Treatment of trigeminal ganglion neurons in vitro with NGF, GDNF or BDNF: effects on neuronal survival, neurochemical properties and TRPV1-mediated neuropeptide secretion

BACKGROUND: Nerve growth factor (NGF), glial cell line-derived neurotrophic factor (GDNF) and brain-derived neurotrophic factor (BDNF) all play important roles in the development of the peripheral sensory nervous system. Additionally, these growth factors are proposed to modulate the properties of the sensory system in the adult under pathological conditions brought about by nerve injury or inflammation. We have examined the effects of NGF, GDNF and BDNF on adult rat trigeminal ganglion (TG) neurons in culture to gain a better understanding of how these growth factors alter the cytochemical and functional phenotype of these neurons, with special attention to properties associated with nociception. RESULTS: Compared with no growth factor controls, GDNF, at 1 and 100 ng/ml, significantly increased by nearly 100% the number of neurons in culture at 5 days post-plating. A significant, positive, linear trend of increasing neuron number as a function of BDNF concentration was observed, also peaking at nearly 100%. NGF treatment was without effect. Chronic treatment with NGF and GDNF significantly and concentration-dependently increased 100 nM capsaicin (CAP)-evoked calcitonin gene-related peptide (CGRP) release, reaching approximately 300% at the highest concentration tested (100 ng/ml). Also, NGF and GDNF each augmented anandamide (AEA)- and arachidonyl-2-chloroethylamide (ACEA)-evoked CGRP release, while BDNF was without effect. Utilizing immunohistochemistry to account for the proportions of TRPV1- or CGRP-positive neurons under each growth factor treatment condition and then standardizing evoked CGRP release to these proportions, we observed that NGF was much more effective in enhancing CAP- and 50 mM K(+)-evoked CGRP release than was GDNF. Furthermore, NGF and GDNF each altered the concentration-response function for CAP- and AEA-evoked CGRP release, increasing the E(max )without altering the EC(50 )for either compound. CONCLUSIONS: Taken together, our results illustrate that NGF, GDNF and BDNF differentially alter TG sensory neuron survival, neurochemical properties and TRPV1-mediated neuropeptide release in culture. In particular, our findings suggest that GDNF and NGF differentially modulate TRPV1-mediated neuropeptide secretion sensitivity, with NGF having a much greater effect on a per neuron basis than GDNF. These findings are discussed in relation to possible therapeutic roles for growth factors or their modulators in pathological pain states, especially as these relate to the trigeminal system

RAG-mediated DNA double-strand breaks activate a cell type-specific checkpoint to inhibit pre-B cell receptor signals

DNA double-strand breaks (DSBs) activate a canonical DNA damage response, including highly conserved cell cycle checkpoint pathways that prevent cells with DSBs from progressing through the cell cycle. In developing B cells, pre–B cell receptor (pre–BCR) signals initiate immunoglobulin light (Igl) chain gene assembly, leading to RAG-mediated DNA DSBs. The pre–BCR also promotes cell cycle entry, which could cause aberrant DSB repair and genome instability in pre–B cells. Here, we show that RAG DSBs inhibit pre–BCR signals through the ATM- and NF-κB2–dependent induction of SPIC, a hematopoietic-specific transcriptional repressor. SPIC inhibits expression of the SYK tyrosine kinase and BLNK adaptor, resulting in suppression of pre–BCR signaling. This regulatory circuit prevents the pre–BCR from inducing additional Igl chain gene rearrangements and driving pre–B cells with RAG DSBs into cycle. We propose that pre–B cells toggle between pre–BCR signals and a RAG DSB-dependent checkpoint to maintain genome stability while iteratively assembling Igl chain genes

Antimicrobial Susceptibility of Escherichia coli and Salmonella spp. Isolates From Healthy Pigs in Australia: Results of a Pilot National Survey

