Efficacy of Milbemax (milbemycin oxime + praziquantel) in the treatment of dogs experimentally infected with Crenosoma vulpis.

Crenosoma vulpis, the fox lungworm, infects wild and domestic canids and is a cause of chronic respiratory disease in dogs in North America and Europe. The objective of this study was to determine the efficacy of milbemycin oxime (0.5mg/kg)/praziquantel (5mg/kg) (Milbemax; Novartis Animal Health, Inc.) against C. vulpis infection in a randomized, blinded, placebo-controlled study using experimentally infected dogs. Sixteen beagles (8 males, 8 females) were each given 100 infective third-stage larvae of C. vulpis. Fecal samples were examined for first-stage larvae by quantitative Baermann examination pre-exposure and at days 21, 28, 35, 42 and 49 post-infection (PI). All of the dogs were shedding larvae in the feces at 21 days PI. The dogs were randomly assigned to one of two groups. At 28 days PI, Group 1 (4 males, 4 females) received placebo only while Group 2 (4 males, 4 females) received a single treatment of milbemycin oxime (0.5mg/kg) and praziquantel (5mg/kg). The 16 dogs were euthanized and necropsied at 49 days PI. Lungs were removed, assessed for gross lesions (graded on a subjective scale 0-3 with 0 being normal) and C. vulpis were collected by lung-flush and counted. Samples of lung tissue were preserved for evaluation of histopathology and the lesions graded on a subjective scale (0-3 with 0 being normal). Gross and histopathology lesions were detected in all 8 untreated Group 1 dogs with mean subjective lesion scores of 1.8 ± 0.7 (range 1-3) and 3.0 ± 0.0 (range 3), respectively. Gross lesions were observed in 3/8 and histopathology lesions in all 8 of the treated Group 2 dogs with mean subjective lesion scores of 0.4 ± 0.5 (range 0-1) and 1.3 ± 0.4 (range 1-2), respectively. The mean (geometric) number for adult C. vulpis recovered in untreated dogs was 48.3 (range 25-70) compared with 0.65 (range 0-2) in animals treated with Milbemax. The resulting efficacy against C. vulpis was 98.7%. The number of C. vulpis was significantly lower for treated dogs than the burden in the untreated group (p=0.0002). A single dose of Milbemax (milbemycin oxime 0.5mg/kg+praziquantel 5mg/kg) was highly effective for the treatment of patent C. vulpis infection in dogs. A dosing interval for the prevention of clinical disease in dogs exposed to natural infections has not been established.

Spin waves and quantum criticality in the frustrated XY pyrochlore antiferromagnet Er2Ti2O7.

We report detailed measurements of the low temperature magnetic phase diagram of Er2Ti2O7. Heat capacity and time-of-flight neutron scattering studies of single crystals reveal unconventional low-energy states. Er3+ magnetic ions reside on a pyrochlore lattice in Er2Ti2O7, where local XY anisotropy and antiferromagnetic interactions give rise to a unique frustrated system. In zero field, the ground state exhibits coexisting short and long-range order, accompanied by soft collective spin excitations previously believed to be absent. The application of finite magnetic fields tunes the ground state continuously through a landscape of noncollinear phases, divided by a zero temperature phase transition at micro{0}H{c} approximately 1.5 T. The characteristic energy scale for spin fluctuations is seen to vanish at the critical point, as expected for a second order quantum phase transition driven by quantum fluctuations.

Characterization of quaternary ammonium oligomers by paired-ion reversed-phase liquid chromatography-mass spectrometry.

The separation of novel permanently charged oligomers was studied using paired-ion reversed-phase liquid chromatography. The polyionene studied is less than 5 kDa in size, but contains three oligomer series with different end-group chemistries. The complexity of this polyionene makes development of a single-dimension separation quite challenging. Separation under critical conditions was employed to fractionate the end-group conformations and then the chain length of the oligomers in each series was confirmed by LC-MS. The oligomers were then used to optimize a single-dimension HPLC separation. Precise modulation of the hydrophobicity of the ion-pair reagent and the stationary-phase chemistry yielded very high resolution one-dimensional separations.

Calcinosis circumscripta in the dog: a retrospective pathological study.

