Skip to content

demylination

The nervous system is composed of a central and peripheral system. The peripheral nervous system is the part of the nervous system that consists of the nerves and bundles of nerve cells on the outside of the brain and spinal cord. A picture is worth a thousand words, look at the picture and let’s fast forward to myelin. Myelin is made of proteins, proteins are made up of amino acids. Groups of amino acids are called peptides, peptides can be as small as two amino acids.

Myelin insulates axons, the axon is a long threadlike part of a nerve cell along which impulses are conducted from the cell body to other cells. If myelin, made up of proteins, is damaged then messages are not transmitted to cells. Myelin proteins are highly similar, conserved, between species. And that is important to the study of polyneuritis equi (PNE).

When scientists (primarily the English and French) were investigating all manner of neurodegenerative diseases in people they got huge amounts of myelin from the spinal cords of cows because all animals have the same protein structure for myelin. This phenomenon is called conservation. Unfortunately, in 1984 an English cow developed strange signs that turned out to be the first recognition of Mad Cow disease.

Let’s digress, I like this part of history and what it gave us. The source of Mad Cow disease was animal feed. Specifically, animal feed contaminated with bits of protein called “prions”. First, disease was linked to sheep--but now some believe human bone might have gotten into the British animal feed. Doctors Alan and Nancy Colchester write that Indian and Pakistani peasants sometimes gather large bones from land and rivers to sell, and that "Hindus believe that it is essential for their remains after death to be disposed of in a river, preferably the Ganges. The ideal is for the body to be burned, but most people cannot afford enough wood for full cremation." During the 1960s and 1970s, the U.K. got a lot of raw material for fertilizers from Bangladesh, Pakistan, and India. Humans were known to have Creutzfeldt-Jakob disease, the infection passed to cows through ground-up bones in animal feed, and then the cows gave it back to people. Fearful of Mad Cow scientists turned to the horse as a source of tissue for their research. This vast body of knowledge gave us a leg up on polyneuritis equi.

There were some distinct differences in the equine protein and the bovine protein. It is molecularly heavier because it has 3 more amino acids. Ok big deal, I’m being thorough. But an unexpected result was horses have more, much more, basic myelin P2 protein (lets call it P2) in their central nervous system tissue. And P2 protein makes up 2-15% of the peripheral nervous system protein. The amount and distribution of P2 is a big deal because disease affecting this protein will be more apparent in horses. Myelin P2’s function is in lipid transport and storage in the cell responsible to myelinate nerves. The take home message here is that it was possible to test these myelin making cells (Schwann cells) for damage. If Schwann cells are damaged myelin production would decrease measurably.

Progressive neurological disease was induced in experimental animals by injecting myelin protein. This was important because a model using laboratory animals allowed controlled experiments. In the case of P2, an animal model for Guillian-Barre syndrome, a demyelinating disease of the human peripheral nervous system, was produced. If Schwann cells are damaged myelin production would decrease measurably in the model animals.

It wasn’t long before a connection was made to neuritis of the cauda equina, now called polyneuritis equi, a neurodegenerative disease in horses. Another similarity between Guillian-Barre and P2 neuritis was paralysis of the trigeminal and facial nerves. Cranial nerve involvement was also recognized in horses.

As an aside, this work was going on in 2005, some purified myelin protein became available. Horse neurologists got some purified spinal protein, injected it into a few horses, but didn’t get disease. Case closed. No more work on cauda equina induction by myelin protein. This small experiment closed the door to a model and produced a bias against this line of research that continues today. Did they use P2? And as you are going to see, different parts of myelin P2 can give different results in laboratory animals.

Back in Europe scientists purified equine myelin P2 and crystallized the protein giving them a highly refined molecule. There was another unexpected result of the P2 experiments that may relate to the small horse study. Myelin P2 was snipped (chemically) into peptides, different peptides and disease depended on the peptides that were used in the experiment. Some peptides did not cause disease whereas the whole purified protein did. The conclusion was that there must be a disease-inducing region of the protein. One peptide caused neuromuscular disease and weakness that would resolve, untreated. Another neuritogenic (disease causing) peptide consistently induced disease. Animals could become desensitized to disease-causing injections of the whole protein P2, but not to the neuritogenic peptide.

