Nickel

For a list of foods and their nickel content visit www.melisa.org This website has a wealth of infomation about nickel and other metal allergies. There are also peer reviewed scientific studies regarding metal allergies and the connection to autoimmune disorders. They provide testing for metal allergies that are non-invasive.

Sharon Hanson

Nickel Allergy Is Found in a Majority of Women with Chronic Fatigue Syndrome and Muscle Pain

And May Be Triggered by Cigarette Smoke and Dietary Nickel Intake

Björn Regland, MD
Olof Zachrisson, MD
Vera Stejskal, PhD
Carl-Gerhard Gottfries, MD

ABSTRACT. Two hundred and four women with chronic fatigue and muscle pain, with no signs of autoimmune disorder, received immune stimulation injections with a Staphylococcus vaccine at monthly intervals over 6 months. Good response was defined as a decrease by at least 50% of the total score on an observer’s rating scale. Nickel allergy was evaluated as probable if the patient had a positive history of skin hypersensitivity from cutaneous exposure to metal objects. The patient’s
smoking habits were recorded. Fifty-two percent of the patients had a positive history of nickel contact dermatitis. There were significantly more good responders among the non-allergic non-smokers (39%) than among the allergic smokers (6%). We also present case reports on nickel-allergic patients who apparently improved after cessation of cigarette smoking and reducing their dietary nickel intake. Our observations indicate that exposure to nickel, by dietary intake or inhalation of cigarette smoke, may trigger systemic nickel allergy and contribute to syndromes of chronic fatigue and muscle pain. [Article copies available for a fee from The Haworth Document Delivery Service: 1-800-342-9678.
E-mail address: Website: Press.com> _ 2001 by The Haworth Press, Inc. All rights reserved.]

KEYWORDS. Nickel allergy, cigarette smoking, dietary nickel, fatigue,
muscle pain

INTRODUCTION
Nickel is a common sensitizing agent responsible for the high prevalence of allergic contact dermatitis. However, the health hazards of nickel allergy with regard to diffuse and general symptomatology, such as chronic fatigue and muscle pain, appear not to be fully understood and are probably underestimated. The prevalence of nickel contact dermatitis among women has increased remarkably and there is a clear relationship between ear piercing and induction of nickel allergy (1). In two Norwegian unselected populations, the prevalence figures for women were reported to be27.5 and 31.1%, respectively (2). Although in the same study the
prevalence for men was 5%, the modern fashion of piercing also anticipates an increasing prevalence among men (3). Nickel allergy is associated with fatigue syndromes with or without autoimmunity (4). In women with chronic fatigue syndrome the prevalence of nickel
contact dermatitis has been reported to be as high as 52% (5). Recently we chanced upon findings regarding nickel allergy in a study set up with quite another purpose. Immune stimulation with a Staphylococcus vaccine was tested in clinical trials of female patients
with chronic fatigue and muscle pain (see 6 for a preliminary report). The patients received subcutaneous injections at monthly intervals. The results are interesting, showing clinical improvement in a substantial number of patients. Moreover, we unexpectedly found that nickel allergy influenced the efficiency of the treatment and that nickel allergy
was interrelated with cigarette smoking.

Original Research 59

The primary incidental finding of nickel sensitization came out of a Memory Lymphocyte Immuno Stimulation Assay (MELISA_) in 16 patients who did not improve or had reacted adversely to the vaccine treatment (7). MELISA_ is an optimized lymphocyte proliferation test (8). The purpose of using MELISA_ in the study was to check whether the unresponsiveness to the vaccine could be due to hypersensitivity to the preservative thiomersal (syn. merthiolate, thimerosal). In addition, reactivity was tested to various metals such as nickel, although the vaccine compound does not contain nickel. The main finding was that 13 of the 16 tested patients (81%) reacted strongly against nickel in vitro (7). Moreover, we found a substantial number of cigarette smokers among the non-responding and MELISA_-positive nickel-allergic patients, whereas the combination of smoking and contact allergy was hardly seen at all in the group of patients rated as good responders.
Thus, we were made attentive to the intriguing possibility that a connection might exist between nickel allergy and cigarette smoking. As nickel and a variety of other metals occur in trace amounts in mainstream cigarette smoke (9), it would be a plausible suggestion that exposure to cigarette smoke may be nickel-sensitizing or, at least, a potential trigger of hyperreactivity in a person already sensitized to nickel.The aim of this study was to further explore the impact of nickel allergy and its interrelation with cigarette smoking in a large number of women with chronic fatigue and muscle pain included in clinical
trials of immune stimulation therapy.

PATIENTS AND METHODS
Two hundred and four women with chronic fatigue and muscle pain, aged between 21 and 73 years (mean _ SD, 48 _ 11), had been included in clinical trials and treated for at least 6 months with subcutaneous injections of a staphylococcus vaccine (Staphypan Berna_) at
monthly intervals. Each woman had a disorder that fulfilled the criteria for both fibromyalgia (10) and chronic fatigue syndrome (11), with no obvious sign of autoimmune disorder. The trials were approved by the Ethics Committee of Göteborg University. Treatment outcome was rated using the Comprehensive Psychopathological Rating Scale (CPRS), an observer’s rating scale previouslydescribed (6).

60 JOURNAL OF CHRONIC FATIGUE SYNDROME

A good responder was defined as a person in whom the symptomatology
evaluated by the CPRS, i.e., the CPRS total score, was decreased by at least 50% 6 months after the initiation of the treatment. Nickel allergy was evaluated as possible/probable if the patient had a positive history of skin hypersensitivity from cutaneous exposure to metal objects such as ear rings (most common), other jewelry, wrist watches and/or clothing fasteners, e.g., jeans buttons.
RESULTS
Table 1 shows the numbers and percentages of good responders in
subgroups of patients divided according to smoking habits and histories
of contact allergy. There were no significant differences in baseline CPRS score between
the nickel-allergic smokers and the others. Among the patients, 52% had a history of possible/probable nickel contact dermatitis and 28% were habitual cigarette smokers.
Thirty-seven percent (36/98) of the non-allergic and 16% (17/106) of the allergic patients were good responders, which is a statistically significant difference according to Fischer’s exact 2-tailed test (p <0.0009). According to the Chi-square test, there were significant differences (p = 0.02) regarding the number of good responders in the subgroups
(Table 1). The most obvious difference was found between non-allergic non-smokers and allergic smokers, which reached statistical sig- TABLE 1. Numbers and percentages of good responders in subgroups of patients divided according to smoking habits and histories of contact allergy

Good responders
Subgroups of patients Number

Yes No
Non-allergic non-smokers 75 29 (39%) 46
Non-allergic smokers 23 7 (30%) 16
Allergic non-smokers 72 15 (21%) 57
Allergic smokers 34 2 (6%) 32
Total 204 53 (26%) 151
Original Research 61
significance according to Fischer’s exact 2-tailed test (p < 0.0005) even
after adjusting for multiple testing (Bon-Ferroni method).
Case Reports
We present two case reports on patients who responded favourably after cessation of cigarette smoking (case 1) and reduced intake of nickel-rich food items (case 2):
Case 1: At baseline, a formerly healthy 36-year-old woman had a troublesome sinusitis with headaches, blurred visual sharpness and a left hand that readily went numb. One month later, she experienced fatigue and diffuse muscle pain. Her mother has fibromyalgia. Routine
laboratory check-outs, including thyroid hormones, were normal. One year after the onset, she was placed on treatment with Staphypan Berna_ and received monthly injections that caused no side effects. However, she was not improving and after 7 months of treatment she
was as fatigued as before. At that time we had learned (see above) that the combination of nickel allergy and cigarette smoking may predict a poor outcome of vaccine treatment. She was known to be nickel-sensitive and used to smoke 15 cigarettes per day. When she heard of ourexperience she became motivated to give up smoking with the aid of nicotine chewing gums. She used to have a high intake of chocolate and liquorice and regularly had oatmeal porridge for breakfast; all of which she dismissed from that time and on. Two months later, she reported that she was healthy and no longer had any of the symptoms she had experienced for more than a year. She no longer had use of analgesics and the improvement held on for more than 6 months. However, after taking up smoking again she relapsed into fatigue and muscle pain symptoms.
Case 2: A 54-year-old non-smoking woman who had had fibromyalgia and chronic fatigue syndrome for many years was treated with Staphypan Berna_. After the injections, she became worse and was bed-ridden with headaches and severe fatigue and pain for a week.
MELISA_ was negative for the preservative thiomersal but strongly positive for nickel. She was not aware of any contact allergy. She had symptoms of irritable bowel syndrome (IBS) but knew of no specific food intolerance. Advised to try a diet she started to avoid food items
known to be high in nickel content such as crustaceans, chocolate, oatmeal products and certain vegetables (mainly broccoli and spinach). This made her experience improved well-being. After 3 months on this type of diet she reported that she had not been in such good
condition for many years. After 6 months she is still improving.
However, when eating nickel-rich food items she experiences an increased
fatigue within two hours and the fatigue may not vanish until
3-4 days later.
DISCUSSION
Metal contact allergy is mostly due to nickel which is ubiquitous in the environment. Sources of human exposure are air, water, food, and tobacco. Food is the main route of uptake followed by cigarette smoke. The dietary intake of nickel is highly variable; the most reported averages are 200-300 micrograms/person/day (12). Consumption of nickel-rich food items may increase the nickel intake from 150 to 900 micrograms/person/day or more (13). Certain food items have very high nickel contents: high levels have been found in legumes, spinach, lettuce, soya beans, oatmeal, and nuts. Certain products, such as baking-
powder and cocoa powder, contain excessive amounts of nickel, perhaps because of leaching of nickel during the manufacturing process. Soft drinking-water and acid beverages may dissolve nickel from pipes and containers. Leaching and corrosion processes may contribute
significantly to the oral nickel intake (13). Immune stimulation with a Staphylococcus vaccine represents a new treatment principle for patients suffering from chronic fatigue and
muscle pain. Our studies have focused on women and the reason is that chronic pain defined as fibromyalgia is a syndrome that almost exclusively affects women. The efficiency of the treatment is evaluated separately (manuscript in preparation). In this paper we report that
immune stimulation differentiated patients who were nickel-allergic smokers from those who were neither nickel-allergic nor smokers. This interesting outcome was thus an incidental observation in a study set up with another purpose. Skin patch testing or an in vitro test such as MELISA_ was not possible to perform in the large number of patients included in the
clinical trials. Therefore we evaluted nickel contact allergy from a positive patient history of metal hypersensitivity. It has been claimed that there is high correlation between patient histories and patch testing results (14). The estimated prevalence of nickel contact dermatitis
in our study is equivalent to that in women with chronic fatigue syndrome
and a positive skin patch test (5). Patch testing is the standard for evaluating nickel contact dermatitis. A number of patients, however, have a positive patch test without a
history of dermatitis (15) and there appears to be considerable intraindividual variation in nickel patch test reactivity (16). With a patch test as reference, dermatologists may consider the sensitivity and specificity of an in vitro lymphocyte proliferation test to be low (17). However, both sensitivity and specificity must be related to the purpose of the
assessment. If the main purpose is to assess nickel hyperreactivity with general symptomatology of ill-being, such as chronic fatigue and muscle pain, an in vitro test of lymphocyte reactivity may prove to be a better reference than a skin test. It might be expected that nickel contact allergy was misdiagnosed in some patients of our study. On the other hand, nickel hyperreactivity along the gastrointestinal route might have been underestimated in patients with no obvious skin contact allergy, as in case report 2. This
notion is supported by several reports indicating that nickel activates T cells from individuals with no history or clinical manifestation of skin diseases (18). Dietary nickel has the potential for triggering hyperreactivity (19). Consequently, one study found that reduction of the dietary intake of nickel may induce long-term improvement of dermatitis in nickel-sensitive
patients (20). In another study a low nickel diet was effective in controlling the symptoms in 39% of nickel-sensitive patients with chronic allergic-like dermatopathies (21). Presumed nickel allergy was a more obvious adverse factor than cigarette smoking with regard to the treatment outcome of the clinical trials in this study, and there was no obvious overrepresentation of habitual smokers in our patient group (28%) in comparison with general populations of adult women (26-31%) in Western societies (22). In a synergistic way, however, cigarette smoking may add to the effects of nickel allergy since mainstream cigarette smoke contains trace amounts of nickel. In this context smoking may be a trigger of nickel hyperreactivity in patients who already have been nickel-sensitized for
other reasons (piercing, etc.). We know of no previous study investigating
the possible interaction between cigarette smoking and nickel allergy (or metal allergy in general).

CONCLUSIONS
The conclusion of this study is that exposure to nickel by dietary intake or by inhalation of cigarette smoke may trigger systemic nickel allergy and result in general symptoms of ill-being. In this way, nickel allergy may contribute to syndromes of chronic fatigue and muscle
pain. Considering the increasing prevalence of women with nickel contact allergy and the increasing rate of female smokers (23), the consequences of the possible interaction between nickel allergy and cigarette smoking may turn out to be a disorder of distinguished selection–
a gender trap with vast health hazards.

REFERENCES
1. Dotterud LK, Falk ES. Metal allergy in north Norwegian schoolchildren and
its relation with ear piercing and atopy. Contact Dermatitis 1994; 31: 308-313.
2. Smith-Sivertsen T, Dotterud LK, Lund E. Nickel allergy and its relationship
with local nickel pollution, ear-piercing, and atopic dermatitis: a population-based
study from Norway. J Am Acad Dermatol 1999; 40: 726-735.
3. Meijer C, Bredberg M, Fischer T et al. Ear piercing, and nickel and cobalt
sensitization, in 520 young Swedish men doing compulsory military service. Contact
Dermatitis 1995; 32: 147-149.
4. Sterzl I, Procházková J, Hrdá P et al. Mercury and nickel allergy: risk factors
in fatigue and autoimmunity. Neuroendocrinology Letters 1999; 20: 221-228.
5. Marcusson JA, Lindh G, Evengård B. Chronic fatigue syndrome and nickel
allergy. Contact Dermatitis 1999; 40: 269-272.
6. Andersson M, Bagby JR, Dyrehag LE, Gottfries CG. Effects of staphylococcus
toxoid vaccine on pain and fatigue in patients with fibromyalgia/chronic fatigue
syndrome. Eur J Pain 1998; 2: 133-142.
7. Stejskal VDM, Cederbrant K, Lindvall A et al. MELISA–an in vitro tool for
the study of metal allergy. Toxic in Vitro 1994; 8: 991-1000.
8. Regland B, Stejskal V, Jahreskog M et al. Nickel allergy in patients with fibromyalgia
and chronic fatigue syndrome. (Abstract) In Proceedings of the 2nd World
Congress on Chronic Fatigue Syndrome and Related Disorders. Brussels, September
9-12, 1999, p. 27.
9. Smith CJ, Livingston SD, Doolittle DJ. An international literature survey of
‘‘IARC Group I carcinogens’’ reported in mainstream cigarette smoke. Food Chem
Toxicol 1997; 35: 1107-1130.
10. Wolfe F, Smythe HA, Yunus MB et al. The American College of Rheumatology
1990 criteria for the classification of fibromyalgia: report of the multicenter criteria
committee. Arthritis Rheum 1990; 33: 160-172.
11. Holmes GP, Kaplan JE, Gantz NM et al. Chronic fatigue syndrome: a working
case definition. Ann Intern Med 1988; 108: 387-389.
12. Grandjean P. Human exposure to nickel. IARC Sci Publ 1984; 53: 469-485.
Original Research 65
13. Flyvholm MA, Nielsen GD, Andersen A. Nickel content of food and estimation
of dietary intake. Z Lebensm Unters Forsch 1984; 179: 427-431 (abstract in English).
14. Christensen OB. Nickel dermatitis. An update. Dermatol Clin 1990; 8: 37-40.
15. Schubert H, Berova N, Czernielewski A et al. Epidemiology of nickel allergy.
Contact Dermatitis 1987; 16: 122-128.
16. Hindsen M, Bruze M, Christensen OB. Individual variation in nickel patch
test reactivity. Am J Contact Dermat 1999; 10: 62-67.
17. Cederbrant K, Hultman P, Marcusson JA et al. In vitro lymphocyte proliferation
as compared to patch test using gold, palladium and nickel. Int Arch Allergy Immunol
1997; 112: 212-217.
18. Lisby S, Hansen LH, Menné T et al. Nickel-induced proliferation of both
memory and naive T cells in patch test-negative individuals. Clin Exp Immunol
1999; 117: 217-222.
19. Möller H. Yes, systemic nickel is probably important! J Am Acad Dermatol
1993; 28: 511-513.
20. Veien NK, Hattel T, Laurberg G. Low nickel diet: an open, prospective trial. J
Am Acad Dermatol 1993; 29: 1002-1007.
21. Antico A, Soana R. Chronic allergic-like dermatopathies in nickel-sensitive
patients. Results of dietary restrictions and challenge with nickel salts. Allergy Asthma
Proc 1999; 20: 235-242.
22. King G, Grizeau D, Bendel R et al. Smoking behaviour among French and
American women. Prev Med 1998; 27: 520-529.
23. Bergen AW, Caporaso N. Cigarette smoking. J Natl Cancer Inst 1999; 91:
1365-1375.
Björn Regland is Assistant Professor, Olof Zachrisson is Medical Doctor, and
Carl-Gerhard Gottfries is Professor, all affiliated with the Department of Psychiatry,
Sahlgrenska University Hospital/Mölndal and Institute of Clinical Neuroscience,
Göteborg, Sweden.
Vera Stejskal is Assistant Professor, Department of Clinical Chemistry, Danderyd
Hospital and Karolinska Institute, Stockholm.
Address correspondence to: Björn Regland, Institute of Clinical Neuroscience,
Sahlgrenska University Hospital, SE-431 80 Mölndal, Sweden (E-mail: bjorn.regland
@ms.se).

The authors express their thanks to Swiss Serum & Vaccine Institute Berne for
providing them with the Staphylococcus vaccine, and to Veronica Nordman for the
performance of in vitro testing.
Journal of Chronic Fatigue Syndrome, Vol. 8(1) 2001_ 2001 by The Haworth Press, Inc. All rights reserved. 57 58 JOURNAL OF CHRONIC FATIGUE SYNDROME

Chronic Fatigue Syndrome, Fibromyalgia, Scleroderma, Lupus, Rheumatoid Arthritis:

The mercury and nickel connection. B. Windham(Ed.) Dec 2002.

I. Introduction.
Chronic fatigue syndrome(CFS) is characterized by fatigue, neurologic symptoms including headaches, brain fog, mood disorders, and motor dysfunction. Spect scans of those with CFS have found that the majority have over 5 times more areas of regional brain damage and reduced blood flow in the cerebral cortex area of the brain(471) than controls. The majority studied were also found to have increased Th2 inflammatory cytokine activity and a blunted DHEA response curve to I.V. ATCH indicative of hypothalamic/adrenal deficiency such as relative glucocorticoid deficiency(472). CFS and Fibromyalgia patients have also been found to commonly have abnormal enzymatic processes that affect the sodium potassium ATPase energy channels(473), which appears to be a major factor in the condition and for which mercury is a known cause(43,288). This also has been found to result in inflammatory processes that cause muscle tissue damage and result in higher levels of urinary excretion of creatine , choline, and glycine in CFS, and higher levels of excretion of choline, taurine, citrate, and trimethyl amine oxide in FM(474). Supplementation of creatine has been found to result in improved muscle mitochondrial function in such patients(502). FM is further characterized by muscle and fibrous tissue pain, and its prevalence has been estimated at greater than 7% in women aged 60-79 years and 3.4% for all women(528). A Swedish study found that in one county, 11.6% of women over 35 surveyed had symptoms of Fibromyalgia, while 5.5% of men reported such symptoms(368).
The main factors determining whether chronic conditions are induced by metals appear to be exposure and genetic susceptibility, which determines individuals immune sensitivity and ability to detoxify metals(405). Very low levels of exposure have been found to seriously affect relatively large groups of individuals who are immune sensitive to toxic metals, or have an inability to detoxify metals due to such as deficient sulfoxidation or metallothionein function or other inhibited enzymatic processes related to detoxification or excretion of metals. A study involving 930 fatigued patients saw more than half (62 percent) test positive for metal allergy. The majority of those who went on to remove the offending metal reported substantial health improvements. When metal particles enter the body (through any number of sources, including dental amalgam fillings) they bind with proteins. This happens to everyone, hypersensitive or not. With hypersensitive people, the new structure is falsely identified by the immune system as a foreign invader. The white blood cells, or lymphocytes, go into attack mode. The activated immune system will up-regulate the activity of certain brain structures (hypothalamus) and adrenal glands (see diagram, right. The brain perceives a warning about danger and prepares for defense against the invader. This stress mode will last as long as the inflammation process is fueled by metals. This will result in fatigue while the attack is being carried out by the lymphocytes. When antibodies are produced to attack the protein, the condition becomes far more serious - possibly leading to neuropsychiatric disorders. For those with chronic conditions, fatigue regardless of the underlying disease is primarily associated with hypersensitivity to inorganic and organic mercury, nickel, and gold(118,313,342,369,382).

