Chris Cheadle

Analysis of Microarray Data Using Z Score Transformation

High-throughput cDNA microarray technology allows for the simultaneous analysis of gene expression levels for thousands of genes and as such, rapid, relatively simple methods are needed to store, analyze, and cross-compare basic microarray data. The application of a classical method of data normalization, Z score transformation, provides a way of standardizing data across a wide range of experiments and allows the comparison of microarray data independent of the original hybridization intensities. Data normalized by Z score transformation can be used directly in the calculation of significant changes in gene expression between different samples and conditions. We used Z scores to compare several different methods for predicting significant changes in gene expression including fold changes, Z ratios, Z and t statistical tests. We conclude that the Z score transformation normalization method accompanied by either Z ratios or Z tests for significance estimates offers a useful method for the basic analysis of microarray data. The results provided by these methods can be as rigorous and are no more arbitrary than other test methods, and, in addition, they have the advantage that they can be easily adapted to standard spreadsheet programs.

Tight junction defects in patients with atopic dermatitis.

BACKGROUND Atopic dermatitis (AD) is characterized by dry skin and a hyperactive immune response to allergens, 2 cardinal features that are caused in part by epidermal barrier defects. Tight junctions (TJs) reside immediately below the stratum corneum and regulate the selective permeability of the paracellular pathway. OBJECTIVE We evaluated the expression/function of the TJ protein claudin-1 in epithelium from AD and nonatopic subjects and screened 2 American populations for single nucleotide polymorphisms in the claudin-1 gene (CLDN1). METHODS Expression profiles of nonlesional epithelium from patients with extrinsic AD, nonatopic subjects, and patients with psoriasis were generated using Illuminas BeadChips. Dysregulated intercellular proteins were validated by means of tissue staining and quantitative PCR. Bioelectric properties of epithelium were measured in Ussing chambers. Functional relevance of claudin-1 was assessed by using a knockdown approach in primary human keratinocytes. Twenty-seven haplotype-tagging SNPs in CLDN1 were screened in 2 independent populations with AD. RESULTS We observed strikingly reduced expression of the TJ proteins claudin-1 and claudin-23 only in patients with AD, which were validated at the mRNA and protein levels. Claudin-1 expression inversely correlated with T(H)2 biomarkers. We observed a remarkable impairment of the bioelectric barrier function in AD epidermis. In vitro we confirmed that silencing claudin-1 expression in human keratinocytes diminishes TJ function while enhancing keratinocyte proliferation. Finally, CLDN1 haplotype-tagging SNPs revealed associations with AD in 2 North American populations. CONCLUSION Collectively, these data suggest that an impairment in tight junctions contributes to the barrier dysfunction and immune dysregulation observed in AD subjects and that this may be mediated in part by reductions in claudin-1.

The Local and Systemic Inflammatory Transcriptome after Acute Kidney Injury

Studies in humans and animal models have demonstrated that acute kidney injury (AKI) has a significant effect on the function of extrarenal organs. The combination of AKI and lung dysfunction is associated with 80% mortality; the lung, because of its extensive capillary network, is a prime target for AKI-induced effects. The study presented here tested the hypothesis that AKI leads to a vigorous inflammatory response and produces distinct genomic signatures in the kidney and lung. In a murine model of ischemic AKI, prominent global transcriptomic changes and histologic injury in both kidney and lung tissues were identified. These changes were evident at both early (6 h) and late (36 h) timepoints after 60-min bilateral kidney ischemia and were more prominent than similar timepoints after sham surgery or 30 min of ischemia. The inflammatory transcriptome (109 genes) of both organs changed with marked similarity, including the innate immunity genes Cd14, Socs3, Saa3, Lcn2, and Il1r2. Functional genomic analysis of these genes suggested that IL-10 and IL-6 signaling was involved in the distant effects of local inflammation, and this was supported by increased serum levels of IL-10 and IL-6 after ischemia-reperfusion. In summary, this is the first comprehensive analysis of concomitant inflammation-associated transcriptional changes in the kidney and a remote organ during AKI. Functional genomic analysis identified potential mediators that connect local and systemic inflammation, suggesting that this type of analysis may be a useful discovery tool for novel biomarkers and therapeutic drug development.

Control of gene expression during T cell activation: alternate regulation of mRNA transcription and mRNA stability.

BackgroundMicroarray technology has become highly valuable for identifying complex global changes in gene expression patterns. The effective correlation of observed changes in gene expression with shared transcription regulatory elements remains difficult to demonstrate convincingly. One reason for this difficulty may result from the intricate convergence of both transcriptional and mRNA turnover events which, together, directly influence steady-state mRNA levels.ResultsIn order to investigate the relative contribution of gene transcription and changes in mRNA stability regulation to standard analyses of gene expression, we used two distinct microarray methods which individually measure nuclear gene transcription and changes in polyA mRNA gene expression. Gene expression profiles were obtained from both polyA mRNA (whole-cell) and nuclear run-on (newly transcribed) RNA across a time course of one hour following the activation of human Jurkat T cells with PMA plus ionomycin. Comparative analysis revealed that regulation of mRNA stability may account for as much as 50% of all measurements of changes in polyA mRNA in this system, as inferred by the absence of any corresponding regulation of nuclear gene transcription activity for these groups of genes. Genes which displayed dramatic elevations in both mRNA and nuclear run-on RNA were shown to be inhibited by Actinomycin D (ActD) pre-treatment of cells while large numbers of genes regulated only through altered mRNA turnover (both up and down) were ActD-resistant. Consistent patterns across the time course were observed for both transcribed and stability-regulated genes.ConclusionWe propose that regulation of mRNA stability contributes significantly to the observed changes in gene expression in response to external stimuli, as measured by high throughput systems.

