The development of canine immunotolerant monoclonal antibodies can accelerate the invention of new medicines for both canine and human diseases. We develop a methodology to clone the naive, somatically mutated variable domain repertoire from canine B cell mRNA using 5'RACE PCR. A set of degenerate primers were then designed and used to clone variable domain genes into archival "holding" plasmid libraries. These archived variable domain genes were then combinatorially ligated to produce a scFv M13 phage library. Next-generation long-read and short-read DNA sequencing methodologies were developed to annotate features of the cloned library including CDR diversity and IGHV/IGKV/IGLV subfamily distribution. A synthetic immunoglobulin G was developed from this scFv library to the canine immune checkpoint receptor PD-1. This synthetic platform can be used to clone and annotate archived antibody variable domain genes for use in perpetuity in order to develop improved preclinical models for the treatment of complex human diseases.
- MeSH
- Programmed Cell Death 1 Receptor immunology MeSH
- Single-Chain Antibodies * immunology genetics MeSH
- Humans MeSH
- Antibodies, Monoclonal immunology genetics MeSH
- Neoplasms immunology therapy MeSH
- Peptide Library MeSH
- Dogs MeSH
- Recombinant Proteins immunology genetics MeSH
- Translational Research, Biomedical MeSH
- Animals MeSH
- Check Tag
- Humans MeSH
- Dogs MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
Trombotická trombocytopenická purpura (TTP) je vzácné, život ohrožující onemocnění patřící do skupiny trombotických mikroangiopatií (TMA). Je charakterizováno přítomností mikroangiopatické hemolytické anemie, těžké trombocytopenie a ischemického postižení cílových orgánů. Podstatou onemocnění je závažný deficit metaloproteázy štěpící von Willebrandův faktor (vWF). Deficit enzymu způsobuje hromadění velkých multimerů vWF, na které se aktivně váží trombocyty, s následným vznikem mikrotrombů v cévním řečišti. Stěžejním vyšetřením pro diagnostiku TTP je prokázání snížené aktivity enzymu ADAMTS13 pod 10 % (0,1 IU/ml). Léčba TTP by měla být zahájena co nejdříve, již při vyslovení podezření na TTP, často ještě před znalostí výsledků aktivity enzymu ADAMTS13. Současný standard léčby akutní ataky získané TTP zahrnuje výměnnou plazmaferézu, imunosupresi a kaplacizumab. O úspěšnosti léčby rozhoduje rychlá diagnostika TTP vč. vyšetření aktivity ADAMTS13 a včasné zahájení komplexní léčby. Nedílnou součástí léčby je také dlouhodobé sledování aktivity onemocnění, vč. monitorace aktivity ADAMTS13.
Thrombotic thrombocytopenic purpura (TTP) is a rare, life-threatening disease belonging to the group of thrombotic microangiopathies (TMA). It is characterised by the presence of microangiopathic haemolytic anaemia, severe thrombocytopenia, and ischaemic end-organ damage. The underlying cause of the disease is a severe deficiency of von Willebrand factor (vWF) cleaving metalloprotease. The enzyme deficiency causes the accumulation of large multimers of vWF to which platelets bind actively, with subsequent formation of microthrombi in the microcirculation. The most important test for the diagnosis of TTP is reduced ADAMTS13 enzyme activity below 10% (0.1 IU/ml). Treatment of TTP should be initiated as early as possible when it is suspected, often before the ADAMTS13 activity results are known. The current standard of care for acute attacks of acquired TTP includes therapeutic plasma exchange, immunosuppression, and caplacizumab. A rapid diagnosis of TTP, including ADAMTS13 activity testing, and early initiation of comprehensive treatment are critical to treatment success. Long-term monitoring of disease activity, including monitoring of ADAMTS13 activity, is also a necessary part of the treatment
- Keywords
- kaplacizumab,
- MeSH
- Adrenal Cortex Hormones therapeutic use MeSH
- Single-Domain Antibodies therapeutic use MeSH
- Humans MeSH
- Antibodies, Monoclonal therapeutic use MeSH
- Plasmapheresis MeSH
- ADAMTS13 Protein metabolism MeSH
- Rituximab administration & dosage therapeutic use MeSH
- Purpura, Thrombotic Thrombocytopenic * diagnosis therapy MeSH
- Check Tag
- Humans MeSH
- Publication type
- Review MeSH
x
x
- Keywords
- studie EXXELERATE,
- MeSH
- Adalimumab pharmacology therapeutic use MeSH
