Clinical and molecular characterization of a re-established line of sheep exhibiting hemophilia A.

BACKGROUND: Large animal models that accurately mimic human hemophilia A (HA) are in great demand for developing and testing novel therapies to treat HA. OBJECTIVES: To re-establish a line of sheep exhibiting a spontaneous bleeding disorder closely mimicking severe human HA, fully characterize their clinical presentation, and define the molecular basis for disease. PATIENTS/METHODS: Sequential reproductive manipulations were performed with cryopreserved semen from a deceased affected ram. The resultant animals were examined for hematologic parameters, clinical symptoms, and responsiveness to human FVIII (hFVIII). The full coding region of sheep FVIII mRNA was sequenced to identify the genetic lesion. RESULTS AND CONCLUSIONS: The combined reproductive technologies yielded 36 carriers and 8 affected animals. The latter had almost non-existent levels of FVIII:C and extremely prolonged aPTT, with otherwise normal hematologic parameters. These animals exhibited bleeding from the umbilical cord, prolonged tail and nail cuticle bleeding time, and multiple episodes of severe spontaneous bleeding, including hemarthroses, muscle hematomas and hematuria, all of which responded to hFVIII. Inhibitors of hFVIII were detected in four treated animals, further establishing the preclinical value of this model. Sequencing identified a premature stop codon and frame-shift in exon 14, providing a molecular explanation for HA. Given the decades of experience using sheep to study both normal physiology and a wide array of diseases and the high homology between human and sheep FVIII, this new model will enable a better understanding of HA and facilitate the development and testing of novel treatments that can directly translate to HA patients.

Hypothalamo-pituitary-adrenocortical axis in stress-susceptible and stress-resistant pigs: endocrine responses to corticotrophin-releasing factor and vasopressin.

Swiss Landrace pigs selected into genetically well-characterized low and high tissue fat lines (f and F respectively) react differently to exogenous and endogenous stressors. Response of the hypothalamo-pituitary-adrenocortical (HPA) axis to i.v. administered ovine corticotrophin-releasing factor (oCRF) and lysine vasopressin (LVP) in young females, intact and pretreated with dexamethasone or metyrapone, leads to the conclusion that different stress susceptibility of the two lines correlates with the sensitivity of pituitary corticotrophs to oCRF stimulation. Total amount of ACTH released after stimulation with submaximal oCRF doses was roughly equal in both lines, and cortisol level is even lower in the f-line, most likely due to the considerably enhanced metabolic clearance rate of cortisol (lower half-life of plasma cortisol compared with F-line). LVP-stimulated ACTH release is comparable with that of oCRF is stronger in the f-line. Combined effect of oCRF and LVP is rather additive than synergistic but the half-life ratio cortisol/ACTH after this stimulation is about four times higher than for stimulation by LVP and oCRF separately. In cases of externally stimulated HPA axis, cortisol plasma concentration tightly cross-correlates with that of ACTH.

[Genomic methods for identification of traits and inherited disorders in farm animals]. / Genomik zur Identifikation von Merkmalen und Erbfehlern beim Nutztier.

In this review we demonstrate the interaction of the blueprint of an individual (the genome, genomic DNA), its phenotype and the environment. The phenotype consists of quantitative (e.g. growth, milk yield) or functional characteristics e.g. fitness, longevity, fertility and disease resistance. The latter characteristics influence the welfare of an animal substantially. As only the genetically determined part of a particular characteristic is transferred from one generation to the next, it is important to know what the genetic variants (alleles) of the parents at one or more gene loci are. New methods in molecular biology have made it possible to localize and characterize important genes which help to breed more efficient and healthy animals. The exact characterization of the phenotype is vital in identifying genes with major effects and therefore the cooperation with experts from veterinary medicine, biochemistry, and biology is indispensable. As well as an overview of available genetic tests in farm animals, we show various examples how to identify the molecular basis of a particular phenotype and how to use the results in practical breeding programs. Genetic diagnosis enables the breeder to identify undesired alleles early and hinders therefore its uncontrolled distribution in the population. In the long term this leads to a smaller number of affected animals and depending on the disease it may help to prevent animals from suffering.

