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Rad govori o promjeni izražaja heat shock proteina gp96 (imunomodulatora) za vrijeme izloženosti stresu

Introduction: Iron (Fe) is essential element for cellular functions but it can also participate in reactions that produce toxic reactive oxygen species. Recent researches show that disturbance of mechanisms that prevent harmful effects of Fe-induced oxidative stress may contribute to demyelinating process in multiple sclerosis (MS). To analyse those mechanisms we evaluated demyelinating changes and expression of metal-regulating proteins hepcidin and metallothionein I/II (MT I/II) in spinal cord (SC) tissue of Fe-overloaded (FeO) male and female Dark Agouti (DA) rats during the experimental autoimmune encephalomyelitis (EAE), an animal model that closely resembles MS. Since MS is more common among women than men, special attention was devoted to gender-related differences. Aim: To analyse the influence of metal-regulating proteins hepcidin and MT I/II on gender-related differences in demyelination process in iron-overloaded EAE rats. Material and methods: Male and female DA rats were treated by intraperitoneal injections of Fe–sucrose or saline for two consecutive weeks. 24 hours after the last injection animals were immunized by bovine brain homogenate in complete Freund's adjuvant. Untreated groups didn’t have any Fe or encephalitogen treatment. Animals were sacrificed 13 days after immunization and results were obtained in SC tissue by Kluver-Barrera staining, by immunohistochemistry and cell-based staining quantification. Results: The results show that male FeO EAE rats developed greater signs of demyelinating plaque formation in SC than female FeO EAE rats. Female EAE rats reacted on FeO by enhanced expression of hepcidin and MT-I/II in SC neurons in comparison to male rats. Conclusion: The data imply that harmful consequences during FeO and autoimmune attack might be partially restrained by Fe-regulated hormone hepcidin and by cytoprotective activities of MTI/II. Expression profiles of these proteins were different in male and female EAE rats exposed to FeO and this might have contributed to gender-related differences in demyelinating process. Funded by the University of Rijeka, Croatia (projects 13.06.1.1.16, 13.06.2.2.61 and 13.06.1.1.10).

Background. Copper chelator cuprizone (CPZ) is neurotoxicant. which selectively disrupts oligodendroglial respiratory chain, leading to oxidative stress and subsequent apoptosis. Demyelination is, however, followed by spontaneous remyelination owing to the activation of intrinsic CNS repair mechanisms. During this process cysteine rich metallothioneins (MT). which through free radical scavenging and intracellular Zn/Cu regulation provide cytoprotection. Furthermore, it has been postulated that secreted MT-I/II might be bind on surface receptors belonging to the family of low- density lipoprotein receptor related proteins (LRP), such as LRP-2/megalin and LRP-1. which in turn activate the signal transduction pathways that support neurite outgrowth and survival. Aim. The goal of this study was to visualize MT/ megalin interaction in the brain tissue of mice affected by CPZ and to determine if this binding leads to the activation of serine/threonine-protein kinase-AKT-l/Protein kinase B signaling cascade. Methods. Experiments were performed in female C57BL/6 mice fed with 0.25% CPZ during 5 weeks. MT/megalin co- localization and interactions were examined by double immunofluorescence and proximity ligation assay (PLA). which enables in situ recognation of two potentially interacting proteins, respectively. The post-translational modifications in target cells were evaluated by the presence of phosphorylated AKT-I (pAKT-1 ; phospho threonine 308). Results. CPZ-induced demyelination was followed by high astrogliosis and enhanced expression of MTs and megalin in white and gray matter of the brain. PLA clearly showed that MT-l/ll interacted with megalin in cortical tissue and in hippocampal subgranular zone of dentate gyrus. Moreover, in most of megalin expressing cortical NeuN+ neurons nuclear expression of pAKT-1 was found.

