Retrospective evaluation of leflunomide as an adjunctive therapy in dogs with non-associative immune-mediated thrombocytopenia: 20 cases (2008-2021).

OBJECTIVE: To describe leflunomide as an adjunctive therapy in the treatment of non-associative immune-mediated thrombocytopenia. MATERIALS AND METHODS: A retrospective study of dogs with a diagnosis of non-associative immune-mediated thrombocytopenia treated with leflunomide March 2008 to September 2021 was conducted. Data collected included signalment, clinical signs, physical examination findings and diagnostic testing performed. Medications administered, duration of hospital stay, time to platelet concentration >150×109/L and adverse events during leflunomide therapy were recorded. Relapses within a year of diagnosis were reported. RESULTS: A total of 20 client-owned dogs met inclusion criteria. Nineteen of 20 dogs (95%) achieved a platelet concentration >150×109/L with leflunomide and prednisone combination therapy and four dogs (21.1%) relapsed during treatment or shortly after treatment. Adverse effects included diarrhoea (n=5), mild lymphopenia (n=9) and mild intermittent anaemia (n=1). A single dog developed hepatotoxicity presumed to be secondary to leflunomide therapy that resolved after drug discontinuation. One dog was treated for aspiration pneumonia during treatment. Two dogs were euthanased while receiving leflunomide. CLINICAL SIGNIFICANCE: Length of hospitalisation, time to platelet recovery, treatment response and relapse rate were comparable with alternative treatment protocols. Most adverse effects did not require leflunomide dose adjustment; however, two dogs died while undergoing leflunomide treatment and there is compelling evidence that one of these dogs experienced fatal infection secondary to immune-suppression. Hepatotoxicity remains a known complication of leflunomide treatment and serial biochemistry testing is recommended.

Chemerin-ChemR23 signaling in tooth development.

Our long-term goal is to identify and characterize molecular mechanisms regulating tooth development, including those mediating the critical dental epithelial-dental mesenchymal (DE-DM) cell interactions required for normal tooth development. The goal of this study was to investigate Chemerin (Rarres2)/ChemR23(Cmklr1) signaling in DE-DM cell interactions in normal tooth development. Here we present, for the first time, tissue-specific expression patterns of Chemerin and ChemR23 in mouse tooth development. We show that Chemerin is expressed in cultured DE progenitor cells, while ChemR23 is expressed in cultured DM cells. Moreover, we demonstrate that ribosomal protein S6 (rS6) and Akt, downstream targets of Chemerin/ChemR23 signaling, are phosphorylated in response to Chemerin/ChemR23 signaling in vitro and are expressed in mouse tooth development. Together, these results suggest roles for Chemerin/ChemR23-mediated DE-DM cell signaling during tooth morphogenesis.

Cocaine's effects on speech sound discriminations and reaction times in baboons.

Three adult baboons were trained using a psychophysical procedure to discriminate between different synthetic vowel sounds [symbol: see text]. Baboons pressed and held a lever down to produce a pulsed train of a single reference vowel that served as the standard stimulus. Animals were trained to release the lever only when this standard vowel sound changed to one of the four remaining comparison vowels. A lever release within 1.5 s of this change in vowel sounds was defined as a correct detection of the change from the standard vowel to one of the comparison vowels, and was reinforced. All baboons readily learned the vowel discriminations and detected vowel changes at the 90-100% correct performance level. Acute IM administration of cocaine prior to test sessions (0.00032-3.2 mg/kg) produced dose-dependent decrements in vowel discriminability. At the same time, cocaine shortened lever release latencies (reaction times) to the vowel stimuli in two of three baboons. The cocaine-induced decrements in vowel discriminability were correlated with the degree to which frequency differences occurred among the different vowels in that lower vowel discriminability scores were found for those vowels with smaller spectral differences from the standard vowel. Further, false alarm rates were not systematically affected by cocaine, indicating that the cocaine-induced decrements in vowel discrimination accuracy occurred in the absence of systematic changes in the reliability of the baboons' discrimination performances.

Identification of a 6-base pair element involved in the sterol-mediated transcriptional regulation of farnesyl diphosphate synthase.

