Mast Cell Sensitization as a Plausible and Researchable Mechanism for Chemical Intolerance

Worldwide observations provide evidence for a two-stage disease process called Toxicant-Induced Loss of Tolerance (TILT), described in this journal in the rst of two related papers. The disease process is initiated by a major exposure event, or a series of lower level exposures (Stage I, Initiation). Subsequently, affected individuals report that common chemical inhalants, foods, and drugs trigger multisystem symptoms (Stage II, Triggering). Given that foods and drugs also are comprised of chemicals, we refer to these intolerances simply as “chemical intolerance” (CI). In this second, companion paper we propose mast cell sensitization and mediator release as a plausible and researchable biological explanation for TILT. we We using across CXCL: (C-X-C growth granulocyte-macrophage colony stimulating factor; HKA: hemokinin-A; interferon; IL: interleukin; MCP: mast upon T Cell Expressed Presumably Secreted; P; SRS-A: of tumor necrosis factor;


Introduction
Chemical, food, and drug intolerances are growing international concerns [1][2][3][4][5]. These intolerances may arise following exposures to building construction or remodeling, pesticides, Gulf War chemicals, combustion products, surgical implants, mold, and many other sources [6]. The exposures may be onetime, acute events; a series of exposures; or long-term, low-level exposures. They often involve particular synthetic chemicals such as an organophosphate pesticide, a combination of synthetic substances, and/or their combustion products. These xenobiotics enter the body via well-known routes: inhalation, ingestion, skin contact, and/or injection/implantation. What remains unclear is why a subset of individuals would subsequently develop persistent intolerances to chemicals, foods, and drugs which never bothered them before and do not bother most people. Over the past 70 years, strikingly similar reports have emerged from patients, doctors, and researchers in different nations supporting the existence of a novel, or at least previously unrecognized, disease process.
There is accumulating evidence for a two-stage causal model which links initiation by chemicals with subsequent chemical, food, food additive, or medicine/drug triggers, called Toxicant-induced Loss of Tolerance [7,8]. The origins of these intolerances have been variously attributed to classical toxicity, allergy, and psychological factors [9][10][11]. Up to now, a precise mechanism has remained elusive.
As with other major categories of exposure-induced illnesses, such as classical toxicity or allergy, those who become ill have been presumed to be more genetically or epigenetically susceptible and/or may have been more heavily or frequently exposed than early evidence indicated. The perplexing question is "What is different about people who develop these intolerances versus those who do not?" In this paper we propose a plausible, parsimonious answer to this question.
In the last decade, our understanding of the evolutionarily ancient mast cell (MC) and its ability to effect a vast range of in ammatory, allergic, and other responses throughout the body has expanded rapidly. Several factors have resulted in the underestimation of the MC's pivotal role in disease: 1) Since the discovery of IgE, allergy's central focus has been on the humoral, as opposed to the cellular, immune system; 2) MCs' typically tiny numbers and their sparse distribution in most tissues have contributed to their anonymity; and 3) MCs are minimally present in circulation, and even where they dominate in peripheral tissues, it has been hard to identify and isolate them.
These sentinel cells guard the perimeters of our skin and other organs, warding off invaders and protecting our internal milieu. The MC is one of the rst responders to most bodily invasions and insults.
They are highly evolved, critical components of the cellular immune system [12]. Largely lying in wait, these warriors spring into action in milliseconds, deploying sophisticated molecular weaponry. If they perceive a major threat, they can release a vast array of mediators all at once. We have long been aware of MCs' ability to precipitate anaphylaxis in response to bee stings, peanuts, and other allergens in previously sensitized individuals. At a bare minimum, the release of histamine by MCs into the surrounding tissues and bloodstream leads to immediately recognizable hives, hypotension, syncope, respiratory arrest, and even death. We now understand, however, that there is an extensive array of other mediators that MCs differentially release in response to varying stimuli. In addition, MCs are sensitizable, that is, they remember past invaders. Receptors decorate their exterior surfaces. These miniature detectors, alone or in varying combinations, can identify an extraordinary array of signals and are capable of precise responses. They are rst to recognize when, where, and what sort of threat is looming.
Over roughly a half-billion years, MCs have evolved their capacity to identify invaders and select precisely which weapons to deploy, in what order, and for how long [12]. Even while an MC is called to action (activated) to launch its pre-formed armaments, it can signal other cells to the join the battle. Meanwhile, behind the frontline, MCs are reloading their weapons and stockpiling new munitions. Thus, our so-called "primitive" immune system is in fact quite sophisticated. It was many decades following the discovery of IgE and its relationships to anaphylaxis and classical allergies (such as to pollens, animal dander, and dust mites) that we learned of the MCs' capacity to respond to a bewilderingly broad range of stimulirevealing a new, alternative pathway for their activation and degranulation, even in the absence of "classic" binding of antigen with multiple molecules of MC-surface-bound IgE.
The fact that chemically intolerant individuals often report immediate symptoms following seemingly insigni cant exposures, such as a whiff of fragrance, has led some to speculate that the mechanism must be neurological. Notably, mast cells may rapidly release, or gradually leak, mediators some of which are pre-formed. In fact, there is no cellular element of the immune system that can react faster than mast cells. Lymphocytes require hours to activate, and neutrophils require minutes, but mast cells can respond to a trigger in sub-second time [13][14][15][16].
The links between our contemporary exposures and our ancient mast cells appear to have been missed.
Since WWII, more and more synthetic organic chemicals have crept into our personal environments. In response to the oil embargo and consequent energy conservation efforts in the 1970s, many U.S. homes and buildings were sealed more tightly, resulting in insu cient fresh air. This has resulted in the accumulation of every sort of indoor air contaminant to higher levels than ever before (e.g., volatile organic chemicals outgassing from new construction and remodeling materials, pesticides, mold, disinfectants, and cleaning agents) [6]. Only now, are we learning that our contemporary exposures may be provoking MCs to release their broad range of in ammatory mediators, resulting in a condition called "Mast Cell Activation Syndrome" (MCAS) [17]. Theoharides et al. [18] propose that the diagnosis "Mast Cell Mediator Disorders (MCMD)" be used whenever unique mast cell mediators are measurable.
We propose mast cell activation (MCA), with mediator release implicit in that process, as a plausible biological mechanism underlying chemical intolerance (CI). If MCA and CI are closely related, they should share similar pathophysiologies and exhibit parallel symptoms and intolerances. In this paper, we explore these parallels.

