P-R-O-C-E-E-D-I-N-G-S

1:04 p.m.

DOCTOR FREAS: Let me just introduce myself to everybody. I am Bill Freas, I'm the Executive Secretary for today's advisory committee meeting, and this is the 20^th meeting of the Allergenic Products Advisory Committee.

We are holding this meeting in Conference Rooms A and B, in Building 29B, on the NIH campus. There's a speaker phone here for public participation, and, of course, the public is more than welcome to participate in our advisory committee meetings.

The entire meeting for today will be open to the public, as announced in the Federal Register on March 10, 2004.

At this time, for the people in the room and for the people on the phone lines, I would like to go around and introduce the committee members. They are our Acting Chair, Doctor Wesley Burks, Chief of Pediatric Allergy and Immunology, Duke University Medical Center. Doctor Burks can you say present?

ACTING DIRECTOR BURKS: Present.

DOCTOR FREAS: Doctor Rebecca Gruchalla is next, Associate Professor of Internal Medicine, University of Texas Southwestern Medical School.

DOCTOR GRUCHALLA: Present.

DOCTOR FREAS: Doctor Melvin Berger, Professor of Pediatrics and Pathology, Case Western Reserve School of Medicine.

DOCTOR BERGER: Present.

DOCTOR FREAS: Doctor Harold Nelson, Senior Staff Physician, National Jewish Medical Center.

DOCTOR NELSON: Present.

DOCTOR FREAS: Our non-voting industry representative, Peter Hauck, Executive Director for Scientific Affairs, Allergen Products Manufacturers Association.

MR. HAUCK: Present.

DOCTOR FREAS: Our acting consumer representative, Ms. Dolores Libera, Director of Publications, Allergy and Asthma Network, and Mothers of Asthmatics, Incorporated, Fairfax, Virginia.

MS. LIBERA: Present.

DOCTOR FREAS: And, we will soon be joined by Doctor Lynelle Granady, Associate Physician with ENT and Allergy Associates, New York, New York.

Unfortunately, Doctor Susan McDonald, from the Johns Hopkins University, could not join us today.

Now, should anybody get dropped from this teleconference, you all have the instructions of how to join us, but just to repeat, you dial 1-888-577-8990, enter a pass code of 25660, and push the # sign, and please rejoin us.

Now, as I said, there are about 20 people in the room. I will not introduce all of them, but there are several key FDA staff that I would like to introduce.

Oh, Doctor Granady, did you just join us?

DOCTOR GRANADY: Yes.

DOCTOR FREAS: Well, thank you very much. We have just been joined by Lynelle Granady, Associate Physician with ENT and Allergy Associates, New York, New York. Thanks for joining us. We are just going around now and introducing some of the key FDA people. There are about 25 people in the room now, I would guess, and I won't introduce everybody, but I would like to acknowledge Doctor Richard Walker, Director, Division of Bacterial, Parasitic and Allergenic Products. At the table is the organizer for this meeting here, Doctor Jay Slater, Chief of Laboratory and Immunobiochemistry. And, also at the table is Doctor Ronald Rabin, Senior Staff Fellow, Laboratory of Immunobiochemistry.

Now, we also have a transcriber here, and I must ask all committee members on the telephone lines to please identify yourselves each and every time you speak, because we are trying to transcribe an official record of this meeting, and we want to be able to attribute the comments to the appropriate individual. So, please identify yourself each and every time you speak.

Now, I'd like to read into the public record the Conflict of Interest Statement written for this meeting. "The following announcement addresses the conflict of interest issues associated with this meeting of the Allergenic Products Advisory Committee on April 2, 2004. The Director of the Center for Biologics Evaluation and Research has appointed Ms. Dolores Libera as a temporary voting member for this meeting. Based on the agenda, it has been determined that there are no specific products being approved at this meeting. The committee participants have been screened for their financial interests. To determine if any conflict of interest existed, the Agency reviewed the agenda and all relevant financial interests reported by the meeting participants. No waivers were required under 18 US Code 208. We would like to note for the record that Mr. Peter Hauck is participating in this meeting as a non-voting industry representative, acting on behalf of regulated industry. Mr. Hauck's appointment is not subject to 18 USC Code 208, he is employed by Alk Abello, Incorporated, and thus has financial interests in his employer. Mr. Hauck also serves as the Executive Director of Scientific Affairs, Allergen Products Manufacturers Association. In addition, in the interest of fairness, FDA is disclosing that his employer, Alk Abello, is a manufacturer of allergen extracts. In the event the discussions involve specific products or firms not on the agenda, for which members have a financial interest, members are reminded of the need to exclude themselves from these discussions. Their exclusion will be noted in the public record. With respect to all other meeting participants, we ask in the interest of fairness that you address any current or previous financial involvement with any firm whose products you wish to comment upon."

