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From: TSS ()
Subject: FDA DHHS TSE ADVISORY COMMITTEE September 18 and 19 2006 ''There is a growing number of human CJD cases, Gambetti''
Date: November 4, 2006 at 7:41 pm PST

Meeting of:

TRANSMISSIBLE

SPONGIFORM ENCEPHALOPATHIES

ADVISORY COMMITTEE

September 18, 2006

Holiday Inn Gaithersburg

Gaithersburg,Maryland

TABLE OF CONTENTS

Page

Administrative Remarks - Executive Secretary 1

Opening Remarks, Glenn Telling 8

Committee Updates:

- US and Worldwide BSE 8

- vCJD epidemiology and transfusion transmission 26

- Draft Guidance for Industry: Amendment (donor 32

deferral for transfusion in France Since 1980)

- Critical Factors Influencing Prion 39

Decontamination Using Sodium Hydroxide

- Human Prions: Clearance and Plasma Lipoproteins 53

TOPIC I: Experimental Clearance of Transmissible

Spongiform Encephalopathy Infectivity on

Plasma Derived FVIII Products 69

TSE Clearance studies for pdFVIII: Study Methods and 70

Clearance Levels

Industry TSE Clearance Studies for pdFVIII 95

Open Public Hearing 116

- Statement by Dave Cavenaugh 118

Open Committee Discussion, Questions for the Committee 122

Committee Updates:

Status of FDA's Initiative on Communication of the 177

Potential Exposure to vCJD Risk

Summary of WHO Consultation on Distribution of 184

Infectivity in Tissues

Open Public Hearing 201

- Statement by Charles Sims 203

- Statement by Paul Brown 209

snip...

To continue on the same theme, I am showing you an update or new information that has recently come out concerning the study by Hilton et al that was published in 2004.

This was a United Kingdom tissue survey where anonymized tonsil and appendix samples were taken from subjects that had undergone surgery between 1996 and 1999 in the United Kingdom.

The samples that were studied were from patients aged 20 to 29. Very interesting and important, three out of the 12,674 samples that were deemed adequate for study were positive, suggesting one in 4,225 people in this age group might actually be infected with variant CJD. All of the positive samples did come from appendices.

Now, what is new about this is that prion protein genotyping was done on two of these samples. In this first sample, there was not enough to do genotype testing, but the tissue was taken and used in a transmission study into mice, and those results are pending. We don't know what has happened to those mice just yet.

In the second subject sampled, the genotype was found to be valine homozygous. So, this was the first report of an infection in a valine homozygous person. The

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second was also a valine homozygous individual.

So, to summarize, variant CJD clinical cases are declining in the United Kingdom. We have had three transfusion transmission infections reported in the United Kingdom, one fairly recently.

I would just like to point out that, out of the 18 identified living recipients of blood from people who came down with variant CJD, recipients that have survived at least five years post-transfusion, now three out of 18 of these people have developed vCJD infection. Two of those are clinical and, as I showed you, one of them was preclinical or subclinical at the time of death.

This implies a fairly efficient transmission by blood. This amounts to about 17 percent. Also, we now know that all three prion protein genotypes at codon 129, the MM, the MV and the VV, are susceptible to infection.

What we don't know is whether people with this genotype ever develop clinical illness. This brings up the continued possibility that there are silent and asymptomatic infections that may never become symptomatic, but may pose a risk of iatrogenic transmission to others.

In particular, we are concerned with blood and plasma, but there are other iatrogenic transmissions to be considered as well. That is all for my update. Thank you all for your attention.

snip...

There is a large percentage of the sheep scrapie which carries selectively the SPRPSC forms, and wouldn't be detected without the availability to detect this PRPSC. We

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saw it in Norway cases and more and more cases in Europe.

There is a growing number of human CJD cases, and they were presented last week in San Francisco by Luigi Gambatti(?) from his CJD surveillance collection.

He estimates that it may be up to 14 or 15 persons which display selectively SPRPSC and practically no detected RPRPSC proteins.

So, this is becoming very important for two reasons. First, practical detection and identification of the prion disease. Second, in a theoretical sense, how is it related to the disease and how important is it in pathogenesis.

snip...

