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Open Access Publications from the University of California

Scholarly Works (8 results)

  • Thesis
  • Peer Reviewed
UC San Francisco Electronic Theses and Dissertations (2008)

Leukostasis, a complication of acute leukemia, occurs when leukemia cells aggregate in and obstruct the microvasculature, leading to profound damage of vital organs like the brain and lungs. The underlying mechanisms are poorly understood but are likely related to the abnormal mechanical properties of leukemia cells. Little improvement has been made in our understanding of leukostasis in the last several decades due in part to a lack of tools to sensitively measure leukemia cell biophysical properties, such as cell stiffness and adhesiveness. Accordingly, no reliable methods currently exist to predict, treat or prevent leukostasis.

Using an atomic force microscopy (AFM) technique I developed to measure leukemia cell deformability, I found that leukemia cells taken from patients with leukostasis were significantly stiffer than leukemia cells taken from asymptomatic patients. To bring single-cell deformability measurements towards more clinical use, I developed a high-throughput microfluidic device to quantify cell deformation through capillary-like microchannels, which is referred to as biophysical flow cytometry. Leukemia cells from leukostasis patients had higher cell transit times and rates of microchannel occlusion than leukemia cells from asymptomatic patients. These results suggest that increased leukemia cell stiffness may be an independent leukostasis risk factor and could be useful for predicting and diagnosing leukostasis.

Using AFM and biophysical flow cytometry, I found that chemotherapy, the standard treatment for leukemia, increases leukemia cell stiffness by two orders of magnitude and therefore, may actually increase leukostasis risk. Toward therapies that reduce the risk associated with altered biophysical properties, I found that the phosphodiesterase inhibitor pentoxifylline substantially decreased leukemia cell stiffness and adhesiveness, and this drug may therefore be a possible therapy or prophylaxis for leukostasis. Overall, these studies show that cell mechanics - and alterations thereof - are relevant in disease pathophysiology. In addition, tools that measure cell mechanics are useful diagnostic or drug discovery platforms for predicting and treating disease states such as leukostasis in acute leukemia.

    Cover page: Cell Mechanics of Leukostasis in Acute Leukemia
    • Article
    • Peer Reviewed
    UC San Francisco Previously Published Works (2017)
    In resource-limited settings and in the military theater, fresh human whole blood is commonly transfused, but infectious risks are a concern. Sophisticated molecular testing for potential infectious agents in the whole blood is often unavailable. To address this unmet need, pathogen reduction technology (PRT) has been developed, and it is an effective approach to inactivate a broad range of pathogens found in human blood. However, studies are needed to determine if it is harmful to blood cells and whether these cells could damage the transfused recipient, including the development of acute lung injury/acute respiratory distress syndrome. In this study, we used a commercial PRT system to treat human whole blood that was then transfused into immunodeficient mice, and the development of acute lung injury was determined. In a model of transfusion-related acute lung injury (TRALI), BALB/c SCID mice developed more robust lung injury when challenged with a MHC Class I monoclonal antibody compared to BALB/c wild-type and NOD/SCID mice. Transfusion of control versus Mirasol PRT-treated whole blood (25% blood volume exchange) into BALB/c SCID mice did not produce lung injury at storage day 1. However, mild lung injury at storage days 14 and 21 was observed without significant differences in lung injury measurements between Mirasol PRT-treated and control groups. The mild storage-dependent acute lung injury correlated with trends for increased levels of cell-free hemoglobin that accumulated in both the control and Mirasol PRT-treated groups. Neutrophil extracellular traps were elevated in the plasma of BALB/c SCID mice in the monoclonal antibody TRALI model, but were not different in mice that received exchange transfusions. In conclusion, exchange transfusion of human whole blood into immunodeficient mice produces mild lung injury that is storage-dependent and not related to pathogen reduction treatment.
      Cover page: Mirasol pathogen reduction technology treatment of human whole blood does not induce acute lung injury in mice
      • Article
      • Peer Reviewed
      UCLA Previously Published Works (2012)
      As the microenvironment of a cell changes, associated mechanical cues may lead to changes in biochemical signaling and inherently mechanical processes such as mitosis. Here we explore the effects of confined mechanical environments on cellular responses during mitosis. Previously, effects of mechanical confinement have been difficult to optically observe in three-dimensional and in vivo systems. To address this challenge, we present a novel microfluidic perfusion culture system that allows controllable variation in the level of confinement in a single axis allowing observation of cell growth and division at the single-cell level. The device is capable of creating precise confinement conditions in the vertical direction varying from high (3 µm) to low (7 µm) confinement while also varying the substrate stiffness (E = 130 kPa and 1 MPa). The Human cervical carcinoma (HeLa) model with a known 3N+ karyotype was used for this study. For this cell line, we observe that mechanically confined cell cycles resulted in stressed cell divisions: (i) delayed mitosis, (ii) multi- daughter mitosis events (from 3 up to 5 daughter cells), (iii) unevenly sized daughter cells, and (iv) induction of cell death. In the highest confined conditions, the frequency of divisions producing more than two progeny was increased an astounding 50-fold from unconfined environments, representing about one half of all successful mitotic events. Notably, the majority of daughter cells resulting from multipolar divisions were viable after cytokinesis and, perhaps suggesting another regulatory checkpoint in the cell cycle, were in some cases observed to re-fuse with neighboring cells post-cytokinesis. The higher instances of abnormal mitosis that we report in confined mechanically stiff spaces, may lead to increased rates of abnormal, viable, cells in the population. This work provides support to a hypothesis that environmental mechanical cues influences structural mechanisms of mitosis such as geometric orientation of the mitotic plane or planes.
        Cover page: Increased Asymmetric and Multi-Daughter Cell Division in Mechanically Confined Microenvironments
        • Article
        • Peer Reviewed
        UC San Francisco Previously Published Works (2016)

