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.

LDL-Binding IL-10 Reduces Vascular Inflammation in Atherosclerotic Mice.

Atherosclerosis is a chronic inflammatory disease associated with the accumulation of low-density lipoprotein (LDL) in arterial walls. Higher levels of the anti-inflammatory cytokine IL-10 in serum are correlated with reduced plaque burden. However, cytokine therapies have not translated well to the clinic, partially due to their rapid clearance and pleiotropic nature. Here, we engineered IL-10 to overcome these challenges by hitchhiking on LDL to atherosclerotic plaques. Specifically, we constructed fusion proteins in which one domain is IL-10 and the other is an antibody fragment (Fab) that binds to protein epitopes of LDL. In murine models of atherosclerosis, we show that systemically administered Fab-IL-10 constructs bind circulating LDL and traffic to atherosclerotic plaques. One such construct, 2D03-IL-10, significantly reduces aortic immune cell infiltration to levels comparable to healthy mice, whereas non-targeted IL-10 has no therapeutic effect. Mechanistically, we demonstrate that 2D03-IL-10 preferentially associates with foamy macrophages and reduces pro-inflammatory activation markers. This platform technology can be applied to a variety of therapeutics and shows promise as a potential targeted anti-inflammatory therapy in atherosclerosis.

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.

Traveler's diarrhea.

ABSTRACT: Traveler's diarrhea (TD) is one of the most common illnesses afflicting the modern traveler. TD refers to a watery diarrhea that varies in severity from tolerable to incapacitating and that develops in individuals during or within 10 days of returning from travel to developing or low-/middle-income countries. Most cases of TD are self-limiting, but in consideration of the ease of international travel, it is important for providers to diagnose and manage TD with the best available evidence.

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.

Targeted Polymersome Delivery of a Stapled Peptide for Drugging the Tumor Protein p53:BCL-2-Family Axis in Diffuse Large B-Cell Lymphoma.

Diffuse large B-cell lymphoma (DLBCL) remains a formidable diagnosis in need of new treatment paradigms. In this work, we elucidated an opportunity for therapeutic synergy in DLBCL by reactivating tumor protein p53 with a stapled peptide, ATSP-7041, thereby priming cells for apoptosis and enhancing their sensitivity to BCL-2 family modulation with a BH3-mimetic, ABT-263 (navitoclax). While this combination was highly effective at activating apoptosis in DLBCL in vitro, it was highly toxic in vivo, resulting in a prohibitively narrow therapeutic window. We, therefore, developed a targeted nanomedicine delivery platform to maintain the therapeutic potency of this combination while minimizing its toxicity via packaging and targeted delivery of a stapled peptide. We developed a CD19-targeted polymersome using block copolymers of poly(ethylene glycol) disulfide linked to poly(propylene sulfide) (PEG-SS-PPS) for ATSP-7041 delivery into DLBCL cells. Intracellular delivery was optimized in vitro and validated in vivo by using an aggressive human DLBCL xenograft model. Targeted delivery of ATSP-7041 unlocked the ability to systemically cotreat with ABT-263, resulting in delayed tumor growth, prolonged survival, and no overt toxicity. This work demonstrates a proof-of-concept for antigen-specific targeting of polymersome nanomedicines, targeted delivery of a stapled peptide in vivo, and synergistic dual intrinsic apoptotic therapy against DLBCL via direct p53 reactivation and BCL-2 family modulation.

Mannose-Decorated Co-Polymer Facilitates Controlled Release of Butyrate to Accelerate Chronic Wound Healing.

