ErbB1-dependent signalling and vesicular trafficking in primary afferent nociceptors associated with hypersensitivity in neuropathic pain.

Effective analgesic treatment for neuropathic pain remains an unmet need, so previous evidence that epidermal growth factor receptor inhibitors (EGFRIs) provide unexpected rapid pain relief in a clinical setting points to a novel therapeutic opportunity. The present study utilises rodent models to address the cellular and molecular basis for the findings, focusing on primary sensory neurons because clinical pain relief is provided not only by small molecule EGFRIs, but also by the anti-EGFR antibodies cetuximab and panitumumab, which are unlikely to access the central nervous system in therapeutic concentrations. We report robust, rapid and dose-dependent analgesic effects of EGFRIs in two neuropathic pain models, matched by evidence with highly selective antibodies that expression of the EGFR (ErbB1 protein) is limited to small nociceptive afferent neurons. As other ErbB family members can heterodimerise with ErbB1, we investigated their distribution, showing consistent co-expression of ErbB2 but not ErbB3 or ErbB4, with ErbB1 in cell bodies of nociceptors, as well as providing evidence for direct molecular interaction of ErbB1 with ErbB2 in situ. Co-administration of selective ErbB1 and ErbB2 inhibitors produced clear evidence of greater-than-additive, synergistic analgesia; highlighting the prospect of a unique new combination therapy in which enhanced efficacy could be accompanied by minimisation of side-effects. Peripheral (intraplantar) administration of EGF elicited hypersensitivity only following nerve injury and this was reversed by local co-administration of selective inhibitors of either ErbB1 or ErbB2. Investigating how ErbB1 is activated in neuropathic pain, we found evidence for a role of Src tyrosine kinase, which can be activated by signals from inflammatory mediators, chemokines and cytokines during neuroinflammation. Considering downstream consequences of ErbB1 activation in neuropathic pain, we found direct recruitment to ErbB1 of an adapter for PI 3-kinase and Akt signalling together with clear Akt activation and robust analgesia from selective Akt inhibitors. The known Akt target and regulator of vesicular trafficking, AS160 was strongly phosphorylated at a perinuclear location during neuropathic pain in an ErbB1-, ErbB2- and Akt-dependent manner, corresponding to clustering and translocation of an AS160-partner, the vesicular chaperone, LRP1. Exploring whether neuronal ion channels that could contribute to hyperexcitability might be transported by this vesicular trafficking pathway we were able to identify Nav1.9, (Nav1.8) and Cav1.2 moving towards the plasma membrane or into proximal axonal locations - a process prevented by ErbB1 or Akt inhibitors. Overall these findings newly reveal both upstream and downstream signals to explain how ErbB1 can act as a signalling hub in neuropathic pain models and identify the trafficking of key ion channels to neuronal subcellular locations likely to contribute to hyperexcitability. The new concept of combined treatment with ErbB1 plus ErbB2 blockers is mechanistically validated as a promising strategy for the relief of neuropathic pain.

Enhanced axonal response of mitochondria to demyelination offers neuroprotection: implications for multiple sclerosis.

Axonal loss is the key pathological substrate of neurological disability in demyelinating disorders, including multiple sclerosis (MS). However, the consequences of demyelination on neuronal and axonal biology are poorly understood. The abundance of mitochondria in demyelinated axons in MS raises the possibility that increased mitochondrial content serves as a compensatory response to demyelination. Here, we show that upon demyelination mitochondria move from the neuronal cell body to the demyelinated axon, increasing axonal mitochondrial content, which we term the axonal response of mitochondria to demyelination (ARMD). However, following demyelination axons degenerate before the homeostatic ARMD reaches its peak. Enhancement of ARMD, by targeting mitochondrial biogenesis and mitochondrial transport from the cell body to axon, protects acutely demyelinated axons from degeneration. To determine the relevance of ARMD to disease state, we examined MS autopsy tissue and found a positive correlation between mitochondrial content in demyelinated dorsal column axons and cytochrome c oxidase (complex IV) deficiency in dorsal root ganglia (DRG) neuronal cell bodies. We experimentally demyelinated DRG neuron-specific complex IV deficient mice, as established disease models do not recapitulate complex IV deficiency in neurons, and found that these mice are able to demonstrate ARMD, despite the mitochondrial perturbation. Enhancement of mitochondrial dynamics in complex IV deficient neurons protects the axon upon demyelination. Consequently, increased mobilisation of mitochondria from the neuronal cell body to the axon is a novel neuroprotective strategy for the vulnerable, acutely demyelinated axon. We propose that promoting ARMD is likely to be a crucial preceding step for implementing potential regenerative strategies for demyelinating disorders.

