Exploring the diversity and evolutionary strategies of prophages in Hyphomicrobiales, comparing animal-associated with non-animal-associated bacteria.

The Hyphomicrobiales bacterial order (previously Rhizobiales) exhibits a wide range of lifestyle characteristics, including free-living, plant-association, nitrogen-fixing, and association with animals (Bartonella and Brucella). This study explores the diversity and evolutionary strategies of bacteriophages within the Hyphomicrobiales order, comparing animal-associated (AAB) with non-animal-associated bacteria (NAAB). We curated 560 high-quality complete genomes of 58 genera from this order and used the PHASTER server for prophage annotation and classification. For 19 genera with representative genomes, we curated 96 genomes and used the Defense-Finder server to summarize the type of anti-phage systems (APS) found in this order. We analyzed the genetic repertoire and length distributions of prophages, estimating evolutionary rates and comparing intact, questionable, and incomplete prophages in both groups. Analyses of best-fit parameters and bootstrap sensitivity were used to understand the evolutionary processes driving prophage gene content. A total of 1860 prophages distributed in Hyphomicrobiales were found, 695 in AAB and 1165 in the NAAB genera. The results revealed a similar number of prophages per genome in AAB and NAAB and a similar length distribution, suggesting shared mechanisms of genetic acquisition of prophage genes. Changes in the frequency of specific gene classes were observed between incomplete and intact prophages, indicating preferential loss or enrichment in both groups. The analysis of best-fit parameters and bootstrap sensitivity tests indicated a higher selection coefficient, induction rate, and turnover in NAAB genomes. We found 68 types of APS in Hyphomicrobiales; restriction modification (RM) and abortive infection (Abi) were the most frequent APS found for all Hyphomicrobiales, and within the AAB group. This classification of APS showed that NAAB genomes have a greater diversity of defense systems compared to AAB, which could be related to the higher rates of prophage induction and turnover in the latter group. Our study provides insights into the distributions of both prophages and APS in Hyphomicrobiales genomes, demonstrating that NAAB carry more defense systems against phages, while AAB show increased prophage stability and an increased number of incomplete prophages. These results suggest a greater role for domesticated prophages within animal-associated bacteria in Hyphomicrobiales.

Enhancement of allyl isothiocyanate-evoked responses of mouse trigeminal ganglion cells by the kokumi substance γ-glutamyl-valyl-glycine (γ-EVG) through activation of the calcium-sensing receptor (CaSR).

Some γ-glutamyl peptides including glutathione (γ-Glu-Cys-Gly) and γ-glutamyl-valyl-glycine (γ-Glu-Val-Gly= γ-EVG) are reported to increase the intensity of basic tastes, such as salty, sweet, and umami, although they have no taste themselves at tested concentrations. The mechanism of action of γ-glutamyl peptides is not clearly understood, but the calcium sensing receptor (CaSR) may be involved. Glutathione and γ-EVG enhance the pungency of some spices, and the present study investigated the effects of γ-EVG on the responses of trigeminal ganglion (TG) cells to thermosensitiveTRP channel agonists. Single-cell RT-PCR revealed that most CaSR-expressing cells co-expressed TRPV1 (sensitive to capsaicin) and TRPA1 (sensitive to allyl isothiocyanate= AITC). Intracellular Ca2+ imaging showed that pretreatment with γ-EVG excited 7% of trigeminal ganglion (TG) cells and increased the amplitude of their responses to AITC, but not to capsaicin or menthol. The enhancing effect of γ-EVG was prevented by a CaSR inhibitor. The results indicate that γ-EVG increases AITC pungency by activating a subset of trigeminal ganglion cells that co-express CaSR and TRPA1.

Sensory Effects of Nicotine and Tobacco.

