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biomarker gifThe secretome is a cell free fluid that concentrates the therapeutic components produced by stem cells, giving them healing powers.  The secretome can be produced from a healthy  person or one’s own cells.  The starting material can come from several sources, blood cells or fat cells are favored. The secretome is thought to give stem cells their effectiveness in various diseases.

The secretome is a collection of “paracrine” soluble factors produced by stem cells that are used for inter-cell communications. The paracrine factors also assist in tissue development, regulate normal functions, and regenerate damaged tissues.  Secretomes can inhibit some cancers. The factors are contained in extracellular vesicles and micro vesicles as well as unbound membrane particles, short bits of proteins (peptides) and cytokines.

These are important factors in degenerative, auto-immune and inflammatory diseases. That makes the secretome relevant to many therapeutic uses that include arthritis, chronic lung disease, multiple sclerosis, cancer, and stroke. Secretome is generally considered safe in humans and animals and is in several clinical trials in the United States. In 2017, a preparation of secretome was deployed in Russia for eye and skin injuries in animals.  Not available in the US, you can order Penapuh drops from an online Russian company.  Check out the 4,087 scientific papers on secretomes to really appreciate this expanding field.

One paper referred to cross-talk between the body’s stem cells and the central nervous system (CNS).  If we could decipher this chatter, we would direct CNS healing in diseases that currently have no treatment! The secretome has piqued our interest for both amyotrophic lateral sclerosis (ALS) and equine neurodegenerative disease.  We have gathered 10 research laboratories across the US, each with unique expertise, into one group,to find hope for ALS patients  Sixty-percent of our efforts use stem cell science to understand disease and how to crack the secretome language. We are holding the pedal to the metal as we explore how to get an effective therapy into desperate people and beloved horses.

wbcstem cellThe illustrations are of white blood cells in the peripheral circulation and a picture of stem cells in the flask after processing.  The stem cells are assayed for several specific markers.  And then they are expanded and some are frozen.  A research step for us is determining if starting cells, the factories to produce the secretome, should come from a healthy person or from a patient already afflicted with a disease. Are cells that have been in a diseased environment primed to respond to a curative mode? Or are cells from healthy sources more capable of sending healing messages to tissues? We have two laboratories asking that one question. The consensus opinion of the group is that a healthy cell should be the starting material.

The next unknown is the cell phenotype produced from the undifferentiated cell and how to appropriately induce the cells to communicate with the diseased body.  Stem cells are induced to produce one cell type. In disease, there are multicellular communications that are disturbed between hundreds of other differentiated cell types.  The source of tissue and cell type that are induced are key issues that must be addressed with firm scientific basis. When cells age in the laboratory, they change their secretome.  What’s old in a flask can simply mean 10 or 15 doublings. The change is a change in their phenotype.  If it takes a long time to get enough cells in the flask, their phenotype won’t be the same as the starting cell. You can already envision the changes if autologous cultures are used in some diseases, autologous means the individual donates the cells. There are obvious advantages to a specific cell background that are modified to an individual disease or unique disease process. However, once we can break the language code, we can tell the cells what we’d like them to say.

These are all issues for licensing the ultimate product for the patient.  The goal is a unified line that can become a licensed therapeutic that is safe and effective for the condition that is treated.  That means a safety study (with a price of half a million dollars for horses and more in humans).  Sometimes, if there is literature to support safety and toxicity, time and money are saved.  A dose must be determined from clinical trials.  That dose is used in a toxicity study that is mandated by regulatory agencies.  Rodent toxicity studies are about a quarter of a million dollars.  The dose titration is done by treating patients within a safety study.  If all goes well, the dose is escalated in a second or even a third trial.  Time and money.

To initiate a clinical trial the product is manufactured.  Before clinical trials are contemplated, one must have an idea of the dose, duration, and frequency of treatment.  That will have a direct effect on the formulation, and formulation studies are expensive. The next decision is packaging.  The ultimate package is cast in stone once the initial exploratory batches are used in trials.  Should it be a liquid, lyophilized, or a tablet? That is determined by the expected stability of the product.  Secretome that is liquid in a vial would need refrigeration or freezing, but consider the longevity (stability) of the final product if it was lyophilized!  When dosing a laboratory animal, a vial with a therapeutic volume of 0.1cc is great, but when scaled up to a human, or horse, one would want to avoid 20 mouse vials for one treatment!  And a horse might require 10 times the human volume, 200 mouse-vials.  Lyophilization can overcome that obstacle. All these steps total close to five million dollars…or more.