Prolonged Analgesia by Spinal Cord Stimulation Following a Spinal Injury Associated With Activation of Adult Neural Progenitors.

OBJECTIVES: Responses of spinal progenitors to spinal cord stimulation (SCS) following spinal cord injury (SCI) in rats were assessed to reveal their potential contribution to SCS-induced analgesia. METHODS: Spinal epidural electrodes were implanted in rats at T12 rostral to a quadrant dorsal horn injury at T13. Further groups additionally received either a microlesion to the dorsolateral funiculus (DLF) or gabapentin (10 mg/kg). SCS was performed at 25 Hz for 10 minutes on day 4 (early SCS) and at 10 Hz for 10 minutes on day 8 (late SCS) after injury. Paw withdrawal threshold (PWT) was measured before injury, 30 minutes before or after SCS, and before cull on day 14, followed by immunostaining assessment. RESULTS: Paw withdrawal thresholds in uninjured animals (51.0 ± 4.0 g) were markedly reduced after SCI (17.3 ± 2.2 g). This was significantly increased by early SCS (38.5 ± 5.2 g, P < 0.01) and further enhanced by late SCS (50.9 ± 1.9 g, P < 0.01) over 6 days. Numbers of neural progenitors expressing nestin, Sox2, and doublecortin (DCX) in the spinal dorsal horn were increased 6 days after SCS by 6-fold, 2-fold, and 2.5-fold, respectively (P < 0.05 to 0.01). The elevated PWT evoked by SCS was abolished by DLF microlesions (48.9 ± 2.6 g vs. 19.0 ± 3.9 g, P < 0.01) and the number of nestin-positive cells was reduced to the level without SCS (P < 0.05). Gabapentin enhanced late SCS-induced analgesia from 37.0 ± 3.9 g to 54.0 ± 0.8 g (P < 0.01) and increased gamma-aminobutyric acid (GABA)-ergic neuronal marker vesicular GABA transporter-positive newborn cells 2-fold (P < 0.01). CONCLUSIONS: Spinal progenitor cells appear to be activated by SCS via descending pathways, which may be enhanced by gabapentin and potentially contributes to relief of SCI-induced neuropathic pain.

Relief of Neuropathic Pain Through Epidermal Growth Factor Receptor Inhibition: A Randomized Proof-of-Concept Trial.

OBJECTIVE: Case reports and a case series have described relief of neuropathic pain (NP) after treatment with epidermal growth factor receptor inhibitors (EGFR-Is). These observations are supported by preclinical findings. The aim of this trial was to explore a potential clinical signal supporting the therapeutic efficacy of EGFR-Is in NP. METHODS: In a proof-of-concept trial using a randomized, double-blind, placebo-controlled design, 14 patients with severe, chronic, therapy-resistant NP due to compressed peripheral nerves or complex regional pain syndrome were randomized to receive a single infusion of the EGFR-I cetuximab and placebo in crossover design, followed by a single open-label cetuximab infusion. RESULTS: The mean reduction in daily average pain scores three to seven days after single-blinded cetuximab infusion was 1.73 points (90% confidence interval [CI] = 0.80 to 2.66), conferring a 1.22-point greater reduction than placebo (90% CI = -0.10 to 2.54). Exploratory analyses suggested that pain reduction might be greater in the 14 days after treatment with blinded cetuximab than after placebo. The proportion of patients who reported ≥50% reduction in average pain three to seven days after cetuximab was 36% (14% after placebo), and comparison of overall pain reduction suggests a trend in favor of cetuximab. Skin rash (grade 1-2) was the most frequent side effect (12/14, 86%). CONCLUSIONS: This small proof-of-concept evaluation of an EGFR-I against NP did not provide statistical evidence of efficacy. However, substantial reductions in pain were reported, and confidence intervals do not rule out a clinically meaningful treatment effect. Evaluation of EGFR-I against NP therefore warrants further investigation.

