Nitric Oxide Modulates Ca2+ Leak and Arrhythmias via S-Nitrosylation of CaMKII.

BACKGROUND: Nitric oxide (NO) has been identified as a signaling molecule generated during ß-adrenergic receptor stimulation in the heart. Furthermore, a role for NO in triggering spontaneous Ca2+ release via S-nitrosylation of CaMKIIδ (Ca2+/calmodulin kinase II delta) is emerging. NO donors are routinely used clinically for their cardioprotective effects on the heart, but it is unknown how NO donors modulate the proarrhythmic CaMKII to alter cardiac arrhythmia incidence. We test the role of S-nitrosylation of CaMKIIδ at the Cysteine-273 inhibitory site and cysteine-290 activating site in cardiac Ca2+ handling and arrhythmogenesis before and during ß-adrenergic receptor stimulation. METHODS: We measured Ca2+-handling in isolated cardiomyocytes from C57BL/6J wild-type (WT) mice and mice lacking CaMKIIδ expression (CaMKIIδ-KO) or with deletion of the S-nitrosylation site on CaMKIIδ at cysteine-273 or cysteine-290 (CaMKIIδ-C273S and -C290A knock-in mice). Cardiomyocytes were exposed to NO donors, S-nitrosoglutathione (GSNO; 150 µM), sodium nitroprusside (200 µM), and ß-adrenergic agonist isoproterenol (100 nmol/L). RESULTS: Both WT and CaMKIIδ-KO cardiomyocytes responded to isoproterenol with a full inotropic and lusitropic Ca2+ transient response as well as increased Ca2+ spark frequency. However, the increase in Ca2+ spark frequency was significantly attenuated in CaMKIIδ-KO cardiomyocytes. The protection from isoproterenol-induced Ca2+ sparks and waves was mimicked by GSNO pretreatment in WT cardiomyocytes but lost in CaMKIIδ-C273S cardiomyocytes. When GSNO was applied after isoproterenol, this protection was not observed in WT or CaMKIIδ-C273S but was apparent in CaMKIIδ-C290A. In Langendorff-perfused isolated hearts, GSNO pretreatment limited isoproterenol-induced arrhythmias in WT but not CaMKIIδ-C273S hearts, while GSNO exposure after isoproterenol sustained or exacerbated arrhythmic events. CONCLUSIONS: We conclude that prior S-nitrosylation of CaMKIIδ at cysteine-273 can limit subsequent ß-adrenergic receptor-induced arrhythmias, but that S-nitrosylation at cysteine-290 might worsen or sustain ß-adrenergic receptor-induced arrhythmias. This has important implications for the administration of NO donors in the clinical setting.

Direct regulation of the cardiac ryanodine receptor (RyR2) by O-GlcNAcylation.

BACKGROUND: O-GlcNAcylation is the enzymatic addition of a sugar, O-linked ß-N-Acetylglucosamine, to the serine and threonine residues of proteins, and is abundant in diabetic conditions. We have previously shown that O-GlcNAcylation can trigger arrhythmias by indirectly increasing pathological Ca2+ leak through the cardiac ryanodine receptor (RyR2) via Ca2+/calmodulin-dependent kinase II (CaMKII). However, RyR2 is well known to be directly regulated by other forms of serine and threonine modification, therefore, this study aimed to determine whether RyR2 is directly modified by O-GlcNAcylation and if this also alters the function of RyR2 and Ca2+ leak. METHODS: O-GlcNAcylation of RyR2 in diabetic human and animal hearts was determined using western blotting. O-GlcNAcylation of RyR2 was pharmacologically controlled and the propensity for Ca2+ leak was determined using single cell imaging. The site of O-GlcNAcylation within RyR2 was determined using site-directed mutagenesis of RyR2. RESULTS: We found that RyR2 is modified by O-GlcNAcylation in human, animal and HEK293 cell models. Under hyperglycaemic conditions O-GlcNAcylation was associated with an increase in Ca2+ leak through RyR2 which persisted after CaMKII inhibition. Conversion of serine-2808 to alanine prevented an O-GlcNAcylation induced increase in Ca2+ leak. CONCLUSIONS: These data suggest that the function of RyR2 can be directly regulated by O-GlcNAcylation and requires the presence of serine-2808.

