Evaluation and Adaptation of Live-Cell Interferometry for Applications in Basic, Translational, and Clinical Research

Cell mass is an important indicator of cell health and status. A diverse set of techniques have been developed to precisely measure the masses of single cells, with varying degrees of technical complexity and throughput. Here, the development of a non-invasive, label-free optical technique, termed Live-Cell Interferometry (LCI), is described. Several applications are presented, including an evaluation of LCI’s utility for assessing drug response heterogeneity in patient-derived melanoma lines and the measurement of CD3+ T cell kinetics during hematopoietic stem cell transplantation. The characterization of mast cells during degranulation, the measurement of viral reactivation kinetics in Kaposi’s Sarcoma, and drug response studies in patient-derived xenograft models of triple-negative breast cancer are also discussed. Taken together, data from these studies highlight LCI’s versatility as a tool for clinical, translational, and basic research applications

Acute Myeloid Leukemia

Acute myeloid leukemia (AML) is the most common type of leukemia. The Cancer Genome Atlas Research Network has demonstrated the increasing genomic complexity of acute myeloid leukemia (AML). In addition, the network has facilitated our understanding of the molecular events leading to this deadly form of malignancy for which the prognosis has not improved over past decades. AML is a highly heterogeneous disease, and cytogenetics and molecular analysis of the various chromosome aberrations including deletions, duplications, aneuploidy, balanced reciprocal translocations and fusion of transcription factor genes and tyrosine kinases has led to better understanding and identification of subgroups of AML with different prognoses. Furthermore, molecular classification based on mRNA expression profiling has facilitated identification of novel subclasses and defined high-, poor-risk AML based on specific molecular signatures. However, despite increased understanding of AML genetics, the outcome for AML patients whose number is likely to rise as the population ages, has not changed significantly. Until it does, further investigation of the genomic complexity of the disease and advances in drug development are needed. In this review, leading AML clinicians and research investigators provide an up-to-date understanding of the molecular biology of the disease addressing advances in diagnosis, classification, prognostication and therapeutic strategies that may have significant promise and impact on overall patient survival

Evaluation of novel therapeutics for HIV prevention and treatment in a humanized mouse model

2011 Fall.Includes bibliographical references.In the absence of an effective HIV-1 vaccine finding alternative therapeutics and preventative methods has become essential. In this regard preventative approaches such as pre-exposure chemo-prophylaxis that employ either topical applied microbicides or systemically administered anti-retroviral drugs show great promise. In these studies, we evaluated two new classes of clinically approved drugs with different modes of action namely, an integrase inhibitor raltegravir and a CCR5 inhibitor maraviroc as potential systemically and topically applied pre-exposure chemo-prophylaxis. Additionally, therapeutic strategies designed to combat HIV/AIDS using siRNAs show considerable promise. However, targeted delivery of these synthetic molecules into infected cells in vivo has been a formidable challenge. In addressing this need we sought to evaluate the efficacy of a chimeric construct consisting of an HIV-1 gp120 specific aptamer with viral neutralization capacity fused to a siRNA with proven efficacy against tat/rev viral transcripts. We also sought to evaluate the efficacy of structurally flexible, cationic PAMAM dendrimers as a siRNA delivery system. For these novel therapeutic strategies to succeed it is important to evaluate them in both in vitro and in vivo. The rhesus macaque has been a valuable research tool for comparative HIV-1 studies. However, aspects of this model render its usefulness limited considering its expensive nature and not utilizing HIV-1 itself. In this regard the recently developed humanized mouse model that permits multi-lineage human hematopoiesis is an excellent alternative to the non human primate model. To generate humanized mice, neonatal Rag2-/-yc-/- or Rag1-/-yc-/- mice were xenografted with human CD34+ hematopoietic stem cells, resulting in a model that can permit HIV-1 infection. Upon infection by HIV-1 chronic viremia develops with a subsequent loss of CD4 T cells. These mice also successfully mimic the predominant mode of HIV-1 transmission via the sexual vaginal route which also results in chronic viremia and helper T cell loss. Thus this small animal model permits the rapid preclinical evaluation of potential candidates for pre-exposure prophylactic (PrEP) efficacy as well as novel RNA-based therapeutics. Here we utilize these humanized mouse models to evaluate the PrEP efficacies of the drugs named above as well as the in vivo efficacy of siRNAs delivered by utilizing a chimeric aptamer construct or a PAMAM dendrimer. Our results showed that both of these approaches using either a chimeric aptamer or a PAMAM dendrimer resulted in suppression of viral loads in vivo and most importantly also resulted in protection from T-cell depletion, making these compounds attractive therapeutic candidates for the treatment of HIV-1 infection. Lastly, using the same humanized mouse model we also successfully tested a gene therapy strategy employing lentiviral vectors having RNA-based anti-HIV-1 constructs convey intracellular immunization against HIV-1 in vivo

