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Abstract Background Plant biotechnology can be leveraged to produce food, fuel, medicine, and materials. Standardized methods advocated by the synthetic biology community can accelerate the plant design cycle, ultimately making plant engineering more widely accessible to bioengineers who can contribute diverse creative input to the design process. Results This paper presents work done largely by undergraduate students participating in the 2010 International Genetically Engineered Machines (iGEM) competition. Described here is a framework for engineering the model plant Arabidopsis thaliana with standardized, BioBrick compatible vectors and parts available through the Registry of Standard Biological Parts (http://www.partsregistry.org). This system was used to engineer a proof-of-concept plant that exogenously expresses the taste-inverting protein miraculin. Conclusions Our work is intended to encourage future iGEM teams and other synthetic biologists to use plants as a genetic chassis. Our workflow simplifies the use of standardized parts in plant systems, allowing the construction and expression of heterologous genes in plants within the timeframe allotted for typical iGEM projects.

Background: Acute pancreatitis remains the most common and morbid complication of endoscopic retrograde cholangiopancreatography (ERCP). The use of rectal indomethacin and pancreatic duct stenting has been shown to reduce the incidence and severity of post-ERCP pancreatitis (PEP), but these interventions have limitations. Recent clinical and translational evidence suggests a role for calcineurin inhibitors in the prevention of pancreatitis, with multiple retrospective case series showing a reduction in PEP rates in tacrolimus users., Methods: The INTRO trial is a multicenter, international, randomized, double-blinded, controlled trial. A total of 4,874 patients undergoing ERCP will be randomized to receive either oral tacrolimus (5 mg) or oral placebo 1-2 h before ERCP, and followed for 30 days post-procedure. Blood and pancreatic aspirate samples will also be collected in a subset of patients to quantify tacrolimus levels. The primary outcome of the study is the incidence of PEP. Secondary endpoints include the severity of PEP, ERCP-related complications, adverse drug events, length of hospital stay, cost-effectiveness, and the pharmacokinetics, pharmacodynamics, and pharmacogenomics of tacrolimus immune modulation in the pancreas., Conclusions: The INTRO trial will assess the role of calcineurin inhibitors in PEP prophylaxis and develop a foundation for the clinical optimization of this therapeutic strategy from a pharmacologic and economic standpoint. With this clinical trial, we hope to demonstrate a novel approach to PEP prophylaxis using a widely available and well-characterized class of drugs., Trial Registration: NCT05252754, registered on February 14, 2022., Competing Interests: Declaration of competing interest Mouen Khashab reports advisory board member and consultant for Boston Scientific. Erwin J.M. van Geenen reports grants from Mylan and Olympus, personal fees from MTW-Endoskopie, outside the submitted work. Vikesh Singh reports personal fees from Abbvie, advisory board participant for Cook Medical, and grants from Orgenesis and Theraly. Venkata Akshintala is co-founder and chief medical officer of Origin Endoscopy Inc. All other authors declare no competing interests., (Copyright © 2022 IAP and EPC. Published by Elsevier B.V. All rights reserved.)

Video 1EUS-directed transenteric ERCP-assisted internalization of percutaneous biliary drain in Roux-en-Y hepaticojejunostomy anatomy., (© 2022 American Society for Gastrointestinal Endoscopy. Published by Elsevier Inc.)

Severe respiratory virus infections feature robust local host responses that contribute to disease severity. Immunomodulatory strategies that limit virus-induced inflammation may be of critical importance, notably in the absence of antiviral vaccines. In this study, we examined the role of the pleiotropic cytokine IL-6 in acute infection with pneumonia virus of mice (PVM), a natural rodent pathogen that is related to respiratory syncytial virus and that generates local inflammation as a feature of severe infection. In contrast to Influenza A, PVM is substantially less lethal in IL-6 -/- mice than it is in wild-type, a finding associated with diminished neutrophil recruitment and reduced fluid accumulation in lung tissue. Ly6C hi proinflammatory monocytes are recruited in response to PVM via a CCR2 -dependent mechanism, but they are not a major source of IL-6 nor do they contribute to lethal sequelae of infection. By contrast, alveolar macrophages are readily infected with PVM in vivo; ablation of alveolar macrophages results in prolonged survival in association with a reduction in virus-induced IL-6. Finally, as shown previously, administration of immunobiotic Lactobacillus plantarum to the respiratory tracts of PVM-infected mice promoted survival in association with diminished levels of IL-6. We demonstrated in this study that IL-6 suppression is a critical feature of the protective mechanism; PVM-infected IL-6 -/- mice responded to low doses of L. plantarum , and administration of IL-6 overcame L. plantarum -mediated protection in PVM-infected wild-type mice. Taken together, these results connect the actions of IL-6 to PVM pathogenesis and suggest cytokine blockade as a potential therapeutic modality in severe infection.