The effect of a reduction in irrational beliefs on Posttraumatic Stress Disorder (PTSD), depression, and anxiety symptoms in a group treatment for post-9/11 Veterans.

Previous research has indicated that a Rational Emotive Behavior Therapy (REBT)-Informed Group focused on changing irrational beliefs to address comorbid depression and anxiety (as well as anger and guilt) in a combat Veteran population diagnosed with Posttraumatic Stress Disorder (PTSD) demonstrated significant reductions in depression and PTSD symptoms at posttreatment. However, mechanisms of change associated with improvement have not been evaluated. REBT theory suggests that a decline in irrational beliefs predicts a decrease in PTSD, depression, and anxiety symptoms. This study aimed to test this tenet of REBT theory in a naturalistic treatment setting. Participants (N = 86) were post-9/11 combat Veterans, engaged in the REBT-Informed Group between October 2016 and February 2020. Results of hierarchical multiple regression analyses indicated that a reduction in irrational beliefs predicted notable decreases in PTSD, depression, and anxiety symptoms controlling for several covariates. This study extends previous research demonstrating the success of the REBT-Informed Group with combat Veterans and gives support to REBT theory regarding the effect of a decline in irrational beliefs. Future directions include replication of findings with Veterans who experienced military sexual trauma (MST), pre-9/11 Veterans, those at other military or Veterans Affairs (VA) medical centers, and civilians to determine generalizability.

Microevolution in response to transient heme-iron restriction enhances intracellular bacterial community development and persistence.

Bacterial pathogens must sense, respond and adapt to a myriad of dynamic microenvironmental stressors to survive. Adaptation is key for colonization and long-term ability to endure fluctuations in nutrient availability and inflammatory processes. We hypothesize that strains adapted to survive nutrient deprivation are more adept for colonization and establishment of chronic infection. In this study, we detected microevolution in response to transient nutrient limitation through mutation of icc. The mutation results in decreased 3',5'-cyclic adenosine monophosphate phosphodiesterase activity in nontypeable Haemophilus influenzae (NTHI). In a preclinical model of NTHI-induced otitis media (OM), we observed a significant decrease in the recovery of effusion from ears infected with the icc mutant strain. Clinically, resolution of OM coincides with the clearance of middle ear fluid. In contrast to this clinical paradigm, we observed that the icc mutant strain formed significantly more intracellular bacterial communities (IBCs) than the parental strain early during experimental OM. Although the number of IBCs formed by the parental strain was low at early stages of OM, we observed a significant increase at later stages that coincided with absence of recoverable effusion, suggesting the presence of a mucosal reservoir following resolution of clinical disease. These data provide the first insight into NTHI microevolution during nutritional limitation and provide the first demonstration of IBCs in a preclinical model of chronic OM.

Inhibition of calcium/calmodulin-dependent kinase II restores contraction and relaxation in isolated cardiac muscle from type 2 diabetic rats.