Butyrate is a key bacterial metabolite that plays an important and complex role in modulation of immunity and maintenance of epithelial barriers. Its translation to clinic is limited by poor bioavailability, pungent smell, and the need for high doses, and effective delivery strategies have yet to realize clinical potential. Here, a novel polymeric delivery platform for tunable and sustainable release of butyrate consisting of a methacrylamide backbone with butyryl ester or phenyl ester side chains as well as mannosyl side chains, which is also applicable to other therapeutically relevant metabolites is reported. This platform's utility in the treatment of non-healing diabetic wounds is explored. This butyrate-containing material modulated immune cell activation in vitro and induced striking changes in the milieu of soluble cytokine and chemokine signals present within the diabetic wound microenvironment in vivo. This novel therapy shows efficacy in the treatment of non-healing wounds through the modulation of the soluble signals present within the wound, and importantly accommodates the critical temporal regulation associated with the wound healing process. Currently, the few therapies to address non-healing wounds demonstrate limited efficacy. This novel platform is positioned to address this large unmet clinical need and improve the closure of otherwise non-healing wounds.

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.

Oral iron replacement vs. IV iron treatment in bariatric surgery patients with anemia.

ABSTRACT: Bariatric surgery has become a recognized tool to reduce weight and resolve or improve comorbid conditions associated with obesity. Patients with obesity are at risk for nutritional deficiencies because of poor-quality diets and the chronic inflammatory state of obesity. Iron deficiency is common in these patients, with incidence rates as high as 21.5% preoperatively and 49% postoperatively. Iron deficiency is often overlooked and not treated, leading to increased complications. This article reviews the risk factors for developing iron-deficiency anemia, diagnosis, and treatment considerations for oral versus IV iron replacement for patients undergoing bariatric surgery.

A curious case of ovarian cysts.

ABSTRACT: Benign mature cystic teratomas are a form of ovarian germ cell tumor that originates from primordial germ cells in the ovaries. Of the three types of teratoma neoplasms, benign mature cystic teratomas (also called dermoid cysts) are the most common. Patients may present with intermittent abdominal or pelvic pain, abdominal enlargement, dysmenorrhea, dyspareunia, or may be asymptomatic. Clinicians should have a high suspicion for benign mature cystic teratomas, which account for more than 20% of all ovarian neoplasms. This article focuses on the clinical symptoms, ovarian growth characteristics, pathophysiology, potential complications, management options, and recurrence of benign mature cystic teratomas.

Lymph Node-Targeted Synthetically Glycosylated Antigen Leads to Antigen-Specific Immunological Tolerance.

Inverse vaccines that tolerogenically target antigens to antigen-presenting cells (APCs) offer promise in prevention of immunity to allergens and protein drugs and treatment of autoimmunity. We have previously shown that targeting hepatic APCs through intravenous injection of synthetically glycosylated antigen leads to effective induction of antigen-specific immunological tolerance. Here, we demonstrate that targeting these glycoconjugates to lymph node (LN) APCs under homeostatic conditions leads to local and increased accumulation in the LNs compared to unmodified antigen and induces a tolerogenic state both locally and systemically. Subcutaneous administration directs the polymeric glycoconjugate to the draining LN, where the glycoconjugated antigen generates robust antigen-specific CD4+ and CD8+ T cell tolerance and hypo-responsiveness to antigenic challenge via a number of mechanisms, including clonal deletion, anergy of activated T cells, and expansion of regulatory T cells. Lag-3 up-regulation on CD4+ and CD8+ T cells represents an essential mechanism of suppression. Additionally, presentation of antigen released from the glycoconjugate to naïve T cells is mediated mainly by LN-resident CD8+ and CD11b+ dendritic cells. Thus, here we demonstrate that antigen targeting via synthetic glycosylation to impart affinity for APC scavenger receptors generates tolerance when LN dendritic cells are the cellular target.

Generation of potent cellular and humoral immunity against SARS-CoV-2 antigens via conjugation to a polymeric glyco-adjuvant.

