mAb against human glycosyl-phosphatidylinositol-linked leucocyte surface Ag CD59 and CD55 immunoprecipitated from detergent lysates of HPB ALL cell line in addition to the respective Ag a common 80-kDa glycoprotein component and (glyco)lipids. The 80-kDa glycoprotein is different from otherwise similar CD44 Ag. The CD59 immunoprecipitate contained also a small amount of the CD55 glycoprotein and the CD55 immunoprecipitate minute amount of the CD59 Ag. These results are interpreted in terms of existence of noncovalent complexes resistant to dissociation by mild detergents and consisting of the 80-kDa glycoprotein, CD59 and CD55 glycoproteins, relatively tightly bound (glyco)lipids and possibly other so far unidentified components. These complexes contain probably also other glycosyl-phosphatidylinositol-linked Ag, as an anti-CD48 mAb immunoprecipitated also an apparently very similar complex. The complexes immunoprecipitated by mAb against the CD55, CD59, and CD48 Ag also contain a protein kinase activity. This type of complexes could not be demonstrated in several other cell types such as RBC, PBMC, and HeLa cells. However, a qualitatively very similar set of components was immunoprecipitated from the murine thymoma EL-4 cell line by an anti-Thy-1 mAb.

• MeSH
• antigeny CD55 MeSH
• antigeny CD59 MeSH
• CD antigeny analýza   MeSH
• fosfatidylinositoly analýza   MeSH
• glykolipidy analýza   MeSH
• glykosylfosfatidylinositoly MeSH
• lidé MeSH
• membránové glykoproteiny analýza   imunologie   MeSH
• membránové lipidy analýza   MeSH
• membránové proteiny analýza   MeSH
• molekulová hmotnost MeSH
• precipitinové testy MeSH
• receptory odpovědné za homing lymfocytů analýza   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• antigeny CD55 MeSH
• antigeny CD59 MeSH
• CD antigeny MeSH
• fosfatidylinositoly MeSH
• glykolipidy MeSH
• glykosylfosfatidylinositoly MeSH
• membránové glykoproteiny MeSH
• membránové lipidy MeSH
• membránové proteiny MeSH
• receptory odpovědné za homing lymfocytů MeSH

Paroxysmal nocturnal hemoglobinuria (PNH) is a rare hematopoietic stem cell disorder resulting from the somatic mutation of the X-linked phosphatidyl-inositol glycan complementation Class A (PIG-A) gene. Depending on the severity of the mutation in the PIG-A gene, there is a partial or absolute inability to make glycosylphosphatidyl-inositol (GPI)-anchored proteins including complement-defense structures such as CD55 and CD59 on RBCs and WBCs. Flow cytometric detection of PNH clones has become the gold standard and has played an increasingly important role in the diagnosis, monitoring, and clinical management of patients with PNH. Recently, a 4-part set of Consensus Guidelines have been published by flow experts in the field to address the key assay-specific considerations for the identification of PNH clones in RBC and WBC, how to report such data and a full validation document for the assays described. Below, we have summarized the most significant aspects of this International effort.

• Klíčová slova
• CD59, FLAER, PNH, aplastic anemia (AA), flow cytometry, myelodysplastic disorder (MDS),
• MeSH
• antigeny CD55 krev   genetika   MeSH
• antigeny CD59 krev   genetika   MeSH
• konsensus MeSH
• lidé MeSH
• membránové proteiny krev   genetika   MeSH
• paroxysmální hemoglobinurie krev   mozkomíšní mok   diagnóza   genetika   MeSH
• průtoková cytometrie metody   normy   MeSH
• směrnice pro lékařskou praxi jako téma MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• přehledy MeSH
• Názvy látek
• antigeny CD55 MeSH
• antigeny CD59 MeSH
• CD59 protein, human MeSH Prohlížeč
• membránové proteiny MeSH
• phosphatidylinositol glycan-class A protein MeSH Prohlížeč

A significant fraction of human glycosyl-phosphatidylinositol-anchored Ag CD59, CD55, CD48, and CDw52 is present in several cell lines tested (HPB-ALL, Jurkat, HL-60, Raji) in very large noncovalent complexes relatively resistant to dissociation by detergents. These complexes also contain some (glyco)lipids, such as these bearing the CD15, CDw17, and CDw65 determinants, and several intracellular components including protein tyrosine kinases and probably several of their potential substrates. Preclearing of the detergent lysates with different antibodies indicated that all these components are present jointly in a common single type of complexes the size of which is around 100 nm (molecular mass in the range of at least tens of thousands kilodaltons) as determined by ultrafiltration and gel chromatography. These results indicate the existence of cell-surface domains, specifically enriched in the above listed components, that may play a critical role in the so far poorly understood phenomenon of cell activation mediated through many different glycosyl-phosphatidylinositol-anchored (glyco)proteins and glycolipids.

