Hen Egg Lysozyme Research Articles

Dendritic Cell-Targeted Nanoparticles Enhance T Cell Activation and Antitumor Immune Responses by Boosting Antigen Presentation and Blocking PD-L1 Pathways.

Dendritic cells (DCs) within the tumor microenvironment (TME) have an insufficient capacity to activate T cells through antigen presentation. Furthermore, the programmed cell-death ligand 1 (PD-L1), abundantly expressed on tumor-associated DCs, binds the programmed cell-death 1 (PD-1)-positive T cells and suppresses their immune function. The binding of PD-L1 to CD80 (B7.1) on the same DC via cis-interactions further prevents T cell costimulation through CD28. Here, we present a strategy to simultaneously promote antigen cross-presentation and block the inhibitory interactions of PD-L1 on DCs to amplify T cell-mediated antitumor responses within the TME. Mesoporous silica nanoparticles (MSNPs) were loaded with clotrimazole (CLT) to boost MHC II-mediated antigen presentation by DCs, surface-modified with mannose to target CD206 on DCs, and then decorated with PD-L1 binding peptide (PDL1bp) to block PD-L1-mediated interactions. PDL1bp was cleaved from the mannosylated and CLT-loaded MSNPs (MSNP-MaN/CLT) under conditions simulating the TME and tethered to PD-L1 to reverse CD80 sequestration on DC2.4 cells. The blocking of PD-L1 by PDL1bp-decorated NPs (MSNP-MaN-PDL1bp) increased the cellular interactions between DC2.4 and EL4 T cells and the amount of IL-2 secretion. The MSNP-MaN/CLT were taken up rapidly by DC2.4 cells, promoted MHC II presentation of hen egg lysozyme (HEL), and increased IL-2 production from HEL antigen-primed 3A9 T cells, which was further enhanced by PDL1bp. In vivo investigation revealed that administration of the CLT-loaded and PDL1bp-functionalized MSNPs remarkably inhibited subcutaneous B16-F10 melanoma tumor growth when compared with anti-PD-L1 therapy. MSNP-MaN-PDL1bp/CLT treatment upregulated the levels of effector molecules such as granzyme B and proinflammatory cytokines (IFNγ and INFα) in the tumor tissue, indicating antitumoral T cell responses. This strategy of utilizing nanoparticles to trigger DC activation while promoting T cell stimulation can be used to amplify the antitumor T cell responses and represents a promising alternative to anti-PD-L1 immunotherapy.

Application of PLGA-PEG-PLGA Nanoparticles to Percutaneous Immunotherapy for Food Allergy.

Compared with oral or injection administration, percutaneous immunotherapy presents a promising treatment modality for food allergies, providing low invasiveness and safety. This study investigated the efficacy of percutaneous immunotherapy using hen egg lysozyme (HEL)-loaded PLGA-PEG-PLGA nanoparticles (NPs), as an antigen model protein derived from egg white, compared with that of HEL-loaded chitosan hydroxypropyltrimonium chloride (CS)-modified PLGA NPs used in previous research. The intradermal retention of HEL in excised mouse skin was measured using Franz cells, which revealed a 2.1-fold higher retention with PLGA-PEG-PLGA NPs than that with CS-modified PLGA NPs. Observation of skin penetration pathways using fluorescein-4-isothiocyanate (FITC)-labeled HEL demonstrated successful delivery of HEL deep into the hair follicles with PLGA-PEG-PLGA NPs. These findings suggest that after NPs delivery into the skin, PEG prevents protein adhesion and NPs aggregation, facilitating stable delivery deep into the skin. Subsequently, in vivo percutaneous administration experiments in mice, with concurrent iontophoresis, demonstrated a significant increase in serum IgG1 antibody production with PLGA-PEG-PLGA NPs compared with that with CS-PLGA NPs after eight weeks of administration. Furthermore, serum IgE production in each NP administration group significantly decreased compared with that by subcutaneous administration of HEL solution. These results suggest that the combination of PLGA-PEG-PLGA NPs and iontophoresis is an effective percutaneous immunotherapy for food allergies.

