Acute and chronic pain in calves after different methods of rubber-ring castration

The goal of the present study was to evaluate the effect of different methods of rubber-ring castration on acute and chronic pain in calves. Sixty-three 4-6 week-old calves were randomly and sequentially allocated to one of five groups: Group RR (traditional rubber ring castration); group BRR (combination of one rubber ring with Burdizzo); group Rcut (one rubber ring applied with the scrotal tissue and rubber ring removed on day 9); group 3RR (three rubber rings placed one above the other around the scrotal neck); and group CO (controls; sham-castrated). All calves received 0.2 mL/kg bodyweight lidocaine 2%, injected into the spermatic cords and around the scrotal neck 15 min before castration. The presence of acute and chronic pain was assessed using plasma cortisol concentrations, response to palpation of scrotal area, time from castration until complete wound healing, and behavioural signs. Calves of group 3RR showed severe swelling and inflammation, and licking of the scrotal area occurred significantly more often than in groups Rcut and CO. Technique 3RR was discontinued for welfare reasons before the end of the study. All castration groups had significantly more pain upon palpation than calves of group CO, but palpation elicited markedly less pain in group Rcut than in the other castration groups. The most rapid healing time and shortest duration of chronic pain after castration was achieved in group Rcut. For welfare reasons, the Rcut technique should be considered as a valuable alternative to traditional rubber ring castration of calves at 4-6 weeks of age.

Acute-on-chronic liver failure is a distinct syndrome that develops in patients with acute decompensation of cirrhosis

BACKGROUND & AIMS Patients with cirrhosis hospitalized for an acute decompensation (AD) and organ failure are at risk for imminent death and considered to have acute-on-chronic liver failure (ACLF). However, there are no established diagnostic criteria for ACLF, so little is known about its development and progression. We aimed to identify diagnostic criteria of ACLF and describe the development of this syndrome in European patients with AD. METHODS We collected data from 1343 hospitalized patients with cirrhosis and AD from February to September 2011 at 29 liver units in 8 European countries. We used the organ failure and mortality data to define ACLF grades, assess mortality, and identify differences between ACLF and AD. We established diagnostic criteria for ACLF based on analyses of patients with organ failure (defined by the chronic liver failure-sequential organ failure assessment [CLIF-SOFA] score) and high 28-day mortality rate (>15%). RESULTS Of the patients assessed, 303 had ACLF when the study began, 112 developed ACLF, and 928 did not have ACLF. The 28-day mortality rate among patients who had ACLF when the study began was 33.9%, among those who developed ACLF was 29.7%, and among those who did not have ACLF was 1.9%. Patients with ACLF were younger and more frequently alcoholic, had more associated bacterial infections, and had higher numbers of leukocytes and higher plasma levels of C-reactive protein than patients without ACLF (P < .001). Higher CLIF-SOFA scores and leukocyte counts were independent predictors of mortality in patients with ACLF. In patients without a prior history of AD, ACLF was unexpectedly characterized by higher numbers of organ failures, leukocyte count, and mortality compared with ACLF in patients with a prior history of AD. CONCLUSIONS We analyzed data from patients with cirrhosis and AD to establish diagnostic criteria for ACLF and showed that it is distinct from AD, based not only on the presence of organ failure(s) and high mortality rate but also on age, precipitating events, and systemic inflammation. ACLF mortality is associated with loss of organ function and high leukocyte counts. ACLF is especially severe in patients with no prior history of AD.

[Anal metastasis from a lung cancer]

We report an anal metastasis from a lung cancer which was diagnosed on symptoms mimicking an acute anal abcess. The diagnosis was based on specific immunohistochemistry.

Derivation and validation of multimarker prognostication for normotensive patients with acute symptomatic pulmonary embolism.

