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NflightPattern recognition is an important process that emphasizes the the identification of data regularities in a given scenario. People are natural pattern-seekers. How many times have you heard things happen in three’s?  Humans are hard wired to recognize sets of three events, even if science proves the events are unrelated!

Students are trained in veterinary school to become observers.  The trained veterinary-observer develops into a diagnostician after years of clinical practice.  The art of practice is a combination of science and observation enhanced by continued questioning of the objective outcomes one effects with treatments.  A clinician adds tools to his/her toolbox over time.

Bioassays are tools that are available in the clinicians toolbox. Our passion is neurodegenerative diseases in horses and people.  We bioassay a lot of samples from laboratory experiments and clinical submissions.  Sometimes, we compare our results to other laboratories by running tests that evaluate similar disease conditions, using different testing platforms, in order understand the differences in case interpretation. Our head-to-head tests (duplicate samples that are run on different platforms) figure into our interpretations.

After results are obtained, recognized patterns are passed along to the field veterinarian. We are not immune to event-bias and overcome that tendency by using algorithms that are coupled with statistical analysis. The systems analysis procedure produces, in a finite number of steps, the answer to questions we pose. For example, is this horse likely to relapse?

Our algorithms sift through the data we get from tests and red flag results to which we should pay attention. A clinician may evaluate a case a year, or perhaps several cases over several years.  Each case presents an individual interpretation of disease that makes field medicine enigmatic.  We enter tens of thousands of results into our algorithm and give the succinct final analysis to the veterinarian. The data to feed the algorithm come from serum bioassays important to the diagnostician.  At the very least, test results offer objective parameters to veterinarians on which to base their treatment decisions. 

There are some new things to consider. And these topics were discussed in our recent Zoom meetings.  We hope you joined in!  To assist with clinical analysis of horses with neuromuscular disease, several bioassays are used.  The term for the assayed molecule is “analyte”. Some of the assays detect antibody against foreign proteins as the analyte and include surface proteins of Sarcocystis neurona, (remember, these are unique to S. neurona and mutually exclusive to serotypes of each neurona species), or Neospora hughesii.  Anti-toxin against Sarcocystis fayeri is an analyte and if disease is stimulated an autoimmune reaction follows.  Two areas of the myelin protein P2 are analytes in our “Sidewinder” panel.  There are bioassays for antigens that include C-reactive protein (CRP) and neurofilaments (NfL) molecules.

The principle difference one should consider between detecting antibodies versus detecting antigens as analytes is time frame for a change in the levels found in the serum.  Some antibodies won’t decrease for months after they are produced against an antigen (foreign agent). Another consideration is that a naïve animal will show a reduction in antibodies much sooner than an animal that is “experienced” with the infection.  That means prior exposure is important information that should be taken into consideration when examining a case.

An animal that is chronically exposed to an organism in the environment will maintain antibodies due to new gut infections and it can be tough to interpret these test results in the face of acute disease. That means prevalence of disease is an important consideration in analysis of these cases. The life-cycle of the organism is an important consideration. Does the organism complete the life-cycle in the host, as in S. fayeri or is it unable to mature, as in S. neurona infections in horses? Does the organism change it’s repertoire of antigens presented during infection, as does S. neurona? Did you consider that one infection, with a particular serotype of S. neurona may not protect against another serotype?  Are antibodies produced against a serotype of S. neurona protective against infection but stimulatory to the inflammation that can become dysregulated?

Antigen molecules, such as CRP, an acute phase protein, are useful. CRP is elevated in inflammation when it is associated with the cytokine IL6.  There are several innate plasma buffer systems that regulate IL6—>CRP, but occasionally the system becomes dysregulated. Chronic dysregulation can lead to an inflammatory condition and chronic inflammation can lead to an autoimmune disease.  The presence of CRP indicates inflammation due to an infective process, however it isn’t specific to one particular organism. CRP is quick to be produced but in our analysis, it doesn’t decline in days.  It can take weeks.  Horses have several conditions that can keep the CRP value elevated; they include encysted parasites or hind gut ulcer disease. 

