Biomedical subjects

M Schlaak

Publications and source records attributed to M Schlaak.

At least 73 records · Page 4Linked to original sources

PubMed1998

Increased expression of proinflammatory chemokines in bronchoalveolar lavage cells of patients with progressing idiopathic pulmonary fibrosis and sarcoidosis.

BACKGROUND: There is increasing evidence that the proinflammatory chemokines interleukin-8 (IL-8) and macrophage inflammatory protein-1 alpha (MIP-1 alpha) are involved in the pathogenesis of interstitial lung diseases. METHODS: We investigated the release of TNF-alpha, IL-8, MIP-1 alpha by cultured bronchoalveolar lavage (BAL) immune cells of patients with idiopathic pulmonary fibrosis (IPF, n = 24), sarcoidosis (SAR, n = 24), and controls (n = 20) by ELISA. Furthermore, mRNA expression of these cytokines in BAL cells immediately frozen after bronchoscopy was determined. The clinical course of the disease was evaluated and the patients were subdivided into groups with progressing or stable disease. RESULTS: TNF-alpha, IL-8, and MIP-1 alpha were significantly elevated in the supernatants of BAL immune cells of IPF and SAR patients with progressing disease compared to controls (p < 0.005 in both diseases) and also when compared to patients with stable disease (IPF p < 0.005, SAR p < 0.05). Interestingly, the release of TNF-alpha, IL-8, and MIP-1 alpha did not differ significantly between IPF patients with stable disease and controls, whereas in SAR patients with stable disease a difference at a low significance level (p < 0.05) was obtained. In IPF and SAR patients with progressing disease, a clear mRNA signal of TNF-alpha, IL-8, and MIP-1 alpha was detected in BAL immune cells not having been stimulated by adherence to plastic, whereas in patients with stable disease or controls only a weak signal was observed. MIP-1 alpha release correlated positively with percentage of BAL eosinophils in IPF and SAR. Furthermore, the percentage of eosinophils in BAL was significantly elevated in the IPF subgroup with progressing disease. CONCLUSIONS: Our data demonstrate that an exaggerated expression of TNF-alpha, IL-8, and MIP-1 alpha in BAL immune cells is characteristic for IPF and SAR patients who show progressing disease.

Adult↗

PubMed1997

Investigation of different recombinant isoforms of grass group-V allergens (timothy grass pollen) isolated by low-stringency cDNA hybridization--antibody binding capacity and allergenic activity.

A cDNA library of timothy grass pollen was screened for homologous isoforms of major group-V allergens by low stringency hybridization with a Phl p 5 (Phleum pratense) probe. After restriction analysis of the 40 clones obtained, 17 were selected for cDNA sequencing. Of these clones, two were unrelated to group-V allergens, six showed high similarity but an incomplete open reading frame and nine had high similarity with a complete open reading frame. Comparison of deduced amino acids of ten complete cDNA clones confirmed the presence of two major isoforms, a and b. Within these two subgroups, only minor sequence variations were observed. Eight isoforms were expressed in Escherichia coli K12 and purified to homogeneity. Although the subgroups a and b could be distinguished by their molecular masses and by binding constants towards monoclonal antibodies, all isoforms turned out to be biochemically similar. Ribonuclease activity as a marker for the biological function of group-V allergens was shown to be in the same range for both subgroups. Analysis of allergenic B-cell responses towards the isoforms in 26 grass pollen allergic patients revealed that the IgE reactivities to the different isoforms were identical for each individual. IgE reactivities and allergenic activities of three isovariants and an allergen of a different group were compared in a selected group of four grass pollen allergic patients by immunoblot, histamine-release and skin-prick tests. The IgE reactivity does not necessarily mirror the allergenic activity of the single molecule, and the variability of allergenic activity between the isovariants does not, in every case, depend on the structural differences of these allergens. We conclude that group-V isoallergens in grass pollen, although they can be structurally different, induce a similar B-cell response but can show variable allergenic activity. Thus, the most allergenic isoform of each important group of allergens should be sufficient for the diagnosis of type-I allergy. Whether the isoallergenic variation has any significant influence on the outcome of immunotherapy in allergic disease still has to be elucidated.

Allergens↗

PubMed1997

Comparison of natural and recombinant isoforms of grass pollen allergens.

