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Alzheimer Immunotherapy Trial Grounded: Time to Reassess Safety and Vaccine Design

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Introduction

Giulio Maria Pasinetti led this live discussion on 5 March 2002. Readers are invited to submit additional comments by using our Comments form at the bottom of the page.
 

 

Transcript:
Live discussion held Tuesday, 5 March 2002.

Participants: Ruth Itzhaki, Einar Sigurdsson, David Morgan, Ken Ugen, Craig Atwood, Flavio Kamenetz, Jon Oscherwitz, Keith Crutcher, MaryAnn, Richard Bowen, Gabrielle Strobel, Juan Troncoso, Alexei Koudinov, Kiminobu Sugaya, Yasuji Matsuoka, John Travis, Glenda Bishop, Steve Snyder, Orest Hurko, Matthew Hejna, Giulio Pasinetti, Ed Spooner, Jodi Leverone, Jimin Zhang, Klee.

Note: The transcript has been edited for clarity and accuracy.

MaryAnn: My mother is only 62, and in the last stage of Alzheimer's. I want to find out more about early onset Alzheimer's. I'm 31 and already pretty forgetful and I get confused easily. I feel like I'm getting some sort of dementia. I'm looking for someone who does early onset Alzheimer's gene testing in my area. I live in Dayton Ohio. Can anyone help me?

Richard Bowen: Mary Ann: The ApoE genotype test is commercially available, but this is for late-onset. PS-1, PS-2, and AβPP are the primary ones for early onset.

Gabrielle: MaryAnn, unless you belong to a family with an inherited AD risk, which I hope not, there is no reliable gene test predicting your risk. We will be discussing Alzheimer vaccination at the top of the hour but feel free to contact me off-line after the chat to point you towards possible sites.

Richard Bowen: With so many folks looking into the genetics of AD it should be fairly easy. You can contact me at rbowenmd@voyagerpc.com and I can see if I can help.

MaryAnn: I want to learn more about the vaccine. I feel that I may need it someday.

Kiminobu Sugaya: MaryAnn, I am not sure if the vaccine is good or not. Hi! Gabrielle, thank you for the nice editing of the previous chat. May I introduce myself, I am Kiminobu Sugaya at the University of Illinois in Chicago. We have a cold day with snow here.

Gabrielle: Hello everyone and welcome. It is 12:00, so let's begin. I am Gabrielle Strobel, managing editor of the Alzheimer Research Forum, for those of you who do not know me yet. I suggest that perhaps everyone first state their full name so we know who's here. Meanwhile we are trying to figure out a technical problem we have with not enough people being able to log on.

Glenda Bishop: Craig Atwood can't log on.

Richard Bowen: Craig probably didn't have any opinions on amyloid anyway…

Gabrielle: Very funny Richard. Is there someone without an opinion on amyloid?

Dave Morgan: I am Dave Morgan in Tampa. Cool but sunny here.

Orest Hurko: My name is Orest Hurko. I am a neurologist in England.

Juan Troncoso: Hi Orest, you are missed here at Hopkins. I am Juan Troncoso from Neuropath at Johns Hopkins.

Yasuji Matsuoka: Hi, all. I'm Yasuji Matsuoka, Nathan Kline Inst/NYU.

Keith Crutcher: Keith Crutcher, logging in from cool sunny Cincinnati.

Ken Ugen: I am Ken Ugen from University of South Florida in Tampa

Steve Snyder: I am Steve Snyder in Bethesda

Glenda Bishop: My name is Glenda Bishop, I am a postdoc in Cleveland Ohio

Flavio Kamenetz: Flavio Kamenetz from Cold Spring Harbor, NY

John Travis: I'm John Travis of Science News in unexpectedly chilly Wash DC

Matthew Hejna: Matthew Hejna, Loyola Univ. Chicago

Einar Sigurdsson: I am Einar Sigurdsson from NYU in Manhattan.

Ed Spooner/Jodi Leverone: Ed Spooner and Jodi Leverone from the Center for Neurologic Diseases in Boston.

Ruth Itzhaki: Ruth Itzhaki is here (with Curtis Dobson and Matthew Wozniak). Manchester, UK

Jon Oscherwitz: I am Jon Oscherwitz from the University of Michigan

Jimin Zhang: Jimin Zhang, a scientist at Genetics Institute, just interested in this vaccine issue.

Richard Bowen: I am VP of scientific and medical affairs of Voyager Pharmaceutical

Glenda Bishop: I would like to give apologies for Steve Robinson from Monash University in Melbourne Australia. The time difference for this talk is a little incompatible for him, but he did submit a comment to the Forum.

Richard Bowen: I wanted to ask Dave Morgan why he thinks these problems were not seen in animals.

Dave Morgan: Richard, Many possibilities. We obviously were looking for it. My first guess is the mice were not old enough. My second is that the mouse immune system is not the same. My big concern is we still have no good idea what the problem in the clinical trial is.

Richard Bowen: Could it be that they don't have plaques?

Orest Hurko: The mice clearly had plaques but no good evidence of tangles.

Dave Morgan: Both mice and early stage humans have plaques (biopsy studies).

Richard Bowen: I would think that the problem is that you are making antibodies to a protein that has high concentrations in CNS. It would be understandable why this would cause encephalitis.

Glenda Bishop: One reason why problems may not have occurred in the mice is that they still have their own amyloid intact as well as having the mutant human AβPP.

Ruth Itzhaki: To Richard and to Gabrielle - We have been told that herpes simplex virus type 1 has been found in "some" CSFs of the patients with inflammation. It could be responsible for the damage. Mice are not natural hosts for this virus.

Richard Bowen: Ruth - good point. HSV can frequently be activated when the immune system is challenged.

Kiminobu Sugaya: General activation of the immune system may activate glia and produce oxidative stress to cause damage to the neurons.

Juan Troncoso: We do not know what the vaccine formulation was; whether patients develop antibodies; if so, what levels and what type of antibodies, etc.

Richard Bowen: It would be nice to know whether there was a correlation between Aβ titer and encephalitis. (Editor: See ARF news story, scroll down to Schenk, for comment regarding antibodies in patients with encephalitis.)

Gabrielle: Ruth: Assuming study subjects had a latent infection, like many of us, and that the vaccine somehow activated it, how can we find out? And what does that mean for any future vaccine trials? Antiviral drugs don't work so well against HSV in CNS, I believe.

