- MeSH
- banky biologického materiálu * organizace a řízení MeSH
- biomedicínský výzkum MeSH
- lidé MeSH
- uchovávání tkání MeSH
- Check Tag
- lidé MeSH
- Geografické názvy
- Česká republika MeSH
Human cytomegalovirus (HCMV) is an important pathogen with multiple immune evasion strategies, including virally facilitated degradation of host antiviral restriction factors. Here, we describe a multiplexed approach to discover proteins with innate immune function on the basis of active degradation by the proteasome or lysosome during early-phase HCMV infection. Using three orthogonal proteomic/transcriptomic screens to quantify protein degradation, with high confidence we identified 35 proteins enriched in antiviral restriction factors. A final screen employed a comprehensive panel of viral mutants to predict viral genes that target >250 human proteins. This approach revealed that helicase-like transcription factor (HLTF), a DNA helicase important in DNA repair, potently inhibits early viral gene expression but is rapidly degraded during infection. The functionally unknown HCMV protein UL145 facilitates HLTF degradation by recruiting the Cullin4 E3 ligase complex. Our approach and data will enable further identifications of innate pathways targeted by HCMV and other viruses.
- MeSH
- cytomegalovirové infekce genetika imunologie virologie MeSH
- Cytomegalovirus genetika imunologie fyziologie MeSH
- DNA vazebné proteiny chemie genetika imunologie MeSH
- imunitní únik MeSH
- lidé MeSH
- proteiny chemie genetika imunologie MeSH
- proteomika MeSH
- stabilita proteinů MeSH
- transkripční faktory chemie genetika imunologie MeSH
- virové proteiny chemie genetika imunologie MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
CD58 is an adhesion molecule that is known to play a critical role in costimulation of effector cells and is intrinsic to immune synapse structure. Herein, we describe a virally encoded gene that inhibits CD58 surface expression. Human cytomegalovirus (HCMV) UL148 was necessary and sufficient to promote intracellular retention of CD58 during HCMV infection. Blocking studies with antagonistic anti-CD58 mAb and an HCMV UL148 deletion mutant (HCMV∆UL148) with restored CD58 expression demonstrated that the CD2/CD58 axis was essential for the recognition of HCMV-infected targets by CD8+ HCMV-specific cytotoxic T lymphocytes (CTLs). Further, challenge of peripheral blood mononuclear cells ex vivo with HCMV∆UL148 increased both CTL and natural killer (NK) cell degranulation against HCMV-infected cells, including NK-driven antibody-dependent cellular cytotoxicity, showing that UL148 is a modulator of the function of multiple effector cell subsets. Our data stress the effect of HCMV immune evasion functions on shaping the immune response, highlighting the capacity for their potential use in modulating immunity during the development of anti-HCMV vaccines and HCMV-based vaccine vectors.
- MeSH
- buněčná imunita * MeSH
- buňky NK imunologie patologie MeSH
- CD8-pozitivní T-lymfocyty imunologie patologie MeSH
- cytomegalovirové infekce genetika imunologie patologie MeSH
- Cytomegalovirus genetika imunologie MeSH
- imunitní únik * MeSH
- lidé MeSH
- proteiny virové fúze genetika imunologie MeSH
- transformované buněčné linie MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
53BP1 is a very well-known protein that is recruited to DNA lesions. The focal accumulation of p53 binding protein, 53BP1, is a main feature indicating the repair of spontaneous or irradiation-induced foci (IRIF). Thus, here, we addressed the question of whether mutations in the TP53 gene, which often affect the level of p53 protein, can change the recruitment of 53BP1 to γ- or UVA-irradiated chromatin. In various TP53 mutants, we observed a distinct accumulation of 53BP1 protein to UV-induced DNA lesions: in R273C mutants, 53BP1 appeared transiently at DNA lesions, during 10-30 min after irradiation; the mutation R282W was responsible for accumulation of 53BP1 immediately after UVA-damage; and in L194F mutants, the first appearance of 53BP1 protein at the lesions occurred during 60-70 min. These results showed that specific mutations in the TP53 gene stand behind not only different levels of p53 protein, but also affect the localized kinetics of 53BP1 protein in UVA-damaged chromatin. However, after γ-irradiation, only G245S mutation in TP53 gene was associated with surprisingly decreased level of 53BP1 protein. In other mutant cell lines, levels of 53BP1 were not affected by γ-rays. To these effects, we conversely found a distinct number of 53BP1-positive irradiation-induced foci in various TP53 mutants. The R280K, G245S, L194F mutations, or TP53 deletion were also characterized by radiation-induced depletion in MDC1 protein. Moreover, in mutant cells, an interaction between MDC1 and 53BP1 proteins was abrogated when compared with wild-type counterpart. Together, the kinetics of 53BP1 accumulation at UV-induced DNA lesions is different in various TP53 mutant cells. After γ-irradiation, despite changes in a number and a volume of 53BP1-positive foci, levels of 53BP1 protein were relatively stable. Here, we showed a link between the status of MDC1 protein and TP53 gene, which specific mutations caused radiation-induced MDC1 down-regulation. This observation is significant, especially with regard to radiotherapy of tumors with abrogated function of TP53 gene.
