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The MP2/MPP ELISA in the differential diagnosis of polyneuritis (PNE)

Updated: Jan 4, 2023

The first time I spoke with Linda Turcotte, in 2015, she told me the tale of her old horse, Bay State Nestle. I thought we could help, he was listing, sidewinding to the right and had some muscle atrophy over his hindquarters. When I told her we might be able to help, she burst into tears. "Ms Turcotte, I just gave you good news! What's wrong?" "Tears of joy", she sobbed, "tears of joy!" He did respond to therapy, we know from our testing that he wasn't at the beginning of the disease process, somewhere in the middle. We just touched base with Linda, she used physical therapy and strengthening exercises and was able to ride him for about 4 years. At 27 years old she lost him to inoperable colic. As we told Linda, he is a horse we still hold in our hearts as does she.

The Sidewinding horse

We call it” sidewinding”. Sidewinding is descriptive of the gait of a hemi-paretic horse. There are other signs of a Sidewinder, paresis of the tail, bladder, rectum, anal and urethral sphincters. There can be loss of feeling around the anal region. Muscle wasting may be the most common observation by an owner. Also, the gait can be uneven. Associated with these signs can be paralysis or paresis of cranial nerves, drooping lip and ears, inability to blink and loss of muscles of mastication. Drooling and dropping feed can be a consequence of chewing-muscle dysfunction. Cranial nerve and behavior issues may precede clinical signs associated with involvement of the sacral and coccygeal nerves.

The pathology of Sidewinders

The pathology associated with a Sidewinder is inflammation in the nerve roots of the cauda equina and other involved peripheral nerves. The cauda equina is the group of nerve roots (nerves that leave the spinal cord between spaces in the bones of the spine to connect to other parts of the body) at the lower end of the spinal cord.

What is seen on histopathology from a Sidewinder is the destruction of the myelin of the affected nerves, demyelination amid inflammation. Demyelination is likely the primary pathophysiological event with degeneration, followed by fibrous tissue replacement of affected nerves. Interestingly, histopathology can also show remyelination! That means the destructive and reparative lesions are juxtaposed. If we slow down demyelination, we can let the horse heal with remyelination, preventing fibrous repair. If nerves are fibrosed, that is a negative game changer, we can’t fix that and that nerve is lost for use.

The science of Sidewinders

Polyneuritis in horses is similar to both Guillian Barre syndrome in people and its laboratory model. Equine polyneuritis was called a T-cell mediated peripheral neuritis by Rostami and Gregorian in 1990. They were able to induce severe experimental autoimmune neuritis with a synthetic peptide that corresponded to the amino acid sequence 53-78 (MPP) of the equine myelin P2 protein. This peptide was neuritogenic, it induced neurodegenerative disease. They also showed that this peptide contains a T-cell epitope, it consists of 8-11 amino acids. Sequence data confirms that this is an IL6 receptor.

We published our observation of amino acid homology between myelin protein P2 and epitopes of Sarcocystis neurona, SAG 6 and SAG 1. If you need a refresher on epitopes, re-read the blog Interpreting S. neurona antibody tests.

There are experimental models used to induce polyneuritis. These models allow scientists to study the process and figure out ways to reverse disease. Injecting equine myelin protein P2 into laboratory animals results in signs of polyneuritis. Horses with clinically and pathologically diagnosed polyneuritis have antibody to P2 protein. These observations led to an ELISA test for diagnosing polyneuritis thirty years ago.

The ELISA tests that detect clinical polyneuritis

Originally the P2 proteins used in ELISA tests were from equine or bovine intradural roots, the tissue was ground up into liquid nitrogen. After some washing and purification steps, the protein was solubilized and concentrated. We improved the antigen for diagnostic testing by making a recombinant equine myelin P2, MP2. At the same time, we made MPP, the sequence that corresponds to the T-cell epitope found on MP2. These two proteins, MP2 and MPP allow us to detect equine myelin protein 2 auto-antibodies by ELISA.

