A metallothionein from disk abalone (Haliotis discus discus): Insights into its functional roles in immune response, metal tolerance, and oxidative stress.

Metallothioneins (MTs) are cysteine-rich metal-binding proteins whose expression is induced by exposure to essential and non-essential metals, making them potential biological markers for assessing metal pollution in various biomonitoring programs. However, the functional properties of these proteins are yet to be comprehensively characterized in most marine invertebrates. In this study, we identified and characterized an MT homolog from the disk abalone (Haliotis discus discus), referred to as disk abalone MT (AbMT). AbMT exhibited the same primary structural features as MTs from other mollusks containing two ß-domains (ß2ß1-form). AbMT protein demonstrated metal-binding and detoxification abilities against Zn, Cu, and Cd, as evidenced by Escherichia coli growth kinetics, metal tolerance analysis, and UV absorption spectrum. Transcriptional analysis revealed that AbMT was ubiquitously expressed in all analyzed tissues and upregulated in gill tissue following challenge with Vibrio parahaemolyticus, Listeria monocytogenes, and viral hemorrhagic septicemia virus (VHSV). Additionally, overexpression of AbMT suppressed LPS-induced NO production in RAW264.7 macrophages, protected cells against H2O2-induced oxidative stress, and promoted macrophage polarization toward the M1 phase. Conclusively, these findings suggest an important role for AbMT in environmental stress protection and immune regulation in disk abalone.

Metallothionein-1 is associated with osteoarthritis disease activity and suppresses proinflammatory cytokines production in synovial cells.

BACKGROUNDS: OA (Osteoarthritis) is a predominant degenerative disease, characterized by the synovial inflammation and cartilage destruction. The pathogenic mechanisms remain mostly unknown. There is an critical require for extra investigations to discover new therapeutic targets to prevent and treat OA disease, as there are currently no effective treatments except for the joint replacement. METHODS: The mRNA and protein levels of Metallothionein-1(MT-1) were quantified by qPCR and ELISA in peripheral blood mononuclear cells (PBMCs), serum and synovial cells (SCs) from erosive inflammatory OA (EIOA) and primary generalized OA (PGOA) patients. Age and sex matched healthy volunteers were recruited as healthy controls (HCs). The correlation between the MT-1 level and OA activity was assessed and the anti-inflammatory effects of MT-1 was determined in vitro. RESULTS: The mRNA and protein levels of MT-1 were significantly increased in the PBMCs and serum of EIOA patients compared with those of PGOA patients and HCs. Serum levels of MT-1 were positively correlated with VAS score, CRP, and ESR in OA patients. And the positive correlations were also identified between the MT-1 and IL-1ß, TNF-α or IL-6 in synovial cells. Furthermore, the recombinant MT-1 protein could significantly inhibit the expression of IL-1ß, TNF-α and IL-6 in PBMCs and SCs from EIOA patients in vitro. CONCLUSION: The data had shown that the MT-1 was up-regulated in EIOA patients and positively correlated with the disease activity. The recombinant MT-1 could suppress the expression of pro-inflammatory cytokines in both PBMCs and synovial cells from EIOA patients. Therefore, the MT-1 might become a novel therapeutic target for OA treatment.

The adaptive immune role of metallothioneins in the pathogenesis of diabetic cardiomyopathy: good or bad.

Diabetes is a metabolic disorder characterized by hyperglycemia, resulting in low-grade systemic inflammation. Diabetic cardiomyopathy (DCM) is a common complication among diabetic patients, and the mechanism underlying its induction of cardiac remodeling and dysfunction remains unclear. Numerous experimental and clinical studies have suggested that adaptive immunity, especially T lymphocyte-mediated immunity, plays a potentially important role in the pathogenesis of diabetes and DCM. Metallothioneins (MTs), cysteine-rich, metal-binding proteins, have antioxidant properties. Some potential mechanisms underlying the cardioprotective effects of MTs include the role of MTs in calcium regulation, zinc homeostasis, insulin sensitization, and antioxidant activity. Moreover, metal homeostasis, especially MT-regulated zinc homeostasis, is essential for immune function. This review discusses aberrant immune regulation in diabetic heart disease with respect to endothelial insulin resistance and the effects of hyperglycemia and hyperlipidemia on tissues and the different effects of intracellular and extracellular MTs on adaptive immunity. This review shows that intracellular MTs are involved in naïve T-cell activation and reduce regulatory T-cell (Treg) polarization, whereas extracellular MTs promote proliferation and survival in naïve T cells and Treg polarization but inhibit their activation, thus revealing potential therapeutic strategies targeting the regulation of immune cell function by MTs.

