Efficacy of RNA interference knockdown using aerosolized short interfering RNAs bound to nanoparticles in three diverse aphid species.

RNA interference (RNAi) has emerged as a promising method for validating gene function; however, its utility in nonmodel insects has proven problematic, with delivery methods being one of the main obstacles. This study investigates a novel method of RNAi delivery in aphids, the aerosolization of short interfering RNA (siRNA)-nanoparticle complexes. By using nanoparticles as a siRNA carrier, the likelihood of cellular uptake is increased, when compared to methods previously used in insects. To determine the efficacy of this RNAi delivery system, siRNAs were aerosolized with and without nanoparticles in three aphid species: Acyrthosiphon pisum, Aphis glycines and Schizaphis graminum. The genes targeted for knockdown were carotene dehydrogenase (tor), which is important for pigmentation in Ac. pisum, and branched chain-amino acid transaminase (bcat), which is essential in the metabolism of branched-chain amino acids in all three aphid species. Overall, we observed modest gene knockdown of tor in Ac. pisum and moderate gene knockdown of bcat in Ap. glycines along with its associated phenotype. We also determined that the nanoparticle emulsion significantly increased the efficacy of gene knockdown. Overall, these results suggest that the aerosolized siRNA-nanoparticle delivery method is a promising new high-throughput and non-invasive RNAi delivery method in some aphid species.

Tazobactam-induced haemolytic anaemia, possibly caused by non-immunological adsorption of IgG onto patient's red cells.

A patient with pneumonia was treated with Tazocin (piperacillin/tazobactam). However, the expected haemoglobin (Hb) increment after transfusion was not achieved. Plasma bilirubin and lactate dehydrogenase were raised. The direct antiglobulin test (DAT) was positive (4+) for immunoglobulin G (IgG) only, but no RBC antibodies were demonstrable in the plasma or an eluate from the patient's RBCs. Drug-induced haemolysis was suspected. After discontinuing Tazocin administration, Hb and bilirubin levels returned to expected values. The patient's plasma gave a positive (3+) indirect antiglobulin reaction only with RBCs pretreated with tazobactam. However, random patient plasmas also gave weak (+/- to 1+) reactions, indicating non-immunological adsorption of IgG onto RBCs rather than specific anti-tazobactam antibodies. Subsequently, plasma samples with varying IgG levels (0.8-89.7 g L(-1)) were tested against RBCs pretreated with tazobactam. The amount of plasma IgG non-immunologically adsorbed onto the drug-coated RBCs was found to correlate directly with the plasma IgG level. The patient had a high plasma IgG level (41.6 g L(-1)) which explains why the antiglobulin test was stronger with the patient's plasma than with random plasma samples. Previous reports (Garratty & Arndt, (1998) British Journal of Haematology, 100, 777-783; Arndt & Garratty (2000) Transfusion, 40, 29S) suggested that non-immunological coating of RBCs with IgG may affect RBC survival; our results would support that suggestion. This is the first reported case of haemolytic anaemia associated with tazobactam.

Salmeterol, a novel, long-acting beta 2-adrenoceptor agonist: characterization of pharmacological activity in vitro and in vivo.

