Cigarette smoke-induced neurogenic inflammation is mediated by α,β-unsaturated aldehydes and the TRPA1 receptor in rodents

Cigarette smoke (CS) inhalation causes an early inflammatory response in rodent airways by stimulating capsaicin-sensitive sensory neurons that express transient receptor potential cation channel, subfamily V, member 1 (TRPV1) through an unknown mechanism that does not involve TRPV1. We hypothesized that 2 alpha,beta-unsaturated aldehydes present in CS, crotonaldehyde and acrolein, induce neurogenic inflammation by stimulating TRPA1, an excitatory ion channel coexpressed with TRPV1 on capsaicin-sensitive nociceptors. We found that CS aqueous extract (CSE), crotonaldehyde, and acrolein mobilized Ca2+ in cultured guinea pig jugular ganglia neurons and promoted contraction of isolated guinea pig bronchi. These responses were abolished by a TRPA1-selective antagonist and by the aldehyde scavenger glutathione but not by the TRPV1 antagonist capsazepine or by ROS scavengers. Treatment with CSE or aldehydes increased Ca2+ influx in TRPA1-transfected cells, but not in control HEK293 cells, and promoted neuropeptide release from isolated guinea pig airway tissue. Furthermore, the effect of CSE and aldehydes on Ca2+ influx in dorsal root ganglion neurons was abolished in TRPA1-deficient mice. These data identify alpha,beta-unsaturated aldehydes as the main causative agents in CS that via TRPA1 stimulation mediate airway neurogenic inflammation and suggest a role for TRPA1 in the pathogenesis of CS-induced diseases.

Neurogenic responses mediated by vanilloid receptor-1 (TRPV1) are blocked by the high affinity antagonist, iodo-resiniferatoxin

(1) Stimulation of the vanilloid receptor-1 (TRPV1) results in the activation of nociceptive and neurogenic inflammatory responses. Poor specificity and potency of TRPV1 antagonists has, however, limited the clarification of the physiological role of TRPV1. (2) Recently, iodo-resiniferatoxin (I-RTX) has been reported to bind as a high affinity antagonist at the native and heterologously expressed rat TRPV1. Here we have studied the ability of I-RTX to block a series of TRPV1 mediated nociceptive and neurogenic inflammatory responses in different species (including transfected human TRPV1). (3) We have demonstrated that I-RTX inhibited capsaicin-induced mobilization of intracellular Ca(2+) in rat trigeminal neurons (IC(50) 0.87 nM) and in HEK293 cells transfected with the human TRPV1 (IC(50) 0.071 nM). (4) Furthermore, I-RTX significantly inhibited both capsaicin-induced CGRP release from slices of rat dorsal spinal cord (IC(50) 0.27 nM) and contraction of isolated guinea-pig and rat urinary bladder (pK(B) of 10.68 and 9.63, respectively), whilst I-RTX failed to alter the response to high KCl or SP. (5) Finally, in vivo I-RTX significantly inhibited acetic acid-induced writhing in mice (ED(50) 0.42 micro mol kg(-1)) and plasma extravasation in mouse urinary bladder (ED(50) 0.41 micro mol kg(-1)). (6) In in vitro and in vivo TRPV1 activated responses I-RTX was approximately 3 log units and approximately 20 times more potent than capsazepine, respectively. This high affinity antagonist, I-RTX, may be an important tool for future studies in pain and neurogenic inflammatory models.

Effect of inclusion of lactulose and Lactobacillus plantarum on the intestinal environment and performance of piglets at weaning

