Mechanisms underlying clinical efficacy of Angiotensin II type 2 receptor (AT(2)R) antagonist EMA401 in neuropathic pain: clinical tissue and in vitro studies

BACKGROUND: The clinical efficacy of the Angiotensin II (AngII) receptor AT(2)R antagonist EMA401, a novel peripherally-restricted analgesic, was reported recently in post-herpetic neuralgia. While previous studies have shown that AT(2)R is expressed by nociceptors in human DRG (hDRG), and that EMA401 inhibits capsaicin responses in cultured hDRG neurons, the expression and levels of its endogenous ligands AngII and AngIII in clinical neuropathic pain tissues, and their signalling pathways, require investigation. We have immunostained AngII, AT(2)R and the capsaicin receptor TRPV1 in control post-mortem and avulsion injured hDRG, control and injured human nerves, and in cultured hDRG neurons. AngII, AngIII, and Ang-(1-7) levels were quantified by ELISA. The in vitro effects of AngII, AT(2)R agonist C21, and Nerve growth factor (NGF) were measured on neurite lengths; AngII, NGF and EMA401 effects on expression of p38 and p42/44 MAPK were measured using quantitative immunofluorescence, and on capsaicin responses using calcium imaging. RESULTS: AngII immunostaining was observed in approximately 75% of small/medium diameter neurons in control (n = 5) and avulsion injured (n = 8) hDRG, but not large neurons i.e. similar to TRPV1. AngII was co-localised with AT(2)R and TRPV1 in hDRG and in vitro. AngII staining by image analysis showed no significant difference between control (n = 12) and injured (n = 13) human nerves. AngII levels by ELISA were also similar in control human nerves (4.09 ± 0.36 pmol/g, n = 31), injured nerves (3.99 ± 0.79 pmol/g, n = 7), and painful neuromas (3.43 ± 0.73 pmol/g, n = 12); AngIII and Ang-(1-7) levels were undetectable (<0.03 and 0.05 pmol/g respectively). Neurite lengths were significantly increased in the presence of NGF, AngII and C21 in cultured DRG neurons. AngII and, as expected, NGF significantly increased signal intensity of p38 and p42/44 MAPK, which was reversed by EMA401. AngII mediated sensitization of capsaicin responses was not observed in the presence of MAP kinase inhibitor PD98059, and the kinase inhibitor staurosporine. CONCLUSION: The major AT(2)R ligand in human peripheral nerves is AngII, and its levels are maintained in injured nerves. EMA401 may act on paracrine/autocrine mechanisms at peripheral nerve terminals, or intracrine mechanisms, to reduce neuropathic pain signalling in AngII/NGF/TRPV1-convergent pathways

Nociceptin/Orphanin FQ receptor expression in clinical pain disorders and functional effects in cultured neurons

The Nociceptin/Orphanin FQ peptide receptor (NOP), activated by its endogenous peptide ligand Nociceptin/Orphanin FQ (N/OFQ), exerts several effects including modulation of pain signalling. We have examined, for the first time, the tissue distribution of the NOP receptor in clinical visceral and somatic pain disorders by immunohistochemistry, and assessed functional effects of NOP and [micro] opioid receptor activation in cultured human and rat dorsal root ganglion (DRG) neurons. Quantification of NOP-positive nerve fibres within the bladder sub-urothelium revealed a remarkable several-fold increase in Detrusor Overactivity (p<0.0001) and Painful Bladder Syndrome patient specimens (p=0.0014), compared to controls. In post-mortem control human DRGs, 75-80% of small/medium neurons (<=50 [micro]m diameter) in the lumbar (somatic) and sacral (visceral) DRG were positive for NOP, and fewer large neurons; avulsion-injured cervical human DRG neurons showed similar numbers. NOP-immunoreactivity was significantly decreased in injured peripheral nerves (p=0.0004), and also in painful neuromas (p=0.025). Calcium imaging studies in cultured rat DRG neurons demonstrated dose-dependent inhibition of capsaicin responses in the presence of N/OFQ, with an IC50 of 8.6 pM. In cultured human DRG neurons, 32% inhibition of capsaicin responses was observed in the presence of 1 pM N/OFQ (p<0.001). The maximum inhibition of capsaicin responses was greater with N/OFQ than [mu]-opioid receptor agonist DAMGO. Our findings highlight the potential of NOP agonists, particularly in urinary bladder overactivity and pain syndromes. The regulation of NOP expression in visceral and somatic sensory neurons by target-derived neurotrophic factors deserves further study, and the efficacy of NOP selective agonists in clinical trials

