Quantitative imaging of living cells by deep ultraviolet microscopy

Thesis (Ph. D.)--Massachusetts Institute of Technology, Biological Engineering Division, 2006.Includes bibliographical references (p. 139-145).Developments in light microscopy over the past three centuries have opened new windows into cell structure and function, yet many questions remain unanswered by current imaging approaches. Deep ultraviolet microscopy received attention in the 1950s as a way to generate image contrast from the strong absorbance of proteins and nucleic acids at wavelengths shorter than 300 nm. However, the lethal effects of these wavelengths limited their usefulness in studies of cell function, separating the contributions of protein and nucleic acid proved difficult, and scattering artifacts were a significant concern. We have used short exposures of deep-ultraviolet light synchronized with an ultraviolet-sensitive camera to observe mitosis and motility in living cells without causing necrosis, and quantified absorbance at 280 nm and 260 nm together with tryptophan native fluorescence in order to calculate maps of nucleic acid mass, protein mass, and quantum yield in unlabeled cells. We have also developed a method using images acquired at 320nm and 340nm, and an equation for Mie scattering, to determine a scattering correction factor for each pixel at 260nm and 280nm. These developments overcome the three main obstacles to previous deep UV microscopy efforts, creating a new approach to imaging unlabeled living cells that acquires quantitative information about protein and nucleic acid as a function of position and time.by Benjamin J. Zeskind.Ph.D

Leveraging existing data sets to generate new insights into Alzheimer’s disease biology in specific patient subsets

To generate new insights into the biology of Alzheimer’s Disease (AD), we developed methods to combine and reuse a wide variety of existing data sets in new ways. We first identified genes consistently associated with AD in each of four separate expression studies, and confirmed this result using a fifth study. We next developed algorithms to search hundreds of thousands of Gene Expression Omnibus (GEO) data sets, identifying a link between an AD-associated gene (NEUROD6) and gender. We therefore stratified patients by gender along with APOE4 status, and analyzed multiple SNP data sets to identify variants associated with AD. SNPs in either the region of NEUROD6 or SNAP25 were significantly associated with AD, in APOE4+ females and APOE4+ males, respectively. We developed algorithms to search Connectivity Map (CMAP) data for medicines that modulate AD-associated genes, identifying hypotheses that warrant further investigation for treating specific AD patient subsets. In contrast to other methods, this approach focused on integrating multiple gene expression datasets across platforms in order to achieve a robust intersection of disease-affected genes, and then leveraging these results in combination with genetic studies in order to prioritize potential genes for targeted therapy

A systematic approach to biomarker discovery; Preamble to "the iSBTc-FDA taskforce on immunotherapy biomarkers"

The International Society for the Biological Therapy of Cancer (iSBTc) has initiated in collaboration with the United States Food and Drug Administration (FDA) a programmatic look at innovative avenues for the identification of relevant parameters to assist clinical and basic scientists who study the natural course of host/tumor interactions or their response to immune manipulation. The task force has two primary goals: 1) identify best practices of standardized and validated immune monitoring procedures and assays to promote inter-trial comparisons and 2) develop strategies for the identification of novel biomarkers that may enhance our understating of principles governing human cancer immune biology and, consequently, implement their clinical application. Two working groups were created that will report the developed best practices at an NCI/FDA/iSBTc sponsored workshop tied to the annual meeting of the iSBTc to be held in Washington DC in the Fall of 2009. This foreword provides an overview of the task force and invites feedback from readers that might be incorporated in the discussions and in the final document

Emerging concepts in biomarker discovery; The US-Japan workshop on immunological molecular markers in oncology

