Cross validated serum small extracellular vesicle microRNAs for the detection of oropharyngeal squamous cell carcinoma.

BACKGROUND: Oropharyngeal squamous cell carcinoma (OPSCC) is often diagnosed at an advanced stage because the disease often causes minimal symptoms other than metastasis to neck lymph nodes. Better tools are required to assist with the early detection of OPSCC. MicroRNAs (miRNAs, miRs) are potential biomarkers for early head and neck squamous cell cancer diagnosis, prognosis, recurrence, and presence of metastatic disease. However, there is no widespread agreement on a panel of miRNAs with clinically meaningful utility for head and neck squamous cell cancers. This could be due to variations in the collection, storage, pre-processing, and isolation of RNA, but several reports have indicated that the selection and reproducibility of biomarkers has been widely affected by the methods used for data analysis. The primary analysis issues appear to be model overfitting and the incorrect application of statistical techniques. The purpose of this study was to develop a robust statistical approach to identify a miRNA signature that can distinguish controls and patients with inflammatory disease from patients with human papilloma virus positive (HPV +) OPSCC. METHODS: Small extracellular vesicles were harvested from the serum of 20 control patients, 20 patients with gastroesophageal reflux disease (GORD), and 40 patients with locally advanced HPV + OPSCC. MicroRNAs were purified, and expression profiled on OpenArray™. A novel cross validation method, using lasso regression, was developed to stabilise selection of miRNAs for inclusion in a prediction model. The method, named StaVarSel (for Stable Variable Selection), was used to derive a diagnostic biomarker signature. RESULTS: A standard cross validation approach was unable to produce a biomarker signature with good cross validated predictive capacity. In contrast, StaVarSel produced a regression model containing 11 miRNA ratios with potential clinical utility. Sample permutations indicated that the estimated cross validated prediction accuracy of the 11-miR-ratio model was not due to chance alone. CONCLUSIONS: We developed a novel method, StaVarSel, that was able to identify a panel of miRNAs, present in small extracellular vesicles derived from blood serum, that robustly cross validated as a biomarker for the detection of HPV + OPSCC. This approach could be used to derive diagnostic biomarkers of other head and neck cancers.

Does gene expression in laryngeal subsites differ between patients with laryngopharyngeal reflux and controls?

OBJECTIVE: To identify laryngeal mRNA gene changes in patients with laryngopharyngeal reflux (LPR). METHOD: Laryngeal biopsies from non-smoking LPR patients (n=10; Reflux Symptom Index (RSI) >12 and a Reflux Finding Score (RFS) >6) and controls (n=9; RSI <12 and RFS <6) were collected from four subsites (true vocal cord, false vocal cord, medial arytenoid and posterior commissure) of the larynx. qRT-PCR analyses were conducted on 20 reflux- and inflammation-related genes, including interleukins 6 and 8, cytokeratins 8 and 14, mucin genes MUC1, MUC2, MUC3B, MUC4, MUC5B, MUC6 and MUC7 and carbonic anhydrase III. Statistical analysis (Mann-Whitney U test) compared gene expression levels between LPR and control groups at each subsite. RESULTS: Site-specific differences in squamous metaplasia and gene expression were noted in LPR patients, with the majority present in the medial arytenoid region. Significant.differences were noted in genes related to mucosal defence and inflammation, including CRNN, CD1d, TGFß-1, MUC2, MUC5B and CDH1. CONCLUSION: Whilst the posterior commissure is commonly identified as the area demonstrating the most significant macroscopic change in LPR, the histological changes and genes assessed here showed more pronounced LPR associated differences in the medial arytenoid. We identified differences in expression of mucin genes, cytokeratin-14 and molecular markers of inflammation. Whilst some of these changes may be metaplasia-related, further evaluation of the mRNA expression of these genes may provide a useful biomarker panel for diagnosis and therapeutic monitoring of LPR.

Cost analysis of injection laryngoplasty performed under local anaesthesia versus general anaesthesia: an Australian perspective.

