Short communications: an ear-attached accelerometer detects effects of regrouping on lying, rumination, and activity times in calves.

The objective of this study was to use a sensor-based accelerometer (ACC) to identify changes in lying, rumination, and activity times in weaned calves during the moving and regrouping process. Overall, 270 healthy Holstein calves (from approximately 16 regrouping events) at the age of approximately 4 months were enrolled and equipped with an ear-attached ACC (SMARTBOW, Smartbow GmbH/ Zoetis LLC). Sensor data were recorded for 5 d before (d -5) until 4 d after moving and regrouping (d 4). The day of regrouping was defined as d 0. Acceleration data were continuously processed by specific algorithms (developed by SMARTBOW) for lying, rumination, and activity. Lying, rumination, and activity times were averaged from d -5 to d -3 to generate a baseline value for each parameter. Parameters on d 0 to d 4 after regrouping were compared to this baseline. All parameters showed significant changes compared with the baseline at d 0. Significant decreases in rumination and inactive times were seen up to d 2. Lying time was significantly lower until d 3. The study results indicate that the ACC can be used to monitor the disruptive effects of regrouping on lying and rumination behaviors. Further research is necessary to elucidate how these changes have an impact on health, performance, and welfare and to evaluate how to reduce these potentially negative effects.

Erratum to "Use of a real-time location system to detect cows in distinct functional areas within a barn" (JDS Commun. 2:217-222).

[This corrects the article DOI: 10.3168/jdsc.2020-0050.].

Monitoring selected behaviors of calves by use of an ear-attached accelerometer for detecting early indicators of diarrhea.

One of the most important diseases in calves worldwide is neonatal calf diarrhea (NCD), which impairs calf welfare and leads to economic losses. The aim of this study was to test whether the activity patterns of calves can be used as early indicators to identify animals at risk for suffering from NCD, compared with physical examination. We monitored 310 healthy female Holstein-Friesian calves on a commercial dairy farm immediately after birth, equipped them with an ear tag-based accelerometer (Smartbow, Smartbow GmbH), and conducted daily physical examinations during the first 28 d of life. The Smartbow system captured acceleration data indicative of standing and lying periods and activity levels (active and inactive), shown as minutes per hour. We categorized calves as diarrheic if they showed fecal scores of ≥3 on a 4-point scale on at least 2 consecutive days. Incidence of diarrhea was 50.7% (n = 148). A mixed logistic regression model showed that lying [odds ratio (OR) = 1.19], inactive (OR = 1.14), and active (OR = 0.92) times, 1 d before clinical identification of diarrhea (d -1), were associated with the odds of diarrhea occurring on the subsequent day. Receiver operating characteristics curve showed that lying time at d -1 was a fair predictor for diarrhea on the subsequent day (area under curve = 0.69). Average lying time on d -1 was 64.8 min longer in diarrheic calves compared with their controls. Median lying and inactive times decreased, and active time increased with age over the study period. The 24-h pattern of behavior indices based on the output of the Smartbow system followed periods of resting and active times, and showed that between 2200 h and 0600 h, calves spent the greatest percentage of time lying and inactive. These results showed that the accelerometer system has the potential to detect early indicators associated with NCD. In future studies, additional data for the development and testing of calf- and event-specific algorithms (e.g., for detecting milk intake, playing behavior) should be collected, which might further improve the early detection of diarrhea in calves.

Use of a real-time location system to detect cows in distinct functional areas within a barn.