INTRODUCTION: Ingestion of nicotine by smoking, vaping, or other means elicits various effects including reward, antinociception, and aversion due to irritation, bitter taste, and unpleasant side effects such as nausea and dizziness. AIMS AND METHODS: Here we review the sensory effects of nicotine and the underlying neurobiological processes. RESULTS AND CONCLUSIONS: Nicotine elicits oral irritation and pain via the activation of neuronal nicotinic acetylcholine receptors (nAChRs) expressed by trigeminal nociceptors. These nociceptors excite neurons in the trigeminal subnucleus caudalis (Vc) and other brainstem regions in a manner that is significantly reduced by the nAChR antagonist mecamylamine. Vc neurons are excited by lingual application of nicotine and exhibit a progressive decline in firing to subsequent applications, consistent with desensitization of peripheral sensory neurons and progressively declining ratings of oral irritation in human psychophysical experiments. Nicotine also elicits a nAChR-mediated bitter taste via excitation of gustatory afferents. Nicotine solutions are avoided even when sweeteners are added. Studies employing oral self-administration have yielded mixed results: Some studies show avoidance of nicotine while others report increased nicotine intake over time, particularly in adolescents and females. Nicotine is consistently reported to increase human pain threshold and tolerance levels. In animal studies, nicotine is antinociceptive when delivered by inhalation of tobacco smoke or systemic infusion, intrathecally, and by intracranial microinjection in the pedunculopontine tegmentum, ventrolateral periaqueductal gray, and rostral ventromedial medulla. The antinociception is thought to be mediated by descending inhibition of spinal nociceptive transmission. Menthol cross-desensitizes nicotine-evoked oral irritation, reducing harshness that may account for its popularity as a flavor additive to tobacco products. IMPLICATIONS: Nicotine activates brain systems underlying reward and antinociception, but at the same time elicits aversive sensory effects including oral irritation and pain, bitter taste, and other unpleasant side effects mediated largely by nicotinic acetylcholine receptors (nAChRs). This review discusses the competing aversive and antinociceptive effects of nicotine and exposure to tobacco smoke, and the underlying neurobiology. An improved understanding of the interacting effects of nicotine will hopefully inform novel approaches to mitigate nicotine and tobacco use.

Effects of pruritogens and algogens on rostral ventromedial medullary ON and OFF cells.

Rostroventromedial medulla (RVM) ON and OFF cells are thought to facilitate and inhibit spinal nociceptive transmission, respectively. However, it is unknown how ON and OFF cells respond to pruritic stimuli or how they contribute to descending modulation of spinal itch signaling. In pentobarbital sodium-anesthetized mice, single-unit recordings were made in RVM from ON and OFF cells identified by their respective increase or decrease in firing that occurred just before nocifensive hindlimb withdrawal elicited by paw pinch. Of RVM ON cells, 75% (21/28) were excited by intradermal histamine, 50% (10/20) by intradermal chloroquine, and 75% (27/36) by intradermal capsaicin. Most chemically responsive units also responded to a scratch stimulus applied to the injected hindpaw. Few ON cells responded to intradermal injection of vehicle (saline: 5/32; Tween 2/17) but still responded to scratching. For OFF cells, intradermal histamine and scratching inhibited 32% (6/19) with no effect of histamine in the remainder. Intradermal chloroquine inhibited 44% (4/9) and intradermal capsaicin inhibited 61% (11/18) of OFF cells. Few OFF cells were affected by vehicles (Tween: 1 inhibited, 7 unaffected; saline: 3 excited, 1 inhibited, 8 unaffected). Both ON and OFF cells that responded to one chemical usually also responded to others, whereas units unresponsive to the first-tested chemical tended not to respond to others. These results indicate that ascending pruriceptive signals activate RVM ON cells and inhibit RVM OFF cells. These effects are considered to facilitate and disinhibit spinal pain transmission, respectively. It is currently not clear if spinal itch transmission is similarly modulated. NEW & NOTEWORTHY The rostroventromedial medulla (RVM) contains ON and OFF cells that are, respectively, excited and inhibited by noxious stimuli and have descending projections that facilitate and inhibit spinal nociceptive transmission. Most RVM ON cells were excited, and OFF cells inhibited, by intradermal injection of the pruritogens histamine and chloroquine, as well as the algogen capsaicin. These results indicate that itchy stimuli activate RVM neurons that presumably give rise to descending modulation of spinal itch transmission.

Innocuous warming enhances peripheral serotonergic itch signaling and evokes enhanced responses in serotonin-responsive dorsal horn neurons in the mouse.

