2 resultados para Clinical consequences

em CaltechTHESIS


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In response to infection or tissue dysfunction, immune cells develop into highly heterogeneous repertoires with diverse functions. Capturing the full spectrum of these functions requires analysis of large numbers of effector molecules from single cells. However, currently only 3-5 functional proteins can be measured from single cells. We developed a single cell functional proteomics approach that integrates a microchip platform with multiplex cell purification. This approach can quantitate 20 proteins from >5,000 phenotypically pure single cells simultaneously. With a 1-million fold miniaturization, the system can detect down to ~100 molecules and requires only ~104 cells. Single cell functional proteomic analysis finds broad applications in basic, translational and clinical studies. In the three studies conducted, it yielded critical insights for understanding clinical cancer immunotherapy, inflammatory bowel disease (IBD) mechanism and hematopoietic stem cell (HSC) biology.

To study phenotypically defined cell populations, single cell barcode microchips were coupled with upstream multiplex cell purification based on up to 11 parameters. Statistical algorithms were developed to process and model the high dimensional readouts. This analysis evaluates rare cells and is versatile for various cells and proteins. (1) We conducted an immune monitoring study of a phase 2 cancer cellular immunotherapy clinical trial that used T-cell receptor (TCR) transgenic T cells as major therapeutics to treat metastatic melanoma. We evaluated the functional proteome of 4 antigen-specific, phenotypically defined T cell populations from peripheral blood of 3 patients across 8 time points. (2) Natural killer (NK) cells can play a protective role in chronic inflammation and their surface receptor – killer immunoglobulin-like receptor (KIR) – has been identified as a risk factor of IBD. We compared the functional behavior of NK cells that had differential KIR expressions. These NK cells were retrieved from the blood of 12 patients with different genetic backgrounds. (3) HSCs are the progenitors of immune cells and are thought to have no immediate functional capacity against pathogen. However, recent studies identified expression of Toll-like receptors (TLRs) on HSCs. We studied the functional capacity of HSCs upon TLR activation. The comparison of HSCs from wild-type mice against those from genetics knock-out mouse models elucidates the responding signaling pathway.

In all three cases, we observed profound functional heterogeneity within phenotypically defined cells. Polyfunctional cells that conduct multiple functions also produce those proteins in large amounts. They dominate the immune response. In the cancer immunotherapy, the strong cytotoxic and antitumor functions from transgenic TCR T cells contributed to a ~30% tumor reduction immediately after the therapy. However, this infused immune response disappeared within 2-3 weeks. Later on, some patients gained a second antitumor response, consisted of the emergence of endogenous antitumor cytotoxic T cells and their production of multiple antitumor functions. These patients showed more effective long-term tumor control. In the IBD mechanism study, we noticed that, compared with others, NK cells expressing KIR2DL3 receptor secreted a large array of effector proteins, such as TNF-α, CCLs and CXCLs. The functions from these cells regulated disease-contributing cells and protected host tissues. Their existence correlated with IBD disease susceptibility. In the HSC study, the HSCs exhibited functional capacity by producing TNF-α, IL-6 and GM-CSF. TLR stimulation activated the NF-κB signaling in HSCs. Single cell functional proteome contains rich information that is independent from the genome and transcriptome. In all three cases, functional proteomic evaluation uncovered critical biological insights that would not be resolved otherwise. The integrated single cell functional proteomic analysis constructed a detail kinetic picture of the immune response that took place during the clinical cancer immunotherapy. It revealed concrete functional evidence that connected genetics to IBD disease susceptibility. Further, it provided predictors that correlated with clinical responses and pathogenic outcomes.

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This thesis is divided into two parts: interacting dark matter and fluctuations in cosmology. There is an incongruence between the properties that dark matter is expected to possess between the early universe and the late universe. Weakly-interacting dark matter yields the observed dark matter relic density and is consistent with large-scale structure formation; however, there is strong astrophysical evidence in favor of the idea that dark matter has large self-interactions. The first part of this thesis presents two models in which the nature of dark matter fundamentally changes as the universe evolves. In the first model, the dark matter mass and couplings depend on the value of a chameleonic scalar field that changes as the universe expands. In the second model, dark matter is charged under a hidden SU(N) gauge group and eventually undergoes confinement. These models introduce very different mechanisms to explain the separation between the physics relevant for freezeout and for small-scale dynamics.

As the universe continues to evolve, it will asymptote to a de Sitter vacuum phase. Since there is a finite temperature associated with de Sitter space, the universe is typically treated as a thermal system, subject to rare thermal fluctuations, such as Boltzmann brains. The second part of this thesis begins by attempting to escape this unacceptable situation within the context of known physics: vacuum instability induced by the Higgs field. The vacuum decay rate competes with the production rate of Boltzmann brains, and the cosmological measures that have a sufficiently low occurrence of Boltzmann brains are given more credence. Upon further investigation, however, there are certain situations in which de Sitter space settles into a quiescent vacuum with no fluctuations. This reasoning not only provides an escape from the Boltzmann brain problem, but it also implies that vacuum states do not uptunnel to higher-energy vacua and that perturbations do not decohere during slow-roll inflation, suggesting that eternal inflation is much less common than often supposed. Instead, decoherence occurs during reheating, so this analysis does not alter the conventional understanding of the origin of density fluctuations from primordial inflation.