Adverse Immune Responses Research Articles

Safety and immunogenicity of a quadrivalent seasonal influenza vaccine adjuvanted with hydroxypropyl-β-cyclodextrin: A phase 1 clinical trial

ObjectivesHydroxypropyl-β-cyclodextrin (HP-β-CyD), an oligosaccharide used as an excipient in pharmaceutical preparation, was recently reported to function as a vaccine adjuvant to co-administered antigens. In this study, we investigated the safety and immunogenicity of a seasonal influenza vaccine adjuvanted with HP-β-CyD (FluCyD-vac) in healthy adults compared with those of a standard seasonal influenza vaccine (Flu-vac). MethodsWe conducted a single-blinded randomized phase 1 clinical trial study, and used two quadrivalent split seasonal influenza vaccines: FluCyD-vac containing 9 μg of HA/strain and 20% w/v of HP-β-CyD, and Flu-vac containing 15 μg of hemagglutinin (HA)/strain only. All participants were randomly assigned to receive a single dose of Flu/CyD-vac or Flu-vac at a ratio of 2:1. We assessed solicited and unsolicited adverse events (AEs) and immune responses using hemagglutination inhibition (HI) titers. In addition, we assessed T-cell function in peripheral blood mononuclear cells (PBMCs), after stimulation with HA vaccine strains, using flow cytometry. ResultsAmong 36 healthy volunteers enrolled in the study (FluCyD-vac, n = 24; Flu-vac, n = 12), FluCyD-vac was well tolerated. Most of the solicited AEs were mild local skin reactions at the injection site. No serious AEs were reported in either group. HI titers 21 days after vaccination with FluCyD-vac were comparable with those of Flu-vac and sufficient to meet international criteria, despite reduced HA antigen doses. When PBMCs were stimulated with the four HA antigens in the vaccine, tumor necrosis factor (TNF)-α-producing CD4+ T cells were enhanced in the FluCyD-vac group. ConclusionFluCyD-vac was well-tolerated and immunogenic, despite containing 40% less HA antigens than Flu-vac. This study showed that HP-β-CyD is a potentially safe, novel adjuvant for human influenza vaccine.Clinical trial registry: UMIN000028530.

Molecular engineering of nanoactuators for neuromodulation

Some of the most advanced techniques for deep-brain stimulation rely on chronic brain implants that can damage fragile tissues and induce adverse immune responses. Herein, researchers report a macromolecular construct as an injectable photothermal nanotransducer for neuromodulation that bypasses these limitations. Some of the most advanced techniques for deep-brain stimulation rely on chronic brain implants that can damage fragile tissues and induce adverse immune responses. Herein, researchers report a macromolecular construct as an injectable photothermal nanotransducer for neuromodulation that bypasses these limitations.

Crystal structure of ultra-humanized anti-pTau Fab reveals how germline substitutions humanize CDRs without loss of binding’

Administration of therapeutic antibodies can elicit adverse immune responses in patients through the generation of anti-drug antibodies that, in turn, reduce the efficacy of the therapeutic. Removal of foreign amino acid content by humanization can lower the immunogenic risk of the therapeutic mAb. We previously developed the ultra-humanization technology “Augmented Binary Substitution” (ABS) which enables single-step CDR germlining of antibodies. The application of ABS to a chicken anti-pTau antibody generated an ultra-humanized variant, anti-pTau C21-ABS, with increased human amino acid content in the CDRs and reduced in-silico predicted immunogenicity risk. Here, we report the high-resolution crystal structure of anti-pTau C21-ABS Fab in complex with the pTau peptide (7KQK). This study examines how ultra-humanization, via CDR germlining, is facilitated while maintaining near-identical antigen affinity (within 1.6-fold). The co-complex structure reveals that the ABS molecule targets the same antigenic epitope, accommodated by structurally-similar changes in the paratope. These findings confirm that ABS enables the germlining of amino acids within CDRs by exploiting CDR plasticity, to reduce non-human amino acid CDR content, with few alterations to the overall mechanism of binding.

Preliminary Study on the Antigen-Removal from Extracellular Matrix via Different Decellularization.

