Native antigen
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Antigen-presenting cells (APCs) are master regulators of the immune response by directly interacting with T cells to orchestrate distinct functional outcomes. Several types of professional APC exist, including conventional dendritic cells, B cells and macrophages, and numerous other cell types have non-classical roles in antigen presentation, such as thymic epithelial cells, endothelial cells and granulocytes. Accumulating evidence indicates the presence of a new family of APCs marked by the lineage-specifying transcription factor retinoic acid receptor-related orphan receptor-γt (RORγt) and demonstrates that these APCs have key roles in shaping immunity, inflammation and tolerance, particularly in the context of host-microorganism interactions. These RORγt+ APCs include subsets of group 3 innate lymphoid cells, extrathymic autoimmune regulator-expressing cells and, potentially, other emerging populations. Here, we summarize the major findings that led to the discovery of these RORγt+ APCs and their associated functions. We discuss discordance in recent reports and identify gaps in our knowledge in this burgeoning field, which has tremendous potential to advance our understanding of fundamental immune concepts.
- MeSH
- antigen prezentující buňky metabolismus MeSH
- endoteliální buňky MeSH
- jaderné receptory - podrodina 1, skupina F, člen 3 * metabolismus MeSH
- lidé MeSH
- lymfocyty * MeSH
- přirozená imunita MeSH
- transportní proteiny metabolismus MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- přehledy MeSH
Salmonella vaccines used in poultry in the EU are based on attenuated strains of either Salmonella serovar Enteritidis or Typhimurium which results in a decrease in S. Enteritidis and S. Typhimurium but may allow other Salmonella serovars to fill an empty ecological niche. In this study we were therefore interested in the early interactions of chicken immune system with S. Infantis compared to S. Enteritidis and S. Typhimurium, and a role of O-antigen in these interactions. To reach this aim, we orally infected newly hatched chickens with 7 wild type strains of Salmonella serovars Enteritidis, Typhimurium and Infantis as well as with their rfaL mutants and characterized the early Salmonella-chicken interactions. Inflammation was characterized in the cecum 4 days post-infection by measuring expression of 43 different genes. All wild type strains stimulated a greater inflammatory response than any of the rfaL mutants. However, there were large differences in chicken responses to different wild type strains not reflecting their serovar classification. The initial interaction between newly-hatched chickens and Salmonella was found to be dependent on the presence of O-antigen but not on its structure, i.e. not on serovar classification. In addition, we observed that the expression of calbindin or aquaporin 8 in the cecum did not change if inflammatory gene expression remained within a 10 fold fluctuation, indicating the buffering capacity of the cecum, preserving normal gut functions even in the presence of minor inflammatory stimuli.
- MeSH
- akvaporiny metabolismus MeSH
- cékum imunologie metabolismus MeSH
- kalbindiny metabolismus MeSH
- kur domácí imunologie MeSH
- nemoci drůbeže imunologie metabolismus MeSH
- O-antigeny metabolismus MeSH
- přirozená imunita MeSH
- Salmonella enterica imunologie metabolismus MeSH
- salmonelová infekce u zvířat imunologie metabolismus MeSH
- séroskupina MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
BACKGROUND: Prostate-specific membrane antigen (PSMA), also known as glutamate carboxypeptidase II (GCPII), is generally recognized as a diagnostic and therapeutic cancer antigen and a molecular address for targeted imaging and drug delivery studies. Due to its significance in cancer research, numerous monoclonal antibodies (mAbs) against GCPII have been described and marketed in the past decades. Unfortunately, some of these mAbs are poorly characterized, which might lead to their inappropriate use and misinterpretation of the acquired results. METHODS: We collected the 13 most frequently used mAbs against GCPII and quantitatively characterized their binding to GCPII by enzyme-linked immunosorbent assay (ELISA) and surface plasmon resonance (SPR). Using a peptide library, we mapped epitopes recognized by a given mAb. Finally, we assessed the applicability of these mAbs to routine experimental setups, including Western blotting, immunohistochemistry, and flow cytometry. RESULTS: ELISA and SPR analyses revealed that mAbs J591, J415, D2B, 107-1A4, GCP-05, and 2G7 bind preferentially to GCPII in native form, while mAbs YPSMA-1, YPSMA-2, GCP-02, GCP-04, and 3E6 bind solely to denatured GCPII. mAbs 24.4E6 and 7E11-C5.3 recognize both forms of GCPII. Additionally, we determined that GCP-02 and 3E6 cross-react with mouse GCPII, while GCP-04 recognizes GCPII and GCPIII proteins from both human and mouse. CONCLUSION: This comparative analysis provides the first detailed quantitative characterization of the most commonly used mAbs against GCPII and can serve as a guideline for the scientific community to use them in a proper and efficient way.