Oxytocin receptors in guinea pig myometrium near term and during labor.

Oxytocin receptors in myometrium of women, rats, and rabbits rise markedly before the onset of labor, suggesting a role in the initiation of labor. In guinea pigs, a previous study reported no such rise by one-point determination of oxytocin binding. The purpose of this study was to use a more rigorous method to determine whether the binding characteristics of myometrial oxytocin receptors change in relation to labor in guinea pigs. Competitive binding studies were carried out in microsomes from inner and outer myometrium between 42 days of gestation and labor. Binding to analogs was also tested. Data were analyzed with affinity spectra and LIGAND. Oxytocin bound to one site with a dissociation constant of 6.3 +/- 0.65 x 10(-9) M. Binding capacity was 1.0 +/- 0.1 x 10(-12) mol/mg protein. The Hill coefficient was near unity. No significant changes occurred with gestation or labor in dissociation constant, binding capacity, or Hill coefficient (all P >/= 0.2, nested ANOVA). Binding capacity was higher in the outer than in the inner layer (1.2 +/- 0.2 vs. 0.8 +/- 0.1 x 10(-12) mol/mg protein, P = 0.02), but the dissociation constants were similar. Differences existed in the dissociation constants of the analogs tested. The main conclusion is that oxytocin receptors are unlikely to have a regulatory role in the initiation of labor in guinea pigs.

Lewis/Fischer rat strain differences in endocrine and behavioural responses to environmental challenge.

The Lewis (LEW) and Fischer (F344) rat strains provide a comparative model of hypothalamic-pituitary-adrenal (HPA) function in which LEW is relatively hypoactive at homeostasis and hyporeactive to environmental challenge. The present study describes a comparison of LEW and F344 rats, males and females, in terms of their corticosterone (CORT) or behavioural responses to a range of behavioural tasks, where each of the tasks used contains a stressor component and has been demonstrated to be sensitive to corticotropin releasing factor (CRF) and/or CORT manipulation: acoustic startle response (ASR), elevated plus maze, schedule-induced polydipsia, and fear-conditioned suppression of drinking. Our aim was to determine to what extent the LEW trait of HPA axis hyporesponsiveness is associated with strain differences in behavioural responsiveness to environmental challenge. As expected, young (2-3 months)-mature (5-10 months) LEW males and females exhibited a lesser CORT response to restraint and novel confinement than did F344 males and females, although in old adulthood (18 months) the CORT stress response was equable in LEW/F344 males and actually higher in LEW than in F344 females. In young-mature adults, the ASR was greater in LEW males than in the other groups; all groups spent a low proportion of time on the open arms of the elevated plus maze; polydipsia was greater in F344 females than in the other groups; and fear-conditioned suppression of drinking was greater in F344 males and females than in LEW males and females. Therefore, relative hyporeactivity of the HPA axis in LEW rats is clearly not associated with uniform behavioural hyporeactivity, including CRF-dependent behaviours. Rather, this study suggests further evidence that environmental reactivity reflects a number of distinct emotional states and underlying neural circuits.

Prenatal stress in rats: effects on plasma corticosterone, hippocampal glucocorticoid receptors, and maze performance.

The present experiments were designed to investigate the effects of maternal stress on cognitive and endocrine parameters in the adult offspring. Pregnant rats were stressed daily during the last week of pregnancy (days 15-19) by restraint, and the performance of their offspring in the Morris water maze was recorded. Plasma corticosterone levels after swimming and the status of hippocampal glucocorticoid receptors (GRs) were determined. During acquisition of the task, prenatally stressed (PS) males - but not females - showed longer escape latencies than non-stressed controls when swimming in cold (10 degrees C) but not in warm (20 degrees C) water. This sex- and prenatal stress-specific difference was even more pronounced during reversal learning of the task. In contrast, PS females - but not males - had higher basal corticosterone levels and a lower density of hippocampal corticosteroid receptors than non-stressed controls. In all animals irrespective of treatment, swimming in the water maze causes an increase of corticosterone that was smaller on day 8 of swimming than on day 1. After swimming in cold water, the rise in corticosterone levels in females was steeper and returned faster to baseline values than after swimming in warm water. A similar pattern could be seen in PS females when compared to their non-stressed controls. The data suggest that prenatal stress impairs spatial learning in males but not in females. Basal and stress-induced increases in corticosterone levels, however, were altered in PS females and not in PS males; i.e., prenatal stress-induced changes in corticosterone secretion were not paralleled by prenatal stress-induced deficits in spatial learning.

