Appetite stimulation with cannabis-based medicine and methods for assessment of glomerular filtration in older patients with medical illness: A study protocol.

BACKGROUND AND AIM: Malnutrition in older patients is linked to poor appetite. Cannabis-based medicine may have orexigenic properties in older patients, but this has to our knowledge never been investigated. In older patients, uncertainty applies to the accuracy of estimated glomerular filtration rate (eGFR) based on creatinine, which is crucial for medication prescribing. In older patients with poor appetite, the study aims (1) to assess the efficacy of Sativex® (8.1-mg delta-9-tetrahydrocannabinol [THC] and 7.5-mg cannabidiol [CBD]) to stimulate appetite and (2) to compare the performance of various GFR-estimates and measured-GFR (mGFR) for determining gentamicin clearance utilizing population pharmacokinetic (popPK) modelling methods. METHODS AND OBJECTIVES: This study is composed of two substudies. Substudy 1 is an investigator-initiated single-center, double-blinded, randomized, placebo-controlled, superiority, cross-over study. Substudy 1 will recruit 17 older patients with poor appetite, who will also be invited to substudy 2. Substudy 2 is a single-dose pharmacokinetics study and will recruit 55 patients. Participants will receive Sativex® and placebo in substudy 1 and gentamicin with simultaneous measurements of GFR in substudy 2. The primary endpoints are as follows: Substudy 1-the difference in energy intake between Sativex® and placebo conditions; substudy 2- the accuracy of different eGFR equations compared to mGFR. The secondary endpoints include safety parameters, changes in the appetite hormones, total ghrelin and GLP-1 and subjective appetite sensations, and the creation of popPK models of THC, CBD, and gentamicin.

Early neonatal lamb mortality: postmortem findings.

An investigation of stillbirth and early neonatal lamb mortality was conducted in sheep flocks in Norway. Knowledge of actual causes of death are important to aid the interpretation of results obtained during studies assessing the risk factors for lamb mortality, and when tailoring preventive measures at the flock, ewe and individual lamb level. This paper reports on the postmortem findings in 270 liveborn lambs that died during the first 5 days after birth. The lambs were from 17 flocks in six counties. A total of 27% died within 3 h after birth, 41% within 24 h and 80% within 2 days. Most lambs (62%) were from triplet or higher order litters. In 81% of twin and larger litters, only one lamb died. The most frequently identified cause of neonatal death was infectious disease (n=97, 36%); 48% (n=47) of these died from septicaemia, 25% (n=24) from pneumonia, 22% (n=21) from gastrointestinal infections and 5% (n=5) from other infections. Escherichia coli accounted for 65% of the septicaemic cases, and were the most common causal agent obtained from all cases of infection (41%). In total, 14% of neonatal deaths resulted from infection by this bacterium. Traumatic lesions were the primary cause of death in 20% (n=53) of the lambs. A total of 46% of these died within 3 h after birth and 66% within 24 h. Severe congenital malformations were found in 10% (n=27) of the lambs, whereas starvation with no concurrent lesions was the cause of death in 6% (n=17). In 16% (n=43) of the lambs, no specific cause of death was identified, lambs from triplet and higher order litters being overrepresented among these cases. In this study, the main causes of neonatal lamb mortality were infection and traumatic lesions. Most neonatal deaths occurred shortly after birth, suggesting that events related to lambing and the immediate post-lambing period are critical for lamb survival.

Carnitine levels in skeletal muscle, blood, and urine in patients with primary carnitine deficiency during intermission of L-carnitine supplementation.