- MeSH
- adenokarcinom diagnóza imunologie metabolismus MeSH
- ELISA MeSH
- imunohistochemie MeSH
- lidé MeSH
- mapování epitopu MeSH
- monoklonální protilátky analýza diagnostické užití imunologie MeSH
- nádorové buněčné linie MeSH
- nádory prostaty diagnóza imunologie metabolismus MeSH
- prostatický specifický antigen imunologie metabolismus MeSH
- průtoková cytometrie MeSH
- western blotting MeSH
- Check Tag
- lidé MeSH
- mužské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- srovnávací studie MeSH
BACKGROUND AND AIMS: The egg protein ovalbumin (OVA) belongs to six most frequent food allergens. We investigated how thermal processing influences its ability to induce allergic symptoms and immune responses in mouse model of food allergy. METHODOLOGY/PRINCIPAL FINDINGS: Effect of increased temperature (70°C and 95°C) on OVA secondary structure was characterized by circular dichroism and by the kinetics of pepsin digestion with subsequent HPLC. BALB/c mice were sensitized intraperitoneally and challenged with repeated gavages of OVA or OVA heated to 70°C (h-OVA). Levels of allergen-specific serum antibodies were determined by ELISA (IgA and IgGs) or by β-hexosaminidase release test (IgE). Specific activities of digestive enzymes were determined in brush border membrane vesicles of jejunal enterocytes. Cytokine production and changes in regulatory T cells in mesenteric lymph nodes and spleen were assessed by ELISA and FACS. Heating of OVA to 70°C caused mild irreversible changes in secondary structure compared to boiling to 95°C (b-OVA), but both OVA treatments led to markedly different digestion kinetics and Tregs induction ability in vitro, compared to native OVA. Heating of OVA significantly decreased clinical symptoms (allergic diarrhea) and immune allergic response on the level of IgE, IL-4, IL-5, IL-13. Furthermore, h-OVA induced lower activities of serum mast cell protease-1 and enterocyte brush border membrane alkaline phosphatase as compared to native OVA. On the other hand h-OVA stimulated higher IgG2a in sera and IFN-γ secretion by splenocytes. CONCLUSIONS: Minor irreversible changes in OVA secondary structure caused by thermal processing changes both its digestion and antigenic epitopes formation, which leads to activation of different T cell subpopulations, induces shift towards Th1 response and ultimately reduces its allergenicity.
- MeSH
- cytokiny krev imunologie MeSH
- imunoglobulin E krev imunologie MeSH
- modely nemocí na zvířatech MeSH
- myši MeSH
- ovalbumin chemie imunologie MeSH
- potravinová alergie krev imunologie MeSH
- prezentace antigenu imunologie MeSH
- regulační T-lymfocyty imunologie MeSH
- sekundární struktura proteinů MeSH
- vysoká teplota MeSH
- zvířata MeSH
- Check Tag
- myši MeSH
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
The frequency of sandfly-host contacts can be measured by host antibody levels against sandfly salivary proteins. Recombinant salivary proteins are suggested to represent a valid replacement for salivary gland homogenate (SGH); however, it is necessary to prove that such antigens are recognized by antibodies against various populations of the same species. Phlebotomus perniciosus (Diptera: Psychodidae) is the main vector of Leishmania infantum (Trypanosomatida: Trypanosomatidae) in southwest Europe and is widespread from Portugal to Italy. In this study, sera were sampled from naturally exposed dogs from distant regions, including Campania (southern Italy), Umbria (central Italy) and the metropolitan Lisbon region (Portugal), where P. perniciosus is the unique or principal vector species. Sera were screened for anti-P. perniciosus antibodies using SGH and 43-kDa yellow-related recombinant protein (rSP03B). A robust correlation between antibodies recognizing SGH and rSP03B was detected in all regions, suggesting substantial antigenic cross-reactivity among different P. perniciosus populations. No significant differences in this relationship were detected between regions. Moreover, rSP03B and the native yellow-related protein were shown to share similar antigenic epitopes, as canine immunoglobulin G (IgG) binding to the native protein was inhibited by pre-incubation with the recombinant form. These findings suggest that rSP03B should be regarded as a universal marker of sandfly exposure throughout the geographical distribution of P. perniciosus.