Transcriptional analysis of kidneys during repair from AKI reveals possible roles for NGAL and KIM-1 as biomarkers of AKI-to-CKD transition

Acute kidney injury (AKI) is being increasingly shown to be a risk factor for chronic kidney disease (CKD), but little is known about the possible mechanistic links. We hypothesized that analysis of the genomic signature in the repair stage after AKI would reveal pathways that could link AKI and CKD. Unilateral renal pedicle clamping for 45 min was performed in male C57BL/6J mice. Mice were euthanized at 3, 10, and 28 days after ischemia-reperfusion injury (IRI). Total RNA was isolated from kidney and analyzed using an Illumina mouse array. Among 24,600 tested genes, 242, 146, and 46 genes were upregulated at days 3, 10, and 28 after IRI, and 85, 35, and 0 genes were downregulated, respectively. Gene ontology analysis showed that gene expression changes were primarily related to immune and inflammatory pathways both early and late after AKI. The most highly upregulated genes late after AKI were hepatitis A virus cellular receptor 1 (Havcr1) and lipocalin 2 (Lcn2), which code for kidney injury molecule-1 (KIM-1) and neutrophil gelatinase-associated lipocalin (NGAL), respectively. This was unexpected since they are both primarily potential biomarkers of the early stage of AKI. Furthermore, increases observed in gene expression in amiloride binding protein 1, vascular cell adhesion molecule-1, and endothelin 1 could explain the salt-sensitive hypertension that can follow AKI. These data suggested that 1) persistent inflammation and immune responses late after AKI could contribute to the pathogenesis of CKD, 2) late upregulation of KIM-1 and NGAL could be a useful marker for sustained renal injury after AKI, and 3) hypertension-related gene changes could underlie mechanisms for persistent renal and vascular injury after AKI.

Stability Regulation of mRNA and the Control of Gene Expression

Microarray technology has become highly valuable for identifying complex global changes in gene expression patterns. Standard techniques measure changes in total cellular poly(A) mRNA levels. The assumption that changes in gene expression as measured by these techniques are directly and well correlated with changes in rates of new gene synthesis form the basis of attempts to connect coordinated changes in gene expression with shared transcription regulatory elements. Yet systematic attempts at this approach remain difficult to demonstrate convincingly. One reason for this difficulty may result from the intricate convergence of both transcriptional and mRNA turnover events which, together, directly influence steady‐state mRNA levels. Recent technical advances have led to the successful scale‐up and application of nuclear run‐on procedures directly to microarrays. This development has allowed a gene‐by‐gene comparison between new gene synthesis in the nucleus and measured changes in total cellular polyA mRNA. Results from these studies have begun to challenge the strict interpretation of changes in gene expression measured by conventional microarrays as being closely correlated with changes in mRNA transcription rate, but rather they tend to support the significant expansion of the role played by changes in mRNA stability regulation to standard analyses of gene expression. Gene expression profiles obtained from both polyA mRNA (whole‐cell) and nuclear run‐on (newly transcribed) RNA across a time course of one hour following the activation of human Jurkat T cells with PMA plus ionomycin revealed that regulation of mRNA stability may account for as much as 50% of all measurements of changes in total cellular polyA mRNA in this system. Stability regulation was inferred by the absence of corresponding regulation of nuclear gene transcription activity for groups of genes strongly regulated at the whole cell level and which were also resistant to inhibition by Actinomycin D pre‐treatment. Consistent patterns across the time course were observed for both transcribed and stability‐regulated genes. It is proposed that the regulation of mRNA stability in response to external stimuli contributes significantly to observed changes in gene expression as measured by high throughput systems.

Differential Expression of Immune-Regulatory Genes Associated with PD-L1 Display in Melanoma: Implications for PD-1 Pathway Blockade