That ends the reading of the Conflict of Interest Statement.

Doctor Burks, I turn the meeting over to you.

ACTING DIRECTOR BURKS: I want to thank everybody for the conference call, and I especially thank Bill for allowing us to do the conference call, rather than having to come to Washington, and thank Jay for helping arrange that.

What we'll do for the next hour and a half or so is to listen to several presentations by Jay and Ron Rabin, and it's on the agenda that you have, and I think each of you have the slides. If you have questions as you are going along, if you'd just identify yourself and ask them, just try to stick to the topic that Jay is addressing at that time, and then he'll leave time for questions at the end of each section to see if we need to say anything else.

So, with that, Jay, we are ready to get started.

DOCTOR SLATER: Terrific, thank you very much, Wes, and thank you all for participating. You know, as you well know in the past this committee has been responsible for valuable contributions, and we look forward to this meeting every year.

Since I'm not in the room with you, I can't point to the slides. We are going to try, as we go along, to identify what slide we are on. All of the slides that we're using today are numbered, and we're going to identify them as we go along every once in a while by number, to make sure we are all literally on the same page.

We are now in the first ? these presentations are also numbered in order. There are five presentation files that you were sent, and it should be pretty easy to decide where we are but I will identify them as we go along ? we are now in the first presentation, the Lab Overview, and after the title slide you can go on to the second slide, please.

This is, basically, what we are going to cover today in our meeting. This is slide two out of ten. We'll start with the Lab Overview, in which we cover some staffing issues, lot release, reference, maintenance activities of our laboratory. That will be fairly brief. We will then get into the meat of the presentation, which involves a review by me and Ron broadly speaking of the research activities in the lab. Then I will focus on two specific areas of both research and regulatory interests. One is our activities in cockroach allergen standardization, and the other is our effort to develop novel methods of determining the potency of allergenic extract, in particular, our very recent and early work on using antibody micro arrays for this purpose.

Next slide, please.

So, quickly, we are going to go through the Lab Overview section, covering staffing, lot release and reference maintenance activities.

Let's go to slide four.

The two principal investigators in our laboratory are myself, I've been here for five years, and Ronald Rabin, who has been with us now for three years. We have three post doctoral fellows, Bo Chi, Jinsong Chang, Nicolette deVore.

Next slide, please.

Those of you that have been here for meetings know that I would normally ask people to stand up, but that would be a little silly under these circumstances, but they are all here and listening to the presentations.

Our research technicians, on slide five, Al Gam, Mona Febus, Marc Alston, Cherry Valeriod and Katia Dobrovolskaia.

Next slide, slide six. This is one of my favorite slides. This is a slide in which I give you an idea of where we are and where we have been, in terms of our staffing. What I include in this slide is only our research technicians, because those are the staff that really are in the forefront of our intersection with the allergen products manufacturers.

And, as you can see, after a couple of years in which our staffing fluctuated up and down we've really been in a very stable pattern for the last three and a half years, with about five research technicians. I think this has really helped us a lot, this stability, in terms of our ability to provide service and to effectively regulate the allergenic products.

Next slide, please.

The routine regulatory activities that our research technicians are involved in include lot release, reference distribution, reference maintenance activities. As you know, for the standardized allergenic extracts we maintain the U.S. Standards of Reference that are used both by us and by all of our manufacturers for effective measurement of the potency of their extracts. And so, we devote quite a lot of effort and expense into maintaining and distributing these references.

Next slide, please. We are now on slide eight.

In the past year, in calendar year 2003, we received 450 protocols which we reviewed prior to the release of Standardized Allergenic Extracts. Also, in the same calendar year we distributed 1,423 vials of reference standards in 96 shipments sent to the manufacturers.