More than 95% of grass pollen allergic patients possess IgE antibodies against grass group I, a heterogeneous group of glycoproteins found in all temperate grasses. We studied the structural variability of the group I allergens in single species and among different grasses. By 2-DE blotting using patients' IgE and monoclonal antibodies, we detected IgE-reactive isoforms with molecular masses between 32 and 37 kDa and focusing in a wide pI ranging from 4.7 to 7.6. While the group I allergens of timothy grass (Phl p 1) were composed of 37 and 35 kDa components, only single isoforms were found for ryegrass (Lol p 1) and velvet grass (Hol l 1): 32 and 34 kDa, respectively. By N-terminal microsequencing we determined single amino acid substitutions in different-sized group I allergens. The post-translational modifications (one N-glycosylation site, two hydroxylated proline residues and seven cysteine residues for potential disulfide formations), which contribute to IgE reactivity, were identical in all. From the cDNA sequences we deduced protein sequence homologies > 90%, a result which might explain the high IgE cross-reactivity among the grasses. In order to test whether recombinant group I grass allergens can act as substitutes for the natural forms, we expressed rPhl p 1 in E. coli and in P. pasteuris. 2-DE immunoblotting again demonstrated a microheterogeneity in molecular mass and pI. While the E. coli products were free from post-translational modifications, rPhl p 1 from Pichia is a heterogeneous glycoprotein fraction with a carbohydrate content of about 15%. This rPhl p 1 is hyperglycosylated compared to the nPhl p 1, which only has a 5% carbohydrate content.

Allergens↗

PubMed1997

Antiinflammatory and antifibrotic properties of colchicine: implications for idiopathic pulmonary fibrosis.

The ancient drug colchicine has repeatedly been proposed as a novel drug for therapy of pulmonary fibrosis. The present study was undertaken to add to the knowledge on colchicine's antiinflammatory and antifibrotic properties and thus help determine its actual rank in the treatment of pulmonary fibrosis. In vitro cell culture experiments with stimulated and unstimulated normal donor peripheral blood mononuclear cells (PMNC) and a human lung fibroblast cell line (WI-38) were used to determine the effects of colchicine on PMNC cytokine release (interleukin-6 and tumor necrosis factor-alpha) as well as on fibroblast proliferation and collagen synthesis rates. Reverse transcriptase polymerase chain amplifications of alpha 1 (III) collagen were done to detect collagen messenger ribonucleic acid (mRNA) expression. Colchicine did not significantly modulate tumor necrosis factor-alpha (TNF-alpha) and interleukin-6 (IL-6) release of PMNC. Colchicine inhibited fibroblast proliferation and total collagen synthesis significantly at concentrations obtainable in serum in vivo. Transcription of the alpha 1 (III) collagen gene into mRNA continued under colchicine. We conclude that colchicine is a potent in vitro inhibitor of fibroblast functions in terms of proliferation and collagen synthesis. The mechanism of collagen inhibition is more likely an inhibition of cellular collagen secretion than a switch off of collagen mRNA transcription. On the other hand, although colchicine is known to inhibit many leukocyte functions, it is a poor inhibitor of cytokines known to be important for fibrogenesis (e.g. IL-6, TNF-alpha, IL-1, platelet-derived growth factor, and transforming growth factor-beta). This makes colchicine, at least from a theoretical standpoint and as concluded from in vitro studies, a preferable candidate for a combined therapeutic strategy.

Cells, Cultured↗

PubMed1997

Shed soluble ICAM-1 molecules in bronchoalveolar lavage cell supernatants and serum of patients with pulmonary sarcoidosis.

The soluble form of intercellular adhesion molecule-1 (sICAM-1) might be a serum parameter of inflammatory activity gauging cellular interactions with possible relevance in sarcoidosis. To address this question we measured sICAM-1 by enzyme-linked immunosorbent assay in serum and shedding of this molecule by bronchoalveolar lavage (BAL) cells in sarcoidosis patients (44 and 40, respectively) and in controls (10 and 19, respectively). Serum concentrations of sICAM-1 (588.3 +/- 72.2 ng/ml) and its spontaneous release by BAL cells (9.9 +/- 1.5 ng/ml) in patients with active sarcoidosis were significantly higher than in those with inactive disease or controls, although no correlation was observed. Significant correlations of sICAM-1 shedding by nonstimulated BAL cells with the serum level of neopterin and of shedding by lipopolysaccharide-stimulated BAL cells with percentage of alveolar macrophages were observed in active sarcoidosis. Kinetic cell culture experiments with peripheral blood mononuclears disclosed a rapid up-regulation of sICAM-1 shedding and tumor necrosis factor-alpha release; however, at 5 h after stimulation a dissociation of their releases was observed. sICAM-1 release was maintained over 2 days, whereas tumor necrosis factor-alpha release peaked at 5 and ceased after 43 h. These results provide evidence that circulating and BAL cell culture-derived sICAM-1 reflect the stage of sarcoid inflammation. Although sICAM-1 in BAL cell supernatants originates from alveolar macrophages; the absence of a correlation with serum sICAM-1 concentration indicates that other cells are additional sources of the circulating pool of this molecule.