Due to the abundance of bioactive components, surficial decoration with cell-derived extracellular matrix (ECM) is a promising strategy to improve the biological functionality of the tissue engineering scaffolds. However, decellularization is necessary to remove antigenic components in the ECM that may trigger adverse immune response. Freeze-thaw (FT) cycles and treatment with Triton X-100/ammonium hydroxide (TN) are two commonly used decellularization methods for ECM, but their effects on both growth factor retention and antigen removal are still controversial. The objectives of this study are to compare the preservation of ECM texture and beneficial ingredients and the removal of cellular antigens by these two methods. First, the constructs combined bone marrow mesenchymal stem cell-derived ECM and poly(lactic-co-glycolic acid) (PLGA) membrane are prepared and decellularized using FT and TN treatments. Moreover, the effects of decellularization on the ultrastructure and the composition of ECM-decorated PLGA membrane are compared by scanning electron microscope observation and protein quantification. Furthermore, the ECM deposited on PLGA is stripped off and then implanted subcutaneously in rats, and the host macrophage and local lymphocyte responses were investigated. Finally, ECM-decorated porous PLGA scaffolds are implanted into rat calvarial defects, and the new bone formation is evaluated. Our results showed that both methods effectively removed DNA. TN treatment partially retained collagen, glycosaminoglycan, bone morphogenetic protein-2, and vascular endothelial growth factor, and better preserved structural integrity than FT treatment. ECM implants decellularized by both methods induced a mild host response after subcutaneous implantation. Although the total content of residual DNA in the two ECMs digested by the DNA enzyme seemed to be similar and very low, the interfaces between implanted materials and natural tissues in the TN group recruited lower numbers of CD68+ macrophages, CD68+CD86+ (M1) macrophages, and CD4+ T lymphocytes than that in FT group, implying that there exist other ECM antigens to influence immune response besides DNA. Furthermore, ECM-decorated scaffolds decellularized by TN treatment induced greater bone formation than that of bare scaffolds in vivo, demonstrating the effective retention of ECM bioactive components after decellularization. This study showed that TN treatment was a more effective and safer decellularization method than FT cycles. Impact statement Decellularization is a prerequisite for extracellular matrix (ECM) application, but there is still no standard for its selection. This study demonstrated that detergent treatment was more effective than freeze-thaw (FT) cycles in removing ECM antigens besides DNA, and the prepared ECM elicited a milder allogenic immune response, which ensured the safety of ECM. Moreover, detergent better preserved the ECM integrity than FT cycles, and effectively retained growth factors, and the decellularized ECM-decorated scaffolds significantly promoted bone repair, which ensured the effectiveness of ECM. This study provides the theoretical and experimental bases for the decellularization strategy of ECM-modified tissue engineering scaffolds.

MO429: The Association of Circulating Monocytes Subsets, NK Cells and Lymphocyte Subpopulations With Conventional and Novel Deformation Indices of Left Ventricular Function in CKD Patients With No Established Cardiovascular Disease