Interaction of the recombinant herpes simplex virus type 1 thymidine kinase with thymidine and aciclovir: a kinetic study.

Herpes Simplex Virus type 1 thymidine kinase (HSV 1 TK) is a key target for antiviral therapy and it phosphorylates a broad spectrum of nucleosides and nucleotides. We report the results from kinetic and inhibition experiments with HSV 1 TK, and show that there is a preferred, but not exclusive, binding order of substrates, i.e. dT binds prior to ATP. Furthermore, the results provide new informations on the mechanism of binding suggesting that HSV1 TK undergoes conformational changes during the catalytic cycle.

Behavioural and hormonal differences between two Lewis rat lines.

Lewis (LEW) is an inbred strain of rats frequently used as an animal model of autoimmune diseases. However, there is evidence that some lines of LEW rats develop autoimmune diseases more readily than do other LEW rat lines. Because the hypothalamus-pituitary-adrenal system is involved in the pathophysiology of these diseases, we compared two LEW lines (SsNHsd and HANRijHsd) in their behavioural and neuroendocrine response to stress. In addition, we studied the psychostimulant effects of acute and repeated amphetamine in these two LEW rat lines. HAN rats were less active in the open field test and showed faster habituation of novelty-induced locomotion. The acoustic startle response was lower in HAN than in SSN rats, whereas prepulse inhibition of the startle response was greater in the HAN than in the SSN LEW subline. Moreover, HAN rats showed impaired acquisition of the two-way active avoidance response relative to SSN rats. The psychostimulant effects of acute amphetamine were smaller in HAN rats. Following repeated injections of amphetamine, behavioural sensitization to the psychostimulant effects of amphetamine was more pronounced in HAN than in SSN rats. Basal concentrations of serum corticosterone did not differ between the two rat lines. Following stress, however, HAN rats showed slightly higher corticosterone secretion than SSN rats. Our results show that two sublines of the LEW inbred strain of rats show profound behavioural differences which are only marginally paralleled by differences at the level of the HPA system.

Partial agonism: mechanisms based on ligand-receptor interactions and on stimulus-response coupling.

Substances eliciting, at very high concentrations, a lower maximal response of a particular biological system than a defined standard, are defined as partial agonists. The convention rests on the definition of a standard substance that achieves a 'full' maximal response; partial agonism being, therefore, relative. Various mechanisms lie behind this phenomenon: 1. Receptor-related mechanisms: the agonist-receptor complex exists in several conformational states from which only one, or only a few, activate the cell signaling pathway. This may occur when the receptor itself, or the agonist, exists in multiple states (e.g., in the form of enantiomers or stereoisomers), or when the agonist-receptor complex changes its conformation (receptor switch: two-state model of receptor activation). Furthermore, a steric hindrance by a 'wrong-way binding' of a part of the agonist's molecules may prevent the full 'correct' occupancy of receptors. 2. Mechanisms based on the efficacy of the stimulus-response coupling. The efficacy is then proportional to the sum of probabilities that receptors in individual states activate the cell-signaling pathway. Doses (concentrations) eliciting the half maximal response (EC50), or similar response sensitivity parameters, are not included in the definition of partial agonism. However, tight correlations exist between maximal response and EC50 in many, but not all, generic groups of agonistically acting substances. These relationships are frequently linear; intercepts and slopes of these 'E, KE plots' are characteristic for individual, putative mechanisms. Dose-response curves of partial agonists are akin to those obtained for a response to a full agonist after a stepwise partial inactivation of receptors by an irreversible inhibitor. Also, the E, KE plots obtained in these instances are similar to those of partial agonists. The receptor reserve, rather vaguely defined in early reports, is therefore closely linked to the phenomenon of partial agonism.

Differential effects of prenatal stress in two inbred strains of rats.