II. Mercury sources and exposure levels.
Amalgam fillings are the largest source of mercury in most people with daily exposures documented to commonly be above government health guidelines(49,79,506,600). This is due to continuous vaporization of mercury from amalgam in the mouth, along with galvanic currents from mixed metals in the mouth that deposit the mercury in the gums and oral cavity
(600). Due to the high daily mercury exposure and excretion into home and business sewers of those with amalgam, dental amalgam is also the largest source of the high levels of mercury found in all sewers and sewer sludge, and thus a significant source of mercury in rivers, lakes, bays, fish, and crops(603). People also get significant exposure from vaccinations, fish, and dental office vapor(600).

When amalgam was placed into teeth of monkeys and rats, within one year mercury was found to have accumulated in the brain, trigeminal ganglia, spinal ganglia, kidneys, liver, lungs, hormone glands, and lymph glands(20). People also commonly get exposures to mercury and other toxic metals such as lead, arsenic, nickel, and aluminum from food, water, and other sources(601). All of these are highly neurotoxic and are documented to cause neurological damage which can result in chronic neurological conditions over time. Mercury induced lipid peroxidation has been found to be a major factor in mercury’s neurotoxicity, along with leading to decreased levels of glutathione peroxidation and superoxide dismustase(SOD)(13,254,489,494-496). Antioxidants have been found to protect against such mercury neurotoxicity(494,572).
Mercury (especially mercury vapor) rapidly crosses the blood brain barrier and is stored
preferentially in the pituitary gland, hypothalamus, thyroid gland, adrenal gland, and occipital cortex in direct proportion to the number and extent of amalgam surfaces (20, many studies referenced in (600)) Thus mercury has a greater effect on the functions of these areas. The range in one study was 2.4 to 28.7 parts per billion(ppb), and one study found on average that 77% of the mercury in the occipital cortex was inorganic(600).
III. Effects of Mercury Exposure
Some of the factors documented to be involved in inflammatory conditions like CFS, FMS, Lupus, Rheumatoid Arthritis, etc and in programmed cell death, apoptosis, of neurons and immune cells in degenerative neurological conditions like ALS, Alzheimer’s, MS, Parkinson’s, etc. include inducement of the inflammatory cytokine Tumor Necrosis Factor-alpha(TNFa) (126), reactive oxygen species and oxidative stress(13,43a,56a,296b), reduced glutathione levels(56,126a,111a), liver enzyme effects and inhibition of protein kinase C and cytochrome P450(43,84,260), nitric oxide and peroxynitrite toxicity (43a,521,524), excitotoxicity and lipid peroxidation(490,496), excess free cysteine levels (56d,111a,33,330),excess glutamate toxicity(13b, 416), excess dopamine toxicity (56d,13a), beta-amyloid generation(462,56a), increased calcium influx toxicity (296b,333,416,432,462c,507) and DNA fragmentation (296,42,114,142) and mitochondrial membrane dysfunction (56de, 416), and autoimmunity (313,342,369,382,405,513).
TNFa(tumor necrosis factor-alpha) is a cytokine that controls a wide range of immune cell response in mammals, including cell death(apoptosis). This process is involved in inflammatory conditions like CFS, FM, RA, Lupus, etc. and in degenerative neurological conditions like ALS, MS, Parkinson’s, rheumatoid arthritis, etc. Cell signaling mechanisms like sphingolipids are part of the control mechanism for the TNFa inflammatory and apoptosis mechanism(126a). glutathione is an amino acid that is a normal cellular mechanism for controlling inflamation and apoptosis. When glutathione is depleted in the brain, reactive oxidative species increase, and CNS and cell signaling mechanisms are disrupted by toxic exposures such as mercury, neuronal cell apoptosis results and neurological damage. Mercury has been shown to induce TNFa, deplete glutathione, and increase glutamate, dopamine, and calcium related toxicity, causing inflammatory effects and cellular apoptosis in neuronal and immune cells(126b,126c). Mercury’s biochemical damage at the cellular level include DNA damage, inhibition of DNA and RNA synthesis (42,114,142,197,296,392); alteration of protein structure (33,111,114,194,252,263,442); alteration of the transport and signaling mechanisms of calcium(333,43b,254,263,416d,462,507); inhibitation of glucose transport(338,254), and of enzyme function and transport/absorption of other essential nutrients (96,198,254,263,264,33,330,331,338,339,347,441,442); induction of free radical formation(13a,43b,54,405,424), depletion of cellular glutathione(necessary for detoxification processes) (56,111,126,424), inhibition of glutathione peroxidase enzyme(13a,442), inhibits glutamate uptake(119,416), induces peroxynitrite and lipid peroxidation damage(521b,56b), causes abnormal migration of neurons in the cerebral cortex(149), immune system damage (111,126,181,194, 226,252,272,316,355); affects dopamine uptake by neuronal synaptosomes(288), inducement of inflammatory cytokines(126,152,181), and induces autoimmunity (181,313,342,369,382,405,etc.).
A direct mechanism involving mercury's inhibition of cellular enzymatic processes by binding with the hydroxyl radical(SH) in amino acids appears to be a major part of the connection to allergic/immune reactive conditions such as:
Lupus (331a,330a,33,113,126,181,234,260d,288a,405,270,226,314,316,263c) and Scleroderma(330a,33,126,181,234,468,405,263c) and
Rheumatoid Arthritis(287,288a,416f,331b, 330a,33,126,181,405,263d,260d), as well as CFS and FMS that are also related to inflammatory cytokine processes and autoimmunity (181,118,313,314,342,369,382,405,126,330,33,263,etc.). One study found that insertion of amalgam fillings or nickel dental materials causes a suppression of the number of T lympocytes(270), and impairs the T 4/T 8 ratio. Low T4/T8 ratio has been found to be a factor in lupus, anemia, MS, eczema, inflammatory bowel disease, and glomerulonephritis. Mercury induced autoimmunity in animals and humans has been found to be associated with mercury's expression of major histocompatibility complex(MHC) class II genes(314,181,226,425c). Both mercuric and methyl mercury chlorides caused dose dependent reduction in immune B cell production(316). B cell expression of IgE receptors were significantly reduced(316,165), with a rapid and sustained elevation in intracellular levels of calcium induced(316,333).
Mercury and other toxic metals also form inorganic compounds with OH, NH2, CL, in addition to the SH radical and thus inhibits many cellular enzyme processes, coenzymes, hormones, and blood cells(405,600). Mercury has been found to impair conversion of thyroid T4 hormone to the active T3 form as well as causing autoimmune thyroiditis common to such patients(369,382). In general, immune activation from toxic metals such as mercury resulting in cytokine release and abnormalities of the hypothalamus pituitary adrenal(HPA) axis can cause changes in the brain, hypocortisolism, fatigue, and severe psychological symptoms (348,369,375,379 382,385,405,118) such as profound fatigue, muscoskeletal pain, sleep disturbances, gastrointestinal and neurological problems as are seen in CFS, Fibromyalgia, and autoimmune thyroiditis. Such hypersensitivity has been found most common in those with genetic predisposition to heavy metal sensitivity(60,313,342,369,405), such as found more frequently in patients with human lymphocyte antingens(HLA DRA) (381-383). A significant portions of the population appear to fall in this category.
Mercury exposure through dental fillings appears to be a major factor in chronic fatigue syndrome(CFS) through its effects on ATP and immune system(lymphocute reactivity, neutraphil activity, effects on T cells and B cells) as well as its promotion of growth of candida albicans in the body and the methylation of inorganic mercury by candida and intestional bacteria to the extremely toxic methyl mercury form, which like mercury vapor crosses the blood brain barrier, and also damages and weakens the immune system (222,225,226,234,235, 265, 293,60,313,314,342,369,404). Mercury vapor or Inorganic mercury have been shown in animal studies to induce autoimmune reactions and disease through effects on immune system T cells(226,268,269,270,314). Chronic immune activation is common in CFS, with increase in activated CD8+ cytotoxic T-cells and decreased NK cells(518). Numbers of suppressor-inducer T cells and NK cells have been found to be inversely correlated with urine mercury levels(270ad). CFS patients usually improve and immune reactivity is reduced when amalgam fillings are replaced (342,383,405).

Mercury lymphocyte reactivity, effects on glutamate in the CNS, and mercury induced hypothyroidism induce CFS type symptoms including profound tiredness, musculoskeletal pain, sleep disturbances, gastrointestinal and neurological problems along with other CFS symptoms and Fibromyalgia (342,346,369). Mercury has been found to be a common cause of Fibromyalgia(293,346, 369,523,527). Glutamate is the most abundant amino acid in the body and in the CNS acts as excitory neurotransmitter (346,386), which also causes inflow of calcium. Astrocytes, a type of cell in the brain and CNS with the task of keeping clean the area around nerve cells and facilitating neurotransmission, have a function of neutralizing excess glutamate by transforming it to glutamic acid. If astrocytes are not able to rapidly neutralize excess glutamate, then a buildup of glutamate and calcium occurs, causing swelling and neurotoxic effects (119,333). Mercury and other toxic metals inhibit astrocyte function in the brain and CNS(119), causing increased glutamate and calcium related neurotoxicity(119,333,226) which are responsible for much of the Fibromyalgia symptoms. This is also a factor in conditions such as CFS, Parkinson's, and ALS(346,416). Animal studies have confirmed that increased levels of glutamate(or aspartate, another amino acid excitory neurotransmitter) cause increased sensitivity to pain , as well as higher body temperature both found in CFS/Fibromyalgia. Mercury and increased glutamate activate free radical forming processes like xanthine oxidase which produce oxygen radicals and oxidative neurological damage(142,346,13). Medical studies and doctors treating Fibromyalgia have found that supplements which cause a decrease in glutamate or protect against its effects have a positive effect on Fibromyalgia. Some that have been found to be effective include Vit B6, methyl cobalamine(B12), L carnitine, choline, ginseng, Ginkgo biloba, vitamins C and E, nicotine, and omega 3 fatty acids(fish and flaxseed oil-GLA,EPA,DHA)(417,229). Other supplements that also have been found to help are magnesium and malic acid(488,489). Avoidance of exictotoxins like MSG and aspartame have been found to eliminate symptoms in some with Fibromyalgia(490).
Clinical tests of patients with chronic neurological conditions, Lupus(SLE), and rheumatoid arthritis have found that the patients generally have elevated plasma cysteine to sulphate ratios, with the average being 500%higher than controls (330,331,600,33e), and in general being poor sulphur oxidizers. This means that these patients have insufficient sulfates available to carry out necessary bodily processes. Mercury has been shown to diminish and block sulphur oxidation and thus reducing glutathione levels which is the part of this process involved in detoxifying and excretion of toxics like mercury(33). Glutathione is produced through the sulphur oxidation side of this process. Low levels of available glutathione have been shown to increase mercury retention and increase toxic effects(111), while high levels of free cysteine have been demonstrated to make toxicity due to inorganic mercury more severe(333,194,33e). Mercury has also been found to play a part in inducing intolerance and neuronal problems through blockage of the P 450 liver enzymatic process(84,33e).

Mercury from amalgam interferes with production of cytokines that activate macrophage and neutrophils, disabling early control of viruses and leading to enhanced infection(131,251). Animal studies have confirmed that mercury increases effects of the herpes simplex virus type 2 for example(131). Mercury damages the immune system and in those with chronic conditions has been found to commonly facilitate infestation by pathogens such as viruses, harmful bacteria, candida, mycoplasma, and parasites(131,251,404,460,470, 473,485). The majority of those tested who have CFS or FMS have been found to have infections of mycoplasma, Human Herpes Virus-6, Cytomeglivirus, or bacterial infections such as intracellular chlamydia(470). Clinics treating these conditions commonly find such pathogens to be a factor in the condition (470,473,485,487,488). Mercury detoxification and treatment of these pathogens results in significant improvement in the majority of those treated (470,485,488,489,230,600).
Mercury exposure causes high levels of oxidative stress/reactive oxygen species(ROS)(13), which has been found to be a major factor in apoptosis and neurological disease (56,250,441,442,443,13) including dopamine or glutamate related apoptosis(288c). Mercury and quinones form conjugates with thiol compounds such as glutathione and cysteine and cause depletion of glutathione, which is necessary to mitigate reactive damage. Such congugates are found to be highest in the brain substantia nigra with similar congugates formed with L-Dopa and dopamine in Parkinson’s disease(56). Mercury depletion of GSH and damage to cellular mitochondria and the increased lipid peroxidation in protein and DNA oxidation in the brain appear to be a major factor in Parkinson’s disease(33,56,442)
It has been well documented by hundreds of medical studies including thousands of tested subjects and by scientific panels that "amalgam fillings" are the number one source of mercury in people and that those with several amalgam fillings often have daily exposures exceeding the Government Health Standards for mercury(600). Thus among those most susceptible, significant neurological and immune effects related to amalgam fillings are common. Symptoms of those with CFS, Fibromyalgia, or thyroid related conditions usually improve significantly after proper amalgam replacement. In thousands of cases undergoing amalgam replacement, the majority recovered or had significant improvement in symptoms for muscular/joint pain/Fibromyalgia
(222,293,317,322,369,440,469,470,523,527, 94), Chronic Fatigue Syndrome(CFS) (8,60,212,230,293,229,222, 232,233,271,313,317,320,342,369, 375,376,382,440,469,470,485), lupus(369,113,222, 229,233,323), autoimmune thyroiditis(369,382), as well as many other conditions(600). Of one group of 86 patients with CFS symptoms, 78% reported significant health improvements after replacement of amalgam fillings within a relatively short period, and the MELISA immune reactivity test found significant reduction in lymphocyte reactivity compared to pre removal tests(342,375). The improvement in symptoms and lymphocyte reactivity imply that most of the Hg induced lymphocyte reactivity is allergenic in nature. Although patch tests for mercury allergy are often given for unresolved oral symptoms, this is not generally recommended as a high percentage of such problems are resolved irrespective of the outcome of a patch test (60,87,90,etc.) Exposure to organochlorine compounds such as DDT/DDE and hexachlorobenzene have also been found to be highly correlated with chronic fatigue.
Nutrition and nutritional support have been found to play significant roles in CFS/FM alleviation. An adequate supply of vitamins and essential minerals as well as antioxidants have been found to benefit such conditions to counteract free radicals and oxidative stress caused by the conditions. Glyconutrients such as Mannatech Ambrotose and Immunostart have also been found to be effective in reducing the effects of CFS and FM(528). Immunostart has been documented to be effective in detoxing toxic metals.
References

(13)(a) S.Hussain et al, “Mercuric chloride induced reactive oxygen species and its effect on antioxidant enzymes in different regions of rat brain”,J Environ Sci Health B 1997 May;32(3):395 409; & P.Bulat, “Activity of Gpx and SOD in workers occupationally exposed to mercury”, Arch Occup Environ Health, 1998, Sept, 71 Suppl:S37-9; & Stohs SJ, Bagchi D. Oxidative mechanisms in the toxicity of metal ions. Free Radic Biol Med 1995; 18(2): 321-36 ; & D.Jay, “Glutathione inhibits SOD activity of Hg”, Arch Inst cardiol Mex, 1998,68(6):457-61 & El-Demerdash FM. Effects of selenium and mercury on the enzymatic activities and lipid peroxidation in brain, liver, and blood of rats. J Environ Sci Health B. 2001 Jul;36(4):489-99. &(b) S.Tan et al, “Oxidative stress induces programmed cell death in neuronal cells”, J Neurochem, 1998, 71(1):95-105; & Matsuda T, Takuma K, Lee E, et al. Apoptosis of astroglial cells [Article in Japanese] Nippon Yakurigaku Zasshi. 1998 Oct;112 Suppl 1:24P-; & Lee YW, Ha MS, Kim YK.. Role of reactive oxygen species and glutathione in inorganic mercury-induced injury in human glioma cells. Neurochem Res. 2001 Nov;26(11):1187-93. & (c)Ho PI, Ortiz D, Rogers E, Shea TB. Multiple aspects of homocysteine neurotoxicity: glutamate excitotoxicity, kinase hyperactivation and DNA damage. J Neurosci Res. 2002 Dec 1;70(5):694-702.

(20) (a) Galic N, Ferencic Z et al, Dental amalgam mercury exposure in rats. Biometals. 1999 Sep;12(3):227-31; & Arvidson B, Arvidsson J, Johansson K,. Mercury deposits in neurons of the trigeminal ganglia after insertion of dental amalgam in rats. Biometals. 1994 Jul;7(3):261-3; & (b)Danscher G, Horsted-Bindslev P, Rungby J. Traces of mercury in organs from primates with amalgam fillings. Exp Mol Pathol. 1990 Jun;52(3):291-9; & L.Hahn et al, Distribution of mercury released from amalgam fillings into monkey tissues”, FASEB J.,1990, 4:5536
(33) (a) Markovich et al, "Heavy metals (Hg,Cd) inhibit the activity of the liver and kidney sulfate transporter Sat 1", Toxicol Appl Pharmacol, 1999,154(2):181 7; & (b)2S.A.McFadden, “Xenobiotic metabolism and adverse environmental response: sulfur- dependent detox pathways”,Toxicology, 1996, 111(1-3):43-65; &(c) S.C. Langley-Evans et al, “SO2: a potent glutathion depleting agent”, Comp Biochem Physiol Pharmocol Toxicol Endocrinol, 114(2):89-98; & (d)Alberti A, Pirrone P, Elia M, Waring RH, Romano C. Sulphation deficit in “low-functioning” autistic children. Biol Psychiatry 1999, 46(3):420-4.
(34) Henriksson J, Tjalve H. Uptake of inorganic mercury in the olfactory bulbs via olfactory pathways in rats. Environ Res. 1998 May;77(2):130-40.
(42) Rodgers JS, Hocker JR, et al, Mercuric ion inhibition of eukaryotic transcription factor binding to DNA. Biochem Pharmacol. 2001 Jun 15;61(12):1543-50; & K.Hansen et al A survey of metal induced mutagenicity in vitro and in vivo, J Amer Coll Toxicol , 1984:3;381 430;
(43) (a)Knapp LT; Klann E. Superoxide induced stimulation of protein kinase C via thiol modification and modulation of zinc content. J Biol Chem 2000 May 22; & P.Jenner,“Oxidative mechanisms in PD”, Mov Disord, 1998; 13(Supp1):24-34;&(b) Rajanna B et al, “Modulation of protein kinase C by heavy metals”, Toxicol Lett, 1995, 81(2-3):197-203: & Badou A et al, “HgCl2-induced IL-4 gene expression in T cells involves a protein kinase C-dependent calcium influx through L-type calcium channels”J Biol Chem. 1997 Dec 19;272(51):32411-8., & D.B.Veprintsev, 1996, Institute for Biological Instrumentation, Russian Academy of Sciences, Pb2+ and Hg2+ binding to alpha lactalbumin”.Biochem Mol Biol Int 1996 ;39(6): 1255 65; & M. J. McCabe, University of Rochester School of Medicine & Dentistry, 2002, Mechanisms of Immunomodulation by Metals, www.envmed.rochester.edu/envmed/TOX/faculty/mccabe.html; & Buzard GS, Kasprzak KS. Possible roles of nitric oxide and redox cell signaling in metal-induced toxicity and carcinogenesis: a review. Environ Pathol Toxicol Oncol. 2000;19(3):179-99
(49) Kingman A, Albertini T, Brown LJ. National Institute of Dental Research, “Mercury concentrations in urine and blood associated with amalgam exposure in the U.S. military population”, J Dent Res. 1998, 77(3):461-71

(54) M.E. Lund et al, “Treatment of acute MeHg poisoning by NAC”, J Toxicol Clin Toxicol, 1984, 22(1):31-49; & Livardjani F; Ledig M; Kopp P; Dahlet M; Leroy M; Jaeger A. Lung and blood superoxide dismustase activity in mercury vapor exposed rats: effect of N acetylcysteine treatment. Toxicology 1991 Mar 11;66(3):289 95. & G.Ferrari et al, Dept. Of Pathology, Columbia Univ., J Neurosci,1995, 15(4):2857-66; & RR. Ratan et al, Dept. of Neurology, Johns Hopkins Univ., J Neurosci, 1994, 14(7): 4385-92;
(56)(a) A.Nicole et al, “Direct evidence for glutathione as mediator of apoptosis in neuronal cells”, Biomed Pharmacother, 1998; 52(9):349-55; & J.P.Spencer et al, “Cysteine & GSH in PD”, mechanisms involving ROS”, J Neurochem, 1998, 71(5):2112-22: & & J.S. Bains et al, “Neurodegenerative disorders in humans and role of glutathione in oxidative stress mediated neuronal death”, Brain Res Rev, 1997, 25(3):335-58;&

