Inhibition of IgE-mediated allergic histamine release from rat peritoneal mast cells by azelastine and selected antiallergic drugs

The ability of azelastine to inhibit IgE-mediated allergic histamine release from the peritoneal mast cells of actively sensitized rats was investigated and compared with selected antiallergic agents. Azelastine added simultaneously with the allergic stimuli (ovalbumin, OA, 10 g/ml + phosphatidylserine, PS, 10 g/ml) or preincubated with cells for 10 min prior to antigen challenge produced similar concentration-dependent inhibition of allergic histamine release. The IC_50s (M) following 10-min preincubation were as follows: azelastine = 4.8; astemizole = 86.3; ketotifen = 112.2; diphenhydramine = 133 and theophylline = 2040.3. At IC₅₀ level azelastine was about 18, 23, 28 and 425 times as effective as astemizole, ketotifen (newer histamine H₁-receptor antagonists), diphenhydramine (a traditional H₁-receptor antagonist), and theophylline (a phosphodiesterase inhibitor), respectively. Sodium cromoglycate in a concentration range or 1–1000 M (0 or 10-min preincubation) failed to exert any inhibitory effect. These data showed that among six drugs tested azelastine is the most potent inhibitor of allergic histamine release from rat peritoneal mast cells.     

Inhibitory effect of lysophosphatidylcholine on the histamine release from rat peritoneal mast cells

Histamine release from isolated rat peritoneal mast cells induced by compound 48/80 (0.5 g/ml) or antigen-antibody reaction was inhibited by lysophosphatidylcholine in a dose-dependent fashion at concentrations up to 4 M. Within the same range of concentration, lysophosphatidylcholine exhibited a membrane-stabilizing action on the model membrane systems decreasing the permeability of lipid bilayer and the fluidity of liposomal membrane in the liquid crystalline state. At concentrations higher than 8 M, lysophosphatidylcholine damaged the cell membrane and subsequently histamine was released. It was assumed that lysophosphatidylcholine may act as an endogenous membrane stabilizer inhibiting histamine release in normal mast cells.     

Adrenaline-stimulated,aspirin-sensitive synthesis of histamine in the rat

The intravenous injection of 40 g/kg of adrenaline raised total rat lung histamine from 5.3±0.7 g, to 8.4±0.7 g and rat skin histamine from 624±51 g to 835±85 within 5 min. These changes were no longer apparent after 10 min. Stomach histamine was unaffected. Blood drawn 2 min after the injection of adrenaline failed to show an increased content of histamine. Rats given 1 mg/kg of compound 48/80, had greatly elevated levels of histamine in blood, but exhibited no increase in lung histamine. This result, as well as the extent of the increase of histamine observed in skin, which cannot be accounted for in any other way, point towards stepped-up local synthesis as the origin of the effect of adrenaline. Aspirin (20 mg/kg, intravenously 10 min prior to adrenaline), prevented increases of skin histamine. Evidence suggesting mast cells as the site of action of adrenaline, is discussed.     

Measurement of urinary Nτ-methylhistamine excretion: Correlation of a newly developed radioimmunoassay (RIA) with gas chromatography mass spectrometry (GCMS)

A newly developed radioimmunoassay (RIA, Y) for the determination of urinary N-methylhistamine concentrations was correlated with gas chromatography mass spectrometry (GCMS, X). In 34 urine samples, with histamine and N-methylhistamine levels within our reference values, the correlation was: Y=1.47X–0.245 mol/l (r=0.92;p-slope 0.0001). In 14 pathological urine samples, derived from patients with mastocytosis and having upper reference values, the correlation was: Y=1.75X–1.02 mol/l (r=0.93;p-slope 0.001). In spite of the greater specificity of the monoclonal antibody for N-methylhistamine compared with that of histamine, relatively high urinary histamine concentrations gave a false positive influence on the RIA results, which was 100% when the histamine/N-methylhistamine ratio was about 19. Clear cases of mastocytosis can be diagnosed, using the RIA-kit, but for a more precise N-methylhistamine value GCMS analyses will remain necessary.     

