Background to our thinking
Serum and plasma contain measurable innate and adaptive responses to diseases. For example, innate inflammatory molecules are produced by the body as a defense against pathogens. Innate molecules act to regulate inflammation.
Adaptive responses include antibodies that are disease-specific. These responses can be against foreign organisms. Sometimes the adaptive response is against “self” and that means this abnormal response is against host proteins and results in an autoimmune disease.
Previously we mentioned a “by-stander” response that could be causative for polyneuritis equi. In PNE the organism responsible for disease is long gone, but the detrimental inflammatory pathway is active. That means it is possible that the adaptive response drives the disease. Antibodies against organisms won’t diagnose polyneuritis—antibodies against myelin protein 2 are present in horses with PNE. Inflammatory biomarkers aren’t specific to diagnose PNE. It may be useful to investigate tissue specific markers that shouldn’t be there. Tissue specific markers can come from degraded or injured cells, for example neurofilament light protein (NfL) is present when there is axon damage. We would like to determine if biomarkers identify subsets of patient populations for our work in equine neurodegenerative diseases.
Uncomplicating plasma/serum biomarkers by purification
it worked with antibodies
In the blog, “The do’s and don’ts of Western Blot testing for EPM, and Sarcocystis sp” we hoped to convey the message that examining a complex cell homogenate by Western Blot was unsatisfactory to clearly identify horses with S. neurona infections. Techniques like immunoelectrophoresis and immunoprecipitation helped refine the complex mix of proteins and allow the detection of antibodies against a protein (or several proteins) found in horses with sarcocystosis. But these techniques weren’t good enough to identify diseased horses. Using the same techniques, we identified and isolated S. neurona-specific proteins and developed diagnostic ELISA tests improveing the specificity and sensitivity of the assays. We can distinguish S. neurona infected horses from those that are not infected and classify the infection by serotype. "EPM" describes an infection in the central nervous system and S. neurona encephalomyelitis (EPM) involves inflammation. Inflammation can be detected after the organism is gone. The disease has become chronic, it is post-S. neurona inflammatory encephalomyelitis.
Damaged axons release neurofilaments into the blood stream
There is considerable interest in neurofilament light (NfL) in human neurodegenerative diseases because it is a circulating marker of axon damage. In our ALS work, we came across NfL as a potential plasma biomarker for progressive ALS. It is dynamic, NfL changes quickly over time. NfL may serve as a potential indicator of effective therapy. Most exciting to us is that NfL is elevated up to 7 years prior to symptoms of clinical ALS, and if detected early, progressive disease may be prevented. That means NfL predicts subclinical disease. We want to know if subclinical axon damage is present in horses and can we prevent disease progression. In humans and mice it is the level of NfL that is critical, what will be useful in horses is a change in NfL over time—perhaps before and after treatment.
We linked plasma/serum MP2 levels in some horses with S. neurona SAG1 infections in a laboratory study. That was a useful first analysis.
Exosomes defined
Plasma contains extracellular vesicles, also called exosomes, which are extremely small lipid enclosed natural nanoparticles that can carry growth factors, cytokines, and nucleic acids. They are involved in cell-to-cell communication. Exosomes can target the peripheral tissue as well as penetrate the brain and spinal cord blood brain barrier. Exosomes carry NfL. Exosomes survive freezing and survive cryopreservation. They can be analyzed, best measured using ultra-sensitive single molecule array. We're are interested in what other markers they carry.
Can NfL be quantitated from serum/plasma?
We want to know if equine plasma exosomes increase the value of NfL as a prognosticator of PNE. Before we put the cart before the horse, there are questions that need answers. Can plasma NfL be accepted as a biomarker for PNE? When we say accepted, we mean accepted by FDA. Can we convince the Agency that a defined population of animals have PNE and the clinical disease is associated with levels of circulating NfL? We already said NfL may predict disease before horses have clinical signs. The Agency focuses on clinical signs. If there are circulating NfLs and no disease, at what point is NfL associated with disease? This conundrum has a solution that is solved at the bench!
You might want to review the blog “Why aren’t there any EPM tests?” Antibodies produced against S. neurona during infection, an event documented in over 80% of the equine population, may be a risk factor for EPM, but not diagnostic of the disease EPM. Ditto for antibodies found in the cervical spinal fluid, CSF. Looking at antibodies in the CSF wasn’t diagnostic because horses with a positive result didn’t match with finding the organism in the horses central nervous system. The gold standard for a definitive diagnosis of EPM is demonstration of the orgaism in the post mortem tissue. Carefully read papers discussing the cohhorts used to validate testes—if the description is inflammatory lesions, the horse probably has anti-MP2 antibody! EPM-diagnostic technology calculates the prediction, percent chance, that a horse sample is similar to a few (less than 20) animals with EPM. EPM is not definitively diagnosed by neurological exam, the diagnosis must be supported with diagnostic tests.
If circulating NfL precedes clinical signs, how can we make the association with disease and validate our assay? Based on the human and mouse data, the levels of NfL may be a key to treating and preventing ALS. Neurofilament light comes from diseased and damaged axons. They increase over time with progressive disease as more axons are damaged. The levels of NfL in mice plateau in late stage disease, in humans with ALS the levels increase and don’t plateau. What we need to determine is when the level of damage (by circulating NfL) is statistically associated with clinical signs of PNE, when does sub-clinical disease become clinical. That is our solution to the diagnostic for PNE conundrum.
Can you help us with our study?
We are investigating the association of NfL levels and clinical signs of polyneuritis with an exploratory study. The proposed study will identify at risk horses. If treatment shows that NfL decreases in concert with alleviating the clinical signs in horses successfully treated for PNE, that would support our position to use NfL clinically. If you would like to participate in our exploratory study, use the contact form on the Clinical Trial page.
What we are looking for are horses with clinical signs of PNE or horses that have had a NfL test within 180 days (6 months). The signs for inclusion in this study are expanded over the PNE study Fordyce Score because the Fordyce Score identifies end-stage disease. There are 15 signs we associate with PNE, 9 are late in the disease process. But, before we get you thinking about the signs, we need to explain how this study works.
There are forms for owners and veterinarians to sign and a clinical score sheet that needs to be completed by the veterinarian. We are investigating how dynamic NfL is before and after treatment; we also need some horses that don't get treated. An at risk horse is one that has multiple treatments for EPM, a horse that relapses after any treatment, or a horse with an unsafe gait and muscle atrophy. A good history helps us and we will ask you questions about the clinical signs you notice and when they appeared.
If you have a horse that may qualify for our exploratory study, contact us and we will send you the eligibility paperwork to get started. The website has an easy Let's Chat button that pings us in real time.
The NfL levels will be assayed in serum and plasma. A second analysis will examine the levels of NfL isolated from plasma and compare samples between the methods.
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