Mercury as a hapten: A review of the role of toxicant-induced brain autoantibodies in autism and possible treatment considerations.

BACKGROUND: Mercury has many direct and well-recognized neurotoxic effects. However, its immune effects causing secondary neurotoxicity are less well-recognized. Mercury exposure can induce immunologic changes in the brain indicative of autoimmune dysfunction, including the production of highly specific brain autoantibodies. Mercury, and in particular, Thimerosal, can combine with a larger carrier, such as an endogenous protein, thereby acting as a hapten, and this new molecule can then elicit the production of antibodies. METHODS: A comprehensive search using PubMed and Google Scholar for original studies and reviews related to autism, mercury, autoantibodies, autoimmune dysfunction, and haptens was undertaken. All articles providing relevant information from 1985 to date were examined. Twenty-three studies were identified showing autoantibodies in the brains of individuals diagnosed with autism and all were included and discussed in this review. RESULTS: Research shows mercury exposure can result in an autoimmune reaction that may be causal or contributory to autism, especially in children with a family history of autoimmunity. The autoimmune pathogenesis in autism is demonstrated by the presence of brain autoantibodies (neuroantibodies), which include autoantibodies to: (1) human neuronal progenitor cells; (2) myelin basic protein (MBP); (3) neuron-axon filament protein (NAFP); (4) brain endothelial cells; (5) serotonin receptors; (6) glial fibrillary acidic protein (GFAP); (7) brain derived neurotrophic factor (BDNF); (8) myelin associated glycoprotein (MAG); and (9) various brain proteins in the cerebellum, hypothalamus, prefrontal cortex, cingulate gyrus, caudate putamen, cerebral cortex and caudate nucleus. CONCLUSION: Recent evidence suggests a relationship between mercury exposure and brain autoantibodies in individuals diagnosed with autism. Moreover, brain autoantibody levels in autism are found to correlate with both autism severity and blood mercury levels. Treatments to reduce mercury levels and/or brain autoantibody formation should be considered in autism.

Altered antigenicity and immunogenicity of human papillomavirus virus-like particles in the presence of thimerosal.

Thimerosal has been widely used as a preservative in human vaccines for decades. Thimerosal, a thiol capping agent with ethyl mercury being the active degradant, could have impacts on the vaccine potency due to potential thiol modification. The effects on the antigenicity and immunogenicity of human papillomavirus (HPV) virus-like particles (VLPs) in the presence of thimerosal was studied. In general, reduced binding activity was observed between HPV antigens and monoclonal antibodies (mAbs) upon thimerosal treatment, accompanied by reduced protein conformational stability. The immunogenicity of a pentavalent vaccine formulation (HPV6, HPV11, HPV16, HPV18 and hepatitis E virus) with or without thimerosal was studied in mice. The functional antibody titres, as well as the binding titres, were determined, showing a substantial decrease for vaccine formulations containing thimerosal for HPV16/18. Similarly, epitope-specific competition assays using specific and functional mAbs as tracers also showed a significant reduction in immunogenicity for HPV16/18 in the presence of thimerosal. Structural alterations in the capsid protein for HPV18 were observed with cryo-electron microscopy and 3-dimensional reconstruction in the comparative structural analysis. The results should alert scientists in formulation development field on the choice for vaccine preservatives, in particular for thiol-containing antigens.

Prospective Single-Center Observational Study of the Allergenic Potential of Mercromina Film and Other Common Antiseptics in Patients With Contact Dermatitis. / Estudio observacional de seguridad, prospectivo y unicéntrico para determinar la capacidad alergogénica de Mercromina Film® y otros antisépticos de uso común en pacientes con dermatitis de contacto.

