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Biomedical subjects

M Schlaak

Publications and source records attributed to M Schlaak.

At least 109 records · Page 6Linked to original sources
PubMed1995

In vitro cultivation of third-stage larvae of Brugia malayi to the young adult stage.

The in vitro cultivation of the filarial nematode Brugia malayi from the infective stage to the fourth and the young adult stage is described. Different culture conditions including cell-free systems and co-culture with different human lymphatic cell lines were compared. Cell-free systems reported by others to promote the in vitro development of the parasites to the adult stage failed to work, i.e. the parasite development stopped at the L4 stage and the larvae died after approximately 3 weeks. Cocultivation with each of the cell lines used enhanced the survival of the parasites. The best results were obtained employing the human T cell leukemia line Jurkat and human dermal fibroblasts as feeder cells in RPMI 1640 supplemented with 10% heat-inactivated human serum. This culture system allowed the cultivation of B. malayi for more than 7 weeks with an average growth of the larvae by factor 6.4 (0.77 +/- 0.035 cm) and a maximum growth by factor 10 (1.2 cm). 69% of the initially cultivated larvae (which corresponded to 100% larvae alive at that time) reached the fourth larval stage after 14 days, and 2.6% of the initially cultivated larvae (which corresponded to 17% of the parasites alive at that day) had reached the young adult stage by day 37 of culture. Parasites remained alive up to 52 days. During the first four weeks of culture, both the length and the periods of moulting of the in vitro cultivated filariae closely resembled those observed with B. malayi in vivo in rodent hosts.

Aedes↗
PubMed1994

Major allergen Phl p Va (timothy grass) bears at least two different IgE-reactive epitopes.

It is established that most grass pollen allergens consist of several isoforms of which the function is mainly still unknown. A number of these allergens belonging to group V have been cloned, sequenced, and expressed. Antigenic sites and IgE-reactive epitopes of the major allergen Phl p Va, are unknown. We have identified the complete cDNA sequence of a Phl p Va isoallergen by immunoscreening of a timothy grass pollen cDNA library and mixed oligonucleotide primed amplification of N-terminal cDNA. Additionally, we found an incomplete isoallergenic cDNA clone of the same protein. Immunoreactivity of the fusion proteins with patients' sera and monoclonal antibodies showed that the clones represent group Va allergens. Comparison of deduced amino acid sequences with published sequences of Lol p V and Poa p IX revealed a homology of 81.1% and 86.9%, respectively. With affinity-purified IgE antibodies recognizing the recombinant fusion protein, we can demonstrate the existence of a common group V IgE-reactive epitope. By construction of both an N-terminal and a C-terminal peptide of the complete Phl p Va and cross-inhibition, we identified at least two different IgE epitopes. Eleven patients showed variable IgE immunoreactivities to both IgE-reactive epitopes.

Allergens↗
PubMed1994

Epitope analysis of isoforms of the major allergen Phl p V by fingerprinting and microsequencing.

The major allergen of timothy grass pollen (Phleum pratense), designated as Phl p V, consists of isoallergenic components of 38 and 32 kDa with pl values of 5.2-7.5 and 4.8-5.9, respectively. The different-sized proteins reveal similarities in IgE reactivity, N-terminal sequence and protein staining. For epitope analysis of these allergens a combination of enzymatic cleavage of electrophoretically separated proteins and immunoblotting techniques with subsequent N-terminal sequencing was performed. After isolation of the components from two-dimensional PAGE gels, proteins were enzymatically cleaved and separated by SDS-PAGE. By endoproteinase Glu-C cleavage six IgE-reactive fragments of each 32 kDa protein and three of each 38 kDa allergen were obtained. Microsequencing of the fragments revealed internal sequences that did not show any similarities between the different-sized allergens. Therefore, we assume only slight structural variations among allergens of similar sizes, whereas the 32 and 38 kDa proteins reveal great differences.

Allergens↗
PubMed1994

Analysis of IgG subclass and IgE response in allergic disease caused by Aspergillus fumigatus by immunoblotting techniques.

