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Die vorliegende Arbeit behandelt die Generierung und Charakterisierung rekombinanter Aktin-modifizierender Fusionstoxine mit dem Ziel der selektiven Inhibition der Chemotaxis humaner neutrophiler Granulozyten. Der klinische Hintergrund dieser Zielsetzung liegt in der Behandlung von Polytraumapatienten. Das Erleiden eines stumpfen Thoraxtraumas verschlechtert die Prognose polytraumatisierter Patienten signifikant. Grund dafür ist vermutlich eine systemische Entzündungsreaktion, die im weiteren Verlauf oftmals zur Entstehung des Acute Respiratory Distress Syndrome‘s (ARDS) führt. Eine Schlüsselrolle in der Pathogenese des ARDS spielen ins Lungenparenchym einwandernde Monozyten und neutrophile Granulozyten. Aus diesem Grund ist die Entwicklung neuartiger pharmakologischer Optionen, welche gezielt die Migration neutrophiler Granulozyten hemmen, von großem wissenschaftlichen und medizinischen Interesse. Ausgangpunkt für das neuartige rekombinante Fusionstoxin ist das clostridiale C2-Toxin, ein binäres Aktin-ADP-ribosylierendes Protein, welches aus der Enzymkomponente C2I und der Translokations- und Bindungskomponente C2IIa besteht. Durch Fusion eines spezifisch an neutrophile Granulozyten und Monozyten bindenden Peptids mit der C2I- Enzymuntereinheit wurde das neuartige P1_C2I-Toxin generiert. Dies erfolgte durch Einfügen der P1-Sequenz in das für C2I-codierende Plasmid mittels erfolgreicher Exponential Megaprimer Polymerasekettenreaktion. Anhand Zytotoxizitäts- und Zellabrundungsassays wurde ein möglicher Vergiftungseffekt von P1_C2I auf CHO-K1- Fibroblasten und epitheliale HeLa- und Vero-Zellen untersucht. Hierbei ergab sich auch über einen längeren Inkubationszeitraum von 24 Stunden mit hohen Toxinkonzentrationen keinen Anhaltspunkt für zytotoxische Effekte von P1_C2I. Aus der Versuchsreihe ging ebenfalls hervor, dass trotz der gentechnischen Modifikation des P1_C2I-Toxins die zelluläre Aufnahme in CHO-, HeLa- und Vero-Zellen bei gleichzeitiger Inkubation mit der Bindungs- und Translokationsdomäne C2IIa unbeeinträchtigt bleibt. Bei der Untersuchung des Effekts von P1_C2I auf humane neutrophile Granulozyten und promyeloischen Leukämiezellen (NB4-Zellen) zeigte sich ein von der Konzentration abhängiger 92 Zusammenfassung Vergiftungseffekt von P1_C2I in der sequentiellen in-vitro-ADP-Ribosylierung. Der direkte Vergleich bei der Vergiftung von PMNs mit P1_C2I bzw. C2I ergab keinen signifikanten Unterschied in der Analyse der ADP-Ribosylierungsstatus von Aktin. Somit ist der beobachtete Vergiftungseffekt nicht auf die gentechnische Modifikation des P1_C2I-Toxins zurückzuführen. Die Ergebnisse dieser Arbeit weisen auf einen bisher nicht näher charakterisierten, vermutlich unspezifischen Aufnahmemechanismus der C2I- Enzymuntereinheit für humane neutrophile Granulozyten und NB4-Zellen bei Inkubation mit höheren Konzentrationen des Toxins hin. Diese Aufnahmemechanismen des C2I-Toxins spielen eine möglicherweise unterschätzte Rolle. Für zukünftige, vom C2I-Toxin abgeleitete pharmakologische Wirkstoffe könnten derartige unspezifische Aufnahmemechanismen potenziell zu zahlreichen, unerwünschten Arzneimittelnebenwirkungen führen. Aus diesem Grund ist die genauere Charakterisierung dieser Aufnahmemechanismen wichtig.

