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Resume Introduction L’analyse des causes d’echec d’une ligamentoplastie du LCA doit permettre de choisir la technique la plus adaptee lors de la reprise chirurgicale afin d’assurer au patient un bon controle de la stabilite rotatoire et de diminuer les risques de ruptures iteratives. Objectif L’objectif de ce travail est d’evaluer les plasties anterolaterales (PAL) dans les reprises de ligamentoplasties du LCA. Hypothese L’hypothese de ce travail etait que les PAL realisees lors des chirurgies de revision du LCA, permettaient d’assurer un bon controle du ressaut sans augmenter les risques de complication. Materiel et methodes Il s’agit d’une etude multicentrique, prospective (sur 24 mois) et retrospective (minimum 3 ans de recul) fondee sur 349 patients, 283 hommes et 66 femmes. Les criteres d’inclusion sont des patients pour lesquels une indication chirurgicale de revision du LCA avec plastie intra-articulaire et PAL a ete retenu suite a un echec de plastie du LCA par autogreffe avec un LCP intact. Les criteres d’exclusion sont les reconstruction du LCA de 1 re intention et les lesions peripheriques mediales et/ou laterale associees. Les patients ont eu une evaluation clinique et radiographique pre- et postoperatoire. Le score IKDC preoperatoire etait en moyenne de 56,5 ± 15,5 avec 96 % de score IKDC C ou D. Resultats Le taux de complications postoperatoires precoces etait de 5 % et postoperatoires tardives de 10,5 %. Au dernier recul, dans 97 % des cas le test de Lachman avait un arret dur. Le ressaut rotatoire etait positif dans 1 % des cas. Le score IKDC subjectif moyen etait de 84,5 ± 13 dont 86,5 % de classe A ou B. Les signes d’arthrose radiographique etaient stables au dernier recul sur les compartiments femorotibial lateral et femoropatellaire, mais en augmentation de 9,7 a 21,2 % sur le compartiment femorotibial medial. Le taux de reruptures etait de 1,2 % et le taux de re-intervention secondaire de 5,4 %. Conclusion La laxite anterieure au dernier recul est similaire aux autres series de revision de la litterature mais le controle du ressaut et le taux de rupture iterative sont inferieurs. Les PAL font partie des techniques a envisager dans l’arsenal therapeutique des chirurgies de reprise du LCA. Type d’etude Etude retrospective et prospective de cohorte niveau IV.

Resume Introduction Les ancillaires personnalisees dans l’arthroplastie totale de genou se definissent comme une technique innovante pour faciliter le geste operatoire. Neanmoins se pose la question encore controversee de la precision de ces guides sur mesure pour aligner les implants. L’objectif de cette etude a ete d’analyser les resultats radiographiques postoperatoires de 50 protheses totales du genou (PTG), en analysant plus particulierement la pente tibiale de l’implant, difficilement controlable en cours d’intervention. Notre hypothese etait que les guides personnalises determinent de facon variable la pente tibiale. Materiel et methode Cinquante PTG d’une serie prospective et continue ont fait l’objet d’une programmation personnalisee Signature® (Biomet) basee sur une IRM. La pente tibiale posterieure planifiee etait de 2°. L’analyse radiologique postoperatoire a ete realisee par un observateur independant a partir de cliches numeriques standardises. Un test de reproductibilite des mesures radiographiques a ete pratique sur 20 cas. La corticale posterieure du tibia a servi de reference pour mesurer la pente tibiale. Une deviation de ± 2° par rapport a la valeur cible a ete consideree comme une imprecision. Resultats La pente tibiale d’implantation etait de 2° ± 2° dans 70 % des cas. Dans 30 % des cas, une imprecision etait rapportee avec 11 pentes tibiales d’implantation excessives (maximum 9°) et 4 pentes inversees, (maximum −6°). L’angle HKA postoperatoire etait de 0° ± 3° dans 88 % des cas avec une deviation maximale en varus de 9°. Conclusion Nos resultats corroborent l’hypothese d’une moins bonne precision sur la pente tibiale en accord avec les donnees recentes issues de la litterature. L’instrumentation specifique a en revanche montre sa capacite a controler l’alignement frontal. Niveau de preuve IV. Etude de cohorte.

Resume Introduction Dans l’arthroplastie totale de genou, la chirurgie assistee par ordinateur se definit depuis plus de 10 ans comme l’outil de precision pour aligner les implants dans le plan frontal. Notre hypothese a ete qu’une imprecision sur les reperes squelettiques pendant la phase d’acquisition et/ou une variabilite de mesure du systeme optique infrarouge pouvaient remettre en question la pertinence des informations numeriques qui guident le geste operatoire. Materiel et methode Une modelisation geometrique d’un systeme de navigation, sans acquisition d’imagerie preoperatoire, a permis de simuler les etapes du processus d’acquisition. Le centre de chaque reflecteur optique et les points anatomiques d’acquisition ont ete bruites aleatoirement de maniere multidirectionnelle dans une sphere d’incertitude dont le diametre a ete prealablement fixe. Trente mille simulations realisees selon la methode statistique de Monte-Carlo ont permis a partir du modele geometrique virtuel et du processus de navigation de calculer la variabilite des referentiels anatomiques qui guident les coupes osseuses. Les variabilites en rotation (α, β et γ) des reperes squelettiques femoral et tibial ont traduit respectivement les erreurs de positionnement des implants en flexion–extension, valgus–varus et rotation. Resultats En considerant les incertitudes du systeme de mesure optique infrarouge 3D et de l’acquisition des points anatomiques, les reperes femoral et tibial ont montre une erreur maximale alpha (flexion–extension), beta (valgus–varus) et gamma (rotation axiale) respectivement de 1,65° (0,9°) ; 1,51° (0,98°) et 2,37° (3,84°). La variabilite du systeme de mesure optique infrarouge n’a pas eu d’influence significative sur les angles d’alignement femoro-tibiaux. Conclusion Sur la base d’une simulation de Monte-Carlo, les systemes de navigation demontrent une certaine vulnerabilite pour le guidage en rotation mais une robustesse pour le controle de l’alignement frontal et coronal. Niveau de preuve de l’etude IV.