- Data Analysis MeSH
- Biological Therapy classification MeSH
- Certolizumab Pegol * pharmacology therapeutic use MeSH
- Tumor Necrosis Factor Inhibitors pharmacology therapeutic use MeSH
- Humans MeSH
- Randomized Controlled Trials as Topic MeSH
- Arthritis, Rheumatoid * diagnosis drug therapy MeSH
- Rheumatoid Factor * analysis MeSH
- Treatment Outcome MeSH
- Check Tag
- Humans MeSH
NMDA receptors (NMDARs) are ionotropic glutamate receptors that play a key role in excitatory neurotransmission. The number and subtype of surface NMDARs are regulated at several levels, including their externalization, internalization, and lateral diffusion between the synaptic and extrasynaptic regions. Here, we used novel anti-GFP (green fluorescent protein) nanobodies conjugated to either the smallest commercially available quantum dot 525 (QD525) or the several nanometer larger (and thus brighter) QD605 (referred to as nanoGFP-QD525 and nanoGFP-QD605, respectively). Targeting the yellow fluorescent protein-tagged GluN1 subunit in rat hippocampal neurons, we compared these two probes to a previously established larger probe, a rabbit anti-GFP IgG together with a secondary IgG conjugated to QD605 (referred to as antiGFP-QD605). The nanoGFP-based probes allowed faster lateral diffusion of the NMDARs, with several-fold increased median values of the diffusion coefficient (D). Using thresholded tdTomato-Homer1c signals to mark synaptic regions, we found that the nanoprobe-based D values sharply increased at distances over 100 nm from the synaptic edge, while D values for antiGFP-QD605 probe remained unchanged up to a 400 nm distance. Using the nanoGFP-QD605 probe in hippocampal neurons expressing the GFP-GluN2A, GFP-GluN2B, or GFP-GluN3A subunits, we detected subunit-dependent differences in the synaptic localization of NMDARs, D value, synaptic residence time, and synaptic-extrasynaptic exchange rate. Finally, we confirmed the applicability of the nanoGFP-QD605 probe to study differences in the distribution of synaptic NMDARs by comparing to data obtained with nanoGFPs conjugated to organic fluorophores, using universal point accumulation imaging in nanoscale topography and direct stochastic optical reconstruction microscopy.SIGNIFICANCE STATEMENT Our study systematically compared the localization and mobility of surface NMDARs containing GFP-GluN2A, GFP-GluN2B, or GFP-GluN3A subunits expressed in rodent hippocampal neurons, using anti-green fluorescent protein (GFP) nanobodies conjugated to the quantum dot 605 (nanoGFP-QD605), as well as nanoGFP probes conjugated with small organic fluorophores. Our comprehensive analysis showed that the method used to delineate the synaptic region plays an important role in the study of synaptic and extrasynaptic pools of NMDARs. In addition, we showed that the nanoGFP-QD605 probe has optimal parameters for studying the mobility of NMDARs because of its high localization accuracy comparable to direct stochastic optical reconstruction microscopy and longer scan time compared with universal point accumulation imaging in nanoscale topography. The developed approaches are readily applicable to the study of any GFP-labeled membrane receptors expressed in mammalian neurons.
- MeSH
- Hippocampus metabolism MeSH
- Immunoglobulin G metabolism MeSH
- Single-Domain Antibodies * metabolism MeSH
- Rabbits MeSH
- Rats MeSH
- Neurons metabolism MeSH
- Receptors, N-Methyl-D-Aspartate * metabolism MeSH
- Mammals MeSH
- Synapses physiology MeSH
- Animals MeSH
- Check Tag
- Rabbits MeSH
- Rats MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
- MeSH
- Certolizumab Pegol * administration & dosage MeSH
- Humans MeSH
- Uveitis, Anterior * epidemiology etiology drug therapy MeSH
- Spondylarthritis drug therapy pathology MeSH
- Check Tag
- Humans MeSH
- Publication type
- Clinical Study MeSH
- Keywords
- kaplacizumab,
- MeSH
- Adrenal Cortex Hormones pharmacology therapeutic use MeSH
- Single-Domain Antibodies pharmacology therapeutic use MeSH
- Middle Aged MeSH
- Humans MeSH
- Rituximab pharmacology therapeutic use MeSH
- Purpura, Thrombotic Thrombocytopenic * diagnosis drug therapy MeSH
- Exchange Transfusion, Whole Blood MeSH
- Treatment Outcome MeSH
- Check Tag
- Middle Aged MeSH
- Humans MeSH
- Female MeSH
- Publication type
- Case Reports MeSH