BACKGROUND: Calcium/calmodulin-dependent kinase II-delta (CaMKIIδ) activity is enhanced during hyperglycemia and has been shown to alter intracellular calcium handling in cardiomyocytes, ultimately leading to reduced cardiac performance. However, the effects of CaMKIIδ on cardiac contractility during type 2 diabetes are undefined. METHODS: We examined the expression and activation of CaMKIIδ in right atrial appendages from non-diabetic and type 2 diabetic patients (n = 7 patients per group) with preserved ejection fraction, and also in right ventricular tissue from Zucker Diabetic Fatty rats (ZDF) (n = 5-10 animals per group) during early diabetic cardiac dysfunction, using immunoblot. We also measured whole heart function of ZDF and control rats using echocardiography. Then we measured contraction and relaxation parameters of isolated trabeculae from ZDF to control rats in the presence and absence of CaMKII inhibitors. RESULTS: CaMKIIδ phosphorylation (at Thr287) was increased in both the diabetic human and animal tissue, indicating increased CaMKIIδ activation in the type 2 diabetic heart. Basal cardiac contractility and relaxation were impaired in the cardiac muscles from the diabetic rats, and CaMKII inhibition with KN93 partially restored contractility and relaxation. Autocamtide-2-related-inhibitor peptide (AIP), another CaMKII inhibitor that acts via a different mechanism than KN93, fully restored cardiac contractility and relaxation. CONCLUSIONS: Our results indicate that CaMKIIδ plays a key role in modulating performance of the diabetic heart, and moreover, suggest a potential therapeutic role for CaMKII inhibitors in improving myocardial function during type 2 diabetes.

A Decision-Support Approach for Provider Scheduling in a Patient-Centered Medical Home.

EXECUTIVE SUMMARY: The patient-centered medical home (PCMH) has been proposed as a viable and medically effective model of primary care delivery. The fundamental principles of a PCMH address increased access and offer new ways to organize a practice. Creating provider schedules able to satisfy the operational and organizational constraints imposed by this type of delivery model is a challenging, complex, and time-consuming task.This article presents a two-step approach for scheduling providers and prospectively assessing the consequences of various schedules on patient throughput without experimenting on the real-world system. The first step uses an original integer programming model to address the tactical planning problem of assigning provider duties to defined shifts while incorporating a number of constraints. Given provider scheduling preferences and availability, a monthly schedule is determined to satisfy the expected demand. The second step consists of quantifying capacity increases that result from adjustments to operating hours, physician availability, and examination room availability. A discrete-event simulation model is used to assess the impact of various proposed operating configurations on patient throughput.

Loss of ascl1a prevents secretory cell differentiation within the zebrafish intestinal epithelium resulting in a loss of distal intestinal motility.

The vertebrate intestinal epithelium is renewed continuously from stem cells at the base of the crypt in mammals or base of the fold in fish over the life of the organism. As stem cells divide, newly formed epithelial cells make an initial choice between a secretory or enterocyte fate. This choice has previously been demonstrated to involve Notch signaling as well as Atonal and Her transcription factors in both embryogenesis and adults. Here, we demonstrate that in contrast to the atoh1 in mammals, ascl1a is responsible for formation of secretory cells in zebrafish. ascl1a-/- embryos lack all intestinal epithelial secretory cells and instead differentiate into enterocytes. ascl1a-/- embryos also fail to induce intestinal epithelial expression of deltaD suggesting that ascl1a plays a role in initiation of Notch signaling. Inhibition of Notch signaling increases the number of ascl1a and deltaD expressing intestinal epithelial cells as well as the number of developing secretory cells during two specific time periods: between 30 and 34hpf and again between 64 and 74hpf. Loss of enteroendocrine products results in loss of anterograde motility in ascl1a-/- embryos. 5HT produced by enterochromaffin cells is critical in motility and secretion within the intestine. We find that addition of exogenous 5HT to ascl1a-/- embryos at near physiological levels (measured by differential pulse voltammetry) induce anterograde motility at similar levels to wild type velocity, distance, and frequency. Removal or doubling the concentration of 5HT in WT embryos does not significantly affect anterograde motility, suggesting that the loss of additional enteroendocrine products in ascl1a-/- embryos also contributes to intestinal motility. Thus, zebrafish intestinal epithelial cells appear to have a common secretory progenitor from which all subtypes form. Loss of enteroendocrine cells reveals the critical need for enteroendocrine products in maintenance of normal intestinal motility.

Systematic investigation of the physicochemical factors that contribute to the toxicity of ZnO nanoparticles.

