Background
Microglia have emerged as key players in the pathogenesis of neurodegenerative conditions such as Alzheimer’s disease (AD), taking on distinct transcriptional and functional states. While ...the ability to profile complex tissues at single‐cell resolution in postmortem brain tissue provides insight into disease‐associated cellular states within the brain, more is required to understand how these changes modulate disease pathogenesis. Building on foundational transcriptomics, the advances in multi‐modal profiling of genomics and proteomics allows for a greater understanding of neuroimmune dysfunction in neurodegenerative disease.
Method
Transcriptomic datasets are often confounded by variability in collection and sequencing methodologies. We have optimized tissue dissociation and cell isolation protocols to avoid many of the pitfalls commonly found in bulk and single‐cell transcriptomic studies. Using these optimized protocols, we have begun assessing paired samples (blood, brain biopsy, and cerebrospinal fluid (CSF), from patients at risk for AD to understand neuroimmune changes in early‐phases of AD. We have also characterized the transcriptional and functional responses of human induced pluripotent stem cell (iPSC) microglia in response to a variety of neurodegenerative brain‐relevant challenges, including amyloid, apoptotic neurons and synaptic debris.
Result
Using scRNA‐seq on fresh mouse and human tissue we have identified a dissociation‐induced signature in microglia that is highly prevalent in current literature, and developed an experimental methodology to prevent this artifact. We have also identified a similar signature in post‐mortem tissue via snRNA‐seq that may be the result of acute‐pre/post‐mortem processes (Marsh et al., 2022). Powerfully, we also have cerebrospinal fluid (CSF) from the same patients, allowing us to correlate proteomic and transcriptomic analyses to determine the connections between disease‐associated transcriptomic cell states and analyte biomarkers. We can then use the iPSC microglia (iMGLs) to understand how altered transcriptomic states and biomarker profiles may alter microglial functions to elucidate mechanisms by which microglia contribute to disease pathogenesis.
Conclusion
Optimization of experimental and analysis methods along with extensive multi‐modal profiling of the same patients will lead to greater understanding of the neuroimmune landscape of neurodegenerative disease to better enable development of novel predictive biomarkers and therapeutic targets.
Identifying and separating a subpopulation of cells from a heterogeneous mixture are essential elements of biological research. Current approaches require detailed knowledge of unique cell surface ...properties of the target cell population. A method is described that exploits size differences of cells to facilitate selective intracellular delivery using a high throughput microfluidic device. Cells traversing a constriction within this device undergo a transient disruption of the cell membrane that allows for cytoplasmic delivery of cargo. Unique constriction widths allow for optimization of delivery to cells of different sizes. For example, a 4 μm wide constriction is effective for delivery of cargo to primary human T‐cells that have an average diameter of 6.7 μm. In contrast, a 6 or 7 μm wide constriction is best for large pancreatic cancer cell lines BxPc3 (10.8 μm) and PANC‐1 (12.3 μm). These small differences in cell diameter are sufficient to allow for selective delivery of cargo to pancreatic cancer cells within a heterogeneous mixture containing T‐cells. The application of this approach is demonstrated by selectively delivering dextran‐conjugated fluorophores to circulating tumor cells in patient blood allowing for their subsequent isolation and genomic characterization.
Mechanical force in a microfluidic device induces transient plasma membrane disruption to deliver payload to the cytoplasm of cells. This platform allows for the use of cell size to facilitate subset selection in a heterogeneous population of cells. The applicability of this method is demonstrated with the identification and analysis of circulating tumor cells of a patient with pancreatic cancer.
Adult age-specific colorectal cancer incidence rates increase exponentially from maturity, reach a maximum, then decline in extreme old age. Armitage and Doll (1) postulated that the exponential ...increase resulted from "n" mutations occurring throughout adult life in normal "cells at risk" that initiated the growth of a preneoplastic colony in which subsequent "m" mutations promoted one of the preneoplastic "cells at risk" to form a lethal neoplasia. We have reported cytologic evidence that these "cells at risk" are fetal/juvenile organogenic, then preneoplastic metakaryotic stem cells. Metakaryotic cells display stem-like behaviors of both symmetric and asymmetric nuclear divisions and peculiarities such as bell shaped nuclei and amitotic nuclear fission that distinguish them from embryonic, eukaryotic stem cells. Analyses of mutant colony sizes and numbers in adult lung epithelia supported the inferences that the metakaryotic organogenic stem cells are constitutively mutator/hypermutable and that their contributions to cancer initiation are limited to the fetal/juvenile period. We have amended the two-stage model of Armitage and Doll and incorporated these several inferences in a computer program CancerFit v.5.0. We compared the expectations of the amended model to adult (15-104 years) age-specific colon cancer rates for European-American males born 1890-99 and observed remarkable concordance. When estimates of normal colonic fetal/juvenile APC and OAT gene mutation rates (∼2-5 × 10(-5) per stem cell doubling) and preneoplastic colonic gene loss rates (∼8 × 10(-3)) were applied, the model was in accordance only for the values of n = 2 and m = 4 or 5.