• MeSH
• antigeny CD59 MeSH
• buněčné linie MeSH
• CD antigeny analýza   MeSH
• fosfatidylinositoly analýza   MeSH
• glykolipidy analýza   MeSH
• glykosylfosfatidylinositoly MeSH
• lidé MeSH
• membránové glykoproteiny analýza   MeSH
• protoonkogenní proteiny analýza   MeSH
• tyrosinkinasa p56(lck), specifická pro lymfocyty MeSH
• tyrosinkinasy analýza   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• antigeny CD59 MeSH
• CD antigeny MeSH
• fosfatidylinositoly MeSH
• glykolipidy MeSH
• glykosylfosfatidylinositoly MeSH
• membránové glykoproteiny MeSH
• protoonkogenní proteiny MeSH
• tyrosinkinasa p56(lck), specifická pro lymfocyty MeSH
• tyrosinkinasy MeSH

Several human leucocyte surface glycoproteins and two lymphoid protein kinases were transiently expressed in monkey COS-7 fibroblastoid cells. All glycosylphospha-tidylinositol (GPI)-anchored proteins (CD14, CD16B, CD48, CD59, CD87 and GPI-anchored versions of CD2 and CD25) and protein tyrosine kinase (PTK) Lck but not transmembrane proteins (CD2, CD4, CD5, CD6, CD8) and PTK ZAP-70 were in part localized in buoyant, lipid-rich, detergent-resistant membrane GPI-microdomains of the COS cells. Endogenous GPI-microdomains of COS cells appear to be, in contrast to those present in leucocytes, essentially devoid of associated PTKs. Our results indicate that GPI-anchor is sufficient to target proteins to these membrane specializations even if expressed ectopically. Moreover, the N-terminal double acylation of the PTK Lck appears to be functional also in COS cells and targets the enzyme to the membrane GPI-microdomains implicated in receptor signalling.

• MeSH
• acylace MeSH
• antigeny CD2 genetika   metabolismus   MeSH
• antigeny CD59 genetika   metabolismus   MeSH
• buněčná membrána metabolismus   MeSH
• COS buňky MeSH
• exprese genu MeSH
• glykosylfosfatidylinositoly metabolismus   MeSH
• leukocyty chemie   MeSH
• lidé MeSH
• membránové lipidy metabolismus   MeSH
• membránové proteiny genetika   metabolismus   MeSH
• protein-tyrosinkináza ZAP-70 MeSH
• receptory interleukinu-2 genetika   metabolismus   MeSH
• rekombinantní proteiny metabolismus   MeSH
• transfekce MeSH
• tyrosinkinasa p56(lck), specifická pro lymfocyty genetika   metabolismus   MeSH
• tyrosinkinasy genetika   metabolismus   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• antigeny CD2 MeSH
• antigeny CD59 MeSH
• glykosylfosfatidylinositoly MeSH
• membránové lipidy MeSH
• membránové proteiny MeSH
• protein-tyrosinkináza ZAP-70 MeSH
• receptory interleukinu-2 MeSH
• rekombinantní proteiny MeSH
• tyrosinkinasa p56(lck), specifická pro lymfocyty MeSH
• tyrosinkinasy MeSH
• ZAP70 protein, human MeSH Prohlížeč

Liposomes functionalized with monoclonal antibodies or their antigen-binding fragments have attracted much attention as specific drug delivery devices for treatment of various diseases including cancer. The conjugation of antibodies to liposomes is usually achieved by covalent coupling using cross-linkers in a reaction that might adversely affect the characteristics of the final product. Here we present an alternative strategy for liposome functionalization: we created a recombinant Fab antibody fragment genetically fused on its C-terminus to the hydrophobic peptide derived from pulmonary surfactant protein D, which became inserted into the liposomal bilayer during liposomal preparation and anchored the Fab onto the liposome surface. The Fab-conjugated liposomes specifically recognized antigen-positive cells and efficiently delivered their cargo, the Alexa Fluor 647 dye, into target cells in vitro and in vivo. In conclusion, our approach offers the potential for straightforward development of nanomedicines functionalized with an antibody of choice without the need of harmful cross-linkers.

