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U. Nebraska Scientist Using Proteomic Tools To Help Understand HIV-Associated Dementia

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Pawel Ciborowski
Assistant professor
University of Nebraska Medical Center
Who: Pawel Ciborowski
 
Position: Assistant professor, department of pharmacology and experimental neuroscience, University of Nebraska Medical Center, 2004 to present
 
Background: Director, mass spectrometry and proteomics core facility, University of Nebraska Medical Center, 2007 to present; director, proteomics program, Center for Neurovirology and Neurodegenerative Disorders, UNMC, 2003 to present; PhD, microbiology, National Institute of Hygiene, Warsaw, Poland, 1977 to 1983
 
At the recent US Human Proteome Organization annual conference outside Washington, DC, Pawel Ciborowski presented a poster on work that he and colleagues are doing profiling sera from patients with HIV-1-associated dementia.
 
While HIV-1 infection is associated with cognitive impairment, the means to diagnose and track disease progression is limited, they said in their poster. In their work, they employed 2 DIGE with DeCyder 6.5 software analysis to analyze differentially expressed proteins in the sera of HIV-1 infected patients.
 
They were able to identify 29 protein spots, corresponding to 17 proteins as being significantly different. Ceruloplasmin, afamin, and “a broad range of components of complement system” were identified. The researchers are currently studying these and other proteins in a larger patient cohort.
 
ProteoMonitor spoke with Ciborowski this week about his research. Below is an edited version of the conversation.
 

 
Describe the work you did in your poster at US HUPO.
 
I am a faculty member and my primary appointment is in the department of pharmacology and experimental neuroscience. However, I’m also a director [in the] mass spectrometry and proteomics core facility. Therefore, on top of my own research that I presented at the conference, I’m involved in many projects, grant proposals, and so on … to different degrees.
 
My research, and that’s what I’ve been doing for the last four [or] five years, was focused on biomarker discovery in HIV-associated neurocognitive disorders. That’s the current term. It’s called HAND.
 
That name evolved over the last several years. You may see some other [names]. I was using HAD, which is HIV-associated dementia. That is the most severe form of neurocognitive disorder associated with infection of the brain by HIV.
 
According to the literature and some epidemiological studies … after [the] introduction of highly active anti-viral retroactive therapy, HAART, or recently the new term, ART, the percentage of people suffering from HIV-associated dementia dropped from 30 to around 7 percent.
 
In other words, HAART did a good job. However, the percentage of people who suffered from neurocognitive disorder or cognitive impairment, whichever term you like, remains the same. What we have [is] less severe cases, but the remaining percentage of neurocognitive impairment … increased.
 
So in this respect, this anti-viral therapy did not fully help.
 
Even though it helped the most severe forms?
 
It helped because it reduced the most severe forms, but not in reducing the number of cases. Some people say that due to the HAART therapy … in extending the life expectancy of HIV-infected people, even if the number of cases of HIV-associated dementia dropped, the prevalence [of dementia] increased.
 
We have people living longer. Many people in the past died even before they developed dementia. Dementia is a [late-stage] disease within the AIDS complex.
 
So anyone with the disease who lives long enough will eventually develop dementia?
 
No, not everybody. We don’t know the reasons for neurocognitive impairment. When there is dementia, there is encephalitis. You can say there is a huge inflammation of the brain, mononuclear phagocytes, they go into the brain. They are neurotoxic, they destroy the neurons, there is dementia, and then death.
 
But now, patients live longer, they don’t have encephalitis, but they still are cognitively impaired. Some of them, and this is an increasing number of cases, are cognitively impaired, which is detected only by rigorous testing.
 
So if you interact with this person, this person might occur to you as normal. But if this person goes through psychiatric, psychological tests, it shows that there is impairment in cognition, but it’s not really visible to the average person on the street.
 
Can you use family history, such as Alzheimer’s or Parkinson’s, to predict which HIV-positive individual is more prone to developing cognitive impairment?
 
No. This is a big problem because we do not have biomarkers that can tell us in laboratory tests whether somebody [who has HIV] is predisposed for cognitive impairment … or how fast the dementia will develop, or if the cognitive impairment will develop, and who will get full-scale dementia, and how effective therapy is, or would be, or how to measure the effectiveness of a therapy.
 
Treating [brain disorders] is particularly hard because antiviral drugs do not cross the blood-brain barrier at a sufficient level to be effective in the brain.
 
So I ask the broad question, ‘Can I use a proteomic approach, [a] proteomic platform, to find the protein level, the molecular correlates of cognitive impairment, and whether these correlates can be found in cerebrospinal fluid or these correlates can be also found in serum?’
 
Some people say that cerebrospinal fluid is the ultra-filtrate of plasma or blood because, after all, what is produced in the brain has to be dumped into blood and then transported to liver or kidney and excreted from the body. But even if the blood-brain barrier is impaired, and we know it is impaired during HIV infection, it is still to some extent a functioning barrier.
 
Initially, I used extreme cases for our proteomic analysis. I took [samples] from HIV-infected, non-demented [patients] and samples from HIV-infected individuals who had full dementia. Going between the two extremes, it helps to discover the differences.
 
What we’re doing right now is trying to apply [what] we’ve learned from these studies … to the cohorts of samples which have more subtle differences. There is an ongoing study [in which] we have samples from individuals who were infected with HIV before the therapy started, and six months [after therapy started].
 
