NgR1 is an NK cell inhibitory receptor that destabilizes the immunological synapse.

The formation of an immunological synapse (IS) is essential for natural killer (NK) cells to eliminate target cells. Despite an advanced understanding of the characteristics of the IS and its formation processes, the mechanisms that regulate its stability via the cytoskeleton are unclear. Here, we show that Nogo receptor 1 (NgR1) has an important function in modulating NK cell-mediated killing by destabilization of IS formation. NgR1 deficiency or blockade resulted in improved tumor control of NK cells by enhancing NK-to-target cell contact stability and regulating F-actin dynamics during IS formation. Patients with tumors expressing abundant NgR1 ligand had poor prognosis despite high levels of NK cell infiltration. Thus, our study identifies NgR1 as an immune checkpoint in IS formation and indicates a potential approach to improve the cytolytic function of NK cells in cancer immunotherapy.

FTO negatively regulates the cytotoxic activity of natural killer cells.

N6 -Methyladenosine (m6 A) is the most abundant epitranscriptomic mark and plays a fundamental role in almost every aspect of mRNA metabolism. Although m6 A writers and readers have been widely studied, the roles of m6 A erasers are not well-understood. Here, we investigate the role of FTO, one of the m6 A erasers, in natural killer (NK) cell immunity. We observe that FTO-deficient NK cells are hyperactivated. Fto knockout (Fto-/- ) mouse NK cells prevent melanoma metastasis in vivo, and FTO-deficient human NK cells enhance the antitumor response against leukemia in vitro. We find that FTO negatively regulates IL-2/15-driven JAK/STAT signaling by increasing the mRNA stability of suppressor of cytokine signaling protein (SOCS) family genes. Our results suggest that FTO is an essential modulator of NK cell immunity, providing a new immunotherapeutic strategy for allogeneic NK cell therapies.

Validity and reliability of itch assessment scales for chronic pruritus in adults: A prospective multicenter study.

BACKGROUND: Several tools can provide a reliable and accurate evaluation of pruritus, including the visual analog scale (VAS), numeric rating scale (NRS), verbal rating scale (VRS), and multidimensional questionnaires such as the Itch Severity Scale (ISS). However, no single method is considered a gold standard. OBJECTIVE: We evaluated the validity and reliability of VAS, NRS, VRS, and ISS and their correlation with a pruritus-specific quality of life instrument, ItchyQoL. METHODS: A total of 419 patients (215 men and 204 women) with chronic pruritus (mean age, 46.58 years) recorded their pruritus intensity on VAS, NRS, VRS, and ISS. Retest reliability was analyzed in a second assessment 3 hours after the initial assessment. All participants answered ItchyQoL. RESULTS: A strong correlation between VAS, NRS, and VRS was found. ISS showed a low intercorrelation validity with these tools. However, ISS was more strongly correlated with ItchyQoL. The retest reliability scores were similar for VAS, NRS, and VRS but lower than the scores obtained for ISS. LIMITATIONS: Limitations include patient heterogeneity and recall bias. CONCLUSION: The assessment of pruritus is challenging because of the subjective symptoms and the multifactorial nature. Therefore, more studies are needed to determine the best strategy to assess itch intensity.

Glass molding of all glass Fresnel lens with vitreous carbon micromold.

An all glass Fresnel lens (AGFL) was fabricated by glass molding with a vitreous carbon (VC) micro mold. In the glass molding process, a glass plate was heated up to its softening temperature and pressed against to the VC mold to replicate the Fresnel pattern. The VC molds having negative shape Fresnel profile were fabricated by carbonization of replicated Furan precursor using a diamond turning machined nickel master. During the carbonization process, the Furan precursor shrank due to the thermal decomposition, and this shrinkage must be compensated to obtain a precise AGFL. In this study, we examined the shrinkage ratio during the carbonization process using a preliminary experiment using the commercially available PMMA Fresnel lens as the master, and fabricated a nickel master with an enlarged Fresnel profile for shrinkage compensation. To verify the compensation method, the surface profiles of the fabricated VC mold and molded AGFL were measured and compared with the designed profile. The deviations between measured and designed profiles were less than 4 µm. In addition, the tip radii of the grooves and draft angle of the molded AGFL were within the acceptable tolerance for CPV applications.