Ruth Itzhaki: : HSV1 DNA could be sought in CSF by PCR, and antibodies to HSV1 proteins in CSF by immunological methods; N.B., HSV1 antibodies are detectable in CSF for very much longer periods than is viral DNA. Future vaccines could be used with antiviral agents, such as acyclovir, and these would stop virus replication. Acyclovir works very well against HSV encephalitis.

Juan Troncoso: Does anyone know how demented the immunized subjects were? Early AD, advanced?

Jon Oscherwitz: They were classified as mild to moderate AD in trial.

Kiminobu Sugaya: On top of that, AβPP may have physiological functions, which may be compromised by the autoimmune problem.

Alexei Koudinov: Good point on physiological role of AβPP.

Dave Morgan: Given that Elan has indicated they will not inject anyone else with this vaccine, I assume the problem is significant. My guess, however, is that passive immunization approaches might still be attempted. (See, for example, ARF news story on one-time antibody injection.)

Yasuji Matsuoka: Do they use adjuvant for vaccination?

Dave Morgan: Yes there was an adjuvant used, but I am not sure what was in it. An article in Nature Medicine indicates the acronym for it, but I was not familiar with it.

Jimin Zhang: Is it possible that the adjuvant caused the inflammation in the trial, not Aβ?

Giulio Pasinetti: {enters}

Dave Morgan: Hi Big Giulio.

Giulio Pasinetti: Hi Dave.

Gabrielle: Welcome, Giulio.

Orest Hurko: It is difficult to do anything but conjecture, since there is very little information available. But am I just not looking in the right places?

Gabrielle: Orest, nothing published in the scientific press that I know of. Elan/AHP is still conducting their own investigation.

Glenda Bishop: Why would you do passive immunization? You are removing the amyloid anyway.

Jon Oscherwitz: Passive immunization would certainly give more control.

Dave Morgan: The advantage of passive is if adverse events occur, they will abate over weeks as the antibodies clear. You cannot unvaccinate someone.

Giulio Pasinetti: Dave, excellent point, this is one of our greatest concerns.

Einar Sigurdsson: Passive immunization may be appropriate for acute disorders but is not very feasible for chronic diseases like AD. Repeated antibody injections may lead to serum sickness.

Alexei Koudinov: I am wondering how many immunologists we have in this auditorium?

Einar Sigurdsson: We need to keep in mind potential toxicity of Ab1-42. There was no reason to use the whole peptide in clinical trials.

Jon Oscherwitz: Why did they use whole peptide? Do we really know the antibody specificities associated with in vivo efficacy?

Glenda Bishop: Passive immunization will remove amyloid anyway, isn't that the problem?

Kiminobu Sugaya: Taking out Aβ is good, but do not forget about the physiological function of AβPP and Aβ.

John Travis: 15 out of 350 people is not stunningly rare. Why wasn't this seen in phase 1--the repeated dosing or a broader population sample that included a susceptible genotype?

Giulio Pasinetti: John, it may be that manipulation of amyloid load or the antibody target (as in the disease) is required to elicit an inflammatory response.

Yasuji Matsuoka: Do they see an elevation of plasma Aβ level after vaccination?

Giulio Pasinetti: Everybody: the reason we are here is to try to put in perspective the evidence that the clinical trials from Elan have been interrupted. What do you think we should do next?

Ruth Itzhaki: Guilio - Get Elan or AHP to seek HSV1 DNA by PCR or antibodies to HSV1 by immunological methods in CSF - or to reveal their results. They have said (in a personal communication) that HSV1 has been detected in "some" of the first four. Our source seems not contactable anymore.

Glenda Bishop: We need to determine why the vaccinations did not work to decide where to go next. Why were the side effects caused, etc?

Juan Troncoso: First of all, we should not abandon the immunotherapy approach because of an initial failure. We need to do more work in animal models, rigorous analyses of immune response in brain, try different adjuvants, and more emphasis on antibody transfer.

Giulio Pasinetti: Juan: this is a point we are trying to make. Far more exhaustive studies are required before we should go forward with clinical trials.

Dave Morgan: One interesting feature of the vaccination in mice that we have recently published is that initially mice have increased microglial activation, but after a number of months of repetitive inoculation it goes away. It will be very interesting to see if the CNS inflammation in these patients resolves over time, and whether they might still show benefit from the treatment.

Giulio Pasinetti: Dave, even if it does so in an experimental model, the cognitive and physiologic consequences in a human are not worth risking.

Gabrielle: If some microglial activation is necessary to remove amyloid, will it be possible to have a therapeutic effect without any inflammation? Can we separate "good" from "bad" inflammation?

Orest Hurko: Dave, isn't microglial activation an inflammatory response?

Glenda Bishop: Yes. Microglial activation is an inflammatory response.

Dave Morgan: I think it's problematic to equate microglia activation with the word inflammation. It depends how one defines inflammation. To me its massive cytokine release with ultimate tissue damage. Microglia have multiple states of activation, some of which are associated with inflammation as defined by these criteria. If microglia activation equals inflammation, it will always be considered bad.

Giulio Pasinetti: Inflammation is a vast term, we need to define features of these responses and relate these to consequences to specific phases of the disease.

Dave Morgan: My general reaction to microglia activation is that some is good and too much is bad. Kind of like red wine.

Giulio Pasinetti: Dave, wine has been shown to prevent AD, however this is not the case for microglial activation. Orest, in a paper in preparation we have been studying microglial activation in the early stages of AD (CDR 0.5 - 1) and we do not show evidence for a strong microglial response.

Juan Troncoso: Do we know whether patients who developed encephalitis after Ab immunization also have systemic immune problems?

Kiminobu Sugaya: The patient has more Aβ and may produce a higher level of antibody against AβPP and Aβ. If we can control the AbPP level rather than completely shutting down the system, it would be helpful.

Glenda Bishop: But if we control AβPP levels, this may affect the beneficial role of AβPP in the brain.

Kiminobu Sugaya: In my study, AβPP regulated stem cell migration and differentiation, which may be necessary for adult neurogenesis. High levels of AβPP cause glial differentiation of stem cells, while low levels of AβPP just increase general differentiation. Thus low level of AβPP is necessary, but high levels of AβPP may produce a problem like AD.

Jon Oscherwitz: Aβ42 generates quite a polyclonal response with many antibody specificities. We probably need to study immunization with more defined sequences.

Kiminobu Sugaya: Some level of microglial activation may be beneficial, but Ab immunization may exceed such a level and we don't have control over how strongly we activate microglia.

