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Biomedical subjects

H Haas

Publications and source records attributed to H Haas.

At least 73 records · Page 4Linked to original sources
PubMed1997

Properties of multinucleated giant cells in a new in vitro model for human granuloma formation.

Multinucleated giant cells (MGCs) are a key feature of granulomas. They have been studied with respect to the mechanism and regulation of their formation, but the function of these cells still remains elusive. A new method for the in vitro generation of granulomas was developed and characterized in which L3 larvae of Nippostrongylus brasiliensis, as a target for the cellular response, were co-incubated with human mononuclear blood cells. The development of epithelioid cells and MGCs was observed and single isolated MGCs were analysed by the reverse transcriptase polymerase chain reaction method. The presence of tumour necrosis factor alpha (TNF alpha), interleukin-1 beta (IL-1 beta), interleukin-6 (IL-6), and inducible nitric oxide synthase (iNOS) transcripts in MGCs was demonstrated. It is proposed that MGCs in the granuloma model may in part represent an active cellular constituent involved in granuloma formation and turnover and in the destruction of the irritant.

Animals↗
PubMed1997

A three-step procedure for the purification of human basophils from buffy coat blood.

OBJECTIVE AND DESIGN: We report a method for basophil purification from buffy coats, which avoids positive selection of the cells and gives rise to good purity, yield and functional integrity of the cells. SUBJECTS: Buffy coat blood (concentrated leukocyte fraction derived from 450 ml venipuncture donations) obtained from healthy blood donors (n = 51). METHODS: Basophils were enriched by a three-step process starting with Ficoll density centrifugation (1.6 +/- 0.1% basophil purity) followed by counter current centrifugal elutriation (17.7 +/- 1.4% basophil purity). The final stage involved negative selection using Dynal immunomagnetic beads directed against CD2, CD14, CD16 and CD19 positive cell contaminants. Functional integrity of which was assessed by comparing the anti-IgE or calcium ionophore A23187 induced histamine release from basophils obtained from each enrichment step. Furthermore, basophil morphology was investigated using light and electron microscopy. RESULTS: The final mean basophil purity of 67.3 +/- 1.4% with a yield of 3.5 +/- 0.5 x 10(6) basophils and a recovery of 21.8 +/- 2.4% was achieved. Net histamine release from basophils stimulated with optimal concentrations of anti-human IgE was 39.1 +/- 6.5% after Ficoll centrifugation, 41.6 +/- 7.7% following elutriation and 35.7 +/- 6.8% from the final purified fraction. Additionally, basophils enriched with our method showed intact morphology by electron microscopy and were functionally active to non-immunological stimulation. CONCLUSIONS: These results compare favourably with previous studies, which have often required the use of positive selection via the Fc epsilon RI receptor, which may result in cell degranulation, or cell sorting, which cannot be applied to large cell numbers. Our method provides a reproducible technique for basophil enrichment when large numbers of functionally intact basophils are required.

Antigen-Antibody Reactions↗
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

Regulated system for heterologous gene expression in Penicillium chrysogenum.

A system for regulated heterologous gene expression in the filamentous fungus Penicillium chrysogenum was established. This is the first heterologous expression system to be developed for this organism. Expression of a recombinant fungal xylanase gene (xylp) and the cDNA for the human tear lipocalin (LCNI) was achieved by placing the encoding sequences under the control of the repressible acid phosphatase gene (phoA) promoter of P. chrysogenum. Secreted recombinant proteins were detected in the growth media of transformed P. chrysogenum cells by means of bioassays, zymogramography, and Western blotting. Levels of transcription and amounts of recombinant proteins secreted varied among transformants, mainly due to the copy number and the integration site of the expression vector on the fungal chromosome.

Carrier Proteins↗
PubMed1996

Reversibility of lower reproductive tract abnormalities in women with Schistosoma haematobium infection after treatment with praziquantel--an interim report.

Little is known whether and to what extent antiparasitic treatment cures female genital schistosomiasis (FGS). Using a standard protocol, of twenty-one women with FGS nine were re-examined at two to nine weeks after they had been treated with praziquantel at a single dose of 40 mg/kg. Symptoms related to pathology of the urinary tract and to a lesser extent of genital pathology subsided in most patients. Schistosoma haematobium ova were no longer detectable in urine of any of the patients post-treatment. Efficiency of chemotherapy against adult worms was confirmed by the disappearance of circulating anodic antigen (CAA) in serum. Sandy patches showed resolution in two of four cases after chemotherapy. Papillomata due to schistosomiasis alone improved, but persisted in mixed infection with human papilloma virus (HPV) or when HPV was the only underlying cause. In one patient ulcera could not be related with certainty to schistosomiasis at admission, but resolved after treatment with parziquantel. Leukoplakia (two cases) was not influenced by chemotherapy, or even increased during follow-up, regardless of whether ova had been detected or not. Although the follow-up period was rather short, time intervals were not standardized, and a relatively small number of patients was investigated, it could be shown that genital pathology due to sequestered S. haematobium ova is, at least partially, reversible already two to nine weeks after killing the adult worms by praziquantel. This is paralleled by a normalization of inflammatory immune responses detectable in histological sections and vaginal lavage.

