[Implementation of biosecurity measures by hoof trimmers in Switzerland]. / Umsetzung von Biosicherheitsmassnahmen durch Klauenpfleger in der Schweiz.

INTRODUCTION: Biosecurity in livestock farming includes all measures preventing pathogen introduction onto a farm (external biosecurity) and pathogen transmission on the farm itself (internal biosecurity). An important risk factor for the dissemination of infectious diseases are specialised external persons working on numerous farms, such as professional hoof trimmers in Switzerland. In the present study, 49 hoof trimmers, participating in the Swiss claw health programme and working as professionals, were questioned regarding their biosecurity measures and observed by two veterinarians during hoof trimming in order to assess the implementation of biosecurity measures by hoof trimmers. Data were processed using a scoring system, in which points were allocated to the different working methods taking into account their assumed transmission potential for infectious diseases such as digital dermatitis (DD) and Salmonellosis. The working method, which complied with the ideal biosecurity measure, was always given a whole point, whereas less optimal working methods were only given an intermediate value or no point. The scoring system helped identify precisely the strengths and weaknesses of the hoof trimmers in terms of biosecurity. The level of implementation of biosecurity measures by hoof trimmers was overall quite low (53 %=average of the overall biosecurity scores of the 49 hoof trimmers). Hoof trimmers which attended specialised training courses tended to have a higher level of implementation of biosecurity measures. The answers given by the hoof trimmers and the observations made by the veterinarians were compared, whereby it was found that hoof trimmers generally evaluated themselves better in regard to biosecurity than veterinarians assessed them. In summary and based on the results of this study, the dissemination of pathogens, such as DD associated treponemes and salmonella is possible during hoof trimming performed by external persons working on numerous farms. Thus, future training and continuing education courses should place emphasis on biosecurity.

INTRODUCTION: Le concept de biosécurité englobe, en lien avec la production animale, toutes les mesures empêchant l'introduction de germes dans une exploitation (biosécurité externe) et la propagation de germes à l'intérieur de l'exploitation (biosécurité interne). Un facteur de risque important pour la propagation de maladies infectieuses est le personnel spécialisé externe travaillant sur plusieurs exploitations, dont font partie les pareurs d'onglons professionnels intervenant sur les exploitations bovines suisses. Dans la présente étude, afin de donner un aperçu de la situation actuelle concernant la mise en oeuvre de mesures de biosécurité par les pareurs d'onglons, 49 pareurs d'onglons participant au programme suisse de santé des onglons, ont été questionnés à ce sujet et observés lors du parage des onglons par des vétérinaires. Le traitement des données a été effectué à l'aide d'un système de notation, attribuant des points aux différentes pratiques de travail selon leur potentiel supposé de transmission des maladies infectieuses que sont la Dermatite digitale (DD) et la Salmonellose. La pratique de travail, qui correspondait à la mesure de biosécurité idéale, obtenait toujours un point entier, alors que les pratiques de travail moins optimales ne recevaient qu'une valeur intermédiaire ou aucun point. Le système de notation a permis de désigner précisément les forces et les faiblesses des pareurs d'onglons en terme de biosécurité. Le niveau de mise en œuvre de mesures de biosécurité par les pareurs d'onglons est de manière générale relativement faible (53 % = moyenne du score de biosécurité générale des 49 pareurs). Les pareurs d'onglons ayant suivi plus fréquemment des formations spécifiques présentaient tendanciellement un niveau de mise en oeuvre de mesures de biosécurité plus élevé. De plus, les réponses des pareurs d'onglons et les observations des vétérinaires ont été comparées. Il a été constaté, que les pareurs d'onglons s'évaluaient généralement meilleurs en matière de biosécurité que les vétérinaires ne les jugeaient. En résumé et en tenant compte des résultats de cette étude, la propagation de germes pathogènes par les pareurs d'onglons dans le cadre de leur activité professionnelle, tels que les tréponèmes associés à la DD et les salmonelles, est possible. Par conséquent, la biosécurité devrait être thématisée en priorité lors des formations et formations continues futures.

[Prevalence of claw disorders in swiss cattle farms]. / Prävalenz von Klauenveränderungen in Schweizer Rindviehbetrieben.

