Moldy Buildings, CIRS, Sick People and Damaged Brains: 25 Years of Research Brought Us to the Cure Word – Part 5

Ritchie C. Shoemaker MD
            Medical Director, Center for Research on Biotoxin Associated Illnesses

David Lark
            Mycologist, MouldLabs, Australia

James C. Ryan, PhD
            Chief Science Officer, ProgeneDx

Editor’s Note: Exposure to mold in water-damaged buildings (WDBs) causes a frustrating number of puzzling symptoms and eventually leads to chronic inflammatory response syndrome (CIRS), as explained in the first article of this five-part series, published in the July 2019 issue. In Parts 2, 3, and 4, the authors explained how to maintain the building envelope, CIRS diagnosis procedures, and a 12-step treatment protocol.


Transcriptomics

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In the annals of medical history, there are advances that have changed both the art and science of the practice of medicine. A few we learned about in high school: Edward Jenner working to prevent smallpox with an inoculation with cowpox and sterilization from Joseph Lister come to mind. Louis Pasteur and the germ theory. Robert Koch and his proof of microbiologic causation, not to mention Semmelweis with his insights into prevention of child bed fever and maternal/fetal loss are revered (now) pioneers. Technical advances included use of radiation for x-ray machines, with CT and MRI scans to follow as the years went by. Certainly, automated blood chemistries, not to mention advances in development of antibiotics, beginning with penicillin and sulfa and extending to the modern armamentarium of effective oral and parenteral bacteria-killers, were great achievements. The new T-cell cancer therapies will soon be next (my opinion).

And yet, even these advances pale in terms of scientific discovery to the work done in the early 2000s in the Human Genome Project. While Watson and Crick get credit for discovery of DNA and working out some of the structures of DNA, it was the ability to identify individual genes that has heralded the advances that we see now in the 21st century. Who knew that there would only be 50,000 genes (20,000 protein-coding and 30,000 non protein-coding)? There is so much complexity of protein interaction, the diversity of diseases, all of which essentially end up having their roots in genes and gene activity. Fifty thousand seems like a small number to me.

The initial 3-5 billion dollars that were spent to sequence the human genome work seems like an overwhelming hurdle that practitioners would have to clear before bringing use of manipulation of gene activity to primary care. In just 10 years, however, automated sequencing devices brought next generation sequencing and RNA Seq to practice—with entire human genome sequencing now costing $5000 and not $5 billion. What an achievement!

It is with the Human Genome Project as a back drop for the work of transcriptomists, including Dr. James Ryan, that has helped us take the next step in not only identifying diagnostic features of illness but also identifying objective biomarkers that will let us follow the results of interventions as a basis for modern therapies. Use of transcriptomics, differential gene activation, permits us to truly see the miracle of monitoring DNA activity. We now know that environmental stimuli rapidly cause a targeted but diverse transcriptomic response. That response is rich in information!

When I use the term “transcriptomics,” please recognize that this is a dynamic field of study with gene activity changing through a variety of mechanisms, including those regulated by non-coding RNA, by ribonuclear proteins, by microRNA, by methylation and demethylation, as well as acetylation and deacetylation. All these controlling elements lead to the function of transcription factors that are doing the work to initiate the copying of individual genes from our DNA, onto another nucleotide backbone, called messenger RNA (mRNA), as a cornerstone of adaptation of our genetic material to metabolic needs.

Transcription factors are not one to one; indeed, a quick read of www.genecards.org will show that for a given gene there can be literally a hundred or more transcription factors that can cause that gene to be activated. By focusing on gene activation, we can then compare gene activity to control patients and understand what “normal” is supposed to be, with that insight also leading to the concept of what gene suppression is. Here we see less activity compared to controls. As an aside, we know that males and females have significant differences in gene activity more than seemingly would be based on reproductive functions alone, but we also see change in activity of genes through time of day and night. The more we learn, the more we don’t know.

In the CIRS world, thanks to the work of Dr. Ryan, we were able to look at 50,000 genes in CIRS cases compared to controls with the discovery of approximately 2000 genes that showed significant differences between cases and controls, including both activation and suppression. For three years we used this 2000 gene registry to analyze complete human gene sequencing, looking at cases with CIRS and controls with RNA Seq.

