Equivalence of as-required salbutamol propelled by propellants 11 and 12 or HFA 134a in mild to moderate asthmatics. German Study Group.

This randomized, double-blind, parallel-group study compared the efficacy and tolerability of as-required salbutamol 100 microg administered from either a chlorofluorocarbon (CFC) pressurized metered dose inhaler (pMDI; Ventolin) or from a non-CFC hydrofluoroalkane (HFA) 134a pMDI (Ventolin CFC-free) in patients with mild to moderate asthma. All patients (n = 423) continued with their standard asthma therapy, and recorded their daily use of study medication, morning and evening peak expiratory flow (PEF) and symptom scores, throughout the 4-week treatment period. Clinic lung function was measured at 2-week intervals. The median daily use of inhaled study medication remained constant at four actuations per day throughout the study in both treatment groups and statistical analysis indicated that the two formulations were equivalent. Small improvements in both treatment groups were reported in mean morning and evening PEF, clinic forced expiratory volume in 1 sec and clinic PEF and there were no significant differences between the two groups. Both formulations were well tolerated. This study indicates that as-required salbutamol 100 microg administered via a HFA 134a pMDI is as effective and safe as the currently available CFC-propelled formulation.

[Kinin generation in acute and chronic airway inflammation].

OBJECTIVE: To investigate the kinin generation pathways in acute and chronic airway inflammation. METHODS: BALF from patients with acute, chronic airway inflammation and healthy controls were collected. Kinins, Plasma kallikrein, alpha 2-macroglobulin and toluenesulphonyl-arginine methyl ester esterase activity (TAME-ea) in BALF were studied. RESULTS: Kinins and TAME-ea values were significantly higher in the BALF of patients with acute and chronic airway inflammation than those in the controls, but there was no significant difference between acute and chronic groups; PK and alpha 2-M values were significantly higher in the acute group than the in chronic one. Gel filtration revealed the highest TAME-ea peak at about 800,000 in the acute group, corresponding with the first alpha 2-M peak, whereas at about 40,000 in chronic bronchitis. The inhibition test of the TAME-ea showed that the TAME-ea peak at 800,000 was mainly due to PK and the TAME-ea peak at 40,000 was mainly due to TK. CONCLUSIONS: The results indicated that in acute airway inflammation kinins seem to be mainly generated by PK, whereas in chronic inflammation kininogenases other than PK--such as TK--seem to be more important.

Kinin generation in acute pneumonia and chronic bronchitis.

Kinins are potent inflammatory mediators, liberated from kininogens by different kininogenases. The aim of this study was to investigate the kinin generation pathways in acute and chronic inflammation of the lower airways. We studied bronchoalveolar lavage fluid (BALF) of patients with acute pneumonia, patients with chronic bronchitis and healthy controls. Kinins were determined by radioimmunoassay (RIA). Plasma kallikrein (pl-Kal), alpha2-macroglobulin (alpha2-M) and toluenesulphonylarginine methyl ester (TAME) esterase activity (TAME-ea) were studied in BALF before and after gel filtration chromatography. Plasma kallikrein and alpha2-M were measured using two newly developed sandwich enzyme-linked immunosorbent assays (ELISAs). TAME-ea was measured by a radiochemical assay. After gel filtration, inhibition of TAME-ea with benzamidine, soy-bean-trypsin inhibitor (SBTI) and aprotinin was performed. Kinins and TAME-ea did not differ significantly between acute pneumonia and chronic bronchitis, whereas pl-Kal and alpha2-M values were significantly higher in acute pneumonia. Gel filtration revealed the highest TAME-ea peak in acute pneumonia corresponding with the first alpha2-M peak at approximately 800 kDa, whereas in chronic bronchitis the highest peak was found at approximately 40 kDa. The inhibition test showed that the TAME-ea peak at approximately 800 kDa was due to pl-Kal and the TAME-ea peak at approximately 40 kDa was mainly due to tissue kallikrein. High peaks of alpha2-M and pl-Kal were found in pneumonia and only small peaks were seen in chronic bronchitis. We conclude that in acute airway inflammation kinins seem to be mainly generated by plasma kallikrein whereas in chronic inflammation, kininogenases other than plasma kallikrein, such as tissue kallikrein, seem to be more important.

