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.     

Perturbation of copper (Cu) homeostasis and expression of Cu-binding proteins in cadmium-resistant lung fibroblasts.

To probe mechanisms of cadmium (Cd) damage to the lung extracellular matrix (ECM), we developed Cd-resistant (CdR) rat lung fibroblasts (RFL6) by incubation with graded concentrations of Cd. CdR cells downregulated lysyl oxidase (LO), a copper (Cu)-dependent enzyme essential for crosslinking of collagen and elastin in the ECM, in conjunction with upregulation of other Cu-binding proteins including Cu,Zn-superoxide dismutase (SOD1), copper chaperone for SOD1 (CCS1), metallothionein (MT), and Menkes P-type ATPase (ATP7A), a Cu transporter in the membrane of the Golgi apparatus, as well as gamma-glutamylcysteine synthetase (gamma-GCS), an enzyme for glutathione biosynthesis. Reduction and loss of cytoplasmic distribution of LO in CdR cells were accompanied by its dislocation with the Menkes P-type ATPase and the endoplasmic reticulum marker. CdR cells displayed a defect in LO catalytic activity but an enhancement in Cu,Zn-SOD catalytic activity consistent with the protein expression levels of these enzymes. Although long-term Cd exposure of cells enhanced the Menkes P-type ATPase protein expression, actually, it reduced Cu-dependent catalytic activity of this enzyme in parallel with the deficiency of LO. The low level of 64Cu bound to the LO fraction and the high level of 64Cu bound to the MT fraction provide direct evidence for limitation of Cu bioavailability for LO existing in the CdR cells. These results suggest that downregulation of LO is linked with upregulation of other Cu-binding proteins and with alteration in Cu homeostasis in the CdR phenotype.     

ApoMT对CdMT肾毒性保护作用研究

本文研究了去除金属离子的金属硫蛋白（ApoMT）对镉金属硫蛋白（CdMT）肾毒性作用的影响。结果发现，与单独给予CdMT比较，ApoMT能降低尿蛋白量和尿碱性磷酸酶（AKP）活性，并能促进尿Cd的排泄。肾组织形态学结果显示肾近曲小管损伤程度明显减轻。提示ApoMT对CdMT肾小管损伤具有保护作用。     

The major 40-kDa glycoprotein in human prostatic fluid is identical to Zn-alpha 2-glycoprotein

A major 40-KDA protein secreted by human prostate was isolated from whole seminal plasma by sequential column chromatography on DEAE-Sepharose CL-6B, concanavalin A(Con A)-Sepharose, and Sephadex G-100. Although the purified preparation still contained minor contaminants, its amino acid composition was found to be identical to the one of a glycoprotein isolated previously from seminal plasma by Lin et al (1983). Antibodies against this protein were produced in rabbits and their use in immunoblotting experiments revealed the presence of the antigen in several tissues including the prostate, the liver, the heart, the kidney, the epididymis, and the testis. A radioimmunoassay confirmed these results and showed that blood serum concentrations of this protein were relatively high in men (81 +/- 3 micrograms/ml), women (68 +/- 3 micrograms/ml), and cord blood of newborns (32 +/- 1 micrograms/ml). The serum concentrations of this protein along with its physicochemical characteristics suggested that it could be identical to Zn-alpha 2-glycoprotein, a human serum protein previously isolated by Burgi and Schmid (1961). This hypothesis was confirmed by a double immunodiffusion analysis using a commercial anti-Zn-alpha 2-glycoprotein antiserum. Finally, in vitro translation of prostatic poly(A) + RNA in rabbit reticulocyte lysate in the presence of canine pancreatic microsomal membranes resulted in the formation of an immunoprecipitable 42-kDa band. These results show that Zn-alpha 2-glycoprotein can be synthesized in the prostate itself. The demonstration of high concentrations of this protein in prostatic tissue and prostatic secretion should facilitate the elucidation of its role in the prostate and in other tissues.     