Inhibitory effect of phloretin on histamine release from isolated rat mast cells

The ability of the flavonoid phloretin to inhibit histamine release from rat mast cells varied considerably with the releasing agent investigated. The response to the combination of the ionophore A23187 and the phorbol ester TPA and to suboptimal concentrations of the ionophore (0.5 M) was potently inhibited (IC₅₀ about 5 M), whereas phloretin was less potent against responses to the ionophore (1 M) IC₅₀ of 17 M), to antigen alone and in combination with TPA (IC₅₀ of 30–50 M), to TPA in the absence of calcium (IC₅₀ of 50 M) and to compound 48/80 in the absence and presence of calcium (IC₅₀ of 60–90 M). The inhibition by phloretin at concentrations above 10M was partly counteracted by glucose (5 mM) indicating effects on oxidative metabolism. The flavonoid quercetin was equally potent in inhibiting histamine release induced by antigen, the ionophore at different concentrations and in combination with TPA (IC₅₀ of 20M). Although not conclusive, the results are consistent with an inhibition of protein kinase C by phloretin at concentrations below 10 M. At higher concentrations unspecific actions become apparent and phloretin therefore seems to be of limited utility as a probe for signal-pathways in cell responses.     

Modulation of lymphocyte activating factor activity (interleukin 1 like activity) and acute phase proteins in pertussis-induced air pouch inflammation by muramyl dipeptide

We have studied the effect of the macrophage activator, muramyl dipeptide (MDP) on immune inflammation induced in the rat six day subcutaneous air pouch. Treated animals received either 100 g or 200 g MDP at the time of challenge and twenty four hours before exudate harvest. Using the thymocyte co-mitogenic assay for lymphocyte activating factor (LAF), 100 g MDP enhanced LAFactivity whereas 200 g caused inhibition. Increased dilution of 200 g exudate in this assay removed this inhibition. Similarly, at the lower dose, MDP caused enhanced production of the acute phase protein alpha 1 glycoprotein, whereas the higher dose had no effect. The present study suggests that macrophage activity can be manipulatedin vivo to produce LAF and naturally occurring inhibitors of LAF. These studies indicate that the stimulation of LAF inhibitors by MDP may be a potential theerapeutic action.     

Histamine as an autocrine growth factor in experimental mammary carcinomas

The antisecretory effect of DS-4574, a mast cell stabilizer with peptidoleukotriene antagonism, on the hypersecretion of gastric acid stimulated by several secretagogues was examined in the pig. Goettingen miniature pigs with chronic gastric fistula were used. Intramuscular injection of carbachol (60 g/kg), tetragastrin (50 g/kg) or histamine (200 g/kg)-induced gastric acid hypersecretion. Intraduodenal administration of DS-4574 (10 and 20 mg/kg) significantly inhibited both the hypersecretion induced by carbachol and that by tetragastrin. On the other hand, DS-4574 (50 mg/kg, intraduodenal) did not suppress histamine-induced hypersecretion. In thein vitro study, no effect on hog gastric K⁺-dependent ATPase activity was found at concentrations of DS-4574 from 10^–7 to 10^–4 M. These results were highly similar to those in the rat. The suppression of histamine release from histamine-containing cells in the gastric mucosa of the rat was concluded to be an antisecretory effect of DS-4574.     