Itch is often triggered by warming the skin in patients with itchy dermatitis, but the underlying mechanism is largely unknown. We presently investigated if warming the skin enhances histamine- or serotonin (5-HT)-evoked itch behavior or responses of sensory dorsal root ganglion (DRG) cells, and if responses of superficial dorsal horn neurons to innocuous warming are enhanced by these pruritogens. In a temperature-controlled environmental chamber, mice exhibited greater scratching following intradermal injection of 5-HT, but not histamine, SLIGRL, or BAM8-22, when the skin surface temperature was above 36°C. Calcium imaging of DRG cells in a temperature-controlled bath revealed that responses to 5-HT, but not histamine, were significantly greater at a bath temperature of 35°C vs. lower temperatures. Single-unit recordings revealed a subpopulation of superficial dorsal horn neurons responsive to intradermal injection of 5-HT. Of these, 58% responded to innocuous skin warming (37°C) prior to intradermal injection of 5-HT, while 100% responded to warming following intradermal injection of 5-HT. Warming-evoked responses were superimposed on the 5-HT-evoked elevation in firing and were significantly larger compared with responses pre-5-HT, as long as 30 min after the intradermal injection of 5-HT. Five-HT-insensitive units, and units that either did or did not respond to intradermal histamine, did not exhibit any increase in the incidence of warmth sensitivity or in the mean response to warming following intradermal injection of the pruritogen. The results suggest that 5-HT-evoked responses of pruriceptors are enhanced during skin warming, leading to increased firing of 5-HT-sensitive dorsal horn neurons that signal nonhistaminergic itch. NEW & NOTEWORTHY: Skin warming often exacerbates itch in patients with itchy dermatitis. We demonstrate that warming the skin enhanced serotonin-evoked, but not histamine-evoked, itch behavior and responses of sensory dorsal root ganglion cells. Moreover, serotonin, but not histamine, enhanced responses of superficial dorsal horn neurons to innocuous warming. The results suggest that skin warming selectively enhances the responses of serotonin-sensitive pruriceptors, leading to increased firing of serotonin-sensitive dorsal horn neurons that signal nonhistaminergic itch.

Thermosensitive transient receptor potential (TRP) channel agonists and their role in mechanical, thermal and nociceptive sensations as assessed using animal models.

INTRODUCTION: The present paper summarizes research using animal models to investigate the roles of thermosensitive transient receptor potential (TRP) channels in somatosensory functions including touch, temperature and pain. We present new data assessing the effects of eugenol and carvacrol, agonists of the warmth-sensitive TRPV3, on thermal, mechanical and pain sensitivity in rats. METHODS: Thermal sensitivity was assessed using a thermal preference test, which measured the amount of time the animal occupied one of two adjacent thermoelectric plates set at different temperatures. Pain sensitivity was assessed as an increase in latency of hindpaw withdrawal away from a noxious thermal stimulus directed to the plantar hindpaw (Hargreaves test). Mechanical sensitivity was assessed by measuring the force exerted by an electronic von Frey filament pressed against the plantar surface that elicited withdrawal. RESULTS: Topical application of eugenol and carvacrol did not significantly affect thermal preference, although there was a trend toward avoidance of the hotter surface in a 30 vs. 45°C preference test for rats treated with 1 or 10% eugenol and carvacrol. Both eugenol and carvacrol induced a concentration-dependent increase in thermal withdrawal latency (analgesia), with no significant effect on mechanosensitivity. CONCLUSIONS: The analgesic effect of eugenol and carvacrol is consistent with previous studies. The tendency for these chemicals to increase the avoidance of warmer temperatures suggests a possible role for TRPV3 in warmth detection, also consistent with previous studies. Additional roles of other thermosensitive TRP channels (TRPM8 TRPV1, TRPV2, TRPV4, TRPM3, TRPM8, TRPA1, TRPC5) in touch, temperature and pain are reviewed.

Intradermal endothelin-1 excites bombesin-responsive superficial dorsal horn neurons in the mouse.

Endothelin-1 (ET-1) has been implicated in nonhistaminergic itch. Here we used electrophysiological methods to investigate whether mouse superficial dorsal horn neurons respond to intradermal (id) injection of ET-1 and whether ET-1-sensitive neurons additionally respond to other pruritic and algesic stimuli or spinal superfusion of bombesin, a homolog of gastrin-releasing peptide (GRP) that excites spinal itch-signaling neurons. Single-unit recordings were made from lumbar dorsal horn neurons in pentobarbital-anesthetized C57BL/6 mice. We searched for units that exhibited elevated firing after id injection of ET-1 (1 µg/µl). Responsive units were further tested with mechanical stimuli, bombesin (spinal superfusion, 200 µg·ml(-1)·min(-1)), heating, cooling, and additional chemicals [histamine, chloroquine, allyl isothiocyanate (AITC), capsaicin]. Of 40 ET-1-responsive units, 48% responded to brush and pinch [wide dynamic range (WDR)] and 52% to pinch only [high threshold (HT)]. Ninety-three percent responded to noxious heat, 50% to cooling, and >70% to histamine, chloroquine, AITC, and capsaicin. Fifty-seven percent responded to bombesin, suggesting that they participate in spinal itch transmission. That most ET-1-sensitive spinal neurons also responded to pruritic and algesic stimuli is consistent with previous studies of pruritogen-responsive dorsal horn neurons. We previously hypothesized that pruritogen-sensitive neurons signal itch. The observation that ET-1 activates nociceptive neurons suggests that both itch and pain signals may be generated by ET-1 to result in simultaneous sensations of itch and pain, consistent with observations that ET-1 elicits both itch- and pain-related behaviors in animals and burning itch sensations in humans.

