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\author[1]{William Reid}%
\author[2]{William K Reid}%
\affil[1]{Affiliation not available}%
\affil[2]{Hematology \& Oncology}%


\vspace{-1em}



  
  \date{May 29, 2024}


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William K. Reid, M.D.\emph{Hematology \& Oncology} 613 Greenwood Rd.
Chapel Hill, N.C. 27514 Cellular: (615)405-5162 Email:
wkrmd2017@gmail.com

Monday, January 25, 2021

Hypothesis: \emph{Mycotoxins Causing Amyotrophic Lateral Sclerosis}

Reviewer's questions:

1. \textbf{Pattern of Cascade through the Neuroaxis in ALS}

2. \textbf{Biomarkers for ALS \& for Mycotoxins}

I. \textbf{ALS Cascade Upper \& Lower Motor Neurons} :

It is intriguing to relate the available data on neurotoxic mycotoxins
with the clinical findings in ALS. ALS is a motor neuron disease with
insidious, progressive weakness and muscle wasting. One of the essential
clues to the diagnosis of ALS is the involvement of both upper and lower
motor neuron disease. The diagnostic strategy presented by Statland et
al 2015\textsuperscript{1} defines upper motor neuron deficits with
spasticity and hyperreflexia and lower motor neuron disease with muscle
wasting, weakness, fasciculations, fibrillations and positive sharp
waves. In ALS there is a mixture of both upper and lower motor neuron
deficits. The pathology of the neurotoxic mycotoxins, especially the
trichothecenes(Dai, C. et al 2019\textsuperscript{2}), closely mimics
the pathology of ALS with oxidative stress, mitochondrial dysfunction
and respiratory chain damage.

\textbf{Intranasal Pathways Bypass of Blood Brain Barrier \& CSF} :

The trichothecenes, T-2 Toxin and Deoxynivalenol, are major contaminants
of the world food supply. If the opportunistic fungi responsible for
these secondary metabolites develop a niche in the body by colonization
or infection, these potent, lipophilic mycotoxins are able to cross
barriers, especially the blood brain barrier and accumulate in the brain
and spinal cord. Marcesca, M. 2013\textsuperscript{3} reports on of
transport of food-associated trichothecenes from the gut to the brain.

There is a more intriguing possibility if there is a focus of a fungal
infection in the upper airway, especially the upper sinus cavity. There
is a growing body of research documenting intranasal delivery of
compounds and drugs to the brain and spinal cord. Thorne, R.G. et al
2004 \textsuperscript{4}report on transport of IGF-1 in the nose of
rats. Thorne, R.G. et al 2008\textsuperscript{5} report on transfer of
interferon-beta in a monkey's nose to brain. There are extensive studies
documenting intranasal absorption of insulin in the objective of
replacing injection requirements. Lochhead, J.J. et al
2019\textsuperscript{6} found insulin had neuroprotective effects
traveling along the trigeminal nerve. Avgerinos, K.L. et al
2018\textsuperscript{7} found intranasal insulin in 293 patients
improved memory in patients with Alzheimer's or mild cognitive deficits.
Intranasal glucagon was effective in adults with type 1 diabetes and
insulin-induced hypoglycemia (Rickels, M.R. et al
2016\textsuperscript{8}). What these studies discovered are pathways
bypassing the blood brain barrier as well as the CSF.

Pardridge, W.M. in 2011\textsuperscript{9} and
2012\textsuperscript{10}UCLA pointed out misconceptions surrounding drug
and compound levels in the CSF, blood and brain. His studies showed that
CSF drug levels were the same as systemic blood levels and were not
equivalent to brain or spinal cord levels. Over the past 10 years the
literature has defined two intranasal pathways that bypass both the CSF
and the blood brain barrier.

The upper sinus cavity is lined by olfactory mucosa that has direct
passage of olfactory nerve endings exposed to the external environment.
There is a second lining of respiratory mucosa fed by the ophthalmic and
maxillary branches trigeminal nerve. Djupesland, P.G. et al
2014\textsuperscript{11} describes the delivery of drugs from the nasal
cavity directly into the brain. Passage is rapid in 5 minutes to 60
minutes passing by convective or bulk flow. Drugs bypass the blood brain
barrier and the CSF.