Analysis of sera from patients suffering from allergic asthma, extrinsic allergic alveolitis (EAA) due to Aspergillus fumigatus or allergic bronchopulmonary aspergillosis (ABPA) by means of IEF immunoprint and Western blot showed characteristic IgG subclasses and IgE responses. Asthmatic patients revealed specific IgE antibodies. IgG subclass reactivities in asthmatics and healthy blood donors were weak and uncharacteristic. Major allergens recognized by asthmatic patients have isoelectric points (pI) of pI 4.4 and 4.8 and molecular weights of 20, 29, 34, 38 and 45 kD. Patients suffering from EAA typically demonstrated strong IgG subclass reactivities with an emphasis to IgG1 and IgG2, while no specific IgE was found. Various major antigens could be identified. Reaction to a pI 6.0 antigen seemed to be characteristic for EAA. ABPA patients showed strong reactivities of all IgG subclasses and of IgE. An intense IgG4 reaction appeared as a typical marker for ABPA. Several major antigens and allergens could be determined. Especially the IgG4 reactivities to proteins with pI 4.6, 5.0, 5.2, 5.4 and 6.5 and molecular weights of 14, 20, 29, 34, 38 and 45 kD seemed to be a characteristic pattern in ABPA. Furthermore, IgE antibodies to culture filtrate components of 34 and 38 kD appeared to be typical in ABPA.

Alveolitis, Extrinsic Allergic↗
PubMed1994

[The Th1/Th2 concept--its importance for regulation of IgE].

T helper type 2 (TH2) cells are a major source of interleukin- (IL-)4 which plays a critical role in the induction of the IgE synthesis. Their counterplayers, the T helper type 1 (TH1) cells produce interferon- (IFN-)gamma which inhibits the IL-4-induced IgE synthesis. Thus, the TH2/TH1 ratio plays an important role in the regulation of the IgE synthesis. Moreover, IL-4 seems to be the crucial factor controlling the switch of T helper precursor (THp) cells to the TH2 phenotype. However, the primary cellular source of IL-4 that skews an immune response towards the TH2 phenotype is not yet known. For future prophylactic and therapeutic interventions, e.g. in allergy, it will be crucial to know the type of cell involved and the factors that are activating this cell, i.e. the exact mechanism that is controlling the switch to the TH2 phenotype.

Animals↗
PubMed1993

Monocyte-mediated restoration of density-dependent growth control and induction of cell death in tumor cell populations.

It is shown in a serum-free system with human cells that during monocyte/tumor cell interactions signals are generated which restore the principles of density-dependent inhibition of growth in tumor cell populations. Tumor cells that fail to comply with these principles are induced to die. This cell death is an active process in target cells involving protein biosynthesis and tumor cell-derived signals.

Cell Death↗
PubMed1993

[Food allergy: identifying and characterizing peanut allergens with patient sera and monoclonal antibodies].

The purpose of this study is to improve the diagnosis of peanut allergy. In order to standardize test substances for in vivo and in vitro diagnostic, the type I allergy-associated single components of peanuts have been identified and characterized with the aid of patients' sera and monoclonal antibodies. For allergen detection IEF-immunoprint-, SDS-PAGE-immunoblot- and 2-D electrophoresis-techniques have been used. A comparison of control sera and patients' sera showed that both contained peanut specific IgG-, IgA- and IgM-antibodies. In contrast, peanut-specific IgE-antibodies were only detectable with patients' sera. In IEF-immunoprint the most intensive IgE-bindings showed up in pl-range from pH 5.5 to 7.5. In SDS-PAGE-immunoblot major allergens could be identified at molecular weight ranges of 17, 30, 48 to 66 and 116 kD. Raising monoclonal antibodies against IgE-reactive components from peanut extract resulted in eight antibody-producing hybridoma cell lines, named PN-a to PN-h. ELISA-inhibition tests revealed common epitopes of monoclonal antibodies and patients' antibodies. Moreover, the monoclonal antibodies were tested to see whether they can be used for detection of hidden peanut allergens.

Allergens↗
PubMed1993

Characterization of grass group I allergens in timothy grass pollen.

Using Phl p V-depleted timothy grass pollen extract (Phleum pratense) as immunogen, we obtained a monoclonal antibody, QG 4, which recognized proteins of 33, 35, and 37 kd as determined by Western blotting. The antibody cross-reacted with pollen proteins of other grass species in the molecular weight range of 30 to 37 kd. By means of two-dimensional polyacrylamide gel electrophoresis blot of timothy grass pollen extract, we demonstrated at least seven protein spots: two of 37 kd with isoelectric points of 6.4 and 6.6; four of 35 kd with isoelectric points of 6.5, 6.8, 7.1, and 7.3; and one of 33 kd with an isoelectric point of 8.5. These protein spots were also detected by patients' pooled serum. Microsequencing of the 20 N-terminal amino acid residues revealed structures with sequence identities up to 90% to the well-established allergen, Lol p I of ryegrass (Lolium perenne). Therefore we assume that the monoclonal antibody QG 4 recognized the corresponding allergen Phl p I in timothy grass pollen.

Allergens↗
PubMed1993

Inhibition of endogenous TNF formation by pentoxifylline.

