Biomedical subjects

T Krieg

Publications and source records attributed to T Krieg.

At least 145 records · Page 8Linked to original sources

PubMed1994

Immunohistochemical demonstration of myoepithelial cells in sweat gland carcinomas.

Although myoepithelial cells are detectable in many benign sweat gland tumours, little is known about their role in sweat gland carcinomas. To specifically demonstrate myoepithelial cells, paraffin sections from 46 sweat gland carcinomas were stained, using a standard avidin-biotin-peroxidase complex method, with the monoclonal alpha-smooth muscle actin antibody 1A4. Myoepithelial cells were not found in adenoid cystic eccrine carcinoma (n = 2), malignant nodular hidradenoma (n = 2), porocarcinoma (n = 4), extramammary Paget's disease (n = 12), sclerosing sweat duct carcinoma (n = 4) or in adenosquamous-mucoepidermoid carcinoma (n = 1). In contrast, myoepithelial cells were demonstrated in two of eight apocrine adenocarcinomas, one of six mucinous eccrine carcinomas and two of seven eccrine adenocarcinomas. In all these tumours myoepithelial differentiation was found in peripheral cells of solid tumour islands, or in basal cells of tubular structures. However, in most areas of the tumours, myoepithelial layers were discontinuous. Cells in the centre of solid tumour nodules, and luminal cells of tubular structures, were negative for alpha-smooth muscle actin. In analogy to breast tumours, in which malignancy and invasiveness correlate with scattered or absent myoepithelial cells, we suggest that disrupted myoepithelial layers in sweat gland carcinomas may be interpreted as a loss of the invasion barrier.

Actins↗

PubMed1994

UVA-induced autocrine stimulation of fibroblast-derived collagenase/MMP-1 by interrelated loops of interleukin-1 and interleukin-6.

Previous work has shown that fibroblast-derived collagenase/matrix-metalloproteinase-1 (MMP-1), responsible for the breakdown of dermal interstitial collagen, was dose-dependently induced in vitro and in vivo by UVA irradiation and this induction was at least partly mediated by interleukin-6 (IL-6). We here provide evidence that UVA-induced IL-1 alpha and IL-1 beta play a central role in the induction of the synthesis both of IL-6 and collagenase/MMP-1. In contrast to the late increase of IL-1 alpha and IL-1 beta mRNA levels at 6 h postirradiation, bioactivity of IL-1 is already detectable at 1 h postirradiation. This early peak of IL-1 bioactivity appears to be responsible for the induction of IL-6 synthesis and together with IL-6 lead to an increase of the steady-state mRNA level of collagenase/MMP-1 as deduced from studies using IL-1 alpha and IL-1 beta antisense oligonucleotides or neutralizing antibodies against IL-1 alpha and IL-1 beta. Besides the early posttranslationally controlled release of intracellular IL-1, a latter pretranslationally controlled synthesis and release of IL-1 perpetuates the UV response. From these data we suggest a UV-induced cytokine network consisting of IL-1 alpha, IL-1 beta and IL-6, which via interrelated autocrine loops induce collagenase/MMP-1 and thus may contribute to the loss of interstitial collagen in cutaneous photoaging.

Aging↗

PubMed1993

Purification of a novel factor which binds to the mouse alpha 2 (I) collagen promoter.

We have identified and purified a DNA binding protein which specifically binds to a segment of the mouse alpha 2 (I) collagen promoter between -420 and -399 bp upstream of the start of transcription. Purification included heparin-agarose and sequence-specific DNA-affinity chromatography, followed by SDS-PAGE and renaturation of the DNA binding activity after elution from SDS-polyacrylamide gel. The DNA binding activity resides in two species of 42 kDa and 40 kDa, respectively. The levels of DNA binding activity of this factor, which has been tentatively designated as ColF1, are considerably higher in nuclear extracts of NIH-3T3 fibroblasts than in nuclear extracts from epidermal cells, lymphoid cells and transformed NIH-3T3 fibroblasts.

3T3 Cells↗

PubMed1993

Inhibition of skin development by overexpression of transforming growth factor beta 1 in the epidermis of transgenic mice.