What we want to know is … how the therapy itself is reflected in the biological ex vivo sample and extrapolate that to the clinical conditions. These studies, which we are doing in collaboration with the University of California, San Diego … are partially blinded, which means that they made a selection of samples, they send these samples to us. We do a proteomics analysis, but they will do the clinical correlates.
 
Biomarker discovery itself, even if you find some proteins differentially expressed, unless you understand how this correlates with the biology of disease, with the pathology of disease, they are not of full value, especially if you [consider] that HIV or AIDS is a very complex disease. It’s not a [monolithic] disease.
 
In AIDS complex, you may have many other opportunistic infections going on. There are a lot of people who are drug abusers, maybe not single drugs … There are a lot of people who are alcohol abusers. HAART therapy may have its own effect, and what we see in plasma, even if HAART is not helping the brain, [maybe] it’s helping on the periphery.
 
In your own research, are you trying to address all these different HIV populations?
 
We are trying to correlate that with some components. And in the paper we published in Virology … [our hypothesis] was that during brain inflammation, there is a recruitment of mononuclear phagocytes to the brain in large numbers.
 
However, not all of them are infected. What we think is that when they are infected or they go over the edge and they become neurotoxic, these macrophages, perivascular macrophages or microglia may produce and secrete neurotoxic substances.
 
Having said that, we set up the in vitro system. When we obtained monocytes from healthy individuals, we infected them with HIV in vitro … and then we looked at [what they produced] using a proteomics platform and a proteomics approach.
 
Then what we are trying to do is correlate that with what we see in either cerebrospinal fluid or … in blood.
 
So that is one approach, to connect that to biology. Dr. Howard Gendelman [at the University of Nebraska] … what he is trying to do is look at the co-cultures. He is culturing infected and non-infected macrophages with astrocytes because … maybe the major damage is caused astrocytes that are stimulated to be neurotoxic.
 
The major role [of astrocytes is] to nourish and protect the brain, but a major question is ‘If astrocytes encounter the HIV-infected macrophage, are they capable of protecting the brain?’
 
And what’s the answer?
 
We don’t have answers. This is all ongoing research.
 
You’re working with both CSF and plasma. What’s your eventual goal: to develop something that would be blood-based or CSF-based?
 
If it is possible to develop a test that is based on blood, that would be fantastic. Not everybody will give you consent to draw cerebrospinal fluid. To draw cerebrospinal fluid is a much more invasive procedure than just to take blood.
 
Most people don’t object [to giving] blood. The question for me is, ‘What does blood represent?’ If there is an ongoing infection, and of course, HIV infected patients have ongoing infections, not only is the brain infected, but so are other organs. You have it in the lungs, in the liver, in the spleen, bone, whatever. You may have symptoms of the inflammation, not necessarily in the brain but also in the periphery, systemically. For example, if you see something related to inflammation in the blood, how do you know that this is related to the development of inflammation in the brain, which will lead to encephalitis and dementia, or [if it] might be something that’s going on systemically in the periphery?
 
That’s why I think that without understanding biology, without linking [the data] with biology, and just going strictly after biomarkers as diagnostic markers, that’s why proteomics is not delivering what people have expected.
 
Are there different neurocognitive disorders associated with HIV in the same way that the general population can suffer from different neurodegenerative diseases such as Alzheimer’s and Parkinson’s disease? 
 
It’s hard to say because there are more and more papers, but so far, I can tell you that most of the papers are cataloging proteins. [They are] trying to link those differentially expressed proteins found in ex vivo samples with the biology of disease, but we are still at the level of cataloging rather than understanding.
 
So we don’t really know.
 
We don’t know the answer. There are, of course, people [working] in Parkinson’s or Alzheimer’s disease, they can tell you ‘Oh this is a biomarker, this is this, this is, this.’
 
However, what we think is that the neurodegenerative process, or neurotoxic process that is going on in the neurodegenerative diseases, may have the same elements. The triggers may be different — the trigger for Parkinson’s disease, the trigger for ALS, the trigger for Alzheimer’s disease, the trigger for HIV cognitive impairment might be different … but part of that which is destroying the brain or destroying neurons might be similar for all of neurodegenerative disorders.
 
We have people who are trying to differentiate biomarkers for different neurodegenerative disorders. Do you foresee that, at some point, we will have to do something similar in this area: that we’ll need to find biomarkers not only for general neurodegenerative disorders for HIV patients, but biomarkers that can differentiate neurodegenerative disorders for this population?
 
First of all, I think that it will be a set of biomarkers. I do not believe it will be one biomarker for Alzheimer’s, one biomarker for Parkinson’s, one biomarker for HIV. I think it will be a set of biomarkers. And I’m trying to look for this set of biomarkers.
 
That will be common to all these diseases?
 
Well, some of them might be common, some of them might be different or common to two out of five. What I would like to accomplish in my work is to add molecular components to clinical diagnosis, not to compete with clinical diagnosis, not to compete with the psychiatric, psychological evaluations, but to help them, to help add molecular components to how it is diagnosed using those tests.
 
You’re trying to find these biomarkers for HIV-associated neurocognitive disorder. If you find them, then what?
 
First of all, if we find biomarkers that can give us a molecular component of effectiveness of a therapy, that would be a very big thing because then we can start correlating the general markers of a disease such as viral load, T-cell count, those markers that are telling us the general condition of a patient with a brain function. And we can say which therapy is the best to target both.
 
There might be drugs developed by pharma … that may be in the pipeline that may not have direct effect on the T-cell count or the viral load, but they might be [helpful] in helping them to cope with infection of the brain.
 
Such biomarkers would a huge help to evaluate treatment regimes and to [individualize treatments].

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