Zusammenfassung Durch Lyophilisation und Losungsmittelextraktion von Lammuterus-Gewebepulver wird ein lagerfahiges und leicht handhabbares Ausgangsmaterial zur Herstellung eines Bindungsreagenzes fur ostrogenwirksame Hormone gewonnen. Die Konzentration an Bindungsstellen fur 17β-Ostradiol ist etwa um den Faktor 10 hoher als bei direkter Extraktion von frischem oder gefrorenem Gewebe bezogen auf den Proteingehalt. Die Affinitat fur 17β-Ostradiol wird durch die Vorbehandlung mit Losungsmitteln erhoht. Im Gewebe enthaltene lipophile Substanzen einschlieslich endogener Hormone werden durch die Losungsmittelbehandlung entfernt. Summary Obtaining proteins of high binding affinity for testing oestrogen receptors by means of a lamb uterine powder extracted with solvent By lyophilization and solvent extraction of powdered lamb uterine tissues a marketable and easily handled binding reagent for oestrogenic hormones was obtained. The concentration of binding sites for 17β-oestradiol is higher by a factor of 10 than that obtained by direct extraction of fresh or frozen tissues, relative to protein content. Affinity for 17β-oestradiol is increased by the pre-treatment and the solvents. Lipophilic substances, including endogenous hormones, in the tissue are removed by the solvent treatment. Resume Obtention de proteines de liaison a forte affinite pour le test de recepteurs des oestrogenes a partir d'une poudre d'uterus d'agneau extraite avec un solvant Un materiel conservable et facilement utilisable pour la preparation d'un reactif de liaison des hormones oestrogenes a ete obtenu a partir d'une lyophilisation et d'une extraction avec un solvant. La concentration en sites de fixation pour 17β-oestradiol est plus elevee d'un facteur d'a peu pres 10 que lors d'une extraction directe a partir d'un tissu frais ou congele rapporte au taux de proteines. L'affinite pour 17β-oestradiol est augmentee avec le pretraitement du solvant. Les substances lipophiles et des hormones endogenes contenues dans le tissu sont eliminees par le traitement avec le solvant. Resumen La obtencion de proteinas fijadoras harto afines para la prueba receptora de estrogenos a partir de polvo uterino de cordera extraido mediante disolventes Por medio de liofilizacion y extraccion con disolventes de tejido uterino pulverizado de cordera, se obtiene un material inicial almacenable y de manipulacion facil para elaborar un reactivo de fijacion para hormonas de accion estrogena. La concentracion en los lugares de fijacion para estradiol-17β es mayor en el factor 10 aprox. que con la extraccion directa a partir de tejido fresco congelado, referida al contenido en proteinas. La afinidad para estradiol-17β se aumenta mediante el pretratamiento con disolventes. Con el tratamiento a base de disolventes, se eliminan las substancias lipofilas, incluyendose las hormonas endogenas contenidas en el tejido.

Zusammenfassung Fur schockgefrorenes und lyophilisiertes Lebergewebe wird eine zeitlich auf 1–2 Stunden verkurzte Homogenisationstechnik bei tiefen Temperaturen beschrieben, die mikroskopisch cytoplasmafreie Zellkerne liefert. Fur die Trennung der subzellularen Partikel wird als neue Methode die Zonalzentrifugation in umorientierenden Gradienten aus Tetrachlorkohlenstoff und Cyclohexan vorgestellt, mit der, gemessen am RNA/DNA-Verhaltnis, sowohl cytoplasmafreie Kernfraktionen als auch stark ribosomenhaltige Cyto-plasmafraktionen definiert und gewonnen werden konnen. Auf die Anwendungsmoglichkeiten der die in vivo Verhaltnisse der Zelle konservierenden Methode wird hingewiesen. Summary Cell fractionation by zonal centrifugation in non-aqueous solutions For shock-frozen and lyophilized liver tissue a shortened (1–2 hours) technique for homogenization at low temperatures is described which yields cell nuclei free from microscopic cytoplasmic material. To separate the subcellular particles a new method comprising zonal centrifugation in gradients of carbon tetrachloride and cyclohexane, with determined RNA/DNA relationship and cytoplasmic nuclear fractions and also markedly ribosome-rich cytoplasm fractions is defined and assessed. The possibilities of using methods which conserve the in vivo relationships of the cells provided by this method are discussed. Resume Fractionnement cellulaire par centrifugation zonale dans un liquide de dilution non aqueux On decrit une technique d'homogeneisation raccourcie de 1–2 heures pour du tissu hepatique surgele et lyophilise a des temperatures basses qui permet d'obtenir des noyaux cellulaires microscopiques exempts de cytoplasme. La nouvelle methode pour la separation des particules subcellulaires consiste en une centrifugation zonale dans des gradients de tetrachlorure de carbone et de cyclohexane qui ont pu etre definis et obtenus avec le rapport mesure ARN/ADN aussi bien qu'avec des fractions de noyaux exemptes de cytoplasme et des fractions cytoplasmiques contenant une grande quantite de ribosomes. On evoque les possibilites d'utilisation concernant la methode de conservation dans les conditions in vivo des cellules. Resumen El fraccionamiento celular mediante centrifugacion zonal en disolventes no acuosos Para tejido hepatico sobrehelado y liofilizado se describe una tecnica de homogeinizacion a temperaturas bajas, reducida en el tiempo a 1–2 horas. La misma permite obtener nucleos celulares microscopicos exentos de citoplasma. Para la separacion de particulas subcelulares se presenta como metodo nuevo la centrifugacion zonal en gradientes reorien-tadores de tetracloruro de carbono y ciclohexano, con el cual se pueden definir y obtener, midiendo la relacion de ARN/ADN, tanto fracciones nucleares exentas de citoplasma como fracciones citoplasmaticas que contienen una cantidad elevada de ribosomas. Se llama la atencion sobre las posibilidades de aplicacion de la tecnica conservadora de las condiciones en las celulas in vivo.

