Hypoxia Modulates Platelet Purinergic Signalling Pathways.

BACKGROUND: Hypoxia resulting from ascent to high-altitude or pathological states at sea level is known to increase platelet reactivity. Previous work from our group has suggested that this may be adenosine diphosphate (ADP)-specific. Given the clinical importance of drugs targeting ADP pathways, research into the impact of hypoxia on platelet ADP pathways is highly important. METHODS: Optimul aggregometry was performed on plasma from 29 lowland residents ascending to 4,700 m, allowing systematic assessment of platelet reactivity in response to several platelet agonists. Aggregometry was also performed in response to ADP in the presence of inhibitors of the two main ADP receptors, P2Y1 and P2Y12 (MRS2500 and cangrelor, respectively). Phosphorylation of vasodilator-stimulated phosphoprotein (VASP), a key determinant of platelet aggregation, was analysed using the VASPFix assay. RESULTS: Hypobaric hypoxia significantly reduced the ability of a fixed concentration of cangrelor to inhibit ADP-induced aggregation and increased basal VASP phosphorylation. However, in the absence of P2Y receptor inhibitors, we did not find evidence of increased platelet sensitivity to any of the agonists tested and found reduced sensitivity to thrombin receptor-activating peptide-6 amide. CONCLUSION: Our results provide evidence of increased P2Y1 receptor activity at high altitude and suggest down-regulation of the P2Y12 pathway through increased VASP phosphorylation. These changes in ADP pathway activity are of potential therapeutic significance to high-altitude sojourners and hypoxic sea level patients prescribed platelet inhibitors and warrant further investigation.

Codeine Addiction and Internet Forum Use and Support: Qualitative Netnographic Study.

BACKGROUND: The use of codeine as an analgesic is well-recognized, but there are increasing concerns that for some individuals continued use may lead to misuse, dependence, and fatalities. Research suggests that those affected may represent a hard-to-reach group who do not engage with formal treatment services. OBJECTIVE: This study sought to explore the experiences of people with self-reported addiction to codeine and, specifically, how a social media forum is used to communicate with others about this issue. METHODS: Using a qualitative netnographic methodology, the social media forum Mumsnet was used, with permission, and searches were undertaken in 2016 of any posts that related to codeine and addiction. A total of 95 relevant posts were identified; a purposive sample of 25 posts was selected to undertake subsequent six-stage thematic analysis and development of emerging themes. These 25 posts were posted between 2003 and 2016 and comprised 757 individual posts. RESULTS: Individuals created posts to actively request help in relation to usually their own, but occasionally their partner's or relative's, problems relating to codeine use and self-reported "addiction." Varying levels of detail were provided in narratives of problematic codeine use. There were both positive and negative descriptions of side effects emerging, problems experiencing withdrawal, and failed attempts to discontinue codeine use. Mainly positive and supportive responses to posts were identified from those with either self-reported health profession experience or lay respondents, who often drew on their own experiences of similar problems. Treatment advice emerged in two main ways, either as signposting to formal health services or to informal approaches and often anecdotal advice about how to taper or use cold turkey techniques. Some posts were more critical of the original poster, and arguments and challenges to advice were not uncommon. Shame and stigma were often associated with users' posts and, while there was a desire to receive support and treatment advice in this forum, users often wanted to keep their codeine use hidden in other aspects of their lives. Distinctly different views emerged as to whether responsibility lay with prescribers or patients. Some users expressed anger toward doctors and their prescribing practices. CONCLUSIONS: This study provides a unique insight into how a public internet forum is used by individuals to confirm and seek support about problematic codeine use and of the ways others respond. The pseudonymous use of internet forums for such information and variation in treatment options suggested by often lay respondents suggest that increased formal support and awareness about codeine addiction are needed. There may be opportunities for providing further support directly on such online forums. Improvements in prescribing codeine and in the over-the-counter supply of codeine are required to prevent problematic use from occurring.

A conserved energetic footprint underpins recognition of human leukocyte antigen-E by two distinct αß T cell receptors.

αß T cell receptors (TCRs) interact with peptides bound to the polymorphic major histocompatibility complex class Ia (MHC-Ia) and class II (MHC-II) molecules as well as the essentially monomorphic MHC class Ib (MHC-Ib) molecules. Although there is a large amount of information on how TCRs engage with MHC-Ia and MHC-II, our understanding of TCR/MHC-Ib interactions is very limited. Infection with cytomegalovirus (CMV) can elicit a CD8+ T cell response restricted by the human MHC-Ib molecule human leukocyte antigen (HLA)-E and specific for an epitope from UL40 (VMAPRTLIL), which is characterized by biased TRBV14 gene usage. Here we describe an HLA-E-restricted CD8+ T cell able to recognize an allotypic variant of the UL40 peptide with a modification at position 8 (P8) of the peptide (VMAPRTLVL) that uses the TRBV9 gene segment. We report the structures of a TRBV9+ TCR in complex with the HLA-E molecule presenting the two peptides. Our data revealed that the TRBV9+ TCR adopts a different docking mode and molecular footprint atop HLA-E when compared with the TRBV14+ TCR-HLA-E ternary complex. Additionally, despite their differing V gene segment usage and different docking mechanisms, mutational analyses showed that the TCRs shared a conserved energetic footprint on the HLA-E molecule, focused around the peptide-binding groove. Hence, we provide new insights into how monomorphic MHC molecules interact with T cells.