And, normal animals that were given blood cells (T-cells) from diseased animals (passive transfer) got disease! The disease was produced from immune cells from animals, not the protein itself. This raised several questions, but we’re going to cut to the chase. It wasn’t a malfunction of the cells that put myelin around axons. The myelinating cells did not change the proteins they made, nor did they change the capacity to remyelinate damaged axons in the face of disease.

What changed was the transfer of sensitized cells to a healthy animal. Researchers found that the sensitized cells induced the production of another protein, an immunoglobulin-binding protein on nerves that increased during the clinical deficit. The net result was now there was an increase in the ability of inflammatory cells to bind nerve cells. The nerve cells became the target of the body’s immune system.

What about that neuritogenic peptide? The neuritogenic peptide of P2, the myelin protein that wraps around axons, turned out to contain an inflammatory receptor that is recognized by the immune system. It participates in inflammatory reactions regulating cells that are involved in cell-to-cell signaling by molecules called cytokines.

In health, the neuritogenic peptide of P2 (and P2) are not exposed to the body’s immune system. When myelin is damaged and P2 is exposed disease ensues. Clinical signs manifest because the exposed peptides sensitize T-cells that stimulate a protein to bind immunoglobulin and make peripheral nerves a target of inflammation.

What’s myelin and why is it important?

For this we need some background.

When we think of the nervous system, we generally think of two parts, first there’s the brain, called the Central Nervous System, CNS. Then there is the part that passes information to and from the brain. We call this the Peripheral Nervous System, PNS.

Nerve impulses from the body are passed along nerves by shifting chemicals. It is a slow process. Some nerves are fine with slow impulses, the nerves that cause your intestines to move food along, for instance. Then there are the nerves that must pass information very quickly.

clip_image002Imagine placing a hand on a hot stove. It wouldn’t do for the information to take a long time to get to the brain then a long time for the brain to communicate to the muscles to move the hand. The quicker the better. To do that, the fast nerves have a method to move the impulses quickly down a nerve. What happens is that the nerve is insulated with a sheath, this allows the impulse to rapidly skip down the nerve jumping long distances across the insulated portions. This insulation is called a myelin sheath.

If the myelin sheath doesn’t work properly, the nerve impulses don’t go quickly. This makes the myelin sheath extremely important. To coordinate complicated actions such as walking, running, and chewing, for examples, muscle groups need to work in complex coordinated patterns. Each muscle or set of muscles needs to contract in the proper sequence and exactly on cue. Any interruption or delay has severe consequences.

Animals and people have these important rapid transit nerves. Unfortunately, this myelin sheath can be damaged. Generally, it is the body’s own defense system that attacks this myelin sheath. It’s the same defense system that helps you conquer disease, and ward off infections. The body has a way of defining what is part of the body, named “self”, and what doesn’t belong, “non-self” which includes invading bacteria, virus, or protozoa. The body routinely attacks any “non-self” invaders and destroys them.

This “self” identification system is usually perfect. Not always, though. If the body misidentifies something that should be “self” as “non-self”, the body attacks the “non-self” with all the resources it has available. In general, it’s the immune system that is called into action. There’s also the inflammatory system which works hand in hand with the immune system to destroy invaders.

So how can this happen?

The body breaks down the invaders into small pieces. The body identifies each of these pieces, classifies them as “non-self” and produces antibodies against these pieces. This way the body can wipe out the invaders by attaching antibodies at many places. Think of an antibody like a hook. Each antibody is very specific and only attaches to the piece that it is meant to attack. The body produces a sea of these antibody hooks. If the target piece exists, the antibody hooks automatically attach and the rest of the immune system uses these hooks to help destroy the invaders.

Now suppose the system that determines “self” and “non-self” gets mixed up- or even duped. Suppose an invader doesn’t get classified as “non-self”. The body will not recognize it as an invader and will not attack it. Some bacteria will take advantage of this, they’ll coat themselves with materials that the body doesn’t recognize as foreign. Parasites disguise themselves by changing the way they present themselves, varying the expression of genes. Sometimes parasites are masters of disguise and hijack “self”-proteins in order to manipulate the immune system. The result is that if the body misinterprets what should be “self” as “non-self”, those antibody hooks will attach to normal tissue and the immune system will attack it.

This can happen with the myelin. There are several ways it can happen. Once it does happen, the immune system will break down the myelin sheaths. When that happens, myelin fragments are released into the blood stream. We test the blood for antibodies against myelin to see if those fragments are present. If they are present, we know that there is a problem.

