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Purpose: The present study aimed to determine the dose-response relationship between targeted nanocarriers released from a novel, sustained release formulation and their ability to specifically deplete cells responsible for the development of posterior capsular opacification (PCO) in month-long, dynamic cell cultures. Methods: Injectable, thermosensitive poly(D,L-lactic-co-glycolic acid)-b-poly(ethylene glycol)-b-poly(D,L-lactic-co-glycolic acid) triblock copolymer hydrogels were loaded with either a low or a high dose of doxorubicin-loaded antibody-targeted nanocarriers (G8:3DNA:Dox). Human rhabdomyosarcoma cells, selected for their expression of PCO marker brain-specific angiogenesis inhibitor 1 (BAI1), were kept under dynamic media flow and received either a low or high dose of nanocarriers. Cells were fixed and stained at predetermined time points to evaluate targeted depletion of BAI1+ cells. Results: A lower dose of nanocarriers in hydrogel depleted BAI1+ cells at a slower rate than the higher dose, whereas both reached over 90% BAI1+ cellular nonviability at 28 days. Both treatment groups also significantly lowered the relative abundance of BAI1+ cells in the population compared with the control group. Conclusions: Controlled release of a lower dose of nanocarriers can still achieve therapeutically relevant effects in the prevention of PCO, while avoiding potential secondary effects associated with the administration of a higher dose.

Introduction: Standard-of-care systemic chemotherapies for pancreatic ductal adenocarcinoma (PDAC) currently have limited clinical benefits, in addition to causing adverse side effects in many patients. One factor known to contribute to the poor chemotherapy response is the poor drug diffusion into PDAC tumors. Novel treatment methods are therefore drastically needed to improve targeted delivery of treatments. Here, we evaluated the efficacy of the 3DNA® Nanocarrier (3DNA) platform to direct delivery of therapeutics to PDAC tumors in vivo., Materials and Methods: A panel of PDAC cell lines and a patient tissue microarray were screened for established tumor-specific proteins to identify targeting moieties for active targeting of the 3DNA. NRG mice with or without orthotopic MIA PaCa-2-luciferase PDAC tumors were treated intraperitoneally with 100 μl of fluorescently labeled 3DNA., Results: Folic acid and transferrin receptors were significantly elevated in PDAC compared to normal pancreas. Accordingly, both folic acid- and transferrin-conjugated 3DNA treatments significantly increased delivery of 3DNA specifically to tumors in comparison to unconjugated 3DNA treatment. In the absence of tumors, there was an increased clearance of both folic acid-conjugated 3DNA and unconjugated 3DNA, compared to the clearance rate in tumor-bearing mice. Lastly, delivery of siLuciferase by folic acid-conjugated 3DNA in an orthotopic model of luciferase-expressing PDAC showed significant and prolonged suppression of luciferase protein expression and activity., Conclusion: Our study progresses the 3DNA technology as a reliable and effective treatment delivery platform for targeted therapeutic approaches in PDAC., Competing Interests: Declaration of Competing Interest All other authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. This work and G.A.M. were supported Code Biotherapeutics through a Sponsored Research Agreement. Materials for 3DNA were supplied by Code Biotherapeutics. A.S., S.Z.B., L.G., and R.G. are all employees of Code Biotherapeutics., (Copyright © 2023. Published by Elsevier Inc.)

Background: Currently, there is no laboratory test that can accurately identify children at risk of developing peanut allergy. Utilizing a subset of children randomized to the peanut avoidance arm of the LEAP trial, we monitored the development of epitope-specific (ses-)IgE and ses-IgG4 from 4-11 months to 5 years of age., Objective: The aim of the study was to evaluate the prognostic ability of epitope-specific antibodies to predict the result of an oral food challenge (OFC) at 5 years., Methods: A Bead-Based Epitope Assay was used to quantitate IgE and IgG4 to 64 sequential (linear) epitopes from Ara h 1-3 proteins at 4-11 months, 1 and 2.5 years of age in 74 subjects (38 of them with a positive OFC at 5 years). Specific IgE (sIgE) to peanut and component proteins was measured using ImmunoCAP. Machine learning methods were used to identify the earliest time point to predict 5-year outcome, developing prognostic algorithms based only on 4-11 month samples, 1-year or 2.5-year, and a combination of them. Data from 74 children were iteratively split 3:1 into training and validation sets, and machine learning models were developed to predict the 5-year outcome. A test set (n = 90) from an independent cohort was used for final evaluation., Results: Elastic-Net algorithm combining ses-IgE and IgE to Ara h 1, 2, 3, and 9 proteins could predict the 5-year peanut allergy status of LEAP participants with an average validation accuracy of 64% at baseline. Samples taken at 1 year accurately predicted a 5-year OFC outcome with 83% accuracy. This performance remained consistent when evaluated on an independent CoFAR2 cohort with an accuracy of 78% for the 1-year model., Conclusion: IgE antibody profiles at 1 year of age are predictive of peanut OFC at 5 years in children avoiding peanuts. If further confirmed, this model may enable early identification of infants who may benefit from early immunotherapeutic interventions., (© 2022 S. Karger AG, Basel.)