This approach was unwieldy and used a long string of sample manipulations, increasing the likelihood of errors. By reducing the number of genes and using a simpler platform, we were able to create a diagnostic test called GENIE. GENIE involves less than 200 genes including some “housekeeping” genes designed to show stability of the test performance. The most important application in use of GENIE in CIRS patients as well as in other illnesses characterized by chronic fatigue had to do with Dr. Ryan’s finding of “hypometabolism.”

What we mean by hypometabolism is stunning in its evolutionary simplicity. Who knew that there were biological warfare elements that one-celled creatures used on other one-celled creatures 3 and 4 billion years ago? These biological warfare elements have different names such as mycotoxins, ribotoxins, ribosomal inhibitory proteins, endotoxins and others. As the names suggest, ribosomes, especially one structural element of the ribosome called the sarcin ricin loop, are attacked by ribotoxins disrupting cellular production of protein. Remember that ribosomes, found in the cytoplasm, can number in the low millions in a human cell. It’s no wonder that ribosome production is one of the cells most energy intense operations.  If a ribotoxin disrupts normal sarcin ricin loop functioning, which they do, the cost to a cell is such that it needs to suppress its metabolic rate to survive the attack.

All living creatures need to make protein; they all use ribosomal machinery to do so. There are two functional units that comprise the ribosome, called the large and small subunits, that wrap around a messenger RNA to then produce a protein by initiating and then elongating an amino acid chain that leads to a protein being created. All known ribosomes of all creatures carry the sarcin ricin loop, a structure that has been ultraconserved throughout evolution. Imagine, a vital piece of the mechanics of a cell not changing in 4 billion years.

Ribosomes are also found in mitochondria, the power house of the cell. Here we have proteins made that are needed for mitochondrial function. Were these “mitoribosomes” attacked by ribotoxins? You bet!

I hope that you have seen that disruption of protein synthetic machinery is a common result of a biological attack. The cell, under attack, has multiple feedback systems built in to help the cell survive. The cost is chronic fatigue and multisystem, multi-symptom illness in humans. Without taking evasive action, the cell would otherwise die.

Energy production systems are also subject to attack. When thinking of mitochondrial function, we think about electron transport chains and production of 36 molecules of ATP for each molecule of glucose, but those thoughts end up being just a small bit of what is involved. Mitochondria, speculated to be free-living bacteria before they were engulfed and kept alive inside the engulfing cell, provide energy to the engulfer. Mitochondria had their own genome. Over time, and 4 billion years is a long time, the mitochondrial genes have migrated (or been migrated) to the nucleus leaving only 37 behind in the mitochondria.

Monitoring gene expression, using transcriptomics, can show the effects of medical interventions, and give insights into a disease.

Going back to the idea that everything that happens in disease and illness is controlled by DNA, the control of mitochondrial function now comes from nuclear DNA. This point cannot be underestimated in that many have felt that treatment of mitochondria with one nostrum or another made sense and yet they were firing at the wrong place. The therapeutic target was in the nucleus and not in the mitochondria itself.

As nature would have it, energy production and protein production are protected in the search for life. By suppressing both mitochondrial gene activity and ribosomal gene activity, the cell can almost “go into hibernation,” or torpor or reduced activity—call it what you want—to “lay low and be still.” This state of reduced metabolism or hypometabolism permits the cell to survive by downregulating its gene activity.

The role of glucose metabolism in this whole concept of reduced cellular metabolism is less well defined. Normally, glucose will be delivered with insulin to bind to the insulin receptor, making a complex on the outside of the cell. That complex will be internalized, surrounded by a cell membrane, in what is called an endosome, like a bubble. If there is insulin receptor substrate available, that bubble can be processed further, (1) releasing glucose to be used for fuel by mitochondria or (2) keeping glucose in cytoplasm where glycolysis, breakdown of sugar, provides a much smaller benefit of just two ATPs per glucose.  It should not be a surprise that the role of sugar delivery becomes one of the feedback systems that guard against having too much sugar metabolism products present at a time since the mitochondria cannot handle sugar break-down products.

Said in a different way, instead of breaking down glucose into pyruvate, used for mitochondrial respiration, suppose that pyruvate reduction will help the cell survive biological attack. Remember that pyruvate is a three-carbon breakdown product of glycolysis; it can be converted to lactic acid, another three-carbon fragment, if pyruvate is not being taken up by the mitochondria. If mitochondria are being attacked and an endless supply of pyruvate supplied, there would be an endless supply of lactic acid made to either poison the cell or change pH-related activities in local tissue because glycolysis sent too much pyruvate towards the mitochondria. What the cell does is to reduce the impact of lactic acid production by preventing excessive amounts of pyruvate to be made in the cell. What an incredible feedback regulation system! Simply shut down glycolysis.