A pharmacologic study on the histamine releasing effect of atracurium and other muscle relaxants in rat isolated ileum

In this study, the effects of histamine, antihistamines (terfenadine and mepyramine), 5-hydroxytryptamine, and muscle relaxants, atracurium, vecuronium and gallamine, on the tone and contractility of rat ileum were studied and compared in vitro.The aim of the present investigation was to measure, pharmacologically, the histamine releasing effect of muscle relaxants, e.g. atracurium, vecuronium and gallamine, by comparing their contractile response in the absence and presence of antihistamines and comparing their mechanical responses with those produced by histamine and 5-hydroxytryptamine (5-HT).The results showed that the antihistamines, triludan (terfenadine) and mepyramine produced opposite effects in rat ileum. Terfenadine (0.1–20 M) produced concentration-dependent contractions in the rat ileum, whereas mepyramine (0.1–10 M) relaxed the muscle, e.g. by 1.2 g tension. Atracurium (0.5–500 M), vecuronium (0.2–200 M), and gallamine (0.1–7.0 M) produced marked contractions (1.5–4.0 g tension) in rat ileum, and these contractions were markedly reduced by mepyramine (1.3 M) or terfenadine (5 M), implicating histamine release in the generation of these contractions. However, there was some residual contraction which was not blocked by mepyramine, but by 5-HT antagonist, methysergide (1 M), indicating that a mechanism other than histamine release may be responsible for the residual contraction, i.e. release of other mediators such as 5-HT, prostaglandins, or calcium. 5-HT (0.5–500 M) and histamine (0.5–500 M) produced contractions in the rat ileum, but 5-HT was more effective than histamine in producing these contractions. Similarly, gall amine was more effective than atracurium and vecuronium in contracting the rat ileum. Since very high concentrations of muscle relaxants were used, it is suggested that in clinical concentrations, the histamine releasing effect of muscle relaxants was minimal, except that of gallamine, which may release histamine event at very low concentrations. The results are discussed in terms of pharmacologic and immunologic implications of drug reactions at the rat intestinal smooth muscle.     

Molecular Characterization of Complement Components (C3, C4, and Factor B) in Human Saliva

A molecular analysis of complement components (C3, C4, and factor B) in human saliva was performed by SDS-PAGE and immunoblotting. Complement C3 was detected as a molecule composed of a 115-kDa -chain linked to a 70-kDa chain by disulfide bonds, and C3 levels ranged from 0.52 to 15.0 /g/ml (n = 15). C4 was detected as a triple-chain molecule (98-kDa chain, 73-kDa chain, and 33-kDa chain) linked by disulfide bonds, and C4 levels ranged from 0.086 to 4.8 g/ml. Factor B was detected as a 100-kDa single chain, and factor B levels ranged from 0.042 to 0.62/g/ml. The sizes and subunit structures of the complement components in human saliva were compatible with those reported in human serum. The results of a hemolytic assay indicated that the complement molecules in human saliva were functionally active. These complement components may participate in the local immune and inflammatory responses in the oral cavity.     

Inhibition of chemiluminescence in granulocytes and alveolar macrophages by azelastine

The effect of azelastine, an orally effective antiasthmatic/antiallergic drug, on the generation of oxygenderived free radicals in phagocytes was investigated using different chemiluminescence-assays. The chemiluminescence (CL) of both human polymorphonuclear granulocytes (PMNL) and guinea-pig alveolar macrophages (AM) was induced either by phorbol myristate acetate (PMA) or zymosan and amplified either by lucigenin or DMNH (7-dimethylamino-naphthalene-1,2-dicarbonic-acidhydrazide). The inhibitory effect of azelastine was dependent on the inducer employed and the condition and type of cells used. Azelastine reduced PMA-induced CL concentration-dependently in both PMNL (IC₃₀=3.9 M) and AM (IC₃₀=9.8 M). In AM zymosan-induced CL was inhibited 21.7% by 10 M azelastine, whereas in PMNL it remained unchanged up to 10 M azelastine. Azelastine has a significantly stronger inhibitory effect (IC₃₀=4.2 M) on oxygen free radical generation in AM primed by fetal calf serum than in unprimed AM. Based on present results it is likely that azelastine inhibitis oxygen-derived free radical generation by interaction with protein kinase C.     

