L. Jäger

Procalcitonin expression in human peripheral blood mononuclear cells and its modulation by lipopolysaccharides and sepsis-related cytokines in vitro.

Procalcitonin (PCT), the precursor of calcitonin, was recently put forward as a diagnostic marker of systemic bacterial infection and sepsis. The major PCT production site in sepsis still remains unclear. Because of a certain association between increased levels of PCT and leukocyte-derived cytokines during sepsis, we assessed the possible expression of PCT in human peripheral blood mononuclear cells (PBMCs) and the modulation of PCT by lipopolysaccharides (LPS) and various sepsis-related cytokines by reverse transcriptase-polymerase chain reaction (RT-PCR) by using a novel primer set and flow cytometric analysis with intracellular staining with antibodies to the PCT components calcitonin and katacalcin. RT-PCR and flow cytometric analysis demonstrated that PBMCs express PCT both on mRNA and on protein levels. LPS and various proinflammatory cytokines (interleukin-1beta (IL-1beta), IL-6, tumor necrosis factor-alpha (TNF-alpha), IL-2) had pronounced stimulatory effects on the expression of PCT mRNA. Under identical experimental conditions the anti-inflammatory cytokine IL-10 had no effect on the expression of mRNA for PCT. Flow cytometric analysis demonstrated increased intracellular amounts of PCT components after LPS stimulation. Thus we demonstrate for the first time that PCT is expressed in PBMCs. This expression is modulated by bacterial LPS and sepsis-related cytokines. Therefore PBMCs may be among the sources of elevated PCT levels in patients with sepsis.

Functional role of CD26 on human B lymphocytes

CD26 is a well-known activation marker on T cells and natural killer (NK) cells [1]. It is identical with the ectopeptidase dipeptidyl peptidase IV (DP IV). The expression of CD26 on B cells has been discussed controversially [2,3]. We have studied the expression of this enzyme on B cells from the peripheral blood of healthy donors and of CVID patients, on cells of the Daudi Burkitt line and the EBV-transformed B-cell lines Jojo and Laz509. DP IV was detected by using anti-CD26 monoclonal antibodies and with help of specific enzyme substrates. Further the influence of specific synthetic DP IV inhibitors on mitogenic activation of purified B cells and DNA synthesis of cell lines was studied. We could show that in both groups 0-5% of freshly isolated CD20-positive B cells do express the CD26 antigen. After stimulation with pokeweed mitogen or St. aureus protein, the fraction of CD26-positive cells was enhanced up to 51% and 36%, respectively. Interestingly, induction of CD26 expression on B cells from CVID patients occurs in a manner similar to the B cells from healthy donors. Treatment of peripheral blood B cells and B-cell lines with highly specific competitive DP IV inhibitors leads to a significant inhibition of DNA synthesis in a dose-dependent manner. These data show that CD26 can be considered to be an activation marker not only of T- and NK cells but also of a main population of B cells, suggesting an involvement of CD26 in B-cell activation.

Investigation of the stability of apple allergen extracts

To determine optimal conditions for allergen preservation, we investigated the influence of different stabilizing additives and of storage temperature on the allergen activity of apple protein preparations, obtained by extraction in phosphate buffer or by precipitation in diacetone alcohol and resolubilization in phosphate buffer in the presence or absence of enzyme inhibitors. For this purpose, the extracts were stored for 6 months either in frozen state at −20° C or in lyophilized state at −20° C, 4° C, or room temperature and were characterized by SDS‐PAGE, immunoblot, ELISA inhibition, and prick test. The highest stability revealed the extracts that were prepared by precipitation in the organic solvent in the presence of enzyme inhibitors, lyophilized, and stored at −20° C. For storage of extract solutions at 4° C, PBS/glycerol and cysteine/sodium citrate/glycerol were found to be the most effective stabilizing additives.

Indoor determinants of Der p 1 and Der f 1 concentrations in house dust are different

Exposure to mite allergens is a major risk factor for sensitization and the development of asthma. Der p 1 and Der f 1 content in homes and probably the proportion of both antigens is highly variable even in the same geographical area.

Katacalcin and calcitonin immunoreactivity in different types of leukocytes indicate intracellular procalcitonin content

PURPOSE Procalcitonin is a new marker of severity of nonviral, in particular, bacterial infections. In respect of sepsis its site of production remains unknown. This study was carried out to determine whether subsets of human leukocytes contain procalcitonin. MATERIALS AND METHODS Blood samples of 17 patients who had demonstrated various degrees of serum procalcitonin levels on the day before evaluation were analyzed for serum procalcitonin by immunoluminometry and for intracellular reaction of monocytes, granulocytes, B-, and T-lymphocytes against katacalcin- or calcitonin-sensitive antibodies. Katacalcin and calcitonin are part of the procalcitonin molecule. Associations of these reactions with serum procalcitonin levels as well as differences between groups with a normal or elevated serum level were analyzed. RESULTS Intracellular antibody reaction against katacalcin was demonstrated in all cell types. We also found a lower rate of intracellular antibody reaction against calcitonin. Associations of serum procalcitonin with the two antibody reactions were demonstrated. Differences in intracellular reactions in the group with elevated serum procalcitonin were seen with both antibodies compared with a normal control. CONCLUSION Intracellular antibody reaction against katacalcin supports the notion that various types of leukocytes contain procalcitonin.