Summary The oestrogen receptor of immature freeze-dried and homogenized lamb uterus is easily extracted from cellular fractions, including nuclei, after centrifugation in cyclohexane/carbon tetrachloride mixtures of different density. Indications for the predominant occurrence of the receptor in nuclei are given. Its affinity to 3H-17β-oestradiol remains unimpaired by this treatment and is comparable to that of receptor fractions prepared by the aqueous technique. Zusammenfassung Zellfraktionierung von Lammuterus in wasserfreiem Medium und intrazellulare Verteilung des Ostrogenrezeptors Nach Homogenisation gefriergetrockneter Lammeruteri sowie Zellfraktionierung durch Zentrifugation in unpolaren Tetrachlorkohlenstoff/Cyclohexan-Gemischen unterschiedlicher Dichte ist der Ostrogenrezeptor aus allen subzellularen Fraktionen leicht zu extrahieren. Der Rezeptor findet sich dabei vorwiegend in den Zellkernen. Seine Affinitat zu 3H-17β-Ostradiol bleibt bei dieser Fraktioniertechnik voll erhalten. Resume Fractionnement cellulaire d'uterus d'agneaux dans un milieu sans eau et repartition intracellulaire des recepteurs d'œstrogene L'extraction du recepteur d'œstrogene a partir de toutes les fractions subcellulaires est facile apres une homogeneisation d'uterus lyophilises d'agneaux et apres un fractionnement cellulaire par une centrifugation dans des melanges non polaires de densite differente fait de tetrachlorure de carbone/cyclohexane. Le recepteur est situe principalement dans les noyaux cellulaires. Son affinite pour 3H-17β-œstradiol demeure intacte avec cette technique de fractionnement. Resumen El fraccionamiento de las celulas de uteros de cordera en medio no acuoso y el compartimiento intracelular del receptor de estrogeno Tras la homogeneizacion de uteros liofilizados de corderas asi como el fraccionamiento celular mediante centrifugacion en mezclas no polares de tetracloruro de carbono/ciclohexano, de densidades diferentes, se puede extraer con facilidad el receptor de estrogeno de todas las fracciones subcelulares. Asi se halla el receptor, sobre todo, en los nucleos celulares. Con esta tecnica de fraccionamiento permanece intacta su afinidad para con 3H-17β-oestradiol.

Resume Les recidives d’entorse apres ligamentoplastie du ligament croise anterieur (LCA) posent le difficile probleme de leur diagnostic etiologique. Les genoux dont le LCA a ete reconstruit presentent frequemment une laxite anterieure residuelle ou controlent parfois insuffisamment la stabilite rotatoire. L’examen ligamentaire des genoux sain et lese juste avant le nouveau traumatisme constitue une double reference a laquelle doit se referer le praticien pour definir les differentes entites anatomocliniques. Le seuil de la laxite anterieure comparative attestant l’incompetence du transplant se situe a 5 mm selon les criteres du groupe examen ligamentaire de la fiche IKDC. Une re-rupture traumatique se differencie en fonction du contexte clinique, d’un echec anatomique precoce ou d’une distension progressive. Un transplant continu et percu en hyposignal a l’IRM doit etre confronte a l’examen clinique, aux mesures instrumentales (KT 1000™) et radiographiques de la laxite avant d’affirmer son integrite. Le macrotraumatisme n’est que revelateur de l’echec en cas d’erreurs techniques liees a un mauvais positionnement des tunnels osseux. Les hypersignaux intra-osseux ou meniscaux persistants apres le premier traumatisme peuvent simuler une lesion recente. Certains facteurs predisposant aux recidives d’entorse ont ete analyses en tenant compte de l’experience des auteurs et des donnees recentes de la litterature.

Resume Introduction Notre etude a evalue la precision de reperes squelettiques mis en place a partir de reconstructions tridimensionnelles in vivo du membre inferieur realisees avec le systeme de radiographie biplanaire basse dose EOS®. Materiel et methodes Une base de donnees de 99 reconstructions de neuf membres inferieurs appartenant a sept patients a ete realisee par quatre operateurs. Huit reperes ont ete definis a partir de regions et points anatomiques du tibia et du femur. Pour chaque os et chaque repere etudie, un repere moyen issu de 11 series consecutives a ete calcule. L’ecart de chaque repere construit au repere moyen correspondant a ete calcule suivant les axes anteroposterieur (X), longitudinal (Y) et mediolateral (Z), en translation (Tx, Ty, Tz) et en rotation (Rx, Ry, Rz). L’incertitude a ete estimee par l’intervalle de confiance a 95 % (IC95 %). Resultats IC 95 % de Tx, Ty, Tz etait respectivement inferieur a 1 mm, 0,5 mm, 1,5 mm pour le femur et 1,5 mm, 0,6 mm, 0,6 mm pour le tibia. Les reperes femoraux utilisant le centre ou le bord posterieur des condyles posterieurs pour construire l’axe mediolateral ont ete retenus avec une incertitude pour les rotations femorales Rx, Ry, Rz, inferieure a 0,3°, 4° et 0,5°. L’ensemble des reperes tibiaux utilisant les bords posterieurs des plateaux a eu une reproductibilite comparable en rotation, 95 % des ecarts Rx, Rz etaient respectivement inferieurs a 0,5° et 1,3°. Pour la rotation tibiale Ry, l’erreur moyenne a ete plus importante (4°) avec une incertitude (IC 95 %) de 11,2°. Les angles 3D d’implantation ont ete mesures sur deux patients en utilisant les reconstructions squelettiques 3D preoperatoires et des modeles geometriques 3D des implants recales sur les pangonogrammes EOS® postoperatoires. Type d’etude : prospective. Niveau IV