Hen egg lysozyme polymorphs mediated by two ionic liquids during batch cooling crystallization

-Lysozyme batch cooling crystallization in the presence of two ionic liquids (ILs), 1-butyl-3-methylimidazolium chloride ([C4mim]Cl), and 1,3-dimethylimidazolium iodide ([C1mim]I). Microscopic observation demonstrated that the crystal morphology with [C1mim]I is needle-like, while that with [C4mim]Cl and no ILs is plate-like. FTIR spectroscopy revealed that new peaks in the range of 1300–400 cm−1 occur with the case of adding [C1mim]I, meaning that the microstructure of lysozyme crystals changed. Raman spectroscopy and X-ray powder diffraction (XRD) pattern further confirmed that crystal forms of lysozyme with adding [C4mim]Cl and no ILs is completely different from that of adding [C1mim]I. Thermogravimetry (TG) and Differential Scanning Calorimeter (DSC) results indicated that addition of [C1mim]I delay the thermal deformation and degradation temperature of lysozyme crystals, suggesting that lysozyme crystals with [C1mim]I possesses higher thermal stability. The change in lysozyme crystal forms can be attributed to changes in interaction among lysozyme molecules and conformation in solution, leading to different assembly mode of molecules and formation of polymorphs. These findings could offer instructions for regulating lysozyme crystal forms and gaining initial insight into screening of potential ILs for developing polymorphs of the protein and other macromolecule crystals.

Immunopotentiating Polyphosphazene Delivery Systems: Supramolecular Self-Assembly and Stability in the Presence of Plasma Proteins.

Studies on the biological performance of nanomedicines have been increasingly focused on the paradigm shifting role of the protein corona, which is imminently formed once the formulation is placed in a complex physiological environment. This phenomenon is predominantly studied in the context of protein adsorption science, while such interactions for water-soluble systems remain virtually unexplored. In particular, the importance of plasma protein binding is yet to be understood for pharmaceuticals designed on the basis of supramolecular architectures, which generally lack well-defined surfaces. Water-soluble ionic polyphosphazenes, clinically proven immunoadjuvants and vaccine delivery vehicles, represent an example of a system that requires supramolecular coassembly with antigenic proteins to attain an optimal immunopotentiating effect. Herein, the self-assembly behavior and stability of noncovalently bound complexes on the basis of a model antigen─hen egg lysozyme─and polyphosphazene adjuvant are studied in the presence of plasma proteins utilizing isothermal calorimetry, asymmetric flow field flow fractionation, dynamic light scattering, and size exclusion chromatography methods. The results demonstrate that although plasma proteins, such as human serum albumin (HSA), show detectable avidity to polyphosphazene, the strength of such interactions is significantly lower than that for the model antigen. Furthermore, thermodynamic parameters indicate different models of binding: entropy driven, which is consistent with the counterion release mechanism for albumin versus electrostatic interactions for lysozyme, which are characterized by beneficial enthalpy changes. In vitro protein release experiments conducted in Franz diffusion cells using enzyme-linked immunoassay detection suggest that the antigen-adjuvant complex stability is not adversely affected by the presence of the most physiologically abundant protein, which confirms the importance of the delivery modality for this immunoadjuvant. Moreover, HSA shows an unexpected stabilizing effect on complexes with high antigen load─an important consideration for further development of polyphosphazene adjuvanted vaccine formulations and their functional assessment.

The Influence of Ionic Liquids on Solubility and Metastable Zone Width of Hen Egg Lysozyme

The Influence of Ionic Liquids on Solubility and Metastable Zone Width of Hen Egg Lysozyme

Novel Fluorometric Assay of Antiglycation Activity Based on Methylglyoxal-Induced Protein Carbonylation.

Advanced glycation end products (AGEs), which can have multiple structures, are formed at the sites where the carbonyl groups of reducing sugars bind to the free amino groups of proteins through the Maillard reaction. Some AGE structures exhibit fluorescence, and this fluorescence has been used to measure the formation and quantitative changes in carbonylated proteins. Recently, fluorescent AGEs have also been used as an index for the evaluation of compounds that inhibit protein glycation. However, the systems used to generate fluorescent AGEs from the reaction of reducing sugars and proteins used for the evaluation of antiglycation activity have not been determined through appropriate research; thus, problems remain regarding sensitivity, quantification, and precision. In the present study, using methylglyoxal (MGO), a reactive carbonyl compound to induce glycation, a comparative analysis of the mechanisms of formation of fluorescent substances from several types of proteins was conducted. The analysis identified hen egg lysozyme (HEL) as a protein that produces stronger fluorescent AGEs faster in the Maillard reaction with MGO. It was also found that the AGE structure produced in MGO-induced in HEL was argpyrimidine. By optimizing the reaction system, we developed a new evaluation method for compounds with antiglycation activity and established an efficient evaluation method (HEL-MGO assay) with greater sensitivity and accuracy than the conventional method, which requires high concentrations of bovine serum albumin and glucose. Furthermore, when compounds known to inhibit glycation were evaluated using this method, their antiglycation activities were clearly and significantly measured, demonstrating the practicality of this method.

