The innate immune stimulant Amplimune® is safe to administer to young feedlot cattle.

BACKGROUND: Infectious disease has a significant impact on livestock production. Availability of alternatives to antibiotics to prevent and treat disease is required to reduce reliance on antibiotics while not impacting animal welfare. Innate immune stimulants, such as mycobacterium cell wall fractions (MCWF), are used as alternatives to antibiotics for the treatment and prevention of infectious disease in a number of species including cattle, horses and dogs. This study aimed to evaluate the safety of Amplimune®, an MCWF-based immune stimulant, for weaner Angus cattle. METHODS: On day -1 and 0, sixty mixed-sex Angus weaner cattle were transported for 6 h before being inducted and housed in a large single pen, simulating feedlot induction conditions. The cattle were assigned to one of six treatment groups (n = 10 per group): 2 mL Amplimune intramuscularly (2IM); 2 mL Amplimune subcutaneously (2SC); 5 mL Amplimune intramuscularly (5IM); 5 mL Amplimune subcutaneously (5SC); 5 mL saline intramuscularly (SalIM) and 5 mL saline subcutaneously (SalSC) on day 0 following transportation. Body temperature, body weight, concentrations of circulating pro-inflammatory cytokines (TNFα, IL-1ß, IL-6 and IL-12) and haematology parameters were measured at various times up to 96 h post-treatment. RESULTS: No adverse effects from Amplimune treatment were observed. Amplimune induced an increase in circulating cytokine TNFα concentrations, total white blood cell count and lymphocyte counts indicative of activation of the innate immune system without causing an excessive inflammatory response. CONCLUSIONS: Results confirm that Amplimune can be safely administered to beef cattle at the dose rates and via the routes of administration investigated here.

LC-MS/MS quantitative analysis of reducing carbohydrates in soil solutions extracted from crop rhizospheres.

A simple, sensitive, and specific analytical method has been developed for the quantitative determination of 15 reducing carbohydrates in the soil solution of crop rhizosphere. Reducing carbohydrates were derivatized with 1-phenyl-3-methyl-5-pyrazolone, separated by reversed-phase high-performance liquid chromatography and detected by electrospray ionization tandem mass spectrometry. Lower limits of quantitation of 2 ng/mL were achieved for all carbohydrates. Quantitation was performed using peak area ratios (analyte/internal standard) and a calibration curve spiked in water with glucose-d(2) as the internal standard. Calibration curves showed excellent linearity over the range 2-100 ng/mL (10-1,000 ng/mL for glucose). The method has been tested with quality control samples spiked in water and soil solution samples obtained from the rhizosphere of wheat and canola and has been found to provide accurate and precise results.

Reducing risk in basin scale CO2 sequestration: a framework for integrated monitoring design.

Injection of CO(2) into geological structures is a key technology for sequestering CO(2) emissions captured from the combustion of fossil fuels. Current projects inject volumes on the order of megatonnes per year. However, injection volumes must be increased by several orders of magnitude for material reductions in ambient concentrations. A number of questions surrounding safety and security of injection have been raised about the large scale deployment of geological CO(2) sequestration. They are site specific and require an effective monitoring strategy to mitigate risks of concern to stakeholders. This paper presents a model-based framework for monitoring design that can provide a quantitative understanding of the trade-offs between operational decisions of cost, footprint size, and uncertainty in monitoring strategies. Potential risks and challenges of monitoring large scale CO(2) injection are discussed, and research areas needed to address uncertainties are identified. Lack of clear guidance surrounding monitoring has contributed to hampering the development of policies to promote the deployment of large scale sequestration projects. Modeling provides an understanding of site specific processes and allows insights into the complexity of these systems, facilitating the calibration of an appropriate plan to manage risk. An integrated policy for risk-based monitoring design, prior to large scale deployment of sequestration will ensure safe and secure storage through an understanding of the real risks associated with large scale injection.

Imaging features of primary lymphoma of bone.