To assess the effect of transforming growth factor beta 1 on the skin in vivo, we have targeted its expression to the epidermis of transgenic mice. To ensure that active TGF-beta 1 was expressed, we used a porcine TGF-beta 1 cDNA with mutations of Cys-223-->Ser and Cys-225-->Ser, which allow constitutive activation. Mice expressing the mutant transforming growth factor beta 1 transgene exhibited a marked phenotype at birth. The skin was very shiny and tautly stretched. These animals were rigid and appeared to be restricted in their ability to move and breathe; death occurred within 24 hr. Histologically, the most prominent features of the skin were a compact orthohyperkeratosis and a reduction in the number of hair follicles. Pulse-labeling studies with 5-bromodeoxyuridine demonstrated a marked reduction in the number of replicating cells in the epidermis and hair follicles. Thus, the macro- and microscopic appearance of these mice, as well as their neonatal lethality, most likely result from inhibition of normal skin development and suppression of epithelial cell proliferation by the overexpression of transforming growth factor beta 1.

Amino Acid Sequence↗

PubMed1993

Mapping of nidogen binding sites for collagen type IV, heparan sulfate proteoglycan, and zinc.

Recombinant nidogen fragments comprising the globular domains G1 plus G2, the rod-like domain, and the rod connected to the globe G3 were prepared from the culture media of transfected human cell clones. In addition, domains G1 and G2 were separated from each other after cleavage with chymotrypsin. The purified fragments were characterized by N-terminal sequences, electrophoresis, electron microscopy, and radioimmunoassays and the cell clones by Northern hybridization. Transfection with a construct comprising a large part of domain G3 showed high mRNA levels but no secreted protein, indicating a protein folding problem. All these fragments were used as soluble and/or immobilized ligands in binding assays. This demonstrated major binding sites on domain G2 for collagen IV and heparan sulfate proteoglycan. Affinity chromatography on zinc- and cobalt-loaded columns showed binding of domains G2 and G3 and the rod. Protein binding, but not metal binding, was abolished by reduction and alkylation of nidogen. This allowed for the isolation of several zinc-binding tryptic peptides, four from G2, two from the rod, and one from the G3 domain. Most of these short peptides contained several histidines that are likely to mediate binding. Zinc inhibited efficiently G3-mediated nidogen binding to laminin at 4 degrees C (IC50 approximately 5 microM) but less at higher temperatures. Similarly, zinc inhibited binding to collagen IV and proteoglycan at low temperatures but not at high (37 degrees C) temperatures. This indicates a complex modulation of nidogen binding to other basement membrane proteins by some, but not all, transition metals. Whether the particularly striking effects shown for zinc are of biological relevance remains to be established.

Amino Acid Sequence↗

PubMed1993

Downregulation of collagen synthesis in fibroblasts within three-dimensional collagen lattices involves transcriptional and posttranscriptional mechanisms.

Culturing human fibroblasts in a three-dimensional collagen matrix leads to a reduction of collagen I by more than 90%, both on the level of mRNA steady-state as well as protein. In order to differentiate changes in de novo transcription and posttranscriptional control, nuclear run on assays and pulse/chase experiments determining mRNA stability were used. Our results indicate that de novo transcription of the COL1A1 gene and pro-alpha 1 (I)collagen mRNA half-life are both decreased by 50% in fibroblasts grown in three-dimensional collagen lattices as compared to monolayer cultures. The extracellular matrix therefore elicits signals which are transduced from the cell surface to the inside of fibroblasts resulting in a specific reprogramming of transcriptional as well as posttranscriptional processes.

Cells, Cultured↗

PubMed1993

Control of fibrosis in systemic scleroderma.

Scleroderma is characterized by an excessive deposition of collagen in all involved organs. This is due to an overproduction of extracellular matrix (ECM) molecules following induction of gene expression, whereas there is no evidence that the composition of the connective tissue matrix is altered. Several in vivo studies and in vitro experiments suggest that a close interaction between inflammatory cells and fibroblasts is required for the initial activation of fibroblasts. TGF-beta presumably plays an important role, but other cytokines, e.g., PDGF or FGF, may also be involved. Many of the ECM molecules have been shown to interact closely with fibroblasts and provide signals that regulate fibroblast metabolism. The cellular response towards those signals is a further aspect of fibrosis that has attracted attention during recent years. The altered expression of receptor proteins on the cell surface of scleroderma fibroblasts for example might explain in part the lack of down-regulation of collagen synthesis in late phases of the disease. This review summarizes the alterations of connective tissue in scleroderma, and discusses the role of cytokines as well as the ECM for the regulation of fibroblast function and their implication for the development of fibrosis.