Abstract BACKGROUND AND AIMS Cardiovascular disease (CVD) and chronic kidney disease (CKD) are closely interrelated. Adverse immune responses have been implicated in the pathogenesis of both CVD and CKD [1, 2]. Monocyte subsets are key in atherogenesis and the inflammatory cascade occurring in heart failure [1, 2]. Likewise, the role of lymphocyte subpopulations including natural killer (NK) cells and CD4 + CD25 + regulatory T cells (Tregs) in the modulation of inflammation and immunity and subsequent cardiovascular implications have received increasing attention [2]. The implication of immune cells subsets in the abnormal myocardial remodeling as universally occurs in CKD patients even without overt CVD, remains to be clarified [3]. The aim of our study was to investigate potential correlations between blood levels of specific immune cells subsets with conventional and novel deformation-related indices of LV function in a cohort of patients with CKD without established CVD. METHOD A total of 35 stable CKD patients (mean age 66 ±12.2 years, 57% males, 20% diabetics) without established CVD were included in the study. Exclusion criteria included the history of malignancy, autoimmune disease and active or chronic infections. The peripheral blood immune cell subsets CD14++CD16-, CD14++CD16 + and CD14 + CD16++ percentage and absolute number of cells out of the total monocytes and NK cells (CD3 + CD16 + 56+), CD3-CD19 + B lymphocytes, CD3 + CD4 + T cells, CD3 + CD8 + T cells and Tregs (CD4 + CD25 + FoxP3+) absolute values and percentage out of the total lymphocytes were measured by flow cytometry. Simultaneously, conventional [left atrial volume index (LAVI), LV mass index (LVMI), LV end-diastolic volume (LVEDV), E/E’] and novel 2D speckle tracking (2DST) echocardiographic indices of LV function [global longitudinal strain (GLS), global circumferential strain (GCS), global radial strain (GRS), TWIST and UNTWIST] were assessed. RESULTS The mean estimated glomerular filtration rate (eGFR) was 28 ±15 mL/min/1.73 m2 (CKD-EPI) and the mean 24-h proteinuria was 2457 ±2399 mg. The percentage of total blood lymphocytes, as well as both percentage and absolute counts of CD4 + T-cells, were positively correlated with the eGFR (P < 0.05 and < 0.01 respectively) whereas. In addition, both NK cells percentage (P < 0.05) and absolute values (P < 0.01) showed a negative association with proteinuria. Regarding classical indices of LV function, NK counts were negatively associated with LVEDV (P < 0.05). An increased percentage of intermediate CD14++CD16 + monocytes was positively associated with the GRS (P < 0.005). Furthermore, the percentage of T-cells and CD8 + T-cells were positively associated with LV TWIST whereas a positive association was found between the percentage of CD8 + T-cells and more negative values of LV UNTWIST (P < 0.05). No specific significant correlations were observed between the rest immune cells subpopulations with either the classical or the novel indices of LV dysfunction. CONCLUSION The results of our study suggest that alterations of immune cells subsets correlate with subclinical markers of LV dysfunction in CKD patients without established CVD. The significance of the implication of the cellular arm of the immune system in the pathophysiological pathways of specific CVD phenotypes in CKD and the utility of immune cells subsets as tools to identify CKD patients who are at the highest risk for developing complications shall be elucidated by future research.

The association between adverse reactions and immune response against SARS-CoV-2 spike protein after vaccination with BNT162b2 among healthcare workers in a single healthcare system: a prospective observational cohort study

ABSTRACT Adverse reactions after vaccination with COVID-19 mRNA vaccines are common; however, the association between adverse reactions and humoral responses is uncertain. To determine whether humoral immune responses after BNT162b2 vaccine administration were associated with local and systemic adverse reactions, we conducted a prospective observational cohort study in a single tertiary referral center. Healthcare workers who received the first dose of BNT162b2 vaccine were recruited. SARS-CoV-2 anti-spike IgG antibody titers were measured three weeks after the second dose and information about adverse reactions after vaccination was collected. Among the 887 participants, 641 (72.3%) were women. The median age was 38 (range, 22–74) years. All but one showed anti-spike IgG levels well above the cutoff, with a median level of 13,600 arbitrary units/mL. Overall, 800 (92.2%) participants reported some reactions after the first dose and 822 (96.3%) after the second dose. Significantly more participants reported systemic reactions after the second dose than after the first dose (P < .01), and 625 (73.6%) reported that reactions were stronger after the second dose. Factors positively associated with elevation of anti-spike IgG levels were history of asthma (24% higher if present, P = .01) and stronger reactions after the second dose (19% higher if experienced, P = .02). The majority of participants showed good humoral responses and reported some adverse reactions after vaccination. Anti-spike IgG levels were significantly higher if adverse reactions after the second dose were stronger than those after the first dose. These findings may help inform current and future vaccine recipients.

AAV-delivered suppressor tRNA overcomes a nonsense mutation in mice.