Electrophysiological characterization of histamine receptor subtypes in sheep cardiac Purkinje fibers

The histamine-receptor-subtype-mediated effects on action potentials of electrically driven and spontaneously active isolated sheep cardiac Purkinje fibers were investigated using H₁-and H₂-selective agonists and antagonists.In electrically stimulated Purkinje fibers, histamine (3 mol/l) increased the action potential plateau height, decreased the action potential duration measured at a repolarization level of –60 mV and enhanced the pacemaker activity. These effects were abolished by the H₂-selective antagonist cimetidine (30 mol/l), but were not impaired by the H₁-selective antagonist dimetindene (0.3 mol/l).In spontaneously active Purkinje fibers, histamine (10 mol/l) increased the spontaneous rate by 24%, the slope of diastolic depolarization by 45% and shortened the duration of the diastole by 32% of the respective control measurements. These effects were blocked by 30 mol/l cimetidine, but remained unchanged in the presence of 0.3 mol/l dimetindene.Concentration-response curves of histamine were shifted to the right by approximately 2 logarithmic units in the presence of 30 mol/l cimetidine, but were not influenced in the presence of 0.3 mol/l dimetindene. The H₂-selective agonist impromidine (0.001–0.3 mol/l) had similar actions as histamine on spontaneously active Purkinje fibers, while the H₁-selective agonist 2-(2-pyridyl-)ethylamine was ineffective. It is concluded that the pronounced stimulatory action of histamine on spontaneous activity in sheep cardiac Purkinje fibers is exclusively mediated by H₂ receptors.Dedicated to Prof. Dr. E. Mutschler on the occasion of his 60th birthday.Supported by Ministerium für Wissenschaft und Forschung, Nordrhein-Westfalen, Projekt-Nr. 40008786.     

Caryospora najadae sp.n. (Apicomplexa: Eimeriidae) from Dahl's whip snake,Coluber najadum (Serpentes: Colubridae)

Oocysts of a newCaryospora species,Caryospora najadae, are described from the feces of a Dahl's whip snake,Coluber najadum, from Israel. The spherical oocysts ofC. najadae measure 31.9(27.9–36.3) m in diameter and lack a micropyle and a oocyst residuum. The oocyst wall is between 1.5–2 m thick. The ovoid sporocysts are 15.2(14.0–16.4) m wide and 21.1(19.9–22.2) m long. A sporocyst residuum, a Stieda body and substieda body are present. The sporulation is completed in about 72 h at 21.1±2° C. Sporozoites are elongate measuring circa 19–21×2–2.5 m.     

Der Phosphocreatingehalt des unbelasteten und des ischämischen Gehirns

Zusammenfassung Die mit unterschiedlichen Methoden gefundenen PC-Werte des Gehirns ergaben bisher Mittelwerte von 2,0–3,5 mol/g Frischgewicht. Es wird an in situ eingefrorenen Gehirnen unter milden Enteiweißungsbedingungen ein mittlerer Gehalt von 4,6 mol/g (Maximalwert 4,98 mol/g) gemessen und damit bewiesen, daß auch im Gehirn 50–60% des Gesamtcreatins als Phosphocreatin vorliegen.Mit 1 TextabbildungDurchgeführt mit Mitteln der Deutschen Forschungsgemeinschaft.     

Intrinsic cholinergic excitation in the rat neostriatum: Nicotinic and muscarinic receptors

Summary An in vitro slice technique was employed to study the receptors involved in intrinsic cholinergic excitation in the rat neostriatum. The locally evoked synaptic potentials were suppressed by antinicotinic agents, mecamylamine (10 M), d-tubocurarine (3 M) or hexamethonium (100 M), but not by the antimuscarinic agent atropine (100 M). If the slices were exposed to an acetylcholinesterase (AChE)-inhibitor (paraoxon 1–20 M, physostigmine 0.1–0.5 M), the synaptic potentials were potentiated. The amplitude of the orthodromic population spike increased, and it was further facilitated when the stimulus frequencies were raised from 1–3 Hz to 10–30 Hz. The frequency facilitation following exposure to an AChE-inhibitor was blocked by atropine (1–100 M). Intracellular recording indicated that a slow depolarizing potential caused the frequency potentiation of the orthodromic discharges. Apparently rat neostriatum is similar to cholinergic systems in sympathetic ganglia and spinal Renshaw cells, in that nicotinic receptors mediate fast excitation and muscarinic receptors mediate slow excitation.     

