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Biomedical subjects

T Krieg

Publications and source records attributed to T Krieg.

At least 73 records · Page 4Linked to original sources
PubMed2000

The absence of nidogen 1 does not affect murine basement membrane formation.

Nidogen 1 is a highly conserved protein in mammals, Drosophila melanogaster, Caenorhabditis elegans, and ascidians and is found in all basement membranes. It has been proposed that nidogen 1 connects the laminin and collagen IV networks, so stabilizing the basement membrane, and integrates other proteins, including perlecan, into the basement membrane. To define the role of nidogen 1 in basement membranes in vivo, we produced a null mutation of the NID-1 gene in embryonic stem cells and used these to derive mouse lines. Homozygous animals produce neither nidogen 1 mRNA nor protein. Surprisingly, they show no overt abnormalities and are fertile, their basement membrane structures appearing normal. Nidogen 2 staining is increased in certain basement membranes, where it is normally only found in scant amounts. This occurs by either redistribution from other extracellular matrices or unmasking of nidogen 2 epitopes, as its production does not appear to be upregulated. The results show that nidogen 1 is not required for basement membrane formation or maintenance.

Animals↗
PubMed2000

Impaired wound healing in embryonic and adult mice lacking vimentin.

It is generally assumed that the vimentin intermediate filament network present in most mesenchymally-derived cells is in part responsible for the strength and integrity of these cells, and necessary for any tissue movements that require the generation of significant tractional forces. Surprisingly, we have shown that transgenic KO mice deficient for vimentin are apparently able to undergo embryonic development absolutely normally and go onto develop into adulthood and breed without showing any obvious phenotype. However, fibroblasts derived from these mice are mechanically weak and severely disabled in their capacity to migrate and to contract a 3-D collagen network. To assess whether these functions are necessary for more challenging tissue movements such as those driving in vivo tissue repair processes, we have analysed wound healing ability in wild-type versus vimentin-deficient embryos and adult mice. Wounds in vimentin-deficient adult animals showed delayed migration of fibroblasts into the wound site and subsequently retarded contraction that correlated with a delayed appearance of myofibroblasts at the wound site. Wounds made to vimentin-deficient embryos also failed to heal during the 24 hour culture period it takes for wild-type embryos to fully heal an equivalent wound. By DiI marking the wound mesenchyme and following its fate during the healing process we showed that this impaired healing is almost entirely due to a failure of mesenchymal contraction at the embryonic wound site. These observations reveal an in vivo phenotype for the vimentin-deficient mouse, and challenge the dogma that key morphogenetic events occurring during development require generation of significant tractional forces by mesenchymal cells.

Age Factors↗
PubMed1999

Tissue inhibitor of matrix metalloproteinase-2 regulates matrix metalloproteinase-2 activation by modulation of membrane-type 1 matrix metalloproteinase activity in high and low invasive melanoma cell lines.

Activation of pro-matrix metalloproteinase (MMP)-2 on the surface of malignant cells by membrane-bound MT1-MMP is believed to play a critical role during tumor progression and metastasis. In this study we present evidence that MT1-MMP plays a key role for the in vitro invasiveness of malignant melanoma. Melanoma cell lines secreted latent MMP-2 when cultured on plastic. However, when cells were grown in floating type I collagen lattices, only high invasive melanoma cells activated proMMP-2. Activation could be inhibited by antibodies against MT1-MMP, by addition of recombinant tissue inhibitor of metalloproteinases (TIMP)-2 and by inhibition of MT1-MMP cleavage. MT1-MMP protein was detected as an inactive protein in all cell lines cultured as monolayers, whereas in collagen gels, active MT1-MMP protein was detected in the membranes of both high and low invasive melanoma cells. Production of TIMP-2 was about 10-fold higher in low invasive cells as compared with high invasive melanoma cells and was further increased in the low invasive cells upon contact to collagen. Thus, in melanoma cells TIMP-2 expression levels might regulate MT1-MMP-mediated activation of proMMP-2. High invasive melanoma cells displayed increased in vitro invasiveness, which was inhibited by TIMP-2. These data indicate the importance of these enzymes for the invasion processes and support a role for MT1-MMP as an activator of proMMP-2 in malignant melanoma.

