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ureaA chemist will tell you that molecules changed the world.  The book, Molecules That Changed The World, by KC Nikolaou and T Montagnon will have you believing it by page 333. The synthesis of urea is credited as not only the earliest contribution to organic synthesis, “but as the single most important blow to the vestigial theory” held in the 1700’s. “Despite using the terms daily, chemists often forget that the classifications of inorganic and organic compounds originally arose from the theory of vitalism, which divided matter into two classes based upon the response of the material to the application of heat.” Plants burn, rocks don’t.

What was apparent, and explained by Nobel prize winner (1902) Emil Fischer, is that principles such as  asymmetry, the intrinsic importance of organic chemistry in understanding biological mechanisms, and the art of extracting, identifying, and synthesizing naturally occurring compounds and their analogues for medicinal purposes are important. These concepts have been the foundation of our understanding how to treat horses with equine protozoal myeloencephalitis and polyneuritis equi.  Our story is similar to the story of Aspirin, the most successful medication in history, because treatments we use are steeped in history, synthesis, and hormone chemistry.

Aspirin’s story is using the medicinal properties of a natural product that is optimized through subtle chemical manipulations, and began 3500 years ago.  Egyptian physicians advocated salicin, in herbal preparations of myrtle bark, as a remedy for rheumatism and back pain. Humans are not the only ones that seek this chemical from the bark of trees such as willow.  Supposedly, forest monkeys and apes nibble on the bark of these trees for pain relief.

The first ever clinical trial used pulverized, dried willow bark that was given in a tea or beer, to 50 patients, and published by Edward Stone in 1763. Advances came in 1828 when Johann Andreas Buchner removed tannins and other impurities obtaining a relatively pure sample which he called salicin.  Ten years passed until Raffaele Piria made the next advance by hydrolyzing salicin, splitting it into its sugar and phenolic components.  Later, he succeeded in oxidizing the hydroxymethyl group of the phenolic fragment to make salicylic acid.  In 1853 another chemist, Charles Frederic Gerhardt prepared acetylsalicylic acid and with that step, Aspirin was born. The best was yet to come.

In 1859 Hermann Kolbe synthesized Aspirin by heating the sodium salt of phenol in the presence of carbon dioxide under pressure and commercialized the process, laying the foundation for todays pharmaceutical industry. What came next is the realization that salicylic acid wasn’t a panacea because there were unpleasant side effects.  The effort was on to modify the chemical structure in order to obtain a derivative that might be devoid of the undesirable side effects such as foul taste, mucosal membrane irritation, vomiting, and ulcerations.aspirin

The breakthrough came in 1897 when a chemist at Bayer, Felix Hoffmann, synthesized acetylsalicylic acid. Bayer now owned the miracle drug they trademarked as Aspirin. Aspirin was used in experiments to determine the mode of action  increasing the knowledge of pain and inflammatory mechanisms. Prostaglandins were identified in 1935 and the cascade of biochemical reactions associated with inflammation were revealed. Finally, in 1971 three scientists linked the ability of Aspirin to inhibit a critical enzyme step in the prostaglandin pathway.

The protagonist Aspirin is heroic.  However, the story isn’t complete without considering the antagonist.  New research spawned the new generation of ‘super-analgesic’ drugs such as Celebrex and Vioxx that didn’t have the side effects of Aspirin.  Yet, unexpected side effects were noticed, like heart attacks and strokes with long term use.  The popular drug Vioxx, with $2.5 billion dollar sales, was removed from the market in 2003.  Isn't it ironic that Aspirin is frequently used to reduce the incidence of myocardial infarction and stroke? The positive effects of Aspirin on heart disease isn’t through the prostaglandin pathway, but by inhibition of another product from the arachidonic acid cascade, thromboxane A2, a hormone discovered in 1975.

Aspirin is a synthetic chemical derived from natures molecule that has profound effects on prostaglandin mediated inflammatory pathways and in an alternate pathway, inhibits the clotting cascade driven by a hormone. Our path, clinical trials using hormones and synthetic chemical-mimics to modulate pathologic inflammatory pathways (non-prostaglandin mediated) are similar to Aspirins story.

Presently, we are concerned with chemical modifications of molecules. As you can see from the work on Aspirin, there are unexpected effects with the slightest modification of a molecules structure.  Structural changes can be non-enzymatic (degradation) and/or enzymatic.

Non-enzymatic modifications are made by facilitating chemical breakdown products by manufacturing processes, storage conditions, or compounding.  Non-enzymatic modifications of a molecule can induce anti-inflammatory effects,  make some drugs inflammatory, and in some cases elicit no effect at all.  Outcomes depend on the modification that is made to the molecule, intentional or not.

We know that a simple amino acid change, such as a valine, in the protein sequence of a hormone can elicit profound effects on receptor binding, increasing potency and duration.  Receptor binding affects the outcome of a treatment.  We are also looking a the effects of chemical modifications on hormone receptor binding. We will put all this together in a story and should have quite a story to tell.

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.





raccoon dogIs anyone untouched by the Covid19 pandemic?  Some of our critical research projects are on hold, mostly due to no PPE for personnel to handle study lab animals and also travel restrictions.  We are fully functional at our lab.  We are using any extra time to evaluate data and share our findings with veterinarians and horse owners through Zoom meetings.  Check out the Facebook meeting posts to find the links and join us in our Zoom room.  Until then, here are some facts that help us focus on what is important. The information is up to date as of mid-April.