The SH3 domain of postsynaptic density 95 mediates inflammatory pain through phosphatidylinositol-3-kinase recruitment.

Sensitization to inflammatory pain is a pathological form of neuronal plasticity that is poorly understood and treated. Here we examine the role of the SH3 domain of postsynaptic density 95 (PSD95) by using mice that carry a single amino-acid substitution in the polyproline-binding site. Testing multiple forms of plasticity we found sensitization to inflammation was specifically attenuated. The inflammatory response required recruitment of phosphatidylinositol-3-kinase-C2alpha to the SH3-binding site of PSD95. In wild-type mice, wortmannin or peptide competition attenuated the sensitization. These results show that different types of behavioural plasticity are mediated by specific domains of PSD95 and suggest novel therapeutic avenues for reducing inflammatory pain.

5-HT2A receptor signalling through phospholipase D1 associated with its C-terminal tail.

The 5-HT2AR (5-hydroxytryptamine-2A receptor) is a GPCR (G-protein-coupled receptor) that is implicated in the actions of hallucinogens and represents a major target of atypical antipsychotic agents. In addition to its classical signalling though PLC (phospholipase C), the receptor can activate several other pathways, including ARF (ADP-ribosylation factor)-dependent activation of PLD (phospholipase D), which appears to be achieved through a mechanism independent of heterotrimeric G-proteins. In the present study we show that wild-type and inactive constructs of PLD1 (but not PLD2) respectively facilitate and inhibit ARF-dependent PLD signalling by the 5-HT2AR. Furthermore we demonstrate that PLD1 specifically co-immunoprecipitates with the receptor and binds to a distal site in GST (glutathione transferase) fusion protein constructs of its C-terminal tail which is distinct from the ARF-interaction site, thereby suggesting the existence of a functional ARF-PLD signalling complex directly associated with this receptor. This reveals the spatial co-ordination of an important GPCR, transducer and effector into a physical complex that is likely to reinforce the impact of receptor activation on a heterotrimeric G-protein-independent signalling pathway. Signalling of this receptor through such non-canonical pathways may be important to its role in particular disorders.

GPUTreeShap: massively parallel exact calculation of SHAP scores for tree ensembles.

SHapley Additive exPlanation (SHAP) values (Lundberg & Lee, 2017) provide a game theoretic interpretation of the predictions of machine learning models based on Shapley values (Shapley, 1953). While exact calculation of SHAP values is computationally intractable in general, a recursive polynomial-time algorithm called TreeShap (Lundberg et al., 2020) is available for decision tree models. However, despite its polynomial time complexity, TreeShap can become a significant bottleneck in practical machine learning pipelines when applied to large decision tree ensembles. Unfortunately, the complicated TreeShap algorithm is difficult to map to hardware accelerators such as GPUs. In this work, we present GPUTreeShap, a reformulated TreeShap algorithm suitable for massively parallel computation on graphics processing units. Our approach first preprocesses each decision tree to isolate variable sized sub-problems from the original recursive algorithm, then solves a bin packing problem, and finally maps sub-problems to single-instruction, multiple-thread (SIMT) tasks for parallel execution with specialised hardware instructions. With a single NVIDIA Tesla V100-32 GPU, we achieve speedups of up to 19× for SHAP values, and speedups of up to 340× for SHAP interaction values, over a state-of-the-art multi-core CPU implementation executed on two 20-core Xeon E5-2698 v4 2.2 GHz CPUs. We also experiment with multi-GPU computing using eight V100 GPUs, demonstrating throughput of 1.2 M rows per second-equivalent CPU-based performance is estimated to require 6850 CPU cores.

