How selective antagonists and genetic modification have helped characterise the expression and functions of vascular P2Y receptors.

Vascular P2Y receptors mediate many effects, but the role of individual subtypes is often unclear. Here we discuss how subtype-selective antagonists and receptor knockout/knockdown have helped identify these roles in numerous species and vessels. P2Y1 receptor-mediated vasoconstriction and endothelium-dependent vasodilation have been characterised using the selective antagonists, MRS2179 and MRS2216, whilst AR-C118925XX, a P2Y2 receptor antagonist, reduced endothelium-dependent relaxation, and signalling evoked by UTP or fluid shear stress. P2Y2 receptor knockdown reduced endothelial signalling and endothelial P2Y2 receptor knockout produced hypertensive mice and abolished vasodilation elicited by an increase in flow. UTP-evoked vasoconstriction was also blocked by AR-C118925XX, but the effects of P2Y2 receptor knockout were complex. No P2Y4 receptor antagonists are available and P2Y4 knockout did not affect the vascular actions of UTP and UDP. The P2Y6 receptor antagonist, MRS2578, identified endothelial P2Y6 receptors mediating vasodilation, but receptor knockout had complex effects. MRS2578 also inhibited, and P2Y6 knockout abolished, contractions evoked by UDP. P2Y6 receptors contribute to the myogenic tone induced by a stepped increase in vascular perfusion pressure and possibly to the development of atherosclerosis. The P2Y11 receptor antagonists, NF157 and NF340, inhibited ATP-evoked signalling in human endothelial cells. Vasoconstriction mediated by P2Y12/P2Y13 and P2Y14 receptors was characterised using the antagonists, cangrelor, ticagrelor, AR-C67085 and MRS2211 or PPTN respectively. This has yet to be backed up by receptor knockout experiments. Thus, subtype-selective antagonists and receptor knockout/knockdown have helped identify which P2Y subtypes are functionally expressed in vascular smooth muscle and endothelial cells and the effects that they mediate.

Characterisation of P2Y receptor subtypes mediating vasodilation and vasoconstriction of rat pulmonary artery using selective antagonists.

Pulmonary vascular tone is modulated by nucleotides, but which P2 receptors mediate these actions is largely unclear. The aim of this study, therefore, was to use subtype-selective antagonists to determine the roles of individual P2Y receptor subtypes in nucleotide-evoked pulmonary vasodilation and vasoconstriction. Isometric tension was recorded from rat intrapulmonary artery rings (i.d. 200-500 µm) mounted on a wire myograph. Nucleotides evoked concentration- and endothelium-dependent vasodilation of precontracted tissues, but the concentration-response curves were shallow and did not reach a plateau. The selective P2Y2 antagonist, AR-C118925XX, inhibited uridine 5'-triphosphate (UTP)- but not adenosine 5'-triphosphate (ATP)-evoked relaxation, whereas the P2Y6 receptor antagonist, MRS2578, had no effect on UTP but inhibited relaxation elicited by uridine 5'-diphosphate (UDP). ATP-evoked relaxations were unaffected by the P2Y1 receptor antagonist, MRS2179, which substantially inhibited responses to adenosine 5'-diphosphate (ADP), and by the P2Y12/13 receptor antagonist, cangrelor, which potentiated responses to ADP. Both agonists were unaffected by CGS1593, an adenosine receptor antagonist. Finally, AR-C118925XX had no effect on vasoconstriction elicited by UTP or ATP at resting tone, although P2Y2 receptor mRNA was extracted from endothelium-denuded tissues using reverse transcription polymerase chain reaction with specific oligonucleotide primers. In conclusion, UTP elicits pulmonary vasodilation via P2Y2 receptors, whereas UDP acts at P2Y6 and ADP at P2Y1 receptors, respectively. How ATP induces vasodilation is unclear, but it does not involve P2Y1, P2Y2, P2Y12, P2Y13, or adenosine receptors. UTP- and ATP-evoked vasoconstriction was not mediated by P2Y2 receptors. Thus, this study advances our understanding of how nucleotides modulate pulmonary vascular tone.

Comparison of early and conventional autogenous secondary alveolar bone graft in children with cleft lip and palate: A systematic review.