Substance P is generated in vivo following nasal challenge of allergic individuals with bradykinin.

BACKGROUND: Bradykinin, a potent inflammatory mediator, is released during allergic and non-allergic rhinitis and asthma in man. Nasal bradykinin challenge induces a dose-dependent plasma leakage into the nasal cavity and relevant symptoms of rhinitis. OBJECTIVE: We now report on substance P generation during nasal bradykinin challenge in vivo. METHODS: The effect of locally applied bradykinin on substance P generation was studied in nine individuals, allergic to grass pollen and six non-allergic controls. In the allergics TAME-esterase activity, histamine and substance P concentrations were measured in nasal lavages and correlated to the clinical symptoms. RESULTS: Substance P concentrations in nasal lavages increased in a dose-dependent fashion during nasal bradykinin challenge in both groups. In the allergic group Substance P-increases correlated with the production of TAME-esterase activity (r = 0.9, P < 0.05) whereas these allergic individuals did not produce any histamine increases. The generation of substance P and the increase of TAME-esterase activity was associated with the onset of clinical symptoms. Correlation of oedema and hypersecretion to substance P were significant by linear regression analysis (r = 0.88, P < 0.005 and r = 0.89, P < 0.02, respectively). Bradykinin induced irritations like burning and itching were short-term and rare. Serial dilutions of nasal washes produced Substance P-RIA displacement curves that paralleled the standard curve and recovery of standard substance P that was added to nasal washes was 76 +/- 4% (mean +/- SEM), n = 8. CONCLUSION: Bradykinin induces in vivo a dose-dependent plasma leakage into the nasal cavity without affecting mast cells, but stimulates nerve endings resulting in the release of the neuropeptide substance P.

Mediator release is altered in immunotherapy-treated patients: a 4-year study.

In recent years, it has been possible to demonstrate mediator release into the nasal secretion after nasal allergen challenge in patients with allergic rhinitis. Using the nasal provocation model, we determined whether the mediator release was altered in immunotherapy-treated patients. Seventeen grass-pollen-allergic patients were examined under controlled, reproducible conditions. Serial challenges with increasing doses of grass pollen produced increasing numbers of clinical symptoms and release of mediators such as kinins, TAME-esterase activity, and histamine. Ten patients received a semidepot perennial grass-pollen extract for 4 years. Seven patients served as controls and did not receive immunotherapy during the observation period. Data from the group of patients receiving immunotherapy over the first year already showed a partially significant decline in the maximal mediator release after nasal allergen challenges compared to the results of pretreated challenges, whereas controls did not show any significant changes. Nasal allergen challenges after termination of 4 years' immunotherapy significantly modified the mediator release compared to pretreatment values (TAME-esterase activity P < 0.05, kinins P < 0.01, and histamine P < 0.01). Decrease of mediator release paralleled the symptom-medication scores and quantitative skin prick test. Finally, we could demonstrate a significant correlation between specific IgG increase and mediator decrease in the treated group.

Determination of substance P in human nasal lavage fluid.

The determination of substance P (SP) concentrations in human nasal lavages can be used to monitor physiological and certain pathophysiological processes in human airway mucosa. But, because of the low concentrations, immunoassays of high sensitivity are needed. Two approaches to improve the sensitivity of the radioimmunological determinations of SP are compared: increasing the sample volume and miniaturizing the assay design. The characterization of SP-like immunoreactivity (SP-LIR) in human nasal lavage was performed by investigating the immunological specificity of the antibody used in the radioimmunoassays and by reversed-phase high-performance liquid chromatography separation of the SP-LIR. SP concentrations in nasal lavages can be reliably measured by each of the two introduced RIA methods. Despite the lower detection limit of the miniaturized immunoassay (0.2 in comparison to 1.3 fmol/incubate) it is advisable to increase the sample volume in order to improve the sensitivity because of the higher precision of the determinations. SP-LIR was found in nasal lavage specimens in concentrations between 2 and 10 fmol/ml and consisted of authentic SP and, to a less extent, SP-sulfoxide.