Can changes in spending on health and social care explain the recent mortality trends in Scotland? A protocol for an observational study.

INTRODUCTION: There have been steady reductions in mortality rates in the majority of high-income countries, including Scotland, since 1945. However, reductions in mortality rates have slowed down since 2012-2014 in these nations; and have reversed in some cases. Deaths among those aged 55+ explain a large amount of these changing mortality trends in Scotland. Increased pressures on health and social care services have been suggested as one factor explaining these changes. This paper outlines a protocol for the approach to testing the extent to which health and social care pressures can explain recent mortality trends in Scotland. Although a slower rate of mortality improvements have affected people of all ages, certain ages have been more negatively affected than the others. The current analyses will be run by age-band to test if the service pressure-mortality link varies across age-group. METHODS AND ANALYSIS: This will be an observational ecological study based on the Scottish population. The exposures of interest will be the absolute (primary outcome) and percentage (secondary outcome) change in real terms per capita spending on social and healthcare services between 2011 and 2017. The outcome of interest will be the absolute (primary outcome) and percentage (secondary outcome) change in age-standardised mortality rate between 2012 and 2018 for men and women separately. The units of analysis will be the 32 local authorities and the 14 territorial health boards. The analyses will be run for both all age-groups combined and for the following age bands: <1, 1-15, 16-44, 45-64, 65-74, 75-84 and 85+.A series of descriptive analyses will summarise the distribution of health and social care expenditure and mortality trends between 2011 and 2018. Linear regression analysis will be used to investigate the direct association between health care spending and mortality rates. ETHICS AND DISSEMINATION: The data used in this study will be publicly available and aggregated and will not be individually identifiable; therefore, ethical committee approval is not needed. This work will not result in the creation of a new data set. On completion, the study will be stored within the National Health Service research governance system. All of the results will be published once they have been shared with partner agencies.

Analgesia mediated by the TRPM8 cold receptor in chronic neuropathic pain.

BACKGROUND: Chronic established pain, especially that following nerve injury, is difficult to treat and represents a largely unmet therapeutic need. New insights are urgently required, and we reasoned that endogenous processes such as cooling-induced analgesia may point the way to novel strategies for intervention. Molecular receptors for cooling have been identified in sensory nerves, and we demonstrate here how activation of one of these, TRPM8, produces profound, mechanistically novel analgesia in chronic pain states. RESULTS: We show that activation of TRPM8 in a subpopulation of sensory afferents (by either cutaneous or intrathecal application of specific pharmacological agents or by modest cooling) elicits analgesia in neuropathic and other chronic pain models in rats, thereby inhibiting the characteristic sensitization of dorsal-horn neurons and behavioral-reflex facilitation. TRPM8 expression was increased in a subset of sensory neurons after nerve injury. The essential role of TRPM8 in suppression of sensitized pain responses was corroborated by specific knockdown of its expression after intrathecal application of an antisense oligonucleotide. We further show that the analgesic effect of TRPM8 activation is centrally mediated and relies on Group II/III metabotropic glutamate receptors (mGluRs), but not opioid receptors. We propose a scheme in which Group II/III mGluRs would respond to glutamate released from TRPM8-containing afferents to exert an inhibitory gate control over nociceptive inputs. CONCLUSIONS: TRPM8 and its central downstream mediators, as elements of endogenous-cooling-induced analgesia, represent a novel analgesic axis that can be exploited in chronic sensitized pain states.

The role of metabolic derangements and glucocorticoid excess in the aetiology of cognitive impairment in type 2 diabetes. Implications for future therapeutic strategies.

