| 1. |
- Bonroy, Carolien, et al.
(författare)
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Detection of antinuclear antibodies : recommendations from EFLM, EASI and ICAP
- 2023
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Ingår i: Clinical Chemistry and Laboratory Medicine. - : Walter de Gruyter. - 1434-6621 .- 1437-4331. ; 61:7, s. 1167-1198
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Tidskriftsartikel (refereegranskat)abstract
- Objectives: Antinuclear antibodies (ANA) are important for the diagnosis of various autoimmune diseases. ANA are usually detected by indirect immunofluorescence assay (IFA) using HEp-2 cells (HEp-2 IFA). There are many variables influencing HEp-2 IFA results, such as subjective visual reading, serum screening dilution, substrate manufacturing, microscope components and conjugate. Newer developments on ANA testing that offer novel features adopted by some clinical laboratories include automated computer-assisted diagnosis (CAD) systems and solid phase assays (SPA).Methods: A group of experts reviewed current literature and established recommendations on methodological aspects of ANA testing. This process was supported by a two round Delphi exercise. International expert groups that participated in this initiative included (i) the European Federation of Clinical Chemistry and Laboratory Medicine (EFLM) Working Group “Autoimmunity Testing”; (ii) the European Autoimmune Standardization Initiative (EASI); and (iii) the International Consensus on ANA Patterns (ICAP).Results: In total, 35 recommendations/statements related to (i) ANA testing and reporting by HEp-2 IFA; (ii) HEp-2 IFA methodological aspects including substrate/conjugate selection and the application of CAD systems; (iii) quality assurance; (iv) HEp-2 IFA validation/verification approaches and (v) SPA were formulated. Globally, 95% of all submitted scores in the final Delphi round were above 6 (moderately agree, agree or strongly agree) and 85% above 7 (agree and strongly agree), indicating strong international support for the proposed recommendations.Conclusions: These recommendations are an important step to achieve high quality ANA testing.
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| 2. |
- Cinege, Gyöngyi, et al.
(författare)
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Distinctive features of Zaprionus indianus hemocyte differentiation and function revealed by transcriptomic analysis
- 2023
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Ingår i: Frontiers in Immunology. - : Frontiers Media S.A.. - 1664-3224. ; 14
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Tidskriftsartikel (refereegranskat)abstract
- Background: Insects have specialized cell types that participate in the elimination of parasites, for instance, the lamellocytes of the broadly studied species Drosophila melanogaster. Other drosophilids, such as Drosophila ananassae and the invasive Zaprionus indianus, have multinucleated giant hemocytes, a syncytium of blood cells that participate in the encapsulation of the eggs or larvae of parasitoid wasps. These cells can be formed by the fusion of hemocytes in circulation or originate from the lymph gland. Their ultrastructure highly resembles that of the mammalian megakaryocytes.Methods: Morphological, protein expressional, and functional features of blood cells were revealed using epifluorescence and confocal microscopy. The respective hemocyte subpopulations were identified using monoclonal antibodies in indirect immunofluorescence assays. Fluorescein isothiocyanate (FITC)-labeled Escherichia coli bacteria were used in phagocytosis tests. Gene expression analysis was performed following mRNA sequencing of blood cells.Results: D. ananassae and Z. indianus encapsulate foreign particles with the involvement of multinucleated giant hemocytes and mount a highly efficient immune response against parasitoid wasps. Morphological, protein expressional, and functional assays of Z. indianus blood cells suggested that these cells could be derived from large plasmatocytes, a unique cell type developing specifically after parasitoid wasp infection. Transcriptomic analysis of blood cells, isolated from naïve and wasp-infected Z. indianus larvae, revealed several differentially expressed genes involved in signal transduction, cell movements, encapsulation of foreign targets, energy production, and melanization, suggesting their role in the anti-parasitoid response. A large number of genes that encode proteins associated with coagulation and wound healing, such as phenoloxidase activity factor-like proteins, fibrinogen-related proteins, lectins, and proteins involved in the differentiation and function of platelets, were constitutively expressed. The remarkable ultrastructural similarities between giant hemocytes and mammalian megakaryocytes, and presence of platelets, and giant cell-derived anucleated fragments at wound sites hint at the involvement of this cell subpopulation in wound healing processes, in addition to participation in the encapsulation reaction.Conclusion: Our observations provide insights into the broad repertoire of blood cell functions required for efficient defense reactions to maintain the homeostasis of the organism. The analysis of the differentiation and function of multinucleated giant hemocytes gives an insight into the diversification of the immune mechanisms.
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| 3. |
- Malina, Tamás, et al.
