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To improve the understanding of the extraction chemistry of An(iii) and Ln(iii) with N-donor ligands 2,6-bis(5-( tert -butyl)-1 H -pyrazol-3-yl)pyridine (C4-BPP) in the presence of 2-bromohexanoic acid was investigated. Extraction studies showed an excellent separation factor of SF Am(III)/Eu(III) ≈ 200 and SF Am(III)/Nd(III) ≈ 60 in comparison with the structurally similar ligand 2,6-bis(5-neopentyl-1 H -pyrazol-3-yl)pyridine C5-BPP (SF Am(III)/Eu(III) ≈ 100), even though C5-BPP showed significantly higher stability constants. Time-resolved laser fluorescence spectroscopy (TRLFS) studies revealed the formation of the ternary 1 : 1 and 1 : 2 complexes [Eu(C4-BPP) n (2-bromohexanoate) m ] (3- m )+ ( n = 1-2) ( and ). [Eu(C4-BPP) 2 (2-bromohexanoate) m ] (3- m )+ was the relevant complex species in solvent extraction. In contrast, Cm(iii) form stable 1 : 3 complexes. The ability of 2-bromohexanoic acid to replace C4-BPP from the inner coordination sphere of Eu(iii) but not from Cm(iii) is due to a more favorable complexation of Cm(iii) over Eu(iii) with C4-BPP. This resulted in a notably more efficient separation of An(iii) and Ln(iii) in comparison with C5-BPP., Competing Interests: No potential conflict of interest was reported by the author(s)., (This journal is © The Royal Society of Chemistry.)

Injurious pecking against conspecifics is a serious problem in turkey husbandry. Bloody injuries act as a trigger mechanism to induce further pecking, and timely detection and intervention can prevent massive animal welfare impairments and costly losses. Thus, the overarching aim is to develop a camera-based system to monitor the flock and detect injuries using neural networks. In a preliminary study, images of turkeys were annotated by labelling potential injuries. These were used to train a network for injury detection. Here, we applied a keypoint detection model to provide more information on animal position and indicate injury location. Therefore, seven turkey keypoints were defined, and 244 images (showing 7660 birds) were manually annotated. Two state-of-the-art approaches for pose estimation were adjusted, and their results were compared. Subsequently, a better keypoint detection model (HRNet-W48) was combined with the segmentation model for injury detection. For example, individual injuries were classified using "near tail" or "near head" labels. Summarizing, the keypoint detection showed good results and could clearly differentiate between individual animals even in crowded situations.

Footpad dermatitis (FPD) is an indicator of animal welfare in turkeys, giving evidence of the animals' physical integrity and providing information on husbandry management. Automated systems for assessing FPD at slaughter can present a useful tool for objective data collection. However, using automated systems requires that they reliably assess the incidence. In this study, the feet of turkeys were scored for FPD by both an automated camera system and a human observer, using a five-scale score. The observer reliability between both was calculated (Krippendorff's alpha). The results were not acceptable, with an agreement coefficient of 0.44 in the initial situation. Therefore, pictures of 3,000 feet scored by the automated system were evaluated systematically to detect deficiencies. The reference area (metatarsal footpad) was not detected correctly in 55.0% of the feet, and false detections of the alteration on the footpad (FPD) were found in 32.9% of the feet. In 41.3% of the feet, the foot was not presented straight to the camera. According to these results, the algorithm of the automated system was modified, aiming to improve color detection and the distinction of the metatarsal footpad from the background. Pictures of the feet, now scored by the modified algorithm, were evaluated again. Observer reliability could be improved (Krippendorff's alpha = 0.61). However, detection of the metatarsal footpad (50.9% incorrect detections) and alterations (27.0% incorrect detections) remained a problem. We found that the performance of the camera system was affected by the angle at which the foot was presented to the camera (skew/straight; p < 0.05). Furthermore, the laterality of the foot (left/right) was found to have a significant effect ( p < 0.001). We propose that the latter depends on the slaughter process. This study also highlights a high variability in observer reliability of human observers. Depending on the respective target parameter, the reliability coefficient (Krippendorff's alpha) ranged from 0.21 to 0.82. This stresses the importance of finding an objective alternative. Therefore, it was concluded that the automated detection system could be appropriate to reliably assess FPD at the slaughterhouse. However, there is still room to improve the existing method, especially when using FPD as a welfare indicator., Competing Interests: RG was employed by Heidemark Mästerkreis GmbH u. Co. JS-L was employed by CLK GmbH. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Stracke, Andersson, Volkmann, Spindler, Schulte-Landwehr, Günther and Kemper.)