Jon Oscherwitz: Kiminobu, perhaps that argues for studying a passive approach to determine the association between antibody level and inflammation.

Kiminobu Sugaya: I think a low level of AβPP is necessary, because in AβPP knockout mice we have not seen any migration of stem cells to the cortex and the lesion did not get repaired. If we can control antibody concentration and determine the level of Ab deposition, we should continue immunization therapy. Otherwise it would be dangerous to do so.

Glenda Bishop: Would you feel comfortable if we tested it on you, Kiminobu?

Kiminobu Sugaya: I am worried about healthy people getting Aβ immunization, because they may continue to eliminate their AβPP, and without AβPP neurogenesis may not occur. Thus I do not want get any immunization in the mean time.

Alexei Koudinov: By the way, why we don't have anybody here from Elan today?

Gabrielle: We invited two representatives from Elan.

Glenda Bishop: When will Elan publish all of its data so that we can understand what is going on?

John Travis: What if Elan doesn't publish its data or conclusions? One could argue the company could discourage others from pursuing the approach and retain a competitive advantage if it doesn't explain the cause. This would only become dangerous/unethical if another company launched a vaccine clinical trial that Elan had reason to fear would be dangerous.

Gabrielle: All here are concerned about Elan not publishing, or at least not fast enough. This is only the latest, not the first, instance of the problem that companies - and academics - are reluctant to publish negative results.

Glenda Bishop: Surely given the topicality of this issue nearly any journal would accept a rapid publication from Elan on this matter.

Orest Hurko: Next Wednesday there is a symposium on Aβ immunization in Paris. I hope Elan will disclose some of the data in that Forum. (Editor: see ARF Ipsen Foundation coverage.)

Yasuji Matsuoka: How about another mode of vaccination therapy? Dave Morgan showed a significant elevation of plasma Aβ after vaccination. Do they see similar change in the clinical trial?

Dave Morgan: Yasuji, it wasn't our group, but Dave Holtzman's that showed the big increase in plasma Aβ. One outcome here is it suggests antibody does get in the brain.

Yasuji Matsuoka: David, excuse my poor memory. Do you see an elevation of plasma Aβ in mice that received passive immunization in your lab? Do you have any idea how much antibody enters the CNS?

Dave Morgan: Yasuji, we have not looked at either issue extensively. Most confirm the antibodies increase plasma Aβ; if we inject into brain we clear Aβ.

Gabrielle: Guilio and Ruth: How about tangle pathology and inflammation? If HSV and Il-6 increase tangle formation, then the vaccine could make the disease much worse. Any way to test this?

Ruth Itzhaki: Gabrielle - we think that HSV1 is normally latent and, as Richard said, can be reactivated by e.g., inflammation and stress. It then becomes an acute infection and causes damage mainly through inflammation. Incidentally, almost all cases of "natural" encephalitis are caused by herpes simplex viruses. The results of the vaccine test suggest that if HSV1 is involved it makes patients worse. We are currently examining the effects of HSV1 infection on cells in culture relating to plaques and NFT.

Alexei Koudinov: I would like to remind the issue that a top journal announced last year that we have to wait for the results of a vaccination study to know whether Aβ is good or bad. We have that now. So, do we limit the discussion to inflammation as a pitfall on the way to the gold, or talk about the whole approach being wrong?

Kiminobu Sugaya: My understanding is that AβPP is necessary for some physiological function of the brain and Aβ production is a byproduct. If it exceeds a certain level it may harm the brain but at low levels, it is not a bad guy.

Glenda Bishop: I don't think that Aβ production is necessarily a byproduct, considering that on its own it can have neurotrophic effects. Craig Atwood says the neurotrophic properties of Aβ have all but been forgotten. Fortunately, or unfortunately, the Elan trial now highlights the importance of this normal physiological property of Aβ.

Alexei Koudinov: And what about its antioxidant capacity at physiological concentrations?

Gabrielle: We are planning an upcoming chat on the physiological function of AβPP. Still a big open question.

Giulio Pasinetti: Do we want to consider combining antiinflammatory therapies with vaccination to keep in check responses coincidental to vaccination? The question is, however, what is the actual mode for clearance induced by vaccination, and what inflammatory cascades are necessary for this response?

Gabrielle: Exactly Guilio, I was going to ask do we have any antiinflammatories that can separate the amyloid-clearing action from the "bad' inflammation. Even experimental ones?

Dave Morgan: Giulio, you are right on. I will send the specific aims for Marcia Gordon's grant!

Gabrielle: Marcia Gordon just has a paper due out this week on one such NSAID. (See ARF news story.)

Giulio Pasinetti: Gabrielle, understanding of vaccination is still in its infancy, and we need to further characterize many facets of these responses before we can tailor antiinflammatory regimens to coincide with this therapy.

Kiminobu Sugaya: Most of the antibodies that worked to eliminate Aβ deposition recognized the N terminal of Aβ, which has a human-specific sequence. That is why in the mice we did not see the side-effect but do in the humans. In the mice, even if the transgene (AβPP) is eliminated by the antibody, they still have their own AβPP system. If inflammation were the only problem, combination of NSAID with vaccination could solve that.

Giulio Pasinetti: Dave, we actually have data demonstrating that both endogenous and exogenous AβPP expression were not influenced by COX-2, only processing of AβPP was altered.

Alexei Koudinov: Am I correct that anti-Aβ-immunization is grounded on the belief that clearing Aβ will cure the disease?

Glenda Bishop: Alex, yes, unfortunately.

Juan Troncoso: Aβ immunization will be grounded as an approach until we know what went wrong in the Elan trial, i.e. adjuvant, Aβ levels, antibody levels, CSF data, etc

Yasuji Matsuoka: Clearance of Aβ is a cure but I am not sure that clearance by microglia is a cure. David Holtzman showed another mode of vaccination therapy, the Aβ sink (see ARF news story). This has been confirmed in a different lab as well.

Yasuji Matsuoka: Dave, do you see more efficient clearance when you inject antibodies into the CNS directly?

Glenda Bishop: Maybe the inflammation side effect we are seeing after vaccination is due to removing the beneficial Aβ.

Orest Hurko: Glenda, do you mean that absence of Aβ would trigger inflammation or that this approach to its clearance results in secondary inflammation?