XAV939, a Wnt/ß-catenin pathway modulator, has inhibitory effects on LPS-induced inflammatory response.

Aim: In this study, we report the anti-inflammatory activity of XAV939, a modulator of the Wnt/ß-catenin pathway. Methods: WNT/ß-catenin pathway and NF-κB signaling pathway were examined in LPS-stimulated human bronchial epithelial cells and effects of XAV939 on these pathways were analyzed. The effect of XAV939 was confirmed in human umbilical vein endothelial cells. Results: LPS-induced expressions of pro-inflammatory genes IL-6, IL-8, TNF-α, IL-1ß, MCP-1, MMP-9, iNOS and COX-2 were significantly and dose-dependently suppressed by XAV939. LPS-induced NF-κB signaling, such as IκB phosphorylation and degradation as well as nuclear translocation of NF-κB, was also suppressed by XAV939. Target DNA binding of NF-κB was significantly and dose-dependently suppressed by XAV939 during LPS-induced inflammatory response. The suppressive effects of XAV939 on NF-κB signaling, target DNA binding of NF-κB and pro-inflammatory gene expression were all rescued by over expression of ß-catenin, which shows that the anti-inflammatory effect of XAV939 is mediated by the modulation of ß-catenin, a central component of the WNT/ß-catenin pathway. Conclusion: The findings of this study showed that XAV939 exerts anti-inflammatory effects through the modulation of the Wnt/ß-catenin pathway.

MicroRNA-150 controls differentiation of intraepithelial lymphocytes through TGF-ß receptor II regulation.

BACKGROUND: Intraepithelial lymphocytes (IELs) in the intestines play pivotal roles in maintaining the integrity of the mucosa, regulating immune cells, and protecting against pathogenic invasion. Although several extrinsic factors, such as TGF-ß, have been identified to contribute to IEL generation, intrinsic regulatory factors have not been determined fully. OBJECTIVE: Here we investigated the regulation of IEL differentiation and the underlying mechanisms in mice. METHODS: We analyzed IELs and the expression of molecules associated with IEL differentiation in wild-type control and microRNA (miRNA)-150 knockout mice. Methotrexate was administered to mice lacking miR-150 and control mice. RESULTS: miR-150 deficiency reduced the IEL population in the small intestine and increased susceptibility to methotrexate-induced mucositis. Evaluation of expression of IEL differentiation-associated molecules showed that miR-150-deficient IELs exhibited decreased expression of TGF-ß receptor (TGF-ßR) II, CD103, CD8αα, and Runt-related transcription factor 3 in all the IEL subpopulations. The reduced expression of TGF-ßRII in miR-150-deficient IELs was caused by increased expression of c-Myb/miR-20a. Restoration of miR-150 or inhibition of miR-20a recovered the TGF-ßRII expression. CONCLUSION: miR-150 is an intrinsic regulator of IEL differentiation through TGF-ßRII regulation. miR-150-mediated IEL generation is crucial for maintaining intestinal integrity against anticancer drug-induced mucositis.

Replication of a glass microlens array using a vitreous carbon mold.

A low-cost fabrication method for a high-surface-quality glass microlens array (MLA) was proposed using a glass molding technique with a vitreous carbon (VC) mold. A VC mold with a high-surface-quality MLA cavity was fabricated, and the glass MLA with a root mean square surface roughness of 4.59 nm was replicated using the VC mold. To obtain the glass MLA with high replication quality, the effects of molding conditions were examined. The surface quality was not degraded during the proposed VC mold fabrication method and glass molding process. The focused beam spot of the glass molded MLA was analyzed; it showed a diffraction-limited characteristic of the glass molded MLA.

Development of a Protein Microarray Chip with Enhanced Fluorescence for Identification of Semen and Vaginal Fluid.