Adult↗

PubMed1997

Mapping of IgE-binding epitopes on the recombinant major group I allergen of velvet grass pollen, rHol l 1.

BACKGROUND: New and more successful approaches to diagnosis and therapy of allergic diseases require a more subtle understanding of the structure and the epitopes on the allergen molecule. OBJECTIVE: This study was done to obtain more information on the structure and the IgE-binding epitopes of a major allergen of velvet grass pollen, Hol l 1. METHODS: We cloned Hol l 1 from a complementary DNA library and performed B-cell epitope mapping with 21 recombinant fragments expressed as fusion proteins in Escherichia coli. The fragments were analyzed by Western blotting with sera from 50 different patients. RESULTS: The patients' sera individually recognized at least four different IgE-binding regions (amino acids 1 to 27, 61 to 76, 84 to 105, and 158 to 240). According to their binding patterns with these epitopes, they were divided into five groups. Most sera (92%) bound to the C-terminal peptide (158 to 240), which consists of more than 80 amino acids, whereas there was virtually no binding to smaller fragments covering this region. In contrast to the C-terminal peptide, the IgE-binding peptides on the N terminus and on the middle region of the molecule were of a smaller size (15 to 30 amino acids). CONCLUSIONS: The major group I allergen of velvet grass bears at least four different IgE-binding epitopes, which were individually recognized by sera from different patients. The C terminus represents the major IgE-binding region and contains at least one discontinuous IgE-binding epitope, whereas the N terminus and middle region of Hol l 1 seem to contain continuous IgE-binding epitopes.

Allergens↗

PubMed1997

Pentoxifylline in treatment of sarcoidosis.

The optimal therapy for sarcoidosis remains unclear. Most patients show a short-term response to corticosteroid therapy, but they have to face the risk of significant side effects. Because tumor necrosis factor alpha (TNF) plays a critical role in granuloma formation and sustenance as well as in the progression of sarcoidosis, we investigated pentoxifylline (POF), which exerts TNF-inhibitory activity, as a therapeutic agent in active pulmonary sarcoidosis. Twenty-three previously untreated patients with documented disease progression during the preceding 3 mo were treated with POF (25 mg/kg daily) and followed for 6 mo of therapy. Two patients were lost to follow-up, and three patients discontinued POF therapy because of gastrointestinal side effects; 18 patients were thus evaluated. Eleven patients improved, seven remained stable and, most importantly, none deteriorated. Lung function tests-DL(CO), Pa(O2) and Pa(O2)[exercise]-were significantly improved in the patient group as a whole and increased in a highly significant manner in those who improved. Three patients with corticosteroid-resistant disease progression were additionally treated with a combination of corticosteroids with POF. In all three patients the combination therapy resulted in an immediate complete decrease of disease activity, even after tapering prednisone to 7.5 mg daily or tapering off corticosteroids. These promising results suggest that POF may improve therapeutic regimens in pulmonary sarcoidosis either by sparing or replacing corticosteroids.

Adult↗

PubMed1997

TCR V beta families in T cell clones from sarcoid lung parenchyma, BAL, and blood.