Trogocytosis drives red blood cell antigen loss in association with antibody-mediated immune suppression

AbstractRed blood cell (RBC) alloimmunization to paternal antigens during pregnancy can cause hemolytic disease of the fetus and newborn (HDFN). This severe and potentially fatal neonatal disorder can be prevented by the administration of polyclonal anti-D through a mechanism referred to as antibody-mediated immune suppression (AMIS). Although anti-D prophylaxis effectively prevents HDFN, a lack of mechanistic clarity has hampered its replacement with recombinant agents. The major theories behind AMIS induction in the hematologic literature have classically centered around RBC clearance; however, antigen modulation/loss has recently been proposed as a potential mechanism of AMIS. To explore the primary mechanisms of AMIS, we studied the ability of 11 different antibodies to induce AMIS, RBC clearance, antigen loss, and RBC membrane loss in the HOD (hen egg lysozyme–ovalbumin–human Duffy) murine model. Antibodies targeting different portions of the HOD molecule could induce AMIS independent of their ability to clear RBCs; however, all antibodies capable of inducing a strong AMIS effect also caused significant in vivo loss of the HOD antigen in conjunction with RBC membrane loss. In vitro studies of AMIS-inducing antibodies demonstrated simultaneous RBC antigen and membrane loss, which was mediated by macrophages. Confocal live-cell microscopy revealed that AMIS-inducing antibodies triggered RBC membrane transfer to macrophages, consistent with trogocytosis. Furthermore, anti-D itself can induce trogocytosis even at low concentrations, when phagocytosis is minimal or absent. In view of these findings, we propose trogocytosis as a mechanism of AMIS induction.

Magnetic amyloid-based biocatalyst for the hydrolysis of urea

The presence of urea in wines and other alcoholic beverages represents a critical problem since it can chemically react with ethanol, which leads to the formation of ethyl carbamate, a carcinogenic agent according to the World Health Organization. Here we report the creation of a biocatalyst for the hydrolysis of urea, which could potentially be used before bottling alcoholic drinks. For this, the effective surface area of streptavidin-labeled magnetic microparticles was amplified by functionalization with biotin-labeled hen egg lysozyme amyloid fibers. Subsequently, by using copper and hydrogen peroxide induced cross-linking of unmodified proteins (CHICUP), soybean urease was immobilized to the fibers. This gave rise to a magnetic biocatalyst with remarkable urease activity, which was maintained even after 10 reuses. We propose that this strategy could be used as a platform for immobilizing other molecules to design and develop a myriad of biocatalysts for the food industry.

Targeted delivery of autoantigen to dendritic cells prevents development of spontaneous uveitis.

Restoration of immunological tolerance to self antigens has been a major drive in understanding the mechanisms of, and developing new treatments for, autoimmune and autoinflammatory disease. Sessile dendritic cells (DC) are considered the main instruments underpinning immunological tolerance particularly the CD205+ (DEC205+) cDC1 subset in contrast to DCIR2+ cDC2 which mediate immunogenicity. Targeting DC using autoantigen peptide-antibody fusion proteins has been a well explored methodology for inducing tolerance. Here we show that subcutaneous (s.c.) inoculation of hen-egg lysozyme (HEL)-DEC205 Ig fusion prevents the development of spontaneous uveoretinitis (experimental autoimmune uveoretinitis, EAU) in a transgenic mouse model generated by crossing interphotoreceptor retinol binding protein (IRBP)-HEL (sTg HEL) with HEL specific TCR (sTg TCR) mice. Prolonged suppression of EAU required injections of HEL-DEC205 Ig once weekly, reflecting the half life of s.c. DC. Interestingly, HEL-DCIR2 Ig also had a suppressive effect on development of EAU but less so than DEC205 Ig while it had minimal effect on preventing the retinal atrophy associated with EAU. In addition, HEL-DEC205 Ig was only effective when administered s.c. rather than systemically and had no effect on EAU induced by adoptive transfer of HEL-activated T cells. These data demonstrate the importance of systemic (lymph node) rather than local (eye) antigen presentation in the development of EAU as well as suggest a potential therapeutic approach to controlling sight-threatening immune-mediated uveitis provided relevant antigen(s) can be identified.