The detection of body fluids has been used to identify a suspect and build a criminal case. As the amount of evidence collected at a crime site is limited, a multiplex identification system for body fluids using a small amount of sample is required. In this study, we proposed a multiplex detection platform using an Ag vertical nanorod metal enhanced fluorescence (MEF) substrate for semen and vaginal fluid (VF), which are important evidence in cases of sexual crime. The Ag nanorod MEF substrate with a length of 500 nm was fabricated by glancing angle deposition, and amino functionalization was conducted to improve binding ability. The effect of incubation time was analyzed, and an incubation time of 60 min was selected, at which the fluorescence signal was saturated. To assess the performance of the developed identification chip, the identification of semen and VF was carried out. The developed sensor could selectively identify semen and VF without any cross-reactivity. The limit of detection of the fabricated microarray chip was 10 times better than the commercially available rapid stain identification (RSID) Semen kit.

WNT/ß-catenin pathway modulates the TNF-α-induced inflammatory response in bronchial epithelial cells.

In this study, TNF-α was found to activate the WNT/ß-catenin pathway in BEAS-2B human bronchial epithelial cells. Levels of phospho-LRP6, Dvl-2, and phospho-GSK-3ß were elevated, while that of Axin was reduced by TNF-α treatment. Nuclear translocation of ß-catenin and the reporter activity of a ß-catenin-responsive promoter were increased by TNF-α treatment. Under the same experimental conditions, TNF-α activated the NF-κB signaling, which includes the phosphorylation and degradation of IκB and nuclear translocation and target DNA binding of NF-κB, and it was found that an inhibitor of NF-κB activation, JSH-23, inhibited TNF-α-induced Wnt signaling as well as NF-κB signaling. It was also found that recombinant Wnt proteins induced NF-κB nuclear translocations and its target DNA binding, suggesting that Wnt signaling and NF-κB signaling were inter-connected. TNF-α-induced modulations of IκB and NF-κB as well as pro-inflammatory cytokine expression were significantly suppressed by the transfection of ß-catenin siRNA compared to that of control siRNA. Transfection of a ß-catenin expression plasmid augmented the TNF-α-induced modulations of IκB and NF-κB as well as pro-inflammatory cytokine expression. These results clearly demonstrated that the WNT/ß-catenin pathway modulates the inflammatory response induced by TNF-α, suggesting that this pathway may be a useful target for the effective treatment of bronchial inflammation.

Fabrication of Glass Microchannel via Glass Imprinting using a Vitreous Carbon Stamp for Flow Focusing Droplet Generator.

This study reports a cost-effective method of replicating glass microfluidic chips using a vitreous carbon (VC) stamp. A glass replica with the required microfluidic microstructures was synthesized without etching. The replication method uses a VC stamp fabricated by combining thermal replication using a furan-based, thermally-curable polymer with carbonization. To test the feasibility of this method, a flow focusing droplet generator with flow-focusing and channel widths of 50 µm and 100 µm, respectively, was successfully fabricated in a soda-lime glass substrate. Deviation between the geometries of the initial shape and the vitreous carbon mold occurred because of shrinkage during the carbonization process, however this effect could be predicted and compensated for. Finally, the monodispersity of the droplets generated by the fabricated microfluidic device was evaluated.

Fabrication of cost-effective surface enhanced Raman spectroscopy substrate using glancing angle deposition for the detection of urea in body fluid.

A surface enhanced Raman spectroscopy (SERS) with glancing angle deposited Ag nanorods structures was developed for the detection of urea in human serum. To maximize the SERS enhancement, the effects of Ag nanorod length on the SERS signal were analyzed. The SERS signals of different concentrations of urea solutions were measured in order to generate a regression model for use in analyzing the amount of urea in body fluid using the SERS substrate. To examine the feasibility of the fabricated SERS substrate, the amount of urea in human serum was measured using the SERS substrate and compared with that determined via conventional blood analysis.

Microfluidics-assisted fabrication of natural killer cell-laden microgel enhances the therapeutic efficacy for tumor immunotherapy.