Medina S, Martinez M, Hernanz A, Antioxidants inhibit the human cortical neuron apoptosis induced by hydrogen peroxide, tumor necrosis factor alpha, dopamine and beta-amyloid peptide 1-42.. Free Radic Res. 2002 Nov;36(11):1179-84. &(b) Pocernich CB, et al. Glutathione elevation and its protective role in acrolein-induced protein damage in synaptosomal membranes: relevance to brain lipid peroxidation in neurodegenerative disease. Neurochem Int 2001 Aug;39(2):141-9; & D. Offen et al, “Use of thiols in treatment of PD”, Exp Neurol, 1996,141(1):32-9; & (c) Pearce RK, Owen A, Daniel S, Jenner P, Marsden CD. Alterations in the distribution of glutathione in the substantia nigra in Parkinson's disease. J Neural Transm. 1997;104(6-7):661-77; & A.D.Owen et al, Ann NY Acad Sci, 1996, 786:217-33; & JJ Heales et al, Neurochem Res, 1996, 21(1):35-39; & X.M.Shen et al, Neurobehavioral effects of NAC conjugates of dopamine: possible relevance for Parkinson’sDisease”, Chem Res Toxicol, 1996, 9(7):1117-26; & Chem Res Toxicol, 1998, 11(7):824-37; & (d) Li H, Shen XM, Dryhurst G. Brain mitochondria catalyze the oxidation of 7-(2-aminoethyl)-3,4-dihydro-5-hydroxy-2H-1,4-benzothiazine-3-carboxyli c acid (DHBT-1) to intermediates that irreversibly inhibit complex I and scavenge glutathione: potential relevance to the pathogenesis of Parkinson's disease. J Neurochem. 1998 Nov;71(5):2049-62; & (e) Araragi S, Sato M. et al, Mercuric chloride induces apoptosis via a mitochondrial-dependent pathway in human leukemia cells. Toxicology. 2003 Feb 14;184(1):1-9.
(60)V.D.M.Stejskal, Dept. Of Clinical Chemistry, Karolinska Institute, Stockholm, Sweden LYMPHOCYTE IMMUNO STIMULATION ASSAY MELISA" & VDM Stejskal et al, "MELISA: tool for the study of metal allergy", Toxicology in Vitro, 8(5):991 1000, 1994. http://www.melisa.org
(79) L.Bjorkman et al, "Mercury in Saliva and Feces after Removal of Amalgam Fillings", Toxicology and Applied Pharmacology, 1997, 144(1), p156-62
(84) J.C.Veltman et al, “Alterations of heme, cytochrome P-450, and steroid metabolism by mercury in rat adrenal gland”, Arch Biochem Biophys, 1986, 248(2):467-78; & A.G.Riedl et al, Neurodegenerative Disease Research Center, King’s College,UK, “P450 and hemeoxygenase enzymes in the basal ganglia and their role’s in Parkinson’s disease”, Adv Neurol, 1999; 80:271-86; & Alfred V. Zamm. Dental Mercury: A Factor that Aggravates and Induces Xenobiotic Intolerance. J. Orthmol. Med. v6#2 pp67-77 (1991).
(87)A. Skoglund, Scand J Dent Res 102(4): 216 222, 1994; and 99(4):320 9,1991(40 cases); & P.O.Ostman et al, "Clinical & histologic changes after removal of amalgma", Oral Surgery, Oral Medicine, and Endodontics, 1996, 81(4):459 465; & S.H.Ibbotson et al, "The relevance of amalgam replacement on oral lichenoid reactions", British Journal of Dermatology, 134(3):420 3, 1996; & J.Bratel et al, "Effect of Replacement of Dental Amalgam on OLR", Journal of Dentistry, 1996, 24(1 2):41 45(. (431 cases)
(90)P.Koch et al, "Oral lesions and symptoms related to metals", Dermatol,1999,41(3):422 430; & "Oral lichenoid lesions,mercury hypersensitity, ...", Contact Dermatitis, 1995, 33(5): 323 328; & S.Freeman et al, "Orallichenoid lesions caused by allergy to mercury in amalgam", Contact Dermatitis, 33(6):423 7, Dec 1995 (Denmark)
(94) F.Berglund, Case reports spanning 150 years on the adverse effects of dental amalgam, Bio-Probe, Inc.,Orlando,Fl,1995;ISBN 0-9410011-14-3(245 cured); & Tuthill JY, "Mercurial neurosis resulting from amalgam fillings", The Brooklyn Medical Journal, December 1898, v.12, n.12, p725-742
(96) A.F.Goldberg et al, “Effect of Amalgam restorations on whole body potassium and bone mineral content in older men”,Gen Dent, 1996, 44(3): 246-8; & (b) K.Schirrmacher,1998, “Effects of lead, mercury, and methyl mercury on gap junctions and [Ca2+]I in bone cells”, Calcif Tissue Int 1998 Aug;63(2):134 9.
(111) (a) Quig D, Doctors Data Lab,"Cysteine metabolism and metal toxicity", Altern Med Rev, 1998;3:4, p262 270, & (b) J.de Ceaurriz et al, Role of gamma glutamyltraspeptidase(GGC) and extracellular glutathione in dissipation of inorganic mercury",J Appl Toxicol,1994, 14(3): 201 ; & W.O. Berndt et al, "Renal glutathione and mercury uptake", Fundam Appl Toxicol, 1985, 5(5):832 9; & Zalups RK, Barfuss DW. Accumulation and handling of inorganic mercury in the kidney after coadministration with glutathione, J Toxicol Environ Health, 1995, 44(4): 385-99; & T.W.Clarkson et al, "Billiary secretion of glutathione metal complexes", Fundam Appl Toxicol, 1985, 5(5):816 31;
(113)T.A.Glavinskiaia et al, "Complexons in the treatment of lupus erghematousus", Dermatol Venerol, 1980,12: 24 28; & A.F.Hall, Arch Dermatol 47, 1943, 610 611.
(118) Tibbling L, Stejskal VDM, et al, Immunolocial and brain MRI changes in patients with suspected metal intoxication", Int J Occup Med Toxicol 4(2):285 294,1995.
(114) (a)M.Aschner et al, “Metallothionein induction in fetal rat brain by in utero exposure to elemental mercury
vapor”, Brain Research, 1997, dec 5, 778(1):222-32; & Baauweegers HG, Troost D. Localization of metallothionein in the mammilian central nervous system.. Biol Signals 1994, 3:181-7. &(b) T.V. O’Halloran, “Transition metals in control Of gene expression”, Science, 1993, 261(5122):715-25; &(c) Matts RL, Schatz JR, Hurst R, Kagen R. Toxic heavy metal ions inhibit reduction of disulfide bonds. J Biol Chem 1991; 266(19): 12695-702; Boot JH. Effects of SH-blocking compounds on the energy metabolism in isolated rat hepatocytes. Cell Struct Funct 1995; 20(3): 233-8.;
(119)(a) L.Ronnback et al, "Chronic encephalopaties induced by low doses of mercury or lead", Br J Ind Med 49: 233-240, 1992; & H.Langauer Lewowicka,” Changes in the nervous system due to occupational metallic mercury poisoning” Neurol Neurochir Pol 1997 Sep Oct;31(5):905 13; &(b) Kim P, Choi BH. “Selective inhibition of glutamate uptake by mercury in cultured mouse astrocytes”, Yonsei Med J 1995; 36(3): 299-305; & Brookes N. In vitro evidence for the role of glutatmate in the CNS toxicity of mercury. Toxicology 1992, 76(3):245-56; & Albrecht J, Matyja E. Glutamate: a potential mediator of inorganic mercury toxicity. Metab Brain Dis 1996; 11:175-84.

(126)(a) Singh I, Pahan K, Khan M, Singh AK. Cytokine-mediated induction of ceramide production is redox-sensitive. Implications to proinflammatory cytokine-mediated apoptosis in demyelinating diseases. J Biol Chem. 1998 Aug 7;273(32):20354-62; & Pahan K, Raymond JR, Singh I. Inhibition of phosphatidylinositol 3-kinase induces nitric-oxide synthase in lipopolysaccharide- or cytokine-stimulated C6 glial cells. J. Biol. Chem. 274: 7528-7536, 1999; & Xu J, Yeh CH, et al, Involvement of de novo ceramide biosynthesis in tumor necrosis factor-alpha/cycloheximide-induced cerebral endothelial cell death. J Biol Chem. 1998 Jun 26;273(26):16521-6; & Dbaibo GS, El-Assaad W, et al, Ceramide generation by two distinct pathways in tumor necrosis factor alpha-induced cell death. FEBS Lett. 2001 Aug 10;503(1):7-12; & Liu B, Hannun YA.et al, Glutathione regulation of neutral sphingomyelinase in tumor necrosis factor-alpha-induced cell death.J Biol Chem. 1998 May 1;273(18):11313-20; & (b)Noda M, Wataha JC, et al, Sublethal, 2-week exposures of dental material components alter TNF-alpha secretion of THP-1 monocytes. Dent Mater. 2003 Mar;19(2):101-5; & Kim SH, Johnson VJ, Sharma RP. Mercury inhibits nitric oxide production but activates proinflammatory cytokine expression in murine macrophage: differential modulation of NF-kappaB and p38 MAPK signaling pathways. Nitric Oxide. 2002 Aug;7(1):67-74; & Dastych J, Metcalfe DD et al, Murine mast cells exposed to mercuric chloride release granule-associated N-acetyl-beta-D-hexosaminidase and secrete IL-4 and TNF-alpha. J Allergy Clin Immunol. 1999 Jun;103(6):1108-14; & (c) Tortarolo M, Veglianese P, et al, Persistent activation of p38 mitogen-activated protein kinase in a mouse model of familial amyotrophic lateral sclerosis correlates with disease progression.. Mol Cell Neurosci. 2003 Jun;23(2):180-92.
(142) Ariza ME; Bijur GN; Williams MV. Lead and mercury mutagenesis: role of H2O2, superoxide dismutase, and xanthine oxidase. Environ Mol Mutagen 1998;31(4):352 61; & M.E. Ariza et al, “Mercury mutagenisis”, Biochem Mol Toxicol, 1999, 13(2):107-12; & M.E.Ariza et al, "Mutagenic effect of mercury", InVivo 8(4):559-63,1994
(152) Langworth et al, “Effects of low exposure to inorganic mercury on the human immune system”, Scand J Work Environ Health, 19(6): 405-413.1993; & Walum E et al, Use of primary cultures to sutdy astrocytic regulatory functions. Clin Exp Pharmoacol Physiol 1995, 22:284-7; & J Biol Chem 2000 Dec 8;275(49):38620-5; & (b)Kerkhoff H, Troost D, Louwerse ES. Inflammatory cells in the peripheral nervous system in motor neuron disease. Acta Neuropathol 1993; 85:560-5; & (c)Appel Sh, Smith RG. Autoimmunity as an etiological factor in amyotrophic lateral sclerosis. Adv Neurol 1995; 68:47-57.
(181)P.W. Mathieson, "Mercury: god of TH2 cells",1995, Clinical Exp Immunol.,102(2):229 30;& & Heo Y, Parsons PJ, Lawrence DA, Lead differentially modifies cytokine production in vitro and in vivo. Toxicol Appl Pharmacol, 196; 138:149-57; & Murdoch RD, Pepys J; Enhancement of antibody and IgE production by mercury and platinum salts. Int Arch Allergy Appl Immunol 1986 80: 405-11.
(181) Mathieson PW, “Mercury: god of TH2 cells”,1995, Clinical Exp Immunol.,102(2):229-30; & (b) Heo Y, Parsons PJ, Lawrence DA, Lead differentially modifies cytokine production in vitro and in vivo. Toxicol Appl Pharmacol, 1996; 138:149-57; & (c) Murdoch RD, Pepys J; Enhancement of antibody and IgE production by mercury and platinum salts. Int Arch Allergy Appl Immunol 1986 80: 405-11;
(194) Lu SC, FASEB J, 1999, 13(10):1169 83, “Regulation of hepatic glutathione synthesis: current concepts and
controversies”; & R.B. Parsons, J Hepatol, 1998, 29(4):595-602; & R.K.Zalups et al,"Nephrotoxicity of inorganic mercury co administered with L cysteine", Toxicology, 1996, 109(1): 15 29.
(198) E.S. West et al, Textbook of Biochemistry, MacMillan Co, 1957,p853;& B.R.G.Danielsson et al,”Ferotoxicity of inorganic mercury: distribution and effects of nutrient uptake by placenta and fetus”, Biol Res Preg Perinatal. 5(3):102-109,1984; & Danielsson et al, Neurotoxicol. Teratol., 18:129-134;
(212)Ziff, M.F., "Documented Clinical Side Effects to Dental Amalgams", ADV. Dent. Res.,1992; 1(6):131 134; & S.Ziff,Dentistry without Mercury, 8th Edition, 1996, Bio Probe, Inc., ISBN 0 941011 04 6; & Dental Mercury Detox, Bio Probe, Inc. http://www.bioprobe.com. (cases:FDA Patient Adverse Reaction Reports 762, Dr.M.Hanson Swedish patients 519, Dr. H. Lichtenberg 100 Danish patients,Dr. P.Larose 80 Canadian patients, Dr. R.Siblerud, 86 Colorado patients,Dr. A.V.Zamm, 22 patients)
(222) M. Daunderer, Handbuch der Amalgamvergiftung, Ecomed Verlag, Landsberg 1998, ISBN 3 609 71750 5 (in German); & "Improvement of Nerve and Immunological Damages after Amalgam Removal", Amer. J. Of Probiotic Dentistry and Medicine, Jan 1991;
(225)S. Yannai et al, "Transformationss of inorganic mercury by candida albicans and saccharomyces cerevisiae", Applied Envir Microbiology,1991, 7:245 247; & N.E.Zorn et al, " A relationship between Vit B 12, mercury
uptake, and methylation", Life Sci, 1990, 47(2):167 73; & W.P.Ridley et al, Environ Health Perspectives, 1977, Aug 19, 43 6; & R.E.DeSimone et al, Biochem Biophys Acta, 1973,May 28; & Yamada, Tonomura"Formation of methyl Mercury Compounds from inorganic Mercury by Chlostridium cochlearium" J Ferment Technol1972 50:159 1660

(226) B.J. Shenker et al, Dept. Of Pathology,Univ. Of Penn. School of Dental Med.,”Immunotoxic effects of mercuric compounds on human lymphocytes and monocytes:Alterations in cell viability” Immunopharmacologicol Immunotoxical, 1992, 14(3):555-77; & M.A.Miller et al, “Mercuric chloride induces apoptosis in human T lymphocytes”, Toxicol Appl Pharmacol, 153(2):250 7 1998; &(b) Rossi AD,Viviani B, Vahter M. Inorganic mercury modifies Ca2+ signals, triggers apoptosis, and potentiates NMDA toxicity in cerebral granule neurons. Cell Death and Differentiation 1997; 4(4):317-24. & Goering PL, Thomas D, Rojko JL, Lucas AD. Mercuric chloride-induced apoptosis is dependent on protein synthesis. Toxicol Lett 1999; 105(3): 183-95;
(230) Rogers S(MD), The E.I. Syndrome: An Rx for Environmental Illness, Keats Publishing, Amazon Books

(234) P.E. Bigazzi, "Autoimmunity and Heavy Metals", Lupus, 1994; 3: 449 453; & Pollard KM, Pearson Dl, Hultman P. Lupus prone mice as model to study xenobiotic induced autoimmunity. Environ Health Perspect 1999;
107(Suppl 5): 729 735; & & Nielsen JB; Hultman P. Experimental studies on genetically determined susceptibility to mercury induced autoimmune response. Ren Fail 1999 May Jul;21(3 4):343 8; & Feighery L, Collins C, Anti-transglutaminase antibodies and the serological diagnosis of coeliac disease. Br J Biomed Sci. 2003;60(1):14-8; & & Mayes MD. Epidemiologic studies of environmental agents and systemic autoimmune diseases. Environ Health Perspect. 1999 Oct;107 Suppl 5:743-8; &. Bigazzi PE. Metals and kidney autoimmunity. Environ Health Perspect. 1999 Oct;107 Suppl 5:753-65
(235) H.J.Hamre, Mercury from Dental Amalga and Chronic Fatigue Syndrom", The CFIDS Chronicle, Fall 1994, p44 47.
(252) B.J.Shenker et al, Dept. of Pathology, Univ. of Pennsylvania, “Immunotoxic effects of mercuric compounds on human lymphoctes and monocytes: Alterations in cellular glutathione content”, Immunopharmacol Immunotoxicol 1993, 15(2-3):273-90.
(254) al-Saleh I, Shinwari N. Urinary mercury levels in females: influence of dental amalgam fillings. Biometals 1997; 10(4): 315-23; & Zabinski Z; Dabrowski Z; Moszczynski P; Rutowski J. The activity of erythrocyte enzymes and basic indices of peripheral blood erythrocytes from workers chronically exposed to mercury vapors. Toxicol Ind Health 2000 Feb;16(2):58 64.
(260) Woods JS et al, Altered porphyrin metabolites as a biomarker of mercury exposure and toxicity”, Physiol Pharmocol, 1996,74(2):210-15, & Strubelt O, Kremer J, et al, Comparative studies on the toxicity of mercury, cadmium, and copper toward the isolated perfused rat liver. J Toxicol Environ Health. 1996 Feb 23;47(3):267-83; & Kaliman PA, Nikitchenko IV, Sokol OA, Strel'chenko EV. Regulation of heme oxygenase activity in rat liver during oxidative stress induced by cobalt chloride and mercury chloride. Biochemistry (Mosc). 2001 Jan;66(1):77-82.; &(d) Kumar SV, Maitra S, Bhattacharya S. In vitro binding of inorganic mercury to the plasma membrane of rat platelet affects Na+-K+-Atpase activity and platelet aggregation. Biometals. 2002 Mar;15(1):51-7.
(263) Kumar AR, Kurup PA. Inhibition of membrane Na+-K+ ATPase activity: a common pathway in central nervous system disorders. J Assoc Physicians India. 2002 Mar;50:400-6; & Kurup RK, Kurup PA. Hypothalamic digoxin, cerebral chemical dominance and myalgic encephalomyelitis. Int J Neurosci. 2003 May;113(5):683-701, & (c) Kurup RK, Kurup PA. Hypothalamic digoxin, hemispheric dominance, and neuroimmune integration. Int J Neurosci. 2002 Apr;112(4):441-62; & (d) Kurup RK, Kurup PA, Hypothalamic digoxin and hemispheric chemical dominance--relation to the pathogenesis of senile osteoporosis, degenerative osteoarthritis, and spondylosis. Int J Neurosci. 2003 Mar;113(3):341-59.
(264) B.R. Danielsson et al, “ ”Behavioral effects of prenatal metallic mercury inhalation exposure in rats”, Neurotoxicol Teratol, 1993, 15(6): 391-6;& A. Fredriksson et al,”Prenatal exposure to metallic mercury vapour and methylmercury produce interactive behavioral changes in adult rats”, Neurotoxicol Teratol, 1996, 18(2): 129-34
(265)K.Lohmann et al, "Multiple Chemical Sensitivity Disorder in patients with neuroltoxic illnesses", Gesundheitswesen, 1996, 58(6):322 31.
(268)J.J.Weening et al, "mercury induced immune complex glomerulopathy", Chap 4, p36 66, VanDendergen, 1980, & P.Duuet et al, "Glomerulonephritis induced by heavy metals", Arch Toxicol. 50:187 194,1982 & Transplantation
Proceedings,Vol XIV(3),1982,482