Automated sensor-based monitoring of cows has become an important tool in herd management to improve or maintain animal health and welfare. Location systems offer the ability to locate animals within the barn for, for example, artificial insemination. Furthermore, they have the potential to measure the time cows spend in important areas of the barn, which might indicate need for improvement in the management of the herd or individuals. In this study, we tested the sensor-based real-time location system (RTLS) Smartbow (SB, Smartbow GmbH) under field conditions. The objectives of this study were (1) to determine the accuracy of the system to predict the location of the cow and the agreement between visual observations and RTLS observations for the total time spent by cows in relevant areas of the barn and (2) to compare the performance of 2 different algorithms (Alg1 and Alg2) for cow location. The study was conducted on a commercial Austrian dairy farm. In total, 35 lactating cows were video recorded for 3 consecutive days. From these recordings, approximately 1 h was selected randomly each day for every cow (3 d × 35 cows). Simultaneously, location data were collected and classified by the RTLS system as dedicated to the alley, feed bunk, or cubicle on a 1-min resolution. A total of 6,030 paired observations were derived from visual observations (VO) and the RTLS and used for the final data analysis. Substantial agreement of categorical data between VO and SB was obtained by Cohen's kappa for both algorithms (Alg1 = 0.76 and Alg2 = 0.78). Similar results were achieved by both algorithms throughout the study, with a slight improvement for Alg2. The ability of the system to locate the cows in the predefined areas was assessed, and the results from Alg2 showed sensitivity, specificity, and positive predictive value of alley (74.0, 91.2, and 76.9%), feed bunk (93.5, 86.2, and 89.1%), and cubicle (90.5, 83.3, and 95.4%) and an overall accuracy of 87.6%.The correlation coefficient (r) between VO and SB for the total time cows spent (within 1 h) in the predefined areas was good to strong (r = 0.82, 0.98, and 0.92 for alley, feed bunk, and cubicle, respectively). These results show the potential of the system to automatically assess total time spent by cows in important areas of the barn for indoor settings. Future studies should focus on evaluating 24-h periods to assess time budgets and to combine technologies such as accelerometers and location systems to improve the performance of behavior prediction in dairy cows.

Comparison of behavioral patterns of dairy cows with natural estrus and induced ovulation detected by an ear-tag based accelerometer.

Dairy farms face many challenges and changes. With increasing herd sizes and fewer farmers or employees per cow, new strategies to maintain or improve reproductive management are required. One of the major challenges is to detect cows in estrus and to estimate the perfect time for artificial insemination (AI). Several estrus and ovulation synchronization programs with timed AI as well as estrus detection aids, e.g., tail-paint, pedometer, accelerometer, and others are available. A combination of ovulation synchronization programs and technical solutions, however, has rarely been tested. This study was designed to gain insights into behavioral patterns of cows subjected to an Ovsynch program and to test if behavioral data could be used to optimize the timing of insemination within an Ovsynch program. In this study, we used an ear-tag based 3D-accelerometer system (SMARTBOW, Smartbow GmbH, Weibern, Austria) to generate data of behavioral patterns, i.e., rumination and activity. In Part 1 of this study, behavioral patterns during the peri-estrus period were compared between cows with physiological estrus and cows subjected to an Ovsynch protocol. On the day before estrus and on the day of estrus/AI, cows with natural estrus showed a clear drop in rumination and "inactivity" and an increase in "high activity", based on an algorithm of the accelerometer system, whereas, cows in the Ovsynch protocol showed only minor changes in behavioral patterns. In Part 2, we analyzed behavioral patterns between synchronized cows that became pregnant after AI and synchronized cows that remained open. As a result, no differences were detected between these two Ovsynch groups before AI. Thus, in this study we found no evidence that behavioral patterns can be used to improve conception rates within an Ovsynch protocol.

Evaluation of an ear-attached accelerometer for detecting estrus events in indoor housed dairy cows.

Precision dairy farming technologies have tremendous potential to improve and support farmers in herd management decisions, particularly in reproductive management. Nowadays, estrus detection in cows is challenging and several supporting tools are available. In this study, a 3D-accelerometer integrated into an ear-tag (SMARTBOW, Smartbow GmbH, Weibern, Austria) was used for the detection of cows in estrus. Movement pattern based on accelerometer data were analyzed and processed by algorithms and machine learning, resulting in estrus alerts. For the evaluation of the system, reproductive performance data of 579 estrus events of multiparous cows were used to retrospectively evaluate the accuracy of estrus alerts generated by the accelerometer-based system and the overall performance of the system. Estrus events were classified as 'gold standard' events, if an estrus followed by AI resulted in pregnancy, and as 'recorded estrus' events, if two estrus events with an interval of 18-25 d were in the herd records, independent of whether estrus was followed by AI or pregnancy. In total, 316 'gold standard' events were matched with estrus alerts generated by the accelerometer-based system, resulting in a sensitivity of 97%. Furthermore, 263 'recorded estrus' events were compared with correct or incorrect estrus alerts by the system. Sensitivity, specificity, positive and negative predictive values, accuracy, and error rate for 'recorded estrus' events were 97%, 98%, 96%, 94%, 96%, and 2%, respectively. In summary, the SMARTBOW system is suitable for an automated detection of estrus events of multiparous cows in indoor housed dairy cows.