Background
Converging lines of evidence support MCA as a plausible unifying explanation for a host of previously unexplained illnesses, including CI.
Mast Cell Activation Syndrome: Mast Cells (MCs) are components of the ancient cellular, or intrinsic, immune system which evolved over 500 million years ago, predating adaptive immunity and immunoglobulins [19]. For a variety of reasons, including the fact that MCs do not circulate in the blood in signi cant numbers, they have been di cult to study. MCs are present-generally in low numbers and sparsely distributed-typically at the interfaces between our tissues and the external environment, including the skin, and the respiratory, gastrointestinal, and genitourinary tracts, as well as in the walls of all vessels-precisely where one would expect a principal host defense effector cell to be sited. These tissue-dwelling cells originate in the bone marrow and then migrate via the bloodstream to reach their target tissues, where they continue to mature and reside for an average of 2-4 years, with little mobility or chemotaxis compared to other leukocytes [20].
Once MCs reach their destinations, they continue to differentiate in ways that are speci c to the tissue and the intrinsic and extrinsic environment, including any chemicals, foods, and drugs encountered. Once triggered, MCs can release more than 1000 distinct mediators [21] resulting in in ammation, allergy-like symptoms, or altered tissue growth and development. A real-time video shows how quickly mast cells degranulate and release mediators (Video S1). Notably, too, these MC mediators can continue to provoke the very MCs that produced them, resulting in a vicious, self-stimulating feedback loop. For example, the presence of activating histamine H1 and H2 receptors on the surface of the MC provides at least part of the rationale for use of both H1 and H2 blockers in MCAS patients, most of whom gain distinct bene ts from each of these therapeutic classes [22][23][24][25].
Fully differentiated MCs bear a wealth of speci c cell-surface receptors (more than 250 [21]), e.g., the KIT transmembrane tyrosine kinase receptor which is the principal MC regulatory element, and the FcεRI receptor for IgE-class immunoglobulin [26,27]. Although IgE antibodies can stimulate FcεRI once they bind antigen [28], MCs respond to a wide variety of environmental cues that can trigger them to release pre-stored and/or newly synthesized mediators. The nal stages of MC differentiation occur in the tissues in which they reside, with different types of MCs present in different tissues. MCs respond by releasing mediators particular to the insult and its anatomic location. Upon triggering, whether by docking of a triggering substance with a MC-surface receptor, or by action of a triggering force (e.g., temperature change, pressure change, speci c wavelength exposure) on a force-sensing element of the MC, MCs can release mediators in a fraction of a second [13,14]. In contrast, neutrophils require minutes, and lymphocytes hours, to activate [29,14]. MCs' ability to respond precisely and rapidly to a vast range of environmental triggers suggests they may possess unique epigenetic and transcriptional capabilities that enable them to adapt rapidly to environmental challenges [30].
Although the proposed diagnostic criteria for MCAS [31][32][33][34]17] differ in some speci c respects, MCAS diagnosis typically rests upon: 1) chronic and/or recurrent symptoms consistent with aberrant MC mediator release; 2) the exclusion of other conditions that might better explain the patient's symptoms; and 3) laboratory evidence of MC activation. Generally accepted laboratory markers of MCAS include elevated levels (in blood or urine, as appropriate for each metabolite) of tryptase or a few other mediators relatively speci c to the MC (e.g., heparin, histamine and its principal immediate urinary metabolite Nmethylhistamine, prostaglandin D2 and its immediate metabolite 11-beta-prostaglandin-F2-alpha, chromogranin A, and leukotriene E4). Clinical experience to date has shown that most patients diagnosed by these criteria respond to MC-targeted treatments. One of the most speci c laboratory tests for MC activation is serum tryptase, although this typically is elevated, and only mildly, in about 9-16% of MCAS patients [34,35]. Further, tryptase is somewhat thermolabile and is constitutively released and thus may not be found elevated in many patients with MC activation [35].
Drugs that inhibit MC degranulation (e.g., cromolyn, ketotifen) or block mediator effects (e.g., welltolerated combinations of histamine type H1 and H2 receptor antagonists administered simultaneously, typically twice daily) often help reduce symptoms. At present, it is not possible to predict which drugs are most likely to help which symptoms in which MCAS patients, thereby requiring both patient and physician to practice great patience, persistence, and a methodical approach for empirically testing the fortunately large array of treatments that have been already found helpful for some MCAS patients. Treatment is palliative, not curative [31][32][33][34]36]. For example, topical diphenhydramine or cromolyn may be helpful for female MCAS patients suffering chronic dyspareunia, vaginitis, or dysfunctional uterine bleeding consequential to their MCA [37]. Although severe morbidity and early mortality may occur, limited data suggest most MCAS patients can expect a normal lifespan.
Chemical Intolerance: Characteristically, CI individuals report multi-system symptoms and new-onset intolerances triggered by structurally unrelated chemicals, foods, and drugs-substances that these individuals say never bothered them previously and do not other most people. Many patients attribute onset of their illness and intolerances to a well-de ned exposure event, such as the Gulf War, disasters like the World Trade Center, indoor air contaminants, or ood or water-damaged buildings resulting in mold and bacterial growth [38]. Different family members or co-workers who become ill frequently exhibit different manifestations, thus confounding physicians and public health investigators [6].
The steps, or process, leading to CI differ from those preceeding infectious diseases or classical toxicity.
Individuals affected by a particular infectious agent or toxicant generally share recognizable constellations of symptoms. This is not the case for CI patients, which explains, in part, why this condition has de ed numerous attempts to establish a consensus case de nition. Indeed, it appears we may be dealing with a new mechanism and category of disease.
Accumulating worldwide reports by patients, physicians, and researchers point to a two-step process [39][40][41][42][43]. Miller [6,44,45] proposed Toxicant-Induced Loss of Tolerance (TILT). This term captures the wide variety of multi-system symptoms and intolerances associated with the condition. TILT develops in two stages: Initiation by a major exposure event, or a series of exposures (Stage I, Initiation), followed by triggering of multisystem symptoms in response to everyday chemical inhalants, foods/food additives, and/or medications/drugs (Stage II, Triggering), often at much lower exposure levels than those found in classical toxicity. Initiating exposures include chemical spills, pesticides, cleaning agents, solvents, combustion products, medications and medical devices, as well as indoor air contaminants associated with materials used in construction or remodeling [6, 44 -46] (see Figures 1 and 2).