Cell Movement↗
PubMed1999

Ultraviolet-B induction of interstitial collagenase and stromelyin-1 occurs in human dermal fibroblasts via an autocrine interleukin-6-dependent loop.

Ultraviolet-B irradiation of human dermal fibroblasts has earlier been shown to induce matrix-degrading metalloproteinases, thus driving connective tissue degradation in photoaging and photocarcinogenesis. Herein, we report that Ultraviolet-B irradiation led to a dramatic increase in specific mRNA and protein levels of interstitial collagenase, stromelysin and interleukin-6. By contrast, the major tissue inhibitor of matrix-degrading metalloproteinases, TIMP-1, was unaffected. Monospecific neutralizing antibodies directed against human interleukin-6 significantly reduced the interstitial collagenase and stromelysin-1 protein levels. Taken together, our data provide the first evidence that Ultraviolet-B induction of interstitial collagenase and stromelysin-1 occurs via the synthesis and release of interleukin-6. Hence, this newly identified autocrine mechanism may contribute to dermal photodamage.

Cells, Cultured↗
PubMed1999

Selective loss of PMA-stimulated expression of matrix metalloproteinase 1 in HaCaT keratinocytes is correlated with the inability to induce mitogen-activated protein family kinases.

Many cell types, including fibroblasts and primary keratinocytes, increase matrix metalloproteinase 1 (MMP-1) production in response to agonists such as growth factors and phorbol esters. However, the spontaneously transformed human keratinocyte cell line HaCaT, although it increases MMP-1 production in response to epidermal growth factor (EGF), does not respond similarly to stimulation with PMA. This phenomenon occurs even though HaCaT cells remain proliferatively responsive to both agonists, suggesting a HaCaT-specific defect in a PMA-mediated signal transduction pathway. Using an inside-out approach to elucidate the source of this defect, we found that EGF, but not PMA, stimulated MMP-1 promoter activity in transiently transfected HaCaT keratinocytes. In addition, an assessment of fibroblast and HaCaT c-fos and c-jun gene expression after exposure to EGF and PMA showed that although both agonists increased the expression of c-fos and c-jun mRNA in fibroblasts, only EGF did so in HaCaT keratinocytes. Finally, we looked at the activation of mitogen-activated protein (MAP) family kinases after stimulation with EGF or PMA and found that both agonists increased the phosphorylation and activation of fibroblast extracellular signal-regulated protein kinase and c-Jun N-terminal kinase, but only EGF activated the same kinase activities in HaCaT cells. Further, the EGF-mediated increase in MMP-1 gene expression was inhibited by the MAP kinase/ERK kinase (MEK)-specific inhibitor PD98059 and the p38 kinase-specific inhibitor SB203580. Our evidence indicates that although HaCaT MAP kinases are functional, they are not properly regulated in response to the activation of protein kinase C, and that the defect that bars HaCaT MMP-1 expression in response to stimulation with PMA lies before MAP kinase activation.

Calcium-Calmodulin-Dependent Protein Kinases↗
PubMed1999

Interactions of fibroblasts with the extracellular matrix: implications for the understanding of fibrosis.

The cellular organization and the compartmentalization in multicellular organisms is mediated by the extracellular matrix (ECM). This structure is composed by a wide variety of different macromolecules which carry distinct domains with defined structural and/or biological activities. Cells are known to interact with these molecules via specific receptors. Following activation, these receptors transduce signals either directly to the intracellular cytoskeleton or via different signalling cascades. Cell-matrix interactions, therefore, not only control the shape and orientation of cells but can also directly regulate cellular functions, including migration, differentiation, proliferation, and the expression of different genes. These cell-matrix interactions have been elucidated in detail for several biological processes, especially morphogenesis and differentiation, but also play an important role during pathological situations, e.g. wound healing and tumor progression. Although much less investigated, similar mechanisms are thought to regulate the biological behavior of fibroblastic cells, the final target cells in fibrosis. The activity of these cells depends in various ways on the presence of ECM molecules. First, some of the molecules are known to bind to and modulate the activity of those growth factors and cytokines, which lead to the activation of fibroblasts during the early phases of fibrosis. Second, deposition of large amounts of ECM molecules alters the environment and the mechanical load on the cells which are embedded in this matrix. Third, ECM molecules directly modulate fibroblast metabolism via certain integrin receptors. This review summarizes recent developments in all three domains. It mainly focuses on the direct role of ECM molecules in the biosynthetic activity of fibroblasts.

