NIH CLINICAL CENTER GRAND ROUNDS
Episode 2009-022
Time: 1:06:23
Recorded September 16, 2009

EPIDEMIOLOGY AND IMMUNOLOGIC RISK FACTORS FOR AGE-RELATED MACULAR DEGENERATION

Dr. Emily Chew, Deputy Director Division of Epidemiology and Clinical Research at the National Eye Institute
Dr. Robert Nussenblatt, Chief of the Laboratory of Immunology at the NEI and acting Scientific Director of the National Center for Complementary and Alternative Medicine.

ANNOUNCER: Discussing Outstanding Science of the Past, Present and Future - this is NIH Clinical Center Grand Rounds.

(Music establishes, goes under VO)

ANNOUNCER: Greetings and welcome to NIH Clinical Center Grand Rounds, recorded September 16, 2009. Today we have two speakers from the National Eye Institute who will address the topic, "Epidemiology and Immunologic Risk Factors for Age-Related Macular Degeneration." Our speakers are Dr. Emily Chew, Deputy Director Division of Epidemiology and Clinical Research at the National Eye Institute, and Dr. Robert Nussenblatt, Chief of the Laboratory of Immunology at the NEI and acting Scientific Director of the National Center for Complementary and Alternative Medicine. We take you now to the Lipsett Ampitheater at the NIH Clinical Center in Bethesda, Maryland, where Dr. David Henderson, Deputy Director of the NIH Clinical Center, will introduce today's program.

HENDERSON: Good afternoon and welcome to Clinical Center Grand Rounds. I'm Dr. David Henderson, Deputy Director of the Clinical Center and I'm happy that you are here. Both of our speakers come from the Eye Institute. Both of them are going to focus on aspects of very interesting disease, macular degeneration.

Our first speaker is Dr. Emily Y. Chew who is deputy director of epidemiology and clinical applications at NEI. Dr. Chew, chief of the division's clinical trials branch, will present a talk entitled epidemiology and immunologic risk factors of aids related macular degeneration. Dr. Chew got her MD degree at the University of Toronto School of Medicine. She interned in internal medicine and completed a residency in ophthalmology. She also completed scholarships in retina at Johns Hopkins and university in the Netherlands. Her research interests are in epidemiological studies in trials of diseases. Dr. Chew is involved in large multicentered clinical trials of diabetic retinopathy and age-related macular degeneration. She currently chairs the Age-Related Eye Disease Study two known as areds2 and the role of nutritional supplements.
Including luetin [indiscernible] and omega polyunsaturated fatty acids in the prevention of age-related macular degeneration and cataracts. She came here in 1987 as a visiting scientist. She serves on editorial boards of investigative up that model -- in up model and American Academy of Ophthalmology.

Our second speaker today is no stranger of this podium as well. Dr. Robert Nussenblatt.
His topic today is new evidence that age-related macular degeneration is an immunologic disease. Dr. Nussenblatt is a graduate of the City College Of New York, received his MD degree at the State University of New York's Downstate Medical Center School of Medicine. He also holds a master of public health degree from Johns Hopkins. He completed his residency training at Bellevue's New York Hospital and his training at up mall ophthalmology at NYU. He established a section of ocular immunoology. He is the senior adviser of deputy director. He co-chairs the oversight committee for the National Electronic Clinical Trial and Research Network, one of the major NIH roadmap initiatives. Dr. Nussenblatt's interests center on immunology and the aging, trying to unlock the mechanism of -- and applying these treatments to patients with blindness diseases. His publications include a standard text on uvitis and it includes the research institute meritorious award medal.

We first introduce Dr. Chew.

CHEW: Thank you for your introduction. It's a great pleasure to be here. Bob and I are going to do a tag team today. This is not two sEPArate lectures but I'm going to set the stage for epidemiology as well as the risk factors and bob's going to talk about the immunological. This is for the understanding of macular degeneration and discuss some of the risk factors associated with AMD and genetics succeptibility of this disease. It is the most common cause of blindness in the U.S. It accounts for more than half of blindness in the USA. In those who are 40 years or older, the prevalence of about 1.5%.
And 1.5 have advanced with 7 or 8% have a type of disease. The thought is it's going to double in 2020 to 3 million people because of the longevity of patients and all of the babe boomers who are growing by leaps and bounds. This increases with increasing age and 15% of women age 80 years and older have advanced AMD.

This is what it looks like. Those with just early AMD, these are small little yellow flecks.
Many of these patients are still asymptomatic and have good vision. And this is picked up by the ophthalmologist on examination of dilation of the fundus. The more advanced stage which is obviously vision impairing -- with new vessels growing, hemorrhage fluid and lipids in this yellow area here. This is a geographic atrophy or central geographic atrophy causing central vision loss. And you see this really a loss of the retinal tissue and you see right down through the large vessels loss of RPE which is a layer underneath the retina.

So those are the types that we're talking about. This is the advanced disease in which vision is affected. Risk factors I alluded to earlier, aging is the most common risk factor.
I'll discuss exposure, smoking, other nutritional factors as well as genetic specific debility.