Tree‐pollen allergy is efficiently treated by short‐term immunotherapy (STI) with seven preseasonal injections of molecular standardized allergens

The efficacy and tolerance of short‐term immunotherapy (STI) by seven preseasonal injections of tree‐pollen allergens (ALK7 FrUhbltihermischung®) was investigated in a double‐blind, placebo‐controlled, multicenter study with 111 rhinoconjunctivitis patients. Nasal and bronchial symptoms simultaneously analyzed, and nasal symptoms as a single end point, but not the overall score of nasal, bronchial, and conjunctival symptoms, showed a significantly lower increase with STI during birch‐pollen exposure (both P= 0.033, n= 105, Mann‐Whitney U‐test). However, a selective analysis with patients from centers with high recruitment figures (nS10 patients, n=29 STI, n=32 placebo) showed a significantly lower increase of nasal, bronchial, and overall symptom score (STI 11.0 vs placebo 18.0, P=0.001, U‐test). STI had equidirected effects on conjunctival, nasal, and bronchial symptoms analyzed as multiple end points, although conjunctival symptoms were not significantly different as a single end point. The seasonal increase in drug use was reduced by 62% in the STI group compared with placebo (P=0.032, Mest), Specific IgG4 increased only after STI (P<0,001); IgE was not significantly different. Eosinophil cationic protein remained unchanged with STI, but significantly increased with placebo in the pollen season (P=Qm3). STI was well tolerated. In conclusion, STI was shown to be efficacious and safe for the treatment of patients with tree‐pollen rhinoconjunctivitis.

Optimization of apple allergen preparation for in vivo and in vitro diagnostics

The aim of our investigation was to obtain a well‐characterized active apple extract suitable for both in vivo and in vitro diagnostics by a technically simple method. For this purpose, apple extracts were prepared by homogenization in potassium phosphate buffer or by precipitation in organic solvents and resolubilization in potassium phosphate buffer in the presence or in the absence of enzyme inhibitors. These extracts were comparatively investigated by means of SDS‐PAGE, two‐dimensional electrophoresis, immunoblotting, RAST inhibition, and prick test. The in vitro investigations indicated that extracts prepared by precipitation in organic solvents (diacetone alcohol) at ‐20°C have a higher allergen activity than those prepared by extraction in aqueous solutions. From the in vivo tests (prick test), it was concluded that application of inhibitors of cytoplasmic enzymes (phenol oxidases, peroxidases, proteases) already during extraction is an essential precondition for active prick test solutions. Correspondingly, the extract obtained by solvent precipitation in the presence of enzyme inhibitors appeared to be most suitable for clinical application.

Purification and Characterization of the Major Allergen from Apple and Its Allergenic Cross-Reactivity with Bet v 1

The major allergen from apple extract was concentrated by anion exchange chromatography and further purified by reverse-phase HPLC. A distinct peak with a high degree of homogeneity was obtained. The isolated protein has a MW of 18 kD and specific IgE-binding capacity (immunoblotting, IgE-binding inhibition). N-terminal amino acid analyses of the allergen allowed 37 cleavages and showed 67.6% identity to Bet v 1, the major allergen of birch pollen. Enzyme immunoassay inhibition studies with serum of birch/apple-allergic patients showed that besides cross-reacting structures to Bet v 1, apple-specific IgE antibodies could exist. Monoclonal antibodies (mAbs) were raised against the 18-kD allergen from apple and characterized by means of immunoblotting and ELISA. Only three of the eight studied mAbs reacted with Bet v 1.

Detection of crossreactive determinants in grass pollen extracts using monoclonal antibodies against group IV and group V allergens.

Monoclonal antibodies (MoAb) to defined allergens of P. pratense were raised. Five of them were selected for detailed studies by means of immunoblotting after SDS–PAGE and IEF of extracts from P. pratense and L. perenne. Three antibodies (1D11, 3B2, 2D1) recognize structures with mol. wt of 29 and 34 kD and pI of 4.4‐7.7, corresponding to group V allergens. Two other MoAbs (2D8, 3C4) are directed against strong basic structures with a mol. wt of 50 kD and pI of 7.7‐9.9 according to group IV allergens. The specificity of antibodies was supported by direct ELISA with purified group V and IV allergens. The isolated allergens were characterized before by SDS‐PAGE and CIE and that allergenicity was detected with sera of patients with allergic rhinitis. Using our selected MoAbs crossreactive epitopes on group V and IV allergens have been excluded. Our antibodies have been used to detect crossreactivity in 14 grass pollen extracts. The evaluation of the pollen extracts has been performed by enzyme immunoassay (EIA) inhibition. One MoAb (3C4) is able to recognize group IV allergens in all grass species analysed whereas the MoAb 2D8 seems to identify group IV structures in selected grasses only. Binding to conserved structures of group V has been proved for MoAb 1D11. Other group V specific MoAbs (2D1, 3B2) identify similar, however incomplete, spectra. These results have been confirmed also by the dot immunobinding assay.

Analysis of Human T Cell Clones Reactive with Group V Grass Pollen Allergens

Twenty-seven T cell clones (TCC) reactive with group V allergens of Phleum pratense (Phlp V) were established from the peripheral blood of 3 patients allergic to grass pollen. Twenty-four TCC showed the helper cell phenotype CD3+, CD4+, CD8-; the remaining clones were CD3+CD4-CD8+. T cell recognition of Phlp V was exclusively HLA-DR restricted. Many of the Phlp V reactive TCC (19 of 27; 70%) were stimulated additionally by other group V allergens isolated from Lolium perenne, Poa pratensis, and Dactylis glomerata. These data indicate the existence of cross-reacting T cell epitopes among group V allergens of different grasses. The Phlp V triggered cytokine production demonstrated in 13 out of 24 CD4+ TCC a Th2-like pattern (high interleukin 4/gamma-interferon ratios) suggesting group V allergens as important targets of grass pollen specific IgE.