So, did we look into sporadic CJD cases. I think that the first step before that, we actually realized that first we have to validate our assay.

We have to show that we have a -- that we can truly detect PRPSC protein and, second, that we can truly measure quantitatively PRPSC protein, and correlate it with the prion infected.

So, in this study, which was actually initiated with Glenn Telling, and whose transgenics he generated in San Francisco, we inoculated three different cases of sporadic CJD in the end point titration experiment in different transgenics to determine end point titers.

At the same time we made homogenase from the brain and tested by CDI, the dilution curve, in parallel. When you see the correlation, there is a very clear overlap.

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It shows one important difference. The 50 percent transmission rate indicating one infectious unit per ml, at that level, CDI has a reading skill of about 20,000, which is about 1,000-fold over the capability of the CDI assay. So, in effect, the CDI is more sensitive than the bioassay in transgenics.

So, one more question was correlating the established procedures of immunohistochemistry and pathology with the infectivity and with the CDI.

So, we blindly tested PRP C protein in those different forms of prion diseases and in 18 different anatomical areas in eight sporadic CJD cases.

We could detect the RPRPSC protein everywhere. In contrast, the immunohistochemistry and localization profiles in many areas the sensitivity of both was not exceeding 20 percent, or was even lower than that.

So, one conclusion. First, the testing has to be in a diagnostic aspect. It has to be in different anatomical areas.

Second, the CJCDI shows absolute diagnostic sensitivity and specificity in all of those anatomical areas.

The second important finding was related to the SPRPSE. When we looked at the concentrations of RPRPSC versus SPRPSE, in all frontal and white matter areas we

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tested, there was more SPRPSE protein over the RPRPSC protein. The RPRPSC protein actually formed about five to 10 percent of the total.

The next presentation is going to be humoral. This is just the first data showing the CDI on the VLDL fraction and plasma of the CJD cases.

When we tested total PRP concentrations in 21 donors and 20 sporadic CJD cases, we didn't find any difference in total PRP or in the SPRP and the PRPSC protein. It is below the threshold.

When we separated VLDL, there was a significant difference in the sense of more total PRP protein in the VLDL from sporadic CJD cases, and most of the total PRP increase was RPRPSC protein, actually.

So, what are the conclusions? I think the apoB containing lipoproteins are strong candidates for carriers of sporadic CJD prions in human plasma, and I will talk about it as the diagnostic implications emerge.

snip...

DR. TELLING: Are there any questions for clarification for Dr. Scott at this time?

MR. BIAS: Dr. Scott, is there a reason that there haven't been any experiments done using human blood of vCJD victims?

DR. SCOTT: That is a very good question. I think that if this was easily available, they definitely would have been done.

I think by the time the patients come to their clinical disease, the ability and the ethical constraints on collecting a lot of blood or plasma from them has been limited.

In the United Kingdom, they are particularly careful to assure that patients have a choice and that their families have a choice.

That is what has caused the limitation. It is not obviously the patient's fault. There aren't very many patients to begin with, but there aren't very many people with this disease at any given time that are in a situation where they might be able to give a large amount of blood or plasma.

i know Dr. Minor is in the audience and I wouldn't want to just call on anybody in the audience at random, but either Dr. Asher or Dr. Minor might have more insight on the availability or the potential availability

89

of variant CJD blood. I guess I will let Dr. Asher, because he is on duty here.

DR. ASHER: I hope that we will hear some thoughts on the issue tomorrow from Dr. Minor who is here, and Dr. Turner who we expect to arrive this evening.

The problem has been this. With sporadic and familial CJD, infectivity has not convincingly been demonstrated in the blood.

So, if you collected it, either by epidemiological look back studies -- and the American Red Cross' study is really now quite extensive -- and a very limited number of studies done at the NIH transfusing whole blood into chimpanzees, none of whom ever became ill with Creutzfeldt Jakob disease.

So, the blood from the forms of Creutzfeldt Jakob disease generally available in the United States, the hypothesis that there is enough infectivity present to be detected at all with any of these assays has not been demonstrated.

