Lipid-Polymer Hybrid Nanoparticles Utilize B Cells and Dendritic Cells to Elicit Distinct Antigen-Specific CD4+ and CD8+ T Cell Responses.

Antigen-presenting cells (APCs) are widely studied for treating immune-mediated diseases, and dendritic cells (DCs) are potent APCs that uptake and present antigens (Ags). However, DCs face several challenges that hinder their clinical translation due to their inability to control Ag dosing and low abundance in peripheral blood. B cells are a potential alternative to DCs, but their poor nonspecific Ag uptake capabilities compromise controllable priming of T cells. Here, we developed phospholipid-conjugated Ags (L-Ags) and lipid-polymer hybrid nanoparticles (L/P-Ag NPs) as delivery platforms to expand the range of accessible APCs for use in T cell priming. These delivery platforms were evaluated using DCs, CD40-activated B cells, and resting B cells to understand the impacts of various Ag delivery mechanisms for generation of Ag-specific T cell responses. L-Ag delivery (termed depoting) of MHC class I- and II-restricted Ags successfully loaded all APC types in a tunable manner and primed both Ag-specific CD8+ and CD4+ T cells, respectively. Incorporating L-Ags and polymer-conjugated Ags (P-Ag) into NPs can direct Ags to different uptake pathways to engineer the dynamics of presentation and shape T cell responses. DCs were capable of processing and presenting Ag delivered from both L- and P-Ag NPs, yet B cells could only utilize Ag delivered from L-Ag NPs, which led to differential cytokine secretion profiles in coculture studies. Altogether, we show that L-Ags and P-Ags can be rationally paired within a single NP to leverage distinct delivery mechanisms to access multiple Ag processing pathways in two APC types, offering a modular delivery platform for engineering Ag-specific immunotherapies.

Semantic Segmentation of Extraocular Muscles on Computed Tomography Images Using Convolutional Neural Networks.

Computed tomography (CT) imaging of the orbit with measurement of extraocular muscle size can be useful for diagnosing and monitoring conditions that affect extraocular muscles. However, the manual measurement of extraocular muscle size can be time-consuming and tedious. The purpose of this study is to evaluate the effectiveness of deep learning algorithms in segmenting extraocular muscles and measuring muscle sizes from CT images. Consecutive CT scans of orbits from 210 patients between 1 January 2010 and 31 December 2019 were used. Extraocular muscles were manually annotated in the studies, which were then used to train the deep learning algorithms. The proposed U-net algorithm can segment extraocular muscles on coronal slices of 32 test samples with an average dice score of 0.92. The thickness and area measurements from predicted segmentations had a mean absolute error (MAE) of 0.35 mm and 3.87 mm2, respectively, with a corresponding mean absolute percentage error (MAPE) of 7 and 9%, respectively. On qualitative analysis of 32 test samples, 30 predicted segmentations from the U-net algorithm were accepted while 2 were rejected. Based on the results from quantitative and qualitative evaluation, this study demonstrates that CNN-based deep learning algorithms are effective at segmenting extraocular muscles and measuring muscles sizes.

Differentiating low and high grade mucoepidermoid carcinoma of the salivary glands using CT radiomics.

BACKGROUND: The purpose of this study is to determine if Haralick texture analysis on CT imaging of mucoepidermoid carcinomas (MEC) can differentiate low-grade and high-grade tumors. METHODS: A retrospective review of 18 patients with MEC of the salivary glands, corresponding CT imaging and pathology report was performed. Tumors were manually segmented and image analysis was performed to calculate radiomic features. Radiomic features were compared between low-grade and high-grade MEC. A multivariable logistic regression model and receiver operating characteristic analysis was performed. RESULTS: A total of 18 patients (mean age, 51, range 9-83 years, 8 men and 10 women) were included. Nine patients had low-grade pathology and nine patients had high-grade pathology. Of the 18 cases, 7 (39%) occurred in the parotid gland and 11 (61%) occurred in minor salivary glands. No individual feature was significantly different between low-grade and high-grade MEC. A logistic regression model including surface regularity, energy and information measure II of correlation was performed and was able to predict high-grade MEC accurately (sensitivity 89%, specificity 68%). The area under the receiver operating characteristic curve was 0.802. CONCLUSIONS: High-grade MEC tend to have a low energy, high correlation texture as well as surface irregularity. Together, these three features may comprise a tumor phenotype that is able to predict high-grade pathology in MECs.

