Imaging chemokine receptor dimerization with firefly luciferase complementation

Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/154639/1/fsb2fj08116749.pd

Applications of bioluminescence imaging to the study of infectious diseases

Bioluminescence imaging (BLI) has emerged as a powerful new method to analyse infectious diseases in animal models. BLI offers real-time monitoring of spatial and temporal progression of infection in the same animal, as opposed to euthanizing a cohort of animals and quantifying colony or plaque forming units at multiple time points. Pathogens or mice are engineered to express genetically encoded luciferase enzymes from bacteria, insects, or the sea pansy. The seminal study showing the feasibility of detecting microbially generated luminescence within a living mouse was published by Contag and colleagues in 1995, using Salmonella typhimurium transformed with the lux operon from Photorhabdus luminescens . Following this, they and others performed many studies of infection by bioluminescent Gram-negative and Gram-positive bacteria. Viruses can also be engineered to encode luciferase. Our laboratory has used bioluminescent reporter viruses to follow HSV and vaccinia pathogenesis; others have used an alphavirus or novirhabdovirus. Recently, even eukaryotic parasites Plasmodium, Leishmania and Toxoplasma have been transformed with luciferase and yielded unique insights into their in vivo behaviour. We expect that both the range of organisms and the molecular events able to be studied by BLI will continue to expand, yielding important insights into mechanisms of pathogenesis.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/73608/1/j.1462-5822.2007.00995.x.pd

Murine alveolar macrophages limit replication of vaccinia virus

AbstractBecause of concerns about zoonotic transmission of monkeypox to humans and the bioterrorism threat posed by orthopoxviruses, there is renewed interest in probing cellular and molecular mechanisms of host defense to these pathogens. In particular, it is essential to understand viral–host interactions in the respiratory tract, which is the route of infection for smallpox and a likely route of transmission for monkeypox. In this study, we analyze functions of alveolar macrophages in poxvirus infection, using a recombinant vaccinia virus expressing firefly luciferase to quantify infection in mice and cell culture. Depletion of alveolar macrophages with liposomal clodronate worsens the overall severity of infection in mice, including greater replication and systemic dissemination of vaccinia as determined by bioluminescence imaging. Absence of alveolar macrophages increases total numbers of granulocytes and granulocytes/monocyte progenitor cells in the lungs during vaccinia infection, indicating that protective effects of alveolar macrophages may be mediated in part by reducing the host inflammation. Alveolar macrophages also limit vaccinia infection in respiratory epithelium, as shown by a co-culture model of cell lines derived from alveolar macrophages and lung epithelium. Collectively, these data demonstrate that alveolar macrophages are key determinants of host defense against local and systemic infection with poxviruses

Biomaterials‐Based Approaches to Tumor Spheroid and Organoid Modeling

Evolving understanding of structural and biological complexity of tumors has stimulated development of physiologically relevant tumor models for cancer research and drug discovery. A major motivation for developing new tumor models is to recreate the 3D environment of tumors and context‐mediated functional regulation of cancer cells. Such models overcome many limitations of standard monolayer cancer cell cultures. Under defined culture conditions, cancer cells self‐assemble into 3D constructs known as spheroids. Additionally, cancer cells may recapitulate steps in embryonic development to self‐organize into 3D cultures known as organoids. Importantly, spheroids and organoids reproduce morphology and biologic properties of tumors, providing valuable new tools for research, drug discovery, and precision medicine in cancer. This Progress Report discusses uses of both natural and synthetic biomaterials to culture cancer cells as spheroids or organoids, specifically highlighting studies that demonstrate how these models recapitulate key properties of native tumors. The report concludes with the perspectives on the utility of these models and areas of need for future developments to more closely mimic pathologic events in tumors.State‐of‐the‐art approaches using natural, synthetic, and composite biomaterials for 3D tumor modeling are presented in this Progress Report. Furthermore, it is discussed how these models uniquely reproduce key properties of native tumors to facilitate basic and applied cancer research and cancer drug discovery efforts.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/142941/1/adhm201700980.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/142941/2/adhm201700980-sup-0001-S1.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/142941/3/adhm201700980_am.pd