RATIONALE Not all patients with acute pulmonary embolism (PE) have a high risk of an adverse short-term outcome. OBJECTIVES This prospective cohort study aimed to develop a multimarker prognostic model that accurately classifies normotensive patients with PE into low and high categories of risk of adverse medical outcomes. METHODS The study enrolled 848 outpatients from the PROTECT (PROgnosTic valuE of Computed Tomography) study (derivation cohort) and 529 patients from the Prognostic Factors for Pulmonary Embolism (PREP) study (validation cohort). Investigators assessed study participants for a 30-day complicated course, defined as death from any cause, hemodynamic collapse, and/or adjudicated recurrent PE. MEASUREMENTS AND MAIN RESULTS A complicated course occurred in 63 (7.4%) of the 848 normotensive patients with acute symptomatic PE in the derivation cohort and in 24 patients (4.5%) in the validation cohort. The final model included the simplified Pulmonary Embolism Severity Index, cardiac troponin I, brain natriuretic peptide, and lower limb ultrasound testing. The model performed similarly in the derivation (c-index of 0.75) and validation (c-index of 0.85) cohorts. The combination of the simplified Pulmonary Embolism Severity Index and brain natriuretic peptide testing showed a negative predictive value for a complicated course of 99.1 and 100% in the derivation and validation cohorts, respectively. The combination of all modalities had a positive predictive value for the prediction of a complicated course of 25.8% in the derivation cohort and 21.2% in the validation cohort. CONCLUSIONS For normotensive patients who have acute PE, we derived and validated a multimarker model that predicts all-cause mortality, hemodynamic collapse, and/or recurrent PE within the following 30 days.

Protective Effect of Focal Adhesion Kinase against Skeletal Muscle Reperfusion Injury after Acute Limb Ischemia.

OBJECTIVES In cardiac muscle, ischemia reperfusion (IR) injury is attenuated by mitochondrial function, which may be upregulated by focal adhesion kinase (FAK). The aim of this study was to determine whether increased FAK levels reduced rhabdomyolysis in skeletal muscle too. MATERIAL AND METHODS In a translational in vivo experiment, rat lower limbs were subjected to 4 hours of ischemia followed by 24 or 72 hours of reperfusion. FAK expression was stimulated 7 days before (via somatic transfection with pCMV-driven FAK expression plasmid) and outcomes were measured against non-transfected and empty transfected controls. Slow oxidative (i.e., mitochondria-rich) and fast glycolytic (i.e., mitochondria-poor) type muscles were analyzed separately regarding rhabdomyolysis, apoptosis, and inflammation. Severity of IR injury was assessed using paired non-ischemic controls. RESULTS After 24 hours of reperfusion, marked rhabdomyolysis was found in non-transfected and empty plasmid-transfected fast-type glycolytic muscle, tibialis anterior. Prior transfection enhanced FAK concentration significantly (p = 0.01). Concomitantly, levels of BAX, promoting mitochondrial transition pores, were reduced sixfold (p = 0.02) together with a blunted inflammation (p = 0.01) and reduced rhabdomyolysis (p = 0.003). Slow oxidative muscle, m. soleus, reacted differently: although apoptosis was detectable after IR, rhabdomyolysis did not appear before 72 hours of reperfusion; and FAK levels were not enhanced in ischemic muscle despite transfection (p = 0.66). CONCLUSIONS IR-induced skeletal muscle rhabdomyolysis is a fiber type-specific phenomenon that appears to be modulated by mitochondria reserves. Stimulation of FAK may exploit these reserves constituting a potential therapeutic approach to reduce tissue loss following acute limb IR in fast-type muscle.

Influence of inspiration to expiration ratio on cyclic recruitment and derecruitment of atelectasis in a saline lavage model of acute respiratory distress syndrome*