Radiomic Model Predicts Lymph Node Response to Induction Chemotherapy in Locally Advanced Head and Neck Cancer.

This study developed a pretreatment CT-based radiomic model of lymph node response to induction chemotherapy in locally advanced head and neck squamous cell carcinoma (HNSCC) patients. This was a single-center retrospective study of patients with locally advanced HPV+ HNSCC. Forty-one enlarged lymph nodes were found from 27 patients on pretreatment CT and were split into 3:1 training and testing cohorts. Ninety-three radiomic features were extracted. A radiomic model and a combined radiomic-clinical model predicting lymph node response to induction chemotherapy were developed using multivariable logistic regression. Median age was 57 years old, and 93% of patients were male. Post-treatment evaluation was 32 days after treatment, with a median reduction in lymph node volume of 66%. A three-feature radiomic model (minimum, skewness, and low gray level run emphasis) and a combined radiomic-clinical model were developed. The combined model performed the best, with AUC = 0.85 on the training cohort and AUC = 0.75 on the testing cohort. A pretreatment CT-based lymph node radiomic signature combined with clinical parameters was able to predict nodal response to induction chemotherapy for patients with locally advanced HNSCC.

Two-Year Outcomes Comparing Anti-VEGF Injections to Laser for ROP Using a Commercial Claims Database.

BACKGROUND AND OBJECTIVE: To report ocular and neurodevelopmental outcomes among infants treated for retinopathy of prematurity (ROP) in a nationwide health insurance claims database. PATIENTS AND METHODS: Retrospective cohort study of 298 infants treated with laser or anti-vascular endothelial growth factor (VEGF) injection identified in the MarketScan database (2011-2017) with 2-year follow-up. RESULTS: A review of claims data found 298 patients with International Classification of Diseases and Common Procedural Technology codes for ROP treatment and 2 years of continuous insurance coverage. Of these, 63 infants received injections and 235 received laser. Overall, the anti-VEGF group had higher rates of underlying neurological comorbidities (35% vs. 23%; P = .05) and thrombocytopenia (17% vs. 8%; P = .02). Most ocular outcomes were similar, including retinal detachment (P = .87). There were higher rates of second procedures after injection (44% vs. 10%; P < .001). Rates of language, motor, and cognitive delays were similar. Rates of cerebral palsy were higher with injections but were not statistically significant after adjusting for comorbidities (odds ratio = 1.88; P = .10). CONCLUSIONS: The prevalence of retinal detachment after 2 years was similar comparing anti-VEGF to laser. Despite the higher rates of underlying neurologic comorbidity in the injection group, there were no differences in language, motor, or cognitive delays. [Ophthalmic Surg Lasers Imaging Retina. 2020;51:486-493.].

Lipid-Mediated Insertion of Toll-Like Receptor (TLR) Ligands for Facile Immune Cell Engineering.