ZnO nanoparticles (NPs) are prone to dissolution, and uncertainty remains whether biological/cellular responses to ZnO NPs are solely due to the release of Zn(2+) or whether the NPs themselves have additional toxic effects. We address this by establishing ZnO NP solubility in dispersion media (Dulbecco's modified Eagle's medium, DMEM) held under conditions identical to those employed for cell culture (37 °C, 5% CO2, and pH 7.68) and by systematic comparison of cell-NP interaction for three different ZnO NP preparations. For NPs at concentrations up to 5.5 µg ZnO/mL, dissolution is complete (with the majority of the soluble zinc complexed to dissolved ligands in the medium), taking ca. 1 h for uncoated and ca. 6 h for polymer coated ones. Above 5.5 µg/mL, the results are consistent with the formation of zinc carbonate, keeping the solubilized zinc fixed to 67 µM of which only 0.45 µM is as free Zn(2+), i.e., not complexed to dissolved ligands. At these relatively high concentrations, NPs with an aliphatic polyether-coating show slower dissolution (i.e., slower free Zn(2+) release) and reprecipitation kinetics compared to those of uncoated NPs, requiring more than 48 h to reach thermodynamic equilibrium. Cytotoxicity (MTT) and DNA damage (Comet) assay dose-response curves for three epithelial cell lines suggest that dissolution and reprecipitation dominate for uncoated ZnO NPs. Transmission electron microscopy combined with the monitoring of intracellular Zn(2+) concentrations and ZnO-NP interactions with model lipid membranes indicate that an aliphatic polyether coat on ZnO NPs increases cellular uptake, enhancing toxicity by enabling intracellular dissolution and release of Zn(2+). Similarly, we demonstrate that needle-like NP morphologies enhance toxicity by apparently frustrating cellular uptake. To limit toxicity, ZnO NPs with nonacicular morphologies and coatings that only weakly interact with cellular membranes are recommended.

Glycosylation-modified antigens as a tolerance-inducing vaccine platform prevent anaphylaxis in a pre-clinical model of food allergy.

The only FDA-approved oral immunotherapy for a food allergy provides protection against accidental exposure to peanuts. However, this therapy often causes discomfort or side effects and requires long-term commitment. Better preventive and therapeutic solutions are urgently needed. We develop a tolerance-inducing vaccine technology that utilizes glycosylation-modified antigens to induce antigen-specific non-responsiveness. The glycosylation-modified antigens are administered intravenously (i.v.) or subcutaneously (s.c.) and traffic to the liver or lymph nodes, respectively, leading to preferential internalization by antigen-presenting cells, educating the immune system to respond in an innocuous way. In a mouse model of cow's milk allergy, treatment with glycosylation-modified ß-lactoglobulin (BLG) is effective in preventing the onset of allergy. In addition, s.c. administration of glycosylation-modified BLG shows superior safety and potential in treating existing allergies in combination with anti-CD20 co-therapy. This platform provides an antigen-specific immunomodulatory strategy to prevent and treat food allergies.

A serine-conjugated butyrate prodrug with high oral bioavailability suppresses autoimmune arthritis and neuroinflammation in mice.

Butyrate-a metabolite produced by commensal bacteria-has been extensively studied for its immunomodulatory effects on immune cells, including regulatory T cells, macrophages and dendritic cells. However, the development of butyrate as a drug has been hindered by butyrate's poor oral bioavailability, owing to its rapid metabolism in the gut, its low potency (hence, necessitating high dosing), and its foul smell and taste. Here we report that the oral bioavailability of butyrate can be increased by esterifying it to serine, an amino acid transporter that aids the escape of the resulting odourless and tasteless prodrug (O-butyryl-L-serine, which we named SerBut) from the gut, enhancing its systemic uptake. In mice with collagen-antibody-induced arthritis (a model of rheumatoid arthritis) and with experimental autoimmune encephalomyelitis (a model of multiple sclerosis), we show that SerBut substantially ameliorated disease severity, modulated key immune cell populations systemically and in disease-associated tissues, and reduced inflammatory responses without compromising the global immune response to vaccination. SerBut may become a promising therapeutic for autoimmune and inflammatory diseases.