Metallothioneins: Emerging Modulators in Immunity and Infection.

Metallothioneins (MTs) are a family of metal-binding proteins virtually expressed in all organisms including prokaryotes, lower eukaryotes, invertebrates and mammals. These proteins regulate homeostasis of zinc (Zn) and copper (Cu), mitigate heavy metal poisoning, and alleviate superoxide stress. In recent years, MTs have emerged as an important, yet largely underappreciated, component of the immune system. Innate and adaptive immune cells regulate MTs in response to stress stimuli, cytokine signals and microbial challenge. Modulation of MTs in these cells in turn regulates metal ion release, transport and distribution, cellular redox status, enzyme function and cell signaling. While it is well established that the host strictly regulates availability of metal ions during microbial pathogenesis, we are only recently beginning to unravel the interplay between metal-regulatory pathways and immunological defenses. In this perspective, investigation of mechanisms that leverage the potential of MTs to orchestrate inflammatory responses and antimicrobial defenses has gained momentum. The purpose of this review, therefore, is to illumine the role of MTs in immune regulation. We discuss the mechanisms of MT induction and signaling in immune cells and explore the therapeutic potential of the MT-Zn axis in bolstering immune defenses against pathogens.

Morphostructural and immunohistochemical study on the role of metallothionein in the detoxification of heavy metals in Apis mellifera L., 1758.

Honey is a valuable food produced by bees from sugary substances that they gather in nature. The transformation the nectar into honey, by bees, is long and complex. Except for honey, where heavy metals are absent or are found only in traces, the bees and their products have always been considered excellent biomarkers of such contaminants. We have assumed that the absence of heavy metals in honey is due to the presence of a detoxification system in the digestive system of bees, which involves metallothioneins, proteins that have a role in the homeostatic control of essential and non-essential metals. We have placed the beehives in three different zones: industrial, urban and rural. Investigations were carried out with ICP-MS method for the detection of heavy metals in the guts of honey bees and honey. The metallothioneins have been identified by Immunohistochemical and Western-blotting analisys. The investigations have shown the presence of heavy metals only in bees guts but not in honey, while the presence of metallothionein has been highlighted only in epithelium of the honey sac, demonstrating the existence of an efficient system of detoxification of heavy metals.

Impact of Influenza A Virus Infection on the Proteomes of Human Bronchoepithelial Cells from Different Donors.

Susceptibility to influenza A virus is determined by a balance of viral and host factors. The genetic background of the host contributes to the severity of disease, but the influenza-related proteomes of cells from different individuals have not been compared. We used high-resolution mass spectrometry to identify proteins in normal human bronchial epithelial (NHBE) cells isolated from three different donors. Infection of each NHBE cell culture with influenza A/California/07/2009 (H1N1) resulted in expression of viral proteins and a variety of host proteins, including interferons, interferon-stimulated genes, and secreted chemokines/cytokines. The expression level of viral proteins corresponded to the level of host proteins that support influenza infection (i.e., pro-viral proteins); however, production of infectious virus was inversely related to the levels of antiviral proteins, suggesting that a balance of pro-viral proteins and the antiviral response controls virus replication. In summary, our results demonstrate that expression levels of pro-viral as well as antiviral factors are different for each donor and suggest that relative quantitation of these factors may provide a way to identify individuals or population groups who are susceptible to severe influenza disease.

Regulatory effects of zinc on cadmium-induced cytotoxicity in chronic inflammation.