During the last decade cytokines were recognized as focal components in acute and chronic inflammatory processes. The growing knowledge about these agents stimulated efforts to pharmacologically control their synthesis and action in clinical situations. Various rational approaches to these issues including selective antibodies or receptor antagonists are at present under clinical investigation. Recently, in our institute evidence was raised that pentoxifylline is able to suppress the synthesis of tumor necrosis factor-alpha in cell cultures, and in vivo, and to protect experimental animals against endotoxin shock. Extended studies in human experimental endotoxemia showed that pentoxifylline decreased circulating TNF without affecting endogenous formation of interleukins. The potency of this drug to interfere with TNF synthesis could also be demonstrated in cases of acute and chronic cytokine release-syndromes such as OKT3 first-dose reaction and severe pulmonary tuberculosis, respectively. In conclusion, we suggest that pentoxifylline may improve therapeutic strategies in septic syndrome and other diseases in which TNF represents a causative pathophysiological factor.

Animals↗
PubMed1993

Successful technique for the selective production of monoclonal antibodies against a major allergenic component in timothy pollen extract.

Monoclonal antibodies (MAbs) were selectively raised against a major allergenic component of 38 kD in timothy grass pollen (Phleum pratense). We used a special prefractionating technique to isolate the 38 kD allergen, because immunizations with crude pollen extract had resulted in a wide variety of antibodies against other determinants, and cross-reactions between different-sized proteins occurred. Pollen extract was separated by Western blotting. Strips of the nitrocellulose membrane containing the allergen were excised, dispersed into allergen bearing particles, and used for immunization of BALB/c mice. Five MAbs were obtained that reacted with the 38 kD allergen. Four of these antibodies (BF 1, DC 9, EB 4, GE 2) exclusively detected the 38 kD protein, while one MAb (EB 6) additionally bound to a 32 kD allergen. Epitope mapping with the 3 IgG 1 antibodies (BF 1, EB 6, DC 9) was performed by ELISA inhibition tests using the purified 38 kD component. The antibodies did not interfere with each other. This confirms that they bind to different sites of the 38 kD molecule. Only a weak inhibitory effect of the MAbs on the binding of patient's IgE was determined, we suppose that the MAbs do not bind to IgE reactive epitopes. For standardization and further characterization of the 38 kD protein by peptide mapping the MAbs will be useful tools.

Allergens↗
PubMed1993

Comparison of four grass pollen species concerning their allergens of grass group V by 2D immunoblotting and microsequencing.

The identification and characterization of allergenic components is a vital step towards improving diagnosis and therapy. Members of the grass family (Poaceae) reveal a high cross-reactivity among each other caused by the close phylogenetical relationship. In order to investigate the variability between allergenic components, we studied the allergen grass group V, one of the major allergens. Pollen extracts of 4 different tribes (timothy grass (Phleum pratense)--Agrostidae, perennial rye grass (Lolium perenne)--Festuceae, meadow velvet (Holcus lanatus)--Aveneae, and rye (Secale cereale)--Triticeae) of the Festucoideae subfamily were separated by 2D PAGE and investigated by immunoblotting using patients' poolserum and monoclonal antibodies (raised against group V allergens of timothy grass pollen). The antibodies identify different allergens in the four grass species. The components vary from 30-50 kDa and pI 4.8-7.0. The eight NH2-terminal amino acids were determined and indicated high similarities between the different components. These results cast doubt on the suitability of classifying allergens into groups based only on their molecular mass, isoelectric point and N-terminal sequence analysis. It suggests to classify allergens according to their IgE-reactive epitopes.

Allergens↗
PubMed1993

[Characterization of allergens of timothy grass (Phleum pratense)].

By means of 2-D electrophoresis and subsequent immunoblotting allergens of timothy grass pollen are separated as single components. Via N-terminal sequencing, it is possible to classify these components as isoallergens of defined groups according to the allergen nomenclature. We are able to identify the allergens as Phl p I, Phl p IV, Phl p V and Phl p VI.

Allergens↗
PubMed1993

[Circadian rhythm in cytokines].

The well-known human circadian rhythm (CR) in body temperature may be due to CR in endogenous pyrogens such as interleukin-1 (IL-1), interleukin-6 (IL-6), and tumor necrosis factor-alpha (TNF). We, therefore, investigated the daily variation in spontaneous and LPS-induced monokine release by peripheral blood cells (whole blood assay) in 10 healthy volunteers. Spontaneous release of monokines (IL-1, IL-6, TNF) showed a simultaneous significant peak at noon, whereas LPS-induced release of IL-1 and IL-6 was strictly correlated with the CR in body temperature with its significant maximum at 8.00 p.m. Since LPS-induced release of TNF showed a significant peak at 4.00 a.m., interleukins and TNF seem to be independently regulated. CR in IL-1 and IL-6 release are suggested to be responsible for the CR in body temperature.

Adult↗