The SARS-CoV-2 virus has caused an unprecedented global crisis, and curtailing its spread requires an effective vaccine which elicits a diverse and robust immune response. We have previously shown that vaccines made of a polymeric glyco-adjuvant conjugated to an antigen were effective in triggering such a response in other disease models and hypothesized that the technology could be adapted to create an effective vaccine against SARS-CoV-2. The core of the vaccine platform is the copolymer p(Man-TLR7), composed of monomers with pendant mannose or a toll-like receptor 7 (TLR7) agonist. Thus, p(Man-TLR7) is designed to target relevant antigen-presenting cells (APCs) via mannose-binding receptors and then activate TLR7 upon endocytosis. The p(Man-TLR7) construct is amenable to conjugation to protein antigens such as the Spike protein of SARS-CoV-2, yielding Spike-p(Man-TLR7). Here, we demonstrate Spike-p(Man-TLR7) vaccination elicits robust antigen-specific cellular and humoral responses in mice. In adult and elderly wild-type mice, vaccination with Spike-p(Man-TLR7) generates high and long-lasting titers of anti-Spike IgGs, with neutralizing titers exceeding levels in convalescent human serum. Interestingly, adsorbing Spike-p(Man-TLR7) to the depot-forming adjuvant alum amplified the broadly neutralizing humoral responses to levels matching those in mice vaccinated with formulations based off of clinically-approved adjuvants. Additionally, we observed an increase in germinal center B cells, antigen-specific antibody secreting cells, activated T follicular helper cells, and polyfunctional Th1-cytokine producing CD4+ and CD8+ T cells. We conclude that Spike-p(Man-TLR7) is an attractive, next-generation subunit vaccine candidate, capable of inducing durable and robust antibody and T cell responses.

Injection of Bone Marrow Aspirate for Glenohumeral Joint Osteoarthritis: A Pilot Randomized Control Trial.

PURPOSE: To compare the efficacy of a single, intra-articular, nonconcentrated bone marrow aspirate (BMA) injection in comparison to cortisone for the treatment of glenohumeral joint osteoarthritis (GHJ OA). METHODS: Inclusion criteria were patients between the ages of 18 and 75 with a diagnosis of GHJ OA on radiograph. Patients were randomized to receive an ultrasound-guided, intra-articular cortisone injection or BMA injection (without concentration). The primary outcome measure was the Western Ontario Osteoarthritis of the Shoulder (WOOS) index at 12 months. Secondary outcome measures were the QuickDASH, EuroQOL 5-dimensions 5-level questionnaire (EQ-5D-5L) and visual analogue scale. RESULTS: The study included 25 shoulders of 22 patients who completed baseline and 12 months' patient-reported outcome measures (12 shoulders received cortisone, 13 shoulders received BMA) after the study was terminated early by changes in Health Canada regulations. Baseline characteristics demonstrated a significant difference in the ages of the 2 groups, with the BMA group being older (61.6 vs 53.8 mean years, P = 0.021). For the BMA group, a significant improvement was seen in the WOOS index (P = 0.002), the QuickDASH (P < 0.001), and the EQ-5D-5L pain dimension (P = 0.004) between baseline and 12 months. No significant difference was seen for any outcome in the cortisone group between baseline and 12 months. No significant difference was demonstrated between changes in the WOOS scores from baseline to 12 months when compared between groups (P = 0.07). However, a significant difference in changes in scores was seen in the QuickDASH (P = 0.006) and the EQ-5D-5L pain scores (P = 0.003) and the EQ-5D-5L health scores (P = 0.032) in favor of BMA. CONCLUSIONS: The results of this study demonstrate that patients with GHJ OA treated with BMA have superior changes in the QuickDASH and EQ-5D-5L pain and health scores but not in the WOOS outcomes measures at 12 months post injection when compared to patients treated with cortisone. However, because of the limited number of patients as a result of the early termination of the study, larger randomized studies are required to confirm these findings. LEVEL OF EVIDENCE: Level II, randomized controlled trial.

Polymersomes Decorated with the SARS-CoV-2 Spike Protein Receptor-Binding Domain Elicit Robust Humoral and Cellular Immunity.

