We combined cell-free ribosome display and cell-based yeast display selection to build specific protein binders to the extracellular domain of the human interleukin 9 receptor alpha (IL-9Rα). The target, IL-9Rα, is the receptor involved in the signalling pathway of IL-9, a pro-inflammatory cytokine medically important for its involvement in respiratory diseases. The successive use of modified protocols of ribosome and yeast displays allowed us to combine their strengths-the virtually infinite selection power of ribosome display and the production of (mostly) properly folded and soluble proteins in yeast display. The described experimental protocol is optimized to produce binders highly specific to the target, including selectivity to common proteins such as BSA, and proteins potentially competing for the binder such as receptors of other cytokines. The binders were trained from DNA libraries of two protein scaffolds called 57aBi and 57bBi developed in our laboratory. We show that the described unconventional combination of ribosome and yeast displays is effective in developing selective small protein binders to the medically relevant molecular target.
The effect of various pretreatments on the bonding of a resin cement to resin-composite CAD/CAM blocks (RCBs) was examined. The surface of dispersed-filler RCBs (DF-RCBs) and a polymer infiltrated ceramic network RCB (PICN-RCB) was roughened using hydrofluoric acid etching (HF) or sandblasting, and followed by silanization and/or universal adhesive (UA) application. Microtensile bond strength (µTBS), surface roughness parameters (arithmetical mean height (Sa); developed interfacial area ratio (Sdr)), and critical surface energy (γc) were determined. For most DF-RCBs, the highest µTBS was obtained using HF+UA. UA application to DF-RCBs resulted in similar or higher µTBS compared to silanization, which indicates that silane treatment is not crucial for DF-RCBs, especially after HF. In contrast, the highest µTBS to PICN-RCB was obtained with silanization. Both roughening pretreatments significantly increased the surface roughness parameters and the γc of all RCBs. The γc was positively correlated with Sa (r=0.756, p<0.001) and Sdr (r=0.837, p<0.001).
- MeSH
- keramika MeSH
- klinické protokoly MeSH
- kyselina fluorovodíková MeSH
- pevnost v tahu MeSH
- povrchové vlastnosti MeSH
- pryskyřičné cementy MeSH
- receptory interleukinů - společná gama-podjednotka MeSH
- silany MeSH
- složené pryskyřice MeSH
- testování materiálů MeSH
- vazba zubní * MeSH
- Publikační typ
- časopisecké články MeSH
Both variants affecting splice sites and those in splicing regulatory elements (SREs) can impair pre-mRNA splicing, eventually leading to severe diseases. Despite the availability of many prediction tools, prognosis of splicing affection is not trivial, especially when SREs are involved. Here, we present data on 92 in silico-/55 minigene-analysed variants detected in genes responsible for the primary immunodeficiencies development (namely BTK, CD40LG, IL2RG, SERPING1, STAT3, and WAS). Of 20 splicing-affecting variants, 16 affected splice site while 4 disrupted potential SRE. The presence or absence of splicing defects was confirmed in 30 of 32 blood-derived patients' RNAs. Testing prediction tools performance, splice site disruptions and creations were reliably predicted in contrast to SRE-affecting variants for which just ESRseq, ΔHZEI-scores and EX-SKIP predictions showed promising results. Next, we found an interesting pattern in cryptic splice site predictions. These results might help PID-diagnosticians and geneticists cope with potential splicing-affecting variants.
- MeSH
- buňky Hep G2 MeSH
- dítě MeSH
- exony MeSH
- HeLa buňky MeSH
- kojenec MeSH
- komplement C1 - inaktivátory genetika MeSH
- lidé MeSH
- messenger RNA genetika MeSH
- mutace MeSH
- předškolní dítě MeSH
- protein Wiskottova-Aldrichova syndromu genetika MeSH
- receptory interleukinů - společná gama-podjednotka genetika MeSH
- rekombinantní fúzní proteiny genetika MeSH
- sestřih RNA * MeSH
- syndromy imunologické nedostatečnosti genetika MeSH
- transkripční faktor STAT3 genetika MeSH
- tyrosinkinasy genetika MeSH
- U937 buňky MeSH
- Check Tag
- dítě MeSH
- kojenec MeSH
- lidé MeSH
- předškolní dítě MeSH
- Publikační typ
- časopisecké články MeSH
Xenograft models represent a promising tool to study the pathogenesis of hematological malignancies. To establish a reliable and appropriate in vivo model of aggressive human B-cell leukemia and lymphoma we xenotransplanted four p53-mutated cell lines and one ATM-mutated cell line into immunodeficient NOD/SCID IL2Rγ-null mice. The cell lines MEC-1, SU-DHL-4, JEKO-1, REC-1, and GRANTA-519 were transplanted intraperitoneally or subcutaneously and the engraftment was investigated using immunohistochemistry and flow cytometry. We found significant differences in engraftment efficiency. MEC-1, JEKO-1 and GRANTA-519 cell lines engrafted most efficiently, while SU-DHL-4 cells did not engraft at all. MEC-1 and GRANTA-519 massively infiltrated organs and the whole intraperitoneal cavity showing very aggressive growth. In addition, GRANTA-519 cells massively migrated to the bone marrow regardless of the transplantation route. The MEC-1 and GRANTA-519 cells can be especially recommended for in vivo study of p53-mutated chronic lymphocytic leukemia and ATM-mutated mantle cell lymphoma, respectively.