High Throughput, Polymeric Aqueous Two‐Phase Printing of Tumor Spheroids

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/109304/1/adfm201401302-sup-0001-S1.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/109304/2/adfm201401302.pd

A Comprehensive Analysis of CXCL12 Isoforms in Breast Cancer1,2

AbstractCXCL12-CXCR4-CXCR7 signaling promotes tumor growth and metastasis in breast cancer. Alternative splicing of CXCL12 produces isoforms with distinct structural and biochemical properties, but little is known about isoform-specific differences in breast cancer subtypes and patient outcomes. We investigated global expression profiles of the six CXCL12 isoforms, CXCR4, and CXCR7 in The Cancer Genome Atlas breast cancer cohort using next-generation RNA sequencing in 948 breast cancer and benign samples and seven breast cancer cell lines. We compared expression levels with several clinical parameters, as well as metastasis, recurrence, and overall survival (OS). CXCL12-α, -β, and -γ are highly co-expressed, with low expression correlating with more aggressive subtypes, higher stage disease, and worse clinical outcomes. CXCL12-δ did not correlate with other isoforms but was prognostic for OS and showed the same trend for metastasis and recurrence-free survival. Effects of CXCL12-δ remained independently prognostic when taking into account expression of CXCL12, CXCR4, and CXCR7. These results were also reflected when comparing CXCL12-α, -β, and -γ in breast cancer cell lines. We summarized expression of all CXCL12 isoforms in an important chemokine signaling pathway in breast cancer in a large clinical cohort and common breast cancer cell lines, establishing differences among isoforms in multiple clinical, pathologic, and molecular subgroups. We identified for the first time the clinical importance of a previously unstudied isoform, CXCL12-δ

Primary myelofibrosis evolving to an aplastic appearing marrow

Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/144664/1/ccr31618.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/144664/2/ccr31618_am.pd

Monitoring implantable immunoisolation devices with intrinsic fluorescence of genipin

Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/149252/1/jbio201800170.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/149252/2/jbio201800170_am.pd

Engineered Fibrillar Fibronectin Networks as Three‐Dimensional Tissue Scaffolds

Extracellular matrix (ECM) proteins, and most prominently, fibronectin (Fn), are routinely used in the form of adsorbed pre‐coatings in an attempt to create a cell‐supporting environment in both two‐ and three‐dimensional cell culture systems. However, these protein coatings are typically deposited in a form which is structurally and functionally distinct from the ECM‐constituting fibrillar protein networks naturally deposited by cells. Here, the cell‐free and scalable synthesis of freely suspended and mechanically robust three‐dimensional (3D) networks of fibrillar fibronectin (fFn) supported by tessellated polymer scaffolds is reported. Hydrodynamically induced Fn fibrillogenesis at the three‐phase contact line between air, an Fn solution, and a tessellated scaffold microstructure yields extended protein networks. Importantly, engineered fFn networks promote cell invasion and proliferation, enable in vitro expansion of primary cancer cells, and induce an epithelial‐to‐mesenchymal transition in cancer cells. Engineered fFn networks support the formation of multicellular cancer structures cells from plural effusions of cancer patients. With further work, engineered fFn networks can have a transformative impact on fundamental cell studies, precision medicine, pharmaceutical testing, and pre‐clinical diagnostics.Fibrillar fibronectin (fFn) networks are freely suspended across porous polymer structures without the use of cells. Engineered fFn networks enable in vivo implantation or in vitro expansion of various cell types including patient breast cancer cells that otherwise fail to survive on tissue culture polystyrene.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/153115/1/adma201904580_am.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/153115/2/adma201904580.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/153115/3/adma201904580-sup-0001-S1.pd

CXCR7 influences leukocyte entry into the CNS parenchyma by controlling abluminal CXCL12 abundance during autoimmunity

During CNS autoimmunity, brain endothelial cell CXCR7 internalizes CXCL12 from the perivascular space, thereby permitting leukocyte migration into the CNS parenchyma