1. Salmeterol, a novel, long-acting beta-adrenoceptor agonist, has been compared with isoprenaline and salbutamol for activity in vitro on a range of beta-adrenoceptor containing preparations from laboratory animals and man, and in vivo for bronchodilator activity in the conscious guinea-pig. 2. Salmeterol, like isoprenaline and salbutamol, relaxed preparations of both guinea-pig trachea (contracted by prostaglandin (PG)F2alpha or electrical stimulation) and human bronchus (contracted by PGF 2 alpha) in a concentration-related fashion. Salmeterol was of similar potency to isoprenaline and more potent than salbutamol on both airway preparations. 3. Relaxant responses of superfused guinea-pig trachea and human bronchus to isoprenaline and salbutamol declined rapidly when the agonists were washed from the tissues, with complete recovery within 10 min, whereas responses to salmeterol were more persistent. In electrically-stimulated guinea-pig trachea preparations, inhibition by salmeterol persisted for periods of up to 12h, despite continuous superfusion with agonist-free medium. However, these persistent responses were rapidly and fully reversed by the beta-adrenoceptor blocking drug, propranolol (0.1 microM). In further studies on guinea-pig trachea, propranolol caused concentration-related parallel, rightward shifts of salmeterol concentration-effect curves, yielding a pA2 of 9.0. The slope of the Schild plot was 1.02. 4. Another beta-adrenoceptor blocking drug, sotalol (10 microM), also fully and rapidly reversed established submaximal responses to salmeterol in superfused guinea-pig trachea. However, after administration of sotalol was stopped, the antagonism waned, and salmeterol responses were reasserted without the addition of further agonist. 5. In the beta 1-adrenoceptor containing preparation, rat left atria, isoprenaline exhibited potent, concentration-related, positive inotropic activity, whereas salbutamol and salmeterol were at least 2000-5000 fold less potent, and appeared to be partial agonists. At a concentration of 72 microM, salmeterol exhibited weak antagonism of isoprenaline-induced increases in atrial force of contraction. This antagonism was less marked than that caused by salbutamol (42 microM).6. On the guinea-pig isolated gastric fundus strip, a putative beta3-adrenoceptor containing preparation, salbutamol and salmeterol had only modest agonist activity, being 20-30 fold less potent than isoprenaline and the selective ,beta3-adrenoceptor agonist, BRL 35135.7. In conscious guinea-pigs, inhaled salmeterol and salbutamol were approximately equipotent in causing dose-related bronchodilatation. Whereas the duration of action of salbutamol at its threshold-effective dose was less than 90min, the responses to a similarly effective dose of salmeterol were well-maintained for at least 6 h.8. Salmeterol is therefore a potent and selective beta2-adrenoceptor agonist with a remarkably long duration of action in isolated superfused airways smooth muscle. It also causes persistent bronchodilatation in vivo, in the guinea-pig, when administered by the inhaled route.

The development of tolerance to antilipolytic agents in rats.

The development of tolerance to the action of certain antilipolytic agents has been investigated in vivo in rats. Tolerance to oral nicotinic acid developed during twice daily dosing for 4 days at 100 and 250 mg/kg but not at 10, 25 or 50 mg/kg. Tolerance induced by high doses of nicotinic acid was no longer detectable after a further week without treatment. Tolerance developed to a dose of 10 mg/kg nicotinic acid when dosing was repeated at hourly intervals for up to 6 hr. Rats made tolerant to nicotinic acid also became tolerant to both 5-methylpyrazole-3-carboxylic acid and to pyridyl-3-tetrazole and rats made tolerant to these antilipolytic agents were also tolerant to nicotinic acid. Rats made tolerant to nicotinic acid still responded to the antilipolytic activity of the prostaglandin analogue, sulprostone. These results suggest that nicotinic acid, pyridyl-3-tetrazole and 5-methylpyrazole-3-carboxylic acid act through a common mechanism or receptor and that the development of tolerance is associated with this receptor or the mechanism by which it is linked to adenylate cyclase.

The development of tolerance to antilipolytic agents by isolated rat adipocytes.

Using an isolated rat epididymal adipocyte system we have studied the development of tolerance to and cross-tolerance between nicotinic acid, 5-methylpyrazole-3-carboxylic acid and pyridyl-3-tetrazole. Preincubating isolated adipocytes with any one of these compounds results in a reduction of the antilipolytic activity of that compound when the cells are exposed to a subsequent challenge dose. Furthermore, preincubation with nicotinic acid, 5-methylpyrazole-3-carboxylic acid or pyridyl-3-tetrazole results in a reduction of the antilipolytic response to challenge with either of the other two compounds. Preincubation of isolated adipocytes with nicotinic acid does not affect the subsequent antilipolytic activity of the PGE2 analogue, sulprostone. Preincubation with sulprostone does not lead to the development of tolerance to its own antilipolytic actions. The results obtained from these studies suggest that nicotinic acid, 5-methylpyrazole-3-carboxylic acid and pyridyl-3-tetrazole exert their antilipolytic activity via a common biochemical pathway which is distinct from that mediating the antilipolytic activity of prostaglandins. These findings also indicate that the development of tolerance occurs prior to the involvement of adenylate cyclase in lipolysis.