With variant Creutzfeldt Jakob disease, as Dot pointed out, the number of patients available has been very small.

In the two cases in the United States, I believe that Dr. Gambetti has a small amount of blood, and I know the Canadian case there is a small amount of plasma

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available, but nothing approaching what would be needed for the kinds of studies that we have been talking about.

I am afraid at the moment we are stuck with blood from endogenous infectivity. We are stuck with blood from animal sources.

DR. GESCHWIND: So, at UCSF we have actually shown that it is pretty feasible to get large volumes of blood from patients with CJD.

We have -- Jiri Safar probably can give you the fact numbers, but probably we have over 50 patients in whom we have gotten 200 to 400 mls of blood.

So, bring in patients from around the country and at certain points when we have funding we have been sending out a nurse to get 200 mls of blood from patients with CJD, and we have been collecting it every two to three months from patients during the course of their disease, depending upon -- we do very strict safety tests that are more conservative than for the Red Cross blood donations, prior to doing this.

So, it is feasible, particularly in patients whom we have diagnosed earlier in the disease course, and in patients who have a slower course.

DR. SCOTT: I think that Dr. Minor also has a comment maybe about the variant CJD cases.

DR. MINOR: Well, I am very jealous of the

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comment that has just been made. I have discussed this extensively with the people at the CJD surveillance unit in Edinburgh, and they won't touch it.

They basically say that the ethical concerns are such that they will not take a unit from people who have variant CJD, no matter who wants it.

I will be talking a little bit about human samples tomorrow in the diagnostic presentation, and the availability of human samples is absolutely tiny, relevant human samples, like within the United Kingdom, is absolutely zero.

There has also been a recent introduction of a thing called the human tissues act, which means that if you don't do it right, you get sent to prison. That has actually been a major inhibitory effect on actually trying to get these kinds of samples.

I am actually very impressed by the fact that you can get those kinds of volumes around. If we could get those kinds of volumes, I think I would put them into diagnostics rather than into plasma fractionation, frankly.

snip...

With the help of NIH, with Michael Nunn and many other people who cooperated, I think that all of those logistical issues -- and that is an answer to Phil Minor more than anybody else -- it can be overcome.

It took time and it was really difficult, but I think that it is feasible to collect a significant amount, two ml, 200 ml, at a session from CJD patients, either variant CJD or sporadic CJD.

So, I think that this is one of the issues which should be discussed tomorrow in more detail, how to organize such a collection and how such a repository should be handled, funded and organized.

DR. TELLING: With the obvious caveat that sporadic and variant CJD may differ radically in their biological properties with respect to infectivity in blood.

DR. SAFAR: Absolutely, yes.

DR. MANUELIDIS: I would like to make one comment about sort of the definitive comment that David made about sporadic CJD.

It was shown in guinea pigs in 1978 that the blood is infectious and the spleen is infectious. It only

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makes sense, really, that it would go to spleen if the sporadic CJD, the agent itself, went through blood.

The second thing is that there were two studies that were published in Lancet, one by our group and one by Tateishi's group, showing that human blood actually transmitted as well.

Now, the Japanese group might have a slightly different variant of their CJD because of the different geographic region.

I think that probably the amount of infectivity is much lower than it is in vCJD, but it is likely to be there, from everything we know about these infections and the fact that spleen is infectious.

DR. ASHER: We agree that in blood of patients with sporadic CJD, infectivity is likely to be there, but the amount would certainly have to be smaller.

snip...

The study was terminated, I believe, five years after these transfusions. It means that during five years these animals got multiple transfusions of blood from squirrel monkeys which were inoculated with variant CJD or sporadic CJD. None of the animals developed the disease, but their organs, I believe are still under evaluation by the CJD surveillance unit. The other part of the study was that blood from chimpanzees, which were infected with sporadic CJD, was

153

taken and separated into components, and these components were inoculated into squirrel monkeys. There was one transfusion from such a chimpanzee which developed sporadic CJD. There was another part, when blood from sporadic CJD patients and from variant CJD patients was separated into components and inoculated into squirrel monkeys. I believe there were also no transmissions.

snip...