The olfactory pathway passes compounds of significant size into the
olfactory bulb. The drugs move outside of the neurons with movement by
pulsatile flow extracellular pathways. The trigeminal pathways from
nasal cavity to brain moves to the midbrain, pons and the lower motor
neurons of the spinal cord. The olfactory pathway feeds into the limbic
system and forebrain.

If there is an infection of the upper nasal cavity involving
opportunistic fungi, such as Fusarium species that release lipophilic,
neurotoxic mycotoxins, it could poison the brain and spinal cord in an
insidious fashion. The pathway from the olfactory bulb to the upper
brain would involve the upper motor neurons. The pathway from the
trigeminal nerve branches would transport drugs to the brainstem and
lower motor neurons (Djupesland, P.G. et al 2014\textsuperscript{11},
Lochhead, J.J. et al 2015\textsuperscript{12}, Ganger, S. \&
Schindowski, J. 2018\textsuperscript{13}). There are studies of giving
chemotherapy intranasally for glioblastoma multiforme (Bruinsmann, F.A.
et al 2019\textsuperscript{14}, van Woensel, M. et al
2013\textsuperscript{15}).

\textbf{Pseudobulbar Affect} :

Intranasal passage of neurotoxins into the brain and spinal cord would
explain the dual injury to upper and lower motor neurons. The olfactory
intranasal pathway leads to the olfactory bulb and limbic system. This
pathway could help to explain pseudobulbar affect. Thakore, N.J. \&
Pioro, E.P. 2017\textsuperscript{16} in Cleveland reported on
pseudobulbar affect found in 209 out of 735 ALS patients. They found an
association with bulbar onset and dysfunction with predominantly upper
motor neuron disease. The ALS patients with pseudobulbar affect were
younger in age with a shorter duration of disease. They found an
association with worse bulbar findings, dysarthria and dysphagia. There
was an association with the use of Baclofen, a surrogate for upper motor
neuron dysfunction.

\textbf{Misconceptions of Blood Brain Barrier \& CSF} :

Pardridge 2011\textsuperscript{9} and 2012\textsuperscript{10}, report
on the literature before 2011 with the misconceptions about CSF drug and
metabolite levels. The intranasal passage of drugs and metabolites to
the brain did not feed into the CSF. This leads to misconceptions when
studies assume CSF levels are equivalent to brain and spinal cord
levels. In order to get accurate brain levels requires Intracerebral
Microdialysis, PET scans or functional MRI (Chefer, V.I. et al
2009\textsuperscript{17}, Lasley, S.M. 2019\textsuperscript{18}).

\textbf{Immunotoxicity of Mycotoxins-Immune Evasion:}

The literature on trichothecenes list their immunotoxicity as possibly
their most significant pathology. T-2 Toxin appears to represent the
most toxic of the trichothecenes. Studies show T-2 toxin impairs the
mouse response to Reovirus with suppressed immunoglobulin and interferon
(Li, M. et al 2006\textsuperscript{19}). Pestka, J.J. et al
2004\textsuperscript{20} found immune suppression by Deoxynivalenol and
other trichothecenes with initial immune stimulation then suppression as
the dose increased. Studies by Obremski, K. et al
2013\textsuperscript{21} showed T-2 Toxin suppressed CD4, CD8 and CD21
in pig ileal Peyer's patches. Wu, Q. et al
2018\textsuperscript{22}report downregulation of interferon, TGF-beta
and Toll-like Receptors(TLR) with immune evasion. This could explain the
recalcitrance of treatment against fungi. It also opens up the
possibility to the use of the new PD-1/PD-L1 pathway inhibitors in ALS
patients.

II. \textbf{Biomarkers for ALS and Mycotoxins} :

\textbf{Biomarkers for ALS} :

There are extensive lists of biomarkers for ALS. Lucas, T.V. \& Bowser,
R. 2017\textsuperscript{23} reviewed the fluid-based biomarkers for ALS
divided into neurofilament proteins, inflammatory markers, mRNA such as
C0orf72 and metabolic biomarkers. It is interesting to compare the
biomarkers for ALS with the biomarkers listed for common mycotoxins. The
pattern of inflammatory biomarkers and metabolites are similar in
mycotoxin toxicity compared to the pathology with ALS.