INTRODUCTION: Although Mercromina Film and other topical antiseptics are widely used, they are not included in the standard series recommended by the Spanish Contact Dermatitis and Skin Allergy Research Group for testing suspected allergic contact dermatitis (ACD). Furthermore, no recent studies have investigated the allergenic potential of merbromin. OBJECTIVE: To determine the allergenic potential of merbromin and compare it with that of other topical antiseptics widely used in clinical practice, including povidone-iodine, chlorhexidine, and eosin. MATERIAL AND METHODS: Prospective single-center observational safety study of 105 patients with suspected ACD seen at the dermatology department of our hospital. RESULTS: Of the 105 patients studied, 1.9% had a positive patch test to merbromin and 12.4% were sensitized to povidone-iodine. The differences in the proportion of patients with ACD to Betadine Solución Dérmica (povidone-iodine) compared with the rest of the antiseptics was statistically significant (McNemar test, P<.05). No adverse reactions were observed in any of the patients. CONCLUSIONS: Based on the patch tests conducted, Mercromina Film has very low allergenic potential. The highest allergenic potential was observed for povidone-iodine.

The patterns and clinical relevance of contact allergen sensitization in a pediatric population with atopic dermatitis.

BACKGROUND/AIM: Data about contact allergen sensitization (CAS) in children with atopic dermatitis (AD) are limited. The purpose of this study was to identify the frequency and patterns of CAS in children with AD by using a ready-to-use patch test system. MATERIALS AND METHODS: After receiving the history of CAS in the patients, the severity of AD and IgE-mediated allergen sensitization were determined. RESULTS: Of 134 children with AD, 33.8% (n = 45) had at least 1 positive reaction. The most frequent positive reaction was to nickel sulfate (NS) (37.8%, 17/45), followed by methylchloroisothiazolinone (20.0%, 9/45) and thimerosal (15.6%, 7/45). The total Scoring Atopic Dermatitis (SCORAD) score was significantly higher in the NS-sensitized group (P = 0.036). The patients with NS sensitization had moderate-severe AD more frequently than those without any reaction (P = 0.020). When the SCORAD score was evaluated in detail, extent of eczema, score of sleep loss, and pruritus were significantly higher in the patients with NS sensitization than those without any reaction (P = 0.002, P = 0.001, and P = 0.002, respectively). CONCLUSION: Our study confirms the necessity of CAS in the management of AD. In particular, NS sensitization should be considered for children with severe AD or larger extent of eczema and trunk involvement.

Pitfalls in anti-influenza T cell detection by Elispot using thimerosal containing pandemic H1N1 vaccine as antigen.

Monitoring T cells in combination with humoral response may be of value to predict clinical protection and cross-protective immunity after influenza vaccination. Elispot technique which measures cytokine produced after antigen-specific T cell stimulation is used routinely to detect and characterize anti-viral T cells. We found that the preservative thimerosal present in most H1N1 pandemic vaccines, induced in vitro abortive activation of T cells followed by cell death leading to false-positive results with the Elispot technique. The size of the spots, usually not measured in routine analysis, appears to be a discriminative criterion to detect this bias. Multi-dose vials of vaccine containing thimerosal remain important for vaccine delivery and our results alert about false-positive results of Elispot to monitor the clinical efficacy of these vaccines. We showed that this finding extends for other T cell monitoring techniques based on cytokine production such as ELISA. Although measuring in vitro immune response using the whole vaccine used for human immunization directly reflects in vivo global host response to the vaccine, the present study strongly supports the use of individual vaccine components for immune monitoring due to the presence of contaminants, such as thimerosal, leading to a bias in interpretation of the results.

El timerosal en la práctica pediátrica / Assisted reproductive techniques and child health