Suppose one has a horse and that horse develops a coordination problem. Suppose it doesn’t walk well, it staggers and falls. If we test the blood and find the myelin fragments, that goes a very long way toward telling us why the horse has a problem and what to do about it.

If myelin is attacked by the immune system, all the myelin is attacked. We won’t generally see a single problem, we’ll see an array of signs-diffuse over the body. We call this array of signs “polyneuritis”. “Poly” means many, neuritis means the nerves are affected. If we’re talking about a horse, we tack on the word “equi” so we know we’re talking about the horse.

Introduction and purpose

http://www.dreamstime.com/stock-image-marionette-myanmar-traditional-knight-horse-sale-shop-mandalay-image57297071Polyneuritis equi (PNE) is an uncommon neurological disease of mature horses and ponies.  Historically, PNE is recognized as untreatable.  Unfortunately, veterinarians aren’t trained to consider the possibility polyneuritis equi underlies the clinical signs seen in horses before untreatable disease manifests.  Our purpose is to present a set of white papers to inform and educate those interested in PNE. Eventually, we will combine the papers into an eBook.

If you are interested in polyneuritis equi, you probably have an afflicted horse or know a person that has a horse suffering from the disease.  Polyneuritis equi is rare, less than 50, 000 horses a year are diagnosed with PNE. That means there isn’t much information out there on this orphan disease and there are few groups conducting studies that will solve problems associated with PNE.

We collected information from published literature and studies that we conduct to write these white papers. Some of our studies were conducted under the most rigorous conditions, blinded and placebo controlled.  Some studies used cases from field veterinarians.  Some field cases could not be used, there just wasn’t enough documentation.  More often, case reports and collaboration allowed us to generalize, statistically evaluate parameters, and the body of data moved our work forward. Reports in the literature are for one or several cases and statistical analysis just isn’t possible.

It is our goal to share our knowledge, bring awareness to this disease in horses and license treatments that can control the clinicals signs of PNE.
http://pathogenes.com/w/polyneuritis-equi-is-an-overlooked-disease-part-1/
http://pathogenes.com/w/polyneuritis-equi-is-an-overlooked-disease-part-2/

Our view is that polyneuritis equi isn’t recognized early in the course of disease, when it is treatable. The current dogma surrounding PNE leaves no option other than humane euthanasia because clinical signs attributed to this disease relate to end-stage pathology. However, polyneuritis equi can have an insidious onset, horses present with subtle clinical signs that can be transient and they are progressive.  We believe these signs are treatable.  Frustratingly, these early signs can accompany other diseases and that makes diagnosis difficult. Scientists are looking at the cellular and molecular aspects of PNE and that gives us hope that we can achieve our goals.

This book is organized into ten stand-alone sections. An extremely brief but relevant overview of the nervous system forms the basis of understanding PNE. The section Relating clinical signs of PNE to pathology explains the underlying pathophysiology of disease.  The Case Reports section is from the few reported cases in literature and current cases we follow. The discussion is approached using a standard scoring system for PNE.  Clinical Scoring system for PNE highlights what may be relevant to evaluating field cases. The section on Considerations for a Differential Diagnosis of PNE explores the current thinking about diseases with similar signs.  This section will highlight the relationship between etiology and pathophysiology of disease.  Research Past and Present is a roadmap to current thinking about PNE. Diagnostic Testing for PNE is written to help alleviate the frustration with using inclusionary and exclusionary diagnostics to get to the root of disease. Treatment and a molecular based approach to treatment gets to our fundamental understanding: PNE is an inflammatory problem. The last sections Summary and How you can be involved in PNE research contains links to important support for the owners of horses with PNE.
http://pathogenes.com/w/consultation/
http://pathogenes.com/w/horse-owner-information/

 

 

 

lab opossum

Neurologic deficits are seen in horses with abnormal gaits, changes in behavior or signs that are limited to the cranial nerves.  These horses are difficult to diagnose because the list of etiologies that result in neurologic deficits is long. The list of disease-causing possibilities is long because animals have a short list of responses to infection and injury.

Long ago lowly slime molds and paramecia selected and perfected a group of chemicals that allowed them to achieve movement and the ability to communicate with other organisms. The chemical signaling mechanism that parasitic protozoa and animals use today is based on these evolutionary successes. Redundancy is built into the chemical structures and the way signals reach the target tissues. Redundant molecules also are used to turn off these systems. The basics of the pathways are common to all living things and they are primal.

