Sodium tetradecyl sulphate foam stability prior to injection: factors affecting liquid reformation.

OBJECTIVE: To study the effect of sclerosant concentration, use of 5-µm filter, use of CO(2) versus air and needle size on the stability of sodium tetradecyl sulphate (STS) foam and observe the dynamics of liquid reformation from foam within the syringe prior to injection. METHOD: Observations of liquid reformation within the syringe following foam preparation using the Tessari method. RESULT: Foam stability varies little with STS concentration between 0.5% and 3%. Needle size has little effect in this study. An in-line filter produces significantly more stable foam, and CO(2) foam is significantly less stable than air foam. Liquid reformation is predictable and does not progress at a constant rate. CONCLUSION: Regardless of the method and details of the foam produced, sclerotherapists should be aware of the dynamics and speed of foam degradation, and reconstitute foam at the first sign of liquid reformation, as this heralds the onset of rapid degradation of the foam. This is particularly relevant for rapidly deteriorating foam produced from CO(2), or without the use of a filter.

Nucleotide oligomerization domain 1 is a dominant pathway for NOS2 induction in vascular smooth muscle cells: comparison with Toll-like receptor 4 responses in macrophages.

BACKGROUND AND PURPOSE: Gram-negative bacteria contain ligands for Toll-like receptor (TLR) 4 and nucleotide oligomerization domain (NOD) 1 receptors. Lipopolysaccharide (LPS) activates TLR4, while peptidoglycan products activate NOD1. Activation of NOD1 by the specific agonist FK565 results in a profound vascular dysfunction and experimental shock in vivo. EXPERIMENTAL APPROACH: Here, we have analysed a number of pharmacological inhibitors to characterize the role of key signalling pathways in the induction of NOS2 following TLR4 or NOD1 activation. KEY RESULTS: Vascular smooth muscle (VSM) cells expressed NOD1 mRNA and protein, and, after challenge with Escherichia coli or FK565, NOS2 protein and activity were induced. Macrophages had negligible levels of NOD1 and were unaffected by FK565, but responded to E. coli and LPS by releasing increased NO and expression of NOS2 protein. Classic pharmacological inhibitors for NF-kappaB (SC-514) and mitogen-activated protein kinase (SB203580, PD98059) signalling pathways inhibited responses in both cell types regardless of agonist. While TLR4-mediated responses in macrophages were specifically inhibited by the pan-caspase inhibitor z-VAD-fmk and the PKC inhibitor Gö6976, NOD1-mediated responses in VSM cells were inhibited by the Rip2 inhibitor PP2. CONCLUSIONS AND IMPLICATIONS: Our findings suggest a selective role for NOD1 in VSM cells, and highlight NOD1 as a potential novel therapeutic target for the treatment of vascular inflammation.

FMRFamide-like peptides in root knot nematodes and their potential role in nematode physiology.

FMRFamide-like peptides (FLPs) are a diverse group of neuropeptides that are expressed abundantly in nematodes. They exert potent physiological effects on locomotory, feeding and reproductive musculature and also act as neuromodulators. However, little is known about the specific expression patterns and functions of individual peptides. The current study employed rapid amplification of cDNA ends-polymerase chain reaction (RACE-PCR) to characterize flp genes from infective juveniles of the root knot nematodes, Meloidogyne incognita and Meloidogyne minor. The peptides identified from these transcripts are sequelogs of FLPs from the free-living nematode, Caenorhabditis elegans; the genes have therefore been designated as Mi-flp-1, Mi-flp-7, Mi-flp-12, Mm-flp-12 and Mi-flp-14. Mi-flp-1 encodes five FLPs with the common C-terminal moiety, NFLRFamide. Mi-flp-7 encodes two copies of APLDRSALVRFamide and APLDRAAMVRFamide and one copy of APFDRSSMVRFamide. Mi-flp-12 and Mm-flp-12 encode the novel peptide KNNKFEFIRFamide (a longer version of RNKFEFIRFamide found in C. elegans). Mi-flp-14 encodes a single copy of KHEYLRFamide (commonly known as AF2 and regarded as the most abundant nematode FLP), and a single copy of the novel peptide KHEFVRFamide. These FLPs share a high degree of conservation between Meloidogyne species and nematodes from other clades, including those of humans and animals, perhaps suggesting a common neurophysiological role which may be exploited by novel drugs. FLP immunoreactivity was observed for the first time in Meloidogyne, in the circumpharyngeal nerve ring, pharyngeal nerves and ventral nerve cord. Additionally, in situ hybridization revealed Mi-flp-12 expression in an RIR-like neuron and Mi-flp-14 expression in SMB-like neurons, respectively. These localizations imply physiological roles for FLP-12 and FLP-14 peptides, including locomotion and sensory perception.