We are using the putative disease-inducing peptides to detect anti-myelin proteins antibodies in horses with suspected peripheral neuropathy. Is there more that we can investigate? Perhaps, yes.

Can we stage the progress of polyneuritis with testing?

Rostami and Gregorian showed that induced cellular immune responses to the MP2 protein was milder than induced responses to the t peptide MPP. They showed that the proliferative activity to myelin protein activity could be lost with repeat challenge in their experiments. The big word for this is the animal becomes amnestic. It no longer works to induce disease. The immune system loses its “memory” of this response. However, activity to the MPP peptide is not affected on multiple challenges.

Each animal has its own timeline in the pathogenesis of polyneuritis. Some horses progress quickly, and some progress more slowly in the disease process. The time of progression may be due to the nature of the antigen exposure eliciting the inflammatory event. We know that 3% of horses react to some vaccine components and these components elicit polyneuritis. We suspect that chronic exposure to S. neurona can cause polyneuritis. We may make the correlation with S. neurona SAG 6 exposure and polyneuritis, given enough samples. We base that hypothesis on the sequence data we generated. We know that perpetuating a parasitemia with some drugs induces polyneuritis. We know that some immune stimulants are a poor idea.

Here is how we think polyneuritis looks through the MP2/MPP-ELISA lens.

As disease is initiated, there are no antibodies to equine myelin 2, the test is MP2-/MPP-. The horse doesn’t have a demyelinating polyneuropathy, it is early in the course of disease. As nerves are inflamed and damaged, the timeline of disease is relative to the individual horse, and there are antibodies produced against MP2, the test results are MP2+/MPP-. The polyneuropathy is now demyelinating.

As disease progresses even more, the test result is MP2+/MPP+. The horse still has a demyelinating polyneuropathy. What is different is that there are antibodies against the MP2 protein and the response is also against the neuritogenic peptide, or IL6r. Remember MPP is a cytokine receptor and causes more severe disease. As disease continues to MP2-/MPP+ there are still auto-antibodies to myelin protein, the neuritogenic region. But now the horse is amnestic to MP2, it still has a demyelinating polyneuropathy. At end stage disease, the test becomes negative for both proteins because nerves are repaired with fibrosis. After nerves are fibrosed, treatment doesn’t work. The nerves are lost to use.

It is good to know when disease is early or late, the test results can be similar. It is good to know when disease is early enough to treat and perhaps prevent from becoming chronic and even progressing to an untreatable disease.

Monitoring NfL may also be useful

As nerves are damaged and disease progresses, we detect axon damage by measuring neurofilament light (NfL) levels. You can refresh your memory by reading Can PNE be diagnosed with NfL found in serum, plasma, or exosomes. Axon damage is a later event in the pathogenesis of this neurodegenerative disease, after auto-antibodies are produced. In cases, we observed, that the higher the MP2/MPP values are, the more likely the NfL test will be positive. That is difficult for field veterinarians to observe, this is a rare disease and they will only see a few cases in a lifetime. We analyze many samples and compare results from hundreds of cases.

Something to remember is that the same T-cell epitopes are present in other places in the body. For example white blood cells have these Il6 receptors. We intend to down-regulate the T-cell receptor with our therapies, in fact, not only IL6r but also TNFα. We want to do this in a precise way because T-cell receptors are uniquely involved in many signaling pathways in health and disease. While immunosuppression may work in the short term, and necessary in specific instances, the remyelination process won’t be preferred. It is important to know what drugs increase receptors, which ones decrease them, and to what extent the receptors are modified by dose of the drug. These are the areas of our research.

Human research indicates that blocking IL6r was protective in multiple sclerosis, an inflammatory demyelinating disease of the central nervous system in people. The work shows that the disease process is facilitated by a subset of T cells called Th17 cells, these cells are highly proinflammatory. Significant immune signaling molecules have biphasic responses. In molecules we investigate, a low dose of drug is anti-inflammatory and a higher dose is proinflammatory. The take home message is to be sure and know the actions of the medications you are giving. And remember that dose is critical.


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