OBJECTIVES: Zinc (Zn) has major effects on immune system activation while Cadmium (Cd) has anti-inflammatory and anti-proliferative effects in several chronic inflammatory contexts. The aim of this work was to investigate by which mechanisms Zn could compete with Cd and eventually counteract its deleterious effects. Rheumatoid arthritis (RA) synoviocytes exposed to cytokines were used as a model of chronic inflammation; osteoarthritis (OA) synoviocytes were used as control. METHODS: Cell/medium fractionation constants were analyzed for different metals by inductively-coupled-plasma mass-spectrometry by comparison to the 70Zn spike. Interleukin-17 (IL-17) and tumor necrosis factor-alpha (TNF-α) were used to mimic inflammation. Gene expression of ZIP-8 importer, metallothioneins-1 (MT-1s) and the ratio between metalloprotease-3 and the tissue inhibitor of metalloproteinases (MMP-3)/TIMP-1) were evaluated after pre-exposure to cytokines and Cd, with or without the addition of exogenous Zn (0.9 ppm). Cell viability was measured by neutral red assay and IL-6 production by ELISA. RESULTS: Synoviocytes selectively absorbed and retained Cd in comparison to Zn. Metal import increased with IL-17/TNF-α exposure, through the enhanced ZIP-8 expression. Zn did not modify ZIP-8 expression, while Cd reduced it (p<0.05). Zn induced a reduction of Cd-induced MT-1s expression, in particular of MT-1X (3-fold), and subsequently the final intra-cellular content of Cd. By reducing Cd accumulation in cells, Zn reversed Cd anti-proliferative and anti-inflammatory effects but preserved the low MMP-3/TIMP-1 ratio induced by Cd, which was enhanced by inflammatory conditions. CONCLUSION: Zinc counteracts the deleterious effect of Cd by reducing its import and accumulation in the cell, without the reactivation of destructive pathways such as MMPs.

Expression, purification of metallothionein genes from freshwater crab (Sinopotamon yangtsekiense) and development of an anti-metallothionein ELISA.

Using the phoA-fusion technology, the recombinant metallothionein (MT) from freshwater crab (Sinopotamon yangtsekiense) has been successfully produced in Escherichia coli. MT purified from the bacterial suspension showed one polypeptide with a molecular weight of 7 kDa by tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis (Tricine-SDS-PAGE). Western-blotting confirmed the polypeptides had a specific reactivity with mouse polyclonal MT anti-serum. Based on the purified MT and MT anti-serum, the reaction parameters for an enzyme-linked immunosorbent assay (ELISA) were developed. The direct coating ELISA showed a higher linear relationship compared to antibody sandwich coating ELISA. The optimal dilution rates of purified MT anti-serum and coating period were shown to be 1:160,000 and 12 hours at 4°C. At 37°C, the appropriate reaction duration of the first antibody and the second antibody were 2 hours and 1 hour, respectively. According to these optimal parameters, the standard linear equation, y = 0.0032x + 0.1769 (R2 = 0.9779, x, y representing MT concentration and OD450 value), was established for the determination of MT concentration with a valid range of 3.9-500 ng/ml. In verification experiments, the mean coefficients of variation of the intra-assay and inter-assay were 3.260% and 3.736%, respectively. According to the result of MT recovery, ELISA with an approaching 100% MT recovery was more reliable and sensitive than the Cd saturation assay. In conclusion, the newly developed ELISA of this study was precise, stable and repeatable, and could be used as a biomarker tool to monitor pollution by heavy metals.

Development of Antigen-Immobilized Metallothionein Sensor that Exploits Gold Nanoparticle-Based Enhancement of Signal.

An antigen-immobilized indirect-competitive immunosensor that detects metallothionein (MT), a potent biomarker of contamination with heavy metals, was developed exploiting enhancement of signal based on an additional binding of gold nanoparticles to an anti-MT antibody through the biotin-avidin interaction. The sensor was constructed by the immobilization of MT at 1 mg/mL on a 9-MHz quartz crystal microbalance and the concentration of the antibody for competitive reaction was optimized as 10 µg/mL based on the degree of sensor response. At this moment, the control response of the sensor obtained with enhancement of signal was 343.8 Hz and was larger than that without enhancement of signal 2.47 fold. The sensor responses decreased gradually with increasing analyte concentrations, and a linear relationship between analyte concentration and sensor response was acquired in the range of 0.005-1 ng/mL MT in double-logarithmic scales with a correlation coefficient (r) of 0.9858. The limit of detection of the present sensor was presumed to be present below 5 pg/mL MT.