PURPOSE: The literature is scarce on studies comparing secondary alveolar bone graft (SABG) performed early at approximately 5-6 years and at the conventional time at 9-11 years. This systematic literature review(SLR) aimed to compare clinical outcomes after two different timings of SABG in children with unilateral and bilateral cleft lip and palate. METHODS: The inclusion criteria were autogenous iliac grafts and the following study designs: case control, cohort, clinical controlled trial (CCT), randomized CCT (RCCT), and previous SLRs. Ovid MEDLINE, Ovid EMBASE, Web of Science, Scopus, Cochrane, ProQuest and Google Scholar were the primary databases. Two calibrated examiners worked independently to select the articles. The MINORS evaluation method for surgical non-RCTs was used to assess for quality. RESULTS: 1,111 articles were retrieved and 19 qualified. Different clinical and radiographic outcomes such as bone level, periodontal status, canine eruption and cleft-side tooth survival were evaluated by different assessment methods such as CBCT volume, computed tomography, periodontal evaluation, panoramic, intraoral radiographs, and Bergland scale. No RCCT or meta-analysis was found. None of the studies received the ideal score, which is 16 for non-comparison studies and 24 for comparison studies. CONCLUSION: Methodological variation, lack of standardization for initial cleft dimension and low-quality level rendered a fair comparison unfeasible. Although further studies are necessary, it can be assumed that early SABG also can be an acceptable option, but this was based on a single study with a reasonable level of evidence.

Using centrally held data to validate carotid surgery outcome data.

BACKGROUND AND PURPOSE: Outcome audit data for peer group comparison must be transparent, objective, and independently reproducible. Personal data sets are difficult to maintain and often lack complete follow-up. Local coding difficulties make initial retrieval of centrally held data unreliable. However starting with a complete list of interventions, reliable identification of patients who have experienced an adverse postoperative event may be possible using record linkages. METHODS: A surgical database, augmented by a hand-search of all theater registries and personal logbooks, identified 378 carotid endarterectomies performed for stroke prevention in symptomatic patients, in a single hospital between 2002 and 2009. A list of the names, unique patient identifiers, and operation dates was sent to the Information Services Division of National Health Service Scotland. Data were requested pertaining to all deaths and potential diagnoses of stroke after surgery. Every identified case was scrutinized. RESULTS: There were 30 (8%) readmissions or transfers of care identified within 30 days of surgery. From this, 12 strokes were identified with another 2 strokes, occurring without readmission, diagnosed in the outpatient clinic. Only 6 of the postoperative strokes were identified during the index admission. There were 2 early deaths resulting in a combined stroke and death rate of 4.2% (95% confidence intervals, 2.4%-6.9%). CONCLUSIONS: These outcome data are similar to the outcomes of the major carotid surgery trials. Record-linked data retrieval seems to be an appropriate starting point for outcome-based audit. This has the potential to generate robust, transparent data for comparison between individuals and centers for a specific procedure.

Acute acalculous cholecystitis-like phenotype in scavenger receptor A knock-out mice.

BACKGROUND: Sepsis is a major health problem in the United States that affects more than three-quarters of a million people every year. Previous studies have shown that scavenger receptor A (Sra), also known as macrophage scavenger receptor 1 (Msr1), is a modifier of interleukin 10 (IL-10) expression after injection of bacterial lipopolysaccharide (LPS). Therefore, we investigated the response to sepsis in Sra knock out mice. MATERIALS AND METHODS: C57BL/6J (B6) (n = 88) and Sra (-/-) mice (n = 88) were subjected to cecal ligation and puncture (CLP) using 18G or 16G needles, sham operation, or non-operated controls. At the end, mice were autopsied for the determination of abnormalities after the procedure. Cytokine gene expression was examined in lung and liver samples by quantitative RT-PCR (qRT-PCR), and circulating cholesterol levels were also measured. RESULTS: Sra (-/-) mice displayed an enlargement of the gallbladder after CLP that was not detected in sham or non-operated mice or in B6 mice (wild-type) after CLP. The enlarged gallbladder resembles a condition of acute acalculous cholecystitis observed in humans. Sra (-/-) mice presented high cholesterol levels in circulation as opposed to wild type B6 mice. Moreover, Sra (-/-) mice exhibited a reduction in IL-10 mRNA levels in lungs compared to wild-type B6 mice after CLP. CONCLUSIONS: The development of acute acalculous cholecystitis may be the combination of pre-existing conditions, such as hypercholesterolemia associated with a defect in Sra (Msr1) and a robust inflammation induced by sepsis.