Evaluation of an ear-tag-based accelerometer for monitoring rumination in dairy cows.

The objective of this study was to evaluate the ear-tag-based accelerometer system Smartbow (Smartbow GmbH, Weibern, Austria) for detecting rumination time, chewing cycles, and rumination bouts in indoor-housed dairy cows. For this, the parameters were determined by analyses of video recordings as reference and compared with the results of the accelerometer system. Additionally, we tested the intra- and inter-observer reliability as well as the agreement of direct cow observations and video recordings. Ten Simmental dairy cows in early lactation were equipped with 10-Hz accelerometer ear tags and kept in a pen separated from herd mates. A total mixed ration was fed twice a day via a roughage intake control system. During the study, cows' rumination and other activities were directly observed for 20 h by 2 trained observers. Additionally, cows were video recorded for 19 d, 24 h a day. After exclusion of unsuitable videos, 2,490 h of cow individual 1-h video sequences were eligible for further analyses. Out of this, one hundred 1-h video sequences were randomly selected and visually and manually classified by a trained observer using professional video analyses software. Based on these analyses, half of the data was used for development (based on data of 50-h video analyses) and testing (based on data of additional 50-h video analyses) of the Smartbow algorithms, respectively. Inter- and intra-observer reliability as well as the comparison of direct against video observations revealed in high agreements for rumination time and chewing cycles with Pearson correlation coefficients >0.99. The rumination time, chewing cycles, as well as rumination bouts detected by Smartbow were highly associated (r > 0.99) with the analyses of video recordings. Algorithm testing revealed in an underestimation of the average ± standard deviation rumination time per 1-h period by the Smartbow system of 17.0 ± 35.3 s (i.e., -1.2%), compared with visual observations. The average number ± standard deviation of chewing cycles and rumination bouts was overestimated by Smartbow by 59.8 ± 79.6 (i.e., 3.7%) and by 0.5 ± 0.9 (i.e., 1.6%), respectively, compared with the video analyses. In summary, the agreement between the Smartbow system with video analyses was excellent. From a practical and clinical point of view, the detected differences were negligible. However, further research is necessary to test the system under various field conditions and to evaluate the benefit of incorporating rumination data into herd management decisions.

Detecting lameness in sows from ear tag-sampled acceleration data using wavelets.

The objective of this study was to develop an automated monitoring system to detect lameness in group-housed sows early and reliably on the basis of acceleration data sampled from ear tags. To this end, acceleration data from ear tags were acquired from an experimental system deployed at the Futterkamp Agriculture Research Farm from May 2012 until November 2013. The developed method performs a wavelet transform for each individual sow's time series of total acceleration. Feature series are then computed by locally estimating the energy, variation and variance in a small moving window. These feature series are then further decomposed into uniform level sets. From these series of level sets, the highest and lowest levels are monitored for lameness detection. To that end, they are split into a past record to serve as reference data representing a sow's expected behaviour. The deviations between the reference and the remaining detection part of current data, termed feature activated, were then utilised to possibly indicate a lameness condition. The method was applied to a sample of 14 sows, seven of which were diagnosed as lame by a veterinarian on the last day of the sampling period of 14 days each. A prediction part of 3 days was set. Feature activated were clearly separable for the lame and healthy group with means of 8.8 and 0.8, respectively. The day-wise means were 1.93, 9.47 and 15.16 for the lame group and 0.02, 1.13 and 1.44 for the healthy group. A threshold could be set to completely avoid false positives while successfully classifying six lame sows on at least one of the 2 last days. The accuracy values for this threshold were 0.57, 0.71 and 0.78 when restricting to data from the particular day. A threshold that maximised the accuracy achieved values of 0.57, 0.79 and 0.93. Thus, the method presented seems powerful enough to suggest that an individual classification from ear tag-sampled acceleration data into lame and healthy is feasible without previous knowledge of the health status, but has to be validated by using a larger data set.