Living donor robotic-assisted kidney transplantation (RAKT) is an alternative to open kidney transplantation (OKT), but experience with this technique is limited in the United States., Methods: A retrospective review of living donor kidney transplants performed between 2016 and 2018 compared RAKT with OKT with regard to recipient, donor, and perioperative parameters. A 1:1 propensity score matching was performed on recipient/donor age, sex, body mass index, race, preoperative dialysis, and calculated panel reactive antibodies., Results: Outcomes of patient survival, graft survival, and postoperative complications were assessed for 139 transplants (47 RAKT and 92 OKT). Propensity score analysis (47:47) showed that RAKT recipients had longer warm ischemic times (49 versus 40 min; P  < 0.001) and less blood loss (100 versus 150 mL; P  = 0.005). Operative time and length of stay were similar between groups. Postoperative serum creatinine was similar during a 2-y follow-up. Post hoc analysis excluding 4 open conversions showed lower operative time with RAKT (297 versus 320 min; P  = 0.04) and lower 30-d (4.7% versus 23.4%; P  = 0.02) and 90-d (7% versus 27.7%; P  = 0.01) Clavien-Dindo grade ≥3 complications., Conclusions: Our findings suggest that RAKT is a safe alternative to OKT., Competing Interests: A.Y. is a consultant and proctor for Intuitive Surgical. The other authors declare no funding or conflicts of interest., (Copyright © 2022 The Author(s). Transplantation Direct. Published by Wolters Kluwer Health, Inc.)

Broiler chickens in Europe are usually raised in a barren environment. Elevated perforated platforms address this problem and can positively influence animal health and welfare. To evaluate the effect of an elevated perforated floor on the behavior of broiler chickens, one of two barns was equipped with a perforated flooring system under the food and water supply. The second barn was used as a control. In total, three fattening periods were observed, with 500 broiler chickens (Ross 308 breed) kept in each barn. To compare the behavior of the birds in these groups, cameras were installed in the two barns. The videos were analyzed by counting the number of birds and observing focal animals while recording their behavior. More animals were observed on the perforated floor than in the littered control area (p < 0.001), but focal animals spent less time on the perforated floor compared to the observed littered area in the control barn (p < 0.05). There were no differences in the length of the recorded behaviors between the treatments. These findings suggest that, in general, the elevated perforated floor is attractive for the animals. However, it does not promote one of the recorded behavior patterns. Our results show that an elevated perforated floor could be an option for structuring broiler barns.

This study aimed to develop a camera-based system using artificial intelligence for automated detection of pecking injuries in turkeys. Videos were recorded and split into individual images for further processing. Using specifically developed software, the injuries visible on these images were marked by humans, and a neural network was trained with these annotations. Due to unacceptable agreement between the annotations of humans and the network, several work steps were initiated to improve the training data. First, a costly work step was used to create high-quality annotations (HQA) for which multiple observers evaluated already annotated injuries. Therefore, each labeled detection had to be validated by three observers before it was saved as "finished", and for each image, all detections had to be verified three times. Then, a network was trained with these HQA to assist observers in annotating more data. Finally, the benefit of the work step generating HQA was tested, and it was shown that the value of the agreement between the annotations of humans and the network could be doubled. Although the system is not yet capable of ensuring adequate detection of pecking injuries, the study demonstrated the importance of such validation steps in order to obtain good training data.

Animal welfare is one of the most challenging issues in modern farm animal husbandry. Animal welfare indicators can be used to monitor welfare on farms or at slaughterhouses, with footpad dermatitis (FPD) being one of the most important indicators used in turkeys. Up to now, the severity of FPD has been measured by evaluating the size of altered lesions on the metatarsal pad of birds. However, such lesions are not only found on the metatarsal pads, but alterations can also occur on the digital pads of the animals, the latter is not included in the European standard scoring systems for turkeys so far. The aim of the present study was to give a detailed outline of alterations on the digital pads of turkeys and associate their occurrence to a standardly used five-point scoring system, which is based on alterations of the metatarsal pad only. Therefore, pictures of 500 feet of turkeys from 16 flocks at the end of the fattening phase were taken, using an automatic camera system. Based on these pictures, alterations on the digits were scored according to different parameters (lesions, swellings, and number of affected digits). Furthermore, detailed measurements were conducted using an imaging software. Results were compared with a standardly used five-point scoring system (standard FPD scoring system), based on the metatarsal pad as reference. Results provide no equivalence in occurrence and severity of alterations on the metatarsal pads compared to those found on the digits. Pathologic alterations on the digits were already present at standard FPD scoring level 0; no differentiation became obvious between the higher scoring levels 2-4. Strong correlations were found when comparing percentage of alterations of the standard FPD scoring system to those of a system including alterations on the digits and the metatarsal pad, using the total foot as a reference (r p = 0.9, p < 0.001). This was the first study conducting a detailed analysis of alterations on the digits of turkeys. In conclusion, results of this study show that including the evaluation of alterations on digits could refine the present FPD scoring system, especially when using FPD as an animal welfare indicator., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Stracke, Volkmann, May, Döhring, Kemper and Spindler.)