The prebiotic lactulose, a probiotic strain of Lactobacillus plantarum (L. plantarum) and a synbiotic combination of these two agents were evaluated as growth promoters in 25–39-day old commercial weaning pigs. Ninety-six weaning pigs were allocated into 32 pens, taking initial weight into account, and distributed into four groups as follows: a control diet (CTR), the same diet supplemented daily with L. plantarum (109 CFU/mL sprayed on top; 20 mL/pig) (LPN); 10 g/kg lactulose (LAC) or a combination of both treatments (SYN). At day 14, eight piglets from each group were euthanized and proximal colon digesta was sampled for luminal pH, short-chain fatty acids (SCFA) and lactic acid concentrations. Deoxyribonucleic acid was extracted from colonic digesta and the microbial community was profiled by terminal restriction fragment length polymorphism analysis (T-RFLP) and qPCR. Blood urea nitrogen (BUN) and acute-phase proteins (Pig-MAP) were measured. Lactulose treatment (LAC) improved feed intake (P<0.05), average daily gain (P<0.01), feed:gain ratio (P<0.05) and reduced BUN (P<0.01). Both, LAC and LPN treatment, decreased the Enterobacteriaceae:Lactobacillus spp. ratio in the colonic luminal contents (P<0.05). Moreover LPN treatment promoted a decrease in the percentage of branched fatty acids (P<0.01) suggesting a reduction in proteolytic microbial activity. Microbial profiling of colonic luminal contents by T-RFLP revealed changes in some microbial species. Terminal restriction fragments (TRFs) compatible with Bifidobacterium thermoacidophilum were more frequently detected in experimental diets compared to CTR (P<0.05). Pigs receiving SYN diet demonstrated the combined positive effects of individual LAC and LPN treatment although we were not able to show a specific increase in the probiotic strain with the inclusion of lactulose. Collectively, these data suggest the combination of lactulose and L. plantarum acts as a complementary synbiotic, but not as a synergistic combination.

Use of pelleted sericea lespedeza (Lespedeza cuneata) for natural control of coccidia and gastrointestinal nematodes in weaned goats

Infection with Eimeria spp. (coccidia) can be devastating in goats, particularly for young, recently-weaned kids, resulting in diarrhea, dehydration, and even death. Feeding dried sericea lespedeza [SL; Lespedeza cuneata (Dum.-Cours.) G. Don.] to young goats has been reported to reduce the effects of internal parasites, including gastrointestinal nematodes (GIN) but there have been no reports of the effects of feeding this forage on Eimeria spp. in goats. Two confinement feeding experiments were completed on recently-weaned intact bucks (24 Kiko-cross, Exp. 1; 20 Spanish, Exp. 2) to determine effects of SL pellets on an established infection of GIN and coccidia. The bucks were assigned to 1 of 2 (Exp. 1) or 3 (Exp. 2) treatment groups based upon the number of Eimeria spp. oocysts per gram (OPG) of feces. In Exp. 1, the kids were fed 1 of 2 pelleted rations ad libitum; 90% SL leaf meal + 10% of a liquid molasses/lignin binder mix and a commercial pellet with 12% crude protein (CP) and 24% acid detergent fiber (n = 12/treatment group, 2 animals/pen). For Exp. 2, treatment groups were fed 1) 90% SL leaf meal pellets from leaves stored 3 years (n = 7), 2) 90% SL pellets from leaf meal stored less than 6 months, (n = 7), and the commercial pellets (n = 6) ad libitum. For both trials, fecal and blood samples were taken from individual animals every 7 days for 28 days to determine OPG and GIN eggs per gram (EPG) and packed cell volume (PCV), respectively. In Exp. 2, feces were scored for consistency (1 = solid pellets, 5 = slurry) as an indicator of coccidiosis. In Exp. 1, EPG (P < 0.001) and OPG (P < 0.01) were reduced by 78.7 and 96.9%, respectively, 7 days after initiation of feeding in goats on the SL pellet diet compared with animals fed the control pellets. The OPG and EPG remained lower in treatment than control animals until the end of the trial. In Exp. 2, goats fed new and old SL leaf meal pellets had 66.2 and 79.2% lower (P < 0.05) EPG and 92.2 and 91.2% lower (P < 0.05) OPG, respectively, than control animals within 7 days, and these differences were maintained or increased throughout the trial. After 4 weeks of pellet feeding in Exp. 2, fecal scores were lower (P < 0.01) in both SL-fed groups compared with control animals, indicating fewer signs of coccidiosis. There was no effect of diet on PCV values throughout either experiment. Dried, pelleted SL has excellent potential as a natural anti-coccidial feed for weaned goats.