Efficiency of a closed-coupled solar pasteurization system in treating roof harvested rainwater.

Many studies have concluded that roof harvested rainwater is susceptible to chemical and microbial contamination. The aim of the study was thus to conduct a preliminary investigation into the efficiency of a closed-coupled solar pasteurization system in reducing the microbiological load in harvested rainwater and to determine the change in chemical components after pasteurization. The temperature of the pasteurized tank water samples collected ranged from 55 to 57°C, 64 to 66°C, 72 to 74°C, 78 to 81°C and 90 to 91°C. Cations analyzed were within drinking water guidelines, with the exception of iron [195.59 µg/L (55°C)-170.1 µg/L (91°C)], aluminum [130.98 µg/L (78°C)], lead [12.81 µg/L (55°C)-13.2 µg/L (91°C)] and nickel [46.43 µg/L (55°C)-32.82 µg/L (78°C)], which were detected at levels above the respective guidelines in the pasteurized tank water samples. Indicator bacteria including, heterotrophic bacteria, Escherichia coli and total coliforms were reduced to below the detection limit at pasteurization temperatures of 72°C and above. However, with the use of molecular techniques Yersinia spp., Legionella spp. and Pseudomonas spp. were detected in tank water samples pasteurized at temperatures greater than 72°C. The viability of the bacteria detected in this study at the higher temperature ranges should thus be assessed before pasteurized harvested rainwater is used as a potable water source. In addition, it is recommended that the storage tank of the pasteurization system be constructed from an alternative material, other than stainless steel, in order for a closed-coupled pasteurization system to be implemented and produce large quantities of potable water from roof harvested rainwater.

Whey or Casein Hydrolysate with Carbohydrate for Metabolism and Performance in Cycling.

The protein type most suitable for ingestion during endurance exercise is undefined. This study compared co-ingestion of either 15 g/h whey or casein hydrolysate with 63 g/h fructose: maltodextrin (0.8:1) on exogenous carbohydrate oxidation, exercise metabolism and performance. 2 h postprandial, 8 male cyclists ingested either: carbohydrate-only, carbohydrate-whey hydrolysate, carbohydrate-casein hydrolysate or placebo-water in a crossover, double-blind design during 2 h of exercise at 60%W max followed by a 16-km time trial. Data were evaluated by magnitude-based inferential statistics. Exogenous carbohydrate oxidation, measured from (13)CO2 breath enrichment, was not substantially influenced by co-ingestion of either protein hydrolysate. However, only co-ingestion of carbohydrate-casein hydrolysate substantially decreased (98% very likely decrease) total carbohydrate oxidation (mean±SD, 242±44; 258±47; 277±33 g for carbohydrate-casein, carbohydrate-whey and carbohydrate-only, respectively) and substantially increased (93% likely increase) total fat oxidation (92±14; 83±27; 73±19 g) compared with carbohydrate-only. Furthermore, only carbohydrate-casein hydrolysate ingestion resulted in a faster time trial (-3.6%; 90% CI: ±3.2%) compared with placebo-water (95% likely benefit). However, neither protein hydrolysate enhanced time trial performance when compared with carbohydrate-only. Under the conditions of this study, ingesting carbohydrate-casein, but not carbohydrate-whey hydrolysate, favourably alters metabolism during prolonged moderate-strenuous cycling without substantially altering cycling performance compared with carbohydrate-only.

Eugenol and carvacrol excite first- and second-order trigeminal neurons and enhance their heat-evoked responses.