Investigation and comparison of patient experiences with removable functional appliances.

OBJECTIVES: To compare patients' experiences with the Invisalign Teen with Mandibular Advancement® (ITMA) and Twin Block (TB) appliances, both initially and after several months of wear. MATERIALS AND METHODS: Sixty-eight patients completed an anonymous survey after at least 2 months of wearing ITMA or TB. Forty-five patients treated with ITMA (18 boys, 27 girls, mean age 13.6 years, SD ± 1.54) and 23 patients treated with TB (13 boys, 10 girls, mean age 10.60 years, SD ± 1.92) were included. RESULTS: More patients using the TB found their appliance to be visually intimidating as compared with patients using the ITMA (21.7% vs 8.9%). TB was more noticeable than the ITMA (69.6% vs 25%). Appliance insertion was more difficult for TB patients (21.8% vs 4.44% for ITMA). After several months, there were more reports of tooth soreness and lip/cheek soreness in the ITMA group. TB patients were more embarrassed even after several months (14.3% vs 0% for ITMA). More TB patients required extra appointments for breakage (50% vs 22.2% for ITMA). Speech, drooling, and jaw and lip/cheek soreness worsened initially for both groups but improved over time. There were no differences between the groups regarding visible facial changes, satisfaction with treatment experience, or time to acclimatize to the appliance. CONCLUSIONS: TB and ITMA patients shared similar experiences for most of the parameters measured, but there were significant differences between the groups regarding appliance wear and management, discomfort, and function.

Quality of life in an adolescent orthodontic population.

OBJECTIVES: To investigate adolescent orthodontic patient experiences and quality of life with fixed appliances compared to Invisalign. MATERIALS AND METHODS: Adolescent patients in active treatment with Invisalign or fixed appliances for a minimum of 6 months were provided with the Child Oral Health Impact Profile-Short Form 19 questionnaire, along with additional items of interest that were assessed separately. Pearson's χ2 test was used to compare responses (P < .05), and unpaired t-tests (P < .05) were used to test for differences in mean satisfaction, quality of life, and domain scores. RESULTS: In total, 74 patients (37 in each treatment group) participated. Overall, no significant differences were noted in the mean quality of life, satisfaction, or domain scores between the two groups. A significant difference was noted in the time taken to adjust to appliances, with the Invisalign group demonstrating faster adaptation. Additionally, the fixed appliance group was 3.8 times more likely to report missing school because of their appliance (95% confidence interval [CI]: 1.2, 12.5) and 2.7 times more likely to report having difficulty eating certain foods (95% CI: 1.1, 7.1). When the sample of females between the ages of 14 and 18 was analyzed, the Invisalign group reported feeling attractive more often than the fixed appliance group. CONCLUSIONS: Both treatment groups were generally very satisfied with their treatment modality. The overall quality of life of adolescent orthodontic patients undergoing treatment with fixed appliances and Invisalign for a minimum of 6 months was similar.

Effect of Low Intensity Pulsed Ultrasound (LIPUS) on Tooth Movement and Root Resorption: A Prospective Multi-Center Randomized Controlled Trial.