The reigning paradigms in allergy, immunology, and occupational/environmental medicine have not widely embraced MCAS or TILT, or agreed upon case de nitions for them [47][48][49]. Although most doctors have some awareness of CI, few are aware of MCAS at all. It follows that the few doctors who evaluate TILT patients are not testing for MCAS. Based upon our prior work, it appears that MCAS and TILT may be part of the same "iceberg," both submerged below a waterline of clinical recognizability [7].
Prevalence of CI and MCAS: By one estimate, 10-17% of the German population may have MCAS [50]. CI prevalence estimates range from 8-33% in population-based surveys [51][52][53]. Hojo et al. [2] in Japan and Steinemann [1] in the U.S. each conducted surveys of chemical intolerance in their respective countries on two separate occasions, a decade apart. According to their research, in just 10 years, substantial increases in CI occurred in both countries.
The Quick Environmental Exposure and Sensitivity Inventory (QEESI), developed by the senior author of this paper, is considered the reference standard for assessing CI and has become a surrogate case de nition used by researchers in more than 15 countries in approximately 80 peer-reviewed studies [1,39,[41][42][43]54]. This validated, self-administrable, 50-item questionnaire is used for: (1) Research-to characterize and compare study populations, and to select subjects and controls; (2) Clinical Evaluations -to obtain pro les of patients' self-reported symptoms and intolerances; the QEESI can be used at intervals to follow symptoms over time or to document responses to treatments or exposure avoidance; and (3) Workplace, Community, or Epidemiological Investigations-to identify those who may be more chemically susceptible and/or measure changes in symptoms and intolerances in exposed versus control groups.
The QEESI has four scales: Symptom Severity, Chemical Intolerances, Other Intolerances, and Life Impact.
Each scale item is scored from 0 to 10 (0 = "not a problem" to 10 = "severe or disabling problem"). There is also a 10-item Masking Index which gauges ongoing exposures (such as to caffeine or tobacco) that can reduce individuals' awareness of their intolerances [55].
Connecting MCAS and TILT: Our understanding of the possible role for MCs in TILT is recent. Both patients with MCAS and those with TILT commonly report symptoms in multiple organ systems and often several systems simultaneously. MCs produce and release scores of chemical signals (generically termed "mediators") that can affect organs, tissues, and systems throughout the body.
TILT encompasses exposures that may have initiated illness and exposures that continue to trigger symptoms. However, until now, TILT has lacked a clear biological mechanism, which MCAS may provide. An understanding of TILT initiation and triggering offers strategies for prevention and intervention, many of which appear applicable to MCAS. Knowledge of the MCAS mechanism has the potential to inform new medical interventions and treatments for TILT. Failure to eliminate or reduce initiators such as pesticides or mold can result in chronic, even lifelong, illness in susceptible people. This suggests persistent MC activation and degranulation. The symptoms and ndings in TILT patients may be best understood in the context of MCs and the mediators they release.
Together, MCAS and TILT satisfy the principle of parsimony (i.e., Occam's Razor). Table 2 summarizes prominent features of each. TILT serves as an umbrella category for these exposure-driven illnesses, while MCAS offers an umbrella mechanism uniting illnesses driven by MC activation.  IL-1α, IL-1β, IL-2, IL-4, IL-5, IL-6, IL-7, IL-9, IL-10, IL-12, IL-13, IL-15, IL-16, IL-18, IL-21, IL-23, IL-25, IL-31,  IL-33, IFN   Historically, physicians have struggled to differentiate between complex, multisystem illnesses and psychiatric disorders, particularly when symptoms involve cognition and mood [70]. Before biological mechanisms are established, there is an initial tendency to attribute complex and unfamiliar conditions like TILT and MCAS to stress and/or psychogenic causes. Unaware of MCAS, some physicians may mistakenly diagnose psychosomatic disorders or even Munchausen syndrome (or, in children, Munchausen syndrome by proxy) in these patients, with adverse consequences for the patients' mental health and further delaying accurate diagnosis and effective treatment.
Both MCAS and TILT have prominent neurological features. For example, organophosphate pesticides, which bind irreversibly to cholinergic receptors in the parasympathetic nervous system, appear to be among the most severe and permanently damaging TILT initiators. Correspondingly, organophosphates have been shown to trigger degranulation in human and animal mast cells [71]. The parasympathetic nervous system also modulates MC activity via a cholinergic pathway [72]. MCs play pivotal roles in regulating cerebral blood ow [73], directly affecting brain function. Notably, both MCAS and TILT patients commonly report cognitive di culties which may be the result of reduced cerebral blood ow due to chemical exposures, such as vehicle exhaust or pesticides [74]. Brain MCs lie close to cerebral blood vessels, nerves, and the meninges, and inhabit the area postrema, choroid plexus, thalamus, hypothalamus, and limbic system, thus affecting memory, mood, and concentration. MCs can migrate between nerve tissue and lymphatics, and appear to contribute to neuroin ammation in many disorders [75][76][77]. Notably, during stress, corticotropin-releasing factor is secreted by the hypothalamus and, together with neurotensin, triggers MCs to release in ammatory and neurotoxic mediators, thereby disrupting the bloodbrain barrier and resulting in neuroin ammation [81]. Referring to ADHD, Song et al.
[63] cite increasing evidence that MCs are involved in brain in ammation and neuropsychiatric disorders. Selective release of in ammatory mediators by MCs, interacting with glial cells and neurons, may activate the hypothalamic-pituitary-adrenal axis and disrupt blood-brain barrier integrity.
This physiology ts the two stages of TILT-initiation and triggering, that is, initiation by a single intense exposure, or repeated lower level exposures (pesticides, implants, drugs, etc.), which immunologically sensitize mast cells in the brain and/or other key sites. Thereafter, both chemically related and chemically unrelated, xenobiotic exposures may readily trigger mediator release by these now "twitchy" mast cells. Anticipated cognitive and mood effects might include: sudden rage (e.g., "road rage"); impulsive, violent, or abusive behaviors; addictive tendencies; mental confusion/fatigue; and/or a sense of depersonalization. MC "twitchiness" renders these cells vulnerable to a host of unrelated exposures that never bothered the person before and do not bother most people. Therefore, it is plausible that MC sensitization and triggering could explain the two stages of TILT.
By the time either MCAS or TILT is suspected, most patients have accumulated numerous diagnoses, each of which may explain a portion of their problems, but none of which account for the full range and duration of their illness. At present, most MCAS cases are classi ed as "idiopathic" rather than primary (i.e., of proven clonal or autoimmune origin) or secondary (i.e., due to some other in ammatory or neoplastic condition). Published preliminary ndings suggest that most "idiopathic" cases likely are If, in fact, TILT and MCAS are closely related conditions, they should share the same underlying pathophysiology and patients should manifest similar symptoms and intolerances. As a rst step in testing this hypothesis, we used the QEESI to explore similarities and differences between TILT and MCAS.