So for early AMD, which basically diagnosed in many different ways, so hence there's a different sort of curve for all of this different population-based studies. Beijing, Barbados, the Japanese study and also in India. You can see that some differences at Barbados have mostly a black population and Beijing has low rate here. This is due to diagnostic and different classifications being used. If you look at the prevalence of advanced AMD that causes vision loss, this certainly increases dramatically over age 65, you start seeing it creep up. And by 80 you see a significant number of people affected by 85. In particular when you look at the blue mountain eye study especially in the 90's, you see a huge number, 20% actually have advanced AMD in those who survived that stage. Some exposure is thought to be very important. Theoretically light exposure could have some damaging effects.

The one study in human studies that we see is the Chesapeake Bay Waterman Study where there's an increase risk of light exposure over 20 years. That's the development of AMD. Many of our studies are not able to rep indicate this. For example, the eye disease case study which is basically we actually ran this large case control study we found it to be one. In other words there was no difference whether light exposure made a difference in terms of increased risk of macular degeneration. Also we found this in age disease study which had 5,000 measures and we did extensive questionnaires and found no association. Smoking is a risk factor. It doubles the risk of macular degeneration in most diseases.

This is based upon population based studies from three consultant informants, Rotterdam in the Netherlands, Beaver Dam in the U.S., Australia. These are all pulled together because of the small number of advanced cases that they have. Consistently we find it around 2.4 with an interval between 1.3 and 4.3 so this is pretty significant. In the Eric study which is a study we worked on here that David mentioned earlier, we found it to be 1.9. So it is obviously an important risk factor we find in most of the studies.
Here is a study from the Mediterranean border. We did a dose response looking at 1 to 19 pack years to as much as 40 pack years. You see with increasing duration and amount of smoking, that the odds ratio increases from 1.8 to 4.8. So again, this points out the importance of smoking.

[indiscernible] structures in the eye are really unknown. These are quite consistent with hypotheses. There's stress that causes AMD. Smokers for example a pack a day will have 70% of the carotene of nonsmokers. Just biologically your smoking decreases your oxidative capability of fighting any oxidative stress. This is also consistent with hypothesis of AMD that smoking may damage the vessels. Interactive smoke is other factors. Smoking genetic factors are associated with the disease. It's hard to say from our analysis.

The age disease study with the clinical trial, and this provides us information on the importance of nutrients and diet in macular degeneration. This is a clinical trial as well as a natural history study. We randomized almost 5,000 patients to a placebo or anti-oxant or combination of the two. These are the numbers this these four categories. Antioxidants we used, vitamin c, (...) 80 milligrams or copper oxide two milligrams.
These are the doses we used and we tested these in the randomized clinical trial. You can see this is a perfect factorial result in that the combination had the lowest rate of progression to advanced AMD.

20% of five years developed advanced AMD compared to the placebo group which was 28%. You see that individually each factor had some effect but the combination did the best. This really translated to a 25% reduction in combination both anti-oxident in zinc -- and patients in category four where they had advanced AMD in one eye and the fellow eye did not have advanced disease. So this is a pretty significant finding in the sense that we can reduce 25%. Obviously it's not a panacea of treatment but important because it is a public health issue and this amount of reduction will actually save a number of people from going blind.

Visual acuity differences or changes are commensurate with the advanced AMD development so at ten years, 44% actually had moderate vision loss compared to 56% in those, in the placebo group. We then also look at the observational aspect of this so that was a randomized trial so we've proven my convictions are important and have a role in macular degeneration. Again we're not certain of the mechanism. So we look then at baseline and we did some dietary questionnaires and we looked as the case control study using the controls are those patients who did not have macular degeneration when they entered a study which is about a thousand measures.

We looked at patients at baseline who had wet AMD, and 118 patients who had geographic atrophy at baseline. And there are degrees could AMD and patients with intermediate druceen. We always compare the cases to the controls. We looked at their dietary questionnaires, we control for other things such as smoking, age and gender, etcetera. So this baseline questionnaire was administered right at baseline with self administered.

It had nine items of foods that they consume and we evaluated that and actually 24 hour recall to validate the actual questionnaire to know how much, give us some estimate of the actual intake. So this is now based upon the baseline data. The dietary luetin and xanthine which is found most commonly in green leafy vegetables and that consists of spinach, collard greens. That's the lowest quintile, you see the differences. So for people who ate the most spinach, collard greens, etcetera, the AMD at baseline was reduced.
So it was protective. .65 means there's a 35% reduction in the risk of AMD at baseline.
This is important because it does not include one. First geographic atrophy when it's also protected even a further reduction, 35% reduction in the risk of having atrophy at baseline. For those people of luetin.

