BACKGROUND: Targeted alpha therapy represents an advanced and rapidly evolving form of precision cancer treatment with increasing importance in recent years. The alpha-emitter 225Ac plays a key role in this clinical development due to its attractive physical and chemical properties. In this context, the macropa chelator has favorable characteristics in terms of labeling conditions and complex stability, making its derivatives exceptionally appealing for 225Ac-labeling of heat-sensitive biomolecules. However, preclinical evaluation of such 225Ac-containing molecules and comprehensive assessment of their pharmacokinetics, dosimetry and radiobiology necessitate a suitable diagnostic counterpart. Due to its attractive radiation properties, 133La represents an adequate positron-emitting radionuclide to form a matched pair with 225Ac for macropa-based radiopharmaceuticals. Herein, we describe the preparation and radiopharmacological characterization of macropa-functionalized, 133La/225Ac-labeled single-domain antibodies (sdAbs) targeting the epidermal growth factor receptor (EGFR) to demonstrate the general suitability of this theranostic pair of radionuclides. RESULTS: The synthesis of a clickable, bicyclononyne-modified macropa chelator and its site-specific conjugation to azide-modified, monovalent and biparatopic sdAbs is presented. Subsequent labeling at room temperature (rt) for 15 min resulted in molar activities of 30 MBq/nmol for 133La and 0.5 MBq/nmol for 225Ac, respectively. In vitro studies using the 133La-labeled sdAbs revealed comparable binding characteristics, but an enhanced cellular internalization of the biparatopic variant compared to its monovalent counterparts. This increased uptake consequently resulted in higher cytotoxicity of the 225Ac-labeled biparatopic conjugate. In vivo PET imaging of the 133La-labeled conjugates indicated comparable uptake and retention of the mono- and biparatopic variants in liver and kidneys, with the former showing slightly higher tumor accumulation. Ex vivo biodistribution studies conducted with 225Ac-labeled conjugates largely confirmed the findings obtained by PET imaging, albeit with a marginally higher tumor accumulation of the biparatopic 225Ac-radioimmunoconjugate. Final histological examinations of tumor and kidney tissues showed DNA damage in the renal cortex of the 225Ac-radioimmunoconjugate-treated mice, but no differences in the number of γ-H2AX-positive cells in the corresponding tumor tissues could be detected. CONCLUSIONS: We present a comprehensive study on the theranostic application of 133La and 225Ac for antibody-based biomolecules and lay the foundation for the future application of this matched pair of radionuclides towards labeling of heat-sensitive, macropa-functionalized radiopharmaceuticals in general. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1186/s41181-025-00354-7.
- Publication type
- Journal Article MeSH
Copper radioisotopes can be used for imaging as well as for therapy and, thus, can form ideal theranostic pairs. The Cu(II) complexes of cross-bridged cyclam (cb-cyclam) derivatives are considered to be highly stable in vivo. However, the complexes are mostly formed under harsh conditions not compatible with sensitive biomolecules. Here, a new class of cb-cyclam derivatives, cross-bridged bis(phosphinate)cyclams ("cb-BPC"), were investigated. Ligands with one or two methylene-bis(phosphinate) -CH2-PO2H-CH2-PO2H(R) (R = H, OH, substituted alkyl) pendant arms were synthesized. Bifunctionalization on the distant phosphorus atom was carried out by employing P-nitrobenzyl (R = CH2-Ph-4-NO2) precursors and/or, for cb-BPC with two bis(phosphinate) pendant arms, by reactions of silyl-phosphites obtained by silylation of their P(O)-H fragments. The reactive bifunctional groups include amine, carboxylate, azide, isothiocyanate, maleimide and/or tetrazine, and also their orthogonally reactive combination in a single molecule of chelator. The cb-BPCs with one bis(phosphinate) arm were not efficiently radiolabelled with 64Cu. The cb-BPCs with two pendant arms were radiolabelled even at room temperature and with only a small excess of chelator, leading to a high specific activity. Radiolabelling was fully comparable with that of analogous bis(phosphinate) derivatives of cyclam and identical radiolabelling of cyclam and cb-cyclam derivatives was observed for the first time. The cb-BPCs with two bis(phosphinate) pendant arms represent a new class of rigid chelators for copper radioisotopes that are easily synthetically modifiable, highly hydrophilic and radiolabelled under mild conditions.
