Low-dose IL-2 prevents murine chronic cardiac allograft rejection: Role for IL-2-induced T regulatory cells and exosomes with PD-L1 and CD73.

To determine the effects and immunological mechanisms of low-dose interleukin-2 (IL-2) in a murine model of chronic cardiac allograft rejection (BALB/c to C57BL/6) after costimulatory blockade consisting of MR1 (250 µg/ip day 0) and CTLA4-Ig (200 µg/ip day 2), we administered low-dose IL-2 (2000 IU/day) starting on posttransplant day 14 for 3 weeks. T regulatory (Treg) cell infiltration of the grafts was determined by immunohistochemistry; circulating exosomes by western blot and aldehyde bead flow cytometry; antibodies to donor MHC by immunofluorescent staining of donor cells; and antibodies to cardiac self-antigens (myosin, vimentin) by ELISA. We demonstrated that costimulation blockade after allogeneic heart transplantation induced circulating exosomes containing cardiac self-antigens and antibodies to both donor MHC and self-antigens, leading to chronic rejection by day 45. Treatment with low-dose IL-2 prolonged allograft survival (>100 days), prevented chronic rejection, and induced splenic and graft-infiltrating CD4+ CD25+ Foxp3 Treg cells by day 45 and circulating exosomes (Foxp3+) with PD-L1 and CD73. MicroRNA 142, associated with the TGFß pathway, was significantly downregulated in exosomes from IL-2-treated mice. In conclusion, low-dose IL-2 delays rejection in a murine model of chronic cardiac allograft rejection and also induces graft-infiltrating Tregs and circulating exosomes with immunoregulatory molecules.

COVID-19 severity from Omicron and Delta SARS-CoV-2 variants.

The Omicron variant of SARS-CoV-2 achieved worldwide dominance in late 2021. Early work suggests that infections caused by the Omicron variant may be less severe than those caused by the Delta variant. We sought to compare clinical outcomes of infections caused by these two strains, confirmed by whole genome sequencing, over a short period of time, from respiratory samples collected from SARS-CoV-2 positive patients at a large medical center. We found that infections caused by the Omicron variant caused significantly less morbidity, including admission to the hospital and requirement for oxygen supplementation, and significantly less mortality than those caused by the Delta variant.

Novel role for tumor suppressor gene, liver kinase B1, in epithelial-mesenchymal transition leading to chronic lung allograft dysfunction.

Epithelial-mesenchymal transition (EMT) has been implicated to play a role in chronic lung allograft dysfunction (CLAD). Liver kinase B1 (LKB1), a tumor suppressor gene, can regulate EMT. However, its role in CLAD development following lung transplantation remains unknown. Using qRT-PCR, biopsies from lung transplant recipients with bronchiolitis obliterans syndrome (BOS) demonstrated significant downregulation of LKB1 (p = .0001), compared to stable biopsies. To determine the role of LKB1 in EMT development, we analyzed EMT in human bronchial epithelial cell line BEAS-2B. Knockdown of LKB1 by siRNA significantly dysregulated mesenchymal markers expression in BEAS-2B cells. Following incubation of human primary bronchial epithelial cell or BEAS-2B cells with exosomes isolated from BOS or stable lung transplant recipients, LKB1 expression was inhibited when incubated with BOS-exosome. Incubation with BOS-exosomes also decreased LKB1 expression and induced EMT markers in air-liquid interface culture method. Our results provide novel evidence that exosomes released from transplanted lungs undergoing chronic rejection are associated with inactivated tumor suppressor gene LKB1 and this loss induces EMT leading to the pathogenesis of CLAD following human lung transplantation.

Strategies for Shared Research Resources for Enhancing Research Sustainability: Communicating Between Stakeholders and Managing Expectations to Maximize Value and Impact.

Shared research resources occupy a unique role in the scientific research landscape. Sometimes called core facilities, shared research resources provide instrumentation, services, and expertise to a wide range of researchers. With dedicated staff maintaining instruments, training users, and supporting collaborations, these resources are well situated to churn out reproducible high-quality data, lead research innovation, create efficiencies, and stimulate economic development all while driving down capital costs for institutions. That being said, in the high-paced disciplines of science with limited resources and competing priorities, these resources are often obligated to demonstrate their worth, especially beyond traditional service delivery models. How can shared research resources quantify and communicate their value and impact to stakeholders for optimal support and sustainability? For best approaches towards value proposition, it is important to understand the various stakeholders in the shared research resource ecosystem, including their needs, expectations, and value systems. This will in turn inform models of support and best approaches for planning, positioning, managing, evaluating, and improving shared research resource output to return the most value to all stakeholders involved. It is imperative that communication is tailored for each unique group of stakeholders, and terminology and expectations are managed accordingly. This work attempts to curate and share approaches and best practices toward this effort, gathered through available literature and focused engagement with various shared research resource stakeholders.

A Shift in Our Thinking: Reframing Shared Research Resources as Investments in Education and Innovation, Not Subsidized Science.

For many researchers, Shared Research Resources are often the most cost-effective means of using state-of-the-art (not to mention expensive) instrumentation. Along with access to the instruments themselves, Shared Research Resources also offer individualized training by highly qualified Shared Research Resource staff-again at deeply discounted costs compared to the operational costs of the facilities. Traditionally, this gap in revenue has been termed a subsidy. But, as with many words, connotation matters, and we posit that this language ought to be changed to reframe our thinking and impart the true impact of Shared Research Resources. We argue here that rather than a subsidy, the revenue gap is better described as an investment. Furthermore, investments of Shared Research Resources lead to positive externalities, including education and innovation.