Finally the large drusen which is really the hallmark of disease is .73% row deduction at the risk of having large drusen who had the highest almost compared to host who never eat it. We did similar results looking at the dietary intake of lipids and in this particular aspect, we're looking at omega 3's which had either DHA or EPA in it. Those are subcomponents of omega 3. The most common source that we looked for in omega 3 were fish products, salmon, etcetera. Neo vascular AMD was baseline. Those people who ate the highest intake of fish or omega 3 compared to those who doesn't it, there's almost no risk of having AMD at baseline.

If we look at DHA which is a component of omega 3 we find the odds ratio {indiscernible] a reduction in the risk of having neo vascular AMD at baseline compared to those who never eat omega 3. High fish consumption. What does that mean? What is higher. It only means two servings a week compared to never eating it. You can see there's almost a 40% reduction in the risk of having neo vascular AMD baseline.

We then looked further into other fats. This is -- acid which is more common in red meats. This is a 54% increased risk of neo vascular AMD in those who had the highest intake of acid which mostly as I said is in red meat. This was published in the archives of opthalmology in 2007.

We then follow our patients and this is at about 6.5 years median follow up. You can see that looking at dietary intake of lipids total DHA and EPP which is the total omega 3's that looking into the future and following patients for about 6.3 years. Those are the highest intake at the lower rate of developing geographic atrophy, central geographic atrophy. About 55% reduction in the risk of central geographic atrophy. With EPA alone you can see a similar odds ratio. So there's a suggestion that this is important for development of AMD. Again, these are observational data. This is not randomized trials so there may be some things that are very different about these people who eat fish.
There are other healthy habits we cannot account for. You can not take these as being recommendations but these are interesting observations of valuational data to suggest for the hypotheses. So we then further looked at the lipids in 12 years of follow up.
You can see this holds true. EPA and advanced AMD gives the highest intake of EPA, the risk of advanced AMD is still reduced by 30% or so. DHA as well.

And combined together it's very similar so its consistency throughout our data for this particular aspect. We now actually have a clinical trial which is ongoing. We have a clinic here at the NEI but headed by one of my colleagues. And it's nationwide. It was 82 clinics. We're looking at luetin 10 milligrams [indiscernible] two milligrams, omega 3 approximately one gram consisting of DHA and EPA at levels of 350 milligrams of DHA and 650 milligrams of EPA. That would be a five year follow up study and this would look very much like the brief study. It's a factorial design. A thousand patients will be randomized into the control group. Another thousand to the advanced and another thousand to the EPA and one thousand to the combination.

Because these are patients who are intermediate AMD and advanced AMD in one eye they will be given the supplement and we'll evaluate that as well. Let's turn our attention to genetics succeptibility. In 2005 was a bumper year for macular degeneration.
We made it on to the science cover and showed that factor h which is on chromosome -- which were published and subsequently two other studies also confirmed the association of common factor h with macular degeneration. This is a real break through in we did a genome wide scan as one of the first genome wide scans done with a small number of patients. We had 96 cases with advanced AMD large drusen with geographic atrophy.
And 50 patients with control group and no drusen. So these are individuals that we did a (....) on and found association the this was candidate gene studies so using different immediate all -- methodology we came to the same conclusion.

The first sign of immunologic. This is showing the drusen factor h and c5 is found in drusen which is the pathway of early signs opposite macular degeneration. The evidence of large (...) when you first start this and it goes on to more advanced disease. Factor h and you'll hear more from bob is the major inhibiter of the pathway.

The first line of defense against bacterial and other microbes sympathized by the pre and choreoid. This is a slide from one of our co-workers at NEI -- and this is found in RPE as well as in choreoid. Obviously it's an important aspect of macular degeneration.

Since 2005 a number of genes have been found. And this up here is in the view that was published. You can see the h factor in chromosome 1s in blue, it's confirmed. The second one which was very important is found in chromosome 10 called the arms 2 hetero one and you'll hear more from Bob as well. It's interesting for the past two decades people have been looking at genetics and linkage studies. These areas did show these areas to be very important.

It's only with the more recent technology that we're able to look at these genes in a more refined manner and to pick out more important genes. So those are the two major genes we're talking about. This needs further work up as to the mechanism which exerts itself.
There's a lot of work needed to be done.

This is probably the first gene announced to be associated with AMD. It's not associate in the (...). Other studies including toll 3 and 4 receptors is not confirmed. The (...) has been confirmed by other studies as well t they're obviously very important is the fact that there are other inflammatory genes in 5 and cofactor b are two important aspects that have been confirmed in a number of studies as well. And c3 which is also very important.

So these really point towards a very important aspect of the immune association. Others that are not -- I've only seen in one study population. We need to look at these further to see the replication.

So this is again taken from a small group of review that was done recently. I think this sort of gives us kind of a basis to consider the pathogenesis of this disease which we really don't know. We don't have good data, we don't have absolute answers to.
But we know this is genetic succeptibility and increasing age and increasing succeptibility. Also we know environmental factors are important. Smoking, diet perhaps in section.