- MeSH
- ATM protein genetika MeSH
- biologické markery MeSH
- chronická lymfatická leukemie genetika metabolismus patologie MeSH
- genový knockout MeSH
- heterografty MeSH
- lidé MeSH
- lymfom z plášťových buněk genetika patologie MeSH
- modely nemocí na zvířatech MeSH
- mutace * MeSH
- myši inbrední NOD MeSH
- myši knockoutované MeSH
- myši SCID MeSH
- myši MeSH
- nádorové buněčné linie MeSH
- nádorový supresorový protein p53 genetika MeSH
- receptory interleukinů - společná gama-podjednotka genetika MeSH
- transplantace heterologní MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- mužské pohlaví MeSH
- myši MeSH
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Mantle cell lymphoma (MCL) is an aggressive type of B-cell non-Hodgkin lymphoma (NHL) associated with poor prognosis. Animal models of MCL are scarce. We established and characterized various in vivo models of metastatic human MCL by tail vein injection of either primary cells isolated from patients with MCL or established MCL cell lines (Jeko-1, Mino, Rec-1, Hbl-2, and Granta-519) into immunodeficient NOD.Cg-Prkdc(scid) Il2rg(tm1Wjl)/SzJ mice. MCL infiltration was assessed with immunohistochemistry (tissues) and flow cytometry (peripheral blood). Engraftment of primary MCL cells was observed in 7 out of 12 patient samples. The pattern of engraftment of primary MCL cells varied from isolated involvement of the spleen to multiorgan infiltration. On the other hand, tumor engraftment was achieved in all five MCL cell lines used and lymphoma involvement of murine bone marrow, spleen, liver, and brain was observed. Overall survival of xenografted mice ranged from 22 ± 1 to 54 ± 3 days depending on the cell line used. Subsequently, we compared the gene expression profile (GEP) and phenotype of the engrafted MCL cells compared with the original in vitro growing cell lines (controls). We demonstrated that engrafted MCL cells displayed complex changes of GEP, protein expression, and sensitivity to cytotoxic agents when compared with controls. We further demonstrated that our MCL mouse models could be used to test the therapeutic activity of systemic chemotherapy, monoclonal antibodies, or angiogenesis inhibitors. The characterization of MCL murine models is likely to aid in improving our knowledge in the disease biology and to assist scientists in the preclinical and clinical development of novel agents in relapsed/refractory MCL patients.
- MeSH
- imunofenotypizace MeSH
- imunohistochemie MeSH
- játra metabolismus MeSH
- Kaplanův-Meierův odhad MeSH
- kostní dřeň metabolismus MeSH
- lidé středního věku MeSH
- lidé MeSH
- lymfom z plášťových buněk farmakoterapie genetika metabolismus MeSH
- modely nemocí na zvířatech * MeSH
- mozek metabolismus MeSH
- myši inbrední NOD MeSH
- myši knockoutované MeSH
- myši SCID MeSH
- myši MeSH
- nádorové buněčné linie MeSH
- nádorové buňky kultivované MeSH
- receptory interleukinů - společná gama-podjednotka nedostatek genetika MeSH
- regulace genové exprese u nádorů * MeSH
- senioři MeSH
- slezina metabolismus MeSH
- transkriptom genetika MeSH
- transplantace heterologní MeSH
- zvířata MeSH
- Check Tag
- lidé středního věku MeSH
- lidé MeSH
- mužské pohlaví MeSH
- myši MeSH
- senioři MeSH
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Altered immune response, including low-grade inflammatory processes, is involved in the pathogenesis of schizophrenia, a chronic psychiatric disorder with complex etiology. Distinct gene variants of a number of pro-inflammatory and chemotactic cytokines together with their receptors associate with this disorder. Interleukin-2 receptor gamma (IL-2RG) represents an important signaling component of many interleukin receptors and so far, no data on the functional state of this receptor in schizophrenia have been reported. The aim of this study was to investigate mRNA expression of the IL2RG gene (IL2RG) in schizophrenia patients in comparison with healthy subjects (controls). Total RNA was isolated from peripheral blood of 66 schizophrenia patients and 99 healthy subjects of Armenian population. The mRNA expression was determined by quantitative real-time polymerase chain reaction (RT-PCR) using PSMB2 as housekeeping gene. IL2RG mRNA expression was upregulated in peripheral blood of patients in comparison with controls (patients vs. controls, median [interquartile range]: 2.080 [3.428-1.046] vs. 0.324 [0.856-0.000], p<0.0001). In conclusion, our findings suggest that over-expression of the IL2RG gene may be implicated in altered immune response in schizophrenia and contribute to the pathomechanisms of this disorder.