The COVID-19 pandemic underscores the need for rapid, safe, and effective vaccines. In contrast to some traditional vaccines, nanoparticle-based subunit vaccines are particularly efficient in trafficking antigens to lymph nodes, where they induce potent immune cell activation. Here, we developed a strategy to decorate the surface of oxidation-sensitive polymersomes with multiple copies of the SARS-CoV-2 spike protein receptor-binding domain (RBD) to mimic the physical form of a virus particle. We evaluated the vaccination efficacy of these surface-decorated polymersomes (RBDsurf) in mice compared to RBD-encapsulated polymersomes (RBDencap) and unformulated RBD (RBDfree), using monophosphoryl-lipid-A-encapsulated polymersomes (MPLA PS) as an adjuvant. While all three groups produced high titers of RBD-specific IgG, only RBDsurf elicited a neutralizing antibody response to SARS-CoV-2 comparable to that of human convalescent plasma. Moreover, RBDsurf was the only group to significantly increase the proportion of RBD-specific germinal center B cells in the vaccination-site draining lymph nodes. Both RBDsurf and RBDencap drove similarly robust CD4+ and CD8+ T cell responses that produced multiple Th1-type cytokines. We conclude that a multivalent surface display of spike RBD on polymersomes promotes a potent neutralizing antibody response to SARS-CoV-2, while both antigen formulations promote robust T cell immunity.

Evaluation of the returning traveler with fever and neurologic symptoms.

ABSTRACT: Exposure to infectious disease increases in tandem with international travel rates. Globally, up to 70% of travelers to developing countries report health problems while traveling, most being self-limiting. Few travelers are ill enough to seek medical care while abroad or upon returning home. Although fever is one of the more common symptoms in these travelers, little attention has been paid to those who return with fever and neurologic symptoms. This article describes conditions that can present with fever and neurologic changes and how to evaluate patients in a timely manner to prevent progression of neural dysfunction and spread of disease in local communities.

Use of Comprehensive Geriatric Assessment in Oncology Patients to Guide Treatment Decisions and Predict Chemotherapy Toxicity.

PURPOSE: Our objective was to review the utility of pretreatment comprehensive geriatric assessment (CGA) and its impact on decision making regarding choice and intensity of oncologic therapeutic regimens for older, frail, or poor-functional-status patients, as well as using this prospective assessment to predict chemotherapy-related toxicities. Database searches were conducted in Medline, PubMed, and Ovid for clinical studies, review articles, and journal publications. Search terms included geriatric assessment, medical oncology, chemotherapy, frailty, toxicity, and functional status. Thirty-seven pertinent articles were retrieved and serve as the basis for this clinical review. OBSERVATIONS: CGA is an important tool for examining aspects of frailty and functional status that are not captured by traditional performance status measures. These findings may then be used in selection of appropriate therapeutic regimens for a given patient that are efficacious and tolerable. Such pretreatment assessments also have been used in predicting therapy-related toxicities. CONCLUSIONS: Frail and older patients are common in oncology practices and are at high risk for therapy-related toxicities because of comorbidities and physiologic changes, presenting a considerable clinical challenge. CGA establishes evidence-based strategies to better assess the functional status of such patients and is predictive for chemotherapy-related toxicities in this vulnerable group. Despite publications on these measures in the oncology literature, there is limited evidence-based research to demonstrate the utility of CGA by practicing oncology providers and how to implement it into practice.

Engineered bridge protein with dual affinity for bone morphogenetic protein-2 and collagen enhances bone regeneration for spinal fusion.

The revolutionizing efficacy of recombinant human bone morphogenetic protein (rhBMP-2) for clinical spinal fusion is hindered by safety issues associated with the high dose required. However, it continues to be widely used, for example, in InFUSE Bone Graft (Medtronic). Here, we developed a translational protein engineering-based approach to reduce the dose and thereby improve the safety of rhBMP-2 delivered in a collagen sponge, as in InFUSE Bone Graft. We engineered a bridge protein with high affinity for rhBMP-2 and collagen that can be simply added to the product's formulation, demonstrating improved efficacy at low dose of rhBMP-2 in two mouse models of bone regeneration, including a newly developed spinal fusion model. Moreover, the bridge protein can control the retention of rhBMP-2 from endogenous collagenous extracellular matrix of tissue. Our approach may be generalizable to other growth factors and collagen-based materials, for use in many other applications in regenerative medicine.