Previous studies identified a 115-base pair (bp) region of the farnesyl diphosphate (FPP) synthase promoter which is involved in the transcriptional regulation of this gene by sterols (Spear, D. H., Kutsunai, S. Y., Correll, C. C., and Edwards, P. A. (1992) J. Biol. Chem. 267, 14462-14469). In the current study we fused a 117-bp fragment, containing this region of interest, upstream of the heterologous minimal promoter of the herpes simplex virus thymidine kinase gene linked to the chloramphenicol acetyltransferase (CAT) reporter gene. Chinese hamster ovary (CHO) cells were stably transfected with this fusion gene and incubated in the absence or presence of sterols. Analysis of CAT mRNA by primer extension indicated that transcription of the fusion gene was under sterol-mediated control. Thus, when cellular sterols were present, the CAT mRNA levels were reduced 2-4-fold. To further localize the FPP synthase sterol-responsive element(s), additional promoter-reporter gene constructs containing either deletions or mutations were constructed and transfected into CHO or CV-1 cells. These studies localized a 6-bp region (ATTGGC) that is required for both transcriptional induction in the absence of sterols and transcriptional repression in the presence of sterols. Gel shift and footprinting analyses demonstrated that nuclear proteins isolated from CHO cells bound to six distinct regions of the promoter between nucleotides -293 to -47. Taken together, these results further define both the cis-acting elements controlling normal transcription of the FPP synthase gene and identify a novel sequence involved in sterol regulation.

Acute and chronic nicotine effects on multiple-schedule behavior: oral and SC routes.

For rats responding on a 3 h FI 4 min FR 20 schedule of food reinforcement, presession SC nicotine doses (0.1-0.8 mg/kg) produced depression in all responding followed by stimulation of FI responding that was dependent upon both time and dose. With daily presession 0.8 mg/kg SC nicotine injections for 9 days, no tolerance to the depressive or stimulatory effects of nicotine occurred. When nicotine solutions were orally self-administered by presession exposure to 3 h of schedule-induced polydipsia, the subsequent FR responding was unaffected, but the degree of FI response stimulation and its duration occurred in a dose-related fashion (1.18-4.10 mg/kg). Prolonged daily sessions of oral nicotine self-administration provide a technique for investigating the effects of chronic exposure to nicotine. The postingestive effects of nicotine reveal stimulatory effects that last for at least 3 h.

Effects of cocaine on simple reaction times and sensory thresholds in baboons.

The effects of chronic, daily administration of cocaine on auditory and visual reaction times and thresholds were studied in baboons. Single intramuscular injections of cocaine hydrochloride (0.1 to 5.6 mg/kg) were given once daily for periods of 10 to 25 days, and were followed immediately by psychophysical tests designed to assess cocaine's effects on simple reaction times as on auditory and visual threshold functions. Consistent reductions in reaction times were frequently observed over the cocaine dose range of 0.32 to 1.0 mg/kg; at higher doses, either decreases or increases in reaction times were observed, depending upon the animal. Lowered reaction times generally occurred immediately following the 1st day's cocaine injection, and continued through all subsequent days during the dose administration period, suggesting little development of tolerance or sensitivity to these reaction-time effects. Reaction-time decreases showed a U-shaped dose-effect function. The greatest decreases in reaction times occurred from 0.32 to 1.0 mg/kg, and produced an average reaction-time decrease of 10 to 12%. Concurrently measured auditory and visual thresholds showed no systematic changes as a function of cocaine dose. Pausing was observed during performance of the psychophysical tasks, with the length of total session pause times being directly related to cocaine dose.

Effects of cocaine on sensory motor function in baboons.

The effects of cocaine on auditory and visual threshold functions and reaction times were studied in baboons. Single IM injections of cocaine HCl (0.001-1.0 mg/kg) were administered once or twice weekly and were followed immediately by psychophysical tests designed to assess cocaine's effects on sensory thresholds and reaction times. Consistent reductions in reaction times were observed in the cocaine dose range of 0.032-0.32 mg/kg. Reaction times were decreased by 5-8% at the more effective cocaine doses. Concurrently measured auditory and visual threshold sensitivities showed no systematic changes at any of the cocaine doses studied.

Molecular cloning and promoter analysis of the rat liver farnesyl diphosphate synthase gene.

