No abstract

The toxicity of two glyphosate formulations (the original formulation of Roundup® and Roundup WeatherMAX®) to six species of North American larval anurans was evaluated by using 96-h static, nonrenewal aqueous exposures. The 96-h median lethal concentration values (LC50) ranged from 1.80 to 4.22 mg acid equivalent (ae)/L and 1.96 to 3.26 mg ae/L for the original formulation of Roundup and Roundup WeatherMAX, respectively. Judged by LC50 values, four species were more sensitive to Roundup WeatherMAX exposures, and two species were more sensitive to the original formulation. Two of six species, Bufo fowleri (p < 0.05, F = 14.89, degrees of freedom [df] = 1) and Rana clamitans (p < 0.05, F = 18.46, df = 1), had significantly different responses to the two formulations tested. Increased sensitivity to Roundup WeatherMAX likely was due to differences in the surfactants or relative amounts of the surfactants in the two formulations. Potency slopes for exposures of the original formulation ranged from 24.3 to 92.5% mortality/mg ae/L. Thresholds ranged from 1.31 to 3.68 mg ae/L, showing an approximately three times difference in the initiation of response among species tested. For exposures of Roundup WeatherMAX, slopes ranged from 49.3 to 84.2% mortality/mg ae/L. Thresholds ranged from 0.83 to 2.68 mg ae/L. Margins of safety derived from a simulated direct overspray were above 1, except for one species in exposures of Roundup WeatherMAX. Laboratory data based on aqueous exposures are conservative because of the lack of environmental ligands; however, these tests provide information regarding the relative toxicity between these two Roundup formulations.

The role of sediment in modifying the toxicity of the original formulation of Roundup® and Roundup WeatherMAX® was examined in aqueous laboratory tests. Six species of anurans (Bufo fowleri, Hyla chrysoscelis, Rana catesbeiana, Rana clamitans, Rana sphenocephala, and Rana pipiens) were exposed at Gosner stage 25 to concentrations of the 2 herbicide formulations in 96-h, static, nonrenewal experiments in the presence and absence of sediment. All species tested had lower median lethal concentration values in water-only exposures of both formulations compared with exposures with sediment. Sediment significantly altered the potency slopes in all tests with the exceptions of H. chrysoscelis and R. clamitans when exposed to the original formulation of Roundup and H. chrysoscelis and R. sphenocephala when exposed to Roundup WeatherMAX. Thresholds were significantly different in all tests, including those in which potency slopes did not differ. Based on water-sediment exposures of the original formulation of Roundup, all 6 species tested had a margin of safety when compared with the predicted environmental concentration of the highest label application rate. Of the 6 species, 5 had a margin of safety when exposed to Roundup WeatherMAX. During incidental exposures in the field, sediments and organic matter present in aquatic systems provide significant sources of environmental ligands. If used according to label instructions, both herbicides should pose minimal risk to anuran amphibians in actual field applications. Environ Toxicol Chem 2014;33:2616-2620. © 2014 SETAC.

Current treatments for autoimmune disorders typically rely on the use of broadly immunosuppressive agents that increase the risk of adverse events in patients, such as cancer and opportunistic infections. Antigen (Ag)-specific immunotherapies have the potential to minimize these risks and improve efficacy by generating Ag-specific suppression to maintain immunohomeostasis. Several studies have established Ag-specific tolerance using red blood cells (RBCs) as a platform for delivery and Ag presentation in the context of eryptosis, a mechanism of RBC clearance. Here, we used the SQZ cell therapy platform to create Agloaded, pro-eryptotic RBCs to induce tolerance. In this approach, cells pass through constricted channels that cause transient permeation of the cell membrane and permit diffusion of cargo before the membrane reseals. In mice, we demonstrated that these highly delivered RBCs are rapidly cleared from circulation by splenic and liver-resident macrophages. The administration of ovalbumin (OVA)-SQZ’d RBCs led to a reduction of OVA-specific T cell proliferation and cytokine production in an OVA immunization model. In an autoimmune diabetes BDC2.5 T cell transfer model, mice treated with RBCs SQZ’d with 1040-p31 peptide mimetope delayed the onset of hyperglycemia in 80% of mice. Additionally, we developed mouse models for adenoassociated virus (AAV)-gene therapy and anti-drug antibody (ADA) to examine immunemediated clearance of exogenous Ag. In the AAV vector-specific model, mice treated with RBCs loaded with AAV-derived peptides abrogated the effector responses of AAV-specific CD8+ T cells upon restimulation. In a model of ADA responses, RBCs SQZ’d with a model drug also led to potent inhibition of ADA responses and preserved drug levels in circulation. In summary, SQZ’d RBCs are a potentially exciting allogeneic cell therapy strategy to induce Ag-specific tolerance across a range of disease mechanisms and indications. Disclosure A. Ramakrishnan: None. T. Lee: None. L.J. Moore: None. A. Vicente Suarez: None. F. Moore: None. H. Bernstein: Employee; Self; SQZ Biotechnologies. Funding JDRF

Antigen-specific CD8+ T cells are critical for mounting an effective immune response against tumors. Generation of antigen-specific T cells require interactions with multiple signals produced by antigen presenting cells (APCs). These signals are comprised of three components: (signal 1) the peptide-MHC complex binding to the T cell receptor, (signal 2) costimulatory molecules on the surface of APCs, and (signal 3) inflammatory cytokines binding to cognate receptors on T cells. To engineer all major cell subsets of human peripheral blood mononuclear cells (PBMCs) to become enhanced APCs (eAPCs), we used Cell Squeeze® technology to deliver multiple mRNAs encoding for non-self-antigens (signal 1), CD86 (signal 2), and/or membrane-bound cytokines (signal 3). The signal 3 molecules, membrane-bound IL-12 (mbIL-12) and membrane-bound IL-2 (mbIL-2), are chimeric proteins designed to increase the localized concentration of the cytokines at the immune synapse and limit off-target effects. Flow cytometry confirmed translation of delivered signal 2/3 mRNAs by all major subsets within PBMCs: T cells, B cells, NK cells, and monocytes. The potency of these SQZ™ eAPCs was assessed in vitro by culturing the eAPCs with antigen-specific T cells for multiple days before measuring the functionality of antigen-specific T cells via intracellular cytokine staining or ELISA. Using this approach, we demonstrate that Cell Squeeze® co-delivery of antigen mRNA and signal 2/3 mRNAs significantly enhances CD8+ T cell responses to a variety of antigens, including CMV pp65, Influenza M1, HPV16 E6, and HPV16 E7. Furthermore, we demonstrate that SQZ™ eAPCs drive significant expansion of antigen-specific CD8+ T cells in a humanized mouse model. Thus, we demonstrate that Cell Squeeze® can deliver multiple mRNAs encoding for signals 1, 2, and 3 to human PBMCs and has the potential to generate enhanced APCs that drive strong CD8+ T cell responses against multiple antigens. The versatility of this approach has the potential to enable rapid exchange of mRNA to encode for other antigens or T cell activation signals. Citation Format: Michael F. Maloney, Emrah Ilker Ozay, Katarina Blagovic, Carolyne Smith, Andrea A. Silva, Amber Martin, Sanjana Manja, Madhav Upadhyay, Lindsay J. Moore, Ryan Stagg, Henry Mack, Christine Trumpfheller, Pablo Umana, Armon Sharei, Howard Bernstein, Scott M. Loughhead. Co-delivery of antigen-encoding mRNA and signal 2/3 mRNAs to PBMCs by Cell Squeeze® technology generates SQZ™ eAPCs that prime CD8+T cells in a humanized mouse model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2853.