorax and above the elbow. The skin lesions appeared during the fourth cycle of chemotherapy with bleomycin. Histologically, incontinence of melanin, focal parakeratosis and a lymphocytic infiltrate with epidermotropism were prominent. By electron microscopic examination metabolically highly active melanocytes were found, with increased number of melanosomes at all stages of maturation and deposits of extracellular melanin in the underlying dermis. The epidermal keratinocytes were unchanged.

Antineoplastic Agents↗

PubMed1992

[Mechanism of the interaction of leukocytes and dermal endothelial cells in cutaneous inflammation].

The infiltration of specific leucocyte populations into inflamed cutaneous tissue is dependent on their interactions with dermal endothelial cells. The dynamic multistep process of vasodilatation, adhesion, transendothelial migration, and penetration through the basal membrane is controlled by a considerable number of soluble mediators and cell adhesion molecules. In this process, dermal endothelium plays a critical role as a gate-keeper who regulates adhesion and penetration of neutrophils, lymphocytes, or monocytes by selection and activation of specific leucocyte subpopulations. In this review, the presently known functions of the involved cell adhesion molecules of the integrin, selectin, and immunoglobulin superfamily, as well as their interactions and their regulation by cytokines and other mediators are summarized.

Cell Adhesion Molecules↗

PubMed1991

Cultivation of human dermal microvascular endothelial cells in vitro: immunocytochemical and ultrastructural characterization and effect of treatment with three synthetic retinoids.

A new reliable and reproducible technique to culture endothelial cells from the small vessels and capillaries of human skin is introduced, and proliferation and differentiation of the growing cells are characterized. The endothelial origin of the culture cells was confirmed by light- and electron microscopy and by labelling with Ulex europaeus Agglutinin I and an antibody against Factor VIII-related antigen. Further immunocytochemical characterization showed that 92-100% of the cells were positive for beta 2-microglobulin and the entire cell population expressed vimentin, whereas cytokeratins, desmin, HLA-DR antigen, Leu 6 and S 100 protein, could not be detected. As vascular endothelium is a common site of inflammation and retinoids have been shown to be of good clinical efficacy in some chronic inflammatory skin diseases, we investigated the influence of etretinate, etretin and isotretinoin on proliferation and antigen expression of our culture cells. All retinoids applied inhibited proliferation of endothelial cells in a dose- and time-dependent manner whereas they induced neither HLA-DR nor intercellular adhesion molecule-1 (ICAM-1). Furthermore, none of the retinoids applied influenced the gamma-interferon-induced expression of these surface antigens on endothelial cells. Our results suggest that the action of retinoids in skin inflammation is not mediated by modulation of HLA-DR or ICAM-1. The cell culture technique described here is an interesting and reliable model for studying the influence of drugs on endothelial cell growth and differentiation in vitro.

Acitretin↗

PubMed1991

A case of classical mycosis fungoides associated with human T-cell lymphotropic virus type I.

A 72-year-old male patient from north-eastern Iran developed the typical clinical and histopathological features of mycosis fungoides with lymphadenopathy, but without any other systemic involvement. Human T-cell lymphotropic virus (HTLV-I) antibodies were detected in the patient's serum by two different ELISAs and by Western blot using purified viral particles from MT-2 culture supernatants. Cultured peripheral blood lymphocytes were positive for labelling with anti-HTLV-I serum. Southern blot hybridization of DNA extracted from a skin tumour and from an involved lymph node revealed integrated proviral DNA with identical restriction patterns. This case supports a relationship between mycosis fungoides and HTLV-I and may indicate a new region of endemic HTLV-I infection.

Aged↗

PubMed1991

Culture of human sebocytes and markers of sebocytic differentiation in vitro.