Molecular basis for potent B cell responses to antigen displayed on particles of viral size.

Multivalent viral epitopes induce rapid, robust and T cell-independent humoral immune responses, but the biochemical basis for such potency remains incompletely understood. We take advantage of a set of liposomes of viral size engineered to display affinity mutants of the model antigen (Ag) hen egg lysozyme. Particulate Ag induces potent 'all-or-none' B cell responses that are density dependent but affinity independent. Unlike soluble Ag, particulate Ag induces signal amplification downstream of the B cell receptor by selectively evading LYN-dependent inhibitory pathways and maximally activates NF-κB in a manner that mimics T cell help. Such signaling induces MYC expression and enables even low doses of particulate Ag to trigger robust B cell proliferation in vivo in the absence of adjuvant. We uncover a molecular basis for highly sensitive B cell responses to viral Ag display that is independent of encapsulated nucleic acids and is not merely accounted for by avidity and B cell receptor cross-linking.

High-Throughput Antibody Discovery Using Barcode Enabled Antigen Mapping (BEAM

Abstract Multi-analyte single cell technologies have potential to greatly accelerate antibody discovery by identifying antigen-specific B cells. Barcode Enabled Antigen Mapping (BEAM) provides a high-throughput, multimodal analysis of antigen-bound B cells (BEAM-Ab) using 10x Genomics Chromium Single Cell Immune Profiling v2 Solution and novel computational approaches to discover B-cell receptor (BCR) sequences for further functional characterization. To demonstrate the antigen sensitivity of BEAM-Ab, we spiked in 5% Hen Egg Lysozyme (HEL) and 5% gp120 transgenic B cells in 90% wild type non-transgenic splenocytes. The cells were screened using a panel of barcoded antigens and then CD19+PE+ (antigen+) B cells were isolated using flow cytometry. Single cell gene expression, BCR, and antigen barcode analysis indicated clonotype specificity to the respective antigens in the sorted cells. Furthermore, we demonstrated BEAM-Ab specificity by analyzing a convalescent COVID patient sample against an antigen panel containing five different COVID antigens and a negative control. Analysis of this COVID sample suggests the clonal expansion of B cells recognizing the ectodomain of the wild type SARS-CoV2 spike protein, receptor binding domain of spike protein, and the ectodomain of the D614G mutant spike protein. We did not observe any clonal expansion to Omicron-specific antigen or the non-specific negative control in our dataset. Our data demonstrates that BEAM-Ab is an effective antibody discovery tool. BEAM-Ab unlocks the ability to rapidly screen large numbers of samples with an accurate single cell resolution and antigen specificity, with the entire workflow generating the candidate sequences just one week after sample processing.

The FcμR regulates IgM-BCR internalization

Abstract The FcμR is a cell surface receptor that can bind to both secreted and membrane-bound IgM. In B cells it co-localizes with IgM-BCR in Golgi vesicles, where it restrains surface BCR-IgM expression. B cell-expressed FcmR is required also for maximal IgG response development after influenza A virus infection, but the mechanisms are unknown. To explore the effects of FcmR expression we generated FcmR-deficient and sufficient BCR-”SwHEL” transgenic C57BL/6 mice. The mice, containing about 20% HEL-specific B cells were immunized either directly with hen egg lysozyme (HEL) or with HEL-ovalbumin (OVA), or we created bone marrow irradiation chimeras in which about 2% of naïve B cells expressed the SwHEL-BCR. Consistent with the influenza infection studies, IgG responses following subcutaneous HEL-OVA immunization were significantly diminished in both systems when B cells lacked FcmR−/− expression compared to controls. Confocal and STED microscopy studies on B cells from these mice were done to quantify IgM-BCR internalization following in vitro exposure to HEL-bodipy. FcmR−/− SwHEL B cells did not show IgM “capping” and retained high amounts of antigen on the cell surface compared to the HEL− FcmR+/+ cells. This reduction in internalization resulted in reduced MHCII-peptide loading, as shown via flow cytometric quantification of surface pMHCII expression using the YAE-antibody. Ongoing studies are defining the subcellular compartments of antigen-processing in the presence and absence of the FcmR. We conclude that the FcmR acts as a non-redundant chaperone that optimizes IgM-BCR antigen internalization and processing. U19AI109962 R01AI148652