INTRODUCTION: The project «Healthy claws - the foundation for the future¼ aims to establish a Swiss national claw health monitoring based on digital recordings by claw trimmers during claw trimming. To assess claw health on the participating farms, between-herd prevalence, within-herd prevalence and cow prevalence of all claw disorders based on the «ICAR Claw Health Atlas¼ were calculated during this study. Claw trimmers underwent an intensive training and examination in order to ensure data quality. To guarantee the representativity of the prevalences, only farm claw trimmings were considered, where ≥ 80 % of the cows in a herd were trimmed. The calculations were based on 7108 cows and 403 heifers from 238 farms, during the period from February 2020 to February 2021. At least one claw disorder was present in 99,2 % of the farms, with 49,6 % of the heifers and 77,7 % of the cows having at least one claw disorder. The high prevalence is seen as a result of all ICAR claw disorders being considered, whereas not all of them are painful and consequently not all of them cause lameness. The absence of lameness assessment limits the evaluation of existing herd problems. High between-herd and cow prevalences were observed for the following claw disorders: heel horn erosion (92,9 %/64,7 %), digital dermatitis (55,9 %/20,7 %), white line disease (81,5 %/17,7 %) and sole hemorrhage (66,4 %/11,6 %). Asymmetric claws, corkscrew claws, scissor claws, horn fissure, interdigital phlegmon, swelling of the coronet and/or bulb and toe necrosis had low prevalences. The proportion of cows treated with a hoof block (0,5 %) was comparatively small in regard of the cows suffering from ulcers (5,6 %) and white line abscesses (2,5 %). The median within-herd prevalence of digital dermatitis was 5,6 %, with a maximal within-herd prevalence of 87,5 %. Despite the contagious nature of digital dermatitis, no increase of between-herd and cow prevalence has been observed in the past ten years throughout Switzerland. Based on this data, the Swiss claw health situation can be monitored, compared over time and improved in the future.

INTRODUCTION: Le projet « Des onglons sains-de bon pied vers l'avenir ¼ vise à établir une surveillance nationale suisse de la santé des onglons basée sur des enregistrements numériques effectués par des ongleurs pendant le parage des onglons. Pour évaluer la santé des onglons dans les exploitations participantes, la prévalence entre les troupeaux, la prévalence au sein du troupeau et la prévalence chez les vaches de tous les troubles des onglons sur la base de l' « ICAR Claw Health Atlas ¼ ont été calculées au cours de cette étude. Les ongleurs ont subi une formation et un examen intensifs afin de garantir la qualité des données. Afin de garantir la représentativité des prévalences, seuls ont été considérés les parages d'exploitations où ≥ 80% des vaches du troupeau ont été parées. Les calculs ont été basés sur 7108 vaches et 403 génisses de 238 élevages, au cours de la période de février 2020 à février 2021. Au moins une lésion des onglons était présente dans 99,2 % des élevages, avec 49,6 % des génisses et 77,7 % des vaches présentant au moins une lésion. La prévalence élevée est considérée comme le résultat de toutes les lésions selon ICAR, alors que toutes ne sont pas douloureuses et par conséquent ne provoquent pas toute de boiterie. L'absence d'évaluation de la boiterie limite l'évaluation des problèmes de troupeau existants. Des prévalences élevées entre les troupeaux et les vaches ont été observées pour les lésions des onglons suivantes : érosion de la corne du talon (92,9%/64,7%), dermatite digitale (55,9%/20,7%), maladie de la ligne blanche (81,5 %/17,7%) et hémorragie de la sole (66,4%/11,6%). Les onglons asymétriques, les onglons en tire-bouchon, les onglons en ciseaux, la fissure de la corne, le phlegmon interdigital, le gonflement de la couronne et/ou la nécrose de la pince avaient de faibles prévalences. La proportion de vaches traitées avec un sabot (0,5 %) était comparativement faible par rapport aux vaches souffrant d'ulcères (5,6%) et d'abcès de la ligne blanche (2,5%). La prévalence médiane intra-troupeau de dermatite digitale était de 5,6%, avec une prévalence intra-troupeau maximale de 87,5%. Malgré le caractère contagieux de la dermatite digitale, aucune augmentation de la prévalence entre troupeaux et vaches n'a été observée au cours des dix dernières années dans toute la Suisse. Sur la base de ces données, la situation sanitaire des onglons en Suisse peut être surveillée, comparée dans le temps et améliorée à l'avenir.

[Evaluation of a novel training course for hoof trimmers to participate in a Swiss national cattle claw health monitoring programme]. / Evaluation eines Ausbildungskonzeptes für Klauenpfleger zur Durchführung eines schweizweiten Klauengesundheitsmonitorings für Rinder.