One very dynamic marker in neurodegenerative disease is neurofilament light (NfL).  Neurofilaments are cytoplasmic neuronal proteins highly expressed in large myelinated axons. The levels of NfL expressed in body fluids are in proportion to the degree of axonal damage (inflammatory, neurodegenerative, traumatic, and cerebrovascular diseases). The utility of NfL is based on the rapid decline of levels, within days, of effective therapy! The difficulty with another measurable neurofilament antigen, heavy chain, is that heavy chains can clump in some cells and clumps aren’t detected in live bioassays.

Our algorithm for suspect cases of EPM first evaluates levels of surface antigens from S. neurona, 1, 5, and 6, as well as CRP. If there is supporting history from bioassay, we can determine if the horse is experienced or naïve.  Further analysis can determine if there is chronic exposure to the parasite in the environment.  Our algorithm gives less attention to N. hughesii and Borrelia infections unless there is a high prevalence of disease (the algorithm uses zip code for the regional association). There is an association with S. fayeri and anti-myelin protein P2 antibody.  For example, 786 horses with circulating S. fayeri anti-toxin and of those, 610 also had circulating anti-myelin protein P2 antibody. This is important to evaluate on a case-by-case basis, but points out that some equine muscular sarcocystosis can result in a demyelinating polyneuropathy.  There are treatment implications to these data.

It may be useful for a clinician to distinguish between demyelinating, (has antibody against MP2), and non-demyelinating polyneuropathy because treatment and prognosis will vary between these presentations.  While NfL responds quickly to successful treatment, this marker can be present in both demyelinating and non-demyelinating polyneuropathies. A panel of assays are useful to determine the pathogenesis of disease.  Take advantage of our neurodegenerative disease panel by downloading our submission form.  The data will be submitted into our algorithm and our interpretation returned to the veterinarian.

 

 

 

 

horseontrailFinding biomarkers that reflect the amount of peripheral nerve damage (peripheral neuropathies) and that the biomarker will quickly drop in value in response to to effective treatment are desired goals.  The tools we need for developing biomarkers for equine neurodegenerative diseases are not available.  These tools include a laboratory model for each neurodegenerative disease, putative treatments, and a money bin.

There is an alternate path leading to biomarker development and that is the horizon we are chasing.  The biomarker quest project identifies natural cases of disease with neurodegeneration followed by evaluating the data from those cases. Sifting through the data is a process of eliminating the negative, selecting the positive, and interpreting the in-between.  I hear a jingle in there somewhere! Generally diseases follow a typical course, or pathogenesis.  Interpreting enough cases points toward the direction we should take and where to concentrate our assets. Often clues to a direction come from previous researchers.

Our assay to detect anti-myelin protein antibodies was described in the literature over 25 years ago.  Researchers used data from horses with polyneuritis equi (PNE) to study human neurodegenerative diseases. Since then novel developments in molecular biology, such as learning how to fold proteins and identifying sequences (genetic code) that are key elements in inflammation, refined the bioassay’s.  The anti-myelin protein antibody tests affirm a specific disease pathogenesis for PNE in humans and horses that involves myelin degeneration, exposure of the immune system to the myelin protein, and production of anti-myelin protein antibodies.  A boon to our work is being able to glean information from human literature and apply it to our horse cases.  An appropriate application from new human neurodegenerative research is an assay for serum or plasma neurofilament light, NfL.

Neurofilaments are the major cytoskeletal proteins of neurons in both the central nervous system (CNS) and peripheral nervous system (PNS).  Neurofilaments form a structure (lattice) composed of light, medium, and heavy chains. Damage to nerves releases fragments of the neurofilament proteins into the central nervous system fluid (CSF) or circulatory system (plasma or serum). The elevation of neurofilaments in the CNS was observed in patients with amyotrophic lateral sclerosis over 20 years ago. Other neurodegenerative diseases also result in the release of NfL.  Abnormal levels of neurofilaments are associated with the disease process and are not necessarily specific for the etiology.

What is interesting is that human patients with demyelinating and axonal forms of an inherited neuropathy exhibit a slowly progressive, axonal degeneration at a constant rate.  Patients with the inherited disease were monitored for NfL over time and the plasma concentration of NfL increased when values were compared to age matched, healthy controls. It was noted that plasma NfL (pNfL) concentration increases with advancing age in some normal subjects.  An increase in disease severity was correlated with pNfL and pNfL discriminated between patients with the disease versus healthy controls. There are sub-types of this inherited, human neurodegenerative disease and NfL was elevated in all forms of disease. In humans, NfL is elevated in several other neurodegenerative diseases.  Because NfL isn’t specific to etiology it wont’ be useful by itself for a diagnosis; however, because it is a dynamic measure of axonal damage, NfL promises utility for monitoring  a response to treatment. There’s a path we intend to follow!