Resume Pour evaluer l’evolution radiologique des arthrodeses cervicales par cage radio-transparente et plaques anterieures, nous avons revu les blesses pris en en charge entre octobre 1999 et juin 2003 pour traumatisme du rachis cervical sans lesion neurologique medullaire. Notre serie comportait 30 patients et 32 arthrodeses. Les lesions se repartissaient en : 1 tear Drop, 8 entorses graves en flexion, 4 luxations bi-articulaires, 4 entorses graves en hyperextension, 1 luxation-fracture, 7 fractures uni-articulaires, 4 fractures-separations du massif articulaire, 3 hernies post-traumatiques. Nous avons pris en compte l’angle inter-somatique, le deplacement anterieur ainsi que la hauteur au milieu du disque intervertebral. La technique chirurgicale consistait en une arthrodese intersomatique utilisant une cage remplie d’os spongieux associee a une plaque anterieure. Il a ete associe 4 temps posterieurs. Tous les malades ont etes revus au moins jusqu’a fusion intersomatique survenue en moyenne au 78e jour. L’angle intersomatique moyen est passe de 11,6° de cyphose a 13° de lordose. Le deplacement anterieur est passe de 3 mm a 0,3 mm. La hauteur de l’espace intersomatique est passee de 5,3 a 8,2 mm. Il n’a pas ete observe de deplacement secondaire, ni de complications. Cette etude montre que l’arthrodese par cages associees a une osteosynthese anterieure peut etre utilisee en traumatologie. Cette technique d’arthrodese inter-somatique est simple, fiable et a faible morbidite pour les lesions traumatiques instables sans lesion neurologique medullaire. Elle permet une reduction quasi anatomique en un delai de fusion faible.

Background Patient-specific cutting guides were recently introduced to facilitate total knee arthroplasty (TKA). Their accuracy in achieving optimal implant alignment remains controversial. The objective of this study was to evaluate postoperative radiographic outcomes of 50 TKA procedures with special attention to posterior tibial slope (PTS), which is difficult to control intraoperatively. We hypothesized that patient-specific cutting guides failed to consistently produce the planned PTS. Material and methods The Signature™ patient-specific cutting guides (Biomet) developed from magnetic resonance imaging data were used in a prospective case-series of 50 TKAs. The target PTS was 2°. Standardised digitised radiographs were obtained postoperatively and evaluated by an independent reader. Reproducibility of the radiographic measurements was assessed on 20 cases. The posterior cortical line of the proximal tibia was chosen as the reference for PTS measurement. Inaccuracy was defined as an at least 2° difference in either direction compared to the target. Results The implant PTS was within 2° of the target in 72% of knees. In the remaining 28%, PTS was either excessive ( n = 10; maximum, 9°) or reversed ( n = 4; maximum, –6°). The postoperative hip-knee-ankle angle was 0° ± 3° in 88% of knees, and the greatest deviation was 9° of varus. Conclusion These findings support our hypothesis that patient-specific instrumentation decreases PTS accuracy. They are consistent with recently published data. In contrast, patient-specific instrumentation provided accurate alignment in the coronal plane. Level of evidence IV, cohort study.

Resume Les auteurs ont evalue au recul moyen de 5,2 ans une serie continue de 50 patients sportifs en pivots repares dans les 8 premiers jours d’une rupture isolee du LCA selon la technique de MacIntosh modifiee utilisant le tractus iliotibial. La section de la cloison intermusculaire laterale a permis une reconstitution du hauban lateral tout en prelevant une bandelette de 40 a 45 mm de largeur. Les resultats globaux selon la fiche de cotation IKDC ont ete comparables a ceux d’autres auto-transplants : 38 % (IKDC A), 46 % (B), 12 % (C), 4 % (D). La laxite residuelle differentielle moyenne a ete mesuree a l’arthrometre KT-1000 a 1,86±1,74 mm. Quarante-huit pour cent des genoux ne presentaient pas de signe de Lachman. Les ressauts ont ete neutralises dans 88 % des cas. Selon les criteres de la fiche de cotation Arpege, nous avons observe 38 % de resultats excellents, 46 % de bons resultats, 12 % de resultats moyens et 4 % de mauvais resultats. Trente-trois patients sur 50 ont conserve au recul le meme niveau d’activite sportive. Un echec anatomique precoce et deux cas de ruptures iteratives traumatiques sont survenus a 25 et 38 mois de recul. Deux flexum irreductibles de plus de +5 et trois deficits en flexion superieurs a 20 ont altere les resultats fonctionnels. Une competitrice ayant pu reprendre ses activites sportives a eu une mobilisation de son genou sous anesthesie generale. Un seul cas de hernie musculaire par dehiscence a ete observe au niveau de la zone prelevee. Le deficit residuel moyen quadricipital a ete a 12 mois de 10 % a 90/s et celui des ischio-jambiers de 1,3 % a la meme vitesse.

The use of a pneumatic tourniquet can induce muscular and neurological complications in the operated limb. The genesis of these injuries could involve an ischaemia/reperfusion phenomenon and a compression under the cuff. We evaluated effects of an antioxidant, acetylcysteine and ischaemic preconditioning on the rhabdomyolysis and postoperative pain following a knee ligamentoplasty using a pneumatic tourniquet.We included 31 patients scheduled for a knee ligamentoplasty randomly assigned in three groups (control, acetylcysteine 1200 mg the day before and 600 mg at the operative day, ischaemic preconditioning).There was a moderate rise in myoglobin and creatinine phosphokinase with no significant difference between the three groups. The muscular functional parameters were similar in all the groups. However, the morphine consumption within the first 48 h was smaller in the treatment groups (0.22 +/- 0.31 mg kg-1 and 0.22 +/- 0.23 mg kg-1 in the preconditioning and antioxidant groups, respectively) than in the control group (0.47 +/- 0.33 mg kg-1, P0.05).Acetylcysteine and ischaemic preconditioning do not decrease the extent of rhabdomyolysis related to the use of a pneumatic tourniquet and do not improve the postoperative muscle recovery. On the other hand, they allow a significant reduction in the postoperative morphine consumption.

