Cloning, sequencing and immunological characterization of Dac g 3, a major allergen from Dactylis glomerata pollen.

Preliminary work showed that a 14-kDa allergen with a pI of 9 was recognized by more than 60% of sera from Dactylis glomerata (Dac g) pollen-allergic individuals. The N-terminal amino acid sequence of this Dac g allergen was determined by Edman degradation and compared with that of Lol p 3, a major allergen of Lolium perenne. A sequence identity of 65% was found, suggesting that the Dac g allergen could be the homologue of Lol p 3 and therefore named Dac g 3. We report the cloning and sequence analysis of a cDNA encoding the Dac g 3 pollen allergen. The recombinant allergen (rDac g 3) expressed in plasmid vector pGEX-2T contained IgE-reactive epitopes found in its natural counterpart, and induced histamine release from basophils of Dac g-allergic individuals, confirming that the recombinant protein has biological properties similar to the pollen extracted allergen. Computer analyses showed that, in spite of a high degree of sequence homology, even closely related allergens such as Dac g 3 and Lol p 3 have dissimilar predictive secondary structures and potential different antigenicity. Because it possesses the properties of the native counterpart, rDac g 3 could be a relevant tool for molecular studies in allergy.

Identification of allergenic epitopes on Der f I, a major allergen of Dermatophagoides farinae, using monoclonal antibodies.

The antigenic and allergenic structure of Der f I, a major allergen of the house dust mite Dermatophagoides farinae (Df) was investigated by means of a panel of 11 selected monoclonal antibodies (mAb) obtained from BALB/c mice immunized with purified Der f I. The species specificity of these mAb, tested with Der f I and Der p I--the homologous allergen from Dermatophagoides pteronyssinus--was generally restricted to Der f I since 10 out of 11 mAb reacted only with this allergen. Epitope specificity of the mAb was determined by both competitive inhibition and sandwich ELISA experiments. The results indicated the presence of at least four non-overlapping, non-repeated antigenic sites on Der f I, which were recognized by one or several mAb (sites A, B, C and D). Comparative epitope specificity studies between human IgE antibodies and mice mAb were performed, on sera and basophils of Df sensitive patients, using different inhibition assays (ELISA and histamine release experiments). The degree of inhibition varied between the patients and upon the assay design. Most of the mAb tested were found to significantly inhibit the binding of human IgE to Der f I (p less than 0.01) when compared with Der p I specific mAb as a control. The mAb reacting with site A was found to be the most potent inhibitor, presenting a mean inhibition of up to 56% in ELISA as well as in histamine release experiments. The results show that both human IgE antibodies and mAb can be directed against identical or closely related epitopes of Der f I. Therefore anti-Der f I mAb constitute immunologic probes in further allergenic epitope and peptide analysis of this major mite allergen.

Molecular characterization of human IgG monoclonal antibodies specific for the major birch pollen allergen Bet v 1. Anti-allergen IgG can enhance the anaphylactic reaction.

We report the molecular characterization of five human monoclonal antibodies, BAB1-5 (BAB1: IgG(1); BAB4: IgG(2); BAB2, 3, 5: IgG(4)), with specificity for the major birch pollen allergen, Bet v 1. BAB1-5 were obtained after immunotherapy and contained a high degree of somatic mutations indicative of an antigen-driven affinity maturation process. While BAB1 inhibited the binding of patients IgE to Bet v 1, BAB2 increased IgE recognition of Bet v 1, and, even as Escherichia coli-expressed Fab, augmented Bet v 1-induced immediate type skin reactions. The demonstration that IgG antibodies can enhance allergen-induced allergic reactions is likely to explain the unpredictability of specific immunotherapy.

Morphological development and neurochemical differentiation of cerebellar inhibitory interneurons in microexplant cultures.