Background: IgE-epitope profiling can accurately diagnose clinical peanut allergy., Objective: We sought to determine whether sequential (linear) epitope-specific IgE (ses-IgE) profiling can provide probabilities of tolerating discrete doses of peanut protein in allergic subjects undergoing double-blind, placebo-controlled food challenges utilizing PRACTALL dosing., Methods: Sixty four ses-IgE antibodies were quantified in blood samples using a bead-based epitope assay. A pair of ses-IgEs that predicts Cumulative Tolerated Dose (CTD) was determined using regression in 75 subjects from the discovery cohort. This epitope-based predictor was validated on 331 subjects from five independent cohorts (ages 4-25 years). Subjects were grouped based on their predicted values and probabilities of reactions at each CTD threshold were calculated., Results: In discovery, an algorithm using two ses-IgE antibodies was correlated with CTDs (rho = 0.61, p < .05); this correlation was 0.51 (p < .05) in validation. Using the ses-IgE-based predictor, subjects were assigned into "high," "moderate," or "low" dose-reactivity groups. On average, subjects in the "high" group were four times more likely to tolerate a specific dose, compared with the "low" group. For example, predicted probabilities of tolerating 4, 14, 44, and 144 or 444 mg in the "low" group were 92%, 77%, 53%, 29%, and 10% compared with 98%, 95%, 94%, 88%, and 73% in the "high" group., Conclusions: Accurate predictions of food challenge thresholds are complex due to factors including limited responder sample sizes at each dose and variations in study-specific challenge protocols. Despite these limitations, an epitope-based predictor was able to accurately identify CTDs and may provide a useful surrogate for peanut challenges., (© 2022 The Authors. Allergy published by European Academy of Allergy and Clinical Immunology and John Wiley & Sons Ltd.)

Purpose: To compare a novel, sustained release formulation and a bolus injection of a targeted nanocarrier for the ability to specifically deplete cells responsible for the development of posterior capsule opacification (PCO) in week-long, dynamic cell cultures. Methods: A novel, injectable, thermosensitive poly(D,L-lactic-co-glycolic acid)-b-poly(ethylene glycol)-b-poly(D,L-lactic-co-glycolic acid) (PLGA-PEG-PLGA) triblock copolymer hydrogel was engineered for the sustained release of targeted, nucleic acid nanocarriers loaded with cytotoxic doxorubicin (G8:3DNA:Dox). Human rhabdomyosarcoma (RD) cells were used due to their expression of brain-specific angiogenesis inhibitor 1 (BAI1), a specific marker for the myofibroblasts responsible for PCO. Under constant media flow, nanocarriers were injected into cell cultures as either a bolus or within the hydrogel. Cells were fixed and stained every other day for 7 days to compare targeted depletion of BAI1 + cells. Results: The formulation transitions to a gel at physiological temperatures, is optically clear, noncytotoxic, and can release G8:3DNA:Dox nanocarriers for up to 4 weeks. In RD cell cultures, G8:3DNA:Dox nanocarriers specifically eliminated BAI1 + cells. The bolus nanocarrier dose showed significantly reduced cell depletion overtime, while the sustained release of nanocarriers showed increased cell depletion over time. By day 7, <2% of BAI1 + cells were depleted by the bolus injection and 74.2% BAI1 + cells were targeted by the sustained release of nanocarriers. Conclusions: The sustained release of nanocarriers from the hydrogel allows for improved therapeutic delivery in a dynamic system. This method can offer a more effective and efficient method of prophylactically treating PCO after cataract surgery.