OBJECTIVE: Our objective was to describe the imaging appearances of primary lymphoma of bone, including conventional radiographic, scintigraphic, CT, and MR imaging features. MATERIALS AND METHODS: We retrospectively reviewed 237 pathologically proven cases of primary lymphoma of bone. Evaluation included patient age, sex, lesion location, and pattern of bone destruction. Pathologic type, periosteal reaction, sequestrum, soft-tissue mass, extension across joints, and pathologic fracture were also noted. RESULTS: The study population included 151 males and 86 females (ratio 1.8:1; range, 2-88 years; mean age, 42 years). Common locations were the distal femoral diametaphysis; proximal metadiaphysis of the tibia, femur, and humerus; and femoral mid shaft. Long bones were involved more often than flat bones (71% versus 22%). Common appearances were a lytic (70%) or mixed-density (28%) lesion with most cases showing a permeative or moth-eaten pattern (74%). Periosteal reaction was seen in 58% of the long bones. Sequestra were found in 37 patients (16%). Soft-tissue masses were present in 113 patients (48%). Extension across joints was seen in nine patients (4%). Pathologic fractures occurred in 53 patients (22%). Radionuclide (n = 56), CT (n = 45), and MR (n = 20) features were usually nonspecific. Pathologic types included non-Hodgkin's (n = 223) and Hodgkin's (n = 14) lymphoma. CONCLUSION: Primary lymphoma of bone most often involves the diametaphysis of a major long bone and has an aggressive pattern of lytic bone destruction and associated soft-tissue mass. CT and MR imaging can suggest the diagnosis, particularly when a large soft-tissue mass and abnormal marrow attenuation or signal intensity is seen without extensive cortical destruction.

Aortic root geometry: pattern of differences between leaflets and sinuses of Valsalva.

BACKGROUND AND AIMS OF THE STUDY: Growing interest in aortic root replacement with the use of stentless auto-, homo- and xenografts, and new developments in aortic valve conservation demand a deeper understanding of the normal aortic root anatomy. METHODS: Ten cryopreserved human aortic roots were pressurized, fixed and measured directly (leaflet free edge and attachment) and using three-dimensional computed tomography imaging software (sinus of Valsalva height and volume). RESULTS: The mean of the measurements of all four parameters yielded a pattern in which the non-coronary sinus (N) structures had the greatest dimensions followed by the right (R) and then the left (L). Non-parametric ANOVA on each of these parameters also showed significant differences among the sinuses yielding a pattern of N > R > L. This pattern determined an angle of tilt between the plane at the base (annulus) and the plane intersecting the sinotubular junction with a mean value of 11 degrees. Linear regression indicated that this angle did not depend on the size of the base (annulus). CONCLUSIONS: The data showed a geometric pattern of the aortic root, with the structures of the non-coronary sinus being the largest followed by the right and then the left. The possible hemodynamic relevance and surgical implications of these findings need to be explored.

Experience with a low profile bipolar, active fixation pacing lead in pediatric patients.

Continued miniaturization of permanent pacing systems has promoted use of this technology in younger and smaller pediatric patients. Intermedics ThinLine 438-10 active fixation pacing leads (4.5 Fr lead body) were implanted in 26 patients (17 males/9 females; 9.9 +/- 6.9 years). Twenty of 26 patients received dual chamber systems, 6 of 26 patients single lead systems. Each patient has been followed 3 months. Pacemaker analysis at implant and 6 months later evaluated pulse width thresholds at 2.5 V (atrial 0.07 +/- 0.02 vs 0.13 +/- 0.02 ms [P = 0.01]; ventricular 0.08 +/- 0.04 ms vs 0.20 +/- 0.04 ms [P = 0.01]); sensing thresholds (atrial 4.1 +/- 0.41 mV vs 4.0 +/- 4.2 mV [P = NS]; ventricular 9.7 +/- 0.72 vs 9.3 +/- 0.94 mV [P = NS]); and impedance (atrial 345 +/- 12 vs 370 +/- 120 O [P = 0.04]; ventricular 412 +/- 17 vs 458 +/- 190 O [P < 0.01]). One volt lead failed with exit block at approximately 6 weeks. The youngest (9 months to 5 years) and smallest (6.5-18.0 kg) ten patients have each shown by venography to have at least mild venous stenosis at the lead(s) insertion site; five patients demonstrated collateral formation around asymptomatic obstruction, with no thrombus formation. The Intermedics 438-10 ThinLine pacing lead has demonstrated good and stable early postimplant electrical parameters. Angiographic evaluation in our smaller patients has shown evidence for asymptomatic venous obstruction.