        Introduction

        Acute traumatic coagulopathy has been associated with shock and tissue injury, and may be mediated via activation of the protein C pathway. Patients with acute traumatic coagulopathy have prolonged PT and PTT, and decreased activity of factors V and VIII; they are also hypocoagulable by thromboelastometry (ROTEM) and other viscoelastic assays. To test the etiology of this phenomenon, we hypothesized that such coagulopathy could be induced in vitro in healthy human blood with the addition of activated protein C (aPC).

        Methods

        Whole blood was collected from 20 healthy human subjects, and was "spiked" with increasing concentrations of purified human aPC (control, 75, 300, 2000 ng/mL). PT/PTT, factor activity assays, and ROTEM were performed on each sample. Mixed effect regression modeling was performed to assess the association of aPC concentration with PT/PTT, factor activity, and ROTEM parameters.

        Results

        In all subjects, increasing concentrations of aPC produced ROTEM tracings consistent with traumatic coagulopathy. ROTEM EXTEM parameters differed significantly by aPC concentration, with stepwise prolongation of clotting time (CT) and clot formation time (CFT), decreased alpha angle (α), impaired early clot formation (a10 and a20), and reduced maximum clot firmness (MCF). PT and PTT were significantly prolonged at higher aPC concentrations, with corresponding significant decreases in factor V and VIII activity.