The aim of this study was to evaluate the possible effect of low intensity pulsed ultrasound (LIPUS) on tooth movement and root resorption in orthodontic patients. Twenty-one patients were included in a split-mouth study design (group 1). Ten additional patients were included with no LIPUS device being used and this group was used as the negative control group (group 2). Group 1 patients were given LIPUS devices that were randomly assigned to right or left side on upper or lower arches. LIPUS was applied to the assigned side that was obtained by randomization, using transducers that produce ultrasound with a pulse frequency of 1.5 MHz, a pulse repetition rate of 1 kHz, and average output intensity of 30 mW/cm2. Cone-beam computed tomography (CBCT) images were taken before and after treatment. The extraction space dimensions were measured every four weeks and root lengths of canines were measured before and after treatment. The data were analyzed using paired t-test. The study outcome showed that the mean rate of tooth movement in LIPUS side was 0.266 ± 0.092 mm/week and on the control side was 0.232 ± 0.085 mm/week and the difference was statistically significant. LIPUS increased the rate of tooth movement by an average of 29%. For orthodontic root resorption, the LIPUS side (0.0092 ± 0.022 mm/week) showed a statistically significant decrease as compared to control side (0.0223 ± 0.022 mm/week). The LIPUS application accelerated tooth movement and minimized orthodontically induced tooth root resorption at the same time.

Characterisation of P2Y2 receptors in human vascular endothelial cells using AR-C118925XX, a competitive and selective P2Y2 antagonist.

BACKGROUND AND PURPOSE: There is a lack of potent, selective antagonists at most subtypes of P2Y receptor. The aims of this study were to characterise the pharmacological properties of the proposed P2Y2 receptor antagonist, AR-C118925XX, and then to use it to determine the role of P2Y2 receptors in the action of the P2Y2 agonist, UTP, in human vascular endothelial cells. EXPERIMENTAL APPROACH: Cell lines expressing native or recombinant P2Y receptors were superfused constantly, and agonist-induced changes in intracellular Ca2+ levels monitored using the Ca2+ -sensitive fluorescent indicator, Cal-520. This set-up enabled full agonist concentration-response curves to be constructed on a single population of cells. KEY RESULTS: UTP evoked a concentration-dependent rise in intracellular Ca2+ in 1321N1-hP2Y2 cells. AR-C118925XX (10 nM to 1 µM) had no effect per se on intracellular Ca2+ but shifted the UTP concentration-response curve progressively rightwards, with no change in maximum. The inhibition was fully reversible on washout. AR-C118925XX (1 µM) had no effect at native or recombinant hP2Y1 , hP2Y4 , rP2Y6 , or hP2Y11 receptors. Finally, in EAhy926 immortalised human vascular endothelial cells, AR-C118925XX (30 nM) shifted the UTP concentration-response curve rightwards, with no decrease in maximum. CONCLUSIONS AND IMPLICATIONS: AR-C118925XX is a potent, selective and reversible, competitive P2Y2 receptor antagonist, which inhibited responses mediated by endogenous P2Y2 receptors in human vascular endothelial cells. As the only P2Y2 -selective antagonist currently available, it will greatly enhance our ability to identify the functions of native P2Y2 receptors and their contribution to disease and dysfunction.

Hibiscus acid from Hibiscus sabdariffa (Malvaceae) has a vasorelaxant effect on the rat aorta.

Hibiscus sabdariffa (Malvaceae) is a plant that is widely recognised for its antihypertensive properties; however the constituent(s) responsible for this biological activity are presently unknown. The aim of this study was to identify the potential compounds that are responsible for the vasorelaxant activity of H. sabdariffa. Thereafter, the mechanisms involved in producing the vasorelaxation were investigated. The plant was extracted consecutively with hexane, ethyl acetate and methanol. The methanolic extract was subjected to bioassay-guided fractionation in order to isolate pure compounds that possessed vasorelaxant activity. The vascular effects of the pure compounds were studied on the rat aorta in vitro using myography techniques. Hibiscus acid produced a concentration-dependent relaxation of the rat aorta pre-contracted with either phenylephrine (3 µM) or KCl (60 mM), irrespective of the presence of the endothelium. When the tissue was pre-contracted with phenylephrine, the concentration required to produce 50% relaxation (IC50), was 0.09 ±â€¯0.01 mg/ml. Hibiscus acid had no effect on the phasic contraction induced by phenylephrine in Ca2+-free physiological solution; but it did affect the component of the contraction that is due to Ca2+ influx. In parallel studies, garcinia acid, a diastereoisomer of hibiscus acid, was found to have an almost identical vasorelaxant effect. The vasorelaxant action of both compounds is most likely due to the inhibition of Ca2+ influx via voltage-dependent Ca2+ channels.