丙氧锗二酸对顺铂毒性的保护作用及其对抗癌效果的影响

 本文研究了丙氧锗二酸（Ge－132）对顺铂毒性及抗癌效果的影响，并对其保护作用与金属硫蛋白（metallothionein，MT）的关系进行了探讨。结果表明Ge－132对顺铂引起的肾脏、血液和致死的毒性具有明显的保护作用。其效果优于次硝酸秘、亚硒醚钠以及氧化锗；Ge－132无诱导MT的作用，经体内抑瘤实验及体外细胞毒性实验表明，Ge－132对顺铂的抑瘤作用无统计学意义的影响。     

Atrophic variants of dermatofibroma and dermatofibrosarcoma protuberans

Dermal atrophy of more than 50% of the locoregional dermis may be the predominant histopathological feature in dermatofibroma and dermatofibrosarcoma protuberans. This may cause diagnostic difficulties. In the present study 26 cases of atrophic dermatofibroma were compared with three cases of atrophic dermatofibrosarcoma protuberans. Clinically, both conditions mostly occurred on the (upper) trunk of females. While atrophic dermatofibroma usually presented as a reddish, umbilicated lesion (0.5–1-cm), often suspected to be a basal cell carcinoma, atrophic dermatofibrosarcoma protuberans showed irregularly arranged tan-brown plaques (3–6 cm). Histologically, atrophic dermatofibroma showed a regular silhouette with a smooth nodular (9/26) or scalloped lower margin with an intervening lace-like pattern of superficial fatty tissue infiltration (17/26) and variable sclerosis; atrophic dermatofibrosarcoma protuberans showed a deep, irregular infiltration of fatty tissue in a lacelike/honeycomb and/ or multilayered pattern, but no sclerosis. Immunohistochemically, atrophic dermatofibroma was mostly negative with QBEnd 10 (CD34; 24/26), variably positive for factor XIIIa (20/26) and metallothionein (11/26). Labelling for factor XIIIa and metallothionein was usually seen in 'early' (metabolically active) lesions, while 'late' sclerotic ones were negative. In contrast to atrophic dermatofibroma all three atrophic dermatofibrosarcoma protuberans showed a consistently uniform profile: CD34 positive, factor XIIIa and metallothionein negative. Our study delineates atrophic dermatofibroma and atrophic dermatofibrosarcoma protuberans as distinct entities clearly distinguishable from each other by clinicopathologic criteria.     

Expression of metallothionein in lung carcinoma: correlation with histological type and grade

AIMS: Over-expression of cellular metallothionein occurs frequently in human tumours but the underlying mechanism remains unknown. The aim of this study was to assess metallothionein expression in cases of lung carcinoma and to correlate it with histopathological parameters. METHODS AND RESULTS: Tumour tissue samples from 89 patients with lung carcinoma were immunostained by the streptavidin-biotin-peroxidase technique, using a monoclonal antibody against both metallothionein-1 and -2 isoforms. Positive matallothionein immunostaining was prominent in 44 out of 89 (49%) and negative in 45 out of 89 (51%) cases of lung carcinoma examined. Metallothionein positivity was prominent in 32 out of 43 (74%) cases of squamous cell lung carcinoma, and in 12 out of 35 (34%) cases of adenocarcinoma, while it was negative in all 11 cases of small-cell lung carcinoma examined, presenting a statistically significant difference between the different histological types. The intensity of metallothionein staining revealed a statistically significant difference between the squamous cell and adenocarcinoma cases examined. The pattern and extent of metallothionein staining in tumour cells and the expression of metallothionein in stromal cells were not correlated with histopathological parameters (type and grade) in metallothionein-positive cases of lung carcinoma examined. No association was found between metallothionein expression and lymph node status in the examined cases of lung carcinoma. CONCLUSIONS: Our findings indicate that expression of metallothionein was evident in squamous cell lung carcinoma and adenocarcinoma, but absent in small-cell lung carcinoma, supporting evidence for participation of this protein in the biological mechanisms underlying the carcinogenic evolution in the lung.     

金属硫蛋白对培养神经细胞迟发性损伤的作用和一氧化氮的表达

本文以新生大鼠原代培养皮质神经元为实验材料，造成迟发性神经元损伤模型．在不同时程内，测试培养液中的细胞乳酸脱氢酶漏出量和用还原型尼克酰胺腺嘌呤二核着苷酸脱氢酶染色反应，观察培养细胞中一氧化氮合酶的表达水平．结果表明，缺血组在缺血与再灌流中，细胞乳酸脱氢酶漏出量明显高于对照组，差异显著（Ｐ＜０．００１）．一氧化氮合酶阳性神经元数量在缺血组的缺血时即明显高于对照组（Ｐ＜０．０１），再灌流后一氧化氮合酶表达仍然强烈，与对照组相比有显著差异（Ｐ＜０．０１）．当损伤细胞再灌流时，同时加入金属硫蛋白后，显示一氧化氮合酶表达减弱，和缺血组相比有显著差异（Ｐ＜０．０５～０．００１），细胞乳酸脱氢酶漏出量在再灌流后的早期近似于对照组．结合文献和本实验结果提示：在迟发性神经元损伤形成过程中一氧化氮起着重要作用；金属硫蛋白对脑缺血后迟发性神经元损伤有一定保护作用，是一种细胞保护剂，可望用于临床，控制缺血再灌流损伤。     

