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Biomedical subjects

G Kunkel

Publications and source records attributed to G Kunkel.

At least 55 records · Page 3Linked to original sources
PubMed1996

Honeybee venom allergy: immunoblot studies in allergic patients after immunotherapy and before sting challenge.

By immunoblot techniques, detailed antibody studies were performed with sera of 20 honeybee-venom-allergic patients during or at the end of specific immunotherapy (median duration: 3 years) and before honeybee sting challenge. Before immunotherapy, all patients had experienced systemic allergic reactions to a honeybee sting, with a mean severity of 3.5 +/- 0.5 according to the Müller classification. After the sting challenge, 10 patients (reactors) reacted again with a systemic allergic reaction, whereas 10 patients (nonreactors) did not. No differences were observed between reactors and nonreactors in total serum IgE and specific IgE to honeybee venom at the time of challenge. For immunoblot, honeybee venom (RELESS) was separated on 7.5-20% SDS-PAGE. For detection of specific IgE, IgG, IgG1, IgG4, and IgM, an alkaline phosphatase-linked second antibody was used. Both groups showed 11 antibody-binding bands: at 52, 46, 40, 31, 18.7, 16.9, 13, 11, 10, 9, and 8 kDa; however, the antibody-binding pattern was individual. The reactors differed from nonreactors in showing intense IgE and less IgG4 binding to at least one single component of the venom extract. For nonreactors, the inverse relationship was observed. The hypothesis, "intensity of IgE > or = IgG4 leads to allergic symptoms", was highly significant (P = 0.00026; chi-square). These immunoblot findings could offer predictive value in distinguishing reactors from nonreactors.

Adolescent↗
PubMed1996

[Bradykinin levels in BAL fluid in patients with sarcoidosis].

In 14 patients with sarcoidosis and in 9 healthy controls the bradykinin level was estimated in broncho-alveolar lavage fluid. The statistically significantly higher bradykinin concentrations was found in sarcoid patients than in controls. No relationship was found between level of bradykinin in BAL and lymphocytes % and no relationship was found between bradykinin level and pulmonary function parameters.

Adult↗
PubMed1996

[Bradykinin level in bronchoalveolar lavage fluid in patients with extrinsic allergic alveolitis].

The presence of kinins in the tracheobronchial tree and their pharmacological properties would suggest that these peptides are involved in inflammatory responses of the airway and lungs. The aim of the study was the estimation the bradykinin level in bronchoalveolar lavage fluid (BAL) in patients with extrinsic allergic alveolitis. The investigated group consisted of 12 patients with farmers lung and a control group, of 9 healthy individuals. The statistically significant higher bradykinin concentration was found in patients BAL fluid than in the controls (675 +/- 472 pg/ml v.s., 220 +/- 57 pg/ml). No relationship was found between bradykinin level and BAL fluid cellularity and pulmonary function parameters. High levels of bradykinin in BAL fluid may suggest the increase of alveolo-capillary membrane permeability.

Adult↗
PubMed1996

Comparison between two automated systems to determine specific IgE: CAP and ELItest.