The many faces of osteosarcoma.

Osteosarcoma is the most common primary malignant tumor of bone in adolescents and young adults. It accounts for approximately 15% of all primary bone tumors confirmed at biopsy. There are numerous types of primary osteosarcoma, including intramedullary (high grade, telangiectatic, low grade, small cell, osteosarcomatosis, and gnathic), surface (intracortical, parosteal, periosteal, and high-grade surface), and extraskeletal. Osteosarcoma may also occur as a secondary lesion in association with underlying benign conditions. The identification of osteoid matrix formation and aggressive characteristics usually allows prospective radiologic diagnosis of osteosarcoma. As with all bone tumors, differential diagnosis is best assessed with radiographs, whereas staging is performed with computed tomography or magnetic resonance imaging. Understanding and recognition of the variable appearances of the different varieties of osteosarcoma allow improved patient assessment and are vital for optimal clinical management including diagnosis, biopsy, staging, treatment, and follow-up.

Effects of dual-chamber pacing for pediatric patients with hypertrophic obstructive cardiomyopathy.

OBJECTIVES: The effects of both temporary and permanent dual-chamber pacing (DCP) were evaluated in symptomatic pediatric patients with hypertrophic obstructive cardiomyopathy (HOCM) unresponsive to medications. BACKGROUND: Permanent DCP pacing can reduce left ventricular outflow tract (LVOT) gradient and relieve symptoms in adult patients with HOCM. METHODS: Ten patients (mean [+/-SD] age 11.1 +/- 6 years, range 1 to 17.5) with HOCM and a Doppler LVOT gradient > or = 40 mm Hg were studied. The seven patients showing hemodynamic improvement during temporary pacing at cardiac catheterization underwent surgical implantation of a permanent DCP system. The effects of permanent pacing were evaluated using a questionnaire, Doppler evaluation, treadmill testing and repeat cardiac catheterization. RESULTS: At initial cardiac catheterization, three patients failed to respond to temporary pacing (inadequate pace capture in two; congenital mitral valve abnormality in one). The remaining seven patients (70%, 95% confidence interval 38.0% to 91.7%, mean age 13 +/- years, range 4 to 17.5) showed a significant reduction (p < 0.05) in LVOT gradient, left ventricular systolic pressure and pulmonary capillary wedge pressure. After pacemaker implantation, these seven patients reported a significant reduction in dyspnea on exertion and exercise intolerance. Serial Doppler evaluation showed a significant reduction in LVOT gradient. Follow-up catheterization at 23 +/- 4 months in six patients (one patient declined restudy) showed a persistent decrease in LVOT gradient (53 +/- 13 vs. 16 +/- 11 mm Hg), left ventricular systolic pressure (149 +/- 16 vs. 108 +/- 14 mm Hg) and pulmonary capillary wedge pressure (18 +/- 2 vs. 12 +/- 4 mm Hg) versus preimplantation values. CONCLUSIONS: Permanent DCP is an effective therapy for selected pediatric patients with HOCM. Rapid atrial rates and intrinsic atrioventricular conduction, as well as congenital mitral valve abnormalities, may preclude effective pacing in certain patients.

Modulation of sexual behaviour in the rat by a potent and selective alpha 2-adrenoceptor antagonist, delequamine (RS-15385-197).