The isolation and characterization of the rat genomic clone encoding the cholesterogenic enzyme farnesyl diphosphate (FPP) synthase is reported. The gene is localized on a 15-kilobase (kb) genomic fragment, spans approximately 12 kb and contains eight exons. Sequences containing from 3.9 kb to 132 base pairs (bp) of the putative promoter were joined to the coding region of the bacterial reporter gene chloramphenicol acetyltransferase (CAT). The CAT activities or CAT mRNA levels of the hybrid genes were determined following either transient transfections into human hepatoma HepG2 cells or stable transfections into Chinese hamster ovary cells. The transient transfections identified a 319-bp fragment that was required for a 4-fold induction in the absence of sterols. Sequence analysis of this region showed it contained five potential copies of the sterol regulatory element (SRE-1) (Smith, J.R., Osborne, T.F., Brown, M.S., Goldstein, J.L., and Gil, G. (1988) J. Biol. Chem. 263, 18480-18487) previously identified in the promoters of the 3-hydroxy-3-methyl-coenzyme A (HMG-CoA) reductase, HMG-CoA synthase, and low density lipoprotein receptor genes. Further mutational and deletion analysis of the FPP synthase promoter-CAT constructs followed by stable transfection and primer extension of the CAT mRNA levels indicated that these potential SRE-1 regulatory elements were not involved in the sterol-mediated transcriptional regulation of the gene. Our analyses have identified a 115-bp region that is required for the transcriptional induction of FPP synthase in the absence of sterols. These results suggest that the FPP synthase gene may be regulated at the transcriptional level by a different mechanism than other sterol regulated genes.

General activity in baboons measured with a computerized, lightweight piezoelectric motion sensor: effects of drugs.

A small, 1-oz activity-monitoring device is described for measuring motor activity continuously for periods of up to 42 days. The monitor employs a piezoelectric sensor that detects extremely small accelerations induced by movements. The monitor can be placed on collars or harnesses (e.g., for rabbits, cats, dogs, nonhuman primates, etc.). The use of the monitor is described within numerous laboratories studying the behavioral pharmacology of drugs in individually caged laboratory baboons. Patterns of daily activity were reliably recorded over periods of several months, and reflected the normal activity patterns of animals. The activity monitor recorded reliable, drug-induced changes in general activity that paralleled the known effects of the same drugs on learned behaviors. Low doses of the stimulants cocaine and d-amphetamine both increased general activity. Marked reductions in general activity were observed following both the administration of delta-9-tetrahydrocannabinol and an antihypertensive drug combination of diuretic and verapamil.

Acute ethanol effects on sensory/motor function in baboons with a history of chronic ethanol ingestion.

Baboons with a history of chronic, daily ethanol ingestion were subsequently studied under conditions that assessed the effects of acute oral self-administration of ethanol on auditory and visual threshold functions and reaction times. During the post-chronic experiment reported herein, the animals consumed specific amounts of ethanol twice weekly (0.1, 0.32, 1.0 or 1.3 g/kg), following which they immediately performed psychophysical tests designed to assess ethanol's effects on sensory thresholds and reaction times. Clear, dose-related increases in reaction times were observed following ethanol doses greater than 0.32 g/kg. Trends within individual threshold functions were consistent with systematic changes in auditory and visual threshold sensitivities of 1-3 dB at the high ethanol doses. Reaction time increases ranged from 25 to 180 ms above baseline levels at the highest dose (a 15% average increase). These general findings however, were in contrast to data obtained in the same animals under conditions of daily, chronic ethanol administration which characteristically showed greater sensory/motor effects of up to twice the magnitude of those observed with single doses.

Acute opioid administration effects on sensory and motor function in baboons: buprenorphine, morphine, and naloxone.

The effects of acute administration of the opioid compounds buprenorphine, morphine, and naloxone were studied on auditory and visual threshold functions and reaction time performances in baboons. Baboons were trained in a reaction time procedure to hold a lever depressed, and release the lever when a signal was presented. Auditory and visual signals were employed in separate sessions. Drug was administered 30min prior to testing. Dose-related increases in visual and auditory thresholds were observed following buprenorphine, with visual thresholds being somewhat more drug-sensitive. Buprenorphine also increased reaction times to both high-intensity and low-intensity stimuli. High doses of morphine increased reaction times to high-intensity auditory and low-intensity visual stimuli; thresholds for both modalities were unaffected by any dose of morphine. Naloxone produced no consistent effects on thresholds or reaction times. False alarm rates were not significantly changed by buprenorphine, morphine, or naloxone.

Cocaine and food as reinforcers: effects of reinforcer magnitude and response requirement under second-order fixed-ratio and progressive-ratio schedules.