The TCR repertoire and the CD4/CD8 ratio of clones from peripheral blood (PB), transbronchial biopsies (TBB), and bronchoalveolar lavage (BAL) of 16 sarcoid patients was analyzed by staining the clones with monoclonal antibodies against nine V beta-families. We observed a striking increase in the CD4/CD8 ratio of the clones from BAL; whereas the CD4/CD8 ratio of the clones from PB was in the normal range. The CD4/CD8 ratio of the TBB-clones had also increased, but this increase did not reach the level of that of the BAL clones. The most prominent changes in the V beta percentages could be detected in the CD4+ subpopulation of the BAL-clones. The most abundant V beta families were V beta 5 in PB and BAL (11.8 and 28.6%, respectively) and V beta 6 in the TBB (12.4%). A similar compartmentalized V beta usage could be demonstrated in one patient with tuberculosis and one patient with HP. The increase in V beta 5, V beta 8, V beta 12, V beta 13.3, and V beta 19 in the BAL and the increase of V beta 5, V beta 6, V beta 13.3, and V beta 19 in the TBB suggest an antigen-driven activation of the T cells in both compartments. Differences in the V beta percentages between BAL and TBB and the lower CD4/CD8 ratio in the TBB, however, demonstrate a relative independence of the two compartments.

Biopsy↗

PubMed1997

Sarcoidosis: TNF-alpha release from alveolar macrophages and serum level of sIL-2R are prognostic markers.

At the time of diagnosis, many sarcoidosis patients have no clinical indication for corticosteroid therapy, and prognostic parameters predicting deterioration are missing. In the present study, we investigated parameters derived from bronchoalveolar lavage (BAL) and serum in 77 patients with recently diagnosed sarcoidosis to test their predictive value. Patients were divided into a group with (Group A, n = 37) and a group without (Group B, n = 40) indications for therapy, and the course of the disease was evaluated after 5.7 +/- 0.4 mo. The CD4+/CD8+ lymphocyte ratio and percentage of BAL lymphocytes were of no predictive value. Release of tumor necrosis factor-alpha (TNF-alpha) from cultured alveolar macrophages (AM) was significantly increased in both groups (Group A = 1,872 +/- 428 pg/ml; Group B = 1,561 +/- 449 pg/ml) as compared with controls (220 +/- 37 pg/ml). In Group B, however, patients with a high level of TNF-alpha release had a significantly greater risk of disease progression than did those with normal TNF-alpha release (43.8% versus 8.3%, respectively). From the serologic parameters investigated, consisting of neopterin, angiotensin converting enzyme (ACE), and soluble interleukin-2 receptor (sIL-2R), only the last was of significant predictive value; 42.1% of sarcoidosis patients in Group B with a high level of sIL-2R experienced disease progression, whereas none of those with a normal level did. We conclude that TNF-alpha release and sIL-2R are suitable parameters for predicting disease progression in sarcoid patients who have no indication for therapy at the time of disease diagnosis.

Adult↗

PubMed1997

Th1/Th2 cell distribution in pulmonary sarcoidosis.

Cytokines released by T lymphocytes activated in the course of sarcoidosis, e.g., interleukin-2 (IL-2) and interferon-gamma (IFN-gamma), indicate the presence of Th1 cells leading to the question of Th1/Th0/Th2 balance in sarcoidosis. The aim of this study was to evaluate Th-like cytokine patterns in different compartments of the body, i.e., peripheral blood, pulmonary parenchyma, and bronchoalveolar lavage (BAL), of patients with pulmonary sarcoidosis by comparing cytokine gene expression of 167 T-cell clones derived from the above-mentioned compartments. Seventy-nine blood, 49 transbronchial biopsy, and 39 BAL clones were analyzed using polymerase chain reaction to identify the respective gene transcripts. The majority of CD4+ and CD8+ blood cells exhibited intermediate cytokine profiles (63.3%) without shifts to either side of the spectrum (6.3% Th1, 5.1% Th2). Lung parenchyma cells shifted to the Th1 side of the spectrum: 26.5% of the cells were of Th1 or of intermediate type (between Th1 and Th0), whereas only 8.1% of the cells were of Th2 or of intermediate type (between Th0 and Th2). CD8+ parenchyma cells were evenly distributed. From BAL only CD4+ clones could be generated with shifts to both ends of the spectrum. Thus, our data provide evidence that in sarcoidosis, at the site of granuloma formation, an accumulation of Th1 cells as well as of intermediate (between Th1 and Th0) cell types occurs, whereas in the alveolar lumen, high numbers of Th1 and Th2 cells with a simultaneous decrease of Th0 cells can be observed.

CD4-Positive T-Lymphocytes↗

PubMed1997

Anti-Borrelia burgdorferi immunoglobulin seroprevalence in pulmonary sarcoidosis: a negative report.