(269)(a)C.J.G.Robinson et al, "Mercuric chloride induced anitnuclear antibodies In mice", Toxic Appl Pharmacology, 1986, 86:159 169. &(b) P.Andres, IgA IgG disease in the intestines of rats ingesting HgCl", Clin Immun Immunopath, 30:488 494, 1984; &(c) F.Hirsch et al, J Immun.,136(9), 3272 3276, 1986 & (d)J.Immun.,136(9):3277 3281; & (e)J Immun., 137(8),1986,2548 & (f)Cossi et al, "Benefiecial effect of human therapeutic IV Ig in mercury indueced autoimune disease" Clin Exp Immunol, Apr, 1991; & (g)El Fawai HA, Waterman SJ, De Feo A, Shamy MY. Neuroimmunotoxicology: Humoral Assesment of Neurotoxicity and Autoimmune Mechinisms. Contact Dermatitis 1999; 41(1): 60 1.
(270)D.W.Eggleston, "Effect of dental amalgam and nickel alloys on T lympocytes",J Prosthet Dent. 51(5):617 623, 1984; & (d) Park SH et al, Effects of occupational metallic mercury vapor exposure on suppressor-inducer(CD4+CD45RA+) T lymphocytes and CD57+CD16+ natural killer cells, Int Arch Occup Environ Health, 2000, 73(8): 537-42.
(272) BJ Shenker,“Low-level MeHg exposure causes human T-cells to undergo apoptosis: evidence of mitochondrial disfunction”, Environ Res, 1998, 77(2):149-159; & O.Insug et al, “Mercuric compounds inhibit human monocyte function by inducing apoptosis: evidence for formation of reactive oxygen species(ROS), development of mitochondrial membrane permeability, and loss of reductive reserve”, Toxicology, 1997, 124(3):211-24;
(287) (Kurup RK, Kurup PA. Hypothalamic digoxin, cerebral chemical dominance, and pathogenesis of pulmonary diseases Int J Neurosci. 2003 Feb;113(2):235-58; & Anner BM, Moosmayer M, Imesch E. Mercury blocks Na-K-ATPase by a ligand-dependent and reversible mechanism.. Am J Physiol. 1992 May;262(5 Pt 2):F830-6; & Walczak-Drzewiecka A, Wyczolkowska J, Dastych J. Environmentally Relevant Metal and Transition Metal Ions Enhance Fc Epsilon RI-Mediated Mast Cell Activation. Environ Health Perspect. 2003 May;111(5):708-13. ); & Hunter I, Cobban HJ, Vandenabeele P, MacEwan DJ, Nixon GF. Tumor necrosis factor-alpha-induced activation of RhoA in airway smooth muscle cells: role in the Ca2+ sensitization of myosin light chain20 phosphorylation. Mol Pharmacol. 2003 Mar;63(3):714-21 )
(288) (a)Hisatome I, Kurata Y, et al; Block of sodium channels by divalent mercury: role of specific cysteinyl residues in the P-loop region. Biophys J. 2000 Sep;79(3):1336-45; & Bhattacharya S, Sen S et al, Specific binding of inorganic mercury to Na(+)-K(+)-ATPase in rat liver plasma membrane and signal transduction. Biometals. 1997 Jul;10(3):157-62; & Anner BM, Moosmayer M, Imesch E. Mercury blocks Na-K-ATPase by a ligand-dependent and reversible mechanism. Am J Physiol. 1992 May;262(5 Pt 2):F830-6. & Anner BM, Moosmayer M. Mercury inhibits Na-K-ATPase primarily at the cytoplasmic side. Am J Physiol 1992; 262(5 Pt2):F84308; & Wagner CA, Waldegger S,et al; Heavy metals inhibit Pi-induced currents through human brush-border NaPi-3 cotransporter in Xenopus oocytes.. Am J Physiol. 1996 Oct;271(4 Pt 2):F926-30; & Lewis RN; Bowler K. Rat brain (Na+ K+)ATPase: modulation of its ouabain sensitive K+ PNPPase activity by thimerosal. Int J Biochem 1983;15(1):5 7
& (b) Rajanna B, Hobson M, Harris L, Ware L, Chetty CS. Effects of cadmium and mercury on Na(+)-K(+) ATPase and uptake of 3H-dopamine in rat brain synaptosomes. Arch Int Physiol Biochem 1990, 98(5):291-6; & M.Hobson, B.Rajanna, “Influence of mercury on uptake of dopamine and norepinephrine”, Toxicol Letters, Dep 1985, 27:2-3:7-14; & & McKay SJ, Reynolds JN, Racz WJ. Effects of mercury compounds on the spontaneous and potassium-evoked release of [3H]dopamine from mouse striatial slices. Can J Physiol Pharmacol 1986, 64(12):1507-14; & Scheuhammer AM; Cherian MG. Effects of heavy metal cations, sulfhydryl reagents and other chemical agents on striatal D2 dopamine receptors. Biochem Pharmacol 1985 Oct 1;34(19):3405 13 ;& K.R.Hoyt et al, “Mechanisms of dopamine-induced cell death and differences from glutamate Induced cell death”, Exp Neurol 1997, 143(2):269-81; & (c)Offen D, et al, Antibodies from ALS patients inhibit dopamine release mediated by L-type calcium channels. Neurology 1998 Oct;51(4):1100-3.
( (291) H.A.Huggins & TE Levy, "cerebrospinal fluid protein changes in MS after Dental amalgam removal", Alternative Med Rev, Aug 1998, 3(4):295 300.
(293)H.Huggins,Burton Goldberg, & Editors of Alternative Medicine Digest,Chronic Fatigue Fibromyalgia & Environmental Illness, Future Medicine Publishing, Inc, 1998, p197.
(296) L.Bucio et al, Uptake, cellular distribution and DNA damage produced by mercuric chloride in a human fetal hepatic cell line. Mutat Res 1999 Jan 25;423(1 2):65 72; & (b) Ho PI, Ortiz D, Rogers E, Shea TB. Multiple aspects of homocysteine neurotoxicity: glutamate excitotoxicity, kinase hyperactivation and DNA damage. J Neurosci Res. 2002 Dec 1;70(5):694-702; &(c) & Snyder RD; Lachmann PJ; Thiol involvement in the inhibition of DNA repair by metals in mammalian cells. Source Mol Toxicol, 1989, 2:2, 117 28; & L.Verschaeve et al, “Comparative in vitro cytogenetic studies in mercury-exposed human lymphocytes”, Muta Res, 1985, 157(2-3): 221-6; & L.Verschaeve,“Genetic damage induced by low level mercury exposure”, Envir Res,12:306-10, 1976.

(305) Soderstrom S, Fredriksson A, Dencker L, Ebendal T, “The effect of mercury vapor on cholinergic neurons in the fetal brain, Brain Research & Developmental Brain Res, 1995, 85:96-108; & Toxicol Lett 1995; 75(1-3): 133-44.; & (b)E.M. Abdulla et al, “Comparison of neurite outgrowth with neurofilament protein levels In neuroblastoma cells following mercuric oxide exposure”, Clin Exp Pharmocol Physiol, 1995, 22(5): 362-3: &(c) Leong CC, Syed NI, Lorscheider FL. Retrograde degeneration of neurite membrane structural integrity of nerve growth cones following in vitro exposure to mercury. Neuroreport 2001 Mar 26;12(4):733-7

(313) V.D.M.Stejskal et al, "Mercury specific Lymphocytes: an indication of mercury allergy in man", J. Of Clinical Immunology, 1996, Vol 16(1);31 40.
(314) M.Goldman et al,1991,"Chemically induced autoimmunity ...",Immunology Today,12:223 ; & K. Warfyinge et al, "Systemic autoimmunity due to mercury vapor exposure in genetically susceptible mice", Toxicol Appl Pharmacol,
1995, 132(2):299 309;& L.M. Bagenstose et al, "Mercury induced autoimmunity in humans", Immunol Res, 1999,20(1): 67 78; &"Mercury induced autoimmunity", Clin Exp Immunol, 1998, 114(1):9 12.
(316)B.J.Shenker et al, Dept. Of Pathology, Univ. Of Pennsylvania School of Dental Medicine, “Immunotoxic effects of mercuric compounds on human lymphocytes and monocytes: Alterations in B-cell function and viability” Immunopharmacol Immunotoxicol, 1993, 15(1):87-112; & J.R.Daum,”Immunotoxicology of mercury and cadmium on B-lymphocytes”, Int J Immunopharmacol, 1993, 15(3):383-94; & Johansson U, et al, "The genotype determines the B cell response in mercury-treated mice", Int Arch Allergy Immunol, 116(4):295-305, (Aug 1998)
(317) S.Zinecker, “Amalgam: Quecksilberdamfe bis ins Gehirn”, der Kassenarzt, 1992, 32(4):23; “Praxiproblem Amalgam”, Der Allgermeinarzt, 1995,17(11):1215-1221. (1800 patients)
(321) R.L.Siblerud, “Relationship between dental amalgam and health”, Toxic Substances Journal, 1990b. 10:425-444; & “Effects on health following removal of dental amalgams”, J Orthomolecular Med,5(2): 95-106, & “Relationship between amalgam fillings and oral cavity health” Ann Dent, 1990, 49(2): 6-10, (86 cured)
(323) Dr. Kohdera, Faculty of Dentistry, Osaka Univ, Internationsl Congress of Allergology and Clinical Immunology, EAACI, Stockholm, June 1994; & Heavy Metal Bulletin, Vol 1, Issue 2, Oct 1994. (160 cases cured eczema,etc.); Tsunetoshi Kohdera, MD, dermatology, allergology, 31 Higashitakada cho Mibu Nakagyo ku Schimazu Clinics Kyoto 604 Japan e mail:smc inet@mbox.kyoto inet.or.jp & G. Ionescu, Biol Med, 1996, (2): 65 68; (these clinics use MELISA test for diagnosis of immune reactivity) Neukirchen (clinic)(Germany, near Czech border). Director; Gruia Ionescu, owns 2 Clinics, cases paid by insurance companies in Germany. Email: Spezialklinik Neukirchen@toolpool.de fax: 0049 9947 10 51 11
(330) Wilkinson LJ, Waring RH. Cysteine dioxygenase: modulation of expression in human cell lines by cytokines and control of sulphate production. Toxicol In Vitro. 2002 Aug;16(4):481-3; & (b) M.T.Heafield et al, "Plasma cysteine and sulphate levels in patients with Motor neurone disease, Parkinson's Disease, and Alzheimer's Disease", Neurosci Lett, 1990, 110(1 2), 216,20; & A.Pean et al, "Pathways of cysteine metabolism in MND/ALS", J neurol Sci, 1994, 124, Suppl:59 61; & Steventon GB, et al; Xenobiotic metabolism in motor neuron disease, The Lancet, Sept 17 1988, p 644-47; & Neurology 1990, 40:1095-98.
(331) C.Gordon et al, “Abnormal sulphur oxidation in systemic lupus erythrmatosus(SLE)”, Lancet, 1992,339:8784,25-6; &(b) P.Emory et al, “Poor sulphoxidation in patients with rheumatoid arthitis”, Ann Rheum Dis, 1992, 51:3,318-20; & Bradley H,et al, Sulfate metabolism is abnormal in patients with rheumatoid arthritis. Confirmation by in vivo biochemical findings. J Rheumatol. 1994 Jul;21(7):1192-6; & (c)T.L. Perry et al, “Hallevorden-Spatz Disease: cysteine accumulation and cysteine dioxygenase defieciency”, Ann Neural, 1985, 18(4):482-489.
(333) A.J.Freitas et al, “Effects of Hg2+ and CH3Hg+ on Ca2+ fluxes in the rat brain”, Brain Research, 1996, 738(2): 257-64; & P.R.Yallapragoda et al,“Inhibition of calcium transport by Hg salts” in rat cerebellum and cerebral cortex”, J Appl toxicol, 1996, 164(4): 325-30; & E.Chavez et al, “Mitochondrial calcium release by Hg+2",J Biol Chem, 1988, 263:8, 3582-; A. Szucs et al,Effects of inorganic mercury and methylmercury on the ionic currents of cultured rat hippocampal neurons. Cell Mol Neurobiol, 1997,17(3): 273-8; & D.Busselberg, 1995, “Calcium channels as target sites of heavy metals”,Toxicol Lett, Dec;82 83:255 61; & Cell Mol Neurobiol 1994 Dec;14(6):675 87; & Rossi AD, et al, Modifications of Ca2+ signaling by inorganic mercury in PC12 cells. FASEB J 1993, 7:1507-14.

(338) (a)W.Y.Boadi et al, Dept. Of Food Engineering and Biotechnology, T-I Inst of Tech., Haifa, Israel, “In vitro effect of mercury on enzyme activities and its accumulation in the first-trimester human placenta”, Environ Res, 1992, 57(1):96-106;& “In vitro exposure to mercury and cadmium alters term human placental membrane fluidity”, Pharmacol, 1992, 116(1): 17-23; & (b)J.Urbach et al, Dept. of Obstetrics & Gynecology, Rambam Medical Center, Haifa, Israel, “Effect of inorganic mercury on in vitro placental nutrient transfer and oxygen consumption”, Reprod Toxicol, 1992,6(1):69-75;& © Karp W, Gale TF et al, Effect of mercuric acetate on selected enzymes of maternal and fetal hamsters” Environmental Research, 36:351-358; & W.B. Karp et al, “Correlation of human placental enzymatic activity with tracemetal concentration in placenta”, Environ Res. 13:470- 477,1977; & (d) Boot JH. Effects of SH blocking compounds on the energy metabolism and glucose uptake in isolated rat hepatocytes. Cell Struct Funct 1995 Jun;20(3):233 8; & H.Iioka et al, “The effect of inorganic mercury on placental amino acid transport”, Nippon sanka Fujinka Gakkai Zasshi, 1987, 39(2): 202-6.
(342) Stejskal VDM, Danersund A, Lindvall A, Hudecek R, Nordman V, Yaqob A et al. Metal specific memory lymphoctes: biomarkers of sensitivity in man. Neuroendocrinology Letters, 1999; 20: 289-298.
(346) Clauw DJ, "The pathogenesis of chronic pain and fatigue syndroms: fibromyalgia" Med Hypothesis, 1995, 44:369 78; & Hanson S, Fibromyalgia, glutamate, and mercury. Heavy Metal Bulletin, Issue 4, 1999, p3 6.
(348) A Kistner, “Quecksilbervergiftung durch Amalgam: Diagnose und Therapie” ZWR, 1995,104(5):412-417; &(b) Villegas J, Martinez R, Andres A, Crespo D. Accumulation of mercury in neurosecretory neurons of mice after long-term exposure to oral mercuric chloride. Neurosci Lett 1999; 271: 93-96; &(c) Kozik MB, Gramza G. Histochemical changes in the neurosecretory hypothalic nuclei as a result of an intoxication with mercury compounds. Acta Histochem Suppl 1980; 22:367-80.
(368) Olin R, Paulander J, Axelsson P; FMS,CFS, and TMS- Prevalences in a Swedish County, An oral examination based study, Preventive Dental Health Care Center, Karlstad, Sweden, 1998.
(369) Sterzl I, Prochazkova J, Stejaskal VDM et al, Mercury and nickel allergy: risk facotrs in fatigue and autoimmunity. Neuroendocrinology Letters 1999; 20:221 228.
(379) MacDonald EM, Mann AH, Thomas HC. Interferons as mediatorors of psychiatric morbidity. The Lancet 1978; Nov 21, 1175 78; & Hickie I, Lloyd A. Are cytokines associated with neuropsychiatric syndrome in humans? Int J Immunopharm 1995; 4:285 294.
(380) Komaroff AL, Buchwald DS. Chronic fatigue syndrom: an update. Ann Rev Med 1998; 49: 1 13; & Buchwald DS, Wener MH, Kith P. Markers of inflamation and immune activation in CFS. J Rheumatol 1997; 24:372 76.
(381) Demitrack MA,Dale JK. Evidence for impaired activation of the hypothalamic pituitary adrenal axis in patients with chronic fatigue sydrome. J Clin endocrinol Metabol 1991; 73:1224 1234; & Turnbull AV, Rivier C. Regulation of the HPA axis by cytokines. Brain Behav Immun 1995; 20:253 75.
(382) Sterzl I, Fucikova T, Zamrazil V. The fatigue syndrome in autoimmune thyroiditis with Polyglanular activation of autoimmunity. Vnitrni Lekarstvi 1998; 44: 456 60; & Sterzl I, Hrda P, Prochazkova J, Bartova J, Reactions to metals in patients with chronic fatigue and autoimmune endocrinopathy. Vnitr Lek 1999 Sep;45(9):527 31
(383) Saito K. Analysis of a genetic factor of metal allergy polymorphism of HLA DR DO gene. Kokubyo Gakkai Zasschi 1996; 63: 53 69; & Prochazkova J, Ivaskova E, Bartova J, Stejskal VDM. Immunogentic findings in patients with altered tolerance to heavy metals. Eur J Human Genet 1998; 6: 175.
(385) Kohdera T, Koh N, Koh R. Antigen specific lympocyte stimulation test on patients with psoriasis vulgaris. XVI International Congress of Allergology and Clinical Immunology, Oct 1997, Cancoon, Mexico; & Ionescu
G. Schwermetallbelastung bei atopischer Dermatitis und Psoriasis. Biol Med 1996; 2:65 68.
(386) Biospectron Lab ....... & Doctors Data Lab ,http://www.doctorsdata.com , inquiries @doctors data.com, & Great Smokies Diagnostic Lab, http://www.gsdl.com; & MetaMatrix Lab, http://www.metamatrix.com
(404) M. E. Godfrey, Candida, Dysbiosis and Amalgam. J. Adv. Med. vol 9 no 2 (1996).
(405) Jenny Stejskal, Vera Stejskal. The role of metals in autoimmune diseases and the link to neuroendocrinology Neuroendocrinology Letters, 20:345 358, 1999.
(411) Puschel G, Mentlein R, Heymann E, 'Isolation and characterization of dipeptyl peptidase IV from human placenta', Eur J Biochem 1982 Aug;126(2):359-65; & Kar NC, Pearson CM. Dipeptyl Peptidases in human muscle disease. Clin Chim Acta 1978; 82(1-2): 185-92; & Seroussi K, Autism and Pervasive Developmental Disorders , 1998, p174,etc.; & Shibuya-Saruta H, Kasahara Y, Hashimoto Y. Human serum dipeptidyl peptidase IV (DPPIV) and its unique properties. J Clin Lab Anal. 1996;10(6):435-40; & Blais A, Morvan-Baleynaud J, Friedlander G, Le Grimellec C. Primary culture of rabbit proximal tubules as a cellular model to study nephrotoxicity of xenobiotics. Kidney Int. 1993 Jul;44(1):13-8