Reinforcer magnitude and fixed-ratio requirement were varied under two second-order schedules. Under one, the first sequence of a fixed number of responses completed after the lapse of a 10-min fixed interval produced reinforcement. Under the second, a second-order progressive-ratio schedule, the fixed number of responses increased after each reinforcement. Either cocaine (0 to 300 micrograms/kg/inj) or food (0 to 5,700 mg/delivery) reinforcers were delivered. Under some conditions, a 2-s illumination of stimulus lights occurred on completion of each ratio sequence. Under the second-order schedule, as cocaine dose or amount of food increased, rates of responding increased; at the highest values, rates of responding decreased. Increases in the ratio requirement from 10 to 170 responses minimally decreased overall response rates. Under the second-order progressive-ratio schedule, increases in dose of cocaine or amount of food increased rates of responding; at the highest amounts of food, rates of responding decreased but response rates at the highest dose of cocaine remained relatively high. The highest ratio requirement that was completed (breaking point) depended on the dose of cocaine but was less dependent on the amount of food. Removing brief-stimulus presentations had a greater effect on completion of ratio requirements with cocaine compared to food.

Methohexital and cocaine self-administration under fixed-ratio and second-order schedules.

Behavior maintained by either cocaine or methohexital was compared under two different schedules of drug delivery. Under a fixed-ratio 10 schedule, each tenth response produced an injection and responding was characterized by pauses alternating with high rates that were sustained until the drug injection. Under a second-order schedule, each tenth response produced a brief visual stimulus, and the first sequence of ten responses emitted after the lapse of a ten-minute interval produced the stimulus and the drug injection. Responding under the second-order schedule was characterized by an overall positive acceleration in responding that consisted of fixed-ratio response patterns terminating in the presentation of a brief stimulus. Under either schedule, each drug maintained maximal rates of responding at intermediate doses. In most respects, rates and patterns of responding depended more on the schedule of drug delivery than on the particular drug maintaining responding.

Testis-specific transcription initiation sites of rat farnesyl pyrophosphate synthetase mRNA.

A variety of rat tissues were screened at low stringency with a rat farnesyl pyrophosphate (FPP) synthetase cDNA. In testis, an FPP synthetase-related RNA was detected that was larger than the liver FPP synthetase mRNA and was present at very high levels comparable with liver FPP synthetase RNA levels obtained from rats fed diets supplemented with cholestyramine and mevinolin. Sequence analysis of testis cDNA clones, together with primer extension and S1 nuclease experiments, indicated that testis FPP synthetase transcripts contain an extended 5' untranslated region. The 5' extension contained one or two out-of-frame upstream ATGs, depending on the site of transcription initiation. Protein in vitro translation studies indicated that the extended 5' untranslated region may play a role in regulating the translation of the FPP synthetase polypeptide in rat testis. Southern blot analysis with a probe containing both testis and liver 5' untranslated sequences provided evidence that both liver and testis transcripts derive from the same gene. The data suggest that an upstream testis-specific promoter results in the abundant production of FPP synthetase transcripts that are translated at low efficiency; another promoter functions in liver and other somatic tissues and directs the regulated synthesis of shorter discrete transcripts.

Monophasic action potential recordings in response to graded hyperkalemia in dogs.

Recent interest in sudden cardiac death during exercise in normal healthy people has highlighted the possible role of swings of extracellular potassium in arrhythmogenesis in conditions other than ischemia. Regional differences in action potential duration and conduction may be important. We have recorded monophasic action potentials (MAPs) from the endocardium and epicardium in nine open-chest dogs during graded intravenous infusion of potassium up to a plasma level of 9 mM. The animals were anesthetized with alpha-chloralose and urethan. Continuous, online arterial potassium monitoring was employed. MAP duration showed a biphasic response with initial shortening up to 7 mM, which tended to be more obvious on the epicardium. Regional activation time was measured as the difference between the onset of depolarization of the endocardial and epicardial MAP. Regional activation time also showed a biphasic response with initial shortening and subsequent delay. The QRS width of the scalar lead II electrocardiogram also showed biphasic changes, and the T wave amplitude progressively decreased. Our results suggest that regional differences in repolarization time may develop in the nonischemic myocardium in response to increased extracellular potassium levels mainly as a result of local changes in regional activation time rather than as a result of a direct effect on action potential duration.

Simultaneous endocardial and epicardial monophasic action potential recordings during brief periods of coronary artery ligation in the dog: influence of adrenaline, beta blockade and alpha blockade.