Can We Agree What Skilled Mindfulness-Based Teaching Looks Like? Lessons From Studying the MBI:TAC

BackgroundThe Mindfulness-Based Interventions: Teaching Assessment Criteria (MBI:TAC) is a widely used tool for assessing fidelity in mindfulness-based program (MBP) research and training. It also supports MBP teacher reflective and skill development. MBI:TAC assessors review MBP teaching and rate the teaching on 6 domains. The MBI:TAC yields individual domain and overall scores, using 6 levels of competence. Although the MBI:TAC is widely used in MBP research and training, research is at an early stage.ObjectiveWe developed and tested a method of training MBI:TAC assessors to use the tool reliably and examined interrater reliability of the tool.MethodsA total of 31 international senior MBP teachers were recruited to join an online training to build their skills in using the MBI:TAC. The training systematically and iteratively built familiarity and skills in assessing the 6 MBI:TAC domains. Qualitative and quantitative data on trainee's experience of the training were gathered. Interrater reliability in using the tool was tested each week of the training. At the end of the training, interrater reliability was tested by asking trainees to individually assess videos that they had not previously seen. Their ratings were compared to benchmark assessments, which had been established via consensus agreement between 4 expert users of the MBI:TAC.ResultsThe training was well received and appreciated, with some challenges experienced in applying the assessment methodology. Participants' ratings became progressively more in line with one another and the benchmark ratings during the training. At the end, interrater reliability was high (ranging from 0.67 to 1.0).ConclusionIt is possible for senior MBP trainers, coming from different regions in the world, to align toward common understandings of the elements of MBP teaching competence and program integrity. An assessor training methodology was tested, and the learning from this project has led to refinements for future delivery

Determining the role of novel metabolic pathways in driving intracranial pressure reduction after weight loss

Idiopathic intracranial hypertension, a disease classically occurring in women with obesity, is characterised by raised intracranial pressure. Weight loss leads to reduction in intracranial pressure. Additionally, pharmacological glucagon-like peptide-1 agonism reduces cerebrospinal fluid secretion and intracranial pressure. The potential mechanisms by which weight loss reduces intracranial pressure are unknown and was the focus for this study.Meal stimulation tests (fasted plasma sample, then samples at 15, 30, 60, 90 and 120 minutes following a standardised meal) were conducted pre- and post-bariatric surgery (early (2 weeks) and late (12 months)) in patients with active idiopathic intracranial hypertension. Dynamic changes in gut neuropeptides (glucagon-like peptide-1, gastric inhibitory polypeptide, and ghrelin) and metabolites (untargeted ultra-high performance liquid chromatography-mass spectrometry) were evaluated. We determined the relationship between gut neuropeptides, metabolites, and intracranial pressure.18 idiopathic intracranial hypertension patients were included (Roux-En-Y gastric bypass n=7, gastric banding n=6, or sleeve gastrectomy n=5). At 2 weeks post-bariatric surgery, despite similar weight loss, Roux-En-Y gastric bypass had a two-fold (50%) greater reduction in intracranial pressure compared to sleeve. Increased meal stimulated glucagon-like peptide-1 secretion was observed after Roux-En-Y gastric bypass (+600 %) compared to sleeve (+319 %). There was no change in gastric inhibitory polypeptide and ghrelin. Dynamic changes in meal stimulated metabolites after bariatric surgery consistently identified changes in lipid metabolites, predominantly ceramides, glycerophospholipids and lysoglycerophospholipids, which correlated with intracranial pressure. A greater number of differential lipid metabolites were observed in the Roux-En-Y gastric bypass cohort at 2 weeks, and these also correlated with intracranial pressure.In idiopathic intracranial hypertension, we identified novel changes in lipid metabolites and meal stimulated glucagon-like peptide-1 levels following bariatric surgery which were associated with changes in intracranial pressure. Roux-En-Y gastric bypass was most effective at reducing intracranial pressure despite analogous weight loss to gastric sleeve at 2 weeks post-surgery and was associated with more pronounced changes in these metabolite pathways. We suggest that these novel perturbations in lipid metabolism and glucagon-like peptide-1 secretion are mechanistically important in driving reduction in intracranial pressure following weight loss in patients with idiopathic intracranial hypertension. Therapeutic targeting of these pathways, for example with glucagon-like peptide-1 agonist infusion, could represent a therapeutic strategy

Appendiceal-sigmoid fistula presenting in a man with ulcerative colitis: a case report