Cells, Cultured↗

PubMed1993

Expression of type VI collagen mRNA during wound healing.

During the highly regulated process of wound healing the expression of the interstitial collagens I and III is increased in a time-dependent fashion. Although ultrastructural and in vitro studies suggest a physiologic role of collagen VI in the organization of extracellular matrix deposition, nothing is known about its role in wound healing. Therefore, we studied collagen VI gene expression during wound healing in humans compared to that of collagens I and III. The presence of specific alpha 1(VI) and alpha 3(VI) mRNA species in scar tissue was demonstrated by Northern blot analysis. Quantification of mRNA expression by dot blot analysis and in situ hybridization indicated that like for the interstitial collagens I and III collagen VI gene expression was increased during wound healing, reaching its maximum 2 weeks after initial insult. In the late phase of wound healing like alpha 1(I) the alpha 1(VI) gene expression was not down regulated significantly. In contrast, a reduction of alpha 3(VI) collagen gene expression was observed, as was for the alpha 1(III) collagen gene, indicating a non-coordinate regulation of these chains. Collagen VI gene expression could be localized to fibroblast-like cells and to endothelial cells of newly formed vessels. Collagen VI gene expression was undetectable in smooth muscle cells and myoepithelial cells of eccrine glands. These results indicate that collagen VI gene expression is regulated in a time-dependent fashion and that fibroblasts and endothelial cells appear to play an important role in collagen VI synthesis during wound healing.

Collagen↗

PubMed1993

Biphasic effects of interleukin-1 alpha on dermal fibroblasts: enhancement of chemotactic responsiveness at low concentrations and of mRNA expression for collagenase at high concentrations.

Fibrotic reactions in the skin are frequently preceded by infiltration of inflammatory cells and subsequent migration of fibroblastic cells. Interleukin-1 is secreted by inflammatory cells and can regulate proliferation and protein synthesis of fibroblasts. Its role in fibroblast chemotaxis has not been elucidated in any detail. Using the well-established Boyden chamber assay for measurement of chemotaxis in vitro, we studied a wide range of recombinant human interleukin-1 alpha concentrations to assess intrinsic chemotactic activity of interleukin-1 alpha and to determine the capacity of this mediator to modify the chemotactic response of fibroblasts to other chemoattractants. This was compared with the interleukin-1 alpha dose required for enhancement of mRNA expression for collagenase. Although interleukin-1 alpha was not chemoattractive for fibroblasts, it specifically augmented migration toward fibroblast-conditioned medium and toward platelet-derived growth factor but not toward epidermal growth factor, fibronectin, or transforming-growth factor-beta. Interleukin-1 alpha did not measurably alter the expression of mRNA for the platelet-derived growth factor receptor or its platelet-derived growth factor-binding characteristics. Doses required to enhance fibroblast chemotaxis were distinctly lower than those required for stimulation of collagenase mRNA expression.

Cell Division↗

PubMed1993

Keratin pattern of acanthosis nigricans in syndromelike association with polythelia, polycystic kidneys, and syndactyly.

BACKGROUND: Acanthosis nigricans (AN) comprises a broad spectrum of etiologic subtypes. The underlying pathomechanisms have not yet been completely clarified. We present a patient affected with a syndromelike AN subtype including disturbed epidermopoiesis as evidenced by immunohistologic findings and in situ hybridization. OBSERVATIONS: A 54-year-old white man contracted AN during childhood. There were connate malformations consisting of webbed toes II/III on the right side and a supernumerary left mammilla. As an adult he developed psoriasis vulgaris, obesity, and latent diabetes mellitus, polycystic kidney and liver disease. With regard to keratin 6 mRNA, and the protein expression of keratin 6/16, KI-67, and proliferating cell nuclear antigen, the AN lesion showed moderate hyperproliferation. A much higher degree of hyperproliferation was evident in psoriatic areas of the patient's skin. In contrast to psoriatic tissue, basal keratinocytes of the AN showed an unusually high expression of keratin 18 and 19 protein. CONCLUSIONS: The observation thus deals with a unique, syndromelike constellation of AN characterized by a particular epidermal pattern of moderate hyperproliferation. A further dysregulation of protein expression in the epidermis is indicated by the demonstration of the rare keratins 18 and 19 in basal keratinocytes of the AN lesion.