BACKGROUND: Primary carnitine deficiency (PCD) is a disorder of fatty acid oxidation with a high prevalence in the Faroe Islands. Only patients homozygous for the c.95A>G (p.N32S) mutation have displayed severe symptoms in the Faroese patient cohort. In this study, we investigated carnitine levels in skeletal muscle, plasma, and urine as well as renal elimination kinetics before and after intermission with L-carnitine in patients homozygous for c.95A>G. METHODS: Five male patients homozygous for c.95A>G were included. Regular L-carnitine supplementation was stopped and the patients were observed during five days. Blood and urine were collected throughout the study. Skeletal muscle biopsies were obtained at 0, 48, and 96 h. RESULTS: Mean skeletal muscle free carnitine before discontinuation of L-carnitine was low, 158 nmol/g (SD 47.4) or 5.4% of normal. Mean free carnitine in plasma (fC0) dropped from 38.7 (SD 20.4) to 6.3 (SD 1.7) µmol/L within 96 h (p < 0.05). Mean T 1/2 following oral supplementation was approximately 9 h. Renal reabsorption of filtered carnitine following oral supplementation was 23%. The level of mean free carnitine excreted in urine correlated (R (2) = 0.78, p < 0.01) with fC0 in plasma. CONCLUSION: Patients homozygous for the c.95A>G mutation demonstrated limited skeletal muscle carnitine stores despite long-term high-dosage L-carnitine supplementation. Exacerbated renal excretion resulted in a short T 1/2 in plasma carnitine following the last oral dose of L-carnitine. Thus a treatment strategy of minimum three daily separate doses of L-carnitine is recommended, while intermission with L-carnitine treatment might prove detrimental.

Isolation of Bacillus anthracis from soil in selected high-risk areas of Zimbabwe.

AIMS: To isolate Bacillus anthracis from cattle carcass burial sites from high-risk districts in Zimbabwe. METHODS AND RESULTS: Soil samples were collected from carcass burial sites from seven areas, including two national game parks. Samples were collected from top 5-10 cm, and for spore extraction, 25 g of soil was suspended in sterile distilled water overnight. Supernatants were filtered through 0.45-µm pore cellulose nitrate, deposits suspended in 5 ml phosphate-buffered saline, aliquoted and heated at temperature regimen of 65, 70, 75 and 80 °C for 15 min. Samples were plated onto PLET agar. B. anthracis isolates were identified using growth morphology and PCR detecting pXO1 and pXO2 virulence plasmids. From samples heated at 75 °C for 15 min, B. anthracis were isolated from 9 of 81 (11.1%) soil samples representing five of the seven sampled areas. CONCLUSIONS: We isolated B. anthracis from soil collected from carcass burial sites. PCR targeting virulence plasmids provided a rapid confirmation of B. anthracis. SIGNIFICANCE AND IMPACT OF THE STUDY: The positive isolation indicated that some carcass burial sites may retain viable spores for at least 12 months after the previous outbreak, which suggests that they may be important sources of B. anthracis and new disease outbreaks.

K(ATP) channel openers in the trigeminovascular system.

BACKGROUND: The ATP-sensitive K(+) (K(ATP)) channel openers levcromakalim and pinacidil are vasodilators that induce headache in healthy people. The neuropeptide calcitonin gene-related peptide (CGRP) induces headache in healthy people and migraine in migraineurs, potentially through a mechanism that involves opening of vascular or neuronal K(ATP) channels and mast cell degranulation. Using rat as a model, we studied the molecular presence of K(ATP) channels in the trigeminovascular system. Furthermore, we examined whether K(ATP) channel openers stimulate the in vitro release of CGRP and whether they degranulate dural mast cells. METHODS: mRNA and protein expression of K(ATP) channel subunits were studied in the trigeminal ganglion (TG) and trigeminal nucleus caudalis (TNC) by qPCR and western blotting. In vitro CGRP release was studied after application of levcromakalim (1 µM) and diazoxide (10 µM) to freshly isolated rat dura mater, TG and TNC. Rat dural mast cells were challenged in situ with levcromakalim (10(-5) M) to study its potential degranulation effect. RESULTS: mRNA and protein of K(ATP) channel subunits Kir6.1, Kir6.2, SUR1 and SUR2B were identified in the TG and TNC. K(ATP) channel openers did not release or inhibit capsaicin-induced CGRP release from dura mater, TG or TNC. They did also not induce dural mast cell degranulation. CONCLUSIONS: K(ATP) channel openers do not interact with CGRP release or mast cell degranulation. Activation of these channels in the CNS is antinociceptive and therefore cannot explain the headache induced by K(ATP) channel openers. Thus, they are likely to induce headache by interaction with extracerebral K(ATP) channels, probably the SUR2B isoforms.

On the neurotoxicity of glutaric, 3-hydroxyglutaric, and trans-glutaconic acids in glutaric acidemia type 1.