- MeSH
- antigeny analýza MeSH
- hmyz - vektory parazitologie fyziologie MeSH
- hmyzí proteiny analýza MeSH
- kousnutí a bodnutí hmyzem epidemiologie etiologie MeSH
- Leishmania infantum fyziologie MeSH
- leishmanióza parazitologie veterinární MeSH
- nemoci psů epidemiologie etiologie MeSH
- Phlebotomus parazitologie fyziologie MeSH
- psi MeSH
- rekombinantní proteiny analýza MeSH
- slinné proteiny a peptidy analýza MeSH
- zvířata MeSH
- Check Tag
- psi MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- Geografické názvy
- Itálie epidemiologie MeSH
- Portugalsko epidemiologie MeSH
Presence of mutated and/or structurally modified (e.g., denatured, aggregated) protein p53 form is associated with several disorders such as Alzheimer's disease, Parkinson's disease, prion diseases, and many types of tumours. The aim of this work was to distinguish native, denatured and aggregated form of full-length p53 by flow injection analysis coupled with electrochemical detector (FIA-ED). Firstly FIA-ED method used for protein native form determination was optimized (detection limit 45.8 amol per 5 mul injection; 3 x S/N). In addition the technique was applied to identify p53 structural forms (denatured and aggregated). It was found out that denatured form provides about three times higher electrochemical response (protein structure unfolding, approach of more electroactive centers - aminoacid residues - towards electrode surface) in comparison with native form. On the other hand, aggregated form offers lower response (steric eclipse of electroactive protein parts) when compared with the signal of native form. The obtained data show that we are not only able to sensitively determine native, denatured, and aggregated structural forms of p53 protein but also to distinguish them.
Background: The Human Cell Differentiation Molecules (HCDM) organizes Human Leukocyte Differentiation Antigen (HLDA) workshops to test and name clusters of antibodies that react with a specific antigen. These cluster of differentiation (CD) markers have provided the scientific community with validated antibody clones, consistent naming of targets and reproducible identification of leukocyte subsets. Still, quantitative CD marker expression profiles and benchmarking of reagents at the single-cell level are currently lacking. Objective: To develop a flow cytometric procedure for quantitative expression profiling of surface antigens on blood leukocyte subsets that is standardized across multiple research laboratories. Methods: A high content framework to evaluate the titration and reactivity of Phycoerythrin (PE)-conjugated monoclonal antibodies (mAbs) was created. Two flow cytometry panels were designed: an innate cell tube for granulocytes, dendritic cells, monocytes, NK cells and innate lymphoid cells (12-color) and an adaptive lymphocyte tube for naive and memory B and T cells, including TCRγδ+, regulatory-T and follicular helper T cells (11-color). The potential of these 2 panels was demonstrated via expression profiling of selected CD markers detected by PE-conjugated antibodies and evaluated using 561 nm excitation. Results: Using automated data annotation and dried backbone reagents, we reached a robust workflow amenable to processing hundreds of measurements in each experiment in a 96-well plate format. The immunophenotyping panels enabled discrimination of 27 leukocyte subsets and quantitative detection of the expression of PE-conjugated CD markers of interest that could quantify protein expression above 400 units of antibody binding capacity. Expression profiling of 4 selected CD markers (CD11b, CD31, CD38, CD40) showed high reproducibility across centers, as well as the capacity to benchmark unique clones directed toward the same CD3 antigen. Conclusion: We optimized a procedure for quantitative expression profiling of surface antigens on blood leukocyte subsets. The workflow, bioinformatics pipeline and optimized flow panels enable the following: 1) mapping the expression patterns of HLDA-approved mAb clones to CD markers; 2) benchmarking new antibody clones to established CD markers; 3) defining new clusters of differentiation in future HLDA workshops.