Cigarette smoke inhibits macrophage sensing of Gram-negative bacteria and lipopolysaccharide: relative roles of nicotine and oxidant stress.

BACKGROUND AND PURPOSE: Smoking cigarettes is a major risk factor for the development of cardiovascular and respiratory disease. Moreover, smokers are more prone to infections. This has been associated with a suppression of the immune system by smoke. However, it is not clear how cigarette smoke affects the ability of immune cells to sense pathogens. Cigarette smoke contains a large number of molecules which may mediate responses on immune cells and of these, nicotine and oxidants have both been identified as inhibitory for the sensing of bacterial lipopolysaccharide (LPS). Nitric oxide synthase (NOS) and tumour necrosis factor (TNF)-alpha are both induced in macrophages on stimulation with Gram negative bacteria or LPS. EXPERIMENTAL APPROACH: We used murine macrophages stimulated with whole heat-killed bacteria or LPS. We measured output of NO (as nitrite) and TNFalpha, NOS protein by Western blotting and cellular oxidant stress. KEY RESULTS: Cigarette smoke extract suppressed the ability of murine macrophages to release NO, but not TNFalpha in response to whole bacteria. Cigarette smoke extract also inhibited nitric oxide synthase II protein expression in response to LPS. The effects of cigarette smoke extract on nitrite formation stimulated by LPS were unaffected by inhibition of nicotinic receptors with alpha-bungarotoxin (100 units ml(-1)). However, the effects of cigarette smoke extract on LPS-induced nitrite formation were mimicked by hydrogen peroxide and reversed by the anti-oxidants N-acetyl cysteine and glutathione. CONCLUSIONS AND IMPLICATIONS: We suggest that cigarette smoke exerts its immunosuppressive effects through an oxidant-dependent and not a nicotine-dependent mechanism.

Dosimetric characterization of the irradiation cavity for accelerator-based in vivo neutron activation analysis.

A neutron irradiation cavity for in vivo activation analysis has been characterized to estimate its dosimetric specifications. The cavity is defined to confine irradiation to the hand and modifies the neutron spectrum produced by a low energy accelerator neutron source to optimize activation per dose. Neutron and gamma-ray dose rates were measured with the microdosimetric technique using a tissue-equivalent proportional counter at the hand irradiation site and inside the hand access hole. For the outside of the cavity, a spherical neutron dose equivalent meter and a Farmer dosemeter were employed instead due to the low intensity of the radiation field. The maximum dose equivalent rate at the outside of the cavity was 2.94 microSv/100 microA min, which is lower by a factor of 1/2260 than the dose rate at the hand irradiation position. The local dose contributions from a hand, an arm and the rest of a body to the effective dose rate were estimated to be 1.73, 0.782 and 2.94 microSv/100 microA min, respectively. For the standard irradiation protocol of the in vivo hand activation, 300 microA min, an effective dose of 16.3 microSv would be delivered.

Screening methods for thyroid hormone disruptors.