The long-term success of fistulating therapies for the treatment of glaucoma is essentially limited by excessive scarring reactions (fibrosis). Cytostatic agents such as mitomycin C can prevent fibrosis, but are often associated with side effects. Specific antifibrotics are not currently in clinical use. Therefore, this study describes a systems biology approach using a dedicated bioinformatics technology platform, with which active substances can be identified and repositioned as antifibrotics.Differential gene expression data of human Tenon fibroblasts (hTF) were collected from untreated hTF and from hTF stimulated with TGF-β1 ("fibrotic fibroblasts") by next-generation sequencing (NGS) and were used as the basis for the drug identification process. These data were filtered with the bioinformatic tool "FocusHeuristics". In comparison with the Connectivity Map database, antifibrotic agents were identified. The evaluation of a potentially promising drug as an antifibrotic was performed at hTF by indirect immunofluorescence in vitro.The analysis of the gene expression data led to the identification of several interaction networks of genes or proteins involved in fibrotic processes. One of these networks contains the cytokine bone morphogenic protein 6 (BMP6), interleukin 6 (IL6) and fibroblast growth factor 1 (FGF1). Another relevant network has been identified around the cluster of differentiation 34 (CD34) gene. The comparison of these data with those of the Connectivity Map allowed the identification of an inhibitory drug. Its evaluation in the fibrotic cell culture model in vitro using indirect immunofluorescence led to a significant reduction in the expression of the fibrotic marker proteins fibronectin and alpha-smooth muscle actin (α-SMA), which confirmed the predicted antifibrotic effect.Systems biological approaches can be used for the identification of antifibrotic drug candidates for the prevention of postoperative fibrosis and should be transferable by the investigating differential gene expression data of further ocular cells or tissues to other ophthalmological fields of application.Der Langzeiterfolg fistulierender Therapiekonzepte zur Behandlung des Glaukoms wird im Wesentlichen durch überschießende Vernarbungsreaktionen (Fibrose) limitiert. Zytostatika wie Mitomycin C können die Fibrose zwar verhindern, sind jedoch häufig mit Nebenwirkungen assoziiert. Spezifisch wirkende Antifibrotika sind derzeit nicht im klinischen Einsatz. Daher beschreibt diese Studie einen systembiologischen Ansatz, mit dem durch eine dedizierte Bioinformatik-Technologieplattform Wirkstoffe identifiziert und als Antifibrotikum repositioniert werden können.Als Basis für den Wirkstoffidentifikationsprozess wurden differenzielle Genexpressionsdaten humaner Tenon-Fibroblasten (hTF) genutzt, die von unbehandelten hTF und von mit Transforming Growth Factor β1 (TGF-β1) stimulierten hTF („fibrotische Fibroblasten“) mittels Next-Generation Sequencing (NGS) erhoben wurden. Diese Daten wurden mit dem bioinformatischen Werkzeug „FocusHeuristics“ gefiltert. Im Vergleich mit der Connectivity-Map-Datenbank wurden der Fibrose entgegenwirkende Wirkstoffe identifiziert. Die Evaluierung eines potenziell erfolgversprechenden Wirkstoffs als Antifibrotikum wurde an hTF mittels indirekter Immunfluoreszenz in vitro durchgeführt.Die Analyse der Genexpressionsdaten führte zur Identifikation mehrerer in fibrotische Prozesse involvierter Interaktionsnetzwerke von Genen bzw. Proteinen. Eines dieser Netzwerke beinhaltet das Zytokin Bone morphogenic Protein 6 (BMP6) sowie Interleukin 6 (IL6) und Fibroblast Growth Factor 1 (FGF1). Ein weiteres relevantes Netzwerk konnte rund um das CD34-Gen (CD34: Cluster of Differentiation 34) identifiziert werden. Der Vergleich dieser Daten mit denen der Connectivity Map ermöglichte die Identifikation eines entsprechend invers wirkenden Wirkstoffs. Dessen Evaluierung im fibrotischen Zellkulturmodell in vitro mittels indirekter Immunfluoreszenz führte zu einer deutlichen Expressionsreduktion der fibrotischen Markerproteine Fibronektin und Alpha-smooth Muscle Actin (α-SMA), womit die vorhergesagte antifibrotische Wirkung bestätigt werden konnte.Systembiologische Ansätze können für die Identifikation von antifibrotischen Wirkstoffkandidaten zur Vermeidung postoperativer Fibrose genutzt werden und sollten sich über die Erfassung differenzieller Genexpressionsdaten weiterer okularer Zellen oder Gewebe auch auf andere ophthalmologische Anwendungsfelder transferieren lassen.

Mit dieser Arbeit konnte das Hitzeschockprotein (Hsp) 70 als ein Wirtszellfaktor identifiziert werden, der an der Aufnahme von binären clostridialen Aktin-Adenosindiphosphat (ADP)-ribosylierenden Toxinen in Säugetierzellen beteiligt ist. Die binären clostridialen Toxine C2 von Clostridium (C.) botulinum, Iota von C. perfringens und C. difficile-Toxin (CDT) von C. difficile bestehen aus einer enzymatisch aktiven A-Komponente und einer Bindungs- und Transportkomponente, der B-Komponente. Mittels der B-Komponenten wird die rezeptorvermittelte Endozytose der Toxine in Säugetierzellen sowie die Translokation der A-Komponente aus den Endosomen in das Zytosol vermittelt. Die A-Komponente besitzt die enzymatische Funktion einer ADP-Ribosyltransferase, durch welche globuläres Aktin (G-Aktin) ADP-ribosyliert wird. Die ADP-Ribosylierung von G-Aktin führt zu einer Zellabrundung bei Zusammenbruch des Zytoskeletts und hat letztendlich den Zelltod zur Folge. So werden die für das jeweilige Toxin typischen enterotoxischen Symptome verursacht. Für die Translokation der A-Komponenten C2I, Ia und CDTa über die endosomale Membran ins Zytosol sind bereits das Chaperon Hsp90 und die Peptidyl-Prolyl-cis/trans-Isomerasen bekannt. Nun ist neben diesen auch Hsp70 als ein Wirtszellfaktor identifiziert worden, der ebenfalls spezifisch an der pH-abhängigen Membrantranslokation beteiligt ist. Dies trägt zum besseren Verständnis des Aufnahmemechanismus dieser Toxine bei und ist von therapeutischer Bedeutung. So stellt Hsp70 eine weitere Möglichkeit dar, um neue Therapieansätze zur Behandlung von Infektionskrankheiten, die durch die clostridialen Aktin-ADP-ribosylierenden Toxine verursacht werden, zu entwickeln.

The ability of tumor cells to leave the primary tumor is prerequisite for metastatic spread. In previous studies, we identified regulator proteins of actin reorganization with essential functions in both synaptogenesis and tumor cell migration.The aim of the studies summarized in this article is to identify signaling pathways associated with actin-related proteins that might represent potential molecular targets for antiinvasive and/or antineoplastic therapies.We used immunohistochemical analyses of protein expression as well as in vitro techniques (cell culture, fluorescence microscopy, RNAi-based knockdown of protein expression, protein biochemistry and in vivo animal experiment substitutes).We show that phosphorylation of Abelson interactor 1 (Abi1) is essential for the adhesion and invasion of colorectal carcinoma cells and might be targeted by the tyrosine kinase inhibitor STI571/Glivec®. HnRNP K, a protein interaction partner of Abi1, is upregulated in malignant melanoma in response to ionizing radiation; this upregulation is impaired upon application of the MEK inhibitor PD98059, enhancing radiosensivity of melanoma. Edelfosin, an alkyl-lipid blocker of the Abi1 interaction partner SK3, inhibits invasion of urothelial carcinoma cells.The studies summarized in this overview confirm a central role for the investigated proteins in tumor cell invasion and resistance to antineoplastic therapies and identify possible molecular targets for novel therapeutic compounds.