Dementia is becoming increasingly common in western societies and carries with it a substantial clinical, social and economic burden. It is now well established that type 2 diabetes is a risk factor for dementia and it is likely that this association has a multifactorial aetiology. There is a relative paucity of data on interventions to improve cognitive function in people with type 2 diabetes. Two small randomized controlled trials have suggested that better glycaemic control, over a relatively short time period, can improve or prevent decline in cognitive function. There is also increasing interest in the link between intracerebral insulin and cognitive impairment. Several studies have suggested that relative and/or absolute deficiency of insulin may occur in Alzheimer's dementia and, although one small randomized trial was essentially negative, randomized trials are currently underway to investigate the impact of thiazolidinediones on cognitive function in dementia. The hypothalamic-pituitary-adrenal axis is also activated in people with type 2 diabetes and there are data linking increased cortisol concentrations with cognitive impairment. Inhibition of the 11 beta-hydroxysteroid dehydrogenase type 1 enzyme, which generates cortisol from inactive cortisone in many tissues including the brain, is an attractive therapeutic target to enhance cognition. Large-scale epidemiological and intervention studies are now underway, which should enhance our understanding and management of cognitive impairment in type 2 diabetes.

A Targeted Mutation Disrupting Mitochondrial Complex IV Function in Primary Afferent Neurons Leads to Pain Hypersensitivity Through P2Y1 Receptor Activation.

As mitochondrial dysfunction is evident in neurodegenerative disorders that are accompanied by pain, we generated inducible mutant mice with disruption of mitochondrial respiratory chain complex IV, by COX10 deletion limited to sensory afferent neurons through the use of an Advillin Cre-reporter. COX10 deletion results in a selective energy-deficiency phenotype with minimal production of reactive oxygen species. Mutant mice showed reduced activity of mitochondrial respiratory chain complex IV in many sensory neurons, increased ADP/ATP ratios in dorsal root ganglia and dorsal spinal cord synaptoneurosomes, as well as impaired mitochondrial membrane potential, in these synaptoneurosome preparations. These changes were accompanied by marked pain hypersensitivity in mechanical and thermal (hot and cold) tests without altered motor function. To address the underlying basis, we measured Ca2+ fluorescence responses of dorsal spinal cord synaptoneurosomes to activation of the GluK1 (kainate) receptor, which we showed to be widely expressed in small but not large nociceptive afferents, and is minimally expressed elsewhere in the spinal cord. Synaptoneurosomes from mutant mice showed greatly increased responses to GluK1 agonist. To explore whether altered nucleotide levels may play a part in this hypersensitivity, we pharmacologically interrogated potential roles of AMP-kinase and ADP-sensitive purinergic receptors. The ADP-sensitive P2Y1 receptor was clearly implicated. Its expression in small nociceptive afferents was increased in mutants, whose in vivo pain hypersensitivity, in mechanical, thermal and cold tests, was reversed by a selective P2Y1 antagonist. Energy depletion and ADP elevation in sensory afferents, due to mitochondrial respiratory chain complex IV deficiency, appear sufficient to induce pain hypersensitivity, by ADP activation of P2Y1 receptors.

The relationship between type 2 diabetes and dementia.

INTRODUCTION: The prevalence of type 2 diabetes and dementia are set to rise inexorably over the next 30-40 years. There are now substantial data to suggest that type 2 diabetes is associated with an increased risk of dementia. SOURCES OF DATA: This is a narrative review using data from individual studies and review articles known to the authors. A Medline search was also undertaken and reference lists were reviewed to identify additional relevant studies. AREAS OF AGREEMENT: Type 2 diabetes is associated with an increased risk of both Alzheimer's and Vascular dementia, although the reality is that many affected individuals have mixed forms of dementia. AREAS OF CONTROVERSY: The mechanisms underpinning this association remain to be clearly delineated. Type 2 diabetes is a complex disorder and so it is likely that multiple different, synergistic processes may interact to promote cognitive decrements. GROWING POINTS: Recent data suggest that glucocorticoids excess and elevated inflammatory markers may also have a role in the aetiology of diabetes-related cognitive impairment. AREAS TIMELY FOR DEVELOPING RESEARCH: Large-scale, prospective epidemiological studies are now required to accurately delineate the pathogenesis of cognitive impairment in people with type 2 diabetes. These are underway and randomized trials of diabetes-specific interventions are also starting to include cognitive function as an outcome measure.

