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Duke s Madan Kwatra Uses Microarrays, Protein Chips on Delirium

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At A Glance:

  • Madan Kwatra
  • Present — associate professor of anesthesiology, pharma- cology; director of third-year study program in pharmacology and molecular therapeutics, Duke University Medical Center, Duke University, Durham, NC.
  • 1969 — BSc, chemistry, Meerut University, India
  • 1973 — MSc, chemistry, Concordia University, Montreal
  • 1977 — PhD, medicinal chemistry, University of Montreal.
  • Postdoc — Quebec Medical Residents Council, Montreal, Canada; senior research fellowship from American Heart Association.
  • Research interest — The Kwatra lab is working to understand molecular components involved in signal transduction through receptors coupled to phosphoinositide hydrolysis, and how these components are affected by aging.

Madan Kwatra, a fresh $2.6 million grant in hand, is preparing to use microarrays to clinically target delirium, a state of psychosis, confusion, or disorientation that affects, some say, as many as 60 percent of elderly patients after major surgery.

Kwatra, a Duke University anesthesiology and pharmacology associate professor, says delirium is an expensive weight on the US medical system, costing some $8 billion a year in additional care. It’s not easily diagnosed and it’s complex. His clinical examination has the simple goal of first finding genomic and proteomic profiles for delirium.

In this research, conducted on circulating blood lymphocytes, which share many of the same responses to neurotransmitters as nerve cells, patient samples will be analyzed by microarray to seek a gene expression profile for delirium; and further study will be conducted to pinpoint protein profiles.

In a previous study, Kwatra’s researchers found that surgery has a genomic component — with nearly 500 genes up-regulated or down-regulated after surgery. With the five-year grant from the National Institute on Aging, part of NIH, he has started to enroll the first of more than 250 surgical patients over the age of 65 in a clinical research effort.

Kwatra spoke to BioArray News about how the study will use microarrays and proteomics to try to identify genes and proteins that may one day serve as markers for post-surgical delirium.

How did you get interested in studying delirium?

A few years back, in 1999, we had a conference here at Duke on surgery and the elderly, focusing on the cognitive effects of non-brain surgery. At that conference, we identified that delirium is one of the major complications of surgery in elderly patients. Paula Trzepacz, who is with Eli Lilly and is an internationally known expert on delirium, gave a lecture on delirium and talked a little bit about the mechanism, which remains poorly studied.

Delirium is a major problem, even in elderly people who don’t undergo surgery but become delirious when they come to the hospital. The cost to society is $8 billion to $9 billion annually. Most of the time, it is unrecognized because these people become confused and then it goes away and then it comes back. Most of the time it is not treated well. And, it’s well documented that people who become delirious have poor outcomes: Most of them don’t end up going home, they end up going to a nursing home. So you need extra care for them. It’s a real medical problem. Our goals are basically to get the cluster of genes and proteins [associated with delirium], which will give us some idea of what is happening, the underlying mechanism.

What do you think is happening with people who are affected by this?

In the literature, some people say those who become delirious have a higher anticholinergic activity, like many drugs have anticholinergic activity, but nobody has really come up with anything. Others say it’s the cytokines, because they go up after surgery tremendously.

What was your early research on this?

I was doing a project where I was looking at changes in G protein signaling, specifically a protein known as GRK2, in lymphocytes. So, I thought ‘why not look at other genes.’ We put in one of the first Affymetrix human gene chips, the first one that came out, which has about 5,600 genes on it. We found out that 486 genes are up- [or] down-regulated after surgery. This was interesting and we decided to use this system to look at delirium because lymphocytes have a complete cholinergic system and should be ideal to study surgery-induced changes in cholinergic signaling. If the hypothesis [is] that cholinergic blockage causes delirium, and if the lymphocytes are a good model, then all genes whose expression is modulated by muscarinic receptor stimulation will be blocked, so we should be able to pick them by this microarray.

We are not only going to look at the mRNA, but also look [to see] if it translates to the protein level, and we have proposed that we look at the serum biochemistry, the proteomics of serum. We are going to use a global proteomics approach to see if these patients have any unique protein expression patterns.

What kind of chips will you use in this work?

We have a DNA microarray facility here at Duke where we did our initial research. They have the Affymetrix system and the software for analysis. Our initial data was obtained from a chip that has 5,600 probes. Then, we got data on the next chip, the U95, which has 12,000 gene [probes] on it. Now, we have chips with even more genes. We haven’t decided which chip we are going to use [next] but I’m sure we are going to use the state of the art, the most comprehensive chip that is available.

How will the analysis for this project be conducted?

We will take blood samples before patients go to surgery. We will isolate serum and lymphocytes. On the second day after surgery, when other studies have shown that the incidence of delirium is at its peak, we’ll do the testing. We will identify patients who become delirious and those who don’t. So, we will have two groups, based on very comprehensive testing.

We have a very big team working on this: We have experts on bioinformatics, surgeons, anesthesiologists, pharmacologists, psychiatrists, and geriatricians. The key aspect is to identify people with delirium accurately. Once we have [delirium] identified, then it becomes the problem of [comparing] normal controls versus the disease and analyzing them by standard methods.

The protein chip analysis of lymphocytes will come after we have done the DNA microarray — to see if the genes that we find are expressed at the protein level. The other part is the proteomics of serum, which is different than the lymphocytes. The serum studies will start immediately. We just bought Ciphergen’s machine and that will be coupled with 2D gel electrophoresis. That’s our staring point. Our goal is basically to examine the serum of patients who become delirious to see if there is a unique protein profile.

What do you plan to use for data analysis?

I think we will go with off-the-shelf technology. I like GeneSpring. We are looking at that, and looking at some software that Duke has developed. I’ll see which one is the best. I will be hiring someone to do the DNA microarray analysis. Dr. [Merlise] Clyde from Duke’s institute of statistics and decision sciences will work with us in data analysis.

Do you have any wishes for improvement on the microarray technology?

I think the technology is good. We are at the age in biochemical research where we have the tools that we need for next five or 10 years. We should be very happy that we have the ability to generate a massive amount of data and the only wish is to have simple tools to analyze the tremendous amount of data that we will be generating.

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