Eosinophils are resident leukocytes of gut mucosa. Here we present a combined flow cytometric-antibiotic protection assay to identify mouse eosinophils capable of bacterial uptake, specifically, Gram-positive Lactobacillus reuteri, in studies performed ex vivo. The assay may be adapted for use in vivo., (Published by Elsevier B.V.)

Although eosinophils as a group are readily identified by their unique morphology and staining properties, flow cytometry provides an important means for identification of subgroups based on differential expression of distinct surface Ags. Here, we characterize an eosinophil subpopulation defined by high levels of expression of the neutrophil Ag Gr1 (CD45 + CD11c - SiglecF + Gr1 hi ). SiglecF + Gr1 hi eosinophils, distinct from the canonical SiglecF + Gr1 - eosinophil population, were detected in allergen-challenged wild-type and granule protein-deficient (EPX -/- and MBP-1 -/- ) mice, but not in the eosinophil-deficient ΔdblGATA strain. In contrast to Gr1 + neutrophils, which express both cross-reacting Ags Ly6C and Ly6G, SiglecF + Gr1 hi eosinophils from allergen-challenged lung tissue are uniquely Ly6G + Although indistinguishable from the more-numerous SiglecF + Gr1 - eosinophils under light microscopy, FACS-isolated populations revealed prominent differences in cytokine contents. The lymphocyte-targeting cytokines CXCL13 and IL-27 were identified only in the SiglecF + Gr1 hi eosinophil population (at 3.9 and 4.8 pg/10 6 cells, respectively), as was the prominent proinflammatory mediator IL-13 (72 pg/10 6 cells). Interestingly, bone marrow-derived (SiglecF + ), cultured eosinophils include a more substantial Gr1 + subpopulation (∼50%); Gr1 + bmEos includes primarily a single Ly6C + and a smaller, double-positive (Ly6C + Ly6G + ) population. Taken together, our findings characterize a distinct SiglecF + Gr1 hi eosinophil subset in lungs of allergen-challenged, wild-type and granule protein-deficient mice. SiglecF + Gr1 hi eosinophils from wild-type mice maintain a distinct subset of cytokines, including those active on B and T lymphocytes. These cytokines may facilitate eosinophil-mediated immunomodulatory responses in the allergen-challenged lung as well as in other distinct microenvironments., (© Society for Leukocyte Biology.)

Pattern recognition receptors (PRRs) engage microbial components in the lung, although their role in providing primary host defense against respiratory virus infection is not fully understood. We have previously shown that Gram-positive Lactobacillus plantarum (Lp) administered to the respiratory tract promotes full and sustained protection in response to an otherwise lethal mouse pneumovirus (PVM) infection, a robust example of heterologous immunity. While Lp engages PRRs TLR2 and NOD2 in ex vivo signaling assays, we found that Lp-mediated protection was unimpaired in single gene-deleted TLR2(-/-) and NOD2(-/-) mice. Here we demonstrate substantial loss of Lp-mediated protection in a double gene-deleted NOD2(-/-)TLR2(-/-) strain. Furthermore, we demonstrate protection against PVM infection by administration of the bi-functional NOD2-TLR2 agonist, CL-429. The bi-functional NOD2-TLR2 ligand CL-429 not only suppresses virus-induced inflammation, it is significantly more effective at preventing lethal infection than equivalent amounts of mono-molecular TLR2 and NOD2 agonists. Interestingly, and in contrast to biochemical NOD2 and/or TLR2 agonists, Lp remained capable of eliciting primary proinflammatory responses from NOD2(-/-)TLR2(-/-) mice in vivo and from alveolar macrophages challenged ex vivo. Taken together, we conclude that coordinate engagement of NOD2 and TLR2 constitutes a key step in the genesis of Lp-mediated protection from a lethal respiratory virus infection, and represents a critical target for modulation of virus-induced inflammatory pathology., (Published by Elsevier B.V.)