Monitoring Protein Complexation with Polyphosphazene Polyelectrolyte Using Automated Dynamic Light Scattering Titration and Asymmetric Flow Field Flow Fractionation and Protein Recognition Immunoassay.

Polyphosphazenes represent a class of intrinsically flexible polyelectrolytes with potent immunoadjuvant activity, which is enabled through non-covalent self-assembly with antigenic proteins by charge complexation. The formation of supramolecular complexes between polyphosphazene adjuvant, poly[di(carboxylatophenoxy)phosphazene] (PCPP), and a model vaccine antigen, hen egg lysozyme, was studied under physiological conditions using automated dynamic light scattering titration, asymmetric flow field flow fractionation (AF4), enzyme-linked immunosorbent assay (ELISA), and fluorescent quenching methods. Three regimes of self-assembly were observed covering complexation of PCPP with lysozyme in the nano-scale range, multi-chain complexes, and larger aggregates with complexes characterized by a maximum loading of over six hundred protein molecules per PCPP chain and dissociation constant in the micromolar range (Kd = 7 × 10-6 mol/L). The antigenicity of PCPP bound lysozyme, when compared to equivalent lysozyme solutions, was largely retained for all complexes, but observed a dramatic reduction for heavily aggregated systems. Routes to control the complexation regimes with elevated NaCl or KCl salt concentrations indicate ion-specific effects, such that more smaller-size complexes are present at higher NaCl, counterintuitive with respect to PCPP solubility arguments. While the order of mixing shows a prominent effect at lower stoichiometries of mixing, higher NaCl salt reduces the effect all together.

C-type lectin receptor agonists elicit functional IL21-expressing Tfh cells and induce primary B cell responses in neonates.

C-type lectin receptor (CLR) agonists emerged as superior inducers of primary B cell responses in early life compared with Toll-like receptor (TLR) agonists, while both types of adjuvants are potent in adults. Here, we explored the mechanisms accounting for the differences in neonatal adjuvanticity between a CLR-based (CAF®01) and a TLR4-based (GLA-SE) adjuvant administered with influenza hemagglutinin (HA) in neonatal mice, by using transcriptomics and systems biology analyses. On day 7 after immunization, HA/CAF01 increased IL6 and IL21 levels in the draining lymph nodes, while HA/GLA-SE increased IL10. CAF01 induced mixed Th1/Th17 neonatal responses while T cell responses induced by GLA-SE had a more pronounced Th2-profile. Only CAF01 induced T follicular helper (Tfh) cells expressing high levels of IL21 similar to levels induced in adult mice, which is essential for germinal center (GC) formation. Accordingly, only CAF01- induced neonatal Tfh cells activated adoptively transferred hen egg lysozyme (HEL)-specific B cells to form HEL+ GC B cells in neonatal mice upon vaccination with HEL-OVA. Collectively, the data show that CLR-based adjuvants are promising neonatal and infant adjuvants due to their ability to harness Tfh responses in early life.

Antigen copy number and antibody dose can determine the outcome of erythrocyte alloimmunization inducing either antibody-mediated immune suppression or enhancement in a murine model.