- MeSH
- 53BP1 metabolismus MeSH
- down regulace MeSH
- jaderné proteiny nedostatek metabolismus MeSH
- lidé MeSH
- mutace * MeSH
- nádorové buňky kultivované MeSH
- nádorový supresorový protein p53 nedostatek genetika metabolismus MeSH
- poškození DNA * MeSH
- trans-aktivátory nedostatek metabolismus MeSH
- ultrafialové záření * MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
The human cytomegalovirus (HCMV) US12 family consists of ten sequentially arranged genes (US12-21) with poorly characterized function. We now identify novel natural killer (NK) cell evasion functions for four members: US12, US14, US18 and US20. Using a systematic multiplexed proteomics approach to quantify ~1300 cell surface and ~7200 whole cell proteins, we demonstrate that the US12 family selectively targets plasma membrane proteins and plays key roles in regulating NK ligands, adhesion molecules and cytokine receptors. US18 and US20 work in concert to suppress cell surface expression of the critical NKp30 ligand B7-H6 thus inhibiting NK cell activation. The US12 family is therefore identified as a major new hub of immune regulation.
- MeSH
- buňky NK imunologie MeSH
- Cytomegalovirus imunologie patogenita MeSH
- imunitní únik MeSH
- imunologické faktory antagonisté a inhibitory MeSH
- interakce hostitele a patogenu * MeSH
- lidé MeSH
- membránové proteiny antagonisté a inhibitory MeSH
- proteomika MeSH
- virové proteiny metabolismus MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Research Support, N.I.H., Extramural MeSH
Monoclonal antibodies are leading agents for therapeutic treatment of human diseases, but are limited in use by the paucity of clinically relevant models for validation. Sporadic canine tumours mimic the features of some human equivalents. Developing canine immunotherapeutics can be an approach for modeling human disease responses. Rituximab is a pioneering agent used to treat human hematological malignancies. Biologic mimics that target canine CD20 are just being developed by the biotechnology industry. Towards a comparative canine-human model system, we have developed a novel anti-CD20 monoclonal antibody (NCD1.2) that binds both human and canine CD20. NCD1.2 has a sub-nanomolar Kd as defined by an octet red binding assay. Using FACS, NCD1.2 binds to clinically derived canine cells including B-cells in peripheral blood and in different histotypes of B-cell lymphoma. Immunohistochemical staining of canine tissues indicates that the NCD1.2 binds to membrane localized cells in Diffuse Large B-cell lymphoma, Marginal Zone Lymphoma, and other canine B-cell lymphomas. We cloned the heavy and light chains of NCD1.2 from hybridomas to determine whether active scaffolds can be acquired as future biologics tools. The VH and VL genes from the hybridomas were cloned using degenerate primers and packaged as single chains (scFv) into a phage-display library. Surprisingly, we identified two scFv (scFv-3 and scFv-7) isolated from the hybridoma with bioactivity towards CD20. The two scFv had identical VH genes but different VL genes and identical CDR3s, indicating that at least two light chain mRNAs are encoded by NCD1.2 hybridoma cells. Both scFv-3 and scFv-7 were cloned into mammalian vectors for secretion in CHO cells and the antibodies were bioactive towards recombinant CD20 protein or peptide. The scFv-3 and scFv-7 were cloned into an ADEPT-CPG2 bioconjugate vector where bioactivity was retained when expressed in bacterial systems. These data identify a recombinant anti-CD20 scFv that might form a useful tool for evaluation in bioconjugate-directed anti-CD20 immunotherapies in comparative medicine.