Methods
The MCAS group consisted of patients of authors LBA and TTD seen between September 2017 and July  Table 3 for all six groups. Fifty-nine percent (59%) of the MCAS clinical group t QEESI criteria for CI, a somewhat higher percentage than among the Gulf War Veterans (49%). Percentages of the other comparison groups meeting CI criteria exceeded 75%, except for controls (7%).  Fig. 3 shows the distribution of total QEESI scores and masking indices by participants in these groups. In every case, controls' scores were signi cantly lower than for the other groups (p < .001). With few exceptions, the CI groups scored signi cantly higher than other groups, whether or not they reported an intiating exposure. Regarding the Chemical Intolerance Scale, scores for the MCAS group were not signi cantly different from the Gulf War Veterans' scores, but were signi cantly lower than scores of all other groups. On the Other Intolerance Scale, the MCAS group scored signi cantly higher than the Gulf War Veterans' group (p < .01); however, the MCAS group's score was statistically equivalent to the other groups' scores. On the Life Impact Scale, the MCAS group's score did not differ signi cantly from the Implant group's, and both were signi cantly higher than the Gulf War Veterans group (p < .01). For the Symptom Severity Scale, the Implant group and CI with known exposure group scored signi cantly higher than the other groups (p < .01). Scores for the CI group without reported exposure and the MCAS groups did not differ signi cantly from each other. The Masking Index (a measure of ongoing exposures) was signi cantly greater among controls compared to the other groups (p < .01), except for the Gulf War Veterans group, whose Masking Index score was not signi cantly different from that of controls. The MCAS group and the CI group with known exposures had similarly low masking scores.
Predicted Probability of CI with Increases in MCAS Scores: Logistic regression appears in Table 4.
Compared to the lowest quartile (Q1), those in the 2nd quartile of MCAS scores were 2.6 times more likely to have CI (p = .027); those in the 3rd quartile of MCAS scores were 6.0 times more likely to have CI (p = .0001); those in the 4th quartile of MCAS scores were 6.2 times more likely to have CI (p = .0001). Figure 4 shows that the probability of CI increases as MCAS scores increase. There is an exponential increase in the probability of CI with increasing MCAS scores, reaching nearly perfect prediction toward the extreme set of MCAS scores. Here we merged four groups: the three exposure groups (chemically-intolerant individuals who reported an intiating exposure, Gulf War Veterans, Implant patients) and the chemically-intolerant individuals who did not report an initiating exposure into one group (TILT group) for purposes of comparison against controls and MCAS patients.
Symptom Severity Scale (Fig. 5) There were no signi cant differences between the TILT and MCAS groups for 8 of the 10 symptom items. For the Neuromuscular and Affective items, the TILT group's scores were slightly higher than those of the MCAS group (p < .04). Both the TILT and MCAS groups reported signi cantly more severe symptoms than did controls (p < .0001).
Chemical Intolerance Scale (Fig. 6) TILT and MCAS groups both had signi cantly higher chemical intolerance scores than did controls (p < .0001). The TILT group's chemical intolerance scores were signi cantly higher than the MCAS group's scores for all items (p < .01).
Other Intolerance Scale (Fig. 7) There were no signi cant differences between the TILT and MCAS groups for 8 of the 10 intolerance items. Only the chlorinated tap water item was scored signi cantly higher by the TILT group (p < .01). Only the Foods/Food Additives item was scored signi cantly higher by the MCAS group (p < .03). Both TILT and MCAS groups scored signi cantly higher than controls (p < .0001).
Life Impact Scale (Fig. 8) Both TILT and MCAS groups scored signi cantly higher than controls (p < .0001) on all Life Impact items. The TILT group consistently scored higher on 9 out of the 10 items on this scale than did the MCAS group (p < .01), with the exception of the diet item where the MCAS group reported the a slightly greater impact of their illness on diet.
Masking Index (Fig. 9) Both TILT and MCAS groups had signi cantly lower masking scores than did controls (p < .0001), meaning that they had fewer ongoing exposures to tobacco smoke, fragrances, or caffeine which tend to hide ("mask") the relationship between symptoms and exposures. However, the MCAS group reported greater use of drugs/medications and gas stoves than did the TILT group (p < .05).