El mercurio no tiene ninguna función fisiológica en el cuerpo humano y está ampliamente distribuido en la naturaleza. Puede ser tóxico por inhalación, ingestión o contacto. El timerosal es una sal orgánica de mercurio usada como antiséptico y antifúngico desde 1928. Desde finales de la década de 1990, el mercurio empezó a ser retirado de los medicamentos y materiales de uso clínico. Se ha hecho un esfuerzo para eliminar el timerosal de las vacunas siguiendo el principio de precaución, pues no hay evidencias científicas que avalen un daño cerebral atribuible al timerosal. No se ha encontrado ninguna asociación entre su uso y el riesgo de desarrollar autismo. Actualmente, en los países de nuestro entorno solo se usa en muy pocas vacunas en envases multidosis, y todas las incluidas en los calendarios oficiales españoles, así como las de uso común en niños fuera de ellos (neumocócicas y rotavirus), están libres de timerosal. No obstante, la OMS ha reiterado que las vacunas que contienen timerosal pueden seguir utilizándose, especialmente en el Tercer Mundo donde, por necesidades logísticas, se utilizan envases multidosis, pues el riesgo ­real de enfermedad y muerte por enfermedades vacunables en quienes no se vacunan es muy superior al riesgo hipotético derivado del su uso. Como con cualquier otro medicamento, pueden presentarse reacciones de hipersensibilidad al timerosal, generalmente locales. Los esfuerzos investigadores sobre determinados problemas neurológicos como el autismo deberían encaminarse a buscar sus verdaderas causas en lugar de a sembrar dudas sobre la seguridad de las vacunas (AU)

Mercury has no physiological function in the human body and is widely distributed in nature. It may be toxic by inhalation, ingestion or contact. Thimerosal is an organic salt of mercury used as an antiseptic and antifungal since 1928. Since the late 1990s, mercury began to be withdrawn from drugs and materials for clinical use. An effort has been made to remove thimerosal from vaccines as a precautionary principle, since there is no scientific evidence to substantiate that brain damage can be attributable to thimerosal. No connection has been found between its use and the risk of developing autism. In the Western World it is only currently used in very few vaccines in multidose containers, whereas all those included in Spanish Schedules, as well as in those commonly used for children outside them (pneumococcal and rotavirus), are free from thimerosal. However, the WHO has reiterated that vaccines containing thimerosal may still be used, especially in the Third World where, for logistical needs, multidose containers are used, as the real risk of illness and death by vaccianble diseases in those who do not get vaccinated is much higher than the hypothetical risk arising from their use. As with any medicine, there may be hypersensitivity reactions to thimerosal, usually local. Research efforts should be directed towards finding the real causes of neurological problems like autism instead of throwing doubts about the safety of vaccines (AU)

Effects of glucan on immunosuppressive actions of mercury.

Global cycling of mercury results in the presence of mercury salts in the environment. The well-established negative effects of mercury on the immune system led us to the study whether natural immunomodulator glucan can overcome the immunosuppressive effects of mercury. Two types of mercury, thimerosal and mercury acetate, were administered in a dose of 2-8 mg/L of drinking water to mice. After 2 weeks, all mice exhibited profound suppression of both cellular (phagocytosis, natural killer cell activity, mitogen-induced proliferation, and expression of CD markers) and humoral (antibody formation and secretion of interleukin-6, interleukin-12, and interferon-gamma) responses. The mice were then fed with a diet containing a standard dose of glucan. Our results showed that simultaneous treatment with mercury and glucan resulted in significantly lower immunotoxic effects of mercury, which suggests that glucans can be successfully used as a natural remedy of low-level exposure to mercury.

Skin reactivity to thimerosal and phenol-preserved Montenegro antigen in Brazil.

A randomized double-blind trial was performed to determine the frequency of positive reactions to the Montenegro antigen (leishmanin) preserved in thimerosal (Merthiolate) 1:10,000 or phenol 0.4%. The respective products were tested separately in 400 young healthy individuals from a non-endemic area for Leishmaniases. Each volunteer received one of the following reagents: merthiolated antigen, phenolated antigen, merthiolated saline, or phenolated saline. The frequency of positive responses to each reagent after the first application was as follows: 0% (phenolated saline), 9.2% (merthiolated saline), 34.6% (antigen in phenolated saline), and 41.1% (antigen in merthiolated saline). After 1 week, volunteers who had tested positive for merthiolated or phenolated antigen were retested with the respective preservative, while negatives were retested with the preservative they had not received during the first test. In all, 331 volunteers who received merthiolated saline during the study, of whom 41 (12.4%) tested positive. Meanwhile, 326 volunteers who received phenolated saline, 4 (1.2%) tested positive. Positive reactions in each group were similar in relation to gross appearance skin reactions. Considering the high frequency of hypersensitivity to thimerosal in the study population, it is recommended that this compound should be replaced as a preservative of the leishmanin antigen. Almost 30% of positive reactions to Montenegro antigen in what is considered a non-endemic region was surprising and will be the object of future studies.