Chlamydia has been implicated. There are cellular processes that are very important and of course we have the immune aspect, the cytokines complex factor b, c3, factor h and others. And so they know the cellular produce that goes on are protein folding aggregation, misfolds and mitochondria metabolism is important. It has the highest metabolic rate and filled with mitochondria. [indiscernible] are implicated as well.
This (...) degradation goes on and the oxygen is inflammatory process has been established to be important. Photo receptor defects then result from this.

[indiscernible] the deeper layers of the retina certainly may undergo oxidative damage.
And then there's of course defects in the cells. It leads to chronic inflammation causing [indiscernible] to be formed and we'll here more about the dysfunction and immune response in the RPE damage that ensues. The photo receptor dysfunction then may then occur. This results in macular generation. Geographic atrophy and neo vascular AMD.
These are very different. They may be genetically, they may have some differences right now. We haven't seen any clear genetic association with one versus the other. The geographic at me is the AMD and very owe vascular AMD may have form of risk factors that are on top of the atrophy or they could be a combination of two.

But certainly this goes on relentlessly. We don't have good treatment for this. We do have treatment for neo vascular AMD at this point and we do have vitamins which hopefully prevent mostly the progression to advanced AMD. So with this, I'm going to conclude. I could take a few questions if you'd like or we could wait until the end.
Which would you rather do, bob?

NUSSENBLATT: At the end.

CHEW: Thank you very much, I'll have Bob come up now.

[applause]

NUSSENBLATT: Well I also want to thank Dave for his very kind introduction.
As Emily said, this is really a continuum. We're not really presenting two different stories. And her presentation as well as her work really has provided all of us with an extraordinary foundation to begin looking at this question from an immunologic point of view. They pointed us in the right way. What I would like to do is present to you some newer ideas and observations concerning the underlying mechanisms of AMD.

There's still observations. The story is filling in slowly and is really becoming quite exciting. And certainly what I would like to do in this part of my presentation, the part of the presentation is to review evidence that supports the notion that AMD in fact is an immunologic disease, immunologically mediated.

I also want to talk to you a little bit about the relationship with the immune system because I think in the end that's going to be the basis of the underlying problem that we see in AMD. And then it in fact then fits into a number of disorders. And finally to review the accepted therapy, and to discuss some new work that has resulted from work that we've done here concerning immunotherapy.

Now let me just start with a definition, and first tell you that this is one of the diseases where further human work is absolutely mandatory because in fact, though we have animal models, they do present real problems. The largest one I guess is that at least in rodents and animals that are usually used for these kinds of studies, there is no macula.
By its very nature we're dealing with animals that are anatomically in the eye very very different and also immune responses are different in mouse as opposed to human.

So having said that, let me give you a definition what we think of interocular inflammatory disease and it's any disorder within the eye that has a major immune component to it's underlying and it can be shown by mechanisms or therapy. That definition was very easy and that is that the definition was that that's uvitis and that's in fact a disease that I see quite a bit of. And that really was it.

Now particularly in this past decade or so we have to win to consider that other diseases such as diabetes, glaucoma and AMD really fit into that definition. I remember early on speaking to my colleagues AMD uvitis is perhaps too great a stretch but there are commonalities in terms of their mechanisms.

A second concept that I just want to bring to your attention is I think a very important one.In medical school or if you've done a Ph.D. and you've learned about the eye, invariably it would say, you would be reading some place that the eye is sequestered and has a privileged relationship with the immune system. Implication of that privilege was that it was not seen, it was somehow ignored but in fact the reverse is true.

This is an old slide that was provided to me by Rachel Casby and we can now fill it in with more il's and numbers. The important issue is that in fact the eye very vigorously interacts with the immune system. Every day. In fact that interaction is rather extraordinary because of the role of organ, of cells that reside in the organ that can interact with the immune system. And this is something that I'm going to come back to because in fact this interaction is critical, I think, to the underlying mechanisms that we see in such diseases as AMD.

Now, what about some of the reasons to consider AMD and immune mediated disease.
Well certainly Emily alluded to many of them. The first perhaps is the question of histopathology in humans. We know this going back to the late 1980's by work done in Australia and then further work done here in the states. Showing that there was an involvement of the immune system. Most particularly macrophages were seen early on in the disease before there was any evidence of large structural changes. So we know from histopathology there is in fact a low grade inflammatory disease. This differs from uvitis where there's a very high grade inflammatory disease. There are whole group of associations. And these associations are just beginning now to be investigated on a functionality level. You can see the list here.

Compliment factor h has to be at the top and I'll refer back to in fact the first three but complement factor h, HGRA, c3 complements 3 and in fact just as an acid complements both c3 and c5 can promote choroidal neo vascularization underneath the eye we're so concerned about. There has been a whole slew of other associations, invariably associated in some way or another with immune system.

I put receptor three in this gray because it had been associated with disease but more recent work has suggested that it is not associated at least with the human form of AMD as opposed to some of the animal models. I mentioned macrophages in passing but there's evidence of auto bodies in patients that have AMD, auto antibodies are part of the retina and I'll finish my comments with therapy.

