Biomedical subjects

N Hunzelmann

Publications and source records attributed to N Hunzelmann.

At least 73 records · Page 4Linked to original sources

PubMed1997

Transforming growth factor-beta reverses deficient expression of type (I) collagen in cultured fibroblasts of a patient with metageria.

Metageria is a generalized form of acrogeria belonging to the group of premature aging syndromes and is characterized by loss of subcutaneous fat, thinning of the dermis, multiple teleangiectasias and mottled hyperpigmentation. The skin changes present suggest that an altered formation of extracellular matrix might be involved in the pathogenesis of this disease. Fibroblasts obtained from the skin of a patient with this disease revealed a marked reduction of type I collagen expression to about 20% of control levels both at the mRNA and protein level. In addition decreased decorin but unchanged type IV collagen and fibronectin mRNA levels were found. Similar although less pronounced changes were observed in fibroblasts obtained from the sister of this patient showing skin changes compatible with acrogeria. To further analyze the deficient expression of type I collagen run on analysis was performed revealing a decrease of transcription of type I collagen. Incubation of the cells with transforming growth factor-beta, a strong inducer of type I collagen and extracellular matrix formation, restored type I collagen expression both at the mRNA and protein level to amounts comparable with normal skin fibroblasts. These results are consistent with a defect in type I collagen transcription that is readily reversed after incubation with transforming growth factor beta. The deficient synthesis of type I collagen and decorin by dermal fibroblasts might thus contribute to an altered formation of the extracellular matrix resulting in the poikilodermic skin changes observed in this patient.

Adult↗

PubMed1997

Systemic scleroderma. Multicenter trial of 1 year of treatment with recombinant interferon gamma.

OBJECTIVE: To confirm significant improvement of the skin score in systemic sclerosis by treatment with interferon gamma in a larger group of patients and to investigate on a molecular level the influence of interferon gamma on collagen type I messenger RNA expression. DESIGN: Open, noncontrolled multicenter study. SETTING: Five outpatient clinics specializing in the care of systemic scleroderma. PATIENTS: Thirty-two patients suffering from the diffuse or limited form of systemic sclerosis and progressive disease were recruited; 20 patients finished the study. INTERVENTION: Each patient received interferon gamma, 50 micrograms subcutaneously 3 times a week for 1 year. MAIN OUTCOME MEASURE: Skin score, collagen type I messenger RNA in skin biopsy specimens. RESULTS: The patients who completed the study showed an unchanged median skin score after 1 year of therapy. In addition, similar collagen type I messenger RNA levels were detected in skin biopsy specimens taken from involved skin before and after therapy in these patients. CONCLUSIONS: Treatment of systemic scleroderma with interferon gamma is associated with stabilization of the skin score and lack of worsening of visceral involvement.

Female↗

PubMed1997

Double-blind, placebo-controlled study of intralesional interferon gamma for the treatment of localized scleroderma.

BACKGROUND: Localized scleroderma is characterized by circumscribed fibrotic plaques and may progress to widespread skin involvement and fibrosis. Interferon gamma (IFN-gamma) has been shown to be a potent inhibitor of collagen synthesis and of the migration and proliferation of dermal fibroblasts. OBJECTIVE: Our purpose was to determine whether IFN-gamma is effective in the treatment of localized scleroderma. METHODS: A double-blind, randomized, placebo-controlled, multicenter study was conducted. Twenty-four patients with progressive lesions received 100 micrograms of IFN-gamma or placebo subcutaneously on 5 consecutive days for 2 weeks followed by 100 micrograms of IFN-gamma or placebo once weekly for 4 weeks. Thereafter patients were observed for 18 weeks. To determine whether improvement could be related to an altered level of collagen messenger RNA (mRNA), biopsy specimens were taken from uninvolved and involved skin before and after therapy. RESULTS: The patients treated with IFN-gamma or placebo showed no significant difference in size or fibrosis of lesions or collagen type I mRNA synthesis. However, a reduction in the number of new lesions was observed in the IFN-gamma-treated group. The biopsy specimens obtained from involved skin showed a moderate increase of type I collagen and a significant decrease in the small proteoglycan decorin mRNA levels. CONCLUSIONS: The results indicate that IFN-gamma is ineffective in the treatment of localized scleroderma, but may inhibit the development of new lesions.

