Industry Tutorials

Friday, May 13

Cytometric Bead Array Cytokine and Gene Expression Profiling of Host Immune Responses to Emerging Infectious Diseases
Presenter: David Kelvin, PhD
Presented by BD Biosciences
Adams Room
12:30-1:30 pm

Severe acute respiratory syndrome (SARS) is a notable example of a recently emerging infectious disease. The highly variable pathogenesis of SARS may be the result of unabated proinflammatory host responses against the SARS coronavirus (CoV). To model host gene expression during SARS CoV infection, we collected longitudinal samples from mild and critical care SARS patients from the onset of symptoms through convalescence or, in some cases, death. Using Cytometric Bead Array (CBA) system (BD Bioscience) and high-density cDNA microarray analysis of peripheral blood RNA, we uncovered multiple gene pathways whose expression patterns associated with different disease courses of SARS. For example, persistent increased expression of the chemokine CXCL10 in the periphery and lungs was linked to uncontrolled immune responses against the SARS CoV. Moreover, signatures of other interferon-associated genes identified deviated adaptive immune responses in patients who were at risk for poor outcome in SARS. In this regard, CBA and gene expression analysis may represent a powerful prognostic tool in differentiating patients on the basis of their host immune responses and immune-mediated injury due to serious infections.

Clinical Genomics - IT Strategies to Enable Translational Research
Presented by IBM
Flying Cloud Room
12:30-1:30 pm

The healthcare and pharmaceutical industries have been buzzing with the promise of personalized medicine since the inception of the human genome project. As this decade unfolds, continued advances in science, technology and information technology will accelerate the translation of research discoveries into clinical practice. In order to achieve this goal of information based medicine, an advanced information infrastructure will be required across the healthcare continuum. This presentation will explore the factors and technologies enabling the move towards information based medicine. Specifically, it will explore the emerging area of Clinical Genomics, where the integration of phenotypic and genotypic data will present a host of opportunities for identifying and validating novel disease markers, enabling more focused clinical research and ultimately transforming the delivery of healthcare.

Saturday, May 14

A Method for Analyzing Gene Expression Using RNA Isolated From Whole Blood
Presented by Agilent Technologies
Adams Room
12:30-1:30 pm

With the recent advances of diagnostic tools such as microarray analysis and quantitative amplification technologies, including real-time reverse transcriptase - polymerase chain reaction (RT-PCR), there is an increasing need for rapid and inexpensive methods to purify high quality RNA. Agilent Technologies has developed a "whole solution" platform for the analysis of differential gene expression experiments, and RNA isolation is often the critical first step for these applications. In many research and clinical assays whole blood is the desired source of cellular RNA. However, isolation of RNA from blood is frequently compromised by instability of the mRNA profile and the presence of interfering compounds such as hemoglobin and heparin. We have developed a method for isolating high- purity, intact cellular RNA from whole blood by combining our Total RNA Isolation Mini Kit with PreAnalytiX PAXgene blood collection tubes. PAXgene tubes enable the collection and stabilization of whole blood samples. Agilent's RNA isolation technology ensures purification of total cellular RNA that is essentially free of interfering components, including genomic DNA, and does not require the use of toxic chemicals such as phenol or chloroform. In preliminary experiments using the RNA as input in Agilent's Low RNA Input Fluorescent Linear Amplification Kit, ample yield of high specific activity cRNA was synthesized. The labeled cRNAs were further utilized in hybridizations to Agilent Human 1A Oligo Microarrays.

Advances in Immune Monitoring and Epitope Discovery: A SARS Case Study
Presenter: Kurtis Bray, Ph.D., Director, Research and Development, Beckman Coulter, Inc.
Presented by Beckman Coulter
Webster Room
7:00-8:00 am

Objective: SARS is a severe infectious disease caused by a virus identified through gene sequencing and serological analysis as a new strain of human Coronavirus. SARS coronovirus (SARS-CoV) causes severe acute respiratory symptoms in patients and results in a high mortality rate. Antigenic peptides recognized by SARS virus specific CTL's are useful tools for studying the CTL responses during infection enabling researchers to better monitor disease and develop T-Cell mediated vaccines. In order to identify potential immunogenic epitopes from the SARS N protein, we used Beckman Coulter's iTopia™ Epitope Discovery System to analyze the protein across 8 MHC Class I alleles. This technology identifies and ranks candidate epitopes across 8 class I alleles based on true experimental binding, affinity and off rate determinations in an in vitro 96-well format

Results: Identification of SARS candidate epitopes were as follows: 6 epitopes for HLA*A0101, 30 epitopes for HLA*A0201, 18 epitopes for HLA*A0301, 28 epitopes for HLA*A1101, 37 epitopes for HLA*A2402, 18 epitopes for HLA*B0702, 7 epitopes for HLA*B0801 and 27 epitopes for HLA*B1501. Based on iTopia rankings of candidate epitopes, SARS iTAg™ MHC Tetramers for two of the candidate epitopes of the A1101 allele were manufactured and used to stain cryopreserved PBMC's from an A1101+ convalescent SARS patient. Both tetramers showed strong staining, confirming the biologic activity of these two epitopes.

Conclusion: Using the iTopia Epitope Discovery System, the SARS N protein (422 amino acids) was screened and potential immunogenic epitopes were identified based on true experimental binding, affinity and off rate determinations. SARS results demonstrate the capacity of the iTopia system to screen large number of MHC Class I restricted peptides and prioritize the epitopes with greatest potential for producing an immune response. Biologic activity of two of the identified epitopes was confirmed by in vitro staining with MHC tetramers of cells from a convalescent SARS patient.

Multiplex Serum Cytokine Analysis for Immunogenicity and Immune Competence
Presenter: Meeta Patnaik, MD, Vice President, Pathway Diagnostics
Presented by Beckman Coulter
Webster Room
12:30-1:30 pm
Lunch Served

Fine Mapping Solutions to the MHC and Beyond
Presenter: Sarah Shaw-Murray PhD
Presented by Illumina
Flying Cloud Room
12:30-1:30 pm

For almost 20 years researchers have been using genome-wide linkage analysis to search for genes conferring susceptibility to inflammatory diseases. The results of these linkage studies have been difficult to replicate. One exception is linkage of most inflammatory disorders to the MHC region. Unfortunately, the MHC region is also one of the most difficult regions to fine map in order to determine the associated gene and the underlying etiologic variant conferring susceptibility to disease. Illumina has developed two high density SNP genotyping products that overcome these challenges. The Illumina MHC panel consists of two multiplexes, each with ~1,200 SNPs that are designed for use with Illumina's GoldenGate® assay and can be used either independently or in conjunction with one another. The first multiplex is "exon-centric" and contains SNPs within 10 kb of coding sequences of genes in the MHC region spanning from ret finger protein (RFP) to motilin (MLN). The second multiplex consists of SNPs evenly spaced across the region with an emphasis on tag SNPs. For genome-wide fine mapping studies, Illumina's new InfiniumTM assay provides a whole genome genotyping solution that enables investigators to more precisely identify inflammatory disease susceptibility genes.

Sunday, May 15

Monitoring Vaccine Response: Standardization of Functional Cell Assays
Presenter: Enrique Rabellino, MD, Medical Officer, Beckman Coulter, Inc.
Presented by Beckman Coulter
Webster Room
7:00-8:00 am

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