Animals↗
PubMed1999

Expression of TGF-beta 1, -beta 2 and -beta 3 in localized and systemic scleroderma.

Scleroderma is a generalized or localized disorder which leads to fibrosis of the affected organs. TGF-beta has been implicated as a causal agent in its pathogenesis. In mammals, TGF-beta comprises a family of three members, beta 1, beta 2 and beta 3. Since cutaneous wound healing is thought to result either in formation of a scar or in scar-free tissue regeneration, depending on the relative amounts of the beta 3 isoform, the expression of all three isoforms was studied in skin biopsies of patients with either localized or systemic scleroderma. mRNA for all three isoforms was detected in inflammatory skin areas of both disease forms, but never in sclerotic or healthy skin. Immunohistochemical analysis confirmed expression of beta1 and beta 2 proteins in inflammatory skin of patients, whereas beta 3 protein appeared to be present in the subepidermal area and also found throughout the dermis of patients and healthy dermis as well.

Adult↗
PubMed1999

The frequency of phospholipase A2 binding of basophilic granulocytes does not decrease during bee-venom-specific immunotherapy.

BACKGROUND: The major allergenic component of bee venom is phospholipase A2 (PLA2). METHODS: In this study, PLA2 was used to analyze and enrich PLA2-binding cells from peripheral blood by high gradient magnetic cell sorting. RESULTS: In normal donors, the frequency of allergen (PLA2)-binding cells among peripheral blood mononuclear cells (PBMC) as determined by flow cytometry is below 0.1%, whereas in bee-venom-allergic patients, PLA2-binding cells are readily detectable at frequencies of up to 2.3%. In severely bee-venom-allergic patients, many basophilic granulocytes are present, as defined by anti-CD9, CD25, and CD38 mAb, comprising up to 95% of the PLA2-binding cells. From blood of allergic and normal donors, about equal absolute numbers of allergen-binding CD19/21-positive B cells can be enriched. Severe anaphylactic reactions (Mueller grade IV) and failure of or adverse reactions during immunotherapy are associated with high numbers of circulating allergen-binding basophils. Interestingly, in the patients studied, the number of PLA2-binding basophilic granulocytes did not markedly change during rush immunotherapy and up to 6 months of maintenance immunotherapy. CONCLUSIONS: The specific and reproducible enrichment of PLA2-binding cells provides a new tool for the analysis and monitoring of effector cells in bee-venom-allergic patients with immediate-type hypersensitivity.

Adolescent↗
PubMed1999

Fibroblast expression of collagen integrin receptors alpha1beta1 and alpha2beta1 is not changed in systemic scleroderma.

The skin of patients with systemic scleroderma (SSc) is characterized by excessive extracellular matrix deposition in the dermis. As collagens represent the major structural component, we used fluorescence-activated cell sorter analysis to study the levels of collagen receptors expressed at the surface of fibroblasts derived from involved skin areas. In contrast to previous reports, no differences in the expression of alpha1, alpha2 or beta1 integrin subunits, which constitute the major collagen receptors on fibroblasts, were detected on SSc fibroblasts as compared with normal control fibroblasts. Variation of cell culture conditions, e. g. passage number (from 2 to 10), seeding density, cell cycle or serum concentration, did not change this result. These observations indicate that any abnormal response of SSc fibroblasts to their matrix environment is not controlled at the level of receptor expression.