Dose and Hg species determine the T-helper cell activation in murine autoimmunity.

Inorganic mercury (mercuric chloride--HgCl(2)) induces in mice an autoimmune syndrome (HgIA) with T cell-dependent polyclonal B cell activation and hypergammaglobulinemia, dose- and H-2-dependent production of autoantibodies targeting the 34 kDa nucleolar protein fibrillarin (AFA), and systemic immune-complex deposits. The organic mercury species methylmercury (MeHg) and ethylmercury (EtHg--in the form of thimerosal) induce AFA, while the other manifestations of HgIA seen after treatment with HgCl(2) are present to varying extent. Since these organic Hg species are converted to the autoimmunogen Hg(2+) in the body, their primary autoimmunogen potential is uncertain and the subject of this study. A moderate dose of HgCl(2) (8 mg/L drinking water--internal dose 148 micro gHg/kg body weight [bw]/day) caused the fastest AFA response, while the induction was delayed after higher (25 mg/L) and lower (1.5 and 3 mg/L) doses. The lowest dose of HgCl(2) inducing AFA was 1.5 mg/L drinking water which corresponded to a renal Hg(2+) concentration of 0.53 micro g/g. Using a dose of 8 mg HgCl(2)/L this threshold concentration was reached within 24 h, and a consistent AFA response developed after 8-10 days. The time lag for the immunological part of the reaction leading to a consistent AFA response was therefore 7-9 days. A dose of thimerosal close to the threshold dose for induction of AFA (2 mg/L drinking water--internal dose 118 micro gHg/kg bw per day), caused a renal Hg(2+) concentration of 1.8 micro g/g. The autoimmunogen effect of EtHg might therefore be entirely due to Hg(2+) formed from EtHg in the body. The effect of organic and inorganic Hg species on T-helper type 1 and type 2 cells during induction of AFA was assessed as the presence and titre of AFA of the IgG1 and IgG2a isotype, respectively. EtHg induced a persistent Th1-skewed response irrespectively of the dose and time used. A low daily dose of HgCl(2) (1.5-3 mg/L) caused a Th1-skewed AFA response, while a moderate dose (8 mg/L) after 2 weeks resulted in a balanced or even Th2-skewed response. Higher daily doses of HgCl(2) (25 mg/L) caused a balanced Th2-Th1 response already from onset. In conclusion, while metabolically formed Hg(2+) might be the main AFA-inducing factor also after treatment with EtHg, the quality of the Hg-induced AFA response is modified by the species of Hg as well as the dose.

Assessment of metallothionein and antibodies to metallothionein in normal and autistic children having exposure to vaccine-derived thimerosal.

Allergic autoimmune reaction after exposure to heavy metals such as mercury may play a causal role in autism, a developmental disorder of the central nervous system. As metallothionein (MT) is the primary metal-detoxifying protein in the body, we conducted a study of the MT protein and antibodies to metallothionein (anti-MT) in normal and autistic children whose exposure to mercury was only from thimerosal-containing vaccines. Laboratory analysis by immunoassays revealed that the serum level of MT did not significantly differ between normal and autistic children. Furthermore, autistic children harboured normal levels of anti-MT, including antibodies to isoform MT-I (anti-MT-I) and MT-II (anti-MT-II), without any significant difference between normal and autistic children. Our findings indicate that because autistic children have a normal profile of MT and anti-MT, the mercury-induced autoimmunity to MT may not be implicated in the pathogenesis of autism.

Patch testing of Singapore children and adolescents: our experience over 18 years.

