Short-term complications in intra- and extra-articular anterior cruciate ligament reconstruction. Comparison with the literature on isolated intra-articular reconstruction. A multicenter study by the French Arthroscopy Society.

INTRODUCTION: Lateral tenodesis (LT) is performed to limit the risk of iterative tear following anterior cruciate ligament (ACL) reconstruction in at-risk patients. By adding an extra procedure to isolated ACL graft, LT reconstruction increases operating time and may complicate postoperative course. The objective of the present study was to evaluate the rate of early complications. The study hypothesis was that associating ALL reconstruction to ACL reconstruction does not increase the complications rate found with isolated ACL reconstruction. MATERIAL AND METHODS: A prospective multicenter study included 392 patients: 70% male; mean age, 29.9 years; treated by associated ACL and LT reconstruction. All adverse events were inventoried. RESULTS: Mean hospital stay was 2 days, with 46% day-surgery. Walking was resumed at a mean 27 days, with an advantage for patients treated by the hamstring technique. The early postoperative complications rate was 12%, with 1.7% specifically implicating LT reconstruction: pain, hematoma, stiffness in flexion and extension, and infection. There was a 5% rate of surgical revision during the first year, predominantly comprising arthrolysis for extension deficit. The 1-year recurrence rate was 2.8%. DISCUSSION: The complications rate for combined intra- and extra-articular reconstruction was no higher than for isolated intra-articular ACL reconstruction, with no increase in infection or stiffness rates. The rate of complications specific to ALL reconstruction was low, at 1.7%, and mainly involved fixation error causing lateral soft-tissue impingement. LEVEL OF EVIDENCE: IV, prospective multicenter study.

Combined intra- and extra-articular grafting for revision ACL reconstruction: A multicentre study by the French Arthroscopy Society (SFA).

BACKGROUND: A careful analysis of the reasons for ACL reconstruction failure is essential to selection of the optimal surgical revision technique designed to ensure good rotational stability and to minimise the risk of re-rupture. OBJECTIVE: To evaluate anterolateral ligament (ALL) stabilisation during revision ACL reconstruction. HYPOTHESIS: ALL stabilisation during revision ACL reconstruction provides good rotational stability without increasing the risk of complications. MATERIAL AND METHODS: This multicentre study included 349 patients, 151 retrospectively and 198 prospectively. There were 283 males and 66 females. Inclusion criteria were an indication for revision ACL reconstruction surgery with combined intra-articular reconstruction and ALL stabilisation after failed autograft ACL reconstruction, and intact PCL. Exclusion criteria were primary ACL reconstruction and concomitant peripheral medial and/or lateral lesions. Each patient underwent a clinical and radiographic evaluation before and after revision surgery. Before revision surgery, the mean IKDC score was 56.5±15.5 and 96% of patients were IKDC C or D. RESULTS: Rates were 5.0% for early and 10.5% for late postoperative complications. Lachmann's test had a hard stop at last follow-up in 97% of patients. The pivot-shift test was positive in 1% of patients. The mean subjective IKDC score was 84.5±13.0 and 86.5% of patients were IKDC A or B. The proportions of patients with radiographic knee osteoarthritis at last follow-up was unchanged for the lateral tibio-femoral and patello-femoral compartments but increased by 9.7% to 21.2% for the medial tibio-femoral compartment. The re-rupture rate was 1.2% and the further surgical revision rate was 5.4%. CONCLUSION: Anterior laxity at last follow-up was consistent with previous studies of revision ACL reconstruction. However, rotational stability and the re-rupture risk were improved. ALL stabilisation is among the techniques that deserve consideration as part of the therapeutic options for revision ACL reconstruction. LEVEL OF EVIDENCE: IV, retrospective and prospective cohort study.

The role of computer assisted navigation in revision surgery for failed anterior cruciate ligament reconstruction of the knee: A continuous series of 52 cases.

INTRODUCTION: The causes of failure of anterior cruciate ligament (ACL) reconstruction mainly involve incorrect tunnel positioning. There is no intraoperative tool allowing the surgeon to test graft biomechanics and to confirm that the new graft is in an optimal position. HYPOTHESIS: Control is improved with computer assisted navigation. MATERIAL AND METHODS: In this retrospective study, revision ACL reconstruction was performed with a new autologous graft in a continuous series of 52 failed ACL reconstructions. A computer assisted navigation system was used intraoperatively in all knees. Evaluation with this system confirmed the position of old and new tunnels as well as intraoperative laxity. RESULTS: Evaluation of tunnel position based on traditional radiological criteria found in the literature significantly underestimated graft biomechanics: 69% of the cases presented with unfavorable graft ansiometry (mean: 13 ± 2.2mm) while the correct position of the tibial tunnel was identified in 64% of cases on radiography and the femoral tunnel in 48%. All new grafts were optimally positioned by the computer assisted navigation system with a mean isometery of 3.2 (± 0.7) mm. Comparative pre- and postoperative evaluation of laxity showed a statistically significant improvement (P < 0.001): preoperative and postoperative Lachman test: 10.5 ± 2 mm and 3 ± 0.5, respectively; global rotational laxity: 24 ± 5° and 37 ± 7° respectively. CONCLUSION: The use of a computer assisted navigation system allows optimal positioning of the graft as well as a predictive assessment of laxity.