• Klíčová slova
• Active targeting, Antibody engineering, Immunoliposome, Liposome functionalization, Recombinant Fab antibody fragment,
• MeSH
• antigen CD48 metabolismus   MeSH
• antigeny CD59 metabolismus   MeSH
• imunoglobuliny - Fab fragmenty chemie   imunologie   metabolismus   MeSH
• Jurkat buňky MeSH
• lidé MeSH
• liposomy chemie   MeSH
• lymfom imunologie   metabolismus   patologie   MeSH
• monoklonální protilátky chemie   imunologie   metabolismus   MeSH
• myši MeSH
• nádorové buňky kultivované MeSH
• peptidové fragmenty imunologie   metabolismus   MeSH
• protein D asociovaný s plicním surfaktantem imunologie   metabolismus   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• antigen CD48 MeSH
• antigeny CD59 MeSH
• CD48 protein, human MeSH Prohlížeč
• CD59 protein, human MeSH Prohlížeč
• imunoglobuliny - Fab fragmenty MeSH
• liposomy MeSH
• monoklonální protilátky MeSH
• peptidové fragmenty MeSH
• protein D asociovaný s plicním surfaktantem MeSH

Abnormalities in ATM and TP53 genes represent important predictive factors in chronic lymphocytic leukemia (CLL); however, the efficacy of CD20 targeting immunotherapy is only poorly defined in the affected patients. Therefore, we tested the in vitro response to ofatumumab (OFA) and rituximab (RTX) in 75 CLL samples with clearly defined p53 or ATM inactivation. Using standard conditions allowing complement-dependent cytotoxicity, i.e., 10 μg/mL of antibodies and 20% active human serum, we observed clear differences among the tested genetic categories: ATM-mutated samples (n = 17) represented the most sensitive, wild-type samples (n = 31) intermediate, and TP53-mutated samples (n = 27) the most resistant group (ATM-mut vs. TP53-mut: P = 0.0005 for OFA and P = 0.01 for RTX). The response correlated with distinct levels of CD20 and critical complement inhibitors CD55 and CD59; CD20 level median was the highest in ATM-mutated and the lowest in TP53-mutated samples (difference between the groups P < 0.01), while the total level of complement inhibitors (CD55 plus CD59) was distributed in the opposite manner (P < 0.01). Negligible response to both OFA and RTX was noted in all cultures (n = 10) tested in the absence of active serum, which strongly indicated that complement-dependent cytotoxicity was a principal cell death mechanism. Our study shows that (1) common genetic defects in CLL cells significantly impact a primary response to anti-CD20 monoclonal antibodies and (2) ATM-mutated patients with currently poor prognosis may potentially benefit from immunotherapy targeting CD20.

• MeSH
• antigeny CD20 účinky léků   imunologie   MeSH
• antigeny CD55 imunologie   MeSH
• antigeny CD59 imunologie   MeSH
• antigeny nádorové účinky léků   imunologie   MeSH
• ATM protein nedostatek   genetika   fyziologie   MeSH
• chemorezistence MeSH
• chronická lymfatická leukemie patologie   MeSH
• dospělí MeSH
• geny p53 * MeSH
• humanizované monoklonální protilátky MeSH
• komplement imunologie   MeSH
• kultivační média MeSH
• lidé středního věku MeSH
• lidé MeSH
• monoklonální protilátky farmakologie   MeSH
• myší monoklonální protilátky farmakologie   MeSH
• nádorové buňky kultivované MeSH
• nádorové proteiny genetika   MeSH
• nádorový supresorový protein p53 nedostatek   fyziologie   MeSH
• oprava DNA genetika   MeSH
• rituximab MeSH
• screeningové testy protinádorových léčiv MeSH
• senioři nad 80 let MeSH
• senioři MeSH
• sérum MeSH
• Check Tag
• dospělí MeSH
• lidé středního věku MeSH
• lidé MeSH
• mužské pohlaví MeSH
• senioři nad 80 let MeSH
• senioři MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• srovnávací studie MeSH
• Názvy látek
• antigeny CD20 MeSH
• antigeny CD55 MeSH
• antigeny CD59 MeSH
• antigeny nádorové MeSH
• ATM protein, human MeSH Prohlížeč
• ATM protein MeSH
• CD59 protein, human MeSH Prohlížeč
• humanizované monoklonální protilátky MeSH
• komplement MeSH
• kultivační média MeSH
• monoklonální protilátky MeSH
• myší monoklonální protilátky MeSH
• nádorové proteiny MeSH
• nádorový supresorový protein p53 MeSH
• ofatumumab MeSH Prohlížeč
• rituximab MeSH
• TP53 protein, human MeSH Prohlížeč