Establishing a national strategy for shared research resources in biomedical sciences.

Contemporary science has become increasingly multi-disciplinary and team-based, resulting in unprecedented growth in biomedical innovation and technology over the last several decades. Collaborative research efforts have enabled investigators to respond to the demands of an increasingly complex 21st century landscape, including pressing scientific challenges such as the COVID-19 pandemic. A major contributing factor to the success of team science is the mobilization of core facilities and shared research resources (SRRs), the scientific instrumentation and expertise that exist within research organizations that enable widespread access to advanced technologies for trainees, faculty, and staff. For over 40 years, SRRs have played a key role in accelerating biomedical research discoveries, yet a national strategy that addresses how to leverage these resources to enhance team science and achieve shared scientific goals is noticeably absent. We believe a national strategy for biomedical SRRs-led by the National Institutes of Health-is crucial to advance key national initiatives, enable long-term research efficiency, and provide a solid foundation for the next generation of scientists.

Flow Assisted Mutation Enrichment (FAME): A highly efficacious and efficient method to enrich Double Knockouts (DKO) after gene editing.

Gene editing has become an essential tool for interrogation of gene function in biomedical research and is also a promising approach for gene therapy. Despite recent progresses, the gene-editing procedure is still a tedious process involving manually isolating large number of single cell colonies to screen for desired mutations. For diploid eukaryotic cells, there is the additional challenge to inactivate both alleles for genes-of-interest, i.e., generating double knockouts (DKOs), for the desired phenotypes or therapeutic effects. In this report, we present a novel method based on Fluorescence Assisted Cell Sorting (FACS) to enrich for DKO cells, using a cell surface marker ß2-microglobulin (B2M) as a basis for negative selection. This method significantly increased percentage of DKOs in isolated cells after gene editing, and in the meantime, significantly improve the efficiency of workflow by automating colony isolation. It would greatly facilitate future biomedical research including potential gene/cell therapies.

Isolation and In Vitro Culture of Murine and Human Alveolar Macrophages.

Alveolar macrophages are terminally differentiated, lung-resident macrophages of prenatal origin. Alveolar macrophages are unique in their long life and their important role in lung development and function, as well as their lung-localized responses to infection and inflammation. To date, no unified method for identification, isolation, and handling of alveolar macrophages from humans and mice exists. Such a method is needed for studies on these important innate immune cells in various experimental settings. The method described here, which can be easily adopted by any laboratory, is a simplified approach to harvesting alveolar macrophages from bronchoalveolar lavage fluid or from lung tissue and maintaining them in vitro. Because alveolar macrophages primarily occur as adherent cells in the alveoli, the focus of this method is on dislodging them prior to harvest and identification. The lung is a highly vascularized organ, and various cell types of myeloid and lymphoid origin inhabit, interact, and are influenced by the lung microenvironment. By using the set of surface markers described here, researchers can easily and unambiguously distinguish alveolar macrophages from other leukocytes, and purify them for downstream applications. The culture method developed herein supports both human and mouse alveolar macrophages for in vitro growth, and is compatible with cellular and molecular studies.

Learning about sounds contributes to learning about words: effects of prosody and phonotactics on infant word learning.

This research investigates how early learning about native language sound structure affects how infants associate sounds with meanings during word learning. Infants (19-month-olds) were presented with bisyllabic labels with high or low phonotactic probability (i.e., sequences of frequent or infrequent phonemes in English). The labels were produced with the predominant English trochaic (strong/weak) stress pattern or the less common iambic (weak/strong) pattern. Using the habituation-based Switch Task to test label learning, we found that infants readily learned high probability trochaic labels. However, they failed to learn low probability labels, regardless of stress, and failed to learn iambic labels, regardless of phonotactics. Thus, infants required support from both common phoneme sequences and a common stress pattern to map the labels to objects. These findings demonstrate that early word learning is shaped by prior knowledge of native language phonological regularities and provide support for the role of statistical learning in language acquisition.

Nitrosyl hydride (HNO) replaces dioxygen in nitroxygenase activity of manganese quercetin dioxygenase.

Quercetin dioxygenase (QDO) catalyzes the oxidation of the flavonol quercetin with dioxygen, cleaving the central heterocyclic ring and releasing CO. The QDO from Bacillus subtilis is unusual in that it has been shown to be active with several divalent metal cofactors such as Fe, Mn, and Co. Previous comparison of the catalytic activities suggest that Mn(II) is the preferred cofactor for this enzyme. We herein report the unprecedented substitution of nitrosyl hydride (HNO) for dioxygen in the activity of Mn-QDO, resulting in the incorporation of both N and O atoms into the product. Turnover is demonstrated by consumption of quercetin and other related substrates under anaerobic conditions in the presence of HNO-releasing compounds and the enzyme. As with dioxygenase activity, a nonenzymatic base-catalyzed reaction of quercetin with HNO is observed above pH 7, but no enhancement of this basal reactivity is found upon addition of divalent metal salts. Unique and regioselective N-containing products ((14)N/(15)N) have been characterized by MS analysis for both the enzymatic and nonenzymatic reactions. Of the several metallo-QDO enzymes examined for nitroxygenase activity under anaerobic condition, only the Mn(II) is active; the Fe(II) and Co(II) substituted enzymes show little or no activity. This result represents an enzymatic catalysis which we denote nitroxygenase activity; the unique reactivity of the Mn-QDO suggests a metal-mediated electron transfer mechanism rather than metal activation of the substrate's inherent base-catalyzed reactivity.