Role of basal cells in nasal polyp epithelium in the pathophysiology of eosinophilic chronic rhinosinusitis (eCRS)

滋賀医科大学博士（医学）Background: Basal cell hyperplasia is commonly observed in nasal polyp epithelium of eosinophilic chronic rhinosinusitis (eCRS). We examined the function and mechanisms of basal cell hyperplasia in the pathophysiology of eCRS. Methods: We found that normal human bronchial epithelial (NHBE) cells obtained basal cell characteristics when cultured with PneumaCult™-Ex Plus Medium. Most of the cells passaged three times expressed basal cell surface markers CD49f and CD271 by flow cytometry, and basal cell nuclear marker p63 by immunohistochemical staining. We named these NHBE cells with basal cell characteristics cultured Basal-like cells (cBC), and NHBE cells cultured with BEGM™ cultured Epithelial cells (cEC). The characteristics of cBC and cEC were examined and compared by RNA sequencing, RT-PCR, ELISA, and cell proliferation studies. Results: RNA sequencing revealed that cBC showed higher gene expression of thymic stromal lymphopoietin (TSLP), IL-8, TLR3, and TLR4, and lower expression of PAR-2 compared with cEC. The mRNA expression of TSLP, IL-8, TLR3, and TLR4 was significantly increased in cBC, and that of PAR-2 was significantly increased in cEC by RT-PCR. Poly(I:C)-induced TSLP production and LPS-induced IL-8 production were significantly increased in cBC. IL-4 and IL-13 stimulated the proliferation of cBC. Finally, the frequency of p63-positive basal cells was increased in nasal polyp epithelium of eCRS, and Ki67-positive proliferating cells were increased in p63-positive basal cells. Conclusions: Type 2 cytokines IL-4 and IL-13 induce basal cell hyperplasia, and basal cells exacerbate type 2 inflammation by producing TSLP in nasal polyp of eCRS.令和6年度doctoral thesi

Proximity-Induced Nucleic Acid Degrader (PINAD) approach to targeted RNA degradation using small molecules

Nature has evolved intricate machinery to target and degrade RNA, and some of these molecular mechanisms can be adapted for therapeutic use. Small interfering RNAs and RNase H-inducing oligonucleotides have yielded therapeutic agents against diseases that cannot be tackled using protein-centered approaches. Because these therapeutic agents are nucleic acid-based, they have several inherent drawbacks which include poor cellular uptake and stability. Here we report a new approach to target and degrade RNA using small molecules, proximity-induced nucleic acid degrader (PINAD). We have utilized this strategy to design two families of RNA degraders which target two different RNA structures within the genome of SARS-CoV-2: G-quadruplexes and the betacoronaviral pseudoknot. We demonstrate that these novel molecules degrade their targets using in vitro, in cellulo, and in vivo SARS-CoV-2 infection models. Our strategy allows any RNA binding small molecule to be converted into a degrader, empowering RNA binders that are not potent enough to exert a phenotypic effect on their own. PINAD raises the possibility of targeting and destroying any disease-related RNA species, which can greatly expand the space of druggable targets and diseases.info:eu-repo/semantics/publishedVersio

Dichloro Butenediamides as Irreversible Site‐Selective Protein Conjugation Reagent

We describe maleic-acid derivatives as robust cysteine-selective reagents for protein labelling with comparable kinetics and superior stability relative to maleimides. Diamide and amido-ester derivatives proved to be efficient protein-labelling species with a common mechanism in which a spontaneous cyclization occurs upon addition to cysteine. Introduction of chlorine atoms in their structures triggers ring hydrolysis or further conjugation with adjacent residues, which results in conjugates that are completely resistant to retro-Michael reactions in the presence of biological thiols and human plasma. By controlling the microenvironment of the reactive site, we can control selectivity towards the hydrolytic pathway, forming homogeneous conjugates. The method is applicable to several scaffolds and enables conjugation of different payloads. The synthetic accessibility of these reagents and the mild conditions required for fast and complete conjugation together with the superior stability of the conjugates make this strategy an important alternative to maleimides in bioconjugation

Sublingual immunotherapy decreases the avidity of antigen-specific IgE in patients with Japanese cedar pollinosis

journal articl

Structure-Guided Approach for the Development of MUC1-Glycopeptide-Based Cancer Vaccines with Predictable Responses