Automatic detection of lameness in gestating group-housed sows using positioning and acceleration measurements.

Lameness is an important issue in group-housed sows. Automatic detection systems are a beneficial diagnostic tool to support management. The aim of the present study was to evaluate data of a positioning system including acceleration measurements to detect lameness in group-housed sows. Data were acquired at the Futterkamp research farm from May 2012 until April 2013. In the gestation unit, 212 group-housed sows were equipped with an ear sensor to sample position and acceleration per sow and second. Three activity indices were calculated per sow and day: path length walked by a sow during the day (Path), number of squares (25×25 cm) visited during the day (Square) and variance of the acceleration measurement during the day (Acc). In addition, data on lameness treatments of the sows and a weekly lameness score were used as reference systems. To determine the influence of a lameness event, all indices were analysed in a linear random regression model. Test day, parity class and day before treatment had a significant influence on all activity indices (P<0.05). In healthy sows, indices Path and Square increased with increasing parity, whereas variance slightly decreased. The indices Path and Square showed a decreasing trend in a 14-day period before a lameness treatment and to a smaller extent before a lameness score of 2 (severe lameness). For the index acceleration, there was no obvious difference between the lame and non-lame periods. In conclusion, positioning and acceleration measurements with ear sensors can be used to describe the activity pattern of sows. However, improvements in sampling rate and analysis techniques should be made for a practical application as an automatic lameness detection system.

A new liquid, intravenous immunoglobulin product (IGIV 10%) highly purified by a state-of-the-art process.

BACKGROUND AND OBJECTIVES: The ultimate goal was to generate an industrial-scale process suitable to produce a high-yield, safe and stable immunoglobulin G (IgG) preparation for intravenous administration, which is ready to use for customer convenience. This new liquid 10% IgG preparation (IGIV 10%) was compared to Gammagard SD, a licenced lyophilized immunoglobulin in biochemical and preclinical testing. MATERIALS AND METHODS: The new process, which includes three dedicated virus clearance steps, is a streamlined combination of the currently applied and well-established manufacturing procedures. The biochemical characterization is done by standard methods focusing on purity, integrity and functionality of the preparation. Efficacy is demonstrated in vivo by mouse protection testing and in vitro by opsonization and protein A affinity chromatography. Pharmacokinetics in rats is evaluated after a single intravenous dose. The anaphylactoid potential is determined in rats and in guinea pigs, while thrombogenicity is assessed in a rabbit model. The influence of the products on vital functions is tested on dogs, while acute toxicity studies are carried out on mice and rats. RESULTS: The biochemical characterization data demonstrate the high purity of monomeric IgG in the product. The mouse protection test showed that the protective activity against systemic bacterial infections of IGIV 10% is at least as good as the reference Gammagard SD. This result is supported by the broad spectrum of antibodies in high titres against bacteria and viruses and the high functional integrity of the IgG molecule (> or = 90% functionally intact IgG) in IGIV 10%. The opsonic activity of all IGIV 10% lots is similar to the one of the reference Gammagard SD. In safety and thrombogenicity studies, no adverse effects of IGIV 10% were observed. Pharmacokinetic studies showed no statistically significant differences between the two products. In the acute toxicity animal studies, IGIV 10% compared favourably to the reference Gammagard SD. CONCLUSIONS: The new manufacturing process enables the production of a highly purified IgG preparation for intravenous administration. The product has an IgG subclass distribution similar to plasma and contains a broad spectrum of functionally intact antibodies. Preclinical studies demonstrate that the liquid IGIV 10% combines excellent qualities of efficacy, safety and tolerability.

Recombinant von Willebrand factor: preclinical development.