Nutrient-deprived conditions in the tumor microenvironment (TME) restrain cancer cell viability due to increased free radicals and reduced energy production. In pancreatic cancer cells a cytosolic metabolic enzyme, wild-type isocitrate dehydrogenase 1 (wtIDH1), enables adaptation to these conditions. Under nutrient starvation, wtIDH1 oxidizes isocitrate to generate α-ketoglutarate (αKG) for anaplerosis and NADPH to support antioxidant defense. In this study, we show that allosteric inhibitors of mutant IDH1 (mIDH1) are potent wtIDH1 inhibitors under conditions present in the TME. We demonstrate that low magnesium levels facilitate allosteric inhibition of wtIDH1, which is lethal to cancer cells when nutrients are limited. Furthermore, the Food & Drug Administration (FDA)-approved mIDH1 inhibitor ivosidenib (AG-120) dramatically inhibited tumor growth in preclinical models of pancreatic cancer, highlighting this approach as a potential therapeutic strategy against wild-type IDH1 cancers., (© 2022. This is a U.S. government work and not under copyright protection in the U.S.; foreign copyright protection may apply.)

Introduction: Molecular studies of hen's egg allergens help define allergic phenotypes, with IgE to sequential (linear) epitopes on the ovomucoid (OVM) protein associated with a persistent disease. Epitope profiles of other egg allergens are largely unknown. The objective of this study was to construct an epitope library spanning across 7 allergens and further evaluate sequential epitope-specific (ses-)IgE and ses-IgG4 among baked-egg reactive or tolerant children., Methods: A Bead-Based Epitope Assay was used to identify informative IgE epitopes from 15-mer overlapping peptides covering the entire OVM and ovalbumin (OVA) proteins in 38 egg allergic children. An amalgamation of 12 B-cell epitope prediction tools was developed using experimentally identified epitopes. This ensemble was used to predict epitopes from ovotransferrin, lysozyme, serum albumin, vitellogenin-II fragment, and vitellogenin-1 precursor. Ses-IgE and ses-IgG4 repertoires of 135 egg allergic children (82 reactive to baked-egg, the remaining 52 tolerant), 46 atopic controls, and 11 healthy subjects were compared., Results: 183 peptides from OVM and OVA were screened and used to create an aggregate algorithm, improving predictions of 12 individual tools. A final library of 65 sequential epitopes from 7 proteins was constructed. Egg allergic children had higher ses-IgE and lower ses-IgG4 to predominantly OVM epitopes than both atopic and healthy controls. Baked-egg reactive children had similar ses-IgG4 but greater ses-IgE than tolerant group. A combination of OVA-sIgE with ses-IgEs to OVM-023 and OVA-028 was the best predictor of reactive phenotype., Conclusion: We have created a comprehensive epitope library and showed that ses-IgE is a potential biomarker of baked-egg reactivity., (© 2021 S. Karger AG, Basel.)

Background: Accurate diagnosis of peanut allergy is a significant clinical challenge. Here, a novel diagnostic blood test using the peanut bead-based epitope assay ("peanut BBEA") was developed utilizing the LEAP cohort and then validated using two independent cohorts., Methods: The development of the peanut BBEA diagnostic test followed the National Academy of Medicine's established guidelines with discovery performed on 133 subjects from the non-interventional arm of the LEAP trial and an independent validation performed on 82 subjects from the CoFAR2 and 84 subjects from the POISED study. All samples were analyzed using the peanut BBEA methodology, which measures levels of IgE to two Ara h 2 sequential (linear) epitopes and compares their combination to a threshold pre-specified in the model development phase. When a patient has an inconclusive outcome by skin prick testing (or sIgE), IgE antibody levels to this combination of two epitopes can distinguish whether the patient is "Allergic" or "Not Allergic." Diagnoses of peanut allergy in all subjects were confirmed by double-blind placebo-controlled food challenge and subjects' ages were 7-55 years., Results: In the validation using CoFAR2 and POISED cohorts, the peanut BBEA diagnostic test correctly diagnosed 93% of the subjects, with a sensitivity of 92%, specificity of 94%, a positive predictive value of 91%, and negative predictive value of 95%., Conclusions: In validation of the peanut BBEA diagnostic test, the overall accuracy was found to be superior to existing diagnostic tests for peanut allergy including skin prick testing, peanut sIgE, and peanut component sIgE testing., (© 2021 The Authors. Allergy published by European Academy of Allergy and Clinical Immunology and John Wiley & Sons Ltd.)

