Development of an immunobiosensor assay for the beta-adrenergic compound zilpaterol

A surface plasmon resonance biosensor method was developed to measure zilpaterol residues in sheep urine. A CM-5 sensor chip previously reacted with ethylenediamine to produce an aminoethyl group was coupled with 4-carboxybutyl zilpaterol activated using EDC/NHS. Five polyclonal and four monoclonal antibodies were screened for their suitability to detect low levels of zilpaterol using the biosensor technology. Total binding was greater for polyclonal than monoclonal antibodies, but a less diluted antibody solution was required for polyclonal antibodies. A fixed antibody concentration and various concentrations of zilpaterol were injected to obtain a standard curve for each antibody to allow for B-0 and IC50 determination. The stability of the assay was assessed by the consistency of B0 in repeated experiments extending at least six hours. A measure of non-specific binding allowed the assessment of the specificity of the antibody-immobilized ligand interaction. The effect of varying concentrations of urine on B-0 and IC50 was evaluated to assess the degree of

Effect of intravenous beta-2 agonist treatment on clinical outcomes in acute respiratory distress syndrome (BALTI-2): a multicentre, randomised controlled trial

BACKGROUND:
In a previous randomised controlled phase 2 trial, intravenous infusion of salbutamol for up to 7 days in patients with acute respiratory distress syndrome (ARDS) reduced extravascular lung water and plateau airway pressure. We assessed the effects of this intervention on mortality in patients with ARDS.
METHODS:
We did a multicentre, placebo-controlled, parallel-group, randomised trial at 46 UK intensive-care units between December, 2006, and March, 2010. Intubated and mechanically ventilated patients (aged =16 years) within 72 h of ARDS onset were randomly assigned to receive either salbutamol (15 µg/kg ideal bodyweight per h) or placebo for up to 7 days. Randomisation was done by a central telephone or web-based randomisation service with minmisation by centre, pressure of arterial oxygen to fractional inspired oxygen concentration (PaO(2)/F(I)O(2)) ratio, and age. All participants, caregivers, and investigators were masked to group allocation. The primary outcome was death within 28 days of randomisation. Analysis was by intention-to-treat. This trial is registered, ISRCTN38366450 and EudraCT number 2006-002647-86.
FINDINGS:
We randomly assigned 162 patients to the salbutamol group and 164 to the placebo group. One patient in each group withdrew consent. Recruitment was stopped after the second interim analysis because of safety concerns. Salbutamol increased 28-day mortality (55 [34%] of 161 patients died in the salbutamol group vs 38 (23%) of 163 in the placebo group; risk ratio [RR] 1·47, 95% CI 1·03-2·08).
INTERPRETATION:
Treatment with intravenous salbutamol early in the course of ARDS was poorly tolerated. Treatment is unlikely to be beneficial, and could worsen outcomes. Routine use of ß-2 agonist treatment in ventilated patients with this disorder cannot be recommended.

Specific role of neuronal nitric-oxide synthase when tethered to the plasma membrane calcium pump in regulating the beta-adrenergic signal in the myocardium

The cardiac neuronal nitric-oxide synthase (nNOS) has been described as a modulator of cardiac contractility. We have demonstrated previously that isoform 4b of the sarcolemmal calcium pump (PMCA4b) binds to nNOS in the heart and that this complex regulates beta-adrenergic signal transmission in vivo. Here, we investigated whether the nNOS-PMCA4b complex serves as a specific signaling modulator in the heart. PMCA4b transgenic mice (PMCA4b-TG) showed a significant reduction in nNOS and total NOS activities as well as in cGMP levels in the heart compared with their wild type (WT) littermates. In contrast, PMCA4b-TG hearts showed an elevation in cAMP levels compared with the WT. Adult cardiomyocytes isolated from PMCA4b-TG mice demonstrated a 3-fold increase in Ser(16) phospholamban (PLB) phosphorylation as well as Ser(22) and Ser(23) cardiac troponin I (cTnI) phosphorylation at base line compared with the WT. In addition, the relative induction of PLB phosphorylation and cTnI phosphorylation following isoproterenol treatment was severely reduced in PMCA4b-TG myocytes, explaining the blunted physiological response to the beta-adrenergic stimulation. In keeping with the data from the transgenic animals, neonatal rat cardiomyocytes overexpressing PMCA4b showed a significant reduction in nitric oxide and cGMP levels. This was accompanied by an increase in cAMP levels, which led to an increase in both PLB and cTnI phosphorylation at base line. Elevated cAMP levels were likely due to the modulation of cardiac phosphodiesterase, which determined the balance between cGMP and cAMP following PMCA4b overexpression. In conclusion, these results showed that the nNOS-PMCA4b complex regulates contractility via cAMP and phosphorylation of both PLB and cTnI.