NfL may be an important biomarker when evaluating a disease known to show no CNS involvement.  In these cases,  changes in concentration of NfL would be attributed to peripheral neuropathies. One caveat is that NfL may be elevated in a T-cell proliferative disorder because neurofilaments are expressed in human T lymphocytes. In human studies, NfH (the heavy chain) was not correlated with disease severity. One proposed reason for the lack of correlation between disease and NfH concentration is that NfH aggregates form and these protein clumps lower detected levels of NfH in fluids.

In humans, and horses with polyneuritis equi,  the gold standard for measuring disease severity for patients is a clinical score. There are several limitations to the clinical score including the scale and a ceiling effect for the most severely affected individuals. It is worth mentioning that a therapeutic benefit for neurodegenerative diseases is to stop progression of disease and is most useful early in the disease process.

A blood biomarker may be more sensitive to multifocal peripheral nerve disease, such as polyneuritis equi.  If proven, that means that when neurofilaments are detected in the serum and disease is supported by the clinical exam, treatment can begin before severe irreparable damage occurs. The most useful interpretation of NfL bioassay is a change in an individual over time or with treatment because intrasubject variability is not expected. The intersubject variability in NfL concentration is a factor we are examining between treated and not treated horses.  Our data may indicate how one horse responds is more meaningful than how groups of horses respond.  That’s why we have our biostatistician on instant redial!

Our goal is to determine the responsiveness of NfL concentration in the clinical course of PNE and relate the assay to demyelination using a second bioassay that detects antibodies against exposed myelin proteins. We have some data to discuss if it relates to a specific case you are working with, the data we have will be at least a year from publication.  If we can help give us a call, we can guide your test selection.

Sometimes we are asked what leads us down a specific road or what inspires an idea.  It is generally the need to find a solution to a problem that is an “outlier”.  An outlier is a case or situation that doesn’t fit the rest of the data.  Everything isn’t a bell-shaped curve, although that is where we start.  As the Covid19 Pandemic sweeps the country, everyone is becoming savvy with interpreting clinical disease and seropositive test results. We thought it would be fun to let you interact with our data as we develop our newest idea.

Our recent work with amyotrophic lateral sclerosis (ALS)  led us to neurofilament light, NfL, a possible biomarker for disease in equine neurodegenerative disease. Serum NfL is a validated marker for ALS as well as a potential pharmacodynamic biomarker that is relevant to ALS therapy development.  It is possible that we could measure NfL in horses and use the result to select candidates for a field study as well as determine a response to treatment.  NfL can change in days with an effective therapy.

We started the analysis of banked serum from some of the cases on which we consulted and will look for patterns.  Our objective is to identify disease conditions and clinically validate serum NfL as a prognostic in our work. When we are satisfied we will send all the data to our biostatistician, the real number cruncher. Of course, he always says we have a biased data set-we look at horses that veterinarians suspect equine protozoal myeloencephalitis or polyneuritis equi.  We have some “gold standard” negative sera and even sera from other countries in which some diseases aren’t present.

What does early, exciting work look like? Here is the result.  Nine sera were selected and examined against many controls.  We detected NfL in horses. A high value in our clinical case analysis was 46.04 ng/ml and the lowest 2.78 ng/ml.  We will select more cases and update our graph so you can follow along! Most likely the graph will get more complicated as we add horses with and without disease, and before and after treatment.

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If we selected a sample from a horse that you submitted you already got the result by email.  If you would like us to add your sample to our early analysis, please give us a call at the lab.  We are all here hunkered down and continuing to offer you cutting edge work.

biomarker gifAn exciting prognostic and potentially pharmacodynamic serum biomarker may be commercially available for horses very soon.  We are borrowing some technological advances from ALS (amyotrophic lateral sclerosis) research.  Recently, we discussed biomarkers that are used in human neurological disease monitoring when we met with researchers at the University of Miami ALS Center (UM ALS).  They have validated neurofilament light as a prognostic and potential pharmacodynamic biomarker for ALS.  Neurofilament light (NFl) was not only useful for identifying disease but changed quickly in response to treatment!  We are investigating the utility of this marker in horses with polyneuritis equi, PNE.