Glenda Bishop: Craig says to Orest: Yes, it's the removal of the amyloid that is the problem because more than 15 studies show that it has neurotrophic properties including antioxidant, metal-binding and sealing features to keep the blood-brain barrier intact. Remove that and you will engender inflammation. (see Atwood comment.)

Dave Morgan: I think the answer to Aβ physiology is partially addressed by the BACE knockouts. They have no Aβ, yet the phenotype is minor so far.

Glenda Bishop: The BACE knockouts also have not been put under stressful conditions, and it is likely that it is these conditions that will require the beneficial effects of Aβ.

Kiminobu Sugaya: Putting them under stress or lesioning the brain may show phenotype.

Kiminobu Sugaya: In the mouse model the phenotype may not show up because they only live 2 years at most. If somebody can give me the BACE knockout mice I can do the same kind of study I did with AβPP knockout mice to find out if AβPP is important.

Glenda Bishop: Kiminobu, Robert Vassar has the BACE KO mice, and he is aware of the need to put these mice under stress. I don't know if he has done it yet though.

Kiminobu Sugaya: Glenda: what kind of stress?

Glenda Bishop: Kiminobu, anything that causes trauma to the brain.

Kiminobu Sugaya: If he is checking only damage, he may not see the effect. Checking the recovery from the damage would be the target.

Glenda Bishop: Is there any other example of a physiological protein being used to treat a disease? Normally this leads to autoimmune disease.

Orest Hurko: Cancer vaccines.

Glenda Bishop: Against differentiated cells?

Steve Snyder: Insulin.

Kiminobu Sugaya: What happens when insulin was imunnosuppressed?

Giulio Pasinetti: Everybody: we all anticipate Elan's findings both of the inflammation and efficacy of the trials, and look forward to future characterization of vaccination in rodent models. Ciao, gotta go.

Richard Bowen: {enters} Sorry I'm late.

KLee: {enters}

Gabrielle: Welcome Richard! Hi KLee. Would you mind introducing yourself? I am managing editor of the Alzforum.

KLee: Hi, I'm an Information Specialist at the Alzheimer's Disease Education and Referral Center. Just trying to stay on top of the news for our callers.

Gabrielle: What about the blood-brain barrier? There are conflicting reports on whether antibodies cross it, on whether it is breached during AD to allow in peripheral inflammatory cells. Can any vaccination approach be safe in light of this uncertainty?

Craig Atwood: Good question! This point seems to be ignored, but obviously if amyloid is being removed, then the integrity of the BBB must be compromised.

Gabrielle: Craig, why? Could amyloid not be removed without injuring the BBB?

Craig Atwood: I don't know if amyloid removal will maintain vessels intact. Physiologically, amyloid probably deposits to maintain regional integrity given its binding to extracellular matrix proteins.

Kiminobu Sugaya: I am not sure if antibodies cross the BBB. It has to be tested in humans.

Steve Snyder: The amyloid "sink" work by the Holtzman group doesn't appear to involve BBB damage. Maybe it's a ligand and receptor story.

Glenda Bishop: Amyloid deposits during head injury when you expect to see BBB disruption.

Einar Sigurdsson: Numerous labs have shown that Aβ crosses the BBB bi-directionally.

Ruth Itzhaki: Regarding a point raised by Trey Sunderland. He suggested that a virus might be involved in AD--coming from the blood after inflammatory damage to the BBB. Elan's finding that the virus is HSV1 in CSF in the vaccine trials is not consistent with this, as HSV1 resides in nerve cells, not in blood cells.

Craig Atwood: Ruth, exactly! But how does the virus get in the nerve cells?

Ruth Itzhaki: The virus might travel from PNS to CNS by retrograde transport or possibly as a new infection via the olfactory tract. We have found the virus in most elderly people including AD patients. In general, most people now accept that HSV1 is present in brain. HSV1 is present in the PNS of nearly all elderly people. It is latent generally but can reactivate under stress or immunosuppression, and in some people causes cold sores (a relatively high proportion of whom have an apoE4 allele).

Orest Hurko: Glenda, (1) Isn't there normally some flux between systemic antibodies and CSF? (2) Could the antibody be depleting amyloid systemically, creating a sink? Is there traffic of CNS amyloid and the systemic circulation?

Keith Crutcher: Is anyone willing to sign on to the possibility that this is the first in a series of failed clinical trials based on an inappropriate drug target? Allen Roses was the most vocal, but not the only, opponent of targeting amyloid deposition to treat this disease. Will there be a point at which the hypothesis might be said to have failed?

Glenda Bishop: I agree with you.

Alexei Koudinov: I would say that the hypothesis was not proved. Ever. Of the 22 of us here, could any supporter of immunization provide me with a reference showing directly a bad role for Aβ in Alzheimer's?

Orest Hurko: Alexei, how about the Elan report itself. Isn't that the most straightforward explanation.

Ken Ugen: As an HIV vaccinologist now working in AD, I would like to point out that the vaccine approach should not be given up. Historically, vaccines against infectious diseases have saved more lives than all other medical interventions combined. If the smallpox vaccine were being tested now it would likely have been pulled because of adverse events. I believe that it may be possible to fix this problem whether it be untoward cellular immune responses, autoimmunity, or adjuvants.

Gabrielle: Ken, what do you say to the detractors (see also Stephen Robinson's comment) that until we know the physiological function of AβPP and Aβ, they are dangerous targets?

Ken Ugen: Gabrielle, nothing in life is perfectly safe.

Kiminobu Sugaya: If it is not perfectly safe I would not get the vaccine.

Kiminobu Sugaya: A vaccine to a virus like HIV is different; it just needs to eliminate the enemy. A vaccine to Aβ is eliminating the functional protein in our body.

Richard Bowen: Ken, have there been any other vaccines produced against ubiquitous proteins that have significant deposits in certain tissues? It seems to me that making a vaccine against Aβ for AD is similar to making a vaccine against uric acid to treat gouty arthritis. The expected result would be a much worsening of the arthritis.

Gabrielle: Ken and Dave, will the failure of this trial make investors and IRBs more hesitant and put a damper on new approaches? Do you feel any of that?

Dave Morgan: Gabrielle, I think all will have additional caution in moving forward with the approach, and reasonably so.

Kiminobu Sugaya: Once we have an antibody against one of our proteins it will stay there for a long time without control, it is not like a drug.

Gabrielle: We are nearing the end of the hour. If anyone wants to make a final statement on where we should go next, now is the time. I would love some suggestions by everyone as to what scientific questions they would like to see answered (We all hope for full disclosure from Elan.) To those who have to leave soon: thanks for joining us, and be back.