We have all of these associations and at least until recently we're lacking in understanding only of the mechanism. I'm not going to suggest we know all the mechanisms but we're beginning to open that door a little bit. Now let's, if I can, start with this question of CSH because in fact again it's a very important one.

It was found and reported and Emily was right in saying 2005 was a bumper crop year for AMD and many individuals reported the association of complement factor h with a variant that was commonly found at least in the caucasian population was associated with macular degeneration. And we found that generally in about 50% of the AMD cases this veers up to 53% but for argument saying 50% versus 29% can grow this variant that's very common in the population was associated with the disease. And I think that there's no question that based on all of these studies that that has to have an important association. However, if you begin now looking at some of the other associations, I think it's worth noting because this is another example and that's HGRA1 which is a member, it's a protease member of the larger family. This is involved in regulating phg beta immune related response involved in the tagness of the beta family. It can relate tg beta production and its function. It has other roles as well.

What is important for this discussion is tgf beta is one of the most important down regulatory cytokines we find in the eye.
I'm going to come back to this theme in a phone about down regulating the immune response. There was an interesting paper that appeared, in fact there was a second one from the same group from a little juggling of the office but the first one that appeared came and reported the association of HGRA one in a group of individuals that lack in large numbers about only 5% of them have this variant, this cha variant that was so strongly associated with AMD in the united states. This work was done in East Asia and in that group, mostly Chinese population, that group a snip in the HGRA one gene was found and associated with a very strong p value. And the conclusion was that in fact with this association, developing what AMD was ten times greater possibility.

That was an interesting observation but I think what really became even more interesting for me is that there was a second article that came out. And there they genotyped 581AMD cases, large number of controls in a caucasian population in Utah. In fact the same snip was associated with AMD in this caucasian population. Remember that's what was associated with csh. Not only did they associate it with the disease but in fact they were able to demonstrate that in drusen three AMD presence there was the presence of this HGRA mutant. And the risk of developing disease was very strongly associated with this particular snip. We're now finding that at least HGRA one as well as csh are associated with AMD so we're finding multiple factors.

So let me stop and just at least from my perspective ask you to continue some points.
First then is well how central is the immune response to AMD. I think already we're saying based at least on the associations and some on the things I mentioned to you which I'll then continue to show you, I think that we can certainly feel that it plays a roll, how central it is to the disease still remains to be seen. And then as Emily alluded to as the wet form and dry form of the disease the same, do they have the same underlying mechanism.

One could argue perhaps the wet form is more extreme part of the dry form because you can find a dry form in one eye and a wet form in the other. As I mentioned to you the studies generally have been associative and then what is the role of csh and if it is what's the role of grh1 and complement. Because complement in fact is something that comes back again and again.

Now before I try to at least begin to answer some of these questions, I think it's appropriate for me to introduce another concept and this is one that we call the down regulatory immune environment. Or die. There's a concept of immune privilege in the eye, that's true but there's an evolutionary adaptation of the eye to protect itself from any excessive inflammation. 22 millimeter org -- organ does not want to have unnecessary inflammatory responses in the eye. There are a number of adaptive mechanisms that try to down regulate the immune response in the eye and there are multiple ones. Certainly one of the best that's seen is a model first developed by Metowar in the 1940's and then really fully developed by Wayne Striline in Boston in which tumors were placed in the eye and one found altered immune responses which in fact can be seen centrally. That is the material is placed in the eye, there is a systemic immune response that is seen. All attempts to down regulate the immune response. So out of that concept that csh may not be exclusively associated with AMD. I want to emphasize exclusively.

Now there's no evidence that suggests the same association is being seen (...) that involve the back of the eye. I would like to introduce again this idea that in fact what we may be seeing in these associations is not exclusively with one disease but could be associated with the underlying down regulatory mechanisms that we see in the eye.
They're either altered or weakened as we grow holder or if we have certain variance.
Only of these important immuno-- we're looking at 48 patients who had corditis. That involves the back of the eye at the level of retinal pigment epithelium the same level which AMD occurs. One of the criticisms of this paper is that their controls were taken from the literature so they didn't redo these controls. On the other hand, this observation noted that the patients who had multifocal corditis, 55% of them had this same allele that was associated with AMD and their report is 53% versus 32% for controls. So essentially the same numbers were found in the patients that had corditis.

We've begun to reproduce this study where we have gotten 96 uvitis patients of all types.
And we have with the help of Dr. Chen in the laboratory and Dr. Chew and others have been able to use and look at now controls that we have in this community. And in fact what we found was that the mean allele frequency of csh, again the same variance that's associated with AMD was 48% versus 35% which is slightly higher than what had been reported in the literature.

When we removed patients that had inflammatory in the eye the numbers go up.
So again we're seeing the association. We're carrying this forward again because we're interested to see whether the same patients may have the variance, the HGRA one variant that's been associated with AMD. It's possible that many of these patients may have both variants which would be very very interesting and provocative and suggested in fact that there is a problem with this down regulatory environment.