The SARS-CoV-2 strain of a novel Coronavirus appeared in Asia in 2019 and is known as Covid2019…Covid19 for short.  SARS is an acronym for severe acute respiratory syndrome caused by a betacoronavirus that is transmitted by contact with fomites.  Fomites are infectious materials transmitted by contact with respiratory droplets or body fluids.  Unfortunately for us this virus is transmitted by airborne particles.  Symptoms include fever, headache, body aches, dry cough, hypoxia (lack of oxygen), and usually pneumonia. For you molecular biologists,  SARS-CoV are enveloped, positive-sense, single-stranded RNA virus that infect the epithelial cells within the lungs. SARS-CoV-1 binds the ACE2 (angiotensin-converting enzyme 2) receptor and infects humans, bats, and palm civets. We learned about SARS-CoV-1 in 2003.  There are seven coronaviruses that infect humans. In the 2003 SARS outbreak 9% of patients with confirmed cases died, the hardest hit population was over 60 and over 50% of these people died.

The evolution of  SARS-CoV is interesting.  There were two different strains of SARS-CoV isolated in China in 2003, indicating there were separate species crossing events.  The virus came from wild animals sold as food in a market in Guangdong, China and it was isolated from asymptomatic masked palm civets.  This virus was able to infect humans, raccoon dogs, ferret badgers, and domestic cats. The virus could not be maintained in tissue culture and it did not infect bats until the virus was altered in 2008 in a laboratory to contain a human receptor binding domain. That indicated to researchers that bats could be asymptomatic and serve as a natural reservoir for the virus. And a note for Julie, the raccoon dog, also known as the mangut, tanuki or neoguri, is a canid indigenous to East Asia. It is the only canid species that can climb trees.

The recent ancestor for all coronavirus existed in 8000 BCE. Some say the virus existed 55 million years ago. They coevolved with birds and bats. Bats are the reservoir for alpha and betacoronavirus  while birds serve the same role for the gamma and deltacoronavirus. The global range allowed evolution and dissemination of this virus family.

The path to human infection is from leaf-nose bats to horseshoe bats, civets, and finally to humans. Bovine coronavirus evolved from rodents and crossed species to equids. It was in the 1890’s when bovine coronavirus jumped to people, and the likely cause of the “flu” pandemic that same year.

Of interest to us is that human corona virus (OC43) causes respiratory infections and is suspected of playing a role in neurological diseases. Mouse hepatitis virus (MHV) is a coronavirus that causes epidemic murine illness that has a high mortality.  Prior to the discovery of SARS-CoV, MHV had been the best-studied coronavirus both in vivo (in the animal) and in vitro (in tissue culture) as well as at the molecular level. Some strains of MHV cause a progressive demyelinating encephalitis in mice which has been used as a murine model for multiple sclerosis.

Human coronaviruses vary in risk factors and disease severity.  Some (MERS-CoV) are deadly to more than 30% of those infected and others just cause an irritating, common cold.The human coronavirus discovered in 2003, SARS-CoV-1 has a unique pathogenesis because it causes both upper and lower respiratory track infections.

Six species of human coronaviruses are known.  One species is subdivided into two different strains, making seven strains of human coronaviruses altogether. Four of these strains continually circulate in the human population and produce the generally mild symptoms of the common cold: OC43, HKU1, HCoV-229E, NL63. These viruses cause about 15% of commons colds (the majority of colds are infections caused by rhinoviruses).

Four coronavirus strains have a seasonal incidence occurring in the winter in temperate climates.  There is no preference towards a particular season in the tropics.  Three strains (two species) produce symptoms that are potentially severe; all three of these are β-CoV strains: MERS-CoV, SARS-CoV-1, SARS-CoV-2. The two SARS-CoV strains have occurred in the last 17 years, both from Chinese wet markets.

How does all this affect us? The Center for Disease Control has updated its guidelines for essential workers that have been exposed to people infected with SARS-CoV-2. Workers that do not feel sick are able to return to work so long as they take their temperature before leaving the workplace, wear a face mask at all times, and practice social distancing.

The department of Homeland Security and Health and Human Services outlines how removing shelter-in-place type restrictions after 30 days would lead to a second, very high peak in the number of cases and deaths.  The number of expected deaths is 300,000. The spike in deaths was projected to occur abut 150 days after lifting the stay-at-home restrictions. We are staying put so you can expect more Zoom meetings.

Its concerning that United States hospitals are seeing a shortage of antibiotics, antivirals, and sedatives required by patients on ventilators.  Increased demand and effects of the pandemic has halted production of some drugs. The University of Minnesota has analyzed the supply chain and identified 156 drugs that could go into shortage in the next 90 days, but they have not released the list.

The numerous studies and information on chloroquine on the outcome of COVID-19 patients are so far inconclusive or halted due to a high number of complications. A vaccine trial, called the Solidarity Vaccine Trial will evaluate multiple candidate vaccines at the same time, against a placebo. The expectation is the researchers will have results in 3-6 months due to high enrollment and an adaptive design. We will be in line for that vaccine when it is ready.

Pathogenes laboratory wishes you and your families well.  We will continue to be here to help you with your horses by testing sera, developing new avenues of research and answering your questions about neurodegenerative diseases. Give us a call or join us through Zoom meetings.

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.


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.