Lactulose and Lactobacillus plantarum, a potential complementary synbiotic to control postweaning colibacillosis in piglets

The potential of a prebiotic oligosaccharide lactulose, a probiotic strain of Lactobacillus plantarum, or their synbiotic combination to control postweaning colibacillosis in pigs was evaluated using an enterotoxigenic Escherichia coli (ETEC) K88 oral challenge. Seventy-two weanlings were fed four diets: a control diet (CTR), that diet supplemented with L. plantarum (2 × 10(10) CFU · day(-1)) (LPN), that diet supplemented with 10 g · kg(-1) lactulose (LAC), or a combination of the two treatments (SYN). After 7 days, the pigs were orally challenged. Six pigs per treatment were euthanized on days 6 and 10 postchallenge (PC). Inclusion of lactulose improved the average daily gain (ADG) (P < 0.05) and increased lactobacilli (P < 0.05) and the percentage of butyric acid (P < 0.02) in the colon. An increase in the ileum villous height (P < 0.05) and a reduction of the pig major acute-phase protein (Pig-MAP) in serum (P < 0.01) were observed also. The inclusion of the probiotic increased numbers of L. plantarum bacteria in the ileum and colon (P < 0.05) and in the total lactobacilli in the colon and showed a trend to reduce diarrhea (P = 0.09). The concentrations of ammonia in ileal and colonic digesta were decreased (P < 0.05), and the villous height (P < 0.01) and number of ileal goblet cells (P < 0.05) increased, at day 10 PC. A decrease in plasmatic tumor necrosis factor alpha (TNF-α) (P < 0.01) was also seen. The positive effects of the two additives were combined in the SYN treatment, resulting in a complementary synbiotic with potential to be used to control postweaning colibacillosis.

Ixodes ricinus (Ixodidae), an occasional phoront on necrophagous and coprophagous beetles in Europe

For ticks, phoretic behaviour using insects associated with vertebrates might offer an alternative strategy to host-seeking. Here we report for the first time the presence of immature stages of the most widespread tick species in Western Europe, Ixodes ricinus (Acari: Ixodidae), on three beetle species belonging to families Silphidae and Geotrupidae (Coleoptera). Specimens were collected while performing fieldwork surveys on insect diversity during the peak of tick’s questing behaviour, therefore, in July and August of 2009 and 2010. The collections took place in two Natural Parks, the Aiako Harria, Guipúzcoa in Northern Spain and Wellington Country Park, Berkshire, in England. The silphid species Nicrophorus vespilloides, together with the geotrupid Trypocopris pyrenaeus were both collected from pig-carcasses and both carried nymphs of I. ricinus; while, the geotrupid Anoplotrupes stercorosus was carrying a tick larva while feeding on Red deer dung. These findings revealed an unnoticed but common relation of ticks not only with decomposed animals but also with insect scavengers. We discuss the rationale of this phenomenon.

Structure and stability of two polymorphs of creatine and its monohydrate

An experimental search for crystalline forms of creatine including a variable temperature X-ray powder diffraction study has produced three polymorphs and a formic acid solvate. The crystal structures of creatine forms I and II were determined from X-ray powder diffraction data plus the creatine formic acid (1 : 1) solvate structure was obtained by single crystal X-ray diffraction methods. Evidence of a third polymorphic form of creatine obtained by rapid desolvation of creatine monohydrate is also presented. The results highlight the role of automated parallel crystallisation, slurry experiments and VT-XRPD as powerful techniques for effective physical form screening. They also highlight the importance of various complementary analytical techniques in structural characterisation and in achieving better understanding of the relationship between various solid-state forms. The structural relationships between various solid-state forms of creatine using the XPac method provided a rationale for the different relative stabilities of forms I and II of creatine with respect to the monohydrate form.