The U.S. Congress has passed legislation requiring the EPA to implement screening tests for identifying endocrine-disrupting chemicals. A series of workshops was sponsored by the EPA, the Chemical Manufacturers Association, and the World Wildlife Fund; one workshop focused on screens for chemicals that alter thyroid hormone function and homeostasis. Participants at this meeting identified and examined methods to detect alterations in thyroid hormone synthesis, transport, and catabolism. In addition, some methods to detect chemicals that bind to the thyroid hormone receptors acting as either agonists or antagonists were also identified. Screening methods used in mammals as well as other vertebrate classes were examined. There was a general consensus that all known chemicals which interfere with thyroid hormone function and homeostasis act by either inhibiting synthesis, altering serum transport proteins, or by increasing catabolism of thyroid hormones. There are no direct data to support the assertion that certain environmental chemicals bind and activate the thyroid hormone receptors; further research is indicated. In light of this, screening methods should reflect known mechanisms of action. Most methods examined, albeit useful for mechanistic studies, were thought to be too specific and therefore would not be applicable for broad-based screening. Determination of serum thyroid hormone concentrations following chemical exposure in rodents was thought to be a reasonable initial screen. Concurrent histologic evaluation of the thyroid would strengthen this screen. Similar methods in teleosts may be useful as screens, but would require indicators of tissue production of thyroid hormones. The use of tadpole metamorphosis as a screen may also be useful; however, this method requires validation and standardization prior to use as a broad-based screen.

The Agricultural Health Study: factors affecting completion and return of self-administered questionnaires in a large prospective cohort study of pesticide applicators.

Response rates were examined in a prospective epidemiologic study of individuals, mostly farmers, from Iowa and North Carolina seeking a pesticide applicator license during the period from 1994 through 1996. In the first year of enrollment 16,535 farmers (representing 77% of eligible farmer applicators) enrolled in the study by completing a 17-page questionnaire administered at a pesticide training session; 47% of the enrolled farmers completed and returned a much longer take-home questionnaire. The characteristics of farmers who completed only the enrollment questionnaire were quite similar to those of farmers who also completed and returned the take-home questionnaire. The most notable difference was the increased age of responders. Thus, the study population might have slightly higher cumulative farm exposures and slightly lower current farm exposures than the base population of all farmer applicators. The lack of evidence for substantial selection bias is reassuring for the Agricultural Health Study, and provides a measure of reassurance for other studies depending on the voluntary completion of self-administered questionnaires.

The Agricultural Health Study.

The Agricultural Health Study, a large prospective cohort study has been initiated in North Carolina and Iowa. The objectives of this study are to: 1) identify and quantify cancer risks among men, women, whites, and minorities associated with direct exposure to pesticides and other agricultural agents; 2) evaluate noncancer health risks including neurotoxicity reproductive effects, immunologic effects, nonmalignant respiratory disease, kidney disease, and growth and development among children; 3) evaluate disease risks among spouses and children of farmers that may arise from direct contact with pesticides and agricultural chemicals used in the home lawns and gardens, and from indirect contact, such as spray drift, laundering work clothes, or contaminated food or water; 4) assess current and past occupational and nonoccupational agricultural exposures using periodic interviews and environmental and biologic monitoring; 5) study the relationship between agricultural exposures, biomarkers of exposure, biologic effect, and genetic susceptibility factors relevant to carcinogenesis; and 6) identify and quantify cancer and other disease risks associated with lifestyle factors such as diet, cooking practices, physical activity, smoking and alcohol consumption, and hair dye use. In the first year of a 3-year enrollment period, 26,235 people have been enrolled in the study, including 19,776 registered pesticide applicators and 6,459 spouses of registered farmer applicators. It is estimated that when the total cohort is assembled in 1997 it will include approximately 75,000 adult study subjects. Farmers, the largest group of registered pesticide applicators comprise 77% of the target population enrolled in the study. This experience compares favorably with enrollment rates of previous prospective studies.