(416)(a) Plaitakis A, Constantakakis E. Altered metabolism of excitatory amino acids, N-acetyl-aspartate and – acetyl-aspartyl-glutamate in amyotrophic lateral sclerosis. Brain Res Bull 1993;30(3-4):381-6 &(b)Rothstein JD, Martin LJ, Kuncl RW. Decreased glutamate transport by the brain and spinal cord in ALS. New Engl J Med 1992, 326: 1464-8:& (c) Leigh Pn. Pathologic mechanisms in ALS and other motor neuron diseases. In: Calne DB(Ed.), Neurodegenerative Diseases, WB Saunder Co., 1997, p473-88; & P.Froissard et al, Universite de Caen, “Role of glutathione metabolism in the glutamate-induced programmed cell death of neuronal cells” Eur J Pharmacol, 1997, 236(1): 93-99; & (d) Kim P, Choi BH. “Selective inhibition of glutamate uptake by mercury in cultured mouse astrocytes”, Yonsei Med J 1995; 36(3): 299-305; & Brookes N. In vitro evidence for the role of glutatmate in the CNS toxicity of mercury. Toxicology 1992, 76(3):245-56; & Albrecht J, Matyja E. Glutamate: a potential mediator of inorganic mercury toxicity. Metab Brain Dis 1996; 11:175-84; &(e) Tirosh O, Sen CK, Roy S, Packer L. Cellular and mitochondrial changes in glutamate-induced HT4 neuronal cell death Neuroscience. 2000;97(3):531-41;
(417) Folkers K et al, Biochemical evidence for a deficiency of vitamin B6 in subjects reacting to MSL Glutamate. Biochem Biophys Res Comm 1981, 100: 972; & Felipo V et al, L carnatine increases the affinity of glutamate for quisqualate receptors and prevents glutamate neurotoxicity. Neurochemical Research 1994, 19(3): 373 377; & Akaike A et al, Protective effects of a vitamin B12 analog(methylcobalamin, against glutamate cytotoxicity in cultured cortical neurons. European J of Pharmacology 1993, 241(1):1 6 .
(432) Sutton KG, McRory JE, Guthrie H, Snutch TP. P/Q-type calcium channels mediate the activity-dependent feedback of syntaxin-1A. Nature 1999, 401(6755):800-4;
(440) Kidd RF. Results of dental amalgam removal and mercury detoxification. Altern Ther Health Med 2000 Jul;6(4):49 55.
(442) Olanow CW, Arendash GW. Metals and free radicals in neurodegeneration. Curr Opin Neurol 1994, 7(6):548- 58; & Kasarskis EJ(MD), Metallothionein in ALS Motor Neurons(IRB #91-22026), FEDRIP DATABASE, National Technical Information Service(NTIS), ID: FEDRIP/1999/07802766.
(462) Olivieri G; Brack C; Muller Spahn F; Stahelin HB; Herrmann M; Renard P; Brockhaus M; Hock C. Mercury induces cell cytotoxicity and oxidative stress and increases beta amyloid secretion and tau phosphorylation in SHSY5Y neuroblastoma cells. J Neurochem 2000 Jan;74(1):231 6; & (b) Tabner BJ, Turnbull S, El-Agnaf OM, Allsop D. Formation of hydrogen peroxide and hydroxyl radicals from A(beta) and alpha-synuclein as a possible mechanism of cell death in Alzheimer's disease and Parkinson's disease. Free Radic Biol Med. 2002 Jun 1;32(11):1076-83; &(c) Ho PI, Collins SC, et al; Homocysteine potentiates beta-amyloid neurotoxicity: role of oxidative stress. J Neurochem. 2001 Jul;78(2):249-53.
(468) Overzet K, Gensler TJ, Kim SJ, Geiger ME, van Venrooij WJ, Pollard KM, Anderson P, Utz PJ. Small nucleolar RNP scleroderma autoantigens associate with phosphorylated serine/arginine splicing factors during apoptosis. Arthritis Rheum 2000 Jun;43(6):1327 36
(469)BrainRecovery.com, the book, by David Perlmutter MD; Perlmutter Health Center, Naples, Florida, www.perlhealth.com/about.htm; &
M.M. Van Benschoten and Associates, Reseda, Calif. Clinic; www.mmvbs.com
(470) Dr. Garth Nicholson, Institute for Molecular Medicine, Huntington Beach, Calif., www.immed.org
& Michael Guthrie, R.Ph. ImmuneSupport.com 07 18 2001 Mycoplasmas – The Missing Link in Fatiguing Illnesses, www.immunesupport.com/library/showarticle.cfm?ID=3066; & New Treatments for Chronic Infections Found in Fibromyalgia Syndrome, Chronic Fatigue Syndrome, Rheumatoid Arthritis, and Gulf War Illnesses, http://www.immed.org/reports/autoimmune_illness/rep1.html ; & Prof. Garth L. Nicolson, Chronic Fatigue Syndrome, Fibromyalgia Syndrome and Other Fatigue Conditions, http://www.immed.org/illness/fatigue_illness_research.html
(471) Schwartz RB, Garada BM, Komaroff AL, Gleit M, Holman BL. Detection of intracranial abnormalities in patients with chronic fatigue syndrome: comparison of MRI and SPECT. Am J Roentgenol, 1994, 162(4):935 41; & Spect Imaging: comparison of findings in patients with CFS, AIDA dementia complex, and major unipolar depression, Am J Roentgenol 1994, 162(4): 943 51; & Ichiso M, Salit IE, Abbey SE. Assessment of regional cerebral perfusion by SPECT in CFS. Nucl Med Commun 1992; 13:767-72.
(472) Patarca Monero R, Klimas NG, Fletcher MA. Immunotherapy of chronic fatigue syndrome. Journal of Chronic Fatigue Syndrome. 2001, 8(1): 3 37; & DeBecker P, De Meirleir K, Joos E, Velkeniers B. DHEA response to I.V. ACTH in patients with CFS. Horm Metab Res 1999, 31(1): 18 21.
(473) De Meirleir K, Bisbal C, Campine I, De Becker, et al. A 37 kDa 1 5A binding proein as a potential biochemical marker for CFS. Am J Med 2000, 108(2): 99 105; & Suhadolnik RJ, Peterson DL, Obrien K, et al, Biochemical evidence for a novel low molecular weight 2 5A dependent Rnase L in CFS. J Interferon Cytokine Res, 1997, 17(7): 377 85; & Chaudhuri A, Watson WS, Pearn J, Behan PO. The symptoms of chronic fatigue syndrome are related to abnormal ion channel function. Med Hypotheses 2000 Jan;54(1):59 63
(474) Richards SCM, Bell J, Cheung, YL, Cleare A, Scott DL. Muscle metabolites detected in urine in FM and CFS suggest ongoing muscle damage. Conference Proceedings of the British Scociety of Rheumatologists, April
2001, Scotland, Abstract 382; http://freespace,virgin.net/david.axford/me_nb_o4.htm.
(485) Hulda Clark, The Cure for all Diseases, 2000, www.drclark.net.

(487) Straus SE et al, CFS and allergy, J Allergy Clin Immunol, 1988, 81(5): 791-95; Straus SE et al, Evidence of Epstein-Barr virus infection in CFS, Annals of Internal Med, 1985, 102(1): 7-16; & Jones JF et al, Annals of Internal Med, Evidence for active Epstein-Barr Virus in patients with chronic unexplained illness, 1985, Annals of Internal Med, 102(1): 1-6; & Tyler AN, Influenza A virus: factor in fibromyalgia?, 1997, 2(2): 82- 86.
(488) Teitelbaum J, Bird B; Effective treatment of CFS, report on 64 patients, J Musculoskeletal Pain, 1995, 3(4): 91-100.
(489) Behan PO et al; Effect of high doses of essential fatty acids on postviral fatigue syndrome, Acta Neurol Scand, 1990, 82: 209-216; & Abraham GE, Flechas JD; Rationale for the use of magnesium and malic acid in fibromyalgis treatment, Journal of Nutritional Medicine, 1992, 3:40-52.
(490) Smith JD, Terpening CM, Schmidt SO, Gums JG. Relief of fibromyalgia symptoms following discontinuation of dietary excitotoxins. Ann Pharmacother 2001 Jun;35(6):702 6.
(490) Shibata N, Nagai R, Kobayashi M. Morphological evidence for lipid peroxidation and protein glycoxidation in spinal cords from sporadic amyotrophic lateral sclerosis patients. Brain Res 2001 Oct 26;917(1):97-104 & Cookson MR, Shaw PJ. Oxidative stress and motor neurons disease. Brain Pathol 1999 Jan;9(1):165 86.
(494) (a)Kobayashi MS, Han D, Packer L. Antioxidants and herbal extracts protect HT-4 neuronal cells against glutamate-induced cytotoxicity. Free Radic Res 2000 Feb;32(2):115-24(PMID: 10653482; & (Bridi R, Crossetti FP, Steffen VM, Henriques AT. The antioxidant activity of standardized extract of Ginkgo biloba (EGb 761) in rats. Phytother Res 2001 Aug;15(5):449-51 ;&(c)Li Y, Liu L, Barger SW, Mrak RE, Griffin WS. Vitamin E suppression of microglial activation is neuroprotective. J Neurosci Res 2001 Oct 15;66(2):163-70.
(496) Doble A. The role of excitotoxicity in neurodegenerative disease: implications for therapy. Pharmacol Ther 1999 Mar;81(3):163 221; & Urushitani M, Shimohama S. N methyl D aspartate receptor mediated mitochondrial Ca(2+) overload in acute excitotoxic motor neuron death: a mechanism distinct from chronic neurotoxicity after Ca(2+) influx. J Neurosci Res 2001 Mar 1;63(5):377 87; & Cookson MR, Shaw PJ. Oxidative stress and motor neurons disease. Brain Pathol 1999 Jan;9(1):165 86
(502) Vielhaber S, Kaufmann J, Kunz WS. Effect of Creatine Supplementation on Metabolite Levels in ALS Motor Cortices. Exp Neurol 2001 Dec;172(2):377-82.
(518) Landay AL, Jessop C, Lenette ET, chronic fatigue syndrome: clinical condition associated with immune activation. Lancet 1991; 338:707-12; & (b)Caliguri M, Murray C, Buchwald D. Phenotypic and functional deficiency of natural killer cells in patients with CFS. J Immunol 1987; 139:3306-13; & Barker E, Fujirmura SF, Fadern MB. Immunologic abnormalities associated with CFS. Clin Infect Dis 1994; 18: 136-41.
(521) Guermonprez L, Ducrocq C, Gaudry-Talarmain YM. Inhibition of acetylcholine synthesis and tyrosine nitration induced by peroxynitrite are differentially prevented by antioxidants. Mol Pharmacol 2001 Oct;60(4):838-46; & & (b)Mahboob M, Shireen KF, Atkinson A, Khan AT. Lipid peroxidation and antioxidant enzyme activity in different organs of mice exposed to low level of mercury. J Environ Sci Health B. 2001 Sep;36(5):687-97. & Miyamoto K, Nakanishi H, et al, Involvement of enhanced sensitivity of N-methyl-D-aspartate receptors in vulnerability of developing cortical neurons to methylmercury neurotoxicity. Brain Res. 2001 May 18;901(1-2):252-8; & (c) Anuradha B, Varalakshmi P. Protective role of DL-alpha-lipoic acid against mercury-induced neural lipid peroxidation. Pharmacol Res. 1999 Jan;39(1):67-80.
(523) CBS Television Network,” 60 Minutes”, television program narrated by Morley Safer, December 12, 1990
(524) Urushitani M, Shimohama S. The role of nitric oxide in amyotrophic lateral sclerosis. Amyotroph Lateral Scler Other Motor Neuron Disord 2001 Jun;2(2):71-81; & Torreilles F, Salman-Tabcheh S, Guerin M, Torreilles J. Neurodegenerative disorders: the role of peroxynitrite.Brain Res Brain Res Rev 1999 Aug;30(2):153-63; & Aoyama K, Matsubara K, Kobayashi S. Nitration of manganese superoxide dismutase in cerebrospinal fluids is a marker for peroxynitrite-mediated oxidative stress in neurodegenerative diseases. Ann Neurol 2000 Apr;47(4):524-7; & Guermonprez L, Ducrocq C, Gaudry-Talarmain YM. Inhibition of acetylcholine synthesis and tyrosine nitration induced by peroxynitrite are differentially prevented by antioxidants. Mol Pharmacol 2001 Oct;60(4):838-46
(527) Cline Medical Center, Vancouver Island, http://www.oceansidemedicine.com/Default.htm

(528) Goldenberg DL;. Fibromyalgia, chronic fatigue syndrome, and myofascial pain. Curr Opin Rheumatol. 1996; 8: 113-123.
(529) GLYCONUTRITIONAL IMPLICATIONS IN FIBROMYALGIA AND CHRONIC FATIGUE SYNDROME http://www.glycoscience.org/glycoscience/start_frames.wm?FILENAME=G005&MAIN=glyconutritionals&SUB=disease
(572) Packer L, Tritschler HJ, Wessel K. Neuroprotection by the metabolic antioxidant alpha-lipoic acid. Free Radic Biol Med 1997;22(1-2):359-78(PMID: 8958163); & McCarty MF. Versatile cytoprotective activity of lipoic acid may reflect its ability to activate signalling intermediates that trigger the heat-shock and phase II responses. Med Hypotheses 2001 Sep;57(3):313-7 & Whiteman M, Tritschler H, Halliwell B. Protection against peroxynitrite-dependent tyrosine nitration and alpha 1-antiproteinase inactivation by oxidized and reduced lipoic acid. FEBS Lett 1996 Jan 22;379(1):74-6(PMID: 8566234); & Patrick L. Mercury toxicity and antioxidants: Part 1: role of glutathione and alpha-lipoic acid in the treatment of mercury toxicity. Altern Med Rev. 2002 Dec;7(6):456-71.
(600) B. Windham, Annotated bibliography: Exposure levels and health effects related to mercury/dental amalgam and results of amalgam replacement, 2002; (over 1500 medical study references documenting mechanism of causality of 40 chronic conditions and over 60,000 clinical cases of recovery or significant improvement of these conditions after amalgam replacement-documented by doctors) www.home.earthlink.net/~berniew1/amalg6.html

(601) B. Windham, Cognitive and Behavioral Effects of Toxic Metal Exposures, 2002; (over 150 medical study references) www.home.earthlink.net/~berniew1/tmlbn.html
(602) The mechanisms by which mercury causes chronic immune and inflamatory condtions, B.Windham(Ed.), 2002, www.home.earthlink.net/~berniew1/immunere.html
(603) B. Windham(Ed.), The environmental effects of dental amalgam affect everyone, 2002,
www.home.earthlink.net/~berniew1/damspr2f.html
*********************

Gerber BO, Pichler WJ. Noncovalent Interactions of Drugs With Immune Receptors May Mediate Drug-induced Hypersensitivity Reactions.

AAPS Journal. 2006; 8(1): E160-E165. DOI: 10.1208/aapsj080119
Noncovalent Interactions of Drugs With Immune Receptors May Mediate Drug-induced Hypersensitivity Reactions
Basil O. Gerber1 and Werner J. Pichler1
1 Division of Allergology, Clinic for Rheumatology and Clinical Immunology/Allergology, Berne, Switzerland
Correspondence to:
Werner J. Pichler
Tel: ++41 +31 632 22 64
Fax: ++41 632 27 47
Email: werner.pichler@insel.ch
Submitted: August 26, 2005; Accepted: December 8, 2005; Published: March 17, 2006
Abstract
Drug-induced hypersensitivity reactions are instructive examples of immune reactions against low molecular weight compounds. Classically, such reactions have been explained by the hapten concept, according to which the small antigen covalently modifies an endogenous protein; recent studies show strong associations of several HLA molecules with hypersensitivity. In recent years, however, evidence has become stronger that not all drugs need to bind covalently to the major histocompatibility complex (MHC)-peptide complex in order to trigger an immune response. Rather, some drugs may bind reversibly to the MHC or possibly to the T-cell receptor (TCR), eliciting immune reactions akin to the pharmacological activation of other receptors. While the exact mechanism is still a matter of debate, noncovalent drug presentation clearly leads to the activation of drug-specific T cells. In some patients with hypersensitivity, such a response may occur within hours of even the first exposure to the drug. Thus, the reaction to the drug may not be the result of a classical, primary response but rather be mediated by existing, preactivated T cells that display cross-reactivity for the drug and have additional (peptide) specificity as well. In this way, certain drugs may circumvent the checkpoints for immune activation imposed by the classical antigen processing and presentation mechanisms, which may help to explain the idiosyncratic nature of many drug hypersensitivity reactions.
Keywords: cross-reactivity, drug hypersensitivity, hapten, prohapten, p-i concept, T-cell receptor, T cells
Introduction
Adverse effects to drugs are a common incidence for the clinician. Most reactions are caused by the pharmacological or toxicological activities of the drug and are generally predictable (type A). However, nonpredictable, idiosyncratic (type B) reactions1,2 may occur as well, amounting to ~15% of all cases. Most of the type-B reactions are mediated by the immune system and thus also termed drug-hypersensitivity reactions. Elicited by different immune mechanisms, they can become manifest as many distinct diseases.3,4 Often, the pathological mechanisms of these immune-mediated adverse effects are not completely clear. Some reactions are of the immediate type and are clearly mediated by antibodies.5 However, more recent studies by different groups clearly show that patients with drug hypersensitivity harbor drug specific T cells in their peripheral blood and in the affected tissues.6-8 The functions of these drug-specific T cells seem to determine the clinical picture of the disease.9
Drug-induced hypersensitivity reactions are fascinating diseases, as a small chemical compound can elicit a strong systemic immune reaction. These diseases can also be seen as “experimental models” of nature, with the physician performing an unintended—albeit instructive—“experiment.” By dissecting the underlying mechanisms, novel insights can be gained from these experiments not only about drug-hypersensitivity reactions in particular,1-3 but also about immune reactions in general.
The Hapten and Prohapten Concept
How do small compounds such as drugs stimulate T cells? An answer to this question is certainly required to understand the side effects observed, and possibly allow for the prediction of these reactions based on the characteristics of the drug. For a long time, the “immunological dogma” postulated that small, low-molecular weight compounds per se are not capable of eliciting an immune response. In order for an immune reaction to occur, APC have to take up and process complex and large antigens and, subsequently, present these to T cells. However, small compounds such as drugs or metal ions were found to be able to trigger an immune response nevertheless. The hapten (and prohapten) model is currently the accepted explanation for these observations. Chemically reactive, small compounds (ie, haptens) bind to proteins or peptides and modify them.10-12 These are then processed and presented as hapten-modified peptides to T cells, which can react with the hapten antigen. Alternatively, haptens may also bind to immune molecules that are involved in the presentation process, such as the major histocompatibility complex (MHC) itself.13,14 Prohaptens are a variation on the same theme: in order to become chemically reactive, they first need to be converted into a hapten by being metabolized into a compound that is chemically reactive.1,2,15,16
Evidence for the Existence of Noncovalent Haptens
The (pro)hapten concept elegantly circumvents the (presumed) blindness of the immune system for low molecular weight compounds by postulating that chemical reactivity and subsequent coupling to a macromolecule carrier is an absolute necessity. As a consequence, drugs and other substances that are incapable of such conjugation with a carrier would not be antigens and could not induce hypersensitivity reactions. However, there is clear clinical, immunological, and biochemical evidence to the contrary (Table 1).
Table 1. The p-i Concept: Evidence Pro and Contra*
________________________________________
PRO
Numerous TCC specific for the parental form of several drugs despite the existence of reactive metabolites7,8,17-21

Glutaraldehyde-fixed APC can still present drug7,8,17-19,22

Washing removes drug and prevents T-cell activation17,22

Kinetics of TCR down-regulation too fast to allow antigen processing18

Kinetics of Ca2+ mobilization too fast to allow antigen processing18

Inhibition of SMX-NO generation by glutathione increases drug presentation23-26

High incidence of unrestricted, drug-reactive clones27

Elevated frequency of alloreactive drug-reactive clones compared with peptide-specific TCC28

Exchange or removal of MHC-class-II-associated peptides does not affect drug presentation29

Kinetics of ERK phosphorylation too fast to allow antigen processing22

CONTRA
Clearly established for chemically reactive drugs10-14

Delayed nature of majority of reactions; induction of a primary response is possible10-14

Strong associations between several MHC-class-I alleles and drug hypersensitivity30-37

________________________________________
*TCC indicates T-cell clone; APC, antigen-presenting cell; TCR, T-cell receptor; SMX-NO, sulfamethoxazole-nitroso; MHC, major histocompatibility complex; and ERK, extracellular signal-related kinase.

The parental, not metabolized forms, of several different drugs are able to stimulate T cells via the T-cell receptor (TCR) in an MHC-dependent way, in particular lamotrigine,7 carbamazepine,8 sulfamethoxazole (SMX),17,18 mepivacaine,19 lidocaine,19,20 p-phenylendiamine,21 and radio-contrast media (RCM),38,39 even though most of them have known metabolites that can, in principle, act as haptens or are known to do so. SMX has been characterized particularly well, as its reactive metabolite SMX-nitroso (SMX-NO), acting as a typical hapten, was available for comparison.23,24 Hypersensitivity to the drug SMX was thought to be a consequence of bioactivation to the hydroxylamine metabolite (SMX-NHOH) and further oxidation to the ultimate, reactive metabolite SMX-NO. The antioxidant glutathione is known to protect cells from reactive metabolites by conjugation and subsequent dissociation to SMX-NHOH and/or SMX.24 However, only a minority of T-cell clones (TCC) derived from SMX-allergic patients reacted with the chemically reactive metabolite.25 Most surprisingly, addition of glutathione to peripheral blood mononuclear cells enhanced rather than reduced the proliferation of T cells in response to SMX-metabolites,26 presumably by transforming SMX-NO back to the “original” antigen, SMX. The response of SMX-NO-specific TCC was abrogated when glutathione was present during the covalent modification of APC. Collectively, these experiments support the concept that some T cells in allergic individuals recognize the noncovalently bound parent drug SMX rather than APC covalently modified by SMX-NO.25,26
For several drugs, the kinetics of in vitro T-cell activation are simply much too fast for any involvement of antigen processing. In the presence of APC, lidocaine and SMX activate T cells quasi immediately as revealed by a rapid and sustained intracellular Ca2+ increase.18 It is impossible to reconcile this timing with an intermediate metabolism and processing step, which needs 60 minutes or longer to occur. Also, the kinetics of TCR down-regulation on drug reactive TCC after encountering the inert drug are similar to the recognition of preprocessed, immunogenic peptides (occurring within the first 30 minutes) and clearly differ from the recognition of proteins, which requires several hours.18 Several other observations argue against processing or covalent binding. For several drugs, specific TCC reacted even if the APC were fixed by glutaraldehyde, excluding the involvement of either processing or intracellular metabolism.7,8,17-19 Further, and at least for SMX, covalent binding is not necessary. Upon pulsing of APC, which removes the drug (incubation of APC with the drug for 1 hour followed by 2 washing steps), no T-cell stimulation was observed, while the hapten SMX-NO, capable of covalently modifying the MHC peptide complex, was still able to stimulate hapten-reactive T cells.17 Many drug-specific TCC were found to be MHC-unrestricted,27 and the frequency of alloreactive TCC is much higher among drug- than peptide-specific TCC from the same donor.28 Last, the MHC-bound peptide seems to be irrelevant for SMX-specific T-cell activation.29
Despite these unusual characteristics, T-cell activation by such drugs is TCR-dependent nevertheless, as recently shown using drug-specific TCR transfectants.22 Two SMX-specific human TCR were introduced into the mouse T-cell hybridoma cell line 54ζ17 (O. Acuto, personal communication, January 2003), according to the method described by Vollmer et al.40 These transfectants expressed drug-specific TCR on the cell-surface and could be stimulated in a specific way in the presence of APC, resulting in interleukin 2 (IL-2) secretion. Key findings with these transfectants, which corroborated the previous observations with drug-specific TCC, were that the drug can be washed away (contrary to haptens covalently bound to carrier molecules), that the presence of APC (MHC) is required for IL-2 production, and that fixed APC are still able to present the drug. Similarly, the kinetics of TCR activation were too fast to involve antigen processing, as antigen-dependent extracellular signal-related kinase (ERK) phosphorylation was detected within 1 minute of SMX exposure.
The P-I Concept: The TCR as the Antigen Binding Molecule?
Clearly, the hapten concept does not suffice to account for all the above observations. As a consequence, we have recently proposed a third model, which is not meant to contradict but rather to supplement the hapten/prohapten concept. Termed the p-i concept, which stands for “direct pharmacological interaction of drugs with immune receptors,”41 it states that certain drugs bind specifically and reversibly to some of the highly variable antigen-specific TCR in a direct way, instead of covalently modifying the MHC-peptide complex, which are the 2 feasible “partners” to accommodate allergy-inducing drugs. Such a drug-TCR interaction would be independent of metabolism and processing and, in fact, mimic drug interactions with other, nonimmunological receptors. While the MHC-peptide complex would not contribute (much) to the binding energy, it would still be necessary for full T-cell activation. Why do we think the TCR to be the more likely candidate for drug binding than the MHC, which is the “traditional” antigen-binding receptor?
The mere idea may appear far-fetched at first, but such a mechanism seems nevertheless feasible in principle, and at least one precedent has already been reported. Divalent Nickel ions (Ni) are generally considered haptens even though they do not bind to proteins covalently but rather by forming reversible coordination complexes.42 Weltzien and coworkers identified and characterized an HLA-DR-promiscuous, Ni-specific TCR in which Ni interacts simultaneously with the MHC and TCR by making contacts with a conserved His81 in the HLA-DR α-chain as well as Tyr29 and Tyr94 in CDR1α of the TCR. Thus, Ni forms a bridge between both receptors, much like a superantigen, even though requiring idiotypic residues in the TCR.13 Ni has 6 coordination sites, of which only 3 are known for this complex at present. Nevertheless, a substantial part of its binding energy will be derived by the 2 (at least) contacts with the TCR of this complex. In fact, Ni binding may represent a “compromise” between how a typical hapten and a small antigen incapable of covalent binding may interact with the MHC and the TCR (see Figure 1 and legend for a detailed explanation).


