Verena Liebers

Health effects due to endotoxin inhalation (review)

Endotoxins are ubiquitous in the environment and represent important components of bioaerosols. High exposure occurs in rural environment and at several workplaces (e.g. waste collecting, textile industry etc.). Adverse effects on human health induced by inhalation of endotoxin are described in several studies. Up to now the endotoxin levels are mainly measured using the Limulus amoebocyte-lysate (LAL) assay. This assay is well established, but for a suitable characterization of bioaerosols more parameters are necessary. Additional information, e.g. concerning the pyrogenic activity of organic dust samples may be delivered by whole blood assay. Whereas on the one hand protection measures at workplaces are demanded to avoid lung function impairment due to endotoxin exposure, on the other hand a protective effect of exposure to microbial agents like endotoxins with regard to allergy development has been observed. On the cellular level toll-like receptor 4 (TLR4) and IL-1 receptor as well as surface molecules like CD14 have been shown to play a pivotal role in the endotoxin activation cascade. In this review we summarize the mechanism of endotoxin recognition and its manifold effects on human health.

Overview on denominated allergens.

Type I allergy is a heightened or altered reactivity ofthe immune system in response to external substances involving immunoglobulins of the IgE class. More than 15% of the population in industrial countries suffer from immediate-type allergic symptoms [1]. Tn recent years, allergy research into the immunological, biochemical and structural characterization of allergens has led to an enormous progress alTecting diagnostics and therapeutics. This article summarizes important aspects of allergy research work on purified allergens. Characterized and denominated allergens have been listed. These data include biochemical and structural allergen characteristics as well as research results on human histocompatibility leucocyte antigens (HLA) restriction of ihe immune response, Tand B-cell epitopes and recombinant allergen expression. Allergens were isolated from a variety of dilTerent species. The taxonomy and relationship of dilTerent vertebrate, invertebrate as well as plant species from which allergens are isolated are also shown.

Lymphocyte proliferation response to extracts from different latex materials and to the purified latex allergen Hev b 1 (rubber elongation factor)

BACKGROUND Type I allergy to latex is a growing problem, especially among health care workers. A detailed study of the peripheral blood cell responses to latex allergens has not been reported. METHODS Peripheral blood mononuclear cells of patients and healthy subjects were isolated and stimulated with protein extracts from latex sap and latex gloves and the purified latex allergen Hev b 1 (rubber elongation factor) at different concentrations to determine the antigen-specific proliferation response. The examined patients were sensitized to latex by occupational exposure (n = 23) and had rhinitis, conjunctivitis, contact urticaria, and/or asthma. Two control groups of nonsensitized subjects were studied: one occupationally exposed to latex (n = 8), and the second, not exposed to latex (n = 8). RESULTS In general, only latex-exposed subjects responded to the different latex antigen preparations. Lymphocyte proliferation responses to latex sap extract were found in 65% of latex-sensitized subjects and in 37.5% of the latex-exposed healthy subjects. Latex glove extract induced a significant proliferative responses in 47.8% of latex-sensitized patients and in 25% of the latex-exposed individuals. Hev b 1 induced lymphocyte proliferation responses in 52% of the latex-sensitized patients and in 25% of the latex-exposed subjects indicating that Hev b 1 is relevant antigen in these latex-sensitized and latex-exposed groups. Peripheral blood mononuclear cells of 39.1% of the latex-sensitized subjects responded to all three allergen preparations (latex sap and latex glove extract, as well as Hev b 1). We could find no correlation between latex-specific IgE level and latex-induced lymphocyte proliferation response. CONCLUSION Our data indicate that the 14 kd protein Hev b 1 is a relevant allergen in health care workers. It can be detected by specific IgE antibodies to Hev b 1, as well as in lymphocyte proliferation assay. In addition, our study suggests that antigen-specific proliferation response to latex is associated with exposure to latex, but not with the level of specific latex IgE. This may be useful for the evaluation and prediction of latex hypersensitivity development.