Autoimmune diseases such as rheumatoid arthritis are caused by immune system recognition of self-proteins and subsequent production of effector T cells that recognize and attack healthy tissue. Therapies for these diseases typically utilize broad immune suppression, which can be effective, but which also come with an elevated risk of susceptibility to infection and cancer. T cell recognition of antigens is driven by binding of T cell receptors to peptides displayed on major histocompatibility complex proteins (MHCs) on the cell surface of antigen-presenting cells. Technology for recombinant production of the extracellular domains of MHC proteins and loading with peptides to produce pMHCs has provided reagents for detection of T cell populations, and with the potential for therapeutic intervention. However, production of pMHCs in large quantities remains a challenge and a translational path needs to be established. Here, we demonstrate a fusion protein strategy enabling large-scale production of pMHCs. A peptide corresponding to amino acids 259-273 of collagen II was fused to the N-terminus of the MHC&#95;II beta chain, and the alpha and beta chains were each fused to human IgG4 Fc domains and co-expressed. A tag was incorporated to enable site-specific conjugation. The cytotoxic drug payload, MMAF, was conjugated to the pMHC and potent, peptide-specific killing of T cells that recognize the collagen pMHC was demonstrated with tetramerized pMHC-MMAF conjugates. Finally, these pMHCs were incorporated into MMAF-loaded 3DNA nanomaterials in order to provide a biocompatible platform. Loading and pMHC density were optimized, and peptide-specific T cell killing was demonstrated. These experiments highlight the potential of a pMHC fusion protein-targeted, drug-loaded nanomaterial approach for selective delivery of therapeutics to disease-relevant T cells and new treatment options for autoimmune disease.

Background: In the LEAP (Learning Early About Peanut Allergy) trial, early consumption of peanut in high-risk infants was found to decrease the rate of peanut allergy at 5 years of age. Sequential epitope-specific (ses-)IgE is a promising biomarker of clinical peanut reactivity., Objective: We sought to compare the evolution of ses-IgE and ses-IgG 4 in children who developed (or not) peanut allergy and to evaluate the immunomodulatory effects of early peanut consumption on these antibodies., Methods: Sera from 341 children (LEAP cohort) were assayed at baseline, 1, 2.5, and 5 years of age, with allergy status determined by oral food challenge at 5 years. A bead-based epitope assay was used to quantitate ses-IgE and ses-IgG 4 to 64 sequential epitopes from Ara h 1 to Ara h 3 and was analyzed using linear mixed-effect models., Results: In children avoiding peanut who became peanut allergic, the bulk of peanut ses-IgE did not develop until after 2.5 years. Minimal increases of ses-IgE occurred after 1 year in consumers, but not to the same epitopes as those in children developing peanut allergy. No major changes in ses-IgE were seen in nonallergic or sensitized children. IgE in sensitized consumers was detected against peanut proteins. ses-IgG 4 increased over time in most children regardless of consumption or allergy status., Conclusions: Early peanut consumption in infants at high risk of developing peanut allergy appears to divert the immunologic response to a presumably "protective" effect. In general, consumers tend to generate ses-IgG 4 earlier and in greater quantities than nonconsumers do, whereas only avoiders tend to generate significant quantities of ses-IgE., (Copyright © 2021 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.)