Characterization of the adenosine receptor responsible for the inhibition of histamine and SRS-A release from human lung fragments.

The inhibitory effects of a range of natural and synthetic derivatives of adenosine on the antigen-induced release of histamine and slow reacting substance of anaphylaxis (SRS-A) from human lung has been studied. The nucleotides ATP, ADP and AMP appear to act by being converted to adenosine. The rank order of inhibitory potency of the synthetic analogues indicates that these compounds act at an extracellular A2/Ra purinoceptor. The xanthines, 1, 3-diethyl-8-phenylxanthine, 8-phenyltheophylline and theophylline antagonized the inhibitory action of N-ethyl-carboxamideadenosine competitively. Theobromine was inactive. This supports the view that the inhibitory receptor is of the A/R type. Hexobendine and dipyridamole, reported to antagonize the uptake of adenosine, failed to modify the response of human lung fragments to adenosine. The P site agonist 2',5' dideoxyadenosine inhibited the release of histamine and SRS-A. This effect was not prevented by the inhibitors of uptake, hexobendine and dipyridamole, nor was it antagonized by 8-phenyltheophylline.

Stimulation of flux through hepatic pyruvate dehydrogenase by 3-mercaptopicolinate.

Isolated hepatocytes from 24-h-starved rats were used to assess the possible effect of the hypoglycaemic agent 3-mercaptopicolinate on flux through the hepatic pyruvate dehydrogenase complex. Increasing the extracellular pyruvate concentration from 1 mM to 2 mM or 5 mM resulted in an increase in flux through pyruvate dehydrogenase and the tricarboxylic acid cycle as measured by 14CO2 evolution from [1-14C]pyruvate and [3-14C]pyruvate. Gluconeogenesis was inhibited by 3-mercaptopicolinate from both 1 mM and 2 mM pyruvate, but significant increases in malate and citrate concentrations only occurred in cells incubated with 1 mM pyruvate. Flux through pyruvate dehydrogenase was stimulated by 3-mercaptopicolinate with 1 mM pyruvate but was unaltered with 2 mM pyruvate. Dichloroacetate stimulated flux through pyruvate dehydrogenase with no effect on gluconeogenesis in the presence of 1 mM pyruvate. There was no effect of 3-mercaptopicolinate, administered in vivo, to 24-h-starved rats on the activity of pyruvate dehydrogenase in freeze-clamped heart or liver tissue, although the drug did decrease blood glucose concentration and increase the blood concentrations of lactate and alanine. Dichloroacetate, administered in vivo to 24-h-starved rats, increased the activity of pyruvate dehydrogenase in freeze-clamped heart and liver, and caused decreases in the blood concentrations of glucose, lactate, and alanine. The results suggest that 3-mercaptopicolinate increases flux through hepatocyte pyruvate dehydrogenase by an indirect mechanism.

The release of histamine from rat mast cells by chymotrypsin.

The release of histamine from rat peritoneal mast cells induced by alpha-chymotrypsin and that induced by antigen have characteristics in common. The kinetics of histamine release initiated by the two agents are similar. Both alpha-chymotrypsin and antigen release histamine in the absence of extracellular calcium, phosphatidyl serine enhances the release, and disodium cromoglycate inhibits both reactions. In contrast, extracellular alpha-chymotrypsin does not induce histamine release from cells isolated from fragments of human lung, human basophils, histamine-containing cells lavaged from the bronchial lumen of the rhesus monkey, fragments of human lung and fragments of guinea pig lung.

Characterization of the receptor mediating the antianaphylactic effects of beta-adrenoceptor agonists in human lung tissue in vitro.