The Edinburgh Type 2 Diabetes Study: study protocol.

BACKGROUND: Risk factors underlying the development and progression of some of the less well-recognised complications of type 2 diabetes, including cognitive impairment and non-alcoholic fatty liver disease, are poorly understood. The Edinburgh Type 2 Diabetes Study was established in 2006 in order to investigate the role of potential risk factors in these complications, as well as to further investigate mechanisms underlying the development and progression of micro and macrovascular disease in type 2 diabetes. METHODS AND DESIGN: The study is designed as a prospective cohort study. Participants recruited at baseline (2006-2007) constitute 1066 men and women aged 60 to 75 years with established type 2 diabetes, living in the Lothian region of central Scotland. Subjects underwent detailed cognitive and physical examination, the latter including measures of micro- and macro-vascular disease, glycaemic control, body fat composition and plasma inflammatory markers, cortisol, lipids and liver function tests. Participants were re-examined after one year with hepatic ultrasonography and additional measures of vascular disease. This paper reports the methods of recruitment to the study and examinations performed at baseline and one year. Follow-up cognitive, vascular and liver assessments are scheduled for 2010-2011 when subjects will have been in the study for 4 years. DISCUSSION: This study will provide a wealth of epidemiological and biomarker data that should be invaluable in the identification of potentially modifiable, causal risk factors for diabetes-related cognitive impairment, liver dysfunction and vascular disease, which can be targeted for the development of preventive and therapeutic interventions.

Neuropathic sensitization of behavioral reflexes and spinal NMDA receptor/CaM kinase II interactions are disrupted in PSD-95 mutant mice.

Chronic pain due to nerve injury is resistant to current analgesics. Animal models of neuropathic pain show neuronal plasticity and behavioral reflex sensitization in the spinal cord that depend on the NMDA receptor. We reveal complexes of NMDA receptors with the multivalent adaptor protein PSD-95 in the dorsal horn of spinal cord and show that PSD-95 plays a key role in neuropathic reflex sensitization. Using mutant mice expressing a truncated form of the PSD-95 molecule, we show their failure to develop the NMDA receptor-dependent hyperalgesia and allodynia seen in the CCI model of neuropathic pain, but normal inflammatory nociceptive behavior following the injection of formalin. In wild-type mice following CCI, CaM kinase II inhibitors attenuate sensitization of behavioral reflexes, elevated constitutive (autophosphorylated) activity of CaM kinase II is detected in spinal cord, and increased amounts of phospho-Thr(286) CaM kinase II coimmunoprecipitate with NMDA receptor NR2A/B subunits. Each of these changes is prevented in PSD-95 mutant mice although CaM kinase II is present and can be activated. Disruption of CaM kinase II docking to the NMDA receptor and activation may be responsible for the lack of neuropathic behavioral reflex sensitization in PSD-95 mutant mice.

Role of the conserved NPxxY motif of the 5-HT2A receptor in determining selective interaction with isoforms of ADP-ribosylation factor (ARF).