On the next slide, slide nine, we give a little context to some of these numbers. You see here the lot release protocols that were submitted by year for the past five years, and what you can see is that 450 is more or less around the same mean where we have been for the last several years, a little bit higher than last year, but definitely in the same range.

The next slide, slide ten, we look at our reference distribution activities. The number of shipments, the number of vials, is down somewhat. It will probably be a couple of years before we know whether this is really a trend. If it is, then I'll be very grateful to our manufacturers. One of the things that we've been requesting for quite a while is that our manufacturers use the reference standards economically. But, this is a trend, and they've been heeding our request, and for that we can all be very thankful.

That's the end of the first presentation.

I'm happy to entertain any questions if there are any about that.

ACTING DIRECTOR BURKS: Does anyone have questions for Jay for this part?

Okay, you can go on to the next part, Jay.

DOCTOR SLATER: Thanks very much.

Okay. We are now in presentation number two, which is entitled, "Research Update." We can go now to the second slide.

The active research projects in the lab are detailed on this slide. Among the projects that I'm doing, we are investing a significant effort in our Cockroach Allergen Standardization Program, which I'll be talking to you about some more later.

Related to this, but not absolutely essential, but something that we consider to be an important contributor to this effort, is our effort to generate cockroach specific IgE and IgG combinatorial libraries. We've really been focusing on the IgG work recently, with an aim towards micro array analysis of allergenic extracts. And again, this work is very preliminary, but it's exciting nonetheless, and I'll be going into that in some more detail.

In addition, you are all familiar with some of the work that we've doing on endotoxins in allergen vaccines. We have some additional data to discuss with you at this ? in fact, in this presentation.

Ron Rabin has two major projects, the MDR proteins and their involvement in T cell activation, and RSV responses in human tonsilar tissue, and he'll be talking about the first of these projects in just a few minutes.

Next slide, please.

These are the publications that have come out of the lab this year. You can see them here. We published some of our first work with the endotoxin content of standardized allergen vaccines. I was also invited to co-author an article in Middleton's Textbook with Dick Lockey and Bob Esch. Ron published a paper in the Journal of Immunology on his work with the CXCR3 induction and CD4 T cell differentiation, and in addition he has a manuscript in preparation about his work which he'll be talking about a little bit more in a few minutes.

Next slide.

At this year's Academy of Allergy Meeting, this Laboratory contributed six abstracts, which were all presented at the meeting, and they are detailed on slide four.

Next slide, please.

Ron Rabin was invited to give two outside presentations, one at the SMI Conference in London in February, 2004, on the relationship between viral respiratory infections and asthma, and another at the most recent Academy of Allergy Meeting in March, on the FDA Food, Drug and Cosmetics Act as it applies to research studies.

Next slide, please.

And, I was invited to give a talk at the State of the Art Analytical Methods for The Characterization of Biological Products Meeting, in June, 2003, in which I talked about allergenic extracts, and I gave four presentations in workshops and courses at the most recent Academy meeting.

Next slide, please.

Ron and I both maintain outside collaborations. I've been collaborating with Larry Arlian, at Wright State University in Dayton, Ohio, as well as with Doctor Patrick Murray, who is the Chief of the Microbiology Service here at the NIH Clinical Center. Ron has active collaborations going on with Peter Collins at NIAID, as well as with Mario Roederer at the Vaccine Research Center, also at NIAID.

Next slide, please.

So, I'd like to spend just a few minutes talking about the endotoxin work, which I first presented to this committee about one year ago, and just to quickly summarize where we left it last year, we were embarked on an activity in which we were, basically, trying to update some very old work that had been done in this laboratory in collaboration with NIH back in the `70s, in which it was first observed that allergen vaccines, especially dust mite vaccine, contained a measurable amount of endotoxin.

Our work focused exclusively on standardized allergen vaccines, in which we found what's really summarized here on slide number eight, and that is that in general, although we were able to detect endotoxin in virtually all the allergen extracts that we examined, we found that the cat and mite extracts had more than the pollen extracts, that the cat pelt had more than cat hair, and what was most striking to us, and what really led us to the rest of what we're going to be presenting today, was our observation that the D. farinae extracts contained significantly more endotoxin than the D. pteronyssinus extracts.