Adolescent↗
PubMed1996

Sequence analysis and expression of the Penicillium chrysogenum nitrate reductase encoding gene (niaD).

The nitrate reductase gene (niaD) of the filamentous fungus Penicillium chrysogenum encodes a protein of 864 amino acids. The derived protein sequence shows 78% and 72% sequence identity to the corresponding Aspergillus niger and A. nidulans proteins, respectively. The coding region of the Penicillium gene is interrupted by six small introns, as deduced by comparison with the niaD sequences of A. niger and A. nidulans, whereby the positions of the introns are perfectly conserved between these three fungal genes. Northern blot analysis indicated a 2.8 kb transcript and showed that expression of this gene is controlled at the level of mRNA accumulation depending on both induction by nitrate and nitrogen metabolite derepression. Induction of transcription of niaD was found to be paralleled by expression of the major nitrogen regulatory gene nre.

Amino Acid Sequence↗
PubMed1996

Structural characterization of the exopolysaccharide produced by Lactobacillus acidophilus LMG9433.

The exopolysaccharide produced by Lactobacillus acidophilus LMG9433 in a semi-defined medium was found to be a charged heteropolymer, with a composition of D-glucose, D-galactose, D-glucuronic acid, and 2-acetamido-2-deoxy-D-glucose in molar ratios of 2:1:1:1. By means of methylation analysis, uronic acid degradation, de-N-acetylation/deamination, partial acid hydrolysis, and 1D/2D NMR studies the polysaccharide was demonstrated to consist of repeating units with the following structure: [Table: see text]

Carbohydrate Conformation↗
PubMed1996

Structural studies of the exopolysaccharide produced by Lactobacillus paracasei 34-1.

The exopolysaccharide produced by Lactobacillus paracasei 34-1 in a semi-defined medium was found to be a heteropolymer, composed of D-galactose, 2-acetamido-2-deoxy-D-galactose, and sn-glycerol 3-phosphate in molar ratios of 3:1:1. By means of deglycerophosphorylation, methylation analysis, and 1D/2D NMR studies (1H, 13C, and 31P) the polysaccharide was shown to consist of repeating units with the following structure: [formula: see text].

Acetylgalactosamine↗
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↗
PubMed1996

Purified human peripheral blood basophils release interleukin-13 and preformed interleukin-4 following immunological activation.

Recent studies have shown that human basophils, like mast cells, generate interleukin (IL)-4 following immunological activation and may thus participate in late-phase allergic and inflammatory processes. Here, we report the capacity of human basophils to release IL-13 within 24 h following stimulation with anti-IgE. Additionally, in 14 out of 31 experiments, we observed that basophils rapidly release performed IL-4 within 5-10 min, as well as newly generated IL-4, which was released 4 h following stimulation of the cells with anti-IgE. In contrast to the biphasic release of IL-4 from the cells, no preformed IL-13 was detected at earlier times (5-30 min). Preformed IL-4 and IL-4 and IL-13 generated de novo were also released after stimulation of the cells with IL-3; an enhanced production of these cytokines was observed using a combination of IL-3 and anti-IgE. We conclude from these data that, by releasing performed IL-4 and IL-4 and IL-13 generated de novo, human basophils may be centrally involved in the orchestration of allergic inflammation by providing a trigger to IL-4-mediated T helper 2 lymphocyte activation, B cell IgE switching, and increased vascular adhesion molecule expression.

Antibodies, Anti-Idiotypic↗
PubMed1996

Methyl mercury reduces voltage-activated currents of rat dorsal root ganglion neurons.

Methyl mercury (MeHg) is a widespread toxicant with major actions on the nervous system. Since the function of neurons depends on voltage gated ion channels, we examined the effects of micromolar concentrations of methyl mercury on voltage-activated calcium, potassium and sodium channel currents of cultured rat dorsal root ganglion (DRG) neurons. The cells, which were obtained from 2-4 day old rat pups, were whole-cell patch-clamped. Currents were separated by selective intra- and extracellular solutions as well as specific depolarizing voltage steps. We did not distinguish between different calcium, potassium or sodium channel subtypes. All three types of voltage-activated currents were irreversibly reduced by MeHg in a concentration dependent manner. Voltage-activated calcium and potassium channel currents were more sensitive to MeHg (Calcium: IC50 = 2.6 +/- 0.4 microM; Potassium: IC50 = 2.2 +/- 0.3 microM) than voltage-activated sodium channels (IC50 = 12.3 +/- 2.0 microM). The Hill coefficients for the reduction of the currents were calculated as approximately 1 for calcium and potassium channel currents and as 1.7 for sodium currents. In the cases of the voltage-activated calcium and sodium channel currents the reduction was clearly use dependent. Higher concentrations of MeHg (> or = 5 microM) resulted in a biphasic change in the holding membrane current at the potential of -80 mV in approximately 25% of the cases.