Screening and confirmatory determination of ractopamine residues in calves treated with growth promoting doses of the beta-agonist

Ractopamine (RCT) is a phenethanolamine member of the family of beta-adrenergic agonists (beta-agonists), This class of compounds have become notable for their properties of enhancing the growth rates of farm animal species but are not licensed for use in Europe. An ELISA procedure employing a polyclonal antibody raised in a goat was developed to detect RCT residues in bovine urine samples, The assay had a high sensitivity (calibration curve mid-point of 22 pg per well), allowing the analysis of urine samples without the need for sample clean-up. In addition, an LC-MS-MS confirmatory procedure was developed which was able to act as a confirmatory procedure for the ELISA results. Four calves were orally treated with RCT (0.1 mg kg(-1) body mass for 17 d) and urine samples collected were assayed by both analytical procedures. It was observed that RCT residues were excreted mainly in the form of glucuronides and deconjugation could be achieved using two different sources of the enzyme beta-glucuronidase (Helix pomatia and Escherichia coli), High concentrations of RCT residues were found throughout the medication period (44-473 ng ml(-1); LC-MS-MS data) and remained present for several days following removal of the drug from the diet, RCT residues were no longer detectable 2 weeks after withdrawal, Good agreement (r(2) = 0.73) was achieved between the ELISA and LC-MS-MS results, especially when sample deconjugation was applied to the urine samples for both sets of analyses, The results show that an effective screening and confirmatory system was devised to detect RCT residues in urine samples taken during treatment and close to withdrawal, However, alternative matrices may have to be selected to allow the illegal use of the substance to be detected following prolonged withdrawal times.

Reliability of blood pressure determination with the Finapres with altered physiological states or pharmacodynamic conditions

The blood pressure waveform is modified on distal propagation by phenomena such as dispersion, reflection and the state of the arterial compliance. The consequent effects are amplification and narrowing of the wave, with an increased systolic, reduced diastolic and essentially unaltered mean blood pressure. The Finapres measures the peripheral pressure using the volume clamp principle; it has not been validated under altered physiological conditions and during pharmacodynamic interventions. We studied simultaneous Finapres and brachial blood pressures (using a conventional oscillometric sphygmomanometer—Vitalmap) in ten normal volunteers at rest, and during dynamic exercise and a cold pressor test. The effects of pharmacodynamic intervention were examined following beta-adrenoceptor blockade with propranolol (160 mg) or beta-adrenoceptor modulation with the beta-adrenoceptor partial agonist celiprolol (400 mg). The Finapres systolic pressure was significantly higher than the brachial value during all three test states. The difference between the systolic pressures measured by the two devices was shown to increase significantly during the cold pressor test, but not during dynamic (supine bicycle) exercise. The Finapres diastolic pressure was significantly higher than the Vitalmap value during exercise and the cold pressor test. The differences between the two methods increased significantly over time. Beta-adrenergic blockade with propranolol or modulation with celiprolol had no significant interaction with the pressure differences between the Finapres and Vitalmap techniques. The results would support the view that the Finapres can provide blood pressure information which is robust under most circumstances. Although this pharmacodynamic intervention did not alter the relationship between the peripheral and central blood pressure, it is important to note that this dynamic relationship is sensitive to circulatory loading conditions and wave transmission characteristics; it is possible that the Finapres could be less reliable in clinical settings where potent vasoactive agents were being administered.