Somatic evolution in healthy and chronically inflamed colon and skin

The human body is made up of trillions of cells which cooperate to reproduce their genetic material. While all the cells are a part of a whole, each is also an individual and will selfishly give rise to a clonal expansion of cells within a tissue given the chance, even to the detriment of the organism. This thesis discusses the evolutionary forces acting on cells within the body, specifically on epithelial cells in the colon and skin. After a general introduction of the evolutionary forces acting on normal cells and the methods used to study them, Chapter 2 focuses specifically on genetic drift within the colon, where clones expand through the process of crypt fission. I apply a statistical framework called Approximate Bayesian Computation to estimate the crypt fission rate in the normal colon and in individuals with Familial adenomatous polyposis (FAP). I estimate the rate of crypt fission to be one every 27 years in the normal colon and one every 13 years in (FAP). In Chapter 3, I describe somatic evolution in the colon under conditions of chronic inflam- mation. I used whole-genome sequencing of individual colonic crypts from patients with inflammatory bowel disease (IBD) to show that the IBD-colon is characterized by a higher mutation burden and larger clonal expansions than the healthy colon. I also show that muta- tions in immune-related genes, including PIGR, ZC3H12A and genes in the interleuking 17 and toll-like receptor pathways, are under positive selection in the colons of IBD patients and may contribute to the disease pathogenesis. In Chapter 4, I focus on the skin. I performed whole-exome sequencing of microbiopsies of epidermis from patients with psoriasis, a second chronic inflammatory disease. In contrast to IBD, I did not find increased mutation burden and clonal spread in psoriasis, except when the skin had been treated with psoralens + UVA (PUVA) phototreatment. The selection landscape of psoriatic skin resembles that of normal skin, and mutations in NOTCH1, FAT1, TP53, PPM1D and NOTCH2 are positively selected. ZFP36L2 was the only gene found to be enriched in mutations that has not been previously reported in normal skin, but it is as yet uncertain if selection of ZFP36L2 mutant cells is a feature specific to psoriatic skin or not. Finally, Chapter 5 discusses my findings in the broader context of cancer and complex-trait genomics. I discuss how a causal relationship between somatic evolution and non-neoplastic diseases may be established and the different ways somatic evolution may affect disease progression for good or ill. I further discuss how to design a study to search for germline determinants of somatic evolution and the need for developing methods to enable such studies to be conducted at scale.Wellcome Trus

Inhibitory Effect of Carbamate on the Cell Division of Rat Bone Marrow

The cytotoxic effect of methomyl carbamate was studied in three groups (control group, 1 % treated group and 10% treated group) and each group contain 2-3 White Wistar rats. The 1% treated group was received 1 % of methomyl LD50, while the 10% treated group was received 10% of methomyl LD50. The administration was orally and repeated three times with 24 hr intervals time. The effect of methomyl was investigated in four experimental run, by analysing the change in cell count in bone marrow after methomyl treatment. It was found that methomyl treatment caused significant reduction in cell number in rat bone marrow indicating that methomyl inhibits cell division. The molecular mechanism of inhibition of cell division was investigated. Analysis of DNA in treated and control animals showed that DNA synthesis is not inhibited upon methomyl treatment. Further investigation of gene products of Ras growth signaling pathway showed that methomyl treatment inhibited the expression of genes that are considered as primary response element (eg. C-Fos) of activation of Ras growth signal transduction pathway. These results indicate that methomyl treatment inhibits cell division by inhibiting, at least partly, Ras-mediated growth signaling pathway. In addition, cytotoxic effect of carbamate on rat was studied by analyzing the expression of two key molecules, cytochrome P-450 and GST proteins which are considered as markers of toxic effect. It was found that cytochrome P-450 gene expression was induced significantly after methomyl treatment while GST level did not change significantly. This suggests that methomyl is a mild toxic agent for mammals and cytochrome P-450 is involved in its metabolism. It might be advisable to use methomyl as insecticide under special precautions

T cell-dependent lysis of CD19-positive leukemia cells mediated by single-chain triplebodies with dual-targeting

