Thomas R. Gaborski, Ph.D. - US grants

DESCRIPTION (provided by applicant): Controlling inflammation is key to improving the viability of engineering and transplanted tissues. With this in mind, our work elucidates the membrane remodeling events that facilitate recruitment of neutrophils to inflamed tissues. The first aim examines the physical mechanisms of adhesion regulation including mobility and localization of integrin and selectin receptors using fluorescence recovery after photobleaching (FRAP), total internal reflection fluorescence (TIRF), and image cross-correlation spectroscopy (ICCS). The complementary nature of these tools will assess the position and mobility of receptors with superior confidence and accuracy over all previous work. Employing these techniques with pharmacological treatments, the second aim tests a hypothesis that adhesion molecules redistribute with increased lateral mobility on activated neutrophils because of transient cytoskeletal release. The third aim examines migratory neutrophils. Using quantitative microscopy and biochemical processing that allows simultaneous visualization of surface receptors and cytoskeleton, this aim hypothesizes that high integrin mobility and low selectin mobility are inherent features of migration and correlated with underlying cytoskeletal structure.

Abstract The main goals of this research program are to; one, research and develop transparent ultrathin nanomembranes and two, utilize these membranes to advance biomedical in vitro model systems through work in the laboratory of the PI and current and future collaborators. Nanomembrane development will include research toward fabricating ultrathin nanoporous silicon dioxide membranes, scaling up their active area, creating unique surface chemistries to promote cellular interaction and integration of sensor technologies. This research program will enable collaborators to visualize endothelial barrier transmigration, produce better in vitro corneal models and visualize motile cilia in a patient derived primary lung model of cystic fibrosis. In addition, nanomembrane development will enable and supply collaborating Investigators with the tools to solve existing challenges and expand their respective fields. A common need is the ability to culture cells in a physiologically relevant model system that can be visualized in real-time. Transparent ultrathin porous membranes can accomplish this for almost any barrier model and co-culture system. SiO2 nanomembranes enable co-cultured cells to be brought within physiological separations distances (~100 nm), while providing glass-like optical transparency and nearly unhindered transport of signaling molecules. Success in developing new human in vitro systems promises to reduce the reliance on animal models, while simultaneously increasing physiological relevance and accelerating drug development. These tissue- and organ-on-a-chips also make feasible live imaging of complex cellular events that require sophisticated and well-orchestrated microenvironments.

Abstract Patients with end-stage kidney and liver disease as well as acute organ failure are unable to maintain the necessary clearance of toxins and require blood-purification techniques or organ transplant. Over 400,000 end-stage renal disease (ESRD) patients receive regular hemodialysis (HD) treatments in the United States. A smaller number receive artificial liver support therapy for detoxification and liver failure. These blood purification techniques place an extremely high financial burden on our medical system with sometimes questionable efficacy and relatively poor quality of life. ESRD treatment alone accounts for 7% of all Medicare spending ($31B). The membrane and adsorption technology behind these treatments has been slow to evolve over the last few decades, limiting the opportunity to make significant improvements. Graphene oxide (GO) has the potential to radically improve and change hemodialysis and liver support systems because GO bilayers are the thinnest possible molecular sieve and nanoscale-spaced GO stacks offer unparalleled adsorptive capacity. The scientific premise behind the use of GO nanoengineered laminates for the clearance of water-soluble and albumin- bound toxins is two-fold. First, prior work has demonstrated that the use of ultrathin nanoporous membranes enables the reduction of laboratory-scale dialyzers by two orders of magnitude compared to conventional polymeric membranes due to dramatically increased permeability, while maintaining size-selectivity. We hypothesize that GO nanoengineered laminate membranes will further reduce required membrane area by at least another order of magnitude based on thinness (<10nm) and increased permeability. Second, albumin-bound toxins have traditionally been removed using anion- exchange columns or porous matrices of activated carbon. Nanospaced GO laminates offer a theoretical limit on surface area within a fixed volume that is likely to exceed conventional adsorbent materials by orders of magnitude. The two aims in the proposal will test both hypotheses. Aim 1 will investigate use of GO to clear water-soluble toxins from plasma, while Aim 2 will investigate the clearance of albumin-bound toxins via albumin dialysis and adsorption to a GO laminate stack. Success in these aims will enable novel device design and treatment flexibility that may include wearable and more efficient therapies with higher quality of life for patients with kidney and liver disease.