Synthetically mannosylated antigens induce antigen-specific humoral tolerance and reduce anti-drug antibody responses to immunogenic biologics.

Immunogenic biologics trigger an anti-drug antibody (ADA) response in patients that reduces efficacy and increases adverse reactions. Our laboratory has shown that targeting protein antigen to the liver microenvironment can reduce antigen-specific T cell responses; herein, we present a strategy to increase delivery of otherwise immunogenic biologics to the liver via conjugation to a synthetic mannose polymer, p(Man). This delivery leads to reduced antigen-specific T follicular helper cell and B cell responses resulting in diminished ADA production, which is maintained throughout subsequent administrations of the native biologic. We find that p(Man)-antigen treatment impairs the ADA response against recombinant uricase, a highly immunogenic biologic, without a dependence on hapten immunodominance or control by T regulatory cells. We identify increased T cell receptor signaling and increased apoptosis and exhaustion in T cells as effects of p(Man)-antigen treatment via transcriptomic analyses. This modular platform may enhance tolerance to biologics, enabling long-term solutions for an ever-increasing healthcare problem.

The protean presentations of XK disease (McLeod syndrome): a case series with new observations and updates on previously reported families.

XK disease is a very rare, multi-system disease, which can present with a wide spectrum of symptoms. This disorder can also be identified pre-symptomatically with the incidental detection of serological abnormalities when typing erythrocytes in peripheral blood, or on other routine laboratory testing. Increasing awareness of this disorder and improved access to genetic testing are resulting in increasing identification of affected patients and families. Here we provide updates to some previously-reported families and patients and provide additional clinical details. We also report four new cases with a variety of presentations, one of whom had a novel mutation.

Old age liaison psychiatry during COVID-19; an audit of pre- and mid-pandemic service provision.

Older adults in acute hospitals are uniquely vulnerable to mental illness during the COVID-19 pandemic. We describe two eighteen-week periods of specialised liaison psychiatry for older inpatients in a large teaching hospital, pre- and mid-pandemic. Service delivery went from almost completely via face-to-face consultation in the Routine period, to majority remote work in the Pandemic period. During the Routine period, 195 patients were assessed, and patients received a mean number of 2.6 consultations (range 1-15). In the Pandemic period, 197 patients were assessed and received 3.1 consultations on average (range 1-19). Patient age trended toward older in the Pandemic period, mean 77 years (SD 6.9) vs 78 years (SD 1.32) in the Routine period. There were more referrals for behavioural disturbance and confusion during the Pandemic period, and more diagnoses of Behavioural and Psychological Symptoms of Dementia, Delirium and Adjustment Disorder during the Pandemic period vs the Routine period.

Neuroplasticity: The Continuum of Change.

Neuroplasticity is a term that is increasingly permeating mainstream discourse and being used by the popular press to simplify descriptions of how the brain changes in response to stimuli such as exercise, sleep, food, drugs of abuse, and medicines, among others. However, it is a complex, multifaceted concept representing a continuum connecting molecular, cellular, and circuit-level changes and their effects on human behavior. In this Viewpoint, we examine neuroplasticity from several perspectives to construct a holistic view of this ambiguous term. By engaging experts across various scientific disciplines, we attempt to provide an easy entry point to the concept of neuroplasticity for readers of ACS Chemical Neuroscience. By highlighting how neuroplasticity changes in both health and disease, we demonstrate that the concept is applicable to both adaptive and maladaptive responses to stimuli, underscoring its significance in chemical neuroscience.

Peripartum Traumas and Mental Health Outcomes in a Low-Income Sample of NICU Mothers: A Call for Family-Centered, Trauma-Informed Care.