The SV-40-transformed MH-S cell line maintains some, but not all, features of primary alveolar macrophages (AMs) from BALB/c mice. We show here that MH-S cells produce inflammatory cytokines IL-6 and CXCL10 in response to challenge with Gram-positive Lactobacillus reuteri, and to TLR2 and NOD2 ligands Pam3CSK4 and MDP, respectively. In contrast, although wild-type AMs are infected in vivo by pneumonia virus of mice (PVM), no virus replication was detected in MH-S cells. Interestingly, the surface immunophenotype of MH-S cells (CD11c(+)Siglec F(-)) differs from that of wild-type AMs (CD11c(+) Siglec F(+)) and is similar to that of immature AMs isolated from granulocyte macrophage-colony stimulating factor (GM-CSF) gene-deleted mice; AMs from GM-CSF(-/-) mice also support PVM replication. However, MH-S cells do not express the GM-CSF receptor alpha chain (CD116) and do not respond to GM-CSF. Due to these unusual features, MH-S cells should be used with caution as experimental models of AMs., (Published by Elsevier B.V.)

Unlabelled: Pneumonia virus of mice (PVM) is a natural rodent pathogen that replicates in bronchial epithelial cells and reproduces many clinical and pathological features of the more severe forms of disease associated with human respiratory syncytial virus. In order to track virus-target cell interactions during acute infection in vivo, we developed rK2-PVM, bacterial artificial chromosome-based recombinant PVM strain J3666 that incorporates the fluorescent tag monomeric Katushka 2 (mKATE2). The rK2-PVM pathogen promotes lethal infection in BALB/c mice and elicits characteristic cytokine production and leukocyte recruitment to the lung parenchyma. Using recombinant virus, we demonstrate for the first time PVM infection of both dendritic cells (DCs; CD11c(+) major histocompatibility complex class II(+)) and alveolar macrophages (AMs; CD11c(+) sialic acid-binding immunoglobulin-like lectin F(+)) in vivo and likewise detect mKATE2(+) DCs in mediastinal lymph nodes from infected mice. AMs support both active virus replication and production of infectious virions. Furthermore, we report that priming of the respiratory tract with immunobiotic Lactobacillus plantarum, a regimen that results in protection against the lethal inflammatory sequelae of acute respiratory virus infection, resulted in differential recruitment of neutrophils, DCs, and lymphocytes to the lungs in response to rK2-PVM and a reduction from ∼ 40% to <10% mKATE2(+) AMs in association with a 2-log drop in the release of infectious virions. In contrast, AMs from L. plantarum-primed mice challenged with virus ex vivo exhibited no differential susceptibility to rK2-PVM. Although the mechanisms underlying Lactobacillus-mediated viral suppression remain to be fully elucidated, this study provides insight into the cellular basis of this response., Importance: Pneumonia virus of mice (PVM) is a natural mouse pathogen that serves as a model for severe human respiratory syncytial virus disease. We have developed a fully functional recombinant PVM strain with a fluorescent reporter protein (rK2-PVM) that permits us to track infection of target cells in vivo. With rK2-PVM, we demonstrate infection of leukocytes in the lung, notably, dendritic cells and alveolar macrophages. Alveolar macrophages undergo productive infection and release infectious virions. We have shown previously that administration of immunobiotic Lactobacillus directly to the respiratory mucosa protects mice from the lethal sequelae of PVM infection in association with profound suppression of the virus-induced inflammatory response. We show here that Lactobacillus administration also limits infection of leukocytes in vivo and results in diminished release of infectious virions from alveolar macrophages. This is the first study to provide insight into the cellular basis of the antiviral impact of immunobiotic L. plantarum., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

We reported previously that priming of the respiratory tract with immunobiotic Lactobacillus prior to virus challenge protects mice against subsequent lethal infection with pneumonia virus of mice (PVM). We present here the results of gene microarray which document differential expression of proinflammatory mediators in response to PVM infection alone and those suppressed in response to Lactobacillus plantarum. We also demonstrate for the first time that intranasal inoculation with live or heat-inactivated L. plantarum or Lactobacillus reuteri promotes full survival from PVM infection when administered within 24h after virus challenge. Survival in response to L. plantarum administered after virus challenge is associated with suppression of proinflammatory cytokines, limited virus recovery, and diminished neutrophil recruitment to lung tissue and airways. Utilizing this post-virus challenge protocol, we found that protective responses elicited by L. plantarum at the respiratory tract were distinct from those at the gastrointestinal mucosa, as mice devoid of the anti-inflammatory cytokine, interleukin (IL)-10, exhibit survival and inflammatory responses that are indistinguishable from those of their wild-type counterparts. Finally, although L. plantarum interacts specifically with pattern recognition receptors TLR2 and NOD2, the respective gene-deleted mice were fully protected against lethal PVM infection by L. plantarum, as are mice devoid of type I interferon receptors. Taken together, L. plantarum is a versatile and flexible agent that is capable of averting the lethal sequelae of severe respiratory infection both prior to and post-virus challenge via complex and potentially redundant mechanisms., (Published by Elsevier B.V.)