Effects of HIV-1 Tat on enteric neuropathogenesis.

The gastrointestinal (GI) tract presents a major site of immune modulation by HIV, resulting in significant morbidity. Most GI processes affected during HIV infection are regulated by the enteric nervous system. HIV has been identified in GI histologic specimens in up to 40% of patients, and the presence of viral proteins, including the trans-activator of transcription (Tat), has been reported in the gut indicating that HIV itself may be an indirect gut pathogen. Little is known of how Tat affects the enteric nervous system. Here we investigated the effects of the Tat protein on enteric neuronal excitability, proinflammatory cytokine release, and its overall effect on GI motility. Direct application of Tat (100 nm) increased the number of action potentials and reduced the threshold for action potential initiation in isolated myenteric neurons. This effect persisted in neurons pretreated with Tat for 3 d (19 of 20) and in neurons isolated from Tat(+) (Tat-expressing) transgenic mice. Tat increased sodium channel isoforms Nav1.7 and Nav1.8 levels. This increase was accompanied by an increase in sodium current density and a leftward shift in the sodium channel activation voltage. RANTES, IL-6, and IL-1ß, but not TNF-α, were enhanced by Tat. Intestinal transit and cecal water content were also significantly higher in Tat(+) transgenic mice than Tat(-) littermates (controls). Together, these findings show that Tat has a direct and persistent effect on enteric neuronal excitability, and together with its effect on proinflammatory cytokines, regulates gut motility, thereby contributing to GI dysmotilities reported in HIV patients.

Cannabinoid receptor-interacting protein 1a modulates CB1 receptor signaling and regulation.

Cannabinoid CB1 receptors (CB1Rs) mediate the presynaptic effects of endocannabinoids in the central nervous system (CNS) and most behavioral effects of exogenous cannabinoids. Cannabinoid receptor-interacting protein 1a (CRIP1a) binds to the CB1R C-terminus and can attenuate constitutive CB1R-mediated inhibition of Ca(2+) channel activity. We now demonstrate cellular colocalization of CRIP1a at neuronal elements in the CNS and show that CRIP1a inhibits both constitutive and agonist-stimulated CB1R-mediated guanine nucleotide-binding regulatory protein (G-protein) activity. Stable overexpression of CRIP1a in human embryonic kidney (HEK)-293 cells stably expressing CB1Rs (CB1-HEK), or in N18TG2 cells endogenously expressing CB1Rs, decreased CB1R-mediated G-protein activation (measured by agonist-stimulated [(35)S]GTPγS (guanylyl-5'-[O-thio]-triphosphate) binding) in both cell lines and attenuated inverse agonism by rimonabant in CB1-HEK cells. Conversely, small-interfering RNA-mediated knockdown of CRIP1a in N18TG2 cells enhanced CB1R-mediated G-protein activation. These effects were not attributable to differences in CB1R expression or endocannabinoid tone because CB1R levels did not differ between cell lines varying in CRIP1a expression, and endocannabinoid levels were undetectable (CB1-HEK) or unchanged (N18TG2) by CRIP1a overexpression. In CB1-HEK cells, 4-hour pretreatment with cannabinoid agonists downregulated CB1Rs and desensitized agonist-stimulated [(35)S]GTPγS binding. CRIP1a overexpression attenuated CB1R downregulation without altering CB1R desensitization. Finally, in cultured autaptic hippocampal neurons, CRIP1a overexpression attenuated both depolarization-induced suppression of excitation and inhibition of excitatory synaptic activity induced by exogenous application of cannabinoid but not by adenosine A1 agonists. These results confirm that CRIP1a inhibits constitutive CB1R activity and demonstrate that CRIP1a can also inhibit agonist-stimulated CB1R signaling and downregulation of CB1Rs. Thus, CRIP1a appears to act as a broad negative regulator of CB1R function.

Racial discrimination and depressive symptoms mediated by conversations about race among students of color.