(författare)
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Individual variability of venom from the European adder (Vipera berus berus) from one locality in Eastern Hungary
- 2017
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Ingår i: Toxicon. - : Elsevier BV. - 0041-0101 .- 1879-3150. ; 135, s. 59-70
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Tidskriftsartikel (refereegranskat)abstract
- We have revealed intra-population variability among venom samples from several individual European adders (Vipera berus berus) within a defined population in Eastern Hungary. Individual differences in venom pattern were noticed, both gender-specific and age-related, by one-dimensional electrophoresis. Gelatin zymography demonstrated that these individual venoms have different degradation profiles indicating varying protease activity in the specimens from adders of different ages and genders. Some specimens shared a conserved region of substrate degradation, while others had lower or extremely low protease activity. Phospholipase A(2) activity of venoms was similar but not identical. Interspecimen diversity of the venom phospholipase A(2)-spectra (based on the components' molecular masses) was detected by MALDI-TOF MS. The lethal toxicity of venoms (LD50) also showed differences among individual snakes. Extracted venom samples had varying neuromuscular paralysing effect on chick biventer cervicis nerve-muscle preparations. The paralysing effect of venom was lost when calcium in the physiological salt solution was replaced by strontium; indicating that the block of twitch responses to nerve stimulation is associated with the activity of a phospholipase-dependent neurotoxin. In contrast to the studied V.b. berus venoms from different geographical regions so far, this is the first V.b berus population discovered to have predominantly neurotoxic neuromuscular activity. The relevance of varying venom yields is also discussed. This study demonstrates that individual venom variation among V.b. berus living in particular area of Eastern Hungary might contribute to a wider range of clinical manifestations of V.b. hems envenoming than elsewhere in Europe.
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| 4. |
- Månsson, Lisa E., et al.
(författare)
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Role of the Lipopolysaccharide-CD14 Complex for the Activity of Hemolysin from Uropathogenic Escherichia coli
- 2007
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Ingår i: Infect. Immun.. ; 75:2, s. 997-1004
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Tidskriftsartikel (refereegranskat)abstract
- Bacterial pathogens produce a variety of exotoxins, which often become associated with the bacterial outer membrane component lipopolysaccharide (LPS) during their secretion. LPS is a potent proinflammatory mediator; however, it is not known whether LPS contributes to cell signaling induced by those microbial components to which it is attached. This is partly due to the common view that LPS present in bacterial component preparations is an experimental artifact. The Escherichia coli exotoxin hemolysin (Hly) is a known inducer of proinflammatory signaling in epithelial cells, and the signal transduction pathway involves fluctuation of the intracellular-Ca2+ concentration. Since LPS is known to interact with Hly, we investigated whether it is required as a cofactor for the activity of Hly. We found that the LPS/Hly complex exploits the CD14/LPS-binding protein recognition system to bring Hly to the cell membrane, where intracellular-Ca2+ signaling is initiated via specific activation of the small GTPase RhoA. Hly-induced Ca2+ signaling was found to occur independently of the LPS receptor TLR4, suggesting that the role of LPS/CD14 is to deliver Hly to the cell membrane. In contrast, the cytolytic effect triggered by exposure of cells to high Hly concentrations occurs independently of LPS/CD14. Collectively, our data reveal a novel molecular mechanism for toxin delivery in bacterial pathogenesis, where LPS-associated microbial compounds are targeted to the host cell membrane as a consequence of their association with LPS.
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| 5. |
- Müller, Claudia M, et al.
(författare)
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Role of histone-like proteins H-NS and StpA in expression of virulence determinants of uropathogenic Escherichia coli.
- 2006
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Ingår i: J Bacteriol. - 0021-9193. ; 188:15, s. 5428-38
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Tidskriftsartikel (refereegranskat)abstract
- The histone-like protein H-NS is a global regulator in Escherichia coli that has been intensively studied in nonpathogenic strains. However, no comprehensive study on the role of H-NS and its paralogue, StpA, in gene expression in pathogenic E. coli has been carried out so far. Here, we monitored the global effects of H-NS and StpA in a uropathogenic E. coli isolate by using DNA arrays. Expression profiling revealed that more than 500 genes were affected by an hns mutation, whereas no effect of StpA alone was observed. An hns stpA double mutant showed a distinct gene expression pattern that differed in large part from that of the hns single mutant. This suggests a direct interaction between the two paralogues and the existence of distinct regulons of H-NS and an H-NS/StpA heteromeric complex. hns mutation resulted in increased expression of alpha-hemolysin, fimbriae, and iron uptake systems as well as genes involved in stress adaptation. Furthermore, several other putative virulence genes were found to be part of the H-NS regulon. Although the lack of H-NS, either alone or in combination with StpA, has a huge impact on gene expression in pathogenic E. coli strains, its effect on virulence is ambiguous. At a high infection dose, hns mutants trigger more sudden lethality due to their increased acute toxicity in murine urinary tract infection and sepsis models. At a lower infectious dose, however, mutants lacking H-NS are attenuated through their impaired growth rate, which can only partially be compensated for by the higher expression of numerous virulence factors.
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