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ε‐poly‐l‐lysine (ε‐PLL) is a natural polypeptide/polycation originating from bacteria. Thanks to its antifungal and antibacterial properties, it is the subject of extensive research in the food and medical industries. ε‐PLL is also used to develop biomaterials in a broad range of applications, such as drug delivery, wound healing, or antimicrobial coatings. Indeed, loading ε‐PLL inside nanoparticles, functionalizing implant surfaces with ε‐PLL, or developing hydrogels based on reactions between ε‐PLL and other polymers can improve the materials properties, leading to biocompatible, antibacterial, and antifungal systems. These characteristics are necessary not only for the development of biomaterials, for their integrity in a biological environment, but also for improving the performances of medical devices. Moreover, ε‐PLL can be used as an alternative to antibiotics as its mechanism of action reduces the bacterial resistance risk compared with antibiotics. Finally, "smart" systems using ε‐PLL may be developed, with controllable material degradation or drug delivery via pH or temperature variations. This review sought to gather the latest research on the development of antimicrobial biomaterials based on the ε‐PLL polypeptide. [ABSTRACT FROM AUTHOR]

Background: Like innate cells, B cells also express Pattern Recognition Receptors (PRRs) to detect danger signal such as tissue damage or pathogen intrusion. Production of specific antibodies by plasma cells results from the activation and differentiation of B cells following three signals: (i) antigen recognition by B Cell Receptors, (ii) recognition of danger and (iii) T-cell help. However, it is unclear whether T-cell help is dispensable for B cell activation and differentiation or not. Few studies have investigated the role of cytosolic PRRs such as NOD1 in B cell differentiation. Methods: We used splenic C57BL6J B cells to evaluate NOD1 expression and then assessed the effect of stimulation with C12-iE-DAP, a NOD1 ligand, with or without CD40L as a T-cell help signal on B-cell responses globally or according to their CD23 expression level. Results: We showed that murine B cells express NOD1 and that the presence of C12-iE-DAP induces activation, proliferation and initiates differentiation in plasma cells even in the absence of a T-dependent signal. Surprisingly, CD23high B cells are more sensitive than CD23low B cells to stimulation. Conclusion: Our results suggest that the NLR pathway could induce antibody development during infections and be exploited to develop more effective vaccination. [ABSTRACT FROM AUTHOR]

Click-based reactions were conducted at the surface of small unilamellar vesicles (SUVs) to provide onto-vesicle chemistry with efficient innovative ready-for-use tools. For that purpose, four amphiphilic molecules were designed to insert into bilayers while presenting a reactive functional head. In this manner, a dioleylglycero-ethoxy-ethoxy-ethoxy-ethanamine (DOG-PEG 4 -NH 2 ) was chosen as a common platform while the reactive amine head was converted into several electrophilic functions. Thus, two dioleylglycerol-based cyclooctyne anchors were prepared: cyclooct-1-yn-3-glycolic acid-based anchor (DOG-COA) and 1-fluorocyclooct-2-ynecarboxylic acid-based anchor (DOG-FCOA). The last one differed from the first one in that a fluorine atom reinforces the electrophilic properties of the unsaturated bond. In addition, a third dioleylglycerol-based triphenylphosphine (DOG-PPh 3 ) was synthesized for the first time. These three innovative amphiphilic anchors were designed to react with any azide-based biomolecule following copper-free Huisgen 1,4-cycloaddition and Staudinger ligation, respectively. A fourth anchor bearing a 3,4-dibromomaleimide ring (DOG-DBM) was also unprecedentedly synthesized, to be further substituted by two thiols. Model reactions conducted in solution with either model biotinyl azide or model biotinyl disulfide gave good to total conversions and excellent isolated yields. The four new anchors were inserted into SUVs whose formula is classically used in in vivo biology. Stability and surface overall electrostatic charge were in the expected range and constant over the study. Then, the functionalized liposomes were ligated to biotin-based reagents and the experimental conditions were finely tuned to optimize the conversion. The biotinyl liposomes were demonstrated functional and totally accessible in an affinity test based on biotin scaffold quantification. Finally, DOG-FCOA’s reactivity was confronted to that of DOG-DBM in a ‘one-pot’ orthogonal reaction. (Biotin-S) 2 and TAMRA-N 3 (tetramethylcarboxyrhodamine azide) were successively conjugated to the liposome suspension in a successful manner. These data implement and reinforce the interest of bioorthogonal click-like reactions onto lipid nanoparticles. [ABSTRACT FROM AUTHOR]