In this study, a new housing system for broiler was tested. This system consisted of a slatted floor area and a littered area with the aim of improving litter quality. Two experimental broiler houses were provided. In house 1, a slatted floor was installed below the drinker and feedlines. Littered areas flanked the slatted floor. Broiler house 2 reflected conditions in commercial systems, consisting of a full littered area. Litter samples were taken at day 11 and at day 32 of the fattening period. Manure samples were taken at day 32. The total bacteria count (TBC), coliforms, Escherichia coli ( E. coli ) and ESBL-producing bacteria were determined. Furthermore, physical parameters (dry matter, water activity, pH) of litter and manure were measured. For statistical analyzes, a generalized linear mixed model (GLIMMIX procedure) was calculated. The floor did not show any significant effect on the bacteria content of the litter. Regarding TBC in litter, the floor showed a tendency for an effect (F = 5.42, p<0.1) with lower contents in house 1. Regarding the manure under the slatted floor, a tendency for a difference between house 1 and house 2 was found for the content of E. coli (F = 5.55, p<0.1) with higher contents in house 1. The floor did not show any significant effect on the physical parameters of litter and manure. The results of this experimental study showed no positive effects on the selected litter parameters, but further studies, especially on-farm experiments are necessary to confirm these results., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2020 The Authors.)

Objectives: During a 'cannot intubate, cannot oxygenate' situation, asphyxia can lead to cardiac arrest. In this stressful situation, two complex algorithms facilitate decision-making to save a patient's life: difficult airway management and cardiopulmonary resuscitation. However, the extent to which competition between the two algorithms causes conflicts in the execution of pivotal treatment remains unknown. Due to the rare incidence of this situation and the very low feasibility of such an evaluation in clinical reality, we decided to perform a randomised crossover simulation research study. We propose that even experienced healthcare providers delay cricothyrotomy, a lifesaving approach, due to concurrent cardiopulmonary resuscitation in a 'cannot intubate, cannot oxygenate' situation., Design: Due to the rare incidence and dynamics of such a situation, we conducted a randomised crossover simulation research study., Setting: We collected data in our institutional simulation centre between November 2016 and November 2017., Participants: We included 40 experienced staff anaesthesiologists at our tertiary university hospital centre., Intervention: The participants treated two simulated patients, both requiring cricothyrotomy: one patient required cardiopulmonary resuscitation due to asphyxia, and one patient did not require cardiopulmonary resuscitation. Cardiopulmonary resuscitation was the intervention. Participants were evaluated by video records., Primary Outcome Measures: The difference in 'time to ventilation through cricothyrotomy' between the two situations was the primary outcome measure., Results: The results of 40 participants were analysed. No carry-over effects were detected in the crossover design. During cardiopulmonary resuscitation, the median time to ventilation was 22 s (IQR 3-40.5) longer than that without cardiopulmonary resuscitation (p=0.028), including the decision-making time., Conclusion: Cricothyrotomy, which is the most crucial treatment for cardiac arrest in a 'cannot intubate, cannot oxygenate' situation, was delayed by concurrent cardiopulmonary resuscitation. If cardiopulmonary resuscitation delays cricothyrotomy, it should be interrupted to first focus on cricothyrotomy., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2019. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Wet litter is the most important cause of footpad dermatitis in poultry, this in turn being a highly relevant animal-related welfare indicator. This field study was subdivided into two experiments. In Experiment 1, the standard diet was supplemented by 0.2% enriched charcoal, being a non-specific absorber and therefore might be promising in reducing faecal moisture. In Experiment 2, the experimental group received a reduced crude protein diet during weeks 6-13, combined with a 0.2% enriched charcoal supplementation. The trials were each conducted with two batches on three farms under on-farm conditions. The animals were observed at 6, 10, 14 and 18 weeks of age to collect data on body weight and different health parameters. The mortality and litter samples were analysed after slaughtering. In Experiment 1, performance and health were not affected despite higher dry matter content of the litter. In Experiment 2, the weight of birds receiving the protein-reduced diet was decreased significantly throughout the experiment. However, the slaughter weight did not differ. The mortality was reduced by 0.5% in the experimental group. Therefore, it was concluded that 0.2% of enriched charcoal is not a valuable feed-additive regarding animal health, while temporary protein reduction might have positive effects., Competing Interests: The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

