ARTICLE IN BRIEF
Investigators develop an animal model of a neuropathy that mirrored the illness that occurred in pork plant workers after exposure to aerosolized brain tissue from pigs.
A strange illness took neurologists and Minnesota pork plant workers by surprise in the fall of 2007, after dozens of employees developed unidentified neuropathic symptoms. In the town of Austin, MN — known to tourists as “Spamtown, USA” — a total of 21 workers had presented with symptoms of pain, prickling and numbness, weakness, headache, difficulty walking; three more pork plant workers with similar complaints were identified in Indiana. All affected workers were working in an area where the pigs' brain cavities were cleared using compressed air.
Neurologists at Mayo Clinic initially hypothesized that the inhalation of aerosolized brain tissue caused the neuropathy symptoms. Now, in a study published online before print on Nov. 25 in the Annals of Neurology, Jeffrey W. Meeusen, PhD, of the department of laboratory medicine and pathology at the Mayo Clinic in Rochester, MN, and colleagues have created an animal model that confirms their hypothesis. The phenomenon, referred to as occupational autoimmune polyradiculoneuropathy, was replicated in laboratory mice using aerosolized pig brain tissue.
P. James Dyck, MD, professor of neurology at Mayo Clinic, one of the study authors, told Neurology Today: “I think this is very exciting work, which has a lot of implications. This neuropathy did happen in a special set of circumstances — exposure to aerosolized brain — that doesn't occur anymore since the plant stopped the removal of pig brains. So, this is not exactly like CIDP [chronic inflammatory polyneuropathy] or other recognized inflammatory neuropathies that occur in humans. Nonetheless, I do think the animal model of neurological disease occurring after exposure to aerosolized brain is very important as it gives us an understanding of inflammatory, immune-mediated neurologic diseases and the mechanisms by which they work.”
Dr. Dyck is currently working on a study, which is following the 24 patients who were initially affected with the occupational neuropathy.
Vanda A. Lennon, MD, PhD, senior author of the study and professor of immunology and neurology at Mayo Clinic, recalled that in 2007 scientists in the Clinical Neuroimmunology Laboratory recognized a strange pattern of autoantibody reactivity common to all of the sick workers from the Minnesota pork plant.
Our next steps were “to analyze the [patient] serums further to see if we could define specific antigens in the nervous system that the patients' antibodies were recognizing. It turns out that they recognize many different antigens. The most prominent ones were cation channel proteins, particularly voltage-gated potassium channel-related proteins. Potassium channel antibodies were detected in highest titer and in 79 percent of the patients,” Dr. Lennon said, adding that they had received patient sera at varying times after their exposure, which affected antibody levels.
For the current study, Dr. Meeusen and colleagues analyzed serum samples from the 24 patients who were subjects of the initial outbreak and were treated at Mayo Clinic. Control serum samples from 85 swine abattoir workers from Austin, MN, were provided by the Minnesota Department of Health.
Five days a week, mice were exposed intranasally to liquefied brain tissue. This was meant to mimic occupational exposure, Dr. Lennon said. “We found after many, many weeks that some of the mice started to produce the signature antibody,” she said. The investigators then doubled the dose and administered it to a new group of mice, twice a day, and “we found that the antibodies came up sooner and reached higher levels, so we were seeing a dose-dependent production of the signature antibody.”
They investigated the serum of the mice using the same tests they had used to test the patient serums in the clinical laboratory. “We found that the mice produced the exact same profile of antibodies that we recognized in the clinical laboratory in the patients. In the mice, 100 percent had the signature antibodies, 100 percent had very high titers of the potassium channel antibodies, 100 percent had myelin basic protein antibody, and 91 percent had calcium channel antibodies. But the titers were far and away highest for the potassium channel antibodies,” Dr. Lennon told Neurology Today.
The animal technician who was anesthetizing the animals twice a day noticed a strange behavior during induction of anesthesia in the mice that were exposed to the aerosolized brain tissue. “The anesthetic agent that we were using was called isoflurane and when we looked into this more closely in the literature, we found that isoflurane can affect potassium channels,” Dr. Lennon said. The most extraordinary thing we found in the literature, she continued, was that the “class of potassium channel that the patients' antibodies were binding to has the name ‘shaker.’ Basic scientists named mutant potassium channels in drosophila flies ‘shaker’ because those flies had a very strange shaking behavior when exposed to isoflurane anesthetic. It was unbelievable that we found this in the drosophila literature.”
Dr. Lennon said, though they could not verify whether the mice would have the same complaints and ailments as their human counterparts, the patients had very diffuse complaints, which were hard to localize. The most consistent finding was that patient MRIs showed swelling of the nerve roots as they exit the spinal cord to go out into the arms, legs, etc, she said.
“Dr. Istvan Pirko, one of the investigators on this project, who is expert in doing MRI on small rodents, was given some of the rodents that were exposed to the aerosolized brain and some that were exposed to mist without brain” without identifying which ones were which, and he identified the exact same swelling of the nerve roots in the mice that were exposed to the brain tissue mist and not in the mice that had the control mist. “We were convinced then that we had certainly solved the problem and this was undoubtedly an autoimmune response initiated by nervous system antigens in healthy brain tissue.
