Respiratory tract specific cell populations, or tissue engineered in vitro grown human lung, have the potential to be used as research tools to mimic physiology, toxicology, pathology, as well as infectious diseases responses of cells or tissues. Studies related to respiratory tract pathogenesis or drug toxicity testing in the past made use of basic systems where single cell populations were exposed to test agents followed by evaluations of simple cellular responses. Although these simple single-cell-type systems provided good basic information related to cellular responses, much more can be learned from cells grown in fabricated microenvironments which mimic in vivo conditions in specialized microfabricated chambers or by human tissue engineered three-dimensional (3D) models which allow for more natural interactions between cells. Recent advances in microengineering technology, microfluidics, and tissue engineering have provided a new approach to the development of 2D and 3D cell culture models which enable production of more robust human in vitro respiratory tract models. Complex models containing multiple cell phenotypes also provide a more reasonable approximation of what occurs in vivo without the confounding elements in the dynamic in vivo environment. The goal of engineering good 3D human models is the formation of physiologically functional respiratory tissue surrogates which can be used as pathogenesis models or in the case of 2D screening systems for drug therapy evaluation as well as human toxicity testing. We hope that this manuscript will serve as a guide for development of future respiratory tract model systems as well as a review of conventional models.

The inability to produce perfusable microvasculature networks capable of supporting tissue survival and of withstanding physiological pressures without leakage is a fundamental problem facing the field of tissue engineering. Microvasculature is critically important for production of bioengineered lung (BEL), which requires systemic circulation to support tissue survival and coordination of circulatory and respiratory systems to ensure proper gas exchange. To advance our understanding of vascularization after bioengineered organ transplantation, we produced and transplanted BEL without creation of a pulmonary artery anastomosis in a porcine model. A single pneumonectomy, performed 1 month before BEL implantation, provided the source of autologous cells used to bioengineer the organ on an acellular lung scaffold. During 30 days of bioreactor culture, we facilitated systemic vessel development using growth factor-loaded microparticles. We evaluated recipient survival, autograft (BEL) vascular and parenchymal tissue development, graft rejection, and microbiome reestablishment in autografted animals 10 hours, 2 weeks, 1 month, and 2 months after transplant. BEL became well vascularized as early as 2 weeks after transplant, and formation of alveolar tissue was observed in all animals ( = 4). There was no indication of transplant rejection. BEL continued to develop after transplant and did not require addition of exogenous growth factors to drive cell proliferation or lung and vascular tissue development. The sterile BEL was seeded and colonized by the bacterial community of the native lung.

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Kaposi sarcoma herpesvirus (KSHV/HHV-8) is a B cell tropic human pathogen, which is present in vivo in monotypic immunoglobulin λ (Igλ) light chain but polyclonal B cells. In the current study, we use cell sorting to infect specific B cell lineages from human tonsil specimens in order to examine the immunophenotypic alterations associated with KSHV infection. We describe IL-6 dependent maturation of naïve B lymphocytes in response to KSHV infection and determine that the Igλ monotypic bias of KSHV infection in vivo is due to viral induction of BCR revision. Infection of immunoglobulin κ (Igκ) naïve B cells induces expression of Igλ and isotypic inclusion, with eventual loss of Igκ. We show that this phenotypic shift occurs via re-induction of Rag-mediated V(D)J recombination. These data explain the selective presence of KSHV in Igλ B cells in vivo and provide the first evidence that a human pathogen can manipulate the molecular mechanisms responsible for immunoglobulin diversity.

We report, for the first time, the development of an organ culture system and protocols to support recellularization of whole acellular (AC) human paediatric lung scaffolds. The protocol for paediatric lung recellularization was developed using human transformed or immortalized cell lines and single human AC lung scaffolds. Using these surrogate cell populations, we identified cell number requirements, cell type and order of cell installations, flow rates and bioreactor management methods necessary for bioengineering whole lungs. Following the development of appropriate cell installation protocols, paediatric AC scaffolds were recellularized using primary lung alveolar epithelial cells (AECs), vascular cells and tracheal/bronchial cells isolated from discarded human adult lungs. Bioengineered paediatric lungs were shown to contain well-developed vascular, respiratory epithelial and lung tissue, with evidence of alveolar-capillary junction formation. Types I and II AECs were found thoughout the paediatric lungs. Furthermore, surfactant protein-C and -D and collagen I were produced in the bioengineered lungs, which resulted in normal lung compliance measurements. Although this is a first step in the process of developing tissues for transplantation, this study demonstrates the feasibility of producing bioengineered lungs for clinical use. Copyright © 2016 John Wiley & Sons, Ltd.