Therapeutic antibodies can undergo a variety of chemical modification reactions in vitro. Depending on the site of modification, either antigen binding or Fc-mediated functions can be affected. Oxidation of tryptophan residues is one of the post-translational modifications leading to altered antibody functionality. In this study, we examined the structural and functional properties of a therapeutic antibody construct and 2 affinity matured variants thereof. Two of the 3 antibodies carry an oxidation-prone tryptophan residue in the complementarity-determining region of the V L domain. We demonstrate the differences in the stability and bioactivity of the 3 antibodies, and reveal differential degradation pathways for the antibodies susceptible to oxidation.

In the present study, we have developed a novel one-arm single chain Fab heterodimeric bispecific IgG (OAscFab-IgG) antibody format targeting the insulin-like growth factor receptor type I (IGF-1R) and the epidermal growth factor receptor (EGFR) with one binding site for each target antigen. The bispecific antibody XGFR is based on the "knob-into-hole" technology for heavy chain heterodimerization with one heavy chain consisting of a single chain Fab to prevent wrong pairing of light chains. XGFR was produced with high expression yields and showed simultaneous binding to IGF-1R and EGFR with high affinity. Due to monovalent binding of XGFR to IGF-1R, IGF-1R internalization was strongly reduced compared with the bivalent parental antibody, leading to enhanced Fc-mediated cellular cytotoxicity. To further increase immune effector functions triggered by XGFR, the Fc portion of the bispecific antibody was glycoengineered, which resulted in strong antibody-dependent cell-mediated cytotoxicity activity. XGFR-mediated inhibition of IGF-1R and EGFR phosphorylation as well as A549 tumor cell proliferation was highly effective and was comparable with a combined treatment with EGFR (GA201) and IGF-1R (R1507) antibodies. XGFR also demonstrated potent anti-tumor efficacy in multiple mouse xenograft tumor models with a complete growth inhibition of AsPC1 human pancreatic tumors and improved survival of SCID beige mice carrying A549 human lung tumors compared with treatment with antibodies targeting either IGF-1R or EGFR. In summary, we have applied rational antibody engineering technology to develop a heterodimeric OAscFab-IgG bispecific antibody, which combines potent signaling inhibition with antibody-dependent cell-mediated cytotoxicity induction and results in superior molecular properties over two established tetravalent bispecific formats., (© 2014 by The American Society for Biochemistry and Molecular Biology, Inc.)

Introduction: Plasma selenium (Se) concentrations are reduced in critically ill surgical patients, and lower plasma Se concentrations are associated with worse outcomes. We investigated whether adjuvant Se supplementation in the form of sodium selenite could improve outcomes in surgical patients with sepsis., Methods: In this retrospective study, all adult patients admitted to a 50-bed surgical ICU with severe sepsis between January 2004 and April 2010 were included and analysed according to whether they had received adjuvant Se supplementation, which was given at the discretion of the attending physician. When prescribed, Se was administered in the form of sodium selenite pentahydrate (Na2SeO3∙5H2O), in which 100 μg of Se corresponds to 333 μg of sodium selenite. A bolus of sodium selenite corresponding to 1,000 μg of Se was injected intravenously through a central venous line for 30 minutes, followed by infusion of 1,000 μg/day for 24 hours for 14 days until ICU discharge or death. We performed logistic regression analysis to investigate the impact of adjuvant Se supplementation on hospital mortality., Results: Adjuvant Se was administered to 413 (39.7%) of the 1,047 patients admitted with severe sepsis. Age and sex were similar between patients who received adjuvant Se and those who did not. Compared with patients who did not receive adjuvant Se supplementation, patients who did had higher scores on the Simplified Acute Physiology Score II, a greater prevalence of cancer upon admission to the ICU and were more commonly admitted after abdominal surgery. Compared with patients who did not receive adjuvant Se, patients who did had higher hospital mortality rates (46% versus 39.1%; P = 0.027), and longer median (interquartile range (IQR)) ICU stays (15 days (6 to 24) versus 11 days (4 to 24); P = 0.01) and hospital lengths of stay (33 days (21 to 52) versus 28 days (17 to 46); P = 0.001). In multivariable analysis, adjuvant Se supplementation was not independently associated with favourable outcome (odds ratio = 1.19, 95% confidence interval = 0.86 to 1.65; P = 0.288)., Conclusions: In this retrospective analysis of a large cohort of surgical ICU patients with severe sepsis, adjuvant Se supplementation in the form of sodium selenite had no impact on in-hospital death rates after adjustment for confounders.