Methylenedioxyamphetamine: neurotoxic effects on serotonergic projections to brainstem nuclei in the rat.

This study employed immunocytochemistry to visualize the neurotoxic effects of methylenedioxyamphetamine hydrochloride (MDA) on serotonergic projections to brainstem structures. Separate groups of animals were injected twice a day, for 4 consecutive days, with: saline; MDA (40 mg/kg/day); or fluoxetine hydrochloride (10 mg/kg) prior to each injection of MDA. In agreement with previous reports, MDA produced a pronounced loss of 5-HT immunoreactivity in the forebrain, most notably in neocortex and hippocampus. However, our results revealed that MDA also produced a loss of 5-HT fibers in brainstem that was as severe as that seen in any region of forebrain. Regions most severely affected included: superior colliculus; superior olivary complex; trigeminal sensory complex and vestibular nuclei. The brains of animals treated with MDA demonstrated a relative absence of fine 5-HT axon terminals within these forebrain and brainstem regions, while thicker axonal elements were still present. The neurotoxic effects of MDA on serotonergic projections to forebrain and brainstem were completely blocked by the prior administration of the 5-HT reuptake inhibitor, fluoxetine. It was suggested that the denervation of the superior colliculus, superior olive and vestibular nuclei could alter visually guided eye movements and the vestibulo-ocular reflex while the loss of serotonergic inputs to the trigeminal sensory complex might be expected to alter tactual reflexes.

Developmental toxicity, reproductive toxicity, and neurotoxicity as regulatory endpoints.

For decades, cancer has been the primary toxicological endpoint used in the assessment of hazard and risk. Regulatory decisions related to the manufacture, transport, and use of a chemical are often based solely on cancer data. Federal policy is now shifting toward more frequent evaluation and application of alternative endpoints of toxicity. Among the endpoints of particular current interest are developmental toxicity, reproductive toxicity, and neurotoxicity. Significant progress has been made in the development of standardized guidelines for testing chemicals for their potential effects on these endpoints. Corresponding guidelines for the assessment of risk on the basis of data on these endpoints are in various stages of development.

Behavioral efficacy of diazepam against nerve agent exposure in rhesus monkeys.

The possibility that nerve agents will be used on the battlefield is real. The traditional therapy against nerve agent exposure consists of pyridostigmine pretreatment and atropine-pralidoxime chloride therapy administered after nerve agent exposure. This therapy regimen is extremely effective in preventing mortality in laboratory animals exposed to multilethal concentrations of nerve agent, yet these animals often display convulsions, brain damage, and behavioral incapacitation. We report here that the addition of diazepam to the traditional therapy for nerve agent (soman) exposure not only decreases the incidence of convulsions, but also attenuates the cognitive impairments of rhesus monkeys trained on a Serial Probe Recognition (SPR) task. Monkeys which received diazepam treatment required only 6 days before their performance on the SPR task returned to presoman exposure levels, compared to nondiazepam-treated monkeys which required 15 days. Moreover, only 1 out of the 5 monkeys which received diazepam treatment suffered tonic-clonic convulsions; in contrast all 5 monkeys which did not receive diazepam treatment experienced severe convulsive episodes. These results suggest that diazepam would be an excellent adjunct to traditional nerve agent therapy to facilitate behavioral recovery from nerve agent intoxication that might be encountered by US military personnel on the battlefield or accidental organophosphate poisoning encountered in industrial or agricultural accidents.

Effects of exercise on behavioral sensitivity to carbamate cholinesterase inhibitors.