“Our findings implicate voltage-gated potassium channels as a significant target of the antibodies,” Dr. Lennon said.
Herbert H. Schaumburg, MD, professor of neurology and a neurotoxicologist at Albert Einstein Medical Center in New York, said that he spoke with neuroimmunologists at the Albert Einstein College of Medicine, and they all agree–d that this was an important study. “They don't have all the answers [in this paper], but our neuroimmunologists were quite impressed.”
“I think this study is a towering achievement,” he said. “This plant [in Minnesota] was located within 100 miles of Mayo Clinic, so they were lucky in that regard. The folks at Mayo responded very quickly and expertly…and developing this animal model is another coup for Dr. Lennon and her colleagues.”
The study showed that the disease the pork plant workers developed was humorally mediated, and not cell-mediated, as something like multiple sclerosis (MS) might be, Dr. Schaumburg said. “I think it would have been interesting had they discussed a bit more about the roots of the nasopharynx to the immune system because that's something that is not widely appreciated except by immunologists. The average clinician doesn't think about it — it stumped me initially, and I have neuroimmunologists right next door….This was the mechanism they [the researchers at Mayo] predicted. And they were absolutely right,” Dr. Schaumburg said.
The last line of this paper, he said, may just be the most important: “Our findings warrant caution in considering therapeutic trials involving autoantigen administration by the oronasal route as ‘tolerization’ therapy for autoimmune neurological disorders,” the authors wrote. This likely refers to therapeutic trials for MS, Dr. Schaumburg said, and this study should be treated as a “cautionary tale” against this usage.
“We're very excited to have an experimental model of potassium channel complex autoimmunity,” Dr. Lennon told Neurology Today. “The potassium channel associates with other neuronal axonal proteins and other synaptic proteins that are all involved with the function of the potassium channel. We're not sure exactly which components of the potassium channel complex the antibodies in the patients and the mice are binding to and we face the same challenge with patients who have spontaneous diseases of the nervous system involving potassium channels.”
“With the animal model,” she said, “we're going to be concentrating largely on the potassium channel story and making a monoclonal antibody probe to help us understand the immunobiology and the immunopathology of potassium channel autoimmunity. We don't expect to again see the disease that the slaughterhouse workers came down with but this has given us terrific insight into what is a spontaneous disease in patients.”
Dr. Lennon added: “Potassium channel autoimmunity is newly recognized over the last three years or so as one of the common disorders that our clinical laboratory encounters. This mouse model will be enormously helpful for studies in that regard.”
THE INITIAL DIAGNOSIS: UNRAVELING A MYSTERY AILMENT
The unraveling of the mystery pork plant ailment required some old-fashioned investigative work. Many of the affected workers were of Hispanic descent, and questionable immigration status, and this caused a delay in identification of the disease. “As they got sick, the workers just disappeared from the production line, and nobody was initially aware that there was an outbreak,” P. James Dyck, MD, professor of neurology at Mayo Clinic, explained.” It was a translator for the workers who finally came to us and said, ‘These people are ill and they all have the similar symptoms,’” Dr. Dyck told Neurology Today.
Many of the workers' symptoms sounded rather vague individually, he said, but when there was one employee after another showing up at our clinic with the same complaints, we recognized that this was unusual and that a reproducible pattern was emerging.
Then, when we started looking at the test results, we saw that there were stereotypical findings; on nerve conductions, there were signs of demyelination. Dr. Dyck said. “We biopsied several of the nerves of these patients and they showed inflammation of the nerve and demyelination of the nerve. Then Dr Lennon and co-workers from the neuroimmunology lab recognized there was a stereotypical biomarker that had not previously been seen on the paraneoplastic panel,” he added.
Drs. Dyck, Daniel H. Lachance, assistant professor of neurology, and other neurologists at Mayo Clinic, first recognized the outbreak, and reported their findings to the Minnesota Department of Health.
All of the Minnesota and Indiana individuals who were affected with this disease worked in the brain-removal section of their respective pork plants, where a tube would be inserted into the foramen magnum, the hole at the base of the animal's skull, and using pressurized air the brain tissue would be forced out, which would aerosolize it. The neurologists at Mayo theorized that the neuropathy symptoms were caused by these particles of pig brain tissue that then lingered in the air and were then inhaled by workers.
“As soon as the company found out, they changed their procedures and stopped exposing people to the brain tissue voluntarily,” said Dr. Dyck.
“So the question was, if you could take animals and take aerosolized brain and put them into an environment where they are breathing brain, could you make an animal model of this? And that's what we did, and that's what Dr. Lennon did,” Dr. Dyck said.
For past reports on the pork plant illness in Neurology Today, see:
* Alert to Neurologists — Help Needed to Help Solve Mysterious Neurological Illness at Minnesota Pork Plant: http://bit.ly/tjXHaS
* Neurologists Asked to Report Neuropathy Cases to CDC: http://bit.ly/tvwNDz
* Antibody Pattern Discovered for Pork Plant Illness: http://bit.ly/utwqPM
* Surveillance Continues on Pork Plant Workers Diagnosed with ‘Mystery’ Neuropathy: http://bit.ly/soQzDd
* Condition of Pork Plant Workers Improving After Developing Neuropathy: http://bit.ly/uU4REg.