Orest Hurko: Goodbye. I hope Elan provides us more data -- some of these speculations could be tested against data that must already exist.

Kiminobu Sugaya: If antibodies cross the BBB and if we can find a good specific antibody against Aβ deposition without compromising the physiological function of AβPP, that would be nice.

Yasuji Matsuoka: If the periphery sink is a primary mode, then we don't have to stick with antibody/immune reaction.

Dave Morgan: Thanks for the chat.

Ruth Itzhaki: We hope that the possibility of HSV1 involvement in AD, further supported by Elan's trial, will encourage the companies to release more information. The trial results must be a great disappointment for patients and their carers, particularly in the absence of information. This is especially important given that safe (off-patent) antiviral drugs are currently available.

Glenda Bishop: I think that until Elan puts out specific information regarding the failure of the trial, it is not possible to decide what should be done next. We need to know whether failure was due to immunizing against Aβ, interactions with AβPP, or problems with the adjuvant. Until then it is dangerous to persist with any vaccination trials. Goodbye.

Alexei Koudinov: What we learn from this is that curing patients is more complicated than getting Aβ aggregated in the test tube.

Keith Crutcher: My concern is that this will go on for a very long time with refinements of the approach substituting for critical testing of the underlying hypotheses.

Gabrielle: Keith, do you have a suggestion for an experiment that would critically test the underlying hypothesis?

Keith Crutcher: I would say that once there is some neuropathology of patients being treated for amyloid deposition and they show loss of amyloid but no improvement (or worsening), that this is the time to seriously question the assumptions about the target.

Alexei Koudinov: The amyloid hypothesis has been on the market for 15 years and gradually become dogma. How long will it take to address Keith's point?

Gabrielle: Keith, this makes sense. It would also be fun to treat tau transgenic mice with the present vaccines.

Keith Crutcher: Yes it would.

Alexei Koudinov: It will not be fun, as tau has clear normal function.

Gabrielle: Alexei, Ruth and Giulio have written in their comments that viral infection, Il-6 expression, and tau pathology may be connected.

Ruth Itzhaki: Gabrielle, thanks for mentioning this. We hope to test this in in vitro shortly.

Alexei Koudinov: I see.

Keith Crutcher: I need to go but I am placing my bets on Ruth's work as an important clue to the etiology of this disease. Cheers to all!

Ruth Itzhaki: Thanks Keith. Your support is appreciated! Goodbye all!

Gabrielle: Ken, in your view, are we squabbling too much about safety problems that are to be expected in a radical new approach? Also, are there reliable methods to make passive immunization safe from serum sickness, i.e. humanize antibodies and the like? Dave Holtzman's "sink" approach sounds attractive.

Ken Ugen: I did not mean for my comments earlier to indicate that I do not think that safety is important. These are significant adverse effects. However, it is possible that further research may circumvent some of these problems. In particular it may be important to do studies in non-human primates. This approach has been fast-tracked in the clinic (much like HIV drugs and vaccine work) because of the serious nature of the disease. There are advantages and disadvantages in doing this. As for passive immune therapy, the way to go is to use human monoclonal antibodies. No serum sickness concerns.

Gabrielle: Ken, are you aware of Beka Solomon's recent Diagnostics paper in which she describes raising anti-aggregating Aβ antibodies with a filamentous phage displaying amino acids 3 to 6 of Aβ, the epitope said to regulate fibril formation? No adjuvant, high titres. What do you think of it? Would that be safer?

Ken Ugen: Yes, I think that this is a potentially good approach since the antibody is only against a fraction of the 42 amino acids in Aβ. This may cut down on adverse events. Also, a truncated peptide displaying only a fraction of the amino acids may be something to try, too, like Einar and Blas Frangione are doing.

Gabrielle: Have a good day everyone. We will fix the access problems we had today. Thank you everyone and goodbye for now.

Kiminobu Sugaya: See you everybody.

Einar Sigurdsson: Goodbye.

Juan Troncoso: Good-bye everyone.

Glenda Bishop: Bye.

Craig Atwood: Goodbye.

Richard Bowen: Goodbye.

Ken Ugen: Goodbye to everyone

Alexei Koudinov: Thanks for today's discussion and talk to you soon again.

Background

Background Text
By Gabrielle Strobel

Last month, the frontrunner in the effort to develop a therapeutic vaccine for Alzheimer disease suffered a widely noted setback when Elan Pharmaceuticals and American Home Products, the companies sponsoring the first human trials, announced their decision to temporarily halt dispensing any more trial drug until they had found out why four patients in France had come down with cerebral inflammation (See ARF story.)

The effort to develop a vaccine is being closely observed, partly because the concept of treating a neurodegenerative disease with a vaccine is radically new and controversial, and in part because the approach has so rapidly moved into the clinic since the first paper describing it appeared three years ago (Schenk et al. 1999). Hopes and stakes are high, and by now, many academic labs and pharmaceutical companies are working on modifications of Elan's original approach.

While the research community is awaiting results of Elan's investigation into the causes of these patients' inflammatory response, the Alzheimer Research Forum is hosting a live discussion into the underlying issues and future directions this incident points to.

Question and Answer session with Blas Frangione, New York University.

Q: What was your initial reaction when you read the press release announcing that Elan Pharmaceuticals was temporarily suspending dosing in their trial of the Aβ vaccine?

A: This was not unexpected. When you immunize humans with something they have, in this case Aβ1-42, you can produce autoimmune disease, that was known. Of course this did not happen in the transgenic animals. So one question was: will it happen in humans?

It might be an antibody against your own protein that induces inflammation. An antibody against Aβ1-42 could conceivably also recognize AβPP. Alternatively, it could be that the protein that was given to the patients intramuscularly went into the brain. We and other groups have shown that the peptide goes into and out of the brain very well. Aβ1-42 could have precipitated in the brain, caused inflammation, and made plaque deposition worse. Which of these two possibilities occurred in these four patients, if any, I do not know. But these certainly can happen, so I was not too surprised.

Q: Could the inflammation be a beneficial part of the reaction to the vaccine? Dave Morgan saw temporary CNS inflammation in vaccinated mice.