Figure 1. A schematic representation of how the TCR and the MHC might accommodate an antigen according to different models. The antigen (metal ion or drug) is depicted as a black ball, covalent bonds are indicated by bold lines, and noncovalent interactions by thin, dashed lines.

For haptens, the majority of the antigen binding energy stems from the interaction with the MHC-peptide complex via few but strong covalent bonds (hapten; note that certain haptens may be strongly associated not only with the MHC but also the TCR43). Ni may interact either like a noncovalent hapten14 or, as depicted here,13 forming equally strong, noncovalent interactions with both MHC and TCR (nickel), while at least some drugs would derive the majority of their binding energy from weak, noncovalent interactions with the TCR (p-i concept). These different modes of interaction represent a continuum of possibilities, with the (pro)hapten mode on one extreme of the spectrum, the p-i-concept mode representing the other extreme, and the Ni mode as an intermediate possibility.
Consider as well that αβ TCR are peptide receptors. It has been known for 30 years that drugs can activate receptors that have peptides or proteins as endogenous receptors, the classical example being the opiate alkaloids. For these as well as many other serpentine receptors, a myriad of compounds are known to bind and evoke many pharmacologically different responses. More recently, nonpeptide agonists have also been found for tyrosine kinases as well as growth factor and cytokine receptors.44 However, apart from a hydrophobic cleft between the CDR3α and CDR3β regions, the TCR does not feature a “suitable” binding pocket or groove for small molecular weight compounds such as peptide receptors. Still, it cannot be excluded categorically that some drugs may bind to a particular TCR, especially given the huge TCR repertoire and the high level of cross-reactivity just from a probabilistic point of view alone.45 It is also worthwhile to remember that the overwhelming majority of low molecular weight, “drug-like” compounds known to bind to differing receptor classes act as antagonists. In analogy to these findings, it seems likely that at least some drugs may not only activate but also block their (drug-specific) TCR.
The P-I Concept: Drug Reactivity Masking as Cross-reactivity to Peptide Antigens?
Even though the majority of drug-induced, T-cell-mediated skin reactions occur only after several days or even weeks of drug exposure, they can sometimes arise within a few hours after administration and/or without previous exposure to the drug (eg, documented for RCM).38,39 RCM are administered in extremely high doses, which might partly explain this observation. However, and notwithstanding the exceptional amounts of drug, the kinetics of such a reaction are much too fast to be explained by the induction of a classical, primary response, which is a prerequisite of the hapten model, as primary responses are mounted in the course of several days. On the other hand, a secondary response of the immune system is generally much faster and can lead to an immune reaction within the time frame observed for some adverse drug reactions. The existence of peptide-specific, preactivated memory T cells already present in the circulation and tissue that are cross-reactive to a particular drug seems an attractive explanation. If a sensitive individual harboring such cells were exposed to sufficient concentrations of the drug, these preactivated T cells would then be stimulated “accidentally” and induce a fast and potentially vigorous response. In line with this notion is the observation that the vast majority of drug-specific TCC have been found to bear αβ TCR, which usually recognize peptides, and that a general stimulation of T cells, as in HIV infection,46 is an important risk factor for drug hypersensitivity. Even more, it seems likely that drug-reactive cells exist even in individuals that are not hypersensitive: in an in vitro study, several blood donors who had never been exposed to SMX nevertheless harbored SMX- and SMX-NO-specific cells in their T-cell repertoire.47
Hence, 2 (yet unproven) hypotheses are inherent to the p-i concept, and experiments unambiguously determining whether or not drug binding to TCR molecules occurs and if TCR double-specific for a drug and a peptide exist are currently under way. If these experiments proved the p-i concept to be correct, they would constitute an important step to show that it may truly be feasible to modulate T-cell-mediated responses in an antigen-specific way by using drugs or other low molecular weight compounds. If the p-i-concept is examined from a different angle, it becomes clear that direct binding of small compounds (such as drugs) to the TCR also implies that the TCR itself may constitute a potential drug target. The underlying assumption for this—namely, the wealth of pharmacological agents acting on many other classes of receptors that often have peptides or proteins as their “real,” endogenous ligands—has already been touched upon in The P-I Concept: The TCR as the Antigen Binding Molecule? It seems possible, albeit probably not feasible at this time, that the “arsenal” of modern drug technology could be used to find small molecules that block or enhance a particular, antigen-specific response of clinical relevance.
Novel Genetic Factors Linked to Drug Hypersensitivity
The idiosyncratic nature of hypersensitivity reactions has prompted an intensive search for genetic factors explaining their occurrence in only a small subset of treated persons.30 In accordance with the (pro)hapten concept, the major emphasis was put on pharmacogenetic factors such as an altered metabolism, as the generation of a more reactive intermediate, able to modify autologous proteins, would have been the most stringent explanation for the occurrence of immunological side effects. However, associations of hypersensitivities with particular pharmacological genotypes remained often tenuous and even controversial,31 such as the slow acetylator phenotype reported to enhance the occurrence of side effects to SMX,32 and the moderate association of certain TNF-α promoter polymorphisms with carbamazepine hypersensitivity.33
More recent studies focusing on immunological rather than metabolic factors have now revealed surprisingly clear associations of certain drug hypersensitivity reactions with HLA-class I alleles. In ~5% of treated patients, abacavir causes a severe hypersensitivity reaction affecting multiple organs. The majority of these patients with drug hypersensitivity carried the HLA-B*5701 allele. This association was strongest in Caucasians,34 and the particular allele was present in 94.4% of patients but in only 1.7% of controls.35 Possibly even more striking is the association of carbamazepine treatment with the appearance of Stevens-Johnson syndrome in Han-Chinese carrying the HLA-B 1502 allele.36 This association is stronger than any other described so far for any HLA marker with a disease. In another case-control association study, the same authors identified HLA-B*5801 as an important genetic risk factor for severe allopurinol-induced cutaneous adverse reactions such as Stevens-Johnson syndrome and toxic epidermal necrolysis.37
It is clear that such strong associations with HLA alleles support an important role for HLA molecules in drug hypersensitivity, and they certainly seem to favor the hapten concept at least for these drugs. However, although the association with HLA alleles is very strong, it is not a sufficient explanation: many patients with HLA-B*5801 are exposed to allopurinol, yet they do not develop hypersensitivity.37 Caucasians do not show the association of HLA-B*5701 and carbamazepine hypersensitivity.34,35 As reported by the authors of these studies, other factors located in this region of chromosome 6 may be important as well (eg, hsp 70 and other genes). Most hypersensitivity reactions involve CD4+ T cells, which are MHC class II restricted.3 Last, not the HLA complex but the TCR, as its counterpart, might be crucial for the reaction, as the positive and negative selection of T cells in the thymus is codetermined by the autologous HLA molecules and peptides that can be presented, thus influencing the antigen repertoire of the individual patient.
Conclusions
Recent studies have shown surprisingly strong associations between particular MHC molecules and several drug-hypersensitivity reactions, lending further credence to the (pro)hapten concept. However, more and more in vitro studies imply that the (pro)hapten model as the sole molecular explanation for drug-induced hypersensitivity may not be sufficient, and that other possibilities should be considered. In fact, evidence is accumulating that certain drugs are able to activate T cells in ways that differ not only from the hapten model but also from other established concepts in immunology. It is becoming increasingly evident that not all drugs need to act as haptens. As far as drugs are concerned, it may be useful to draw inspiration from concepts of classical pharmacology, which have been established for greatly differing receptor classes. The potential reward may not only be a better understanding of drug-induced hypersensitivity reactions, but also novel means for immunomodulatory therapies.
References
1. Park BK, Pirmohamed M, Kitteringham NR. Role of drug disposition in drug hypersensitivity: a chemical, molecular, and clinical perspective. Chem Res Toxicol. 1998;11:969-987.
PubMed DOI: 10.1021/tx980058f
2. Naisbitt DJ, Gordon SF, Pirmohamed M, Park BK. Immunological principles of adverse drug reactions: the initiation and propagation of immune responses elicited by drug treatment. Drug Saf. 2000;23:483-507.
PubMed
3. Pichler WJ. Delayed drug hypersensitivity reactions. Ann Intern Med. 2003;139:683-693.
PubMed
4. Manfredi M, Severino M, Testi S, et al. Detection of specific IgE to quinolones. J Allergy Clin Immunol. 2004;113:155-160.
PubMed DOI: 10.1016/j.jaci.2003.09.035
5. Pichler WJ, Schnyder B, Zanni MP, Hari Y, von Greyerz S. Role of T cells in drug allergies. Allergy. 1998;53:225-232.
PubMed
6. Yawalkar N, Egli F, Hari Y, Nievergelt H, Braathen LR, Pichler WJ. Infiltration of cytotoxic T cells in drug-induced cutaneous eruptions. Clin Exp Allergy. 2000;30:847-855.
PubMed DOI: 10.1046/j.1365-2222.2000.00847.x
7. Naisbitt DJ, Farrell J, Wong G, et al. Characterization of drug-specific T cells in lamotrigine hypersensitivity. J Allergy Clin Immunol. 2003;111:1393-1403.
PubMed DOI: 10.1067/mai.2003.1507
8. Naisbitt DJ, Britschgi M, Wong G, et al. Hypersensitivity reactions to carbamazepine: characterization of the specificity, phenotype, and cytokine profile of drug-specific T cell clones. Mol Pharmacol. 2003;63:732-741.
PubMed DOI: 10.1124/mol.63.3.732
9. Pichler WJ. Drug-induced autoimmunity. Curr Opin Allergy Clin Immunol. 2003;3:249-253.
PubMed DOI: 10.1097/00130832-200308000-00003
10. Padovan E, Bauer T, Tongio MM, Kalbacher H, Weltzien HU. Penicilloyl peptides are recognized as T cell antigenic determinants in penicillin allergy. Eur J Immunol. 1997;27:1303-1307.
PubMed
11. Cavani A, Hackett CJ, Wilson KJ, Rothbard JB, Katz SI. Characterization of epitopes recognized by hapten-specific CD4+ T cells. J Immunol. 1995;154:1232-1238.
PubMed
12. Cavani A, Mei D, Guerra E, et al. Patients with allergic contact dermatitis to nickel and nonallergic individuals display different nickel-specific T cell responses: evidence for the presence of effector CD8+ and regulatory CD4+ T cells. J Invest Dermatol. 1998;111:621-628.
PubMed DOI: 10.1046/j.1523-1747.1998.00334.x
13. Gamerdinger K, Moulon C, Karp DR, et al. A new type of metal recognition by human T cells: contact residues for peptide-independent bridging of T cell receptor and major histocompatibility complex by nickel. J Exp Med. 2003;197:1345-1353.
PubMed DOI: 10.1084/jem.20030121
14. Lu L, Vollmer J, Moulon C, Weltzien HU, Marrack P, Kappler J. Components of the ligand for a Ni++ reactive human T cell clone. J Exp Med. 2003;197:567-574.
PubMed DOI: 10.1084/jem.20021762
15. Griem P, Wulferink M, Sachs B, Gonzalez JB, Gleichmann E. Allergic and autoimmune reactions to xenobiotics: how do they arise? Immunol Today. 1998;19:133-141.
PubMed DOI: 10.1016/S0167-5699(97)01219-X
16. Knowles SR, Uetrecht J, Shear NH. Idiosyncratic drug reactions: the reactive metabolite syndromes. Lancet. 2000;356:1587-1591.
PubMed DOI: 10.1016/S0140-6736(00)03137-8
17. Schnyder B, Mauri-Hellweg D, Zanni M, Bettens F, Pichler WJ. Direct, MHC-dependent presentation of the drug sulfamethoxazole to human alphabeta T cell clones. J Clin Invest. 1997;100:136-141.
PubMed
18. Zanni MP, von Greyerz S, Schnyder B, et al. HLA-restricted, processing- and metabolism-independent pathway of drug recognition by human alpha beta T lymphocytes. J Clin Invest. 1998;102:1591-1598.
PubMed
19. Zanni MP, von Greyerz S, Hari Y, Schnyder B, Pichler WJ. Recognition of local anesthetics by alphabeta+ T cells. J Invest Dermatol. 1999;112:197-204.
PubMed DOI: 10.1046/j.1523-1747.1999.00484.x
20. Zanni MP, Mauri-Hellweg D, Brander C, et al. Characterization of lidocaine-specific T cells. J Immunol. 1997;158:1139-1148.
PubMed
21. Sieben S, Kawakubo Y, Al Masaoudi T, Merk HF, Blomeke B. Delayed-type hypersensitivity reaction to paraphenylenediamine is mediated by 2 different pathways of antigen recognition by specific alphabeta human T-cell clones. J Allergy Clin Immunol. 2002;109:1005-1011.
PubMed DOI: 10.1067/mai.2002.123872
22. Depta JP, Altznauer F, Gamerdinger K, Burkhart C, Weltzien HU, Pichler WJ. Drug interaction with T-cell receptors: T-cell receptor density determines degree of cross-reactivity. J Allergy Clin Immunol. 2004;113:519-527.
PubMed DOI: 10.1016/j.jaci.2003.11.030
23. Cribb AE, Spielberg SP. Sulfamethoxazole is metabolized to the hydroxylamine in humans. Clin Pharmacol Ther. 1992;51:522-526.
PubMed
24. Naisbitt DJ, Vilar FJ, Stalford AC, Wilkins EG, Pirmohamed M, Park BK. Plasma cysteine deficiency and decreased reduction of nitrososulfamethoxazole with HIV infection. AIDS Res Hum Retroviruses. 2000;16:1929-1938.
PubMed DOI: 10.1089/088922200750054657
25. Schnyder B, Burkhart C, Schnyder-Frutig K, et al. Recognition of sulfamethoxazole and its reactive metabolites by drug-specific CD4+ T cells from allergic individuals. J Immunol. 2000;164:6647-6654.
PubMed
26. Burkhart C, von Greyerz S, Depta JP, et al. Influence of reduced glutathione on the proliferative response of sulfamethoxazole-specific and sulfamethoxazole-metabolite-specific human CD4+ T cells. Br J Pharmacol. 2001;132:623-630.
PubMed DOI: 10.1038/sj.bjp.0703845
27. Zanni MP, von Greyerz S, Schnyder B, Wendland T, Pichler WJ. Allele-unrestricted presentation of lidocaine by HLA-DR molecules to specific alphabeta+ T-cell clones. Int Immunol. 1998;10:507-515.
PubMed DOI: 10.1093/intimm/10.4.507
28. von Greyerz S, Bultemann G, Schnyder K, et al. Degeneracy and additional alloreactivity of drug-specific human alpha beta(+) T cell clones. Int Immunol. 2001;13:877-885.
PubMed DOI: 10.1093/intimm/13.7.877
29. Burkhart C, Britschgi M, Strasser I, et al. Non-covalent presentation of sulfamethoxazole to human CD4+ T cells is independent of distinct human leucocyte antigen-bound peptides. Clin Exp Allergy. 2002;32:1635-1643.
PubMed DOI: 10.1046/j.1365-2222.2002.01513.x
30. Pirmohamed M, Park BK. Cytochrome P450 enzyme polymorphisms and adverse drug reactions. Toxicology. 2003;192:23-32.
PubMed DOI: 10.1016/S0300-483X(03)00247-6
31. Alfirevic A, Stalford AC, Vilar FJ, Wilkins EG, Park BK, Pirmohamed M. Slow acetylator phenotype and genotype in HIV-positive patients with sulphamethoxazole hypersensitivity. Br J Clin Pharmacol. 2003;55:158-165.
PubMed DOI: 10.1046/j.1365-2125.2003.01754.x
32. Pirmohamed M, Lin K, Chadwick D, Park BK. TNFalpha promoter region gene polymorphisms in carbamazepine-hypersensitive patients. Neurology. 2001;56:890-896.
PubMed
33. Mallal S, Nolan D, Witt C, et al. Association between presence of HLA-B*5701, HLA-DR7, and HLA-DQ3 and hypersensitivity to HIV-1 reverse-transcriptase inhibitor abacavir. Lancet. 2002;359:727-732.
PubMed DOI: 10.1016/S0140-6736(02)07873-X
34. Martin AM, Nolan D, Gaudieri S, et al. Predisposition to abacavir hypersensitivity conferred by HLA-B*5701 and a haplotypic Hsp70-Hom variant. Proc Natl Acad Sci USA. 2004;101:4180-4185.
PubMed DOI: 10.1073/pnas.0307067101
35. Chung WH, Hung SI, Hong HS, et al. Medical genetics: a marker for Stevens-Johnson syndrome. Nature. 2004;428:486.
PubMed DOI: 10.1038/428486a
36. Hung SI, Chung WH, Liou LB, et al. HLA-B*5801 allele as a genetic marker for severe cutaneous adverse reactions caused by allopurinol. Proc Natl Acad Sci USA. 2005;102:4134-4139.
PubMed DOI: 10.1073/pnas.0409500102
37. Christiansen C, Pichler WJ, Skotland T. Delayed allergy-like reactions to X-ray contrast media: mechanistic considerations. Eur Radiol. 2000;10:1965-1975.
PubMed DOI: 10.1007/s003300000543
38. Christiansen C. Late-onset allergy-like reactions to X-ray contrast media. Curr Opin Allergy Clin Immunol. 2002;2:333-339.
PubMed DOI: 10.1097/00130832-200208000-00007
39. Pirmohamed M, Park BK. Genetic susceptibility to adverse drug reactions. Trends Pharmacol Sci. 2001;22:298-305.
PubMed DOI: 10.1016/S0165-6147(00)01717-X
40. Vollmer J, Weltzien HU, Dormoy A, Pistoor F, Moulon C. Functional expression and analysis of a human HLA-DQ restricted, nickel-reactive T-cell receptor in mouse hybridoma cells. J Invest Dermatol. 1999;113:175-181.
PubMed DOI: 10.1046/j.1523-1747.1999.00646.x
41. Pichler WJ. Pharmacological interaction of drugs with antigen-specific immune receptors: the p-i concept. Curr Opin Allergy Clin Immunol. 2002;2:301-305.
PubMed DOI: 10.1097/00130832-200208000-00003
42. Thierse HJ, Gamerdinger K, Junkes C, Guerreiro N, Weltzien HU. T-cell receptor (TCR) interaction with haptens: metal ions as non-classical haptens. Toxicology. 2005;209:101-107.
PubMed DOI: 10.1016/j.tox.2004.12.015
43. Stockl J, Majdic O, Fischer G, Maurer D, Knapp W. Monomorphic molecules function as additional recognition structures on haptenated target cells for HLA-A1-restricted, hapten-specific CTL. J Immunol. 2001;167:2724-2733.
PubMed
44. Beeley NR. Can peptides be mimicked? Drug Discov Today. 2000;5:354-363.
PubMed DOI: 10.1016/S1359-6446(00)01528-2
45. Mason D. A very high level of cross-reactivity is an essential feature of the T-cell receptor. Immunol Today. 1998;19:395-404.
PubMed DOI: 10.1016/S0167-5699(98)01299-7
46. Pirmohamed M, Park BK. HIV and drug allergy. Curr Opin Allergy Clin Immunol. 2001;1:311-316.
PubMed DOI: 10.1097/01.all.0000011032.69243.10
47. Engler OB, Strasser I, Naisbitt DJ, Cerny A, Pichler WJ. A chemically inert drug can stimulate T cells in vitro by their T-cell receptor in non-sensitized individuals. Toxicology. 2004;197:47-56.
PubMed DOI: 10.1016/j.tox.2003.12.008


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Relating nickel-induced tissue inflammation to nickel release in vivo.