Standardization of whole blood assay for determination of pyrogenic activity in organic dust samples

To characterize bioaerosol exposure at workplaces standardized methods are necessary. Activity of endotoxin, one component of organic dust, can be quantified with the Limulus-Amoebocyte Lysat test (LAL test). Further information with respect to pyrogenic activity of the organic dust can be achieved by measuring cytokine release of human blood after stimulation with the dust or its extract (whole blood assay). The aim of our study was the standardization of the whole blood assay (WBA) while using cryo-preserved human blood (Qualis Laboratorium) and to compare the outcome of the different cytokines determined by incubation of the blood cells with extracts from dust samples collected at various workplaces. Cytokine release (IL-1 beta, IL-6, IL-8, TNF-alpha, MCP-1) was measured by ELISA after stimulation of fresh blood from ten donors as well as cryo-preserved human blood. In both cases blood was stimulated with E. coli endotoxin as well as with 30 dust filter extracts from various workplaces. All dust filter extracts were investigated in the WBA using cryo-preserved blood as well as with LAL test. E. coli endotoxin stimulated the release of IL-1 beta, IL-6, IL-8, TNF-alpha and MCP-1 in a dose-dependent manner in fresh as well as cryo-preserved human whole blood. 200 pg/ml E. coli endotoxin induced maximal cytokine release in cryo-preserved blood (mean value for IL-1 beta 2509+/-418 pg/ml; n=13 experiments) whereas fresh blood of single donors reached a maximum release when stimulated with 50 ng/ml endotoxin (mean value of ten donors 1125+/-553 pg/ml IL-1beta). Using cryo-preserved blood the coefficient of variation (CV) regarding the interassay variability was below 21% for all cytokines measured. Regarding 26 dust sample extracts correlation coefficient r2 for LAL test and WBA was between 0.90 and 0.93 (Pearson) for IL-1 beta, IL-6, IL-8 and TNF-alpha whereas correlation for MCP-1 was lower (r(2)=0.59). Two dust sample extracts which showed similar reactivity patterns in LAL test as well as in WBA with respect to IL-1 beta, IL-6, IL-8 and TNF-alpha could be differentiated by measuring MCP-1. In conclusion, cryo-preserved blood pools are suitable to standardize WBA. Combination of different outcome variables like IL-1 beta and MCP-1 improve the characterization from the inflammatory potency of workplace related dust samples.

Increased gamma/delta-positive T-cells in blood and bronchoalveolar lavage of patients with sarcoidosis and hypersensitivity pneumonitis

A small population of T-cells does not express the conventional T-cell receptor (TCR), characterized by the alpha and beta polypeptide chains (alpha/beta TCR) but two polypeptides termed gamma and delta (gamma/delta TCR). Changes in gamma/delta TCR expression may be relevant as the cause or consequence of several diseases. Our study was undertaken to determine and compare the distribution of T-cells expressing gamma/delta TCR in blood and bronchoalveolar lavage (BAL) of patients with sarcoidosis, hypersensitivity pneumonitis (HP), idiopathic pulmonary fibrosis (IPF), and of healthy controls. In addition, the association between gamma/delta TCR of blood T-lymphocytes and accessory molecules was analysed. Using direct immunofluorescence with the anti-gamma/delta TCR and anti-CD3 monoclonal antibodies (MoAbs) followed by flow cytometric analysis, the blood of patients with pulmonary sarcoidosis, HP, IPF and of healthy controls was analysed. To reveal the association between gamma/delta TCR of blood T-lymphocytes and the accessory molecules, expression of CD4, CD8 and CD25 were determined. Calculating the percentage and the total number of CD3+ gamma/delta TCR cells in blood, the data indicated a significant increase of gamma/delta T-cells in individuals with pulmonary sarcoidosis and HP, compared to healthy controls and IPF patients. In sarcoidosis patients with elevated CD3+ gamma/delta TCR levels, significantly lower CD4/CD8 ratios were observed. In addition, our data demonstrate a correlation between the decrease of CD4+ cells in blood and the amplified appearance of gamma/delta TCR expression in sarcoidosis patients, but not in HP patients.(ABSTRACT TRUNCATED AT 250 WORDS)