Eugenol and carvacrol from clove and oregano, respectively, are agonists of the warmth-sensitive transient receptor potential channel TRPV3 and the irritant-sensitive transient receptor potential ankyrin (TRPA)-1. Eugenol and carvacrol induce oral irritation that rapidly desensitizes, accompanied by brief enhancement of innocuous warmth and heat pain in humans. We presently investigated if eugenol and carvacrol activate nociceptive primary afferent and higher order trigeminal neurons and enhance their heat-evoked responses, using calcium imaging of cultured trigeminal ganglion (TG) and dorsal root ganglion (DRG) neurons, and in vivo single-unit recordings in trigeminal subnucleus caudalis (Vc) of rats. Eugenol and carvacrol activated 20-30% of TG and 7-20% of DRG cells, the majority of which additionally responded to menthol, mustard oil and/or capsaicin. TG cell responses to innocuous (39°) and noxious (42 °C) heating were enhanced by eugenol and carvacrol. We identified dorsomedial Vc neurons responsive to noxious heating of the tongue in pentobarbital-anesthetized rats. Eugenol and carvacrol dose-dependently elicited desensitizing responses in 55% and 73% of heat-sensitive units, respectively. Responses to noxious heat were briefly enhanced by eugenol and carvacrol. Many eugenol- and carvacrol-responsive units also responded to menthol, cinnamaldehyde and capsaicin. These data support a peripheral site for eugenol and carvacrol to enhance warmth- and noxious heat-evoked responses of trigeminal neurons, and are consistent with the observation that these agonists briefly enhance warmth and heat pain on the human tongue.

Behavioral model of itch, alloknesis, pain and allodynia in the lower hindlimb and correlative responses of lumbar dorsal horn neurons in the mouse.

We have further developed a behavioral model of itch and pain in the lower hindlimb (calf) originally reported by LaMotte et al. (2011) that allows comparisons with responses of lumbar dorsal horn neurons to pruritic and noxious stimuli. Intradermal (id) microinjection of the pruritogens histamine, SLIGRL-NH2 (agonist of PAR-2 and MrgprC11) and chloroquine (agonist of MrgprA3) into the calf of the lower limb elicited significant biting and a small amount of licking directed to the injection site, over a 30-min time course. Following id injection of histamine, low-threshold mechanical stimuli reliably elicited discrete episodes of biting (alloknesis) over a longer time course; significantly less alloknesis was observed following id injection of SLIGRL-NH2. Capsaicin injections elicited licking but little biting. Following id injection of capsaicin, low-threshold mechanical stimuli elicited discrete hindlimb flinches (allodynia) over a prolonged (>2h) time course. In single-unit recordings from superficial lumbar dorsal horn neurons, low-threshold mechanically evoked responses were significantly enhanced, accompanied by receptive field expansion, following id injection of histamine in histamine-responsive neurons. This was not observed in histamine-insensitive neurons, or following id injection of saline or SLIGRL-NH2, regardless of whether the latter activated the neuron or not. These results suggest that itch-responsive neurons are selectively sensitized by histamine but not SLIGRL-NH2 to account for alloknesis. The presently described "calf" model appears to distinguish between itch- and pain-related behavioral responses, and provides a basis to investigate lumbar spinal neural mechanisms underlying itch, alloknesis, pain and allodynia.

Scratching inhibits serotonin-evoked responses of rat dorsal horn neurons in a site- and state-dependent manner.

Scratching inhibits pruritogen-evoked responses of neurons in the superficial dorsal horn, implicating a spinal site for scratch inhibition of itch. We investigated if scratching differentially affects neurons depending on whether they are activated by itchy vs. painful stimuli, and if the degree of inhibition depends on the relative location of scratching. We recorded from rat lumbar dorsal horn neurons responsive to intradermal (id) microinjection of serotonin (5-hydroxytryptamine, 5-HT). During the response to 5-HT, scratch stimuli (3mm, 300 mN, 2 Hz, 20s) were delivered at the injection site within the mechanosensitive receptive field (on-site), or 4-30 mm away, outside of the receptive field (off-site). During off-site scratching, 5-HT-evoked firing was significantly attenuated followed by recovery. On-site scratching excited neurons, followed by a significant post-scratch decrease in 5-HT-evoked firing. Most neurons additionally responded to mustard oil (allyl isothiocyanate). Off-site scratching had no effect, while on-site scratching excited the neurons. These results indicate that scratching exerts a state-dependent inhibitory effect on responses of spinal neurons to pruritic but not algesic stimuli. Moreover, on-site scratching first excited neurons followed by inhibition, while off-site scratching immediately evoked the inhibition of pruritogen-evoked activity. This accounts for the suppression of itch by scratching at a distance from the site of the itchy stimulus.