Adult↗
PubMed1999

Adaptive antioxidant response of manganese-superoxide dismutase following repetitive UVA irradiation.

In response to the attack of reactive oxygen species, the skin has developed a complex antioxidant defense system including among others the manganese-superoxide dismutase (MnSOD). MnSOD dismutates the superoxide anion (O2*-) derived from the reduction of molecular oxygen to hydrogen peroxide (H2O2), which is detoxified by glutathione peroxidase to water and molecular oxygen. We have addressed the question whether MnSOD is inducible upon UVA irradiation and whether repetitive UV exposure, as practiced for the light-hardening during phototherapy of various photodermatoses, can even enhance the adaptive antioxidant response. Single exposure of four different strains of fibroblasts to UVA irradiation resulted in a dose- and time-dependent increase in specific MnSOD mRNA levels. Interestingly, repetitive UVA exposure at days 1, 2, and 3 at a dose rate of 200 kJ per m2 resulted in a 5-fold induction of specific MnSOD mRNA levels following the third UVA exposure. Similar results were obtained for MnSOD activity. This adaptive response in terms of upregulation of the antioxidant enzyme MnSOD correlates with the protection against high UV doses, if cells were preexposed to sublethal UV doses. Importantly, MnSOD substantially differed between the tested individuals in both mRNA and activity levels. Taken together, we here provide evidence for the increasing induction of MnSOD upon repetitive UVA irradiation that may contribute to the effective adaptive UVA response of the skin during light hardening in phototherapy. Interindividual differences in the inducibility of MnSOD might account for differences in the susceptibility to develop photodermatologic disorders related to photosensitivity, photoaging, and skin cancer. The molecular basis for interindividual differences in the inducibility of antioxidant enzymes remains to be elucidated.

Adaptation, Physiological↗
PubMed1999

Particle-mediated gene transfer of PDGF isoforms promotes wound repair.

Several techniques for cutaneous gene transfer have been investigated for either in vitro or in vivo applications. In the present study, we investigated whether the direct delivery of platelet-derived growth factor cDNA into skin results in improvement in tissue repair. Cutaneous transfections were carried out in rats using a particle-bombardment device (Accell). As revealed by reverse transcriptase-polymerase chain reaction, transgene expression in vivo was transient, with low level expression by day 5. When compared with wounds transfected with a control cytomegalovirus-luciferase plasmid, wounds transfected with platelet-derived growth factor A or B in the MFG vector showed a significant increase in wound tensile strength 7 and 14 d after transfection. At both time points platelet-derived growth factor A transfected wounds exhibited the highest increase in tensile strength over controls, resulting in a 3.5-fold increase at day 7 and a 1.5-fold increase at day 14. The degree of stimulation was not remarkably different between wounds transfected with platelet-derived growth factor B, which is predominantly cell associated, or a truncation mutant, platelet-derived growth factor B211, which is predominantly secreted. These findings demonstrate that in vivo gene transfer by particle bombardment can be used to improve the tissue repair response. This approach provides a robust tool to assess the biologic activity of various proteins and will aid in the development of therapeutic cutaneous gene delivery.

Animals↗
PubMed1999

Inhibition of allergic contact dermatitis to DNCB but not to oxazolone in interleukin-4-deficient mice.

The role of interleukin-4 as a regulator of immune responses in the skin is investigated with regard to the outcome of contact hypersensitivity reaction in interleukin-4-deficient BALB/C mice. In previous studies conflicting results were obtained concerning the role of interleukin-4 in contact hypersensitivity reactions supporting either a proinflammatory or rather an inhibitory function of this cytokine. Interleukin-4 deficient BALB/C mice sensitized to 2,4-dinitrochlorobenzene showed after challenge a significant reduction in magnitude and duration of the contact hypersensitivity response in comparison with wild-type mice. This attenuation was accompanied by a significant reduction of edema and cellular infiltrates in the dermis and a lacking induction of IL-10 mRNA expression in skin. Also, adoptive transfer experiments revealed that BALB/C mice failed to exhibit contact hypersensitivity after injection of lymph node cells obtained from sensitized interleukin-4 deficient mice. To examine further the role of the contact allergen used to induce the contact hypersensitivity response, mice were also sensitized and challenged with Oxazolone. Here a similar magnitude and duration of contact hypersensitivity in both the interleukin-4 deficient mice and BALB/C control mice was observed. This indicates that the contact hypersensitivity response to 2,4-dinitrochlorobenzene and Oxazolone may partly evolve on different pathways being dependent and independent of interleukin-4. Our results clearly show that the complete loss of endogenous interleukin-4 expression in BALB/C mice is associated with an impaired manifestation of contact hypersensitivity response to 2,4-dinitrochlorobenzene, implying an important proinflammatory function of this cytokine.