Glutaric acidemia type 1 (GA1) is an autosomal recessively inherited deficiency of glutaryl-CoA dehydrogenase. Accumulating metabolites, 3-hydroxyglutaric (3-OH-GA), glutaric (GA), and trans-glutaconic (TG) acids, have been proposed to be involved in the development of the striatal degeneration seen in children with GA1 via an excitotoxic mechanism. We have studied the extent to which 3-OH-GA, GA, and TG are neurotoxic and whether neurotoxicity is caused by an excitotoxic mechanism in which 3-OH-GA, GA, or TG overactivates N-methyl-D-aspartate (NMDA) receptors. In cultured mouse neocortical neurons, all three compounds were weakly neurotoxic, possibly through activation of NMDA receptors. However, further studies in the rat cortical wedge preparation and with NMDA receptors expressed in Xenopus oocytes could not confirm an interaction of the compounds with NMDA receptors. It is concluded that the metabolites 3-OH-GA, GA, and TG are only weak neurotoxins and that the neurodegenerative cascade destroying the striatum in patients with GA1 involves mainly mechanisms other than excitoxicity.

Mycoplasma canis and urogenital disease in dogs in Norway.

Mycoplasmas identified as Mycoplasma canis were isolated from nine dogs with clinical signs of urogenital disease in Norway over a period of 20 months. Some of the dogs had been treated unsuccessfully with antibiotics, and three were euthanased as a result of severe persistent disease. Seven of the dogs had a urinary tract infection, one had chronic purulent epididymitis and one had chronic prostatitis. Overt haematuria was frequently observed among the dogs with cystitis. M canis was isolated in pure culture from seven of the dogs and in mixed culture from the other two. In three cases the mycoplasma was cultivated only from urinary sediment, and it was typically obtained in smaller numbers than would be considered indicative of a urinary tract infection. In contrast with most mycoplasmas, the M canis isolated from all the dogs grew on ordinary blood agar plates used for routine bacteriological cultivation. Specific mycoplasma media were not used and the presence of other Mycoplasma or Ureaplasma species cannot be excluded.

Role of GluR2 expression in AMPA-induced toxicity in cultured murine cerebral cortical neurons.

alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPA-R)-mediated neurotoxicity was studied in relation to subunit expression and the presence of Ca(2+)-permeable receptor channels. AMPA-mediated toxicity had two components: 1) a direct AMPA-R-mediated component, which was not due to Ca(2+) influx through voltage-gated Ca(2+) channels, reversal of the Na(+)/Ca(2+) exchanger or release of calcium from dantrolene-sensitive intracellular Ca(2+) stores, and 2) a minor, indirect component involving activation of NMDA receptor channels, because of glutamate release and removal of the Mg(2+) block of the NMDA receptor on AMPA-R stimulation. The involvement of Ca(2+) influx through AMPA-R was also examined. The number of neurons possessing Ca(2+)-permeable AMPA-R increased during culture development, concurrently with an increasing susceptibility for AMPA-induced toxicity during development. GluR2(R) levels also increased during development, and channel blockers of Ca(2+)-permeable AMPA-R lacking the GluR2(R) subunit (spermine and philanthotoxin) failed to prevent neurotoxicity or increases in [Ca(2+)](i). Thus, the direct AMPA-R-mediated toxicity may be explained by initiation of cell death by Ca(2+) fluxing through AMPA-R containing GluR2(R). The components of direct AMPA-R-mediated toxicity are proposed to be 1) toxicity mediated by GluR2(R)-lacking AMPA-R and 2) toxicity mediated by low-Ca(2+)-permeability AMPA-R containing GluR2(R).

Localization and pharmacological characterization of voltage dependent calcium channels in cultured neocortical neurons.

The physiological significance and subcellular distribution of voltage dependent calcium channels was defined using calcium channel blockers to inhibit potassium induced rises in cytosolic calcium concentration in cultured mouse neocortical neurons. The cytosolic calcium concentration was measured using the fluorescent calcium chelator fura-2. The types of calcium channels present at the synaptic terminal were determined by the inhibitory action of calcium channel blockers on potassium-induced [3H]GABA release in the same cell preparation. L-, N-, P-, Q- and R-/T-type voltage dependent calcium channels were differentially distributed in somata, neurites and nerve terminals. omega-conotoxin MVIIC (omega-CgTx MVIIC) inhibited approximately 40% of the Ca(2+)-rise in both somata and neurites and 60% of the potassium induced [3H]GABA release, indicating that the Q-type channel is the quantitatively most important voltage dependent calcium channel in all parts of the neuron. After treatment with thapsigargin the increase in cytosolic calcium was halved, indicating that calcium release from thapsigargin sensitive intracellular calcium stores is an important component of the potassium induced rise in cytosolic calcium concentration. The results of this investigation demonstrate that pharmacologically distinct types of voltage dependent calcium channels are differentially localized in cell bodies, neurites and nerve terminals of mouse cortical neurons but that the Q-type calcium channel appears to predominate in all compartments.

