Neonatal Administration of Thimerosal Causes Persistent Changes in Mu Opioid Receptors in the Rat Brain

Thimerosal added to some pediatric vaccines is suspected in pathogenesis of several neurodevelopmental disorders. Our previous study showed that thimerosal administered to suckling rats causes persistent, endogenous opioid-mediated hypoalgesia. Here we examined, using immunohistochemical staining technique, the density of μ-opioid receptors (MORs) in the brains of rats, which in the second postnatal week received four i.m. injections of thimerosal at doses 12, 240, 1,440 or 3,000 μg Hg/kg. The periaqueductal gray, caudate putamen and hippocampus were examined. Thimerosal administration caused dose-dependent statistically significant increase in MOR densities in the periaqueductal gray and caudate putamen, but decrease in the dentate gyrus, where it was accompanied by the presence of degenerating neurons and loss of synaptic vesicle marker (synaptophysin). These data document that exposure to thimerosal during early postnatal life produces lasting alterations in the densities of brain opioid receptors along with other neuropathological changes, which may disturb brain development

Mercury exposure, malaria, and serum antinuclear/antinucleolar antibodies in amazon populations in Brazil: a cross-sectional study

BACKGROUND: Mercury is an immunotoxic metal that induces autoimmune disease in rodents. Highly susceptible mouse strains such as SJL/N, A.SW, B10.S (H-2(s)) develop multiple autoimmune manifestations after exposure to inorganic mercury, including lymphoproliferation, elevated levels of autoantibodies, overproduction of IgG and IgE, and circulating immune complexes in kidney and vasculature. A few studies have examined relationships between mercury exposures and adverse immunological reactions in humans, but there is little evidence of mercury-associated autoimmunity in humans. METHODS: To test the immunotoxic effects of mercury in humans, we studied communities in Amazonian Brazil with well-characterized exposures to mercury. Information was collected on diet, mercury exposures, demographic data, and medical history. Antinuclear and antinucleolar autoantibodies (ANA and ANoA) were measured by indirect immunofluorescence. Anti-fibrillarin autoantibodies (AFA) were measured by immunoblotting. RESULTS: In a gold mining site, there was a high prevalence of ANA and ANoA: 40.8% with detectable ANoA at ≥1:10 serum dilution, and 54.1% with detectable ANA (of which 15% had also detectable ANoA). In a riverine town, where the population is exposed to methylmercury by fish consumption, both prevalence and levels of autoantibodies were lower: 18% with detectable ANoA and 10.7% with detectable ANA. In a reference site with lower mercury exposures, both prevalence and levels of autoantibodies were much lower: only 2.0% detectable ANoA, and only 7.1% with detectable ANA. In the gold mining population, we also examined serum for AFA in those subjects with detectable ANoA (≥1:10). There was no evidence for mercury induction of this autoantibody. CONCLUSIONS: This is the first study to report immunologic changes, indicative of autoimmune dysfunction in persons exposed to mercury, which may also reflect interactions with infectious disease and other factors

Sometimes Sperm Whales (Physeter macrocephalus) Cannot Find Their Way Back to the High Seas: A Multidisciplinary Study on a Mass Stranding