This study investigated the frequency of antimicrobial non-susceptibility (defined as the frequency of isolates with minimum inhibitory concentrations above the CLSI susceptible clinical breakpoint) among E. coli and Salmonella spp. isolated from healthy Australian finisher pigs. E. coli (n = 201) and Salmonella spp. (n = 69) were isolated from cecal contents of slaughter-age pigs, originating from 19 farms distributed throughout Australia during July-December 2015. Isolates underwent minimum inhibitory concentration (MIC) susceptibility testing to 11 antimicrobials. The highest frequencies of non-susceptibility among respective isolates of E. coli and Salmonella spp. were to ampicillin (60.2 and 20.3%), tetracycline (68.2 and 26.1%), chloramphenicol (47.8 and 7.3%), and trimethoprim/sulfamethoxazole (33.8 and 11.6%). Four E. coli isolates had MICs above the wild-type epidemiological cut-offvalue for ciprofloxacin, with two isolates from the same farm classified as clinically resistant (MICs of > 4 μg/ml), a noteworthy finding given that fluoroquinolones (FQs) are not legally available for use in Australian food-producing animals. Three of these four E. coli isolates belonged to the sequence type (ST) 10, which has been isolated from both humans and production animals, whilst one isolate belonged to a new ST (7573) and possessed qnrS1. This study shows that non-susceptibility to first line antimicrobials is common among E. coli and Salmonella spp. isolates from healthy slaughter age pigs in Australia. However, very low levels of non-susceptibility to critically important antimicrobials (CIAs), namely third generation cephalosporins and fluoroquinolones were observed. Nevertheless, the isolation of two ciprofloxacin-resistant E. coli isolates from Australian pigs demonstrates that even in the absence of local antimicrobial selection pressure, fluoroquinolone-resistant E. coli clonal lineages may enter livestock production facilities despite strict biosecurity

Characterisation of methicillin-resistant and methicillin-susceptible isolates of Staphylococcus pseudintermedius from cases of canine pyoderma in Australia

Meticillin-resistant Staphylococcus pseudintermedius (MRSP) has recently emerged as a worldwide cause of canine pyoderma. In this study, we characterized 22 S. pseudintermedius isolates cultured from 19 dogs with pyoderma that attended a veterinary dermatology referral clinic in Australia in 2011 and 2012. Twelve isolates were identified as MRSP by mecA real-time PCR and phenotypic resistance to oxacillin. In addition to β-lactam resistance, MRSP isolates were resistant to erythromycin (91.6 %), gentamicin (83.3 %), ciprofloxacin (83.3 %), chloramphenicol (75 %), clindamycin (66 %), oxytetracycline (66 %) and tetracycline (50 %), as shown by disc-diffusion susceptibility testing. Meticillin-susceptible S. pseudintermedius isolates only showed resistance to penicillin/ampicillin (90 %) and tetracycline (10 %). PFGE using the SmaI restriction enzyme was unable to type nine of the 12 MRSP isolates. However the nine isolates provided the same PFGE pulsotype using the Cfr91 restriction enzyme. Application of the mec-associated direct repeat unit (dru) typing method identified the nine SmaI PFGE-untypable isolates as dt11cb, a dru type that has only previously been associated with MRSP sequence type (ST)45 isolates that possess a unique SCCmec element. The dt11cb isolates shared a similar multidrug-resistant antibiogram phenotype profile, whereas the other MRSP isolates, dt11a, dt11af (dt11a-associated) and dt10h, were resistant to fewer antibiotic classes and had distinct PFGE profiles. This is the first report of MRSP causing pyoderma in dogs from Australia. The rapid intercontinental emergence and spread of multidrug-resistant MRSP strains confirms the urgent need for new treatment modalities for recurrent canine pyoderma in veterinary practice

Genomic analysis of phylogenetic group B2 extraintestinal pathogenic E. coli causing infections in dogs in Australia

This study investigated the prevalence of extraintestinal pathogenic E. coli (ExPEC)-associated sequence types (STs) from phylogenetic group B2 among 449 fluoroquinolone-susceptible dog clinical isolates from Australia. Isolates underwent PCR-based phylotyping and random amplified polymorphic DNA analysis to determine clonal relatedness. Of the 317 so-identified group B2 isolates, 77 underwent whole genome sequencing (WGS), whereas the remainder underwent PCR-based screening for ST complexes (STc) STc12, STc73, STc372, and ST131. The predominant ST was ST372 according to both WGS (31 % of 77) and ST-specific PCR (22 % of 240), followed by (per WGS) ST73 (17 %), ST12 (7 %), and ST80 (7 %). A WGS-based phylogenetic comparison of ST73 isolates from dogs, cats, and humans showed considerable overall phylogenetic diversity. Although most clusters were species-specific, some contained closely related human and animal (dog > cat) isolates. For dogs in Australia these findings both confirm ST372 as the predominant E. coli clonal lineage causing extraintestinal infections and clarify the importance of human-associated group B2 lineage ST73 as a cause of UTI, with some strains possibly being capable of bi-directional (i.e., dog-human and human-dog) transmission

Companion animals are spillover hosts of the multidrug-resistant human extraintestinal Escherichia coli pandemic clones ST131 and ST1193