Gene therapy is a potentially curative medicine for many currently untreatable diseases, and recombinant adeno-associated virus (rAAV)is the most successful gene delivery vehicle for in vivo applications1-3. However, rAAV-based gene therapy suffers from several limitations, such as constrained DNA cargo size and toxicities caused by non-physiological expression of a transgene4-6. Here we show that rAAV delivery of a suppressor tRNA (rAAV.sup-tRNA) safely and efficiently rescued a genetic disease in a mouse model carrying a nonsense mutation, and effects lasted for more than 6 months after a single treatment. Mechanistically, this was achieved through a synergistic effect of premature stop codon readthrough and inhibition of nonsense-mediated mRNA decay. rAAV.sup-tRNA had a limited effect on global readthrough at normal stop codons and did not perturb endogenous tRNA homeostasis, as determined by ribosome profiling and tRNA sequencing, respectively. By optimizing the AAV capsid and the route of administration, therapeutic efficacy in various target tissues was achieved, including liver, heart, skeletal muscle and brain. This study demonstrates the feasibility of developing a toolbox of AAV-delivered nonsense suppressor tRNAs operating on premature termination codons (AAV-NoSTOP) to rescue pathogenic nonsense mutations and restore gene function under endogenous regulation. As nonsense mutations account for 11% of pathogenic mutations, AAV-NoSTOP can benefit a large number of patients. AAV-NoSTOP obviates the need to deliver a full-length protein-coding gene that may exceed the rAAV packaging limit, elicit adverse immune responses or cause transgene-related toxicities. It therefore represents a valuable addition to gene therapeutics.

Characterizing the transition from immune response to tissue repair after myocardial infarction by multiparametric imaging

Persistent inflammation following myocardial infarction (MI) precipitates adverse outcome including acute ventricular rupture and chronic heart failure. Molecular imaging allows longitudinal assessment of immune cell activity in the infarct territory and predicts severity of remodeling. We utilized a multiparametric imaging platform to assess the immune response and cardiac healing following MI in mice. Suppression of circulating macrophages prior to MI paradoxically resulted in higher total leukocyte content in the heart, demonstrated by increased CXC motif chemokine receptor 4 (CXCR4) positron emission tomography imaging. This supported the formation of a thrombus overlying the injured region, as identified by magnetic resonance imaging. The injured and thrombotic region in macrophage depeleted mice subsequently showed active calcification, as evidenced by accumulation of 18F-fluoride and by cardiac computed tomography. Importantly, macrophage suppression triggered a prolonged inflammatory response confirmed by post-mortem tissue analysis that was associated with higher mortality from ventricular rupture early after occlusion and with increased infarct size and worse chronic contractile function at 6 weeks after reperfusion. These findings establish a molecular imaging toolbox for monitoring the interplay between adverse immune response and tissue repair after MI. This may serve as a foundation for development and monitoring of novel targeted therapies that may include immune modulation and endogenous healing support.

Evaluation of native and non-native biomaterials for engineering human skin tissue.

A variety of human skin models have been developed for applications in regenerative medicine and efficacy studies. Typically, these employ matrix molecules that are derived from non‐human sources along with human cells. Key limitations of such models include a lack of cellular and tissue microenvironment that is representative of human physiology for efficacy studies, as well as the potential for adverse immune responses to animal products for regenerative medicine applications. The use of recombinant extracellular matrix proteins to fabricate tissues can overcome these limitations. We evaluated animal‐ and non‐animal‐derived scaffold proteins and glycosaminoglycans for the design of biomaterials for skin reconstruction in vitro. Screening of proteins from the dermal‐epidermal junction (collagen IV, laminin 5, and fibronectin) demonstrated that certain protein combinations when used as substrates increase the proliferation and migration of keratinocytes compared to the control (no protein). In the investigation of the effect of components from the dermal layer (collagen types I and III, elastin, hyaluronic acid, and dermatan sulfate), the primary influence on the viability of fibroblasts was attributed to the source of type I collagen (rat tail, human, or bovine) used as scaffold. Furthermore, incorporation of dermatan sulfate in the dermal layer led to a reduction in the contraction of tissues compared to the control where the dermal scaffold was composed primarily of collagen type I. This work highlights the influence of the composition of biomaterials on the development of complex reconstructed skin models that are suitable for clinical translation and in vitro safety assessment.

High-stability conducting polymer-based conformal electrodes for bio-/iono-electronics