Von Willebrand factor (vWF) is a multimeric glycoprotein (GP) that attracts platelets to the site of vascular injury, mediates platelet-platelet interaction, and stabilizes factor VIII (FVIII) in the circulation. Quantitative and qualitative defects of vWF result in von Willebrand disease (vWD), manifested by modest to severe bleeding episodes. Substitution therapy, with plasma-derived FVIII/vWF complex concentrates, is used for patients suffering the more severe forms of vWD. Efficacy of these preparations is often unsatisfactory because inadvertent proteolytic degradation during the manufacturing process causes them to lack the hemostatically most active high-molecular-weight multimers. In contrast, recombinant vWF (r-vWF), which is constitutively expressed at high yields in Chinese hamster ovary (CHO) cells and secreted into the conditioned medium under perfusion fermentation in "protein-free" medium, has high-molecular-weight multimers of extraordinary structural integrity. Functional analysis has shown that r-vWF promotes ristocetin cofactor-mediated platelet aggregation, collagen interaction and FVIII binding, and platelet-collagen adhesion under shear stress. Infusing vWF-deficient animals with r-vWF corrected vWF concentration and reduced blood loss, subsequently stabilizing endogenous FVIII associated with the reduction of bleeding time. Compared with plasma-derived vWF preparations, r-vWF was found to have a prolonged half-life, further enhancing the potential value of r-vWF as a therapeutic agent for treating patients suffering from vWD.

Blockade of CD40/CD40 ligand interactions prevents induction of factor VIII inhibitors in hemophilic mice but does not induce lasting immune tolerance.

Patients with severe hemophilia A frequently develop neutralizing anti-factor VIII antibodies after replacement therapy with factor VIII (FVIII). In a search for new strategies to induce immune tolerance against FVIII in these patients, we used a murine model of hemophilia A to investigate the importance of CD40/CD40 ligand (CD40L) interactions for the initiation of the anti-FVIII immune response. We focused our attention in particular on the induction of neutralizing anti-FVIII antibodies and the Th1/Th2 polarization of FVIII-specific T cells. The development of anti-FVIII antibodies was analyzed by ELISA systems (detection of total anti-FVIII antibodies) and Bethesda assays (determination of neutralizing anti-FVIII antibodies). Factor VIII-specific T cells were characterized by multiparameter flow cytometry and cytokine ELISAs for the detection of cytokine production in splenic CD4+ T cells after in vitro restimulation with FVIII. Hemophilic mice received four doses of FVIII and anti-CD40L antibody MR1 (24 h before FVIII). Subsequently mice received four doses of FVIII only. The induction of neutralizing anti-FVIII antibodies in hemophilic mice after treatment with human FVIII could be prevented completely by a blockade of CD40/CD40L interactions using MR1. Furthermore, FVIII-specific T-cell responses that included both Th1 and Th2 cells were suppressed when mice were treated with FVIII and MR1. The initial blockade of CD40/CD40L interactions was, however, not sufficient to induce a lasting immune tolerance against FVIII. The immune suppression was abolished and both neutralizing anti-FVIII antibodies and FVIII-specific T cells developed when treatment with FVIII was continued after the omission of MR1. In addition, there were no alterations in the Th1/Th2 polarization induced by the initial blockade of CD40/CD40L interactions.

Factor Xa and prothrombin: mechanism of action of FEIBA.

A complex consisting of activated factor X (FX) (enzyme) and prothrombin (substrate), both highly purified from human plasma and virus inactivated, was formulated, characterised biochemically as well as in animal studies, and given the name Partial Prothrombinase (PPT). In vitro, PPT shortened the clotting time of a high-titre human factor VIII (FVIII) inhibitor plasma in a manner similar to that of the activated prothrombin complex concentrate FEIBA and triggered coagulation in plasma samples in which factor V (FV) is present. In vivo, the ability of PPT to activate coagulation in both chimpanzees and baboons was equivalent to that of FEIBA. PPT also triggered coagulation in a von Willebrand factor(vWF)-deficient dog and controlled bleeding in rabbits with antibody-induced haemophilia A. Thus, studying the mechanism of action of PPT also explains the therapeutic principle of FEIBA.

Evidence for extracellular processing of pro-von Willebrand factor after infusion in animals with and without severe von Willebrand disease.