<p>Abstract</p> <p>Introduction</p> <p>Ulcerative colitis is a chronic disease characterized by diffuse mucosal inflammation limited to the colon. It mostly affects young adults, yet a large number of middle-aged and older patients with ulcerative colitis have also been reported.</p> <p>Case presentation</p> <p>A 58-year-old Caucasian man presented to our hospital in August 2006 with continuous and diffuse abdominal pain, meteorism, fever and bloody diarrhea. He had a two-year history of ulcerative colitis. Our patient was treated with intravenous medical therapy. As his condition worsened, he underwent surgery. An explorative laparotomy revealed that the entire colon was distended and pus was found around an appendiceal-sigmoid fistula.</p> <p>Conclusions</p> <p>Therapy for ulcerative colitis <b/>is a rapidly evolving field, with many new biological agents under investigation that are likely to change therapeutic strategies radically in the next decade. Indications for surgery are intractability (49%), stricture, dysplasia, toxic colitis, hemorrhage and perforation. To the best of our knowledge, this is the first case of an appendiceal-sigmoid fistula in a patient affected by ulcerative colitis reported in the literature. Fistulae between the appendix and the sigmoid tract are rarely reported in cases of diverticular disease and appendicitis.</p

Nerve and vascular biomarkers in skin biopsies differentiate painful from painless peripheral neuropathy in type 2 diabetes

Painful diabetic peripheral neuropathy can be intractable with a major impact, yet the underlying pain mechanisms remain uncertain. A range of neuronal and vascular biomarkers was investigated in painful diabetic peripheral neuropathy (painful-DPN) and painless-DPN and used to differentiate painful-DPN from painless-DPN. Skin biopsies were collected from 61 patients with type 2 diabetes (T2D), and 19 healthy volunteers (HV). All subjects underwent detailed clinical and neurophysiological assessments. Based on the neuropathy composite score of the lower limbs [NIS(LL)] plus seven tests, the T2D subjects were subsequently divided into three groups: painful-DPN (n = 23), painless-DPN (n = 19), and No-DPN (n = 19). All subjects underwent punch skin biopsy, and immunohistochemistry used to quantify total intraepidermal nerve fibers (IENF) with protein gene product 9.5 (PGP9.5), regenerating nerve fibers with growth-associated protein 43 (GAP43), peptidergic nerve fibers with calcitonin gene-related peptide (CGRP), and blood vessels with von Willebrand Factor (vWF). The results showed that IENF density was severely decreased (p < 0.001) in both DPN groups, with no differences for PGP9.5, GAP43, CGRP, or GAP43/PGP9.5 ratios. There was a significant increase in blood vessel (vWF) density in painless-DPN and No-DPN groups compared to the HV group, but this was markedly greater in the painful-DPN group, and significantly higher than in the painless-DPN group (p < 0.0001). The ratio of sub-epidermal nerve fiber (SENF) density of CGRP:vWF showed a significant decrease in painful-DPN vs. painless-DPN (p = 0.014). In patients with T2D with advanced DPN, increased dermal vasculature and its ratio to nociceptors may differentiate painful-DPN from painless-DPN. We hypothesized that hypoxia-induced increase of blood vessels, which secrete algogenic substances including nerve growth factor (NGF), may expose their associated nociceptor fibers to a relative excess of algogens, thus leading to painful-DPN

Establishing a Metabolite Extraction Method to Study the Metabolome of Blastocystis Using NMR

Blastocystis is an opportunistic parasite commonly found in the intestines of humans and other animals. Despite its high prevalence, knowledge regarding Blastocystis biology within and out-side the host is limited. Analysis of the metabolites produced by this anaerobe could provide in-sights that can help map its metabolism and determine its role in both health and disease. Due to its controversial pathogenicity, these metabolites could define its deterministic role in microbi-ome’s “health” and/or subsequently resolve Blastocystis’ potential impact in gastrointestinal health. A common method for elucidating the presence of these metabolites is through 1H nuclear magnetic resonance (NMR). However, there are currently no described benchmarked methods available to extract metabolites from Blastocystis for 1H NMR analysis. Herein, several extrac-tion solvents, lysis methods and incubation temperatures were compared for their usefulness as an extraction protocol for this protozoan. Following extraction, the samples were freeze-dried, re-solubilized and analysed with 1H NMR. The results demonstrate that carrying out the proce-dure at room temperature using methanol as an extraction solvent and bead bashing as a lysis technique provides a consistent, reproducible and efficient method to extract metabolites from Blastocystis for NMR