Wataha JC; O'Dell NL; Singh BB; Ghazi M; Whitford GM; Lockwood PE
Department of Oral Rehabilitation, Medical College of Georgia School of Dentistry, Augusta, Georgia 30912-1260, USA. watahaj@mail.mcg.edu
Nickel has a number of adverse biological effects that have made the use of nickel in biomedical implants controversial. Yet information about the distribution of nickel in tissues around nickel-containing implants is scarce. The purpose of the current study was to use a laser ablation technique, combined with inductively coupled mass spectroscopy, to assess the spatial distribution of nickel around nickel-containing implants in vivo. Polyethylene, pure nickel wire, or a nickel-containing alloy (Ni-Cr) were implanted subcutaneously into rats for 7 days. The tissues were analyzed for Ni content and inflammation at 1-mm intervals up to 5 mm away from the implants. The sham surgery sites and the polyethylene caused mild to moderate inflammation 1-2 mm from the implant site with no detectable nickel in the tissue. The nickel wire caused severe inflammation up to 5 mm away from the implant site with necrosis for 1 mm around the implant. Nickel concentrations reached 48 microg/g near the implants, falling exponentially to undetectable levels at 3-4 mm from the implants. The Ni-Cr wire caused inflammation equivalent to polyethylene, with less than 4 microg/g of nickel present in the tissue for 1-2 mm around the implants. The current study showed that the laser-ablation technique was well suited for the analysis of soft tissues for metal-ion content, and that the nickel distribution in tissues correlated well with overt tissue inflammation.

Biocompatibility aspects of NiTi alloy components

Biocompatibility evaluation of nickel-titanium shape memory metal alloy:
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Chapter 2. Review of the literature
2.7. Biocompatibility aspects of NiTi alloy components
It is necessary to review the biocompatibility of NiTi alloy components for several reasons: 1) There is only little knowledge about the biocompatibility of NiTi. 2) Components may dissolve from NiTi due to corrosion. 3) Alloy components may form some compounds which have their own effects and toxicity. 4) Nickel may have deleterious effects. 5) Titanium may have some deleterious effects, especially in a particular form.
The corrosion resistance of the alloy and the toxicity of the individual metals that make up the alloy are the main determinants of biocompatibility. The properties and biocompatibility of NiTi have their own characteristics, which are different from those of nickel or titanium alone. Due to corrosion, however, nickel and titanium ions may dissolve from NiTi. To understand the possible host effects of NiTi, it is very important to understand the effects of its components. The local and systemic toxicity, carcinogenic effects, immune response, and teratogenic aspects of nickel will be reviewed in detail below. This matter is essential because of the high nickel content of the NiTi alloy. Titanium, the other component of NiTi, will be discussed briefly.
2.7.1. Nickel: absorption and elimination
Nickel is received into the body via the lungs, oral intake and skin. The average oral intake from the diet is estimated to be 150 microgram/person/day and may increase up to 900 micrograms/person/day or more (Flyvholm et al. 1984). Only a minor amount (1%) of the nickel from food is adsorbed into body from the intestine, but one fourth of the nickel from drinking water is adsorbed (Sunderman et al. 1989).
In blood, nickel is mainly bound to the albumin fraction, but also to many other proteins of serum (Nielsen et al. 1994). The serum and blood values vary within < 1-5 µg/l (Iyengar et al. 1994, Andreassi et al. 1998).
Most of the nickel is eliminated into urine (90%) and some into feces. The elimination half-life of nickel is quite rapid (Sunderman et al. 1989), but the elimination of different nickel compounds may be radically different (Oller et al. 1997).
2.7.2. Nickel in tissues
There has been great variation in the concentrations of nickel in human tissues reported in the literature. Standard reference values are still missing. The older methods of measurement and sample processing have involved many sources of error. There is also some variation in the concentrations between different animal species and humans due to metabolic and other factors. The suggested normal nickel concentrations in human tissues are (microgram/kg of dry weight): 173 in lung, 62 in kidney, 54 in heart, 50 in liver, 44 in brain, 37 in spleen and 34 in pancreas (Rezuke et al. 1987).
Increased nickel concentrations have been found in tissues adjacent to stainless steel implant materials (116 and 1200 mg/L) as well as in some distant organs (Michel et al. 1978, Bergman et al. 1980, Poehler 1983). The maximum rate of Ni release due to corrosion in patients with implants made of Ni alloys is estimated to be 20 g/kg/day (Black 1981). Infection may raise the peri-implant nickel concentrations (Hierholzer et al. 1984).
2.7.3. Nickel as an essential trace element
Nickel is one of the trace elements essential for vertebrates, including humans. Nickel deficiency in goats, rats and chicks has been found to have many deleterious effects and pathological consequences. These include general disorders, such as reduced growth, weight loss and increased perinatal mortality (Anke et al. 1984). Skin changes, including altered skin pigmentation, parakeratosis and uneven hair development, have been reported (Szilagyi et al. 1991).
Nickel deficiency impairs the metabolism of iron, fats, glucose, and glycogen. It may disturb the incorporation of calcium into the skeleton and decrease the length:width ratios of chick tibias and femurs. Animals with nickel deficiency have been found to have depressed activity of several enzymes in the heart, liver and kidneys as well as degeneration of cardiac and skeletal muscle (Szilagyi et al. 1991). Changes in the liver have also been reported. These include differences in the rough endoplasmic reticulum, decreased liver cholesterol and triacylglycerol accumulation (Nielsen et al. 1975, Nielsen et al. 1984, Stangl et al. 1996).
2.7.4. Toxicity and carcinogenicity of nickel
The chemical toxicity of metal inside the body is closely related to the concentration of released ions and wear particles, the toxicity of these elements and the toxicity of the formed compounds. Even a poisonous substance has no toxic effects in small concentrations, while nutritious substances cause adverse responses when present in excessive amounts. It is difficult to know the exact concentrations of metallic compounds released from implanted material, because there are many factors affecting them, such as implantation time and the local conditions (PH, fretting, etc.).
The high nickel content of NiTi (54 % by weight) may cause biocompatibility problems if deleterious amounts if nickel dissolve from it. The toxicity of nickel has been studied using in vitro and in vivo nickel salts, solid nickel or particulate form nickel (Putters et al. 1992, Takamura et al. 1994).
The problem with using metal salts is that the toxicity of different nickel salts vary notably. The benefit of this method is that we know the exact composition of the nickel salt, and it also permits the use of very high concentrations. The benefits and weaknesses of using nickel powder are that the particle itself may have toxic, irritating and even carcinogenic effects. This has been documented with alloys normally non-toxic, such as titanium (Zhang et al. 1998, Maloney et al. 1998). Another problem associated with reading in vitro results is that different cells have different toxic responses. The benefit of using solid nickel is that solid nickel in vitro usually correlates in situation in vivo, but we cannot be sure what kind of compounds have the effect we observe. The benefit of solid and particle material testing is that metal alloys can also be tested. Also, in vitro methods can never simulate the in vivo environment completely, and these results can only be considered suggestive.
Nickel is known to have toxic effects with cellular damage in cell cultures at high concentrations (Putters et al. 1992). It also appears to be harmful to bone in tissue cultures, but less so than cobalt or vanadium, which are also routinely used in implant alloys (Gerber et al. 1980). The toxicity of metal salts in cell cultures has shown decreasing toxicity in the order cobalt > vanadium > nickel > chromium > titanium > iron (Yamamoto et al. 1998). In vitro tests have also shown cobalt, nickel and chromium to have a potency for carcinogenicity.
Pure nickel implanted intramuscularly or inside bone has been found to cause severe local tissue irritation and necrosis (Laing et al. 1967) and to have high carcinogenic and toxic potencies. The tumors that retained nickel were malignant fibrous histiocytomas or fibrosarcomas (Takamura et al. 1994). Inhaled Ni3S2 caused adenomas and carcinomas of the lungs in rats, but nickel oxide and sulphate did not (Oller et al. 1997).
Due to the corrosion of the implants, small amounts of metal ions may also be released into distant organs. Systemic toxicity may be caused by the accumulation, processing, and subsequent reaction of the host to corrosion products (Bergman et al. 1980, Lugowski et al. 1991, Ishimatsu et al. 1995).
When high-dose nickel salts were injected into mice, accumulation and some deleterious effects were seen in the liver, kidney and spleen (Pereira et al. 1998).
We do not know what compounds form inside the body after the implantation of nickel-containing alloys. However, it is likely that NiCl and NiO compounds may form in the body environment, while the most toxic and carcinogenic compounds, e.g. Ni3S2, are not likely to occur. The underlying mechanism of the carcinogens of nickel is still unclear (Hartwig et al. 1994, Oller et al. 1997).
In vivo, Ni2+ ions may cross the cell membrane using the Mg2+ ion transport system. Since the concentration of Mg2+ inside and outside the cell is in the millimolar range, the levels of soluble nickel needed to compete with Mg2+ for its uptake must be at least in the millimolar range. Additionally, once Ni2+ is inside the cell, it binds to cytoplasmic ligands and it does not accumulate in the cell nucleus at the concentrations needed to have a genetic effect (Abbracchio et al. 1982a, Abbracchio et al. 1982b). In addition, soluble Ni2+ is rapidly cleared in vivo, which is why no direct efficient delivery of Ni2+ to the target site within the cell nucleus may occur to cause carcinogenic effects in vivo (Oller et al. 1997). Thus, carcinogenesis seems to be related to some nickel compounds rather than Ni2+ ions.
Another way in which nickel may be harmful is the effect of phagocytosed nickel compound particles. Some of the characteristics of nickel compounds that increase their ability to be endocytosed include crystalline nature, negative surface charge, 2–4 µm range particle size, and low solubility (Sunderman et al. 1987). Ni3S2 and NiO, which show otherwise low in vivo solubility may act by this mechanism (Dunnick et al. 1995). It was shown early on that endocytosis by target cells was likely to play an important role in the transforming potential of nickel compounds (Costa et al. 1980). When the nickel compound particles are endocytosed by the target cells, the endocytic vesicles are acidified by fusion with lysosomes and Ni2+ is released. Deleterious changes, such as the formation of oxygen radicals and DNA damage and the inactivation of tumor supressor genes, may occur (Klein et al. 1991a, Klein et al. 1991b).
Pathological alterations of nickel metabolism have been recognized in several human diseases. The diverse clinical manifestations of nickel toxicology include (1) acute pneumonitis from inhalation of nickel carbonyl, (2) chronic rhinitis and sinusitis from inhalation of nickel aerosols, (3) cancers of nasal cavities and lungs in nickel workers, and (4) dermatitis and other hypersensitive reactions from cutaneous and parental exposures to nickel alloys (Sunderman 1977).
2.7.5. Nickel-containing biomaterial alloys in humans
Neoplasms associated with clinical implants are very rare. They may be related more to the physical than the chemical configuration of the implant. The mechanism of tumor formation is not understood, but it appears to be related to the implant fibrous capsule (Schoen 1996). Occasional reports on humans have been published, which report the development of malignant fibrous histiocytomas and osteosarcomas at the site of a prosthetic replacement or previous internal fixation. Most of these (> 80%) have been related to the cobalt-chromium alloy, some to stainless steel or other nickel-containing alloys, and none to titanium (Rock 1998).
The low toxicity of a constituent does not exclude the possibility of deleterious effects. As local or systemic toxicity is usually dose-dependent, reactions caused by the immune response may activate at much lower thresholds (Remes et al. 1992).
Nickel is the major cause of allergic contact dermatitis (Peltonen 1979). Epidemiological studies have shown a sensitization frequency up to 20 % in young females and 10 % in the elderly (Menne 1996). Two to four percent of males are sensitized. Most cases of nickel allergy may be related to skin contact with nickel-containing metallic items. The significant biological parameter is not the nickel concentration in the alloy or the coating, but the amount released to the skin during exposure to human sweat. A threshold of 0.5 microgram/cm2/week has been established, at which only a minor part of nickel-sensitive subjects will react (Menne 1996).
When implants containing perceptible amounts of nickel, for example, stainless steel implants (nickel content 10-14 %), are clinically used inside the body, no sensitization or immune disorders commonly occur (Christensen 1990, Gawkrodger 1993). Why could it be used even in patients with nickel contact dermatitis?
Allergic contact dermatitis is a cell-mediated immune response caused by Ni2+ ions. In fact, the nickel ion itself is too small to act as an antigen. It binds with a carrier protein and acts as a hapten. The nickel-protein complex activates Langerhans’ cells in the skin, which presents an antigen to T-lymphocytes. Memory T-cells develop. When circulating in the body, these memory cells are able to start cell-mediated immune reactions upon meeting the same allergen again.
The antigenic determinants created by nickel as well as the mechanisms of recognition by specific T-cell clones have not been elucidated (Moulon et al. 1995). T-cells detect haptens as structural entities attached covalently or by complexion to self-peptides anchored in the binding grooves of major histocompatibility antigens (MHC proteins) (Weltzien et al. 1996).
Two major types of hapten-specific T-cell receptors have been identified: one reacting to hapten regardless of the chemical composition of the carrier peptide, and the other contacting hapten and peptide via two apparently independent contact sites (Martin et al. 1994). The present study suggests that the presence of specific CD8+ T-cells and a distinct pattern of cytokine release (e.g. augmented production of interleukin-10) by CD4+ T-cells may be important elements in determining whether a hapten induces allergy or a silent immune response (Cavani et al. 1998). T lymphocytes are critical effectors in the pathogenesis of contact hypersensitivity. Nickel-specific CD4+ T helper cells have been extensively characterized. The characterization of nickel-specific cytotoxic CD8+ T-cells with different requirements for nickel-specific target lysis may have important implications for the development or control of human contact hypersensitivity reactions to nickel in vivo (Moulon et al. 1998).
The intercellular adhesion molecule-1 (ICAM-1), the vascular cell adhesion molecule-1 (VCAM-1), and the endothelial leukocyte adhesion molecule-1 (ELAM-1, E-selectin) are endothelial surface molecules that play a role in leukocyte recruitment to sites of inflammation during, for instance, contact hypersensitivity. NiCl2 and, to a lesser extent, CoCl2 were found to up-regulate ICAM-1, VCAM-1, and ELAM-1 expression on cultured human umbilical vein endothelium. Both Ni2+ and Co2+ , which frequently induce simultaneous contact sensitivity, have the ability to directly up-regulate endothelial adhesion molecules. This shared property may represent an adjuvant mechanism that promotes sensitization and elicitation events in contact hypersensitivity to these haptens (Goebeler et al. 1993). It was observed recently that Ni ions can either promote or suppress the expression of the intercellular adhesion molecule 1 (ICAM-1) on endothelial cells, depending on their concentration and probably the time of exposure. ICAM-1 is known to be involved in the recruitment of inflammatory cells from the bloodstream. Ni ions could promote the expression of ICAM-1 at concentrations high enough to suppress cell metabolic activity. At lower concentrations, they suppress ICAM expression (Wataha et al. 1997).
Control of the allergic reaction also requires inhibitory systems which prevent the immune response from causing systemic damage. To control the reactions, several kinds of suppressor T-cells are generated at different levels (Barnetson et al. 1993). Unresponsiveness to oral exposure (oral tolerance) to nickel is due the action of these suppressor cells (van Hoogstraten et al. 1992, Ishii et al. 1993). This is also the presumptive explanation for why sensitization and immune disorders from metallic prostheses are very unusual, although, for example, the stainless steel used in implants contains perceptible amounts of nickel (Bjurholm et al. 1990, Gawkrodger 1993, Milavec-Puretic et al. 1998).
2.7.6. Titanium
It is generally accepted that pure titanium is extremely well tolerated by local tissues and induces neither toxic nor inflammatory reactions (Branemark et al. 1969, Toth et al. 1985, Linder et al. 1988, Pfeiffer et al. 1994). The normal tissue concentration of titanium in humans is 0.2 ppm. Around the titanium implants no clinical tissue toxicity has been observed even at local concentrations higher than 2000 ppm (Hildebrand et al. 1998). In optimal situations, titanium is able to osseointegrate with bone, thus forming a direct contact with bone at the light microscopy level (Branemark et al. 1969). The good bone contact may be due to the ability of titanium to form a Ca-P rich layer on its surface (Hanawa 1991). Titanium is bacteriostatic (Elagli et al. 1992) and does not significantly activate or inhibit different enzyme systems specific to toxic reactions, e.g. β - glucuronidase, lactate dehydrogenase, glucose-6-phosphate dehydrogenase and acid phosphatase (Elagli et al. 1995). The good biocompatibility and corrosion resistance are due to the naturally forming stable titanium oxide (TiO2) film on titanium surfaces (Zitter et al. 1987, Kasemo et al. 1991).
Particles from titanium arise from the passivation layer of the implant, but they are not titanium ions, but mostly insoluble titanium oxides or suboxides, which are recognized to be biologically inert. Indeed, the passivation layer is immediately reformed after abrasion because of the high oxidizability of titanium. This behavior protects the alloy and prevents the formation of chemical compounds other than oxides (Hildebrand et al. 1998). Tissue discoloration due to titanium oxide particles is sometimes seen around pure titanium implants, but this seems to have no clinical consequences (Onodera et al. 1993, Rosenberg et al. 1993). Experiments with laboratory animals and some limited analyses of human tissues have also revealed evidence of titanium release into distant tissues (Schliephake et al. 1993, Jorgenson et al. 1997).
Wear particles produced by abrasion appear especially in the vicinity of articular prostheses and implants with certain mobility, e.g. uncemented total hip replacements. These particles may induce multiple tissue reactions, including osteolysis, degradation of normal bone structure, severe macrophagic reactions, granuloma, fibrotic capsules and chronic inflammation, which may cause destabilization and loosening of prostheses and implants (Santavirta et al. 1991, Santavirta et al. 1993, Rubash et al. 1998). Particle size and composition are of essential importance in that process. Deleterious reactions have been reported with Ti-6Al-4V based prostheses (Nasser et al. 1990, Rubash et al. 1998), but not with pure titanium implants.
In vitro, pure titanium particles have also been shown to have some effects on cells. Low concentrations may stimulate fibroblast proliferation, while high concentrations may be toxic. At high particle concentrations, titanium caused a decrease in proteolytic and collagenolytic activity in the culture medium. Titanium also elevated the lysosomal enzyme marker, hexosaminidase, except at high concentrations (Maloney et al. 1993).
Titanium ostheosynthesis plates have been observed to be totally recovered by newly formed bone tissue after an exposure period of 3-4 years. The retrieval of such implants becomes particularly difficult (Hildebrand et al. 1998).