- MeSH
- antigeny povrchové * metabolismus MeSH
- buňky NK metabolismus MeSH
- CD antigeny metabolismus MeSH
- leukocyty MeSH
- lidé MeSH
- monoklonální protilátky MeSH
- přirozená imunita * MeSH
- průběh práce MeSH
- průtoková cytometrie metody MeSH
- referenční standardy MeSH
- reprodukovatelnost výsledků MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
BACKGROUND: Optimised purification steps for concentrating trace target native antigens are needed. Combining the p-aminobenzamidine ligand with protease inactivation enables partial purification of mite non-protease allergens lacking proteases. OBJECTIVE: We sought to analyse in detail proteins obtained using this method from eight species of synanthropic acaridid mites and tested IgE reactivity using pooled human sera. MATERIALS AND METHODS: Proteins affinity bound to p-aminobenzamidine as a ligand were identified by MALDI TOF/TOF. After electroblotting, the proteins were visualised using the fluorescent SYPRO-Ruby protein blot stain, and IgE reactivity was further analysed using pooled human sera collected from patients allergic to house dust mites. RESULTS: MS/MS identification confirmed previous results that no proteases were purified. Protein patterns corresponding to the allergens Der f 7, Der f 30 and actins indicated that these proteins are purified using p-aminobenzamidine and are present across a wide spectrum of acaridid mites. When using Dermatophagoides farinae, apolipophorins (Der f 14), chitinase-like Der f 15 and 18, 70-kDa heat shock protein, and a Der f Alt a10 allergen homolog (gi|37958173) were also detected. The target antigens tropomyosins and paramyosins showed similar IgE binding among the mite species tested. IgE reactivity with miscellaneous D. farinae antigen was also observed. CONCLUSIONS: Partial purification of mite non-protease antigens using a strategy combining p-aminobenzamidine with protease inactivation was verified by 1D-E and 2D-E analyses. IgE binding to p-aminobenzamidine-purified native non-protease mite antigens was tested using pooled sera. This preliminary study allows for further work on individual serum samples, allowing confirmation of immunoreactivity.
The autoimmune regulator (Aire) serves an essential function for T cell tolerance by promoting the "promiscuous" expression of tissue antigens in thymic epithelial cells. Aire is also detected in rare cells in peripheral lymphoid organs, but the identity of these cells is poorly understood. Here, we report that Aire protein-expressing cells in lymph nodes exhibit typical group 3 innate lymphoid cell (ILC3) characteristics such as lymphoid morphology, absence of "classical" hematopoietic lineage markers, and dependence on RORγt. Aire+ cells are more frequent among lineage-negative RORγt+ cells of peripheral lymph nodes as compared with mucosa-draining lymph nodes, display a unique Aire-dependent transcriptional signature, express high surface levels of MHCII and costimulatory molecules, and efficiently present an endogenously expressed model antigen to CD4+ T cells. These findings define a novel type of ILC3-like cells with potent APC features, suggesting that these cells serve a function in the control of T cell responses.
- MeSH
- adhezní molekula epiteliálních buněk metabolismus MeSH
- antigen prezentující buňky imunologie MeSH
- antigeny CD11 metabolismus MeSH
- fenotyp MeSH
- genetická transkripce MeSH
- jaderné receptory - podrodina 1, skupina F, člen 3 metabolismus MeSH
- lymfatické uzliny cytologie MeSH
- lymfocyty imunologie metabolismus MeSH
- MHC antigeny II. třídy metabolismus MeSH
- myši inbrední BALB C MeSH
- myši knockoutované MeSH
- myši MeSH
- přirozená imunita MeSH
- regulace genové exprese MeSH
- transkripční faktory genetika metabolismus MeSH
- zvířata MeSH
- Check Tag
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