        Conclusion

        A phenotype of acute traumatic coagulopathy can be induced in healthy blood by the in vitro addition of aPC alone, as evidenced by viscoelastic measures and confirmed by conventional coagulation assays and factor activity. This may lend further mechanistic insight to the etiology of coagulation abnormalities in trauma, supporting the central role of the protein C pathway. Our findings also represent a model for future investigations in the diagnosis and treatment of acute traumatic coagulopathy.
          Cover page: Inducing Acute Traumatic Coagulopathy In Vitro: The Effects of Activated Protein C on Healthy Human Whole Blood
          • Article
          • Peer Reviewed
          UC Berkeley Previously Published Works (2014)
          As platelets aggregate and activate at the site of vascular injury to stem bleeding, they are subjected to a myriad of biochemical and biophysical signals and cues. As clot formation ensues, platelets interact with polymerizing fibrin scaffolds, exposing platelets to a large range of mechanical microenvironments. Here, we show for the first time (to our knowledge) that platelets, which are anucleate cellular fragments, sense microenvironmental mechanical properties, such as substrate stiffness, and transduce those cues into differential biological signals. Specifically, as platelets mechanosense the stiffness of the underlying fibrin/fibrinogen substrate, increasing substrate stiffness leads to increased platelet adhesion and spreading. Importantly, adhesion on stiffer substrates also leads to higher levels of platelet activation, as measured by integrin αIIbβ3 activation, α-granule secretion, and procoagulant activity. Mechanistically, we determined that Rac1 and actomyosin activity mediate substrate stiffness-dependent platelet adhesion, spreading, and activation to different degrees. This capability of platelets to mechanosense microenvironmental cues in a growing thrombus or hemostatic plug and then mechanotransduce those cues into differential levels of platelet adhesion, spreading, and activation provides biophysical insight into the underlying mechanisms of platelet aggregation and platelet activation heterogeneity during thrombus formation.
            Cover page: Platelet mechanosensing of substrate stiffness during clot formation mediates adhesion, spreading, and activation
            • Article
            • Peer Reviewed
            UC San Francisco Previously Published Works (2018)
            This 9th Symposium on Hemostasis is an international scientific meeting held biannually in Chapel Hill, North Carolina. The meeting is in large measure the result of the close friendship between the late Dr. Harold R. Roberts of UNC Chapel Hill and Dr. Ulla Hedner of Novo Nordisk. When Novo Nordisk was developing the hemophilia therapy that would become NovoSeven, they sponsored a series of meetings to understand the basic biology and clinical applications of factor VIIa. The first meeting in Chapel Hill was held April 4-6, 2002 with Dr. Roberts as the organizer. Over the years, the conference emphasis has expanded from discussions of factor VIIa and tissue factor to additional topics in hemostasis and thrombosis. This year's meeting includes presentations by internationally renowned speakers that discuss the state-of-the-art on an array of important topics, including von Willebrand factor, engineering advances, coagulation and disease, tissue factor biology, therapeutic advances, and basic clotting factor biology. Included in this review article are illustrated abstracts provided by our speakers, which highlight the main conclusions of each invited talk. This will be the first meeting without Dr. Roberts in attendance, yet his commitment to excellent science and his focus on turning science to patient care are pervasively reflected in the presentations by our speakers.
              Cover page: Advances in Clinical and Basic Science of Coagulation: Illustrated abstracts of the 9th Chapel Hill Symposium on Hemostasis
              • Article
              • Peer Reviewed
              UC San Francisco Previously Published Works (2024)
              Translational medicine has been a buzzword for over two decades. The concept was intended to be lofty, to reflect a new bench-to-bedside approach to basic and clinical research that would bridge fields, close gaps, accelerate innovation, and shorten the time and effort it takes to bring novel technologies from basic discovery to clinical application. Has this approach been successful and lived up to its promise? Despite incredible scientific advances and innovations developed within academia, successful clinical translation into real-world solutions has been difficult. This has been particularly challenging within the pulmonary field, because there have been fewer U.S. Food and Drug Administration-approved drugs and higher failure rates for pulmonary therapies than with other common disease areas. The American Thoracic Society convened a working group with the goal of identifying major challenges related to the commercialization of technologies within the pulmonary space and opportunities to enhance this process. A survey was developed and administered to 164 participants within the pulmonary arena. This report provides a summary of these survey results. Importantly, this report identifies a number of poorly recognized challenges that exist in pulmonary academic settings, which likely contribute to diminished efficiency of commercialization efforts, ultimately hindering the rate of successful clinical translation. Because many innovations are initially developed in academic settings, this is a global public health issue that impacts the entire American Thoracic Society community. This report also summarizes key resources and opportunities and provides recommendations to enhance successful commercialization of pulmonary technologies.
                Cover page: Challenges and Opportunities for Commercializing Technologies in the Pulmonary Arena: An Official American Thoracic Society Report.
                • Article
                • Peer Reviewed
                UCLA Previously Published Works (2023)
                Extracellular vesicles (EVs) carry diverse bioactive components including nucleic acids, proteins, lipids and metabolites that play versatile roles in intercellular and interorgan communication. The capability to modulate their stability, tissue-specific targeting and cargo render EVs as promising nanotherapeutics for treating heart, lung, blood and sleep (HLBS) diseases. However, current limitations in large-scale manufacturing of therapeutic-grade EVs, and knowledge gaps in EV biogenesis and heterogeneity pose significant challenges in their clinical application as diagnostics or therapeutics for HLBS diseases. To address these challenges, a strategic workshop with multidisciplinary experts in EV biology and U.S. Food and Drug Administration (USFDA) officials was convened by the National Heart, Lung and Blood Institute. The presentations and discussions were focused on summarizing the current state of science and technology for engineering therapeutic EVs for HLBS diseases, identifying critical knowledge gaps and regulatory challenges and suggesting potential solutions to promulgate translation of therapeutic EVs to the clinic. Benchmarks to meet the critical quality attributes set by the USFDA for other cell-based therapeutics were discussed. Development of novel strategies and approaches for scaling-up EV production and the quality control/quality analysis (QC/QA) of EV-based therapeutics were recognized as the necessary milestones for future investigations.
                  Cover page: Current challenges and future directions for engineering extracellular vesicles for heart, lung, blood and sleep diseases
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