BACKGROUND AND OBJECTIVE: The ELItest is a newly developed system to measure specific IgE based on allergen bound to paper rings and an alkaline phosphatase conjugated second antibody detection system. It was compared to the CAP system, a method based on allergen conjugated to an encapsulated cellulose polymer and a beta-galactosidase conjugated fluorescence detection system. METHODS: Sera of 300 patients with positive history and positive skin-prick tests to common allergens (birch, timothy-grass, cat dander, dermatophagoides ptronyssinus, wasp venom) and 30 negative controls were tested in both systems. Serial dilutions of high titre sera were measured; inter- and intraassay coefficients of variation (cv) were determined. RESULTS: The CAP system proved to be more sensitive (92.3%) compared to ELItest (84%) but marginally less specific (94.7% for CAP versus 96.7% for ELItest). Intraassay cv were slightly lower in the ELItest (7.2% CAP versus 6.4% ELItest), whereas the interassay cv was roughly twice as high for ELItest (20.1%) than for the CAP system (11.4%). Linearity over an 8-fold dilution was good in both tests (r2 0.979 ELItest versus 0.996 CAP), although ELItest levelled off at higher allergen concentrations. Similarly, correlation analysis between both systems revealed that ELItest consistently measured lower values, especially at higher concentrations of specific IgE. The slope of the linear regression line of a log/log plot of measured IgE concentrations was significantly lower than 1 in birch, cat and wasp; the y-intersect was significantly lower than 0 in all analysed allergens. CONCLUSION: These results suggest that the ELItest system for the measurement of specific IgE is not quite as reproducible and sensitive as the CAP system but slightly more specific, and that higher concentrations of specific IgE are measured lower in the ELItest. One potential reason might be that the amount of allergen bound to a paper ring might be smaller than that bound to a cellulose polymer, but further experiments are necessary to prove this hypothesis.

Adolescent↗
PubMed1995

Effect of theophylline on beta-adrenergic receptor density and cAMP content in bovine aortic smooth muscle cells.

Activation of vascular beta-adrenergic receptors prevents an increase in vascular permeability caused by free radicals or inflammatory peptides. Methylxanthines seem to have similar protective effects on vascular endothelium. In the present study we investigated the effect of theophylline on the beta-adrenergic receptor expression and cAMP concentrations in cultured endothelial and smooth muscle cells from bovine aorta. Comparable values for beta-receptor density and binding affinity were detected in both cell types. Isoproterenol induced significant downregulation of beta-receptors in endothelial (BAEC: -60.5%) and smooth muscle cells (BASMC: -52.5%; P < 0.01). Incubation of endothelial cells with theophylline (4 micrograms/ml and and 40 micrograms/ml) for 24 hours did not affect beta-receptor expression, whereas in smooth muscle cells the beta-receptor density was reduced for -31.5% and -28.7%, respectively. In endothelial cells a transient effect on cAMP concentrations was observed after stimulation with isoproterenol (1 microM), but no effect was found in theophylline treated endothelial cells. Stimulation of intact smooth muscle cells with isoproterenol and theophylline (4 micrograms/ml and 40 micrograms/ml) resulted in a significant increase of cAMP concentrations after 60 and 240 minutes. The present data suggest a novel, celltype specific effect of theophylline on the beta-adrenergic receptor expression in vascular smooth muscle cells in vitro.

Adrenergic beta-Agonists↗
PubMed1995

Gelatin sponge-supported histoculture of human nasal mucosa.

Considerable progress has recently been made in the understanding of airway inflammation by cell culture assays and in vivo provocation studies. Inasmuch as ethical considerations limit experimental work in humans, physiologically relevant in vitro models are required to better understand cellular and molecular tissue interactions in human nasal mucosa. Here we describe a human nasal mucosa culture model utilizing a simple gelatin sponge-supported histoculture system at the air-liquid interface. Viable mucosa was preserved for at least 48 h, as shown by morphology and immunohistochemical staining with Ki-67 as marker for proliferation. Pro-inflammatory mediators (kinins, histamine, thromboxane B2, prostaglandin F2 alpha, and substance P) are detectable in serum-containing as well as serum-free culture medium. Incubation with 10(-8) M substance P increases the number of degranulated mast cells after 48 h by 26% (P < 0.01). In this model, biochemical responses can be correlated with histologic alterations of the target tissue. Inflammatory parameters can be examined and compared in various patient groups and different stimulators/inhibitors. This culture method provides a valuable research tool for analyzing all compartments present in nasal mucosa under physiologically relevant conditions, and for studying complex interactions and responses of mucosal cell populations in their natural tissue environment.

Blood↗
PubMed1995

Activation of adenylate cyclase and phosphodiesterase inhibition enhance neutral endopeptidase activity in human endothelial cells.