Although proteolytic processing of pro-von Willebrand factor (pro-vWF) resulting in free propeptide and mature vWF is known to be initiated intracellularly, vWF released from endothelial cells may contain a high proportion of incompletely processed pro-vWF. Because pro-vWF is only rarely detectable in normal human plasma, we investigated whether extracellular processing of pro-vWF is possible. A recombinant preparation (rpvWF) containing both pro-vWF and mature vWF subunits was infused into 2 pigs and 1 dog with severe von Willebrand disease, 2 mice with a targeted disruption of the vWF gene, and 2 healthy baboons. Total vWF antigen (vWF:Ag), free propeptide, and pro-vWF were measured using enzyme-linked immunosorbent assay techniques in blood samples drawn before and after infusion. vWF:Ag increased promptly. No pro-vWF could be detected when the first postinfusion sample was drawn after 30 minutes (pigs) or 60 minutes (mice), but pro-vWF was detectable for short periods when postinfusion samples were drawn after 15 minutes (dog) or 5 minutes (baboons). In contrast, free propeptide was increased at the first timepoint measured, suggesting that it was generated from the pro-vWF in the rpvWF preparation. vWF multimers were analyzed in the rpvWF preparation and in plasma samples drawn before and after infusion of rpvWF using ultra-high resolution 3% agarose gels to allow separation of homo- and hetero-forms of the vWF polymers. Within 30 minutes after infusion in the pigs, 1 hour in the dog and the mice, and within 2 hours in the baboons, the multimer pattern had changed to that typically seen in mature vWF. These data indicate that propeptide cleavage from unprocessed vWF can occur extracellularly in the circulation. The enzyme or enzymes responsible for this cleavage in plasma remain to be identified.

Effects of alpha 1-acid glycoprotein in different rodent models of shock.

It was the aim of the present study to investigate the effects of the acute phase protein alpha 1-acid glycoprotein in different models of shock. The human plasma preparation used was without effect on mortality in lipopolysaccharide-injected mice when administered in two different doses (1 or 0.33 g/kg i.v.) and according to different treatment schedules. The same preparation significantly increased survival rate (48 h) in rats with septic peritonitis. This effect was seen when alpha 1-acid glycoprotein (200 mg/kg i.v.) was given 15 min prior to and 24 h after cecal puncture. All other dose regimes tested were without significant effect on survival rate. A hemorrhagic/hypovolemic shock model (including a defined trauma) in rats resuscitated with 200 mg/kg alpha 1-acid glycoprotein resulted in significantly higher values of mean arterial blood pressure, cardiac output and stroke volume when compared to corresponding values obtained after resuscitation with Ringer's solution or 200 mg/kg albumin i.v. (free of alpha 1-acid glycoprotein; placebo formulation). Taking all other possible mechanisms of alpha 1-acid glycoprotein into consideration, the partially protective effects of the preparation are explained by enhancing the capillary barrier function and thereby maintaining perfusion of vital organs.

In vivo characterization of recombinant von Willebrand factor in dogs with von Willebrand disease.

Hereditary von Willebrand factor (vWF ) deficiency in Dutch Kooiker dogs, which have undetectable levels of vWF, causes spontaneous hemorrhage of mucosal surfaces similar to the clinical picture of von Willebrand disease in humans. Therefore, we used this canine model to study the in vivo effects of a new recombinant von Willebrand factor (rvWF ) preparation containing all species of vWF multimers compared with a rvWF fraction containing only low molecular weight multimers (LMW-rvWF ) and with a plasma-derived factor VIII/vWF concentrate (pdvWF ). In the vWF-deficient dogs, the half-life of vWF:Ag was 21.6 and 22.1 hours for rvWF, 7.7 hours for pdvWF, and 9 hours for LMW-rvWF; in vivo recovery of vWF:Ag was 59%, 64%, and 70% for rvWF, 33% for pdvWF and 92% for LMW-rvWF; in vivo recovery of RCoF was 78%, 110%, and 120% for rvWF, and 25% for pdvWF. Both rvWF and pdvWF caused increases in factor VIII, which were sustained even when vWF:Ag had decreased to nearly undetectable levels and only monomeric or dimeric species were detectable on agarose gels. At the dosages used, no effect was seen on bleeding time, but the rate of blood flow from cuticle wounds was reduced after a single bolus administration of rvWF. The rvWF was able to control a severe nose bleed in one dog.