Collagen↗

PubMed1997

Cutaneous adverse reaction to ciprofloxacin: demonstration of specific lymphocyte proliferation and cross-reactivity to ofloxacin in vitro.

Ciprofloxacin (CPFX) is a widely used fluoroquinolone antibiotic, inducing cutaneous adverse drug reactions in about 1 to 2% of the treated patients. Conclusive diagnosis of drug allergy, however, still remains a major problem in daily clinical practice. Here, we present 2 patients with drug allergy to CPFX. In both cases the clinical suspicion for CPFX as the causative agent was confirmed in vitro by means of the lymphocyte transformation test, whereas epicutaneous patch tests remained negative. In vivo, a small percentage of the drug is biotransformed to the three major metabolites desethylene-, sulfo- and oxociprofloxacin. Though structurally closely related to their mother compound, these metabolites failed to induce in vitro lymphocyte proliferation in both patients. On the other hand, in vitro crossreactivity to ofloxacin, another fluorinated quinolone, could be demonstrated, which to our knowledge has not previously been reported.

Aged↗

PubMed1996

Quantitative analysis of alpha 1 (I) procollagen transcripts in vivo by competitive polymerase chain reaction.

Due to the limited amount of RNA obtainable from punch biopsies, few data exist on the human alpha 1 (I) procollagen mRNA steady state level in vivo. Therefore, we established a competitive PCR method to quantitate this mRNA in human biopsies. In our approach, the target template and the standard share the same sequence except for a 69 bp deletion, thus competing for the same primer pair and subsequently amplifying at the same rate. Titration of a competitor DNA dilution series against an unknown cDNA sample then allows quantitation of the separated and nonradioactively detected PCR products after densitometrical analysis. Almost identical results were obtained by quantitations of the same total RNA by competitive PCR as well as Northern blot analysis.

Base Sequence↗

PubMed1996

Regulation of collagen expression by interleukin-1 beta is dependent on donor age.

Cultured skin fibroblasts derived from old (> 60 years) donors differ in various morphological and functional aspects from cells obtained from young (< 20 years) donors. We were interested in whether fibroblasts obtained from old and young donors differ in their cellular response to interleukin-1 beta, a cytokine which has been shown to affect the synthesis of extracellular matrix proteins in human dermal fibroblasts. Therefore the expression of interstitial collagen (type I), minor collagen (type VI) and interstitial collagenase genes was investigated, using fibroblasts derived from either old or young donors. Fibroblasts were incubated in the absence or presence of interleukin-1 beta (5 units/ml, 50 units/ml, 500 units/ml) for 48 h. Total RNA was isolated and mRNA levels were determined by Northern and dot blot analysis. Comparing the synthetic response of fibroblasts obtained from young and old donors, we observed a marked increase of the inhibition of type I and type VI collagen expression and the stimulation of interstitial collagenase in the aged fibroblasts. These results suggest that physiological ageing in human fibroblasts is associated with an altered responsiveness to interleukin-1 beta.

Adolescent↗

PubMed1996

Co-ordinate induction of collagen type I and biglycan expression in keloids.