OBJECTIVE: To conduct a cost analysis of injection laryngoplasty performed in the operating theatre under local anaesthesia and general anaesthesia. METHODS: The retrospective study included patients who had undergone injection laryngoplasty as day cases between July 2013 and March 2016. Cost data were obtained, along with patient demographics, anaesthetic details, type of injectant, American Society of Anesthesiologists score, length of stay, total operating theatre time and surgeon procedure time. RESULTS: A total of 20 cases (general anaesthesia = 6, local anaesthesia = 14) were included in the cost analysis. The mean total cost under general anaesthesia (AU$2865.96 ± 756.29) was significantly higher than that under local anaesthesia (AU$1731.61 ± 290.29) (p < 0.001). The mean operating theatre time, surgeon procedure time and length of stay were all significantly lower under local anaesthesia compared to general anaesthesia. Time variables such as operating theatre time and length of stay were the most significant predictors of the total costs. CONCLUSION: Procedures performed under local anaesthesia in the operating theatre are associated with shorter operating theatre time and length of stay in the hospital, and provide significant cost savings. Further savings could be achieved if local anaesthesia procedures were performed in the office setting.

Hyaluronic acid for post sinus surgery care: systematic review and meta-analysis.

BACKGROUND: Wound healing after endoscopic sinus surgery may result in adhesion formation. Hyaluronic acid may prevent synechiae development. A systematic review was performed to evaluate the current evidence on the clinical efficacy of hyaluronic acid applied to the nasal cavity after sinus surgery. METHODS: Studies using hyaluronic acid as an adjunct treatment following endoscopic sinus surgery for chronic rhinosinusitis were identified. The primary outcome was adhesion formation rates. A meta-analysis was performed on adhesion event frequency. Secondary outcome measures included other endoscopic findings and patient-reported outcomes. RESULTS: Thirteen studies (501 patients) met the selection criteria. A meta-analysis of adhesion formation frequency on endoscopy demonstrated a lower risk ratio in the hyaluronic acid intervention group (42 out of 283 cases) compared to the control group (81 out of 282) of 0.52 (95 per cent confidence interval = 0.37-0.72). Hyaluronic acid use was not associated with any significant adverse events. CONCLUSION: Hyaluronic acid appears to be clinically safe and well tolerated, and may be useful in the early stages after sinus surgery to limit adhesion rate. Further research, including larger randomised controlled trials, is required to evaluate patient- and clinician-reported outcomes of hyaluronic acid post sinus surgery.

Long-term quality-of-life outcomes following treatment for adult obstructive sleep apnoea: comparison of upper airway surgery, continuous positive airway pressure and mandibular advancement splints.

OBJECTIVE: Long-term quality-of-life (QOL) outcomes, complications and clinical effectiveness in patients undergoing treatment with upper airway surgery (UAS), continuous positive airway pressure (CPAP) and mandibular advancement splints (MAS) for adult obstructive sleep apnoea (OSA). DESIGN: Retrospective cohort study. SETTING: Multidisciplinary OSA clinic in University teaching hospital. PARTICIPANTS: Consecutive, simultaneously treated patients with OSA undergoing UAS (n = 83), CPAP (n = 83) and MAS (n = 79). MAIN OUTCOME MEASURES: Glasgow Benefit Inventory (GBI), Snoring Severity Scale (SSS), Epworth Sleepiness Score (ESS) and side-effects in all three groups were recorded at a mean of 34.5 months following start of treatment and compared via anova with Bonferroni's adjustment for pairwise comparisons. RESULTS: Upper airway surgery demonstrated a statistically significant QOL benefit over MAS. All three groups showed a significant improvement in SSS with CPAP significantly better than MAS, but equivalent to UAS. Uncomplicated UAS provided a greater QOL outcome than compliant MAS, non-compliant CPAP (P < 0.05) and comparable to compliant CPAP. Patients undergoing UAS with recorded complications still reported equivalent QOL outcomes to compliant CPAP and MAS, suggesting these surgical complications are relatively minor compared to the QOL benefit of OSA treatment. CONCLUSION: Upper airway surgery showed a significant improvement in QOL outcomes compared to non-compliant CPAP or MAS and equivalent benefit to compliant CPAP. This study strongly supports the role for contemporary UAS in OSA when CPAP is not or no longer an option.

