S. Norn

Sensitive glass microfibre-based histamine analysis for allergy testing in washed blood cells. Results compared with conventional leukocyte histamine release assay

The new microfibre method for allergy testing is based on basophil histamine release after challenge with suspected allergens in samples of 50 μl washed blood cells. Released histamine is bound to microfibres and measured after removal of interfering substances by washing. The microfibre method was compared with the conventional leukocyte histamine release assay in 18 allergic patients tested with 10 different allergens. It was found that the same individuals responded with histamine release to the same allergens in both assays, and the number of responders was almost identical. Also the dose‐response curves and the cell sensitivity were almost identical, which further substantiated identity between the results obtained by the new microfibre method and the conventional assay. A comparison between the microfibre method and in vivo provocation tests showed good agreement when comparing the number of positive and negative responses in these test. The new method overcomes the problems in allergy testing, where only small amounts of blood are available and many tests have to be carried out.

A new method for detecting histamine release

Glass microfibres have been found to bind histamine with high affinity and selectivity. A new test for measuring basophil hisamine release has been developed using the glass microfibres as a solid phase. Glass microfibres are crushed and fixed to the bottom of microtitre plates with a water-soluble glue. Histamine release is performed in the glass microfibre-prepared microtitre plates by challenging 100 μl washed blood with 20 μl antigen per well for 90 min at 37°C. Released histamine is bound with high affinity to the glass microfibres, since 90% of histamine in the solution is adsorbed to the fibres. After incubation the microtitre plate is washed with H2O to remove cells and interfering substances. Fibre-bound histamine is detected by the fluorometrico-phthaldialdehyde method. The sensitivity of the assay is 0.63 ng histamine, 2 HCl and the histamine standard curve is linear up to at least 5 ng histamine, 2 HCl. Optimal conditions for the new assay are described. After challenge with anti-Ige a comparison with the conventional histamine release from Ficoll-Hypaque-isolated leukocytes showed almost identical results.

A SIMPLIFIED METHOD FOR MEASURING BASOPHIL HISTAMINE RELEASE AND BLOCKING ANTIBODIES IN HAY FEVER PATIENTS. BASOPHIL HISTAMINE CONTENT AND CELL PRESERVATION

A simplified method for measuring basophil histamine release in grass pollen hay fever patients has been developed. Leukocytes were challenged in vitro with extracts of Phleum pratense (timothy) and the release of histamine was determined indirectly as the residual histamine in the cell sediment. Several steps to purify histamine thus became superfluous and histamine was directly conjugated with o-phthaldialdehyde to form a fluorophore. The simplified method showed a basophil histamine content which was in accordance with results obtained by more specific methods. No difference in basophil histamine content was found between normal and allergic persons. For the histamine liberation assay blood could be adequately preserved for transport for 48 h at room temperature by adding cell culture medium. Basophil histamine release technique allows evaluation of cell sensitivity for determination of the degree of allergy as well as the level of blocking antibodies.

Gastric mucosal cytokine responses in Helicobacter pylori-infected patients with gastritis and peptic ulcers. Association with inflammatory parameters and bacteria load

Helicobacter pylori is an important pathogen in gastroduodenal inflammation and ulceration. Several mechanisms have been proposed to explain its role. We studied the cytokine production patterns in situ in gastric mucosal biopsies from H. pylori-positive and H. pylori-negative patients with dyspepsia. Immunohistochemistry with monoclonal antibodies was used. The study showed enhanced expression of interleukin (IL) -8, IL-10 and interferon-gamma (IFN-gamma) in H. pylori infection and a significant association was found between these cytokines and the following parameters: bacteria load, chronic inflammation and activity. These parameters were significantly correlated with the cell markers CD19 and CD56. The study indicates a dual effect of H. pylori on the Th1 response, i.e. a stimulation of the response verified by increased IFN-gamma and a feed-back verified by an increase of the counterinflammatory IL-10, which may dampen the inflammatory and cytotoxic effect of the Th1 response. Furthermore, the study confirms the connection between increase of IL-8 and inflammatory activity in gastric mucosa in H. pylori infection.

Bacterial Histamine Release by Immunological and Non-Immunological Lectin-Mediated Reactions

The mechanisms of bacteria‐induced histamine release were examined in vitro in human leukocytes and rat mast cells. Three types of bacterial responders were found. In persons with IgE‐bearing basophilocytes bacterial histamine release could be triggered by two different mechanisms, an IgE‐dependent mechanism where removal of IgE abolished the release and a non‐immunological mechanism where this was not the case. In responders with no IgE‐bearing cells bacterial histamine release was caused by a non‐immunological mechanism. The non‐immunological mechanism was further substantiated by release in isolated mast cells from germ‐free rats. These experiments suggest a direct interaction between bacteria and target cell, and experiments with multi‐washed bacteria and bacteria cell wall preparations indicate the possibility of the bacteria wall interacting with the target cell. It is probable that the non‐immunological mechanism depends on lectin‐mediated reactions, since bacteria‐induced histamine release was inhibited by lectin‐binding sugars as is release caused by plant lectins.

Serum IgE specific to indoor moulds, measured by basophil histamine release, is associated with building-related symptoms in damp buildings.