The cerebellar cortex comprises a rather limited variety of interneurons, prominently among them inhibitory basket and stellate cells and Golgi neurons. To identify mechanisms subserving the positioning, morphogenesis, and neurochemical maturation of these inhibitory interneurons, we analyzed their development in primary microexplant cultures of the early postnatal cerebellar cortex. These provide a well-defined, patterned lattice within which the development of individual cells is readily accessible to experimental manipulation and observation. Pax-2-positive precursors of inhibitory interneurons were found to effectively segregate from granule cell perikarya. They emigrate from the core explant and avoid the vicinity of granule cells, which also emigrate and aggregate into small clusters around the explant proper. This contrasts with the behavior of Purkinje neurons, which remain within the explant proper. During migration, a subset of Pax-2-positive cells gradually acquires a GABAergic phenotype, and subsequently also expresses the type 2 metabotropic receptor for glutamate, or parvalbumin, markers for Golgi neurons and basket or stellate cells, respectively. The latter eventually orient their dendrites such that they take a preferentially perpendicular orientation relative to granule cell axons. Both the neurochemical maturation of basket/stellate cells and the specific orientation of their dendrites are independent of their continuous contact with radially oriented glia or Purkinje cell dendrites projecting from the core explant. Numbers of parvalbumin-positive basket/stellate cells and the prevalence of glutamate-positive neurites, which form a dense network preferentially within cell clusters containing granule cell perikarya and their dendrites, are subject to regulation by chronic depolarization. In contrast, brain-derived neurotrophic factor results in a drastic decrease of numbers of basket/stellate cells. These findings document that granule cell axons (parallel fibers) are the major determinant of basket/stellate cell dendritic orientation. They also show that the neurochemical maturation of cerebellar interneurons is sensitive to regulation by activity and neurotrophic factors.

In vitro inhibition, by loratadine and descarboxyethoxyloratadine, of histamine release from human basophils, and of histamine release and intracellular calcium fluxes in rat basophilic leukemia cells (RBL-2H3).

The effect of the H1-antihistamine drug loratadine and its active metabolite descarboxyethoxyloratadine upon histamine release was examined on anti-immunoglobulin E (IgE) triggered human basophils and 2,4-dinitrophenyl (DNP) triggered rat basophilic leukemia (RBL-2H3) cells. In both experimental systems, dose-dependent inhibition of histamine release was observed at descarboxyethoxyloratadine and loratadine doses above 2 and 7 microM, respectively. In the RBL-2H3 experimental system, inhibition by loratadine increased when the concentration of extracellular Ca2+ was reduced from 1.8 to 0.45 mM. We further investigated the effect of loratadine and descarboxyethoxyloratadine on the increase in cytosolic calcium concentration (Ca2+)i, an early step in biochemical events leading to exocytosis. The effect of these two drugs upon (Ca2+)i changes was measured using the fluorescent probe fura-2 loaded into RBL-2H3 cells passively sensitized with DNP-specific IgE. Both drugs inhibited, in a dose-dependent manner (2.5-25 microM), the (Ca2+)i rise induced by DNP-BSA challenge in sensitized RBL cells, a process observed in both the presence and absence of extracellular Ca2+. Loratadine also inhibited the Mn2+ influx into these cells, thus reflecting the Ca2+ influx. These results suggest that loratadine and descarboxyethoxyloratadine impair the increase in (Ca2+)i following cell activation by decreasing both the influx of extracellular Ca2+ and the release of Ca2+ from intracellular stores.

Prevention of puerperal lactation with Parlodel long-acting (Parlodel LA).

Thirty women who did not want to breast-feed postpartum were treated with Parlodel LA. Immediately after birth 50 mg Parlodel LA were injected i.m. by means of a double-chamber syringe. The aim was to investigate in an open study the clinical efficacy and tolerability of Parlodel LA in the prevention of postpartum lactation. The overall efficacy was very good in 29 women (96.6%) and good in 1 woman (3.4%). In 27 women no symptoms of tenderness, engorgement or milk secretion were found (90%). In two cases slight and moderate mammary pain were reported during a few days, engorgement in only one case during one day and in three cases a slight milk let-down was seen for 1-2 days. In all cases the breast symptoms ceased spontaneously and no rebound lactation occurred in any of the women treated with Parlodel LA. The general tolerability was very good in 29 cases (96.6%) and good in one case (3.4%). No adverse effects were reported in any case. No obvious changes in blood pressure were noticed after the injection. In general a slight decrease occurred in systolic and/or diastolic blood pressure post-partum well within the range of non-treated healthy postpartum women. There were also no signs of orthostatic hypotension or changes in pulse rate accompanying the slight decrease in blood pressure. The local tolerability at the injection site was very good in 23 women (76.6%). Seven had short-lasting pain, slight in 6 cases and moderate in 1. Slight redness was reported in one case.(ABSTRACT TRUNCATED AT 250 WORDS)

Voxel-based morphometry and voxel-based relaxometry in multiple system atrophy-a comparison between clinical subtypes and correlations with clinical parameters.