Cell-based immunotherapies have tremendous potential to treat many diseases, such as activating immunity in cancer or suppressing it in autoimmune diseases. Most cell-based cancer immunotherapies in the clinic provide adjuvant signals through genetic engineering to enhance T cell functions. However, genetically encoded signals have minimal control over dosing and persist for the life of a cell lineage. These properties make it difficult to balance increasing therapeutic efficacy with reducing toxicities. Here, we demonstrated the potential of phospholipid-coupled ligands as a non-genetic system for immune cell engineering. This system provides simple, controlled, non-genetic adjuvant delivery to immune cells via lipid-mediated insertion into plasma membranes. Lipid-mediated insertion (termed depoting) successfully delivered Toll-like receptor (TLR) ligands intracellularly and onto cell surfaces of diverse immune cells. These ligands depoted into immune cells in a dose-controlled fashion and did not compete during multiplex pairwise loading. Immune cell activation could be enhanced by autocrine and paracrine mechanisms depending on the biology of the TLR ligand tested. Depoted ligands functionally persisted on plasma membranes for up to 4 days in naïve and activated T cells, enhancing their activation, proliferation, and skewing cytokine secretion. Our data showed that depoted ligands provided a persistent yet non-permanent adjuvant signal to immune cells that may minimize the intensity and duration of toxicities compared to permanent genetic delivery. Altogether, these findings demonstrate potential for lipid-mediated depoting as a universal cell engineering approach with unique, complementary advantages to other cell engineering methods.

Normative measurements of parotid lymph nodes on CT imaging.

PURPOSE: The goal of this study is to characterize the normal size of parotid lymph nodes among healthy adult patients on CT. METHODS: This was a single-center retrospective observational study of 543 patients who underwent maxillofacial CT scans between January 2019 and July 2019. The long and short axis diameters of the largest lymph nodes in the bilateral superficial parotid glands were measured. RESULTS: Among the 543 patients, 407 subjects with a mean age of 47.0 ± 18.4 years had a total of 719 detectable intraparotid lymph nodes. The mean patient age was 47.0 ± 18.4 years. Of all 719 measured intraparotid lymph nodes, the measured long and short axis diameter means were 4.4 ± 1.4 mm and 3.3 ± 1.1 mm, respectively. In our study, 96% (689/719) of all lymph nodes had a long axis diameter of 7 mm or less and 93% (671/719) of all lymph nodes had a short axis diameter of 5 mm or less. Younger patients had significantly larger lymph nodes than older patients in both long axis (4.5 vs 4.3 mm; P = 0.03) and short axis (3.4 vs 3.1 mm, P = 0.01) measurements. CONCLUSION: Our findings suggest 5 mm as an upper limit of normal for the short axis diameter of superficial intraparotid lymph nodes.

Short-Term Non-Infectious Outcomes After a Pars Plana Intravitreal Antibiotic-Steroid Injection of Triamcinolone, Moxifloxacin, and Vancomycin During Cataract Surgery versus a Standard Postoperative Topical Regimen.

PURPOSE: To compare short-term non-infectious clinical outcomes after cataract surgery with an intraoperative pars plana intravitreal antibiotic-steroid (IVAS) injection of triamcinolone, moxifloxacin, and vancomycin (TMV) versus a standard postoperative topical regimen. PATIENTS AND METHODS: A retrospective comparative case series of 1058 eyes (control = 487, treatment = 571) undergoing cataract surgery were included. Endpoints included best-corrected visual acuity (BCVA), intraocular pressure (IOP), and the unplanned use of anti-inflammatory topical medication in the postoperative period. The follow-up period ranged from 1 to 6 months. RESULTS: A final monocular BCVA of 20/25 or better was achieved in 78.8% and 87.4% of eyes in the control and treatment groups, respectively (p = 0.001). The overall incidence of an IOP spike (Δ ≥ 10 mm Hg) was not significantly different between the two groups (0.4% versus 1.9%, p = 0.027). The rates of persistent anterior chamber inflammation (PACI), rebound anterior chamber inflammation (RACI), and cystoid macular edema (CME) in the control and treatment groups were 8.0% vs 2.6% (p < 0.001), 6.4% vs 2.6% (p = 0.003), and 3.9% vs 4.7% (p = 0.511), respectively. The use of an IVAS injection of TMV conferred an increased risk of CME (odds ratio [OR] = 3.21, 95% confidence interval [CI] = 1.42 to 7.23) but no significant effect on the risk of PACI (OR = 0.34, 95% CI = 0.10 to 1.14) or RACI (OR = 0.52, 95% CI = 0.18 to 1.54) when compared to a topical regimen. CONCLUSION: An intraoperative IVAS injection after uncomplicated cataract surgery may be as safe and effective as a standard topical regimen in terms of postoperative IOP and anterior chamber inflammatory events, respectively. However, the efficacy of a TMV formulation for CME prophylaxis appears to be unsatisfactory. Future studies with prospective and randomized designs are needed to further evaluate this technique.

