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Biomedical subjects

Michael Roth

Publications and source records attributed to Michael Roth.

32 records · Page 2Linked to original sources
PubMed2004

Extracellular matrix proteins modulate asthmatic airway smooth muscle cell proliferation via an autocrine mechanism.

BACKGROUND: Airway remodeling is a key feature of persistent asthma and includes alterations in the extracellular matrix protein profile around the airway smooth muscle (ASM) and hyperplasia of the ASM. We have previously shown that nonasthmatic ASM cells in culture produce a range of extracellular matrix protein proteins and that asthmatic ASM cells proliferate faster than cells from nonasthmatic patients. OBJECTIVE: In this study, we compared the profile of extracellular matrix proteins produced by nonasthmatic and asthmatic ASM cells. We also examined the influence of these extracellular matrix protein proteins and conditioned medium derived from nonasthmatic or asthmatic ASM cells on the proliferation of nonasthmatic and asthmatic ASM cells. METHODS: Extracellular matrix proteins were measured by ELISA; proliferation of ASM cells was measured by tritiated thymidine incorporation. RESULTS: Production of perlecan and collagen I by the cells from asthmatic patients were significantly increased. In contrast, laminin alpha1 and collagen IV were decreased. Chondroitin sulfate was detectable only in the cells from nonasthmatic patients. Compared with nonasthmatic extracellular matrix proteins, proteins from asthmatic cells enhanced ASM cell proliferation. Conditioned medium from asthmatic ASM cells did not induce greater proliferation compared with conditioned medium from nonasthmatic cells. CONCLUSIONS: The data show that the profile of extracellular matrix protein components is altered in asthmatic cells and that this altered profile and not soluble mediators secreted from the ASM cells has the potential to influence the proliferation of these cells. These changes are likely to contribute to the airway wall remodeling that occurs in asthma.

Adult↗
PubMed2004

[Paroxetine versus clomipramine in female adolescents suffering from anorexia nervosa and depressive episode--a retrospective study on tolerability, reasons for discontinuing the antidepressive treatment and different outcome measurements].

OBJECTIVES: So far, there have only been few studies concerning the question of indication and efficacy of antidepressive medication in children and adolescents with anorexia nervosa and depressive episode in the course of an inpatient treatment. In addition, there is a lack of studies comparing the tolerability and efficacy of different antidepressants given to anorectic patients of this particular age group. This study compares paroxetine, a specific SRI, with clomipramine, a TCA with SRI activity, concerning the frequency and quality of adverse side effects, the frequency and the reasons for discontinuating the antidepressive treatment and different outcome measurements. METHODS: 83 female patients, aged 10.9 to 18.1 years, who underwent an inpatient treatment at the Departement of Child and Adolescent Psychiatry and Psychotherapy at the University of Wuerzburg, Germany, were enrolled in this retrospective study. All of them met the ICD-10 criteria for anorexia nervosa and depressive episode and received an antidepressant medication with clomipramine or paroxetine. We collected data from basic documentation, treatment reports, and the multiaxial classification (MAS). Outcome measurements were the duration of treatment (days) and the increase of body weight (kg/m2). RESULTS: The discontinuation of the antidepressive treatment due to adverse side effects or a lack of efficacy was significantly more frequent with clomipramine than paroxetine (33.3 vs. 15.4%). The increase of body weight (2.8 vs. 2.6 kg/m2) was similar in both groups, but the duration of treatment was significantly shorter under paroxetine (71.9 vs. 96.5 days). CONCLUSIONS: A shorter duration of treatment, faster increase of body weight, lower percentage of dicontinuating the antidepressive medication and last but not least economic reasons lead to the conclusion, that paroxetine should be preferred in female adolescents with anorexia nervosa and depressive episode. However, prospective studies are needed to confirm our findings.

Adolescent↗
PubMed2003

Exo1: a new chemical inhibitor of the exocytic pathway.