Evaluation of Quantification Methods of Occupational Endotoxin Exposure

Endotoxin has been identified as important component of organic-dust exposure and is suspected as main cause of work-related adverse health effects in dusty areas. Although the determination of endotoxin levels by using the Limulus amoebocyte lysate (LAL) assay is internationally accepted, reliability and variation of values measured with this test remain a point of discussion. Therefore, the purpose of the study was to determine the influence of different parameters on endotoxin activity measured in airborne samples. This study thus analyzed: (a) dust filter extraction procedures, (b) storage of samples, (c) usage of different commercially available LAL assays, and (d) results of the whole blood assay (WBA) compared to the LAL test. Using a parallel sampler, 120 filters were loaded with dust at 4 different occupational settings and extracted in 2 labs using a standardized protocol. Parameters like Tween in the extraction medium, extraction volume, centrifugation speed, and material of tubes used for extraction were tested. The LAL test and the WBA were able to determine the differences in dust load of filters obtained from the settings investigated. In addition, results varied significantly with modifications in extraction procedures. Using Tween for filter extraction mainly influenced the resulting endotoxin activity. In addition, LAL test differences according to manufacturer of LAL test, extraction volume, and whether the samples are freshly processed or frozen also resulted in significant variations in the endotoxin levels. In conclusion, a reliable assessment of exposure to endotoxin activity is only possible if standard operation procedures (SOPs) for sampling and determination are established.

Chironomidae hemoglobin allergy in Japanese, Swedish, and German populations

Hemoglobins of the Diptera (insect) family Chironomidae have been identified as causative allergens in asthmatic patients. In this study, 229 Japanese, 17 Taiwanese, and 92 Swedish sera from atopic patients were tested for antibodies against Chi t I, the hemoglobin from the European midge species Chironomus thummi, and against crude extracts from the Japanese midges Tokunagayusurika akamusi (T. akamusi) and Cricotopus sylvestris (Cr. sylvestris). Nearly 40% of patients showed a positive reaction to Cr. sylvestris extract, which contains no hemoglobin. This result is probably due to the presence of other partially cross‐reacting allergens than hemoglobin. Nearly all tested Japanese serum samples showed cross‐reactivity between Chi t I and Cr. sylvestris, a finding which is evidence for common epitopes in both midge species. Furthermore, an overall good correlation between the amounts of IgE antibodies against Chi I I and Chi 1 I component III was found in sera from Swedish, Japanese, and German patients.

Concentration of Bioaerosols in Composting Plants Using Different Quantification Methods

BACKGROUND Bioaerosols (organic dusts) containing viable and non-viable microorganisms and their metabolic products can lead to adverse health effects in exposed workers. Standard quantification methods of airborne microorganisms are mainly based on cultivation, which often underestimates the microbial burden. The aim of the study was to determine the microbial load in German composting plants with different, mainly cultivation-independent, methods. Second purpose was to evaluate which working areas are associated with higher or lower bioaerosol concentrations. METHODS A total of 124 inhalable dust samples were collected at different workplaces in 31 composting plants. Besides the determination of inhalable dust, particles, and total cell numbers, antigen quantification for moulds (Aspergillus fumigatus, Aspergillus versicolor, Penicillium chrysogenum, and Cladosporium spp.) and mites was performed. Concentrations of β-glucans as well as endotoxin and pyrogenic activities were also measured. The number of colony forming units (cfu) was determined by cultivation of moulds and actinomycetes in 36 additional dust samples. RESULTS With the exception of particle numbers, concentrations of all determined parameters showed significant correlations (P < 0.0001; r Spearman: 0.40-0.80), indicating a close association between these exposure markers. Colony numbers of mesophilic moulds and actinomycetes correlated also significantly with data of cultivation-independent methods. Exposure levels showed generally large variations. However, all parameters were measured highest in dusty working areas like next to the shredder and during processing with the exception of Cladosporium antigens that were found in the highest concentrations in the delivery area. The lowest concentrations of dust, particles, antigens, and pyrogenic activity were determined in wheel loader cabins (WLCs), which were equipped with an air filtration system. CONCLUSION It was possible to assess the microbial load of air in composting plants with different quantification methods. Since allergic and toxic reactions may be also caused by nonliving microorganisms, cultivation-independent methods may provide additional information about bioaerosol composition. In general, air filtration reduced the bioaerosol exposure shown in WLCs. Due to the fact that the mechanical processing of compost material, e.g. by shredding or sieving is associated with the generation of high bioaerosol concentrations, there is still a need of improved risk assessment and state-of-the-art protective measures in composting plants.