1. The contributions of alpha 2-adrenoceptors and 5-HT1A receptors to sexual behaviour in the rat have been re-evaluated by use of a highly potent and selective alpha 2-adrenoceptor antagonist, delequamine (RS-15385-197), yohimbine, idazoxan and the partial agonist at 5-HT1A receptors, 8-hydroxy-2(di-n-propylamino)-tetralin (8-OH-DPAT). 2. In a model where naive male rats were introduced to oestrogen-progesterone primed, sexually receptive female rats, delequamine (0.4-6.4 mg kg-1, p.o.) dose-relatedly increased the sexual behaviour score over the entire dose-range whereas yohimbine was effective at only one dose, 2 mg kg-1, p.o.. Idazoxan was active only at 2.5 and 5 mg kg-1, p.o. Yohimbine, but neither delequamine nor idazoxan, decreased ejaculation latency. 8-OH-DPAT (0.1 and 0.25 mg kg-1, s.c.) reduced the time, and the number of intromissions to ejaculation without affecting other parameters. A combination of delequamine (0.4 mg kg-1, p.o.) and 8-OH-DPAT (0.1 mg kg-1 s.c.) increased the percentage of rats mounting, intromitting and ejaculating, and reduced ejaculation latency and the number of intromissions. 3. In orchidectomized, sexually experienced rats exposed to sexually receptive females, delequamine, idazoxan and yohimbine increased the number of rats mounting, and there was a tendency to increase the number of animals intromitting, but no effect on ejaculatory behaviour. 4. In ovariectomized female rats brought to low level receptivity by priming with low dose injections of oestradiol benzoate and progesterone, delequamine, at 1.6 and 6.4 mg kg-1 p.o., increased lordosis, while yohimbine, at 2, 4 and 8 mg kg-1 p.o., reduced lordotic responses to sexually experienced males in a dose-dependent manner. 8-OH-DPAT at 0.1, 0.25 mg kg-1, s.c. reduced lordosis in a dose-dependent manner. 5. These findings may be explained on the basis that yohimbine is an alpha 2-adrenoceptor antagonist with affinity for 5-HT1A receptors and that the effects of 5-HT1A receptors may modulate the sexual behaviour responses to alpha 2-receptor antagonism in some models. Thus, in contrast to yohimbine, the highly-selective alpha 2-adrenoceptor antagonist, delequamine, was very effective in increasing the behavioural score in male and female rats over a wide dose-range.

Encapsulated liquid crystals as probes for remote thermometry.

A temperature probe based on the magnetic resonance properties of an encapsulated liquid crystal has been investigated. Large changes in magnetic resonance signals occur as the liquid crystal undergoes a phase transition from an anisotropic (nematic) state to the isotropic liquid. The low latent heat of such phase transitions allows for rapid phase changes during a hyperthermia treatment. Transition temperatures can be tailored by adding suitable compounds such as analogues of the liquid crystal or various solvents. Encapsulation is required to maintain the integrity of the liquid crystal, particularly for applications in vivo. Results of preliminary studies designed to demonstrate the technical feasibility of the concept are presented.

Potent, long-acting luteinizing hormone-releasing hormone antagonists containing new synthetic amino acids: N,N'-dialkyl-D-homoarginines.

A new series of unnatural amino acids has been prepared and incorporated into antagonistic analogues of luteinizing hormone-releasing hormone (LH-RH), on the basis of the hypothesis that stabilization of a proposed phospholipid membrane interaction might yield analogues with high potency and a prolonged duration of action. Thus a series of NG,NG'-dialkyl-D-homoarginine analogues [H-D-hArg(R2)-OH; R = Me, Et, Pr, i-Pr, Bu, hexyl, cyclohexyl, (Et, Me2NPr)] was conveniently prepared by semisynthesis from D-Lys using the appropriate dialkylcarbodiimide. A number of the analogues that were prepared by using these new amino acid analogues exhibited very high potency and a prolonged duration of action. One of the most potent members of the series, [N-Ac-D-Nal(2)1,D-pCl-Phe2,D-Trp3,D-hArg(Et2)6,D-Ala10 ]LH-RH (detirelix), had an ED50 of 0.7 microgram in the rat antiovulatory assay when administered at noon on proestrus and only 2.5 micrograms when administered 24 h earlier, at noon on diestrus II. This antagonist is undergoing detailed biological and clinical evaluation.

Prolonged controlled-release of nafarelin, a luteinizing hormone-releasing hormone analogue, from biodegradable polymeric implants: influence of composition and molecular weight of polymer.