The complexity of these interactions of energy and protein are accentuated by mitochondrial translocases, proteins that are coded for by nuclear genes. These proteins provide a protein import system across the mitochondrial inner and outer membranes. If the translocase genes are suppressed, fewer necessary mitochondrial proteins will be transported into the mitochondria. By suppressing translocase activity, there is an additional mechanism to prevent excessive mitochondrial activation at a time of metabolic stress from external environmental attackers.

There are other modalities of feedback interaction that are remarkable. Some seem so simplistic now that Dr. Ryan has identified what they are for us based on his review of next generation sequencing and review of existing literature.  As Dr. Ryan would say, “I didn’t invent this, this was here, we have known all the time that these pathways were functioning in everyone’s body.”

Taking a step back, we see the complications that come from reduction of cellular metabolism. Where does chronic fatigue come from? Does this come from protein abnormalities or from energy abnormalities or both? Where does injury to grey matter nuclei that we see on NeuroQuant come from? Could there be a role for mitoribosomes in normal survival? What about in cardiac myositis? Could there be a role for reduction of activity of portions of cells that control contractility? You bet!

As Dr. Ryan expands his work, he has quietly set the stage for a massive rethinking of what chronic fatiguing illnesses are, what we do to diagnose them, and what we do to correct them. Dr. Ryan has shown what I would like to call a “CIRS curve,” in which initial reductions of activity, gene suppression is a better term, for ribosomal RNA large and small subunits is evident. ATP synthesis (there are more new terms to come), mitoribosome large and small subunits, NAD-ubiquinone scaffolding for electronic transport chain systems inside mitochondria, together with translocase functions of inner and outer mitochondrial membranes all are involved in hypometabolism. When these are all suppressed, as we see in patients who have not been treated, i.e., they are “naïve to treatment,” with use of the first of the 11 steps of the Shoemaker protocol, there will be correction of these evidences of suppression of gene activity. Indeed, there is an overshoot to exceed control values at the end of the first eleven steps. This overshoot is corrected by use of VIP, leading to the last step of the Shoemaker pathway which is one of restoration and normalcy. Over time, this restoration will stay constant and there will be no change when patients go off VIP.

What the CIRS curve means is that we can look at patients, whether they began with post-Lyme syndrome or CIRS-WDB or ciguatera or possibly traumatic brain injury or many others, that will predictably lead to identify metabolic abnormalities that let us not only establish the stage of therapy where people are but also what is left to be done.

These are exciting times for those in the chronic fatigue world because we finally have reached an objective, testable abnormality that defines the illness for the first time ever. If one thinks that we have finally reached the “Holy Grail” of disease management, maybe so. I do too. But the reality is that our knowledge is woefully incomplete. Yet our duty is to make the best use of the best data available to help our patients. So, let us enjoy the Holy Grail idea for today. Tomorrow is another day.

What We Learn from GENIE

The key indication for GENIE is to verify whether a patient has hypometabolism. Taken together, all the elements of abnormalities of nuclear encoded mitochondrial genes are the keys to mitochondrial functioning that can’t be evaluated by any mechanism other than transcriptomics.

We have an additional series of biomarkers that involve immune functions as well. The first are CIRS biomarkers. These are genes that have importance for CIRS patients. These markers are used as guides for diagnosis and monitoring sequential therapy as we are looking for transcriptomic cure. Here the word “cure” means that the biomarkers would be returned by therapies to equal levels seen in controls.

The vital importance of apoptosis, otherwise called programmed cell death, means that the cycle of cell life and death can go awry. When there is disruption of the enzymes that should be marking a cell to be killed by cytotoxic T-lymphocytes or natural killer cells, the cell is programmed to die but die safely. What can happen is that instead of dutifully packaging all of the intracellular contents before the cell is burst apart, when the cell enters defective apoptosis it will release into circulation free DNA as well as organelles such as Golgi body, endoplasmic reticulum, and mitochondria. These elements are intensely inflammatory, giving rise to the suggestion that defective apoptosis can be a form of endogenous inflammation added to exogenously induced inflammation in CIRS. We need more data to support this idea but right now the hypothesis remains tantalizing.