Abstract:Objective: To study the relationship between basophil histamine release (HRT) to indoor moulds, indicating specific IgE, and building-related symptoms (BRS), asthma, and hay fever in individuals working in damp and mouldy buildings.¶Methods: A cross-sectional study was performed among 86 school staff members, who on average had worked 143 months (range: 3-396) in moist buildings with mould growth in the constructions. A questionnaire concerning mucous membrane symptoms, facial skin symptoms, central nervous system symptoms, hay fever, and asthma was fulfilled by the participants, and blood samples were taken. Eight mould species growing on building constructions were identified and cultivated to obtain allergenic materials for testing. The presence in serum of IgE specific to moulds was verified by histamine release test ( HRT ) based on passive sensitization of basophil leukocytes. The validity of the method was confirmed by parallel testing of patients allergic to grass- and birch pollen and by the shift from positive to negative response after removal of serum IgE and by using sham sensitization.¶Results: The prevalence of most BRS was between 32% and 62%. Positive HRT, showing serum IgE specific to one or more of the moulds, was observed in 37% of the individuals. The highest frequency of positive HRT was found to Penicillium chrysogenum and then to Aspergillus species, Cladosporium sphaerospermum and Stachybotrys chartarum. A significant association was found between most BRS and positive HRT, whereas no association was observed between positive HRT to moulds and self reported hay fever or asthma.¶Conclusion: Positive HRT to indoor moulds, showing the presence in serum of IgE specific to the fungi, was found to be related to BRS in individuals working in damp and mouldy buildings. Whether the association is of causal character is a question for further studies. The test may be useful in the evaluation and study of possible mould induced BRS.¶

Volatile organic compounds from the indoor mould Trichoderma viride cause histamine release from human bronchoalveolar cells.

F. O. Larsen , P. Clementsen, M. Hansen, N. Maltbæk, T. Ostenfeldt-Larsen, K. F. Nielsen, S. Gravesen, P. Stahl Skov and S. Norn Department of Pharmacology, University of Copenhagen, The Panum Institute, Blegdamsvej 3, DK-2200 Copenhagen N, Denmark, Fax +45 35 32 76 10 Department of Pulmonary Medicine, Gentofte Hospital, University of Copenhagen, Niels Andersens Vej 65, DK-2900 Hellerup, Denmark Department of Biotechnology, Technical University of Denmark, DK-2800 Lyngby, Denmark The Danish Building Research Institute, Dr. Neergaardsvej 15, DK-2970 Hørsholm, Denmark Reference Laboratory, Henrik Harpestrengs Vej 4, DK-2100 Copenhagen Ø, Denmark

Chlamydia pneumoniae and possible relationship to asthma. Serum immunoglobulins and histamine release in patients and controls.

Chlamydia pneumoniae (C.pn.) is claimed to be of importance for the development of bronchial asthma in previously healthy individuals. This is a new and speculative theory. Earlier studies have mainly focused on C.pn. and exacerbation of asthma. If this new theory were true, one would expect titres of C.pn.‐specific IgG to be higher or more common in patients compared with controls. It would also seem probable that pathobiological mechanisms as found in connection with other microorganisms could be demonstrated, i.e. presence of C.pn.‐specific IgE and the capability of C.pn. to induce or enhance histamine release from basophil leukocytes. We therefore examined C.pn.‐specific IgE, IgG and IgM in sera from 22 adults with bronchial asthma and 25 healthy controls. IgE was verified by passive sensitization of basophils from umbilical cord blood. The prevalence of IgE was approx. 69% and IgG approx. 23% in both groups. IgG‐titres were between 1:16 and 1:64 in both groups. No IgM was found. Further, C.pn. could neither induce nor enhance histamine release from basophil leukocytes of patients or controls. We conclude that patients with bronchial asthma and healthy controls do not differ in relation to 1) C.pn.‐specific IgE in sera, 2) the capability of C.pn. to induce or enhance histamine release from basophil leukocytes, since no such effect was found, or 3) previous C.pn. infection judged by the presence of specific IgG antibodies. Our results cannot support the theory that C.pn. is a cause of adult‐onset asthma.

Inhibitory Effect of Calcium Antagonists on Histamine Release from Human Leukocytes.: In Vivo and in Vitro Experiments

A study was made of the influence of calcium antagonists on human basophil histamine release induced in vitro by specific antigen, anti‐IgE or the calcium ionophore A23187. Both verapamil, nifedipine, and nimodipine were found to inhibit the release, and a similar effect was also observed after peroral administration of verapamil and nifedipine. The inhibitory effect of the drugs on histamine release seems to depend on interaction with calcium at different sites. The anti‐allergic effect might explain the improvement found with calcium antagonists in exercise‐induced asthma.

Influence of bacterial endotoxins on basophil histamine release: potentiation of antigen- and bacteria-induced histamine release

The histamine‐releasing capability of lipopolysaccharides (LPS) was examined in human leukocyte suspensions. LPS alone did not release histamine, but was found to enhance the histamine release caused by anti‐IgE. Also the IgE‐mediated histamine release caused by specific antigens (allergens or bacteria) in sensitized individuals was enhanced by LPS. The potentiating effect of LPS was observed in grass pollen and dog dander allergic patients as well as in patients sensitized to E. coli or Staph, aureus bacteria. No potentiation was obtained by exposure to unspecific allergens or bacteria to which the persons were not sensitized. Bacteria can release histamine by immunological or nonimmunological mechanisms, and only the immunological histamine release was found to be potentiated by LPS. It is speculated that endotoxins reinforce release of histamine caused by allergens in allergic patients or by bacteria in persons sensitized to these microorganisms.