A phenotypic screen was used to search for drug-like molecules that can interfere with specific steps in membrane traffic. 2-(4-Fluorobenzoylamino)-benzoic acid methyl ester (Exo1), identified in this screen, induces a rapid collapse of the Golgi to the endoplasmic reticulum, thus acutely inhibiting the traffic emanating from the endoplasmic reticulum. Like Brefeldin A (BFA), Exo1 induces the rapid release of ADP-ribosylation factor (ARF) 1 from Golgi membranes but has less effect on the organization of the trans-Golgi network. Our data indicate that Exo1 acts by a different mechanism from BFA. Unlike BFA, Exo1 does not induce the ADP-ribosylation of CtBP/Bars50 and does not interfere with the activity of guanine nucleotide exchange factors specific for Golgi-based ARFs. Thus, Exo1 allows the fatty acid exchange activity of Bars50 to be distinguished from ARF1 activity in the control of Golgi tubulation.

Adenosine Diphosphate Ribose↗
PubMed2003

Urokinase potentiates PDGF-induced chemotaxis of human airway smooth muscle cells.

We investigated the chemotactic action of PDGF and urokinase on human airway smooth muscle (HASM) cells in culture. Cells were put in collagen-coated transwells with 8-micro m perforations, incubated for 4 h with test compounds, then fixed, stained, and counted as migrated nuclei by microscopy. Cells from all culture conditions showed some basal migration (migration in the absence of stimuli during the assay), but cells preincubated for 24 h in 10% FBS or 20 ng/ml PDGF showed higher basal migration than cells quiesced in 1% FBS. PDGF(BB), PDGF(AA), and PDGF(AB) were all chemotactic when added during the assay. PDGF chemotaxis was blocked by the phosphatidyl 3'-kinase inhibitor LY-294002, the MEK inhibitor U-0126, PGE(2), formoterol, pertussis toxin, and the Rho kinase inhibitor Y-27632. Urokinase alone had no stimulatory effect on migration of quiescent cells but caused a dose-dependent potentiation of chemotaxis toward PDGF. Urokinase also potentiated the elevated basal migration of cells pretreated in 10% FBS or PDGF. This potentiating effect of urokinase appears to be novel. We conclude that PDGF and similar cytokines may be important factors in airway remodeling by redistribution of smooth muscle cells during inflammation and that urokinase may be important in potentiating the response.

Becaplermin↗
PubMed2003

Expression of connective tissue growth factor in asthmatic airway smooth muscle cells.

There is strong evidence to implicate transforming growth factor-beta in the remodeling that occurs in asthma, as levels are increased in bronchial lavage fluid and gene expression is increased in bronchial tissue. Transforming growth factor-beta is also known to increase the release of collagen from airway smooth muscle. Here we identify for the first time a possible mechanism for the effects of transforming growth factor-beta. Transforming growth factor-beta specifically induces mRNA and protein for connective tissue growth factor in airway smooth muscle, and moreover, we report that the connective tissue growth factor response is greater in airway smooth muscle cultured from patients with asthma compared with patients without asthma. This occurs at both the level of mRNA (37.53 +/- 11.62- and 13.59 +/- 3.12-fold increase at 24 hours compared with time 0, respectively, p < 0.02) and protein production (67.57 +/- 27.80- and 3.58 +/- 0.6-fold increase at 24 hours compared with time 0, respectively, p < 0.03). The differential connective tissue growth factor response to transforming growth factor-beta in asthmatic airway smooth muscle identifies a potential role for connective tissue growth factor in the remodeling that is characteristic of severe persistent asthma.

Adolescent↗
PubMed2003

Chlamydia pneumoniae infections in asthma: clinical implications.