Micro-ribonucleic acids in head and neck cancer: an introduction.

BACKGROUND AND METHODS: Head and neck cancer is the sixth most common cancer worldwide. Advances in management have not greatly altered overall survival. Over the last decade, there have been significant scientific advances in our knowledge of cell cycle regulation and the complex oncogenic processes. MicroRNAs are small, non-coding RNAs which are integral to the regulation of gene expression and which play a part in carcinogenesis. The literature on the role of microRNA in head and neck cancer is reviewed. OBJECTIVE: To introduce the role and significance of microRNAs in head and neck cancer. RESULTS: The possibilities of incorporating microRNAs into clinical practice are discussed, including their potential role in diagnosis, prognosis, prediction of metastatic spread, therapy and tumour surveillance. CONCLUSION: Discoveries in expression profiling of microRNA in head and neck oncology promise advancements in the diagnosis, prognosis and therapy of these cancers.

Lysozyme expression is increased in the sinus mucosa of patients with chronic rhinosinusitis.

BACKGROUND: The presence of fungi and bacteria in the paranasal sinuses may contribute to ongoing inflammation. Lysozyme is an innate immune peptide with bactericidal and fungicidal activity. The expression of lysozyme in chronic rhinosinusitis (CRS) is poorly understood and deficiencies in lysozyme expression may contribute to the ongoing inflammation in CRS patients. OBJECTIVE: Determine lysozyme expression in sinus mucosa of normal and CRS patients with (CRSwNP) and without (CRSsNP) nasal polyps. METHODOLOGY: Sinus mucosa specimens (n = 82) were processed for standard histology, immunohistochemical localisation of lysozyme, immunofluorescent localisation of fungi, and qPCR analysis of lysozyme expression. RESULTS: CRS specimens displayed high-levels of lysozyme immunoreactivity in many of the abundant serous cells. Moderate levels were detected in some epithelial cells and inflammatory cells. Low levels were detected in some subepithelial glands of control specimens. No difference in immunoreactivity was detected between CRSwNP and CRSsNP specimens. Fungal elements were not visualised in any sinus specimen. qPCR analysis demonstrated variable lysozyme expression between individuals. CONCLUSIONS: Lysozyme protein expression is increased in patients with CRS, suggesting a defect in lysozyme expression is not responsible for the microbial colonisation often associated with CRS. The functional activity of lysozyme in CRS patients needs to be further investigated.

Biomarkers and laryngopharyngeal reflux.

Laryngopharyngeal reflux is a controversial but increasingly made diagnosis used in patients with a collection of often non-specific laryngeal symptoms. It is a clinical diagnosis, and its pathophysiology is currently poorly understood. Previous reflux research has focused on injurious agents, acid, pepsin and biomarker expression. Failure of intrinsic defences in the larynx may cause changes in laryngeal epithelia, particularly alterations in carbonic anhydrases and E-cadherin. Carbonic anhydrase III levels vary in the larynx in response to laryngopharyngeal reflux, depending on location. Expression of E-cadherin, a known tumour suppressor, is reduced in the presence of reflux. Mucin expression also varies according to the severity of reflux. Further research is required to define the clinical entity of laryngopharyngeal reflux, and to identify a definitive mechanism for mucosal injury. Understanding this mechanism should allow the development of a comprehensive model, which would enable future diagnostic and therapeutic interventions to be developed.

A novel preparation to study rat pancreatic spinal and vagal mechanosensitive afferents in vitro.

