Antibodies guard against a wide range of substances that can combine to form plaques, such as those found in Alzheimer's patients, are found in the blood and cerebrospinal fluid of healthy people. Levels of these antibodies decline with age and, in Alzheimer's patients, with increasing progression of the disease.

These findings by Stanford University School of Medicine researchers and outside collaborators, to be published online July 6 in the journal Proceedings of the National Academy of Sciences, raise the possibility that many of us are carrying antibodies in our blood that could be playing a role in staving off or slowing the progression of Alzheimer's disease. This seems to be true even when we are young and healthy and would presumably have had little or no exposure to the substances that build up in the brain to cause this disorder.

Alzheimer's disease is characterized by the build-up of amyloid plaques in the brain. These are large accumulations of a protein breakdown product, or peptide, called A-beta. Many experiments have shown that immunization with A-beta can reduce the formation of amyloid plaques. Clinical trials now underway are exploring whether this can safely produce cognitive benefits in Alzheimer's patients, while other trials are treating patients directly with antibodies to A-beta. These are designed to stop the build up of plaques in the brain, and thus prevent Alzheimer's.

But A-beta is a slippery character. A number of different modified, mutated, or metabolized forms of this peptide may also be involved in the disease. Furthermore, it is believed that more than the plaques themselves (which are also found in the brains of people with no Alzheimer's symptoms) cause the disease. It is the smaller masses of only a few A-beta molecules, called oligomers, which are most toxic to neurons.

"Other studies have found antibodies against A-beta, but nobody has ever done a large-scale analysis using hundreds of different samples and almost a hundred different peptides to look for what's already in people's bodies," said the paper's first author, Markus Britschgi, PhD, who works in the laboratory of Tony Wyss-Coray, PhD.

Britschgi, Wyss-Coray, and their colleagues used a microarray technology in which a large number of different peptides are affixed to pixels on a microchip, which signals binding of those peptides by antibodies. This technology was developed in a neighboring lab by another co-author of the paper, William Robinson, MD, assistant professor of rheumatology.

The researchers customized the microarrays containing close to 100 different peptides apiece, including A-beta and several of its other forms. The peptides were displayed in various degrees of accumulation. Ranging from single molecules to small assemblages, or oligomers, to denser masses called fibrils.

Since A-betas are not the only cause of the plaque build ups which lead to the disease, other peptides made from different amino-acids than A-betas were included on the chip. These sequences are unrelated to A-betas but are capable of combining to form other, rare plaque-associated dementias.

The researchers incubated these chips with blood samples from more than 250 individuals, who were between 21 and 89 years old, some with Alzheimer's disease and others without it.

They observed antibodies attacking many forms and collection-states of A-beta in both healthy and diseased subjects' blood, with antibodies to oligomers showing the most immunoreactivity. Researchers then showed that overall levels of these antibodies decline with age and, in Alzheimer's patients, with advancing stages of the disease.

"This was the first study to show an age-related decline in the levels of these antibodies," said Britschgi.

A follow-up experiment showed that the same antibodies, whether isolated from plasma of either Alzheimer's patients or healthy control groups, were able to protect freshly cultured mouse neurons in a dish from destruction by A-beta, which is typically highly toxic to these neurons.

Furthermore, the researchers studied samples from Vervet Monkeys, who, like humans, develop A-beta-derived brain plaques as they age. Earlier experiments have shown that immunizing older monkeys with A-beta substantially cleared their plaques.

In this study, researchers obtained blood samples extracted from those monkeys before and after immunization. They then compared levels and diversity of relevant antibodies in pre-inoculation and post-inoculation samples. They observed several such antibodies in the pre-immunized samples, as well as significant post-immunization increases in levels of several different antibodies.

In both monkeys and human subjects the investigators' microarrays also detected antibodies to some mutated peptides. These are associated with rare dementias which have brain plaques bearing a striking resemblance to those of Alzheimer's with a notable exception. Oddly, these dementia-related peptides' amino-acid sequences are nothing like that of A-beta's. Yet, antibodies against them occur even in the blood of healthy study subjects who have never been diagnosed with any of those rare dementias.

Wyss-Coray and Britschgi think this may mean that at least some of the antibodies they've isolated target not plaque-generating peptides' amino-acid sequences, but rather some common shape these molecules assume in the early, oligomeric stages of their combining, when they are most toxic.

The methods the researchers used could lead to improved monitoring of clinical trials of immunotherapeutic treatments for Alzheimer's disease, Wyss-Coray said. "With our microarray, you could easily look for antibodies to hundreds of different peptides. It would be possible to see whether certain types of antibodies correlate better with cognitive benefits than others do."

Another possibility, said Wyss-Coray, is to try immunizing Alzheimer's patients with peptides that, unlike A-beta, have amino-acid sequences different from any of those occurring naturally in the human body. Due to their three-dimensional similarity with A-beta, as the peptides begin to aggregate they would generate antibodies to A-beta oligomers as well. This would reduce the possibility of an autoimmune reaction to such a vaccine.