Preserving the chemical and structural integrity of therapeutic antibodies during manufacturing and storage is a major challenge during pharmaceutical development. Oxidation of Fc methionines Met252 and Met428 is frequently observed, which leads to reduced affinity to FcRn and faster plasma clearance if present at high levels. Because oxidation occurs in both positions simultaneously, their individual contribution to the concomitant changes in pharmacokinetic properties has not been clearly established. A novel pH-gradient FcRn affinity chromatography method was applied to isolate three antibody oxidation variants from an oxidized IgG1 preparation based on their FcRn binding properties. Physico-chemical characterization revealed that the three oxidation variants differed predominantly in the number of oxMet252 per IgG (0, 1, or 2), but not significantly in the content of oxMet428. Corresponding to the increase in oxMet252 content, stepwise reduction of FcRn affinity in vitro, as well as faster clearance and shorter terminal half-life, in huFcRn-transgenic mice were observed. A single Met252 oxidation per antibody had no significant effect on pharmacokinetics (PK) compared with unmodified IgG. Importantly, only molecules with both heavy chains oxidized at Met252 exhibited significantly faster clearance. In contrast, Met428 oxidation had no apparent negative effect on PK and even led to somewhat improved FcRn binding and slower clearance. This minor effect, however, seemed to be abrogated by the dominant effect of Met252 oxidation. The novel approach of functional chromatographic separation of IgG oxidation variants followed by physico-chemical and biological characterization has yielded the first experimentally-backed explanation for the unaltered PK properties of antibody preparations containing relatively high Met252 and Met428 oxidation levels.

Bispecific antibodies (bsAbs) that bind to cell surface antigens and to digoxigenin (Dig) were used for targeted small interfering RNA (siRNA) delivery. They are derivatives of immunoglobulins G (IgGs) that bind tumor antigens, such as Her2, IGF1-R, CD22, and LeY, with stabilized Dig-binding variable domains fused to the C-terminal ends of the heavy chains. siRNA that was digoxigeninylated at its 3'end was bound in a 2:1 ratio to the bsAbs. These bsAb-siRNA complexes delivered siRNAs specifically to cells that express the corresponding antigen as demonstrated by flow cytometry and confocal microscopy. The complexes internalized into endosomes and Dig-siRNAs separated from bsAbs, but Dig-siRNA was not released into the cytoplasm; bsAb-targeting alone was thus not sufficient for effective mRNA knockdown. This limitation was overcome by formulating the Dig-siRNA into nanoparticles consisting of dynamic polyconjugates (DPCs) or into lipid-based nanoparticles (LNPs). The resulting complexes enabled bsAb-targeted siRNA-specific messenger RNA (mRNA) knockdown with IC(50) siRNA values in the low nanomolar range for a variety of bsAbs, siRNAs, and target cells. Furthermore, pilot studies in mice bearing tumor xenografts indicated mRNA knockdown in endothelial cells following systemic co-administration of bsAbs and siRNA formulated in LNPs that were targeted to the tumor vasculature.Molecular Therapy - Nucleic Acids (2012) 1, e45; doi:10.1038/mtna.2012.39; published online 18 September 2012.

Bispecific antibodies that bind cell-surface targets as well as digoxigenin (Dig) were generated for targeted payload delivery. Targeting moieties are IgGs that bind the tumor antigens Her2, IGF1R, CD22, or LeY. A Dig-binding single-chain Fv was attached in disulfide-stabilized form to C termini of CH3 domains of targeting antibodies. Bispecific molecules were expressed in mammalian cells and purified in the same manner as unmodified IgGs. They are stable without aggregation propensity and retain binding specificity/affinity to cell-surface antigens and Dig. Digoxigeninylated payloads were generated that retain full functionality and can be complexed to bispecific antibodies in a defined 21 ratio. Payloads include small compounds (Dig-Cy5, Dig-Doxorubicin) and proteins (Dig-GFP). Complexed payloads are targeted by the bispecifics to cancer cells and because these complexes are stable in serum, they can be applied for targeted delivery. Because Dig bispecifics also effectively capture digoxigeninylated compounds under physiological conditions, separate administration of uncharged Dig bispecifics followed by application of Dig payload is sufficient to achieve antibody-mediated targeting in vitro and in vivo.