Pharmacological profile of store-operated Ca(2+) entry in intrapulmonary artery smooth muscle cells.

Store-operated Ca(2+) entry (SOCE) plays an important role in the contraction and proliferation of pulmonary artery smooth muscle cells (PASMCs). The aim of this study was to characterise the pharmacological properties of the SOCE pathway in freshly isolated PASMCs from rat lung and to determine whether this Ca(2+) entry pathway is sensitive to nitric oxide donor drugs. Following depletion of Ca(2+) from the sarcoplasmic reticulum, by treating cells with thapsigargin, re-addition of Ca(2+) produced an increase in cytosolic fluo-4 fluorescence that was sustained for the period that extracellular Ca(2+) was present. Thapsigargin also increased the rate of quench of fura-2 fluorescence, confirming that SOCE was activated. The SOCE pathway was not affected by nifedipine or verapamil; however, it was inhibited by the divalent cations Ni(2+) (10 microM) and Cd(2+) (10 microM) by 47+/-5% and 49+/-5% respectively. SOCE was also inhibited 42+/-5% by 2-aminoethoxydiphenyl borate (2-APB; 75 microM) and 58+/-4% by Gd(3+) (10 microM), although La(3+) (100 microM) had little effect. None of the NO donors examined, including sodium nitroprusside, glyceryl trinitrate, and 2-(N,N-diethylamino)-diazenolate-2-oxide had any effect on SOCE. Thus, the pulmonary vasorelaxation produced by NO does not involve direct inhibition of SOCE in PASMCs. Western blot and immunocytochemistry using antibodies directed against specific TRPC subunits detected the presence of TRPC1, 3, and 6 in pulmonary artery and the pharmacological profile of SOCE in PASMCs favours a role for TRPC1 in mediating the underlying channels that are activated by store depletion.

A case of Clostridium septicum spontaneous gas gangrene.

Severe skin and soft tissue infections (SSTIs) are often life-threatening emergencies that require a rapid diagnosis. Gas gangrene is one of the most fulminant types of SSTI and is usually caused by Clostridium perfringens' contamination of an open wound. Although gas gangrene is usually associated with fecally contaminated wounds, "spontaneous" cases occur and are most commonly caused by Clostridium (C.) septicum. We report a case of spontaneous gas gangrene caused by C. septicum that only became manifest while the patient was being monitored in the emergency department. We also review the diagnosis and treatment aspects of this entity.

The role of the C-terminal tail in protease-activated receptor-2-mediated Ca2+ signalling, proline-rich tyrosine kinase-2 activation, and mitogen-activated protein kinase activity.

C-terminal truncation mutants were made to investigate the role of the C-terminus in coupling proteinase-activated receptor-2 (PAR-2) to various signalling pathways. Membrane expression of the delta15, delta34, delta43, and delta34-43 mutants was similar; however, expression of deltatail was lost, as was agonist-mediated internalisation of deltatail, delta43, and delta34-43. Additionally, trypsin and SLIGKV-stimulated [3H]IP accumulation was abrogated in cells transiently expressing delta43 or delta34-43 truncations, but remained unaffected in cells expressing delta34 or delta15. PAR-2 agonist-stimulated intracellular Ca(2+) mobilisation and PYK-2 activity were also abolished by deltatail, delta43, and delta34-43 mutants. However, trypsin-stimulated stress-activated protein kinases (SAPKs) or extracellular signal-regulated kinase (ERK) activities were unaffected by the delta34-43 mutation, although activity was abrogated following delta43 or deltatail truncations, suggesting that Ca(2+) mobilisation, PYK-2, or receptor internalisation are not requied for activation of SAPKs or ERK. These studies identify a novel sequence within the PAR-2 C-terminus essential for InsP(3) generation and PYK-2 activity but not mitogen-activated protein kinase (MAPK) activation.

Polarisation and functional characterisation of hepatocytes derived from human embryonic and mesenchymal stem cells.