Imbalanced secondary mucosal antioxidant response in inflammatory bowel disease

Intestinal mucosal damage in the inflammatory bowel diseases (IBD) Crohn's disease (CD) and ulcerative colitis (UC) involves reactive oxygen metabolites (ROMs). ROMs are neutralized by endogenous antioxidant enzymes in a carefully balanced two-step pathway. Superoxide dismutases (SODs) convert superoxide anion to hydrogen peroxide (H(2)O(2)), which is subsequently neutralized to water by catalase (CAT) or glutathione peroxidase (GPO). Remarkably changed expression levels of the three isoforms of SOD in paired non-inflamed and inflamed mucosae from CD and UC patients have been previously reported in comparison to normal control mucosa. Most notable was the strong up-regulation of Mn-SOD in inflamed epithelium. It was hypothesized that in order to provide optimal protection against ROM-mediated damage, these changes should be coordinately counterbalanced by an increased H(2)O(2)-neutralizing capacity. Therefore, the same tissue samples were used to assess the levels, activities, and/or localization of the most prominent mucosal H(2)O(2)-related antioxidants CAT, GPO, glutathione (GSH), myeloperoxidase (MPO), and metallothionein (MT). Quantitative measurements showed that in both CD and UC patients, intestinal inflammation was associated with increased activities of CAT, GPO, and MPO, whereas the mucosal GSH content was unaffected and the concentration of MT was decreased. Despite this overall increase in mucosal H(2)O(2)-metabolizing enzyme capacity, immunohistochemical analysis revealed a differentially disturbed antioxidant balance in IBD epithelium and lamina propria. In the lamina propria, the risk of direct H(2)O(2)-mediated damage seemed to be restrained by the increasing numbers of CAT- and MPO-positive monocytes/macrophages and neutrophils that infiltrated the inflamed areas. On the other hand, MPO overexpression might increase the lamina propria levels of hypochlorous acid, a stable ROM with multiple pro-inflammatory effects. In the epithelium, the number of cells that expressed CAT remained unchanged during inflammation and GPO was found in only a very low and constant number of epithelial cells. In addition, the inflamed epithelium displayed decreased expression of the hydroxyl radical (OH(*)) scavenger MT. In view of the high epithelial SOD levels in inflamed IBD epithelium, it is speculated that the efficient removal of excess H(2)O(2) is hampered in these cells, thereby increasing not only the risk of detrimental effects of H(2)O(2) directly, but also those of its extremely reactive derivatives such as OH(*). Taken together, the results suggest an imbalanced and inefficient endogenous antioxidant response in the intestinal mucosa of IBD patients, which may contribute to both the pathogenesis and the perpetuation of the inflammatory processes.     

锌对小鼠组织MT-1基因转录及MT蛋白表达的影响

目的 :研究锌对小鼠各脏器金属硫蛋白 (MT)基因表达的影响。方法 :将小鼠随机分为注锌组和对照组 ,注锌后不同时间或不同剂量锌注射后 16 h断头处死动物 ,取一定量肝、脑、胸腺及脾脏组织制成匀浆 ,采用 Northern blot及 10 9Cd- hem饱和法测定组织 MT基因转录及蛋白表达的变化。结果 :时程研究表明 ,锌 10 mg/ kg腹腔注射后 2 h,小鼠肝脏及脾脏 MT- 1m RNA水平明显升高 ,4 h达峰值 ,分别为对照组的 5 .30和 2 .92倍 ,2 4 h恢复至正常水平 ;其 MT蛋白表达迟于 MT- 1m RNA水平升高 ,注锌后 8～ 2 4 h肝脏及脾脏 MT蛋白含量均比对照组明显升高 (P<0 .0 5或 P<0 .0 0 1) ,2 4 h达峰值 ,72 h恢复至正常水平。注射不同剂量锌后小鼠肝脏及脾脏 MT- 1m RNA水平 (4h)和 MT蛋白含量 (16 h)均呈剂量依赖性增加。小鼠腹腔注射锌 2 .5～ 10 mg/ kg 16 h,小鼠肝脏或脾脏 MT含量或锌5～ 10 mg/ kg注射后脾脏 MT含量均显著高于对照组 (P<0 .0 1或 P<0 .0 0 1)。但注射锌后脑及胸腺MT- 1m RNA水平及 MT蛋白含量均无明显变化经。结论 :锌诱导小鼠组织 MT合成具有明显的组织器官特异性。