Targeted tumor therapy with multispecific antibody formats bears great potential to improve the efficacy of cancer immunotherapy: The simultaneous interaction of antibody derivatives with immune effector cells and multiple tumor-associated antigens is expected to increase cancer cell selectivity, to block cancer cell survival mechanisms and to hamper immune escape. For this purpose a large number of bi- and multispecific molecular platforms have been developed including the single-chain triplebody format. Triplebodies are composed of three antibody-derived single-chain variable fragments interconnected by flexible glycine-serine peptide linkers. They are used for re-targeting of cytotoxic immune effector cells towards cancer cells, which are bound bivalently by the triplebody. In the present work the triplebody-mediated engagement of T cells for the lysis of B lymphoid leukemia cells was established. A prototype with specificity for B lymphoid differentiation antigen CD19 and T cell trigger antigen CD3-epsilon – triplebody 19-3-19 – was shown to activate T lymphocytes at picomolar concentrations and to engage them for the efficient, serial lysis of target antigen-positive cancer cells. The triplebody 19-3-19 also induced T cell proliferation, which can lead to the partial regeneration of a patient’s immune effector cell pool. In these capacities the triplebody 19-3-19 was comparable to the bispecific T cell engager (BiTE®) blinatumomab, which is approved for the treatment of relapsed or refractory acute precursor B lymphoid leukemia in the USA and in the European Union since late 2014/2015. Furthermore, it was shown with the trispecific triplebody 33-3-19 that dual targeting of CD19 and myeloid surface marker CD33 on biphenotypic leukemia blasts results in selective lysis of these target cells. The CD19 and CD33 double-positive blasts were 145-fold more sensitive to treatment with the triplebody 33-3-19 than CD19 single-positive cells. Parts of the author’s work also contributed to the functional characterization of two previously developed NK cell-recruiting triplebodies – SPM-1 (19-16-19) and SPM-2 (33-16-123) – which are candidates for clinical development. The results of this thesis project have established the triplebody format as a molecular platform, which can be employed for the recruitment of any cytotoxic effector cell population as required in a particular therapeutic setting. Furthermore, the improved target cell selectivity that was achieved in vitro with the dual-targeting triplebody 33-3-19 adds weight to the concept of improved therapeutic efficacy of multispecific antibodies

Multi-Scale Modeling of the Innate Immune System: A Dynamic Investigation into Pathogenic Detection

Having a well-functioning immune system can mean the difference between a mild ailment and a life-threatening infection; however, predicting how a disease will progress has proven to be a significant challenge. The dynamics driving the immune system are governed by a complex web of cell types, signaling proteins, and regulatory genes that have to strike a balance between disease elimination and rampant inflammation. An insufficient immune response will induce a prolonged disease state, but an excessive response will cause unnecessary cell dead and extensive tissue damage. This balance is usually self-regulated, but medical intervention is often necessary to correct imbalances. Unfortunately, these therapies are imperfect and accompanied by mild to debilitating side-effects caused by off-target effects. By developing a detailed understanding of the immune response, the goal of this dissertation is to predict how the immune system will respond to infection and determine how new potential therapies could overcome these threats. Computational modeling provides an opportunity to synthesize current immunological observations and predict response outcomes to pathogenic infections. When coupled with experimental data, these models can simulate signaling pathway dynamics that drive the immune response, incorporate regulatory feedback mechanisms, and model inherent biological noise. Taken together, computational modeling can explain emergent behavior that cannot be determined from experiment alone. This dissertation will unitize two computational modeling techniques: ordinary differential equations (ODEs) and agent-based modeling (ABMs). Ultimately, they are combined in a novel way to model cellular immune responses across multiple length scales, creating a more accurate representation of the pathogenic response. TLR4 and cGAS signaling are prominent in a number of diseases and dysregulations including---but not limited to---autoimmunity, cancer, HIV, HSV, tuberculosis, and sepsis. These two signaling pathways are so prevalent because they are activated extremely early and help drive the downstream immune signaling. Modeling how cells dynamically regulate these pathways is critical for understanding how diseases circumvent feedback mechanisms and how new therapies can restore immune function to combat disease progression. By using ODE and ABM techniques, these studies aim to incrementally expand our knowledge of innate immune signaling and understand how feedback mechanisms control disease severity