Functional polymorphisms in the P2X7 receptor gene are associated with osteoporosis.

UNLABELLED: The P2X(7) receptor is an ATP-gated cation channel. We investigated the effect of both loss-of-function and gain-of-function polymorphisms in the P2X(7) receptor gene on BMD and risk of vertebral fractures and found that five polymorphisms and haplotypes containing three of these polymorphisms were associated with BMD and fracture risk. INTRODUCTION: The P2X(7) receptor is an ATP-gated cation channel. P2X(7) receptor knockout mice have reduced total bone mineral content, and because several functional polymorphisms have been identified in the human P2X(7) receptor gene, we wanted to investigate the effect of these polymorphisms on BMD and risk of vertebral fractures in a case-control study including 798 individuals. METHODS: Genotyping was carried out using TaqMan assays. BMD was measured using dual energy X-ray absorptiometry, and vertebral fractures were assessed by lateral spinal X-rays. RESULTS: The rare allele of a splice site polymorphism, 151 + 1: G-T, was associated with increased fracture risk and reduced BMD in women. Two other loss-of-function polymorphisms, Glu496Ala and Gly150Arg, were also associated with BMD. The Glu496Ala variant allele was associated with decreased lumbar spine BMD in women and decreased total hip BMD in men. The 150Arg allele was associated with decreased total hip BMD in women and men combined. The minor allele of the gain-of-function polymorphism, Ala348Thr, was associated with reduced fracture risk and increased BMD at all sites in men. The Gln460Arg variant allele, which has been associated with increased receptor function in monocytes, was associated with increased total hip BMD in women. With the exception of His155Tyr for which we found conflicting results in men and women, our results are consistent with the phenotype of the knockout mouse. Analysis of a haplotype containing Ala348Thr, Gln460Arg, and Glu496Ala showed that the effects of the haplotypes on BMD and fracture were driven by Ala348Thr in men and by Gln460Arg and Glu496Ala in women. CONCLUSION: In conclusion, we found that functional polymorphisms in the P2X(7) receptor gene and haplotypes containing three of these polymorphisms are associated with osteoporosis.

Optimization of encapsulation of a synthetic long peptide in PLGA nanoparticles: low-burst release is crucial for efficient CD8(+) T cell activation.

Overlapping synthetic long peptides (SLPs) hold great promise for immunotherapy of cancer. Poly(lactic-co-glycolic acid) (PLGA) nanoparticles (NPs) are being developed as delivery systems to improve the potency of peptide-based therapeutic cancer vaccines. Our aim was to optimize PLGA NP for SLP delivery with respect to encapsulation and release, using OVA24, a 24-residue long synthetic antigenic peptide covering a CTL epitope of ovalbumin (SIINFEKL), as a model antigen. Peptide-loaded PLGA NPs were prepared by a double emulsion/solvent evaporation technique. Using standard conditions (acidic inner aqueous phase), we observed that either encapsulation was very low (1-30%), or burst release extremely high (>70%) upon resuspension of NP in physiological buffers. By adjusting formulation and process parameters, we uncovered that the pH of the first emulsion was critical to efficient encapsulation and controlled release. In particular, an alkaline inner aqueous phase resulted in circa 330 nm sized NP with approximately 40% encapsulation efficiency and low (<10%) burst release. These NP showed enhanced MHC class I restricted T cell activation in vitro when compared to high-burst releasing NP and soluble OVA24, proving that efficient entrapment of the antigen is crucial to induce a potent cellular immune response.

Cross-sensitization of histamine-independent itch in mouse primary sensory neurons.