Multiple system atrophy (MSA) is a neurodegenerative disease affecting basal ganglia, brainstem, cerebellum, and intermediolateral cell columns of the spinal cord. Clinically, a cerebellar (MSA-C) and a parkinsonian variant of MSA (MSA-P) are distinguished. We used voxel-based morphometry (VBM) and voxel-based relaxometry (VBR) in 48 MSA patients (32 MSA-C, 16 MSA-P) and 46 controls. In MSA-C, VBM revealed gray matter loss in cerebellum, right thalamus, both putamina and several cortical regions including insular cortex. Gray matter loss in the cerebellum and insular cortex was correlated with disease duration and severity. There was white matter loss in the brainstem, which was correlated with disease duration and severity. VBR analysis in MSA-C showed decreased relaxation rate R2 in cerebellum, pontine brainstem and cortical regions including insular cortex. In MSA-P, gray matter was reduced in cerebellum, dorsal midbrain, both putamina, and several cortical regions including insular cortex. A correlation with disease duration and severity was detected only for some small cortical areas. Direct comparison of MSA-C and MSA-P showed differences only in infratentorial brain regions where structural abnormalities were more pronounced in MSA-C than in MSA-P. In MSA-C, there was a stronger reduction of gray matter in the basal parts of the cerebellum, of white matter in the brainstem and of the relaxation rate R2 in the cerebellum and brainstem.

[Intracellular histamine content in human basophils and individual variation in allergic persons during densensitization]. / Taux d'histamine intra cellulaire dans les basophiles humains et variabilité individuelle chez des sujets allergiques au cours de la désensibilisation

A positive correlation has been found to exist between the percentage of basophiles and the histamine content in a suspension of washed human leukocytes obtained from allergic and healthy donors. The mean value of histamine per basophile was 1,9 +/- 0,3 pg. The leukocyte histamine content was measured as a function of time in a non-allergic person and during the desensitization period of allergic patients.

Inhibitory effect of wheat germ agglutinin on mouse mast cell adhesion to fibronectin.

BACKGROUND: Mast cells play a critical role in allergic and inflammatory responses. The interactions between these cells and extracellular matrix components influence the distribution of mast cells in tissues and their biological responsiveness. It has been reported that the lectin wheat germ agglutinin (WGA) inhibits mast cell mediator release. We decided to investigate whether adhesion to fibronectin (FN), another mast cell function, which is upregulated following FcepsilonRI cross-linking, is also inhibited by WGA. METHODS: Mouse bone-marrow-derived mast cell line MCP5/L was used. For FcepsilonRI-dependent mast cell activation, MCP5/L cells were sensitized with mouse IgE antibodies. WGA was added to cell suspensions simultaneously with a challenging agent and, after an appropriate incubation period, beta-hexosaminidase release and adhesion to FN were determined. RESULTS: Both FcepsilonRI cross-linking-dependent mast cell adhesion to FN and mediator release were dose-dependently inhibited by WGA; however, the lectin concentrations required to induce maximum inhibition of adhesion were significantly lower. Furthermore, WGA inhibited phorbol-myristate-acetate- and A-23187-mediated mast cell adhesion to FN, i.e. processes that do not engage FcepsilonRI. The effect of WGA on FcepsilonRI-mediated secretion was reversed by GlcNAc. In contrast, combination of GlcNAc and NeuNAc or N, N'-diacetylchitobiose was required to reverse the inhibitory effect of WGA on mast cell adhesion. CONCLUSION: The characteristics of WGA-mediated inhibition of MCP5/L mast cell adhesion to FN suggest that mast cell integrins are targets of the inhibitory action of WGA and the sugar moieties on these receptors might be important for receptor function.

Production of a monoclonal antibody against a major allergen of Dactylis glomerata pollen (Dg1).

A mouse monoclonal antibody was produced against the most frequent allergen Ag Dg1ief, purified from the Dactylis glomerata grass pollen by isoelectric focusing. Using the immunoprint technique, this monoclonal antibody detected Ag Dg1ief in D. glomerata pollen and some cross-reacting molecules in four other grass pollens. This hybridoma was grown in ascitic fluid. An immunosorbent was made from it and, by affinity chromatography, was used to purify monoclonal Ag Dg1m from the pollen extract. Ag Dg1m (6,000 to 8,000 daltons) appeared to be 4 to 5 times smaller than Ag Dg1ief. It appeared as a weak allergen in passive cutaneous anaphylaxis in rats, but was more potent in triggering histamine release on human basophils from grass-pollen sensitive patients. These results suggest that Ag Dg1m could be a part of the Ag Dg1ief used to induce production of this monoclonal antibody.