Calcinosis circumscripta is an uncommon syndrome of ectopic idiopathic, dystrophic, metastatic or iatrogenic mineralization characterized by deposition of calcium salts in soft tissues. This paper is a retrospective study of 77 canine cases. The age of dogs in the study varied from 4 months to 15 years and 55% were <1 year old, 74% <2 years old and 88% <4 years old. Several pure and mixed, typically large breed dogs were affected so that 28.6, 13 and 9% were German Shepherd, Rottweiler and Labrador Retriever respectively. The size of lesions varied from 2 mm to 13 cm in diameter but most lesions were between 0.5 cm and 3 cm in diameter. Lesions were solitary in 82% of the affected dogs, and occurred most commonly on the hind feet (50%) and tongue (23%). With multiple lesions there was no apparent body symmetry. Microscopically, most lesions were well-defined single or multiple variably sized aggregates of amorphous to granular, lightly to darkly basophilic material with or without peripheral granulomatous reaction and surrounded by varying amounts of fibrous connective tissue. Additionally, three small nodular masses in the wall of the jejunum of a dog were diagnosed as calcinosis circumscripta. This is the first reported case of idiopathic intestinal calcinosis circumscripta in the dog.

Pharmacokinetics and tolerability of intravenous trecovirsen (GEM 91), an antisense phosphorothioate oligonucleotide, in HIV-positive subjects.

Trecovirsen, a 25-mer antisense phosphorothioate oligonucleotide targeted at the gag site of the HIV gene, was administered to HIV-positive volunteers as an i.v. infusion. Single doses ranged from 0.1 to 2.5 mg/kg in an ascending escalation in cohorts of 6 to 12 subjects. Plasma trecovirsen concentrations and pharmacokinetic parameters could be assessed at doses > or = 0.3 mg/kg. Peak plasma concentrations and AUC values increased disproportionately with increasing dose while elimination half-life increased and plasma clearance decreased, indicating a saturable process over this dose range. The only significant adverse event observed was an isolated, transitory increase in activated partial thromboplastin time at doses > or = 2.0 mg/kg that was related to plasma trecovirsen concentrations and is attributed to the polyanionic character of the molecule. Thus, trecovirsen administration was well tolerated in single i.v. doses up to 2.5 mg/kg.

Organized cell swimming motions in Bacillus subtilis colonies: patterns of short-lived whirls and jets.

The swimming motions of cells within Bacillus subtilis colonies, as well as the associated fluid flows, were analyzed from video films produced during colony growth and expansion on wet agar surfaces. Individual cells in very wet dense populations moved at rates between 76 and 116 microm/s. Swimming cells were organized into patterns of whirls, each approximately 1,000 microm2, and jets of about 95 by 12 microm. Whirls and jets were short-lived, lasting only about 0.25 s. Patterns within given areas constantly repeated with a periodicity of approximately 1 s. Whirls of a given direction became disorganized and then re-formed, usually into whirls moving in the opposite direction. Pattern elements were also organized with respect to one another in the colony. Neighboring whirls usually turned in opposite directions. This correlation decreased as a function of distance between whirls. Fluid flows associated with whirls and jets were measured by observing the movement of marker latex spheres added to colonies. The average velocity of markers traveling in whirls was 19 microm/s, whereas those traveling in jets moved at 27 microm/s. The paths followed by markers were aligned with the direction of cell motion, suggesting that cells create flows moving with them into whirls and along jets. When colonies became dry, swimming motions ceased except in regions close to the periphery and in isolated islands where cells traveled in slow whirls at about 4 microm/s. The addition of water resulted in immediate though transient rapid swimming (> 80 microm/s) in characteristic whirl and jet patterns. The rate of swimming decreased to 13 microm/s within 2 min, however, as the water diffused into the agar. Organized swimming patterns were nevertheless preserved throughout this period. These findings show that cell swimming in colonies is highly organized.

Kinetics of phosphorothioate oligonucleotide metabolism in biological fluids.