Racial discrimination is associated with adverse mental health outcomes among Students of Color. In order to address racial tensions, it is important to consider students' dialogues about race. The current study tested whether having positive and negative conversations about one's ethnic-racial group mediated the relation between racial discrimination at T1 and depressive symptoms 5 months later at T2 among 94 college Students of Color. Findings indicated that greater racial discrimination at T1 was associated with more frequent negative conversations about race at T2 (b = .38, p = .00), which was, in turn, associated with greater depressive symptoms at T2 (b = 2.73, p = .04); this pathway demonstrated significant mediation. However, positive conversations about race was not a significant mediator in this association. The current study highlights the importance of focusing on racial conversations after racial discrimination in order to minimize adverse effects on mental health among Students of Color.

The role of ß-arrestin2 in the mechanism of morphine tolerance in the mouse and guinea pig gastrointestinal tract.

ß-Arrestin2 has been reported to play an essential role in analgesic tolerance. Analgesic tolerance without concomitant tolerance to constipation is a limiting side effect of chronic morphine treatment. Because tolerance to morphine develops in the mouse ileum but not the colon, we therefore examined whether the role of ß-arrestin2 in the mechanism of morphine tolerance differs in the ileum and colon. In both guinea pig and mouse, chronic in vitro exposure (2 h, 10 µM) to morphine resulted in tolerance development in the isolated ileum but not the colon. The IC(50) values for morphine-induced inhibition of electrical field stimulation contraction of guinea pig longitudinal muscle myenteric plexus shifted rightward in the ileum from 5.7 ± 0.08 (n = 9) to 5.45 ± 0.09 (n = 6) (p < 0.001) after morphine exposure. A significant shift was not observed in the colon. Similar differential tolerance was seen between the mouse ileum and the colon. However, tolerance developed in the colon from ß-arrestin2 knockout mice. ß-Arrestin2 and extracellular signal-regulated kinase 1/2 expression levels were determined further by Western blot analyses in guinea pig longitudinal muscle myenteric plexus. A time-dependent decrease in the expression of ß-arrestin2 and extracellular signal-regulated kinase 1/2 occurred in the ileum but not the colon after 2 h of morphine (10 µM) exposure. Naloxone prevented the decrease in ß-arrestin2. In the isolated ileum from guinea pigs chronically treated in vivo with morphine for 7 days, neither additional tolerance to in vitro exposure of morphine nor a decrease in ß-arrestin2 occurred. We conclude that a decrease in ß-arrestin2 is associated with tolerance development to morphine in the gastrointestinal tract.

Measuring sexual relationship power equity among young women and young men South Africa: Implications for gender-transformative programming.

INTRODUCTION: Measures used to assess equitable relationship dynamics, including the sexual relationship power scale (SRPS) have previously been associated with lower HIV-risk among young women, and reduced perpetration of intimate partner violence among men. However, few studies describe how the SRPS has been adapted and validated for use within global youth sexual health studies. We examined gender-specific psychometric properties, reliability, and validity of a SRPS used within a South African youth-engaged cohort study. METHODS: Young men and women (16-24 years) enrolled in community-based cohorts in Durban and Soweto (2014-2016) reporting a primary partner at 6-month follow-up completed a 13-item (strongly agree/agree/disagree/strongly disagree) South African adaptation of Pulerwitz's SRPS (range 13-52, higher scores indicating greater sexual relationship power [SRP] equity). SRPS modifications were made using gender-specific exploratory factor analyses (EFAs), removing items with factor loadings <0.3. Cronbach alphas were conducted for full and modified scales by gender. Using modified scales, unadjusted and adjusted regression models examined associations between 1. relevant socio-demographic and relationship determinants and SRP equity, and 2. SRP equity and sexual relationship related outcomes. All models adjusted for education, age, site, and current employment. RESULTS: 235 sexually-active youth (66% women, median age = 20) were included. Mean scores across all 13 scale items were 2.71 (SD 0.30) for women and 2.70 (SD 0.4) for men. Scale Cronbach's alphas were 0.63 for women and 0.64 for men. EFAs resulted in two gender-specific single-factor SRPS. Modified SRPS Cronbach alphas increased to 0.67 for women (8-items) and 0.70 for men (9-items). After adjusting for age, site and current employment, higher education remained associated with SRP equity across genders. In adjusted models, correlates of SRP equity included primary partnerships that were age-similar (<5 years older) and <2 years in length for women and living in Soweto and younger age for men. Greater SRP equity among women was also independently associated with no recent partner violence. CONCLUSIONS: Results highlight important gender differences in SRP equity measures and associations, highlighting the critically need for future research to examine gendered constructions of SRP equity in order to accurately develop, validate and use appropriate measures within quantitative surveys.