Substance P enhances antigen-evoked mediator release from human nasal mucosa.

Exogenous substance P (10-80 nmol/ml) induced a dose-dependent increase in nasal symptoms in asymptomatic allergics with rhinitis (n = 15) and controls (n = 8), but did not release any mediators. However, comparing the antigen-evoked release of mediators into nasal secretions with that of a substance P-pretreated antigen challenge, we found a significant enhancement of kinins, TAME esterase activity (p < 0.05-0.01), and histamine (p < 0.001, NS) 10-20 min after antigen challenge. These results suggest 1) that substance P-induced increase in nasal congestion is mediated through direct neurokinin receptor activation independently of mast cell activation, and 2) that during the allergic reaction there is a substance P-mast cell interaction that enhances the mediator response to nasal allergen challenge.

Contralateral differences among biomarkers determined by a modified nasal lavage technique after unilateral antigen challenge.

The concentration of biomarkers from vessels and inflammatory cells in nasal lavage fluid reflects the degree of hyperresponsiveness in patients with allergic rhinitis. The lavage has usually been performed of both nasal cavities together after prewashings and administration of decongestants. To improve the technique, we introduced a modification involving lavage of the nasal cavities separately without any prewashings or decongestants. We challenged 20 rhinitic subjects sensitive to timothy unilaterally with timothy extract. In nasal lavages performed before, immediately after, and 6 h after the challenge, we determined the concentrations of albumin, histamine, bradykinin, TAME (N-alpha-tosyl-L-arginine methyl ester)-esterase, and leukotriene C4 (LTC4). In eight subjects, the procedure was repeated 1 and 2 weeks later. After the challenge, albumin, bradykinin, TAME-esterase, and LTC4 in the nasal lavage fluid increased on the ipsilateral side but not on the contralateral side. Histamine did not increase after antigen challenge. After 6 h, the biomarkers were not increased. The concentrations of biomarkers did not differ between sides before the challenge and not between visits. Thus, the modified nasal lavage technique is reliable and improved compared to previous methods because it involves reproducible determinations of different biomarkers, and it is simple and easy to perform.

Tissue kallikrein activity and kinin release in human endothelial cells.

The kininogenase, tissue kallikrein (EC 3.4.21.8), has been identified in different blood vessels. The enzyme was mainly found in vascular smooth muscle cells. It is not known whether it is present and functionally active in vascular endothelial cells. The following study investigates the presence of tissue kallikrein in endothelial cells from human umbilical veins and pulmonary arteries. Tissue kallikrein was demonstrated in three ways: 1) by immunostaining in endothelial cells; 2) by measurement of tissue kallikrein activity using a colorimetric assay; 3) by the measurement of kinin release in intact and homogenised endothelial cells with a radioimmunoassay. Immunostaining demonstrated the presence of tissue kallikrein in endothelial cells from human umbilical veins and endothelial cells from human pulmonary arteries. Tissue kallikrein-like activity, measured by the degradation of D-Val-cyclohexyl-Ala-Arg-4-nitraniline, was 3.57 +/- 0.5 mU/10(6) endothelial cells from human umbilical veins and 7.52 +/- 0.84 mU/10(6) endothelial cells from human pulmonary arteries. Intracellular kinin concentrations were 424 +/- 83 pg/10(6) cells in endothelial cells from human umbilical veins and 576 +/- 146 pg/10(6) cells in endothelial cells from human pulmonary arteries, and they increased in a time-dependent manner after homogenisation. The increase was abolished by aprotinin (1000 kIU), an inhibitor of tissue kallikrein in both cell types. Addition of exogenous kallikrein (5 mU) to homogenised cells led to a five fold increase of kinin concentrations after five minutes, indicating a sufficient resource of cellular kininogen. Removal of extracellularly bound kininogen by washing with dextran sulphate (100 mg/l) resulted in an approximately 75% reduction of the cellular kinin release.(ABSTRACT TRUNCATED AT 250 WORDS)

[Circadian rhythm in allergic inflammation]. / Zirkadiane Rhythmiken bei der allergischen Entzündung.

