Presented by Ruth Itzhaki
Molecular Neurobiology Lab., Dept. of Optometry & Vision Sciences University of Manchester Institute of Science & Technology (UMIST) Manchester M60 1QD, UK.

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Our results show that herpes simplex type 1 virus (HSV1) in brain of apoE4 carriers is a strong risk factor for AD (see Internet Conference Poster). We found several years ago, using PCR, that HSV1 is present in a high proportion of brains of elderly people and of AD patients (our 1st relevant paper was published in 1991). This result was broadly substantiated several years later by two quite large studies on brain* - from normals but not AD patients - in other labs, and is now, I think, generally accepted. We have since found that the combination of herpes simplex virus type 1 in brain and possession of an apoE4 allele provides a strong risk for AD (accounting for almost 2/3 of the patients whose brains we examined), and that neither factor on its own is a risk. We found a striking parallelism in the PNS in that cold sore sufferers had a much higher apoE4 allele frequency than did non- sufferers. These results were published in The Lancet (349, 241-4, 1997, see abstract) and since have been independently confirmed by a Japanese group (Itabashi, et al., Lancet 349, 1102, 1997). They raise the future possibility of immunisation against the virus, and of slowing down the progression of the disease by the use of anti-viral agents. We hope that other groups will follow the Japanese in repeating the work but there are great practical difficulties in carrying out PCR to detect very low levels of virus in tissue. It is far harder than using PCR for genotyping, and we have found that it takes almost a year to train a person who has not done PCR before -and would probably take several months for somebody who has used PCR merely for genotyping.

The precautions that we take are:

1) Dissection of brains is carried out using fresh scalpel blades, gloves and working surface for each specimen, to prevent cross-contamination.

2) Any type of homogeniser used for dispersing the brain specimens needs to be most scrupulously cleaned between each specimen. We check by doing a blank homogenisation between specimens, from time to time.

3)We always use primers for a cell gene in the same tube as the primers for the viral sequence, and would accept the results only if the cell sequence is amplified.

4) We use an HSV1-positive and an HSV1-negative standard, prepared respectively from virus-infected or uninfected Vero cells; in these cases also we use primers for a cell gene (HPRT, which is present also in these monkey cells). An alternative positive would be brain DNA from a person who died from herpes simplex encephalitis.

5) If we find an apparently HSV1-negative brain DNA specimen, we mix it with a known HSV1-positive specimen and then do a PCR again to check that there is no interference with amplification of the positive specimen, i.e., to find if the negative specimen is truly negative, rather than having a contaminant which interferes with amplification.

6) We always do a reagent blank.

7) It is necessary to keep HSE specimens, or plasmids with a viral sequence, very strictly segregated from the test brain specimens.

I stress that these are essential precautions and checks: we know of at least two groups who have done the work in a very perfunctory way and who could not do the first stage - reproducible detection of HSV1 in a proportion of brains - and so could not possibly proceed to the next stage - searching for a correlation of HSV1 in brain and apoE4 in AD. We would be happy to give further advice or to train someone in these methods.

*In these studies, brains from young and old people were used; the young were likely to be virus-negative, as we found, so the over-all proportion which were virus-positive was lower than the value we found for aged normals.

Additional References:
Baringer and Pisani, Ann. Neurol. 36, 823-829, 1994. Abstract.
Gordon et al., Clin. Diag. Virol. 6, 33-40, 1997.

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