Polymersomes decorated with SARS-CoV-2 spike protein receptor binding domain elicit robust humoral and cellular immunity.

A diverse portfolio of SARS-CoV-2 vaccine candidates is needed to combat the evolving COVID-19 pandemic. Here, we developed a subunit nanovaccine by conjugating SARS-CoV-2 Spike protein receptor binding domain (RBD) to the surface of oxidation-sensitive polymersomes. We evaluated the humoral and cellular responses of mice immunized with these surface-decorated polymersomes (RBDsurf) compared to RBD-encapsulated polymersomes (RBDencap) and unformulated RBD (RBDfree), using monophosphoryl lipid A-encapsulated polymersomes (MPLA PS) as an adjuvant. While all three groups produced high titers of RBD-specific IgG, only RBDsurf elicited a neutralizing antibody response to SARS-CoV-2 comparable to that of human convalescent plasma. Moreover, RBDsurf was the only group to significantly increase the proportion of RBD-specific germinal center B cells in the vaccination-site draining lymph nodes. Both RBDsurf and RBDencap drove similarly robust CD4+ and CD8+ T cell responses that produced multiple Th1-type cytokines. We conclude that multivalent surface display of Spike RBD on polymersomes promotes a potent neutralizing antibody response to SARS-CoV-2, while both antigen formulations promote robust T cell immunity.

Soluble N-Acetylgalactosamine-Modified Antigens Enhance Hepatocyte-Dependent Antigen Cross-Presentation and Result in Antigen-Specific CD8+ T Cell Tolerance Development.

Hepatocytes compose up to 80% of the total liver and have been indicated as important players in the induction of immunologic tolerance in this organ. We show that hepatocytes possess the molecular machinery required for the cross-presentation of extracellular antigens. Using a derivative of the model antigen ovalbumin (OVA) covalently modified with a polymer containing multiple N-acetylgalactosamine residues (pGal-OVA) that enhance extracellular antigen uptake by mimicking the glycome of apoptotic debris, we show efficient hepatocyte-dependent induction of cross-tolerance of both adoptively transferred OT-I cells and endogenous OVA-specific CD8+ T lymphocytes, for example inducing tolerance to OVA-expressing skin transplants. Our study confirms that hepatocytes are capable of inducing peripheral tolerogenesis and provides proof of concept that they may be a valuable candidate for in vivo targeted tolerogenic treatments.

Persistent antigen exposure via the eryptotic pathway drives terminal T cell dysfunction.

Although most current treatments for autoimmunity involve broad immunosuppression, recent efforts have aimed to suppress T cells in an antigen-specific manner to minimize risk of infection. One such effort is through targeting antigen to the apoptotic pathway to increase presentation of the antigen of interest in a tolerogenic context. Erythrocytes present a rational candidate to target because of their high rate of eryptosis, which facilitates continual uptake by antigen-presenting cells in the spleen. Here, we develop an approach that binds antigens to erythrocytes to induce sustained T cell dysfunction. Transcriptomic and phenotypic analyses revealed signatures of self-tolerance and exhaustion, including up-regulation of PD-1, CTLA4, Lag3, and TOX. Antigen-specific T cells were incapable of responding to an adjuvanted antigenic challenge even months after antigen clearance. With this strategy, we prevented pathology in a mouse experimental autoimmune encephalomyelitis model. CD8+ T cell education occurred in the spleen and was dependent on cross-presenting Batf3+ dendritic cells. These results demonstrate that antigens associated with eryptotic erythrocytes induce lasting T cell dysfunction that could be protective in deactivating pathogenic T cells.