Rosemarie Hunziker

A targeted glucocorticoid receptor antisense transgene increases thymocyte apoptosis and alters thymocyte development

The exquisite sensitivity of thymocytes to steroid-induced apoptosis, the steroidogenic potential of thymic epithelial cells, and the ability of steroid synthesis inhibitors to enhance antigen-specific deletion of thymocytes in fetal thymic organ cultures suggest a role for glucocorticoids in thymocyte development. To address this further, transgenic mice that express antisense transcripts to the glucocorticoid receptor (GR) specifically in immature thymocytes were generated. The consequent hyporesponsiveness of thymocytes to glucocorticoids was accompanied by a reduction in thymic size, primarily owing to a decrease in the number of CD4+CD8+ cells. While an enhanced susceptibility to T cell receptor (TCR)-mediated apoptosis appeared to be partially responsible for this reduction, thymocyte loss could also be detected before thymocytes progressed to the CD4+CD8+ TCR alpha beta-expressing stage. These results suggest that glucocorticoids are necessary for survival and maturation of thymocytes, and are consistent with a role for steroids in both the transition from CD4-CD8- to CD4+CD8+ cells and the survival of CD4+CD8+ cells stimulated via the TCR.

Split tolerance to the MHC class I molecule H-2Dd in animals transgenic for its soluble analog

To determine whether the function of MHC molecules in tolerance and education is related to cell surface expression, we have produced two strains of transgenic mice in the C57Bl/6 background that express soluble analogs of the H-2D(d) class I protein. The transgenes were stably integrated and genetically transmitted in a Mendelian fashion. Messenger RNA for the hybrid genes was detected in all tissues analyzed in a class I-like pattern of expression, with the highest levels in lymphoid tissues. All mice bearing the transgenes expressed relatively high levels (0.1 mg/ml) of the encoded protein in their serum as assessed by Western blotting and enzyme-linked immunosorbent assay (ELISA). Gel filtration chromatography showed that the soluble H-2D(d) protein exists as a heterodimer with beta2-microglobulin and as higher order multimers in serum. Lymphoid cells from the transgenic mice showed no cell surface expression of the soluble class I protein in indirect immunofluorescence assays. Splenocytes from two independently derived transgenic lines generated primary cytotoxic and proliferative responses directed against membrane H-2D(d) antigens. Mice of both strains rejected tail skin from donors that differed from the B6 background at the H-2D(d) locus only, but with delayed kinetics compared to nontransgenic littermate controls. Mice expressing the transgenic protein on immunization did not produce antibodies that recognized soluble H-2D(d) in ELISA, whereas B6 mice generated strong antibody responses to challenge with splenocytes bearing cell surface H-2D(d). Thus, transgenic mice expressing soluble H-2D(d) were partially tolerant to stimulation by membrane-bound H-2D(d). As with the activation of T-cells, the induction and maintenance of immunologic tolerance apparently displayed different requirements depending upon the T-cell subpopulation involved.

Fitting tissue chips and microphysiological systems into the grand scheme of medicine, biology, pharmacology, and toxicology:

Microphysiological systems (MPS), which include engineered organoids (EOs), single organ/tissue chips (TCs), and multiple organs interconnected to create miniature in vitro models of human physiological systems, are rapidly becoming effective tools for drug development and the mechanistic understanding of tissue physiology and pathophysiology. The second MPS thematic issue of Experimental Biology and Medicine comprises 15 articles by scientists and engineers from the National Institutes of Health, the IQ Consortium, the Food and Drug Administration, and Environmental Protection Agency, an MPS company, and academia. Topics include the progress, challenges, and future of organs-on-chips, dissemination of TCs into Pharma, children’s health protection, liver zonation, liver chips and their coupling to interconnected systems, gastrointestinal MPS, maturation of immature cardiomyocytes in a heart-on-a-chip, coculture of multiple cell types in a human skin construct, use of synthetic hydrogels to create EOs that form neural tissue models, the blood–brain barrier-on-a-chip, MPS models of coupled female reproductive organs, coupling MPS devices to create a body-on-a-chip, and the use of a microformulator to recapitulate endocrine circadian rhythms. While MPS hardware has been relatively stable since the last MPS thematic issue, there have been significant advances in cell sourcing, with increased reliance on human-induced pluripotent stem cells, and in characterization of the genetic and functional cell state in MPS bioreactors. There is growing appreciation of the need to minimize perfusate-to-cell-volume ratios and respect physiological scaling of coupled TCs. Questions asked by drug developers are followed by an analysis of the potential value, costs, and needs of Pharma. Of highest value and lowest switching costs may be the development of MPS disease models to aid in the discovery of disease mechanisms; novel compounds including probes, leads, and clinical candidates; and mechanism of action of drug candidates. Impact statement Microphysiological systems (MPS), which include engineered organoids and both individual and coupled organs-on-chips and tissue chips, are a rapidly growing topic of research that addresses the known limitations of conventional cellular monoculture on flat plastic – a well-perfected set of techniques that produces reliable, statistically significant results that may not adequately represent human biology and disease. As reviewed in this article and the others in this thematic issue, MPS research has made notable progress in the past three years in both cell sourcing and characterization. As the field matures, currently identified challenges are being addressed, and new ones are being recognized. Building upon investments by the Defense Advanced Research Projects Agency, National Institutes of Health, Food and Drug Administration, Defense Threat Reduction Agency, and Environmental Protection Agency of more than

Editorial: Scaffolds for Regenerative Medicine: A Special Issue of the Annals of Biomedical Engineering

200 million since 2012 and sizable corporate spending, academic and commercial players in the MPS community are demonstrating their ability to meet the translational challenges required to apply MPS technologies to accelerate drug development and advance toxicology.

Class I MHC/Peptide/ β 2-Microglobulin Interactions:The Basis of Cytotoxic T-Cell Recognition

The field of tissue engineering and regenerative medicine (TE/RM) still faces considerable scientific, technical and regulatory challenges in translating its approaches from bench to bedside. But the multiple benefits of replacing tissues and organs lost to disease or trauma are tantalizingly clear and, given recent exciting advances in the field, may be within our grasp. Design of an engineered tissue intended to become a functional transplant has historically depended on integration of three different scientific thrusts: relevant cell type(s), a biocompatible supporting structure, and bioactive molecules. Recent advances in understanding and controlling cell biology and development have focused much attention on the control of cell fate, mostly via timely application of specific growth factors. Yet, this emerging understanding of how cells relate to each other and their local environment demands a synthesis of our growing understanding of that environment. Bioactive scaffolds are widely regarded as key players in functional maturation of engineered tissue constructs as well as in tissue regeneration in situ, that can accomplish a plethora of different functions including guiding cellular and tissue responses, controlling cell migration and homing, delivering bioactive molecules and providing structural support and architecture for developing tissues. This special issue of the Annals of Biomedical Engineering was conceived as a vehicle to address recent progress in scaffold design and to serve as a resource for the community with respect to the current state-of-the-art in the field. The reviews have been loosely organized around four broad themes: ‘‘building blocks and physical properties’’, ‘‘cell-host interactions’’, ‘‘assembling the parts’’, and ‘‘perspectives on translation’’. The editors recruited expertise across a spectrum of areas from the basic elements (cells, biomaterials, and bioactive molecules), to tools and approaches for directing and guiding tissue assembly and monitoring the tissue Rosemarie Hunziker, PhD, Director, Tissue Engineering/ Regenerative Medicine and Biomaterials Programs, National Institute of Biomedical Imaging and Bioengineering (NIBIB), National Institutes of Health (NIH).

Host MHC class I molecules modulate in vivo expression of a NK cell receptor.

The past decade has witnessed a major revolution in our thinking and understanding of the molecular basis of T-cell recognition. In this brief review we outline the historical development of this knowledge and how it has drawn upon advances in cellular immunology, virology, genetics, molecular biology, and structural biology. The current status of our view of the molecular details is summarized, and an outline of critical molecular and cellular questions for the future is presented.