Background: Peanut allergy is characterized by the development of IgE against peanut antigen., Objective: We sought to evaluate the evolution of epitope-specific (es)IgE and esIgG 4 in a prospective cohort of high-risk infants to determine whether antibody profiles can predict peanut allergy after age 4 years., Methods: The end point was allergy status at age 4 + years; samples from 293 children were collected at age 3 to 15 months and 2 to 3 and 4 + years. Levels of specific (s)IgE and sIgG 4 to peanut and component proteins, and 50 esIgE and esIgG 4 were quantified. Changes were analyzed with mixed-effects models. Machine learning algorithms were developed to identify a combination of antigen- and epitope-specific antibodies that using 3- to 15-month or 2- to 3-year samples can predict allergy status at age 4 + years., Results: At age 4 + years, 38% of children were Tolerant or 14% had Possible, 8% Convincing, 24% Serologic, and 16% Confirmed allergy. At age 3 to 15 months, esIgE profiles were similar among groups, whereas marked increases were evident at age 2 and 4 + years only in Confirmed and Serologic groups. In contrast, peanut sIgE level was significantly lower in the Tolerant group at age 3 to 15 months, increased in Confirmed and Serologic groups but decreased in Convincing and Possibly Allergic groups over time. An algorithm combining esIgEs with peanut sIgE outperformed different clinically relevant IgE cutoffs, predicting allergy status on an "unseen" set of patients with area under the curves of 0.84 at age 3 to 15 months and 0.87 at age 2 to 3 years., Conclusions: Early epitope-specific plus peanut-specific IgE is predictive of allergy status at age 4 + years., (Copyright © 2020 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.)

Background: Egg-white ovomucoid, that is, Gal d 1, is associated with IgE-mediated allergic reactions in most egg-allergic children. Epitope-specific IgE levels have been correlated with the severity of egg allergy, while emerging evidence suggests that other antibody isotypes (IgG 1 , IgG 4 , IgA, and IgD) may have a protective function; yet, their epitope-specific repertoires and associations with atopic comorbidities have not been studied., Methods: Bead-based epitope assay (BBEA) was used to quantitate the levels of epitope-specific (es)IgA, esIgE, esIgD, esIgG 1 , and esIgG 4 antibodies directed at 58 (15-mer) overlapping peptides, covering the entire sequence of ovomucoid, in plasma of 38 egg-allergic and 6 atopic children. Intraclass correlation (ICC) and coefficient of variation (CV) were used for the reliability assessment. The relationships across esIgs were evaluated using network analysis; linear and logistic regressions were used to compare groups based on egg allergy status and comorbidities., Results: BBEA had high reliability (ICC >0.75) and low variability (CV <20%) and could detect known IgE-binding epitopes. Egg-allergic children had lower esIgA 1 (P = .010) and esIgG 1 (P = .016) and higher esIgE (P < .001) and esIgD (P = .015) levels compared to the atopic controls. Interestingly, within the allergic group, children with higher esIgD had decreased odds of anaphylactic reactions (OR =0.48, P = .038). Network analysis identified most associations between esIgE with either esIgG 4 or esIgD; indicating that IgE-secreting plasma cells could originate from either sequential isotype switch from antigen-experienced intermediate isotypes or directly from the IgD + B cells., Conclusions: Collectively, these data point toward a contribution of epitope-specific antibody repertoires to the pathogenesis of egg allergy., (© 2020 EAACI and John Wiley and Sons A/S. Published by John Wiley and Sons Ltd.)

The tryptophan catabolizing enzyme indoleamine 2,3-dioxygenase 2 (IDO2) has been identified as an immunomodulatory agent promoting autoimmunity in preclinical models. As such, finding ways to target the expression of IDO2 in B cells promises a new avenue for therapy for debilitating autoimmune disorders such as rheumatoid arthritis. IDO2, like many drivers of disease, is an intracellular protein expressed in a range of cells, and thus therapeutic inhibition of IDO2 requires a mechanism for targeting this intracellular protein in specific cell types. DNA nanostructures are a promising novel way of delivering small molecule drugs, antibodies, or siRNAs to the cytoplasm of a cell. These soluble, branched structures can carry cell-specific targeting moieties along with their therapeutic deliverable. Here, we examined a 3DNA nanocarrier specifically targeted to B cells with an anti-CD19 antibody. We find that this 3DNA is successfully delivered to and internalized in B cells. To test whether these nanostructures can deliver an efficacious therapeutic dose to alter autoimmune responses, a modified anti-IDO2 siRNA was attached to B-cell-directed 3DNA nanocarriers and tested in an established preclinical model of autoimmune arthritis, KRN.g7. The anti-IDO2 3DNA formulation ameliorates arthritis in this system, delaying the onset of joint swelling and reducing total arthritis severity. As such, a 3DNA nanocarrier system shows promise for delivery of targeted, specific, low-dose therapy for autoimmune disease., Competing Interests: Declaration of conflicting interests:The author(s) declared the following potential conflicts of interest with respect to the research, authorship, and/or publication of this article: J.B. is Director of Academic Collaborations and Marketing Communications, T.S. is a Research and Development Scientist, and R.G. is Founder and Chief Scientific Officer of Genisphere LLC, the commercial manufacturer of 3DNA. L.M.-N. and L.M.F.M. declare no conflict of interest., (© The Author(s) 2020.)