Animals↗
PubMed1999

Serum xylosyltransferase: a new biochemical marker of the sclerotic process in systemic sclerosis.

UDP-D-xylose:proteoglycan core protein beta-D-xylosyltransferase (EC2.4.2.26) is the initial enzyme in the biosynthesis of chondroitin sulfate and dermatan sulfate proteoglycans in fibroblasts and chondrocytes. Secretion of xylosyltransferase into the extracellular space was determined in cultured human dermal fibroblasts. A more than 6-fold accumulation of xylosyltransferase activity in cell culture supernatant was observed (day 1, 0.6 microU per 106 cells; day 9, 4.1 microU per 106 cells); however, intracellular xylosyltransferase activity remained at a constant level (0.4 microU per 106 cells). Exposure of human chondrocytes to colchicine led to a 3-fold decreased level of xylosyltransferase and chondroitin-6-sulfate concentration in cell culture. Specific xylosyltransferase activity and chondroitin-6-sulfate concentration decreased in a concentration-dependent manner and in parallel in culture medium and accumulated 5-fold in cell lysates indicating that xylosyltransferase is secreted simultaneously into the extracellular space with chondroitin sulfate proteoglycans. Xylosyltransferase activities were determined in serum samples of 30 patients with systemic sclerosis. Xylosyltransferase activities in female (mean value 1.28 mU per liter, 90% range 1.10-1.55 mU per liter) and male patients (mean 1.39 mU per liter, 90% range 1.16-1. 57 mU per liter) with systemic sclerosis were significantly increased in comparison with blood donors of a corresponding age. Furthermore, xylosyltransferase activity was correlated with the clinical classification of systemic sclerosis. Female patients with diffuse cutaneous systemic sclerosis showed higher serum xylosyltransferase activities than patients with limited systemic sclerosis. These results confirm that the increase of proteoglycan biosynthesis in sclerotic processes of scleroderma is closely related to an elevated xylosyltransferase activity in blood and demonstrate the validity of xylosyltransferase as an additional diagnostic marker for determination of sclerotic activity in systemic sclerosis.

Adult↗
PubMed1999

A disease severity scale for systemic sclerosis: development and testing.

OBJECTIVE: To develop and test a severity scale for individual organ involvements in systemic sclerosis (SSc, scleroderma). METHODS: An international study group completed the following tasks: (1) developed a glossary of terms including all pertinent variables for 9 potentially affected organ systems; (2) collected prospective data to determine the feasibility and practicality of each proposed variable; (3) revised the initial list of variables; (4) determined the association of each variable with mortality (a proxy for morbidity) using 579 patients in an existing comprehensive longitudinal scleroderma databank; (5) developed a severity grading scale for each organ system by discussion and consensus; and (6) externally validated the scale using an independent group of 680 patients from the same databank. RESULTS: Nine organ-specific severity scales were developed from 0 (no documented involvement) to 4 (endstage disease). The data required for scale completion are relatively easy and practical for all physicians to obtain. CONCLUSION: This preliminary severity scale will be useful for assessing disease severity status in individual patients both at one point in time and longitudinally. The severity scale will assist in the design and conduct of clinical trials and the comparison of study populations with one another. The scale will serve as a framework for developing a scleroderma disease activity index.

Humans↗