BACKGROUND: Mass strandings of sperm whales (Physeter macrocephalus) remain peculiar and rather unexplained events, which rarely occur in the Mediterranean Sea. Solar cycles and related changes in the geomagnetic field, variations in water temperature and weather conditions, coast geographical features and human activities have been proposed as possible causes. In December 2009, a pod of seven male sperm whales stranded along the Adriatic coast of Southern Italy. This is the sixth instance from 1555 in this basin. METHODOLOGY/PRINCIPAL FINDINGS: Complete necropsies were performed on three whales whose bodies were in good condition, carrying out on sampled tissues histopathology, virology, bacteriology, parasitology, and screening of veins looking for gas emboli. Furthermore, samples for age determination, genetic studies, gastric content evaluation, stable isotopes and toxicology were taken from all the seven specimens. The animals were part of the same group and determined by genetic and photo-identification to be part of the Mediterranean population. Causes of death did not include biological agents, or the "gas and fat embolic syndrome", associated with direct sonar exposure. Environmental pollutant tissue concentrations were relatively high, in particular organochlorinated xenobiotics. Gastric content and morphologic tissue examinations showed a prolonged starvation, which likely caused, at its turn, the mobilization of lipophilic contaminants from the adipose tissue. Chemical compounds subsequently entered the blood circulation and may have impaired immune and nervous functions. CONCLUSIONS/SIGNIFICANCE: A multi-factorial cause underlying this sperm whales' mass stranding is proposed herein based upon the results of postmortem investigations as well as of the detailed analyses of the geographical and historical background. The seven sperm whales took the same "wrong way" into the Adriatic Sea, a potentially dangerous trap for Mediterranean sperm whales. Seismic surveys should be also regarded as potential co-factors, even if no evidence of direct impact has been detected

Primary biliary cirrhosis

Primary biliary cirrhosis (PBC) is an immune-mediated chronic cholestatic liver disease with a slowly progressive course. Without treatment, most patients eventually develop fibrosis and cirrhosis of the liver and may need liver transplantation in the late stage of disease. PBC primarily affects women (female preponderance 9–10:1) with a prevalence of up to 1 in 1,000 women over 40 years of age. Common symptoms of the disease are fatigue and pruritus, but most patients are asymptomatic at first presentation. The diagnosis is based on sustained elevation of serum markers of cholestasis, i.e., alkaline phosphatase and gamma-glutamyl transferase, and the presence of serum antimitochondrial antibodies directed against the E2 subunit of the pyruvate dehydrogenase complex. Histologically, PBC is characterized by florid bile duct lesions with damage to biliary epithelial cells, an often dense portal inflammatory infiltrate and progressive loss of small intrahepatic bile ducts. Although the insight into pathogenetic aspects of PBC has grown enormously during the recent decade and numerous genetic, environmental, and infectious factors have been disclosed which may contribute to the development of PBC, the precise pathogenesis remains enigmatic. Ursodeoxycholic acid (UDCA) is currently the only FDA-approved medical treatment for PBC. When administered at adequate doses of 13–15 mg/kg/day, up to two out of three patients with PBC may have a normal life expectancy without additional therapeutic measures. The mode of action of UDCA is still under discussion, but stimulation of impaired hepatocellular and cholangiocellular secretion, detoxification of bile, and antiapoptotic effects may represent key mechanisms. One out of three patients does not adequately respond to UDCA therapy and may need additional medical therapy and/or liver transplantation. This review summarizes current knowledge on the clinical, diagnostic, pathogenetic, and therapeutic aspects of PBC

Effect of thimerosal on the murine immune system : especially induction of systemic autoimmunity