Web-based interventions for prevention and treatment of perinatal mood disorders: a systematic review.

BACKGROUND: Perinatal depression is strikingly common with a prevalence of 10-15%. The adverse effects of perinatal depression on maternal and child health are profound with considerable costs. Despite this, few women seek medical attention. E-health, providing healthcare via the Internet is an accessible and effective solution for the treatment of depression in the general population. We aimed to conduct a systematic review of web-based interventions for the prevention and treatment of mood disorders in the perinatal period, defined as the start of pregnancy to 1 year post-partum. METHODS: Six databases were searched until 26(th) March 2015. Two researchers independently screened articles for eligibility. Of the 547 screened articles, four met the inclusion criteria. These included three randomised-controlled trials and one feasibility trial, with total data from 1274 participants. MOOSE and PRISMA guidelines were adhered to for the conduct and reporting of the systematic review. RESULTS: All studies were conducted in the post-partum period. All reported an improvement in maternal mood following intervention. A significant improvement in depressive symptoms was measured using validated rating scales, such as the Edinburgh Postnatal Depression Scale (EPDS), either at post-treatment or follow-up which ranged from 3 to 12 months post study completion. For the two RCTs utilising the EPDS, the EPDS score reductions were (mean ± SEM) 8.52 ± 0.22 (Range 19.46 to10.94) and 9.19 ± 0.63 (Range, 20.24 to 11.05) for treatment groups and 5.16 ± 0.25 (Range 19.44 to 14.28) and 6.81 ± 0.71 (Range 21.07 to 14.26) for comparator groups. However attrition within studies ranged from 13 to 61%. One study was rated as 'good' quality. CONCLUSIONS: Preliminary data suggests web-based therapies for perinatal depression delivered in the post-partum period may play a role in improving maternalmood but more studies are needed, particularly with interventions delivered antenatally. Further research is needed to address the limitations of the existing evidence base.

Effect of penetrating brain injury on aquaporin-4 expression using a rat model.

Cerebral edema (CE) is a frequent and potentially lethal consequence of various neurotraumas, including penetrating brain injury (PBI). Aquaporin-4 (AQP4) water channel is predominantly expressed by astrocytes and plays an important role in regulating water balance in the normal and injured brain. Using a rat model of PBI, we show that AQP4 immunoreactivity was substantially increased in the peri-injury area at both 24 and 72 h after PBI. The increase in AQP4 expression was paralleled by increased GFAP expression. The two proteins were co-expressed by peri-vascular astrocytes, whereas reactive astroglia identified by their stellar morphology did not express AQP4 at either time points after injury. Western analysis confirmed the increase in AQP4 immunoreactivity observed in the injured tissue. The apparent increase in AQP4 immunoreactivity was likely due to de novo AQP4 protein synthesis, as most of the increased AQP4 immunoreactivity was found in the soluble (cytosolic) fraction. Our results demonstrate dynamic spatial and temporal changes in AQP4 expression that contribute to the molecular pathophysiology of PBI.

Cullin 5 gene expression in the rat cerebral cortex and hippocampus following traumatic brain injury (TBI).

Cullin-5 (Cul-5), a member of the cullin gene family of scaffold proteins of E3 ubiquitin-ligase complexes, has a role in proteolysis and cell cycle regulation. We recently demonstrated that cul-5 mRNA is ubiquitously expressed in the central nervous system. The present study used quantitative real time polymerase chain reaction and western blotting to measure changes in cul-5 mRNA and Cul-5 protein expression, respectively, in the injured CNS in response to traumatic brain injury (TBI). cul-5 mRNA levels were significantly decreased in the ipsilateral rat cerebral cortex on Days 1 and 7, but not on Day 3 following TBI. In the ipsilateral hippocampus, cul-5 mRNA was significantly reduced on Day 1 after TBI. Cul-5 protein levels were significantly decreased in the ipsilateral rat cerebral cortex on Days 1-7 post-TBI while levels were significantly lower in the ipsilateral hippocampus on Days 3-7 post-TBI. Since Cul-5 is ubiquitously expressed in eukaryotic cells and is linked to proteasome-mediated protein degradation, it may have a role in CNS cell fate determination under conditions of traumatic stress.