Overexpression of pruritogens and their precursors may contribute to the sensitization of histamine-dependent and -independent itch-signaling pathways in chronic itch. We presently investigated self- and cross-sensitization of scratching behavior elicited by various pruritogens, and their effects on primary sensory neurons. The MrgprC11 agonist BAM8-22 exhibited self- and reciprocal cross-sensitization of scratching evoked by the protease-activated receptor-2 (PAR-2) agonist SLIGRL. The MrgprA3 agonist chloroquine unidirectionally cross-sensitized BAM8-22-evoked scratching. Histamine unidirectionally cross-sensitized scratching evoked by chloroquine and BAM8-22. SLIGRL unidirectionally cross-sensitized scratching evoked by chloroquine. Dorsal root ganglion (DRG) cells responded to various combinations of pruritogens and algogens. Neither chloroquine, BAM8-22 nor histamine had any effect on responses of DRG cell responses to subsequently applied pruritogens, implying that their behavioral self- and cross-sensitization effects are mediated indirectly. SLIGRL unilaterally cross-sensitized responses of DRG cells to chloroquine and BAM8-22, consistent with the behavioral data. These results indicate that unidirectional cross-sensitization of histamine-independent itch-signaling pathways might occur at a peripheral site through PAR-2. PAR-2 expressed in pruriceptive nerve endings is a potential target to reduce sensitization associated with chronic itch.

Topical hindpaw application of L-menthol decreases responsiveness to heat with biphasic effects on cold sensitivity of rat lumbar dorsal horn neurons.

Menthol is used in pharmaceutical applications because of its desired cooling and analgesic properties. The neural mechanism by which topical application of menthol decreases heat pain is not fully understood. We investigated the effects of topical menthol application on lumbar dorsal horn wide dynamic range and nociceptive-specific neuronal responses to noxious heat and cooling of glabrous hindpaw cutaneous receptive fields. Menthol increased thresholds for responses to noxious heat in a concentration-dependent manner. Menthol had a biphasic effect on cold-evoked responses, reducing the threshold (to warmer temperatures) at a low (1%) concentration and increasing threshold and reducing response magnitude at high (10%, 40%) concentrations. Menthol had little effect on responses to innocuous or noxious mechanical stimuli, ruling out a local anesthetic action. Application of 40% menthol to the contralateral hindpaw tended to reduce responses to cooling and noxious heat, suggesting a weak heterosegmental inhibitory effect. These results indicate that menthol has an analgesic effect on heat sensitivity of nociceptive dorsal horn neurons, as well as biphasic effects on cold sensitivity, consistent with previous behavioral observations.

[The relevance of the sagittal profile in cervical artificial discs]. / Die Bedeutung des sagittalen Profils bei der zervikalen Endoprothetik.

We report on the results of 246 Bryan cervical discs, which were implanted between June 2002 and September 2010 in 146 patients. Of the patients 74 (128 prostheses) could be followed up for more than 1 year and the average follow-up period was 2.6 years. Of the patients 18 were operated on at one level (group 1), 77 prostheses were multilevel surgery (group 2) and with 33 patients arthroplasty was combined with fusion (hybrid, group 3). The global lordosis remained unchanged during follow-up and a recurrence of kyphosis was evident in group 3. The overall mobility improved in all 3 subgroups and 2 cases (group 3) fused. With 5 patients the prosthesis had to be removed and the segment had to be fused in the postoperative course. As a conclusion a meticulous preoperative planning as well as a subtle surgical technique is the main prerequisite for long-lasting mobility of the Bryan prosthesis.

The expression and regulation of bone-acting cytokines in human peripheral adipose tissue in organ culture.

The humoral cross-talk between bone and fat is an area of increasing interest. We investigated the expression and regulation of the bone-acting cytokines; bone morphogenetic protein 2 (BMP2), connective tissue growth factor (CTGF), osteoprotegerin (OPG), and transforming growth factor beta (TGFB1). Subcutaneous adipose tissue was aspirated from lean, healthy women. Tissue samples were incubated with interleukin 1-ß (IL1-ß), tumor necrosis factor-α (TNF-α), cortisol, troglitazone, IL1-ß + troglitazone, or vehicle. Gene expression in the adipose tissue was analyzed using qPCR and protein levels in the incubation media were analyzed using ELISA. OPG expression and secretion was diminished by 40.8% and 43.1% respectively, by cortisol, and OPG expression was diminished by 67.5% by troglitazone (p<0.05). The proinflammatory cytokines IL1-ß and TNF-α significantly increased the expression of CTGF (p<0.05) by 65.1% and 101.3%, respectively, and the expression and secretion of OGP by 62.3-165.8% (p<0.05). This interleukin 1-ß mediated increase in CTGF- and OPG expression and secretion was ameliorated by troglitazone. Troglitazone and related drugs are known to have adverse effects on bone. We suggest that this could be mediated via altered cytokine production in adipose tissue. Moreover, obese individuals have a low-grade inflammation in their adipose tissue and have higher bone mineral density than lean individuals. We suggest that this inflammation may increase the expression and secretion of OPG and CTGF and thereby increase BMD. In conclusion, bone acting cytokines are produced in the adipose tissue and may affect bone through endocrine mechanisms.