Class 1 integrons mediate antibiotic resistance in the fish pathogen Aeromonas salmonicida worldwide.

The presence of class 1 integrons was investigated in 38 sulfonamide-resistant strains of Aeromonas salmonicida subsp. salmonicida, atypical A. salmonicida and Escherichia coli conjugants with R plasmids originating from A. salmonicida. The strains originated from Finland, France, Japan, Norway, Scotland, Switzerland, and the United States. Additional resistance determinants in strains with class 1 integrons were also determined. Of 21 strains containing a class 1 integron, 19 had a single gene cassette, 1 strain had two cassettes, and 1 strain was found to lack an integrated gene cassette. In the integrons with single cassettes, aadA2 was present in eight strains, dfr16 in five strains, and aadA1 and dfrIIc in three strains each. In the integron with two cassettes, qacG and orfD were present. Tetracycline resistance was observed in 20 of the integron-positive strains, caused by the determinants Tet A and Tet E, in which Tet A frequently was associated with Tn1721. Class 1 integrons seem to be important in mediating antibiotic resistance also in the marine environment. The gene cassettes reported in this study are all described in bacteria associated with humans, and this demonstrates once more how the common gene pool is shared between organisms belonging to different environments.

Functional Tn5393-like transposon in the R plasmid pRAS2 from the fish pathogen Aeromonas salmonicida subspecies salmonicida isolated in Norway.

Tn5393c containing strA-strB was identified as part of R plasmid pRAS2 from the fish pathogen Aeromonas salmonicida subsp. salmonicida. This is the first time an intact and active transposon in the Tn5393 family has been reported in an ecological niche other than an agricultural habitat.

Effects of taurine depletion on cell migration and NCAM expression in cultures of dissociated mouse cerebellum and N2A cells.

Cultures of dissociated cerebellum from 5- to 6-day-old mice as well as of the N2A neuronal cell line were exposed to guanidino ethane sulfonate (GES, 2-5 mM) to reduce the cellular taurine content. Control cultures were kept in culture medium or medium containing 2-5 mM GES plus 2-5 mM taurine to restore the intracellular taurine content. Taurine depletion led to changes in the expression of certain splice variants of NCAM mRNA such as the AAG and the VASE containing forms, while no differences were seen in the expression of the three forms of NCAM protein. In the N2A cells taurine depletion led to a decreased migration rate of the cells. The results suggest that the reduced migration rate of neurons caused by taurine depletion may be correlated to changes in expression of certain adhesion molecules such as NCAM. Moreover, taurine appears to be involved in regulation of transcription processes.

Molecular pharmacology of the AMPA agonist, (S)-2-amino-3-(3-hydroxy-5-phenyl-4-isoxazolyl)propionic acid [(S)-APPA] and the AMPA antagonist, (R)-APPA.

The heterocyclic analogue of (S)-glutamic acid, (S)-2-amino-3-(3-hydroxy-5-methyl-4-isoxazolyl)propionic acid [(S)-AMPA] is a potent and selective AMPA receptor agonist, whereas the enantiomeric compound, (R)-AMPA, is virtually inactive. We have previously characterized (RS)-2-amino-3-(3-hydroxy-5-phenyl-4-isoxazolyl)propionic acid [(RS)-APPA] as a partial AMPA receptor agonist showing about 60% of the efficacy of (RS)-AMPA. This partial agonism produced by (RS)-APPA is, however, only apparent, since resolution of (RS)-APPA has now been shown to provide the full AMPA receptor agonist, (S)-APPA, whereas (R)-APPA is a non-N-methyl-D-aspartic acid (non-NMDA) receptor antagonist showing preferential AMPA blocking effects. In agreement with classical theories for competitive interaction between agonists and antagonists, the efficacy of depolarizations produced by (S)-APPA in the rat cortical wedge preparation was shown to be progressively reduced with increasing molar ratios of (R)-APPA/(S)-APPA. These compounds and the competitive antagonists (RS)-2-amino-3-(3-carboxymethoxy-5-methyl-4-isoxazolyl)propionic acid [(RS)-AMOA], 6-cyano-7-nitroquinoxalin-2,3-dione (CNQX) and 6-nitro-7-sulfamoylbenzo(f)quinoxalin-2,3-dione (NBQX) were also tested in [3H]AMPA and [3H]CNQX binding systems, the latter ligand being used in the absence or presence of thiocyanate ions. On the basis of these studies it is suggested that (RS)-AMPA and the AMPA agonist (S)-APPA interact with a high-affinity receptor conformation, whereas the competitive antagonists (RS)-AMOA and (R)-APPA, derived from these agonists, preferentially bind to a low-affinity AMPA receptor conformation.(ABSTRACT TRUNCATED AT 250 WORDS)