As I noted last year, it's not clear that any of these endotoxin amounts are physiologically significant, but, nonetheless, we felt that we needed to examine this further to characterize the nature of this endotoxin content.

And, we wondered about several different possibilities. We wondered about the degree of bioburden that might be present. We wondered about whether there might be an endogenous heat-stable and debinding activator in D. farinae, and we wondered whether there was actually endogenous endotoxin neutralizing protein in D. pteronyssinus.

Just to very quickly go through what else we found, next slide please. The next slide is a graphical representation of the corrected endotoxin content of the different classes of vaccines. Note that this is on a log scale. The significant differences are shown, and this, basically, replicates what I had in the previous slide.

Next slide, please.

So, our plan was to investigate the differences between D. farinae and D. pteronyssinus and to attempt to identify some of these organisms.

Next slide, please. We are now on slide 11.

Our first approach, which consumed most of the year, was our attempt to actually culture organisms out of these different mite preparations. We were fortunate enough to be able to obtain fresh mite and egg materials from three different sources. These mites were all washed, free of media, using two different methods. One was sieving, in which the mites were caught on a fine mesh sieve, and the growth medium was washed off with sterile water. Another method we used was with sucrose gradients, and both of these were quite effective, at least visually, at removing all traces of contaminating material from the mites or the eggs. And then, we submitted the material for culture to Pat Murray's lab over at NIH.

Next slide, please.

This slide really summarizes several months' worth of culture work, and the bottom line is, we were able to culture organisms, both gram positive and gram negative, out of both of these sources, and there was no really big difference between the two, either quantitatively or qualitatively.

You'll note that we obtained E. cloacae, R. pickitii and acinetobacter from both species of dust mites. We also have some unidentified gram negative rods, but, basically, our culturing techniques really did not show us any large differences between the two species of mites.

Next slide, please.

Possible problems with this approach include the fact that the culture data were really non-quantitative, and that, in fact, the bioburdens between the two species might be very different. We are actually working on quantifying this now.

The culture techniques, we should note, of course, have been optimized for human pathogens, and it's not likely that what we are dealing with in these dust mites are, in fact, human pathogens, although that's a possibility.

The organisms may be in privileged sites, and the optimal conditions for extraction of the organisms may be uncertain. And, in addition, endosymbiotic organisms are notoriously difficult to culture.

So, we tried a different approach, even while we are continuing with the culturing at the same time.

Next slide, please.

Approach number two has been used by other investigators to identify endosymbiotic organisms, and that's actually to look for characteristic DNA of the bacteria from DAN that's extracted from the mites. In this approach, we extract genomic DNA from fresh washed mite material, and then amplify with 16S ribosomal RNA primers. As you know, 16 S ribosomal RNA is specific for bacteria, and the sequencing has been well identified. In fact, this approach of amplifying 16S ribosomal RNA is a well-established technique to try to track bacteria.

We identified individual clones with restriction fragment analyses. We sequenced and then we attempt ? the idea was to attempt to identify predominant organisms.

Slide 15, next slide, please.

What you can see here is just an acerose gel that shows that we were, in fact, successful at extracting what appeared to be reasonably good quality DNA from both species of mites. This was using standard techniques.

Next slide, please.

These are the 16S ribosomal primers that we used. The sequences were based on an article from 1991 by Weisburg in the Journal of Bacteriology, and these primers have, in fact, been used in multiple experiments by many investigators since that time. It's the upper case bases that are specific for the 16S RNA, the lower case segments of the primers are tails that include, as you can see, specific restriction enzyme sites for easy insertion and characterization.

Next slide, please.

On the next slide you can see that we were able to obtain 1.6 KB TCR fragments of DNA using this amplification method from both D. pteronyssinus and D. farinae. Now, you'll note that we used two different primer pairs. One primer pair, primer pair No. 1, really is relatively non-specific, and would be expected to generate results from most eubacteria, and primer pair No. 2 would allegedly be more specific for enterics.

But, you can see that we were able to obtain fragments from both organisms. Qualitatively, there appears to be somewhat more from D. farinae, but as you know without good internal controls it's impossible to quantify TCR results.

Next slide, please.