The interaction between exercise and drug response has not been studied extensively. The present study examined the relationship between both acute (15 minute) and chronic (10 week) treadmill exercise and behavioral response to the carbamates physostigmine and pyridostigmine. Rats trained on an operant task under a multi-component FR30 schedule were used to evaluate the interaction between exercise and performance following drug administration. The direct effects of both 10 weeks of exercise conditioning and a moderate exercise challenge, as well as the interaction between two were assessed. Results obtained with physostigmine show that acute exercise increased behavioral sensitivity. Chronic exercise resulted in behavioral tolerance. These results are consistent with previously reported studies of centrally acting compounds. In contrast, pyridostigmine, which has little or no central activity, produced no behavioral changes. This result was constant over exercise conditions.

A study of proteins in the auditory system of rabbits using two-dimensional gels: identification of glial fibrillary acidic protein and vitamin D-dependent calcium binding protein.

Two-dimensional gel electrophoresis and computerized optical densitometry were employed to compare the relative content of proteins across major auditory brain regions in rabbits. Areas examined included the dorsal and ventral cochlear nuclei which receive the primary afferents from the organ of Corti, the lateral superior olivary nucleus which has strong reciprocal relationships with the cochlear nucleus, and the successively more rostral projections of the auditory pathways to inferior colliculus, medial geniculate and auditory cortex. Twelve proteins demonstrated significant decreases and 5 proteins significant increases in content at successively more rostral levels of the auditory system, including 2 proteins which were highly localized to the cochlear nuclei and 2 proteins greatest in amounts in the auditory cortex. One protein which was localized to the cochlear nuclei and lateral superior olive (molecular weight (MW) = 50.3, isoelectric point (pI) = 5.7) was identified as the glial fibrillary acidic protein by reaction of specific antisera on blots. Antisera to the vitamin D-dependent calcium binding protein reacted specifically with one protein (MW = 27.2, pI = 4.8) which was greatest in amount in the lateral superior olive (LSO) versus other auditory regions examined. The significance of these findings rests in the potential for identifying specific markers for cellular elements that are important in auditory function and which might be lost as a consequence of developmental abnormalities or other traumas.

Asymmetric uptake of 2-deoxy-D-[14C]glucose in the dorsal cochlear nucleus during Pavlovian conditioning in the rabbit.

Uptake of 2-deoxy-D-[14C]glucose was measured during Pavlovian conditioning of the rabbit's nictitating membrane response by both qualitative autoradiography and by quantitative measurement of radioactivity in samples of brain tissue. Conditioning was accomplished by pairing a tone stimulus delivered to both ears with an air-puff stimulus delivered to the right eye. Infusion of 2-deoxy-D-[14C]glucose during the first day of conditioning when there was no evidence of acquisition or during the 7th day of conditioning when animals demonstrated 68% conditioned responses resulted in a significantly greater uptake of radioactivity by the caudal portions of the left as compared with the right dorsal cochlear nucleus. Similar changes were not observed in other auditory and non-auditory nuclei. Rabbits that had acquired conditioned responses across 6 days of training and were exposed only to the tone-conditioned stimulus on the 7th day of testing exhibited 69% conditioned responses but no asymmetry in the uptake of 2-deoxy-D-[14C]glucose. Control animals receiving unpaired presentations of tone and air puff or no stimulation did not acquire conditioned responses and did not demonstrate asymmetric uptake of radioactivity in the dorsal cochlear nucleus. These results indicate that the asymmetric uptake of radioactivity by the dorsal cochlear nucleus did not result from the effects of stimulation per se or the prior occurrence of learning but was due to the explicit pairing of the tone stimulus with the asymmetric delivery of the air puff. It would appear that the caudal dorsal cochlear nucleus not only serves as a signal transducer for auditory stimuli but also receives inputs from other sensory systems thus allowing it to both recognize when an auditory stimulus is followed by a biologically significant event and to transmit such information to other brain regions that are, in turn, responsible for learning.

Chronic exercise produces tolerance to muscarinic antagonists in rats.