Chlamydia pneumoniae is an intracellular pathogen that has been suggested to play a role in the pathology of asthma. However, so far none of the studies have provided clear evidence for a causative role of C. pneumoniae infections in asthma, although there is little doubt that chronic C. pneumoniae infection does aggravate asthma and should be treated. The diagnosis of C. pneumoniae infection is still a matter of concern for it is dependent on trained skilled personnel and can vary significantly between different diagnostic laboratories. This fact is also one of the major problems encountered when comparing epidemiological studies investigating the possible role of C. pneumoniae infections and their impact on the pathogenesis of other diseases. With regard to therapy, long-term treatment with macrolides is the best available method to eradicate C. pneumoniae. Successful therapy for C. pneumoniae, however, can also be complicated by the high possibility of de novo infection as epidemiological studies have shown that the prevalence of antibodies to C. pneumoniae increases with age in all populations studied. In the northern hemisphere the prevalence of C. pneumoniae is also affected by seasonal conditions. It is too early to draw any conclusions from the equatorial belt countries. The available data on C. pneumoniae in tropical countries indicate a much faster infection rate during early adulthood with 100% serological prevalence at an age greater than 25 years. This data, if confirmed, would argue against C. pneumoniae causing asthma since the asthma prevalence in those countries does not increase in a parallel pattern. An alternative interpretation of most studies could be that the increased rate of C. pneumoniae infections in patients with asthma results from a modified susceptibility towards the microorganism, due to yet unknown changes of the host cell's physiology. It should be kept in mind that increased prevalence of C. pneumoniae infection is not restricted to asthma. Further studies are needed to understand the role of C. pneumoniae, especially of chronic infection, in the pathogenesis of inflammatory diseases with a specific focus on the effect that the microorganism triggers in the infected host cell. Only when we understand what C. pneumoniae does to its host cell will we be able to judge its impact on the overall status of an affected patient, and this knowledge will help us to develop a successful therapy.

Asthma↗
PubMed2003

Hypoxia modulates the effects of transforming growth factor-beta isoforms on matrix-formation by primary human lung fibroblasts.

Chronic hypoxia is implicated in lung fibrosis, which is characterized by enhanced deposition of extracellular matrix (ECM) molecules. Transforming growth factor-beta (TGF-beta) plays a key role in fibroblast homeostasis and is involved in disease states characterized by excessive fibrosis, such as pulmonary fibrosis. In this study, we investigated if hypoxia modulates the effects of TGF-beta on the expression of gelatinases: matrix metalloproteinase (MMP)-2 and MMP-9, interstitial collagenases: MMP-1 and MMP-13, tissue inhibitors of MMP (TIMP), collagen type I and interleukin-6 (IL-6). Primary human lung fibroblasts, established from tissue biopsies, were cultivated under normoxia or hypoxia in the presence of TGF-beta1, TGF-beta2 or TGF-beta3. Gelatinases were assessed by gelatin zymography and collagenases, TIMP, collagen type I and IL-6 by ELISA. Under normoxia fibroblasts secreted MMP-2, collagenases, TIMP, collagen type I and IL-6. TGF-betas significantly decreased MMP-1 and increased TIMP-1, IL-6 and collagen type I. Hypoxia significantly enhanced MMP-2, and collagenases. Compared to normoxia, the combination of TGF-beta and hypoxia reduced MMP-1, and further amplified the level of TIMP, IL-6, and collagen type I. Thus, in human lung fibroblasts hypoxia significantly increases the TGF-betas-induced secretion of collagen type I and may be associated to the accumulation of ECM observed in lung fibrosis.

Collagen Type I↗
PubMed2003

Chlamydia pneumoniae activates epithelial cell proliferation via NF-kappaB and the glucocorticoid receptor.

Chlamydia pneumoniae is an obligate intracellular eubacterium and a common cause of acute and chronic respiratory tract infections. This study was designed to show the effect of C. pneumoniae on transcription factor activation in epithelial cells. The activation of transcription factors by C. pneumoniae was determined in human epithelial cell lines (HL and Calu3) by electrophoretic DNA mobility shift assay, Western blotting, and luciferase reporter gene assay. The activation of transcription factors was further confirmed by immunostaining of C. pneumoniae-infected HL cells and mock-infected controls. The effect of transcription factors on C. pneumoniae-induced host cell proliferation was assessed by [(3)H]thymidine incorporation and direct cell counting in the presence and absence of antisense oligonucleotides targeting transcription factors or the glucocorticoid receptor (GR) antagonist RU486. The activation of the GR, CCAAT-enhancer binding protein (C/EBP), and NF-kappaB was induced within 1 to 6 h by C. pneumoniae. While the interleukin-6 promoter was not activated by C. pneumoniae, the GR-driven p21((Waf1/Cip1)) promoter was increased 2.5- to 3-fold over controls 24 h after infection. C. pneumoniae dose-dependently increased the DNA synthesis of the host cells 2.5- to 2.9-fold, which was partly inhibited either by RU486 or by NF-kappaB antisense oligonucleotides. Furthermore, we provide evidence that heat-inactivated C. pneumoniae does not cause a significant increase in cell proliferation. Our results demonstrate that C. pneumoniae activates C/EBP-beta, NF-kappaB, and the GR in infected cells. However, only NF-kappaB and the GR were involved in C. pneumoniae-induced proliferation of epithelial cells.