- MeSH
- antigeny CD20 * chemie genetika imunologie metabolismus MeSH
- buněčné linie MeSH
- epitopy imunologie MeSH
- exprese genu MeSH
- hybridomy imunologie metabolismus MeSH
- jednořetězcové protilátky imunologie farmakologie MeSH
- klonování DNA MeSH
- lehké řetězce imunoglobulinů genetika MeSH
- lidé MeSH
- molekulární sekvence - údaje MeSH
- myši MeSH
- peptidová knihovna MeSH
- peptidy chemie metabolismus MeSH
- psi MeSH
- rekombinantní fúzní proteiny farmakologie MeSH
- sekvence aminokyselin MeSH
- sekvenční seřazení MeSH
- specificita protilátek imunologie MeSH
- těžké řetězce imunoglobulinů genetika MeSH
- tvorba protilátek imunologie MeSH
- vazba proteinů imunologie MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- myši MeSH
- psi MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Publikační typ
- abstrakt z konference MeSH
Vývoj rekombinantních terapeutických protilátek je v poslední době jednou z nejrychleji se rozvíjejících disciplín aplikovaného biomedicínského výzkumu. Rekombinantní monoklonální protilátky nalézají stále větší uplatnění v biologické terapii řady závažných lidských chorob a jsou v současné době nenahraditelnou součástí komplexní protinádorové terapie. Terapeutické protilátky využívané v klinické praxi prošly značným vývojem. Z prvních protilátek produkovaných v myších, které jako vedlejší účinek indukovaly silnou imunitní odpověď, byly metodami rekombinantní DNA a genové manipulace vyvinuty plně lidské protilátky s výrazně omezenými vedlejšími účinky a zároveň se zvýšenou specifitou a efektivitou. V této práci jsou shrnuty základní poznatky o terapeutických monoklonálních protilátkách, jejich historický vývoj a přehled metodických přístupů vedoucích k vývoji účinnějších, ale také bezpečnějších protilátek.
Development of recombinant therapeutic antibodies is recently one of the fastest growing disciplines of applied biomedical research. Recombinant monoclonal antibodies are increasingly applied in biological therapy of many serious human diseases and are currently an irreplaceable part of a comprehensive cancer therapy. First mouse therapeutic antibodies had only limited applicability due to the strong immune response; however, technological advances enabled engineering of antibodies with increased specificity and efficacy, and on the other hand with reduced adverse effects due to lower antigenicity. This review provides a summary of knowledge about recombinant therapeutic antibodies, their mechanism of action and approaches how to improve their efficacy. Key words: antineoplastic agents – immunoglobulins – humanized monoclonal antibodies – therapeutic antibodies – recombinant antibodies This study was supported by the European Regional Development Fund and the State Budget of the Czech Republic (RECAMO, CZ.1.05/2.1.00/03.0101), MEYS – NPS I – LO1413 and MH CZ – DRO (MMCI, 00209805). The authors declare they have no potential conflicts of interest concerning drugs, products, or services used in the study. The Editorial Board declares that the manuscript met the ICMJE “uniform requirements” for biomedical papers. Submitted: 20. 4. 2015 Accepted: 26. 6. 2015
- Klíčová slova
- terapeutické protilátky, protinádorová léčiva,
- MeSH
- farmaceutická chemie * MeSH
- humanizované monoklonální protilátky * chemie MeSH
- imunoglobuliny genetika MeSH
- lidé MeSH
- monoklonální protilátky dějiny chemie MeSH
- nádory farmakoterapie MeSH
- protilátky genetika MeSH
- protinádorové látky * MeSH
- rekombinantní DNA MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- práce podpořená grantem MeSH
- přehledy MeSH
- Publikační typ
- abstrakt z konference MeSH