We then did a substantial amount of DNA sequencing, and these are the sequences that we recovered. Now, the fact is that from both organisms of dust mites the most predominant recovery is the first organisms ? the first two organisms that are indicated on this slide, Bartonella or Rochalimaea species, specifically, Bartonella henselae and Bartonella quintana. Just to refresh your memories, Bartonella organisms are responsible ? are the pathogens in cat scratch fever and in trench fever, which we don't normally see very much nowadays, although cat scratch fever we certainly see. Bartonella is also associated with infections in immune compromised individuals. There has been a series of reports of it causing endocarditis in immune compromised individuals, such as HIV positive patients and homeless individuals. It's also responsible for bacillary angiomatous in HIV positive patients.

Needless to say, if there were Bartonella in our dust mites they would not survive into the final extract. There's no evidence whatever of live Bartonella in our extracts at all. But, there is evidence, based on this very preliminary study, that Bartonella species may be in this endosymbiotic with some of our dust mite species.

Next slide, please.

Just a quick review, Bartonella endotoxin has, in fact, been fairly well described, both functionally and structurally. It's of interest from a scientific point of view, because it has a high degree of LAL reactivity, but in both human cells and in rat cells it has minimal inflammatory responses, and there's actually minimal activation of ? like receptors 2 and 4, and the two publications very recent are indicated in the bottom of this slide.

Next slide, please.

Obviously, this is a very early finding, and we are not ready yet to publish it in a peer review journal, because we really need to verify the source of the endotoxin. We need to do high-fidelity PCR and verify some of this sequencing data. We need to get some additional mite sources, and we need to attempt to profile the LPS structurally, which we actually can do with colleagues here at CBER.

We are also in the process of putting together internal standards so that we can quantify the DNA in our source materials. Obviously, if there's just as much signal from D. farinae and D. pteronyssinus, this may be of interest, but it certainly does not explain the finding of much more LAL reactive endotoxin in D. farinae extracts.

And, we are going to also need to look physiologically at the effects of this endotoxin on immune responses. These are all studies that are in progress.

That's the end of my presentation. We are going to go on next to Ron Rabin's research presentation, but I'd like to give you an opportunity to ask questions before we do that.

DOCTOR GRANADY: This is Lynelle Granady.

DOCTOR SLATER: Hi, Lynelle.

DOCTOR GRANADY: Hi. This is actually really very, very interesting. I have a quick question about the initial cultures, if you were able to culture the bacteria from the extracts or culture small amounts of bacteria from the extracts?

DOCTOR SLATER: No, no, I'm sorry, let me stop you. You can go on with the question afterwards, but we cultured nothing at all from the extracts. They are sterile. We cultured this from mites, live mites.

DOCTOR GRANADY: Oh, okay, from the live mites.

DOCTOR SLATER: Yes.

DOCTOR GRANADY: Now, is it possible to ? how do you determine which ? I don't know how to exactly ask the question ? but how ? is it possible to actually measure endotoxin from that same ? from the live mites, or not really?

DOCTOR SLATER: Yes, we've actually done that. Well, I mean, measuring the ? you have to sort of mulch up the mites to measure the endotoxins.

DOCTOR GRANADY: Right.

DOCTOR SLATER: You kill them in the process of doing it.

DOCTOR GRANADY: Right.

DOCTOR SLATER: But, we've done that. In our studies that we presented last year, we were able to show that endotoxin was not only present in the extract, but also in the source material, which is the live mites. So, it's present in both.

Are you asking whether it's possible to identify which organism the endotoxin comes from?

DOCTOR GRANADY: Yes.

DOCTOR SLATER: Yes, the answer is yes. We haven't really successfully done that yet, but there are a number of papers in which endotoxins can be profiled, both crudely on SDS-PAGE, and also much more sophisticated using HPLC and gas chromatography. We haven't done that yet, although we had intended to do that, we just sort of got sidetracked a little bit, but that's definitely something that we are interested in.

The problem, Lynelle, is that no one has really ever attempted to do this on a mixed culture, and that would be a little bit hard to sort out. The profiling activities, in general, are on pure cultures or vaccines, and my guess is that ? we are certainly planning on doing this, but my guess is that the data are going to be a little bit messy, just because they are mixed.

DOCTOR GRANADY: I see.