Signaling pathways are also common to many tissues and they are ubiquitous throughout the body. An infectious agent will set off  a protective primal response in an animal. That response is called “innate immunity,” and this immunity results in signs that are non-specific to a single agent.  Vertebrates have many checks-and-balances regulatory pathways that control these common systems most of the time.  Sometimes the pathways become unregulated. As animals evolved to efficiently use the chemicals (cytokines) involved in protection against infections, parasitic microbes exploited the same systems. These highly successful disease-causing organisms use some pretty nifty  methods to evade or even hijack the innate immune signaling cascade.

The result is that an animal with neurologic disease has a limited repertoire of responses to several insults and they all look the same to the clinician.  We have accrued a vast amount of scientific knowledge about some diseases.  The diagnosis of these diseases is verified by  objective methods.  Some diseases have classically presenting signs that allow a veterinarian to easily rule them in or out. Of course, field experience is valuable in recognizing some classic signs. The diagnostician thins the list of possible causes of disease to the short list.

The short list for neurologic diseases that are found in horses often don’t have definitive diagnostics and that leads to using exclusion to help diagnose the cause. A diagnosis of exclusion  is a diagnosis of a medical condition reached by a process of elimination, which may be necessary if the presence cannot be established with complete confidence from the history, examination or testing. Such elimination of other reasonable possibilities is a major component in performing a differential diagnosis.

Diagnosis by exclusion tends to occur where scientific knowledge is scarce, specifically where the means to verify a diagnosis by an objective method is absent. As a specific diagnosis cannot be confirmed, a fall back position is to exclude that group of known causes that may cause a similar clinical presentation.  Polyneuritis equi (PE) is such a disease.  We have, along with our regulatory partners, put together a list of definitive tests and some that are exclusionary to pinpoint the tests most likely to give us the target population for our polyneuritis equi study.
http://pathogenes.com/w/polyneuritis-equi-is-an-overlooked-disease-part-1/
http://pathogenes.com/w/polyneuritis-equi-is-an-overlooked-disease-part-2/

The PE horse has evidence of neurologic disease that can be identified by neurological exam and those that are treatable have serum antibody against some specific proteins.  These are inclusionary criteria for our study.  The horse may or may not have antibodies against parasitic protozoa. To keep our study uncomplicated, we exclude horses with antibody against S. neurona, a parasitic protozoa that causes equine protozoal myeloencephalitis (EPM). Other exclusionary criteria are no recent history of trauma, no  recent respiratory infection (or a current vaccination for EHV-1 can satisfy this one).  Vaccination for rabies will exclude rabies as a cause.  Of course,  a horse would need to have a normal vitamin E level ( > 1.5 micrograms/ml serum).

Some treatments will exclude horses from our study.  They include anti-inflammatory agents within 3 days and anti-protozoal agents within a time frame that the anti-protozoal is expected to exert its effect.  Each expected effect from licensed anti-protozoal medication is different, some 30 days and some beyond 90 days.

Diagnostics are a major frustration for owners of horses with suspected EPM.  Owners spend many dollars on exclusionary diagnostics and don’t have an answer at the end of it.  Unfortunately, that is the nature of EPM. It is also the nature of Sarcocystis infections to trigger innate immune inflammatory cytokine responses that produce the signs associated with neurological diseases.  That means the horse can have two active pathological processes!  Because the nature of these diseases and the repair mechanisms that are associated with them use similar pathways, it is unlikely that there will be definitive diagnostics.  A good neurological examination by a veterinarian familiar with neurological disease and judicious use of diagnostics to form a short list  is the most successful path to health.
See: http://pathogenes.com/w/the-biology-of-sarcocystis/

Recently JAVMA published a letter to the editor and we’d like to give the author kudos!  He was disappointed that after years of using a particular product, a clinical trial revealed the drug was not effective.  He placed his faith on recommendations from trusted and respected colleagues, board certified specialists and continuing education speakers. They didn’t base their opinions on clinical data and that led to years of using a drug for pain in dogs that was just shown to have a complete lack of effect.  We all recognize the importance of new scientific evidence, lack of funding for many projects, and the burden on the people that provide treatment recommendations to know and understand the supporting data- or lack thereof-before dissemination of their opinion.  We concur with the statement in response to the letter that  many clinicians, if they looked at the information used in the daily treatment of patients, would likely be shocked to find out just how little clinical data are truly available to support current recommendations, or how many opinions are not based on experience or understanding-just a conflict of opinion.