- Publication type
- Journal Article MeSH
BACKGROUND: The research is focused on sensitive biomarkers in multiple sclerosis (MS). OBJECTIVE: The aim of the study was to assess the relationship between plasma neurofilament light chain (pNfL) and disease activity as defined by the concept NEDA (no evident disease activity), including brain volumetry, in a cohort of MS patients treated with disease-modifying treatment (DMT). METHODS: Levels of pNfL (Single Molecule Array (SIMOA) technology) were examined in 95 RRMS (relapsing-remitting multiple sclerosis) patients and analyzed in relationship to NEDA-3 status and NEDA-BVL (brain volume loss; NEDA-3 extended by brain volumetry) during the last 12 months. The statistical model was developed using logistic regression analysis, including the independent variables: demographic, clinical, and magnetic resonance imaging (MRI) data. Dependent variables were NEDA-3 and NEDA-BVL status. RESULTS: The mean age of the study participants (n = 95, 62% females) was 37.85 years (standard deviation (SD) = 9.62) and the median disability score was 3.5 (2.5-4.1). Receiver operating characteristics (ROC) analysis showed that pNfL predicts NEDA-3 (the sensitivity and specificity of the model were 92% and 78%, respectively, p < 0.001) and NEDA-BVL status (the sensitivity and specificity were 80% and 65%, respectively, p < 0.001). CONCLUSION: The results show that pNfL levels are a useful biomarker of disease activity determined by NEDA-BVL status, including brain MRI-volumetry in patients with RRMS.
- MeSH
- Adult MeSH
- Intermediate Filaments MeSH
- Humans MeSH
- Magnetic Resonance Imaging MeSH
- Brain diagnostic imaging MeSH
- Multiple Sclerosis, Relapsing-Remitting * diagnostic imaging MeSH
- Multiple Sclerosis * diagnostic imaging MeSH
- Check Tag
- Adult MeSH
- Humans MeSH
- Male MeSH
- Female MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
Plant phospholipase Ds (PLDs), essential regulators of phospholipid signaling, function in multiple signal transduction cascades; however, the mechanisms regulating PLDs in response to pathogens remain unclear. Here, we found that Arabidopsis (Arabidopsis thaliana) PLDδ accumulated in cells at the entry sites of the barley powdery mildew fungus, Blumeria graminis f. sp hordei Using fluorescence recovery after photobleaching and single-molecule analysis, we observed higher PLDδ density in the plasma membrane after chitin treatment; PLDδ also underwent rapid exocytosis. Fluorescence resonance energy transfer with fluorescence lifetime imaging microscopy showed that the interaction between PLDδ and the microdomain marker AtREMORIN1.3 (AtREM1.3) increased in response to chitin, indicating that exocytosis facilitates rapid, efficient sorting of PLDδ into microdomains upon pathogen stimulus. We further unveiled a trade-off between brefeldin A (BFA)-resistant and -sensitive pathways in secretion of PLDδ under diverse conditions. Upon pathogen attack, PLDδ secretion involved syntaxin-associated VAMP721/722-mediated exocytosis sensitive to BFA. Analysis of phosphatidic acid (PA), hydrogen peroxide, and jasmonic acid (JA) levels and expression of related genes indicated that the relocalization of PLDδ is crucial for its activation to produce PA and initiate reactive oxygen species and JA signaling pathways. Together, our findings revealed that the translocation of PLDδ to papillae is modulated by exocytosis, thus triggering PA-mediated signaling in plant innate immunity.plantcell;31/12/3015/FX1F1fx1.