The purpose of this study was to examine changes in the polar fatty acid (PL FAs) profile in mice spleen after a one-third partial hepatectomy (PHx) and a diet enriched with olive and corn oil. Fatty acids (FAs) were determined by gas chromatography (GC) after previous fractionation of polar fatty acids by solid-phase extraction using an aminopropyl (NH2) column. The data were analysed using the nonparametric Kruskal-Wallis test and linear regression analysis. A diet supplemented with corn oil (FCO) increased palmitic acid, while an olive oil-enriched diet (FOO) increased arachidonic and docosahexaenoic acid in the spleen PL FAs during PHx. Based on the FAs profile of PL FAs in the spleen during PHx, in the FCO diet group stearoyl CoA desaturase (SCD1) activity showed a positive correlation (R=0.58) with 18:2n-6 as the major FAs in corn oil, while in the FOO group, SCD1 and elongase- 6 (Elovl6) activities positively correlated (R=0.84, R=0.55, respectively) with 18:1n-9 as the major FAs in olive oil. To conclude, despite the beneficial effect of diet, lipid homeostasis in the spleen was regulated more by PHx than the n-6 and n-9 diet.

Background. Copper chelator cuprizone (CPZ) is neurotoxicant, which selectively disrupts oligodendroglial respiratory chain, leading to oxidative stress and subsequent apoptosis. Demyelination is, however, followed by spontaneous remyelination owing to the activation of intrinsic CNS repair mechanisms. To the later contribute also cysteine rich metallothioneins (MT), which through free radical scavenging and intracellular Zn/Cu regulation provide cytoprotection. Besides, it has been postulated that secreted MT-I/II might be bind on surface receptors belonging to the family of low-density lipoprotein receptor related proteins (LRP), such as LRP-2/megalin and LRP-1, which in turn activate the signal transduction pathways that support neurite outgrowth and survival. Aim. The goal of this study was to visualize MT/ megalin interaction in the brain tissue of mice affected by CPZ and to determine if this binding leads to the activation of serine/threonine-protein kinase-AKT-1/Protein kinase B signaling cascade. Methods. Experiments were performed in female C57BL/6 mice fed with 0.25% CPZ during 5 weeks. MT/megalin co-localization and interactions were examined by double immunofluorescence and proximity ligation assay (PLA), which enables in situ recognation of two potentially interacting proteins, respectively. The post-translational modifications in target cells were evaluated by the presence of phosphorylated AKT-1 (pAKT-1 ; phospho threonine 308). Results. CPZ-induced demyelination was followed by high astrogliosis and enhanced expression of MTs and megalin in white and gray matter of the brain. PLA clearly showed that MT-I/II interacted with megalin in cortical tissue and in hippocampal subgranular zone of dentate gyrus. Moreover, in most of megalin expressing cortical NeuN+ neurons nuclear expression of pAKT-1 was found. Conclusion. The data imply that astrocyte-derived MT-I/II modulates cell signaling and neuronal repair through direct contact with megalin and suggest that internalization of MT-I/II and accelerated phosphorylation of AKT-1 contribute to activation of signal transduction pathways that protect cortical neurons and neuronal progenitors against toxic effects of CPZ

Introduction. Demyelinating autoimmune diseases, such as multiple sclerosis and experimental autoimmune encephalomyelitis (EAE) are often followed by cognitive deficits associated with the inflammation, neuronal injury and altered neurogenesis within the hippocampus. Pathogenic mechanisms include the appearance of "danger signals" that contribute to neuronal dyshomeostasis and activation o f innate and adaptive immunity, as well as those that provide neuroprotection and ensure the restoration of tolerance. Aim. To estimate the expression profiles of endoplasmic reticulum (ER)-resident chaperon gp96 and its receptors - CD91 and Toll- like receptor 2 (TLR2) in hippocampi of DA rats during the course of EAE, since these pathways are involved in unfolded protein response (UPR) and F.R-associated degradation of the misfolded proteins, as well as in activation of immune response. Methods: Rats were immunized with BBH in complete Freund's adjuvant (CFA) or only with CFA. The expressions of gp96, CD91 and TLR2 were estimated on days 12 and 22 after immunization by PCR. Western blot and immunohistochemistry. Results: Granular cells constitutively expressed gp96. but its mRNA decreased in early phase of EAE and arose during remission of disease. CD9I and TLR2 genes expression increased in both phases of EAE. but protein expression of CD91 was greater in acute than in late phase of EAE. In contrast. TLR2 protein expression increased particularly in remission phase of EAE. Moreover, in late phase of EAE gp96 was co-localized with CD91 in granular neurons, as well as with TLR2 in numerous doublecortin positive neuroblasts in SGZ of dentate gyros Conclusions: The data point to time-dependent expression of gp96 during EAE and its receptors and imply that during an autoimmune reaction gp96 may through UPR and CD91 and TLR2 signaling affect the reestablishment of hippocampal integrity and adult neurogenesis.