The Myo/Nog cell lineage was discovered in the chick embryo and is also present in adult mammalian tissues. The cells are named for their expression of mRNA for the skeletal muscle specific transcription factor MyoD and bone morphogenetic protein inhibitor Noggin. A third marker for Myo/Nog cells is the cell surface molecule recognized by the G8 monoclonal antibody (mAb). G8 has been used to detect, track, isolate and kill Myo/Nog cells. In this study, we screened a membrane proteome array for the target of the G8 mAb. The array consisted of >5,000 molecules, each synthesized in their native confirmation with appropriate post-translational modifications in a single clone of HEK-293T cells. G8 mAb binding to the clone expressing brain-specific angiogenesis inhibitor 1 (BAI1) was detected by flow cytometry, re-verified by sequencing and validated by transfection with the plasmid construct for BAI1. Further validation of the G8 target was provided by enzyme-linked immunosorbent assay. The G8 epitope was identified by screening a high-throughput, site directed mutagenesis library designed to cover 95-100% of the 954 amino acids of the extracellular domain of the BAI1 protein. The G8 mAb binds within the third thrombospondin repeat of the extracellular domain of human BAI1. Immunofluorescence localization experiments revealed that G8 and a commercially available BAI1 mAb co-localize to the subpopulation of Myo/Nog cells in the skin, eyes and brain. Expression of the multi-functional BAI1 protein in Myo/Nog cells introduces new possibilities for the roles of Myo/Nog cells in normal and diseased tissues., Competing Interests: This work was supported by an anonymous donation for the Myo/Nog cell program project to MG-W and AB-N. Funding was also provided by Genisphere, LLC, https://genisphere.com/. Employees of Genisphere participated in study design, data collection and analysis, the decision to publish and preparation of the manuscript. A provisional patent has been submitted describing uses of the G8 monoclonal antibody. Related products are in development. No income is derived from these products. This does not alter our adherence to PLOS ONE policies on sharing data and materials.

Identification of allergenic IgE epitopes is instrumental for the development of novel diagnostic and prognostic methods in food allergy. In this work, we present the quantification and validation of a Bead-Based Epitope Assay (BBEA) that through multiplexing of epitopes and multiple sample processing enables completion of large experiments in a short period of time, using minimal quantities of patients' blood. Peptides that are uniquely coupled to beads are incubated with serum or plasma samples, and after a secondary fluorophore-labeled antibody is added, the level of fluorescence is quantified with a Luminex reader. The signal is then normalized and converted to epitope-specific antibody binding values. We show that the effect of technical artifacts, i.e. well position or reading order, is minimal; and batch effects - different individual microplate runs - can be easily estimated and eliminated from the data. Epitope-specific antibody binding quantified with BBEA is highly reliable, reproducible and has greater sensitivity of epitope detection compared to peptide microarrays. IgE directed at allergenic epitopes is a sensitive biomarker of food allergy and can be used to predict allergy severity and phenotypes; and quantification of the relationship between epitope-specific IgE and IgG4 can further improve our understanding of the immune mechanisms behind allergic sensitization.