The aetiology of sarcoidosis is still unknown. An infectious microorganism as causal agent for this disease could not be identified, but high titres of antibodies against Borrelia burgdorferi were detected in Chinese studies implying a causality with this disease. These findings, however, could not be reproduced by other researchers. The aim of this study was, therefore, to evaluate the possible role of these spirochetes in the pathogenesis of sarcoidosis by serological examinations. Sixty sera of patients suffering from sarcoidosis were examined for anti-B. burgdorferi immunoglobulin by enzyme-linked immunosorbent assay (ELISA). ELISAs for these antibodies show a high sensitivity, but a low specificity; therefore, a specific immunoblot was used to confirm positive results. Initially, 8% of the patients were reactive in the ELISA, and 20% of these could be confirmed by immunoblot. Therefore, the prevalence for B. burgdorferi antibodies in sarcoidosis patients was 1.6%. This result did not differ significantly from the prevalence of B. burgdorferi antibodies in 1,000 regular blood donors of the city of Hamburg (7% reactive in the ELISA, 38% confirmed via immunoblot, prevalence 2.7%). The hypothesis of causality between a B. burgdorferi infection and sarcoidosis cannot be confirmed by this data.

Antibodies, Bacterial↗

PubMed1997

Accessory function of alveolar macrophages from patients with sarcoidosis and other granulomatous and nongranulomatous lung diseases.

BACKGROUND: Accessory function (AF) is one way antigen presenting cells generate sufficient secondary signals for optimal T-cell proliferation and IL-2 production. In general, alveolar macrophages (AM) are inferior accessory cells in comparison to monocytes whereas in sarcoidosis AF of AM is increased. METHODS: We compared the accessory index (AI) of AM and peripheral blood monocytes (PBM) of 41 patients with inactive sarcoidosis (SAR I, n = 12); active sarcoidosis with new or progressing symptoms (SAR II, n = 19), active sarcoidosis with spontaneous remission (SAR III, n = 10), tuberculosis (TB, n = 12), hypersensitivity pneumonitis (HP, n = 12), Wegener's disease (WD, n = 2), undefined alveolitis (UA, n = 8) and chronic obstructive pulmonary disease (COPD, n = 6) by employing the histoincompatibility-insensitive Jurkat cells as indicator cells. RESULTS: Compared with the controls (1.08 +/- 0.3) AMs of all groups but SAR I (AI: 0.96 +/- 0.42) exhibited significantly increased AIs (SAR II: 3.6 +/- 3.9; SAR III: 3.2 +/- 2.4; TB: 2.8 +/- 2.2; HP: 3 +/- 2; UA: 2.7 +/- 2.3; COPD: 3.1 +/- 2.2; p < 0.05 for all comparisons). Only in HP, AI of PBM was significantly increased compared with controls (3 +/- 1.5, 1.3 +/- 0.5, respectively; p < 0.001). Alveolar macrophages from patients with arcoidosis, TB, and HB express the costimulatory molecule CD80 on their surface and anti-CD80 antibodies inhibited the IL-2 release of Jurkat cells in this system to 59 +/- 27%. CONCLUSIONS: Our data demonstrate that AM from patients with various diseases have the capability to act as competent accessory cells and that the reported accessory function of these cells is at least in part mediated by the expression of CD80.

Adult↗

PubMed1997

[Pentoxifylline inhibits experimental bleomycin-induced fibrosing alveolitis].

