Inflammation and the Host Response to Injury

As we all learnt in this class inflammation is one of fundamental protection mechanisms in human biology. This is how we protect ourselves from foreign invaders as well as abnormal cell inside of our body. But gone awry inflammation that spreads beyond the primary locus or fails to subside poses serious chronic and acute health risks for millions of people.

Each year about 60 million Americans sustain injuries. Trauma accounts for more than 12% of all medical spending in America. After the first 24 hours following the injury the primary cause of death is Multiple Organ Failure (MOF). It presents as an excessive inflammatory response directed towards patient’s own tissues. It is closely related to Systemic Inflammatory Response Syndrome (SIRS) and Sepsis. Sepsis alone accounts for up to 215,000 American’s lifes every year, according to the National Institute of General Medical Sciences. We referred to SIRS in Immu 7630 course as cytokine storm.

Growing evidence suggests that genetic factors drive key aspects of an individual’s inflammatory outcome. Scientists studying inflammation are trying to identify the genes that drive inflammation as well as biomarkers from throughout the course of inflammation. Genomic studies, in addition to their proteomic and metabolomic cousins, aim to resolve an age-old mystery: namely, why some patients recover readily from inflammation while others suffer and die from it. Ideally, new gene based discoveries will provide diagnostic biomarkers to predict who among these patients will react poorly to inflammation and why. If doctors could reliably predict this outcome in advance, they might tailor antibiotics and other treatment options to a patient’s own inflammatory system, potentially saving lives.

Among the numerous programs moving inflammation research forward is an effort funded by a National Institute of General Medical Sciences “glue grant,” so named because it “glues together” multidisciplinary efforts to tackle biomedical questions beyond the means of any one research group. This program, called Inflammation and the Host Response to Injury, strives to determine why patients can have dramatically different outcomes after traumatic injuries and burns.

UCD and Denver Health Medical Center are part of this multicenter research study.

In order to find immuno-inflammatory biomarkers for developing MOF Glue Grant is using genomic and proteomic analyses:


mRNA Isolation for Protein Coding

The Glue Grant investigators nationwide are isolating messenger RNA (mRNA) from blood and other available tissues. Isolation of the mRNA molecule is key to finding expressed genes in the vast expanse of the human genome. Since mRNA is very unstable outside of a cell, we are using a special enzyme called reverse transcriptase to convert it to complementary DNA (cDNA) which is a much more stable compound than mRNA and is presumed to represent the sequences of the genes being expressed. Sequences contained in the cDNA are used to generate expressed sequence tags (ESTs) that code for the protein in question or represent non-translated regions. ESTs are powerful tools for gene mapping and gene discovery. In the search for known genes, they greatly reduce the time needed to locate a gene. ESTs provide sequences that can be generated rapidly and inexpensively, in that only one sequencing experiment is needed per each cDNA generated and ESTs do not have to be confirmed for sequencing errors.

Only a fraction of genes are turned on, and it is the subset that is "expressed" that confers the unique properties to each cell type. Gene expression is the term used to describe the transcription of the information contained within the DNA into mRNA molecules that are then translated into the proteins that perform most of the critical functions of cells. Gene expression is a highly complex and tightly regulated process that allows a cell to respond dynamically both to environmental stimuli and its own changing needs. This mechanism acts as both an "on/off" switch to control which genes are expressed in a cell as well as a "volume control" that increases or decreases the level of expression of particular genes as necessary.

Microarray technology is applied to the samples to collect gene expression data. This technology works through the ability of a given mRNA molecule to bind specifically to, or hybridize to, the DNA template from which it originated. By using an array that contains many DNA samples, we can determine - in a single experiment - the expression levels of thousands of genes within a cell by measuring the amount of mRNA bound to each site on the array. The amount of mRNA bound to the spots on the microarray is precisely measured, thus generating a profile of gene expression in the cell. Microarray expression analysis are performed on the samples to determine the level, or volume, at which a certain gene is expressed. The arrays used in this analysis are called expression chips or "Gene Chips". We use the expression chips to detect expression patterns, that is, whether or not a particular gene is being expressed more or less under certain circumstances, and to examine changes in gene expression over a given period of time.

Detection and Analyses of SNPs

A single nucleotide polymorphism (SNP) is a small genetic alteration that can occur in a patient's DNA sequence. An SNP variation occurs when a single nucleotide (adenine, cytosine, thymine, or guanine) is replaced by one of the other three nucleotides. Each patient's genetic material contains a unique genetic pattern that is composed of several different genetic variations, including SNPs. It is believed that SNPs can serve both as a biological marker for pinpointing a disease or condition and may be directly associated with or causative of a certain disease or condition.

Proteomic Analyses

Cytokine mediators, leukocyte phenotypes, and cell signaling intermediates (e.g., protein kinases, NF-ê B family) will be analyzed from blood and plasma of trauma patients. Of particular interest are investigations of changes in pro-inflammatory and anti-inflammatory cytokines in isolated populations of cells from these patients. There are several cytokines of interest that include TNF, IL-1, IL-6, IL-8, IL-10, IL-12, IL-18, IL-1ra, p55, and p75.

Glue Grant study is still underway and the final results are yet to be analyzed…

For more information please visit www.gluegrant.org or search PubMed for “Inflammation and the host response to injury: a Large-Scale Collaborative Project”.