Structural basis for a major histocompatibility complex class Ib-restricted T cell response.

In contrast to antigen-specific immunity orchestrated by major histocompatibility complex (MHC) class Ia molecules, the ancestrally related nonclassical MHC class Ib molecules generally mediate innate immune responses. Here we have demonstrated the structural basis by which the MHC class Ib molecule HLA-E mediates an adaptive MHC-restricted cytotoxic T lymphocyte response to human cytomegalovirus. Highly constrained by host genetics, the response showed notable fine specificity for position 8 of the viral peptide, which is the sole discriminator of self versus nonself. Despite the evolutionary divergence of MHC class Ia and class Ib molecules, the structure of the T cell receptor-MHC class Ib complex was very similar to that of conventional T cell receptor-MHC class Ia complexes. These results emphasize the evolutionary 'ambiguity' of HLA-E, which not only interacts with innate immune receptors but also has the functional capacity to mediate virus-specific cytotoxic T lymphocyte responses during adaptive immunity.

Progesterone differentially regulates pro- and anti-apoptotic gene expression in cerebral cortex following traumatic brain injury in rats.

Although the administration of progesterone has been shown to be neuroprotective in experimental models of traumatic brain injury (TBI), the mechanisms for this beneficial effect are still poorly understood. The present study examined the effects of progesterone on mRNA and protein levels of the Bcl-2 apoptosis regulatory genes, bax, bad, bcl-2, and bcl-x(L), in cerebral cortex after TBI. Male Sprague-Dawley rats were subjected to either sham surgery or lateral fluid percussion brain injury of moderate severity (2.4-2.6 atm). Within 1 h post-surgery, progesterone (4 mg/kg) or vehicle (corn oil) administration was initiated for 1-7 days postoperatively. Our results indicate that bax and bad mRNA levels and Bax and Bad protein expression in the ipsilateral, injured cerebral cortex were significantly elevated post-TBI, while mRNA levels of bcl-2 and bcl-x(L) or Bcl-2 and Bcl-x(L) protein expression were not changed. Under the sham-treated condition, progesterone significantly increased mRNA levels of the anti-apoptotic gene, bcl-2, but down-regulated pro-apoptotic gene expression (bax and bad) in cerebral cortex. After TBI, progesterone treatment reduced bax and bad mRNA levels in the ipsilateral cerebral cortex of TBI rats, and decreased Bax and Bad protein levels. In addition, bcl-2 and bcl-x(L) mRNA levels, as well as Bcl-2 and Bcl-x(L) protein expression, were increased by progesterone in TBI injured cortex. These data indicate that one of the neuroprotective mechanisms of progesterone may be related to its differential regulation of apoptotic signals.

Traumatic brain injury in the rat using the fluid-percussion model.

Traumatic brain injury is a leading cause of death and disability, particularly among young adults. During closed head trauma, the injury process is initiated by the impact of the brain against the inner table of the calvarium. Subsequently, there is prompt initiation of a complex biochemical, cellular, and physiological injury cascade that may take days to complete. From a functional standpoint, this culminates in neurologic dysfunction and, if severe, death. This unit describes an impact-induced brain trauma model in rats which replicates nonpenetrating head injury. It does not model either penetrating or ischemic brain injuries.

Low doses of dizocilpine block the development and subsequent expression of locomotor sensitization to nicotine in rats.

RATIONALE AND OBJECTIVES: We attempted to determine if the effects of the glutamate NMDA receptor blocker dizocilpine (MK-801) on nicotine locomotor sensitization were due to a blockade of the development of sensitization or to state-dependency. METHODS AND RESULTS: In experiment 1, repeated co-administration of a high dose of dizocilpine (0.25 mg/kg) along with 0.4 mg/kg nicotine enhanced locomotion, failed to alter the development of locomotor sensitization to nicotine, but completely blocked the subsequent expression of sensitization to a challenge injection of nicotine alone. However, repeated injections of this dose of dizocilpine alone produced locomotion and sensitization that was equivalent to that produced by the dizocilpine/nicotine combination. In four separate replications in experiments 2 and 3, co-administration of a low dose of dizocilpine (0.075 mg/kg), which did not produce sensitization to itself, blocked both the development of nicotine sensitization and its subsequent expression in response to a challenge injection of nicotine. Moreover, this repeated dizocilpine/nicotine administration did not affect the subsequent development of sensitization to nicotine alone (experiment 3). Suggesting that these effects of dizocilpine may be confined to the development of sensitization, challenge injections of dizocilpine failed to block the capacity to express previously nicotine-sensitized locomotion (experiment 2). CONCLUSIONS: Co-administration of a low dose of dizocilpine can block the development of locomotor sensitization to repeated injections of nicotine without producing state-dependency. Thus, NMDA receptor activation appears to be critical for the development, but not the subsequent expression, of nicotine locomotor sensitization.