Immunotherapy aiming at enhancing innate and acquired host immunity is a promising approach for cancer treatment. The invariant NKT (iNKT) cell ligand α-galactosylceramide (α-GalCer) holds great promise in cancer therapy, although several concerns limit its use in clinics, including the uncontrolled response it promotes when delivered in a nonvectorized form. Therefore, development of delivery systems to in vivo target immune cells might be a valuable option to optimize iNKT cell-based antitumor responses. Using dendritic cell (DC)-depleted mice, DC transfer experiments, and in vivo active cell targeting, we show that presentation of α-GalCer by DCs not only triggers optimal primary iNKT cell stimulation, but also maintains secondary iNKT cell activation after challenge. Furthermore, targeted delivery of a-GalCer to CD8α+ DCs, by means of anti-DEC205 decorated nanoparticles, enhances iNKT cell-based transactivation of NK cells, DCs, and γδ T cells. We report that codelivery of α-GalCer and protein Ag to CD8α+ DCs triggers optimal Ag-specific Ab and cytotoxic CD8+ T cell responses. Finally, we show that targeting nanoparticles containing α-GalCer and Ag to CD8α+ DCs promotes potent antitumor responses, both in prophylactic and in therapeutic settings. Our data may have important implications in tumor immunotherapy and vaccine development. [ABSTRACT FROM AUTHOR]

Abstract: The activity of synthetic interfering nucleic acids (siRNAs) relies on the capacity of delivery systems to efficiently transport nucleic acids into the cytosol of target cells. The pyridylthiourea-grafted 25KDa polyethylenimine (πPEI) is an excellent carrier for siRNA delivery into cells and it was extensively investigated in this report. Quantification of the siRNA-mediated gene silencing efficiency indicated that the πPEI specific delivery activity at the cell level may be measured and appears relatively constant in various cell lines. Delivery experiments assaying inhibitors of various entry pathways or concanamycin A, an inhibitor of the H+/ATPase vacuolar pump showed that the πPEI/siRNA polyplexes did not require any specific entry mode but strongly relied on vacuolar acidification for functional siRNA delivery. Next, πPEI polyplexes containing a siRNA targeting the transcription factor HIF-1α, known to be involved in tumor progression, were locally injected into mice xenografted with a human glioblastoma. A 55% reduction of the level of the target mRNA was observed at doses comparable to those used in vitro when the πPEI delivery activity was calculated per cell. Altogether, our study underscores the usefulness of “simple”/rough cationic polymers for siRNA delivery despite their intrinsic limitations. The study underscores as well as that bottom–up strategies make sense. The in vitro experiments can precede in vivo administration and be of high value for selection of the carrier with enhanced specific delivery activity and parallel other research aiming at improving synthetic delivery systems for resilience in the blood and for enhanced tissue-targeting capacity. [Copyright &y& Elsevier]

The mRNA vaccine technology has promising applications to fight infectious diseases as demonstrated by the licensing of two mRNA-based vaccines, Comirnaty® (Pfizer/BioNtech) and Spikevax® (Moderna), in the context of the Covid-19 crisis. Safe and effective delivery systems are essential to the performance of these vaccines and lipid nanoparticles (LNPs) able to entrap, protect and deliver the mRNA in vivo are considered by many as the current "best in class". Nevertheless, current mRNA/LNP vaccine technology has still some limitations, one of them being thermostability, as evidenced by the ultracold distribution chain required for the licensed vaccines. We found that the thermostability of mRNA/LNP, could be improved by a novel imidazole modified lipid, DOG-IM4, in combination with standard helper lipids. DOG-IM4 comprises an ionizable head group consisting of imidazole, a dioleoyl lipid tail and a short flexible polyoxyethylene spacer between the head and tail. Here we describe the synthesis of DOG-IM4 and show that DOG-IM4 LNPs confer strong immunization properties to influenza HA mRNA in mice and macaques and a remarkable stability to the encapsulated mRNA when stored liquid in phosphate buffered saline at 4 °C. We speculate the increased stability to result from some specific attributes of the lipid's imidazole head group. [Display omitted] [ABSTRACT FROM AUTHOR]