Discussion
For decades, both MCAS and CI patients have been misunderstood, marginalized, and often referred for mental health evaluation [6, 34,86]. Symptoms in these patients arising from MC-neuronal interactions often are viewed as neuropsychiatric in origin, with mental health practitioners assigning diagnostic labels such as somatic symptom disorder or conversion disorder. Many psychiatric diagnoses are not etiologic and thus fail to de ne the potential role of environmental exposures/xenobiotics in initiating and perpetuating illness [78].
Some have alleged that these conditions could not be real because they involve too many symptoms in too many organ systems, with no known etiology or unifying mechanism [6,34]. Others have invoked olfactory-limbic sensitization [87] and neurogenic in ammation [60] as potential unifying mechanisms. The result has been a stalemate in diagnosis, research, and treatment. Our results imply that environmental agents could initiate or escalate TILT/CI, resulting in chronic, aberrant MC mediator release in potentially every tissue of the body. MCAS and TILT both involve sensitization, i.e., response ampli cation over time, initiated and triggered by a host of chemicals, foods, and drugs. Mast cell activation and mediator release may be driven via the "classic" IgE-mediated route to MC activation, or potentially via the panoply of non-IgE-mediated MC activation mechanisms now known to exist. We propose MC activation and mediator release as a plausible biological mechanism for chemical intolerance or TILT and suggest how treatment may be informed by successful therapies for MCAS.
Similarities between MCAS and TILT: In Figs. 5-8 we see that all comparison groups had statistically higher scores than did controls on the QEESI scales. We also see that the MCAS and TILT groups share strikingly similar patterns of symptoms and intolerances to structurally diverse chemicals, foods, and drugs.