Thank you.

DOCTOR SLATER: Thank you.

ACTING DIRECTOR BURKS: Anybody else have questions for Jay?

All right, go ahead.

DOCTOR RABIN: Hi, this is Ron Rabin, and what I'm going to discuss with you is a project, and show you some data of a manuscript that's pretty much written. We are waiting for one experiment and then, you know, the final touches and getting it out.

If you'll to slide number two then. So, my interest in the multi-drug resistant proteins came from a serendipitous observation that I made while I was still a Fellow in Josh Farber's lab. And, as some of you may know, I have a reasonable amount of experience in flow cytometry, and spent some time in that laboratory looking at calcium flux in response to chemokines, and as a relationship to chemokine receptor expression on lymphocytes.

And, as sort of a side project I began to look at responses in ? in patients with Wegener's granulomatosis with a friend of mine. We were, basically, just playing, and in the process of looking at this I noticed that the patient cells had lost a lot ? or had a lot less of the calcium probe, which is ENDO 1, in their cells than cells from the healthy controls that we were using in the experiment. This was consistent from experiment to experiment, and so I wondered, I thought about that and wondered whether or not because these patients sort of had ? were in a state of inflammation, or activation, in a very general term, whether or not that affects this probe concentration, and the probe concentration then would reflect gene expression of some sort of a probe transporter, which might then modulate activation and give incite into a pharmacologic target. So, that was sort of the fantasy.

And, the cartoon that I'm showing you is drawing what I basically observed. So, when one does calcium studies on a flow cytometer with ENDO 1 you are looking at a ratio, which is essentially the slope of fluorescent's emission into violet on the Y axis or blue on the X axis, and you could see at normal the cells were kind of high up on the X/Y plot there, and the Wegener's granulomatosis patients that slope was down in the corner. But, the slope ? the lines were in different positions, but the slopes were the same, and it's the slope that reflects the calcium. So, it wasn't an issue of calcium, it was an issue of probe concentration, and that's really the bottom line.

And so, if you go to slide number three, that brought me to the MDR family, because the MDR family are proteins that transport substances across cellular membranes, against a concentration gradient, in an energy-dependent manner.

They are in a larger group of what are called ABC proteins, ATP Binding Cassette proteins, that contain distinctive nucleotide binding domains, and their genes are highly conserved across species. The first member that was identified was MDR1, which I'm sure many of you have also heard of identified as P-glycoprotein. And, its substrates are large hydrophobic cations, including such molecules as Doxorubicin and Ritonavir, and even the calcium channel blocker Verapamil.

A little bit later, the MDR-associated Resistant Protein-1 was identified, and now there are nine proteins that carry that prefix of MRP, and it has some homology to MDR, but it's actually more closely related to the Cystic Fibrosis transport protein.

MDR ? MRP1 transports organic anions and it also transports glutathione and glutathione conjugates, and it transports ? in fact, it is the mechanism by which cells export LTC₄, so it does have a physiologic function, and it is the transporter of many of these florescent molecules that we use as probes in cellular biology, because the molecules themselves are organic anions. So, my attention was then turned to MRP1, slide four.

Just to give you a little bit of context of these MDR family proteins, or the MRPs, you can see there's an identity amongst them or between them, the low, and they are widely distributed along the human genome. Many of them do transport conjugated glutathione, and some of them have known physiologic substrates and some of them don't, and there's various new level of distribution of expression, as you can see.

Slide five, please.

So, the first thing that we did was just to look at the basic ? if the basic hypothesis had any truth to it, then we would see two things. We might see a difference in expression of MRP1 in mammary cells versus naive T cells, and we should see a difference with activation. And, this 35 cycle RT-PCR on slide five demonstrates that that, in fact, is the case. So, if you look at the set of ? the area MRP1 there, you'll notice that the band is much brighter on CD4 memory versus naive, CD8 memory versus naive is also a little bit brighter, it seems to be expressed to some degree, and then K cells, B cells, and a little bit in monocytes.

To further drive home the preferential expression, these cells were ? the memory and naive cells were sorted by flow cytometry, using two markers in addition to CD4 and CD8, but we know for a fact that that isn't ? you don't get pure naive cells, and really one way to get absolutely pure naive cells is to use cord CD4 T cells. And there on the far right lanes you can see that there's no expression of MRP1 in cord CD4 T cells, and that after three days with anti-CD3 and anti-CD28 there is expressions regulated.