Background.Exogenous activation of pulmonary invariant natural killer T (iNKT) cells, a population of lipid-reactive [alpha][beta] T lymphocytes, with use of mucosal [alpha]-galactosylceramide ([alpha]-GalCer) administration, is a promising approach to control respiratory bacterial infections. We undertook the present study to characterize mechanisms leading to [alpha]-GalCer-mediated protection against lethal infection with Streptococcus pneumoniae serotype 1, a major respiratory pathogen in humans. Methods and Results.[alpha]-GalCer was administered by the intranasal route before infection with S. pneumoniae. We showed that respiratory dendritic cells (DCs), most likely the CD103(+) subset, play a major role in the activation (IFN-[gamma] and IL-17 release) of pulmonary iNKT cells, whereas alveolar and interstitial macrophages are minor players. After challenge, S. pneumoniae was rapidly (4 hours) eliminated in the alveolar spaces, a phenomenon that depended on respiratory DCs and neutrophils, but not macrophages, and on the early production of both IFN-[gamma] and IL-17. Protection was also associated with the synthesis of various interferon-dependent and IL-17-associated genes as revealed by transcriptomic analysis. Conclusions.These data imply a new function for pulmonary CD103(+) DCs in mucosal activation of iNKT cells and establish a critical role for both IFN-[gamma] and IL-17 signalling pathways in mediating the innate immune response to S. pneumoniae. [ABSTRACT FROM AUTHOR]

Background.Exogenous activation of pulmonary invariant natural killer T (iNKT) cells, a population of lipid-reactive αβ T lymphocytes, with use of mucosal α-galactosylceramide (α-GalCer) administration, is a promising approach to control respiratory bacterial infections. We undertook the present study to characterize mechanisms leading to α-GalCer-mediated protection against lethal infection with Streptococcus pneumoniae serotype 1, a major respiratory pathogen in humans.Methods and Results.α-GalCer was administered by the intranasal route before infection with S. pneumoniae. We showed that respiratory dendritic cells (DCs), most likely the CD103+ subset, play a major role in the activation (IFN-γ and IL-17 release) of pulmonary iNKT cells, whereas alveolar and interstitial macrophages are minor players. After challenge, S. pneumoniae was rapidly (4 hours) eliminated in the alveolar spaces, a phenomenon that depended on respiratory DCs and neutrophils, but not macrophages, and on the early production of both IFN-γ and IL-17. Protection was also associated with the synthesis of various interferon-dependent and IL-17–associated genes as revealed by transcriptomic analysis.Conclusions.These data imply a new function for pulmonary CD103+ DCs in mucosal activation of iNKT cells and establish a critical role for both IFN-γ and IL-17 signalling pathways in mediating the innate immune response to S. pneumoniae. [ABSTRACT FROM AUTHOR]

Human gingival fibroblasts (HGFs) cell sheets have apotentialuse for in vivo wound healing due to the ability of HGFs to adopta contractile phenotype which is typically expressed during extracellularmatrix tissue remodeling. For this purpose, we developed a chemicallydetachable platform based on poly(allylamine hydrochloride)/poly(styrenesulfonate) multilayer film built on a sacrificial precursor film whichserved as a substrate for HGF cell layer formation. The sacrificialprecursor film, based on disulfide-containing polycation and polyanion,is degradable under mild conditions compatible for cell sheet detachment.Cellular viability and cell phenotype analysis of HGF show that thedesigned platform promotes cell phenotype switch into contractilephenotype, maintained after cell sheet lift-off. This contractilephenotype is acquired by fibroblasts during in vivo wound healingand tissue remodeling. HGFs cell sheet fragments, obtained by thisdetachment process, could be cultured later on showing a good retentionof the typical spindle-shape of differentiated cells after 10 daysof culture. HGFs cell sheets have great potential applications asautologous substrates for tissue repair and cellular synthetic platformsfor research on connective tissue diseases or evaluation of noveltherapeutic agents. [ABSTRACT FROM AUTHOR]

Abstract: Invariant Natural Killer T (iNKT) cells have potent immunostimulatory activities that could be exploited for human therapies. The high-affinity CD1d antigen α-galactosylceramide analogue KRN7000 (KRN) activates a cascade of anti-tumor effector cells and clinical studies have already had some initial success. To improve the efficacy of the treatment, strategies that aim to vectorize KRN would be valuable. In this study, we intended to characterize and compare the effect of KRN encapsulated in poly(lactic-co-glycolic acid) (PLGA)-based nanoparticles (NPs, 90nm) and microparticles instead of macroparticles (MPs, 715nm) on the iNKT cell response. Our data show that whatever the size of the particles, vectorized KRN induced potent primary activation of iNKT cells in vitro and in vivo. We show that endocytosis of PLGA-based particles by dendritic cells is mediated by a clathrin-dependent manner and that this event is important to stimulate iNKT cells. Finally, we report that KRN vectorized in NPs and MPs exhibited different behaviours in vivo in terms of iNKT cell expansion and responsiveness to a recall stimulation. Collectively, our data validate the concept that KRN encapsulated in PLGA-based particles can be used as delivery systems to activate iNKT cells in vitro and in vivo. [Copyright &y& Elsevier]