Posterior tibial slope accuracy with patient-specific cutting guides during total knee arthroplasty: A preliminary study of 50 cases.

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.

Influence of the optical system and anatomic points on computer-assisted total knee arthroplasty.

BACKGROUND: For over a decade, computer-assisted orthopaedic surgery for total knee arthroplasty has been accepted as ensuring accurate implant alignment in the coronal plane. HYPOTHESIS: We hypothesised that lack of accuracy in skeletal landmark identification during the acquisition phase and/or measurement variability of the infrared optical system may limit the validity of the numerical information used to guide the surgical procedure. METHODS: We built a geometric model of a navigation system, with no preoperative image acquisition, to simulate the stages of the acquisition process. Random positions of each optical reflector center and anatomic acquisition point were generated within a sphere of predefined diameter. Based on the virtual geometric model and navigation process, we obtained 30,000 simulations using the Monte Carlo statistical method then computed the variability of the anatomic reference frames used to guide the bone cuts. Rotational variability (α, ß, γ) of the femoral and tibial landmarks reflected implant positioning errors in flexion-extension, valgus-varus, and rotation, respectively. RESULTS: Taking into account the uncertainties pertaining to the 3D infrared optical measurement system and to anatomic point acquisition, the femoral and tibial landmarks exhibited maximal alpha (flexion-extension), beta (valgus-varus), and gamma (axial rotation) errors of 1.65° (0.9°); 1.51° (0,98°), and 2.37° (3.84°), respectively. Variability of the infrared optical measurement system had no significant influence on femoro-tibial alignment angles. CONCLUSION: The results of a Monte Carlo simulation indicate a certain level of vulnerability of navigation systems for guiding position in rotation, contrasting with robustness for guiding sagittal and coronal alignments. LEVEL OF EVIDENCE: Level IV.

A risk-benefit analysis of alternating low-pressure CO2 insufflation and fluid medium in arthroscopic knee ACL reconstruction.

PURPOSE: 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. METHODS: 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. RESULTS: 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. CONCLUSION: 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.

Skeletal landmarks for TKR implantations: evaluation of their accuracy using EOS imaging acquisition system.

INTRODUCTION: Lower extremity alignment remains one essential objective during total knee replacement. Implants positioning analysis requires selecting reliable skeletal landmarks. Our objective was to in vivo evaluate the precision of the implemented skeletal landmarks. This evaluation was based on multiple three-dimensional (3D) computer reconstructions of the lower extremity derived from an EOS biplanar low-dose X-ray system acquisition. A 3D angle measurement protocol was used. HYPOTHESIS: Currently defined landmarks carry a tolerable uncertainty margin, which can still probably be further improved. MATERIAL AND METHODS: Nine lower extremity 3D computer reconstructions were obtained from an EOS protocol based on seven simultaneous A-P and lateral views performed in standing position. A database was established by four operators; finally, building up a total of 99 in vivo 3D reconstructions of these nine lower extremities. Specific algorithms were used for such 3D reconstructions of lower extremities based on bone points and pre-identified contours on X-ray. Four femoral landmarks and four tibial landmarks were thus defined. For each bone and each landmark studied, a mean landmark for the 11 consecutive series elements was established. The deviation from each constructed landmark to the corresponding mean landmark was calculated based on the anteroposterior (x), longitudinal (y) and mediolateral axes (z), in translation (Tx, Ty, Tz) and in rotation (Rx, Ry, Rz). Uncertainty was estimated by the 95% confidence interval (95% CI). RESULTS: The landmarks located at the middle of the segment joining the center of each posterior condyle and at the barycenter of the plateaux showed a greater reliability; these landmarks uncertainty (95% CI) of Tx, Ty, Tz was less than 1, 0.5, 1.5 mm for the femur and 1.5, 0.6, 0.6 mm for the tibia, respectively. The femoral landmarks using the center or posterior edge of the posterior condyles to define the mediolateral axis were retained; for rotations Rx, Ry, and Rz, uncertainty remained less than 0.3, 4, and 0.5 degrees. All of the tibial landmarks had a comparable reliability in rotation, 95% of the Rx and Rz deviations were under 0.5 and 1.3 degrees, respectively, with a mean error less than 1 degrees . For the tibial rotation Ry, the mean error was greater (4 degrees), with uncertainty (95% CI) at 11.2 degrees. All tibial translations showed a mean error of 1 mm. The 3D implantation angles were measured on two patients using preoperative 3D skeletal reconstructions and 3D geometric models of the implants repositioned on postoperative EOS knee X-rays. DISCUSSION: The posterior condyles are rarely involved in the arthritic wear process, making them an anatomic landmark of choice in the analysis of the femoral component positioning. The femoral landmarks using the posterior condyles were sufficiently reliable for clinical use. However, the posterior contours of the tibial plateaux were less precise. The knees should be staggered from an anteroposterior perspective on the EOS lateral images so that they can be visualized separately. The anatomic zones on which the skeletal landmarks are based are usually removed by the bone cuts, making it preferable to save the preoperative computer reconstructions to analyze the postimplantation 3D reconstruction. CONCLUSION: The lower extremity skeletal landmarks precision relates to the quality of the corresponding 3D reconstructions. Except for tibial rotation, all the translation and rotation parameters were estimated within a mean error margin inferior to 1.2 mm and 1.3 degrees, respectively. Making the reconstruction algorithms more robust would render certain anatomic zones even more precise. Biplanar low-dose EOS X-ray system is a tool of the future to generate 3D knee X-rays that can improve the evaluation and follow-up of total knee arthroplasty patients.