There are significant bioperiodicities for hormonal, neural, cellular, and humoral factors as well as for mediators. A combination of these findings is an explanation for the increased hyperreactivity in patients with allergic diseases during the night. In the morning hours between 2 and 6 a.m., the histamine concentration shows a peak, adrenaline and cyclic AMP have their minimum, while cortisol secretion is just ascending. Circadian variations are also seen with respect to the density of beta-receptors. Thromboxane A2 shows a peak during the night, PGE2 is depressed, a finding also in favour of bronchial constriction. Total plasma protein IgA, IgM, IgG and IgE show a distinct bioperiodicity with a minimum during the night, cellular elements like T11, T4 and B-lymphocytes, and Leu8 have a maximum. The nocturnal symptom exacerbation must be given the fullest attention in choosing the time of administration of the appropriate medication.

ELISA for the neuropeptide degrading endopeptidase 3.4.24.11 in human serum and leukocytes.

We describe the development of a new ELISA for the detection of neural endopeptidase 3.4.24.11 (NEP). Neutral endopeptidase 3.4.24.11 was determined in preparations of human granulocytes, mononuclear cells (MNC), and in serum. Human recombinant NEP was used as reference. Specificity of the mAbs was tested using APAAP, FACS analysis, and Western blot analysis. Lysis of the blood cells was performed by incubating the cells with 0.4% Tween-20 and repeated freezing cycles. The minimal detectable dose for recombinant NEP was 15 pg/ml. The recovery was 94 +/- 9%. The NEP was detectable in 15 out of 20 serum samples of 20 volunteers (mean +/- SEM, 245 +/- 88 pg/ml, n = 20)) and in all granulocyte preparations (1176 +/- 138 pg/10(7) cells, n = 20)). The results were reproducible among replicates (CV = 3 +/- 1%, n = 40), dilutions (CV = 8 +/- 2%, n = 5), and assays (CV = 12 +/- 4%, n = 5). With this new ELISA, a simple and reproducible method for the measurement of NEP 3.4.24.11 is described.

Effect of angiotensin-converting-enzyme inhibition on bradykinin metabolism by vascular endothelial cells.

The degradation of bradykinin by angiotensin-converting-enzyme (ACE) activity in cultured human endothelial cells was studied by direct measurement of bradykinin and by its effect on the release of endothelium-derived relaxing factors. The half-life of exogenous bradykinin (10,000 pg/ml) was calculated from the decay of the bradykinin concentration as 46 +/- 2 min in cell monolayers, 133 +/- 15 min in conditioned medium, and 24 +/- 2 min in homogenates. Most of the bradykinin-degrading activity in cell monolayers could be inhibited in a concentration-dependent manner by the ACE inhibitors lisinopril, ramiprilat, and captopril. Bradykinin-degrading activity was released into the culture medium containing one-fourth of the bradykinin-degrading activity found in the presence of cell monolayers. In cell homogenates higher unspecific bradykinin-degrading activities were present. The functional consequence of bradykinin degradation was demonstrated by the potentiating effect of ramiprilat on the generation of endothelium-derived relaxing factors nitric oxide and prostacyclin from endothelial cells. The study supports the concept of increased vasodilatory effects of bradykinin during ACE inhibition.

Ramiprilat increases bradykinin outflow from isolated hearts of rat.