Dendrite morphogenesis is a complex but well-orchestrated process. Various studies reported the involvement of alteration in dendrite morphology in different brain disorders, including neuropsychiatric disorders. Initially, beta B2-crystallin (gene symbol: Crybb2/CRYBB2) has been described as a structural protein of the ocular lens. Mutations of the corresponding gene, Crybb2, lead to cataract. Recent studies in mice suggested that mutations in Crybb2 cause alterations in hippocampal morphology and function, albeit its function in hippocampal neuron development remained elusive. In the current study, we found that Crybb2 contributes to dendritogenesis in vitro and in vivo. Furthermore, screening of previous data on differential expression-arrays, we found Tmsb4X up-regulated in Crybb2 mutants mouse brain. Additionally, Tmsb4X was co-expressed with Crybb2 at actin-enriched cell ruffles. Over-expression of Tmsb4X in cultured hippocampal neurons inhibited dendritogenesis, which phenocopied Crybb2 knockdown. The current study uncovers a new function of Crybb2 in brain development, especially in dendritogenesis, and the possible interplay partner Tmsb4X involved in this process. (C) 2018 Elsevier Inc. All rights reserved.

Photodynamic inactivation (PDI) may be a potential treatment alternative in therapy-resistant infectious keratitis. PDI may eliminate the microorganisms from the infected cornea by damage caused through free oxygen radicals, or even by supporting different stages of activation of keratocytes and inflammatory cell response. The purpose of this study was to determine the impact of PDI on activation of human keratocytes in culture.Primary human keratocytes were isolated by digestion in collagenase A (1 mg/mL) from human corneal buttons, and cultured in DMEM/Ham's culture medium supplemented with 10% foetal calf serum. Keratocytes underwent illumination (670 nm) for 13 minutes following exposure to 0, 50, 150 and 250 nMol/ml concentrations of the photosensitizer chlorin e6 (Ce6) in the culture medium. Twenty-four hours after treatment CD34 and α-smooth-muscle actin expression of the cells was analysed using flow-cytometry (FACS).Using Ce6 or illumination only, α-smooth-muscle actin expression of the cells did not change significantly. Twenty-four hours after PDI the percentage of CD34-positive keratocytes did not change significantly using 50-250 nM Ce6, however, the percentage of α-smooth-muscle actin-positive keratocytes decreased significantly at 250 nM Ce6 (p = 0.01).As a short-term effect, PDI seems to inhibit myofibroblastic transformation of keratocytes, but does not have an impact on activation of CD34-positive keratocytes.With this impact PDI possibly may reduce the antimicrobial defence of keratocytes.

Complications deriving from arterial aneurysm and dissection without signs of atherosclerosis are rare clinical entities. In recent literature case reports show a descriptive similarity of pathological findings summarized as segmental arterial mediolysis (SAM).The purpose of this study was to answer the question whether, among 16 patients suffering from SAM histological findings corresponded and assess causality.In a prospective prognostic trial sixteen patients were enrolled between 1st January 2008 and 31st October 2011. Inclusion criteria were a lack of atherosclerosis, age under 60 and clinical findings. Most of these sixteen patients were treated as emergency cases of life-threatening blood loss or organ system ischemia. Thirteen of the patients were male, 3 female and their average age was 44 (28-59) years. Localisation of the segmental aneurysm or dissection showed a broad variability from central to renovisceral and peripheral lesions. Imaging diagnostics (e.g., US and CT-A) were complemented by exclusion of positive family history, connective tissue diseases and autoimmune or inflammative disorders. In 8 patients with open vascular reconstructions, it was possible to obtain a biopsy from the target lesion to analyse morphological and immunochemical expression levels (e.g., MMP1-12, vWF, vSMC or CD 68).None of the patients died nor had described familiar associations. Even the examination of twins with sCAD showed no coincidence. Differential diagnostic findings were excluded. All patients agreed to undergo human genetic screening. The 8 biopsy tissues showed homogeneously mediolysis with focal and increasingly confluent lesions. Main findings were that the vessel wall layering was destroyed and that capillarisation was initiated from the adventitial layer. Furthermore, all patients suffered from hypertension associated to the SAM, or developed it during surveillance.SAM is a rare, life-threatening diagnosis and has to be taken into consideration in young patients with aneurysm and dissection of unusual locations. Rare vascular diseases should have a forum in future investigations which might highlight molecular genetic triggers and associated diseases, e.g., hypertension and aortic type B dissection.

The influence of silicone implants on the formation of a periprosthetic capsule can be well examined in animal studies. New implant materials have been developed to reduce capsular contracture. In order to evaluate the capsule formation, Wilflingseder et al. developed a histological score system. Because of new knowledge in the development of capsular contracture, the Wilflingseder classification is no longer appropriate. Current references are not considered so that a modification is required.In a randomised, experimental animal study 31 mini-implants were implanted into the dorsum of female Wistar rats [17 smooth, 10 mL saline-filled silicone implants (Group A) and 14 titanium coated silicone implants (Group B)]. After 12 (group A/B12) or 36 (group A/B36) weeks, surgical removal of the implants with subsequent histomorphological and immunohistochemical examination of periprosthetic capsule formation was performed by 2 independent investigators in a double-blind manner.An analysis of the studies showed that the inner synovia metaplasia and the infiltration by inflammatory cells such as lymphocytes, histiocytes, plasma cells and granulocytes are of crucial importance in the development of a fibrotic capsule. The occurrence of these factors correlated significantly with each other and influenced also significantly the capsule architecture depending on implant surface. An adjustment of the existing Wilflingseder classification system was evaluated. The current rating system contains the following parameters: capsule thickness and cell layers of the capsule, the thickness of the inner synovial metaplasia, collagen structure, presence of histiocytes and the incidence of inflammatory cells. According to this classification, titanium-coated implants show an advantage in terms of the formation of capsular contracture.In 1974 Wilflingseder et al. developed a classification system for capsular contracture which is no longer appropriate, since current histological and immunohistochemical findings are not mentioned. Our study presents a new system which includes the latest insights into the development of capsular contracture and provides an objective classification of histological changes. Furthermore, we were able to show that titanium-coated implants are a promising approach in the reduction of capsular contracture.