- MeSH
- Arabidopsis genetics immunology metabolism microbiology MeSH
- Ascomycota pathogenicity MeSH
- Brefeldin A immunology metabolism MeSH
- Cell Membrane metabolism MeSH
- Chitin immunology metabolism MeSH
- Cyclopentanes metabolism MeSH
- Exocytosis drug effects immunology MeSH
- Phospholipase D genetics metabolism MeSH
- Phosphatidic Acids metabolism MeSH
- Plant Diseases immunology microbiology MeSH
- Oxylipins metabolism MeSH
- Hydrogen Peroxide metabolism MeSH
- Immunity, Innate * drug effects MeSH
- Arabidopsis Proteins metabolism MeSH
- Qa-SNARE Proteins metabolism MeSH
- R-SNARE Proteins metabolism MeSH
- SNARE Proteins genetics metabolism MeSH
- Reactive Oxygen Species metabolism MeSH
- Signal Transduction immunology physiology MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
... Composed of Two Interconnected Arms: Innate Immunity and Adaptive Immunity • Immune Cells and Molecules ... ... • Class I Molecules Consist of One Large Glycoprotein Heavy Chain Plus a Small Protein Light Chain ... ... • Class II Molecules Consist of Two Nonidentical Membrane-Bound Glycoprotein Chains • Class I and ... ... of MHC Genes • The MHC Locus Encodes the Three Major Classes of MHC Molecules -- 250 -- 250 -- The ... ... Role and Expression Pattern of MHC Molecules • MHC Molecules Present Both Intracellular and Extracellular ...
Eight edition různé stránkování : barevné ilustrace ; 28 cm
- MeSH
- Allergy and Immunology MeSH
- Immune System MeSH
- Publication type
- Textbook MeSH
- Conspectus
- Patologie. Klinická medicína
- Učební osnovy. Vyučovací předměty. Učebnice
- NML Fields
- alergologie a imunologie
DNA double stranded breaks (DSBs) are the most serious type of lesions introduced into chromatin by ionizing radiation. During DSB repair, cells recruit different proteins to the damaged sites in a manner dependent on local chromatin structure, DSB location in the nucleus, and the repair pathway entered. 53BP1 is one of the important players participating in repair pathway decision of the cell. Although many molecular biology details have been investigated, the architecture of 53BP1 repair foci and its development during the post-irradiation time, especially the period of protein recruitment, remains to be elucidated. Super-resolution light microscopy is a powerful new tool to approach such studies in 3D-conserved cell nuclei. Recently, we demonstrated the applicability of single molecule localization microscopy (SMLM) as one of these highly resolving methods for analyses of dynamic repair protein distribution and repair focus internal nano-architecture in intact cell nuclei. In the present study, we focused our investigation on 53BP1 foci in differently radio-resistant cell types, moderately radio-resistant neonatal human dermal fibroblasts (NHDF) and highly radio-resistant U87 glioblastoma cells, exposed to high-LET 15N-ion radiation. At given time points up to 24 h post irradiation with doses of 1.3 Gy and 4.0 Gy, the coordinates and spatial distribution of fluorescently tagged 53BP1 molecules was quantitatively evaluated at the resolution of 10⁻20 nm. Clusters of these tags were determined as sub-units of repair foci according to SMLM parameters. The formation and relaxation of such clusters was studied. The higher dose generated sufficient numbers of DNA breaks to compare the post-irradiation dynamics of 53BP1 during DSB processing for the cell types studied. A perpendicular (90°) irradiation scheme was used with the 4.0 Gy dose to achieve better separation of a relatively high number of particle tracks typically crossing each nucleus. For analyses along ion-tracks, the dose was reduced to 1.3 Gy and applied in combination with a sharp angle irradiation (10° relative to the cell plane). The results reveal a higher ratio of 53BP1 proteins recruited into SMLM defined clusters in fibroblasts as compared to U87 cells. Moreover, the speed of foci and thus cluster formation and relaxation also differed for the cell types. In both NHDF and U87 cells, a certain number of the detected and functionally relevant clusters remained persistent even 24 h post irradiation; however, the number of these clusters again varied for the cell types. Altogether, our findings indicate that repair cluster formation as determined by SMLM and the relaxation (i.e., the remaining 53BP1 tags no longer fulfill the cluster definition) is cell type dependent and may be functionally explained and correlated to cell specific radio-sensitivity. The present study demonstrates that SMLM is a highly appropriate method for investigations of spatiotemporal protein organization in cell nuclei and how it influences the cell decision for a particular repair pathway at a given DSB site.
