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It will soon be our 20th Anniversary at Pathogenes!  We’ve paved new ground and published most of our work because I grew up in the “publish or perish” era.  Now days science is all about technology transfer, it’s “patent or perish”! You may have participated in some things we invented that didn’t get off the ground, looking at down-regulation of molecules in response to S. neurona infection. I never cease to be amazed that this assay predicted the degree of illness a horse would have in a few months. It was expensive and cumbersome to run and ultimately discarded.  How about our horse-side antibody assay?  Who could predict that would strike out?  In a mere 15 minutes you knew if antibodies were present in serum or CSF.
I loved that one. Here is what it looked like:Dipstick assay

We took some side trips, identifying a cell that could support the replication of millions of PRRS virus in vitro, we grew 7 species of Eimeria in cell cultures resulting in a vaccine that would eliminate all those hen houses, and developed monoclonal antibodies, one  against S. suis and provided a local University with a much needed pig-herd vaccine. We collaborated with folks in Germany (looking at pigeon sarcocystosis), Norway (an acquired polyneuritis in Norwegian horses) and Canada. Sarcocystosis and Toxoplasmosis in dogs and cats didn’t escape our interest. But our heart belongs to people dealing with neuromuscular diseases in horses. We remember Amy and Ty from years ago, their picture shows the bond we have with our horses.

We have a paper, in press, that blazes a new trail for others to follow.  The paper explains the rationale for detecting S. fayeri toxin in horses, if you want the punch line--we detect cysts in live horses! We aren’t giving up our quest for new treatments or determining how disease progresses in horses, mouse or man. Horses have always led the field in neuromuscular research, spinal nerves were an important source of myelin for research in Myasthenia Gravis and Guillain Barre syndrome.  As most of you know, we got our ideas and direction from the work done in the 1980’s.  Wish we could say it was our idea and our hard work, but really, we  just pieced together a logical story from published literature.

Our current  directions are identifying the inherited genes that predict late onset ataxia in horses and defining the expressed protein environment in response to specific treatments given to animals with neurologic disease.  We are interested in the molecular targets of drugs in the nervous system and why they act for weeks after a dose.  We are surprisingly close to figuring that one out! You'll need special tubes to participate in these studies, contact us to find out if you can be a part of this research.

What stands out in our work is our ability to give clinicians information about their case and how it compares to hundreds of similar cases across the country.  We document all the information derived from our assays and connect the data to the feedback we get from you.  It’s time to catch up and let us know about your horse.  Even if our last contact was years and years ago, you are still in our system and gentle on our minds. Why, we may even have a picture of your horse if you sent one! We’d like to hear from you.  Here are some useful links:

To give us your update please use this form:  https://docs.google.com/forms/d/e/1FAIpQLSc_4HNIEtaCYI4qgt8X99OHypTSOhaJYv0iXO4kXJZ0hqIUPA/viewform?usp=sf_link

A veterinarian can give us information about a horse with neurological disease: https://docs.google.com/forms/d/e/1FAIpQLSeLU8t3ROPE9nrPjhSuReg4C2ZpmsCrYPyzmMQ8yuwVgMfong/viewform?usp=sf_link

A horse owner can give us information about a horse with neurological disease: https://docs.google.com/forms/d/e/1FAIpQLScB0jNG9dHpnNc2AhOIn8KpNVxMFc4QZ2YvSIJHyrh86hZAFQ/viewform?usp=sf_link

If your horse was treated we’d like a post-treatment update: https://docs.google.com/forms/d/e/1FAIpQLSevDKLdGqXFdr_JrH2W8h_4xbgBYJAxXH4Ydt-_vKUjlffy5A/viewform?usp=sf_link

If you suspect polyneuritis equi this form is appropriate:https://docs.google.com/forms/d/e/1FAIpQLScv4wQlg1pW13VPCuZHaG3yGsxQDnF5C2FrHkjgCH7mrT6Swg/viewform?usp=sf_link

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.

We’ve been reading a lot about amyotrophic lateral sclerosis (ALS) lately.  One thing is abundantly clear and that is there will be no single treatment to cure this disease.  There are inherited forms of ALS, fALS, and then there are sporadic cases, sALS. Our take on the overall picture of ALS is that no matter the inciting event at some point there is a final common pathway…that is inflammation.

Dogs get late onset fALS.  We suspect horses get fALS as well.  Is someone looking for it?  It would be very rare and most likely, with no treatment options, the horse would be euthanized before a clinician would think of ALS. Horses get subclinical inflammation presenting as a peripheral neuropathy.  After some time passes the neuromuscular disease progresses to the lower motor neurons (polyneuritis equi) and then it affects the upper motor neurons if the horse lives long enough. Diagnosis is the big issue here, to recognize a case of equine ALS.