Paramecium tetraurelia possesses more actin isoforms than most other cells. With monospecific antibodies against actin subfamily 4 members, we could label cleavage furrow, nascent food vacuoles, oral apparatus, cilia, cell surface and macronucleus. Expression as green fluorescent protein- (GFP-) fusion protein now allowed us to localize more stringently actin4, e.g., in the macronucleus, particularly when enhanced with anti-GFP antibodies. Posttranscriptional gene silencing of actin4 resulted in disturbances at sites where actin4 has been localized. Cell division was impaired already early on, occasionally resulting in deformed cells. Both micro- and macronuclear development during vegetative cell fission were disturbed. Over longer periods, actin4 silencing entailed reduced phagocytotic activity, paralleled by accumulation of “acidosomes” (late endosomes) near the cytopharynx where they normally fuse with nascent phagosomes. In addition, near the cell surface, extensively misshapen “terminal cisternae” (early endosomes) occurred. In deformed cells, both constitutive endocytosis and stimulated trichocyst exocytosis were impaired. Thus, actin4 exerts pleiotropic effects at widely different sites of the Paramecium cell and disturbances generally coincide with sites where actin4 is normally enriched. Evidently the loss of actin4 cannot easily be compensated for by any other of the large number of actin isoforms occurring in a Paramecium cell.

Myofibroblastic sarcomas or myofibrosarcoma, are extremely rare malignant neoplasms of myofibroblasts. They are characterized by the pattern of cells and special immunohistochemical markers such as vimentin, desmin and alpha-smooth-muscle actin.The case of a patient with a history of frequently relapsing papillomas of the larynx is reported. Chronic laryngitis with focal low-grade dysplasia of the squamous epithelium was diagnosed approximately 1 year after the first treatment of the papillomas. After approximately 2 years the pathologist diagnosed the rare myofibroblastic sarcoma of the larynx. The patient underwent laryngectomy due to the spread of the tumor with a bilateral selective neck dissection. The patient is at present still free of recurrence and metastases.There is a great danger of misjudging a myofibroblastic sarcoma as an inflammatory myofibroblastic tumor and consequently to delay the urgently needed treatment. Therefore, an overview of the present state of knowledge about diagnosis and treatment of myofibroblastic sarcomas will be given based on this case report.

In the therapy for peripheral arterial occlusive disease there remain inadequacies in the use of alloplastic material concerning thrombogenicity and biological compliance. In the 1960s, Sparks tried to combine the advantages of alloplastic prostheses with those of autologous reconstructions by using incorporated prostheses. No extensive myointimal hyperplasia was noted, but besides infections aneurysmatic dilatation were limiting factors in clinical practice.The incorporation of modern alloplastic prostheses without connection to circulation concerning the thickness of neointima as well as the percentage of smooth muscle cells was examined in a dog model.The thickness of the neointima increased significantly in Dacron grafts with a peak on day 70 (p = 0.022), additionally a significantly greater percentage of smooth muscle cells was noted in Dacron grafts after 44 and 58 days (p = 0.008, p = 0.036).Due to the decreased thickness of the incorporating matrix as well as the lower percentage of smooth muscle cells, PTFE grafts should be preferred for peripheral arterial revascularisation.

Leiomyomas are by far the most frequent mesenchymal uterine neoplasms. Leiomyoma variants refer to a particular histological differentiation and growth pattern, respectively. To assess malignancy, an algorithm is used based on the presence or absence of cellular atypia, tumor cell necrosis and mitosis. In addition, vascular invasion is a criterion for malignancy. Ischemic or infarct type necrosis is not considered a criterion for malignancy. The differential diagnosis of leiomyosarcoma includes cellular and mitotically active leiomyoma and leiomyoma with extensive infarct type necrosis (apoplectic leiomyoma). The term smooth muscle tumor of uncertain malignant potential (STUMP) should be reserved for tumors with uncertainty regarding cell type, type of necrosis and mitotic index, as well as for special cases of myxoid and epithelioid smooth muscle neoplasms. Immunohistochemically, the expression of desmin and caldesmon is indicative of smooth muscle tumors, while stromal tumors can express smooth muscle actin and, rarely, desmin in addition to CD10.

The transcription factor serum response factor (SRF) plays a crucial role in the development of several organs. However, its role in the skin has not been explored. Here, we show that keratinocytes in normal human and mouse skin expressed high levels of SRF but that SRF expression was strongly downregulated in the hyperproliferative epidermis of wounded and psoriatic skin. Keratinocyte-specific deletion within the mouse SRF locus during embryonic development caused edema and skin blistering, and all animals died in utero. Postnatal loss of mouse SRF in keratinocytes resulted in the development of psoriasis-like skin lesions. These lesions were characterized by inflammation, hyperproliferation, and abnormal differentiation of keratinocytes as well as by disruption of the actin cytoskeleton. Ultrastructural analysis revealed markedly reduced cell-cell and cell-matrix contacts and loss of cell compaction in all epidermal layers. siRNA-mediated knockdown of SRF in primary human keratinocytes revealed that the cytoskeletal abnormalities and adhesion defects were a direct consequence of the loss of SRF. In contrast, the hyperproliferation observed in vivo was an indirect effect that was most likely a consequence of the inflammation. These results reveal that loss of SRF disrupts epidermal homeostasis and strongly suggest its involvement in the pathogenesis of hyperproliferative skin diseases, including psoriasis.