The organic mercury compound ethylmercurithiosalicylate (thimerosal), an antiseptic and a preservative, has recently raised public health concern due to its presence in vaccines globally. Thimerosal dissociates in the body to thiosalicylate and ethyl mercury (EtHg), which is partly converted to inorganic mercuric mercury (Hg2+). The immunosuppressive, immunostimulatory, and de novo autoimmunogen effect of thimerosal in mice, as well as the accelerating/aggravating effect on spontaneous systemic autoimmunity including dose-response aspects were the subject of this thesis. Thimerosal perorally (590 μg Hg/kg body weight (bw)/day) to genetically susceptible (H-2s) mice caused immunosuppression during the first week with reduction of the total number of splenocytes, T- and B-cells. The suppression lasted 2 weeks for CD4+ cells, but was superseded by a strong immunostimulation/proliferation including T- as well as B-cells, and polyclonal B-cell activation (PBA). Antinuclear antibodies targeting the 34-kDa nucleolar protein fibrillarin (AFA) appeared after 10 days, followed by renal mesangial and systemic vessel wall immune-complex (IC) deposits. The Lowest Observed Adverse Effect Level (LOAEL) was in the order AFA = glomerular and splenic vessel wall deposits < hyperimmunoglobulinemia < PBA. The LOAEL for AFA was 118 μg Hg/kg bw/day. The LOAEL for the different parameters of this thimerosal-induced systemic autoimmune condition (HgIA) was 3-11-fold higher compared with HgIA induced by HgCl2. The thimerosal-induced HgIA shared with HgCl2 a significant dose-response relationship, and requirement for: T-cells, the costimulatory factor CD28, the IFN-γ/IFN-γ-receptor pathway,but not IL-4. The mRNA expression in lymph nodes of IL-2, IFN-γ, IL-4, and IL-15 was significantly increased but not delayed compared with HgCl2. Treatment with the ubiquitous organic Hg compound methyl Hg using equimolar doses of Hg (533 μg Hg/kg bw/day) caused a transient immunosuppression, followed by a weak immunostimulation and AFA. The IgG AFA isotypes induced by the organic Hg compounds MeHg and EtHg were stable and dominated by a Th1-like pattern over a broad time- and dose range. Treatment with inorganic HgCl2 caused a dose- and time-dependent pattern of IgG AFA isotypes. Low doses favored a Th1-like pattern, a high dose a balanced or Th2-like pattern. Middle-range doses showed initially a Th1-like pattern which gradually evolved into a balanced or Th2-like pattern. The qualitative difference in IgG AFA isotypes between organic and inorganic Hg may be due to differences in activation and/or suppression of T-helper cell subsets or factors influencing the Th1/Th2-function. Speciation of the renal Hg2+ concentration and comparison with the threshold dose for induction of AFA by HgCl2 showed that even with the lowest doses of thimerosal and MeHg used in this thesis, the AFA response might from a dose threshold point of view have been caused by conversion of the organic Hg species to Hg2+. Primary treatment with inorganic Hg (HgCl2) accelerates/aggravates murine systemic autoimmunity, both spontaneous (genetic) and induced by other means. This capacity was assessed for thimerosal over a broad dose range using the (NZB X NZW)F1 hybrid mouse model. Significantly increased antinuclear antibodies (ANA) was seen after 4-7 weeks treatment (LOAEL 147 μg Hg/kg bw/day), and the response was dose-dependent up to 13 weeks. Renal mesangial and systemic vessel walls deposits similar to those in de novo HgIA were present after 7 weeks treatment. Twenty-two to 25 weeks treatment with thimerosal caused, in a dose-dependent fashion (LOAEL 295 μg Hg/kg bw/day), relocalization of the spontaneously developing glomerular IC deposits from the capillary vessel walls to the mesangium, which attenuated histological kidney damage and proteinuria, and increased survival. Thimerosal caused systemic vessel wall IC-deposits over a broad dose range: the Low Observed Adverse Effect Level (LOAEL) for renal and splenic vessel wall IC deposits was 18 and 9 μg Hg/kg bw/day, respectively. The No Observed Adverse Effect Level (NOAEL) could not be determined for the latter, since deposits were present even with the lowest dose used. Thimerosal causes in genetically susceptible mice an initial, transient immunosuppression which is superseded by a strong immunostimulation and systemic autoimmunity, sharing many characteristics with the HgIA induced by inorganic HgCl2. The IgG AFA isotype pattern is however qualitatively different, and the threshold dose substantially higher. In contrast, long-term treatment with thimerosal induces systemic vessel wall IC-deposits also using doses below those needed to induce HgIA de novo in H-2s mice

Autoantibodies in outbred Swiss Webster mice following exposure to gold and mercury