Identification of two metallothioneins as novel inhalative coffee allergens cof a 2 and cof a 3.

BACKGROUND: Dust of green coffee beans is known to be a relevant cause for occupational allergic disorders in coffee industry workers. Recently, we described the first coffee allergen (Cof a 1) establishing an allergenic potential of green coffee dust. OBJECTIVE: Our aim was to identify allergenic components of green coffee in order to enhance inhalative coffee allergy diagnosis. METHODS: A Coffea arabica pJuFo cDNA phage display library was created and screened for IgE binding with sera from allergic coffee workers. Two further coffee allergens were identified by sequence analysis, expressed in E. coli, and evaluated by Western blots. The prevalence of sensitization to recombinant Cof a 1, Cof a 2, and Cof a 3 and to commercially available extract was investigated by ELISA (enzyme-linked immunosorbent assay) respectively CAP (capacity test) screening in 18 sera of symptomatic coffee workers. RESULTS: In addition to the previously described chitinase Cof a 1, two Coffea arabica cysteine-rich metallothioneins of 9 and 7 kDa were identified and included in the IUIS Allergen Nomenclature as Cof a 2 and Cof a 3. Serum IgE antibodies to at least one of the recombinant allergens were found in 8 out of 18 symptomatic coffee workers (44%). Only 2 of the analysed sera (11%) had reacted previously to the commercial allergy test. CONCLUSIONS: In addition to the previously described Cof a 1 we have identified two further coffee proteins to be type I coffee allergens (Cof a 2 and Cof a 3) which may have a relevant potential for the specific diagnosis and/or therapy of coffee allergy.

Metallothionein immunoexpression in non-syndromic and syndromic keratocystic odontogenic tumour.

BACKGROUND: To compare the metallothionein (MT) immunoexpression in non-syndromic and syndromic keratocystic odontogenic tumour (KOT), to correlate MT with cellular proliferation, and to evaluate the influence of inflammation in MT. STUDY DESIGN: Fourteen cases of KOT were submitted to immunohistochemistry for MT and Ki-67 analysis. The lesions were grouped according to their grade of inflammation, and statistical analysis was performed. RESULTS: MT was higher in non-syndromic KOT than in syndromic KOT (p<0.05). No statistical difference in Ki-67 could be identified; however, an inverse correlation was observed between MT and Ki-67 in both lesions. When analysing inflammation, non-syndromic KOT showed no differences in either MT or Ki-67. CONCLUSIONS: The MT immunophenotype of syndromic KOT was different from non-syndromic KOT. MT might not be involved in the proliferation control of both KOT. MT and Ki-67 immunoexpressions proved to be unaffected by inflammation in non-syndromic KOT.

Zinc-binding metallothioneins are key modulators of IL-4 production by basophils.

Zinc (Zn) is an essential nutrient, and Zn deficiency causes immunodeficiency and skin disorders. Basophils express FcɛRI on their surface and release multiple mediators after receptor cross-linking, including large amounts of IL-4. However, the mechanisms involved in the FcɛRI-mediated regulation of basophil IL-4 production are currently unclear. Here, we show that the Zn-binding metallothionein (MT) proteins are essential for the FcɛRI-induced basophil production of IL-4. Basophils from MT-I/II(-/-) mice produced significantly less FcɛRI-induced IL-4 than did wild-type basophils. The MTs were involved in maintaining intracellular Zn levels, thereby regulated the calcineurin activity and nuclear factor of activated T-cell (NFAT)-mediated IL-4 production. These results suggest that the MT-dependent control of Zn homeostasis is a novel mechanism for regulating basophil IL-4 production.

Paraquat reduces natural killer cell activity via metallothionein induction.