The management of pancreatic pain is a significant clinical problem so understanding of how sensory signals are generated in pancreatic tissue is fundamental. We aimed to characterize mechanosensitive and chemosensitive properties of pancreatic spinal and vagal afferents in vitro. Spinal and vagal afferent preparations from Sprague-Dawley rats were established incorporating the left splanchnic nerve or vagus nerves respectively. The common bile duct was cannulated for distension of the pancreatic duct with fluid. Nerve discharge evoked by blunt probing, duct distension or electrical stimulation was obtained from teased nerve bundles using standard extra-cellular recording. Discharge from 197 spinal afferent bundles was recorded, of which 57% displayed spontaneous activity. Blunt probing revealed 61 mechanosensitive receptive fields which were associated primarily with arteries/blood vessels (33/61) and the parenchyma (22/61). All mechanosensitive responses were slowly adapting, with 33% continuing to discharge after termination of the stimulus and 60% displaying a response threshold <10 g. Application of chemical mediators (bradykinin, histamine, 5-hydroxytryptamine, cholecystokinin octapeptide) evoked a response from 31/57 units, with 33% excitatory and 23% inhibitory. Spontaneous discharge was recorded from 72% of 135 vagal bundles. Mechanosensitive receptive fields were not identified in the pancreas but were evident in adjacent organs. No spinal or vagal afferent response to duct distension was obtained. In conclusion, pancreatic mechanosensitive spinal afferents are common, in contrast to pancreatic mechanosensitive vagal afferents indicating that pancreatic sensory innervation is predominantly spinal. Chemosensitive spinal afferent nerve endings are present in the pancreas and respond to a variety of inflammatory and physiological mediators.

Selective iNOS inhibition enhances spontaneous gallbladder motility in the Australian possum.

Gallbladder inflammation is a common and painful disease. Inducible nitric oxide synthase (iNOS) plays a major role in inflammatory diseases, and iNOS inhibitors are being developed as therapeutic agents. Reports are inconsistent regarding iNOS expression in normal gallbladder. The aim of this study was to determine the effect of iNOS inhibition on spontaneous gallbladder motility. mRNA extracted from normal possum gallbladders was analysed by PCR. Gallbladder contractility was evaluated using a highly selective iNOS inhibitor AR-C102222AA (AR-C) in in vitro muscle strips (0.1-10 000 microm) and in vivo (0.1-30 micromol kg(-1)) experiments. Gene expression analysis revealed the presence of iNOS mRNA in normal gallbladder (n = 3). In vitro, AR-C (0.1-1000 micromol L(-1)) produced a concentration-dependent increase in spontaneous gallbladder contractile activity and basal tension (P < 0.05; n = 6). The maximum effect was a 1.8-fold increase in activity and 2.1-fold increase in basal tension. Pretreatment of muscle strips with tetrodotoxin (1 micromol L(-1)) did not block the AR-C-induced response (n = 5). In vivo, AR-C (30 micromol kg(-1), i.v.) increased gallbladder contraction frequency (P < 0.05; n = 8). These data suggest that iNOS is continually expressed in the normal gallbladder, which presumably releases low levels of nitric oxide and in turn may modulate spontaneous gallbladder motility. AR-C may be a beneficial treatment for patients suffering from acute cholecystitis.

Exogenous purines induce differential responses in the proximal and distal regions of the possum sphincter of Oddi.

1. The aim of this study was to compare the effect of exogenous ATP and adenosine on spontaneous motility of the proximal and distal regions of the possum sphincter of Oddi (SO). 2. ATP or adenosine (1 microm-1 mm) was applied to distal-SO or proximal-SO muscle rings in organ baths in the absence or presence of tetrodotoxin (TTX) or P1/P2 antagonists. 3. Both ATP and adenosine altered spontaneous activity, predominantly in proximal-SO rings. 4. Exogenous ATP induced a bi-phasic response consisting of a brief TTX-sensitive excitatory component, and a longer-lasting TTX-insensitive inhibitory component. 5. The excitatory ATP response likely involves P2X receptors, whereas the late inhibitory response likely involves P2Y receptors. 6. Exogenous adenosine decreased spontaneous SO activity, via a TTX-insensitive mechanism. 7. Exogenous purines modulate SO motility, acting primarily in the proximal region of the SO, via neural and non-neural mechanisms and multiple purine receptor subtypes.

Exogenous adenosine triphosphate and adenosine stimulate proximal sphincter of oddi motility via neural mechanisms in the anesthetized Australian possum.