Postpartum depression (PPD), postpartum anxiety (PPA), and post-traumatic stress disorder (PTSD) among birthing people have increased substantially, contributing to adverse maternal/infant dyad outcomes, with a high prevalence in the neonatal intensive care unit (NICU). Despite calls for trauma-informed care in the NICU and high rates of post-traumatic stress, little research has examined the rates of or the relationships between peripartum mood and adverse child experiences (ACEs) in NICU mothers or evaluated which peripartum traumas are most distressing. This study employed structural equation modeling (SEM) to explore whether peripartum-related traumas and NICU-related stressors mediated the associations between ACEs and mental health outcomes in 119 lower-income, racially diverse mothers in a Level IV NICU. Mental health concerns were prevalent and highly comorbid, including 51.3% PPA, 34.5% PPD, 39.5% post-traumatic stress, and 37% with ≥4 ACEs. The majority (53.8%) of mothers endorsed multiple peripartum traumas; NICU admission was the most common trauma (61%), followed by birth (19%), pregnancy (9%), and a medical event in the NICU (9%). Our SEMs had good fit and demonstrated that ACEs predicted peripartum distress. Trauma-informed care efforts should employ transdiagnostic approaches and recognize that women commonly present to the NICU with childhood trauma history and cumulative peripartum traumas.

Nitric Oxide modulates spontaneous Ca2+ release and ventricular arrhythmias during ß-adrenergic signalling through S-nitrosylation of Calcium/Calmodulin dependent kinase II.

Rationale: Nitric oxide (NO) has been identified as a signalling molecule generated during ß-adrenergic receptor (AR) stimulation in the heart. Furthermore, a role for NO in triggering spontaneous Ca2+ release via S-nitrosylation of Ca2+/calmodulin kinase II delta (CaMKIIδ) is emerging. NO donors are routinely used clinically for their cardioprotective effects in the heart, but it is unknown how NO donors modulate the pro-arrhythmic CaMKII to alter cardiac arrhythmia incidence. Objective: We test the role of S-nitrosylation of CaMKIIδ at the Cys-273 inhibitory site and Cys-290 activating site in cardiac Ca2+ handling and arrhythmogenesis before and during ß-AR stimulation. Methods and Results: We measured Ca2+-handling in isolated cardiomyocytes from C57BL/6J wild-type (WT) mice and mice lacking CaMKIIδ expression (CaMKIIδ-KO) or with deletion of the S-nitrosylation site on CaMKIIδ at Cys-273 or Cys-290 (CaMKIIδ-C273S and -C290A knock-in mice). Cardiomyocytes were exposed to NO donors, S-nitrosoglutathione (GSNO; 150 µM), sodium nitroprusside (SNP; 200 µM) and/or ß-adrenergic agonist isoproterenol (ISO; 100 nM). WT and CaMKIIδ-KO cardiomyocytes treated with GSNO showed no change in Ca2+ transient or spark properties under baseline conditions (0.5 Hz stimulation frequency). Both WT and CaMKIIδ-KO cardiomyocytes responded to ISO with a full inotropic and lusitropic Ca2+ transient response as well as increased Ca2+ spark frequency. However, the increase in Ca2+ spark frequency was significantly attenuated in CaMKIIδ-KO cardiomyocytes. The protection from ISO-induced Ca2+ sparks and waves was mimicked by GSNO pre-treatment in WT cardiomyocytes, but lost in CaMKIIδ-C273S cardiomyocytes that displayed a robust increase in Ca2+ waves. This observation is consistent with CaMKIIδ-C273 S-nitrosylation being critical in limiting ISO-induced arrhythmogenic sarcoplasmic reticulum Ca2+ leak. When GSNO was applied after ISO this protection was not observed in WT or CaMKIIδ-C273S but was apparent in CaMKIIδ-C290A. In Langendorff-perfused isolated hearts, GSNO pre-treatment limited ISO-induced arrhythmias in WT but not CaMKIIδ-C273S hearts, while GSNO exposure after ISO sustained or exacerbated arrhythmic events. Conclusions: We conclude that prior S-nitrosylation of CaMKIIδ at Cys-273 can limit subsequent ß-AR induced arrhythmias, but that S-nitrosylation at Cys-290 might worsen or sustain ß-AR-induced arrhythmias. This has important implications for the administration of NO donors in the clinical setting.