Human sebocytes obtained as explants after in vitro culture of isolated sebaceous glands were recently shown to maintain in part a sebocytic differentiation. The aim of this study was to further identify markers of sebocytic differentiation in vitro. Therefore, the morphology of cultured human sebocytes, and their differentiation with lipid storing and expression of cellular proteins were investigated by microscopy, electron microscopy, study of cell kinetics, cytochemistry and immunocytochemistry, and were compared to cultured human keratinocytes obtained from the same skin specimens. At first, sebocytes in all stages of sebocytic differentiation were detected in vitro. Abundant cytoplasmic lipids and the absence of desmosomes were identified as their ultrastructural characteristics. Secondly, an increasing number of sebocytes storing lipids was detected during cell proliferation. Sebocytes contained up to 4 times more lipids than keratinocytes in vitro. Squalene and increased quantities of wax/sterol esters could be extracted from secondary sebocyte cultures. Thirdly, the monoclonal antibodies 6B10 (keratin 4), RPN1162 (keratin 7), and OM-1 labeled only sebocytes in vitro. Furthermore, sebocytes presented a marked expression of keratin 19 in comparison to keratinocytes, as detected with CK 4.62, and a lack of RPN1161 (keratins 1 and 2) expression, which was typically found to be expressed in cultured keratinocytes. The culture of human sebocytes possessing several characteristics of sebocytic differentiation in vivo offers unique possibilities in investigating direct effects on sebaceous cell growth, differentiation and their regulation.

Antibodies, Monoclonal↗

PubMed1990

Tumor necrosis factor-alpha inhibits cell proliferation and induces class II antigens and cell adhesion molecules in cultured normal human keratinocytes in vitro.

The effects of recombinant human tumor necrosis factor-alpha (TNF) on cell proliferation, cell morphology, and on the expression of class II alloantigens and intercellular adhesion molecule-1 (ICAM-1) were assessed in human keratinocytes cultured in serum-free medium. TNF inhibited cell proliferation in a time- and dose-dependent manner with a minimum effective dose of 10 U/ml and a 50% inhibitory dose of 100 U/ml. However, TNF did not induce cell death, and the growth inhibition induced by TNF was completely reversible after its withdrawal. In vitro combination of TNF with interferon-alpha (IFN-alpha) and IFN-beta resulted in additive growth inhibitory effects, while IFN-gamma enhanced the TNF mediated growth inhibition in a synergistic way. Furthermore, TNF altered the morphology of the growing keratinocytes inducing the appearance of a fusiform, fibroblast-like population. Also, treatment with TNF over 6 days markedly induced the expression of ICAM-1 on the cultured keratinocytes with a minimal effective dose of 10 U/ml, while HLA-DR was only moderately expressed after 1,000 U/ml. TNF did not induce HLA-DQ, but reduced the IFN-gamma induced expression of HLA-DR and HLA-DQ. By immunoelectron microscopy, an intense membrane-bound labeling for ICAM-1 was found after treatment with TNF, clearly pronounced in areas of microvillous membrane protrusions. These results indicate that epidermal keratinocytes are a major target for various biological effects of TNF. We also found that TNF differentially modulates IFN-gamma-induced effects, thus suggesting its potential role in the regulation of inflammatory skin disorders.

Cell Adhesion Molecules↗

PubMed1990

Effects of rIFN alpha, beta, and gamma on the morphology, proliferation, and cell surface antigen expression of human dermal microvascular endothelial cells in vitro.

The influence of recombinant human interferon alpha 2a (rIFN alpha), recombinant human interferon beta 1 (rIFN beta), and recombinant human interferon gamma (rIFN gamma) on human dermal microvascular endothelial cells (HDMEC) cultured in vitro was studied in various rIFN concentrations (0.1 IU/ml-10(4) IU/ml) over 2, 3, 4, 6, 8, and 10 d. Cell morphology and ultrastructure, cell proliferation, expression of class II alloantigens (HLA-DR and HLA-DQ), and intercellular adhesion molecule-1 (ICAM-1) were investigated using an in vitro technique established in our laboratory. All rIFN tested induced alterations of typical HDMEC morphology; the cells became spindle-shaped and fibroblastoid, although they maintained their endothelial cell marker expression. Also, all IFN dose- and time-dependently inhibited the proliferation of HDMEC in vitro (rIFN alpha greater than beta greater than gamma), whereby rIFN alpha exerted the strongest growth-inhibitory effect. Alkaline phosphatase anti-alkaline phosphatase (APAAP) immunocytochemistry of the cultured cells showed dose- and time-dependent stimulation of ICAM-1 and class II antigen expression only by rIFN gamma (HLA-DR greater than HLA-DQ), rIFN alpha and beta did not exert any immunomodulatory activity on HDMEC in vitro. These results indicate that HDMEC are an important target for the action of IFN. Besides growth inhibition, it seems that rIFN gamma in particular may be involved in the modulation of leucocyte adhesion and trafficking by altering the immunophenotype of the endothelial cell population.