Analysis of b-cell epitopes in the n-terminal region of Chi t I component III using monoclonal antibodies

The hemoglobins of the midge Chironomus thummi thummi (Chi t I) are known to cause immediate-type hypersensitivity reactions in humans. Further knowledge of the antigenic sites of such allergens will provide new therapeutic approaches. The aim of our study was to identify and characterize linear B-cell epitopes of the hemoglobin component III of Chi t I (136 amino acid residues). Using the antigenic index algorithm of Jameson and Wolf (Jameson and Wolf (1988) Comput. Appl. Biosci. 4, 181-186), three linear binding sequences of this allergen molecule were predicted. Two mouse monoclonal antibodies (mAbs 3 and 6) raised against purified Chi t I component III were investigated by ELISA for their binding to nine synthetic peptides 19-21 residues in length, covering nearly the whole sequence of component III. MAb 6 recognized only one peptide (11-30) while mAb 3 bound to both N-terminal peptides (1-19 and 11-30), suggesting that the antibody binding site is located in the overlapping region. This assumption could be confirmed in ELISA with solid phase-bound recombinant peptides (RP) as well as in inhibition studies with free tryptic peptides indicating that identification of these linear B-cell epitopes is neither influenced by the method of peptide production nor by the kind of used immunoassay. To define the essential amino acid residues we investigated mAbs with solid phase-bound overlapping octamers. In the case of mAb 3, amino acids experimentally identified as essential for antibody binding (aa 13-17) are identical with those residues predicted as a B-cell epitope with the antigenic index of Jameson and Wolf.

T-cell receptor repertoire expression in workers with occupational asthma due to platinum salt

There is a high incidence of asthma, rhinitis, conjunctivitis and contact urticaria in workers of precious metal refineries. Symptoms are closely associated with sensitization to halogenated platinum compounds, as assessed by skin-prick test. The aim of the present study was to examine the molecular mechanisms involved by describing the T-cell receptor (TCR) repertoire distribution of peripheral blood mononuclear cells (PBMCs) without and after in vitro stimulation with sodium hexachloroplatinate. PBMCs of 17 sensitized subjects with work-related asthma and a positive skin-prick test result to sodium hexachloroplatinate and of 15 nonexposed subjects were isolated and TCR expression determined by flow cytometry. Furthermore, the sodium hexachloroplatinate-mediated in vitro effects on the frequency of Vbeta-expressing T-cells, the proliferation response and the expression of cell surface molecules like CD71, CD25, CD95 and HLA-DR were studied. CD3-positive lymphocytes of platinum salt-sensitized workers had a significantly higher frequency of Valpha2a+, Vbeta11+ and Vbeta21.3+ T-cells than controls (p<0.01, p<0.01 and p<0.001 respectively). In vitro stimulation of PBMCs from platinum salt-sensitized as well as control subjects with sodium hexachloroplatinate increased the percentage of CD3-positive cells bearing specific TCRs, especially Vbeta5.3, Vbeta6.7, Vbeta8a, Vbeta20 and Vbeta21.3. This effect was time- and dose-dependent. The present results indicate that the frequencies of Valpha2a-, V11 and Vbeta21.3-bearing blood T-cells and platinum salt-induced lymphocyte proliferation are strongly enhanced in subjects who suffer from asthma due to platinum salt. In addition, in vitro stimulation with sodium hexachloroplatinate modulates the frequencies of certain T-cell receptor-bearing T-cells.