Inaugural cutaneous metastasis is exceptional in osteosarcoma. We report a case of a woman with osteosarcoma of the right knee who presented two tumefactions of the scalp at the time of diagnosis. Magnetic resonance imaging identified a high intensity T2 signal in the femur and also a high-intensity signal in the soft tissues. The head scan confirmed the presence of two subcutaneous tumors. Biopsy of the two sites demonstrated the metastatic nature of the cutaneous lesions. The patient was given preoperative and postoperative chemotherapy. Wide resection followed by prosthesis reconstruction was performed. Pulmonary metastases developed and led to two thoracotomy procedures. The patient has remained in remission 22 months. Six cases of cutaneous metastases of osteosarcoma have been reported in the literature. The cutaneous metastases were secondary in five of six cases, occurring after other metastatic localizations had been discovered. The present case is the first published observation of synchronous cutaneous metastases. The duration of remission suggests that cutaneous localizations should be treated like other inaugural metastatic localizations with intensive chemotherapy and wide resection of the primary tumor.

The successful separation of beta-lactoglobulin from other bovine whey proteins was performed by ceramic hydroxyapatite chromatography with a fluoride ion gradient in phosphate buffer as displacement agent. The method was applied to acid whey originating from milk of healthy as well as of mastitic cows. beta-Lactoglobulin was completely eluted in one peak at a fluoride concentration of about 0.6 mol/l. The purity of beta-lactoglobulin in this fraction was at least 96% if whey from healthy milk was processed. Co-eluted contaminants are traces of immunoglobulin G, serum albumin and lactoferrin. In case of mastitic whey the proportion of beta-lactoglobulin is diminished as the amounts of immunglobulin G, serum albumin and lactoferrin are increased within this fraction. Size exclusion chromatography on Superdex 75 pg effectively removed contaminants resulting in a purity for beta-lactoglobulin from normal whey of approximately 99%. The yield of beta-lactoglobulin from physiological whey was 50-55% referring to the fraction highly enriched with beta-lactoglobulin by hydroxyapatite chromatography. In case of mastitic milk the higher amounts of contaminants were also removed successfully by size exclusion chromatography.

A direct and an indirect antibody enzyme-linked immunosorbent assay for duck yolk IgY and duck serum IgY was developed and tested on egg yolk and serum of ducks vaccinated with Riemerella anatipestifer (Ra). Tests were performed either with primary antibodies labelled with horseradish peroxidase or with alkaline phosphatase-labelled secondary antibodies reacting with specifically bound rabbit anti-duck IgY antibodies, respectively. Ra-specific IgYs in egg yolk from three ducks increased rapidly at day 8 after the first of two vaccinations. In two ducks, the IgY titre persisted on a high plateau for 3 months. The concentration of Ra-specific IgYs in the serum of the progeny of vaccinees decreased between day 3 and day 10 after hatching. The fraction of total IgYs decreased less but also significantly. It was shown that antibodies were vertically transmitted and therefore protect offspring against Ra infection at least during the first week after hatching. The test design with anti-IgY rabbit antibodies is further suitable to detect other specific antibodies if respective antigens were fixed on solid phases.

The whey protein pattern of milk from animals affected by mastitic inflammation was resolved by two-dimensional gel electrophoresis (2D-PAGE) and compared to milk from unaffected cows. Inflammation caused the appearance of four spots aligned at a molecular weight level of 26 kDa and over a pH-region of 5.0 to 6.4. The spots excised from 2D gels were treated with chymotrypsin and the resulting peptides analyzed by MALDI-TOF mass spectrometry and RP-HPLC. All four spots yielded highly similar chymotryptic peptide mass fingerprints as well as chromatographic peak patterns. A database search could identify the four spots as isoforms of the bovine prostaglandin D synthase (PGD-S). In one of the isoforms a defined cysteine residue was shown to be oxidized to a sulfonic acid.

Summary The usefulness of soluble markers in indirectly differentiating inflamed quarters from healthy quarters of 30 Holstein Friesian cows was tested over a follow-up period of 20 weeks. In comparison to N-acetyl-β-D-glucosaminidase (NAGase), lactate dehydrogenase (LDH), Na+, K+ and Cl− the somatic cell counts (SCC) were determined in foremilk samples. The soluble parameters were measured by means of an autoanalyzer system (Beckman CX5CE) equipped with solid state ion electrodes. Due to different types of inflammatory reactions it is considered to be advantageous to perform two tests in parallel for assessing the mammary gland status. Cell-damaging processes should best be detected by measuring elevated LDH activity, whereas the impairment of the blood—udder barrier is recorded by the respective elevation of the indicators Na+ and Cl−. Among all variables, SCC included, the soluble parameters showed the least dispersions, whereas those of SCC were the most unfavourable in this respect. Moreover, NAGase/LDH and Na+/Cl− among all variables had the highest correlation coefficients of 0.85 and 0.86, respectively. Threshold values as decision limits are proposed. Additionally, an inter-quarter comparison and evaluation improved the diagnostic value of each indirect mastitis parameter analyzed. By means of discriminant analysis, it is shown that the distribution of inflammation within the udders of this herd was unequal.

The purpose of this study was to test the hypothesis that the surgical technique using alternating fluid irrigation and carbon dioxide (CO2) gas medium as a means of knee joint distension during arthroscopy is a safe and effective method, compared to traditional fluid arthroscopy. This prospective randomized comparative study involved 94 patients undergoing the same arthroscopic ACL reconstruction surgical procedure except for the medium used to distend the joint: In a first group of 48 patients (“ACL gas” group), CO2 insufflation was mainly used, at a low pressure of 40–50 mmHg, alternating with a fluid medium for knee joint washout only. In the second group of 46 patients, classical arthroscopic joint distension by fluid was used, with a pump pressure of 50–70 mmHg. Early pre- and post-operative complications, duration of surgery, intraoperative monitoring data and particularly the end-tidal carbon dioxide (EtCO2) as a marker of CO2 blood diffusion were prospectively collected. EtCO2 and other monitoring data changes before and after tourniquet release were not different between the two groups. Tourniquet time was significantly longer when using fluid rather than gas. We observed 3 cases of small and localized subcutaneous emphysema, resolving completely within hours. Hematoma and reflex sympathetic dystrophy occurred more often in the “ACL fluid” group, with no statistical significance. Low-pressure CO2 knee joint insufflation proved to be a safe technique capable of improving surgical comfort compared with the use of fluid alone. The CO2 blood diffusion measured by end-tidal carbon dioxide monitoring during ACL arthroscopic reconstruction was similar with or without gas insufflation. Randomized controlled trial, Level I.