[Indication for and results of the application of glycerol-preserved homologous split-thickness skin following burns]. / Indikation und Ergebnisse der Anwendung glycerol-konservierter homologer Spalthaut nach Verbrennungen.

Since 1983 the treatment of second-degree burns with glycerol-conserved allogenic split-thickness skin grafts is published. In case of actual problems with AIDS the gathering of split-thickness skin was modified and a new method for gaining split-thickness skin of organ-donors created to prevent the virus transfer. Because of these high security in contagiousness the skin graft transplantation with allogenic glycerol-conserved donor-skin in case of second-degree-burns was standardized in the Berlin Burn Center. To prevent the formation of granulation tissue after second-degree burns a tangential debridement of necrotic corium will be done in 1/10 mm thick slices on the third posttraumatic day and the defects covered with 1:1.5 mesh grafts extended. The results of 267 patients in 1988 and 1989, who were treated in accordance to this regimen, will be demonstrated. In 11% of cases the alloplastic skin graft in adults was not rejected 6 month after transplantation and in 2% of cases the depth of burn was not estimated properly and hypertrophic scars had to be excised and covered with autogenous split-thickness skin. General second-degree-burn healed after debridement and covering with allogeneic split-thickness skin without formation of hypertrophic scars.

Bactericidal activity of disinfectants on Listeria.

The bactericidal activity on Listeria spp. of nine disinfectants used in the food industry was studied by previously published methods. The disinfectants were diluted to the test concentration in sterile standard hard water. Various types of chemical agents were evaluated, including phenolic compounds, alcohols, quaternary ammonium compounds, surface-active agents, aldehydes and disochlorine tablets. The following strains isolated from cheese were studied: Listeria innocua, L. welshimeri, L. monocytogenes 1/2a, 1/2b, 1/2c and 4b. The results show that the listerias are not particularly resistant to disinfectants but the efficacy of some agents is affected by organic matter.

In vitro histamine release from human basophils triggered by a purified allergen from Dermatophagoides farinae: bimodal aspect of the dose response curve.

Histamine release experiments were performed over a 10(6)-fold range of allergen concentrations on basophils of patients sensitive to Dermatophagoides farinae. Two extracts of D. farinae were used, a partially purified one (Df 80d) containing several allergens and a highly purified component (Ag11). This histamine release patterns obtained with the complex allergen mixture Df 80d gave broad histamine release curves presenting an initial increase, followed by a decrease or a plateau, with occasionally a small dip. The Ag11-induced histamine release curves presented two peaks (bimodal pattern), separated by a more or less pronounced inhibition dip. No non-specific histamine releasing activity was observed with the basophils of two controls. The possible theoretical models for the bimodal histamine release patterns are discussed.

Cellular histamine release, specific and total serum IgE levels in hay fever patients and controls.

The amount of histamine released from blood leucocytes by allergen, the amount of allergen required to release 50% histamine and the total IgE and IgE specific for Dactylis glomerata are compared in nine hay fever patients and five control individuals. The variation with time of total IgE, of specific IgEe and of the percentage of histamine released at a fixed allergen concentration, corresponding to the cellular sensitivity, is shown for one control individual with positive in vitro and skin tests, but without clinical symptoms. For four patients with a low total IgE level the evolution of these parameters during a 1 year treatment period is presented.

Study of adenine aminohydrolase in the yeast, Schizosaccharomyces pombe.

Observation of the growth of some adenineless mutants of Schizosaccharomyces pombe on six substituted purine analogs leads to the hypothesis that an enzyme is present which catalyzes the conversion of these analogs into hypoxanthine. The enzyme adenase (adenine aminohydrolase, EC 3.5.4.2) has been found to be active in cell-free extracts of S. pombe. Results are reported which are in agreement with the hypothesis that this enzyme is responsible for the in vivo utilization of 6-chloropurine. This evidence comes mainly from a study of adenine aminohydrolase in two mutants selected for partial inability to grow on 6-chloropurine.

Monoclonal anti-human IgE: histamine release capacity and effect on in vitro sensitization of human basophils.