We have taken a different path with our work.   Rather than patenting the intellectual property (IP) and  then licensing the technology, on completing research we decided to make our work available to those who needed it, and open the discussion.  Has it cost us in many ways?  Yes.  But then, I grew up in the scientific community at a different time.  Once upon a time, that would be the 1970’s, we shared data and discovery.  I was a lab rat back then, spending many hours with the fluorescent microscope-looking at how Chinese hamster ovary cells responded to ATP stimulation.  OK, I get it.  But the experience was good.  We shared samples and “stuff” that moved knowledge forward.  I used that experience to look at Leptospira and Moon Blindness, another immune mediated condition, for my Masters thesis.  Then off to veterinary school.  While I was practicing veterinary medicine in the field over the next 20 years, things changed.  There was the biochemical revolution that spawned the field of molecular biology, and PCR, and proteomics.  Suddenly things like PCR, had monumental value.  Everything was DNA and  genes.  Heck, whole organisms are patented-Neospora comes to mind, (quick update, the patent office won’t do that anymore).  To use the IP, one has to have a license and pay royalties.  The revolution was in the 80”s to the 90’s, the patent for PCR was filed on June 17, 1987.

When I returned in 1999 to work on my PhD, in molecular biology of course, everything went through the University’s Office of Licensing and Technology (OLT).  Universities recognized the value of IP.  No more sharing.  No more shared discovery.  Heck, recently I wanted to use a video to help veterinarians get CSF taps using a standing procedure.  I was told there would be a largish up front fee followed by a royalty due for each use…one fee for every veterinarian that viewed the tape. It was explained to me the video is like a Beatle’s song-and they wanted to hold my hand.  I made my own video and freely gave it out hoping to get veterinarians to collect CSF and support our Orogin trial. Oh, we were criticized, one veterinarian wanted the video removed and not shared because all those horse owners would be getting CSF taps on their own horses.

We persevered, partially due to the words of one of my PhD professors, Ellis Greiner, “Science is self-correcting”.  If I continued to do work, collaborate with those with true curiosity and an open mind, we would eventually see the day when our ideas would receive acknowledgement and open discussion.  Because that is what science is, open discussion of ideas. To that end we publish our work, in peer reviewed journals, and submit our papers and abstracts for presentation at scientific meetings.  We collected and shared samples, (DNA, RNA, organisms), and imparted ideas, traveled to NIH and Washington and beyond; we teleconferenced, (with Germany, the Netherlands, Japan, and even Kentucky), with those who  were open to the burden of review.  There was plenty of criticism from those who didn’t take the time to understand, review and hold an open discussion.  We carefully persevered, leaving breadcrumbs.  Sure, we patented some IP.  And made some IP  public so that  others can’t patent it.  The IP is available to everyone.

I think the long awaited day is dawning.  The discussion at the upcoming EPM special information session will have a panel discussion on the evidence of the role of S. neurona in infection and disease.  Is it a disease of low parasite burden-host mediated pro-inflammatory response or is disease a reflection of high parasite burden resulting in direct injury to the CNS? they ask.  We are presenting two papers, the most important is our recent data on polyneuritis equi.  Our work is important because our evidence supports, and may identify, one inflammatory pathway for disease.  The story we tell is one of relapsing disease and hope of treatment.  Our story identifies a protagonist molecule and the hero horse heading off with all four legs moving in proper order.  We look forward to SIG participants that will critically review what we have done.  We welcome suggestions or guidance on our errant interpretations. Perhaps some will return to their laboratories and repeat our work. Or collaborate without IP on the mind.  They may claim no funding-we are used to that because we are self-funded.  No big grants, although we’d gladly accept one.*  We are optimistic that this may be the moment.  We’ll let you know Dr. Greiner, and if our time has not come, we will continue to drop those breadcrumbs.

 

image

*Disclaimer: We are a for profit company.  We do not accept tax-exempt donations. Nor do we accept generously offered dowry’s from grateful horse-owning newly weds-although we gratefully appreciated the gesture.