Recently, interest in cancer immunotherapy has increased over traditional anti-cancer therapies such as chemotherapy or targeted therapy. Natural killer (NK) cells are part of the immune cell family and essential to tumor immunotherapy as they detect and kill cancer cells. However, the disadvantage of NK cells is that cell culture is difficult. In this study, porous microgels have been fabricated using microfluidic channels to effectively culture NK cells. Microgel fabrication using microfluidics can be mass-produced in a short time and can be made in a uniform size. Microgels consist of photo cross-linkable polymers such as methacrylic gelatin (GelMa) and can be regulated via controlled GelMa concentrations. NK92 cell-laden three-dimensional (3D) microgels increase mRNA expression levels, NK92 cell proliferation, cytokine release, and anti-tumor efficacy, compared with two-dimensional (2D) cultures. In addition, the study confirms that 3D-cultured NK92 cells enhance anti-tumor effects compared with enhancement by 2D-cultured NK92 cells in the K562 leukemia mouse model. Microgels containing healthy NK cells are designed to completely degrade after 5 days allowing NK cells to be released to achieve cell-to-cell interaction with cancer cells. Overall, this microgel system provides a new cell culture platform for the effective culturing of NK cells and a new strategy for developing immune cell therapy.

Modulating Lipid Nanoparticles with Histidinamide-Conjugated Cholesterol for Improved Intracellular Delivery of mRNA.

Recently, mRNA-based therapeutics, including vaccines, have gained significant attention in the field of gene therapy for treating various diseases. Among the various mRNA delivery vehicles, lipid nanoparticles (LNPs) have emerged as promising vehicles for packaging and delivering mRNA with low immunogenicity. However, while mRNA delivery has several advantages, the delivery efficiency and stability of LNPs remain challenging for mRNA therapy. In this study, an ionizable helper cholesterol analog, 3ß[L-histidinamide-carbamoyl] cholesterol (Hchol) lipid is developed and incorporated into LNPs instead of cholesterol to enhance the LNP potency. The pKa values of the Hchol-LNPs are ≈6.03 and 6.61 in MC3- and SM102-based lipid formulations. Notably, the Hchol-LNPs significantly improve the delivery efficiency by enhancing the endosomal escape of mRNA. Additionally, the Hchol-LNPs are more effective in a red blood cell hemolysis at pH 5.5, indicating a synergistic effect of the protonated imidazole groups of Hchol and cholesterol on endosomal membrane destabilization. Furthermore, mRNA delivery is substantially enhanced in mice treated with Hchol-LNPs. Importantly, LNP-encapsulated SARS-CoV-2 spike mRNA vaccinations induce potent antigen-specific antibodies against SARS-CoV-2. Overall, incorporating Hchol into LNP formulations enables efficient endosomal escape and stability, leading to an mRNA delivery vehicle with a higher delivery efficiency.

Design optimization of structural parameters for highly sensitive photonic crystal label-free biosensors.

The effects of structural design parameters on the performance of nano-replicated photonic crystal (PC) label-free biosensors were examined by the analysis of simulated reflection spectra of PC structures. The grating pitch, duty, scaled grating height and scaled TiO2 layer thickness were selected as the design factors to optimize the PC structure. The peak wavelength value (PWV), full width at half maximum of the peak, figure of merit for the bulk and surface sensitivities, and surface/bulk sensitivity ratio were also selected as the responses to optimize the PC label-free biosensor performance. A parametric study showed that the grating pitch was the dominant factor for PWV, and that it had low interaction effects with other scaled design factors. Therefore, we can isolate the effect of grating pitch using scaled design factors. For the design of PC-label free biosensor, one should consider that: (1) the PWV can be measured by the reflection peak measurement instruments, (2) the grating pitch and duty can be manufactured using conventional lithography systems, and (3) the optimum design is less sensitive to the grating height and TiO2 layer thickness variations in the fabrication process. In this paper, we suggested a design guide for highly sensitive PC biosensor in which one select the grating pitch and duty based on the limitations of the lithography and measurement system, and conduct a multi objective optimization of the grating height and TiO2 layer thickness for maximizing performance and minimizing the influence of parameter variation. Through multi-objective optimization of a PC structure with a fixed grating height of 550 nm and a duty of 50%, we obtained a surface FOM of 66.18 RIU-1 and an S/B ratio of 34.8%, with a grating height of 117 nm and TiO2 height of 210 nm.

Understanding nucleic acid sensing and its therapeutic applications.