Abnormalities, Multiple↗

PubMed1992

Effect of transforming growth factor-beta on collagen type VI expression in human dermal fibroblasts.

Steady-state mRNA levels and protein synthesis of collagen type VI were determined after stimulation of human dermal fibroblasts with transforming growth factor-beta (TGF beta). While there was a 227% increase in the alpha 3(VI) subunit mRNA at maximal TGF-beta concentration, alpha 1(VI) and alpha 2(VI) subunit mRNA levels remained unchanged. Concomitantly collagen type VI immuno-reactive material increased up to 172% of controls in cell culture medium and cell layer extracts. Regulation of alpha 3(VI) gene expression is therefore critical for the control of collagen type VI synthesis and determines the deposition of collagen type VI heterotrimeric molecules.

Blotting, Northern↗

PubMed1992

Interleukin-6 expression by fibroblasts grown in three-dimensional gel cultures.

We investigated the expression and biological activity of interleukin-6 (IL-6) by human fibroblasts cultured as monolayers and within three-dimensional type I collagen lattices. In the course of contracting the gel to a dense tissue-like structure, the cells upregulated their levels of IL-6 mRNA as well as IL-6 biological activity. While there was little mRNA and protein activity (6,500 U/ml) in monolayer cultures, fibroblasts in the 3D system showed a 13-fold increase in IL-6 mRNA on day 3. IL-6 protein was increased 6-fold (38,000 U/ml) on day 4. Stimulation of fibroblast cultures with IL-1 alpha resulted in enhanced IL-6 production in both systems, but the fibroblasts embedded into the 3D network continued to exhibit higher levels.

Blotting, Northern↗

PubMed1992

In situ localization of IgG in epidermolysis bullosa acquisita by immunogold technique.

BACKGROUND: Epidermolysis bullosa acquisita (EBA) is an immunologically mediated mechanobullous dermatosis. Recently, serum antibodies in patients with EBA have been reported to bind to the carboxyl terminus of type VII collagen, the disease antigen. However, the precise localization of immunoglobulins in diseased skin has not been demonstrated. OBJECTIVE: In the present study skin samples taken from a 59-year-old man with EBA were processed for immunoelectron microscopy. METHODS: A preembedding immunogold ultrastructural technique was applied. RESULTS: Gold particles, indicative of IgG binding, were deposited within the anchoring plaques and within the lamina densa in some perilesional skin sections. In a similar distribution, IgG-containing amorphous aggregates were detected within the basement membrane zone of perilesional and lesional skin. In some places these immunoglobulin deposits covered the whole region of anchoring fibrils. CONCLUSION: These results demonstrate the precise localization of IgG within the basement membrane zone of EBA skin that corresponds to the carboxyl terminus of collagen VII in normal skin.

Diagnosis, Differential↗

PubMed1992

Increased adhesion of fibroblasts from patients with scleroderma to extracellular matrix components: in vitro modulation by IFN-gamma but not by TGF-beta.

A characteristic feature of systemic scleroderma is fibrosis of the skin and eventually of internal organs resulting from an overproduction of collagen and other connective tissue components by the resident fibroblasts. The balance between the cells and the amount of the surrounding extracellular matrix is then altered. Because cellular metabolism depends to a large extent on cellular contacts and communications with connective tissue molecules, we have therefore investigated the interactions with extracellular matrix components of fibroblasts obtained from skin of patients affected with scleroderma. In comparison to fibroblasts from healthy skin, all fibroblasts from scleroderma patients had an increased adhesion capacity to collagens I, IV, VI, fibronectin, and laminin. In addition, whereas adhesion of control fibroblasts was stimulated by a pre-treatment with transforming growth factor-beta, adhesion patterns of scleroderma fibroblasts remained unchanged. However, pre-incubation of the cells with interferon-gamma decreased the adhesion of both scleroderma and control fibroblasts.

Cell Adhesion↗

PubMed1992

The effect of interferon-gamma on the invasiveness of HT-180 cells.

