Over the past several years, a diverse group of physicians and other laboratory scientists have developed various recommendations and guidelines regarding best practices for PNH testing. This manuscript is based on these previous recommendations as well as various other relevant publications of experts in the area of PNH testing. The goal is to provide flow cytometry laboratories with an updated consensus approach to analysis and reporting of PNH results and to address the most common analytical challenges for accurate reporting of this rare disease. A comprehensive case library is included in this section. © 2017 International Clinical Cytometry Society.
- MeSH
- analýza dat MeSH
- erytrocyty metabolismus MeSH
- glykosylfosfatidylinositoly metabolismus MeSH
- konsensus MeSH
- leukocyty metabolismus MeSH
- lidé MeSH
- paroxysmální hemoglobinurie diagnóza metabolismus MeSH
- průtoková cytometrie metody normy MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- MeSH
- biochemická analýza krve MeSH
- elektrochemické techniky * MeSH
- glykosylfosfatidylinositoly diagnostické užití MeSH
- lidé MeSH
- preeklampsie * diagnóza MeSH
- receptor 1 pro vaskulární endoteliální růstový faktor izolace a purifikace MeSH
- těhotenství MeSH
- Check Tag
- lidé MeSH
- těhotenství MeSH
- ženské pohlaví MeSH
- MeSH
- diferenciální diagnóza MeSH
- elektrochemické techniky * MeSH
- glykosylfosfatidylinositoly diagnostické užití MeSH
- hospitalizace MeSH
- lidé MeSH
- preeklampsie * diagnóza MeSH
- receptor 1 pro vaskulární endoteliální růstový faktor izolace a purifikace MeSH
- těhotenství MeSH
- ultrasonografie dopplerovská MeSH
- Check Tag
- lidé MeSH
- těhotenství MeSH
- ženské pohlaví MeSH
BACKGROUND: The accumulation of the misfolded forms of cellular prion protein, i.e. prions (PrPSc), in the brain is one of the crucial characteristics of fatal neurodegenerative disorders, called transmissible spongiform encephalopathies (TSEs). Cellular prion protein is normally linked to the cell surface by the glycosylphosphatidylinositol (GPI) anchor. There is accumulating evidence that the GPI-anchorless prion protein may act as an accelerator of formation and propagation of prions. In the TSE affected human brain we have previously discovered a novel GPI-anchorless prion protein fragment, named PrP226*, which ends with the tyrosine 226. This fragment can be labeled specifically by the monoclonal antibody V5B2. METHODS: We developed a DELFIA based assay for quick and sensitive detection of the PrP226* fragment in human brain tissue homogenates. By calculating the ratio between the signals of native (N) and denatured (D) samples applied to the assay we were able to observe significant difference between 24 TSE affected brains and 10 control brains. The presence of PrP226* in brain tissue was confirmed by western blot. RESULTS: Our results demonstrate that PrP226* is present in small quantities in healthy human brain, whereas in degenerated brain it accumulates in prion aggregates, proportionally to PrPSc. Samples with high D/N ratio generally comprised more proteinase K resistant PrP, while no correlation was found between the quantity of PrP226* and standard classification of Creutzfeldt-Jakob disease (CJD). CONCLUSIONS: In the present study we show that the PrP226* fragment accumulates in prion aggregates and after being released from them by a denaturation procedure, could serve as a proteinase K digestion independent biomarker for human TSEs. The PrP226* assay described in this paper offers a tool to follow and study this unique anchorless PrP fragment in various parts of human brain and possibly also in other tissues and body fluids.
- MeSH
- Creutzfeldtova-Jakobova nemoc * patologie MeSH
- ELISA MeSH
- endopeptidasa K farmakologie MeSH
- glykosylfosfatidylinositoly metabolismus MeSH
- lidé MeSH
- mozek * metabolismus účinky léků MeSH
- proteiny 14-3-3 metabolismus MeSH
- PrPSc proteiny metabolismus účinky léků MeSH
- statistika jako téma MeSH
- teplota MeSH
- Check Tag
- lidé MeSH
- mužské pohlaví MeSH
- ženské pohlaví MeSH
Mutations of the TMPRSS6 gene, encoding the serine protease matriptase-2, lead to iron-refractory iron deficiency anemia. Matriptase-2 is a potent negative regulator of hepcidin. Based on in vitro data, it has recently been proposed that matriptase-2 decreases hepcidin synthesis by cleaving membrane hemojuvelin, a key protein of the hepcidin-regulatory pathway. However, in vivo evidence for this mechanism of action of matriptase-2 is lacking. To investigate the hemojuvelin-matriptase-2 interaction in vivo, an immunoblot assay for liver membrane hemojuvelin was optimized using hemojuvelin-mutant mice as a negative control. In wild-type mice, two hemojuvelin-specific bands of 35kDa and 20kDa were detected in mouse liver membrane fraction under reducing conditions; under non-reducing conditions, a single band of approximately 50kDa was seen. Phosphatidylinositol-specific phospholipase C treatment confirmed binding of the detected protein to the cell membrane by a glycosylphosphatidylinositol anchor, indicating that the major form of mouse liver membrane hemojuvelin is a glycosylphosphatidylinositol-bound heterodimer. Unexpectedly, comparison of liver homogenates from Tmprss6+/+ and Tmprss6-/- mice revealed significantly decreased, rather than increased, hemojuvelin heterodimer content in Tmprss6-/- mice. These data do not provide direct support for the concept that matriptase-2 cleaves membrane hemojuvelin and may indicate that, in vivo, the role of matriptase-2 in the regulation of hepcidin gene expression is more complex.