Assessment of bleeding for the evaluation of therapeutic preparations in small animal models of antibody-induced hemophilia and von Willebrand disease.

Three small animal models of bleeding are described and used to evaluate the effects of preparations intended for therapy of human bleeding disorders. We modified techniques for the assessment of bleeding to be able to reproducibly quantify blood loss and rate of blood flow in addition to the measurement of bleeding time. Temporary hemophilia was induced in a rabbit model by injection of high titer human inhibitor plasma [> or = 1000 Bethesda units (BU)/ml]. A decrease in rabbit FVIII from normal values to below the limit of detection was observed within 30 min, cuticle bleeding time changed from normal (approx. 10 min) to steady state bleeding (> 30 min), and the rate of blood flow increased from 4 to > 30 microliters blood/min. Infusion of an activated prothrombin complex concentrate, (FEIBA STIM4, Immuno) at doses between 75 and 150 U/kg normalized the rate of blood flow, while infusion of FVIII/vWF concentrate resulted in partial correction. Administration of FVIIa, both recombinant and plasma-derived, failed to correct bleeding, however. In an analogous murine model, FVIII/ vWF inhibitor plasma was obtained by immunizing goats with a purified human FVIII/vWF complex. This plasma cross-reacted with mouse vWF in vitro. Injection of the anti-FVIII/vWF inhibitor plasma into mice caused a decrease in vWF antigen, in some animals with a complete loss of vWF multimers comparable to severe von Willebrand disease. A specific anti-vWF inhibitor plasma obtained by immunization of goats with recombinant vWF was used in a further murine model, resulting in a gradual but substantial decrease in FVIII as well as in intensive bleeding. The infusion of a FVIII/vWF concentrate (IMMUNATE, IMMUNO) normalized the rate of blood flow in both murine models. The same assessment methods were used to characterize bleeding in a natural mouse model of von Willebrand disease (strain RIIIS/J). The use of quantitative techniques of assessment of blood loss and rate of blood flow appears to be a helpful tool for characterizing hemorrhagic situations and evaluating the capacity of therapeutic preparations to correct hemostatic defects.

Studies on the effect of human lys-plasminogen in a rat model of global cerebral ischemia.

Human lys-plasminogen and the corresponding formulation buffer were tested in a rat model of global cerebral ischemia (clamping of both carotid arteries, withdrawal of 5 ml blood for 30 min). The two main parameters, tested in different experimental set-ups, were 1. brain edema (water content) 23.5 h after reperfusion and 2. assessment of neurological deficits 24, 48 and 72 h after reperfusion. In some groups of animals of the first set-up, brains were examined histologically for microvascular fibrin deposits. In a separate group of animals the fibrinolytic plasma activity of rats treated with 500 CU/kg lys-plasminogen was studied. Concerning brain water content lys-plasminogen completely antagonized the formation of brain edema when given with 500 caseolytic Units (CU)/kg i.v. with blood reperfusion and was still effective when given 30 min later. 200 CU/kg i.v. given with blood reperfusion as well as 500 CU/kg i.v. given 60 min after blood reperfusion proved ineffective. In none of the brains investigated microvascular fibrin deposits were found. In experiments with assessment of neurological deficits, animals treated with 500 CU/kg lys-plasminogen i.v. showed almost no disabilities (like sham operated animals) when compared to ischemic (positive) controls which were rather severely handicapped. The formulation buffer of lys-plasminogen, tested in an equivalent volume, was without any effect in both set-ups. No fibrinolytic activity was found in plasma samples of rats up to 240 min after treatment with 500 CU/kg lys-plasminogen i.v. It is concluded from these experiments that human lys-plasminogen has a protective effect in rats against the sequelae of global cerebral ischemia which is not related to the well-known fibrinolytic potential but might be a separate quality.