So, those are the reasons why the World Health Organization remains concerned, hosts these consultations. It is gratifying that, except for blood, no other new class of medical products has been recognized as transmitting CJD in the past 10 years, but here have been now over 360 recognized iatrogenic transmissions.

One of the things that was attempted at the consultation was to simplify, for those of us who are more public health oriented, the nomenclature regarding abnormal forms of the prion protein.

Jiri Safar discussed this this morning, the various forms of abnormal prion protein protease resistant, protease sensitive, degradation products of different lengths.

We didn't get into the five or maybe six subclasses of prion proteins in the Cohen classification scheme or the four, maybe it is five now, in the Parker Gambetti scheme. It became so difficult just to talk to each other that it was proposed that, for our purposes, the term PRPTSE be used for all the abnormal forms of prion proteins, regardless of their molecular nature, and let the

191

specialists, in the end of days, figure out what those forms were.

So, for our purposes, we used that instead of PRPSC or PRPRES, although UK authorities, before the end of the meeting, had started referring to PRPD for disease related PRP. So, go figure. At least for the rest of this talk, we will use PRPTSE .

I went through the proceedings and I selected some points that I thought were of particular interest to this group today, and I list them on the next three or four slides:

Naturally affected cattle, infectivity detected by mouse assay demonstrated only in brain, spinal cord, retina and a pool of nictitating membranes, but not in pools of lymph nodes.

Infectivity was detected in some peripheral nerves, and a solitary muscle of a single case of BSE in a German cow. That is a greater concern in Europe where there is more BSE than the maximum of one case per million, we hope, that USDA estimates here. There was only infectivity found in a semi-tendonoisis muscle using only an extremely sensitive transgenic mouse that Martin Groship(?) has developed.

Still, it would be disturbing if meat itself was intrinsically infected rather than neural tissue, because

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contamination has been the thing that is of greatest concern for the safety of the food supply.

Cattle experimentally exposed to oral route BSE infectivity detected in distal ilium throughout the course of disease after six months in the palatine tonsil, and only in cattle assay, demonstrating that the mouse assay, except for some transgenic mice, is considerably less sensitive, and this is the part that got amputated from your handout, BSE experimentally transmitted via the oral route to sheep and goats and one, possibly two, goats now recognized in Europe to have been infected under field conditions.

No sheep has been recognized, but they both got fed similar, presumably some similar feeds having presumably some level of contamination.

Under specific experimental conditions, brains of some TSE infected rodents may be infectious by bioassay, while TSE remains undetected. That problem has been mentioned here today.

Immunoassays detected PRPTSE in brain of BSE in cattle at least thee months before onset of illness. No immunological method yet validated as sufficiently sensitive to detect PRPTSE in the blood of infected animals or humans, although promising initial results were reported by several groups of investigators and we will be hearing

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tomorrow reports by all six of those groups who reported last year, plus a report by Dr. safar.

Transfusion experiments have not been conducted in cattle, although studies using small amounts of blood and spleen of cattle assayed in mice and cattle, in operating cerebrally, failed to detect infectivity.

The regulatory conclusion to that, a conservative regulatory approach would assume that bovine serum might potentially contain TSE infectivity, presumably, in small amounts.

Ruminant blood, blood derivatives such as fetal calf serum in cell culture, and bovine serum albumen have not been identified as a source of infection, but should be properly collected to reduce the risk.

However, blood of sheep with both experimental BSE and natural scrapie can be infectious and, because scrapie and BSE agents behave similarly in sheep and goals, the blood of small ruminants should either be avoided in preparing biologicals, or selected very carefully.

I think because sheep and goat blood are used as a source of immune sera, some quite useful sera, the second criteria is more feasible, at least on a worldwide basis.

