At the 2008 Days of Molecular Modeling Conference in Sweden, biomedical researcher Trevor Marshall sat on the edge of his chair listening intently to a talk presented by Adriano Aguzzi of the University Hospital of Zurich. Aguzzi was discussing research that confirmed much of what Marshall had long suspected to be true about prions – small, potentially infectious molecules that are hypothesized to be made only of protein.

The protein structure of a prion.

Prions have been implicated as the cause of a number of diseases in a variety of mammals, including bovine spongiform encephalopathy (BSE, also known as “mad cow disease”) in cattle, and Creutzfeldt-Jakob disease (CJD) in humans. All thus-far hypothesized prion diseases affect the structure of the brain or other neural tissue, and all are considered untreatable or fatal by mainstream medicine.

Although prions have been studied to some extent in the lab for decades, very little research has delved into their actions when inside the human body (in vivo). Thus, many theories put forth about how prions might cause or contribute to neurological disease have been largely speculative.

That is until Aguzzi’s work. At DMM, he presented a series of excellent experiments that studied the actions of prions inside the tissues of animals or human beings. His data confirms that after prions enter the body, they are able to pass through essentially all of the body’s barriers such as the mucosal barrier and the blood brain barrier. They can also bypass both the innate and adaptive immune system. This means that, if a person or animal consumes food containing prions, the small protein molecules can easily pass from the gut all the way up to the brain. As Aguzzi describes, the timing at which prions make their way from the gut to the brain is incredibly precise. For example, when his team exposed a large group of mice to prions in their food, the prions reached the brain in 220 days (plus or minus 3 days) in every single one of the rodents studied.

Years ago, Aguzzi was puzzled by the fact that, if his team infected the lymphatic systems of healthy mice with prions, the prions did not migrate from the lymph system to the brain, meaning that the mice remained healthy. However, when the team wiped out the ability of the mice to secrete immune cells called activated B-lymphocytes, prions in the lymph system were suddenly able to migrate to the brain and cause disease.

Dr. Adriano Aguzzi

This information percolated in Aguzzi’s head, and it was not until decades later that he suddenly realized that, in many infectious states, B-lymphocytes migrate away from the lymph system in order to deal with pathogens in other organs. Under these conditions, so many B-lymphocytes migrate away from the lymph system that the system resembles that of the mice whose B-lymphocytes had been completely knocked out. In somewhat of a “eureka moment,” he realized that such B cell migration is a telltale sign that the host is suffering from a chronic inflammatory disease.

The implications of this connection? It is now accepted that prions are not infectious by themselves, but are only infectious in the presence of chronic inflammation.

Since it is now increasingly understood that chronic inflammatory diseases are the result of infection with an intraphagocytic, metgenomic microbiota (L-form, biofilm, and other persistent bacteria, collectively called the Th1 pathogens), Aguzzi’s work strongly implies that it is only when prions infect a person or animal that harbors the Th1 pathogens that they become effective infectious agents.

Such thinking contradicts previous assumptions about prions, in which the small protein molecules were considered capable of causing infection on their own. Whereas, before Aguzzi’s work, it was simply assumed that prions could fold into tightly packed beta sheets (in which their polymers are connected by hydrogen bonds) on their own, it is now understood that chronic inflammation must be present if such folding is to occur and lead to disease. The altered structure of a folded prion is extremely stable and accumulates in infected tissue, causing cell death and tissue damage. Such stability means that prions are resistant to denaturation caused by chemical and physical agents, making disposal and containment of the particles very difficult.

Indeed, this new view on prions was confirmed by a study in which Aguzzi’s team induced chronic hepatitis in mice. The disease caused the animals’ livers to become inflamed. The mice were subsequently fed prions, and when the rodents’ organs were dissected after death, the team found that the prions had spread directly from the gut to the inflamed tissue in the liver. When the same experiment was performed on a group of healthy mice without hepatitis, no prions were found in the rodent livers after death.

Marshall has several theories about how the Th1 pathogens might interact with prions in order to facilitate their ability to cause disease. Perhaps the Th1 pathogens transcribe enzymes which can actively fold prions into the specific shapes in which they become infectious. Or perhaps proteins, peptides, or lipids from the Th1 pathogens transform human enzymes or proteins into forms which tend to fold the prions and allow them to damage tissues. Nobody knows for sure at this point.

This has led Marshall to believe that prions are just one of the artifacts produced by the [Th1] pathogens. “Something has to make them change shape in the first place, even if they ’snap shut’ after that. Prions may well propagate the damage being done by the Th1 pathogens more quickly, especially if they are injected, ingested, etc. but, unless the Th1 pathogens are there, the prions are not infectious, and do not spread to the brain,” he states.

A number of other experiments conducted by Aguzzi support the hypothesis that prions become pathogenic only after interaction with the Th1 pathogens. Recently Aguzzi’s team travelled to Slovenia in order to research the effects of prions on sheep, animals that can develop a prion-induced disease called scrapie.