Purpose: Blocking the immunosuppressive PD-1/PD-L1 pathway has antitumor activity in multiple cancer types, and PD-L1 expression on tumor cells and infiltrating myeloid cells correlates with the likelihood of response. We previously found that IFNG (interferon-gamma) was overexpressed by tumor-infiltrating lymphocytes in PD-L1+ versus PD-L1(−) melanomas, creating adaptive immune resistance by promoting PD-L1 display. This study was undertaken to identify additional factors in the PD-L1+ melanoma microenvironment coordinately contributing to immunosuppression. Experimental Design: Archived, formalin-fixed paraffin-embedded melanoma specimens were assessed for PD-L1 protein expression at the tumor cell surface with IHC. Whole-genome expression analysis, quantitative (q)RT-PCR, IHC, and functional in vitro validation studies were used to assess factors differentially expressed in PD-L1+ versus PD-L1(−) melanomas. Results: Functional annotation clustering based on whole-genome expression profiling revealed pathways upregulated in PD-L1+ melanomas, involving immune cell activation, inflammation, and antigen processing and presentation. Analysis by qRT-PCR demonstrated overexpression of functionally related genes in PD-L1+ melanomas, involved in CD8+ T-cell activation (CD8A, IFNG, PRF1, and CCL5), antigen presentation (CD163, TLR3, CXCL1, and LYZ), and immunosuppression [PDCD1 (PD-1), CD274 (PD-L1), and LAG3, IL10]. Functional studies demonstrated that some factors, including IL10 and IL32-gamma, induced PD-L1 expression on monocytes but not tumor cells. Conclusions: These studies elucidate the complexity of immune checkpoint regulation in the tumor microenvironment, identifying multiple factors likely contributing to coordinated immunosuppression. These factors may provide tumor escape mechanisms from anti–PD-1/PD-L1 therapy, and should be considered for cotargeting in combinatorial immunomodulation treatment strategies. Clin Cancer Res; 21(17); 3969–76. ©2015 AACR.

Renal ischemia-reperfusion leads to long term infiltration of activated and effector-memory T lymphocytes

It is well-established that significant ischemia-reperfusion injury during kidney transplantation results in increased incidence of long-term fibrosis and rejection. To test for a role of T cell infiltration and activation following ischemic injury, we induced both bilateral and unilateral renal ischemia in mice, followed by reperfusion, and then isolated mononuclear cells. Analysis of these cells by flow cytometry showed that 2 weeks after bilateral ischemia there was a significant increase of CD8(+) T cells. Furthermore, both CD4(+) and CD8(+) T cells infiltrated the injured kidney 6 weeks after unilateral ischemia. These T cells had increased expression of CD69(+) and CD44(hi)CD62L(-), markers of activation and effector-memory, respectively. CD4(+)NK1.1(+) and CD19(+) B cells were decreased in percentage both 6 and 11 weeks after bilateral or unilateral injury. There was a significant upregulation of IL-1beta, IL-6, TNF-alpha, IFN-gamma, MIP-2, and RANTES expression, measured by real-time PCR, 6 weeks after unilateral renal ischemia, further indicating T cell activation. Depletion of CD4(+) and CD8(+) T cells before ischemia caused less medullary damage and reduced kidney IFN-gamma expression, whereas their depletion following ischemia increased kidney IL-1beta; however, depletion of these cells had no effect on histological damage to the kidney. Our study demonstrates that moderate or severe kidney ischemia induces long-term T lymphocyte infiltration and cytokine/chemokine upregulation, leading to kidney structural changes.

Antisense DNAs as multisite genomic modulators identified by DNA microarray

Antisense oligodeoxynucleotides can selectively block disease-causing genes, and cancer genes have been chosen as potential targets for antisense drugs to treat cancer. However, nonspecific side effects have clouded the true antisense mechanism of action and hampered clinical development of antisense therapeutics. Using DNA microarrays, we have conducted a systematic characterization of gene expression in cells exposed to antisense, either exogenously or endogenously. Here, we show that in a sequence-specific manner, antisense targeted to protein kinase A RIα alters expression of the clusters of coordinately expressed genes at a specific stage of cell growth, differentiation, and activation. The genes that define the proliferation-transformation signature are down-regulated, whereas those that define the differentiation-reverse transformation signature are up-regulated in antisense-treated cancer cells and tumors, but not in host livers. In this differentiation signature, the genes showing the highest induction include genes for the G proteins Rap1 and Cdc42. The expression signature induced by the exogenously supplied antisense oligodeoxynucleotide overlaps strikingly with that induced by endogenous antisense gene overexpression. Defining antisense DNAs on the basis of their effects on global gene expression can lead to identification of clinically relevant antisense therapeutics and can identify which molecular and cellular events might be important in complex biological processes, such as cell growth and differentiation.

von Hippel-Lindau protein-mediated repression of tumor necrosis factor alpha translation revealed through use of cDNA arrays.

ABSTRACT Based on evidence that the von Hippel-Lindau (VHL) tumor suppressor protein is associated with polysomes and interacts with translation regulatory factors, we set out to investigate the potential influence of pVHL on protein translation. To this end, renal cell carcinoma (RCC) cells that either lacked pVHL or expressed pVHL through stable transfection were used to prepare RNA from cytosolic (unbound) and polysome-bound fractions. Hybridization of cDNA arrays using RNA from each fraction revealed a subset of transcripts whose abundance in polysomes decreased when pVHL function was restored. The tumor necrosis factor alpha (TNF-α) mRNA was identified as one of the transcripts that preferentially associated with polysomes in pVHL-deficient cells. Additional evidence that the TNF-α mRNA was a target of translational repression by pVHL was obtained from reporter gene assays, which further revealed that pVHLs inhibitory influence on protein synthesis occurred through the TNF-α 3′-untranslated region. Our findings uncover a novel function for the pVHL tumor suppressor protein as regulator of protein translation.