INTRODUCTION: The main goal of the resources project «Healthy claws - the foundation for the future¼ is to establish a Swiss national claw health monitoring programme for cattle, similar to what has already been established in other countries (e. g. Finland, Sweden). So far in the course of the project, 30 claw trimmers have been trained to use a digital recording software. An appropriate training concept and information to the work environment of Swiss claw trimmers are necessary to ensure that the recorded claw health data is of good quality. The newly developed training programme for claw trimmers was evaluated using the 30 first trained claw trimmers of the project. The training consisted of group events and individual schooling sessions, during which the claw trimmers were trained to recognise, classify and digitally record foot and claw disorders according to the «ICAR claw health atlas¼. At the end of the training programme, demographic and work relevant data about the claw trimmers was collected and their abilities when using the recording software, as well as recognising and classifying foot and claw disorders using pictures or live animals, were evaluated. The demographic and work relevant data collected at the end of the training showed an ageing of the profession (43% of the participants were over 50 years old), a lack of full-time claw trimmers (23% of the claw trimmers worked 90% or 100%), a neglect of locomotion scoring during claw trimming (no locomotion scoring standing from 60% and in movement from 33% of the participants) and a broad use of the Swiss claw trimming method (90% of the claw trimmers indicate using this method). An average Cohens kappa value of 0,74, and thus an overall substantial agreement with the first author, respectively the ICAR Atlas, was achieved for the identification and classification of foot and claw disorders. It was also noted, that all the participants were capable of using the recording software in practice after their schooling. The calculation of Cohens kappa values helps to recognise claw trimmers which fall below the limiting value and therefore show an insufficient agreement. These claw trimmers can thus be excluded from the scientific evaluation or undergo further training. It was concluded that the described training concept is suitable to establish a national claw health monitoring programme.

INTRODUCTION: L'objectif principal du projet «Des onglons sains ­ de bon pied vers l'avenir¼ est la mise en place d'un programme national suisse de surveillance de la santé des onglons des bovins, similaire à ce qui a déjà été mis en place dans d'autres pays (par ex. Finlande, Suède). Jusqu'à présent, au cours du projet, 30 pareurs d'onglons ont été formés à l'utilisation d'un logiciel de documentation électronique. Un concept de formation approprié et des informations sur l'environnement de travail des pareurs suisses sont nécessaires pour garantir la bonne qualité des données enregistrées. Le programme de formation nouvellement mis au point a été évalué à l'aide des 30 premiers pareurs formés. La formation a consisté en des formations de groupe et des formations individuelles, au cours desquelles les pareurs ont été formés à reconnaître, classifier et enregistrer électroniquement les maladies des onglons selon l'«Atlas ICAR santé des onglons¼. À la fin de la formation, des données démographiques et professionnelles concernant les pareurs ont été collectées et leurs capacités à utiliser le logiciel d'enregistrement, à reconnaître et classifier les maladies des onglons à l'aide d'images ou sur des animaux vivants, ont été évaluées. Les données démographiques et professionnelles collectées à l'issue de la formation ont entre autre montré un vieillissement de la profession (43% des participants avaient plus de 50 ans), un manque d'ongleurs à temps plein (23% des ongleurs travaillent à 90% resp. 100%), une négligence de la notation de la motricité lors du parage des onglons (pas de notation de la motricité stationnaire chez de 60% et en mouvement chez33% des participants) et une large utilisation de la méthode suisse de parage des onglons (90% des ongleurs indiquent utiliser cette méthode). Une valeur moyenne du coefficient kappa de Cohens de 0,74, et donc une concordance globale avec le premier auteur, respectivement l'Atlas ICAR, a été obtenue pour l'identification et la classification des maladies des onglons. Il a également été constaté que tous les participants étaient capables après leur formation d'utiliser le logiciel d'enregistrement dans la pratique. Le calcul de valeurs kappa de Cohens permet de reconnaître les pareurs qui descendent en dessous d'une valeur limite et présentent donc une concordance insuffisante. Ces pareurs peuvent ainsi être exclus de l'évaluation scientifique ou suivre une formation complémentaire. On peut en conclure que le concept de formation décrit convient pour la mise en place d'un programme national de surveillance de la santé des onglons.

[Improvement of claw health of cattle in Switzerland]. / Verbesserung der Klauengesundheit beim Rind in der Schweiz.

INTRODUCTION: The modern technique of cattle hoof care was founded by E. Toussaint Raven in 1977. Environmental risk factors on cattle claws altered in the past 43 years. The change from free ranging to indoor housing, the intensified feeding and the breeding towards traits of high performance have significantly increased the mechanical and chemical stress on the claws. In modern free-stalls, dairy cows are required to walk on hard flooring to feed, drink and get milked. Good hoof health is a basic requirement for cattle welfare. Professional and regular hoof trimming is still considered the most effective measure to promote hoof health in dairy cattle. In order to meet today's requirements and to promote claw health, the Swiss Hoof Trimmers Association (SKV), in collaboration with the Vetsuisse faculties, Universities of Berne and Zurich, and the Bovine Health Service (RGD, Bern) developed and described the Swiss technique of functional claw trimming. The aim was to establish a consistent method, which takes into account the size and bodyweight of the modern cow, the anatomical and physiological characteristics of their claws and includes adaptations counteracting very relevant diseases such as digital dermatitis. The result is a workflow described and illustrated with coloured pictures and consisting of five individual steps based on the technique of E. Toussaint Raven, Additionally, the upcoming Swiss national resource project on long-term improvement of claw health is presented in some detail. The key point of this project is the electronic documentation of clinical findings by the trained professional claw trimmers. This data will later (i) be used to assess the foot health of Swiss cows, (ii) allow to determine the prevalence of foot diseases of cattle in Switzerland and (iii) to monitor the effect of the implementation of foot health concepts. The aim of this work is to combine the findings from science and the practical experience of hoof trimmers in one method, to standardize the applied hoof care in Switzerland and to adapt it to today's hoof health requirements.