SARS-CoV-2 infection during pregnancy leads to an increased risk of adverse pregnancy outcomes. Although the placenta itself can be a target of virus infection, most neonates are virus free and are born healthy or recover quickly. Here, we investigated the impact of SARS-CoV-2 infection on the placenta from a cohort of women who were infected late during pregnancy and had tested positive for SARS-CoV-2 by qRT-PCR at delivery. SARS-CoV-2 genomic and subgenomic RNA was detected in 23 out of 55 placentas (41%). Three placentas with high virus content were obtained from mothers who presented with severe COVID-19 and whose pregnancies resulted in adverse outcomes for the fetuses, including intrauterine fetal demise, stillbirth, and a preterm delivered baby still in newborn intensive care. Examination of the placental samples with high virus content showed efficient SARS-CoV-2 infection, using RNA in situ hybridization to detect genomic and replicating viral RNA, and immunohistochemistry to detect SARS-CoV-2 nucleocapsid protein. Infection was restricted to syncytiotrophoblast cells that envelope the fetal chorionic villi and are in direct contact with maternal blood. The infected placentas displayed massive infiltration of maternal immune cells including macrophages into intervillous spaces, potentially contributing to inflammation of the tissue. Ex vivo infection of placental cultures with SARS-CoV-2 or with SARS-CoV-2 spike (S) protein pseudotyped lentivirus targeted mostly syncytiotrophoblast and, to a lesser extent, endothelial cells. Infection was reduced by using blocking antibodies against ACE2 and against Neuropilin 1, suggesting that SARS-CoV-2 may utilize alternative receptors for entry into placental cells.

Introduction: Autoimmune diseases such as systemic lupus erythematosus, rheumatoid arthritis, and multiple sclerosis affect millions of people worldwide. Interferon regulatory factor 5 (IRF5) contains polymorphisms associated with these autoimmune diseases. Two of these functional polymorphisms are found upstream of the IRF5 gene. rs2004640, which is a single nucleotide polymorphism and the CGGGG insertion/deletion (indel) were studied. IRF5 uses four different promoters for its four first exons: 1A, 1B, 1C, and 1D. Each promoter was analyzed, including functional differences due to the autoimmune-risk polymorphisms.Results: IRF5 promoters were analyzed using ChIP-Seq data (ENCODE database) and the FactorBook database to define transcription factor binding sites. To verify promoter activity, the promoters were cloned into luciferase plasmids. Each construct exhibited luciferase activity. Exons 1A and 1D contain putative PU.1 and NFkB binding sites. Imiquimod, a Toll-like receptor 7 (TLR7) ligand, was used to activate these transcription factors. IRF5 levels were doubled after imiquimod treatment (p < 0.001), with specific increases in the 1A promoter (2.2-fold, p = 0.03) and 1D promoter (2.8-fold, p = 0.03). A putative binding site for p53, which affects apoptosis, was found in the promoter for exon 1B. However, site-directed mutagenesis of the p53 site showed no effect in a reporter assay.Conclusion: The IRF5 exon 1B promoter has been characterized, and the responses of each IRF5 promoter to TLR7 stimulation have been determined. Changes in promoter activity and gene expression are likely due to specific and distinct transcription factors that bind to each promoter. Since high expression of IRF5 contributes to the development of autoimmune disease, understanding the source of increased IRF5 levels is key to understanding autoimmune etiology.

The rs2004640 single nucleotide polymorphism and the CGGGG copy-number variant (rs77571059) are promoter polymorphisms within interferon regulatory factor 5 (IRF5). They have been implicated as susceptibility factors for several autoimmune diseases. IRF5 uses alternative promoter splicing, where any of 4 first exons begin the mRNA. The CGGGG indel is in exon 1A's promoter; the rs2004640 allele creates a splicing recognition site, enabling usage of exon 1B. This study aimed at characterizing alterations in IRF5 mRNA due to these polymorphisms. Cells with risk polymorphisms exhibited ~2-fold higher levels of IRF5 mRNA and protein, but demonstrated no change in mRNA stability. Quantitative PCR demonstrated decreased usage of exons 1C and 1D in cell lines with the risk polymorphisms. RNA folding analysis revealed a hairpin in exon 1B; mutational analysis showed that the hairpin shape decreased translation 5-fold. Although translation of mRNA that uses exon 1B is low due to a hairpin, increased IRF5 mRNA levels in individuals with the rs2004640 risk allele lead to higher overall protein expression. In addition, several new splice variants of IRF5 were sequenced. IRF5's promoter polymorphisms alter first exon usage and increase transcription levels. High levels of IRF5 may bias the immune system toward autoimmunity.