The long-term effects of prenatal stress (three times daily restraint stress during the last week of gestation) on the behavioral response to stress, as assessed by novelty-induced locomotion, performance in the forced swim test, and the acquisition of a two-way active avoidance, were investigated in two inbred strains of rats, Fischer 344 (F344/NHsd/Zur) and Lewis (LEW/SsNHsd/Zur). Additional measures included birth weights, pain threshold on the hot plate, and basal and stress-induced corticosterone secretion. In all of the behavioral paradigms strain differences were found: LEW rats showed poorer acquisition of avoidance conditioning, displayed higher levels of activity on the open plate, less immobility time in the forced swim test, and lower pain thresholds in the hot-plate test compared with F344 rats. LEW rats had higher birth weights after prenatal stress, whereas F344 rats were lighter. Following prenatal stress the pattern of behavioral effects obtained in LEW rats in stress-related tests could be interpreted as improved coping abilities with stress, i.e., improved acquisition of active avoidance, less immobility in the forced swim test, and reduced novelty-induced locomotion. Prenatal stress was much less effective in inducing long-term behavioral changes in F344 rats, yielding only one effect, namely, enhanced novelty-induced locomotion in female F344 rats. Pain thresholds were increased as a consequence of prenatal stress, irrespective of strain and gender. Basal and stress-induced corticosterone release differed in the two strains, with LEW rats showing less stress-induced corticosterone release. Prenatal stress did not, however, affect basal or stress-induced corticosterone release. The results suggest that prenatal stress exerts long-term effects on behavior, which depend on the genetic background.

A rapid assay for tyrosine hydroxylase activity, an indicator of chronic stress in laboratory and domestic animals.

Tyrosine hydroxylase (TH) (EC 1.14.16.2) activity has been frequently employed as a marker of adrenomedullar catecholamine-synthesizing capacity and, thus, as an indicator of chronic stress exposure in various animal species. We have developed a thin layer chromatography (TLC) procedure for its assay in adrenal glands of rats and large animals that reduces some of the drawbacks of currently employed methods, thereby facilitating routine use. Preparation of tissue samples was adapted for rats and pigs. The activity of the enzyme is expressed as the rate of the TH-catalysed tyrosine hydroxylation to 3,4-dihydroxyphenyl-alanine (DOPA) using tritium-labeled tyrosine, in the presence of cofactors and a DOPA decarboxylase inhibitor. The subsequent separation of the radioactive product (DOPA) from the substrate (tyrosine) is accomplished by TLC on silicagel plates, in a n-butanol/acetic acid/water solvent system (4:1:1). Radioactivity in the scraped zones, in which DOPA has been detected by means of an internal standard, is measured by beta-counting. An advantage of this procedure is its simplicity, reliability, and convenience for routine assays. Levels of endogenous adrenal tyrosine (HPLC assay) are considerably higher in pig (2.5-5 nmol/mg protein) than in rat (0.15 nmol/mg protein); their effects upon assay results being, in both cases, negligible. Michaelis constants estimated by this procedure amounted to 0.9 mmol l(-1) (at 0.7 mM DMPH4) for pig, and 1.1 mmol l(-1) (at 1.5 mM DMPH4) for rat.

Effects of neurohypophyseal hormone analogues on blood clotting factor VIII and fibrinolytic activity in sheep.