In this study, we describe novel tetravalent, bispecific antibody derivatives that bind two different epitopes on the HIV coreceptor CCR5. The basic protein formats that we applied were derived from Morrison-type bispecific antibodies: whole IgGs to which we connected single-chain antibodies (scFvs) via (Gly4Ser)n sequences at either the C or N terminus of the light chain or heavy chain. By design optimization, including disulfide stabilization of scFvs or introduction of 30-amino-acid linkers, stable molecules could be obtained in amounts that were within the same range as or no less than 4-fold lower than those observed with monoclonal antibodies in transient expression assays. In contrast to monospecific CCR5 antibodies, bispecific antibody derivatives block two alternative docking sites of CCR5-tropic HIV strains on the CCR5 coreceptor. Consequently, these molecules showed 18- to 57-fold increased antiviral activities compared to the parent antibodies. Most importantly, one prototypic tetravalent CCR5 antibody had antiviral activity against virus strains resistant to the single parental antibodies. In summary, physical linkage of two CCR5 antibodies targeting different epitopes on the HIV coreceptor CCR5 resulted in tetravalent, bispecific antibodies with enhanced antiviral potency against wild-type and CCR5 antibody-resistant HIV-1 strains.

The 3Rs - Replacement, Reduction and Refinement - have become increasingly important in designing animal experiments. The Pavlov sling is thought to be a non-invasive method to restrain dogs for examinations. The aim of our study was to investigate whether laboratory Beagle dogs that had been trained to tolerate restraint by a Pavlov sling are stressed by this procedure and, furthermore, to analyze their behavior during this period. Five male and five female Beagle dogs were used, each three years of age. Animals were restrained in the Pavlov sling for 30 min on six days with an interval of at least two days. The following behaviors were recorded every minute for each session: postures of body, head, and ears, as well as state of eyes, tail, legs, and mouth. Additionally, the animals were observed for the occurrence of particular stress signs, including body shaking, sweating of the paws, increased saliva production, piloerection, blinking of eyes, snout licking, yawning, and panting. As an indicator for stress, salivary cortisol levels were measured before, during, and after each session. Our results show that for most behavioral parameters, e.g., body, leg, head, tail, and ear posture, the frequency of changes between different behavior patterns, as well as cortisol concentration, were not influenced by restraint in the Pavlov sling. Therefore, the Pavlov sling does not seem to be perceived as a stressful situation by the Beagle dogs. Our study demonstrates that under certain conditions the use of the Pavlov sling in trained dogs can substitute for more ordinary methods of immobilization, e.g., the use of narcotics.

The role of matrix metalloproteinases in the degradative events invoked in the cartilage and bone of arthritic joints has long been appreciated and attempts at the development of proteinase inhibitors as potential therapeutic agents have been made. However, the spectrum of these enzymes orchestrating connective tissue turnover and general biology is much larger than anticipated. Biochemical studies of the individual members of the matrix metalloproteinase family are now underway, ultimately leading to a more detailed understanding of the function of their domain structures and to defining their specific role in cellular systems and the way that they are regulated. Coupled with a more comprehensive and detailed study of proteinase expression in different cells of joint tissues during the progress of arthritic diseases, it will be possible for the future development and application of highly specific proteinase inhibitors to be directed at specific key cellular events.

Double (plp-/-mag-/-) and triple (plp-/-mbp-/-mag-/-) null-allelic mouse lines deficient in proteolipid protein (PLP), myelin-associated glycoprotein (MAG), and myelin basic protein (MBP) were generated and characterized genetically, biochemically, and morphologically including their behavioral capacities. The plp-/-mag-/- mutant develops a rapidly progressing axon degeneration in CNS with severe cognitive and motor coordinative deficits but has a normal longevity. CNS axons of the plp-/-mbp-/-mag-/- mouse are hypomyelinated and ensheathed by "pseudomyelin" with disturbed protein and complex lipid composition. The shiverer trait in the plp-/-mbp-/-mag-/- similar to the plp-/-mbp-/- mutant is significantly ameliorated, and its lifespan is considerably prolonged. The longevity of these dysmyelinosis mouse mutants recommends them as suitable models for the long-term evaluation of stem cell therapeutic strategies.