OBJECTIVE Cyclic recruitment and derecruitment of atelectasis can occur during mechanical ventilation, especially in injured lungs. Experimentally, cyclic recruitment and derecruitment can be quantified by respiration-dependent changes in PaO2 (ΔPaO2), reflecting the varying intrapulmonary shunt fraction within the respiratory cycle. This study investigated the effect of inspiration to expiration ratio upon ΔPaO2 and Horowitz index. DESIGN Prospective randomized study. SETTING Laboratory investigation. SUBJECTS Piglets, average weight 30 ± 2 kg. INTERVENTIONS At respiratory rate 6 breaths/min, end-inspiratory pressure (Pendinsp) 40 cm H2O, positive end-expiratory pressure 5 cm H2O, and FIO2 1.0, measurements were performed at randomly set inspiration to expiration ratios during baseline healthy and mild surfactant depletion injury. Lung damage was titrated by repetitive surfactant washout to induce maximal cyclic recruitment and derecruitment as measured by multifrequency phase fluorimetry. Regional ventilation distribution was evaluated by electrical impedance tomography. Step changes in airway pressure from 5 to 40 cm H2O and vice versa were performed after lavage to calculate PO2-based recruitment and derecruitment time constants (TAU). MEASUREMENTS AND MAIN RESULTS In baseline healthy, cyclic recruitment and derecruitment could not be provoked, whereas in model acute respiratory distress syndrome, the highest ΔPaO2 were routinely detected at an inspiration to expiration ratio of 1:4 (range, 52-277 torr [6.9-36.9 kPa]). Shorter expiration time reduced cyclic recruitment and derecruitment significantly (158 ± 85 torr [21.1 ± 11.3 kPa] [inspiration to expiration ratio, 1:4]; 25 ± 12 torr [3.3 ± 1.6 kPa] [inspiration to expiration ratio, 4:1]; p < 0.0001), whereas the PaO2/FIO2 ratio increased (267 ± 50 [inspiration to expiration ratio, 1:4]; 424 ± 53 [inspiration to expiration ratio, 4:1]; p < 0.0001). Correspondingly, regional ventilation redistributed toward dependent lung regions (p < 0.0001). Recruitment was much faster (TAU: fast 1.6 s [78%]; slow 9.2 s) than derecruitment (TAU: fast 3.1 s [87%]; slow 17.7 s) (p = 0.0078). CONCLUSIONS Inverse ratio ventilation minimizes cyclic recruitment and derecruitment of atelectasis in an experimental model of surfactant-depleted pigs. Time constants for recruitment and derecruitment, and regional ventilation distribution, reflect these findings and highlight the time dependency of cyclic recruitment and derecruitment.

Clinical and haematological predictors of antibiotic prescribing for acute cough in adults in Swiss practices - an observational study.

BackgroundAcute cough is a common problem in general practice and is often caused by a self-limiting, viral infection. Nonetheless, antibiotics are often prescribed in this situation, which may lead to unnecessary side effects and, even worse, the development of antibiotic resistant microorganisms worldwide. This study assessed the role of point-of-care C-reactive protein (CRP) testing and other predictors of antibiotic prescription in patients who present with acute cough in general practice.MethodsPatient characteristics, symptoms, signs, and laboratory and X-ray findings from 348 patients presenting to 39 general practitioners with acute cough, as well as the GPs themselves, were recorded by fourth-year medical students during their three-week clerkships in general practice. Patient and clinician characteristics of those prescribed and not-prescribed antibiotics were compared using a mixed-effects model.ResultsOf 315 patients included in the study, 22% were prescribed antibiotics. The two groups of patients, those prescribed antibiotics and those treated symptomatically, differed significantly in age, demand for antibiotics, days of cough, rhinitis, lung auscultation, haemoglobin level, white blood cell count, CRP level and the GP¿s license to self-dispense antibiotics. After regression analysis, only the CRP level, the white blood cell count and the duration of the symptoms were statistically significant predictors of antibiotic prescription.ConclusionsThe antibiotic prescription rate of 22% in adult patients with acute cough in the Swiss primary care setting is low compared to other countries. GPs appear to use point-of-care CRP testing in addition to the duration of clinical symptoms to help them decide whether or not to prescribe antibiotics.

Stress-induced modulation of NF-κB activation, inflammation-associated gene expression, and cytokine levels in blood of healthy men

Acute psychosocial stress stimulates transient increases in circulating pro-inflammatory plasma cytokines, but little is known about stress effects on anti-inflammatory cytokines or underlying mechanisms. We investigated the stress kinetics and interrelations of pro- and anti-inflammatory measures on the transcriptional and protein level. Forty-five healthy men were randomly assigned to either a stress or control group. While the stress group underwent an acute psychosocial stress task, the second group participated in a non-stress control condition. We repeatedly measured before and up to 120min after stress DNA binding activity of the pro-inflammatory transcription factor NF-κB (NF-κB-BA) in peripheral blood mononuclear cells, whole-blood mRNA levels of NF-κB, its inhibitor IκBα, and of the pro-inflammatory cytokines interleukin (IL)-1ß and IL-6, and the anti-inflammatory cytokine IL-10. We also repeatedly measured plasma levels of IL-1ß, IL-6, and IL-10. Compared to non-stress, acute stress induced significant and rapid increases in NF-κB-BA and delayed increases in plasma IL-6 and mRNA of IL-1ß, IL-6, and IκBα (p's<.045). In the stress group, significant increases over time were also observed for NF-κB mRNA and plasma IL-1ß and IL-10 (p's<.055). NF-κB-BA correlated significantly with mRNA of IL-1β (r=.52, p=.002), NF-κB (r=.48, p=.004), and IκBα (r=.42, p=.013), and marginally with IL-6 mRNA (r=.31, p=.11). Plasma cytokines did not relate to NF-κB-BA or mRNA levels of the respective cytokines. Our data suggest that stress induces increases in NF-κB-BA that relate to subsequent mRNA expression of pro-inflammatory, but not anti-inflammatory cytokines, and of regulatory-cytoplasmic-proteins. The stress-induced increases in plasma cytokines do not seem to derive from de novo synthesis in circulating blood cells.