BACKGROUND: The diagnosis of paroxysmal nocturnal hemoglobinuria (PNH) relies on flow cytometric demonstration of loss of glycosyl-phosphatidyl inositol (GPI)-anchored proteins from red blood cells (RBC) and white blood cells (WBC). High-sensitivity multiparameter assays have been developed to detect loss of GPI-linked structures on PNH neutrophils and monocytes. High-sensitivity assays to detect PNH phenotypes in RBCs have also been developed that rely on the loss of GPI-linked CD59 on CD235a-gated mature RBCs. The latter is used to delineate PNH Type III (total loss of CD59) and PNH Type II RBCs (partial loss of CD59) from normal (Type I) RBCs. However, it is often very difficult to delineate these subsets, especially in patients with large PNH clones who continue to receive RBC transfusions, even while on eculizumab therapy. METHODS: We have added allophycocyanin (APC)-conjugated CD71 to the existing CD235aFITC/CD59PE RBC assay allowing simultaneous delineation and quantification of PNH Type III and Type II immature RBCs (iRBCs). RESULTS: We analyzed 24 medium to large-clone PNH samples (>10% PNH WBC clone size) for PNH Neutrophil, PNH Monocyte, Type III and Type II PNH iRBCs, and where possible, Type III and Type II PNH RBCs. The ability to delineate PNH Type III, Type II, and Type I iRBCs was more objective compared to that in mature RBCs. Additionally, total PNH iRBC clone sizes were very similar to PNH WBC clone sizes. CONCLUSIONS: Addition of CD71 significantly improves the ability to analyze PNH clone sizes in the RBC lineage, regardless of patient hemolytic and/or transfusion status.

• Klíčová slova
• CD71, PNH clones, immature red blood cells, paroxysmal nocturnal hemoglobinuria,
• MeSH
• antigeny CD59 metabolismus   MeSH
• buněčná diferenciace MeSH
• CD antigeny krev   fyziologie   MeSH
• diferenciální diagnóza MeSH
• erytrocyty metabolismus   patologie   MeSH
• glykoforin metabolismus   MeSH
• imunofenotypizace přístrojové vybavení   metody   normy   MeSH
• kohortové studie MeSH
• leukocyty patologie   MeSH
• lidé MeSH
• monocyty metabolismus   patologie   MeSH
• neutrofily metabolismus   patologie   MeSH
• paroxysmální hemoglobinurie krev   klasifikace   diagnóza   patologie   MeSH
• počet leukocytů přístrojové vybavení   metody   MeSH
• průtoková cytometrie přístrojové vybavení   metody   normy   MeSH
• receptory transferinu krev   fyziologie   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• antigeny CD59 MeSH
• CD antigeny MeSH
• CD59 protein, human MeSH Prohlížeč
• CD71 antigen MeSH Prohlížeč
• glykoforin MeSH
• GYPA protein, human MeSH Prohlížeč
• receptory transferinu MeSH

Paroxysmal nocturnal hemoglobinuria (PNH) is a rare but often debilitating disease which may lead to death in up to 35% of patients within 5 years if unrecognized and untreated. Detection of PNH and assessment of PNH clone size in RBC and WBC lineages by flow cytometric analysis has increased in importance due to the availability of novel therapies. These therapies typically block the hemolysis of red blood cells and thus significantly lower the morbidities and mortality associated with this disease. This chapter describes validated, state-of-the-art, high-sensitivity flow cytometric methodologies based on latest published testing guidelines for PNH.

