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, you are familiar with CRP.
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. The trouble with this disease pathogenesis is the organism responsible for disease is long gone, but the detrimental inflammatory pathway is active. Antibodies against organisms won’t diagnose PNE. Inflammatory biomarkers aren’t specific to diagnose PNE. However, it may be useful to investigate tissue specific markers that shouldn’t be there. Tissue specific markers can come from degraded or injured cells. Biomarkers in horses at risk for PNE are in high demand. We would like to determine if biomarkers can identify sub-sets of patient populations for our work in equine neurodegenerative diseases.
Cell-free DNA is possible
but too complicated because it isn’t tissue specific
We are working with researchers at Emory University to examine and map cytosine methylation (of DNA) in pathologic pathways recognized in ALS. That approach may show diagnostic markers, and hopefully lead to therapies. To reach those goals they look at circulating cell-free DNA (cfDNA), small fragments of DNA molecules circulating in the bloodstream. The cfDNA is released from dead cells and contains important genomic information that can potentially be associated with disease status. The issue is that the cfDNA is a mixture of DNA released from dead cells from different organs. Their challenge is developing computational methods to predict the source of the cfDNA.
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. Using techniques like immunoelectrophoresis and immunoprecipitation helped refine the milieu of interesting adaptive antibodies. But these techniques weren’t good enough to select diseased horses. However, using those techniques we were able to identify and isolate S. neurona-specific proteins and develop them into diagnostic ELISA tests that gave us much higher specificity and sensitivity. We can distinguish infected horses from those that are not infected and classify the infection by serotype. But EPM is a syndrome that involves inflammation.
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 disease. Because it is dynamic, it 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, disease may be prevented. If one monitored at risk horses with NfL, we may be able to prevent disease. In humans and mice it is the level of NfL that is critical.
We linked plasma/serum NfL levels in some horses with clinical signs of PNE, but not Sarcocystosis, in our sample population. That is a useful first analysis. We are wondering if we could improve our sensitivity and specificity for a PNE biomarker by concentrating the sample. It turns out the body may do this for us.
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.
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. First, 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. If there are circulating NfLs and no disease, at what point are the clinical signs 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 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. EPM-diagnostic technology is at the level of calculating the prediction, percent chance, that a horse sample is similar to a few (less than 20) animals with a known syndrome.
If the levels of NfL precede clinical signs, how can we make the association with disease and validate our assay? We believe, based on the human and mouse data, that the levels of NfL are a key to treating and preventing this heartbreaking disease. 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. That is our solution to the diagnostic for PNE conundrum.
Can you help us with our study?
We can investigate the association of NfL levels and clinical signs of PNE with an exploratory study. The proposed study will need to identify at risk horses. The identified population will be followed over time. We have a treatment for PNE. If treatment can show that NfL decreases in concert with alleviating the clinical signs in horses successfully treated for PNE, that would support our position. 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. The signs for inclusion in this study are expanded over the 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 that the owner and veterinarian will sign. We need to know how dynamic the biomarker is with and without treatment. We will be contacting your veterinarian with some offers of free NfL testing if we think your horse is at risk or has had NfL levels tested in the past. An at risk horse is one that has multiple treatments for EPM, a horse that relapses after 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 treatment, we can talk to you and send the eligibility paperwork to get started.
The NfL levels will be assayed using a commercial kit comparing serum and plasma. A second analysis will examine the levels of exosomes isolated from plasma on a validated ultra-sensitive single molecule array. The samples will be compared between the methods.
Our study population is fifteen animals with multiple assessments. If you want to participate, please use the contact form on the clinical trial page found on our website.