We aimed to determine if exogenous adenosine triphosphate or adenosine modulated sphincter of Oddi motility and involved neural mechanisms. Sphincter of Oddi motility was recorded in anesthetized possums by manometry. Adenosine triphosphate or adenosine (1 microM-10 mM) was applied topically to the sphincter before and after pretreatment with tetrodotoxin, hexamethonium, atropine, or Nomega-nitro-L-arginine methyl ester. Sphincter contraction amplitude and frequency were quantified. Adenosine triphosphate induced a concentration-dependent increase in proximal sphincter contraction amplitude and frequency (P < 0.05). This response was reduced by tetrodotoxin and atropine but enhanced by hexamethonium and Nomega-nitro-L-arginine methyl ester. Adenosine concentration dependently increased proximal sphincter contraction amplitude (P < 0.05) only. This response was reduced by tetrodotoxin, atropine, and Nomega-nitro-L-arginine methyl ester, whereas hexamethonium had no effect. We conclude that exogenous adenosine triphosphate and adenosine stimulate proximal sphincter of Oddi motility via neural mechanisms, involving cholinergic motor neurons. Adenosine triphosphate may further modulate sphincter motility via nicotinic and nitrergic pathways.

Pancreatobiliary afferent recordings in the anaesthetised Australian possum.

The sensory innervation to the pancreatobiliary system is poorly characterized. Afferent signals from the gastrointestinal tract and biliary tree are transmitted to the central nervous system via the vagus and spinal nerves. We aimed to record afferent discharge in order to characterize the vagal and splanchnic afferent signals from the possum upper gastrointestinal tract, biliary tree and pancreas. In 21 anaesthetised possums nerve fibres were teased from the vagus or splanchnic nerve for multi-unit recording. Mechanical stimuli consisted of balloon distension of the gallbladder and duodenum (2-7 ml) and fluid distension (0-20 mm Hg) of the bile or pancreatic ducts. Approximately 60% of fibres from all nerves displayed spontaneous discharge. Spinal afferent responses to mechanical stimuli were infrequent (n=13). Increased discharge occurred in response to duodenal (12/99 fibres) or gallbladder (7/96 fibres) distension, but not to bile duct (0/73 fibres) or pancreatic duct (0/51 fibres) distension. Vagal afferent responses to distension of the duodenum or stomach (5-30 ml) were more common (n=8). Increased discharge was recorded in response to duodenal (49/134 fibres), or gastric (22/70 fibres) distension. Responses to gallbladder distension were less frequent (6/99 fibres) and as with the spinal afferent no response to bile duct (0/66) or pancreatic duct (0/70) distension were recorded. We conclude that mechanosensitive afferents in the pancreatobiliary system are relatively rare, particularly within the ducts, and/or that they are adapted to monitor stimuli other than luminal distension.

The sphincter of Oddi: understanding its control and function.

The most common functional disorders of the biliary tract and pancreas are associated with disordered motility of the sphincter of Oddi (SO). The SO is a neuromuscular structure located at the junction of the bile and pancreatic ducts with the duodenum. The primary functions of the SO are to regulate the delivery of bile and pancreatic juice into the duodenum, and to prevent the reflux of duodenal contents into the biliary and pancreatic systems. Disordered motility of the SO leads to the common and painful clinical conditions of SO dysfunction and acute pancreatitis. In order to understand normal SO motility, studies have been performed addressing SO function, control of spontaneous SO activity, responses to bioactive agents, SO innervation, and reflexes with other gastrointestinal organs. These studies have led to the current understanding of how the SO functions and may permit the development of targeted therapy for SO dysfunction and acute pancreatitis. This review summarizes the current knowledge regarding the control and regulation of SO motility, highlighting laboratory based and clinical research performed over the last 5 years.

Electrical activation of common bile duct nerves modulates sphincter of Oddi motility in the Australian possum.