Compared to conventional rigid electronics, polymer-based soft electronics conformal to organisms of irregular shapes have emerged as the next-generation devices, especially benefiting long-term bio-interface interactions that avoid mechanical mismatch and consequent adverse immune responses. Highly conductive poly(3,4-ethylenedioxythiophene):poly(styrenesulfonate) (PEDOT:PSS) has become a promising candidate for building soft conductors/electrodes due to its good conductance, tunable mechanical stiffness, good biocompatibility and facile fabrication into various structures. However, their high instability towards alkaline, reductants and applied voltage has not yet been fully addressed, which inevitably leads to deteriorated performance in complex physiological environments or weather conditions (e.g., humidity). Such intolerances are rooted in unstable electronic/molecular structures of PEDOT caused by de-doping. Besides the low electrical stability, PEDOT:PSS films also exhibit an impaired overall conductance due to its phase separation into PSS-rich and PEDOT-rich domains. Herein, a general and effective coating strategy is proposed, based on a mechanism of simultaneous molecular rejection and electron conjugation, to improve the stability and boost the conductance. Specifically, a reduced-graphene-oxide (rGO) thin layer can not only protect PEDOT: PSS from being de-doped by alkali, bio-reductants and applied voltage through molecular rejection, to maintain its conductivity and ensure stable functions, but also further boost the overall conductance through a bridging effect with its large conjugated domain. This strategy is compatible with various material fabrication techniques, including blade-coating, dip-coating and extrusion-based printing techniques, enabling the fabrication of conductors/electrodes with different structures. Finally, the advantages of excellent stability and high conformability of the composite films as soft conductors have been demonstrated through practical applications in tissue stimulation, electrophysiological recording and proprioceptive hydrogel skins, exhibiting great promise in bio/iono-electronics and human–machine interactions.

Isolation and Characterization of Plasma-Derived Exosomes from the Marine Fish Rock Bream (Oplegnathus fasciatus) by Two Isolation Techniques

Exosomes are important mediators of intercellular communication and modulate many physiological and pathological processes. Knowledge of secretion, content, and biological functions of fish exosomes during pathological infection is still scarce due to lack of suitable standardized isolation techniques. In this study, we aimed to isolate exosomes from the plasma of marine fish, rock bream (Oplegnathus fasciatus), by two isolation methods: differential ultracentrifugation (UC) and a commercial membrane affinity spin column technique (kit). Morphological and physicochemical characteristics of the isolated exosomes were determined by these two methods, and the efficiencies of the two methods were compared. Exosomes isolated by both methods were in the expected size range (30–200 nm) and had a characteristic cup-shape in transmission electron microscopy observation. Moreover, more intact exosomes were identified using the kit-based method than UC. Nanoparticle tracking analysis demonstrated a heterogeneous population of exosomes with a mean particle diameter of 114.6 ± 4.6 and 111.2 ± 2.2 nm by UC and a kit-based method, respectively. The particle concentration obtained by the kit method (1.05 × 1011 ± 1.23 × 1010 particles/mL) was 10-fold higher than that obtained by UC (4.90 × 1010 ± 2.91 × 109 particles/mL). The kit method had a comparatively higher total protein yield (1.86 mg) and exosome protein recovery (0.55 mg/mL plasma). Immunoblotting analysis showed the presence of exosome marker proteins (CD81, CD63, and HSP90) in the exosomes isolated by both methods and suggests the existence of exosomes. However, the absence of cytotoxicity or adverse immune responses to fish and mammalian cells by the exosomes isolated by the UC procedure indicates its suitability for functional studies in vitro. Overall, our basic characterization results indicate that the kit-based method is more suitable for isolating high-purity exosomes from fish plasma, whereas UC has higher safety in terms of yielding exosomes with low toxicity. This study provides evidence for the existence of typical exosomes in rock beam plasma and facilitates the selection of an efficient exosome isolation procedure for future applications in disease diagnosis and exosome therapy as fish medicine.

Aggregation Time Machine: A Platform for the Prediction and Optimization of Long-Term Antibody Stability Using Short-Term Kinetic Analysis.

Monoclonal antibodies are the fastest growing class of therapeutics. However, aggregation limits their shelf life and can lead to adverse immune responses. Assessment and optimization of the long-term antibody stability are therefore key challenges in the biologic drug development. Here, we present a platform based on the analysis of temperature-dependent aggregation data that can dramatically shorten the assessment of the long-term aggregation stability and thus accelerate the optimization of antibody formulations. For a set of antibodies used in the therapeutic areas from oncology to rheumatology and osteoporosis, we obtain an accurate prediction of aggregate fractions for up to three years using the data obtained on a much shorter time scale. Significantly, the strategy combining kinetic and thermodynamic analysis not only contributes to a better understanding of the molecular mechanisms of antibody aggregation but has already proven to be very effective in the development and production of biological therapeutics.