- MeSH
- dospělí MeSH
- lidé středního věku MeSH
- lidé MeSH
- messenger RNA krev genetika MeSH
- receptory interleukinů - společná gama-podjednotka genetika MeSH
- receptory interleukinu-2 genetika MeSH
- schizofrenie genetika imunologie patofyziologie MeSH
- studie případů a kontrol MeSH
- Check Tag
- dospělí MeSH
- lidé středního věku MeSH
- lidé MeSH
- mužské pohlaví MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
IL-2 and IL-15 are structurally relative cytokines that share two receptor subunits, CD132 (γ(c) chain) and CD122 (β chain). However, the expression pattern and physiological role of IL-2 and IL-15 private receptor α chains CD25 and IL-15Rα, respectively, are strikingly different. CD25, together with CD122 and CD132, forms a trimeric high affinity IL-2 receptor that is expressed and functions on cells acquiring an IL-2 signal. Conversely, IL-15Rα is expressed and binds IL-15 with high affinity per se already in the endoplasmic reticulum of the IL-15 producing cells and it presents IL-15 to cells expressing CD122/CD132 dimeric receptor in trans. Thus, while IL-2 is secreted almost exclusively by activated T cells and acts as a free molecule, IL-15 is expressed mostly by myeloid cells and works as a cell surface-associated cytokine. Interestingly, the in vivo biological activity of IL-2 can be dramatically increased through complexing with certain anti-IL-2 mAbs; such IL-2/anti-IL-2 mAbs immunocomplexes selectively stimulate the proliferation of a distinct population of immune cells, depending on the clone of the anti-IL-2 mAb used. IL-2/S4B6 mAb immunocomplexes are highly stimulatory for CD122(high) populations (memory CD8(+) T and NK cells) and intermediately also for CD25(high) populations (Treg and activated T cells), while IL-2/JES6-1 mAb immunocomplexes enormously expand only CD25(high) cells. Although IL-2 immunocomplexes are much more potent than IL-2 in vivo, they show comparable to slightly lower activity in vitro. The in vivo biological activity of IL-15 can be dramatically increased through complexing with recombinant IL-15Rα-Fc chimera; however, IL-15/IL-15Rα-Fc complexes are significantly more potent than IL-15 both in vivo and in vitro. In this review we summarize and discuss the features and biological relevance of IL-2/anti-IL-2 mAbs and IL-15/IL-15Rα-Fc complexes, and try to foreshadow their potential in immunological research and immunotherapy.
- MeSH
- buňky NK cytologie účinky léků imunologie MeSH
- CD8-pozitivní T-lymfocyty cytologie účinky léků imunologie MeSH
- imunoglobuliny - Fc fragmenty chemie MeSH
- imunokomplex genetika imunologie farmakologie MeSH
- interleukin-15 genetika imunologie farmakologie MeSH
- interleukin-2 genetika imunologie farmakologie MeSH
- lidé MeSH
- monoklonální protilátky chemie MeSH
- myši MeSH
- receptor interleukinu-15 - alfa-podjednotka genetika imunologie MeSH
- receptor interleukinu-2 - alfa-podjednotka genetika imunologie MeSH
- receptory interleukinů - společná gama-podjednotka genetika imunologie MeSH
- regulace genové exprese MeSH
- regulační T-lymfocyty cytologie účinky léků imunologie MeSH
- signální transdukce MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- přehledy MeSH