Antigens, Surface↗

PubMed1990

Effects of recombinant tumor necrosis factor-alpha on cultured microvascular endothelial cells derived from human dermis.

We investigated the effects of recombinant human tumor necrosis factor-alpha (TNF) on cell proliferation and on expression of MHC class II antigens and intercellular adhesion molecule ICAM-1 in human dermal microvascular endothelial cells (HDMEC) derived from human foreskin. Second-passage HDMEC were treated with 0.1-10,000 U/ml TNF for up to 6 d, and cell growth was assessed by cell counts and a recently developed fluorogenic assay using 4-methylumbelliferyl heptanoate as a substrate. APAAP immunocytochemistry was performed using monoclonal antibodies against HLA-DR, HLA-DQ, and ICAM-1. TNF did not markedly inhibit the growth of HDMEC but induced expression of HLA-DR (1,000 U/ml and more) and of ICAM-1 (1 U/ml and more). Combination with interferon-gamma led to synergistic ICAM-1 induction. These results demonstrate a profound effect of TNF on the activation of dermal microvascular endothelial cells and suggest a major role of TNF in the mediation of leucocyte adhesion to endothelial cells of the skin microvasculature with possible implications for the initiation and maintenance of inflammatory skin processes.

Cell Adhesion Molecules↗

PubMed1990

Initial hyperproliferation and incomplete terminal differentiation of cultured human keratinocytes from lesional and uninvolved psoriatic skin.

Human epidermal keratinocytes (KCs) were isolated from lesional and from uninvolved skin of 8 patients with chronic plaque-like psoriasis and from the normal skin of 8 healthy volunteers. Primary KC cultures, grown on 3T3 cell feeder layers, were examined over a period of 4 weeks and their plating efficiency, colony growth area, DNA synthesis and ultrastructural cell differentiation were evaluated. Psoriatic KCs formed colonies one day earlier than non-psoriatic controls and proliferated faster during the first 2 weeks, as assessed by the mean colony growth area and 3H-thymidine incorporation. After 4 weeks, however, no significant differences were observed between the in vitro proliferation parameters of normal and psoriatic KCs. At the ultrastructural level, cultures of lesional psoriatic KCs consisted of more cell layers with adherent transitional cells and incomplete formation of cornified envelopes, even after 4 weeks, while KCs from uninvolved psoriatic skin were characterized by a transient delay of in vitro maturation. These results indicate that the characteristic hyperproliferation of psoriatic KCs may only be maintained over a short period of primary culture, whereas defective terminal differentiation of lesional psoriatic KCs was maintained throughout the culture period.

Biopsy↗

PubMed1990

[Preliminary findings on lectin binding in human follicular epithelium].

Glycoconjugates in the cell membranes of the outer and the inner root sheath of human anagen hair follicles were histochemically characterized by means of the following lectins: UEA-I, DBA, PNA, WGA, SBA, RCA, and Con A (avidin-biotin technique, counter-staining with methyl green). We observed intense labeling of the outer root sheath with UEA-I, DBA, Con A, and PNA, whereas the other markers were only weakly positive. VEA-I, DBA and SBA labeling was confiried to the upper two thirds of the outer root sheath. As to the inner root sheath, we found intense labeling with UEA-I, WGA, RCA, and SBA; all other lectins were only weakly represented. Our results suggest that the cell membranes of the outer and inner root sheaths of human hair follicles are characterized by the prevalence of different glycoconjugates; moreover, labeling with UEA-I, SBA, and DBA is obviously related to the process of differentiation within the follicle.

Cell Differentiation↗

PubMed1990

[Kyrle disease in juvenile diabetes mellitus and chronic renal failure].

We report on a 32-year-old female patient with chronic diabetes mellitus, type I, and chronic renal failure, who developed the typical clinical picture of hyperkeratosis follicularis et parafollicularis in cuteum penetrans (Kyrle's disease) within one year. Histological examination revealed a defective epidermal differentiation with hyper- and parakeratosis as well as premature keratinization as early as in the epidermal basal cell layer. Studies on lectin binding showed that the glycosylation process was impaired in both the epidermis and the basement membrane zone of the lesional skin. In addition, electron microscopic investigation revealed diabetic microangiopathy of the dermal vessels as well as marked ultrastructural alterations of the dermo-epidermal basal lamina. These findings confirm the association of diabetes mellitus with Kyrle's disease previously described; they make us suggest that Kyrle's disease might be characterized by a defective differentiation of the epidermis and the dermo-epidermal junction--due to some alteration of the underlying glycosylation processes--rather than by a local disorder of keratinization. Regarding the clinical manifestation of the disease, both diabetes mellitus and chronic renal failure may play a part as precipitating factors.