Symptom Severity Scale
For eight of the 10 symptom items there was no signi cant difference between the MCAS and TILT groups. There was only a slight increase in severity in Affective and Neuromuscular symptoms in the TILT group compared to the MCAS group. Mediators released by mast cells in the central nervous system may explain the depression, irritability, and loss of motivation patients in both groups commonly report.

Chemical Intolerance Scale
The same classes of chemicals appear to trigger symptoms in the MCAS group as in the TILT group. However, for every chemical item, the TILT group reported signi cantly more severe symptoms than did the MCAS group. Notably, it appears (Fig. 6) that the most problematic triggers for many MCAS patients are fragrances, which also pose major problems for CI individuals [88]. Fragrances are often used in poorly ventilated places such as restrooms or airplanes, triggering signi cant symptoms in growing numbers of "TILTed" and other susceptible individuals. Finding fragrance-free workplaces, schools, housing, churches, etc., can be a near-impossible quest. Fragrances have deleterious effects on cognition, mood, and breathing, and must become a principal focus for environmental and public health agencies that deal with air pollutants. Prior to the discovery of IgE, who would have guessed that just a few molecules of latex, penicillin, or peanuts could trigger allergic reactions in sensitized individuals?
Belatedly, we are learning that extraordinarily low-level exposures to synthetic chemicals, such as fragrances, may trigger mast cell mediator release in previously sensitized individuals.

Other Intolerance Scale
It is striking for the Other Intolerance Scale, there were no signi cant differences between the TILT and MCAS groups for eight of the 10 items. Only the Chlorinated Tap Water item was scored signi cantly higher by the TILT group. Many CI patients nd that the smell and taste of chlorinated tap water objectionable and use ltered or bottled water.