Study Of Mri Signal In The Presence Of Discrete Spherical Magnetic Particles

Simulating signal behavior in Magnetic Resonance imaging (MRI) is often a necessary step in being able to understand how signal relates to certain physiological parameters. One such parameter of interest in the body is magnetic susceptibility since it is related to iron content. The bulk magnetic susceptibility of an object is a property that describes how magnetized it becomes when placed in an external magnetic field. When the bulk susceptibility of an object arises from the presence of discrete magnetic inclusions, the MRI phase signal inside the object can no longer be determined analytically by assuming it has a continuous susceptibility. This phase will depend on the microstructure of the inclusions and requires either simulations or some other analytical modeling which makes assumptions about the microstructure. Under static dephasing conditions, if the discrete inclusions are spherical particles and randomly dispersed, then a known frequency shift will affect the phase signal. It has also recently been shown that this shift can vary depending on the volume fraction and clustering of the particles. The main focus dissertation is to demonstrate that spherical particles inside an object can lead to non-linear phase behavior which is not describable by a signal frequency shift, while the phase outside the object behaves as if it were continuous. This makes the phase outside the object a more reliable source for susceptibility quantification, as it does not depend on the microstructure of the object. This dissertation consists of three major research projects. The first explores different static dephasing simulation model parameters to predict MRI phase from different quasi-random arrangements of spherical particles. Guidelines are established on the required size of the modeled particles and how many are needed per simulated MRI voxel to obtain precise and accurate results. It is also shown how restricting the randomness of particles affects the simulated voxel phase and R2\u27 values. The second research project uses these guidelines to simulate long cylinders made up of discrete spherical particles. Both random and quasi-random particle arrangements were used. Input parameters for these simulations were taken from experimental phantom data which also consisted of cylinders that contain mixtures of nanoparticles and polystyrene beads, separately. Phase inside the cylinders, bulk susceptibility quantified from phase outside them, and R2\u27 were compared between simulation and experiment. In most cases, the averaged phase inside the simulated and experimental cylinders agree with the theoretical shift for static dephasing regime, while one experimental case agrees better with the quasi-random arrangement. The predicted large variation of phase values from having low numbers or particles per voxel was seen in experiment. The R2\u27 from simulations was generally higher than the quantified R2* from experiment. Bulk susceptibilities of simulated and experimental cylinders were in good agreement and shown to be insensitive to particle arrangement. This supports the reliability of using outside phase for quantification. In the third research project, this concept of using outside phase as an accurate reflection of bulk susceptibility was applied to clusters of iron-tagged stem cells. It was shown how the magnetic moment of the cluster should can be used to determine the number of cells there

Understanding Gene Regulation In Development And Differentiation Using Single Cell Multi-Omics

Transcriptional regulation is a major determinant of tissue-specific gene expression during development. My thesis research leverages powerful single-cell approaches to address this fundamental question in two developmental systems, C. elegans embryogenesis and mouse embryonic hematopoiesis. I have also developed much-needed computational algorithms for single-cell data analysis and exploration. C. elegans is an animal with few cells, but a striking diversity of cell types. In this thesis, I characterize the molecular basis for their specification by analyzing the transcriptomes of 86,024 single embryonic cells. I identified 502 terminal and pre-terminal cell types, mapping most single cell transcriptomes to their exact position in C. elegans’ invariant lineage. Using these annotations, I find that: 1) the correlation between a cell’s lineage and its transcriptome increases from mid to late gastrulation, then falls dramatically as cells in the nervous system and pharynx adopt their terminal fates; 2) multilineage priming contributes to the differentiation of sister cells at dozens of lineage branches; and 3) most distinct lineages that produce the same anatomical cell type converge to a homogenous transcriptomic state. Next, I studied the development of hematopoietic stem cells (HSCs). All HSCs come from a specialized type of endothelial cells in the major arteries of the embryo called hemogenic endothelium (HE). To examine the cellular and molecular transitions underlying the formation of HSCs, we profiled nearly 40,000 rare single cells from the caudal arteries of embryonic day 9.5 (E9.5) to E11.5 mouse embryos using single-cell RNA-Seq and single-cell ATAC-Seq. I identified a continuous developmental trajectory from endothelial cells to early precursors of HSCs, and several critical transitional cell types during this process. The intermediate stage most proximal to HE, which we termed pre-HE, is characterized by increased accessibility of chromatin enriched for SOX, FOX, GATA, and SMAD binding motifs. I also identified a developmental bottleneck separates pre-HE from HE, and RUNX1 dosage regulates the efficiency of the pre-HE to HE transition. A distal enhancer of Runx1 shows high accessibility in pre-HE cells at the bottleneck, but loses accessibility thereafter. Once cells pass the bottleneck, they follow distinct developmental trajectories leading to an initial wave of lympho-myeloid-biased progenitors, followed by precursors of HSCs. During the course of both projects, I have developed novel computational methods for analyzing single-cell multi-omics data, including VERSE, PIVOT and VisCello. Together, these tools constitute a comprehensive single cell data analysis suite that facilitates the discovery of novel biological mechanisms