There are other points that I would like to mention to you which I think are rather provocative. We thought of complement always being involved in the enate immune system and it certainly is, a very important central one that goes back to non-vertebras.
We've seen recently in the laboratory and others have seen in nice is that complement may play an important role in acquired immunity particularly with t cells. These are some of the bits of evidence that I think are important.

One of the expression of c5a the receptor for c5a is increased in patients who have AMD.
C5a is a very interesting molecule because it protect these cells from under going apoptosis so they live longer and interesting enough c 5a and c3a have been elevated in the serums of patients who have AMD. And what is very provocative is that we think that c5a induces the production of aisle 17 in t cells. Inflammatory cytokine that we can see in the patients who have AMD. This is evidence of the receptor expression in the patients, the AMD patients versus controlled and the enhancement of aisle 17 cells seen and cd4 component, cd8 also make aisle 17 but we're looking at the cd4's positive cells.
And there's a rather marked increase from .6% of cdc4 cells to 2.2 which from a human perspective is an important increase in these cells. So complement may be playing a role in immunity as well as the acquired immune system.

Now, when I mention to you about the down regulatory interocular environment, this is a list and it's not complete of the factors that can down regulate the immune response.
Tgf beta is also a part of this but as you can see from the cellular to the cell surface and soluble markers even to the physical, even to the presence of light, we see that we can down regulate the immune response. So in fact, one of the concepts that's coming out of many of the studies that we've done is there is this balance. Obviously the organism wants to be able to respond appropriately from an immune perspective. It needs to protect itself. On the other hand, it also wants to down regulate any of the unnecessary ones or to minimize the inflammatory response.

And so the concept that has been emerging from a this is that with this balance that we see here, with these vast numbers of mechanisms almost like a d40 -- like a Boeing 747 with multiple back up mechanisms if there are trigger factors such as aging, such as snips or oxidative stress.

There's a diminution of this underlying mechanism that lien leads to a change in the kinds of immune profile we're seeing in the eyes. More inflammatory things are occurring and we've seen in the vitreous of the patient but there's a change from the down regulatory to this pro inflammatory environment. Now, let me jump a little bit then to the question of markers of disease. And drusen remains really the hallmark of this disorder and the question is what role do they play. The straight answer is we don't know. You can find almost any component of the immune system within the drusen and one thing that, one possibility could be that it's an attempt by the eye to roll off active immune components bulb but but that's pure hypotheses on my part.

The other part of macrophage application while I'm using this m2 to m1 switch because this has really been reported in mice, the concept is are we finding a switch of macrophage or monocyte like cells some that reside in the eye from a more passive role to one where there's pro inflammatory responses and I would draw your attention again to the fact they are autoimmune -- antibodies produced. There has to be antigens so we know this is occur. We know this occurs in the uvitic eyes can [indiscernible] Except for the presence of drusen but we really don't have good ways to detect who is at high risk or not and certainly we're hoping that maybe antibodies or tc markers will provide us with that. But we don't at the moment.

Bruceen is very interesting because one study I would like to quickly tell you about here by Anita Chen one of our colleagues in the eye institute, the concept arose from the fact in the uvitic population we almost never see drusen, we just don't see it. And she looked at a large number of individuals ultimately 126 individuals who had gradable on this photograph, we take pictures of the back of the eye. Uvitis patients are suppressed and who are 55 years or older. Then we would expect that the overall prevalence would be at least about 6%. In fact as you grow older, the amount of patients who get these drusen increases dramatically.

What's very interesting in our average population of individuals whose age was 70, we found only one patient who had large drusen and in fact this is way below what we would expect in the general population. What's rather interesting, and this is a photograph of this individual, an 83 year old patient with large drusen, she's African American with Native American background. This is usually not associated with large drusen but she had it.

So the evidence this is a photograph of a 70 year old, 73 year old individual with no drusen. She has some changes here due to her inflammatory disease but this was the most common finding. Suggesting perhaps that either uvitis can alter the expression of drusen or the immunosuppression itself may somehow get rid of the drusen. Ones that argue either way at this point for argument sake I'm going to argue it was immunosuppression.

I want to mention the role of macrophages still remains a very important area that still needs to be looked at aggressively. There is one report in the literature by Carl Chalky when he was here at the NEI and with Scott Cousins who was in Miami at the time looking at activated monocyte of the peripheral blood of patients who had AMD.
Those monocytes defined by high tnf alpha expression were associated with those individuals who had can cordoidal activation that means at least in humans it's playing an important role. This yet needs to be determined.

Finally in terms of therapy what do we know. Certainly Emily has presented to you the elegant studies, these antioxidants and zinc have very important in dealing with antioxidants. The ARADS study going on as well. Through no accepted therapy for the dry form of the disease. That's the majority of the individuals we deal with.

Now in terms of the wet form or the neo vascular form, in fact there is anti-therapy has been accepted by the FDA and is now used (...). Avastin used for systemic therapy for tumors to decrease neo vascular component of tumors has been also used and there is now an ongoing randomized study, the contract study that is going to look at these two methodologies. How do you give this anti-veg f therapy you get injections in the eye, sometimes every month. Some patients break through.