Proteoglycans are macromolecules displaying structural roles as well as regulatory functions in the maintenance of the extracellular matrix. Biglycan/PG-I and decorin/PG-II are two small proteoglycans that are structurally related but differ considerably in their localization in vivo and behaviour in vitro. Decorin and, to a minor extent, biglycan, can be located at the surface of type I collagen fibrils and have been shown to influence collagen fibrillogenesis. However, the physiological role of biglycan in the dermis is not known. Biopsies obtained from keloids were bisected and processed for total RNA extraction and immunohistochemistry. Northern blot analysis of total RNA obtained from keloids with high growth tendency in vivo showed a marked induction of biglycan and collagen alpha 1(I)mRNA expression in comparison with total RNA obtained from normal skin or keloids with little growth tendency. In contrast, decorin mRNA expression remained largely unaltered. Studying these biopsies by immunohistochemistry, decorin expression in the dermis was unaltered comparing normal and keloid tissue, whereas a markedly increased staining for biglycan was observed in the keloid tissue, which was most pronounced in the nodular formations, and was a characteristic feature of keloids. The altered expression of biglycan in keloid tissue might be involved in the abnormal regulation of extracellular matrix deposition either through the binding of growth factors or by influencing the three-dimensional organization of collagen fibres or associated molecules.

Biglycan↗

PubMed1995

Isolation and characterization of allergen-binding cells from normal and allergic donors.

BACKGROUND: Flow cytometry of the immune system so far has been limited to the analysis of subpopulations according to lineage markers. The cells involved in a particular immune response could not be assayed due to their low frequency. Here we show the potential of antigen-specific high gradient magnetic cell sorting to enrich cells for visualisation in multiparameter cytometry, functional studies and immortalization. OBJECTIVES: The aim of this study was the development of an efficient technology for staining and isolation of antigen-binding cells from human peripheral blood. In particular, allergen-specific cells from normal and allergic donors should be analysed and compared to develop a cellular diagnosis of allergy. STUDY DESIGN: The rare antigen-specific cells were sorted by high-gradient magnetic cell sorting with MACS. Haptenized phospholipase A2 (PLA2), the major allergen of bee venom, or haptenized ParoI, the major allergenic component of Parietaria officinalis, were used as antigens. The cells from normal and allergic donors, binding to the allergen were characterized phenotypically by immuno-fluorescence. Allergen-specific B-cells were immortalized by EBV transformation. RESULTS AND CONCLUSION: Allergen-specific cells can be enriched from blood of both allergic and normal donors to purities of up to 75%, by high gradient magnetic cell sorting. The specificity of labelling with allergen was confirmed by establishing allergen-specific EBV-transformed B-cell lines from the sorted cells. Clear differences exist in the cellular composition of allergen-binding cells from normal compared to allergic donors. In normal donors the allergen-binding cells are B-cells expressing CD19 and CD21. In allergic donors, in addition to allergen-binding B-cells, occurring in about equal absolute numbers as in normal donors, basophilic granulocytes are labeled by allergen. These cells express CD38, CD9 and CD25 on their surface, and stain for IgE.

Allergens↗

PubMed1995

Altered immunohistochemical expression of small proteoglycans in the tumor tissue and stroma of basal cell carcinoma.

Small proteoglycans have been shown to act as receptors for matrix molecules or growth factors and to influence the attachment and the migration of cells. We therefore report here on the immunocytochemical expression of three small proteoglycans, i.e., decorin, biglycan, and the recently described PG-100, in normal human skin and in basal cell carcinoma. In normal human skin, staining for decorin revealed expression throughout the dermis with an increased signal in the papillary dermis, whereas no expression was observed in the epidermis. Biglycan and PG-100 were mainly detected in the epidermis, with biglycan being expressed only in suprabasal layers. In addition, biglycan could be detected in a narrow zone below the basement membrane. In tissue specimens obtained from 12 basal cell carcinomas, the expression of biglycan and PG-100 was absent or strongly down-regulated in the tumor tissue. Tumor cells thus displayed a staining pattern similar to that found on the basal cells of normal human skin. In the stroma surrounding the tumor, however, the expression of biglycan and to a lesser degree decorin was increased when compared with normal human dermis. The increased deposition appears to be due to an increased synthesis of these molecules, as total RNA extracted from basal cell carcinoma tissue revealed an induction of biglycan and decorin mRNA. This study indicates that the expression of proteoglycans in basal cell carcinoma tumor cells and in tumor stroma is altered from that in normal skin.