Biomarkers are molecules that can be measured in serum and it is feasible that neurodegenerative diseases such as polyneuritis equi (PNE), equine motor neuron disease (EMND) and equine protozoal myeloencephalitis (EPM) could be identified using a simple serum test. Particularly important is NFl may respond quickly to appropriate therapy.  In mice and men, NFl levels decrease in days. No one has yet published data from horses with PNE.

The UM ALS Center addressed several issues. Which assay should be selected? Do NFl and pNfH (phosphorylated neurofilament heavy) yield the same information or should both be measured? And how do levels relate to disease progression? The UM ALS group found that others made statements about prognostic and potential pharmacodynamic utility of neurofilaments.  The studies were single center, measured either NFl or pNfH, but not both, used only a single assay to quantify NF levels, evaluated blood or CSF (but not both) and examined either survival or decline in function, but not both.  In a UM ALS paper, the authors used a multi-center study with head-to-head comparison to three different assays in serum and CSF and also examined the utility of these markers for determining a treatment response.

Particularly exciting was the ability of serum NFl to predict the clinical course of disease. Similar to other assays, absolute values vary between patients. That means it is not useful to hang your hat on one value.  Serum NFl levels remain stable in individual subjects when measured over time. In patients with neurodegenerative disease the values increase significantly above values seen in non-diseased people.  NFl values increase and plateau in some species.  That is contrasted to levels that continue to rise in other species.  An efficacious treatment will result in NFl values that decrease. What we heard was that NFl would be clinically useful if there are detectable changes (decrease) in levels following exposure to a therapeutic agent.

To move these markers forward in human disease there needs to be an effective therapeutic to test (in motor neuron diseases). They propose using NFl in phase-2 human trials to reduce sample size and most promising, using serum NFl as a pharmacodynamic biomarker. They found the potential utility of serum pNfH more “nuanced”. It didn't add prognostic value to readily available clinical parameters and serum NFl. The bottom line was that serum NFl, but not serum pNfH, may be considered a clinically validated prognostic biomarker for human ALS. And NFl may have value as a potential pharmacodynamic biomarker that should be incorporated into ongoing and future phase-2 drug trials.  Again, they await an effective treatment for ALS.

What can we horse owners and veterinarians take from this work?  We don’t know until we look at a sample size that can be statistically meaningful.  We are accepting serum samples from horses with possible PNE (the veterinarian must complete the PNE examination form).  We will determine the levels of NFl before and after a treatment. The NFl values will be reported the day after we receive the sample.  Give us a call for details on this exciting program!

The CLIFS notes on neurofilaments

NflightA neurofilament makes up part of the cytoskeleton that is specific to neurons. Neurofilaments are similar to cytoskeletal elements in other cells, but they are made up of a different set of proteins. They are found in the axons, the long extensions of neurons that transmit nerve impulses away from the cell body toward other cells.

A neurofilament is an intermediate filament that is made up of at least two of the three different types of specialized protein subunits. These three types are called Neurofilament light (NFl), Neurofilament medium (NF-M) and Neurofilament heavy (pNfH); each neurofilament consists of NFl and either NF-M or pNfH. When neurons or axons degenerate, neurofilament proteins are released into the blood or cerebrospinal fluid. Immunoassays of neurofilament proteins in cerebrospinal fluid and plasma can thus serve as indicators of axonal damage in neurological disorders.

Here is a picture of a muscle cyst in a horse.  It is caused by a parasitic protozoa and is commonly found on post mortem exam.  You may be surprised to know that we can detect these cysts without killing the horse!

Sarcocysts in horse muscle are the final destination of infections caused by Sarcocystis fayeri.  This is a very common pathogenic protozoal infection called EMS (equine muscular sarcocystosis).  More than 80% of the horses in the United States are infected.  Muscle cysts aren’t generally  associated with clinical signs although disease is recognized in thin, debilitated, or stressed horses.  These infections are sometimes associated with muscle loss and weakness in a performance horse.  Rarely, they are associated with death. `The clinician  differentiates EMS from other neurologic diseases because it affects muscles, is slowly progressive, and doesn’t respond to common treatments. When infected muscles are ingested S. fayeri cysts release an enterotoxin that can make dogs and humans sick.  Consuming raw horse meat is the cause with different outcomes, both dogs and people can get enteritis from the toxin.  When the cysts are consumed by dogs the parasites can finish the life cycle and the dog  infects more horses. People get food poisoning, or enteritis, but the parasite can’t complete the life cycle.