Endothelial neutral endopeptidase (EC 3.4.24.11, NEP) contributes to the inactivation of vasoactive and inflammatory peptides such as f-Met-Leu-Phe, substance P, atrial natriuretic peptide, and bradykinin. The aim of the present study was to investigate the cellular regulation of NEP expression in human endothelial cells, focusing on the role of cyclic nucleotides and cellular phosphodiesterases (PDE). Activation of adenylate cyclase by forskolin or prostaglandin E1 (PGE1) induced an increase of NEP activity and NEP protein after 24 h of incubation. This effect was mimicked by two activators of protein kinase A, dibutyryl-cAMP and 8-bromo-cAMP. The nonspecific PDE inhibitor, 3-isobutyl-1-methylxanthine (200 microM), increased NEP activity up to 192%. The activator of guanylate cyclase, sodium nitroprusside (SNP), did not affect NEP activity but completely inhibited the 3-isobutyl-1-methylxanthine-mediated increase of NEP activity. The PDE-III inhibitors motapizone (100 microM) and enoximone (100 microM) enhanced NEP activity up to 188% and 213%, the PDE-IV inhibitor rolipram (3 microM) up to 162%, and the combined PDE-III/IV inhibitor zardaverine (1 microM) up to 176% of control values. The present data provide evidence for a cAMP-mediated increase of NEP activity in human endothelial cells.

Adenylyl Cyclases↗
PubMed1995

[Evaluation of bradykinin levels and activation of Tame-esterase in Bronchial alveolar lavage fluid of patients with bronchial asthma].

Airway inflammation is a prominent feature of chronic airway disease as asthma and chronic bronchitis. Multiply cells released mediators and neurotransmitters which are likely to be involved in their origination. The purpose of this study was to establish the levels of kinin, albumin, TAME-esterase activity in BAL fluid of symptomatic and asymptomatic asthmatic patients and to determinate the relationship among mediators. There were significant increases in the mean concentrations of kinin, HSA, TAME-esterase activity in BAL fluid from patients with asthma, chronic bronchitis, compared with the controls (p < 0.005). Kinin mean concentration was in asthmatics 5313, 2 ng/ml, in chronic bronchitis patients 6796.2 ng/ml, versus 468.1 ng/ml in control group. TAME-esterase activity in investigated group was as follow asthmatics 12666 cmp, CB 15131, 3 cmp, versus 3695, 5 cmp in controls. We observed good correlation of kinin and TAME-esterase with HSA in BAL fluid suggest vascular origin of the mediators. The presence of kinins, TAME-esterase in BALs from symptomatic asthmatics and patients with chronic bronchitis provide strong evidence that kinins are involved in this group of lower airway diseases.

Adult↗
PubMed1994

[Epidemiologic aspects of allergic respiratory tract diseases].

There is no doubt today, that allergic diseases of the airways have a considerable genetic background. However it is also evident, that extrinsic factors are more or less responsible for the manifestation of these diseases. Epidemiological studies are an important tool to develop programs for effective prevention measurements. This is necessary to decrease the morbidity and with that also mortality. In principle allergic airway diseases have a very good prognostic outlook, since our present symptomatic therapy as well as the specific immunotherapy are very effective in treating these patients. Another aspect, which has to be considered, is the social economical factor, which signals, that increased mortality is to a great extend dependent on medical judgement and appropriate treatment, especially in the poorer patient population. Another reason for the increased mortality might be the inadequate education of therapists to deal with this type of disease.

Asthma↗
PubMed1994

Inhibition of IgE- and non-IgE-mediated histamine release from human basophil leukocytes in vitro by a histamine H1-antagonist, desethoxycarbonyl-loratadine.