This is a retrospective epidemiologic study of allergic contact dermatitis in children and adolescents in Singapore who had undergone patch testing from January 1, 1986 to December 31, 2003. A total of 2,340 patients below the age of 21 years had undergone patch testing. Of these, 1,063 (583 girls, 480 boys) were positive to one or more allergens. The most common allergens were: nickel (40%), thimerosal (15%), colophony (9%), lanolin (8%), cobalt (8%), fragrance mix (5%), and neomycin (4%). Each of these allergens will be discussed individually. This is the first study with over 1000 patients on this subject from Asia.

Immunosuppressive and autoimmune effects of thimerosal in mice.

The possible health effects of the organic mercury compound thimerosal (ethylmercurithiosalicylate), which is rapidly metabolized to ethylmercury (EtHg), have recently been much debated and the effect of this compound on the immune system is largely unknown. We therefore studied the effect of thimerosal by treating A.SW (H-2s) mice, susceptible to induction of autoimmunity by heavy metals, with 10 mg thimerosal/L drinking water (internal dose ca 590 microg Hg/kg body weight/day) for up to 30 days. The lymph node expression of IL-2 and IL-15 mRNA was increased after 2 days, and of IL-4 and IFN-gamma mRNA after 6 and 14 days. During the first 14 days treatment, the number of splenocytes, including T and B cells as well as Ig-secreting cells decreased. A strong immunostimulation superseded after 30 days treatment with increase in splenic weight, number of splenocytes including T and B cells and Ig-secreting cells, and Th2- as well as Th-1-dependent serum immunoglobulins. Antinucleolar antibodies (ANoA) targeting the 34-kDa nucleolar protein fibrillarin, and systemic immune-complex deposits developed. The H-2s strains SJL and B10.S also responded to thimerosal treatment with ANoA. The A.TL and B10.TL strain, sharing background genes with the A.SW and B10.S strain, respectively, but with a different H-2 haplotype (t1), did not develop ANoA, linking the susceptibility to H-2. Thimerosal-treated H-2s mice homozygous for the nu mutation (SJL-nu/nu), or lacking the T-cell co-stimulatory molecule CD28 (B10.S-CD28-/-), did not develop ANoA, which showed that the autoimmune response is T-cell dependent. Using H-2s strains with targeted mutations, we found that IFN-gamma and IL-6, but not IL-4, is important for induction of ANoA by thimerosal. The maximum added renal concentration of thimerosal (EtHg) and inorganic mercury occurred after 14 days treatment and was 81 microg Hg/g. EtHg made up 59% and inorganic mercury 41% of the renal mercury. In conclusion, the organic mercury compound thimerosal (EtHg) has initial immunosuppressive effects similar to those of MeHg. However, in contrast to MeHg, thimerosal treatment leads in genetically susceptible mice to a second phase with strong immunostimulation and autoimmunity, which is T-cell dependent, H-2 linked and may at least partly be due to the inorganic mercury derived from the metabolism of ethyl mercury.

A generalized reaction to thimerosal from an influenza vaccine.

BACKGROUND: Thimerosal is a preservative commonly used in ophthalmic solutions, otic drops, and vaccines because of its bactericidal property. OBJECTIVE: To report a case of a generalized reaction to thimerosal in a patient who received an influenza vaccine. METHODS: We describe a patient who developed a generalized maculopapular eruption after receiving a thimerosal-containing influenza vaccine. Patch testing was performed to determine if there was an allergy to thimerosal. RESULTS: Patch testing confirmed a T-cell-mediated sensitivity to thimerosal. CONCLUSIONS: Physicians need to be aware that thimerosal is found in many products, including vaccinations. Clinicians should also be aware that allergic reactions occur with exposure to thimerosal even in vaccines. To our knowledge, this is the first case report in the literature of a generalized reaction to thimerosalfrom an influenza vaccine.

Detection of antinuclear and antilaminin antibodies in autistic children who received thimerosal-containing vaccines.