The release of the peptide hormone nafarelin, 5-oxo-L-prolyl-L-histidyl-L-tryptophyl-L-seryl-L-tyrosyl-3-(2-naphthyl)- D-alanyl-L-leucyl-L-arginyl-L-prolylglycinamide, a potent luteinizing hormone-releasing hormone (LHRH) agonist, from implants of the biodegradable copolymer poly(d,l-lactide-co-glycolide) (PLGA) has been studied both in vivo and in vitro. The release has a triphasic profile typical for bulk-eroding monolithic controlled-release systems, characterized by a secondary phase of lower release preceded and followed by phases of higher release. The primary factor controlling the peptide release profile is polymer erosion, which in turn may be controlled by modifying physical properties of the polymer such as the molecular weight or the ratio of the more hydrophobic lactic acid monomer to the less hydrophobic glycolic acid monomer. The duration of the secondary phase has been found to be directly proportional to the molecular weight of the copolymer, and the total duration as well as the duration of the secondary phase are both directly proportional to the monomer ratio. A system has been identified in which the secondary phase is sufficiently reduced to provide essentially continuous efficacy in the rat for greater then eight months, with partially effective levels of release of nafarelin continuing beyond 15 months.

Long-term reversible suppression of oestrus in bitches with nafarelin acetate, a potent LHRH agonist.

Adult cyclic beagle bitches were treated for up to 18 months with nafarelin acetate via subcutaneously implanted osmotic pumps, starting during the first week of a pro-oestrous vaginal discharge. The imminent ovulation appeared to be unaffected by treatment, but doses of 8 or 32 micrograms analogue/day reduced the integrated luteal progesterone values. No new oestrus was detected in 3 bitches during 18 months of treatment with 32 micrograms/day, which resulted in mean plasma levels of 0.4 ng analogue/ml. A return to oestrus was observed in all 3 bitches between 3 and 18 weeks after cessation of treatment: 2 of the bitches mated at those times and produced normal litters. Another 2 bitches were similarly treated with 32 micrograms analogue/day; they were mated at the oestrus at start of treatment and dosing was continued for about 63 days. One of the bitches conceived and produced a normal litter. Nafarelin acetate treatment begun during anoestrus resulted in an induced heat 1-2 weeks after the start of treatment. The induced heat consisted of pro-oestrous vaginal discharge, oestrous vaginal cytology, and ovulation (judged by increased circulating levels of progesterone). Three bitches mated at the induced heat and treated for the normal duration of gestation did not litter. Nafarelin treatment of 3 bitches before puberty did not induce signs of oestrus and prevented the occurrence of oestrus through 18 months of treatment. The first oestrus in these bitches occurred 3.5-4 months after cessation of treatment, but mating at that time did not result in pregnancy. These studies have established the feasibility of and dosage requirement for the use of the LHRH agonist as a contraceptive in the bitch.

Suppression of luteal progesterone secretion in the stumptailed macaque by an antagonist analogue of luteinizing hormone releasing hormone.

The dependence of progesterone secretion from the corpus luteum on pituitary gonadotrophin was examined in the cyclic stumptaile macaque by studying the effects of a single s.c. injection of a potent LH releasing hormone (LHRH) antagonist, [N-Ac-D-Nal(2)1, D-pCl-Phe2, D-Trp3, D-hArg(Et2)6, D-Ala10] LHRH. A dose of 100 micrograms antagonist/kg administered on days 9/10 of the luteal phase in three monkeys caused a marked temporary suppression of serum concentrations of LH and progesterone during the following 32h but levels still remained detectable and after 2 days serum hormone concentrations returned to the normal luteal-phase range. When the same animals were treated with 300 micrograms antagonist/kg at the same period during a subsequent cycle, serum LH levels were close to or at the limits of detection of the bioassay for the next 48h and progesterone concentrations declined steadily, reaching non-detectable values by 48h. In two monkeys the progesterone levels remained suppressed and they menstruated prematurely; in the third monkey the progesterone concentration rose to just above baseline and menstruation occurred at the expected time. Administration of 300 micrograms antagonist/kg on days 6/7 of the luteal phase in a further three monkeys also suppressed progesterone concentrations but not to baseline values, and after 2 days a normal progesterone profile was regained. These results suggest that the corpus luteum of the stumptailed macaque is largely dependent on pituitary gonadotrophin support during the mid to late luteal phase.