Loratadine, a new nonsedating histamine H1-antagonist, has been shown to inhibit immunologic release of inflammatory mediators in addition to its H1-receptor blocking properties. After oral administration, the agent is metabolized primarily to desethoxycarbonyl-loratadine (DCL). The basic piperidine, DCL, is readily soluble in water, whereas the nonbasic urethane, loratadine, is insufficiently soluble in water for some in vitro investigations. Therefore we used the metabolite, DCL, to study its influence on in vitro leukocyte histamine release (LHR) in 24 allergic and 22 nonallergic subjects. IgE-mediated and calcium ionophore A23187-induced LHR were inhibited by DCL in a dose-dependent fashion (values of drug concentration to induce 30% inhibition after stimulation with inhalant antigen, anti-IgE, concanavalin A, and calcium ionophore A23187 were 6, 8, 5, and 11 mumol/L, respectively). Higher concentrations of DCL caused mediator release in all subjects (n = 45, 30 mumol/L DC: 11% +/- 2% LHR, 100 mumol/L DCL: 35% +/- 1% LHR), abolishing any inhibitory effect of the drug. Rapid onset of inhibition by 10 mumol/L DCL was found in kinetic studies (n = 10). The inhibition of anti-IgE-induced histamine secretion was synergistically increased by simultaneous preincubation of DCL with the potent histamine H2-agonist, FRA-19. Additional data indicate that the inhibition of LHR by DCL might involve biochemical events that occur after cellular Ca++ influx because LHR induced by N-formyl-methionyl-leucyl-phenylalanine or the phorbol ester, 12-O-tetradecanoyl phorbol-12-acetate, was not significantly affected by DCL.

Basophils↗
PubMed1994

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.

Blotting, Western↗
PubMed1994

Measurement of allergen-specific IgD and correlation with allergen-specific IgE.

A new method for the measurement of allergen-specific IgD (as-IgD) was developed by modifying the ImmunoCAP assay (Pharmacia), and amplification of the signal with a goat anti-human/rabbit anti-goat detection system. The assay was sensitive enough to measure as-IgD in serum samples. The specificity of the assay was examined using inhibition tests with excess corresponding and non-corresponding allergens. For the different allergens inhibition rates between 56% (house dust mite) and 88% (cat) could be achieved. Non-corresponding allergens did not inhibit the as-IgD binding. Total IgE and allergen-specific IgE (as-IgE) was measured using the ImmunoCAP system. Total IgD was measured using a sandwich ELISA. As-IgD was measured in serum samples from 51 atopic and 23 non-atopic subjects, and the correlation with as-IgE was examined. As-IgD was detected in both atopics and non-atopics but at higher levels in atopics. As-IgD against birch pollen and timothy pollen allergen was found to be increased in atopics with IgE directed against these allergens compared to atopics without IgE against these allergens (P < 0.02 and P < 0.03). As-IgD against birch pollen allergen was higher in atopics with IgE specific to this allergen than in non-atopics (P < 0.02). In contrast to total IgE and total IgD, significant correlations were observed between as-IgD and as-IgE against timothy pollen (r = 0.34, P < 0.04), birch pollen (r = 0.38, P < 0.05) and cat dander allergen (r = 0.52, P < 0.01). The observed correlations between as-IgD and IgE suggest that IgD and IgE may be similarly regulated, and thus the measurement of as-IgD may give further insight into the regulation of IgE.

Adolescent↗
PubMed1994

Comparative efficacy of azelastine nasal spray and terfenadine in seasonal and perennial rhinitis.

The efficacy and tolerability of intranasal azelastine (0.14 mg/nostril twice daily) and oral terfenadine (60 mg twice daily) were compared under double-blind conditions in two 6-week, multicenter, parallel-group studies, including 167 patients suffering from seasonal and 52 patients suffering from perennial allergic rhinitis. In both studies, patients were symptomatic on entry and showed significant improvement on both treatments within the first 8 d of therapy, showing little further improvement with continued treatment. Symptoms most pronounced on entry--nasal itching, rhinorrhea, sneezing, and nasal obstruction--responded best to treatment (response rates 80-90%). Objective signs such as mucosal swelling and conjunctivitis improved in a manner parallel to symptoms. In perennial rhinitis, azelastine showed a trend to a superior relief of rhinorrhea and nasal obstruction, whereas terfenadine showed a trend toward better control of sneezing and nasal itchiness. No clinically relevant or statistically significant differences between treatments could be identified. The incidence of adverse effects of possible causal relationship to therapy was low. The most frequent effects in azelastine-treated patients were related to application site disorders, e.g., nasal irritation. Results indicate that with the dose used azelastine nasal spray is an effective treatment for both seasonal and perennial allergic rhinitis.