Autism, a neurodevelopmental disorder, may involve autoimmune pathogenesis. Since mercury is potentially a risk factor for autoimmunity, we conducted a study of mercury-induced antinuclear and antilaminin antibodies in autistic and normal children who had been pre-administered with thimerosal-containing vaccines. Laboratory analysis by different immunoassays showed that the serum level of these two autoimmune markers did not significantly differ between autistic and normal children. This finding suggests that the mercury as in thimerosal-containing vaccines is likely not related to autoimmune phenomenon in autism.

Eyedrop-induced allergy: clinical evaluation and diagnostic protocol.

BACKGROUND: In the last few years, adverse reactions to mydriatic eyedrops have been investigated. However, is not still available a standardized protocol, capable of identify the pathogenetic mechanism. In the light of these findings we have evaluated the reliability of a protocol with well established concentration of specific allergens. METHODS: The diagnostic method includes the application of patch test series Gruppo Italiano Ricerca Dermatiti da Contatto e Ambientali (GIRDCA), medicaments, corticosteroids, local anesthetics, main eyedrops' excipients, pure active principles and extemporaneous preparations with mydriatic eyewashes. At the same time, skin prick test with a solution of atropine sulfate at 1 per thousand and an intradermal test with injection of atropine at 0.01 per thousand were carried out. RESULTS: After patch tests were removed, we detected seven positiveness to the phenylephrine, two to the benzalkonium chloride, one to thiomersal, one to the ethylendiamine and one to the atropine sulfate 1 per thousand. With intradermal tests we obtained three positiveness in patients who reported adverse reactions to atropine. CONCLUSIONS: Our results show that phenylephrine is frequently responsible for allergic conjunctivitis (53.8%). In the case of atropine, even though the limited number of patients suggests to perform more extensive studies, it emerges that our diagnostic protocol is safe and might be able to screen allergic reactions in the field of ophthalmopathies.

Homozygous gene deletions of the glutathione S-transferases M1 and T1 are associated with thimerosal sensitization.

OBJECTIVE: Thimerosal is an important preservative in vaccines and ophthalmologic preparations. The substance is known to be a type IV sensitizing agent. High sensitization rates were observed in contact-allergic patients and in health care workers who had been exposed to thimerosal-preserved vaccines. There is evidence for the involvement of the glutathione system in the metabolism of thimerosal or its decomposition products (organomercury alkyl compounds). Thus detoxification by polymorphically expressed glutathione S-transferases such as GSTT1 and GSTM1 might have a protective effect against sensitization by these substances. METHODS: To address this question, a case control study was conducted, including 91 Central European individuals with a positive patch-test reaction to thimerosal. This population was compared with 169 healthy controls and additionally with 114 individuals affected by an allergy against para-substituted aryl compounds. The latter population was included in order to test whether possible associations were due to substance-specific effects, or were a general feature connected with type IV immunological diseases. Homozygous deletions of GSTT1 and GSTM1 were determined by polymerase chain reaction. RESULTS: Glutathione S-transferase M1 deficiency was significantly more frequent among patients sensitized to thimerosal (65.9%, P = 0.013) compared with the healthy control group (49.1%) and the "para-compound" group (48%, P = 0.034). Glutathione S-transferase T1 deficiency in the thimerosal/mercury group (19.8%) was barely elevated versus healthy controls (16.0%) and the "para-compound" group (14.0%). The combined deletion (GSTT1-/GSTM1-) was markedly more frequent among thimerosal-sensitized patients than in healthy controls (17.6% vs. 6.5%, P = 0.0093) and in the "para-compound" group (17.6% vs. 6.1%, P =0.014), revealing a synergistic effect of these enzyme deficiencies (healthy controls vs. thimerosal GSTM1 negative individuals, OR = 2.0 [CI = 1.2-3.4], GSTT1-, OR = 1.2 [CI = 0.70-2.1], GSTM1/T1-, OR = 3.1 [CI = 1.4-6.5]). CONCLUSIONS: Since the glutathione-dependent system was repeatedly shown to be involved in the metabolism of thimerosal decomposition products, the observed association may be of functional relevance.