Therapy of idiopathic pulmonary fibrosis (IPF) is directed at 1) inhibition of alveolitis and tissue damage, and 2) inhibition of matrix deposition. We and others have identified pentoxifylline (POF) as a promising drug in achieving these aims. For further clarification, we established a model of bleomycin-induced fibrosing alveolitis. Fisher 344 rats (n = 7 per group) were given bleomycin intratracheally once (0.7 U/100 g bw) and treated with POF (1.5 or 3 mg/kg bw per day i.p.), prednisolone (15 mg/kg bw i.m. per day), or sodium chloride solution (NaCL). The extent of inflammatory reactions was determined after 8 days by differentiation of cells of broncho-alveolar lavage (BAL) and by quantification of proliferating cells in lung interstitium subsequent to staining of the Ki-67 antigen. POF inhibited neutrophil alveolitis in BAL and reduced the amount of proliferating cells in the lungs significantly while prednisolone and NaCL did not. Both POF and prednisolone exerted a positive influence on postoperative weight loss as well as on lung weight increase subsequent to bleomycin instillation. The postoperative body weight loss and the lung weight increase after bleomycin instillation are most likely due to an inflammatory reaction subsequent to operation and bleomycin deposition. Tumor necrosis factor alpha (TNF-alpha) has been shown to be a key cytokine in bleomycin-induced fibrosing alveolitis as well as in IPF; it also exerts catabolizing effects. Since both POF and prednisolone are known to effectively inhibit proinflammatory cytokines and, among those, TNF-alpha, nonspecific antiinflammatory effects probably explain the benefits. Additionally, however, this study proved POF to be more effective in inhibition of BAL neutrophils and number of proliferating cells in lung interstitium. Further, it has been shown that POF, but not prednisolone, inhibits activation of neutrophil granulocytes and formation of reactive oxygen species. Thus we believe that the mechanism of action of xanthines might contribute to therapy of IPF. For further clarification, a prospective clinical study of POF in IPF therapy has been initiated.

Animals↗

PubMed1997

Spontaneous and interleukin-2-modulated cytokine release by bronchoalveolar cells in pulmonary malignancy.

In a recent phase I study of inhalative, human natural interleukin-2 (hnIL-2) treatment of pulmonary metastases from previously resected solid tumors (mainly renal carcinoma), we have reported that this treatment resulted in an increased accessory function of alveolar macrophages (AM) [1]. Encouraged by these data, we investigated the influence of hnIL-2 inhalation on proinflammatory cytokines spontaneously released by AM. Bronchoalveolar lavage was performed in four groups, each of four patients, before and after 2 weeks of daily inhalation of 0, 200,000, 600,000 and 1,200,000 IU of hnIL-2, respectively. Bronchoalveolar cells were cultured without stimulation to allow spontaneous release over a period of 24 h, into the supernatant. Concentrations of tumor necrosis factor-alpha (TNF-alpha), IL-6, IL-8 and macrophage inflammatory protein-1alpha (MIP-1alpha) were determined by the ELISA technique. Before hnIL-2 inhalation, we measured the following spontaneous cytokine release: TNF-alpha: 1,115.4 +/- 469.1 pg/ml, IL-6: 267.5 +/- 67.7 pg/ml cells, IL-8: 137.8 +/- 40.5 ng/ml, MIP-1alpha: 9.5 +/- 6.8 ng/ml. Inhalation of hnIL-2 did not result in any significant changes in these cytokines. Comparing TNF-alpha release in healthy controls (250.6 +/- 46.7 pg/ml) with that of tumor patients (1,115.4 +/- 469.1 pg/ml), we observed significantly (p < 0.05) elevated TNF-alpha levels in the patient group, which did not change significantly in response to IL-2 inhalation. Our data demonstrate that the activation of AM previously observed after hnIL-2 inhalation is not directly related to a hnIL-2-induced cytokine release by bronchoalveolar cells.

Adult↗

PubMed1996

[Differential diagnosis of berylliosis/sarcoidosis in a dental technician].

HISTORY AND CLINICAL FINDINGS: Sarcoidosis was diagnosed in a dental technician when he was aged 21 years. Two years later prednisolone treatment was started and continued for 14 years because of nonproductive cough with progressive reduction in vital capacity and CO transfer capacity. Subsequently the risk of exposure to beryllium-containing dust in dental laboratories became known. In his case exposure had started before sarcoidosis had been diagnosed and had continued for 16 years. Changes typical of sarcoidosis (unproductive cough and dyspnoea; bihilar lymphadenopathy and reticulonodular marking in the chest radiogram) were now present, at the age of 40 years. INVESTIGATIONS: Vital and diffusion capacities were diminished, serum levels of angiotensin-converting enzyme (141 U/I) and of neopterin (5.8 mg/l) were increased. Beryllium-lymphocyte transformation test of peripheral mononuclear cells after invitro culture with beryllium sulphate gave a raised stimulation index, and the intracutaneous beryllium sensitisation test was positive. This indicated sensitisation to beryllium even 9 years after exposure had ceased. CONCLUSION: Even after exposure to beryllium has ended, proof of exposure together with clinical and radiological findings typical of sarcoidosis and beryllium sensitisation can provide the diagnosis of berylliosis.

