Zur Toxicität von Tetrahydrofuran

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Auto‐ and cross‐reactivity to thioredoxin allergens in allergic bronchopulmonary aspergillosis

Background: Thioredoxins are cross‐reactive allergens involved in the pathogenesis of atopic eczema and asthma. Cross‐reactivity to human thioredoxin can contribute to the exacerbation of severe atopic diseases. Methods: Human thioredoxin, Asp f28 and Asp f29, two thioredoxins of Aspergillus fumigatus, and thioredoxin of Malassezia sympodialis were cloned and produced as recombinant proteins. Allergenicity and cross‐reactivity to thioredoxins in allergic bronchopulmonary aspergillosis patients were assessed by enzyme‐linked immunosorbent assay (ELISA), inhibition ELISA, immunoblot analysis, proliferation assays and skin tests. Molecular homology modelling was used to identify conserved, surface‐exposed amino acids potentially involved in immunoglobulin E (IgE)‐binding. Results: All thioredoxins, including the human enzyme, bind IgE from patients with allergic bronchopulmonary aspergillosis and induce allergen‐specific proliferation in peripheral blood mononuclear cells and positive skin reactions in thioredoxin‐sensitized patients. Inhibition experiments showed that the thioredoxins are cross‐reactive indicating humoral immune responses based on molecular mimicry. To identify structural surface elements involved in cross‐reactivity, the three‐dimensional structures were modelled based on solved thioredoxin structures. Analysis of the molecular surfaces combined with sequence alignments allowed identification of conserved solvent exposed amino acids distantly located in the linear sequences which cluster to patches of continuous surface areas. The size of the surface areas conserved between human and fungal thioredoxins correlates well with the inhibitory potential of the molecules in inhibition ELISA indicating that the shared amino acids are involved in IgE‐binding. Conclusions: Conserved, solvent exposed residues shared between different thioredoxins cluster to continuous surface regions potentially forming cross‐reactive conformational B‐cell epitopes responsible for IgE‐mediated cross‐reactivity and autoreactivity.     

Isolation and characterisation of Ralstonia solanacearum strains from Solanaceae crops in Ethiopia

Eighty one isolates of Ralstonia solanacearum -like bacteria on triphenyl tetrazolium chloride (TTC) medium were collected from different Solanaceae crops (i.e. potato, tomato and pepper plants and potato tubers) at various sites in Ethiopia. Of these, 62 strains were identified as R. solanacearum based on their cultural characteristics on TTC medium, tomato pathogenicity bioassay, carbon source utilisation patterns and a specific PCR-based assay. By Hayward's classification method, based on carbon source utilisation, 19 of the 62 R. solanacearum strains were identified as biovar I and 43 strains were identified as biovar II. The biovar I strains exhibited a high growth rate at high temperatures (37 degrees C). Whereas the growth rate of biovar II strains was greatest at lower temperatures (22 degrees C). Biovar I strains had broader host range than biovar II strains, which were limited to potato, tomato, and eggplant. To our knowledge, this is the first report of R. solanacearum biovar I in Ethiopia. The existence of biovar I strains in Ethiopia raises concerns because they have a broader host range than biovar II strains.     

Characterization of 107 genomic DNA reference materials for CYP2D6, CYP2C19, CYP2C9, VKORC1, and UGT1A1: a GeT-RM and Association for Molecular Pathology collaborative project

Pharmacogenetic testing is becoming more common; however, very few quality control and other reference materials that cover alleles commonly included in such assays are currently available. To address these needs, the Centers for Disease Control and Prevention's Genetic Testing Reference Material Coordination Program, in collaboration with members of the pharmacogenetic testing community and the Coriell Cell Repositories, have characterized a panel of 107 genomic DNA reference materials for five loci (CYP2D6, CYP2C19, CYP2C9, VKORC1, and UGT1A1) that are commonly included in pharmacogenetic testing panels and proficiency testing surveys. Genomic DNA from publicly available cell lines was sent to volunteer laboratories for genotyping. Each sample was tested in three to six laboratories using a variety of commercially available or laboratory-developed platforms. The results were consistent among laboratories, with differences in allele assignments largely related to the manufacturer's assay design and variable nomenclature, especially for CYP2D6. The alleles included in the assay platforms varied, but most were identified in the set of 107 DNA samples. Nine additional pharmacogenetic loci (CYP4F2, EPHX1, ABCB1, HLAB, KIF6, CYP3A4, CYP3A5, TPMT, and DPD) were also tested. These samples are publicly available from Coriell and will be useful for quality assurance, proficiency testing, test development, and research.     

C-reactive protein selectively enhances the intracellular generation of reactive oxygen products by IgG-stimulated monocytes and neutrophils.