The administration of anti-D for the prevention of hemolytic disease of the fetus and newborn is one of the most successful clinical uses of the phenomenon of antibody-mediated immune suppression (AMIS). However, despite adequate prophylaxis, failures can still occur in the clinic and are poorly understood. Recently, the copy number of red blood cell (RBC) antigens has been shown to influence immunogenicity in the context of RBC alloimmunization; however, its influence on AMIS remains unexplored. RBCs expressing approximately 3,600 and approximately 12,400 copy numbers of surface-bound hen egg lysozyme (HEL), named respectively HELmed -RBCs and HELhi -RBCs, and selected doses of a polyclonal HEL-specific IgG were transfused into mice. Recipient HEL-specific IgM, IgG, and IgG subclass responses were evaluated by ELISA. Antigen copy number affected the antibody dose required for AMIS induction with higher antigen copy numbers requiring larger doses of antibody. For instance, 5μg of antibody caused AMIS for HELmed -RBCs but not HELhi -RBCs, while 20 μg induced significant suppression for both HEL-RBCs. Overall, increasing amounts of the AMIS-inducing antibody were associated with a more complete AMIS effect. In contrast, the lowest tested doses of the AMIS-inducing IgG led to evidence of enhancement at the IgM and IgG levels. The results demonstrate that the relationship between antigen copy number and antibody dose can influence the outcome of AMIS. Further, this work suggests that the same antibody preparation can induce both AMIS and enhancement but that the outcome may depend on the quantitative interrelationship of antigen-antibody binding.

Food protein-derived amyloids do not accelerate amyloid β aggregation

The deposition of proteins in the form of amyloid fibrils is closely associated with several serious diseases. The events that trigger the conversion from soluble functional proteins into insoluble amyloid are not fully understood. Many proteins that are not associated with disease can form amyloid with similar structural characteristics as the disease-associated fibrils, which highlights the potential risk of cross-seeding of disease amyloid by amyloid-like structures encountered in our surrounding. Of particular interest are common food proteins that can be transformed into amyloid under conditions similar to cooking. We here investigate cross-seeding of amyloid-β (Aβ), a peptide known to form amyloid during the development of Alzheimer’s disease, by 16 types of amyloid fibrils derived from food proteins or peptides. Kinetic studies using thioflavin T fluorescence as output show that none of the investigated protein fibrils accelerates the aggregation of Aβ. In at least two cases (hen egg lysozyme and oat protein isolate) we observe retardation of the aggregation, which appears to originate from interactions between the food protein seeds and Aβ in aggregated form. The results support the view that food-derived amyloid is not a risk factor for development of Aβ pathology and Alzheimer’s disease.

Colitis in a transgenic mouse model of autoimmune uveitis may be induced by neoantigen presentation in the bowel

Undifferentiated uveitis (intraocular inflammation, IOI) is an idiopathic sight-threatening, presumed autoimmune disease, accountable for ~ 10% of all blindness in the developed world. We have investigated the association of uveitis with inflammatory bowel disease (IBD) using a mouse model of spontaneous experimental autoimmune uveoretinitis (EAU). Mice expressing the transgene (Tg) hen egg lysozyme (HEL) in the retina crossed with 3A9 mice expressing a transgenic HEL-specific TCR spontaneously develop uveoretinitis at post-partum day (P)20/21. Double transgenic (dTg TCR/HEL) mice also spontaneously develop clinical signs of colitis at ~ P30 with diarrhoea, bowel shortening, oedema and lamina propria (LP) inflammatory cell infiltration. Single (s)Tg TCR (3A9) mice also show increased histological LP cell infiltration but no bowel shortening and diarrhoea. dTg TCR/HEL mice are profoundly lymphopenic at weaning. In addition, dTg TCR/HEL mice contain myeloid cells which express MHC Class II-HEL peptide complexes (MHCII-HEL), not only in the inflamed retina but also in the colon and have the potential for antigen presentation. In this model the lymphopenia and reduction in the absolute Treg numbers in dTg TCR/HEL mice is sufficient to initiate eye disease. We suggest that cell-associated antigen released from the inflamed eye can activate colonic HEL-specific T cells which, in a microbial micro-environment, not only cause colitis but feedback to amplify IOI.

Antigen-specific IgG subclass composition in recipient mice can indicate the degree of red blood cell alloimmunization as well as discern between primary and secondary immunization.

Despite the vast antigen disparity between donor and recipient red blood cells (RBCs), only 2%-6% of transfusion patients mount an alloantibody response. Recently, RBC antigen density has been proposed as one of the factors that can influence alloimmunization, however, there has been no characterization of the role of antigen density along with RBC dose in primary and secondary immunization. To generate RBCs that express distinct antigen copy numbers, different quantities of hen egg lysozyme (HEL) were coupled to murine RBCs. The HEL-RBCs were subsequently transfused into recipient mice at different RBC doses and their HEL-specific IgM, IgG, and IgG subclass response was evaluated. Productive immune responses could be generated through a high copy number antigen transfused at low RBC doses or a low copy number transfused at high RBC doses. Further, primary but submaximal humoral immunization predominantly induced the IgG2b and IgG3 subclasses. In contrast, a maximal primary immunization or a secondary immunization induced all four IgG subclasses. Our results confirm the existence of an antigen threshold for productive immune responses but indicate that a high antigen copy number alone might not be enough to induce a response, but rather a combination of both antigen copy number and cell dosage may determine the outcome of immunization. Further, this study provides a proof of concept that the IgG subclass composition can be an indicator of the level of RBC alloimmunization as well as discern between primary and secondary immunization at least in this murine model.