1 The rank order of potency of six beta-adrenoceptor agonists as inhibitors of the anaphylactic release of histamine from fragments of passively sensitized human lung in vitro was (--)-isoprenaline greater than (--) -adrenaline greater than (+/-)-salbutamol greater than (--)-noradrenaline greater than R0363 greater than H133/22. 2 The beta-adrenoceptor antagonists, propranolol, atenolol and H35/25, blocked the response to both (--)-isoprenaline and (+/-)-salbutamol competitively. Each antagonist gave similar pA2 values with both agonists. pA2 values were consistently at the high end of the range expected for interaction at a beta 2-adrenoceptor. 3 Practolol did not antagonize isoprenaline in a competitive manner but was a competitive antagonist of salbutamol with a pA2 at the high end of the range expected for interaction at a beta 2-adrenoceptor. 4 Data obtained with agonists are consistent with the receptor being of the beta 2-subtype. Data obtained with antagonists indicate a consistently higher affinity for the receptor than observed for the beta 2-subtype in other tissues but do not suggest a novel beta-adrenoceptor subtype on the mast cell of the human lung.

Histamine-containing cells from bronchial lavage of macaque monkeys. Time course and inhibition of anaphylactic histamine release.

Bronchial lavage of rhesus and cynomolgus monkeys provided leucocyte suspensions with viable histamine-containing cells (HCC) as 1--8% of the white cell population. These HCC released histamine or challenge with antiserum to human IgE. HCC from 2 monkeys with pulmonary and cutaneous hypersensitivity to Ascaris antigen (AA) released histamine on challenge with AA. The extent of histamine release was related to the concentration of antigen and antiserum, and histamine release was complete within 10 min of challenge. (+/-)Salbutamol and (-)isoprenaline were potent inhibitors of anaphylactic histamine release from HCC, but disodium cromoglycate and AH 9679 were relatively poor inhibitors. The HCC system combines the reproducibility of a cell suspension with a response to drugs similar to that of human lung fragments.

A comparison of the anti-anaphylactic activities of salbutamol and disodium cromoglycate in the rat, the rat mast cell and in human lung tissue.

1 Salbutamol and disodium cromoglycate were compared for anti-anaphylactic activity against passive anaphylaxis in rat skin and peritoneum in vivo and in rat mast cells and human lung fragments in vitro.2 Salbutamol administered intravenously to rats inhibited cutaneous anaphylaxis, but also inhibited cutaneous responses to histamine and 5-hydroxytryptamine. Salbutamol administered intraperitoneally inhibited the release of slow reacting substance of anaphylaxis (SRS-A) but not the release of histamine in the peritoneum. It was a very weak inhibitor of histamine release from rat mast cells in vitro.3 Disodium cromoglycate administered intravenously to rats inhibited cutaneous anaphylaxis. Disodium cromoglycate administered intraperitoneally to rats inhibited the release of histamine and, to a lesser extent, SRS-A in the peritoneum. It was an effective but short-acting inhibitor of histamine release from rat mast cells in vitro.4 Salbutamol was a potent inhibitor of the anaphylactic release of histamine and SRS-A from fragments of human lung.5 Disodium cromoglycate was a weak inhibitor of the anaphylactic release of histamine and SRS-A from fragments of human lung. The inhibition was variable and not dose-related.6 The concentration of salbutamol required to inhibit anaphylaxis in human lung is of the same order as that required to relax human bronchial muscle. It is suggested that salbutamol may be more effective in allergic asthma if given in a prophylactic regimen.

Benzopyrones. 14. Synthesis and antiallergic properties of some N-tetrazolylcarboxamides and related compounds.

A series of chromones containing an acidic group has been synthesized and screened for the ability to inhibit passive cutaneous anaphylaxis and the release of histamine from mast cells of the rat. Many of the chromones contain the N-(5-tetrazolyl)carboxamido group, a novel source of acidity. Others contain a carboxyl, C-(5-tetrazolyl), 5-(4H)-oxotetrazolinyl, or N-(5-tetrazolyl)sulfonamido function. The compounds were compared with cromolyn sodium (sodium cromoglycate) and many were found to be powerful inhibitors of anaphylaxis. The most potent was 7-methoxy-4-oxo-N-(5-tetrazolyl)-4H-1-benzopyran-2-carboxamide (15). Structure-activity relationships among the chromones and also some related compounds are discussed.