Supported by the Office of International Affairs, National Cancer Institute (NCI), the "US-Japan Workshop on Immunological Biomarkers in Oncology" was held in March 2009. The workshop was related to a task force launched by the International Society for the Biological Therapy of Cancer (iSBTc) and the United States Food and Drug Administration (FDA) to identify strategies for biomarker discovery and validation in the field of biotherapy. The effort will culminate on October 28th 2009 in the "iSBTc-FDA-NCI Workshop on Prognostic and Predictive Immunologic Biomarkers in Cancer", which will be held in Washington DC in association with the Annual Meeting. The purposes of the US-Japan workshop were a) to discuss novel approaches to enhance the discovery of predictive and/or prognostic markers in cancer immunotherapy; b) to define the state of the science in biomarker discovery and validation. The participation of Japanese and US scientists provided the opportunity to identify shared or discordant themes across the distinct immune genetic background and the diverse prevalence of disease between the two Nations

Comparing the Biological Impact of Glatiramer Acetate with the Biological Impact of a Generic

<div><p>For decades, policies regarding generic medicines have sought to provide patients with economical access to safe and effective drugs, while encouraging the development of new therapies. This balance is becoming more challenging for physicians and regulators as biologics and non-biological complex drugs (NBCDs) such as glatiramer acetate demonstrate remarkable efficacy, because generics for these medicines are more difficult to assess. We sought to develop computational methods that use transcriptional profiles to compare branded medicines to generics, robustly characterizing differences in biological impact. We combined multiple computational methods to determine whether differentially expressed genes result from random variation, or point to consistent differences in biological impact of the generic compared to the branded medicine. We applied these methods to analyze gene expression data from mouse splenocytes exposed to either branded glatiramer acetate or a generic. The computational methods identified extensive evidence that branded glatiramer acetate has a more consistent biological impact across batches than the generic, and has a distinct impact on regulatory T cells and myeloid lineage cells. In summary, we developed a computational pipeline that integrates multiple methods to compare two medicines in an innovative way. This pipeline, and the specific findings distinguishing branded glatiramer acetate from a generic, can help physicians and regulators take appropriate steps to ensure safety and efficacy.</p></div

Flow chart of process for comparing a branded medicine to a generic, and model of key differences between GA and generic.

<p>(A) Overview of the methods for analyzing gene expression data to compare the immunological impact of GA to that of generic. After processing, direct differences are identified by multiple parametric methods, non-parametric methods, as well as ANOVA-based pattern analysis, and variability analysis. The genes identified by these methods are analyzed using a variety of enrichment-based methods, which result in hypotheses that are then verified through additional methods. (B) The key hypotheses emerging from our studies involve the greater heterogeneity in the generic’s biological impact compared to GA’s, and the fact that GA appears to more effectively upregulate FoxP3 expression and promote tolerance-inducing Tregs, while generic appears to upregulated myeloid lineage cells such as monocytes and macrophages which may impair tolerance. Given these findings, it is reasonable to hypothesize that GA may suppress harmful cytotoxic cells more effectively than generic, and this hypothesis warrants further investigation.</p

The biological impact of GA is significantly more consistent than that of generic.

<p>Among probes with variability induced by activation, 4-fold more probes had significant variation by F-test across 11 generic-activated samples from 5 batches, when compared to the number of probes with significant variation by F-test across 34 GA-activated samples from 30 batches (A). Defining tolerance as the percentage of samples with expression levels falling within the range between the maximum and minimum expression levels induced by reference standard for that probe, for any given tolerance threshold the number of probes failing to meet this this threshold is displayed for both generic and GA (B), showing that in almost all cases more probes fail to meet tolerance following induction by generic.</p

GA induces Tregs more effectively than generic.

<p>(A) GA induces significantly higher expression of FoxP3 than generic. FoxP3 is a key marker of Tregs, and (B) another key Treg marker Gpr83 shows a similar pattern of expression. (C) Both FoxP3 and Gpr83 are low in the same samples as indicated by scatter plot, further strengthening the case that generic fails to induce a strong Treg response in some patients. (D) As further evidence of the difference in FoxP3 induction, GSEA analysis found a significantly stronger upregulation of FoxP3 target genes in GA-activated samples than in generic-activated samples. (E) GSEA analysis also found a significant enrichment of Treg-specific genes among the genes with higher expression in GA than in generic. <i>NS = not significant.</i></p