To establish that bradykinin is formed in the heart we measured bradykinin in the venous effluent from rat isolated hearts perfused with Krebs-Henseleit buffer. In addition, we examined the effect on bradykinin outflow of the angiotensin converting enzyme (ACE) inhibitor, ramiprilat. From rat isolated normoxic hearts a bradykinin outflow of 0.85 +/- 0.1 ng ml-1 perfusate g-1 wet weight was measured. Perfusion with ramiprilat increased the bradykinin concentration to 2.8 +/- 0.3 ng ml-1 perfusate g-1 wet weight. During ischaemia bradykinin outflow maximally increased 8.2 fold to 7.0 +/- 0.5 ng ml-1 perfusate g-1, and in ramiprilat-perfused hearts 5.8 fold to 16.0 +/- 1.8 ng ml-1 perfusate g-1. In the reperfusion period bradykinin outflow normalized to values measured in the respective pre-ischaemic period. The presents data show that bradykinin is continuously formed in the rat isolated heart. Ischaemia increases bradykinin outflow from the heart. Presumably by inhibiting degradation of kinins, ACE inhibition significantly increased the bradykinin concentration during normoxia, ischaemia and reperfusion.

Substance P and beta-endorphin-like immunoreactivity in lavage fluids of subjects with and without allergic asthma.

Six atopic subjects with grass pollen allergy and six nonallergic healthy volunteers were enrolled into this study. Substance P-like immunoreactivity (SP-LIR) and beta-endorphin-like immunoreactivity (beta E-LIR) were determined in bronchoalveolar lavage (BAL) and nasal lavage (NAL) fluids before and after allergen (grass pollen) provocation. A significant increase in the baseline concentration of SP-LIR and beta E-LIR was seen in BAL of allergic subjects. In NAL of allergic subjects an increased baseline concentration of SP-LIR was found (beta E-LIR not detectable). After allergen provocation there was a rise of SP-LIR and beta E-LIR in BAL fluids of allergic subjects immediately after provocation. In NAL fluids of allergic subjects allergen challenge resulted in a rise of SP-LIR within 10 minutes. Allergen provocation did not influence SP-LIR and beta E-LIR concentration in BAL and NAL in nonallergic controls. The demonstrated higher baseline levels of SP-LIR and beta E-LIR as well as the increase after provocation in the BAL and NAL of allergic subjects but not in nonallergic controls support the hypothesis that these neuropeptides contribute to allergic reactions in airways of humans.

Topical vasoconstrictor (oxymetazoline) does not affect histamine-induced mucosal exudation of plasma in human nasal airways.

Mucosal exudation of almost unfiltered plasma proteins, plasma-derived mediators and fluid has recently been advanced as a major respiratory defence mechanism. Oxymetazoline chloride is a commonly used decongestant agent. By reducing blood flow it may reduce mucosal exudation and thus compromise the mucosal defence capacity. This study examines the effect of topically applied oxymetazoline on histamine-induced plasma exudation into human nasal airways. Twelve normal volunteers participated in a double-blind, randomized, cross-over and placebo-controlled study with pretreatment with a single dose oxymetazoline chloride (5 micrograms or 50 micrograms; a dose previously known to reduce nasal mucosal blood flow by almost 50%) prior to the histamine challenge sequence. Nasal lavages were performed every 10 min for 140 min, and three histamine challenges were performed at 30-min intervals during this period. The concentrations of two exudative indices, N-alpha-tosyl-L-arginine methyl ester (TAME)-esterase activity and albumin, were measured in the nasal lavage fluids. Nasal symptoms (sneezing, nasal secretion and blockage) were assessed by a scoring technique. Histamine induced all three symptoms with correlatively raised levels of the biochemical markers for plasma exudation. Oxymetazoline chloride caused a significant decrease in nasal stuffiness, but did not influence the other nasal symptoms or the histamine-induced plasma exudation. It is concluded that histamine-induced plasma exudation is not influenced by topical oxymetazoline. Thus, an important airway defence reaction such as plasma exudation may be little affected by topical alpha-adrenoreceptor-mediated vasoconstriction.(ABSTRACT TRUNCATED AT 250 WORDS)

Bradykinin degrading activity in cultured human endothelial cells.