• Klíčová slova
• Aplastic anemia (AA), CD59, FLAER, Glycophosphatidylinositol (GPI)-anchored protein, Myelodysplastic syndrome (MDS), Paroxysmal nocturnal hemoglobinuria (PNH),
• MeSH
• antigeny CD59 imunologie   MeSH
• erytrocyty imunologie   MeSH
• imunofenotypizace metody   MeSH
• leukocyty imunologie   MeSH
• lidé MeSH
• paroxysmální hemoglobinurie krev   imunologie   MeSH
• průtoková cytometrie metody   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• antigeny CD59 MeSH

The role of detergent-insensitive membrane domains (DIMs) in desensitisation of the G protein-coupled receptor-mediated hormone response was studied in clone E2M11 of HEK293 cells which stably express high levels of both thyrotropin-releasing hormone (TRH) receptors and G(11)alpha G protein. DIMs were prepared by flotation in equilibrium sucrose density gradients and characterised by a panel of membrane markers representing peripheral, glycosylphosphatidylinositol-bound as well as integral membrane proteins (caveolin, CD29, CD55, CD59, CD147, the alpha subunit of Na, K-ATPase) and enzyme activities (alkaline phosphatase, adenylyl cyclase). Caveolin-containing DIMs represented only a small fraction of the overall pool of G(q)alpha/G(11)alpha-rich domains. Prolonged stimulation of E2M11 cells with TRH resulted in dramatic depletion of G(q)alpha/G(11)alpha from all DIMs, which was paralleled by a concomitant G(q)alpha/G(11)alpha increase in the high-density gradient fractions containing the bulk-phase membrane constituents soluble in 1% Triton X-100. Distribution of membrane markers was unchanged under these conditions. Membrane domains thus represent a substantial structural determinant of the G protein pool relevant to desensitisation of hormone action.

• MeSH
• adenylátcyklasy metabolismus   MeSH
• alkalická fosfatasa metabolismus   MeSH
• antigeny CD29 metabolismus   MeSH
• antigeny CD55 metabolismus   MeSH
• antigeny CD59 metabolismus   MeSH
• buněčná membrána účinky léků   metabolismus   MeSH
• buněčné linie MeSH
• centrifugace - gradient hustoty MeSH
• detergenty farmakologie   MeSH
• hormon uvolňující thyreotropin farmakologie   MeSH
• imunoblotting MeSH
• kaveolin 1 MeSH
• kaveoliny * MeSH
• lidé MeSH
• membránové proteiny metabolismus   MeSH
• oktoxynol farmakologie   MeSH
• proteiny vázající GTP - alfa-podjednotky Gq-G11 MeSH
• proteiny vázající GTP chemie   metabolismus   MeSH
• sodíko-draslíková ATPasa metabolismus   MeSH
• terciární struktura proteinů MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• adenylátcyklasy MeSH
• alkalická fosfatasa MeSH
• antigeny CD29 MeSH
• antigeny CD55 MeSH
• antigeny CD59 MeSH
• CAV1 protein, human MeSH Prohlížeč
• detergenty MeSH
• hormon uvolňující thyreotropin MeSH
• kaveolin 1 MeSH
• kaveoliny * MeSH
• membránové proteiny MeSH
• oktoxynol MeSH
• proteiny vázající GTP - alfa-podjednotky Gq-G11 MeSH
• proteiny vázající GTP MeSH
• sodíko-draslíková ATPasa MeSH

Membrane domains are highly specialized parts of the cell plasma membrane, carrying on and augmenting the incoming signals. To study their structural and functional properties, it is crucial to find the least damaging mode of their isolation. Using two different cell lines, epithelial HEK cells (clone E2M11) and S49 lymphoma cells, three methods of membrane domain isolation (i.e., detergent extraction, alkaline treatment, and "drastic" homogenization) were tested for similarity and reproducibility by 2-D electrophoresis. Our data show that the protein composition of membrane domains obtained by different isolation methods is similar and that approximately 60% of the spots are present in all membrane domain preparations. Furthermore, the same degree of similarity of 2-D profiles of the most intensively silver stained spots found in membrane domains of the two cell lines derived from different tissues suggests that the composition of a large part of membrane domains proteins is conservative. We suggest that these proteins may either be involved in the organization of membrane domain structure or represent the conservative component of signal transduction machinery.

• MeSH
• 2D gelová elektroforéza MeSH
• antigeny CD44 analýza   imunologie   MeSH
• antigeny CD59 analýza   imunologie   MeSH
• buněčná membrána metabolismus   MeSH
• centrifugace - gradient hustoty MeSH
• detergenty chemie   MeSH
• kultivované buňky MeSH
• lidé MeSH
• membránové proteiny izolace a purifikace   metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• antigeny CD44 MeSH
• antigeny CD59 MeSH
• detergenty MeSH
• membránové proteiny MeSH