\textbf{Biomarkers of Mycotoxins} :

Mycotoxins have garnered increasing scrutiny given their prevalence in
food and human environmental exposure. Vidal, A. et al
2018\textsuperscript{24} did a comprehensive review of mycotoxin
biomarkers. The development of ultra-high-performance liquid
chromatography-high resolution mass spectroscopy has improved the
accuracy and sensitivity of detecting food borne mycotoxins. The main
mycotoxins found were trichothecenes from Fusarium species,
Deoxynivalenol, one of the most frequently occurring fungal contaminants
in food and feed worldwide. Nivalenol, Fusarenon-X, T-2 Toxin and
Diacetoxyscirpenol are biomarkers of Fusarium species in food.

Fumonisins are secondary metabolites of Fusarium species found mainly in
maize. They cause hepatotoxicity, nephrotoxicity and are possibly
carcinogenic. They are notable for causing neurologic disease such as
Equine Leukoencephalopathy as well as swine pulmonary edema, esophageal
cancer and neural tube defects.

Ochratoxin A and Citrinin are products of Penicillium and Aspergillus
found in cereals. They are possibly carcinogenic, but notable for
nephrotoxicity along with neurotoxicity and immunotoxicity.

Zearalenone is a Fusarium metabolite known as a powerful estrogen mimic
causing birth defects and breast cancer.

Aflatoxins are secondary metabolites of Aspergillus species. Of all the
mycotoxins in human food, aflatoxins have the strongest association with
cancer. Sterigmatocystin is precursor of aflatoxin produced by
Aspergillus species. It has been associated with cancer in humans.

\textbf{Mitochondrial Dysfunction \& Biomarkers} :

Mitochondrial dysfunction is a major finding in both ALS and with
poisoning by trichothecenes neurotoxins. Kreb cycle intermediates in
urine rise. Erythrocyte Protoporphyrins and Urine Fractionated
Porphyrins are abnormal in a pattern suggesting a toxic exposure.

\textbf{Immunologic Biomarkers} :

One of the most prominent abnormal findings in both ALS and mycotoxin
exposure is the suppression of the immune system. Immune deficits would
be potential early clues to help follow the course of ALS or mycotoxin
exposure. Suggested blood tests would be, IgG Subclass, IgA, IgM, IgE

Lymphocyte Mitogen Proliferation

Cytokines, IL-6, IL-2, Interferon-gamma

\textbf{Immune Evasion} :

Studies for PD-1/PD-L1 pathway abnormalities could uncover evidence of
immune evasion. This could explain the recalcitrance of ALS to
treatments.

\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_ \_\_\_\_\_\_\_

William K. Reid, M.D. Date

1. Statland JM, Barohn RJ, McVey AL, Katz JS, Dimachkie MM. Patterns of
Weakness, Classification of Motor Neuron Disease, and Clinical Diagnosis
of Sporadic Amyotrophic Lateral Sclerosis. \emph{Neurol
Clin.}2015;33(4):735-748.

2. Dai C, Xiao X, Sun F, et al. T-2 toxin neurotoxicity: role of
oxidative stress and mitochondrial dysfunction. \emph{Arch
Toxicol.}2019;93(11):3041-3056.

3. Maresca M. From the gut to the brain: journey and pathophysiological
effects of the food-associated trichothecene mycotoxin
deoxynivalenol.\emph{Toxins (Basel).} 2013;5(4):784-820.

4. Thorne RG, Pronk GJ, Padmanabhan V, Frey WH, 2nd. Delivery of
insulin-like growth factor-I to the rat brain and spinal cord along
olfactory and trigeminal pathways following intranasal
administration.\emph{Neuroscience.} 2004;127(2):481-496.

5. Thorne RG, Hanson LR, Ross TM, Tung D, Frey WH, 2nd. Delivery of
interferon-beta to the monkey nervous system following intranasal
administration. \emph{Neuroscience.} 2008;152(3):785-797.

6. Lochhead JJ, Kellohen KL, Ronaldson PT, Davis TP. Distribution of
insulin in trigeminal nerve and brain after intranasal
administration.\emph{Sci Rep.} 2019;9(1):2621.

7. Avgerinos KI, Kalaitzidis G, Malli A, Kalaitzoglou D, Myserlis PG,
Lioutas VA. Intranasal insulin in Alzheimer's dementia or mild cognitive
impairment: a systematic review. \emph{J Neurol.} 2018;265(7):1497-1510.