Predictive Model for Adverse Events and Immune Response Based on the Production of Antibodies After the Second-Dose of the BNT162b2 mRNA Vaccine.

The BNT162b mRNA vaccine for coronavirus disease 2019, which is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), mimics the immune response to natural infection. Few studies have predicted the adverse effects (AEs) after the second-dose vaccination. We present a predictive model for AEs and immune response after the second-dose of the BNT162b mRNA vaccine. To predict AEs, 282 healthcare workers (HCWs) were enrolled in this prospective observational study. The classification and regression tree (CART) model was established, and its predictive efficacy was assessed. To predict immune response, 282 HCWs were included in the analysis. Moreover, the factors affected by anti-SARS-CoV-2 spike protein RBD antibody (s-IgG) were evaluated using serum samples collected 2 months after the second-dose vaccination. The s-IgG level was assessed using Lumipulse G1200. Multiple regression analyses were conducted to evaluate variables associated with anti-s-IgG titer levels. The most common AEs after the second-dose vaccination were pain (87.6%), redness (17.0%) at the injection site, fatigue (68.8%), headache (53.5%), and fever (37.5%). Based on the CART model, headache after the first-dose vaccination and age < 30 years were identified as the first and second discriminators for predicting the headache after the second-dose vaccination, respectively. In the multiple linear regression model, anti-s-IgG titer levels were associated with age, female sex, and AEs including headache and induration at the injection site after the second-dose vaccination. Headache after the first-dose vaccination can be a predictor of headache after the second-dose vaccination, and AEs are indicators of immune response.

Tailoring the dissolution rate and in vitro cell response of silicon nitride coatings through combinatorial sputtering with chromium and niobium.

Ceramic coatings have been widely investigated as a means to reduce wear and metallic ion release from joint implants. Silicon nitride-based coatings have been a topic of interest specifically due to their solubility in aqueous solutions. This could imply a reduced adverse immune response since the generated debris would dissolve. However, there are concerns regarding the dissolution rate and adhesion of these silicon nitride-based coatings. This study attempts to address the concern of dissolution rate as well as coating adhesion of silicon nitride coatings. We hypothesized that alloying with chromium and niobium would affect the adhesion, dissolution rate, and the resulting ion release and cell response to the coatings. A combinatorial approach was used to deposit sputtered coatings with compositional gradients both with and without a CrN interlayer. Compositional gradients were achieved for all the investigated elements: Si (38.6-46.9 at%), Nb (2.2-4.6 at%) and Cr (1.9-6.0 at%). However, while the presence of an interlayer reduced the delamination during adhesion testing, the differences in composition in the top coating did not affect the adhesion. Nor did the top coating's composition affect the surface roughness or the coatings' inherent mechanical properties (elastic modulus and hardness). All coating compositions were associated with a low Co release from the underlying metal and points with a higher Cr content (4.3-6.0 at%) gave an overall lower release of Si, Cr and Nb ions, possibly due to the formation of a stable oxide, which reduced the dissolution rate of the coating. Optimum chromium contents were furthermore found to give an enhanced in vitro fibroblast cell viability. In conclusion, the results indicate a possibility to tailor the ion release rate, which lends promise to further investigations such as tribocorrosive tests towards a future biomedical application.

A novel afterglow nanoreporter for monitoring cancer therapy.

Rationale: Immunogenic cell death (ICD)-associated immunogenicity evoked through reactive oxygen species (ROS) is an efficient way to fight against the immune-dysfunctional microenvironment, so as to provoke potent anti-tumor immunity. However, the unknown ROS dose during cancer therapies may induce adverse immune responses (e.g., insufficient ICD, toxicity toward normal tissues or immune system).Methods: Herein, we developed a pyrido pyrazine - thiophene based semiconducting polymer as novel near-infrared (NIR) organic afterglow nanoparticles for the real-time visualization of self-generated ROS, during photodynamic-mediated immunogenic cell death. Specifically, we introduced the strong “acceptor” (pyrido pyrazine) into thiophene based semiconducting polymer to redshift emission wavelength, and further modulate the “donor” to afford more afterglow reaction sites and reducing ΔEst, so as to enhance luminescence intensity.Results: The semiconducting polymer-based afterglow nanoparticles exhibit strong afterglow emission with longer-wavelength emission (> 800 nm), compared with the reported organic afterglow nanoparticles (e.g., MEHPPV, PFODBT or Chlorin, < 690 nm), which endows this afterglow nanoparticles with a greatly improvement of signal to noise ratio. Moreover, the photodynamic effect of this afterglow nanoparticles can induce immunogenic cell death of cancer cells and further cause immune responses in mice.Conclusions: The NIR afterglow signal presents a good relationship with ROS generation, immunogenic cell death and outcome of treatment. Therefore, it was able to provide a non-invasive tool for predicting the degree of ICD that occurs during ROS-mediated cancer therapy and may contribute to precise immunotherapy.