INTRODUCTION: La technique moderne du soin des pieds des bovins et du parage des onglons a été fondée par E. Toussaint Raven en 1977. Au cours des 43 dernières années, le passage d'une détention au pâturage à une détention à l>intérieur, l'intensification de l'alimentation et la sélection vers des caractéristiques de hautes performances ont provoqué une augmentation sensible du stress mécanique et chimique exercé sur les onglons. Dans les stabulations libres modernes, les vaches laitières doivent se déplacer activement sur un sol dur pour se nourrir, boire et se faire traire. Pour cela, une marche indolore est une exigence de base. Un parage professionnel et régulier est toujours considéré comme la mesure la plus efficace pour promouvoir la santé des onglons chez les bovins laitiers. Afin de répondre aux exigences actuelles et de promouvoir la santé des onglons, l' Association suisse des pareurs d'onglons (ASPO), en collaboration avec les facultés Vetsuisse des universités de Berne et de Zurich et le Service sanitaire bovin (SSB, Bern) a développé et décrit une technique suisse de parage fonctionnel des onglons. L'objectif était d'établir une méthode cohérente, qui tienne compte de la taille et du poids corporel de la vache moderne, des caractéristiques anatomiques et physiologiques de ses onglons et qui inclue en même temps des innovations pour lutter contre des maladies très importantes telles que la dermatite digitée. Le résultat est un processus de travail composé de cinq étapes individuelles adaptées selon E. Toussaint Raven, décrites et illustrées dans un tableau avec des illustrations en couleurs. En outre, un projet de ressource nationale suisse sur l'amélioration à long terme de la santé des onglons est présenté en détail. Dans ce cadre, la documentation électronique des découvertes pathologiques faites pendant le parage des onglons par des ongleurs professionnels et formés représente le cœur de ce projet. Ces données seront ensuite utilisées (i) pour évaluer la santé des pieds des vaches suisses, (ii) pour permettre de déterminer la prévalence des maladies des pieds des bovins en Suisse et (iii) pour surveiller l'effet de la mise en œuvre des concepts de santé des onglons. Le but de ce travail est de combiner les résultats de la recherche et l'expérience pratique des ongleurs en une seule méthode, de standardiser les soins pratiqués en Suisse et de les adapter aux exigences actuelles en matière de santé des onglons.

The past and presence of gene targeting: from chemicals and DNA via proteins to RNA.

The ability to target DNA specifically at any given position within the genome allows many intriguing possibilities and has inspired scientists for decades. Early gene-targeting efforts exploited chemicals or DNA oligonucleotides to interfere with the DNA at a given location in order to inactivate a gene or to correct mutations. We here describe an example towards correcting a genetic mutation underlying Pompe's disease using a nucleotide-fused nuclease (TFO-MunI). In addition to the promise of gene correction, scientists soon realized that genes could be inactivated or even re-activated without inducing potentially harmful DNA damage by targeting transcriptional modulators to a particular gene. However, it proved difficult to fuse protein effector domains to the first generation of programmable DNA-binding agents. The engineering of gene-targeting proteins (zinc finger proteins (ZFPs), transcription activator-like effectors (TALEs)) circumvented this problem. The disadvantage of protein-based gene targeting is that a fusion protein needs to be engineered for every locus. The recent introduction of CRISPR/Cas offers a flexible approach to target a (fusion) protein to the locus of interest using cheap designer RNA molecules. Many research groups now exploit this platform and the first human clinical trials have been initiated: CRISPR/Cas has kicked off a new era of gene targeting and is revolutionizing biomedical sciences.This article is part of a discussion meeting issue 'Frontiers in epigenetic chemical biology'.

Subcutaneous immunotherapy with purified Der p1 and 2 suppresses type 2 immunity in a murine asthma model.