From ciguatera to mold to Lyme, coagulation abnormalities are routinely seen when transcriptomics are assessed. The coag abnormalities involve both suppression and activation of a series of genes that independently interact with platelet function together with coagulation pathways. We now know much more about coagulation problems in CIRS than just the well documented abnormalities in von Willebrand’s profile. One of the observations for years has been the elevated levels of d-dimer in cases of CIRS as an unexplained finding in CIRS cases. I can’t tell you in how many people I have chased after elevated d-dimer levels, looking for evidence of intravascular clotting, without finding a source.

Now that we have gene activity, we are looking at a different mechanism for this non-specific rise of d-dimer. At first glance, it seems odd that enhanced clotting would lead to enhanced bleeding. Missing is the role of enhanced lysis of sub-clinical clots, hence the increased d-dimer. With correction of inflammation, the transcriptomic basis for d-dimer formation, namely enhanced clotting, resolves.

Defensins are substances made by white blood cells used non-specifically to combat ongoing bacterial or viral infection. With elevated defensins, there usually will be an infectious basis. Defensins are not activated without basis.

Granzymes are intimately involved with apoptosis. If granzymes are elevated, there will be activation of signaling for natural killer cells and cytotoxic T-lymphocytes to sort out and then kill the targeted cells. “No cell lives forever” is an old expression but has a lot to do with granzyme function.

For years, I have had wonderful Sunday afternoon discussions with clinicians regarding methylation.   Methylation seems to be an active subject for discussion among many alternative providers. Epigenetics, anyone? The idea is that by putting a methyl group on a gene, there will be an effect on the gene activity. But that idea does not include the role of demethylation reversing the activation (or suppression) the methyl group could create. There is an additional role-modulating gene activity for a two-carbon chain attachment, that being acetylation and deacetylation. This two-carbon subunit is more frequently attached to histones controlling the structure of the insulating proteins around DNA. We can’t look at epigenetic change without thinking about methylation and demethylation—and acetylation and deacetylation. Some of these gene-changing properties of a single carbon group (or two-carbon group) can be long lasting, others are short lasting. It is another mechanism of regulation of gene activity.

Cytokine changes are very difficult to measure in blood. The reason for this oddity is that these pro-inflammatory humoral factors can be bound by the cell that makes them or by the adjacent cell. The only measure we see in blood test results is the so-called “endocrine” function of the cytokine floating unbound in blood. Assays, including the multiple cytokine assays, will tell us about the endocrine function of cytokines but not the autocrine or paracrine. Gene activity from GENIE tells us activity immediately, without concern about false elevation or false suppression of endocrine values.

In all the discussion about what an objective test for Lyme disease is, NeuroQuant rises to the top in that it shows a distinctive pattern whether the patient has had Lyme for six months or six years. Finally, thanks to the work of Bouquet, et al,1 we also have an additional distinctive marker for Lyme, a transcriptomic series of genes that show us what occurs in Lyme before antibiotics and Lyme after antibiotics. We worked independently of Bouquet’s group to now come up with a Post-CIRS marker for what we see in Lyme patients after antibiotics are done and CIRS therapy is initiated. These are exciting times for Lyme!

As briefly mentioned previously, for years I have looked at HLA being the marker for who has increased relative risk for CIRS illness. The number 24% for total at-risk HLA in the US population has come up repeatedly both in my practice as well2 as in practices of many other physicians. We know that HLA has a lot to do with antigen presentation and immune response based on its location of chromosome 6. The idea has been that if there is a problem with antigen presentation, represented by HLA, then there will be a defect in antibody formation. No antigen presentation, no antibody. No antibody, no protection for repeat illness with re-exposure.

Absence of protection from relapse with re-exposure is a cardinal finding in CIRS.

We now have an additional marker for defective antigen presentation, namely the gene abnormality seen in the T-cell synapse with antigen presenting cells. What this means is that when a pathogen is taken into an antigen presenting cell, the professional antigen presenting cell (1) breaks down the pathogen into small fragments in the lysosome; (2) is then processed through the endoplasmic reticulum; and (3) loaded onto a major histocompatibility (HLA) receptor; (4) which is then taken to the cell membrane, creating a signal that permits a naïve T-cell to home in and attach to this tasty morsel of this antigen ready for recognition and T-cell processing.

The first step for antigen presentation, after antigen processing, is formation of a synapse between the dendritic cell and the T-cell. The genes involved in this vitally important synapse are ones that are routinely found to be suppressed in untreated CIRS. Fortunately, treatment, especially with VIP, corrects the T-cell synapse abnormalities. The problem is not just HLA!