A: Could be, but you cannot test that in humans. It creates this ethical dilemma that when someone has a cerebral inflammation you cannot go on vaccinating for months to find out if it is a beneficial or a detrimental reaction to the trial vaccine. Remember that transgenic mice are not humans. These sorts of data in animals do not reflect very well what will happen in humans. This is a general phenomenon, and a big problem. Elan was perfectly correct in stopping dosing for now.

Usually when you vaccinate, especially in children, you do get a local inflammation at the injection site. It lasts a few days and goes away. In this case it is probably not that simple. The press release did not explain for how long the inflammation went on. That is an important question. Has this gone on for months? We do not know. Also, the inflammation here was probably not local. It was in the brain, which means that it went through the blood-brain-barrier.

Q: What's most important now?

A: The important thing is to find out why this happened. For example, if it is an autoimmune response, these patients will have a high titer of antibody against the injected antigen. You can find that out with a spinal tap and blood sample, which you react with the injected antigen.

If, however, this is an aggravation of plaques, they can find out with MRI or other methods. It is possible to find out in the next few months. It is very important to study these two mechanisms, and the third possibility that desired improvements would happen via a temporary inflammation.

Q: It is difficult for a drug company to be first to develop a new approach, and vaccination is a radically new therapeutic concept in Alzheimer's disease. Competitors who are a year or more behind can learn from the stumbles of the leader and refine their method. Do you think these other approaches waiting in the wings stand a better chance?

A: Yes I do. Of course I am partial to an approach we published earlier this year (see ARF news story). Personally, I worried about Elan's approach from the beginning. When they published the initial paper on injecting Aβ1-42 more than two years ago (Schenk et al., 1999), I was very surprised they did it in that way. At least in vitro, 1-42 aggregates in 20 minutes and also is very toxic, killing cells within a few days.

That is why we decided not to use the carboxy terminal of Aβ, which aggregates and produces the in-vitro toxicity. We only use the antigenic determinant, which is known to be in the amino terminus of the peptide between position 1 and 28. That way we hoped to reduce toxicity.

Elderly patients generally have a poor immune response, so we added polylysine to increase the antigenicity. Polylysine has two properties: it diminishes the probability of Aβ being converted into a β-pleated form, which is the toxic, fibrillogenic material, and secondly it increases antigenicity, which has been shown in guinea pigs and mice. At this point there is really no good scientific argument to use Aβ1-42, because in our paper we have the same antibody titer as the Elan group.

Q: Some advocate passive immunization, that is, injection with anti-Ab antibodies, as a better approach?

A: I disagree. Passive immunization works well for hepatitis B and other acute diseases, including even prion disease. Because of the short half-life of immunoglobulin G, you have to inject antibody every three weeks. But if you do that for months, you can induce serum sickness or an anaphylactic reaction. AD is a chronic process that takes10 or 20 years, and passive immunization for such long time is difficult.

An exception would be if you used an approach called one-chain antibody, which means you make a small, roughly 100-residue piece of antibody that contains the antigen-recognition site. Those you can inject for long periods of time. Or you could make a humanized antibody, there are many options to pursue.

Q: Can CNS inflammation appear suddenly?

A: Making the diagnosis of cerebral inflammation in a patient with AD is not simple. Patients cannot express themselves well, they do not understand what you are asking them and don't necessarily describe their symptoms clearly. Only the caregiver sees that something is wrong. Then you need to do a spinal tap, or MRI to see edema in brain. It does not become apparent in one week.

Comments

  1. Vaccine Disrupting "Scab"? Aβ as a Vascular Sealant That Protects Against Hemorrhage
    The question whether removing amyloid-β from the brain is therapeutic has been raised again by the recent interruption of the Elan/AHP trial due to clinical signs of inflammation. Numerous papers suggest that Aβ has neuroprotective properties (reviewed in Atwood et al., 2001), but this literature is being overlooked in the great rush to find better treatments. Aβ's protective functions include metal chelation, antioxidant activity and, perhaps most significantly in the present context, sealant properties that we think help to maintain the integrity of the blood-brain barrier and parenchymal structures (Atwood et al., 1998).

    Consistent with Aβ's proposed role as an antioxidant and its role in maintaining structural integrity under stress conditions are data showing that Aβ binds copper under acidotic conditions, and that it possesses hydrophobic and hydrophilic regions, which both span the plasma membrane and bind to extracellular matrix molecules (Atwood et al., 1998; Chan et al., 1999; Kontush et al., 2001; Narindrasorasak et al., 1991). Together with Aβ's ability to aggregate under inflammatory conditions, these properties make it an excellent candidate for a molecule that could form an intracranial 'scab'. The aggregated Cu,Zn-Aβ would serve as a superoxide-scavenging, solid-phase matrix, which disassembles when Zn and Cu levels lower as tissue damage resolves (Pluta et al., 1999). This may explain the acute-phase generation and rapid cortical deposition of Aβ in stroke and following head trauma (Roberts, et al., 1994), an important physiological response to limit loss of terminally differentiated neurons (Smith et al., 2000; Perry et al., 2000).

    If indeed Aβ acts as a seal to maintain the blood-brain barrier, then we would expect its removal to cause leakage of serum components into the brain, resulting in an immune or autoimmune response characterized by inflammation. A severe consequence would be the occurrence of mini-strokes. Notably, this breakdown of the blood-brain barrier is consistent with the presence of a virus within the cerebrospinal fluid, as was reported in some of the four patients under investigation. The adverse effects of Aβ vaccination has halted the current clinical trial, but a more balanced assessment of the current literature would have cautioned against such trials in the first place.

    References:

    Atwood CS, Moir RD, Huang X, Scarpa RC, Bacarra NM, Romano DM, Hartshorn MA, Tanzi RE, Bush AI. Dramatic aggregation of Alzheimer abeta by Cu(II) is induced by conditions representing physiological acidosis. J Biol Chem. 1998 May 22;273(21):12817-26. PubMed.

    Chan AC, Chow CK, Chiu D. Interaction of antioxidants and their implication in genetic anemia. Proc Soc Exp Biol Med. 1999 Dec;222(3):274-82. PubMed.

    Kontush A, Berndt C, Weber W, Akopyan V, Arlt S, Schippling S, Beisiegel U. Amyloid-beta is an antioxidant for lipoproteins in cerebrospinal fluid and plasma. Free Radic Biol Med. 2001 Jan 1;30(1):119-28. PubMed.