Activity of trimethoprim/ sulfamethoxazole plus polymyxin B against multiresistantStenotrophomonas maltophilia

The inhibitory activity of eight antibiotics and the inhibitory and bactericidal activities of combinations of trimethoprim/sulfamethoxazole (TMP/SMX) plus three fixed concentrations of polymyxin B (0.01 g/ml, 0.1 g/ml and 0.5 g/ml) against 30 multiresistant strains ofStenotrophomonas maltophilia were tested. Polymyxin B at 0.01 g/ml modified the inhibitory activity of TMP/SMX against only 40% of strains. At 0.1 g/ml and 0.5 g/ml, polymyxin B enhanced the inhibitory activity of TMP/SMX activity against all strains. Polymyxin B enhanced the bactericidal activity of TMP/SMX only at concentrations near the minimum inhibitory concentration of polymyxin B alone.     

Inhibition of14C-Aminopyrine accumulation in isolated rabbit gastric glands by the H2-receptor antagonist HOE 760 (TZU-0460)

Acid secretion in isolated rabbit gastric glands was measured by means of the¹⁴C-aminopyrine accumulation technique. Hoe 760 (TZU-0460) and Hoe 062, the desacetylated compound of Hoe 760, caused a concentration-dependent reduction of histamine (100 M) induced aminopyrine-accumulation. The IC₅₀-values were 3.16±0.84 M (n=5) and 1.58±0.6 M (n=6) for Hoe 760 and Hoe 062, respectively. In comparison an IC₅₀ of 9.0±0.72 M (n=6) was obtained for cimetidine and 3.3±1.4 M (n=5) for ranitidine. The IC₅₀-values of ranitidine, Hoe 760 and Hoe 062 were significantly different (p<0.05) from cimetidine. The addition of increasing concentrations of Hoe 760 to the histamine concentration-response curve caused a parallel rightward shift. The transformation of these concentration-response curves according to Arunlakshana and Schild indicated that this inhibition was caused by a competitive antagonism of the histamine receptor on the parietal cell. In agreement with these findings the dbc-AMP stimulated aminopyrine accumulation remained unaffected by the H₂-receptor antagonists.     

The effect of different calcium antagonists on histamine-and carbachol-induced acid secretion in the isolated mouse stomach

Earlier experiments suggested that the action of muscarinic agonists in gastric acid secretion might be partly mediated by endogenous histamine release. This possibility could be tested by use of various calcium antagonists provided that they show different activity on histamine- and carbachol-induced secretion.In the present work, three calcium antagonists, verapamil, nifedipine, and sodium nitroprusside were used on the isolated mouse stomach where acid secretion induced by histamine and by carbachol was measured.Verapamil (100 M) did not affect either histamine or carbachol-induced acid secretion. Nifedipine (100 M) reduced histamine-induced acid secretion, whereas the effect of carbachol remained unchanged. Sodium nitroprusside (10–100 M) reduced both histamine- and carbachol-induced acid secretion.The differences in action of calcium antagonists in histamine- and carbachol-induced acid secretion could exclude the possibility that the effect of carbachol is partly mediated by histamine release from mast cells present in the mouse stomach.     