We know that the gene expression for complement remains important in many elements. By including a gene marker, we can look at the 33-member protein system in a different fashion compared to trying to make sense of changes in individual proteins. Complement interactions can be numbingly complicated!

In the world of PTSD, while we think we have identified a biomarker in NeuroQuant, we just don’t have an adequate number of cases. There is a gene reported to be involved with ACTH and cortisol metabolism that has an association reported in the literature suggesting that it is important in recognition of PTSD. This gene is part of GENIE. Initial results are promising; data is in its infancy.

The cytoskeleton of a cell is based on microtubular formation. These microtubules are dependent on their genes called tubulins. If there are tubulin abnormalities, like what we see in genes found in plants where benomyl (an azole anti-fungal) had been used, abnormalities can tell us about microtubule problems in day-to-day life of the patient.

There are additional genes as part of GENIE, including those looking at insulin signaling; those looking at anti-inflammatory nuclear transcription factors; those looking at activation of MAP kinases as well as B-cell markers for the synapse between T-cells and B-cells. 

Taken together, the information we glean from GENIE cannot be accessed by any diagnostic mechanism. GENIE, therefore, is providing us with information about abnormalities of physiology based on gene activation. CIRS remains the teacher for other illnesses to follow. We have identified the origins of abnormalities in transcriptomics, followed through with abnormalities of proteomics, followed through with hormonal disruption and multiple layers of dysregulation of proteomic activity.

All these changes can be shown to be related to (1) dysregulation of (2) dysregulation of (3) gene regulation. That’s right; at least three layers of defective regulation of gene activity are found in illnesses such as CIRS.

From where I sit today, there is no limit to what questions we can ask of the transcriptomic findings accumulated to date.  Sometimes the more important features of a new paradigm aren’t simply what is newly found to be true, but what was incorrect about older ideas. We return to Aldous Huxley telling us, “The key to understanding is casting out false knowledge.” We are “casting out” every day, it seems. Let us not forget that only a few ideas in sciences survive the passage of time.

Simple applications of the “casting out” from transcriptomics let us see that viral reactivation is not likely to be a root cause of CFS, despite antibody testing that appears significant. Another is the diagnosis of “mycotoxin illness,” already been exposed as flawed earlier. With the ability to define the expected differential gene activation associated with mycotoxin exposure from the literature, we can flesh out what is likely associated with pathologic changes after mycotoxin exposure and what is not. Remember that in CIRS-WDB we see suspected endotoxin effects in over 50% of cases, closely followed in suspected incidence by actinomycetes. Mycotoxin findings are a distant third.

The enormity of importance of hypometabolism in assessment of a unified cause of fatigue and a transcriptomic mechanism to show correction of that cause brings hope to those searching for answers to fundamental questions, such as “When will I get my life back?” Or, “When can I walk into a new restaurant without fear that the restaurant was once a WDB from a prior flood indoors?” Or, “Are my children condemned to this kind of life due to my HLA?”

We must also consider the role of several compensatory metabolic mechanisms once hypometabolism has been initiated. If mitochondrial injury from a ribotoxin attack on mitochondrial ribosomes (mitoribosomes) is present, the cell won’t be able to shuttle its normal amount of the fuel source pyruvate, created by glycolysis, into mitochondria. Excess pyruvate not taken into the mitochondria would otherwise be converted to lactic acid, an intracellular poison. How does the cell avoid dying from lactic acid? Simple, reduce glycolysis! Curiously, in the presence of interferon gamma, one of the enzymes that does the work in glycolysis (GAPDH) also interacts with ribosomal protein L13a and a transfer RNA (EPRS) to form a protein complex called GAIT.  The GAIT complex will bind to a specific set of messenger RNA in the cell to curb inflammation. “Surviving hypometabolism” is getting complicated. And there is more.

We are building a database to attempt to show what role the insulin receptor has in hypometabolism. We have interesting findings on insulin receptor substrate 2; the data show great promise.

The sustained finding of genes that predispose to defective apoptosis also holds great promise.  We see one particular gene repeatedly in patients with abnormalities in the caspase-driven mechanism of programmed cell death. If the dying cell, programmed to be lysed by natural killer cells and cytotoxic T cells, fails to safely “package” its intracellular materials that are intensely inflammagenic before lysis, bad things will happen. Face it, if cellular contents, especially DNA, are released freely into circulation, we will have an endogenous source of inflammatory response. As Pogo would tell us, “We have found the enemy and he is us.”