8. Rickels MR, Ruedy KJ, Foster NC, et al. Intranasal Glucagon for
Treatment of Insulin-Induced Hypoglycemia in Adults With Type 1
Diabetes: A Randomized Crossover Noninferiority Study. \emph{Diabetes
Care.} 2016;39(2):264-270.

9. Pardridge WM. Drug transport in brain via the cerebrospinal
fluid.\emph{Fluids Barriers CNS.} 2011;8(1):7.

10. Pardridge WM. Drug transport across the blood-brain barrier. \emph{J
Cereb Blood Flow Metab.} 2012;32(11):1959-1972.

11. Djupesland PG, Messina JC, Mahmoud RA. The nasal approach to
delivering treatment for brain diseases: an anatomic, physiologic, and
delivery technology overview. \emph{Ther Deliv.} 2014;5(6):709-733.

12. Lochhead JJ, Wolak DJ, Pizzo ME, Thorne RG. Rapid transport within
cerebral perivascular spaces underlies widespread tracer distribution in
the brain after intranasal administration. \emph{J Cereb Blood Flow
Metab.} 2015;35(3):371-381.

13. G\selectlanguage{ngerman}änger S, Schindowski K. Tailoring Formulations for Intranasal
Nose-to-Brain Delivery: A Review on Architecture, Physico-Chemical
Characteristics and Mucociliary Clearance of the Nasal Olfactory
Mucosa.\emph{Pharmaceutics.} 2018;10(3).

14. Bruinsmann FA, Richter Vaz G, de Cristo Soares Alves A, et al. Nasal
Drug Delivery of Anticancer Drugs for the Treatment of Glioblastoma:
Preclinical and Clinical Trials. \emph{Molecules.} 2019;24(23).

15. van Woensel M, Wauthoz N, Rosière R, et al. Formulations for
Intranasal Delivery of Pharmacological Agents to Combat Brain Disease: A
New Opportunity to Tackle GBM? \emph{Cancers
(Basel).}2013;5(3):1020-1048.

16. Thakore NJ, Pioro EP. Laughter, crying and sadness in ALS. \emph{J
Neurol Neurosurg Psychiatry.} 2017;88(10):825-831.

17. Chefer VI, Thompson AC, Zapata A, Shippenberg TS. Overview of brain
microdialysis. \emph{Curr Protoc Neurosci.} 2009;Chapter 7:Unit7.1.

18. Lasley SM. The Use of Intracerebral Microdialysis to Elucidate
Environmentally Induced Neurotoxic Mechanisms. \emph{Curr Protoc
Toxicol.} 2019;80(1):e72.

19. Li M, Cuff CF, Pestka JJ. T-2 toxin impairment of enteric reovirus
clearance in the mouse associated with suppressed immunoglobulin and
IFN-gamma responses. \emph{Toxicol Appl Pharmacol.} 2006;214(3):318-325.

20. Pestka JJ, Zhou HR, Moon Y, Chung YJ. Cellular and molecular
mechanisms for immune modulation by deoxynivalenol and other
trichothecenes: unraveling a paradox. \emph{Toxicol
Lett.}2004;153(1):61-73.

21. Obremski K, Podlasz P, Zmigrodzka M, et al. The effect of T-2 toxin
on percentages of CD4+, CD8+, CD4+ CD8+ and CD21+ lymphocytes, and mRNA
expression levels of selected cytokines in porcine ileal Peyer's
patches. \emph{Pol J Vet Sci.} 2013;16(2):341-349.

22. Wu Q, Wu W, Franca TCC, Jacevic V, Wang X, Kuca K. Immune Evasion, a
Potential Mechanism of Trichothecenes: New Insights into Negative Immune
Regulations. \emph{Int J Mol Sci.} 2018;19(11).

23. Vu LT, Bowser R. Fluid-Based Biomarkers for Amyotrophic Lateral
Sclerosis. \emph{Neurotherapeutics.} 2017;14(1):119-134.

24. Vidal A, Mengelers M, Yang S, De Saeger S, De Boevre M. Mycotoxin
Biomarkers of Exposure: A Comprehensive Review. \emph{Compr Rev Food Sci
Food Saf.} 2018;17(5):1127-1155.

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