Aquaporin1 (AQP1) is a water channel protein which facilitates water flux across cell membranes. AQP1 is found in epithelial and endothelial cells in various tissues. There is increasing evidence that AQP1 is expressed in malignant tumours and that it may play a role in tumour angiogenesis, cell migration and metastasis. We studied the immunohistochemical expression of AQP1 in a cohort of 203 invasive breast carcinomas with long-term follow up. AQP1 expression was detected in 11 cases (5.4%), and showed a significant correlation with high tumour grade, medullary-like histology, "triple-negativity", as well a cytokeratin 14 and actin expression. In univariate analysis, AQP1 was associated with a significantly poorer prognosis. Multivariate analysis showed that AQP1 expression has an independent predictive value for outcome if stratified by age, tumour size, lymph node status, histological grade and ER status. AQP1 expression in invasive breast carcinomas is associated with a basal-like phenotype and poor prognosis.

Human fetal bladder development starts in the 9th to 10th week of gestation. Smooth muscle cell formation is induced by Hedgehog proteins and their signaling in the surrounding mesenchyma of the fetal bladder cavity. Bladder wall thickness increases significantly with advancing gestation, mediated by linear growth patterns irrespective of the related sex. lt is the aim of this article to address new insights into bladder development to enhance our knowledge about normal and pathological conditions that occur when developmental processes are disturbed.

We present the clinical and histopathological findings in the unusual case of a recurrent corneal tumour in a child. Light and ultrastructural observations as well as immunohistochemistry indicate a benign reactive process. However, the prognosis regarding visual function is poor due to the infiltrative nature of the lesion and its tendency to recur. A possible diagnosis of nodular fasciitis is discussed.

M3-specific inhibitors are currently preferred for anticholinergic therapy of OAB. However, not all of the patients profit from this regimen. This might reflect a heterogeneity of the patient group. The aim of this work is to define subgroups of patients with specific alterations of receptor expression and to profile the receptor expression individually. These receptor profiles might be used for the development of evidence-based "tailored" therapies.Detrusor probes from bladder carcinoma patients (BCa, n=9 F, n=7 male) and interstitial cystitis patients (IC, n=9 female) were examined using confocal immunofluorescence and PCR.M2, M3, P2X1-3, and H1-3 mRNAs were demonstrated in detrusor tissue. As revealed by immunofluorescence, the M2 receptor expression was significantly higher in female compared to male BCa tissues. In addition, the M2 receptor was further upregulated in IC vs BCa in female detrusor.IC patients showed specific alterations of their receptor profile. Individual receptor profiles might be used to optimize medicinal therapies.

Myofibroblasts play a pivotal role in numerous pathological alterations. Clarification of the structure and function and of the cellular plasticity of this cell type in the bladder may lead to new insights into the pathogenesis of lower urinary tract disorders.Bladder biopsies from patients with bladder carcinoma and interstitial cystitis were used to analyse the morphology and receptor expression using confocal immunofluorescence and electron microscopy. Cytokine effects and coupling behavior were tested in cultured myofibroblasts and detrusor smooth muscle cells.Myofibroblasts are in close contact with the suburothelial capillary network. They express Cx43 and form functional syncytia. The expression of muscarinic and purinergic receptors is highly variable. Dye coupling experiments showed differences to detrusor myocytes.Upregulation of smooth muscle cell alpha-actin and/or transdifferentiation into smooth muscle cells may contribute to the etiology of urge incontinence. A multi-step model is presented as a working hypothesis.

Besides cell proliferation, transdifferentiation of lens epithelial cells (LECs) to myofibroblasts is one of the mechanisms of secondary cataract formation. This process is characterized by increased expression of alpha-smooth muscle actin (alpha-SMA). This study investigated the influence of bFGF, TGF-beta2, EGF and IGF-1 on the expression of alpha-SMA in porcine LECs.Porcine LECs were cultured for 7 days in serum-free medium without or with 1 to 50 ng/ml bFGF, TGF-beta2, EGF or IGF-I. Alpha-SMA was detected immunocytochemically with a mouse monoclonal antibody, and the relative numbers of alpha-SMA-positive cells were calculated. Statistical analysis was performed using Student's unpaired t-test.The ratio of alpha-SMA-positive cells cultured for 7 days in serum-free medium was 36 +/- 11.9 % (mean +/- SD). BFGF significantly reduced this ratio in a dose-dependent manner to 11.2 +/- 7.3 % at a concentration of 50 ng/ml (p0.0001). EGF reduced the ratio significantly to 25.1 +/- 15.7 % (p = 0.05) when 50 ng/ml were applied. IGF-1 (10 ng/ml) reduced the relative numbers of transdifferentiated cells to 16.8 +/- 5.8 %, but the reduction was not statistically significant (p = 0.0787). TGF-beta2 (50 ng/ml) slightly increased the relative number of alpha-SMA-positive cells to 44.2 +/- 13.8 %. However, this increase was not significant (p = 0.1202) during a culture period of 7 days.BFGF and EGF significantly reduced the expression of alpha-SMA by LECs while TGF-beta and IGF-1 had no statistically significant effect. These results suggest that bFGF and EGF do not primarily induce secondary cataract formation by the mechanism of cell transdifferentiation.