Purpose: Posterior capsule opacification (PCO) is a vision-impairing disease that occurs in some adults and most children after cataract surgery. Contractile myofibroblasts contribute to PCO by producing wrinkles in the lens capsule that scatter light. Myofibroblasts in the lens originate from Myo/Nog cells named for their expression of the MyoD transcription factor and bone morphogenetic protein inhibitor noggin. In this study we tested the effects of depleting Myo/Nog cells on development of PCO., Methods: Myo/Nog cells were eliminated by injecting the G8 antibody conjugated to 3DNA nanocarriers for the cytotoxin doxorubicin (G8:3DNA:Dox) during cataract surgery in rabbits. The severity of PCO was scored by slit lamp analysis, gross and histologic observation, and immunofluorescence localization of α-smooth muscle actin., Results: G8:3DNA:Dox specifically induced cell death in Myo/Nog cells in the lens. None of the lenses administered G8:3DNA containing 9 to 36 μM doxorubicin developed greater than trace levels of central PCO and few myofibroblasts were present on the capsule. Less than 9% of these lenses exhibited greater than mild levels of peripheral PCO. Doxorubucin itself reduced PCO; however, myofibroblasts and wrinkles were abundant in the lens, and off-target effects were observed in the ciliary processes and cornea., Conclusions: Myo/Nog cells are the primary source of myofibroblasts in the lens after cataract surgery. Targeted depletion of Myo/Nog cells has potential for preventing PCO and preserving vision.

Background: In a recent trial of milk oral immunotherapy (MOIT) with or without omalizumab in 55 patients with milk allergy treated for 28 months, 44 of 55 subjects passed a 10-g desensitization milk protein challenge; 23 of 55 subjects passed the 10-g sustained unresponsiveness (SU) challenge 8 weeks after discontinuing MOIT., Objective: We sought to determine whether IgE and IgG 4 antibody binding to allergenic milk protein epitopes changes with MOIT and whether this could predict the development of SU., Methods: By using a novel high-throughput Luminex-based assay to quantitate IgE and IgG 4 antibody binding to 66 sequential epitopes on 5 milk proteins, serum samples from 47 subjects were evaluated before and after MOIT. Machine learning strategies were used to predict whether a subject would have SU after 8 weeks of MOIT discontinuation., Results: MOIT profoundly altered IgE and IgG 4 binding to epitopes, regardless of treatment outcome. At the initiation of MOIT, subjects achieving SU exhibited significantly less antibody binding to 40 allergenic epitopes than subjects who were desensitized only (false discovery rate ≤ 0.05 and fold change > 1.5). Based on baseline epitope-specific antibody binding, we developed predictive models of SU. Using simulations, we show that, on average, IgE-binding epitopes alone perform significantly better than models using standard serum component proteins (average area under the curve, >97% vs 80%). The optimum model using 6 IgE-binding epitopes achieved a 95% area under the curve and 87% accuracy., Conclusion: Despite the relatively small sample size, we have shown that by measuring the epitope repertoire, we can build reliable models to predict the probability of SU after MOIT. Baseline epitope profiles appear more predictive of MOIT response than those based on serum component proteins., (Copyright © 2018 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.)

Background: The majority of children with cow's milk allergy (CMA) tolerate baked milk. However, reactivity to fermented milk products such as yogurt/cheese has not been previously evaluated. We sought to determine whether children with CMA could tolerate yogurt/cheese and whether a patient's IgE and IgG4-binding pattern to milk protein epitopes could distinguish clinical reactivity., Methods: Four groups of reactivity were identified by Oral food challenge: baked milk reactive, fermented milk reactive, whole milk reactive, and outgrown. sIgE and sIgG4 binding to milk protein epitopes were assessed with a novel Luminex-based peptide assay (LPA). Using machine learning techniques, a model was developed to predict different degrees of CMA., Results: The baked milk reactive patients demonstrated the highest degree of IgE epitope binding, which was followed sequentially by fermented milk reactive, whole milk reactive, and outgrown. Data were randomly divided into two groups with 75% of the data utilized for model development (n = 68) and 25% for testing (n = 21). All 68 children used for training were correctly classified with models using IgE and IgG4 epitopes. The average cross-validation accuracy was much higher for models using IgE plus IgG4 epitopes by LPA (84.8%), twice the performance of the serum component proteins assayed by UniCAP (41.9%). The performance of the model on "unseen data" was tested using the 21 withheld patients, and the accuracy of IgE was 86% (AUC = 0.89) while of IgE+IgG4 model was 81% (AUC = 0.94)., Conclusion: Using a novel high-throughput LPA, we were able to distinguish the diversity of IgE/IgG4 binding to epitopes in the varying CMA phenotypes. LPA is a promising tool to predict correctly different degrees of CMA., (© 2018 EAACI and John Wiley and Sons A/S. Published by John Wiley and Sons Ltd.)