Three doses of two different mixtures of PCDD homologues and congeners were administered on day 9 of pregnancy to C57BL/6J (B6) or DBA/2J (D2) mice. Doses given to D2 mice were tenfold higher than those given to B6 mice. In addition, 2,3,7,8-TCDD (15 μg/kg and 150 μg/kg b.w. resp.) was tested separately as well as in combination with the applied doses of the PCDD mixtures. On day 18 of pregnancy the mice were sacrificed, organ weight data were taken, and the number of fetuses with cleft palates was determined. Liver weight was significantly increased in all treated D2 mice, however, in B6 mice only when 2,3,7,8-TCDD was co-administered. The incidence of cleft palates were dose-dependent in the fetuses of D2 mice. In B6 mice this malformation occured either after the administration of 2,3,7,8-TCDD alone or with 2,3,7,8-TCDD added to the different PCDD mixtures. The concentration of the 2,3,7,8-substituted PCDDs analysed in the liver and white adipose tissue reflect the doses and the kind of mixture given orally. However, in the liver of B6 mice, organ dosages (expressed as I-TEQ) obviously do not correspond to the administered increasing dosages. It is concluded that there is some additive effect for TCDD and the 2,3,7,8-substituted congeners.

In an in vivo/in vitro assay system (Massa et al., 1990) we have detected the carcinogenic activity of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). The carcinogenic potential measured in this system is concentration-dependent. Experiments with other carcinogenic compounds have revealed that TCDD at low doses can act as co-carcinogen. At higher concentrations TCDD induces TNF-α production.

We have developed a host-mediated assay system for the detection of the transforming action of chemical carcinogens on peritoneal macrophages. Directly as well as indirectly acting carcinogenic substances administered intraperitoneally to NMRI mice could be examined in this way. Resident macrophages were recovered by peritoneal lavage from treated and untreated mice and were cultured in soft agar. After 5-6 days normal and transformed cells could be distinguished. Statistical analysis comparing cells, for example, from alpha-naphthylamine or diphenylhydantoin-treated animals with those from control mice proved that the test is positive at least on a significance level of 5% using the t-test. Further substances revealing a cell-transformation potential were benzene, benz(a)pyrene, 2,3,7,8-tetrachlorodibenzodioxin, N-nitrosodimethylamine, ethidium bromide, aflatoxin B1,N-methyl-N-nitrosourea, 1-methyl-3-nitro-1-nitrosoguanidine, 2-naphthylamine, dieldrin, suramin and trichloroethylene. A weak transforming potential was found for chlorambucil as well as for tetrachloroethylene. With toluene or azidothymidine no cell transformation could be observed. Several immortal cell lines could be established form NMRI mice treated with alpha-naphthylamine or N-methyl-N-nitrosourea. Athymic nu/nu mice injected subcutaneously with these cells developed tumors, establishing the oncogenic potential of these cell lines.

The host-mediated in vitro/in vivo assay system was used to evaluate the tumorigenic potential of the aromatic nitro compound 2-nitroanisole (2-NA). After intraperitoneal administration of the compound, resident macrophages were recovered by peritoneal lavage from treated and untreated mice and cultured in soft agar. 2-NA was shown to be carcinogenic, and the tumorigenic potential was evaluated. Additionally, by establishment of a transformed peritoneal macrophage cell line, the underlying molecular mechanism of 2-NA's carcinogenic effect was studied.

Prostaglandin D synthase (L-PGDS) is a major glycosylated polypeptide in cerebrospinal fluid (CSF). The overexpression of L-PGDS in inflamed bovine mammary glands indicates its role as biomarker. No diagnostic tool for the quantitative detection of L-PGDS in cows has been reported. Immunometric ELISA tests might help to identify inflamed bovine tissue. The isolation of pure bovine L-PGDS, which is required for the generation of monoclonal antibodies, is an important prerequisite for a diagnostic ELISA test. Our goal was to identify a suitable technique to generate pure L-PGDS from bovine substrates. In the present study a two-step method for the purification of bovine CSF using ceramic hydroxyapatite chromatography followed by size exclusion chromatography is described. Subsequently, the identification of bovine L-PGDS was demonstrated by Western blot analysis and the high grade of the pure product was shown by 2-D PAGE. The yield of purified L-PGDS was 6.8 mg/l bovine CSF. L-PGDS from bovine CSF is shown to consist of multiple isoforms identical in molecular mass and p I values to those in previously described secretions of inflamed bovine mammary glands. In addition, the method was successfully applied to the purification of L-PGDS from human CSF.

12-O-retinoylphorbol-13-acetate (RPA), an incomplete tumor promoter of the phorbol ester type and protein kinase C (PKC) activator, consists of two characteristic structural elements: the phorbol body and the retinoyl ester chain. Therefore, possible binding of the incomplete tumor promoter RPA to the human transport protein retinol-binding-protein (RBP) has been examined by molecular modeling methods and experimental binding studies. The calculated prediction of binding properties was primarily based on a comparative geometrical approach. It was shown that the beta-ionone-ring of RPA was not altered within the binding pocket of RBP (molecular modeling) compared to retinoic acid (X-ray crystallographic data). The torsion angle C5'-C6'-C7'-C8', determining the conformation of the RBP-beta-ionone-ring relative to the isoprene tail, is rotated by 42 degrees for RPA compared to retinol and to retinoic acid, respectively. Combining all the results from force field calculations, MD simulations and geometrical comparisons, the conclusion could be drawn that RPA should be able to bind to RBP. This interaction should be less strong than that with its natural ligand retinol or with retinoic acid. This prediction was proven experimentally. RPA was able to compete with retinoic acid for binding at RBP in human plasma. The binding properties were investigated using 3H-labeled retinoic acid in homologous and heterologous competition studies in a one-dimensional native polyacrylamide gel electrophoresis system. An approximately 2000-fold weaker binding of RPA to RBP as compared to retinoic acid was determined experimentally, confirming the prediction of the molecular modeling approach. The characteristic behaviour of RPA as an incomplete promoter, due to possible binding to PKC and RBP, is discussed.