Animals↗

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↗

PubMed1992

Autocrine growth limitation of human papillomavirus type 16-harboring keratinocytes by constitutively released tumor necrosis factor-alpha.

TNF-alpha is known to exert antitumor and antiviral effects and to participate in the regulation of the immune response. In our study we demonstrate that human rTNF-alpha specifically blocks growth of SK-v keratinocyte cell line harboring and expressing human papillomavirus type 16 (HPV16) sequences. This inhibitory effect was shown by [3H]TdR incorporation and cell counting. Binding experiments with 125I-TNF-alpha showed that SK-v cells express about 10,000 single class TNF-alpha R per cell with affinity constant of about 0.7 nM. Binding of 125I-TNF-alpha could be inhibited by htr-9 mAb recognizing a 55/60-kDa type I TNF-alpha R but not by utr-1 mAb recognizing 75/80-kDa type II TNF-alpha R or irrelevant mAb specific for HPV16E7 protein. Addition of anti-TNF-alpha antibodies to SK-v cell culture resulted in significant (p < 0.05), dose-dependent stimulation of their proliferation. SK-v cells constitutively expressed TNF-alpha mRNA, and SK-v CM contained TNF-alpha, as demonstrated by Northern blot analysis, a specific ELISA, Western blot analysis, and a bioassay with TNF-alpha-sensitive L-M cells. HPLC gel filtration of SK-v cell CM showed that the factor cytotoxic for L-M cells coeluted with immunoreactive TNF-alpha. These results demonstrate that HPV16-harboring SK-v cells constitutively express and release immunoreactive and biologically active TNF-alpha that in turn may exert an autocrine growth inhibitory effect. This phenomenon could represent one of the self-limiting mechanisms controling growth of HPV-induced neoplasia.

Cell Division↗

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

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

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

Excessive collagen formation in fibrolamellar carcinoma of the liver: a morphological and biochemical study.

We have studied the excessive deposition of extracellular matrix in a patient with fibrolamellar carcinoma of the liver. The collagen matrix was predominantly composed of collagens I, III, and V. Since specific mRNAs for collagens I and III were detected by in situ hybridization, we also provide evidence that the fibroblastoid stromal cells were the major source of this collagen. Occasionally, also tumor cells could be shown to express collagen III-mRNA. Furthermore, some tumor cells showed positive signals for TGF-beta 1, while isolated stromal cells expressed interleukin-6. This cytokine expression may probably be related to the altered control of collagen gene expression.

Adolescent↗

PubMed1991

Cytokines alter mRNA steady state levels for basement membrane proteins in human skin fibroblasts.

Keratinocytes and fibroblasts synthesize basement membrane proteins and even contribute to the formation of basement membrane structures following injury or tissue damage. Under these conditions many cellular functions are regulated by mediators e.g. transforming growth factor-beta, tumor necrosis factor alpha, interferon-gamma or interleukin-1 alpha. We therefore describe here their influence on synthesis of basement membrane proteins in human skin fibroblasts. A comparative analysis of mRNA steady levels coding for BM-40, nidogen, laminin B1 and B2 chains and collagen IV in fibroblasts, in primary human keratinocytes and a epidermal cell line grown in monolayer culture demonstrated that the highest amounts were present in human fibroblasts. Interferon-gamma reduces all mRNA steady state levels dose dependently in comparison to the control, while transforming growth factor-beta simultaneously induces BM-40, alpha 1 and alpha 2 (IV) collagen mRNAs. TGF-beta, however, has no effect on nidogen and laminin mRNA levels. Interleukin-1 alpha and tumor necrosis factor alpha do not affect the mRNA levels of most basement membrane proteins. However, the alpha 1 (IV) collagen mRNA is upregulated by both cytokines to 300%. These data demonstrate a specific control of the expression of several basement membrane proteins by cytokines and indicate that fibroblasts could contribute to basement membrane formation during wound healing and tissue repair.

Basement Membrane↗