Until recently the only definitive diagnosis for EMS was post-mortem examination.  The cysts are identified by histopathology using muscles from the tongue, esophagus, or skeletal muscles.  To recognize disease a cyst has to be present on that particular slide so it can sometimes be missed.  More often the cysts that are observed on slides are unrelated to the cause of death. Recently, Japanese scientists did an exhaustive evaluation of a case of food poisoning following raw horse meat ingestion by picnic goers.  They elucidated  the molecular signature of S. fayeri toxin. We were able to use that signature to identify S. fayeri infections in living horses by using an assay for anti-toxin. An advantage to the assay is that it can also differentiate sarcocystosis caused by S. neurona or S. fayeri. S. neurona infection in horses doesn’t terminate in the production of sarcocysts so the molecular signature of the toxin and surface antigens 1, 5, and 6 are uniquely different.  S. fayeri does have shared molecular markers with S. neurona, these are the surface antigens called 2, 3 and 4. The toxin is common to other cyst forming pathogenic protozoa but fortunately the only one of significance to the horse is S. fayeri.

We compared the histologic diagnosis of EMS in 32 horses to serum bioassay and found the results were very similar.  The histopathologist used 3 slides (one each from a different tissue in a horse) and we used a serum sample to bioassay for anti-toxin.  The diagnosis was correct in 78% of the horses by histopathology and 74% of the horses with the serum analysis.  The obvious advantage is that the bioassay is in the living horse.  There are several reasons that the analysis differs between the tests.  The toxin is present before cysts are formed so the serum test will detect infected horses before cysts could be seen on histopathology.  The animal will have cysts in the muscle about 77 days after the initial infection so the first phase of the infection isn’t detected on post mortem exam.  It is possible that some cysts don’t produce the toxin.  When the toxin isn’t produced differences in pathogenicity between strains may exist. Cysts that don’t produce the toxin won’t be detected by the serum test.  This isn’t a limitation on the serum test because the toxin is responsible for the clinical signs.

Another test that is useful for neurodegenerative disorders is the anti-myelin protein antibody test.  These tests were developed over thirty years ago specifically using horse samples and tissues. The horse tissues were used in myasthenia gravis (MG) research, a neurodegenerative disease in people, anti-myelin protein antibody is a hallmark of MG. It was prudent for researchers to substitute horse tissue instead of bovine tissue when Mad Cow disease was identified in Europe, this was great for equine researchers!  The molecular signature for myelin protein was identified and diseased horses were soon recognized as positive reactors when they had a disease called polyneuritis equi.

The anti-myelin protein assay  improved when myelin protein was mapped for epitopes.  Epitopes are reactive sites on proteins and and are the signal that reagents in tests detect. Another improvement in disease detection came along with the molecular biology revolution. Scientists realized that myelin needed to be folded properly.  When proteins are denatured they are like string, epitopes sit side by side.  Visualize a string, the two ends are far apart. However, when the protein (or a string)  is properly folded, conformational epitopes are recognized in the assay.  If you fold your string in half the two ends can form a unique epitope where the ends touch.

Another advance in assay development was determining that an inflammatory cytokine receptor was one of those epitopes found on the end of the folded myelin.  That was a useful discovery to figure out how the disease worked.  When the mechanism of disease in known, that is the pathogenesis of disease, a treatment can be designed. ,Fortunately the inflammation recruited by the cytokine doesn’t destroy the myelin producing cells it is active on the myelin proteins.  That means when the inflammatory disease is properly treated, and treatment is instituted early enough,  the nerves are repaired.  If the disease is untreated the nerves will be protected from inflammation by scar tissue.  Scar tissue won’t allow the proper function of the nerve. The assay can detect antibody against the myelin (diseased nerves) before there is irreparable damage. It is necessary to know what kind of inflammatory pathways are triggered, there are different types of inflammatory pathways that require different drugs.

Bioassays are useful when they detect markers that are characterized and understood.  They can be life-saving.