The in vitro stability and metabolism of GEM[91, a 25mer phosphorothioate antisense oligonucleotide complementary to the gag mRNA region of HIV-1, was investigated using capillary electrophoresis (CE). The in vitro degradation of the parent compound at 37 degrees C was followed over the course of 120 h in human plasma. A CE method using laser-induced fluorescence detection was able to detect 5'-end intact metabolites including the parent compound extracted from biological fluids. Because the primary metabolic pathway is believed to be via 3'-exonuclease activity, the results of this study were compared with the stability of the compound in a solution containing 3'-exonuclease. The numerical solution of sequential first-order reactions was used to obtain kinetic parameters. Exonuclease digestion of the parent compound, as measured using an automated CE-UV instrument, yielded striking similarities between the two in vitro systems as well as between in vitro and in vivo systems.

A nonradioisotope approach to study the in vivo metabolism of phosphorothioate oligonucleotides.

A 25-mer phosphorothioate oligodeoxynucleotide (GEM 91) complementary to the gag gene mRNA of HIV-1 virus was administered intravenously (i.v.) at a dose of 10 mg/kg/day for 8 weeks or 25 mg/kg single dose subcutaneously (SC) to adult Rhesus monkeys. No radioactive markers were used. A capillary gel electrophoresis (CGE) method with UV detection was used to determine the concentration of GEM 91 in plasma and the metabolite profile. The metabolite profile was virtually the same following a single dose of either 10 mg/kg i.v. or 25 mg/kg SC. A different metabolite profile was observed after 4 or 8 weeks of multiple i.v. doses of 10 mg/kg/day. The extract was subjected to matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOFMS) for positive identification. Mass spectrometry confirmed the major metabolic pathway in vivo to be via 3'-end exonuclease activity. The extract was then subjected to a hybridization-assisted ligation reaction in which only 5'-end intact metabolites were labeled. Analysis by CGE with laser-induced fluorescence (LIF) detection allowed each of these metabolites to be quantified with a limit of detection of 1 ppb (ng/ml). MALDI-TOFMS identified components digested from both ends of the DNA. This study demonstrates that the combination of quantitative CGE-LIF and MALDI-TOFMS yields a powerful and unique approach to study the metabolism of phosphorothioate oligonucleotides.

Perceptions of physical fitness and exercise activity among older adults.

The purpose of this research was to examine older adults' perceptions of physical fitness and exercise. This qualitative study was divided into four stages as described by Berg (1989): identification of the concept of the study; development of the interview guide; collection of data; and data analysis. Twenty-three older adults, aged 63-82 years (9 females, 14 males), participated in the interviews. Transcripts were analysed using content analysis. Three major themes emerged as the participants viewed physical fitness in terms of: functional independence ('being able to do'); holism ('mind-body works together'); and age reference (-for people my age'). Nine elements which impeded or enhanced physical activity were identified. Implications for education, research and practice are discussed.

Ultrastructural changes in ovarian follicles of monkeys administered hexachlorobenzene.

OBJECTIVE: To test hexachlorobenzene (HCB), an environmental pollutant, for its potential toxicity to the ovary. DESIGN: Nonhuman primates were orally administered the pollutant at doses between 0.01 and 10 mg of HCB/kg of body weight to test viability of ovarian follicles. At the end of dosing period, the monkeys received a compound that contained follicle-stimulating and luteinizing hormones to stimulate development of follicles that would be examined by electron microscopy. ANIMALS: Twenty, 6- to 13-year-old cynomolgus monkeys were randomly assigned to 5 groups. PROCEDURE: The HCB, in concentrations of 0.01, 0.1, 1.0, and 10.0 mg/kg, was orally administered with glucose in gelatin capsules for 13 weeks. Monkeys receiving capsules containing glucose only served as controls. After the 13th week, monkeys were given a compound that contained follicle-stimulating and luteinizing hormones daily during days 2 to 7 from the start of menses. On day 8 of the menstrual cycle, 5,000 IU of human chorionic gonadotropin was administered, and 35 to 38 hours later, 1 ovary from each monkey was obtained during laparotomy. Approximately 1-mm cubes of tissue from each ovary were harvested, fixed by immersion in buffered 2% glutaraldehyde, and processed for transmission electron microscopy. RESULTS: Ultrastructure of ovarian follicles was altered in the monkeys administered HCB. Lesions were observed in the follicles from monkeys given the lowest concentrations of HCB, and comprised condensed mitochondria in the developing ova and follicular cells that contained nuclei with deep indentations and abnormal accumulation of cytoplasmic lipid droplets. Alterations, such as herniation of the ooplasm, degeneration of the follicular cells, and appearance of abnormal spaces between follicular cells were observed in the follicles from monkeys of the 0.1 to 1.0 mg of HCB/kg dosage groups. The most relevant alterations were seen in the mitochondrion, an organelle that appeared to be most sensitive to the compound. Mitochondria were condensed, with abnormal intracristal spaces in the lower-dosage groups, and were markedly degenerated in the 10 mg/kg group. The effect of HCB were dose-related. CONCLUSION: The HCB is a reproductive system toxicant, and its damaging effects may be a result of augmentation of lipid peroxidation, especially in the primary follicle, which abnormally affects cellular membranes and thus, impairs their permeability.