It has been shown that tumour necrosis factor-alpha (TNF-alpha) induces the gene expression of collagenase and enhances the invasiveness of many cell types. However, we have previously demonstrated that interferon-gamma (IFN-gamma) induces the chemotactic response of cells and we have studied the in vitro effects of both cytokines on invasive migration using a human fibrosarcoma cell line (HT-1080). Invasive migration occurred with HT-1800 cells through a basement membrane equivalent (matrigel) and collagen type I gel. Pre-incubation of cells with increasing concentrations of IFN-gamma resulted in a dose-dependent reduction of this invasive migration. TNF-alpha considerably enhanced the invasiveness of HT-1080 cells and of fibroblasts. This effect could be significantly diminished by the pre-incubation of cells with IFN-gamma. Inhibition of invasiveness did not appear to be due to an altered binding to the barriers or altered collagenolytic activity of these cells, as shown by attachment and collagenase assays. These data support the concept that IFN-gamma can reduce the invasiveness of transformed cells which contributes to its in vivo anti-neoplastic effect.

Cell Adhesion↗

PubMed1992

Ultrastructural immunogold studies in two cases of linear IgA dermatosis. Are there two distinct types of this disease?

It has been suggested that patients with homogeneous linear IgA deposits at the basement membrane zone constitute a distinct bullous disorder called linear IgA dermatosis (LAD) of adults or children. The results of the present ultrastructural immunogold study in two patients with LAD suggest that LAD is not a single disease entity. LAD in a 10-year-old girl was found to be ultrastructurally similar to an IgA-type pemphigoid. IgA was detected in the uppermost lamina lucida underlying the basal cell plasma membrane. In a second patient, an 86-year-old man, IgA deposits were present within the lamina densa and the anchoring plaques. The distribution of IgA in this patient was ultrastructurally identical with that of IgG in epidermolysis bullosa acquisita skin and with that of the non-collagenous globular terminus of collagen VII within the basement membrane zone of normal skin. By using the immunogold technique, we could distinguish two distinct types of LAD according to the IgA binding sites in the diseased skin. We suggest that different labelling patterns may correspond to different clinical pictures.

Aged↗

PubMed1992

Treatment of systemic sclerosis with gamma-interferon.

Numerous drugs have been recommended for the treatment of systemic sclerosis, but without any significant effect on the fibrotic stage of this disorder. Because recombinant gamma-interferon (gamma-IFN) is a potent and selective inhibitor of fibroblast proliferation and collagen production by human dermal fibroblasts in vitro, we assessed the effects of gamma-IFN treatment on the skin and on pulmonary function in patients with systemic sclerosis. Fourteen patients entered the study, and nine completed the 12-month trial. Fifty micrograms/day of gamma-IFN was administered subcutaneously 3 days per week. At the end of the 12-month treatment period a significant improvement was observed in total skin score, and blood gas analysis showed a significant increase in Pa O2 during therapy with gamma-interferon. Other clinical parameters (dysphagia, Raynaud's phenomenon, cardiac involvement) were not altered significantly. No serious adverse effects were noted. These results suggest a beneficial effect of gamma-IFN on the cutaneous fibrotic abnormalities and on lung fibrosis in systemic sclerosis.

Adult↗

PubMed1992

Altered regulation of collagen metabolism in scleroderma fibroblasts grown within three-dimensional collagen gels.

In systemic scleroderma (SSc) excessive deposition of collagen leads to fibrosis of various tissues including the skin. Previous studies have demonstrated that scleroderma fibroblasts in explant monolayer cultures are heterogeneous with respect to their levels of collagen synthesis. The critical role played by the extracellular matrix (ECM) in the modulation of fibroblast metabolism prompted us to study the regulation of collagen synthesis in scleroderma fibroblasts grown within three-dimensional collagen gels, a culture system representing more physiological conditions than monolayer cultures. Normal fibroblasts grown in this system dramatically reduce their collagen synthesis as compared to monolayer cultures. Quantification of total protein and collagen synthesis showed that scleroderma fibroblasts did not demonstrate the down regulation of collagen synthesis as observed in control fibroblasts, resulting in a much higher collagen synthesis in scleroderma fibroblasts compared to controls. However, also in this system scleroderma fibroblasts were heterogeneous in their response to the collagenous lattice. Ten strains were investigated, of which 3 were indistinguishable from controls, while 7 maintained higher levels of collagen production. In addition, our data showed that the changes in collagen synthesis on the protein level were accompanied by respective up- or downregulation on the mRNA level. These results indicate that an altered response to the surrounding ECM is an important factor in the disturbed regulation of connective tissue synthesis in scleroderma fibroblasts observed in vivo.

Adult↗