Thesis: M. Eng, Massachusetts Institute of Technology, Department of Biological Engineering, 2016.
Cataloged from PDF version of thesis.
Includes bibliographical references (pages 61-65).
Amitotic ...cells with large, hollow bell-shaped nuclei, or metakaryotic stem cells, are the post-embryonic stem cells of the fetal organs from about the fourth week post conception through physical maturity. These metakaryotic stem cells, after acquiring necessary genetic, and possibly other events, are also the stem cells of precancerous, cancerous and metastatic lesions of carcinogenesis. Furthermore, our lab has discovered that metakaryotic stem cells, both in fetal development and tumor growth, use a peculiar mode of DNA synthesis and segregation that involves inter alia expression of large amounts of RNA polymerase beta during DNA synthesis. It was hypothesized that an inhibitor of DNA polymerase beta would be toxic to metakaryotic stem cells at concentrations lower than necessary to kill eukaryotic non stem cells. The polymerase beta small-molecule inhibitor chosen for this study was pamoic acid, a napthoic acid derivative. With it we determined the relative sensitivity of metakaryotes and eukaryotic cells in the human colorectal cancer cell culture, HT- 29mes, that expresses characteristics expected of colorectal cancer metastases. We conclude that, at 300 muM and above pamoic acid does not selectively kill metakaryotes in cell culture below that concentration which kills eukaryotes. Rather, pamoic acid acts in a similar fashion as X-rays: eukaryotic non-stem cells are killed at lower doses than those that kill metakaryotic stem cells. Treatment of pamoic acid with these concentrations causes concomitant declines in colony-formation potential for both metakaryotes and eukaryotes alike. At lower overall survival levels, surviving colonies appear to have arisen from metakaryotic cells, not eukaryotic cells as evidenced by the presence of visible metakaryotic cells in most colonies and the ability of such colonies to support continuous growth upon passaging. We conclude with the possibility that this specific polymerase beta inhibitor is not an effective metakaryocide in culture, insofar as they are not selectively toxic for these stem cells in the HT-29mes colorectal cancer cell line.
by Tushar Vinod Kamath.
M. Eng
Melioidosis is gaining recognition as an emerging infectious disease with diverse clinical manifestations and high-case fatality rates worldwide. However, the molecular epidemiology of the disease ...outside the endemic regions such as northeast part of Thailand and northern Australia remains unclear.
Clinical data and B. pseudomallei isolates obtained from 199 culture-confirmed cases of melioidosis diagnosed during 2006-2016 in South India were used to elucidate the host and pathogen specific variable virulence determinants associated with clinical presentations and disease outcome. Further, we determined the temporal variations and the influence of ecological factors on B.pseudomallei Lipopolysaccharide (LPS) genotypes causing infections. Severe forms of the disease were observed amongst 169 (85%) patients. Renal dysfunction and infection due to B.pseudomallei harboring BimABm variant had significant associations with severe forms of the disease. Diabetes mellitus, septicemic melioidosis and infection due to LPSB genotype were independent risk factors for mortality. LPSB (74%) and LPSA (20.6%) were the prevalent genotypes causing infections. Both genotypes demonstrated temporal variations and had significant correlations with rainfall and humidity.
Our study findings suggest that the pathogen specific virulence traits under the influence of ecological factors are the key drivers for geographical variations in the molecular epidemiology of melioidosis.
Background & Aims Alcoholic hepatitis is a cause of major morbidity and mortality that lacks effective therapies. Both experimental and clinical evidence indicate that the multifunctional cytokine ...tumor necrosis factor-α (TNF-α) contributes to pathogenesis and clinical sequelae of alcoholic hepatitis. A pilot study demonstrated that the TNF-α-neutralizing molecule etanercept could be an effective treatment for patients with alcoholic hepatitis. Methods Forty-eight patients with moderate to severe alcoholic hepatitis (Model for End-Stage Liver Disease score ≥15) were enrolled and randomized to groups that were given up to 6 subcutaneous injections of either etanercept or placebo for 3 weeks. Primary study end points included mortality at 1- and 6-month time points. Results There were no significant baseline differences between the placebo and etanercept groups in demographics or disease severity parameters including age, gender, and Model for End-Stage Liver Disease score. The 1-month mortality rates of patients receiving placebo and etanercept were similar on an intention-to-treat basis (22.7% vs 36.4%, respectively; OR, 1.8; 95% CI, 0.5–6.5). The 6-month mortality rate was significantly higher in the etanercept group compared with the placebo group (57.7% vs 22.7%, respectively; OR, 4.6; 95% CI, 1.3–16.4; P = .017). Rates of infectious serious adverse events were significantly higher in the etanercept group compared with the placebo group (34.6% vs 9.1%, respectively, P = .04). Conclusions In patients with moderate to severe alcoholic hepatitis, etanercept was associated with a significantly higher mortality rate after 6 months, indicating that etanercept is not effective for the treatment of patients with alcoholic hepatitis.
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