So it is certainly a huge improvement in what we've had but still it may be that we're dealing with the expression of the disease as opposed to the underlying disease.
Now there have been several studies even going back to the Australians actually in the 1990's suggesting the use of steroids. This is one study and I have put many many suggests suggesting that perhaps the use of steroids in this case combined with other medication might have some effectiveness. They haven't been overwhelming.
More recently again in that bumper year of 2005 and then the same group published a couple years later, suggested that anti-tns therapy, the use of remicade was useful in cautions the regression of cordoidal vascularization given systemically or injected in the eye.

I'll mention the results we've had in a very small randomized pilot study where we gave systemic immunosuppression and randomized patients with AMD and the results we found there.

Now before I do that let me just emphasize to you what are we treating.
Well, this is an example of wet AMD. This is a patient who has had a cordoidal neo vascularization and a bleed. This is what we would see if we did an angiogram.
As the (...) angiogram continues on we're injecting die into the arm vein and we take rapid photographs at the back of the eye and so this initial one is very early on 26 seconds into the study, this is a minute, a little over a minute and this is five minutes. You can see this membrane underneath the retina, it becomes larger, it can hyper fluoresce more and the typical appearance what we see with these membranes. Why we're afraid of them they distort the back of the eye, they bleed, they cause scarring and ultimately a decrease in vision. That is the end point of the study I want to mention to you. Randomization scheme. Sorry this is fuzzy.

These were patients who needed constant injections of their anti-vegf therapy. They would then randomize to one of four arms. This is a very small study, a pilot study of only 1 patients. Let me emphasize this to (...) one group received a quizamad plus standard of chair which was the an joe genic therapy and the other one [indiscernible] and the other was observation which was the standard of care. The injection of vegf and nothing else.

What we saw and I have to emphasize very small study was that the monthly rates of anti-vegf injections decrease by about half in the patients who receive the quizrad and [indiscernible] we did not see that in patients [indiscernible] we also compared the rates of injections that they had received before starting the study the same thing held true.
So in fact this would suggest, it doesn't prove that immunotherapy can alter the course of this disease. Now. Some of the immunologists in the audience I want to mention something very quickly. The quizmad we saw found in patients who have multiple sclerosis. Those patients who received this medication which is dedicated against the receptor have a marked increase in their cd56 bright cells, pure nk cells that become really very dramatic in the periphery. We studied these. They have down regulatory components, aspects to their nature but we don't know their real role.

Let me mention what about these therapeutic considerations. Again we've not proved anything, we've at least shown perhaps one way we could go, there is no therapy yet for the dry form of the disease. That remains a big problem.

Now one thing that we need to consider with any therapies it needs to be early on in the disease. We want to identify this before there's any structural change in the back of eye.
Perhaps the presence of large drusen or if we can find other markers. The problem is that while this is for us a very exciting proof of concept study. You can't immunosuppress patients for years or even decades. Because of adverse events. It's just not a practical way to deal with this.

Another very big question is, is this disease so systemic that you have to treat it systemically or can you as most opt -- ophthalmologists would like to do is inject it in the eye or use other methods. There's no answer to that, it's still an open question.

So in conclusion, I would like to leave you with the following concepts. That immune mechanic subpoenas can be central to AMD mechanisms itself. CSH as well as other molecules may not be exclusively associated with AMD rather with this down regulatory immune environment that seems to be very critical for many many things that we see in the eye.

The role of complement appears to be beyond that of innate immunity and have effect on t cells. The role of macrophages are going to be extremely important but they remain to be really better defined. Immunotherapy does seem to alter the course of the disease with small studies but the challenge is long term therapy. We can't do what we did to large numbers of patients long term. But I think what is really important to note from my perspective is AMD seems to be falling into the category of Alzheimer's and multiple sclerosis where these were once thought to be degenerative.

I thank you very much for your attention. I'm sure Emily and I would like to answer any questions.

[applause]

Let me if I can just finish by thank my extramural colleagues and my very special NEI colleagues listed here. We have an immunoretinal group working together which really makes things a lot easier and of course all the patients that have provided us with support and also most very importantly with their material. Thank you. I will be happy to take any questions if there are any. Can I ask a question to the audience.

QUESTION: Patients who are on long term anti-inflammatory are long term immunosuppressant therapies for other disorders observational for the frequency of drusen often the frequentity of AMD.

NUSSENBLATT: In the Eric study we looked at risk narcotics and one of them was [indiscernible] anti-inflammatory for example. Mesnt we were able to show it was almost .2 and that was one that we've had and we tried to look at again and that was longer term. We haven't really looked at that very carefully so there are suggestions.
There is a paper on aspirin, randomize trial of aspirin in physician studies for example.
So we worked with the group there and we found it was not significant but it was very close. So there are interesting clues from other studies.