The role of angiotensin-converting enzyme (ACE), neutral endopeptidase 24.11 (NEP), and other peptidases in the endothelial degradation of bradykinin was investigated in cultured human umbilical vein endothelial cells (HUVEC). The major part of the kininase II activity on intact cells was attributed to ACE activity, the minor part to NEP activity. Amastatin, as aminopeptidase inhibitor, and DL-2-mercaptomethyl-3-guanidinoethyl-thiopropionic acid (MGTA), an inhibitor of kininase I, did not affect endothelial kininase activity. The decline of the bradykinin concentrations in the supernatant of intact endothelial monolayer indicated a total kininase activity of 289 +/- 27 fmol/min/dish. The calculated activity of ACE was 223 fmol/min/dish and the neutral endopeptidase activity was 51 fmol/min/dish. Thus, ACE and neutral endopeptidase are the main kininases in the degradation of bradykinin by intact endothelial cells. In contrast to the intact endothelial monolayers, in homogenates additional kininase activity was found which was not affected by either ACE and NEP inhibitors nor by amastatin and MGTA.

Bradykinin and other inflammatory mediators in BAL-fluid from patients with active pulmonary inflammation.

We evaluated the levels of bradykinin, albumin, TAME-esterase activity, histamine, PGD2 and LTC4 in bronchoalveolar lavage fluid from asthmatics and from patients with pneumonia, sarcoidosis, fibrosis, and chronic bronchitis. Compared with the results of healthy volunteers and atopic asymptomatic asthmatics the bradykinin levels and TAME-esterase activity were significantly elevated. In all other groups, histamine was additionally elevated in asymptomatic asthmatics, whereas albumin was elevated in symptomatic asthmatics and fibrosis patients, and decreased in chronic bronchitis and pneumonia patients. Following local intrabronchial allergen challenge of mild grass pollen asthmatics out of season bradykinin levels increased significantly, correlated with albumin, histamine and TAME-esterase activity. In contrast to the increased mediator concentrations in the early phase reaction there was no change of BAL cells in asthmatics compared to baseline and healthy volunteers. The presence of bradykinin in the bronchoalveolar space of patients with active pulmonary inflammations and bradykinin generation in asthmatics as a result of intrabronchial allergen challenge provides strong evidence that kinins are involved in inflammatory disorders of the lower airways.

Kinin generation following methacholine nasal airway challenge of non-allergic subjects.

Following nasal challenges with the muscarinic neurotransmitter methacholine in 14 patients with chronic non-allergic rhinitis, kinin generation was induced. This occurrence was inhibited by pretreatment with anticholinergic agents (ipratropium bromide) and by an overdose of neurotransmitter, indicating that a muscarinic receptor is stimulating the liberation of kinin. These observations suggest that patients with chronic non-allergic rhinitis exhibit cholinergic neurotransmitter-induced kinin generation.

Tryptase in nasal lavage fluid after local allergen challenge. Relationship to histamine levels and TAME-esterase activity.

The activation of mast cells is generally considered to be an important trigger mechanism in the immediate allergic response. This study focused on the determination of three markers of mast cell activation after an allergen challenge. Nasal allergen challenges were performed in 25 subjects with seasonal allergic rhinitis using three allergen doses increasing in 10-fold steps in a standardised nasal lavage model for the subsequent recovery of the markers of mast cell activation. The levels of histamine and tryptase in the nasal lavage fluid were determined using radioimmunoassays, while the TAME-esterase activity was determined using a radiochemical technique. The nasal symptoms obtained on challenge were assessed using a scoring technique. The allergen challenge resulted in significant increases in the levels of all three markers, tryptase, histamine and TAME-esterase. In the individual measurements after the challenges there was a highly significant correlation between the TAME-esterase levels and the tryptase levels (r = 0.71; P less than 0.001), while the generation of histamine and tryptase was not significantly correlated. When comparing the cumulative generation of the three markers, significant correlations were found between all three. Allergen challenges in six non-allergic controls using the same technique did not result in any increase in tryptase levels. The findings suggest that the determination of tryptase in nasal lavage fluid may be a valuable indicator of mast cell activation in the upper airways.