- MeSH
- Tumor Suppressor p53-Binding Protein 1 metabolism MeSH
- Microscopy, Confocal methods MeSH
- Cells, Cultured MeSH
- Humans MeSH
- Cell Line, Tumor MeSH
- Recombinational DNA Repair * MeSH
- Protein Transport MeSH
- Single Molecule Imaging methods MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
... Protein 299 -- Control of Gene Expression 369 -- WAYS OF WORKING WITH CELLS 439 -- Analyzing Cells, Molecules ... ... - INTERNAL ORGANIZATION OF THE CELL 565 -- Membrane Structure 565 -- Membrane Transport of Small Molecules ... ... -- Together in Cells 44 -- Some Polar Molecules Form Acids and Bases in Water 45 -- A Cell Is Formed ... ... Molecules Can Be Touched, Imaged, and Moved Using -- Atomic Force Microscopy 548 -- Superresolution ... ... Can Overcome -- Diffraction-Limited Resolution 549 -- Superresolution Can Also be Achieved Using Single-Molecule ...
Sixth edition xxxiv, 1430 stran v různém stránkování : ilustrace (převážně barevné) ; 29 cm
- MeSH
- Cells * MeSH
- Molecular Biology MeSH
- Conspectus
- Biochemie. Molekulární biologie. Biofyzika
- NML Fields
- molekulární biologie, molekulární medicína
- NML Publication type
- učebnice vysokých škol
... can readily be cleaved into functionally distinct fragments. 130 -- 4-4 The immunoglobulin molecule ... ... is flexible, especially at the hinge region. 131 -- 4-5 The domains of an immunoglobulin molecule have ... ... structures. - 141 -- 4-13 Peptides are stably bound to MHC molecules, and also serve to stabilize the ... ... MHC molecule on the cell surface. 142 -- 4-14 MHC class I molecules bind short peptides of 8-10 amino ... ... vesicles. 211 -- 6-8 A specialized MHC class Il-like molecule catalyzes loading of MHC class II molecules ...
8th ed. xix, 868 s. : il., tab.
- MeSH
- Immunity MeSH
- Immune System MeSH
- Immunotherapy MeSH
- Publication type
- Monograph MeSH
- Conspectus
- Patologie. Klinická medicína
- NML Fields
- alergologie a imunologie
- biologie
... Hurt Us 4 -- Viruses Are the Ultimate Parasites 6 -- Changes in Cells Underlie Evolution 6 -- Even Single ... ... Cells Can Have Sex 7 -- We Develop from a Single Cell 8 -- Stem Cells, Fundamental to Forming Tissues ... ... and Organs, Offer Medical Opportunities 8 -- |Q The Molecules of a Cell 9 -- Small Molecules Carry Energy ... ... May Be Good, Bad, or Indifferent 13 -- The Work of Cells 14 -- Cells Build and Degrade Numerous Molecules ... ... 625 -- Signaling Molecules Can Act Locally or at a Distance 625 -- Binding of Signaling Molecules Activates ...
6th ed. xxxvii, 1150 s. : il., tab. ; 29 cm
- MeSH
- Cell Biology MeSH
- Molecular Biology MeSH
- Publication type
- Monograph MeSH
- Conspectus
- Biochemie. Molekulární biologie. Biofyzika
- NML Fields
- biologie
- cytologie, klinická cytologie
... 47 -- High-Resolution Imaging of the ACh Receptor 47 -- Open and Closed States of the ACh Receptor 48 ... ... 53 -- Pore Formation in Voltage-Activated Channels 54 -- High-Resolution Imaging of a Potassium Channel ... ... 101 -- Threshold and Refractory Period 102 -- Gating Currents 103 -- Activation and Inactivation of Single ... ... Analysis of the End-Plate Potential 209 -- Quantum Content at Neuronal Synapses 211 -- Number of Molecules ... ... -- The Identification of Transmitters 244 -- Neurotransmitters as Messengers 245 -- Transmitter Molecules ...
4th ed. XIX,580,A-8,B-5,C-8,G-7,BB-60,I-10 : obr.,tab.,grafy.