Adult↗

PubMed1989

Effects of azelaic acid on proliferation and ultrastructure of mouse keratinocytes in vitro.

The effects of azelaic acid (C9-dicarboxylic acid, AZA) on the proliferation and ultrastructure of neonatal NMRI mouse keratinocyte cultures were studied to clarify the mechanisms of AZA action on normal and diseased epidermis. The dose- and time-dependency of the drug effects on DNA synthesis was examined by 3H-thymidine incorporation into DNA and by autoradiography. Electron microscopy was used to detect the target cell organelles of the drug. Azelaic acid decreased DNA synthesis in a dose- and time-dependent manner with a 50% inhibitory concentration of 20 mM. The inhibition of DNA synthesis was already observed after 1 h of treatment, reached its maximum after 4 h, and was stable for 24 h. A complete reversibility of the inhibitory effects was observed within 2 h after discontinuation of the treatment, and, interestingly, a rebound effect occurred with a temporary increase of DNA synthesis. Furthermore, treatment with AZA reduced the RNA and protein synthesis of the cells. Electron microscopic evaluation of treated cultures showed early marked damage of the mitochondria, followed by dilation of the rough endoplasmic reticulum (RER). These alterations were completely reversible after discontinuation of the treatment. Our findings show that AZA exerts a dose- and time-dependent, reversible antiproliferative effect on keratinocytes, acting primarily on mitochondria and RER. The antiproliferative action of AZA could explain its beneficial effect in some skin disorders characterized by alteration of keratinocytic differentiation.

Animals↗

PubMed1989

A rapid fluorometric assay for the determination of keratinocyte proliferation in vitro.

In the present study we describe a simple technique for the determination of keratinocyte proliferation in vitro, based on the hydrolysis of a fluorogenic substrate by cell esterases. Normal and transformed human keratinocytes were grown in microtiter plates and were incubated with 4-methylumbelliferyl heptanoate after 3, 5, and 7 days. The fluorescence was quantified using an automatic fluorescence detection unit. The fluorescence showed a strong correlation with the cell number at various growth phases. In addition, the method reliably detected the growth inhibitory effect of recombinant interferon gamma on human keratinocytes. The fluorometric assay is a simple, fast and reliable method to assess cell number in keratinocyte cultures.

Cell Count↗

PubMed1989

Effects of azelaic acid on sebaceous gland, sebum excretion rate and keratinization pattern in human skin. An in vivo and in vitro study.

The effects of azelaic acid (AZA) on the epidermis of 47 individuals (12 with normal skin, 15 with seborrheic skin and 20 suffering from acne) and on in vitro cultured keratinocytes are reported. Topical application of a 20% AZA cream significantly improved the lesions of acne patients, but failed to induce clinically detectable changes in normal or seborrheic epidermis. Complementary investigations clearly showed that AZA treatment failed to induce specific changes in sebum composition, excretion rate, or in the size of sebaceous glands, but modified epidermal keratinization. Keratohyalin granules and tonofilament bundles were reduced in size and number, mitochondria were swollen and the rough endoplasmic reticulum of malpighian keratinocytes enlarged. The infundibular epidermis of acne individuals showed marked reduction of the horny layer thickness, widening of the horny cell cytoplasm, transitional corneal cells, normalization of filaggrin distribution, and the comedo contained few bacteria and spores. In vitro, AZA exerted marked time- and dose-dependent antiproliferative cytostatic effects on cultured keratinocytes, with a 50% inhibitory dose of 20 mM, decreased some keratinocyte proteins (highly soluble fractions S2, keratohyalin macroaggregate R2, and non-cross-linked fibrous protein S4) and a 95 kD and a 35 kD protein of the cytosolic fraction. Mitochondria were frequently damaged and the rough endoplasmic reticulum enlarged. Our results indicate that AZA is an antikeratinizing agent, displaying antiproliferative cytostatic effects on keratinocytes and modulating the early and terminal phases of epidermal differentiation.

Acne Vulgaris↗