Upcoming investigations are focused on correlation of abnormal NeuroQuant findings with early dementia. By looking at tau in spinal fluid and simultaneous transcriptomics, we hope to bridge the gap between unknown gene activity in brain tissue and known activity in blood cells.  We can’t use brain tissue for gene expression studies, but we may have a biomarker in blood to correlate to NeuroQuant abnormalities and cognitive decline, as one of the genes on our GENIE, found overexpressed in CIRS patients, is also found in beta amyloid plaques.

The commonly found differential activation of coagulation genes that are responsive to VIP provides another reachable window for intervention. Stay tuned on this topic!

I would be remiss if I didn’t mention treatment of dilated cardiomyopathy in a patient with at least four documented bouts with acute Lyme disease. As her ejection fraction by MUGA bottomed out at 11%, a referral to a cardiac transplant unit at the University of Maryland was made. RNA Seq using whole blood, not cardiac cells, showed a complex pattern of gene disruption, including adrenoceptors and those involved with the calcium/sodium pump, the basis for contractility. Addition of high dose VIP after antibiotics and CIRS RX resulted in correction of heart failure, reduction of LVIDD from 9.2 centimeters to 6.8 in less than one year. I was elated but Dr. Ryan quietly reminded me that a N=1 study, as this one is, must be shown in many others because we can’t confirm that cardiac myocyte findings would parallel the WBC findings of correction of genes involved in contractility. My N=1 case success is one more than anything I have seen from the Lyme-dilated cardiomyopathy world.

Even still, the patient is fully active now, without symptoms of heart failure, off all meds. An unexpected spin off in this trial was confirmation of negative effects from carvedilol that followed a dose/response of adrenoceptors. This beta blocker is widely used in heart failure, sometimes with serious adverse effects. Transcriptomics showed us the way in this case; future titration of carvedilol dose to adrenoceptor expression makes sense if the WBC genes hold up as a model for cardiac myocytes.

Summary

The story of CIRS could fit into Thomas Kuhn’s Structure of a Scientific Revolution. What began as an anomaly, an isolated observation of fish kills and human illness from exposure to Pfiesteria, a dinoflagellate, has expanded over the past twenty-three years to an integrated paradigm of an entirely new illness concept that for the first time provides a supported, evidence-based explanation for countless chronic fatiguing illnesses. It is possible that there is no “modern illness” paradigm that has more supporting biomarkers than CIRS. Beginning with exposure assessments, especially use of HERTSMI-2 for CIRS-WDB, cluster analysis of symptoms, and labs ranging to proteomics and transcriptomics, and including volumetric studies of brain injury, stress echocardiogram measurements and VO2 max, the diagnosis and treatment is buttressed by association studies, prospective re-exposure trials using a published protocol (SAIIE), and randomized clinical trials. Using published protocols, we have corrected proteomics, transcriptomics, and grey matter nuclear atrophy.

What this density of objective biomarkers provides is confirmation of diagnosis and treatment, backed by nearly 40 published papers and clinical use by thousands of physicians. As the research basis of CIRS continues to expand, we feel that CIRS will provide the basis to look for new approaches to inflammatory illnesses of our era, especially atherosclerosis, obesity, diabetes, and chronic pain.

And one parting thought: hope for cure is here. Hope now rests on hard clinical trials that show us the way to help those trapped by WDB, among other causes of CIRS.  The answers to causes of chronic fatigue are apparent; effective gene-based therapies also are apparent. 

In data we will find our answers for today’s hope and tomorrow’s standard of care.

References

  1. Bouquet J, et al. Longitudinal transcriptome analysis reveals a sustained differential gene expression signature in patients treated for acute Lyme disease. MBio. 2016;7.1: e00100-16.
  2. Shoemaker R, Rash J, Simon E. Sick Building Syndrome in Water Damaged Buildings: Generalization of the Chronic Biotoxin Associated Illness Paradigm to Indoor Toxigenic Fungi. Bioaerosols, Fungi, Bacteria, Mycotoxins and Human Health. In: Bioaerosols, Fungi, Bacteria, Mycotoxins and Human Health: Patho-Physiology, Clinical Effects, Exposure Assessment, Prevention and Control in Indoor Environments and Work. Fungal Research Group Foundation, Inc, 2006. pp. 52–63. E. Johanning, editor.

We are grateful for technical assistance in preparation of this manuscript provided by Debbie Waidner and JoAnn Shoemaker.