BACKGROUND: Sphincter of Oddi (SO) motility is regulated by extrinsic and intrinsic nerves. The existence of neural circuits between the SO and the proximal extrahepatic biliary tree has been reported, but they are poorly understood. Using electrical field stimulation (EFS), we determined if a neural circuit exists between the common bile duct (CBD) and the SO in anaesthetized Australian brush-tailed possums. METHODS: The gallbladder, cystic duct or CBD were subjected to EFS with a stimulating electrode. Spontaneous SO phasic waves were measured by manometry. RESULTS: EFS at sites on the distal CBD (12-20 mm proximal to the SO), but less commonly at more proximal CBD, evoked a variety of responses consisting of an excitatory and/or inhibitory phase. Bi-phasic responses consisting of an excitation followed by inhibition were the most common. Tri-phasic responses were also observed as well as excitation or inhibition only. These evoked responses were blocked by topical application of local anaesthetic to the distal CBD or transection of the CBD. EFS at sites on the gallbladder body, neck or cystic duct did not consistently evoke an SO response. Pretreatment with atropine or guanethidine reduced the magnitude of the evoked response by about 50% (p<0.05), pretreatment with hexamethonium had no consistent effect and pretreatment with a nitric oxide synthase inhibitor increased the response. DISCUSSION: A neural circuit(s) between the SO and the distal CBD modulates SO motility. Damage to this area of the CBD during bile duct exploration surgery could adversely affect SO motility.

Highly selective iNOS inhibition and sphincter of Oddi motility in the Australian possum.

AIM: Inducible nitric oxide synthase (iNOS) plays a major role in acute pancreatitis. Selective inhibitors of iNOS are being developed as therapeutic agents. Sphincter of Oddi (SO) dysfunction may cause pancreatitis and nitric oxide is necessary for SO relaxation. A new highly selective iNOS inhibitor, AR-C102222AA (AR-C), is evaluated together with the established iNOS inhibitor, L-N(6)-(1-iminoethyl)lysine (L-NIL), and the selective neuronal nitric oxide synthase (nNOS) blocker S-methyl-l-thiocitrulline (SMTC). METHODS: In anaesthetized Australian Brush-tailed possums, the effect of topical, i.v. or i.a. administration of these drugs was evaluated on spontaneous SO motility, blood pressure (BP) and pancreatic vascular perfusion. SO motility was recorded by manometry and pancreatic vascular perfusion by laser Doppler fluxmetry. Also, the effect of SMTC and AR-C on electrical field stimulation (EFS)-induced non-cholinergic non-adrenergic (NANC) SO relaxation in vitro was evaluated. RESULTS: Infusion of AR-C (0.1-30 micromol kg(-1)) increased SO contraction frequency (P = 0.026) only at the two highest doses. L-NIL infusion (0.15 to 14.7 micromol kg(-1)) also increased SO contraction frequency at 8.8 micromol kg(-1) (P < 0.05) and reduced SO contraction amplitude at the two highest doses (P < 0.05). SMTC injections (0.5 nmol-2.4 micromol) produced a dose-dependent increase in SO contraction frequency (P = 0.009), but no effect was seen on the other parameters. In vitro SMTC (40-400 microm) inhibited EFS-induced NANC relaxation in a dose-dependent manner (P < 0.0005). In contrast AR-C (10-500 microm) had no effect on EFS-induced NANC relaxation (P > 0.05). CONCLUSIONS: At low doses, AR-C does not effect SO motility or EFS-induced NO mediated relaxation. However, high doses of AR-C and L-NIL in vivo influenced SO motility by inhibiting nNOS activity and these effects need be considered in relation to therapeutic doses of this agent.

Endogenous endothelin increases gallbladder tone and leads to acute cholecystitis in the Australian possum.

Endothelins are bioactive peptides produced by gallbladder epithelial cells. We aimed to determine the role of endothelins in acute cholecystitis. Escherichia coli lipopolysaccharide vs saline (sham) was instilled into the gallbladder lumen of Australian possums. Some animals received the non-selective endothelin antagonist, tezosentan. At 4 or 24 h, plasma and gallbladder endothelins and white blood cell count (WBCC) were determined. Acute cholecystitis was assessed using a histopathology score. In other animals gallbladder tone was determined. At 4h, a dose-dependent 60-fold increase in gallbladder endothelin level occurred (P = 0.001) but other parameters remained comparable with sham animals. Epithelial cells were endothelin-immunoreactive. At 24 h, the WBCC rose (P < 0.007), and severe cholecystitis developed. Gallbladder but not plasma endothelin levels remained elevated. Tezosentan pre-treatment resulted in a histologically normal gallbladder, but the WBCC and gallbladder endothelin levels were elevated. Lipopolysaccharide or saline instillation also caused a time-dependent increase in gallbladder tone over 4 h (P < 0.001), but not in control animals. This increase was reduced by tezosentan treatment. Gallbladder endothelin production is an early event in acute cholecystitis, increases gallbladder tone and plays a crucial role in the inflammatory process.