BACKGROUND: Allergen-specific immunotherapy can induce long-term suppression of allergic symptoms, reduce medication use, and prevent exacerbations of allergic rhinitis and asthma. Current treatment is based on crude allergen extracts, which contain immunostimulatory components such as ß-glucans, chitins, and endotoxin. Use of purified or recombinant allergens might therefore increase efficacy of treatment. AIMS: Here, we test application of purified natural group 1 and 2 allergens from Dermatophagoides pteronyssinus (Der p) for subcutaneous immunotherapy (SCIT) treatment in a house dust mite (HDM)-driven mouse model of allergic asthma. MATERIALS AND METHODS: HDM-sensitized mice received SCIT with crude HDM extract, a mixture of purified Der p1 and 2 (DerP1/2), or placebo. Upon challenges, we measured specific immunoglobulin responses, allergen-induced ear swelling response (ESR), airway hyperresponsiveness (AHR), and inflammation in bronchoalveolar lavage fluid (BAL) and lung tissue. RESULTS: ESR measurement shows suppression of early allergic response in HDM-SCIT- and DerP1/2-SCIT-treated mice. Both HDM-SCIT and DerP1/2-SCIT are able to suppress AHR and eosinophilic inflammation. In contrast, only DerP1/2-SCIT is able to significantly suppress type 2 cytokines in lung tissue and BAL fluid. Moreover, DerP1/2-SCIT treatment is uniquely able suppress CCL20 and showed a trend toward suppression of IL-33, CCL17 and eotaxin levels in lung tissue. DISCUSSION: Taken together, these data show that purified DerP1/2-SCIT is able to not only suppress AHR and inflammation, but also has superior activity toward suppression of Th2 cells and HDM-induced activation of lung structural cells including airway epithelium. CONCLUSIONS: We postulate that treatment with purified natural major allergens derived from HDM will likely increase clinical efficacy of SCIT.

Bidirectional modulation of endogenous EpCAM expression to unravel its function in ovarian cancer.

BACKGROUND: The epithelial cell adhesion molecule (EpCAM) is overexpressed on most carcinomas. Dependent on the tumour type, its overexpression is either associated with improved or worse patient survival. For ovarian cancer, however, the role of EpCAM remains unclear. METHODS: Cell survival of ovarian cancer cell lines was studied after induction or repression of endogenous EpCAM expression using siRNA/cDNA or artificial transcription factors (ATF) consisting of engineered zinc-fingers fused to either a transcriptional activator or repressor domain. RESULTS: Two ATFs were selected as the most potent down- and upregulator, showing at least a two-fold alteration of EpCAM protein expression compared with control. Downregulation of EpCAM expression resulted in growth inhibition in breast cancer, but showed no effect on cell growth in ovarian cancer. Induction or further upregulation of EpCAM expression decreased ovarian cancer cell survival. CONCLUSION: The bidirectional ATF-based approach is uniquely suited to study cell-type-specific biological effects of EpCAM expression. Using this approach, the oncogenic function of EpCAM in breast cancer was confirmed. Despite its value as a diagnostic marker and for immunotherapy, EpCAM does not seem to represent a therapeutic target for gene expression silencing in ovarian cancer.

Transcription factors and molecular epigenetic marks underlying EpCAM overexpression in ovarian cancer.

BACKGROUND: The epithelial cell adhesion molecule (EpCAM) is overexpressed on carcinomas, and its downregulation inhibits the oncogenic potential of multiple tumour types. Here, we investigated underlying mechanisms of epcam overexpression in ovarian carcinoma. METHODS: Expression of EpCAM and DNA methylation (bisulphite sequencing) was determined for ovarian cancer cell lines. The association of histone modifications and 16 transcription factors with the epcam promoter was analysed by chromatin immunoprecipitation. Treatment with 5-Aza-2'-deoxycytidine (5-AZAC) was used to induce EpCAM expression. RESULTS: Expression of EpCAM was correlated with DNA methylation and histone modifications. Treatment with 5-AZAC induced EpCAM expression in negative cells. Ten transcription factors were associated with the epcam gene in EpCAM expressing cells, but not in EpCAM-negative cells. Methylation of an Sp1 probe inhibited the binding of nuclear extract proteins in electromobility shift assays; such DNA methylation sensitivity was not observed for an NF-κB probe. CONCLUSION: This study provides insights in transcriptional regulation of epcam in ovarian cancer. Epigenetic parameters associated with EpCAM overexpression are potentially reversible, allowing novel strategies for sustained silencing of EpCAM expression.

Pompe disease: current state of treatment modalities and animal models.

Pompe disease is a rare autosomal recessive lysosomal storage disease caused by deficiency of acid-alpha-glucosidase (GAA). This deficiency results in glycogen accumulation in the lysosomes, leading to lysosomal swelling, cellular damage and organ dysfunction. In early-onset patients (the classical infantile form and juvenile form) this glycogen accumulation leads to death. The only therapy clinically available is enzyme replacement therapy, which compensates for the missing enzyme by i.v. administration of recombinant produced enzyme. The development of clinically relevant animal models gained more insight in the disease and allowed evaluation of recombinant enzyme therapy. Several therapies are currently under investigation for Pompe disease, including gene therapy. This review gives an overview of the available knockout mouse models, of the in vitro and in vivo studies performed using recombinant produced enzyme. Furthermore, it describes current therapeutic approaches for Pompe disease as well as experimental therapies like gene correction therapy.