Global Health and Emergency Care: Defining Clinical Research Priorities.

OBJECTIVES: Despite recent strides in the development of global emergency medicine (EM), the field continues to lag in applying a scientific approach to identifying critical knowledge gaps and advancing evidence-based solutions to clinical and public health problems seen in emergency departments (EDs) worldwide. Here, progress on the global EM research agenda created at the 2013 Academic Emergency Medicine Global Health and Emergency Care Consensus Conference is evaluated and critical areas for future development in emergency care research internationally are identified. METHODS: A retrospective review of all studies compiled in the Global Emergency Medicine Literature Review (GEMLR) database from 2013 through 2015 was conducted. Articles were categorized and analyzed using descriptive quantitative measures and structured data matrices. The Global Emergency Medicine Think Tank Clinical Research Working Group at the Society for Academic Emergency Medicine 2016 Annual Meeting then further conceptualized and defined global EM research priorities utilizing consensus-based decision making. RESULTS: Research trends in global EM research published between 2013 and 2015 show a predominance of observational studies relative to interventional or descriptive studies, with the majority of research conducted in the inpatient setting in comparison to the ED or prehospital setting. Studies on communicable diseases and injury were the most prevalent, with a relative dearth of research on chronic noncommunicable diseases. The Global Emergency Medicine Think Tank Clinical Research Working Group identified conceptual frameworks to define high-impact research priorities, including the traditional approach of using global burden of disease to define priorities and the impact of EM on individual clinical care and public health opportunities. EM research is also described through a population lens approach, including gender, pediatrics, and migrant and refugee health. CONCLUSIONS: Despite recent strides in global EM research and a proliferation of scholarly output in the field, further work is required to advocate for and inform research priorities in global EM. The priorities outlined in this paper aim to guide future research in the field, with the goal of advancing the development of EM worldwide.

Physical and Psychological Effects of a 12-Session Cancer Rehabilitation Exercise Program.

BACKGROUND: The positive effects of regular exercise for cancer survivors are becoming increasingly apparent. However, comprehensive examination of the benefits of modest levels of physical activity is somewhat lacking. OBJECTIVES: This study aimed to test the hypothesis that participating in a 12-session exercise program will improve depression, fatigue, aerobic endurance, muscular strength, and quality of life (QOL) in patients with cancer. METHODS: A group of 20 older adult women with a prior cancer diagnosis were evaluated during a 6- to 10-week exercise program that occurred twice weekly. The majority of patients had breast cancer (n = 14), but treatment status varied (11 were currently undergoing treatment, and 9 were post-treatment). Each patient completed initial and exit assessments, which consisted of three physical function tests and three psychosocial questionnaires. Patient charts contained the initial and final assessment scores and personal demographics. FINDINGS: Analyses of pre- and postprogram data using paired t tests revealed that 12 exercise sessions (each lasting about an hour) significantly improved six-minute walk test, 30-second sit-and-stand test, hand grip strength test (dominant and nondominant hand), and overall QOL scores in patients. As a result, moderate levels of exercise have a beneficial effect in this population.

Pain Assessment for the Dementing Elderly (PADE): reliability and validity of a new measure.