[Experience with Poly Ether Ether Ketone (PEEK) cages and locking plate for anterior cervical fusion in the treatment of spine trauma without cord injury]. / Résultats radiologiques des arthrodèses par cages radio-transparentes et plaque antérieure dans le traitement des traumatismes du rachis cervical sans lésions médullaires.

PURPOSE OF THE STUDY: The purpose of this study was to determine whether spinal fusion using radiotransparent cages can be an appropriate treatment for traumatic injury of the cervical spine. MATERIAL AND METHODS: This series included 30 patients aged 17-84 years (average 46 years) treated between October 1999 and June 2003 for traumatic injury of the cervical spine without neurological deficit or cord injury. There were two bifocal cases so that the study concerned 32 fusions. Injuries were: tear drop (n=1), serious flexion sprain (n=8), biarticular dislocation (n=4), serous hyperextension sprain (n=4), dislocation-fracture (n=1), uniarticular fracture (n=7), fracture-separation of the facet joints (n=4), post-traumatic herniation (n=3). For each injury, we measured pre and postoperatively and at last follow-up: the intersomatic angle, anterior displacement, and height of the intersomatic space at the center of the intervertebral disc. All x-rays were read twice, by two independent investigators. In the event of disagreement, the x-rays were read again by a senior surgeon and the main author of this article. Anterior fusion was achieved using a Poly Ether Ether Ketone (PEEK) (32%) and knitted carbon (68%) cage (cologne, Ostapek, Nexis) filled with cancellous bone harvested percutaneously from the iliac crest. The cage was associated with an anterior titanium plate fixation (Senegas, Euros and Orion, Medtronic). A posterior approach was associated if further stability was required (n=4 fusions). All patients were reviewed at minimum five months follow-up. Intersomatic fusion was verified on the standard x-rays (plus stress images and computed tomography at three months). Fusion was considered to be achieved if continuous bone lines crossed the graft and angle measurements remained stable, with the cage in the same position on successive examinations. RESULTS: One patient died from lung cancer five months after spinal fusion. All other patients survived with a mean follow-up of 24 months. Fusion was achieved in all cases, at mean 78 days. The mean intersomatic angle increased from 12 degrees kyposis preoperatively to 13 degrees lordosis postoperatively at last follow-up. Anterior displacement of the fractured vertebral body was 3 mm preoperatively and 0.3 mm postoperatively. Height in the middle of the intersomatic space was 5.3 mm preoperatively and 8.2 mm postoperatively. There were no cases of secondary displacement. DISCUSSION: This study demonstrated that fusion with an intersomatic cage associated with anterior plating can be used in spine trauma victims, providing an outcome as good as in patients with degenerative disease. This method enables nearly anatomic reduction without secondary displacement and fusion in a short delay (which can be explained by the mechanical properties of the assembly and by the use of pure cancellous graft from the iliac crest). There is very little morbidity in our experience. For us, this technique is more reliable than fusion using a tri-cortical iliac crest graft. CONCLUSION: The use of an intersomatic cage is a simple, reliable technique for intersomatic spinal fusion with little morbidity for unstable traumatic injury of the spine without spinal cord injury.