Nucleic acid sensing is involved in viral infections, immune response-related diseases, and therapeutics. Based on the composition of nucleic acids, nucleic acid sensors are defined as DNA or RNA sensors. Pathogen-associated nucleic acids are recognized by membrane-bound and intracellular receptors, known as pattern recognition receptors (PRRs), which induce innate immune-mediated antiviral responses. PRR activation is tightly regulated to eliminate infections and prevent abnormal or excessive immune responses. Nucleic acid sensing is an essential mechanism in tumor immunotherapy and gene therapies that target cancer and infectious diseases through genetically engineered immune cells or therapeutic nucleic acids. Nucleic acid sensing supports immune cells in priming desirable immune responses during tumor treatment. Recent studies have shown that nucleic acid sensing affects the efficiency of gene therapy by inhibiting translation. Suppression of innate immunity induced by nucleic acid sensing through small-molecule inhibitors, virus-derived proteins, and chemical modifications offers a potential therapeutic strategy. Herein, we review the mechanisms and regulation of nucleic acid sensing, specifically covering recent advances. Furthermore, we summarize and discuss recent research progress regarding the different effects of nucleic acid sensing on therapeutic efficacy. This study provides insights for the application of nucleic acid sensing in therapy.

NK cells encapsulated in micro/macropore-forming hydrogels via 3D bioprinting for tumor immunotherapy.

BACKGROUND: Patients face a serious threat if a solid tumor leaves behind partial residuals or cannot be completely removed after surgical resection. Immunotherapy has attracted attention as a method to prevent this condition. However, the conventional immunotherapy method targeting solid tumors, that is, intravenous injection, has limitations in homing in on the tumor and in vivo expansion and has not shown effective clinical results. METHOD: To overcome these limitations, NK cells (Natural killer cells) were encapsulated in micro/macropore-forming hydrogels using 3D bioprinting to target solid tumors. Sodium alginate and gelatin were used to prepare micro-macroporous hydrogels. The gelatin contained in the alginate hydrogel was removed because of the thermal sensitivity of the gelatin, which can generate interconnected micropores where the gelatin was released. Therefore, macropores can be formed through bioprinting and micropores can be formed using thermally sensitive gelatin to make macroporous hydrogels. RESULTS: It was confirmed that intentionally formed micropores could help NK cells to aggregate easily, which enhances cell viability, lysis activity, and cytokine release. Macropores can be formed using 3D bioprinting, which enables NK cells to receive the essential elements. We also characterized the functionality of NK 92 and zEGFR-CAR-NK cells in the pore-forming hydrogel. The antitumor effects on leukemia and solid tumors were investigated using an in vitro model. CONCLUSION: We demonstrated that the hydrogel encapsulating NK cells created an appropriate micro-macro environment for clinical applications of NK cell therapy for both leukemia and solid tumors via 3D bioprinting. 3D bioprinting makes macro-scale clinical applications possible, and the automatic process shows potential for development as an off-the-shelf immunotherapy product. This immunotherapy system could provide a clinical option for preventing tumor relapse and metastasis after tumor resection. Micro/macropore-forming hydrogel with NK cells fabricated by 3D bioprinting and implanted into the tumor site.

A simulation model considering sidewall deposition for the precise prediction of the performance of label-free photonic-crystal biosensors.

Photonic-crystal (PC) structures are used as biosensors that detect the changes in the surrounding refractive index due to biomolecular interactions. In this study, a method of modeling a PC structure that considers the sidewall deposition effect during high-index layer deposition was proposed to precisely predict the performance of a label-free PC biosensor. A PC composed of nanoreplicated grating and a TiO2 high-index layer was fabricated. To replicate nanograting, a silicon mold with a 450 nm pitch and a 100 nm grating height was fabricated via photolithography and reactive ion etching. A mold cavity was coated with a self-assembled monolayer, and UV replication was performed. To realize the PC structure, a TiO2 high-index layer was deposited using the E-beam evaporation system. To design the simulation model considering the sidewall deposition effect, the cross-sectional surface profile of each PC layer was measured. Finally, the changes in the transmission spectrum of the fabricated PC structure with different buffer solutions were measured and compared with the simulated results obtained from rigorous coupled wave analysis to examine the prediction accuracy of the proposed simulation model.