The acute phase protein, C-reactive protein (CRP), when heat-aggregated (Agg-CRP), potentiates immunoglobulin G (IgG) Fc receptor-mediated luminol-enhanced chemiluminescence (CL) in human monocytes and neutrophils. Luminol-CL is a sensitive measure of phagocyte respiratory burst activity; however, the nature of oxidative products contributing to the light emission and their site of generation remain incompletely defined. To more precisely describe the oxidative burst of monocytes and neutrophils to Agg-CRP, superoxide anion release was measured by cytochrome c reduction. In addition, the extracellular release of hydrogen peroxide was distinguished from hydrogen peroxide generation using a phenol red oxidation assay. Finally, a flow cytometric determination of dichlorofluorescein (DCFH) oxidation was employed as an index of intracellular peroxide production. Although Agg-CRP alone did not stimulate hydrogen peroxide generation by either monocytes or neutrophils, it significantly enhanced hydrogen peroxide generation in response to heat-aggregated IgG (Agg-IgG). In contrast, Agg-CRP did not enhance the extracellular release of either hydrogen peroxide or superoxide anion from Agg-IgG-stimulated cells. The capacity of Agg-CRP to enhance selectively intracellular oxidative product generation was confirmed when measuring DCFH oxidation in Agg-IgG-stimulated cells. To evaluate whether this selective enhancement of intracellular oxidative events could be attributed, at least in part, to a scavenging effect of Agg-CRP, a cell-free oxygen radical-generating system was employed. Agg-CRP did not significantly diminish the lucigenin-amplified CL response induced by the xanthine/xanthine oxidase reaction. These results indicate that although Agg-CRP enhances the intracellular generation of reactive oxygen intermediates by monocytes and neutrophils, extracellular release of those products is not influenced by cell interaction with Agg-CRP. It is tempting to speculate that CRP can selectively boost the microbicidal activities of monocytes and neutrophils within an inflammatory site by amplifying the intracellular generation of reactive oxygen products without increasing damage to surrounding normal tissues.     

Electron microscopic and antigenic studies of uncharacterized viruses. II. Evidence suggesting the placement of viruses in the familyBunyaviridae

Summary This is the second of three papers describing the use of electron microscopy and antigenic analyses intended to characterize and place in taxa more than 60 previously unclassified viruses. The first paper of the series describes the viruses we classified as provisional members of the familiesArenaviridae, Paramyxoviridae, orPoxviridae; another paper, published separately, discusses theRhabdoviridae. In this paper we report that electron microscopy provided sufficient evidence to place 17 of these viruses (Belem, Erve, Estero Real, Mojui dos Campos, Nyando, Odrenisrou, Okola, Pacora, Para, Santarem, Tanga, Telok Forest, Termeil, Thiafora, Thottapalayam, Wanowrie, and Yacaaba) in the familyBunyaviridae and to support the observations of others that Yogue and Kasokero viruses are members of this virus family. Subsequent antigenic studies allowed us to place some of these viruses in recognized antigenic groups and to establish new antigenic groups for others.     

TNFalpha decreases gluconeogenesis in hepatocytes isolated from 10-day-old rats

Gluconeogenesis decreases during septic shock, but its mechanism is not well known. Tumor necrosis factor alpha (TNF-alpha), which is a key cytokine in septic shock, can increase GLUT1 gene expression and glucose uptake in muscles and fatty tissues. TNF-alpha does not alter the metabolism of hepatocytes in which GLUT2 is the predominant glucose transporter. However, GLUT1 is the predominant glucose transporter in hepatocytes of 10-d-old rats. Thus, we hypothesized that TNF-alpha might increase glucose uptake and glycolysis in those cells, and decrease gluconeogenesis. In the present study, hepatocytes isolated from 10-d-old rats were incubated with TNF-alpha at the concentrations of 0, 0.98, 9.8, 98, and 980 ng/mL to evaluate TNF-alpha effects on gluconeogenesis and glucose uptake. TNF-alpha increased glucose uptake (41.1 +/- 8 to 114 +/- 21.4 micromol/10(6) cells at the concentration of 980 ng/mL of TNF-alpha) in a dose-dependent manner, and decreased gluconeogenesis (98.2 +/- 8.2 to 1.1 +/- 3.2 micromol/10(6) cells at the concentration of 980 ng/mL of TNF-alpha) in a dose-dependent manner. The decrease of glucokinase mRNA and GLUT1 mRNA abundance correlated with glucose uptake (r = 0.988 and 0.997, respectively), and the decrease of phosphoenolpyruvate carboxykinase mRNA abundance correlated with the decrease of gluconeogenesis (r = 0.972). The decrease of gluconeogenesis by TNF-alpha correlated with the increase of glucose uptake (r = -0.988). We concluded that TNF-alpha reciprocally suppressed gluconeogenesis in hepatocytes isolated from 10-d-old rats.     

Cutaneous malignant melanoma and neurofibromatosis type 1

Neurofibromatosis 1 (NF1) is a genetically transmitted disease occurring approximately once in 3000 live births and resulting from mutations of the NF1 gene that encodes a protein named neurofibromin, a negative regulator of the ras-dependent pathway. An excess of neoplasia especially tumours of neuroectodermal origin is classically observed. The occurrence of malignant melanoma in patients with NF1 has already been described in scattered clinical reports but little is known as to the characteristics of melanoma arising in NF1 patients. A multicentric retrospective study was conducted on a panel of French referring centres for a period of 13 years to identify patients with both melanoma and NF1. Patients with mucosal or ocular melanoma were excluded. The diagnosis of malignant melanoma was based on specific histology whereas NF1 was identified according to the criteria proposed by the NIH Consensus Conference. All patient fulfilling criteria for both melanoma and NF1 were investigated using a common procedure recording clinical and histological data along with prognostic factors for the two diseases. Eleven patients were identified with both diseases. The clinical pattern of NF1 was quite similar to the classical form of the disease, but some unusual features were present as regards to the melanoma: a sex-ratio of 10 women for one man and an average age lower than expected (median age=33 years) for melanoma occurrence. Among prognostic factors, median thickness was high compared to large series of melanoma in the literature (3.20 versus 1.5 mm). Another neoplasia occurred in three patients. An increase in melanoma incidence in patients with NF1 remains hypothetical but our small series of malignant melanoma arising in NF1 patients displays a large female preponderance, a higher thickness than expected and a frequent association with a second neoplasia. The peculiar female proneness for cancer whatever its localization and the risk of multiple neoplasias have already been reported in NF1 patients and could be true for malignant melanoma as well.