Effective monitoring for ractopamine residues in samples of animal origin by SPR biosensor and mass spectrometry

Ractopamine (RCT) is a member of the beta-2-agonist (beta-agonist) family. It is licensed for use as an animal growth promoter in more than 20 countries worldwide, including the United States and Canada, but is either not licensed or prohibited by over 150 others, including those within the European Union. The issue of the use of RCT in livestock bound for human consumption has risen to prominence recently following the decision by The People's Republic of China to ban the import of pork from a number of processing plants after finding traces of RCT in shipments from the U.S.A.

Consequences of unlocking the cardiac myosin molecule in human myocarditis and cardiomyopathies

Myocarditis, often initiated by viral infection, may progress to autoimmune inflammatory heart disease, dilated cardiomyopathy and heart failure. Although cardiac myosin is a dominant autoantigen in animal models of myocarditis and is released from the heart during viral myocarditis, the characterization, role and significance of anti-cardiac myosin autoantibodies is poorly defined. In our study, we define the human cardiac myosin epitopes in human myocarditis and cardiomyopathies and establish a mechanism to explain how anti-cardiac myosin autoantibodies may contribute to heart disease. We show that autoantibodies to cardiac myosin in sera from myocarditis and dilated cardiomyopathies in humans targeted primarily epitopes in the S2 hinge region of cardiac myosin. In addition, anti-cardiac myosin antibodies in sera or purified IgG from myocarditis and cardiomyopathy targeted the beta-adrenergic receptor and induced antibody-mediated cAMP-dependent protein kinase A (PKA) cell signaling activity in heart cells. Antibody-mediated PKA activity in sera was abrogated by absorption with anti-human IgG. Antibody-mediated cell signaling of PKA was blocked by antigen-specific inhibition by human cardiac myosin or the beta-adrenergic receptor but not the alpha adrenergic receptor or bovine serum albumin. Propranolol, a beta blocker and inhibitor of the beta-adrenergic receptor pathway also blocked the antibody-mediated signaling of the beta-adrenergic receptor and PKA. The data suggest that IgG antibody against human cardiac myosin reacts with the beta-adrenergic receptor and triggers PKA signaling in heart cells. In summary, we have identified a new class of crossreactive autoantibodies against human cardiac myosin and the beta-adrenergic receptor in the heart. In addition, we have defined disease specific peptide epitopes in the human cardiac myosin rod S2 region in human myocarditis and cardiomyopathy as well as a mechanistic role of autoantibody in the pathogenesis of disease.

Acoustic rhinometry in the dog:a novel large animal model for studies of nasal congestion

The aim of this project was to develop and pharmacologically characterize an experimental dog model of nasal congestion in which nasal patency is measured using acoustic rhinometry. Solubilized compound 48/80 (0.3-3.0%) was administered intranasally to thiopental anesthetized beagle dogs to elicit nasal congestion via localized mast cell degranulation. Compound 48/80-induced effects on parameters of nasal patency were studied in vehicle-treated animals, as well as in the same animals pretreated 2 hours earlier with oral d-pseudoephedrine or chlorpheniramine. Local mast cell degranulation caused a close-related decrease in nasal cavity volume and minimal cross-sectional area (Amin) together with a highly variable increase in nasal secretions. Maximal responses were seen at 90-120 minutes after 48/80 administration. Oral administration of the adrenergic agonist, d-pseudoephedrine (3.0 mg/kg), significantly antagonized all of the nasal effects of compound 48/80 (3.0%). In contrast, oral administration of the histamine H1 receptor antagonist chlorpheniramine (10 mg/kg) appeared to reduce the increased nasal secretions but was without effect on the compound 48/ 80-induced nasal congestion (i.e., volume and Amin). These results show the effectiveness of using acoustic rhinometry in this anesthetized dog model. The observations that compound 48/80-induced nasal congestion was prevented by d-pseudoephedrine pretreatment, but not by chlorpheniramine, suggest that this noninvasive model system may provide an effective tool with which to study the actions of decongestant drugs in preclinical investigations.