Administration, Intranasal↗
PubMed1994

[Circadian rhythm in allergic inflammation].

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.

Asthma↗
PubMed1993

Measurement of membrane bound IgD and IgM on B lymphocytes.

An assay system for the determination of the membrane bound IgD (mIgD) and IgM (mIgM) on B lymphocytes was developed by the combination of two new ELISA methods with the results of flow cytometry after labeling with specific antibodies. The mIgD and mIgM of B lymphocytes were prepared by incubating mononuclear cells (MNCs) in Tween 20 containing buffer and repeated freeze/thaw cycles. Optimal results were achieved with 0.2-0.4% Tween 20 and two freezing cycles. With biotin-streptavidin amplification the sensitivity of the ELISA was 30 microU/ml for IgD and 0.5 ng/ml for IgM. In healthy persons 3.5 +/- 0.5 mU mIgD were detected on 10(6) IgD+ cells and 57.1 +/- 5.9 ng mIgM on 10(6) IgM+ cells. The mIgD/mIgM ratio was 0.065 +/- 0.005 mU/ng. The developed ELISA systems utilize only commercially available reagents and therefore provide a convenient reproducible tool for determining membrane bound IgD and IgM on B lymphocytes.

Adult↗
PubMed1993

Predominance of the major allergen (Alt a I) in Alternaria sensitized patients.

Thirty-nine patients sensitized to Alternaria were evaluated using titrated skin-prick test (SPT), histamine release studies (HR), inhibition of RAST and immunoblotting studies. To determine the relevance of the major allergen, Alt a I, specific rabbit antibodies against Alt a I and Alt a B were used. The antibodies were preincubated at different concentrations: (i) with the Alternaria allergen dose required for maximum response in the HR assay (10 BU/ml) and (ii) with the Alternaria antigen coupled to RAST paper discs (1000 BU/disc). Dose dependent inhibition of histamine release (n = 30, mean = 80% +/- 4%, IC30 = 0.69 micrograms/ml) and of RAST (n = 7, IC30 = 4.4 micrograms/ml) was found in all patients sensitized to Alternaria as indicated by allergen induced HR. The greater the response to Alternaria in HR, the higher the antibody concentrations necessary for inhibition (P < 0.05). Immunoblot experiments (n = 25) using SDS-PAGE showed in all cases IgE- and IgG binding at approximately 28 kD, which is the size reported for the major allergen, Alt a I. In two cases, slight IgE binding at 45 and 66 kD was also found, while in two other patients, only IgE binding at 66 kD was seen. Our findings emphasize the major importance of Alt a I in patients sensitized to Alternaria.

Adolescent↗
PubMed1993

Effect of azelastine on substance P content in bronchoalveolar and nasal lavage fluids of patients with allergic asthma.

The study was carried out to investigate the effect of azelastine on the Substance P (SP) concentration in bronchoalveolar (BAL) and nasal (NAL) lavage obtained from atopic grass pollen asthmatics and non-atopic healthy subjects. In BAL and NAL fluids there was a significant elevation in the baseline concentration of SP between asthmatics and volunteers. Allergen provocation induced a rise of SP in BAL and NAL in asthmatics, but not in volunteers. Azelastine pre-treatment resulted in a significant reduction of SP in baseline concentration of SP in BAL and NAL from asthmatics. An elevation of SP in BAL or NAL fluids after allergen provocation was not seen in asthmatics pretreated with azelastine. Azelastine did not influence the SP concentration in BAL and NAL of volunteers.

Adolescent↗