Exposure to heavy metals may have toxic effects on several human organs causing morbidity and mortality. Metals may trigger or exacerbate autoimmunity in humans. Inbred mouse strains with certain H-2 haplotypes are susceptible to xenobiotic-induced autoimmunity; and their immune response to metals such as mercury, gold, and silver have been explored. Serum antinuclear antibodies (ANA), polyclonal B-cell activation, hypergammaglobulinemia and tissue immune complex deposition are the main features of metal-induced autoimmunity in inbred mice. However, inbred mouse strains do not represent the genetic heterogeneity in humans. In this study, outbred Swiss Webster (SW) mice exposed to gold or mercury salts showed immune and autoimmune responses. Intramuscular injection of 22.5 mg/kg.bw aurothiomalate (AuTM) induced IgG ANA in SW mice starting after 5 weeks that persisted until week 15 although with a lower intensity. This was accompanied by elevated serum levels of total IgG antibodies against chromatin and total histones. Exposure to gold led to development of serum IgG autoantibodies corresponding to H1 and H2A histones, and dsDNA. Both gold and mercury induced polyclonal B-cell activation. Eight mg/L mercuric chloride (HgCl2) in drinking water, caused IgG antinucleolar antibodies (ANoA) after 5 weeks in SW mice accompanied by immune complex deposition in kidneys and spleen. Serum IgG antibodies corresponding to anti-fibrillarin, and anti-PM/Scl-100 antibodies, were observed in mercury-exposed SW mice. Gold and mercury trigger systemic autoimmune response in genetically heterogeneous outbred SW mice and suggest them as an appropriate model to study xenobiotic-induced autoimmunity.Funding Agencies|County Council of Ostergotland; ALF grant Region Ostergotland; Diagnostikcentrum Linkoping; Linkoping University</p

Bank1 and NF-kappaB as key regulators in anti-nucleolar antibody development

<div><p>Systemic autoimmune rheumatic disorders (SARD) represent important causes of morbidity and mortality in humans. The mechanisms triggering autoimmune responses are complex and involve a network of genetic factors. Mercury-induced autoimmunity (HgIA) in mice is an established model to study the mechanisms of the development of antinuclear antibodies (ANA), which is a hallmark in the diagnosis of SARD. A.SW mice with HgIA show a significantly higher titer of antinucleolar antibodies (ANoA) than the B10.S mice, although both share the same MHC class II (H-2). We applied a genome-wide association study (GWAS) to their Hg-exposed F2 offspring to investigate the non-MHC genes involved in the development of ANoA. Quantitative trait locus (QTL) analysis showed a peak logarithm of odds ratio (LOD) score of 3.05 on chromosome 3. Microsatellites were used for haplotyping, and fine mapping was conducted with next generation sequencing. The candidate genes <i>Bank1</i> (B-cell scaffold protein with ankyrin repeats 1) and <i>Nfkb1</i> (nuclear factor kappa B subunit 1) were identified by additional QTL analysis. Expression of the <i>Bank1</i> and <i>Nfkb1</i> genes and their downstream target genes involved in the intracellular pathway (<i>Tlr9</i>, <i>Il6</i>, <i>Tnf</i>) was investigated in mercury-exposed A.SW and B10.S mice by real-time PCR. <i>Bank1</i> showed significantly lower gene expression in the A.SW strain after Hg-exposure, whereas the B10.S strain showed no significant difference. <i>Nfkb1</i>, <i>Tlr9</i>, <i>Il6</i> and <i>Tnf</i> had significantly higher gene expression in the A.SW strain after Hg-exposure, while the B10.S strain showed no difference. This study supports the roles of <i>Bank1 (</i>produced mainly in B-cells) and <i>Nfkb1</i> (produced in most immune cells) as key regulators of ANoA development in HgIA.</p></div

Gene expression of <i>Il6</i> and <i>Tnf</i>.

<p>Experimental design same as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0199979#pone.0199979.g004" target="_blank">Fig 4</a>. Total RNA expression of <i>Il6</i> in <b>A</b>) A.SW and <b>B</b>) B10.S mice followed by <i>Tnf</i> expression in <b>C)</b> A.SW and <b>D)</b> B10.S mice. Figure represents fold difference (y-axis) and presented as median ± interquartile range for each group (Kruskal Wallis, Dunn's multiple comparisons test). Significant difference (* p < 0.05, ** p < 0.01) between groups in each strain. Endogenous controls and statistical analysis same as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0199979#pone.0199979.g004" target="_blank">Fig 4</a>.</p