Paraquat (PQ), one of the most widely used herbicides, has been used for several decades in agriculture. Some studies suggest that PQ has effects on the immune system. Moreover, previous studies have shown that PQ imparted some immunosuppressive effects. In the present study, cytotoxicity assays using splenic NK cells from mice treated for 28 days with PQ (at 0.2, 1, and 5 mg/kg) were performed to determine whether PQ altered the function of NK cells. Given that PQ was expected to induce an immunosuppressive effect, it was hypothesized that a gene involved in cellular metal ion homeostasis, metallothionein-1 (MT-1), could play an important role in this outcome. This belief was based on the fact that MT1 encodes a protein responsible for zinc ion homeostasis, and that a reduction in free zinc ion levels impairs NK cell function. The results showed that PQ treatments led to increased MT expression in several organs (liver, kidneys, testes) and in splenocytes, caused a reduction of both free zinc ions in sera and in free intracellular zinc, and reduced the expression of GATA-3, a zinc-finger transcription factor important for maturation and activity of T-cells and NK cells. These results provide a basis for a new molecular mechanism to describe potential immunosuppressive effects of PQ in vivo.

[Preparation and characterization of monoclonal antibodies against metallothionein of freshwater crab (Sinopotamon henanense)].

OBJECTIVE: To prepare a monoclonal antibody (mAb) against metallothionein (MT) of freshwater crab (Sinopotamon henanense) and characterize its immunologic properties. METHODS: Two recombinant MT of S.henanense, namely SUMO-MT and His-MT, were produced by SUMO fusion system and phoA secretion expression system in E.coli. SUMO-MT was used as an antigen to immunize BALB/c mice. By means of the cell fusion technique, multiple cell subcloning, repeated screening with His-MT as detecting antigen, the hybridomas specifically secreting mouse mAb against the MT of S. henanense were generated. The titers of mAbs were measured by indirect ELISA and the specificity of the mAbs was evaluated by Western blotting and Dot-ELISA. RESULTS: Two hybridoma cell lines designated mAb-MT2 and mAb-MT3 with the property of secreting mAb against the MT continuously and steadily were successfully obtained. Their immunoglobulin subclass was IgG1. The titers of the ascites fluid were 1:500 000 and 1:1000 000, respectively. Western blot analysis confirmed that the two mAbs both reacted with recombinant SUMO-MT and His-MT with good sensitivity. The Dot-ELISA demonstrated that the two mAbs reacted specifically not only with recombinant MT but also natural MT. CONCLUSION: The mAbs against MT of S. henanense with high specificity were successfully prepared.

Role of metallothioneins as danger signals in the pathogenesis of colitis.

Inflammatory bowel diseases (IBDs) are recurrent intestinal pathologies characterized by a compromised epithelial barrier and an exaggerated immune activation. Mediators of immune cell infiltration may represent new therapeutic opportunities. Metallothioneins (MTs) are stress-responsive proteins with immune-modulating functions. Metallothioneins have been linked to IBDs, but their role in intestinal inflammation is inconclusive. We investigated MT expression in colonic biopsies from IBDs and acute infectious colitis patients and healthy controls and evaluated MT's role in experimental colitis using MT knockout mice and anti-MT antibodies. Antibody potential to target extracellular MT and its mechanism was tested in vitro. Biopsies of patients with active colitis showed infiltration of MT-positive cells in a pattern that correlated with the grade of inflammation. MT knockout mice displayed less severe acute dextran sulphate sodium (DSS)-induced colitis compared to congenic wild-type mice based on survival, weight loss, colon length, histological inflammation and leukocyte infiltration. Chronic DSS-colitis confirmed that Mt1 and Mt2 gene disruption enhances clinical outcome. Blockade of extracellular MT with antibodies reduced F4/80-positive macrophage infiltration in DSS- and trinitrobenzene sulphonic acid-colitis, with a tendency towards a better outcome. Whole-body single-photon emission computer tomography of mice injected with radioactive anti-MT antibodies showed antibody accumulation in the colon during colitis and clearance during recovery. Necrotic and not apoptotic cell death resulted in western blot MT detection in HT29 cell supernatant. In a Boyden chamber migration assay, leukocyte attraction towards the necrotic cell supernatant could be abolished with anti-MT antibody, indicating the chemotactic potential of endogenous released MT. Our results show that human colitis is associated with infiltration of MT-positive inflammatory cells. Since antibody blockade of extracellular MT can reduce colitis in mice, MT may act as a danger signal and may represent a novel target for reducing leukocyte infiltration and inflammation in IBD patients.

RNA-Seq analysis reveals candidate genes for ontogenic resistance in Malus-Venturia pathosystem.