There is a continuing need to ensure that all regulatory authorities with limited resources have ready access to reliable and up to date information when

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assessing TSE risks and evaluating medicine product safety. Those were one of the main reasons for the consultation.

full text 223 pages ;

http://www.fda.gov/ohrms/dockets/ac/06/transcripts/1006-4240t1.htm

http://www.fda.gov/ohrms/dockets/ac/06/transcripts/2006-4240t1.pdf

Meeting of:

TRANSMISSIBLE

SPONGIFORM ENCEPHALOPATHIES

ADVISORY COMMITTEE

September 19, 2006

Holiday Inn Gaithersburg

Gaithersburg, Maryland

TABLE OF CONTENTS

Page

Opening Remarks 1

Topic II: Possible Criteria for Approval of

a Donor Screening Test for vCJD

Donor Screening Test Issues: Sensitivity, Specificity 4

and Confirmatory Testing

Algorithm for Approval of Human TSE Tests in Europe 13

Available Reference Materials 35

Research Updates from Test Developers: 69

- Prionics - A. Raeber 70

- University of Texas - C. Soto 78

- Microsens Biotechnologies - S. Wilson 88

- BioMerieux - Dr. Van Driesische 94

- Adlyfe - K. Lohman 98

- Chiron - D. Peretz 109

- UC San Francisco - J. Safar 117

Questions for Presenters from Committee 128

Open Public Hearing 144

- Arllene Carr-Greer 145

Open Committee Discussion 148

snip...

DR. SOTO: The other point is, you kind of hinted in your last slide that the ideal test should give three positives in 10,000 cases of the United Kingdom and none in the United States. This is based on what?

DR. MINOR: I tried to hint at that by saying it may give -- it is based on the appendix studies that Marc

59

referred to.

It may be that if you screen through 10,000 blood donors you will find three people who are incubating variant CJD. If you do it in the United States, you shouldn't find any.

DR. SOTO: That is what I thought, that it was based on the appendix. You have to realize that the tests that people are developing today are much more sensitive than the system used to detect in appendix. So, you could expect much higher numbers.

DR. EPSTEIN: Thank you, and thank you very much, Dr. Minor. I ask this question with trepidation. What is the thinking in the United Kingdom regarding obtaining post mortem blood samples from people who have expired with vCJD.

One would think that the ethical questions are at least different and that that might be a potential source of human infectious material.

DR. MINOR: The answer to your question is, I don't know. I am, thank God, a PhD, not an MD. So, I know nothing about ethics at all.

I have discussed this with the guys up at the CJD surveillance unit about getting blood at all. They seem to be quite reluctant to do this.

I mean, I am not quite sure why. I guess you

60

could say that they would have to get permission from the next of kin, and the next of kin would be suffering enough at the time and they don't want to add to the suffering, I suppose. I am really not quite sure what the ethical position actually is. I am really not sure.

snip...

DR.TURNER: I am sorry, but just to try and clarify for colleagues the current ethics position in the United Kingdom, and that is that we are taking some samples from patients with clinical variant CJD.

They are 50 ml blood samples which are then separated according to a standardized protocol. That clearly has gone through a research ethics committee process in the United Kingdom. We have been doing that for some time.

I agree with the comments of colleagues around the table that it may well be that we can move on from that now and take sequential samples from such individuals. I think that would be extremely helpful.

Whether the ethics committee would tolerate it or whether, indeed, it is clinically acceptable to think about taking whole blood units from such individuals, I think, is debatable. We would have to have a think about that.

With regard to the 5,000 or the 10,000 blood donor samples, that also clearly has gone through an ethics approval process.

The reason for us blinding and unlinking those is that we think we probably will come up with positives and that would put us in a terribly difficult moral position, of knowing whether we should go back to those individuals,

69

whether we should defer them as blood donors at what stage.

We come across such problems with HTLV assays, for example. We don't want to particularly go there at this stage. So, that is really the reason for keeping them unlinked.

It means that we would not be able to go back to that individual. Nevertheless, there is a whole blood unit available, albeit that it is aliquoted.

For example, we could go back to those individuals, extract DNA from their leukocytes and look at their codon 129 genotype, for example. Thank you.

snip...

DR. MANUELIDIS: Just two very fast questions. One is, did you do any of the samples blind? The second question is, you showed us a slide, and I didn't catch it all -- maybe you could explain it -- you had some other species like mouse.

138

I was wondering whether these were as sensitive, as robust, let's say, as the hamster 263K, and maybe you could say a little bit more about blood of different species, or the 263K versus some of the other samples.