Topically-applied collagen-binding serum albumin-fused interleukin-4 modulates wound microenvironment in non-healing wounds.

Non-healing wounds have a negative impact on quality of life and account for many cases of amputation and even early death among patients. Diabetic patients are the predominate population affected by these non-healing wounds. Despite the significant clinical demand, treatment with biologics has not broadly impacted clinical care. Interleukin-4 (IL-4) is a potent modulator of the immune system, capable of skewing macrophages towards a pro-regeneration phenotype (M2) and promoting angiogenesis, but can be toxic after frequent administration and is limited by its short half-life and low bioavailability. Here, we demonstrate the design and characterization of an engineered recombinant interleukin-4 construct. We utilize this collagen-binding, serum albumin-fused IL-4 variant (CBD-SA-IL-4) delivered in a hyaluronic acid (HA)-based gel for localized application of IL-4 to dermal wounds in a type 2 diabetic mouse model known for poor healing as proof-of-concept for improved tissue repair. Our studies indicate that CBD-SA-IL-4 is retained within the wound and can modulate the wound microenvironment through induction of M2 macrophages and angiogenesis. CBD-SA-IL-4 treatment significantly accelerated wound healing compared to native IL-4 and HA vehicle treatment without inducing systemic side effects. This CBD-SA-IL-4 construct can address the underlying immune dysfunction present in the non-healing wound, leading to more effective tissue healing in the clinic.

Synthetically mannosylated antigens induce antigen-specific humoral tolerance and reduce anti-drug antibody responses to immunogenic biologics.

Immunogenic biologics trigger an anti-drug antibody (ADA) response in patients, which reduces efficacy and increases adverse reactions. Our laboratory has previously shown that targeting protein antigen to the liver microenvironment can reduce antigen-specific T cell responses; herein, we present a strategy to increase delivery of otherwise immunogenic biologics to the liver via conjugation to a synthetic mannose polymer (p(Man)). This delivery leads to reduced antigen-specific T follicular helper cell and B cell responses resulting in diminished ADA production, which is maintained throughout subsequent administrations of the native biologic. We found that p(Man)-antigen treatment impairs the ADA response against recombinant uricase, a highly immunogenic biologic, without a dependence on hapten immunodominance or control by Tregs. We identify increased TCR signaling and increased apoptosis and exhaustion in T cells as effects of p(Man)-antigen treatment via transcriptomic analyses. This modular platform may enhance tolerance to biologics, enabling long-term solutions for an ever-increasing healthcare problem.

Synthetically glycosylated antigens for the antigen-specific suppression of established immune responses.

Inducing antigen-specific tolerance during an established immune response typically requires non-specific immunosuppressive signalling molecules. Hence, standard treatments for autoimmunity trigger global immunosuppression. Here we show that established antigen-specific responses in effector T cells and memory T cells can be suppressed by a polymer glycosylated with N-acetylgalactosamine (pGal) and conjugated to the antigen via a self-immolative linker that allows for the dissociation of the antigen on endocytosis and its presentation in the immunoregulatory environment. We show that pGal-antigen therapy induces antigen-specific tolerance in a mouse model of experimental autoimmune encephalomyelitis (with programmed cell-death-1 and the co-inhibitory ligand CD276 driving the tolerogenic responses), as well as the suppression of antigen-specific responses to vaccination against a DNA-based simian immunodeficiency virus in non-human primates. Our findings show that pGal-antigen therapy invokes mechanisms of immune tolerance to resolve antigen-specific inflammatory T-cell responses and suggest that the therapy may be applicable across autoimmune diseases.