Abstract: Success of synthetic interfering nucleic acids (siRNAs)-based therapy relies almost exclusively on effective, safe and preferably nanometric delivery systems which can be easily prepared, even at high concentrations. We prepared by chemical synthesis various self-assembling polymers to entrap siRNAs into stable polyplexes outside cells but with a disassembly potential upon sensing endosomal acidity. Our results revealed that pyridylthiourea-grafted polyethylenimine (πPΕΙ) followed the above-mentioned principles. It led to above 90% siRNA-mediated gene silencing in vitro on U87 cells at 10nM siRNA concentration and did not have a hemolytic activity. Assembly of siRNA/πPΕΙ at high concentration was then studied and 4.5% glucose solution, pH 6.0, yielded stable colloidal solutions with sizes slightly below 100nm for several hours. A single injection of these concentrated siRNA polyplexes into luciferase-expressing human glioblastoma tumors, which were subcutaneously xenografted into nude mice, led to a significant 30% siRNA-mediated luciferase gene silencing 4days post-injection. Our results altogether substantiate the potential of self-assembling cationic polymers with a pH-sensitive disassembly switch for siRNA delivery in vitro and also in vivo experiments. [Copyright &y& Elsevier]

One important function of conventional dendritic cells (cDC) is their high capacity to capture, process and present Ag to T lymphocytes. Mouse splenic cDC subtypes, including CD8α+ and CD8α- cDC, are not identical in their Ag presenting and T cell priming functions. Surprisingly, few studies have reported functional differences between CD4- and CD4+ CD8α- cDC subsets. We show that, when loaded in vitro with OVA peptide or whole protein, and in steady-state conditions, splenic CD4- and CD4+ cDC are equivalent in their capacity to prime and direct CD4+ and CD8+ T cell differentiation. In contrast, in response to α-galactosylceramide (α-GalCer), CD4- and CD4+ cDC differentially activate invariant Natural Killer T (iNKT) cells, a population of lipid-reactive non-conventional T lymphocytes. Both cDC subsets equally take up α-GalCer in vitro and in vivo to stimulate the iNKT hybridoma DN32.D3, the activation of which depends solely on TCR triggering. On the other hand, and relative to their CD4+ counterparts, CD4- cDC more efficiently stimulate primary iNKT cells, a phenomenon likely due to differential production of co-factors (including IL-12) by cDC. Our data reveal a novel functional difference between splenic CD4+ and CD4- cDC subsets that may be important in immune responses. [ABSTRACT FROM AUTHOR]

Abstract: In an effort to develop new biomaterial coatings, it was shown that polyelectrolyte multilayers constitute a very powerful tool to render surfaces biologically active. The challenge is to multi-functionalize surfaces in a controlled way. We show here, for the first time, that it is possible to functionalize multilayer films simultaneously with two molecules acting in totally different ways on cells, namely plasmid DNA (pDNA), pre-complexed with poly(ethyleneimine) (PEI), and a peptide molecule, NDPMSH. This peptide, grafted to poly(l-glutamic acid) (PGA) was used as a signal molecule for melamona cells B16-F1 and for its ability to enhance gene delivery in a receptor-independent manner. The PGA–NDPMSH chains are embedded in poly-(allylamine hydrochloride)/poly-(sodium 4-styrene sulfonate) multilayers and the pDNA–PEI complexes are deposited on top of the films. It is shown that melanoma cells (B16-F1) are efficiently transfected after 24h of contact with functionalized films. When brought in contact with Huh-7 cells that do not express the peptide receptors, these films trigger significantly the transfection rate, showing that it is possible to enhance the transfection process by incorporating specific peptides into multilayer films. Moreover, transfected cells sorted by flow cytometry produce melanin, demonstrating both activation via the peptide signaling pathway and cell transfection. [Copyright &y& Elsevier]

Summary: Unnatural oligomeric scaffolds designed to adopt defined secondary structures (e.g., helices), while retaining the chemical diversity of amino acid side chains, are of practical value to elaborate functional mimetics of bioactive α-polypeptides. Enantiopure N,N′-linked oligoureas as short as seven residues long have been previously shown to fold into a stable helical structure, stabilized by 12- and 14-membered H-bonded rings. We now report that eight-residue oligoureas designed to mimic globally amphiphilic α-helical host-defense peptides are effective against both gram-negative and gram-positive bacteria (including methicillin-resistant Staphylococcus aureus [MRSA]) and exhibit selectivity for bacterial versus mammalian cells. Circular dichroism (CD) spectroscopy studies suggest enhanced helical propensity of oligoureas in the presence of phospholipid vesicles. The utility of this new class of nonpeptidic foldamers for biological applications is highlighted by high resistance to proteolytic degradation. [Copyright &y& Elsevier]