An SV40 large T-antigen immortalized human umbilical vein endothelial cell line for anti-endothelial cell antibody detection.

OBJECTIVE: Anti-endothelial cell antibodies in serum of patients with different inflammatory diseases can be detected by a whole cell enzyme-linked immunosorbant assay, using primary cultures of human umbilical vein endothelial cells. To avoid repeated isolation, it would be of great value if an immortal endothelial cell line could be used to perform anti-endothelial cell antibody assays. METHODS: In this study endothelial cells from human umbilical and iliac veins and arteries were transfected with a plasmid containing the Simian Virus 40 large T-antigen. Endothelial cell line(s) derived from this procedure were compared with human umbilical vein endothelial cells in the anti-endothelial cell antibody assay. RESULTS: After transfection, clones of homologous cell populations showed an extended lifespan, before entering a period of crisis. In one human umbilical vein endothelial cell clone a subpopulation of cells escaped crisis and became immortal (EVLC2). Telomerase was activated in this endothelial cell line, resulting in maintenance of the telomere length. There was a significant correlation between anti-endothelial cell antibody testing on human umbilical vein endothelial cells and on the cell line EVLC2. CONCLUSION: The Simian Virus 40 large T-antigen immortalized human umbilical vein endothelial cell line EVLC2 may be useful for the detection of anti-endothelial cell antibodies.

Telomere length in breast cancer patients before and after chemotherapy with or without stem cell transplantation.

High-dose chemotherapy and peripheral blood stem cell transplantation (PBSCT) may accelerate telomere length loss in haematopoietic stem cells. As data including pre-and post-treatment samples are lacking, we studied leukocyte telomere length and telomerase activity before and after treatment in breast cancer patients randomized to receive 5 adjuvant courses FEC (5-FU, epirubicin and cyclophosphamide) (n = 17), or 4 x FEC followed by high-dose cyclophosphamide, thiotepa, carboplatin and autologous PBSCT (n = 16). Haemoglobin, MCV, leukocyte-and platelet numbers were assessed prior to (t(0)), 5 months after (t(1)) and 9 months after chemotherapy (t(2)); these parameters were decreased at t(1)and t(2)compared to t(0)(high-dose: all parameters; standard-dose: leukocytes and platelets), and all parameters were lower after high-dose than standard-dose treatment at t(1). Paired individual leukocyte samples of t(0)and t(1)showed telomere length change (determined by telomere restricted fragment (TRF) assay) ranging from +0.8 to -2.2 kb, with a decreased TRF length in 9 patients of both groups. Telomerase activity (determined by TRAP assay) was below detection limit in leukocyte samples of t(0)and t(1). Thus, standard-and high-dose chemotherapy negatively affect haematological reconstitution in this setting. In individual patients, telomere length can be remarkably changed following haematological proliferative stress after treatment.

The epithelial glycoprotein 2 (EGP-2) promoter-driven epithelial-specific expression of EGP-2 in transgenic mice: a new model to study carcinoma-directed immunotherapy.

The human pancarcinoma-associated epithelial glycoprotein-2 (EGP-2), a M(r) 38,000 transmembrane antigen also known as 17-1A or Ep-CAM, is commonly used for targeted immunotherapy of carcinomas because it is strongly expressed by most carcinomas. EGP-2 is, however, also expressed in most normal epithelia. To evaluate anti-EGP-2-directed treatment-associated effects on tumors and on EGP-2-positive normal tissue, we generated EGP-2-expressing transgenic mice. A 55-kb DNA fragment consisting of the 14-kb genomic coding sequence of the human EGP-2 gene with approximately 10-kb-upstream and approximately 31-kb-downstream sequences was isolated and used to direct EGP-2 expression in an epithelium-specific manner. In the EGP-2 transgenic mice, EGP-2 appeared to be specifically expressed in all of those epithelial tissues that also express EGP-2 in humans, whereas all of the other tissues were negative. The specific in vivo localization of the i.v. administered anti-EGP-2 monoclonal antibody MOC31 was studied in EGP-2-positive and -negative tumors induced s.c. in this EGP-2 transgenic mouse model. Immunohistochemical analysis showed specific localization of MOC31 in the EGP-2-positive tumors but not in the EGP-2-negative tumors. No anti-EGP-2 monoclonal antibody localization was observed in any of the EGP-2-positive normal mouse tissues, which indicated a limited in vivo accessibility. In conclusion, an EGP-2 transgenic mouse model has been generated that expresses the EGP-2 antigen as in humans and, therefore, can serve as a model to evaluate the efficacy and safety of a variety of anti-EGP-2-based immunotherapeutic modalities in both tumors and normal tissue.

Scanning electron microscopic analysis of endothelial cell coverage and quality in large vessels from multi-organ donors: effects of preservation on endothelial cell integrity.