Scrotal (n = 66), inguinal (n = 7) and abdominal (n = 36) testes from pigs differing in age (from 1 day to 4 years) were collected after slaughtering or by castration, fixed in 4% formalin and examined by light microscopy. The expression of vimentin, desmin, alpha-actin and PCNA was detected by immunohistochemical technique. Histologically no differences between scrotal and cryptorchid testes are obvious during the prepubertal period, until the age of 10-12 weeks, but with the onset of puberty degenerative alterations (Sertoli-cell-only morphology, giant cells) in dystopic testes occur. Additionally the differentiation of the interstitial cells is disturbed in abdominal testes resulting in a deviating expression pattern of alpha-actin. The Sertoli cells show different distribution patterns (basal, perinuclear, apical) of vimentin depending on the position of the testis and the age of the animal.

Dilated cardiomyopathy is one of the leading causes of heart failure and a primary cause for heart transplantation in patients below the age of 40 years. Despite major advances in diagnostic procedures such as examination of myocardial biopsies, the etiology remains unknown in many patients. Chronic inflammation or myocarditis and chronic alcohol abuse are considered two main etiologic factors in dilated cardiomyopathy. A third causal factor, namely genetic transmission of the disease, is at least as common as myocardial inflammation or toxic damage. Several prospective studies of relatives of patients with dilated cardiomyopathy proved that about 25-30% of all cases are of familial etiology. The most common mode of inheritance is autosomal dominant. Less frequently is the disease inherited as an X-chromosomal trait. Autosomal recessive and mitochondrial transmission is rare. The penetrance is highly variable and age dependent. Many relatives of patients with DCM show only minor cardiac abnormalities and it is unknown whether they progress to full cardiomyopathy in later life. Examination of families has identified so far eight disease genes, namely the dystrophin, tafazzin, cardiac actin, desmin, lamin A/C, delta- sarcoglycan, cardiac beta-myosin heavy chain, and cardiac troponin T gene. Certain mutations in lamin A/C cause conduction system disease and dilated cardiomyopathy, whereas other mutations cause in addition skeletal muscle myopathy. Dystrophin mutations are the cause of the rare X-linked dilated cardiomyopathy without skeletal muscle involvement and a progressive course in young men. Other mutations in the dystrophin gene, mainly deletions, are the cause of the muscular dystrophy Becker and Duchenne which also present with dilated cardiomyopathy. Mutations of the desmin, delta-sarcoglycan, the cardiac actin and beta-myosin heavy chain as well as the troponin T gene are known to cause autosomal dominant-dilated cardiomyopathy without other abnormalities. The infantile X-linked DCM is caused by mutations of the tafazzin gene. The onset of the disease is typically within the first year of life and death occurs usually in childhood. Most patients may in addition be characterized by skeletal myopathy, short stature, neutropenia and abnormal mitochondria, also referred to as Barth syndrome. Knowledge of the DCM disease genes led to the new hypothesis that dilated cardiomyopathy is a disease of the myocardial force generation or force transmission. Many more disease loci are known but the responsible disease genes are not yet identified. Better understanding of the expression and function of disease genes may eventually result in new diagnostic and therapeutic tools in order to improve the prognosis of this severe disorder.

Undifferentiated small-cell hepatoblastoma (HB) is a rare malignant tumor of childhood. The cell of origin is supposed to be a pluripotential, probably entodermal, stem-cell. Differential diagnosis of this type of HB is difficult among the group of small round and blue cell malignant tumors of children. The immunohistochemically determined coexpression of cytokeratin 8, 18, and 19 and of vimentin and actin, regularly in the absence of alpha-fetoprotein expression may be diagnostically helpful. We present the case of an undifferentiated small-cell HB of a 15-month-old girl with agenesis of the right kidney. As morphological peculiarity the tumor presented disseminated histiocytic giant cells.

Contraction of the capsule of the ocular lens is based upon proliferation and contraction of transformed lens epithelial cells. It is assumed that these processes can be assisted by postoperative intraocular inflammation. Previously, we reported that lens epithelial cell proliferation is enhanced by lymphocyte-conditioned medium (LCM). In this study we investigated the effect of LCM as well as of a culture medium conditioned by pigmented ciliary epitheilal cells (CBCM) on the expression of the smooth-muscle alpha-actin of the contractile cytoskeletal elements.Explants of the anterior lens capsule of freshly enucleated bovine eyes were cultured in serum-free LCM and CBCM for 3 days, followed by fixation. Smooth-muscle alpha-actin was identified by indirect immunoflorescence. Explants cultured in serum-free bFGF-containing and TGF-beta containing medium served as control.Lens epithelial cells expressed smooth-muscle alpha-actin under the influence of LCM or TGF-beta. No smooth muscle alpha-actin could be detected under the influence of CBCM or bFGF.Our results demonstrate that secreted molecules of activated lymphocytes are able to induce the transformation of lens epithelial cells into contractile myofibroblasts and may be involved in the post-operative contraction of lens capsules.

Myositis proliferans is a reactive, intramuscular soft tissue disease characterized by fibroblast and myofibroblast proliferation, showing similarities to the phase-like development seen in the general pathology of wound healing and hypertrophic scars. Immunohistochemically, a combined expression of vimentin and alpha-sm actin is seen in the spindle-shaped cell formations. The decisive histological preparations is supported by immunohistochemical techniques, especially in the differentiation from sarcoma. If a definite diagnosis is made, incomplete excision may suffice.

Epiretinal tissue proliferations occurring during the evolution of ischemic microangiopathies or preretinal diseases are tough to cause retinal detachment by traction mechanisms. Cellular migration/proliferations and finally contraction are tough to be the pathogenic element. Myofibroblasts are contractile cells having features intermediate between those of the fibroblasts and smooth muscle. We conducted a study to explore whether such cells are present in preretinal membranes.8 membranes, preelevated during vitrectomy for proliferative vitreoretinopathy or diabetic proliferative vitreoretinopathy, were analysed with immunostaining technique searching for alpha-actine smooth muscle, desmine, which are specific markers for myofibroblasts and TGF-beta1, that is considered as the mean factor promoting the transformation of fibroblasts into myofibroblasts.All the histological preparation showed abundant staining with antibody against alpha-actine smooth muscle, desmine and TGF-beta1.Myofibroblasts are one of the major cellular element of preretinal membranes. They are scattered throughout the membrane and seem to account for their contractile properties.