[Inaugural cutaneous metastases of an osteosarcoma: a case report]. / Métastases sous-cutanées révélatrices d'un ostéosarcome du fémur: à propos d'un cas.

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.

Purification of prostaglandin D synthase by ceramic- and size exclusion chromatography.

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 pI 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.

Effects of acetylcysteine and ischaemic preconditioning on muscular function and postoperative pain after orthopaedic surgery using a pneumatic tourniquet.

BACKGROUND AND OBJECTIVE: 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. METHODS: 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). RESULTS: 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, P <0.05). CONCLUSIONS: 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.

Tumorigenic potential and the molecular mechanism of the carcinogenic effect exerted by 2-nitroanisole.

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.

[Acute anterior cruciate ligament repair with combined intra- and extra-articular reconstruction using an iliotibial band with the modified MacIntosh technique: a five-year follow-up study of 50 pivoting sport athletes]. / Reconstruction précoce intra et extra-articulaire du ligament croisé antérieur utilisant le tractus iliotibial selon la technique de MacIntosh modifiée: résultats chez 50 patients pratiquant un sport avec pivots au recul de 5 ans.

PURPOSE OF THE STUDY: Comparisons have been often made between bone-tendon-bone plasty and hamstring tendon four-strand plasty. Whether a lateral tenodesis should be associated with the intra-articular reconstruction and the appropriate time between the accident and the repair remain two topics of debate. We present results obtained in a consecutive series of 50 pivoting sport atheletes reviewed retrospectively. These patients had been treated within eight days of trauma with a modified MacIntosh technique using an iliotibial band. This technique enables lateral tenodesis and reconstruction of the central pivot with only one harvesting site. We searched for responses to two questions: are our results comparable to those in other published series? could this operation be warranted as an emergency procedure? MATERIAL AND METHODS: Fifty patients from a consecutive retrospective series of 62 patients (eight lost to follow-up and four excluded from the analysis) were reviewed by an independent observer at mean follow-up of 5.2 years (range 54.4 to 86.4 months). The ARPEGE and IKDC scoring systems were used. An isokinetic assessment was obtained in 38 patients at one year. The reconstruction technique used an iliotibial band measuring 40-45 mm in width. The lateral reconstruction consisted in section then translation of the lateral intermuscular partition. RESULTS: The overall outcome was scored as follows: IKDC A 38%, B 46%, C 12%, D 4%. Mean residual differential laxity (KT 1000) was 1.86+/-1.74 assessed manually with a negative Lachman in 48% of knees. 88% of the positive tests had been neutralized. Early anatomic failure was noted in two knees with recurrent traumatic tears at 25 and 38 months. Using the ARPEGE scoring system, outcome was excellent in 38%, good in 46%, fair in 12% and poor in 4%. At last follow-up, the level of sports activities was unchanged in 33 patients. Irreducible flexion measuring more than 5 degrees was noted in two patients, and a deficit in flexion greater than 20 degrees in three. One female athlete who had resumed her former sports level presented mobilization under narcosis. None of the patients complained of pain at the harvesting site. There was one case of muscle herniation proximally by wound dehiscence. Mean residual deficit of the quadriceps, measured at twelve months, was 10% at 90 degrees /s; mean residual deficit of the hamstrings at the same speed was 1.3%. DISCUSSION: Our overall results as measured with the IKDC scoring system were comparable with those observed in series using other autologous transplants. The risk of stiffness is greater with early reconstruction, suggesting emergency repair should be considered with caution. Recovery of muscle force demonstrates one of the advantages of using the iliotibial band which does not injure the extensor-flexor system of the knee joint. The fact that none of the patients complained of pain at the harvesting site is a favorable element for rehabilitation and resumed sports activities.

12-O-retinoylphorbol-13-acetate (RPA) behaves like a retinoid-derivative in binding to retinol-binding-protein: a correlation with its specific action as an incomplete tumor promoter?

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.

Improved purification of beta-lactoglobulin from acid whey by means of ceramic hydroxyapatite chromatography with sodium fluoride as a displacer.

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.

Development and application of an ELISA for the detection of duck antibodies against Riemerella anatipestifer antigens in egg yolk of vaccinees and in serum of their offspring.

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.

PCDDs/PCDFs, PCBs, and organochlorine pesticides in eggs of Eurasian sparrowhawks (Accipiter nisus), hobbies (Falco subbuteo), and northern goshawks (Accipiter gentilis) collected in the area of Berlin-Brandenburg, Germany.

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.

Lipocalin-type prostaglandin D synthase in milk: a new biomarker for bovine mastitis.

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.

Peritoneal macrophages from 17 alpha-ethinylestradiol-treated NMRI mice secrete transformation-specific low molecular weight proteins.

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.