The Effect of Pharmacological Dilation on Calculation of Targeted and Ideal IOL Power Using Multivariable Formulas.

BACKGROUND: To examine the effect of pharmacologic dilation on biometric parameters measured by the Lenstar LS 900, and whether these changes affect the power of the calculated intraocular lens (IOL) using multivariable formulas in an undilated versus pharmacologically dilated state. METHODS: Prospective study of 98 phakic eyes from 53 patients. Axial length (AL), central corneal thickness (CCT), anterior chamber depth (ACD), lens thickness (LT), and keratometry (K) readings were measured. The first set of measurements was taken prior to dilation. After dilation (pupil diameter ≥ 6.0 mm), a second set of measurements was taken. The Barrett, Olsen, Hill-RBF, Haigis, SRK/T, and Holladay I formulas were used to calculate IOL power before and after dilation. Two calculation methods were used: method A used a commonly available IOL targeted to achieve the lowest myopic spheroequivalent residual refraction; method B calculated ideal IOL power for emmetropia. RESULTS: Statistically significant increases were seen in CCT (p < 0.01), ACD (p < 0.01), and AL (p < 0.01) whereas a statistically significant decrease was seen in LT (p < 0.01) post dilation. Using method A, the percentage of eyes which would have received an IOL with 0.5 D or 1.0 D of higher power, if post-dilation measurements were used, were 25.5%, 30.6%, 20.4%, and 23.5% for Barrett, Olsen, Hill-RBF, and Haigis, respectively. Using method B, only Haigis and Olsen had a statistically significant increase in ideal IOL power. CONCLUSIONS: Pharmacologic dilation can be associated with an increase in non-custom IOL dioptric power when using multivariable formulas, which may lead to a myopic surprise.

Heat Stress Impacts on Lactating Cows Grazing Australian Summer Pastures on an Automatic Robotic Dairy.

The objective of this study was to measure the impacts of summer heat events on physiological parameters (body temperature, respiratory rate and panting scores), grazing behaviour and production parameters of lactating Holstein Friesian cows managed on an Automated Robotic Dairy during Australian summer. The severity of heat stress was measured using Temperature-Humidity Index (THI) and impacts of different THIs-low (≤72), moderate (73-82) and high (≥83)-on physiological responses and production performance were measured. There was a highly significant (p ≤ 0.01) effect of THI on respiratory rate (66.7, 84.7 and 109.1/min), panting scores (1.4, 1.9 and 2.3) and average body temperature of cows (38.4, 39.4 and 41.5 °C), which increased as THI increased from low to moderate to high over the summer. Average milk production parameters were also significantly (p ≤ 0.01) affected by THI, such that daily milk production dropped by 14% from low to high THI, milk temperature and fat% increased by 3%, whilst protein% increased by 2%. The lactation stage of cow had no significant effect on physiological parameters but affected (p ≤ 0.05) average daily milk yield and milk solids. Highly significant (p ≤ 0.01) positive correlations were obtained between THI and milk temperature, fat% and protein% whilst the reverse was observed between THI and milk yield, feed intake and rumination time. Under moderate and high THI, most cows sought shade, spent more time around watering points and showed signs of distress (excessive salivation and open mouth panting). In view of the expected future increase in the frequency and severity of heat events, additional strategies including selection and breeding for thermotolerance and dietary interventions to improve resilience of cows need to be pursued.

A randomized, controlled trial of video supplementation on the cataract surgery informed consent process.