Myo/Nog cells, named for their expression of MyoD and noggin, enter the eye during early stages of embryonic development. Their release of noggin is critical for normal morphogenesis of the lens and retina. Myo/Nog cells are also present in adult eyes. Single nucleated skeletal muscle cells designated as myofibroblasts arise from Myo/Nog cells in cultures of lens tissue. In this report we document the presence of Myo/Nog cells in the lens, ciliary body and on the zonule of Zinn in mice, rabbits and humans. Myo/Nog cells were rare in all three structures. Their prevalence increased in the lens and ciliary body of rabbits 24 h following cataract surgery. Rabbits developed posterior capsule opacification (PCO) within one month of surgery. The number of Myo/Nog cells continued to be elevated in the lens and ciliary body. Myo/Nog cells containing alpha smooth muscle actin and striated muscle myosin were present on the posterior capsule and overlaid deformations in the capsule. Myo/Nog cells also were present on the zonule fibers and external surface of the posterior capsule. These findings suggest that Myo/Nog contribute to PCO and may use the zonule fibers to migrate between the ciliary processes and lens., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Posterior capsule opacification (PCO) occurs in some adults and most children following cataract surgery. The fibrotic form of PCO arises, in part, from migratory, contractile myofibroblasts that deform the lens capsule and impair vision. In short-term cultures of human anterior lens tissue, myofibroblasts emerge from Myo/Nog cells that are identified with the G8 monoclonal antibody and by their expression of the MyoD transcription factor and bone morphogenetic protein inhibitor noggin. In this study, we tested the hypothesis that targeted depletion of Myo/Nog cells with the G8 monoclonal antibody (mAb) conjugated to three-dimensional DNA nanocarriers intercalated with doxorubicin (G8:3DNA:Dox) would prevent the accumulation of myofibroblasts in long-term, serum- and growth factor-free cultures of human lens tissue obtained by capsulorhexis. The mAb:nanocarrier complex was internalized into acidic compartments of the cell. G8:3DNA:Dox killed nearly all Myo/Nog cells without affecting the lens epithelial cells. In 30-day cultures, all G8-positive cells expressed noggin, and subpopulations had synthesized MyoD, sarcomeric myosin, and alpha smooth muscle actin ( α -SMA). Myo/Nog cells responded to scratching of the lens epithelium by accumulating around the edges of the wound. Treatment with two doses of G8:3DNA:Dox completely eliminated G8+/ α -SMA+ cells throughout the explant. These experiments demonstrate that Myo/Nog cells are the source of myofibroblasts in long-term cultures of anterior human lens tissue and mAb:3DNA nanocarriers specifically and effectively deliver cytotoxic cargo to a subpopulation of cells without off-target effects. G8:3DNA:Dox has the potential to reduce PCO following cataract surgery., (Copyright © 2017 by The American Society for Pharmacology and Experimental Therapeutics.)

Drug delivery strategies help cope with drawbacks of classical pharmaceuticals, including increasing their solubility, diminishing side effects and improving biodistribution. A multitude of vehicles have been envisioned to this end, encompassing a variety of materials, architectures, and functionalities. DNA-built carriers are relatively new, yet promising devices to help overcome some of the current barriers in drug delivery. Its easy fabrication, reproducibility and tunability make DNA a unique material for building devices ranging from macro-scale depot systems to nano-scale vehicles tailored as nanoparticles, containers, dendrimers, tubes, etc. Although this DNA application is relatively novel and knowledge is building up, current advances suggest great potential. Intracellular delivery is a promising option, whereby access of cargoes (from small molecules to large biotherapeutics) to the cytosol has been demonstrated in many cell types, without apparent side effects or need for additional actuators. This is paradoxical since "natural" DNA cannot access cells and nucleic acids are among the most difficult cargoes to be delivered within cells; yet this property arises from engineering DNA into particular configurations. Their biocompatibility and safety also holds potential, since this natural material is biodegradable and nucleotides are biological metabolites. Using modified nucleotides and properly designing their sequence, along with classical chemical means, make it feasible to tune the stability and degradation rates of DNA devices. Taken together, although much research is still necessary to understand the in vivo behaviour of DNA-based vehicles, the design parameters ruling their optimization, and the biological pathways regulating their function, DNA represents a unique material to help in future drug delivery applications.