Life Impact Scale
Notably, the TILT group consistently scored higher than the MCAS group on nine of the 10 Life Impact items. One reason for this may be that individuals with TILT/CI have greater di culty tolerating exposures commonly encountered in social activities, which can be attributed to, for example, fragrances that people wear, fragrance-emitting devices, cleaning products, candles, tra c exhaust, and various indoor and outdoor air contaminants. Even outdoor events like concerts or ballgames can entail exposures to tobacco smoke, insect repellants, BBQ smoke, mold, and chlorinated pools/spas. With such restrictions, it is understandable that depression, irritability, and loss of motivation may develop over time.
Many become frustrated by healthcare providers who do not understand chemical intolerance. Costly therapies often yield little or no improvement and potentially worsen their condition. CI patients, their families, friends, employers, and colleagues grow weary. Notably, both the MCAS and TILT groups and their doctors frequently recognize adverse food reactions, and patients in both groups identify speci c food intolerances.
Masking Index "Masking" results from overlapping responses to chemical inhalants, foods, and drugs, as well as an individual's tendency to addict or habituate to these substances. Masking obscures the relationship between symptoms and triggers, literally hiding the cause-and-effect relationship between them from both patients and clinicians [89].
The control group endorsed more Masking items than did the TILT and MCAS groups, consistent with our prior studies [84,90]. People without CI or MCAS may be more apt to use alcohol, tobacco, and caffeine for their stimulatory effects. Our controls reported less di culty with chemicals in the environment, perhaps because they were more masked and/or they were more biologically tolerant. The MCAS group reported greater use of drugs/medications which could re ect the fact that MCAS is more commonly treated with medications to prevent MC degranulation and/or to block MC mediator effects. Individuals with CI often experience so many adverse drug reactions that they avoid most prescription and over-thecounter drugs, favoring alternative therapies such as herbs, homeopathy, or acupuncture [91].
Current TILT/CI Treatment: Trigger identi cation and avoidance, rather than medications, are mainstays for treating the symptoms of CI. Likewise, these are the rst steps for managing MCAS. There are two principal ways to protect a patient from problem exposures: either remove the exposure from the patient or remove the patient from the exposure [6]. Various prophylactic medications/supplements (e.g. cromolyn) or desensitization procedures may bene t some patients.  Fig. 10). Likewise, administering the QEESI can help researchers and clinicians gauge the success of their interventions, whether medications, avoidance, or other.
Environmental Interventions: The most effective method for identifying potential problem exposures is for an indoor air quality professional who understands CI to conduct an environmental audit/investigation.
An experienced evaluator can assess air ows, migration of air contaminants from an attic, basement, or attached garage; identify sources of water/humidity that foster mold or bacterial growth; and locate exposure sources, including use and storage of pesticides, fragrances, cleaning, personal care, and laundry products; and document the presence of vehicle exhaust, petroleum, glues, solvents, and tobacco smoke.
Interestingly, the MCAS group reported greater use of gas stoves than did the TILT group (58% vs 25% respectively), perhaps suggesting an important source and intervention for MCAS patients who use gas stoves. Historically, as early as the 1960s, removing gas appliances has been a principal recommendation for CI individuals [95].

Dietary Interventions
Both TILT and MCAS patients report adverse reactions to foods. Because food intolerances are common among these patients, but often hard to identify, their diets deserve special attention. We recommend assistance from dieticians who understand food intolerances, food addiction, and elimination diets. The majority of these adverse food reactions are food intolerances as opposed to classical, IgE-mediated food allergies (e.g., to peanuts) that are discoverable through skin or blood testing. In contrast, food intolerances often are "hidden" or "masked." The gold standard for identifying food intolerances involves "unmasking," that is, the rigorous elimination of suspect foods for 4 to 7 days, followed by judicious reintroduction of single foods, one-at-a-time, under close medical and dietary supervision. For a detailed description of masking, unmasking, and food intolerances, see references [96][97][98][99][100].
Foods themselves may be triggers, but food additives and chemical residues on foods are also potential triggers. For example, some patients exclusively eat organic food to avoid pesticide residues. There are growing concerns over a related chemical, glyphosate, introduced in 1974, now globally the most widely used herbicide since the development of genetically engineered, herbicide-tolerant crops. Contact allergies with glyphosate have been documented, and recently, glyphosate has been shown to be associated with allergic reactions in both human and animal studies [101][102][103].