OBJECTIVES: To establish the reliability and validity of a measure to assess pain in individuals with advanced dementia. DESIGN: Sixty-five residents of long-term care facilities were assessed using a new rating tool, the Pain Assessment for the Dementing Elderly (PADE), in two separate studies: (1) Residents were assessed simultaneously by two different raters, at Time 1 and 2, to establish interrater reliability, stability, and internal consistency. (2) Validity was established by assessing the correlation between an agitation scale and the PADE; by comparing groups with pain as a significant clinical factor (as assessed by an independent rater) versus not a significant factor, and by assessing individuals receiving versus not receiving psychoactive medications. SETTING: Four different long-term care facilities, three skilled nursing facilities, and a locked dementia assisted-living facility. PARTICIPANTS: Twenty-five residents of long-term care facilities with advanced levels of dementia in Study 1, and 40 residents with similar level of dementia in Study 2; 42% of the total sample were rated as having significant painful conditions. MEASUREMENTS: For Study 1, the PADE was administered; for Study 2, the PADE and the Cohen-Mansfield Agitation Inventory (CMAI) were administered. RESULTS: Reliability coefficients were adequate (interrater = 0.54-0.95; stability = 0.70-0.98; and internal consistency = 0.24-0.88). Validity coefficients were likewise encouraging, with the PADE demonstrating the expected relationship with a measure of agitation. The PADE also differentiated between groups that were independently judged to suffer clinically problematic pain versus those who were not. CONCLUSION: The PADE is a reliable and valid tool to assess pain in dementing elderly residents of long-term care facilities.

Morphine dependence in single enteric neurons from the mouse colon requires deletion of ß-arrestin2.

Chronic administration of morphine results in the development of tolerance to the analgesic effects and to inhibition of upper gastrointestinal motility but not to colonic motility, resulting in persistent constipation. In this study we examined the effect of chronic morphine in myenteric neurons from the adult mouse colon. Similar to the ileum, distinct neuronal populations exhibiting afterhyperpolarization (AHP)-positive and AHP-negative neurons were identified in the colon. Acute morphine (3 µM) decreased the number of action potentials, and increased the threshold for action potential generation indicative of reduced excitability in AHP-positive neurons. In neurons from the ileum of mice that were rendered antinociceptive tolerant by morphine-pellet implantation for 5 days, the opioid antagonist naloxone precipitated withdrawal as evidenced by increased neuronal excitability. Overnight incubation of ileum neurons with morphine also resulted in enhanced excitability to naloxone. Colonic neurons exposed to long-term morphine, remained unresponsive to naloxone suggesting that precipitated withdrawal does not occur in colonic neurons. However, morphine-treated colonic neurons from ß-arrestin2 knockout mice demonstrated increased excitability upon treatment with naloxone as assessed by change in rheobase, number of action potentials and input resistance. These data suggest that similar to the ileum, acute exposure to morphine in colonic neurons results in reduced excitability due to inhibition of sodium currents. However, unlike the ileum, dependence to chronic exposure of morphine develops in colonic neurons from the ß-arrestin2 knockout mice. These studies corroborate the in-vivo findings of the differential role of neuronal ß-arrestin2 in the development of morphine tolerance/dependence in the ileum and colon.

An in-vitro preparation of isolated enteric neurons and glia from the myenteric plexus of the adult mouse.

The enteric nervous system is a vast network of neurons and glia running the length of the gastrointestinal tract that functionally controls gastrointestinal motility. A procedure for the isolation and culture of a mixed population of neurons and glia from the myenteric plexus is described. The primary cultures can be maintained for over 7 days, with connections developing among the neurons and glia. The longitudinal muscle strip with the attached myenteric plexus is stripped from the underlying circular muscle of the mouse ileum or colon and subjected to enzymatic digestion. In sterile conditions, the isolated neuronal and glia population are preserved within the pellet following centrifugation and plated on coverslips. Within 24-48 hr, neurite outgrowth occurs and neurons can be identified by pan-neuronal markers. After two days in culture, isolated neurons fire action potentials as observed by patch clamp studies. Furthermore, enteric glia can also be identified by GFAP staining. A network of neurons and glia in close apposition forms within 5-7 days. Enteric neurons can be individually and directly studied using methods such as immunohistochemistry, electrophysiology, calcium imaging, and single-cell PCR. Furthermore, this procedure can be performed in genetically modified animals. This methodology is simple to perform and inexpensive. Overall, this protocol exposes the components of the enteric nervous system in an easily manipulated manner so that we may better discover the functionality of the ENS in normal and disease states.