    Narindrasorasak S, Lowery D, Gonzalez-DeWhitt P, Poorman RA, Greenberg B, Kisilevsky R. High affinity interactions between the Alzheimer's beta-amyloid precursor proteins and the basement membrane form of heparan sulfate proteoglycan. J Biol Chem. 1991 Jul 15;266(20):12878-83. PubMed.

    Pluta R, Barcikowska M, Misicka A, Lipkowski AW, Spisacka S, Januszewski S. Ischemic rats as a model in the study of the neurobiological role of human beta-amyloid peptide. Time-dependent disappearing diffuse amyloid plaques in brain. Neuroreport. 1999 Nov 26;10(17):3615-9. PubMed.

    Smith MA, Joseph JA, Perry G. Arson. Tracking the culprit in Alzheimer's disease. Ann N Y Acad Sci. 2000;924:35-8. PubMed.

    Roberts GW, Gentleman SM, Lynch A, Murray L, Landon M, Graham DI. Beta amyloid protein deposition in the brain after severe head injury: implications for the pathogenesis of Alzheimer's disease. J Neurol Neurosurg Psychiatry. 1994 Apr;57(4):419-25. PubMed.

    Perry G, Nunomura A, Raina AK, Smith MA. Amyloid-beta junkies. Lancet. 2000 Feb 26;355(9205):757. PubMed.

    View all comments by Craig Atwood

  2. The release from Elan certainly raises more questions for me than it answers. For example, what is the "marker of CNS inflammation" it is referring to?

    Our first experiments in mice anticipated exactly the outcome that the Aß vaccine would provoke brain inflammation and lead to premature memory deficits (Morgan et al., 2000). We did not find the memory deficits.

    In more recent work, we measured brain inflammation by microglia activation in response to the vaccine (Wilcock et al, 2001). There, we find a slow development of an inflammatory reaction, perhaps related to the slow rise in antibody titers, that was maximal in mice given five inoculations, roughly one per month. Interestingly, in mice given nine inoculations, the microglial activation had abated (it was 50% lower, but this was not statistically significant).

    We can only hope that the same will prove true in the human trials. It may be that, initially, the vaccine causes an inflammatory reaction that normalizes after the microglia succeed in phagocytosing the amyloid deposits.

    A vaccine trial continues whether you wish it to or not. You cannot reverse the vaccination (which is why we have suggested passive immunization for clinical trials). Still, this will likely permit us to determine if the same course of reaction occurs in humans.

    Of course, it is entirely possible the inflammatory reaction in these four people is unrelated to the vaccine. Without more details, I cannot evaluate the likelihood of that outcome.

    We would all like to see the vaccine work. Still, I am confident that if this one does not succeed, one of the half dozen other approaches currently underway will eventually prove beneficial.

    References:

    Morgan D, Diamond DM, Gottschall PE, Ugen KE, Dickey C, Hardy J, Duff K, Jantzen P, DiCarlo G, Wilcock D, Connor K, Hatcher J, Hope C, Gordon M, Arendash GW. A beta peptide vaccination prevents memory loss in an animal model of Alzheimer's disease. Nature. 2000 Dec 21-28;408(6815):982-5. PubMed.

    Wilcock DM, Gordon MN, Ugen KE, Gottschall PE, DiCarlo G, Dickey C, Boyett KW, Jantzen PT, Connor KE, Melachrino J, Hardy J, Morgan D. Number of Abeta inoculations in APP+PS1 transgenic mice influences antibody titers, microglial activation, and congophilic plaque levels. DNA Cell Biol. 2001 Nov;20(11):731-6. PubMed.

    View all comments by Dave Morgan

  3. Ruth Itzhaki, Matthew Wozniak, Curtis Dobson, University of Manchester, UK.

    Inflammatory Consequences: Benevolent or Virulent?
    The main point by Pasinetti et al. is that there is an intriguing paradox: prevention of inflammation appears to be beneficial in respect to development of Alzheimer disease (AD), but the inflammation caused by vaccination with Aβ is beneficial too, at least in mice. The protection anti-inflammatory agents afford presumably is due to the prevention of the induction of pro-inflammatory cascades-microglial activation and generation of free radicals-which are thought to cause neuronal injury. Passive or active immunization by intraperitoneal injection of Aβ antibodies or of Aβ peptides appears to remove Aβ plaques by activating pro-inflammatory microglial cells via immunoglobulin receptor signaling.

    Whether vaccination is beneficial also for humans is yet to be determined, and in fact there has recently been an interesting development during the progress of a clinical trial: four patients treated with the AN-1792 vaccine, a synthetic version of Aβ, have suffered from inflammation of the brain. The company press release notes that viral infection can cause inflammation, and furthermore that in some of these patients (presumably two or three of the four examined), a virus - herpes simplex virus type 1 (HSV1) (D.Schenk, personal communication) - was found in cerebrospinal fluid (CSF).

    HSV1 has long been known to reside in a latent state in the PNS in most humans, and in our lab we showed, using polymerase chain reaction, that it is present also in the brain in a high proportion of the elderly, including AD patients [5,6], although not in the brain of most younger people (Wozniak, Lin, Cairns, Mann, Itzhaki, in preparation). In the virus' dormant state, only its genome is present, there is only one set of transcripts-those termed latency-associated transcripts-and no viral proteins have yet been detected. The latent virus can reactivate in the peripheral nervous system (PNS) as a result of inflammation and other forms of stress, and this results in an acute infection, i.e. synthesis of whole virus, which causes cold sores in some people.

    We have proposed that a similar process can occur in the central nervous system (CNS) and that the consequence is a localized encephalitis. In fact, we now have direct evidence that reactivation does occur in the CNS and we infer that the whole viral genome must therefore be present. We have detected antibodies to HSV1 in CSF that are not due to leakage across the blood-CSF barrier (Wozniak, Klapper, Combrinck, Esiri, Wilcock, Itzhaki, in preparation). For an in-depth discussion of our hypothesis, visit our entry in the Alzheimer Hypotheses/Online Forums section.

    Presumably, in the clinical trial, the vaccine caused inflammation that reactivated HSV1 residing in the brain. HSV may, in turn, have caused damage in the way that viruses normally do-through the inflammatory response. Following our finding that HSV1 in brain, together with carriage of the type 4 allele of the apolipoprotein E gene (apoE-e4), is a strong risk factor for AD (and that apoE-e4 is a risk for cold sores), we suggested that the extent of damage caused by HSV1 is greater-or the extent of subsequent repair is less-in the presence of the apoE4 isoform [2,4,7]. We attributed the protective action of anti-inflammatory agents to their preventing reactivation episodes caused by inflammation.