Animals↗
PubMed1996

[Non-specific tuberculin reactivity due to sensitization to non-tuberculous mycobacteria (NTM) in children not vaccinated with BCG. Diagnostic value of a comparison of intradermal tests with tuberculin and NTM antigens].

Sensitisation to non tuberculous mycobacteria (NTM) may provoke a non specific tuberculin reaction and can cause problems in the interpretation of tuberculin tests in subjects who have not been vaccinated with BCG. The practice of testing for antigens to NTM (sensitins) should be useful for pointing to the right diagnosis. Our study was carried out on 24 asymptomatic children aged between 11 months and 8 years 9 months; these children were considered to be sensitive to NTM based on comparative cutaneous tests (reactions to sensitins > or = 5 mm and > or = 3 mm vs tuberculin) and were selected from a group of 53 children who had either initially achieved a positive tuberculin reaction or who had undergone conversion on subsequent prevaccination testing. The tests were carried out with tuberculin RT 23, tuberculin Merieux and sensitins from M. avium and M. intracellulare. They had been repeated two to four months later in cases of a response to one of the different reactants. At the first examination the tuberculin reactions were most often weak, the Merieux tuberculin induced a stronger response than the RT23 tuberculin. A dominant response for one or both sensitins was present in 18 cases. The tuberculin reactions became weaker whilst the sensitins remained relatively stable. The difference became significant in the six remaining cases. Although tests for tuberculin and sensitins have some diagnostic value which is necessarily imprecise, comparative analysis of the cutaneous reactions to the different antigens may contribute to avoid an erroneous interpretation for tuberculin allergy in asymptomatic young children without any risk factors for tuberculous infection. It is important to identify those subjects having non specific tuberculin reactivity induced by sensitisation to NTM in order to avoid non justified chemoprophylaxis and also to be able to vaccinate these children with BCG.

Antigens↗
PubMed1995

Cloning, structural organization and regulation of expression of the Penicillium chrysogenum paf gene encoding an abundantly secreted protein with antifungal activity.

An abundantly secreted, highly basic 12-kDa protein (PAF) was purified from the culture medium of Penicillium chrysogenum (Pc). Based on the N-terminal amino acid (aa) sequence of the protein, an oligodeoxyribonucleotide probe was derived and used for amplification of the encoding cDNA by PCR. This cDNA fragment encodes a Cys-rich preproprotein of 92 aa which appears to be processed to a mature product of 55 aa. The deduced aa sequence of the preproprotein reveals 42.6% identity to an antifungal protein (AFP) of Aspergillus giganteus. Agar diffusion tests confirmed that the Pc protein exhibits antifungal activity. In order to investigate the promoter region and the structural organization of the paf gene, a genomic 6-kb fragment was isolated and partially sequenced. Comparison of the nucleotide sequence of the genomic fragment and the cDNA clone revealed the presence of a coding region of 279 bp which is interrupted by two introns of 76 and 68 bp in length. In the promoter region, a typical TATA box, a motif resembling the fungal carbon catabolite repression element, as well as several putative GATA factor binding motifs, were found. Northern blot analysis indicated that the regulation of paf expression occurs at the level of mRNA transcription and is under control of carbon catabolite and nitrogen metabolite repression regulatory circuits.

Amino Acid Sequence↗
PubMed1995

The structure of the exopolysaccharide produced by Lactobacillus helveticus 766.

The exopolysaccharide produced by Lactobacillus helveticus 766 in skimmed milk was found to be composed of D-glucose and D-galactose in a molar ratio of 2:1. Linkage analysis and 1D/2D NMR studies (1H and 13C) performed on the native polysaccharide, and on oligosaccharides obtained from a partial acid hydrolysate, showed the polysaccharide to consist of hexasaccharide repeating units with the following structure: [formula: see text]

Carbohydrate Sequence↗
PubMed1995

Determination of the structure of the exopolysaccharide produced by Lactobacillus sake 0-1.

The exopolysaccharide produced by Lactobacillus sake 0-1 in a semi-defined medium was found to have an average molecular mass of 6 x 10(6) Da and a composition of D-glucose, L-rhamnose, and sn-glycerol 3-phosphate (3:2:1). The polysaccharide is partially O-acetylated. By means of partial acid hydrolysis, O-deacetylation, deglycerophosphorylation, methylation analysis, and 1D/2D NMR (1H, 13C, and 31P) studies the polysaccharide was shown to be composed of repeating units with the following structure: [formula: see text]

Carbohydrate Sequence↗