Carboxylato‐ and squarato‐bridged dinuclear/tetranuclear Cu(II) complexes: Synthesis, magnetic property, and protein binding studies

Two new Cu(II) complexes [Cu2(L)2(mb)](ClO4)·0.5H2O (1) and [Cu4(L)4(sq)(H2O)2](ClO4)2·2H2O (2) (HL = 2‐[(2‐morpholine‐4‐yl‐ethylimino)‐methyl]‐phenol); mb = 4‐methyl benzoate; sq2− = C4O42− squarate dianion) have been synthesized and characterized by single crystal X‐ray analysis, electrospray ionization mass spectrometry, and infrared spectroscopic technique. The structural determination showed the Schiff base acting as tridentate ligand coordinating the copper(II) atoms by the phenoxo, imino and morpholine nitrogen donors to give comparable Cu(L) entities in both complexes 1 and 2. Complex 1 is a dinuclear cationic species built by a 4‐methylbenzoate anion coordinating two Cu(L) moieties. The two Cu(II) centers are chemically equivalent exhibiting a distorted square pyramidal geometry. Complex 2 is tetranuclear and it is formed by a centrosymmetric squarate core connected to four Cu(L) complex units to give a cationic complex counterbalanced by perchlorate anions. Of the two independent Cu(II) atoms, Cu1 exhibits a square pyramidal coordination sphere, whereas Cu2 is better described as square planar. The crystal packing of complex 2 shows a polymeric arrangement through H‐bonding. Magnetic measurements show antiferromagnetic coupling in both the complexes. The interaction of complexes with hen egg lysozyme has been investigated by using UV–vis absorption and 2D/3D fluorescence spectroscopic techniques and the calculated values of binding constants, Kbin, are (5.9 ± 0.11) × 104, (17.39 ± 0.04) × 104, and (10.1 ± 0.07) × 104 M−1 for HL, 1, and 2, respectively.

Hen Egg Lysozyme Alleviates Static Mechanical Pain Via NRF1-Parkin-TACAN Signaling Axis in Sensory Neurons

Mechanical allodynia impinges on the life quality of patients. Hen Egg Lysozyme (HEL) is a substance extracted from eggs that is commonly used to inhibit bacterial activity. The role of HEL in regulating and treating pain is unclear. Here, we find that HEL selectively attenuates static mechanical allodynia of mice induced by complete Freund's adjuvant (CFA), spinal nerve ligation (SNL) and chemotherapeutic agent. RNA-seq screening reveals that CFA significantly reduces the expression of Parkin in dorsal root ganglion (DRG) neurons of mice, while pre-administration of HEL increases the expression of Parkin and remits the static mechanical allodynia induced by Parkin-siRNA. Moreover, HEL increases the interaction between nuclear respiratory factor 1 (NRF1) and histone acetyltransferase P300 and then enhances the NRF1 mediated histone acetylation in prkn promoter region in DRGs of mice. Further, Parkin interacts with mechanotransducing ion channel TACAN (Tmem120a) and knockdown of Parkin significantly increases the membrane trafficking of TACAN in sensory neurons of mice. While pre-administration of HEL inhibits the increased membrane trafficking of TACAN in sensory neurons of mice induced by Parkin-siRNA. In addition, pre-given of HEL also significantly attenuates the static mechanical allodynia induced by overexpression of TACAN in mice, and the effect of HEL can be blocked by Parkin-siRNA. This indicates that HEL increases the expression of Parkin through epigenetic mechanisms and then decreases TACAN membrane trafficking in sensory neurons to relieve static mechanical hypersensitivity. Therefore, we reveal a novel function of HEL, which is a potential substance for the treatment of static mechanical pain.