Adult hepatocytes are polarised with their apical and basolateral membranes separated from neighbouring cells by tight junction proteins. Although efficient differentiation of pluripotent stem cells to hepatocytes has been achieved, the formation of proper polarisation in these cells has not been thoroughly investigated. In the present study, human embryonic stem cells (hESCs) and human mesenchymal stem cells (hMSCs) were differentiated to hepatocyte-like cells and the derived hepatocytes were characterised for mature hepatocyte markers. The secretion of hepatic proteins, expression of hepatic genes and the functional hepatic polarisation of stem cell-derived hepatocytes, foetal hepatocytes and the HepG2 hepatic cell line were evaluated and the different lines were compared. The results indicate that hESC-derived hepatocytes are phenotypically more robust and functionally more efficient compared with the hMSC-derived hepatocytes, suggesting their suitability for toxicity studies.

The Cl(-) channel blocker niflumic acid releases Ca(2+) from an intracellular store in rat pulmonary artery smooth muscle cells.

The effect of the Cl- channel blockers niflumic acid (NFA), 5-nitro-2-(3-phenylpropylamino)-benzoic acid (NPPB), 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS), and anthracene-9-carboxylic acid (A-9-C), on Ca2+ signalling in rat pulmonary artery smooth muscle cells was examined. Intracellular Ca2+ concentration ([Ca2+]i) was monitored with either fura-2 or fluo-4, and caffeine was used to activate the ryanodine receptor, thereby releasing Ca2+ from the sarcoplasmic reticulum (SR). NFA and NPPB significantly increased basal [Ca2+]i and attenuated the caffeine-induced increase in [Ca2+]i. These Cl- channel blockers also increased the half-time (t1/2) to peak for the caffeine-induced [Ca2+]i transient, and slowed the removal of Ca2+ from the cytosol following application of caffeine. Since DIDS and A-9-C were found to adversely affect fura-2 fluorescence, fluo-4 was used to monitor intracellular Ca2+ in studies involving these Cl- channel blockers. Both DIDS and A-9-C increased basal fluo-4 fluorescence, indicating an increase in intracellular Ca2+, and while DIDS had no significant effect on the t1/2 to peak for the caffeine-induced Ca2+ transient, it was significantly increased by A-9-C. In the absence of extracellular Ca2+, NFA significantly increased basal [Ca2+]i, suggesting that the release of Ca2+ from an intracellular store was responsible for the observed effect. Depleting the SR with the combination of caffeine and cyclopiazonic acid prevented the increase in basal [Ca2+]i induced by NFA. Additionally, incubating the cells with ryanodine also prevented the increase in basal [Ca2+]i induced by NFA. These data show that Cl- channel blockers have marked effects on Ca2+ signalling in pulmonary artery smooth muscle cells. Furthermore, examination of the NFA-induced increase in [Ca2+]i indicates that it is likely due to Ca2+ release from an intracellular store, most probably the SR.

Radiation-induced aortoesophageal fistula: an unusual case of massive upper gastrointestinal bleeding.

Aortoesophageal fistula (AEF) is an unusual cause of massive upper gastrointestinal bleeding. Thoracic aortic aneurysm is the most common etiology of primary AEF followed by, respectively, foreign body ingestion, esophageal malignancy, and postsurgical fistulization. Radiation-induced damage to the great vessels is well recognized and some authors in the past have suggested that AEF may be caused by radiotherapy. However, previous case reports of radiation-induced AEF involved patients who received radiotherapy for esophageal carcinoma, and precise histopathologic differentiation between AEF secondary to esophageal malignancy and that induced by radiation was difficult. We present here the unique case of a patient with a non-esophageal carcinoma who received radiotherapy before the development of an AEF, thus providing further evidence for the role of radiation injury in the development of this condition. As well, we discuss current opinion regarding etiology, clinical presentation, diagnosis, and management of this entity.

On a wing and a prayer: medical emergencies on board commercial aircraft.

Medical emergencies sometimes arise in the isolated and confined environment of a commercial aircraft. Because a physician passenger may be on board in 40% to 90% of all commercial flights, it follows that this physician may be asked to render assistance to an acutely ill passenger. Although data suggest that the incidence of such emergencies is low, the potential for serious events necessitates a degree of familiarity with the nature of emergencies in the air and with the options available to the travelling physician.