Medical Interventions
Notably, in medical history, striking recoveries or responses to particular medications have led doctors to infer particular mechanisms. Such may be the case for TILT if the knowledge gleaned from treating MCAS patients is applied to those suffering from CI or TILT. Up to now, there has been no demonstrable biological mechanism for CI/TILT, making it di cult to develop evidence-based treatments.
After trigger identi cation and avoidance strategies are implemented, potential medical interventions for CI may include many of those used to treat MCAS. Many authors report success with pharmacological prophylaxis for MCAS, including the agents that prevent MC degranulation and/or reduce tissue in ammation caused by MC mediators. [50,104,105]. For example, impressive clinical responses to H1/H2 blockers and mast cell stabilizers like cromolyn or quercetin have been observed in MCAS patients [31,34,[79][80][81]104]. Similarly, cromolyn might bene t CI/TILT patients. Cromolyn, however, is poorly absorbed from mucosal surfaces and therefore must be applied fairly frequently to speci c surfaces (e.g., the gastrointestinal tract, nasal mucosa, and respiratory tract) to maximize its bene t. Patients may not tolerate usual commercial preparations of cromolyn or H1/H2 blockers and may require compounded formulations with no excipients or coloring.
[81] report that luteolin may lessen the tissue in ammation underlying "brain fog." Interestingly, low-dose benzodiazepines help some MCAS patients. This may be due to the presence of benzodiazapine receptors on not only neurons but also MCs [107][108], suggesting a pharmaceutical therapy targeting both the nervous and immune systems.
Various kinds of pain in various sites affect at least three-quarters of MCAS patients, and experienced physicians recommend that potential causes be evaluated and treated speci cally [79,34,13]. Wirz and Molderings [50] reviewed analgesic drug options for MC-mediated acute and chronic pain.
Pharmacotherapy for TILT/CI is by no means a simple process and still requires minimizing chemicals, foods, and medications known to precipitate adverse reactions. Furthermore, drug excipients, diluents, and food additives, including dyes and preservatives, are known to provoke symptoms in both MCAS and TILT patients and must be suspected whenever a new medication or food formulation has been introduced into the patient's regimen shortly before symptoms appear or escalate. The vast majority of medications contain so-called 'inactive ingredients' that are not listed on the label but can trigger symptoms in sensitized MCAS patients or people with TILT/CI [109]. These excipients can include "inert" llers, binders, dyes, and preservatives in oral medications. Other possibilities include adhesives used for patch medications and plasticizers that leach from intravenous bags and tubing. Similarly, silver nanoparticles applied to plastics to prevent infection can trigger MC activation in some patients [110].
Other Implications for Clinical Practice Mast cell degranulation and mediator release suggest an elegant explanation for TILT's numerous "unexplained" symptoms and for a host of so-called "idiopathic" illnesses sharing features of TILT. These include Gulf War Syndrome, Breast Implant Illness, some mold-related illnesses, and various other exposure-induced conditions ( Figs. 1 and 2). Likewise, researchers and clinicians who wish to understand TILT-related, or overlapping conditions including bromyalgia, chronic fatigue syndrome, depression, irritable bowel syndrome, asthma, eczema, attention de cit/hyperactivity disorder, or autism spectrum disorders [111,112] need to take exposure histories that include asking when illness began or was exacerbated, whether an initiating event occurred, and whether other people (or animals) were exposed or affected.
We have observed that some of the most severely "TILTed" individuals were "initiated" by exposure to organophosphates (OPs) [90]. Groups exposed to OPs and at risk for TILT include agricultural workers, sheep dippers, occupants exposed to pesticides, Gulf War soldiers, and airline crew members exposed to "fume events" during which engine lubricants (OPs) bleed into cabin air [6,113]. OPs irreversibly bind acetylcholinesterase. The enzyme paraoxonase, or PON1, helps determine a person's ability to detoxify organophosphates [114] and may explain why certain individuals are particularly susceptible to both TILT and to MCAS.

Implications for Regulatory Agencies/Authorities
Any policymakers or regulatory bodies dealing with chemicals, foods, and/or drugs need to understand TILT and mast cell sensitization.

Directions for Future Research
With this new understanding of the role of mast cells in TILT, important questions arise concerning individual susceptibility differences that may be due to prior exposures, genetics, epigenetics, and nutrition. Mast cell activation and mediator release suggest a rational and parsimonious explanation for both the initiation and triggering phases of TILT.
Given the overlap between the TILT and MCAS populations, and the fact that mast cell mediators could explain much about TILT, future research should examine the following questions: (1) What proportion of the TILT population harbors detectable MC activation as determined by a rigorous diagnostic MCAS workup? (2) Do patients with TILT have somatic MC regulatory gene mutations as already found in most MCAS patients? (3) If so, are there recurrent mutations re ecting differing clonality patterns (e.g., in KIT) [26] characterizing differing subsets of TILT patients, perhaps even " ngerprinting" particular initiating exposures, and (4) Would speci c treatments targeting MCs or their mediators prove helpful for the TILT population as a whole or for certain subsets?

Conclusion
Mast cell activation and mediator release appear to explain decades of observations by physicians and their patients who report multi-system symptoms and intolerances following a wide variety of exposures.
We have demonstrated that as the likelihood of patients' having CI/TILT increases, their likelihood of having MCAS similarly increases, to a near-perfect correspondence at the high ends of these scales. An association is, of course, not proof of causation. Nevertheless, the strikingly similar symptom and intolerance patterns for the MCAS and TILT patients suggest a shared biological mechanism which we posit to be mast cell sensitization by xenobiotics and subsequent degranulation with re-exposure.
Traditional referral pathways send TILT/CI patients and their healthcare providers on a "wild goose chase," diverting them from addressing the environmental (TILT) and biological (mast cell) origins of patients' conditions. Faced with patients suffering from complex conditions affecting multiple organ systems with symptoms that wax and wane unpredictably, clinicians should ask themselves two questions: 1) Could MCs be causing these problems? 2) Could environmental exposures be driving MC activation and mediator release?
Understanding the connection between TILT and mast cells has the potential to reveal hidden links between environmental exposures and illness, and improve practice in medicine, psychology, public health, environmental health, and regulatory toxicology.

Declarations
Authors' contributions: CSM conceived and designed this work. LBA and TTD provided insight and guidance regarding mast cells, and were responsible for the acquisition of the clinical data. RFP acquired the archived data, and analyzed and interpreted the statistical results. NAA provided content related to chemical intolerance and regulatory concerns in Europe and the U.S. All authors contributed substantially to the drafting and revisions of the manuscript and approved the submitted version. All authors are