The increase in blood clotting factor VIII (antihaemophilic factor, F-VIII) and fibrinolytic activity induced by the administration of neurohypophyseal hormone analogues, was assayed in sheep. Peptides with high selectivity for vasopressin V1, V2 or myometrial oxytocin receptors in the dose range of 0.1-10 micrograms/kg body weight were investigated. The main conclusions are as follows. The time-course of the F-VIII plasma levels following the administration of the peptides was biphasic, with one surge at about 20 min, a rebound phase, and another increase with the maximum at 60-90 min. The time-course of the fibrinolytic response, expressed as biological activity of plasminogen activator in the plasma euglobulin fraction, displayed a single maximum within 60 min. The baseline responses were reached within 90-120 min. Responses were expressed as integrals of the time-concentration curves in a predetermined time range (90-120 min). F-VIII and plasminogen activator enhancing effects seemed to be tightly linked to the specific vasopressin V2 receptor activities. [Val4,D-Arg8]Vasopressin displayed higher plasminogen activator activities than the standard substance, deamino[D-Arg8]vasopressin. The vasotocin analogue [Phe2,Orn8]oxytocin, a specific vasopressin V1 receptor agonist, also displayed high antihaemophilic and fibrinolytic potencies, expressed in terms of ED50 values, but did not reach the same maximal response as vasopressin V2 receptor agonists. Oxytocin and its highly selective uterotonic analogue, [Thr4,Gly7]oxytocin, displayed low antihaemophilic, and virtually no plasminogen activating potencies. Surprisingly, vasopressin V2 and V1V2 receptor antagonists studied in our experiments showed both enhanced F-VIII and fibrinolytic responses. Dose-response curves frequently displayed a decrease of the F-VIII, and sometimes also decreased fibrinolytic responses, at higher peptide doses. Strong decreases of the packed cell volume (haematocrit) and somewhat lower decreases of the total plasma protein concentration were observed shortly after administration of the peptides.

Steroid receptors in the rat hippocampus: a note to the methodology of their binding assay.

Mineralocorticoid (MR) and glucocorticoid (GR) receptors in the rat hippocampus are linked to several cognitive functions of the animal and seem to play an important role in the response to various stressors. Their assessment by binding experiments brings about problems associated with their intracellular compartmentalization, and in particular with the separation of the bound and free ligands. Adrenalectomy 24 h before sacrificing is commonly used to clear the circulating adrenal steroids, and to facilitate their dissociation from hippocampal MR and GR. We have successful attempted to use dialysis to these purposes and thus, to avoid a potential surgical stress. Without dialysis, only GR can be measured in the cytosol from intact rats, while the corresponding pellet contains MR as a component of the cell nuclei. The bound ligand fraction was separated by filtration on polyethyleneimine pretreated glass fiber filters as suggested earlier. The method has clear-cut preferences compared to any alternative used up to now. Discrimination between the two receptor types can be optimally achieved in a cross-displacement experiment in which two labeled ligands possessing various affinities to individual receptors (in our case: corticosterone and aldosterone, or their synthetic analogs) are displaced with the two corresponding nonlabelled ligands from their receptors. Computations can be carried out with LIGAND software which yield accurate values of binding parameters.

Thermodynamic parameters of ligand-receptor interactions: computation and error margins.

A semiempirical relationship describing the temperature function of ligand-receptor dissociation constants (Kd), derived from heat capacities of the system in equilibrium, is suggested for computation of the standard enthalpy (delta H degree) and standard entropy (delta S degree) changes in equilibrium. The use of the linear expression (called Gibbs-van't Hoff equation) may lead to inaccurate results when heat capacity Cp displays a considerable temperature dependence. The accuracy of Kd, delta H degree and delta S degree has been studied by simulation experiments. In the case of Kd, deviations of computed from "true" values are determined by both the accuracy of experimental data used for its estimation, and by the shape of the binding isotherm (for instance, by Hill coefficients or by the presence of low affinity sites). As a rule, if errors of bound ligand measurements are greater than 20 per cent, Kd estimates ought to be considered as less reliable. However, computations of delta H degree and delta S degree that use such Kd values, are more correct, probably due to an error compensation. The suggested nonlinear temperature function of Kd enables an estimate of the heat capacity of the system and its temperature dependence.

Early high-affinity neutralizing anti-viral IgG responses without further overall improvements of affinity.

Affinity maturation of IgG antibodies in adaptive immune responses is a well-accepted mechanism to improve effector functions of IgG within 2 weeks to several months of antigen encounter. This concept has been defined mainly for IgG responses against chemically defined haptens. We have evaluated this concept in a viral system and analyzed neutralizing IgG antibody responses against vesicular stomatitis virus (a close relative of rabies virus) with a panel of monoclonal antibodies obtained early (day 6 or 12) and late (day 150) after hyperimmunization. These neutralizing IgG antibodies recognize a single major antigenic site with high affinities (Ka of 10(8)-10(10) liter.mol-1) and with rapid on-rates already on day 6 of a primary response and with no evidence for further antigen dose- and time-dependent overall improvement of affinity. This type of IgG response is probably representative for viruses or bacterial toxins which are crucially controlled by neutralizing antibodies.