Abstract Long-term cognitive impairment affects more than 70% of sepsis survivors, but the underlying mechanisms remain unknown. Though widely hypothesized, evidence of blood-brain barrier (BBB) dysfunction in septic patients is limited by practical barriers to diagnostic studies in critically ill subjects. While BBB breakdown and cognitive impairment are seen in animal models of sepsis, the complexity of sepsis in vivo and differences between animal and human responses means that animal models cannot unambiguously identify the circulating factors that cause brain injury in human sepsis. Therefore, we propose to develop the µSiM-hNVU as an `on-chip' platform featuring a human iPSC-derived neurovascular unit (NVU; brain microvascular endothelial cells, pericytes and astrocytes). The `blood side' will allow the flow-based introduction of blood- borne cells and molecules with known or hypothesized roles in sepsis related brain injury, and the `brain side' will feature iPSC-derived microglial cells serving as a reporter of the brain inflammatory status. The human NVU will be built on a device platform ? the µSiM ? featuring ultrathin silicon nanomembranes that provide for unhindered solute exchange between `blood' and `brain' compartments and glass-like optical quality for live cell imaging and high-resolution microscopy. In the R61 phase, the device platform will be advanced for ease-of- use including `plug-and-play' modules for flow and barrier measurements (TEER, diffusion), and compatibility with a small-volume, digital-ELISA assay for secreted proteins. The µSiM-hNVU will be validated with functional assays of blood-brain barrier (BBB) function, protein expression studies, and transcriptional analysis. We will also build a iPSC NVU in which each cellular component of the NVU carries the ApoE4 allele. The expression of the ApoE4 lipoprotein drives BBB dysfunction by a known pathway and increases the risk of cognitive impairment in humans and animals experiencing brain inflammation. We will use the ApoE4-NVU as a `diseased BBB on a chip? which we hypothesize will show enhanced vulnerabilities to candidate mechanisms of brain injury identified by our team and others. Specifically, we will test the hypotheses that 1) pre-activated monocytes invade the brain and drive microglial activation; 2) the damage associated molecular pattern (DAMP) complex S100A8/A9 drive BBB breakdown to promote leukocyte infiltration and neuroinflammation; and 3) circulating factors that degrade endothelial glycocaylx (e.g., heparinase) or contribute to systemic inflammation (cell-free hemoglobin) promote CNS infiltration of leukocytes and subsequent neuroinflammation.

PROJECT SUMMARY Sepsis is not only the most expensive condition treated in US hospitals, but also a leading cause of death. Sepsis occurs when host pro-inflammatory immune responses become abnormally elevated due to a dysregulated or aberrant host-response to infection. Diagnostic methods for sepsis can vary between hospitals, but often involve scoring systems (e.g. APACHE II and SOFA) that grade the severity of illness in patients. Many of the altered physiological parameters measured by these scoring systems are not necessarily specific to sepsis, which makes it difficult to diagnose sepsis in early stages. Timing of a patient?s sepsis diagnosis is often predictive of their clinical outcome, underlining the need for a more definitive molecular diagnostic test. However, a recent study found that in the majority (70.1%) of sepsis cases, a specific causal organism could not be determined, likely due to aggressive antibiotics or localized infections. Bacterial outer membrane vesicles (OMVs) are attractive diagnostic biomarkers because of: A) their abundance and ability to circulate throughout the body and pass tissue barriers more easily than bacteria themselves; B) their robustness - unlike their bacterial cell ?parent,? OMVs can withstand the inundation of broad-spectrum antibiotics; and C) their unique features that could allow for differentiation between bacterial species. The objective of this proposal is two-fold: Identify whether bacterial OMVs could be a molecular diagnostic biomarker for sepsis and develop a rapid approach to isolate them from patient plasma. We seek to develop a straightforward high-purity and rapid separation technology that effectively isolates and purifies OMVs from biofluids, including plasma. We will implement a modified tangential flow filtration approach, similar to those used by biopharma for high-capacity and high- yield purification, with a new nanopocket membrane that effectively captures and releases bacterial OMVs with minimal loss. Success of these aims will pave the way for the future development of a one-step, point-of-care diagnostic test for sepsis using bacterial OMVs as the molecular biomarker, as well a more complex diagnostic test that more accurately quantifies OMV levels in the patient?s biofluids to help direct early treatment and reduce mortality. This project will be led by an interdisciplinary team of experts in identifying potential biomarkers in E. coli; membranes, materials, and bioseparations; and a critical care physician scientist.