Previous work has shown that exercise can modify behavioral sensitivity to antimuscarinic compounds. The present study examined the effect of 10 weeks of endurance exercise on atropine and scopolamine potency. The behaviorally disruptive effects of these compounds were evaluated in rats trained to respond under a MULT TO FR30 schedule of reinforcement for food reward. Following 10 weeks of endurance exercise, atropine and scopolamine dose response curves were significantly altered. The ED50 values were increased 10 and 40-fold, respectively. Tolerance to atropine or scopolamine has been reported previously only in response to chronic drug administration. The present data demonstrate that non-drug factors can significantly influence behavioral response to muscarinic antagonists.

Exercise-induced changes in schedule controlled behavior.

This experiment was designed to measure the direct effect of acute exercise on performance of an operant task in rats. Treadmill exercise was manipulated along two dimensions: speed and duration. Separate groups of rats (n = 12) were tested under a multi-component time-out fixed-ratio (MULT TO FR) schedule following four exercise treatments. The first group of animals (Group A) ran at a constant speed for four different periods of time. A second group (Group B) ran for a constant period of time at four different treadmill speeds. For both groups, running took place just before operant test sessions. Operant responding on test days was compared with operant responding on the immediately preceding day. Both exercise duration and exercise speed had significant effects on operant performance.

Changes in drug sensitivity following acute and chronic exercise.

A number of factors are known to influence drug sensitivity. These include biological variables such as genetics, age, endocrine status and gender, as well as environmental variables such as operant schedules, ambient temperature and sleep deprivation. Additional factors function as either biological or environmental variables in different situations. For example, chronic drug administration can produce tolerance and cross tolerance and function as a biological variable. Acute administration of the same compound can function as an environmental variable. The present study examined exercise as both a biological and an environmental variable influencing drug sensitivity. Chronic exercise leads to relatively long term changes in physical fitness level, and functions as a biological variable. Fitness level did not influence drug sensitivity when physically conditioned animals and non-exercised control subjects were compared under rested conditions. Mild acute exercise, an environmental variable, increased sensitivity to muscarinic antagonists in the control subjects but not in the exercise trained animals. These results indicate that exercise state should be considered as an environmental variable capable of influencing drug response and that biological fitness level modifies this effect.

Sensitivity of inbred mice to methylxanthines is not determined by plasma xanthine concentration.

Male CBA/J and SWR/J mice were tested with doses of caffeine, theophylline and 8-p-sulfophenyltheophylline (xanthine). Caffeine produced dose-related decreases in locomotor activity and colonic temperatures in SWR/J mice. However, caffeine produced increases in locomotor activity and failed to lower the body temperature of CBA/J mice. Theophylline produced a decrease in body temperature of SWR/J mice. Comparison of brain caffeine levels demonstrated no difference in brain pharmacokinetics. The peripherally active xanthine failed to alter body temperature at the same molar dose as that of theophylline. These data clearly demonstrate that genetic differences in the effects of methylxanthine are due to inherent differences in the central nervous system sensitivity of the two strains. The data further indicate that while differences in xanthine metabolism may occur in inbred mice, these differences are not a major factor in the acute, peak plasma level, effects of xanthines.

Comparative behavioral effects of CNS cholinesterase inhibitors.

Phenylmethanesulfonyl fluoride, methanesulfonyl fluoride, and physostigmine were compared on the efficacy with which each could suppress methylphenidate-induced stereotyped gnawing, an extrapyramidal motor behavior. Whereas physostigmine produced powerful suppression of the stereotypy, the sulfonyl fluorides did not produce any clear behavioral effect. Biochemical experiments conducted with the behavioral tests demonstrated that the sulfonyl fluorides produced inhibition of whole brain, caudate, cortex, cerebellum, hippocampus and brain stem cholinesterate equal to that produced by physostigmine. The reason for the marked discrepancy between the behavioral effect of physostigmine and the sulfonyl fluorides is unknown. It is, however, clear that the effect of the various drugs on extrapyramidal motor behaviors is not a simple function of the degree to which each inhibited CNS cholinesterase.