Polymorphisms of the peroxisome proliferator-activated receptor γ (PPARγ) gene are associated with osteoporosis.

UNLABELLED: Stimulation of PPARγ turns mesenchymal stem cells into adipocytes instead of osteoblasts. We investigated the effect of polymorphisms in the PPARγ gene on BMD and fracture risk in two Danish cohorts and found opposing effects of certain SNPs and haplotypes in the two cohorts probably owing to environmental factors. INTRODUCTION: Stimulation of PPARγ causes development of mesenchymal stem cells to adipocytes instead of osteoblasts leading to decreased osteoblast number and BMD. The aim of this study was to examine the effect of PPARG polymorphisms on BMD and fracture risk in two Danish cohorts: AROS, a case-control population comprising 809 individuals and DOPS, a population comprising 1,716 perimenopausal women allocated to hormone therapy or not at baseline and followed for 10 years. On the basis of linkage disequilibrium between SNPs throughout the gene and previous studies we chose 10 polymorphisms for investigation. METHODS: In AROS, individuals heterozygous for the polymorphisms rs12497191, rs4135263, and rs1151999 had an increased risk of vertebral fractures (OR = 1.48-1.76, p = 0.005-0.04) compared with individuals homozygous for the common allele. In DOPS, individuals heterozygous for rs1151999 had an increased BMD at the hip sites (p ≤ 0.02). An interaction between rs1151999 and diet was found on BMD in both cohorts. RESULTS: For the polymorphism rs1152003 there was an interaction with body weight on BMD at all sites in both cohorts (p ≤ 0.07). Stratified analyses revealed that in the high weight group in AROS individuals homozygous for the variant allele had a decreased BMD (p ≤ 0.02), whereas the same pattern was found in the low weight group in DOPS (p ≤ 0.03). A number of haplotype associations were found as well, the direction of which was opposite in the two cohorts. CONCLUSION: Our study suggests an association SNPs in PPARG and haplotypes thereof and BMD and fracture risk. The effect however appears to be modifiable by environmental factors.

Polymorphisms in the ALOX12 gene and osteoporosis.

UNLABELLED: ALOX12 produces ligands for PPARγ thereby turning mesenchymal stem cells into adipocytes instead of osteoblasts. We investigated the effect of polymorphisms in the ALOX12 gene on BMD and fracture risk in two Danish cohorts and found four polymorphisms and a haplotype thereof to be associated with BMD and fracture risk. INTRODUCTION: Stimulation of the PPARγ with ligands produced by the ALOX enzymes drives mesenchymal stem cells in an adipocyte direction at the expense of osteoblasts leading to decreased osteoblast number and BMD. Previously, polymorphisms in the ALOX12 gene have been associated with osteoporosis. METHODS: We examined the effect of ALOX12 polymorphisms on BMD and the risk of fractures in two Danish cohorts: AROS, a case-control population comprising 809 individuals and DOPS, a population comprising 1,716 perimenopausal women allocated to hormone therapy or not at baseline and followed for up to 10 years. On the basis of linkage disequilibrium (LD) between SNPs throughout the gene and previous genetic association studies we chose ten polymorphisms for investigation. Genotyping was carried out using the Sequenom MassARRAY genotyping system and TaqMan assays. RESULTS: In AROS, individuals heterozygous for the polymorphisms rs3840880, rs9897850, rs2292350 and rs1126667 had a 3.0-4.7% decreased lumbar spine BMD (p = 0.02-0.06) and an increased risk of vertebral fractures (p < 0.05) compared with individuals homozygous for either allele. In DOPS, none of the individual SNPs were associated with BMD or incident fractures. In both cohorts, the above-mentioned SNPs comprised an LD-block (pairwise D´ = 1.0, r (2) = 0.45-0.97). A haplotype comprising all the common alleles (frequency 9%) was associated with decreased bone loss at the hip (p < 0.05) and decreased incidence of osteoporotic fractures (p < 0.05) in DOPS and increased femoral neck BMD in AROS (p < 0.05). CONCLUSION: Our study suggests that genetic variants in ALOX12 may influence BMD and fracture risk.