- MeSH
- anemie z nedostatku železa genetika metabolismus MeSH
- buněčná membrána genetika metabolismus MeSH
- dimerizace MeSH
- elektroforéza v polyakrylamidovém gelu MeSH
- fosfolipasa C fosfoinositidové signalizace metabolismus MeSH
- glykosylfosfatidylinositoly chemie metabolismus MeSH
- játra metabolismus patologie MeSH
- kationické antimikrobiální peptidy genetika metabolismus MeSH
- membránové proteiny nedostatek genetika metabolismus MeSH
- myši knockoutované MeSH
- myši MeSH
- polymerázová řetězová reakce MeSH
- regulace genové exprese MeSH
- serinové endopeptidasy nedostatek genetika MeSH
- signální transdukce genetika MeSH
- tkáňové extrakty chemie MeSH
- železo metabolismus MeSH
- zvířata MeSH
- Check Tag
- mužské pohlaví MeSH
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- MeSH
- glykosylfosfatidylinositoly diagnostické užití MeSH
- lidé MeSH
- membránové proteiny diagnostické užití MeSH
- prediktivní hodnota testů MeSH
- preeklampsie diagnóza krev MeSH
- receptor 1 pro vaskulární endoteliální růstový faktor izolace a purifikace MeSH
- těhotenství MeSH
- Check Tag
- lidé MeSH
- těhotenství MeSH
- ženské pohlaví MeSH
The exposure of phosphatidylserine (PS) on the cell surface is a general marker of apoptotic cells. Non-apoptotic PS externalization is induced by several activation stimuli, including engagement of immunoreceptors. Immune cells can also be activated by aggregation of glycosylphosphatidylinositol-anchored proteins (GPI-APs). However, it is unknown whether cell triggering through these proteins, lacking transmembrane and cytoplasmic domains, also leads to PS externalization. Here we show that engagement of GPI-APs in rodent mast cells induces a rapid and reversible externalization of PS by a non-apoptotic mechanism. PS externalization triggered by GPI-AP-specific monoclonal antibodies was dependent on the activity of H(+)-ATP synthase and several other enzymes involved in mast cell signaling but was independent of cell degranulation, free cytoplasmic calcium up-regulation, and a decrease in lipid packing as determined by merocyanine 540 binding. Surprisingly, disruption of actin cytoskeleton by latrunculin B or plasma membrane integrity by methyl-beta-cyclodextrin had opposite effects on PS externalization triggered through GPI-AP or the high affinity IgE receptor. We further show that PS externalization mediated by GPI-APs was also observed in some other cells, and its extent varied with antibodies used. Interestingly, effects of different antibodies on PS externalization were additive, indicating that independent stimuli converge onto a signaling pathways leading to PS externalization. Our findings identify the cell surface PS exposure induced through GPI-AP as a distinct mechanism of cell signaling. Such a mechanism could contribute to "inside-out" signaling in response to pathogens and other external activators and/or to initiation of other functions associated with PS externalization.
- MeSH
- apoptóza genetika účinky záření MeSH
- beta-cyklodextriny farmakologie MeSH
- bicyklické sloučeniny heterocyklické farmakologie MeSH
- biologický transport fyziologie účinky léků MeSH
- buňky NIH 3T3 MeSH
- degranulace buněk fyziologie účinky léků MeSH
- financování organizované MeSH
- fluorescenční barviva farmakologie MeSH
- fosfatidylseriny metabolismus MeSH
- glykosylfosfatidylinositoly metabolismus MeSH
- krysa rodu rattus MeSH
- lidé MeSH
- mastocyty metabolismus MeSH
- mitochondriální protonové ATPasy metabolismus MeSH
- myši MeSH
- nádorové buněčné linie MeSH
- potkani Wistar MeSH
- pyrimidinony farmakologie MeSH
- receptory IgE metabolismus MeSH
- signální transdukce fyziologie účinky léků MeSH
- thiazolidiny farmakologie MeSH
- zvířata MeSH
- Check Tag
- krysa rodu rattus MeSH
- lidé MeSH
- mužské pohlaví MeSH
- myši MeSH
- zvířata MeSH