Engineering of the Recombinant Expression and PEGylation Efficiency of the Therapeutic Enzyme Human Thymidine Phosphorylase.

Human thymidine phosphorylase (HsTP) is an enzyme with important implications in the field of rare metabolic diseases. Defective mutations of HsTP lead to mitochondrial neurogastrointestinal encephalomyopathy (MNGIE), a disease with a high unmet medical need that is associated with severe neurological and gastrointestinal complications. Current efforts focus on the development of an enzyme replacement therapy (ERT) using the Escherichia coli ortholog (EcTP). However, bacterial enzymes are counter-indicated for human therapeutic applications because they are recognized as foreign by the human immune system, thereby eliciting adverse immune responses and raising significant safety and efficacy risks. Thus, it is critical to utilize the HsTP enzyme as starting scaffold for pre-clinical drug development, thus de-risking the safety concerns associated with the use of bacterial enzymes. However, HsTP expresses very poorly in E. coli, whereas its PEGylation, a crucial chemical modification for achieving long serum persistence of therapeutic enzymes, is highly inefficient and negatively affects its catalytic activity. Here we focused on the engineering of the recombinant expression profile of HsTP in E. coli cells, as well as on the optimization of its PEGylation efficiency aiming at the development of an alternative therapeutic approach for MNGIE. We show that phylogenetic and structural analysis of proteins can provide important insights for the rational design of N’-terminus-truncation constructs which exhibit significantly improved recombinant expression levels. In addition, we developed and implemented a criteria-driven rational surface engineering strategy for the substitution of arginine-to-lysine and lysine-to-arginine residues to achieve more efficient, homogeneous and reproducible PEGylation without negatively affecting the enzymatic catalytic activity upon PEGylation. Collectively, our proposed strategies provide an effective way to optimize enzyme PEGylation and E. coli recombinant expression and are likely applicable for other proteins and enzymes.

Hydroxyapatite composited PEEK with 3D porous surface enhances osteoblast differentiation through mediating NO by macrophage.

The adverse immune response mediated by macrophages is one of the main factors that are prone to lead poor osseointegration of polyetheretherketone (PEEK) implants in clinic. Hence, endowing PEEK with immunomodulatory ability to avoid the adverse immune response becomes a promising strategy to promote bone repair. In this work, sulfonation and hydrothermal treatment were used to fabricate a 3D porous surface on PEEK and hydroxyapatite (HA) composited PEEK. The HA composited PEEK with 3D porous surface inhibited macrophages polarizing to M1 phenotype and downregulated inducible nitric oxide synthase protein expression, which led to a nitric oxide concentration reduction in culture medium of mouse bone marrow mesenchymal stem cells (mBMSCs) under co-culture condition. The decrease of nitric oxide concentration could help to increase bone formation-related OSX and ALP genes expressions and decrease bone resorption-related MMP-9 and MMP-13 genes expressions via cAMP–PKA–RUNX2 pathway in mBMSCs. In summary, the HA composited PEEK with 3D porous surface has the potential to promote osteogenesis of PEEK through immunomodulation, which provides a promising strategy to improve the bone repair ability of PEEK.

43P Assessment of biomarker kinetics for ADG106 (anti-CD137 agonist) as monotherapy or combined with toripalimab

ADG106, a human anti-CD137 IgG4 agonist antibody engineered using Adagene’s NEObody™ technology, binds specifically and with high affinity to a unique and conserved epitope of CD137 on activated human T cells. ADG106 blocks CD137 ligand binding and stimulates CD4+ and CD8+ T cell proliferation and proinflammatory interferon-gamma (IFN-γ) release while displaying a low risk for adverse immune responses. We present here studies of changes in immune activation associated pharmacodynamic (PD) markers, including soluble CD137 (sCD137) following treatment with ADG106 alone or combined with toripalimab, an anti-PD-1 antibody.