Base Sequence↗
PubMed2002

Interaction between glucocorticoids and beta2 agonists on bronchial airway smooth muscle cells through synchronised cellular signalling.

BACKGROUND: Increased airway smooth muscle bulk is a pathological feature of asthma. Asthma is well controlled by the combined inhalation of glucocorticoids and beta2-adrenoceptor agonists. The basic molecular mechanism of the interaction of the two drugs on proliferation of airway smooth muscle cells is yet to be identified. Our aim was to elucidate how glucocorticoids and beta2 agonists affect the growth of human bronchial airway smooth muscle cells. METHODS: We assessed the effect of formoterol and budesonide on the activation and function of transcription factors by immunohistochemistry, western blotting, DNA mobility shift assay, and a luciferase reporter gene assay. The effect of the drugs and the involvement of specific transcription factors on cell proliferation was ascertained by direct cell count and confirmed by thymidine incorporation. FINDINGS: Both classes of drugs (10(-8) mol/L) activated C/EBP-alpha and the glucocorticoid receptor with different kinetic profiles, and inhibited proliferation. The combination of lower doses of drugs (10(-12) to 10(-9) mol/L) resulted in a synchronised activation of the transcription factors and an enhanced antiproliferative effect. The action of the drugs alone or in combination on transcription-factor activity and proliferation was suppressed by either depletion of C/EBP-alpha or in the presence of a glucocorticoid-receptor blocker. INTERPRETATION: Our findings could provide one explanation for the interaction of beta2 agonists and glucocorticoids at a molecular level, and indicate that the concentration of inhaled glucocorticoids can be reduced when combined with beta2 agonists, minimising the side-effects of the drugs.

Adrenergic beta-Agonists↗
PubMed2002

Asthma and the CCAAT-enhancer binding proteins: a holistic view on airway inflammation and remodeling.

Asthma is an airway disease with increasing prevalence characterized by intermittent reversible airway obstruction, airway inflammation, and airway wall remodeling. The disease is generally triggered by inhalation of allergens, but nonallergic asthma triggers are quite common. The pathogenesis of asthma is well documented, and a great deal of research has been carried out to elucidate the underlying mechanisms. A multitude of articles have focused on cells alleged to be involved in the pathogenesis, including circulating cells from the immunologic compartment (ie, eosinophils and T lymphocytes) and resident cells, such as fibroblasts, airway smooth muscle cells, and, more recently, the airway epithelium. Despite the enormous amount of research, it is still unclear what exactly causes asthma. A general feature of most studies is an enhanced activation status of asthmatic cells, suggesting a general defect with respect to regulation of cellular responses. Here we discuss the ubiquitous transcription factor family of CCAAT-enhancer binding proteins (C/EBPs) and its involvement in inflammation and proliferation. We propose that an imbalance of C/EBP isoform expression might lead to an enhanced activity of asthmatic cells and provide an overall hypothesis that both airway inflammation and remodeling can be conceived as the result of an imbalance of C/EBP isoform expression.

Adrenal Cortex Hormones↗
PubMed2002

Interaction of C/EBPalpha and the glucocorticoid receptor in vivo and in nontransformed human cells.