Ontogenic scab resistance in apple leaves and fruits is a horizontal resistance against the plant pathogen Venturia inaequalis and is expressed as a decrease in disease symptoms and incidence with the ageing of the leaves. Several studies at the biochemical level tried to unveil the nature of this resistance; however, no conclusive results were reported. We decided therefore to investigate the genetic origin of this phenomenon by performing a full quantitative transcriptome sequencing and comparison of young (susceptible) and old (ontogenic resistant) leaves, infected or not with the pathogen. Two time points at 72 and 96 hours post-inoculation were chosen for RNA sampling and sequencing. Comparison between the different conditions (young and old leaves, inoculated or not) should allow the identification of differentially expressed genes which may represent different induced plant defence reactions leading to ontogenic resistance or may be the cause of a constitutive (uninoculated with the pathogen) shift toward resistance in old leaves. Differentially expressed genes were then characterised for their function by homology to A. thaliana and other plant genes, particularly looking for genes involved in pathways already suspected of appertaining to ontogenic resistance in apple or other hosts, or to plant defence mechanisms in general. IN THIS WORK, FIVE CANDIDATE GENES PUTATIVELY INVOLVED IN THE ONTOGENIC RESISTANCE OF APPLE WERE IDENTIFIED: a gene encoding an "enhanced disease susceptibility 1 protein" was found to be down-regulated in both uninoculated and inoculated old leaves at 96 hpi, while the other four genes encoding proteins (metallothionein3-like protein, lipoxygenase, lipid transfer protein, and a peroxidase 3) were found to be constitutively up-regulated in inoculated and uninoculated old leaves. The modulation of the five candidate genes has been validated using the real-time quantitative PCR. Thus, ontogenic resistance may be the result of the corresponding up- and down-regulation of these genes.

Granulocyte macrophage-colony stimulating factor induced Zn sequestration enhances macrophage superoxide and limits intracellular pathogen survival.

Macrophages possess numerous mechanisms to combat microbial invasion, including sequestration of essential nutrients, like zinc (Zn). The pleiotropic cytokine granulocyte macrophage-colony stimulating factor (GM-CSF) enhances antimicrobial defenses against intracellular pathogens such as Histoplasma capsulatum, but its mode of action remains elusive. We have found that GM-CSF-activated infected macrophages sequestered labile Zn by inducing binding to metallothioneins (MTs) in a STAT3 and STAT5 transcription-factor-dependent manner. GM-CSF upregulated expression of Zn exporters, Slc30a4 and Slc30a7; the metal was shuttled away from phagosomes and into the Golgi apparatus. This distinctive Zn sequestration strategy elevated phagosomal H⁺ channel function and triggered reactive oxygen species generation by NADPH oxidase. Consequently, H. capsulatum was selectively deprived of Zn, thereby halting replication and fostering fungal clearance. GM-CSF mediated Zn sequestration via MTs in vitro and in vivo in mice and in human macrophages. These findings illuminate a GM-CSF-induced Zn-sequestration network that drives phagocyte antimicrobial effector function.

The analysis of metallothionein immunoreactivity in stromal fibroblasts and macrophages in cases of uterine cervical carcinoma with respect to both the local and distant spread of the disease.