Multiple receptor populations: binding isotherms and their numerical analysis.

This paper reviews present models and methods of parameter estimation in relationships describing receptor-ligand interactions in equilibrium, as used in the author's laboratory. The state-of-the-art and the present experience can be summarized as follows: 1) Binding isotherms (relationships of bound and free/total ligand concentrations) are superpositions of several elementary terms describing the ligand binding to individual binding sites (receptors) present in the biological material investigated. The "nonspecific binding" is usually represented by a linear term. 2) The elementary terms are most frequently described by a rectangular hyperbola, Hill (power) function, or a rational function (binding of several ligand molecules to one molecule of receptor). Heterologous displacement requires specific functions which, however, can be transformed into one of the elementary terms. 3) Parameters (binding capacities, dissociation constants, Hill coefficients, etc.) can most reliably be estimated by nonlinear regression methods. However, these methods frequently fail to yield physically relevant values if initial estimates are far from "real" values, or if the data are strongly scattered. Some of the available routines (e.g., LIGAND) offer manifold tools to solve these difficulties. 4) The "affinity spectrum", a relationship between binding capacities and equilibrium constants, shows the presence of individual binding sites in the binding system in question. The spectrum can be constructed either by Fourier analysis, or by a stepwise procedure (computation of binding capacity for several dissociation constants). The former way of analysis is demanding; software tools are rare. 5) The "STEP" routine based on Hill/Scatchard linearization routines yields profiles similar to affinity spectra, but offers, in addition, values of Hill coefficients of individual binding populations. Values obtained can be used as initial estimates for nonlinear regression. 6) Selection of a suitable model, its testing, numerical procedures, statistical estimates, etc. frequently entail severe difficulties which are approached in the available software packages in different ways, none of them is usually optimal.

Kinetic analysis of ligand-receptor association data that display an overshoot phenomenon.

A binding overshoot was frequently observed in the time course of association of diazepam with rat brain membrane receptors shortly after the start of the interaction. Such time profiles most likely reflect the "receptor switch" mechanism, assuming an equilibrium between two forms of a receptor (R and R*) that possess different affinities to the ligand (L) in question. Similar effects could be caused by the presence of a slowly dissociating competitor. The kinetics of these mechanisms were verified by simulation of theoretical time courses. A computer program for simulation of the time course, and estimation of rate constants of the individual reaction steps, was developed and is described in this communication. It employs the Euler-Cauchy integration for simulation of theoretical time courses. Optimised estimates of the rate constants were computed by simultaneous random variation of parameters within a pre-set interval. Stable solutions can be obtained for this system, thus enabling evaluation of equilibrium constants defined by the model. The source code is available in Turbo-Pascal. It can be used, after re-writing the rate equations, for fitting of similar kinetic models to suitable experimental data.

Carrier detection of ovine hemophilia A using an RFLP marker, and mapping of the factor VIII gene on the ovine X-chromosome.

Ovine hemophilia A is an X-linked recessive bleeding disorder. For diagnostic purposes, restriction fragment length polymorphism (RFLP) analysis in the region of the factor VIII (F-VIII) gene was carried out using human F-VIII gene probes. The probe St14, known to detect a highly polymorphic region that is closely linked to the F-VIII gene in humans, hybridized nonspecifically with DNA from sheep. Searching for intragenic RFLPs, the entire 9.0-kb coding sequence of the human F-VIII was used as a probe. Using the 1.8-kb SstI/KpnI F-VIII cDNA probe for hybridization, an MspI-RFLP with allelic bands of 5.8 kb (A1) and 4.2 kb (A2) was detected. A1 was in linkage phase with the mutated allele responsible for hemophilia A. The F-VIII locus in the sheep genome was assigned to the long arm of the X-chromosome in the region Xq24-q33, using in situ hybridization with a 3-kb human F-VIII cDNA probe to QFQ banded sheep metaphase chromosomes.