Escherichia coli sequence types 131 (ST131) and 1193 are multidrug-resistant extraintestinal pathogens that have recently spread epidemically among humans and are occasionally isolated from companion animals. This study characterized a nationwide collection of fluoroquinolone-resistant (FQ) E. coli isolates from extraintestinal infections in Australian cats and dogs. For this, 59 cat and dog FQ clinical E. coli isolates (representing 6.9% of an 855-isolate collection) underwent PCR-based phylotyping and whole-genome sequencing (WGS). Isolates from commensal-associated phylogenetic groups A (14/59, 24%) and B1 (18/59, 31%) were dominant, with ST224 (10/59, 17%), and ST744 (8/59, 14%) predominating. Less prevalent were phylogenetic groups D (12/59, 20%), with ST38 (8/59, 14%) predominating, and virulence-associated phylogenetic group B2 (7/59, 12%), with ST131 predominating (6/7, 86%) and no ST1193 isolates identified. In a WGS-based comparison of 20 cat and dog-source ST131 isolates with 188 reference human and animal ST131 isolates, the cat and dog-source isolates were phylogenetically diverse. Although cat and dog-source ST131 isolates exhibited some minor sub-clustering, most were closely related to human-source ST131 strains. Furthermore, the prevalence of ST131 as a cause of FQ infections in Australian companion animals was relatively constant between this study and the 5-year-earlier study of Platell et al. (2010) (9/125 isolates, 7.2%). Thus, although the high degree of clonal commonality among FQ clinical isolates from humans vs. companion animals suggests the possibility of bi-directional between-species transmission, the much higher reported prevalence of ST131 and ST1193 among FQ clinical isolates from humans as compared to companion animals suggests that companion animals are spillover hosts rather than being a primary reservoir for these lineages

Aging and estrogen alter endothelial reactivity to reactive oxygen species in coronary arterioles

Endothelium-dependent, nitric oxide (NO)-mediated vasodilation can be impaired by reactive oxygen species (ROS), and this deleterious effect of ROS on NO availability may increase with aging. Endothelial function declines rapidly after menopause, possibly because of loss of circulating estrogen and its antioxidant effects. The purpose of the current study was to determine the role of O2− and H2O2 in regulating flow-induced dilation in coronary arterioles of young (6-mo) and aged (24-mo) intact, ovariectomized (OVX), or OVX + estrogen-treated (OVE) female Fischer 344 rats. Both aging and OVX reduced flow-induced NO production, whereas flow-induced H2O2 production was not altered by age or estrogen status. Flow-induced vasodilation was evaluated before and after treatment with the superoxide dismutase (SOD) mimetic Tempol (100 μM) or the H2O2 scavenger catalase (100 U/ml). Removal of H2O2 with catalase reduced flow-induced dilation in all groups, whereas Tempol diminished vasodilation in intact and OVE, but not OVX, rats. Immunoblot analysis revealed elevated nitrotyrosine with aging and OVX. In young rats, OVX reduced SOD protein while OVE increased SOD in aged rats; catalase protein did not differ in any group. Collectively, these studies suggest that O2− and H2O2 are critical components of flow-induced vasodilation in coronary arterioles from female rats; however, a chronic deficiency of O2− buffering by SOD contributes to impaired flow-induced dilation with aging and loss of estrogen. Furthermore, these data indicate that estrogen replacement restores O2− homeostasis and flow-induced dilation of coronary arterioles, even at an advanced age

Treatment of trigeminal ganglion neurons in vitro with NGF, GDNF or BDNF: effects on neuronal survival, neurochemical properties and TRPV1-mediated neuropeptide secretion-8

<p><b>Copyright information:</b></p><p>Taken from "Treatment of trigeminal ganglion neurons in vitro with NGF, GDNF or BDNF: effects on neuronal survival, neurochemical properties and TRPV1-mediated neuropeptide secretion"</p><p>BMC Neuroscience 2005;6():4-4.</p><p>Published online 24 Jan 2005</p><p>PMCID:PMC548274.</p><p>Copyright © 2005 Price et al; licensee BioMed Central Ltd.</p>tor conditions (n = 6). Panel A illustrates the maximum relative effect of capsaicin under each condition matched to the maximum evoked-CGRP release for that given condition. Panel B depicts the CAP-evoked CGRP release data in terms of CGRP released in fmoles in the presence or absence of NGF or GDNF to illustrate the magnitude of increases in Eand the biphasic nature of the capsaicin-evoked CGRP concentration-response function