DR. SOTO: We have defined a process of going very much from the scientific concept to later concentrating more on the development.

So, focusing on the scientific aspect, we have been more -- most of the experiments I showed you were not blind.

We are now working with blind samples provided by others, but for us the most important part is first developing, being confident with the technology, and then we will do all the blind studies.

The efficiency of the test depends on how much experience we have with them or how much organization we have done.

So far I can say that the very best that we have is the hamster 263K, but we have also several other strains of hamster, and we have also amplified mouse.

We have a very, very high efficiency compared to the hamster 263K. We have been able to amplify at least six or seven different strains of mice, also CWD, showing similar levels of efficiency.

The others are a little lower, like sheep or

139

cattle or human, but it is only because we have really spent much less time on those.

I think it is just a matter of organization. You have to do some of the experimental conditions of strength of sonication, incubation time, et cetera, to reach the maximum efficiency.

DR. CERVENAKOVA: I do have a question, actually, and a comment. My question is, when you showed your proof of presence of PRPres in buffy coat samples, did you try to inoculate these particular samples into the animals to see what the infectivity level was, if there is a correlation between those two.

Your data actually are in some dissonance with data from multiple broader studies, and our studies in mice, when the infectivity rises during the incubation period toward clinical stage.

My second, I would answer the question that you didn't answer. It was the third question about the levels of infectivity not PRP in blood.

If you take per ml of component -- I am talking about buffy coat and plasma -- it is derived that there is approximately 10 times less of infectivity in plasma, per ml of plasma, than in buffy coat. If you take the volume of plasma, it is more infectivity in plasma than in buffy coat.

140

DR. SOTO: Right, yes, that is the third question. I knew I was forgetting one question. The infectivity studies from the work of Larisa and Bob Rohrer, from PRPSC, I can tell you that we have the same thing, that we have much more, at least 10 times more, infectivity per unit of volume in buffy coat than in plasma.

Larisa's question, no, we did not inject the samples. I was not expecting to get this result. I was expecting to get what you get when you look at infectivity. Now, I regret not to have taken samples and infected it into animals as well.

snip...

DR. MANUELIDIS: Two comments. First, I go back to this one. In terms of -- you know, we used to do a great number of biopsies as well as autopsy material.

In terms of immune histochemistry, resistant PRP, I don't think that anybody could tell a difference between all the cases that we got. They were instantaneously frozen, that we did PRPres in the brain with PK versus autopsies that were as long as 20, 40 days later.

Similarly, the experiments that I have done with some of those frozen brains that were biopsied versus autopsied, there is no difference, at least in sporadic CJD, because that is what we look at in humans, and the amount of PRPres in the human brain in the biopsy versus

158

the autopsy material.

There may be a difference, but I still think that one of the great valuable resources that we have, and I would like to encourage people to do it is, patients and patients' families are very, very nice about donating blood, about getting a lot of resource material.

The problem has been the lack of autopsies, the lack of facilities at the site in the states to be able to help facilitate this.

People call me up all the time from all sorts of states, and I say, try to contact somebody in that state, or I give them something like the prion surveillance.

Obviously, if it is Connecticut, it is not a problem for me, but then it becomes a problem of who is going to pay for this, who is going to pay for the transportation.

I have gone and drawn blood myself from patients who are terminal, but by the time they get around to getting me there, it is usually impossible.

I think the patients and the patients' families really want to help and I think we should help them facilitate that, because these are valuable samples.

Then an autopsy will come through and say, brain only. I have to take out the brain because everybody is so terrified about it.

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I say, why can't I get the spleen or why can't I get blood. It is simply because the families don't know that these are important samples and the autopsy people don't want to do a complete autopsy.

There is a whole series of events that play into us not getting stuff, aside from the fact that the autopsy rate is extremely low compared to when I was a medical student.

snip...full text 184 pages......TSS

http://www.fda.gov/ohrms/dockets/ac/06/transcripts/2006-4240t2.pdf

http://www.fda.gov/ohrms/dockets/ac/06/transcripts/2006-4240t2.htm

TSS




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