The expression of ferritin, lactoferrin, transferrin receptor and solute carrier family 11A1 in the host response to BCG-vaccination and Mycobacterium tuberculosis challenge

Iron is an essential cofactor for both mycobacterial growth during infection and for a successful protective immune response by the host. The immune response partly depends on the regulation of iron by the host, including the tight control of expression of the iron-storage protein, ferritin. BCG vaccination can protect against disease following Mycobacterium tuberculosis infection, but the mechanisms of protection remain unclear. To further explore these mechanisms, splenocytes from BCG-vaccinated guinea pigs were stimulated ex vivo with purified protein derivative from M. tuberculosis and a significant down-regulation of ferritin light- and heavy-chain was measured by reverse-transcription quantitative-PCR (P ≤0.05 and ≤0.01, respectively). The mechanisms of this down-regulation were shown to involve TNFα and nitric oxide. A more in depth analysis of the mRNA expression profiles, including genes involved in iron metabolism, was performed using a guinea pig specific immunological microarray following ex vivo infection with M. tuberculosis of splenocytes from BCG-vaccinated and naïve guinea pigs. M. tuberculosis infection induced a pro-inflammatory response in splenocytes from both groups, resulting in down-regulation of ferritin (P ≤0.05). In addition, lactoferrin (P ≤0.002), transferrin receptor (P ≤0.05) and solute carrier family 11A1 (P ≤0.05), were only significantly down-regulated after infection of the splenocytes from BCG-vaccinated animals. The results show that expression of iron-metabolism genes is tightly regulated as part of the host response to M. tuberculosis infection and that BCG-vaccination enhances the ability of the host to mount an iron-restriction response which may in turn help to combat invasion by mycobacteria.

Nuclear accumulation of myocyte muscle LIM protein is regulated by heme oxygenase 1 and correlates with cardiac function in the transition to failure

Impaired mechanosensing leads to heart failure and we have previously shown that a decreased ratio of cytoplasmic to nuclear CSRP3/Muscle LIM protein (MLP ratio) is associated with a loss of mechanosensitivity. Here we tested whether passive or active stress/strain was important in modulating the MLP ratio and determined whether this correlated with heart function during the transition to failure. We exposed cultured neonatal rat myocytes to 10% cyclic mechanical stretch at 1 Hz, or electrically paced myocytes at 6.8 V (1 Hz) for 48 h. The MLP ratio decreased 50% (P < 0.05, n = 4) only in response to electrical pacing, suggesting impaired mechanosensitivity. Inhibition of contractility with 10 μM blebbistatin resulted in a ∼3 fold increase in the MLP ratio (n = 8, P < 0.05), indicating that myocyte contractility regulates nuclear MLP. Inhibition of histone deacetylase (HDAC) signaling with trichostatin A increased nuclear MLP following passive stretch, suggesting that HDACs block MLP nuclear accumulation. Inhibition of heme-oxygenase1 (HO-1) activity with PPZII blocked MLP nuclear accumulation. To examine how mechanosensitivity changes during the transition to heart failure, we studied a guinea pig model of angiotensin II infusion (400 ng/kg/min) over 12 weeks. Using subcellular fractionation we showed that the MLP ratio increased 88% (n = 4, P < 0.01) during compensated hypertrophy, but decreased significantly during heart failure (P < 0.001, n = 4). The MLP ratio correlated significantly with the E/A ratio (r = 0.71, P < 0.01 n = 12), a clinical measure of diastolic function. These data indicate for the first time that myocyte mechanosensitivity as indicated by the MLP ratio is regulated primarily by myocyte contractility via HO-1 and HDAC signaling.

An adhesive elastomeric supramolecular polyurethane healable at body temperature

In this paper, we report the synthesis and healing ability of a non-cytotoxic supramolecular polyurethane network whose mechanical properties can be recovered efficiently (> 99%) at the temperature of the human body (37 ºC). Rheological analysis revealed an acceleration in the drop of the storage modulus above 37 ºC, on account of the dissociation of the supramolecular polyurethane network, and this decrease in viscosity enables the efficient recovery of the mechanical properties. Microscopic and mechanical characterisation has shown that this material is able to recover mechanical properties across a damage site with minimal contact required between the interfaces and also demonstrated that the mechanical properties improved when compared to other low temperature healing elastomers or gel-like materials. The supramolecular polyurethane was found to be non-toxic in a cytotoxicity assay carried out in human skin fibroblasts (cell viability > 94% and non-significantly different compared to the untreated control). This supramolecular network material also exhibited excellent adhesion to pig skin and could be healed completely in situ post damage indicating that biomedical applications could be targeted, such as artificial skin or wound dressings with supramolecular materials of this type.