Endothelial cell integrity (coverage and quality) of large donor vessels is important because these vessels are used for vascular reconstructions in solid-organ transplantation. Disruption of the endothelial cell monolayer will initiate blood coagulation and may lead to thrombosis of large vessels, often resulting in the loss of the transplanted organ. Iliac arteries and veins, removed from 10 heart-beating multi-organ donors at the end of the donor procedure, were analyzed using scanning electron microscopy at three different time points of preservation. Endothelial cell coverage and quality were determined immediately after removal from the donor, after 10 h (time of transplantation) and 7 d storage in 'University of Wisconsin' cold preservation solution (UW). Endothelial cell coverage decreased during the preservation of arteries, but was maintained in veins. Storage of the veins for 7 d in plastic bags showed a decreased endothelial cell coverage compared to storage in glass vials. Early removal of the blood vessels and proper storage, free floating and in clean UW, may improve maintenance of the endothelial cell integrity. These findings may be important in order to reduce the risk of thrombosis and, consequently, organ failure after transplantation. Furthermore, vessels with maintained endothelial cell integrity after 7 d may be used for in vitro research.

Telomerase activity in needle biopsies from prostate cancer and benign prostates.

BACKGROUND: Telomerase activation is thought to be essential for the immortality of cancer cells. It may be a prognostic factor in small volume well differentiated prostate cancers and hence a guide for the aggressiveness of the approach. The length of the chromosome tips (telomeres) are maintained by a specific enzyme (telomerase) independently of the normal cell division cycle. Although telomerase is not expressed in most normal human tissues, it is expressed in most human tumours. For the detection of telomerase in small prostate needle biopsy samples a recently developed telomeric repeat amplification protocol (TRAP) assay was used. The aim of the present study was: to measure telomerase activity in human prostate samples, and to evaluate the applicability of this assay on specimens from a prostate biopsy. MATERIALS AND METHODS: From 36 patients referred for lower urinary tract symptoms (LUTS) or suspicion of having prostate cancer a total of 288 prostate biopsy samples were obtained (8 in each patient). When the digital rectal examination was abnormal and/or when the PSA level was elevated in L.U.T.S., or asymptomatic patients' tissue samples were obtained by transrectal ultrasound (TRUS) guided biopsies. Samples were tested for telomerase activity by a modified TRAP and forwarded for histology. RESULTS: In 19 out of 36 patients prostate cancer was diagnosed on histology. In 11 of these 19 tumours substantial telomerase activity was detected, whereas only very low telomerase activity existed in 2 of 17 samples from benign prostatic hypertrophy (BPH) patients. In this small series the relative telomerase activity in prostate cancer correlated with histopathological grade. CONCLUSIONS: Our results show the applicability of a TRAP assay to measure telomerase activity in small needle biopsied prostate samples. In poorly differentiated and metastatic cancer we observed that levels of telomerase activity were high. To establish accuracy and to distinguish the 'relative good from the ugly' further study is needed.

The ubiquitin-activating enzyme E1-like protein in lung cancer cell lines.

The UBE1L gene isolated from the chromosome 3p21 region has an extremely reduced level of mRNA in lung cancer. Sequence analysis showed a 45% homology to the human ubiquitin-activating enzyme E1 at the amino acid level. To further characterize the protein product, we generated UBE1L protein-specific antibodies. Immunoblot analysis revealed a full-length gene product of approximately 112 kDa. Assessment of the level and distribution pattern of the UBE1L protein in normal and tumor tissue using the generated antibodies showed that the UBE1L protein was present in normal lung cells and non-lung cancer cell lines, but was undetectable in all 14 human lung cancer cell lines analyzed. This difference in expression of the UBE1L protein between normal lung tissue and lung tumor-derived cell lines suggests a possible involvement of an E1-like protein in the origin and/or progression of lung tumors.

Characterization of immortalized human umbilical and iliac vein endothelial cell lines after transfection with SV40 large T-antigen.

Most in vitro studies of human endothelial cells have relied on cells derived from human umbilical veins (HUVEC); however, heterogeneity of primary cultured endothelial cells can make critical interpretation of results difficult. Several endothelial cell lines have been produced to serve as a more constant source of endothelial cells. In this study, we characterized the endothelial cell lines EVLB3 and EVLC2 derived from HUVEC, and EVLK1 and EVLK2 derived from human iliac vein endothelial cells (HIVEC). These cell lines maintained the typical endothelial cell cobblestone morphology and appeared to be growth factor independent. They lost PECAM-1 and von Willebrand factor, GP96 was reduced to the level of vascular smooth muscle cells (SMC), but aSMC-actin was far less than in vascular SMC. Antigen levels of tissue-type plasminogen activator (tPA) and plasminogen activator inhibitor (PAI-1) were comparable with young endothelial cells, and mRNA was present for tPA, PAI-1, tissue factor (TF), tissue factor pathway inhibitor and thrombomodulin. This study revealed that mRNA and protein expression of coagulation and fibrinolytic factors was influenced by the stage of cell confluence. No differences could be detected between the endothelial cell lines derived from HUVEC and HIVEC. These cell lines may be a useful tool for studies on cellular interactions of fibrinolytic components or exploring the regulation of TF expression.