IMMUNOTOXICOLOGY

Joseph G. Vos, Henk van Loveren
The functions of the immune system are to protect the body from invading infectious agents and to provide immune surveillance against arising tumour cells. It has a first line of defence that is non-specific and that can initiate effector reactions itself, and an acquired specific branch, in which lymphocytes and antibodies carry the specificity of recognition and subsequent reactivity towards the antigen.Immunotoxicology has been defined as “the discipline concerned with the study of the events that can lead to undesired effects as a result of interaction of xenobiotics with the immune system. These undesired events may result as a consequence of (1) a direct and/or indirect effect of the xenobiotic (and/or its biotransformation product) on the immune system, or (2) an immunologically based host response to the compound and/or its metabolite(s), or host antigens modified by the compound or its metabolites” (Berlin et al. 1987).When the immune system acts as a passive target of chemical insults, the result can be decreased resistance to infection and certain forms of neoplasia, or immune disregulation/stimulation that can exacerbate allergy or auto-immunity. In the case that the immune system responds to the antigenic specificity of the xenobiotic or host antigen modified by the compound, toxicity can become manifest as allergies or autoimmune diseases.Animal models to investigate chemical-induced immune suppression have been developed, and a number of these methods are validated (Burleson, Munson, and Dean 1995; IPCS 1996). For testing purposes, a tiered approach is followed to make an adequate selection from the overwhelming number of assays available. Generally, the objective of the first tier is to identify potential immunotoxicants. If potential immunotoxicity is identified, a second tier of testing is performed to confirm and characterize further the changes observed. Third-tier investigations include special studies on the mechanism of action of the compound. Several xenobiotics have been identified as immunotoxicants causing immunosuppression in such studies with laboratory animals.The database on immune function disturbances in humans by environmental chemicals is limited (Descotes 1986; NRC Subcommittee on Immunotoxicology 1992). The use of markers of immunotoxicity has received little attention in clinical and epidemiological studies to investigate the effect of these chemicals on human health. Such studies have not been performed frequently, and their interpretation often does not permit unequivocal conclusions to be drawn, due for instance to the uncontrolled nature of exposure. Therefore, at present, immunotoxicity assessment in rodents, with subsequent extrapolation to man, forms the basis of decisions regarding hazard and risk.Hypersensitivity reactions, notably allergic asthma and contact dermatitis, are important occupational health problems in industrialized countries (Vos, Younes and Smith 1995). The phenomenon of contact sensitization was investigated first in the guinea pig (Andersen and Maibach 1985). Until recently this has been the species of choice for predictive testing. Many guinea pig test methods are available, the most frequently employed being the guinea pig maximization test and the occluded patch test of Buehler. Guinea pig tests and newer approaches developed in mice, such as ear swelling tests and the local lymph node assay, provide the toxicologist with the tools to assess skin sensitization hazard. The situation with respect to sensitization of the respiratory tract is very different. There are, as yet, no well-validated or widely accepted methods available for the identification of chemical respiratory allergens although progress in the development of animal models for the investigation of chemical respiratory allergy has been achieved in the guinea pig and mouse.Human data show that chemical agents, in particular drugs, can cause autoimmune diseases (Kammüller, Bloksma and Seinen 1989). There are a number of experimental animal models of human autoimmune diseases. Such comprise both spontaneous pathology (for example systemic lupus erythematosus in New Zealand Black mice) and autoimmune phenomena induced by experimental immunization with a cross-reactive autoantigen (for example the H37Ra adjuvant induced arthritis in Lewis strain rats). These models are applied in the preclinical evaluation of immunosuppressive drugs. Very few studies have addressed the potential of these models for assessment of whether a xenobiotic exacerbates induced or congenital autoimmunity. Animal models that are suitable to investigate the ability of chemicals to induce autoimmune diseases are virtually lacking. One model that is used to a limited extent is the popliteal lymph node assay in mice. Like the situation in humans, genetic factors play a crucial role in the development of autoimmune disease (AD) in laboratory animals, which will limit the predictive value of such tests.
The Immune System
The major function of the immune system is defence against bacteria, viruses, parasites, fungi and neoplastic cells. This is achieved by the actions of various cell types and their soluble mediators in a finely tuned concert. The host defence can be roughly divided into non-specific or innate resistance and specific or acquired immunity mediated by lymphocytes (Roitt, Brostoff and Male 1989).Components of the immune system are present throughout the body (Jones et al. 1990). The lymphocyte compartment is found within lymphoid organs (figure 33.14). The bone marrow and thymus are classified as primary or central lymphoid organs; the secondary or peripheral lymphoid organs include lymph nodes, spleen and lymphoid tissue along secretory surfaces such as the gastrointestinal and respiratory tracts, the so-called mucosa-associated lymphoid tissue (MALT). About half of the body's lymphocytes are located at any one time in MALT. In addition the skin is an important organ for the induction of immune responses to antigens present on the skin. Important in this process are epidermal Langerhans cells that have an antigen-presenting function. ___________________________________________________________________________
Figure 33.14 Primary and secondary lymphoid organs and tissues

___________________________________________________________________________
Phagocytic cells of the monocyte/macrophage lineage, called the mononuclear phagocyte system (MPS), occur in lymphoid organs and also at extranodal sites; the extranodal phagocytes include Kupffer cells in the liver, alveolar macrophages in the lung, mesangial macrophages in the kidney and glial cells in the brain. Polymorphonuclear leukocytes (PMNs) are present mainly in blood and bone marrow, but accumulate at sites of inflammation.
Non-specific defence
A first line of defence to micro-organisms is executed by a physical and chemical barrier, such as at the skin, the respiratory tract and the alimentary tract. This barrier is helped by non-specific protective mechanisms including phagocytic cells, such as macrophages and polymorphonuclear leukocytes, which are able to kill pathogens, and natural killer cells, which can lyse tumour cells and virus-infected cells. The complement system and certain microbial inhibitors (e.g., lysozyme) also take part in the non-specific response.
Specific immunity
After initial contact of the host with the pathogen, specific immune responses are induced. The hallmark of this second line of defence is specific recognition of determinants, so-called antigens or epitopes, of the pathogens by receptors on the cell surface of B- and T-lymphocytes. Following interaction with the specific antigen, the receptor-bearing cell is stimulated to undergo proliferation and differentiation, producing a clone of progeny cells that are specific for the eliciting antigen. The specific immune responses help the non-specific defence presented to the pathogens by stimulating the efficacy of the non-specific responses. A fundamental characteristic of specific immunity is that memory develops. Secondary contact with the same antigen provokes a faster and more vigorous but well-regulated response.The genome does not have the capacity to carry the codes of an array of antigen receptors sufficient to recognize the number of antigens that can be encountered. The repertoire of specificity develops by a process of gene rearrangements. This is a random process, during which various specificities are brought about. This includes specificities for self components, which are undesirable. A selection process that takes place in the thymus (T cells), or bone marrow (B cells) operates to delete these undesirable specificities.Normal immune effector function and homeostatic regulation of the immune response is dependent upon a variety of soluble products, known collectively as cytokines, which are synthesized and secreted by lymphocytes and by other cell types. Cytokines have pleiotropic effects on immune and inflammatory responses. Cooperation between different cell populations is required for the immune response-the regulation of antibody responses, the accumulation of immune cells and molecules at inflammatory sites, the initiation of acute phase responses, the control of macrophage cytotoxic function and many other processes central to host resistance. These are influenced by, and in many cases are dependent upon, cytokines acting individually or in concert.Two arms of specific immunity are recognized-humoral immunity and cell-mediated or cellular immunity:Humoral immunity. In the humoral arm B-lymphocytes are stimulated following recognition of antigen by cell-surface receptors. Antigen receptors on B-lymphocytes are immunoglobulins (Ig). Mature B cells (plasma cells) start the production of antigen-specific immunoglobulins that act as antibodies in serum or along mucosal surfaces. There are five major classes of immunoglobulins: (1) IgM, pentameric Ig with optimal agglutinating capacity, which is first produced after antigenic stimulation; (2) IgG, the main Ig in circulation, which can pass the placenta; (3) IgA, secretory Ig for the protection of mucosal surfaces; (4) IgE, Ig fixing to mast cells or basophilic granulocytes involved in immediate hypersensitivity reactions and (5) IgD, whose major function is as a receptor on B-lymphocytes.Cell-mediated immunity. The cellular arm of the specific immune system is mediated by T-lymphocytes. These cells also have antigen receptors on their membranes. They recognize antigen if presented by antigen presenting cells in the context of histocompatibility antigens. Hence, these cells have a restriction in addition to the antigen specificity. T cells function as helper cells for various (including humoral) immune responses, mediate recruitment of inflammatory cells, and can, as cytotoxic T cells, kill target cells after antigen-specific recognition.
Mechanisms of ImmunotoxicityImmunosuppression
Effective host resistance is dependent upon the functional integrity of the immune system, which in turn requires that the component cells and molecules which orchestrate immune responses are available in sufficient numbers and in an operational form. Congenital immunodeficiencies in humans are often characterized by defects in certain stem cell lines, resulting in impaired or absent production of immune cells. By analogy with congenital and acquired human immunodeficiency diseases, chemical-induced immunosuppression may result simply from a reduced number of functional cells (IPCS 1996). The absence, or reduced numbers, of lymphocytes may have more or less profound effects on immune status. Some immunodeficiency states and severe immunosuppression, as can occur in transplantation or cytostatic therapy, have been associated in particular with increased incidences of opportunistic infections and of certain neoplastic diseases. The infections can be bacterial, viral, fungal or protozoan, and the predominant type of infection depends on the associated immunodeficiency. Exposure to immunosuppressive environmental chemicals may be expected to result in more subtle forms of immunosuppression, which may be difficult to detect. These may lead, for example, to an increased incidence of infections such as influenza or the common cold.In view of the complexity of the immune system, with the wide variety of cells, mediators and functions that form a complicated and interactive network, immunotoxic compounds have numerous opportunities to exert an effect. Although the nature of the initial lesions induced by many immunotoxic chemicals have not yet been elucidated, there is increasing information available, mostly derived from studies in laboratory animals, regarding the immunobiological changes which result in depression of immune function (Dean et al. 1994). Toxic effects might occur at the following critical functions (and some examples are given of immunotoxic compounds affecting these functions):
· development and expansion of different stem cell populations (benzene exerts immunotoxic effects at the stem cell level, causing lymphocytopenia)
· proliferation of various lymphoid and myeloid cells as well as supportive tissues in which these cells mature and function (immunotoxic organotin compounds suppress the proliferative activity of lymphocytes in the thymic cortex through direct cytotoxicity; the thymotoxic action of 2,3,7,8-tetrachloro-dibenzo-p-dioxin (TCDD) and related compounds is likely due to an impaired function of thymic epithelial cells, rather than to direct toxicity for thymocytes)
· antigen uptake, processing and presentation by macrophages and other antigen-presenting cells (one of the targets of 7,12-dimethylbenz(a)anthracene (DMBA) and of lead is antigen presentation by macrophages; a target of ultraviolet radiation is the antigen-presenting Langerhans cell)
· regulatory function of T-helper and T-suppressor cells (T-helper cell function is impaired by organotins, aldicarb, polychlorinated biphenyls (PCBs), TCDD and DMBA; T-suppressor cell function is reduced by low-dose cyclophosphamide treatment)
· production of various cytokines or interleukins (benzo(a)pyrene (BP) suppresses interleukin-1 production; ultraviolet radiation alters production of cytokines by keratinocytes)
· synthesis of various classes of immunoglobulins IgM and IgG is suppressed following PCB and tributyltin oxide (TBT) treatment, and increased after hexachlorobenzene (HCB) exposure).
· complement regulation and activation (affected by TCDD)
· cytotoxic T cell function (3-methylcholanthrene (3-MC), DMBA, and TCDD suppress cytotoxic T cell activity)
· natural killer (NK) cell function (pulmonary NK activity is suppressed by ozone; splenic NK activity is impaired by nickel)
· macrophage and polymorphonuclear leukocyte chemotaxis and cytotoxic functions (ozone and nitrogen dioxide impair the phagocytic activity of alveolar macrophages).
Allergy
Allergy may be defined as the adverse health effects which result from the induction and elicitation of specific immune responses. When hypersensitivity reactions occur without involvement of the immune system the term pseudo-allergy is used. In the context of immunotoxicology, allergy results from a specific immune response to chemicals and drugs that are of interest. The ability of a chemical to sensitize individuals is generally related to its ability to bind covalently to body proteins. Allergic reactions may take a variety of forms and these differ with respect to both the underlying immunological mechanisms and the speed of the reaction. Four major types of allergic reactions have been recognized: Type I hypersensitivity reactions, which are effectuated by IgE antibody and where symptoms are manifest within minutes of exposure of the sensitized individual. Type II hypersensitivity reactions result from the damage or destruction of host cells by antibody. In this case symptoms become apparent within hours. Type III hypersensitivity, or Arthus, reactions are also antibody mediated, but against soluble antigen, and result from the local or systemic action of immune complexes. Type IV, or delayed-type hypersensitivity, reactions are effected by T-lymphocytes and normally symptoms develop 24 to 48 hours following exposure of the sensitized individual.The two types of chemical allergy of greatest relevance to occupational health are contact sensitivity or skin allergy and allergy of the respiratory tract.Contact hypersensitivity. A large number of chemicals are able to cause skin sensitization. Following topical exposure of a susceptible individual to a chemical allergen, a T-lymphocyte response is induced in the draining lymph nodes. In the skin the allergen interacts directly or indirectly with epidermal Langerhans cells, which transport the chemical to the lymph nodes and present it in an immunogenic form to responsive T-lymphocytes. Allergen-activated T-lymphocytes proliferate, resulting in clonal expansion. The individual is now sensitized and will respond to a second dermal exposure to the same chemical with a more aggressive immune response, resulting in allergic contact dermatitis. The cutaneous inflammatory reaction which characterizes allergic contact dermatitis is secondary to the recognition of the allergen in the skin by specific T-lymphocytes. These lymphocytes become activated, release cytokines and cause the local accumulation of other mononuclear leukocytes. Symptoms develop some 24 to 48 hours following exposure of the sensitized individual, and allergic contact dermatitis therefore represents a form of delayed-type hypersensitivity. Common causes of allergic contact dermatitis include organic chemicals (such as 2,4-dinitrochlorobenzene), metals (such as nickel and chromium) and plant products (such as urushiol from poison ivy).Respiratory hypersensitivity. Respiratory hypersensitivity is usually considered to be a Type I hypersensitivity reaction. However, late phase reactions and the more chronic symptoms associated with asthma may involve cell-mediated (Type IV) immune processes. The acute symptoms associated with respiratory allergy are effected by IgE antibody, the production of which is provoked following exposure of the susceptible individual to the inducing chemical allergen. The IgE antibody distributes systemically and binds, via membrane receptors, to mast cells which are found in vascularized tissues, including the respiratory tract. Following inhalation of the same chemical a respiratory hypersensitivity reaction will be elicited. Allergen associates with protein and binds to, and cross-links, IgE antibody bound to mast cells. This in turn causes the degranulation of mast cells and the release of inflammatory mediators such as histamine and leukotrienes. Such mediators cause bronchoconstriction and vasodilation, resulting in the symptoms of respiratory allergy; asthma and/or rhinitis. Chemicals known to cause respiratory hypersensitivity in man include acid anhydrides (such as trimellitic anhydride), some diisocyanates (such as toluene diisocyanate), platinum salts and some reactive dyes. Also, chronic exposure to beryllium is known to cause hypersensitivity lung disease.
Autoimmunity
Autoimmunity can be defined as the stimulation of specific immune responses directed against endogenous “self” antigens. Induced autoimmunity can result either from alterations in the balance of regulatory T-lymphocytes or from the association of a xenobiotic with normal tissue components such as to render them immunogenic (“altered self”). Drugs and chemicals known to incidentally induce or exacerbate effects like those of autoimmune disease (AD) in susceptible individuals are low molecular weight compounds (molecular weight 100 to 500) that are generally considered to be not immunogenic themselves. The mechanism of AD by chemical exposure is mostly unknown. Disease can be produced directly by means of circulating antibody, indirectly through the formation of immune complexes, or as a consequence of cell-mediated immunity, but likely occurs through a combination of mechanisms. The pathogenesis is best known in immune haemolytic disorders induced by drugs:
· The drug can attach to the red-cell membrane and interact with a drug-specific antibody.
· The drug can alter the red-cell membrane so that the immune system regards the cell as foreign.
· The drug and its specific antibody form immune complexes that adhere to the red-cell membrane to produce injury.
· Red-cell sensitization occurs due to the production of red-cell autoantibody.A variety of chemicals and drugs, in particular the latter, have been found to induce autoimmune-like responses (Kamüller, Bloksma and Seinen 1989). Occupational exposure to chemicals may incidentally lead to AD-like syndromes. Exposure to monomeric vinyl chloride, trichloroethylene, perchloroethylene, epoxy resins and silica dust may induce scleroderma-like syndromes. A syndrome similar to systemic lupus erythematosus (SLE) has been described after exposure to hydrazine. Exposure to toluene diisocyanate has been associated with the induction of thrombocytopenic purpura. Heavy metals such as mercury have been implicated in some cases of immune complex glomerulonephritis.
Human Risk Assessment
The assessment of human immune status is performed mainly using peripheral blood for analysis of humoral substances like immunoglobulins and complement, and of blood leukocytes for subset composition and functionality of subpopulations. These methods are usually the same as those used to investigate humoral and cell-mediated immunity as well as nonspecific resistance of patients with suspected congenital immunodeficiency disease. For epidemiological studies (e.g., of occupationally exposed populations) parameters should be selected on the basis of their predictive value in human populations, validated animal models, and the underlying biology of the markers (see table 33.6). The strategy in screening for immunotoxic effects after (accidental) exposure to environmental pollutants or other toxicants is much dependent on circumstances, such as type of immunodeficiency to be expected, time between exposure and immune status assessment, degree of exposure and number of exposed individuals. The process of assessing the immunotoxic risk of a particular xenobiotic in humans is extremely difficult and often impossible, due largely to the presence of various confounding factors of endogenous or exogenous origin that influence the response of individuals to toxic damage. This is particularly true for studies which investigate the role of chemical exposure in autoimmune diseases, where genetic factors play a crucial role. ___________________________________________________________________________
Table 33.6 Classification of tests for immune markers

Test category
Characteristics
Specific tests
Basic-generalShould be included with general panels
Indicators of general health and organ system status
Blood urea nitrogen, blood glucose, etc.
Basic-immuneShould be included with general panels
General indicators of immune statusRelatively low costAssay methods are standardized among laboratoriesResults outside reference ranges are clinically interpretable
Complete blood countsSerum IgG, IgA, IgM levelsSurface marker phenotypes for major lymphocyte subsets
Focused/reflex Should be included when indicated by clinical findings, suspected exposures, or prior test results
Indicators of specific immune functions/eventsCost variesAssay methods are standardized among laboratoriesResults outside reference ranges are clinically interpretable
Histocompatibility genotypeAntibodies to infectious agentsTotal serum IgEAllergen-specific IgEAutoantibodiesSkin tests for hypersensitivityGranulocyte oxidative burstHistopathology (tissue biopsy)
Research Should be included only with control populations and careful study design
Indicators of general or specific immune functions/eventsCost varies; often expensiveAssay methods are usually not standardized among laboratoriesResults outside reference ranges are often not clinically interpretable
In vitro stimulation assaysCell activation surface markersCytokine serum concentrationsClonality assays (antibody, cellular, genetic)Cytotoxicity tests ___________________________________________________________________________ As adequate human data are seldom available, the assessment of risk for chemical-induced immunosuppression in humans is in the majority of cases based upon animal studies. The identification of potential immunotoxic xenobiotics is undertaken primarily in controlled studies in rodents. In vivo exposure studies present, in this regard, the optimal approach to estimate the immunotoxic potential of a compound. This is due to the multifactoral and complex nature of the immune system and of immune responses. In vitro studies are of increasing value in the elucidation of mechanisms of immunotoxicity. In addition, by investigating the effects of the compound using cells of animal and human origin, data can be generated for species comparison, which can be used in the “parallelogram” approach to improve the risk assessment process. If data are available for three cornerstones of the parallelogram (in vivo animal, and in vitro animal and human) it may be easier to predict the outcome at the remaining cornerstone, that is, the risk in humans.When assessment of risk for chemical-induced immunosuppression has to rely solely upon data from animal studies, an approach can be followed in the extrapolation to man by the application of uncertainty factors to the no observed adverse effect level (NOAEL). This level can be based on parameters determined in relevant models, such as host resistance assays and in vivo assessment of hypersensitivity reactions and antibody production. Ideally, the relevance of this approach to risk assessment requires confirmation by studies in humans. Such studies should combine the identification and measurement of the toxicant, epidemiological data and immune status assessments.To predict contact hypersensitivity, guinea pig models are available and have been used in risk assessment since the 1970s. Although sensitive and reproducible, these tests have limitations as they depend on subjective evaluation; this can be overcome by newer and more quantitative methods developed in the mouse. Regarding chemical-induced hypersensitivity induced by inhalation or ingestion of allergens, tests should be developed and evaluated in terms of their predictive value in man. When it comes to setting safe occupational exposure levels of potential allergens, consideration has to be given to the biphasic nature of allergy: the sensitization phase and the elicitation phase. The concentration required to elicit an allergic reaction in a previously sensitized individual is considerably lower than the concentration necessary to induce sensitization in the immunologically naïve but susceptible individual.As animal models to predict chemical-induced autoimmunity are virtually lacking, emphasis should be given to the development of such models. For the development of such models, our knowledge of chemical-induced autoimmunity in humans should be advanced, including the study of genetic and immune system markers to identify susceptible individuals. Humans that are exposed to drugs that induce autoimmunity offer such an opportunity.