Two female and three male patients with acquired "tufted angioma" are presented. The age of these patients ranged from 10 to 62 years. Two lesions were sited in the head and neck region, two in the upper extremities, and one on the trunk. Clinically, the angiomatous lesions appeared as elevated plaques, flat lesions with papular and macular areas, or erythematous plaques with small nodules. In four cases a biopsy was done, and in one case the tumour was excised. Histologically, the neoplasms were characterized by irregularly distributed vascular tufts in the dermis, and, in one case, in the upper subcutis. The vascular tufts were composed of plump endothelial cells and spindle-shaped pericytes surrounded by crescent-shaped vascular spaces. The positive staining for CD 31 and for CD 34 and alpha-smooth muscle actin, and the negative staining of endothelial cells for factor VIII underline both the existence of two cellular components in tufted angioma and the immaturity of endothelial cells. Evidence of regular mitotic figures in two cases and increased proliferative activity in three out of four cases tested, emphasize the neoplastic nature of slowly growing tufted angioma. Benign tufted angioma is a distinct entity in the spectrum of capillary haemangiomas and must be distinguished from other vascular neoplasms.

Fibrovascular polyps of the esophagus account for 0.5-1% of all esophageal tumors. These are usually pedunculated lesions that originate below the upper esophageal sphincter. Etiology is unknown. Histological examination typically shows a fibrovascular stroma, which is covered by non-keratinizing squamous epithelium. Seventy-five percent of all patients are male, and are usually between 40 and 70 years of age. Most fibrovascular polyps cause few but non-specific symptoms. Occasionally they are regurgitated into the oral cavity and may then give rise to choking episodes and even fatal aspiration. Fibrovascular polyps are best diagnosed by endoscopy and/or radiography. Differential diagnosis includes carcinoma and intramural myoma. Once diagnosis is made, surgical removal, is indicated by either transoral or lateral cervical approaches. With adequate treatment prognosis is excellent.

This is a report on a patient of 74 years of age presenting with ear pain and bleeding granulations on his tympanic membrane. X-ray of the mastoid revealed partial destruction of the bone structures and reduced pneumatisation. A tumour that was mainly located in the temporal bone was surgically removed and proved to be a leiomyosarcoma. This is the first case of a leiomyosarcoma of the temporal bone to be described. Differential diagnosis and therapy of this tumour are discussed.

An example of a benign haemorrhagic spindle cell tumour of the lymph node (synonym: intranodal myofibroblastoma) is presented, which was first described 5 years ago and has not as yet been documented in the German literature. This rare benign mesenchymal neoplasm of the lymph node occurs almost exclusively in the inguinal region. Diagnostic criteria include its composition of myofibroblastic cells (with immunoreactivity for vimentin and actin), which may show nuclear palisading, the presence of peripheral haemorrhage zones and the occurrence of characteristic amianthoid structures, which have the ultrastructural appearance of dense meshworks of collagen fibers. Knowledge of this tumour entity is important for differential diagnosis of primary and secondary malignant mesenchymal neoplasias of the lymph node.

Chondroid syringoma belongs to the group of so-called mixed tumors, like pleomorphic adenomas of the lacrimal and salivary glands. The histogenesis of this tumour is still disputed, in particular with respect to its stromal component. The distribution of cytokeratins (CKs), CEA, EMA, vimentin, S-100 protein, desmin and actin [alpha-smooth muscle actin (alpha-SMA)] was investigated by immunohistological examination of paraffin sections from a chondroid syringoma of the apocrine type. The neoplastic formations have been classified into tubuloalveolar structures, solid nests/aggregations and stromal cells of varying morphology. The inner-most cells of tubuloalveolar structures were characterized by marked expression of CKs (KL1 and MNF116), CEA and EMA, while in the outer ones there was moderate expression of vimentin, S-100 was expressed to a lesser extent and KL1, weakly but there was marked and consistent expression of MNF116. Whereas the solid nests expressed vimentin, S-100 protein, MNF116 markedly and KL1 weakly, the stromal cells were consistently positive for vimentin, S-100 protein and, focally, CKs and alpha-SMA. Anti-alpha-SMA specifically detects myoepithelial cells. In addition, the partly overlapping immunoreactivity of the intermediate filaments, membrane proteins and proteins in the different structures may indicate a common clonal origin of all neoplastic cells in chondroid syringoma.

Alterations in gene expression are frequently encountered in malignant tissues. Quantification of the often quite subtle changes in mRNA content is routinely performed by northern blot hybridization. Practical experience shows that the methodology is subject to considerable error, necessitating the use of an internal mRNA standard for quantification. Many different control mRNAs have been employed over the years, but no systematic study has proven their applicability to comparisons made between tumor cells and normal tissues. The validity of these control RNAs, usually housekeeping genes, must be established to exclude the possibility that they themselves are expressed at different levels in tumor and non-tumor cells. Our experience has revealed that serious discrepancies may arise when incorrect standard genes are used to compare tumor and normal tissues.

Minor salivary gland neoplasms are extremely rare in the nasopharynx. We herein report a case of a 68-year-old man with a rapidly expanding myoepithelial carcinoma of the nasopharynx. The classification of this carcinoma as well as the difficulty in effective treatment are discussed A review of the literature is given.