A novel regulatory macrophage induced by a helminth molecule instructs IL-10 in CD4+ T cells and protects against mucosal inflammation

Immunomodulation is a common feature of chronic helminth infections and mainly attributed to the secretion of bioactive molecules, which target and modify host immune cells. In this study, we show that the helminth immunomodulator AvCystatin, a cysteine protease inhibitor, induces a novel regulatory macrophage (Mreg; AvCystatin-Mreg), which is sufficient to mitigate major parameters of allergic airway inflammation and colitis in mice. A single adoptive transfer of AvCystatin-Mreg before allergen challenge suppressed allergen-specific IgE levels, the influx of eosinophils into the airways, local and systemic Th2 cytokine levels, and mucus production in lung bronchioles of mice, whereas increasing local and systemic IL-10 production by CD4(+) T cells. Moreover, a single administration of AvCystatin-Mreg during experimentally induced colitis strikingly reduced intestinal pathology. Phenotyping of AvCystatin-Mreg revealed increased expression of a distinct group of genes including LIGHT, sphingosine kinase 1, CCL1, arginase-1, and costimulatory molecules, CD16/32, ICAM-1, as well as PD-L1 and PD-L2. In cocultures with dendritic cells and CD4(+) T cells, AvCystatin-Mreg strongly induced the production of IL-10 in a cell-contact-independent manner. Collectively, our data identify a specific suppressive macrophage population induced by a single parasite immunomodulator, which protects against mucosal inflammation.

Comparative proteomic analysis of lung tissue from guinea pigs with leptospiral pulmonary haemorrhage syndrome (LPHS) reveals a decrease in abundance of host proteins involved in cytoskeletal and cellular organization

Leptospiral pulmonary haemorrhage syndrome (LPHS) is a particularly severe form of leptospirosis. LPHS is increasingly recognized in both humans and animals and is characterized by rapidly progressive intra-alveolar haemorrhage leading to high mortality. The pathogenic mechanisms of LPHS are poorly understood which hampers the application of effective treatment regimes. In this study a 2-D guinea pig proteome lung map was created and used to investigate the pathogenic mechanisms of LPHS. Comparison of lung proteomes from infected and non-infected guinea pigs via differential in-gel electrophoresis revealed highly significant differences in abundance of proteins contained in 130 spots. Acute phase proteins were the largest functional group amongst proteins with increased abundance in LPHS lung tissue, and likely reflect a local and/or systemic host response to infection. The observed decrease in abundance of proteins involved in cytoskeletal and cellular organization in LPHS lung tissue further suggests that infection with pathogenic Leptospira induces changes in the abundance of host proteins involved in cellular architecture and adhesion contributing to the dramatically increased alveolar septal wall permeability seen in LPHS. BIOLOGICAL SIGNIFICANCE The recent completion of the complete genome sequence of the guinea pig (Cavia porcellus) provides innovative opportunities to apply proteomic technologies to an important animal model of disease. In this study, the comparative proteomic analysis of lung tissue from experimentally infected guinea pigs with leptospiral pulmonary haemorrhage syndrome (LPHS) revealed a decrease in abundance of proteins involved in cellular architecture and adhesion, suggesting that loss or down-regulation of cytoskeletal and adhesion molecules plays an important role in the pathogenesis of LPHS. A publically available guinea pig lung proteome map was constructed to facilitate future pulmonary proteomics in this species.