An EGP-2/Ep-CAM-expressing transgenic rat model to evaluate antibody-mediated immunotherapy.

The human pancarcinoma-associated epithelial glycoprotein-2 (EGP-2), also known as 17-1A or Ep-CAM, is a 38-kDa transmembrane antigen, commonly used for targeted immunotherapy of carcinomas. Although strongly expressed by most carcinomas, EGP-2 is also expressed in most simple epithelia. To evaluate treatment-associated effects and side-effects on tumor and normal tissue respectively, we generated an EGP-2-expressing transgenic Wistar rat. To express the cDNA of the EGP-2 in an epithelium-specific manner, the 5' and 3' distal flanking regions of the human keratin 18 (K18) gene were used. EGP-2 protein expression was observed in the liver and pancreas, whereas EGP-2 mRNA could also be detected in lung, intestine, stomach and kidney tissues. In this rat, EGP-2-positive tumors can be induced by injecting a rat-derived carcinoma cell line transfected with the GA733-2 cDNA encoding EGP-2. Transgenic rats were used to study specific in vivo localization of an i.v. anti-EGP-2 monoclonal antibody, MOC31, applied i.v. Immunohistochemical analyses showed the specific localization of MOC31 in s.c. induced EGP-2-positive tumors, as well as in the liver. In contrast, in EGP-2-transgenic rats, MOC31 did not bind to EGP-2-negative tumors, the pancreas, or other normal tissues in vivo. In conclusion, an EGP-2-transgenic rat model has been generated that serves as a model to evaluate the efficacy and safety of a variety of anti-EGP-2-based immunotherapeutic modalities.

Regulation of spontaneous and TNF/IFN-induced IL-6 expression in two human ovarian-carcinoma cell lines.

Autocrine and paracrine production of interleukin-6 (IL-6) is considered to be involved in the ongoing proliferation of ovarian-cancer cells. In view of the variability in IL-6 expression between various ovarian-cancer cells, we questioned whether differences in IL-6-gene regulation might be observed in ovarian tumor cells with and without IL-6 expression. The CAOV-3 cell line spontaneously secreted IL-6, which was enhanced by tumor necrosis factor-alpha (877 +/- 89 vs. 8,452 +/- 1,762 pg/ml, x +/- sd, p < 0.01). The electrophoretic mobility shift assay (EMSA) demonstrated that basic IL-6 expression was associated with DNA binding of activator protein-1 (AP-1) and nuclear factor IL-6 (NF-IL6). Nuclear factor kappa-B (NF-KB), which consisted mainly of p65-NF-KB was induced in response to TNF-alpha stimulation. A2780 cells did not express IL-6, either spontaneously or after stimulation with TNF-alpha. EMSAs, showed spontaneous AP-1 but no NF-IL6 or NF-KB DNA binding. TNF-alpha stimulation enhanced AP-1 and induced NF-KB but no NF-IL6 DNA binding in these cells. NF-IL6 protein, however, was detected in nuclear extracts of these cells by Western blotting. In contrast, IL-6-promoter transfection studies showed no difference in promoter activation between CAOV-3 and A2780. This study reveals that differential IL-6-gene expression observed in ovarian-cancer cell lines is independent of NF-IL6 activation.

Transfection of small numbers of human endothelial cells by electroporation and synthetic amphiphiles.

OBJECTIVES: This study compared the efficiency of electroporation and synthetic amphiphiles. (SAINT-2pp/DOPE) in transfecting small numbers of human endothelial cells. METHODS AND RESULTS: Optimal transfection conditions were tested and appeared to be 400 V and 960 microF for electroporation and a 10:1 ratio for concentrations of SAINT-2pp/DOPE: plasmid. Using these conditions, cell concentrations were lowered step-wise and we were able to transfect as few as one thousand cells with both methods. For detection of transfection of a small number of cells a sensitive assay was needed (Luciferase). A plasmid containing the neomycin resistance gene was used to determine the transfection rate expressed in colony forming units by counting colonies after selection. At low plasmid concentrations this transfection rate was within the same range for both electroporation and SAINT-2pp/DOPE transfection. Fluorescent in situ hybridisation of metaphase chromosomes of transfected endothelial cells using the plasmid as a probe showed that stable integration was possible with both methods. CONCLUSIONS: Electroporation and a synthetic amphiphile, SAINT-2pp, provide the possibility of transfecting small numbers of cells resulting in stable integration of low plasmid concentrations. The availability of this technology is important in order to obtain functional endothelial cell lines from various human blood vessels for research purposes.