phone boothFormula and packaging are set. Manufacturing is complete.  Off to regulatory officials to decide on the best path to get into trials.  Phenotype selection is based on cell culture conditions that were worked out when we learned to speak their language. We give them the messages so they can don their superhero costumes and churn out the paracrine factors that are expected to ameliorate disease.  This is what we are working on.  If you’d like to participate, please use the following link. And keep an eye out for our progress, we will report it here. Click here to donate.

horse menuAn unfortunate legacy of COVID-19 is the critical importance that better diagnostics tools could have played to mitigate the virus’ impact on human health and the world’s economy. And the lost lives.  As widely known in the industry, lab services account for less than 3% of total U.S. healthcare spending today, even though their test results impact a majority of all clinical decisions regarding patient care. Just as importantly, diagnostic testing saves horses lives.

The following case scenarios illustrate the folly of skipping diagnostic testing

A patients gums are very pale and anemia is a likely diagnosis.  In this case, the course of action is not directed with diagnostic point of care (POC) tests to provide actionable information, treatment commences with an iron supplement.  The intent is to “observe a treatment response”. You can take your pick of species in this example, be it goat, horse, or human.

If you picked a goat the most likely cause of anemia is parasitic and no amount of iron supplement will save the patient. The correct treatment is an effective anti-parasitic agent.  The anti-parasitic drug must be selected based on the parasite target--be it strongyle or coccidia.  An incorrect guess as to the parasite is just as lethal as treating parasites with iron. Picking the diagnostics from the a la carte menu is the best option.

If you selected horse as the example, a cause of anemia might be blood loss that is due to a guttural pouch mycosis. An iron supplement could help with chronic anemia, but supplements won’t prevent the eventual acute onset of lethal exsanguination—these horses often die because they hemorrhage from the nose.  Instituted early, surgery and medical treatment with various antifungal preparations can be effective.

Chronic anemia can be due to an inability of the bone marrow to produce red cells and differentiated from anemia due to blood loss.  Toxins, metabolic diseases-such as renal disease, and cancer can result in a lack of red cell production in humans and animals.

Diagnostic tests are selected and yield enhanced directed information, ultimately improving patient care and outcomes. Diagnostic tests save money.

What drove some people to rely on treatment response in dealing with equine sarcocystosis, a cause of equine protozoal myeloencephalitis (EPM) instead of pursuing diagnostic testing?  Perhaps it was confusing early messaging from leading experts in the field. The Western Blot was unable to distinguish between infection and disease. The Western Blot test failed to distinguish between species such as neurona, fayeri, and falcatula.  Experts failed to get the message out that antibodies vary in response to exposure, infection, and central nervous system disease.  An important lost message was that different tests measure different things.  It wasn’t the testing that failed, it was the messaging on what information the tests could reveal that missed the mark.

It took many years before three serotypes of Sarcocystis neurona were recognized. Once the serotypes were discernable, it was no longer necessary to use non-specific detection of apicomplexans that required reducing data to a probability.  And then there are the confusing experiments that absolved S. falcatula of infecting horses.  Failing to recognize that S. neurona, serotype SAG 6, is antigenically (highly) similar to S. falcatula made these infections indistinguishable with the available testing. And then there is the question of relapses.  Ineffective treatment led to the myth that most horses  with EPM will relapse. However, relapses are often a manifestation of another disease.  A disease that is a bystander to S. neurona…and not treatable with anti-protozoal agents.

The effects of an infection with Sarcocystis are innate immune responses that result in a peripheral neuropathy.  There are other causes of peripheral neuropathies.  The bystander mechanism, inflammation, results in non-demyelinating or demyelinating disease.  The pathophysiology of polyneuritis is a spectrum, from initiation to end-stage disease. Diagnostic tests can indicate the difference between non-demyelinating and demyelinating disease and direct appropriate treatments. With careful interpretation it is possible to stage the disease and that can help with clinical decisions.

The diagnostic menu for equine disease is constantly being developed and refined.  Each test gives a clue to the disease process and most importantly, diagnostics help predict the effect of a drug on the disease process.  A diagnostic test is used in conjunction with patient history, clinical signs, and clinician experience.  A diagnostic test can save money, time, and often lives. Once familiar with testing options, (https://pathogenes.com/w/epm-testing/),  rational treatments can be planned. If you need help reading the menu, give us a call.

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.