Belonging to the family of steroid hormones, glucocorticoids are essential for development and survival of vertebrates. The cellular response to glucocorticoids is attributed to the glucocorticoid receptor, which functions as a transcription factor. However, the majority of glucocorticoid-modulated genes lack a DNA binding site for the glucocorticoid receptor, raising the question of which mechanism mediates the responses to glucocorticoids. It has been suggested that besides direct DNA binding of the glucocorticoid receptor, interaction with members of other transcription factor families modulates the effect of the glucocorticoid receptor. However, the significance of such transcription factor interaction is not clear. In cultured human mesenchymal cells and peripheral blood leukocytes of human volunteers treated with glucocorticoids, we detected the formation of a complex between the GR and the CCAAT/enhancer binding protein alpha. In in vitro experiments, this interaction turned out to be responsible for the inhibitory action of glucocorticoids on lymphocytic and mesenchymal cell proliferation. Our results suggest that complex formation of the GR with C/EBPalpha accounts for a novel pathway of glucocorticoid action.

CCAAT-Enhancer-Binding Protein-alpha↗
PubMed2002

Hypoxia differentially enhances the effects of transforming growth factor-beta isoforms on the synthesis and secretion of glycosaminoglycans by human lung fibroblasts.

Interstitial lung diseases associated with hypoxia, such as lung fibrosis, are characterized by enhanced production of transforming growth factor-beta (TGF-beta) and increased deposition of extracellular matrix (ECM) molecules, including glycosaminoglycans (GAGs). In this study, we investigated the effect of hypoxia (3% O(2)) on TGF-beta-induced GAG synthesis by primary human pulmonary fibroblasts, established from lung biopsies. Total GAG synthesis was assessed by the incorporation of [(3)H]glucosamine into GAGs associated with the cell layer (cells and ECM) or secreted in the medium. GAGs were isolated and purified by gel filtration, fractionated by electrophoresis on cellulose acetate membranes, and characterized using GAG-degrading enzymes. GAG molecules identified in the cell layer and the medium were: hyaluronic acid, and chondroitin, dermatan, and heparan sulfates. All TGF-beta isoforms time dependently induced [(3)H]glucosamine incorporation into GAGs of the cell layer or the medium. Characterization of individual GAG molecules indicated that this was attributed to dermatan and heparan sulfates in the cell layer and to hyaluronic acid and chondroitin and dermatan sulfates in the medium. Hypoxia enhanced the effect of all TGF-beta isoforms, particularly that of TGF-beta3, on the secretion of hyaluronic acid and chondroitin and dermatan sulfates. In the cell layer, hypoxia stimulated only the effect of TGF-beta2-induced [(3)H]glucosamine incorporation into GAGs. Our data indicate that hypoxia differentially enhances the effect of TGF-beta isoforms on the secretion and deposition of GAGs and may hasten ECM remodeling associated with the pathogenesis of lung fibrosis.

Cell Culture Techniques↗
PubMed2002

Identification of a novel IL-6 isoform binding to the endogenous IL-6 receptor.

Interleukin (IL)-6 is a multifunctional cytokine showing a wide variety of biologic functions on various tissues. Extracellular IL-6 signals through heterohexameric complex formation with IL-6 receptor-alpha (IL-6Ralpha) and IL-6 receptor-beta (IL-6Rbeta). In analogy to cytokines IL-2 and IL-4, we investigated the expression of IL-6 splice variants in lung tissue and cultivated fibroblasts. In human lung specimens, four different IL-6 transcripts were characterized as follows: native IL-6; IL-6 missing either exon 2 (IL-6Delta2), exon 4 (IL-6Delta4), or missing both; and exons 2 and 4 (IL-6Delta2,4). Only native IL-6 and IL-6Delta4 encoded for proteins of ~ 26 and 17 kD, respectively. Although the overall structure and most functional sites of the IL-6Delta4 protein were predicted to be maintained, IL-6Delta4 was found to lack two amino acids necessary for IL-6/IL-6 homodimerization as well as two of the six amino acids required for interaction with IL-6Rbeta. Receptor mobility shift assays confirmed that the new isoform formed a stable complex with IL-6Ralpha; however, no interaction with IL-6Rbeta was observed. Thus, IL-6Delta4 is likely to compete with native IL-6 for IL-6Ralpha binding but fails to transmit IL-6Rbeta-mediated signaling.

Alternative Splicing↗