A 20-year-old white man demonstrated a 2-3 mm in diameter ball like, grey white vascularized tumor at 12h in the iris stroma adjacent to the sphincter pupillae OD. The tumor was excised by sector iridectomy. Light microscopically the tumor consisted of densely packed spindle-shaped cells with oval nuclei with granular eosinophilic cytoplasm. Electronmicroscopically numerous 9nm-filaments in parallel configuration were found, fusiform dense formations, basal membranes and pinocytotic vesicles. Positive immunohistochemical reaction for Actin, Desmin and Vimentin, negative for S100.

Spontaneous sound signals emitted from the inner ear were first recorded by Kumpf and Hoke in 1970. Kemp reported phenomena of sounds which were emitted by the ear responding to acoustic stimulation. These "otoacoustic emissions" are supposed to be generated in the outer hair cells. Active contractions of the actin and myosin in these cells produce a frequency specific cochlear amplifier mechanism. Although the clinical value of spontaneous otoacoustic emissions is yet unclear, as well as role in tinnitus, the recording of click-evoked otoacoustic emissions has now become diagnostic routine. Click-evoked otoacoustic emissions cannot be recorded in ears with cochlear mid-frequency hearing lossesor = 25 dB. However, the use of sinus tones or distortion products as stimuli promises more frequency-specific results. Most important for the quality of measurement is complete closure of the external ear canal, correct positioning of the recording probe, maximal suppression of background noise and sufficient compliance of the patient, especially when testing children. Middle ear effusions also prevent recording. Responses to a standard of 260 stimulations are averaged and identified as "true" emissions by their sufficient reproducibility and characteristic pattern in frequency analysis. Currently, the recording of click-evoked otoacoustic emissions can be used to detect early discrete lesions of the outer hair cells. Their use as a screening tool concerning cochlear hearing disorders is already possible in newborn children. Isolated central hearing disorders still cannot be detected by this diagnostic procedure.

Leiomyosarcomas arise rarely in the head and neck. We report a 41-year-old woman with an extremely rare leiomyosarcoma of the floor of the mouth extending to the oropharynx. This article stresses the value of imaging methods such as CT, MRI and ultrasound for preoperative diagnosis, and emphasises histopathological findings. The therapeutic principles and prognostic factors of leiomyosarcomas are illustrated on the basis of this case.

This report describes a 50-year-old male patient with fibroma of tendon sheath. The tumour had grown rapidly, and clinical examination of it led to the suspicion of a giant cell tumour of tendon sheath and a vascular glomus tumour. Microscopical investigation then allowed diagnosis of the tumour as a fibroma of tendon sheath, which was characterized by spindle-shaped and stellate-shaped fibroblasts, a fibrocollagenous, partly myxoid stroma, and slit-like vessels. Immunohistochemically, most tumour cells expressed vimentin and smooth muscle actin, but not desmin. Thus, fibroma of tendon sheath can be regarded as a synovial tumour with myofibroblastic differentiation. Histologically, fibroma of tendon sheath must be differentiated from other tumours, such as giant cell tumour of tendon sheath and histiocytoma, but also from nodular fasciitis and sarcomas.

Vasoactive agents which elevate either cGMP or cAMP inhibit platelet activation by pathways sharing at least one component, the 46/50 kDa vasodilator-stimulated phosphoprotein (V ASP). V ASP is stoichiometrically phosphorylated by both cGMP-dependent and cAMPdependent protein kinases in intact human platelets, and its phosphorylation correlates very well with platelet inhibition caused by cGMP- and cAMP-elevating agents. Here we report that in human platelets spread on glass, V ASP is associated predominantly with the distal parts of radial micro filament bundles and with microfilaments outlining the periphery, whereas less V ASP is associated with a central microfilamentous ring. V ASP is also detectable in a variety of different cell types including fibroblasts and epithelial cells. In fibroblasts, V ASP is concentrated at focal contact areas, along microfilament bundles (stress fibres) in a punctate pattern, in the periphery of protruding lamellae, and is phosphorylated by cGMP- and cAMP-dependent protein kinases in response to appropriate stimuli. Evidence for the direct binding of V ASP to F -actin is also presented. The data demonstrate that V ASP is a novel phosphoprotein associated with actin filaments and focal contact areas, i.e. transmembrane junctions between microfilaments and the extracellular matrix.

The ADP-ribosylation of actin is the pathobiochemical mechanism by which various clostridial toxins affect the eukaryotic target cell. The toxins are binary in structure and consist of a binding component and an enzyme component with ADP-ribosyltransferase activity. Probably endocytosis-mediated the binding component transfers the clostridial ADP-ribosyltransferase into the target cell, whereupon G-actin but not F-actin is ADP-ribosylated. The ADP-ribosylated actin is incapable of polymerization but binds to the barbed ends of actin filaments to inhibit polymerization of non-modified actin. Thereby the ADP-ribosylation of actin destroys the cellular architecture of the microfilament network. The ADP-ribosylating toxins are novel tools to study the physiological functions of actin.

Primary leiomyosarcoma of the heart is very rare, and in most cases the diagnosis is performed during postmortem examinations. We report on a 71-year-old woman with a large leiomyosarcoma of the right atrium. The preoperative diagnosis of cardiac tumor was made by 2-D echocardiography, transesophageal echocardiography, computed tomography, and MR-imaging, and was confirmed by histological and immunhistological findings of the resected part.

Using fluorescence microscopy, cytoskeletal actin filaments were found in the terminal blood vessels of chicken intestine. Direct detection with Tetramethylrhodaminyl-Phalloidin showed a high concentration of actin filaments in the tunica media and a low concentration in the endothelium. Actin filaments were also present in the endothelial cells of capillaries and to a higher degree also in the pericytes. The wall of the veins in the villi, which were concentrically surrounded by bundles from the tunica muscularis mucosae, had a greater number of actin filaments than the wall of the venules present in the villi. The discussion touches on the possibility that the microfilaments play a role in blood regulation, especially in the capillaries.