The sporadic form of ALS seems more insidious…the sub-clinical pathways probably take a long time to surface into clinical disease.  The dysregulated systems that are forefront in human fALS are being targeted with specific small molecules-it is unlikely they will target sALS patients.  Even after years of failed attempts to find the cure for ALS the approach is the same: one path-one drug. New molecules come along, and so far they failed, even if they showed promise in mice that develop ALS.

The newest platform is skipping the animal step and moving right into people with promising drugs or small molecules. The entry criteria for the studies using novel small molecules targeting fALS are not available to sALS.  The treatments are super expensive. These approaches will never translate into an equine therapy.

Back to our hypothesis that it will take multiple drugs to combat presumptive equine ALS (eALS).  And this is a huge problem.  If one decided on a cocktail that was effective, the licensing process for the therapy would be impossible to get through FDA.  The effectiveness trial is mind boggling…we are suggesting the cocktail may involve 5 drugs at a minimum. The safety trial alone would cost at least a million dollars, our one-drug safety trial was nearly a half-million alone.

How does one start to evaluate therapeutic cocktails for a rare disease such as ALS?   Initially an animal model of the disease is necessary.  And a non-subjective test to evaluate if the disease is present in the animal.  And then one needs to identify the drugs that could be beneficial based on a firm understanding of the disease process. After all this is complete, it would be possible to approach FDA.

After our experiences with a simple and direct treatment using two well known drugs with specific actions for a specific disease with a defined and useful animal model, we can absolutely say the task for licensing a putative five drug ALS cocktail is insurmountable.

Ah, but a man’s reach should exceed his grasp, Or what’s a heaven for? (Robert Browning)

We have an approach that just may be doable.  And that is finding single therapies that hit multiple targets.  Our idea will rest upon finding a useful diagnostic test to ascertain effectiveness.  We aren’t afraid to try our approach in ALS models and compare the results to multiple drugs that target dysfunctional ALS pathways. Of course, testing multiple drugs together is a huge step that is outside the box thinking.  Out of the box thinking is what it will take to tackle ALS.

Our reach is big.  We’ll let you know how it goes.

We have been a bit quiet lately and that’s because a friend is very sick.  He has amyotrophic lateral sclerosis (ALS). In order to help him, the Pathogenes team needed to catch up on the particulars of this most horrible disease.  There is no cure for ALS and quite frankly, no useful treatments.  It has taken us a few months to read stacks of papers and gather a team of experts. And form a plan.  Our plan isn’t simple and it isn’t easy.  It’s complicated and can change.  As we formulate and fine tune our approach to stopping the progression of ALS our experts review our work for scientific accuracy and feasibility. We don’t mind that no one has heard of what we propose.  We will test our hypothesis and march forward. 

You can find a lot of information on ALS on the Web and we won’t repeat it here.  You can check out our ALS tab for some links we found useful. It is worth pointing out that there is familial ALS (fALS) in which genetics plays a big part.  Only 10-15% of patients with ALS (PALS)…these folks are acronym heavy…get fALS.  Most people get spontaneous ALS (sALS), close to 90%.  There are different camps and controversies concerning the pathways involved in disease and how to approach reversing the progress of motor neuron (MN) death, but an overriding theme of those that investigate ALS is the compassion and sharing of information.  Basically, ALS is associated with an enzyme mutation (superoxide dismutase, SOD) and/or other mutations that cause MN death.  Motor neurons make muscles work. You need motor neurons to breathe.

What stirred us to take a break from our bench work and communicate with our EPM-centric following is learning that dogs get spontaneous ALS!   Dog-ALS is associated with the enzyme superoxide dismutase, the mutation is in SOD1.  The onset of dog disease is late in life.  That means there are ALS cases in people, dogs, and genetically engineered mice.  Horsey people realize there are unknown causes of spontaneous neuromuscular disease in horses that cause them to be wobbly.  These horses can progress until they can’t get around and are euthanized.  In some cases there is no diagnosis and no treatment.  We reviewed two of these diseases.

Equine Motor Neuron Disease (EMD) is an acquired neurodegenerative disease in horses affecting the lower motor neurons of adult horses. The disease is characterized by the onset of abnormal nerve function and muscle wasting resulting from the deterioration of motor neurons and myopathy. Horses from 15 months to 25 years old can get EMD. EMD is considered to be a multifactorial disease, however a dietary deficiency in vitamin E is considered to be a major predisposing factor in its development. This is largely related to when horses have a decreased antioxidant capacity leading  to accumulation of free radials and that results in oxidative damage to the ventral motor neuron cells. Could a decreased function of horse-SOD be a factor?