Structured oral interview. One way to identify family physicians' educational needs.

OBJECTIVE: To design and test a structured oral interview that would elicit information on the educational needs of physicians in order to help them plan individualized continuing education. DESIGN: Seven different sets of problems were prepared, each including 40 cases, of which 26 are common. Each pilot test candidate was interviewed by two physician-interviewers during a 1-day session. After each answer, candidates were told the predetermined correct answer. PARTICIPANTS: Six candidates were selected at random from among Montreal physicians aged 50 and older with no hospital privileges. All had to have no history of professional complaints or prosecution and to be unknown to the interviewers. MAIN OUTCOME MEASURES: Inter-rater reliability and perceived difficulty of the cases. RESULTS: Candidates rated the interview process and cases used pertinent, credible, and not too difficult. Candidates' performance level was about 50%. Agreement between interviewers averaged 91.2%. CONCLUSIONS: A structured oral interview appears to be a credible instrument for helping determine practising physicians' deficiencies in clinical knowledge and reasoning.

Quantitative analysis of phosphorothioate oligonucleotides in biological fluids using direct injection fast anion-exchange chromatography and capillary gel electrophoresis.

The analysis of antisense phosphorothioate DNA (SODN) in human plasma via direct injection using anion-exchange high-performance liquid chromatography (AE-HPLC) is presented. The method relies on the ability to selectively extract phosphorothioate DNA from undigested serum, plasma and urine on anion-exchange resins. The automated HPLC method can analyze a sample every 5 min with a limit of detection of 50 ng/ml (ppb). The DNA was collected, desalted and analyzed by capillary gel electrophoresis. Due to the high resolving power of this technique, a qualitative assessment of enzymatic degradation of the antisense oligonucleotide can be made.

Solid-phase derivatization reactions for biomedical liquid chromatography.

Polymeric reagents have been developed for performing off- and on-line derivatizations of numerous organic analytes in HPLC-detection modes. Such reagents utilize ionic or covalent attachment of labile tags that possess specific detector enhancement properties: ultraviolet, electrochemical, fluorescence, and so forth. Specific synthetic procedures have evolved to generate various linkages of the tag to the underlying, polymeric support, usually involving activated ester connections (leashes). The polymer itself may play a number of roles in the nature of the overall reactions, such as hydrophobic-hydrophillic exclusion, pore size restriction, stabilization of the attachment leashes, and protection of the tags from hydrolysis in aqueous media. The basic, underlying chemistry of polymeric reagents has evolved to the point where it is possible to engineer the polymer support itself, the attachment leash, and the various tags that are then transferred to the analyte molecules. These procedures have now reached the stage of commercialization and practical applicability for real-world drugs and bioorganics in complex biofluid type samples. Polymer supported reagents can now be used for direct injection of biofluids with solid-phase (hydrophobic) extraction of the analytes of interest, followed by sample cleanup, derivatization, elution onto the HPLC column, peak compression, gradient HPLC elution, multiple detection, and final data interpretation with quantitation. This review summarizes much or most of what has been described in the scientific literature over the past decade in the various areas where polymeric reagents are being used for derivatization in HPLC and in capillary electrophoresis as well.

MR evaluation of clinically suspected adnexal masses.