Adult↗

PubMed1996

Crystallization and preliminary diffraction data of a major pollen allergen. Crystal growth separates a low molecular weight form with elevated biological activity.

Group V major allergen Phl p 5b of timothy grass pollen induces allergic rhinitis and bronchial asthma in 90% of grass pollen-allergic patients. In addition to its allergenicity ribonuclease activity has recently been attributed to this 29-kDa protein. The allergen was expressed in Escherichia coli and subsequently purified. Spontaneous conversion of these preparations to a mixture of various forms with molecular sizes between 10 and 29 kDa was consistently observed. Surprisingly, crystals could be grown from this heterogenous preparation. Single crystals, redissolved and analyzed by SDS-polyacrylamide gel electrophoresis and immunoblot, yielded one distinct low molecular weight protein, which was identified by amino acid sequencing as the C-terminal 13-kDa portion of the allergen. Histamine release assays with single crystal solutions using basophils of an allergic patient demonstrated allergenicity comparable with that of the holo-allergen. By contrast, RNase activity of the crystallized C-terminal form was 23 times higher than that of the full-length parent allergen. Crystals were used to collect preliminary diffraction data; the space group was evaluated to I4122 with cell dimensions of a = 87.7 A, b = 87.7 A, and c = 59.6 A. We conclude that preferential crystal growth of the 13-kDa form is indicative of a compact conformation of this particular C-terminal portion of the allergen. Thus, we show here that protein crystallization is not only a prerequisite for structural analyses, but it also can provide a unique separation technique to localize the functional domain of a major allergen.

Allergens↗

PubMed1996

Induction of cell death by cytokines in cell cycle-synchronous tumor cell populations restricted to G1 and G2.

In the present work, cytokine-mediated induction of cell death was investigated by flow cytometry in cell cycle-synchronous human tumor cell populations gained by centrifugal elutriation or by cell cycle blockade with mimosine and aphidicolin. Attention was payed to the question of whether the effector phase of cell death takes place in the same phase of the cell cycle in which the death signal is received. Another point of interest was the question whether synchronization of cell populations with respect to the cell cycle leads to increased synchronicity of the death phase. The results demonstrate that supernatants from monocyte/tumor cell interaction cultures containing tumor necrosis factor-alpha, interferons, and interleukins-1 and -6 or appropriate combinations of pure cytokines cause cell cycle arrest predominantly in G1 and to a lesser extent in G2. Cell death is initiated from both arrest points. Cytokine-treated G1 cells do not enter S phase. They die within the same G1 phase in which they receive the death signal. In contrast, a high proportion of cytokine-treated G2 cells pass through mitosis and are arrested and die in the subsequent G1 phase, whereas only a smaller proportion of cells are arrested and die in G2. The synchronicity of the death phase cannot be increased by the diverse methods of cell cycle synchronization applied. Interestingly, aurintricarboxylic acid, an agent known for inhibitory effects on nucleolytic activities and other protein/nucleic acid interactions, not only prevents cell death, but also cell cycle arrest.

Aphidicolin↗

PubMed1996

Human basophils release interleukin-4 after stimulation with Schistosoma mansoni egg antigen.

The elevated interleukin (IL)-4 and IgE production in Schistosoma mansoni infection seems to be induced essentially by the egg stage of the parasite. The underlying mechanism, however, is not known. Since basophils from human peripheral blood can produce IL-4, we asked, whether soluble S. mansoni egg antigens (SEA) would trigger basophils to release IL-4. Basophils from healthy human donors (n = 32) without prior history of schistosomiasis were incubated with SEA in the presence of IL-3. In all donors, IL-4 was produced at different concentrations. The IL-4 production was dependent on the dose of SEA, was correlated with the purity of the basophil preparation, and the IL-4 concentration in the culture supernatant was maximal 5 h after stimulation with SEA. In addition to its IL-4-stimulatory effect, SEA triggered basophils to degranulate, thereby releasing histamine and sulfidoleukotrienes. Stripping of receptor-bound IgE from basophils inhibited both SEA- and anti-IgE-induced, but not ionomycin-induced IL-4 production. Moreover, resensitization of stripped basophils with stripping supernatants or human serum restored SEA-induced IL-4 production. This suggests that IgE is involved in the mechanism of IL-4 induction by SEA. Since IL-4 is induced in basophils from nonexposed donors, basophils may play a role as an early source of IL-4 in S. mansoni infection.

Animals↗