    Another relevant finding is that certain vaccines appear to protect against AD development. A study of 3,682 cognitively normal people and 183 people newly diagnosed with AD found that after adjustment for age, sex, and education, past exposure to vaccines against diphtheria or tetanus, poliomyelitis, and influenza was associated with lower risk of AD [11]. This raises the possibility that viruses other than HSV1 might be involved in AD. Alternatively, it could be that the vaccines reduce the frequency of inflammatory events as a consequence of preventing infection with the target virus. They also support the possibility that vaccination against HSV1 might be feasible in future, as do our findings that a vaccine of mixed viral glycoproteins protects HSV1-infected mice from establishment of latency by the virus in brain [8].

    Pasinetti et al. point out the need for considering possible effects of Ab immunization therapy on neurofibrillary tangles (NFT) and neuronal loss, especially as these features do not occur in animal models of AD. Also, the authors discovered that a raised Il-6 level in severe AD correlates with level of NFT pathology [9]. Thus, immunization might increase the numbers of NFT. Incidentally, it may be highly relevant that the main cytokine induced by HSV1 infection is IL-6 [1,10], and that NFT have been described in two viral disorders [3]: subacute sclerosing panencephalitis, caused by measles virus, and herpes simplex encephalitis. A further major difference between animal models and humans is that, unlike humans, mice and other animals are not natural hosts for HSV1. This might explain the absence of NFT and neuronal loss in Aβ-immunized mice.

    Pasinetti et al. suggest that selective anti-inflammatory agents that interfere only with harmful pro-inflammatory responses might be developed. Alternatively, if the putative effects of the vaccine on HSV1 reactivation are confirmed as the cause of the inflammation in the AN-1792 trial, usage of antiviral agents in combination with the vaccine should perhaps be considered.

    Clearly, further information about disease states, the role of specific cytokines at each stage, and the mechanisms involved in plaque clearance, as well as modification of the antigen, might enable conditions to be chosen which resolve the dilemma.

    References:

    Baker M, Noisakran S, Gebhardt BM, Kriesel JD, Carr DJ. The relationship between interleukin-6 and herpes simplex virus type 1: implications for behavior and immunopathology. Brain Behav Immun. 1999 Sep;13(3):201-11. PubMed.

    Dobson CB, Itzhaki RF. Herpes simplex virus type 1 and Alzheimer's disease. Neurobiol Aging. 1999 Jul-Aug;20(4):457-65. PubMed.

    Esiri MM. Potential for HSV-1 vaccination to reduce risk of HSV-1 encephalitis and/or Alzheimer's disease?. Neurobiol Aging. 2001 Sep-Oct;22(5):711-3; discussion 717-9. PubMed.

    Itzhaki RF, Lin WR, Shang D, Wilcock GK, Faragher B, Jamieson GA. Herpes simplex virus type 1 in brain and risk of Alzheimer's disease. Lancet. 1997 Jan 25;349(9047):241-4. PubMed.

    Jamieson GA, Maitland NJ, Wilcock GK, Craske J, Itzhaki RF. Latent herpes simplex virus type 1 in normal and Alzheimer's disease brains. J Med Virol. 1991 Apr;33(4):224-7. PubMed.

    Jamieson GA, Maitland NJ, Wilcock GK, Yates CM, Itzhaki RF. Herpes simplex virus type 1 DNA is present in specific regions of brain from aged people with and without senile dementia of the Alzheimer type. J Pathol. 1992 Aug;167(4):365-8. PubMed.

    Lin WR, Graham J, MacGowan SM, Wilcock GK, Itzhaki RF. Alzheimer's disease, herpes virus in brain, apolipoprotein E4 and herpes labialis. . Alzheimer's Reports. 1998;1:173-8.

    Lin WR, Jennings R, Smith TL, Wozniak MA, Itzhaki RF. Vaccination prevents latent HSV1 infection of mouse brain. Neurobiol Aging. 2001 Sep-Oct;22(5):699-703. PubMed.

    Luterman JD, Haroutunian V, Yemul S, Ho L, Purohit D, Aisen PS, Mohs R, Pasinetti GM. Cytokine gene expression as a function of the clinical progression of Alzheimer disease dementia. Arch Neurol. 2000 Aug;57(8):1153-60. PubMed.

    Oshima M, Azuma H, Suzutani T, Ikeda H, Okuno A. Direct and mononuclear cell mediated effects on interleukin 6 production by glioma cells in infection with herpes simplex virus type 1. J Med Virol. 2001 Mar;63(3):252-8. PubMed.

    Verreault R, Laurin D, Lindsay J, De Serres G. Past exposure to vaccines and subsequent risk of Alzheimer's disease. CMAJ. 2001 Nov 27;165(11):1495-8. PubMed.

    View all comments by Curtis Dobson

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References

News Citations

  1. Human Aβ Vaccine Snagged by CNS Inflammation
  2. Early-Stage Alternative Vaccine Reported: Better Antibody Response with Liposomes?
  3. More Cases of Brain Inflammation in Vaccine Trial
  4. Conference Coverage: IPSEN Foundation
  5. NO-Releasing NSAID Reduces b-Amyloid, Activates Microglia
  6. Two Ways to Attack Amyloid: Metal Chelator and Antibody

Webinar Citations

  1. Alzheimer Immunotherapy Trial Grounded: Time to Reassess Safety and Vaccine Design

Paper Citations

  1. Frenkel D, Solomon B. Towards Alzheimer's beta-amyloid vaccination. Biologicals. 2001 Sep-Dec;29(3-4):243-7. PubMed.

Other Citations

  1. see ARF news story

External Citations

  1. Schenk et al. 1999
  2. free Acrobat Reader

Further Reading

Papers

  1. Flax JD, Aurora S, Yang C, Simonin C, Wills AM, Billinghurst LL, Jendoubi M, Sidman RL, Wolfe JH, Kim SU, Snyder EY. Engraftable human neural stem cells respond to developmental cues, replace neurons, and express foreign genes. Nat Biotechnol. 1998 Nov;16(11):1033-9. PubMed.
  2. Brüstle O, Choudhary K, Karram K, Hüttner A, Murray K, Dubois-Dalcq M, McKay RD. Chimeric brains generated by intraventricular transplantation of fetal human brain cells into embryonic rats. Nat Biotechnol. 1998 Nov;16(11):1040-4. PubMed.