In this study we have shown that N376 to D mutation in the conserved NPxxY motif within the carboxy terminal tail domain (CT) of the 5-HT2A receptor alters the binding preference of GST-fusion protein constructs of the CT domain from ARF1 to an alternative isoform, ARF6. These findings were corroborated by experiments investigating co-immunoprecipitation of the wild type (WT) and N376D mutant of the 5-HT2A receptor with ARF1 or 6 or dominant negative ARF1/6 constructs co-expressed in COS7 cells. In functional assays of 5-HT-induced phospholipase D (PLD) activation responses of the WT receptor were inhibited by a dominant negative mutant of ARF1 but not ARF6, whereas responses of the N376D mutant were strongly inhibited by negative mutant ARF6. No equivalent effect of the ARF mutants was seen on phospholipase C activation. In experiments assaying 5-HT-induced increases in [35S]GTPgammaS binding to ARF 1/6 immunoprecipitates as a measure of ARF activation, increased ARF6 activation was seen only with the mutant receptor. When cellular PLD responses of other NPxxY- or a DPxxY-containing GPCRs were measured in the presence of dominant negative ARF1/6 constructs, the majority, but not all, fitted the pattern exemplified by the 5-HT2A receptor and its N376D mutant. These data suggest that the presence of the N or a D in this highly conserved motif is an important, but not exclusive, determinant of which ARF isoform interacts with the GPCR.

EP2 receptor antagonism reduces peripheral and central hyperalgesia in a preclinical mouse model of endometriosis.

Endometriosis is an incurable gynecological disorder characterized by debilitating pain and the establishment of innervated endometriosis lesions outside the uterus. In a preclinical mouse model of endometriosis we demonstrated overexpression of the PGE2-signaling pathway (including COX-2, EP2, EP4) in endometriosis lesions, dorsal root ganglia (DRG), spinal cord, thalamus and forebrain. TRPV1, a PGE2-regulated channel in nociceptive neurons was also increased in the DRG. These findings support the concept that an amplification process occurs along the pain neuroaxis in endometriosis. We then tested TRPV1, EP2, and EP4 receptor antagonists: The EP2 antagonist was the most efficient analgesic, reducing primary hyperalgesia by 80% and secondary hyperalgesia by 40%. In this study we demonstrate reversible peripheral and central hyperalgesia in mice with induced endometriosis.

Informing Investment to Reduce Inequalities: A Modelling Approach.

BACKGROUND: Reducing health inequalities is an important policy objective but there is limited quantitative information about the impact of specific interventions. OBJECTIVES: To provide estimates of the impact of a range of interventions on health and health inequalities. MATERIALS AND METHODS: Literature reviews were conducted to identify the best evidence linking interventions to mortality and hospital admissions. We examined interventions across the determinants of health: a 'living wage'; changes to benefits, taxation and employment; active travel; tobacco taxation; smoking cessation, alcohol brief interventions, and weight management services. A model was developed to estimate mortality and years of life lost (YLL) in intervention and comparison populations over a 20-year time period following interventions delivered only in the first year. We estimated changes in inequalities using the relative index of inequality (RII). RESULTS: Introduction of a 'living wage' generated the largest beneficial health impact, with modest reductions in health inequalities. Benefits increases had modest positive impacts on health and health inequalities. Income tax increases had negative impacts on population health but reduced inequalities, while council tax increases worsened both health and health inequalities. Active travel increases had minimally positive effects on population health but widened health inequalities. Increases in employment reduced inequalities only when targeted to the most deprived groups. Tobacco taxation had modestly positive impacts on health but little impact on health inequalities. Alcohol brief interventions had modestly positive impacts on health and health inequalities only when strongly socially targeted, while smoking cessation and weight-reduction programmes had minimal impacts on health and health inequalities even when socially targeted. CONCLUSIONS: Interventions have markedly different effects on mortality, hospitalisations and inequalities. The most effective (and likely cost-effective) interventions for reducing inequalities were regulatory and tax options. Interventions focused on individual agency were much less likely to impact on inequalities, even when targeted at the most deprived communities.

A role of the ubiquitin-proteasome system in neuropathic pain.