PURPOSE: To assess the effects of the American Academy of Ophthalmology's 2015 patient education video on patient information retention and anxiety preoperatively, on the day of surgery and postoperatively. METHODS: This is a prospective, surgeon-blinded randomized controlled trial at the University of Chicago Medical Center. Ninety-one patients with a diagnosis of first-eye cataract were randomized into either a video or control group. Subjects in both groups received face-to-face discussion with the surgeon and an informational brochure at the preoperative evaluation. Participants in the video group then viewed a four-minute educational video at the preoperative evaluation and on the day of surgery. Both groups completed an information retention quiz and a state anxiety assessment at the preoperative visit, on the day of surgery, and on the postoperative week one visit. Subject understanding of cataract surgery was measured using a twelve-question multiple choice quiz. State anxiety was measured by State Trait Anxiety Inventory-Y1 survey score. RESULTS: Participants in the video group did not score significantly higher on the information retention quiz compared with the control group at the preoperative evaluation (8.7 ± 2.4 vs 7.7 ± 2.5, P = 0.07), but did so on the day of surgery (11.2 ± 0.8 vs 8.4 ± 1.7, P < 0.001) and postoperative week 1 visit (10.8 ± 1.5 vs 9.0 ± 2.0, P < 0.001). Subjects in the video group were significantly less anxious on the day of surgery (26.4 ± 5.1 vs 41.1 ± 10.3, P < 0.001). CONCLUSIONS: Video supplementation to the traditional informed consent process demonstrated an improvement in patient understanding of cataract surgery at multiple timepoints and decreased anxiety on the day of surgery.

Enhancing Humoral Responses Against HIV Envelope Trimers via Nanoparticle Delivery with Stabilized Synthetic Liposomes.

An HIV vaccine capable of eliciting durable neutralizing antibody responses continues to be an important unmet need. Multivalent nanoparticles displaying a high density of envelope trimers may be promising immunogen forms to elicit strong and durable humoral responses to HIV, but critical particle design criteria remain to be fully defined. To this end, we developed strategies to covalently anchor a stabilized gp140 trimer, BG505 MD39, on the surfaces of synthetic liposomes to study the effects of trimer density and vesicle stability on vaccine-elicited humoral responses in mice. CryoEM imaging revealed homogeneously distributed and oriented MD39 on the surface of liposomes irrespective of particle size, lipid composition, and conjugation strategy. Immunization with covalent MD39-coupled liposomes led to increased germinal center and antigen-specific T follicular helper cell responses and significantly higher avidity serum MD39-specific IgG responses compared to immunization with soluble MD39 trimers. A priming immunization with liposomal-MD39 was important for elicitation of high avidity antibody responses, regardless of whether booster immunizations were administered with either soluble or particulate trimers. The stability of trimer anchoring to liposomes was critical for these effects, as germinal center and output antibody responses were further increased by liposome compositions incorporating sphingomyelin that exhibited high in vitro stability in the presence of serum. Together these data highlight key liposome design features for optimizing humoral immunity to lipid nanoparticle immunogens.

Human AML activates the aryl hydrocarbon receptor pathway to impair NK cell development and function.

Acute myeloid leukemia (AML) can evade the mouse and human innate immune system by suppressing natural killer (NK) cell development and NK cell function. This is driven in part by the overexpression of microRNA (miR)-29b in the NK cells of AML patients, but how this occurs is unknown. In the current study, we demonstrate that the transcription factor aryl hydrocarbon receptor (AHR) directly regulates miR-29b expression. We show that human AML blasts activate the AHR pathway and induce miR-29b expression in NK cells, thereby impairing NK cell maturation and NK cell function, which can be reversed by treating NK cells with an AHR antagonist. Finally, we show that inhibition of constitutive AHR activation in AML blasts lowers their threshold for apoptosis and decreases their resistance to NK cell cytotoxicity. Together, these results identify the AHR pathway as a molecular mechanism by which AML impairs NK cell development and function. The results lay the groundwork in establishing AHR antagonists as potential therapeutic agents for clinical development in the treatment of AML.