No one knows what causes Equine degenerative myeloencephalopathy (EDM) that is a diffuse, degenerative disease which primarily causes damage to the horse’s spinal cord. EDM is considered to be an advanced form of neuron-axon dystrophy. EDM may have a genetic basis. Horses can develop EDM and equine motor neuron disease (EMD) at the same time and in association with an underlying vitamin E deficiency. Horses with EDM show clinical signs of a general proprioceptive  ataxia-“I don’t know where my feet are” and an abnormal base-wide stance while at rest. Horses will usually start to show signs of EDM when they are 6 to 12 months old. Horses with mild cases of EDM may present as performance-related problems. At first the condition produces subtle signs, being nothing more than  "clumsy" but ataxia slowly progresses as clinical signs are usually slow and insidious. Ataxia signs will become more apparent and worsen over time. Paralysis and spastic muscular movements will become more evident, until late stages where the horse is unable to get up from laying down without assistance. The only way to get a definite diagnosis that a horse has EDM is by conducting post-mortem examination shortly after death.

We’d like to test horses for EMD and EDM for antibody against neurofilaments.  It’s a serum test.   Once we have some results we will share them with everyone.  If we can demonstrate that horses also have a form of ALS, and why shouldn’t they?, we can start looking at treatments in this species.  If you have a horse with a diagnosis of EMD or EDM send us serum and we will test it.  Be sure and have a firm diagnosis.  Not just a “This is on the differential” or “It’s nothing I’ve seen before!”. Testing is expensive and we’re proposing to pay for it.  We need to know and have proof that several diseases have been ruled out.  You can email us for the form to send in a sample for this specific testing until we put a submission form up on our site. We want late cases as well as cases that are early.

The funny thing is that the deeper we delve into ALS and our approach to treatment, some paths seem to converge. As the ALS community gets closer to understanding the pathophysiology of disease we are finding common roads.  All roads seem to be leading to Rome after all. 

At some later date when we have some good news for your ALS friends we will ask you to share what we find.  Until then, we are going back to work.

Jim

 

Muscle fasciculations are visible, fine and fast contractions of fine muscle fibers that occur spontaneously and intermittently. Injury to central or peripheral nerves can result in muscle fasciculations. The pathophysiology is different for different sites of the injury. It is thought that most fasciculations have a location away from (distal origin) the motor nerve in normal animals as well as patients with motor neuron diseases.

Fasciculations are known to be associated with hypersensitivity of denervated muscles and they are observed in some diseases such as amyotrophic lateral sclerosis in people. In horses the fine tremor of the face, muzzle, or lips is best associated with West Nile Virus infection.

Other triggers of fasciculations are progressive spinal muscular atrophy, neuromuscular junction disorders, electrolyte disorders, systemic diseases and some medications. Even healthy animals can have fasciculations-these are usually located in the forearm or the eye-lids. Fasciculations were thought to be a prelude to the onset of a progressive or lethal disease that involved the lower motor neuron. However, benign fascicular syndrome has been described in young healthcare professionals.

In normal individuals physical exercise, stress, fatigue, and caffeine abuse can cause or aggravate twitchy muscles. A diagnosis of benign fascicular syndrome can be diagnosed after five years. A benign diagnosis is made when there isn’t a progression to motor neuron disease and that takes 5 years.

Muscle tremors are abnormal and are motor disorders, although fasciculations are not classified as motor disorders. Some genetic diseases of the cerebellum associated with motor disorders are accompanied by fasciculations. A specific type of fasciculation with cramping occurs in peripheral axonal excitability. This occurs when adjacent neurons (to the damaged neuron) begin to re innervate partially denervated muscles. Sometimes in genetic disease, fasciculations affect the tongue as well as the trunk and limb muscles. The lower motor neurons are involved in these cases.

Rare cases of fasciculations occur when muscles fail to relax. Failure of muscle fiber relaxation can occur with neoplasia, immune-mediated disease, heredity, and degenerative disease. In these cases, the pathophysiology is hyperexcitability of the peripheral nerves and consequent continuous muscle fiber activity. Continuous activity occurs when potassium channels are dysfunctional. Potassium channels can be damaged when there is an antibody response against proteins in this structure. Cramps, stiffness, delayed muscle relaxation and excessive sweating can be seen clinically. Motor neuron disease is associated with fasciculations in people. These diseases are detected using EMG’s, electromyography, this machine measures the muscle response or electrical activity in response to nerve stimulation of a specific muscle. During the test, one or more small needles (also called electrodes) are inserted through the skin into the muscle.

Systemic disease, drugs and heavy metal toxicity (like lead) can also induce fasciculations. Low blood levels of phosphate (hypophosphatemia) and calcium disorders (secondary to hyperparathyroidism) sometimes result in fasciculations. Calcium disorders occur with some renal diseases. Neostigmine, a drug, can increase fasciculations in cats by increasing the concentration of acetylcholine (a direct effect) in the concentration of acetylcholine at the neuromuscular junction. Some anesthetic agents work using the same pathway at the neuromuscular junction and have the same results. Mercury toxicity should be considered in peripheral neuropathies of unknown origin that are also accompanied by tremor, ataxia, and depression.

There is no specific treatment for the muscle contractions because fasciculations are a symptom of an underlying condition. It is necessary to identify the origin of the underlying condition and that condition is the therapeutic target.