Neuropathic pain (characterized by hyperalgesia and allodynia to mechanical and thermal stimuli) causes cellular changes in spinal dorsal horn neurons, some of which parallel those in synaptic plasticity associated with learning. Ubiquitin C-terminal hydrolase (UCH) appears to play a key role in long-term facilitation in Aplysia. The cooperation of UCH with the proteolytic enzyme complex known as the proteasome is required for the degradation of a number of signaling molecules within the cell that may remove normal restraints on synaptic plasticity. We have used electrophysiology, in situ hybridization histochemistry, semiquantitative RT-PCR, Western blotting, and in vivo behavioral reflex analysis to investigate the ubiquitin-proteasome system in a model of neuropathic pain. In neuropathic animals, ionophoretic application of selective proteasome inhibitors attenuated dorsal horn neuron firing evoked by normally innocuous brush or cold stimuli and by noxious mustard oil stimuli. In control animals, only mustard oil-evoked responses were inhibited. Intrathecal administration of proteasome inhibitors attenuated hyperalgesia and allodynia in neuropathic rats. Expression of UCH-L1 (a rat homolog of Aplysia neuronal UCH and of the human UCH-L1, also known as PGP 9.5) and its mRNA were selectively increased within the ipsilateral dorsal horn of neuropathic rats, supporting the idea of a role for the ubiquitin-proteasome system in nociceptive processing. Proteasome inhibitors selectively attenuate allodynic and hyperalgesic responses in neuropathic pain, with some reduction in normal nociceptive, but not non-nociceptive responses, and potentially represent a novel therapeutic strategy for neuropathic pain.

NMDA receptor antagonist treatment at the time of nerve injury prevents injury-induced changes in spinal NR1 and NR2B subunit expression and increases the sensitivity of residual pain behaviours to subsequently administered NMDA receptor antagonists.

Spinal NMDA receptors (NMDA R) are important in neuropathic sensitisation and acute administration of antagonists can provide temporary attenuation of sensitisation. If establishment of the chronic pain state could be prevented by brief administration of such agents at or around the time of nerve injury (pre-emptive analgesia) it might be possible to avoid many of the unacceptable side effects associated with repeated administration of these or other antagonists. Several reports describe aspects of effective pre-emptive analgesia from NMDA R antagonists in animal models of neuropathic pain. The first aim of the present study was to make a direct comparison of changes in mechanical allodynia, cold allodynia and thermal hyperalgesia following nerve injury, demonstrating their increasing degree of susceptibility to pre-emptive NMDA R antagonist treatment. Secondly, we used immunoblotting and immunohistochemistry to investigate the effects of nerve injury on NMDA receptor subunit expression, revealing increased expression of NR2B, but not NR2A and reduced NR1 in the superficial dorsal horn. These changes were attenuated following NMDA receptor antagonist pre-treatment. Thirdly, we investigated the pharmacological properties of residual mechanical allodynia and cold allodynia that remained after pre-emptive treatment and revealed a greater sensitivity to NMDA R antagonists. These findings indicate that in addition to a marked suppression of thermal hyperalgesia and cold allodynia, pre-emptive treatment with NMDA R antagonist causes a lasting change in spinal NMDA R complexes such that remaining mechanical allodynia should be more effectively targeted by NMDA R antagonists.

Functional interactions between native Gs-coupled 5-HT receptors in HEK-293 cells and the heterologously expressed serotonin transporter.

In HEK-293 cells, serotonin (5-hydroxytryptamine, 5-HT) was found to induce cAMP production showing pharmacological characteristics consistent with the 5-HT(7) receptor. The presence of 5-HT(7) (and 5-HT(6)) receptor mRNA was confirmed by RT-PCR. Stable HEK-293 cell lines expressing either wild-type or haemagglutinin (HA)-tagged human 5-HT transporter (SERT) were selected and SERT function was confirmed using [3H]5-HT transport. The presence of SERT caused a 10-fold reduction in the potency of 5-HT-induced cAMP production compared to control cells. Downstream signalling by 5-HT(6/7) receptors could be detected as 5-HT-induced protein kinase A activation and phosphorylation of MAP kinase and CREB using phospho-specific antibodies. SERT inhibitors reversed the reduction in potency of 5-HT-induced cAMP production caused by the presence of SERT, resulting in a concentration-dependent left shift in EC(50) values but also a progressive decrease in the maximal response. Thus, when antidepressants were used to block SERT activity, 5-HT receptor signalling was effectively clamped within a mid-range.