OBJECTIVE: Our goal was to determine the sensitivity, specificity, predictive value, and accuracy of pelvic MRI in the prospective evaluation of women with a clinically suspected pelvic mass. MATERIALS AND METHODS: One-hundred three patients with clinically suspected pelvic masses were prospectively evaluated by pelvic MRI. Masses were analyzed for size, location, morphological characteristics, and signal behavior. Masses were classified as benign, malignant, or indeterminate and in every case an attempt was made to generate a specific diagnosis according to previously reported characteristic MR appearances. Surgical follow-up was obtained within 3 months of MRI examination for all patients, excepting 11 patients with typically appearing leiomyomas. RESULTS: One hundred twenty pelvic masses were confirmed. Magnetic resonance was 100% sensitive and 99% specific in prospectively diagnosing dermoids, 96% sensitive and 100% specific in diagnosing subserosal leiomyomas, and 92% sensitive and 91% specific in diagnosing endometriomas. CONCLUSION: When physical examination or ultrasound examination is inconclusive, pelvic MRI can aid in the evaluation of women with a suspected pelvic mass. By identifying these common benign gynecologic conditions, unnecessary surgery can be avoided.

Automated HPLC analyses of drugs of abuse via direct injection of biological fluids followed by simultaneous solid-phase extraction and derivatization with fluorescence detection.

An automated system is described for the simultaneous extraction and derivatization of nucleophilic compounds from various biological media. The method includes the use of a solid-phase reagent containing a 9-fluorenylacetate activated ester. The reagent is based on a controlled pore, polystyrene divinylbenzene support prepared through a silica template procedure. An X-Y-Z robotic arm equipped with a needle is used in conjunction with a syringe pump for aspirating and dispensing samples and standards into the HPLC system. A precolumn cartridge containing the solid-phase reagent is put on-line in place of the fixed-volume injection loop. Injections of biological fluids such as urine or plasma with minimal sample treatment and handling are made directly into this reactor. The analytes are derivatized as they are extracted, allowing virtually unlimited sample volumes to be injected. The polymeric cartridge can be used for up to 100 injections without accruing unacceptable reductions in sensitivity. A detection limit of 500 p.p.t. (parts per trillion) of amphetamine in urine was achieved with this system.

Stabilization of reactive species within polystyrene divinylbenzene polymer networks.

The effect of cross-linking, surface area, and porous nature of modified polystyrene-divinylbenzene (STY-DVB) reagents has been investigated. The supports were prepared via two techniques and modified to contain various chemical functionalities. These reagents were used in an on-line reactor for automated derivatization of amines in HPLC. The reproducibility of the response vs the physical nature of the porous support and the chemical functionality was determined. The ability to stabilize highly reactive acylating reagents toward high concentrations of aqueous base was found to be a complex interaction of pore size distribution, percent cross-linking, surface area, and absolute loading of the analytical reagent on the porous support.

Quantitative analysis of phosphorothioate oligonucleotides in biological fluids using fast anion-exchange chromatography.

Phosphorothioate oligonucleotides are potentially useful as anti-viral drugs. Classical DNA extraction methods are not as effective on short single-stranded DNA as with longer double-stranced chains. The classical method of phenol-chloroform extraction followed by ethanol precipitation is difficult to quantify, thus monitoring of the pharmacological disposition of these compounds is subject to error. A method has been devised and validated for extraction and analysis of modified oligonucleotides from biological fluids such as urine and serum based on protein kinase digestion and phenol-chloroform extraction. Due to the high native ultraviolet absorbance of the oligomers, detection limits in the low ppb range were obtained without derivatization.

Immobilized isocyanates for derivatization of amines for chiral recognition in liquid chromatography with UV detection.

Solid-phase derivatization reagents containing a 3,5-dinitrophenyl moiety for the derivatization of amines are described. The reagents are useful for Pirkle-type recognition of stereochemical composition of amines and related nucleophiles. The ability to stabilize 3,5-dinitrophenylisocyanate by covalent immobilization on a polymeric support varied greatly with the support chosen. The kinetics of the activated dinitrophenylcarbamate and the use of this reagent for enhanced recognition of optical purity is shown and compared to previous findings with a similar polymeric activated ester.