Local differences in the time course of recovery of excitability during the early phase of myocardial ischaemia are important in the genesis of arrhythmias. Catecholamines are known to encourage the formation of arrhythmias and adrenergic blockade is a recognised therapeutic regime. The purpose of this study was to compare the effect of short periods of coronary artery ligation on endocardial and epicardial repolarisation time, to assess any disparity between the two surfaces, and investigate the influence of catecholamines and adrenergic blockade. Simultaneous left ventricular endocardial and epicardial monophasic action potentials (MAPs) were recorded during short periods of left anterior descending coronary artery (LAD) ligation in 9 open chested dogs. Recordings were made during two 90 s periods of LAD ligation. Two further ligations were made during infusion of adrenaline (1 microgram.kg-1.min-1). Subsequently ligations were made after beta blockade with propranolol (0.25 mg.kg-1) and then in the presence of a combination of alpha blockade (phentolamine, 0.15 mg.kg-1) and beta blockade. MAP duration was measured at 90% repolarisation. LAD ligation produced a marked shortening of MAP duration epicardially with only minimal shortening endocardially, which resulted in a highly significant difference between the repolarisation times on the two surfaces. The disparity between surfaces tended to be augmented by adrenaline and was significantly minimised by either beta blockade alone or in combination with alpha blockade. Our results show rapid development of substantial regional differences in repolarisation time between endocardium and epicardium in response to "ischaemia".(ABSTRACT TRUNCATED AT 250 WORDS)

Characterization of the enzymatic defect in late-onset muscle phosphofructokinase deficiency. New subtype of glycogen storage disease type VII.

Human phosphofructokinase (PFK) exists in tetrameric isozymic forms, at least in vitro. Muscle and liver contain homotetramers M4 and L4, respectively, whereas red cells contain five isozymes composed of M (muscle) and L (liver) type subunits, i.e., M4, M3L, M2L2, and ML3, and L4. Homozygous deficiency of muscle PFK results in the classic glycogen storage disease type VII characterized by exertional myopathy and hemolytic syndrome beginning in early childhood. The genetic lesion results in a total and partial loss of muscle and red cell PFK, respectively. Characteristically, the residual red cell PFK from the patients consists of isolated L4 isozyme; the M-containing hybrid isozymes are completely absent. In this study, we investigated an 80-yr-old man who presented with a 10-yr history of progressive weakness of the lower limbs as the only symptom. The residual red cell PFK showed the presence of a few M-containing isozymes in addition to the predominant L4 species, indicating that the genetic lesion is a "leaky" mutation of the gene coding for the M subunit. The presence of a small amount of enzyme activity in the muscle may account for the atypical myopathy in this patient.

Pyrophosphate-driven proton transport by microsomal membranes of corn coleoptiles.

Corn (Zea mays L. cv Trojan T929) coleoptile membranes were fractionated on isopycnic sucrose density gradients. Two peaks of ATP-driven H(+)-transport activity, corresponding to the previously characterized tonoplast (1.07 grams per cubic centimeter) and Golgi (1.13 grams per cubic centimeter) fractions (Chanson and Taiz, Plant Physiol 1985 78: 232-240) were localized. Coincident with these were two peaks of inorganic pyrophosphate (PPi)-driven H(+)-transport. At saturating (3 millimolar) concentrations of Mg(2+):ATP, the rate of proton transport was further enhanced by the addition of 3 millimolar PPi, and the stimulation was additive, i.e. equal to the sum of the two added separately. The specific PPi analog, imidodiphosphate, antagonized PPi-driven H(+)-transport, but had no effect on ATP-driven transport. Moreover, PPi-dependent proton transport in both tonoplast-enriched and Golgi-enriched fractions was strongly promoted by 50 millimolar KNO(3), unlike the ATP-dependent H(+)-pumps of the same membranes. Taken together, the results indicate that PPi-driven proton transport is mediated by specific membrane-bound H(+)-translocating pyrophosphatases. Both potassium and a permanent anion (NO(3) (-) > Cl(-)), were required for maximum activity. The PPi-driven proton pumps were totally inhibited by N,N'-dicyclohexylcarbodiimide, but were insensitive to 100 millimolar vanadate. The PPi concentration in coleoptile extracts was determined using an NADH oxidation assay system coupled to purified pyrophosphate:fructose 6-phosphate 1-phosphotransferase (EC 2.7.1.90). The total pyrophosphate content of corn coleoptiles was 20 nanomoles/gram fresh weight. Assuming a cytoplasmic location, the calculated PPi concentration is sufficient to drive proton transport at 20% of the maximum rate measured in vitro for the tonoplast-enriched fraction, and 10% of the maximum rate for the Golgi-enriched fraction.