Concentrations of polychlorinated dibenzo-p-dioxins (PCDDs), polychlorinated dibenzofurans (PCDFs), polychlorinated biphenyls (PCBs), and organochlorine (OC) pesticides were measured in unsuccessfully hatched eggs of three different kinds of predatory birds: 3 eggs of sparrowhawks, 7 eggs of hobbies, and 16 eggs of goshawks collected in the German region of Berlin-Brandenburg. Using toxic equivalency factors for birds, eggs of hobbies contained mean concentrations of 478 pg TEQ/g fat and 551 pg TEQ/g fat contributed by PCDD/Fs and coplanar PCBs, respectively. For sparrowhawks the respective TCDD equivalents were 424 and 1278 pg/g fat; those for goshawks were 211 and 935 pg/g fat. The mean value of the summed concentrations of the PCB congeners 28, 52, 101, 138, 153, and 180 amounted to 11 microg/g fat in eggs of hobbies, 9.5 microg/g fat for sparrowhawks, and 16.1 microg/g fat for goshawks. Of the analyzed organochlorine pesticides the concentration of p,p'-DDE was highest (up to 273 microg/g); the concentration of the other OCs, with the exception of methoxychlor in eggs of goshawks (highest level of 10.6 microg/g fat), were negligible. In all cases the calculated toxicity of the coplanar PCBs surpassed the toxicity of the PCDD/Fs, at least by a factor of two. Due to the higher concentration of the coplanar PCB 77 compared to PCB 126 in eggs of hobbies, it is concluded that its metabolic excretion in this species is much lower than in sparrowhawks and goshawks.

12-O-Tetradecanoyl-phorbol-13-acetate (TPA) has been shown to induce a broad variety of morphological and metabolic changes in mouse skin. These include gross changes in cell growth as well as subtle changes regarding gene expression. Changes in the induction of specific proteins have been described, but it is not yet clear, if there exists a strict causal correlation between the induction of expression of specific proteins and the phenomenon of tumor promotion. At least some of these proteins might also be correlated with an accompanying hyperproliferative effect. This has been shown for protein p10, a 10 kDa protein not found in untreated adult mouse epidermis. The expression of this protein can be induced de novo after the topical administration of TPA. It is also shown, that p10 is expressed in untreated neonatal murine epidermis. Due to the induction of this protein by irritation, hyperproliferation-causing tumor promoters and due to the observable abundance in neonatal epidermis, this protein seems not strictly related to tumor promotion, but rather to proliferation and differentiation.

Analysis of protein secretion was performed for a macrophage-like cell line, which was established from the peritoneal cells of NMRI mice treated with 17 alpha-ethinylestradiol. The protein secretion pattern was investigated by computerized analysis of high resolution two-dimensional gel electrophoresis (2-D PAGE) and compared to that of control macrophages, intraperitoneally activated by bacterial lipopolysaccharide. The transformed cells encode a number of low molecular weight proteins (10-20 kDa), which were not observed in control cells under identical experimental conditions. In conclusion the transformation of peritoneal macrophages by 17 alpha-ethinylestradiol leads to an upregulation of polypeptides, in particular of low molecular weight proteins. A high similarity between the induced low molecular weight protein secretion by macrophages of 17 alpha-ethinylestradiol-treated and that of 2,3,7,8-tetrabromodibenzo-p-dioxin-treated mice was found.

Systematic screening of different organs has shown that induction of particular proteins by 12-0-tetradecanoyl-phorbol-13-acetate is specific for mouse skin. Different compartments of skin showed all the same expression. For experimental reasons we focused the analysis performed thereafter on the epidermis. As an end- point of mouse skin tumorigenesis papilloma and carcinoma were also screened for changes in protein expression. We focused our efforts on the characterization and identification of selected, de novo expressed proteins induced by various tumor promoters in murine epidermis. The analysis presented here consists of functional characterization. Having found differences in protein expression in TPA-treated murine epidermis compared with acetone-treated control mice, we then tried to correlate the expression to the signal transduction pathway which the tumor promoter exerts its activity through. For this reason we screened protein expression patterns after topical administration of mechanistically differently acting tumor promoters, which were inflammatives not bearing tumor promoting activity anti-promoters or a combination of tumor promoters with modulators of tumor promotion. The epidermis of untreated newborn mice served as a model for high cellular proliferation and differentiation. Alterations were compared with changes observed after tumor promoter application.

Treatment of murine epidermis with the tumor promotor 12-O-tetradecanoylphorbol-13-acetate (TPA) shows characteristic and significant changes in protein expression analyzed by 2D PAGE, compared to that of acetone-treated mouse epidermis. Of the seven de novo expressed proteins in TPA treated murine epidermis, one is a 44 kDa protein (p44) located nearby actin, and six proteins are in the low-molecular range between 10-20 kDa (p10, pY, pCa, p1, p2 and p3). Interestingly, the incomplete promoting and inflammative hyperplasiogen 12-O-retinoylphorbol-13-acetate (RPA) and the non-promoting but inflammative and hyperplasiogenic calcium-ionophore A23187 induced the same pattern of proteins observed in the pidermis of mice treated with TPA, with minor quantitative differences. In all cases, p10 expression was quantitative the most abundant. Partial sequencing of this protein has led to the conclusion that it is a novel protein with no such sequences in the database comparisons using FASTA and TFASTA computer programs of Genetics Computer Group. The data presented here do not strictly support the functional role of de novo induced proteins to tumor promotion, but show a causal relationship to hyperplasiogenic potency of TPA, RPA and A23187.