Mucin-1 (MUC1) glycopeptides are exceptional candidates for potential cancer vaccines. However, their autoantigenic nature often results in a weak immune response. To overcome this drawback, we carefully engineered synthetic antigens with precise chemical modifications. To be effective and stimulate an anti-MUC1 response, artificial antigens must mimic the conformational dynamics of natural antigens in solution and have an equivalent or higher binding affinity to anti-MUC1 antibodies than their natural counterparts. As a proof of concept, we have developed a glycopeptide that contains noncanonical amino acid (2S,3R)-3-hydroxynorvaline. The unnatural antigen fulfills these two properties and effectively mimics the threonine-derived antigen. On the one hand, conformational analysis in water shows that this surrogate explores a landscape similar to that of the natural variant. On the other hand, the presence of an additional methylene group in the side chain of this analog compared to the threonine residue enhances a CH/π interaction in the antigen/antibody complex. Despite an enthalpy–entropy balance, this synthetic glycopeptide has a binding affinity slightly higher than that of its natural counterpart. When conjugated with gold nanoparticles, the vaccine candidate stimulates the formation of specific anti-MUC1 IgG antibodies in mice and shows efficacy comparable to that of the natural derivative. The antibodies also exhibit cross-reactivity to selectively target, for example, human breast cancer cells. This investigation relied on numerous analytical (e.g., NMR spectroscopy and X-ray crystallography) and biophysical techniques and molecular dynamics simulations to characterize the antigen–antibody interactions. This workflow streamlines the synthetic process, saves time, and reduces the need for extensive, animal-intensive immunization procedures. These advances underscore the promise of structure-based rational design in the advance of cancer vaccine development

Structure-Guided Approach for the Development of MUC1-Glycopeptide-Based Cancer Vaccines with Predictable Responses

Mucin-1(MUC1)glycopeptidesareexceptionalcandidatesforpotentialcancervaccines.However,theirautoantigenicnatureoftenresultsinaweakimmuneresponse.Toovercomethisdrawback,wecarefullyengineeredsyntheticantigenswithprecisechemicalmodifications.Tobeeffectiveandstimulateananti-MUC1response,artificialantigensmustmimictheconforma-tionaldynamicsofnaturalantigensinsolutionandhaveanequivalentorhigherbindingaffinitytoanti-MUC1antibodiesthantheirnaturalcounterparts.Asa proofofconcept,wehavedevelopeda glycopeptidethatcontainsnoncanonicalaminoacid(2S,3R)-3-hydroxynorvaline.Theunnaturalantigenfulfillsthesetwopropertiesandeffectivelymimicsthethreonine-derivedantigen.Ontheonehand,conformationalanalysisinwatershowsthatthissurrogateexploresalandscapesimilartothatofthenaturalvariant.Ontheotherhand,thepresenceofanadditionalmethylenegroupinthesidechainofthisanalogcomparedtothethreonineresidueenhancesa CH/interactionintheantigen/antibodycomplex.Despiteanenthalpyentropybalance,thissyntheticglycopeptidehasabindingaffinityslightlyhigherthanthatofitsnaturalcounterpart.Whenconjugatedwithgoldnanoparticles,thevaccinecandidatestimulatestheformationofspecificanti-MUC1IgGantibodiesinmiceandshowsefficacycomparabletothatofthenaturalderivative.Theantibodiesalsoexhibitcross-reactivitytoselectivelytarget,forexample,humanbreastcancercells.Thisinvestigationreliedonnumerousanalytical(e.g.,NMRspectroscopyandX-raycrystallography)andbiophysicaltechniquesandmoleculardynamicssimulationstocharacterizetheantigenantibodyinteractions.Thisworkflowstreamlinesthesyntheticprocess,savestime,andreducestheneedforextensive,animal-intensiveimmunizationprocedures.Theseadvancesunderscorethepromiseofstructure-basedrationaldesignintheadvanceofcance

Structure-Based Design of Potent Tumor-Associated Antigens: Modulation of Peptide Presentation by Single-Atom O/S or O/Se Substitutions at the Glycosidic Linkage.

GalNAc-glycopeptides derived from mucin MUC1 are an important class of tumor-associated antigens. α- O-glycosylation forces the peptide to adopt an extended conformation in solution, which is far from the structure observed in complexes with a model anti-MUC1 antibody. Herein, we propose a new strategy for designing potent antigen mimics based on modulating peptide/carbohydrate interactions by means of O → S/Se replacement at the glycosidic linkage. These minimal chemical modifications bring about two key structural changes to the glycopeptide. They increase the carbohydrate-peptide distance and change the orientation and dynamics of the glycosidic linkage. As a result, the peptide acquires a preorganized and optimal structure suited for antibody binding. Accordingly, these new glycopeptides display improved binding toward a representative anti-MUC1 antibody relative to the native antigens. To prove the potential of these glycopeptides as tumor-associated MUC1 antigen mimics, the derivative bearing the S-glycosidic linkage was conjugated to gold nanoparticles and tested as an immunogenic formulation in mice without any adjuvant, which resulted in a significant humoral immune response. Importantly, the mice antisera recognize cancer cells in biopsies of breast cancer patients with high selectivity. This finding demonstrates that the antibodies elicited against the mimetic antigen indeed recognize the naturally occurring antigen in its physiological context. Clinically, the exploitation of tumor-associated antigen mimics may contribute to the development of cancer vaccines and to the improvement of cancer diagnosis based on anti-MUC1 antibodies. The methodology presented here is of general interest for applications because it may be extended to modulate the affinity of biologically relevant glycopeptides toward their receptors