Hydroxylated analogues of 5-aminovaleric acid as 4-aminobutyric acidB receptor antagonists: stereostructure-activity relationships.

The (R) and (S) forms of 5-amino-2-hydroxyvaleric acid (2-OH-DAVA) and 5-amino-4-hydroxyvaleric acid (4-OH-DAVA) were designed as structural hybrids of the 4-aminobutyric acidB (GABAB) agonist (R)-(-)-4-amino-3-hydroxybutyric acid [(R)-(-)-3-OH-GABA] and the GABAB antagonist 5-aminovaleric acid (DAVA). (S)-(-)-2-OH-DAVA and (R)-(-)-4-OH-DAVA showed a moderately potent affinity for GABAB receptor sites in rat brain and showed GABAB antagonist effects in a guinea pig ileum preparation. The respective enantiomers, (R)-(+)-2-OH-DAVA and (S)-(+)-4-OH-DAVA, were markedly weaker in both test systems. All four compounds were weak inhibitors of GABAA receptor binding in rat brain, and none of them significantly affected synaptosomal GABA uptake. Based on molecular modeling studies it has been demonstrated that low-energy conformations of (R)-(-)-3-OH-GABA, (S)-(-)-2-OH-DAVA, and (R)-(-)-4-OH-DAVA can be superimposed. These conformations may reflect the shapes adopted by these conformationally flexible compounds during their interaction with GABAB receptors. The present studies emphasize the similar, but distinct, constraints imposed on agonists and antagonists for GABAB receptors.

Japanese tooth positions and their relation to panoramic radiography.

1. Tooth positions did not vary between ages and sexes in Japanese. 2. As the arches lengthened posteriorly, anterior tooth positions showed little variation. 3. Japanese tooth positions lie within the range shown for Caucasoid and Negroid persons. 4. Japanese tooth positions have a shorter anteroposterior dimension than their Caucasoid and Negroid counterparts. 5. The relationships of Japanese positions to the Panorex, Panelipse, and Orthopantomograph are similar to those found for Negroid and Caucasoid persons.

Relations between tooth positions and focal troughs of panoramic machines.

A comparison of the established focal troughs of three panoramic machines with positions of the teeth in the dental arches of 240 patients. The patients, divided into twelve cells, were selected on the basis of race, sex, age, number of teeth, and occlusion. Occlusal registrations were made in fast-set acrylic. Tooth centers were identified and, with the midline-incisal point as a reference point, various composite drawings of tooth positions of patients were made. The total composite of all teeth revealed the striking similarity of the arches. Superimposition on the focal troughs showed that the focal trough of the Panorex easily encompassed all tooth positions. The focal trough of the Orthopantomograph encompassed all tooth positions, although tooth centers lie on the edges of the trough. The focal trough of the GE-3000 with profile index 10 extended so far buccally that many teeth are positioned lingual to the trough. The focal trough of the GE-3000, when reduced uniformly to profile index 7.5, encompasses all tooth positions although some positions fall quite near the borders of the trough.

A study of the focal troughs of three panoramic dental x-ray machines. Part I. The area of sharpness.

A comparison of three panoramic x-ray machines revealed that the focal trough of the panorex curved inward in the posterior region, that of the GE-3000 remained straight, and that of the Orthopantomograph flared outward. The panorex was found to have the widest trough in the anterior region; the GE-3000 had the widest trough in the posterior regions.