Comparison of disk diffusion,the E test,and detection ofmecA for determination of methicillin resistance in coagulase-negative staphylococci

The aim of this study was to find a reliable, fast, and simple alternative to the methicillin disk method for determination of methicillin resistance in coagulase-negative staphylococci, since results of this method are often difficult to read due to growth within the zone of inhibition. The sensitivity of 319 strains of coagulase-negative Staphylococci to a 5 g methicillin disk on Mueller-Hinton agar using an incubation period of 48 h was compared with that of 1 (1 g and 5 g oxacillin disks on Mueller-Hinton agar with or without 2% NaCl, using an incubation period of 24 h. The detection ofmecA (MecAgen) by the polymerase chain reaction was used as a standard. Minimum inhibitory concentrations were determined by means of the E test. Of the 225mecA-positive strains, 190, 215, and 193 were resistant to 5 g methicillin, 1 g oxacillin and 5 g oxacillin disks on Mueller-Hinton agar, respectively, and 216, 218, and 223 were resistant on Mueller-Hinton agar with 2% NaCl. Of the 94mecA-negative strains, 89, 93, and 94 were susceptible to 5 g methicillin, 1 g oxacillin, and 5 g oxacillin disks on Mueller-Hinton agar, respectively, and 92, 93, and 94 were susceptible on Mueller-Hinton agar with 2% NaCl. Using breakpoints of 2 g/ml for oxacillin resistance and 8 g/ml for methicillin resistance, the E test yielded sensitivities of 99.6 and 99.1% and specificities of 97.9 and 98.9% after 48 h of incubation. The 5 g oxacillin disk was faster and easier to read than the methicillin disk and correlated better with detection ofmecA than the methicillin disk or the 1 g oxacillin disk.     

The effects of TRH analogues on cerebral ischaemia produced by middle cerebral artery occlusion in the rat

Summary This study examined the effects of two stabilised analogues of TRH, RX 77368 and CG 3509, in a rat cerebral ischaemia model produced by unilateral occlusion of the middle cerebral artery. The analogues were given intraventricularly after artery occlusion. The extent of the cortical ischaemia was evaluated after 10 days by somatosensory evoked potential (SEP) recording, followed by tetrazolium staining of brain slices for NADH-diaphorase activity. RX 77368 (2×10 g; 15 min, 24 h) significantly improved the survival rate, protected the SEP and reduced the area of infarct. In contrast, neither a smaller dose of RX 77368 (2×3 g) nor a 4 h delay in the treatment had any significant beneficial effects. Although CG 3509 (2×10 g) resulted in an apparent improvement in survival, its overall effects were not statistically significant. The findings indicate that stabilised TRH analogues may have beneficial effects when given to animals with focal cerebral ischaemia.     

2 μm plasmid copy number in different yeast strains and repartition of endogenous and 2 μm chimeric plasmids in transformed strains

By using the renaturation kinetics technique we tried to get informations about the maintenance of the 2 m plasmid in yeast cells. For this purpose we determined the 2 m plasmid copy number: in various yeast strains, in a special set of mutants, in cells treated with ethidium bromide and cycloheximide and in different yeast strains obtained by transformation with 2 m chimeric plasmids.According to the strain used the proportion of 2m DNA varied from 1.1% to 3.9%, which corresponds to 24 to 88 2 m molecules per haploid genome. The particular multiresistant mutant, where the frequent loss of oligomycine resistance is correlated with the loss of extractible covalently closed circular DNA, contained 39 2 m copies per haploid genome. In the partial revertant oligomycine sensitive all the 2 m DNA sequences were lost. (Less than 0.1 copy per haploid genome.)Ethidium bromide did not affect the 2 m copy number while cycloheximide induces an increase of 36%.When a strain containing 88 2 m DNA copies per haploid genome is transformed with 2 m chimeric plasmids there is no significative change in the total number of plasmid: 36 copies of endogenous and 44 of chimeric plasmid per haploid genome. When 2 m chimeric plasmids were introduced in our 2 m-less strain despite the stability of the transformants, there is only 8 copies per haploid genome.