IgE-anti-IgE complexes were formed by preincubation of a human myeloma IgE with a monoclonal anti-human IgE antibody at different concentrations. Binding of IgE onto the anti-IgE inhibited the histamine release capacity of anti-IgE from basophils. The IgE cell-binding capacity was altered by the IgE/anti-IgE ratio in the preincubation buffer. Heating of the complexes gave a partial recovery of the anti-IgE degranulation capacity, suggesting that the monoclonal anti-IgE is specific for an epitope present on the D epsilon 2 domain.

Basophil sensitivity and reactivity to monoclonal anti-human IgE after in vitro sensitization with human myeloma IgE.

Leucocytes from human peripheral blood were acid eluted and sensitized in vitro with increasing concentrations of radiolabelled myeloma IgE. This sensitization step was performed with or without 30% IgE depleted serum. After the IgE binding, cells were washed and submitted to a challenge with monoclonal anti-IgE for the determination of the cellular sensitivity and reactivity in a histamine release assay. A sample of each of the sensitized cells was analyzed for its radioactivity and the number of basophils quantified, thus allowing the determination of the mean number of IgE molecules per basophil. Raising the IgE concentrations in the sensitization procedure led to an increase of the IgE on the basophil membrane, and to a concomitant elevation of the cell sensitivity. The presence of serum during the binding of IgE onto the cells lowers slightly the binding of IgE to the basophils but decreases strongly the cellular reactivity.

Relationship between specific circulating IgE, basophil cell-bound IgE and histamine release induced by purified allergens of Dermatophagoides farinae.

In this study we analysed the presence and proportions of IgE specific for Df 6 and Df 11, two purified allergens of Dermatophagoides farinae. The characterisation of serum IgE and cell-bound IgE for 3 D. farinae-sensitive patients were performed by CRIE, RAST and PRIST assays. Furthermore the basophils from these same patients were studied by histamine release assays in the presence of Df 6 and Df 11. All the individual patient's cell and serum IgE samples displayed the presence of IgE antibodies specific for Df 6 and Df 11, but the relative quantities of the IgE for these two specificities were characteristic of each patient. The ratios (Specific IgE for Df 11) : (Specific IgE for Df 6) (ratio 11 : 6) were similar in the serum and on the cells for an individual patient. As judged by histamine release assays, the basophil sensitivities towards Df 6 and Df 11 were very different from one atopic patient to another. Moreover cell sensitivities reflected the proportion of Df 6- and Df 11-specific IgE antibodies found in the serum and in the cell eluate.

Thermoinactivation of human IgE: antigenic and functional modifications.

The thermoinactivation kinetics of IgE were studied in experimental models revealing the antigenic properties and the basophil-sensitizing capacity of these immunoglobulins. A pool of human sera containing anti-Dactylis glomerata (Dg) IgE was heated from 5 min up to 4 hr at 56 degrees. The IgE antigenicity was tested by two polyclonal 125I-labelled anti-IgE antibodies; one anti-IgE was specific of the whole Fc epsilon region, while the other had a specificity restricted to the D epsilon 2 domain. Radioimmunoassays showed that the D epsilon 2 epitopes were more rapidly altered than the D epsilon 1 epitopes. The capacity of IgE to bind to basophil Fc epsilon receptors was assayed by passive sensitization experiments. Basophil sensitivity towards the Dg pollen extract was tested by histamine release experiments in the presence of this allergen. A progressive decrease in cell sensitivity was observed when IgE samples used for cell sensitization were heated for longer than 5 min. Thermoinactivation kinetics of IgE revealed an unexpected increase in the apparent quantity and biological activity of IgE heated for 5 min at 56 degrees. This fact could be due to auto-anti-IgE antibodies linked to the unheated IgE and which interfere with the biological activities of IgE and their quantification.

Immunoglobulin-G-dependent stimulation of guinea pig lung mast cells and macrophages.

Alveolar macrophages and mast cells isolated from guinea pig lung were passively sensitized with IgG1, IgG2, or serum obtained from guinea pigs actively sensitized with ovalbumin. The release of histamine by mast cells and of thromboxane A2 by alveolar macrophages upon ovalbumin challenge indicated that both antibodies and serum were capable of sensitizing these cells with similar effectiveness. Heating the serum at 56 degrees C for 4 h to inactivate IgE did not modify the antigen-dependent response of lung cells. These results suggest a predominant role for IgG in the allergic response of the guinea pig through the activation of different cell types such as lung mast cells and alveolar macrophages.