A2A and A3 receptors mediate the adenosine-induced relaxation in spontaneously active possum duodenum in vitro.

1. The aim of this study was to define the P1 purinergic receptors that regulate spontaneous or adenosine-induced duodenal motor activity. 2. Spontaneous contractile activity was recorded isometrically from possum longitudinal duodenal muscle strips. Adenosine (0.5 micro M-1 mM) was administered noncumulatively and repeated after pretreatment with a P1 antagonist or tetrodotoxin (TTX, 1 micro M), (n=4-7). Antagonists used were: A(1), 8-cyclopentyl-1,3-dipropylxanthine (DPCPX, 10 nM); A(2A), 8-(3-chlorostyryl)caffeine (CSC, 10 micro M); A(2B), 3-isobutyl-8-pyrrolidinoxanthine (IPDX, 10 micro M); A(3), 9-chloro-2-(2-furanyl)-5-[(phenylacetyl) amino][1,2,4]-triazolo(1,5-c)quinazoline (MRS1220, 10 micro M). Changes in activity are expressed as percentage of baseline. Statistical analysis utilised nonparametric tests. 3. Adenosine (n=34) induced a long-lasting, concentration-dependent decrease in activity by 55.6+/-3.2% area under curve (AUC), 47.3+/-4.0% contraction amplitude, 31.6+/-3.6% basal tension and 10.4+/-1.7% contraction frequency (all P<0.001). The adenosine-induced decrease in contraction amplitude was blocked by CSC (P<0.01) or inhibited by MRS1220 (P<0.03) pretreatment, but not modified by TTX, DPCPX or IPDX pretreatment. 4. Adenosine antagonists modified spontaneous contractile activity. Pretreatment with DPCPX or CSC increased basal tension, whereas IPDX or MRS1220 pretreatment decreased contractile activity. 5. In conclusion, exogenous adenosine reduced duodenal longitudinal motor activity via A(2A) and A(3) receptors. Our findings suggest that endogenous purines may modulate spontaneous duodenal motor activity.

Receptor-mediated targeting of spray-dried lipid particles coformulated with immunoglobulin and loaded with a prototype vaccine.

PURPOSE: Spray-dried lipid-based microparticles (SDLM) serve as a platform for delivery of a wide variety of compounds including peptides, proteins, and vaccines to the respiratory mucosa. In the present study, we assessed the impact of IgG-mediated targeting to phagocytic cells of inactivated influenza virus formulated in SDLM, on subsequent immune responses. METHODS: SDLM were produced containing inactivated influenza virus strain A/WSN/32/H1N1 (WSN), with or without IgG. Using phagocytic antigen presenting cells (APC) and a T cell hybridoma (TcH) line specific for a dominant influenza virus epitope, we compared the in vitro responses elicited by ligand-formulated (SDLM-IgG-WSN) and non-ligand particles (SDLM-WSN). The effect of including the IgG ligand in the formulation was further characterized by measuring the immune responses of rodents vaccinated with SDLM. RESULTS: SDLM-IgG-WSN were internalized in an Fc receptor (FcR)-dependent manner by phagocytic APC that were then able to effectively present a dominant, class II-restricted epitope to specific T cells. While SDLM-WSN elicited a lower response than administration of plain inactivated virus in saline, the level of the T cell response was restored both in vitro and in vivo by incorporating the APC FcR ligand, IgG, in the SDLM. CONCLUSIONS: Incorporation of FcR ligand (IgG) in SDLM restored the limited ability of formulated virus to elicit T-cell immunity, by receptor-mediated targeting to phagocytes.