Cigarette Smoke Exposure Increases Myeloid Cell Production and Lung Bacterial Clearance in Mice

Pneumonia is a leading cause of hospitalization in patients with chronic obstructive pulmonary disease (COPD). Although most COPD patients are smokers, the effects of cigarette smoke exposure on clearance of lung bacterial pathogens and on immune and inflammatory responses are incompletely defined. Here, clearance of Streptococcus pneumoniae and Pseudomonas aeruginosa and associated immune responses were examined in mice exposed to cigarette smoke or following smoking cessation. Mice exposed to cigarette smoke for 6 weeks or 4 months demonstrated decreased lung bacterial burden compared to air-exposed mice when infected 16-24 hours post-exposure. When infection was performed after smoke cessation, bacterial clearance kinetics of mice previously exposed to smoke reversed to comparable levels as those of control mice suggesting that the observed defects were not dependent on adaptive immunological memory to bacterial determinants found in smoke. Comparing cytokine levels and myeloid cell production prior to infection in mice exposed to cigarette smoke relative to mice never exposed or following smoke cessation revealed that reduced bacterial burden was most strongly associated with higher levels of IL-1β and GM-CSF in the lungs and with increased neutrophil reserve and monocyte turnover in the bone marrow. Using serpinb1a-deficient mice with reduced neutrophil numbers and treatment with G-CSF showed that increased neutrophil numbers contribute only in part to the effect of smoke on infection. Our findings indicate that cigarette smoke induces a temporary and reversible increase in clearance of lung pathogens, which correlates with local inflammation and increased myeloid cell output from the bone marrow.

Prognostic value of PCSK9 levels in patients with acute coronary syndromes.

AIMS Proprotein convertase subtilisin kexin 9 (PCSK9) is an emerging target for the treatment of hypercholesterolaemia, but the clinical utility of PCSK9 levels to guide treatment is unknown. We aimed to prospectively assess the prognostic value of plasma PCSK9 levels in patients with acute coronary syndromes (ACS). METHODS AND RESULTS Plasma PCSK9 levels were measured in 2030 ACS patients undergoing coronary angiography in a Swiss prospective cohort. At 1 year, the association between PCSK9 tertiles and all-cause death was assessed adjusting for the Global Registry of Acute Coronary Events (GRACE) variables, as well as the achievement of LDL cholesterol targets of <1.8 mmol/L. Patients with higher PCSK9 levels at angiography were more likely to have clinical familial hypercholesterolaemia (rate ratio, RR 1.21, 95% confidence interval, CI 1.09-1.53), be treated with lipid-lowering therapy (RR 1.46, 95% CI 1.30-1.63), present with longer time interval of chest pain (RR 1.29, 95% CI 1.09-1.53) and higher C-reactive protein levels (RR 1.22, 95% CI 1.16-1.30). PCSK9 increased 12-24 h after ACS (374 ± 149 vs. 323 ± 134 ng/mL, P < 0.001). At 1 year follow-up, HRs for upper vs. lower PCSK9-level tertiles were 1.13 (95% CI 0.69-1.85) for all-cause death and remained similar after adjustment for the GRACE score. Patients with higher PCSK9 levels were less likely to reach the recommended LDL cholesterol targets (RR 0.81, 95% CI 0.66-0.99). CONCLUSION In ACS patients, high initial PCSK9 plasma levels were associated with inflammation in the acute phase and hypercholesterolaemia, but did not predict mortality at 1 year.

Organ inflammation in porcine Escherichia coli sepsis is markedly attenuated by combined inhibition of C5 and CD14.

Sepsis is an infection-induced systemic inflammatory syndrome, potentially causing organ failure. We previously showed attenuating effects on inflammation, thrombogenicity and haemodynamics by inhibiting the Toll-like receptor co-factor CD14 and complement factor C5 in a porcine Escherichia coli-induced sepsis model. The present study explored the effect on organ inflammation in these pigs. Tissue samples were examined from the combined treatment group (n = 8), the positive (n = 8) and negative (n = 6) control groups after 4h of sepsis. Inflammatory biomarkers were measured using ELISA, multiplex and qPCR analysis. Combined inhibition of C5 and CD14 markedly attenuated IL-1β by 31-66% (P < 0.05) and IL-6 by 54-96% (P < 0.01) in liver, kidney, lung and spleen; IL-8 by 65-100% in kidney, lung, spleen, and heart (P < 0.05) and MCP-1 by 46-69% in liver, kidney, spleen and heart (P < 0.05). Combined inhibition significantly attenuated tissue factor mRNA upregulation in spleen (P < 0.05) and IP-10 mRNA upregulation in four out of five organs. Finally, C5aR mRNA downregulation was prevented in heart and kidney (P < 0.05). Combined inhibition of C5 and CD14 thus markedly attenuated inflammatory responses in all organs examined. The anti-inflammatory effects observed in lung and heart may explain the delayed haemodynamic disturbances observed in septic pigs receiving combined inhibition of C5 and CD14.