A modifiable universal cotinine-chimeric antigen system of NK cells with multiple targets.

Natural killer (NK) cells are immune effector cells with outstanding features for adoptive immunotherapy. Immune effector cells with chimeric antigen receptors (CARs) are promising targeted therapeutic agents for various diseases. Because tumor cells exhibit heterogeneous antigen expression and lose cell surface antigen expression during malignant progression, many CARs fixed against only one antigen have limited efficacy and are associated with tumor relapse. To expand the utility of CAR-NK cells, we designed a split and universal cotinine-CAR (Cot-CAR) system, comprising a Cot-conjugator and NK92 cells (α-Cot-NK92 cells) engineered with a CAR containing an anti-Cot-specific single-chain variable fragment and intracellular signaling domain. The efficacy of the Cot-CAR system was assessed in vitro using a cytolysis assay against various tumor cells, and its single- or multiple- utility potential was demonstrated using an in vivo lung metastasis model by injecting A549-Red-Fluc cells. The α-Cot-NK92 cells could switch targets, logically respond to multiple antigens, and tune cytolytic activation through the alteration of conjugators without re-engineering. Therefore the universal Cot-CAR system is useful for enhancing specificity and diversity of antigens, combating relapse, and controlling cytolytic activity. In conclusion, this universal Cot-CAR system reveals that multiple availability and controllability can be generated with a single, integrated system.

Development of a two-chamber process for self-assembling a fluorooctatrichlorosilane monolayer for the nanoimprinting of full-track nanopatterns with a 35 nm half pitch.

The technology of depositing a uniform and stable anti-adhesion layer on a wafer-scale nanostamp is a critical issue in the industrialized nanoimprinting process. The deposition of an anti-adhesion layer involves O2 plasma treatment to modify the stamp surface and the reaction of the monomers with the surface. Although an automated one-chamber system was developed for uniform and stable anti-adhesion layer coating, unwanted molecules are irregularly deposited on a sample during the O2 plasma treatment due to the contamination of the chamber, leading to the degradation of the anti-adhesion properties. In this paper, a two-chamber self-assembled monolayer (SAM) deposition system was proposed to prevent the degradation of the anti-adhesion properties due to contamination. To examine the effectiveness of the proposed system, the contact angles and chemical compositions of the SAM-coated silicon mold prepared using the one- and two-chamber systems were measured and compared. Finally, 4-in nanoimprinting of 35-nm-half-pitch full-track nanopatterns was conducted using a SAM-coated silicon nanomold prepared using the one- and two-chamber systems, and the replication quality was examined.

Fabrication of Ag nanorods on micropost array for a metal-enhanced fluorescence substrate with a high signal-to-background ratio.

Selective fabrication of metallic nanostructures at the spotting area is required to increase the signal-to-background noise ratio (SBR) of the metal-enhanced fluorescence (MEF) substrate. As a simple and cost-effective fabrication method for MEF substrate with high SBR, a glancing angle deposition (GLAD) process of Ag material on the UV-imprinted micropost array (50 µm in height, 300 µm in diameter, and 600 µm in pitch) was proposed to selectively fabricate Ag nanorods on the top of micropost structure (spotting area). Ag nanorod formation at the bottom of the micropost decreased as the deposition angle in Ag GLAD increased. A deposition angle of 89° and deposition thickness of 500 nm were selected as the optimum GLAD conditions to maximize the SBR. The optimum Ag nanorods on micropost array (AgNMPA) MEF substrate provided 71-fold fluorescence signal enhancement and 25-times higher SBR than the bare glass substrate. It also provided 7-times higher SBR than the Ag nanorod MEF substrate, which has a similar Ag nanorod structure but is not selectively formed. The detection limit of AgNMPA was 16- and 4-times lower than that of the amine-functionalized glass substrate and commercial epoxy slide, respectively. Although the fluorescence signal of AgNMPA was similar to that of Ag nanorod substrate, the detection limit was 2-times lower because of the low signal standard deviation caused by the low background noise and clear spot shape.