Analysis of frontal cortex samples from the brain of a patient who died of non-cerebral causes (upper row); patient suffering from Creutzfeldt-Jakob Disease, CJD (lower row). Prion protein deposits are visible in the samples of the patient with CJD. Photo from Nature Reviews Neuroscience by Aguzzi.

Aguzzi proceeded to separate sheep into two groups. One group had a chronic viral inflammatory condition called mastitis, while the other group did not. When the milk from both groups of sheep was examined, prions were only secreted in the milk of those sheep who had mastitis. In these cases, macrophages were also secreted in the milk, some or all of which were certainly infected by the Th1 pathogens. This solidified the hypothesis that inflammation of the mammary glands (which occurs in mastitis) is necessary if prions are to infect the mammary glands and end up in an animal’s milk.

A second study by the team examined the milk content of healthy cows that had been infected with the BSE prions that cause mad cow disease. Since the cows were healthy and did not suffer from any inflammatory conditions (they had been kept in what Aguzzi describes as “five star hotels for cows”), the BSE prions were not found in the milk of healthy cows, nor did the cows actually develop mad cow disease.

Results of the above studies were confirmed by yet another experiment that tested the urine content of mice for prions. As Aguzzi describes humorously, several researchers on his team spent two years of their lives collecting rodent urine samples. Some of the rodents were made to suffer from chronic kidney inflammation called nephritis. When prions were introduced into the inflamed kidneys of these mice, they were excreted in the urine. But if prions were introduced into rodents that did not suffer from nephritis, the animals’ urine remained prion-free.

Further research by the team showed that, if inflammation is induced in any excretory organ of the body, prions are excreted in whatever substance the organ excretes.

But perhaps the most exciting aspect of Aguzzi’s research is the fact that his team has developed a florescent stain, called a Luminescent Conjgated Polymers (LCP) stain, that is able to illuminate the polymers created by inflammation. According to Marshall, this stain may be capable of identifying not just prions, but also the protein biofilms (made of protein polymers) that protect the Th1 pathogens in the cytoplasm of infected cells.

Because LCP is made of flexible polymers itself, when it binds bacterial polymers of different shapes, it emits different wavelengths of light depending on the geometry of the polymer under study. Thus, scientists can learn to associate different color wavelengths with bacterial polymers of certain shapes and sizes. For example, during his presentation, Aguzzi shows a slide in which a protein polymer stained with LCP is emitting two different colors. Because of the color difference, he hypothesizes that each end of the protein has a different structure.

The color of the wavelengths emitted by the LCP stain also change in response to the strength between the bonds of certain molecules, or the pH of a a particular environment. The varied spectrums of light emitted by the LCP stain in response to a certain protein or bacterial polymer can also be conveniently compressed into a chart that effectively represents the polymer’s shape and properties.

Indeed, thanks to the stain, Aguzzi presented two slides that Marshall believes show the Th1 pathogens inside various cells. One slide shows the protein polymers indicative of the Th1 pathogens inside the cells of patients with Parkinson’s disease. Another stain reveals amyloid protein in the heart. Amyloid proteins are insoluble fibrous proteins that, according to Marshall, are created by the Th1 pathogens.

If further research proves that Aguzzi’s stain is indeed able to reveal the biofilm surrounding the Th1 pathogens, the stain may allow Autoimmunity Research Foundation to conduct a study that could definitively show the presence of the Th1 pathogens in the blood of people with Th1 disease, as well as the absence of the Th1 pathogens in the blood of patients who complete the Marshall Protocol.

“[Aguzzi's stain] can use spectra to distinguish polymers. This may well be the diagnostic (screening) tool we have been looking for,” states Marshall.

Cellular inclusions in Parkinson’s Disease, which look quite similar to the Th1 pathogens when viewed under phase contrast microscopy.

Essentially, the stain could be applied to a sample of blood. Th1 bacterial proteins would show up as bright spots. By measuring the amount of flourescence given off by the blood, the extent of bacterial load could be estimated. For example, a particular number of photons could be correlated with X number of bacteria. It’s very likely that such staining would reveal that even the blood of people considered to be healthy harbors a certain number of Th1 pathogens, and that nobody is spared from the effects of these persistent bacterial forms.

Clearly, if the flourescent stain used by Aguzzi’s team can identify the Th1 pathogens, the implications of such a discovery are far-reaching. However, the fact that prions cannot cause infection on their own, but only in the presence of inflammation, also offers great hope for the elimination of diseases caused by prions.

If the Marshall Protocol is used to wear away at a host’s Th1 bacterial load, then the person or animal should reach a point at which the Th1 pathogens can no longer facilitate the folding of prions into infectious agents. So by effectively reducing bacteria-induced inflammation, the MP may make render prions harmless.

Does that mean the MP might be able to prevent mad cow disease? Perhaps it’s possible that if the MP were ever adapted to treat animals, it could stop the accumulation of infectious agents that would foster chronic inflammation and the spread of prions.