Eradication of large established tumors in mice by combination immunotherapy that engages innate and adaptive immune responses.

Checkpoint blockade with antibodies specific for cytotoxic T lymphocyte-associated protein (CTLA)-4 or programmed cell death 1 (PDCD1; also known as PD-1) elicits durable tumor regression in metastatic cancer, but these dramatic responses are confined to a minority of patients. This suboptimal outcome is probably due in part to the complex network of immunosuppressive pathways present in advanced tumors, which are unlikely to be overcome by intervention at a single signaling checkpoint. Here we describe a combination immunotherapy that recruits a variety of innate and adaptive immune cells to eliminate large tumor burdens in syngeneic tumor models and a genetically engineered mouse model of melanoma; to our knowledge tumors of this size have not previously been curable by treatments relying on endogenous immunity. Maximal antitumor efficacy required four components: a tumor-antigen-targeting antibody, a recombinant interleukin-2 with an extended half-life, anti-PD-1 and a powerful T cell vaccine. Depletion experiments revealed that CD8+ T cells, cross-presenting dendritic cells and several other innate immune cell subsets were required for tumor regression. Effective treatment induced infiltration of immune cells and production of inflammatory cytokines in the tumor, enhanced antibody-mediated tumor antigen uptake and promoted antigen spreading. These results demonstrate the capacity of an elicited endogenous immune response to destroy large, established tumors and elucidate essential characteristics of combination immunotherapies that are capable of curing a majority of tumors in experimental settings typically viewed as intractable.

A Progenitor Cell Expressing Transcription Factor RORγt Generates All Human Innate Lymphoid Cell Subsets.

The current model of murine innate lymphoid cell (ILC) development holds that mouse ILCs are derived downstream of the common lymphoid progenitor through lineage-restricted progenitors. However, corresponding lineage-restricted progenitors in humans have yet to be discovered. Here we identified a progenitor population in human secondary lymphoid tissues (SLTs) that expressed the transcription factor RORγt and was unique in its ability to generate all known ILC subsets, including natural killer (NK) cells, but not other leukocyte populations. In contrast to murine fate-mapping data, which indicate that only ILC3s express Rorγt, these human progenitor cells as well as human peripheral blood NK cells and all mature ILC populations expressed RORγt. Thus, all human ILCs can be generated through an RORγt(+) developmental pathway from a common progenitor in SLTs. These findings help establish the developmental signals and pathways involved in human ILC development.

Paper and flexible substrates as materials for biosensing platforms to detect multiple biotargets.

The need for sensitive, robust, portable, and inexpensive biosensing platforms is of significant interest in clinical applications for disease diagnosis and treatment monitoring at the point-of-care (POC) settings. Rapid, accurate POC diagnostic assays play a crucial role in developing countries, where there are limited laboratory infrastructure, trained personnel, and financial support. However, current diagnostic assays commonly require long assay time, sophisticated infrastructure and expensive reagents that are not compatible with resource-constrained settings. Although paper and flexible material-based platform technologies provide alternative approaches to develop POC diagnostic assays for broad applications in medicine, they have technical challenges integrating to different detection modalities. Here, we address the limited capability of current paper and flexible material-based platforms by integrating cellulose paper and flexible polyester films as diagnostic biosensing materials with various detection modalities through the development and validation of new widely applicable electrical and optical sensing mechanisms using antibodies and peptides. By incorporating these different detection modalities, we present selective and accurate capture and detection of multiple biotargets including viruses (Human Immunodeficiency Virus-1), bacteria (Escherichia coli and Staphylococcus aureus), and cells (CD4(+) T lymphocytes) from fingerprick volume equivalent of multiple biological specimens such as whole blood, plasma, and peritoneal dialysis effluent with clinically relevant detection and sensitivity.