Morphine decreases enteric neuron excitability via inhibition of sodium channels.

Gastrointestinal peristalsis is significantly dependent on the enteric nervous system. Constipation due to reduced peristalsis is a major side-effect of morphine, which limits the chronic usefulness of this excellent pain reliever in man. The ionic basis for the inhibition of enteric neuron excitability by morphine is not well characterized as previous studies have mainly utilized microelectrode recordings from whole mount myenteric plexus preparations in guinea pigs. Here we have developed a Swiss-Webster mouse myenteric neuron culture and examined their electrophysiological properties by patch-clamp techniques and determined the mechanism for morphine-induced decrease in neuronal excitability. Isolated neurons in culture were confirmed by immunostaining with pan-neuronal marker, ß-III tubulin and two populations were identified by calbindin and calretinin staining. Distinct neuronal populations were further identified based on the presence and absence of an afterhyperpolarization (AHP). Cells with AHP expressed greater density of sodium currents. Morphine (3 µM) significantly reduced the amplitude of the action potential, increased the threshold for spike generation but did not alter the resting membrane potential. The decrease in excitability resulted from inhibition of sodium currents. In the presence of morphine, the steady-state voltage dependence of Na channels was shifted to the left with almost 50% of channels unavailable for activation from hyperpolarized potentials. During prolonged exposure to morphine (two hours), action potentials recovered, indicative of the development of tolerance in single enteric neurons. These results demonstrate the feasibility of isolating mouse myenteric neurons and establish sodium channel inhibition as a mechanism for morphine-induced decrease in neuronal excitability.

Initial predictors associated with outcome in injured multiple traumatic limb amputations: a Kandahar-based combat hospital experience.

INTRODUCTION: Improvised explosive devices (IEDs) are the defining mechanism of injury during Operation Enduring Freedom. This is a retrospective analysis of initial management for IED blast injuries presenting with bilateral, traumatic, lower-extremity (LE) amputations with and without pelvic and perineal involvement. METHODS: A database of trauma admissions presenting to a North Atlantic Treaty Organization (NATO) Role 3 combat hospital in southern Afghanistan over a 7-month period was created to evaluate the care of this particular injury pattern. Patients were included if they were received from point of injury with at least bilateral traumatic LE amputations and had vital signs with initial resuscitation efforts. RESULTS: Thirty-two presented with double LE amputations (36%) and nine with triple amputations (10%). After excluding 10 patients who failed to meet the inclusion criteria, 22 patients were analysed. The mean age was 29 years, and the average ISS and admission haemoglobin were 22 and 11.3mgl(-1), respectively. Patients received an average of 54 units of blood products and underwent 1.6 operations with a mean operative time of 142.5min. The pattern of injury was associated with an increase in the total blood products required for resuscitation (pelvis n=12, p=0.028, gastrointestinal tract (GI) n=14, p=0.02, perineal n=15, p=0.036). There was no relationship between ISS or admission haemoglobin and the need for massive transfusion. Low Glasgow Coma Scale (GCS) was associated with increased 30-day mortality. Hollow viscus injury and operative hemipelvectomy were also associated with mortality. CONCLUSIONS: Early 30-day follow-up demonstrated that IED injuries with bilateral LE amputations with and without pelvic and perineal involvement are survivable injuries. Standard measures of injury and predictors of survival bore little relationship to observed outcomes and may need to be re-evaluated. Long-term follow-up is needed to assess the extent of functional recovery and overall morbidity and mortality.

Cannabinoid CB1 receptor-interacting proteins: novel targets for central nervous system drug discovery?