Xiphophorine fish from wild populations are insusceptible to develop neoplasia. In contrast, certain backcrosses of Xiphophorus maculatus (platyfish) with Xiphophorus helleri (swordtail) as the recurrent parent, produce offspring that develop neoplasia in a Mendelian fashion. We concentrated our research on melanoma. The starting signal for the development of melanoma comes from an accessory v-erbB-related oncogene, x-erbB*, which is highly homologous to the human EGF receptor gene, and is part of a platyfish-specific tumor gene-complex designated as Tu. Normally, the platyfish is protected from its own Tu by Tu-specific regulatory gene systems. The swordtail has neither evolved the Tu-complex nor the regulatory gene systems. Therefore, the backcross procedure dismantles the regulatory gene systems thus permitting Tu-directed melanoma formation. x-erbB* derived from the platyfish, together with the swordtail-derived oncogenes src, sis, pdgf-r, ras, myc, erbA are expressed or overexpressed in the melanoma, and inositol lipid turnover is considerably elevated. x-src and inositol lipid turnover have also been found elevated in the healthy tissues (eg brain) of the tumourous fish. To construct a fish strain which is highly susceptible to mutagenic carcinogens, we introduced a particular regulatory gene, ie an oncosuppressor gene (Bs), into the genome of the animals developing Mendelian inherited melanoma, by introgressive breeding. Bs prevents the strain from germ line-inherited melanoma but, following carcinogen-induced impairment in a somatic cell gives rise to the Tu-directed development of clonal melanoma in a particular fish, x-src activity and inositol lipid turnover are elevated in the tumor but, in contrast to the animals bearing the inherited melanoma, are not elevated in the brain. Promoting carcinogens (tumor promoters) do not induce melanoma in this strain. Similarly, in order to breed a fish strain susceptible to tumor promoters, we introduced a regulatory gene (the oncostatic gene g, golden) coding for a pre-transformational arrest of pigment cell differentiation in the stem cell stage of the fish that develop the Mendelian inherited melanoma. The new strain is incapable of developing melanoma. Its x-src activity and inositol lipid turnover is elevated in the brain indicating that the biochemical processes which were found to be correlated with the hereditary melanoma formation, are running in the new strain without the occurrence of melanoma. Following treatment with tumor promoters that overcome the arrest of pigment cell differentiation, melanoma develops within a very short latent period.

Xiphophorine fish from wild populations are insusceptible of developing neoplasia. In contrast, certain backcrosses of Xiphophorus maculatus (platyfish) with Xiphophorus helleri (swordtail) as the recurrent parent produce offspring that develop neoplasia in a Mendelian fashion. We concentrated our research on melanoma. To construct a fish strain which is highly susceptible to mutagenic carcinogens, a particular regulatory gene, ie an oncosuppressor gene (Bs), was introduced into the fish developing the Mendelian inherited melanoma by introgression. Bs prevents the progeny from developing melanoma. However, Bs can be impaired by carcinogen-induced somatic mutation which gives rise to the development of clonal melanoma. Activity of the oncogene x-src (measured on pp60x-src kinase activity) and inositol lipid turnover is elevated in the tumor but, in contrast to the animals bearing the inherited melanoma, not in the brain. Tumor promoters do not induce melanoma in this strain. Similarly, in order to breed a fish strain which is highly susceptible to tumor promoters we introduced a regulatory gene, for instance an oncostatic gene (g) coding for a pretransformational arrest of pigment cell differentiation in the stem cell stage of the fish that develop the Mendelian inherited melanoma. The new strain is incapable of developing melanoma. Its x-src kinase activity and inositol lipid turnover is elevated in the brain, indicating that the biochemical processes which were found to be correlated with the hereditary melanoma formation, operate without the occurrence of melanoma. Following treatment of these animals with tumor promoters, melanoma develops within a very short latent period. Our tester strain can discriminate between tumor-initiating and tumor-promoting activities of agents of unknown carcinogenic potential.

Like other aryloxyisobutyrate derivates nafenopin is considered to lower the level of triglycerides in plasma due to a decreased synthesis of triglycerides in the liver. Moreover nafenopin is an extremely potent inducer of peroxisome proliferation and hepatocarcinogenesis. Since peroxisome proliferating agents have not been found to be genotoxic either in vitro nor in vivo studies (Warren et al 1980) these compounds will be act by other mechanism.

The synthesis of rRNA in cultured human liver cells grown in the presence of L-triiodothyronine (1.6 x 10(-5)M) was measured by means of polyacrylamide gel electrophoresis using a double labelling technique. The rate of accumulation of rRNA in the cytoplasm as well as the turnover of rRNA in ribosomes remained unchanged in comparison to control cells regardless of growth stage or cell population density. L-triiodothyronine, however, induced an increased synthesis of rpreRNA when added in the early growth phase of sparse cultures. This induction was time dependent and occurred temporarily, but it was never followed by a similar accumulation of mature rRNA. Therefore, in this system two control mechanisms counteracting each other were effective: the increased rpreRNA synthesis indicates a transcriptional control, and the wastage of odd rpreRNA, however, a post-transcriptional control.

The German Chemicals Act requires that chemicals are tested for behavioral toxicity at stage 2 of the testing procedure, i.e. if more than 1000 annual tons are produced. For this purpose a guideline was developed according to which data on behavioral toxicity are to be collected, which are based on cageside observations during longterm exposure. The protocol covers outer appearance, as well as motor, sensory, autonomic and central nervous system functions. Data are to be reported in tabular form and should be evaluated by taking all aspects of the toxicological profile into account.

The content of ribosomal DNA in mice liver at the beginning as well as near the end of the hematopoietic period was measured by RNA/DNA-hybridization in solution. At both stages the amount of ribosomal DNA was the same and comparable to that of postnatal liver.