PROBLEM: The tumor microenvironment is made up of tissue that is responsible for the growth and progression of the tumor as well as its ability to initiate metastases. The cancer cells on the front of the tumor together with the macrophages and fibroblasts help to constitute the aggressive phenotype of the tumor. The presence of this aggressive phenotype is indicated by the local infiltration of cancer cells and by the development of lymph node metastases. In cases of uterine cancer, the extent of the local and distant spread of the disease is crucial for determining the type of therapeutic strategy to be applied - surgery alone, surgery followed by radio-chemotherapy, or radio-chemotherapy alone. In the interest of trying to improve the patient's quality of life, different studies supporting the therapeutic model of surgery alone have been conducted. While the cancer cells on the tumor front together with the macrophages and the fibroblasts help to constitute the aggressive phenotype of the tumor, metallothionein (MT) has been shown to have both pro-proliferative and anti-apoptotic activities and to participate in microenvironment remodeling. The aim of the current study was to determine the levels of MT immunoreactivity in the uterine cervical cancer cells as well as in the stromal fibroblasts and macrophages of the tumor microenvironment with respect to the depth of the local invasion and the extent of the distant metastases, so that its potential predictive value as a therapeutic strategy for cervical cancer can be ascertained. METHODS: We determined the levels of immunoreactivity of MT in a total of 57 carcinoma tissue samples (in the tumor front, in its central part, and in the macrophages and fibroblasts present within the tumor microenvironment). The patients from whom the samples derived were divided into groups with respect to the presence of lymph node metastases (distant spread) and to the depth of invasion (local spread) in relation to the FIGO stage. RESULTS: Metallothionein immunoreactivity was observed in uterine cervical cancer cells; it was also identified in the fibroblasts and macrophages found within the microenvironments of the tumors of patients suffering from the disease. The MT immunoreactivity level significantly increased within the whole cancer nest in relation to the FIGO stage (intensity of the local spread of the disease). Similarly, the infiltration of MT-positive CAFs and TAMs statistically significantly increased in relation to the FIGO stage. CONCLUSION: The level of MT immunoreactivity found in the fibroblasts and macrophages within the tumor microenvironment seems to be indicative of the intensity of the remodeled cervical tumor microenvironment, and this in turn may be related to the local advancement of the disease. Moreover, it appears that the intensity of the metallothionein immunoreactivity in the immunoreactivity profile of the cervical tumor may be linked to both the depth of the local invasion and the extent of the distant advancement of the disease.

Development of a high sensitivity ELISA for the assay of metallothionein.

We have developed a competitive ELISA using a polyclonal antibody that showed specificity to both metallothionin (MT)-1 and MT-2 isoforms in human and animal specimens. The advantage of this ELISA depends on the characteristics of the polyclonal antibody. The NH2 terminal peptide of MT with acetylated methionine was shown to be the epitope of this antibody. The reactivity of this ELISA system with liver, kidney, and brain extracts worked very well for MT1,2 in wild type mice. Extracts of MT-3 knock-out mice also react well this ELISA, as expected, very low in MT 1,2 but in MT1/2 knock-out mice. Detection limits, the ranges of linearity, and reliability coefficients of MT quantification of the ELISA were suitable for determination of MT. From the preliminary study of normal reference ranges, we found that normal MT levels were between approximately 10-30 ng/ml in human serum. We expect in the future to detect cases with low (MT deficiency) and high serum MT concentrations in patients with various diseases, such as brain/liver disorders, and cancers, using this MT ELISA.

Glucocorticoids regulate metallothionein-1/2 expression in rat choroid plexus: effects on apoptosis.

The choroid plexus (CP) participates in the synthesis, secretion and regulation of the cerebrospinal fluid, in the removal of its toxic compounds and in the regulation of the availability of essential metal ions to the brain. It expresses and secretes metallothioneins 1/2 (MT-1/2) which are key components in the maintenance of the central nervous system metal homeostasis and have anti-apoptotic properties, thereby protecting the brain. Glucocorticoids regulate MT-1/2 expression in several brain regions, but within the choroid plexuses (CPs) it remains unknown. Glucocorticoid levels increase in response to stress with implications in apoptosis. Further, CP expresses glucocorticoid (GR) and mineralocorticoid receptors (MR) turning it into likely glucocorticoid responsive structure. Data prompted us to study the regulation of MT-1/2 expression in response to glucocorticoids in the rat CP, and to investigate its implications in apoptosis. MT-1/2 protein and mRNA expression analysis showed that hydrocortisone up-regulates MT-1/2 expression in rat choroid plexus (RCP) cell line and in primary cultures of choroid plexus epithelial cells (CPEC) cultures via GR and MR. Also, incubation of RCP cells with hydrocortisone significantly diminished apoptosis, an effect eliminated by the addition of a MT-1/2 antibody. Moreover, induction of psychosocial stress, with concomitant rise of corticosterone levels, increased MT-1/2 expression in liver and in CP of male and female rats, with an exception observed in CP from males subjected to acute stress in which down-regulation in MT-1/2 expression occurred. Altogether, the results obtained demonstrated that stress/glucocorticoids regulate MT-1/2 expression in rat CP, with implications on apoptosis.