The main pharmacological effects of marijuana, as well as synthetic and endogenous cannabinoids, are mediated through G-protein-coupled receptors (GPCRs), including CB(1) and CB(2) receptors. The CB(1) receptor is the major cannabinoid receptor in the central nervous system and has gained increasing interest as a target for drug discovery for treatment of nausea, cachexia, obesity, pain, spasticity, neurodegenerative diseases and mood and substance abuse disorders. Evidence has accumulated to suggest that CB(1) receptors, like other GPCRs, interact with and are regulated by several other proteins beyond the established role of heterotrimeric G-proteins. These proteins, which include the GPCR kinases, beta-arrestins, GPCR-associated sorting proteins, factor associated with neutral sphingomyelinase, other GPCRs (heterodimerization) and the novel cannabinoid receptor-interacting proteins: CRIP(1a/b), are thought to play important roles in the regulation of intracellular trafficking, desensitization, down-regulation, signal transduction and constitutive activity of CB(1) receptors. This review examines CB(1) receptor-interacting proteins, including heterotrimeric G-proteins, but with particular emphasis on non-G-protein entities, that might comprise the CB(1) receptosomal complex. The evidence for direct interaction with CB(1) receptors and potential functional roles of these interacting proteins is discussed, as are future directions and challenges in this field with an emphasis on the possibility of eventually targeting these proteins for drug discovery.

FAAH-/- mice display differential tolerance, dependence, and cannabinoid receptor adaptation after delta 9-tetrahydrocannabinol and anandamide administration.

Repeated administration of Delta(9)-tetrahydrocannabinol (THC), the primary psychoactive constituent of Cannabis sativa, induces profound tolerance that correlates with desensitization and downregulation of CB(1) cannabinoid receptors in the CNS. However, the consequences of repeated administration of the endocannabinoid N-arachidonoyl ethanolamine (anandamide, AEA) on cannabinoid receptor regulation are unclear because of its rapid metabolism by fatty acid amide hydrolase (FAAH). FAAH(-/-) mice dosed subchronically with equi-active maximally effective doses of AEA or THC displayed greater rightward shifts in THC dose-effect curves for antinociception, catalepsy, and hypothermia than in AEA dose-effect curves. Subchronic THC significantly attenuated agonist-stimulated [(35)S]GTP gamma S binding in brain and spinal cord, and reduced [(3)H]WIN55,212-2 binding in brain. Interestingly, AEA-treated FAAH(-/-) mice showed less CB(1) receptor downregulation and desensitization than THC-treated mice. Experiments examining tolerance and cross-tolerance indicated that the behavioral effects of THC, a low efficacy CB(1) receptor agonist, were more sensitive to receptor loss than those of AEA, a higher efficacy agonist, suggesting that the expression of tolerance was more affected by the intrinsic activity of the ligand at testing than during subchronic treatment. In addition, the CB(1) receptor antagonist, rimonabant, precipitated a markedly reduced magnitude of withdrawal in FAAH(-/-) mice treated subchronically with AEA compared with mice treated repeatedly with THC. The findings that repeated AEA administration produces lesser adaptive changes at the CB(1) receptor and has reduced dependence liability compared with THC suggest that pharmacotherapies targeting endocannabinoid catabolic enzymes are less likely to promote tolerance and dependence than direct acting CB(1) receptor agonists.

A pilot study showing an association between platelet hyperactivity and the severity of peripheral arterial disease.

Peripheral arterial disease (PAD) is a chronic fibroproliferative inflammatory condition associated with progressive vascular stenosis. We set out to determine the relationship between spontaneous stirring-induced platelet aggregation in whole blood and the severity of lower-limb PAD, represented by the ankle-brachial pressure index (ABPI). ABPI was determined pre- and post-exercise in 31 subjects (20 men and 11 women) with established PAD. Platelets counts were determined in EDTA blood (total count) and in citrated whole blood after stirring in the absence of added ADP (spontaneous aggregation) and in the presence of added ADP (ADP-induced) for 3 min at 37 degrees C. Aggregation was calculated as a percentage of the total platelet count. Spontaneous platelet aggregation showed an inverse correlation with pre-exercise ABPI (r = -0.32; P < 0.05) and ADP-induced aggregation correlated inversely with post-exercise ABPI (r = -0.34; P < 0.05). These results indicate that platelet hyperactivity is associated with increasing severity of PAD. Increased platelet aggregation may result in thromboembolic events in the affected limb.