Dose-response studies on male reproductive parameters in dogs with nafarelin acetate, a potent LHRH agonist.

Adult male beagle dogs were administered daily subcutaneous injections of either 0.5 or 2.0 micrograms/kg of a potent LHRH agonist, nafarelin acetate, for 44 days. Although there was a rise in the circulating levels of the gonadotropins and of testosterone following the early injections of agonist, continued treatment caused a marked decline in acute response and basal levels of both LH and testosterone and smaller decreases in the acute FSH response. The decline in LH and testosterone was accompanied by decreases in testicular volume, ejaculated sperm count, sperm motility, ejaculate volume, and duration of ejaculation. The decline in these parameters was more rapid at 2.0 micrograms/kg than at 0.5 micrograms/kg. The profile of responses to 2.0 micrograms/kg could be superimposed on that previously shown for the injection of 10.0 micrograms/kg. At the end of treatment, prostate weights were 36% and 68% of vehicle-treated controls for high- and low-dose animals, respectively. Spermatogenesis was absent in the testes of all agonist-treated animals. Over the dose range tested, the dose-response on all parameters was characterized by a slower evolution to the same maximal effect, rather than by a partial effect. If these data can be extrapolated to man, they would suggest that administration of higher dose levels of LHRH agonists than presently reported should be explored.

Controlled release of a luteinizing hormone-releasing hormone analogue from poly(d,l-lactide-co-glycolide) microspheres.

The performance in vivo of nafarelin acetate, a potent analogue of luteinizing hormone-releasing hormone, microencapsulated in poly(d,l-lactide-co-glycolide), was evaluated. The influence of polymer composition and molecular weight on the estrus-suppressing activity of the microspheres in female rats was determined. Compound release was shown to be effected by polymer erosion rather than by diffusion. A triphasic release of compound was observed, which was adjusted by altering the critical parameters of the polymer. A mechanism for the release of the compound was proposed. The primary release phase was compound loss by diffusion from the surface of the microspheres. The secondary phase of subeffective rates of release occurred concomitantly with polymer hydrolysis and a decrease in its molecular weight, although it remained insoluble. Dissolution of low-molecular weight fragments and erosion of the bulk of the polymer then initiated the tertiary phase of release of compound.

Luteinizing hormone-releasing hormone antagonists containing very hydrophobic amino acids.

In a continuation of our studies on the effects of hydrophobic substitutions in analogues of luteinizing hormone-releasing hormone (LH-RH), we have synthesized LH-RH antagonists containing the very hydrophobic amino acid 3-(2-naphthyl)-D-alanine (D-Nal(2)). The D-Nal(2) substitution was found to be effective when incorporated in positions 3 and 6. The most potent analogue containing two D-Nal(2) residues was [N-Ac-Pro,D-pF-Phe,D-Nal(2)]LH-RH (ED50 = 2.2 micrograms, rat antiovulatory assay, propylene glycol-saline vehicle). This analogue also demonstrates that the N-Ac-Pro substitution is as effective as the more costly N-Ac-delta-Pro modification. Analogues containing D-Nal(2) in combination with the hydrophilic D-Arg residue in position 6 were prepared. Neither N-Ac-Pro at position 1 nor D-Nal(2) at position 3 was effective in combination with D-Arg. N-Ac-D-Nal(2) at position 1 gave a highly potent antagonist ([N-Ac-D-Nal(2),D-pF-Phe,D-Trp,D-Arg]LH-RH; ED50 = 2.4 micrograms) that exhibited a prolonged duration of action (ED50 = 9.0 micrograms, corn oil vehicle, dosing on diestrus II).

Nasal absorption of nafarelin acetate, the decapeptide [D-Nal(2)6)]LHRH, in rhesus monkeys. I.

Nafarelin acetate, [D-Nal(2)6]LHRH, a highly potent superagonist of luteinizing hormone-releasing hormone, was given intranasally to six female rhesus monkeys. Absorption was rapid and very reproducible, with peak levels occurring at 15-30 min and a bioavailability of approximately 2% relative to a subcutaneous dose. The nasal dose response was highly nonlinear. The nonlinearity was apparently associated with the absorption phase, since elimination profiles at all doses were similar.