Distribution of Immune Cells Including Macrophages in the Human Cochlea.

Background: The human cochlea was earlier believed to lack capacity to mount specific immune responses. Recent studies established that the human cochlea holds macrophages. The cells appear to surveil, dispose of, and restore wasted cells to maintain tissue integrity. Macrophage activities are believed to be the central elements in immune responses and could swiftly defuse invading microbes that enter via adjacent infection-prone areas. This review updates recent human studies in light of the current literature and adds information about chemokine gene expression.Materials and Methods: We analyzed surgically obtained human tissue using immunohistochemistry, confocal microscopy, and multichannel super-resolution structured illumination microscopy. The samples were considered representative of steady-state conditions. Antibodies against the ionized calcium-binding adaptor molecule 1 were used to identify the macrophages. CD68 and CD11b, and the major histocompatibility complex type II (MHCII) and CD4 and CD8 were analyzed. The RNAscope technique was used for fractalkine gene localization.Results: Many macrophages were found around blood vessels in the stria vascularis but not CD4 and CD8 lymphocytes. Amoeboid macrophages were identified in the spiral ganglion with surveilling “antennae” projecting against targeted cells. Synapse-like contacts were seen on spiral ganglion cell bodies richly expressing single CXC3CL gene transcripts. Branching neurite-like processes extended along central and peripheral axons. Active macrophages were occasionally found near degenerating hair cells. Some macrophage-interacting T lymphocytes were observed between the scala tympani wall and Rosenthal's canal. CD4 and CD8 cells were not found in the organ of Corti.Conclusions: The results indicate that the human cochlea is equipped with macrophages and potentially lymphocytes, suggesting both an innate and adaptive immune capacity. A rich expression of fractalkine gene transcripts in spiral ganglion neurons suggest an essential role for auditory nerve protection, as has been demonstrated experimentally. The findings provide further information on the important role of the immune machinery present in the human inner ear and its potential to carry adverse immune reactions, including cytotoxic and foreign body responses. The results can be used to form a rationale for therapies aiming to modulate these immune activities.

Synthetic Amorphous Silica Nanoparticles Promote Human Dendritic Cell Maturation and CD4+ T-Lymphocyte Activation.

Innate immune cells such as dendritic cells (DCs) sense and engulf nanomaterials potentially leading to an adverse immune response. Indeed, as described for combustion-derived particles, nanomaterials could be sensed as danger signals, enabling DCs to undergo a maturation process, migrate to regional lymph nodes and activate naive T lymphocytes. Synthetic amorphous silica nanoparticles (SAS-NPs) are widely used as food additives, cosmetics, and construction materials. This work aimed to evaluate in vitro the effects of manufactured SAS-NPs, produced by thermal or wet routes, on human DCs functions and T-cell activation. Human monocyte-derived DCs (moDCs) were exposed for 16 h to 3 endotoxin-free test materials: fumed silica NPs from Sigma-Aldrich (no. S5505) or the JRC Nanomaterial Repository (NM-202) and colloidal LudoxTMA NPs. Cell viability, phenotypical changes, cytokines production, internalization, and allogeneic CD4+ T-cells proliferation were evaluated. Our results showed that all SAS-NPs significantly upregulated the surface expression of CD86 and CD83 activation markers. Secretions of pro-inflammatory cytokines (CXCL-8 and CXCL-12) were significantly enhanced in a dose-dependent manner in the moDCs culture supernatants by all SAS-NPs tested. In an allogeneic coculture, fumed silica-activated moDCs significantly increased T-lymphocyte proliferation at all T-cell: DC ratios compared with unloaded moDCs. Moreover, analysis of coculture supernatants regarding the production of T-cell-derived cytokines showed a significant increase of IL-9 and IL-17A and F, as well as an upregulation of IL-5, consistent with the pro-inflammatory phenotype of treated moDCs. Taken together, these results suggest that SAS-NPs could induce functional moDCs maturation and play a role in the immunization process against environmental antigens.