Mechanisms of apoptosis in bleomycin-induced lung injury: Implications for pulmonary fibrosis

Chronic inflammation leading to pulmonary fibrosis develops in response to environmental pollutants, radiotherapy, or certain cancer chemotherapeutic agents. Studies have shown that several cell types accumulate during the inflammatory process, but little information is known about what actually triggers and stimulates persistent inflammation culminating in fibrosis. As a first step in defining the events that precipitate inflammation in the lung, the biological mechanism(s) mediating apoptosis and cellular targets must be identified. The purpose of this study was to determine the molecular mechanism(s) of bleomycin-induced apoptosis in the lung using mice deficient in genes that we hypothesized to play a key role in apoptosis. Intratracheal administration of bleomycin led to caspase-mediated DNA fragmentation characteristic of apoptosis. The effects of bleomycin were associated with translocation of p53 from the cytosol to the nucleus only in alveolar macrophages that had been exposed to the drug in vivo, suggesting that the lung microenvironment regulated p53 activation. Experiments with a thiol antioxidant (N-acetylcysteine) in vivo and nitric oxide donors in vitro confirmed that reactive oxygen species were required for p53 activation. A specific role for NO was demonstrated in experiments with iNOS−/− macrophages, which failed to demonstrate nuclear p53 localization after in vivo bleomycin exposure. Strikingly, rates of bleomycin-induced apoptosis were at least two-fold higher in iNOS−/− and p53−/− C57BL/6 mice compared to wild-type controls. Laser Scanning Cytometry (LSC) analysis revealed that bleomycin exposure resulted in a 2-fold induction in Fas and FasL expression in wild-type mice but not iNOS−/− or p53−/− mice. Experiments using gld mice confirmed that the Fas/FasL pathway was the primary mechanism of bleomycin-induced apoptosis in the lung. LSC-mediated analysis indicated that bleomycin exposure resulted in a 2-fold induction in Bax expression in iNOS−/− and P53−/− mice but not wild-type mice. Furthermore, LSC analysis revealed that bleomycin exposure induced a 3-fold increase in thrombospondin expression in wild-type mice. However, thrombospondin was not expressed in either the iNOS−/− or p53−/− mice, implicating a thrombospondin-mediated apoptotic cell clearance mechanism in the lung. Together, these results demonstrate that iNOS and p53 positively regulate apoptosis via the Fas/FasL pathway and mediate a novel apoptosis-suppressing pathway in the lung. ^

Adenosine mediated lung mast cell degranulation in the mouse

Adenosine has been implicated to play a role in inflammatory processes associated with asthma. Most notable is adenosine's ability to potentiate mediator release from mast cells. Mast cells are bone marrow derived inflammatory cells that can release mediators that have both immediate and chronic effects on airway constriction and inflammation. Most physiological roles of adenosine are mediated through adenosine receptors. Four subtypes of adenosine receptors have been identified, A1, A2A, A2B and A 3. The mechanisms by which adenosine can influence the release of mediators from lung tissue mast cells is not understood due to lack of in vivo models. Mice deficient in the enzyme adenosine deaminase (ADA) have been generated. ADA controls the levels of adenosine in tissues and cells, and consequently, adenosine accumulates in the lungs of ADA-deficient mice. ADA-deficient mice develop features seen in asthmatics, including lung eosinophilia and mucus hypersecretion. In addition, lung tissue mast cell degranulation was associated with elevated adenosine in ADA-deficient lungs and can be prevented by ADA enzyme therapy. We established primary murine lung mast cell cultures, and used real time RT-PCR and immunofluorescence to demonstrate that A 2A, A2B and A3 receptors are expressed on murine lung mast cells. Studies using selective adenosine receptor agonists and antagonists and A3 receptor deficient (A3−/−) mast cells suggested that activation of A3 receptors could induce mast cell mediator release in vitro. Furthermore, this mediator release was associated with increases in intracellular Ca++ that appeared to be mediated through a Gi and PI3K pathway. In addition, nebulized A3 receptor agonist directly induced lung mast cell degranulation in wild type mice while having no effect in A3−/− mice. These results demonstrate that the A3 receptor plays an important role in adenosine mediated murine lung mast cell degranulation. Therefore, the A3 adenosine receptor and its signaling pathways may represent novel therapeutic targets for the treatment and prevention of asthma. ^