CHEW: To support dr. Nussenblatt's last point, Sulky Schmidt has been talking for years about [indiscernible] which is consistent with your very last point.

QUESTION: I wanted to ask about the difficulty of finding (...) normal controls. One would think to hear both of you that one could just pluck a few 75 year olds off the street and find wonderful age matched controls. That is not the case and I would like for you to talk about how to do that in the future.

NUSSENBLATT: The AREDS was a study when we tried to recruit controls because we're looking at controls especial lay for the progression of cataract. So it was hard to find very pure controls, people who don't have drusen in their eye. So it's been said that drusen in age 40 and above you're going to have a little drusen. You're going to see that, it's very common. So drusen might be part of just normal aging. It's going to get to a certain amount like that, large size, large extent involved. So what's special about these people who have gotten that old with macular degeneration and who don't have cataracts etcetera, that's a special group. We have DNA on that group and they are more difficult to find. There's no question when you get to a certain age that drusen is very common and to find pure controls, it is difficult. How different are they. So the DNA that we looked at in terms of factor age suggested 39% versus 50%. So there are some differences but again they are difficult to come by.

CHEW: I guess I want to follow up that as a reviewer one usually hopes to see several times as many controls in this study as you have cases. In both of the AMD papers that I've reviewed, we have a plexity of controls relative to cases. But you know, you've brought up a really difficult issue because you're quite right. It's very hard to find controls. And I guess that's the challenge for setting these human problems but you're absolutely right. It's really a problem, no question.

QUESTION: I have two questions. Emily you mentioned about 50% of females in 80 years old and over, 15%. Can you comment on compared to the men?

CHEW: I just took -- there really isn't a big gender difference between men and women.
I was just saying if you get to be 80, 15% of women, they live longer than men but there really isn't a big gender difference when you look at it. If you look at it in a certain way, the analysis are done, if you look at age of the variables or if you look at my categories, 50 to 60, 60 to 70 you might see differences but by and large there isn't a big difference.
If you look at hormones, it's been evaluated. It's somewhat a mixed bag. The hormone replacement therapy that was very common prior to the randomized trials. We looked at that and in some of the analysis when we just do it by it sell, it might be a factor that might be protective for macular generation but when you do the actual adjusted analysis for age and sex and everything else, they seem to go away. At least [indiscernible] in this case for the hormonal replacement. It isn't a clear risk factor. We certainly did not find a study to be a risk factor [indiscernible] hormone.

QUESTION: Thank you. My second question about this [indiscernible] so this changes on the animal models but on the human models. Do you think this is a similar case for uvitis?

NUSSENBLATT: Well, I can't answer the role of -- I don't know. I can only answers that in a genasense. Talking about uvitis, we see from a systems point of view that mechanisms are extremely similar. However when we begin now to be more granular and the way interpret and look at these things we're finding there are significant differences. But for the toll like receptor I don't know. There was a recent article, Emily was a coauthor on that showing the tlr, the toll like receptor three was not associated with human AMD. That could very well be. I think we're going to find more and more as we become, we hope a little more intelligent in our interpretation that there's going to be divergence of observation which will make it more difficult to interpret animal data.

QUESTION: I was wondering if there was a correlation between the development of arthritis and the dry form of uvitis. I also was wondering -- oh, the dry form of AMD.

CHEW: We have studies we've looked at in terms of associations. We have not found that and I don't know people look very specifically. If you look at large doses of aspirin being taken for arthritis you might think that was the reason, that was is association that was protective. But we really don't have any clear answer on that at all. So there might be some indirect looks at people taking large doses for whatever reason. They didn't really ask. We did ask for arthritis and arthritis by itself was not a risk factor when we looked at it in the study.

QUESTION: There isn't a correlation between a patient having AMD and having arthritis? I was also wondering now, with the use of growth factors in getting cells mobilized, if there's an increase incidence of AMD developing in those patients?

NUSSENBLATT: keep in mind that AMD by definition is when patients are over the age of 55. So we may have to wait a while.

QUESTION: Has that been looked at?

NUSSENBLATT: I don't think that's been looked at.

HENDERSON: Thank you very much for coming. Thank you.

[applause]

ANNOUNCER: You've been listening to NIH Clinical Center Grand Rounds, recorded September 16, 2009. You can see a closed-captioned videocast of this lecture by logging onto http://videocast.nih.gov -- click the "Past Events" link -- or by clicking the "View Videocast" link on the podcast homepage at www.cc.nih.gov/podcast. The NIH CLINICAL CENTER GRAND ROUNDS podcast is a presentation of the NIH Clinical Center, Office of Communications, Patient Recruitment and Public Liaison. For more information about clinical research going on every day at the NIH Clinical Center, log on to http://clinicalcenter.nih.gov. From America's Clinical Research Hospital, this has been NIH CLINICAL CENTER GRAND ROUNDS. In Bethesda, Maryland, I'm Bill Schmalfeldt at the National Institutes of Health, an agency of the United States Department of Health and Human Services.