Now, I'm showing you a number of the members of the family for two reasons, one to call attention that this isn't just some sort of general thing, non-specific event, because MRP3, for example, is not expressed in any context, and MRP54 doesn't really appear to be affected by memory subsets in any event, but then there are some molecules that seem to have this same level or the same pattern of expression, such as MRP4 and MDR1.

Next slide, please.

So, we started ? we set about using a small molecule inhibitor of MRP1, that is well known in the literature, called MK-571. MK-571, as many of you might know, is sort of the granddaddy of singular or monteleukast, and so it is a leukotron, it is a ? LTR1 antagonist at very low concentrations, but I'm sure related to the fact that MRP1 is the exporter of LTC4 at higher concentrations MK-571 does block this pump. And so, we first just looked at ? we used the molecule TSST, the superantigen, to stimulate our T cells for, I think, all of the studies that I'm showing you here, because in certain times we thought we might want to look at those responding cells, and since TSST-1 is fairly specific for V beta 2, we can follow the responding cells if we wish.

And so, on the left these cells have been stimulated with the superantigen overnight, and you can see that they look like cells, they look as cells generally look when they've been stimulated overnight with some sort of superantigen, they tend to cluster and become aggregates. And, on your right, with the MK-571, they are just in a homogenous suspension.

They are mostly alive by the way.

Next slide.

So, slide number seven then shows some ? a little bit more specific characterizations of the inhibition here, and so these cells now were stimulated and we looked at them four hours after stimulation with TSST-1 in the presence of absence of this inhibitor MK-571, and on the far left you can see on the X axis that there's a subset of cells that express CD69 after activation and then a subset of those are staining positive for interferon gamma. In the presence of 50 micromolar, we've cut that down ? the MK-571, we've lost a lot of expression of the interferon gamma and CD69 both, and it's practically all ablated at 125 micromolar.

Next experiment.

To look at the cytokine profiles a little bit more closely, we did experiments such as demonstrated on slide number eight here, which shows interferon gamma on the X axis and IL-4 on the Y axis, and you'll see a very clear dose-dependent decrease in interferon gamma, as the dose of MK-571 goes up. What you don't see is that IL-4, or what you also see, I guess, is that IL-4 is similarly ? is not affected, okay, and that has struck us, you know, as very odd.

Now, we've tried to make sure that this is, in fact, the case, because, as you know, there's a lot of ? there's a lot more ? you know, the numbers are better for gamma than 4, and so we wanted to be sure we weren't playing with a number game, but, in fact, this does look like the case. It does look like at least in the context of these experiments MK-571 inhibiting MRP1 doesn't seem to affect IL-4. And, at the time I was a bit baffled by that, and I have a number of thoughts about that that I can share with you towards the end.

Next slide.

The specificity ? but, at any rate, to look at the gamma at least, this is ? we did a number of experiments now, so these are experiments similar to and including the one that I showed you on the previous slide, showing the dose response, and on the upper half of the panel there is the cumulative normalized response to interferon gamma of all these, and you can see that this is a statistically significant, dose-dependent decrease, and that this dose-dependent decrease in cellular expression of interferon gamma protein is at least ? is reflected by a decrease in gene expression, as shown by the TaqMan assay below.

Next slide, please.

We looked at a bunch of other cytokines using the BD biosciences cytokine bead assay, and so this is now secreted into the supernatant, and the interferon gamma, as you can see, is no surprise, it's reflected by supernatant concentration, as is TNF and IL-10, and to some degree IL-2. What's kind of interesting about IL-2, and it may give us some incite into the mechanism that this is working, is that you can see that the curves are a bit different, and it appears that IL-2 is not as easily affected by the MRP1 inhibition. But, it is affected, nonetheless.

And, this last point at 125 micromolar was excluded from the regression line, because a queue test demonstrated that it was clearly different than the previous five points.

Next slide, please.

Well, we wanted ? I was curious about whether or not this inhibition was cell specific, was T cell specific or not, and so one of the things that I did was, we looked at monocyte-derived macrophages, and we stimulate