Inflammatory demyelinating diseases such as multiple sclerosis and experimental autoimmune encephalomyelitis (EAE) are often followed by cognitive deficits associated with the neuronal injury, synaptic loss and altered neurogenesis within the hippocampus. Changes depend on the genetic and epigenetic factors that ensure the cellular and environmental homeostasis and regulate the interactions of immunocompetent, glial and neural cells. Owing to high impact of stress proteins on these processes, in this study we compared the protein content of interleukin-6, transforming growth factor-β1, metallothioneins I/II (MTs) and glycoprotein 96 (gp96) in the hippocampus of DA and AO rats that differ in the susceptibility to the induction of EAE, and tested the relationship of MTs and gp96 to granule neurons, glial cells and neural progenitors in different subfields of dentate gyrus. Rats were immunized with bovine brain homogenate emulsified in complete Freund’s adjuvant or only with CFA. The data showed that acute attack of EAE in DA rats was followed by accumulation of IL-6, TGF-β1 and MTs proteins, by increased expression of MTs in molecular and granular cell layer, by reduced expression of gp96/granular cell, by apoptosis and by microgliosis with appearance of Iba-1+ cells, co-expressing MT I/II and gp96. Furthermore, in subgranular zone (SGZ) of DA rats an augmented number of GFAP+ precursors, but decreased number of doublecortin (DCX)+ neuroblasts and immature NeuN+ neurons were found, implying that in DA rats the neurogenesis was delayed or reduced. Besides, in SGZ of both strains several DCX+ and NeuN+ cells co-expressing gp96 and MT I/II were found.

Background/Objectives: Iron is an essential element for cell functions, such as cell proliferation, respiration, folate metabolism and DNA synthesis, having a crucial role in metabolic pathways involved in electron transfer and ATP production. The redox potential of Fe2+/Fe3+ favors its use in a number of protein complexes, but ferrous iron via the Fenton reaction may induce the production of extremely reactive hydroxyl radicals that damage DNA, proteins and lipids. Design/Method: Using experimental autoimmune encephalomyelitis, as an animal model of multiple sclerosis (MS) in this study we analyzed the effects of iron overload on kinetics of disease, iron status and lipid peroxidation. For this purpose female and male DA rats were treated by iron sucrose (75 mg/kg bw/day) or with saline solution during two weeks before the sensitization with bovine brain homogenate in complete Freund's adjuvant. Serum ferritin and tissue contents of iron and malondialdehyde (MDA) were determined in the central nervous system and in the liver on day 13 after immunization. Results: Iron overload in female rats accelerated the onset and first peak of disease, increased the content of ferritin and induced an accumulation of Fe (liver and brain) and MDA (liver). In contrast, in male rats it accelerated the second relapse, augmented iron content (liver) and MDA (spinal cord and brain), and increased the mortality rate. Conclusions: The data point to sexual dimorphism in mechanisms that regulate peripheral and brain iron homeostasis and imply that men and women during MS might be differentially vulnerable to exogenous iron overload.

Background/Objectives: Inflammatory, demyelinating diseases such as multiple sclerosis and experimental autoimmune encephalomyelitis (EAE) are often followed by cognitive deficits associated with the neuronal injury, synaptic loss and altered neurogenesis within the hippocampus. Changes depend on the genetic and epigenetic factors that ensure the cellular and environmental homeostasis and regulate the interactions among immunocompetent, glial and neural cells. Owing to known participation of cytokines and stress proteins in maintenance of this balance, in this study we compared the expression profile of IL-6, TGF-b, metallothionein I/II (MTs) and glycoprotein 96 in the hippocampus of DA and AO rats that differ in the susceptibility to the induction of EAE and estimated the relationship of gp96 and MTs to adult neurogenesis occurring in subgranular zone (SGZ) of hippocampus. Design/Method: EAE was induced by immunization of rats by bovine brain homogenate emulsified in complete Freund's adjuvant. Results have shown that first attack of disease in DA rats was followed by upregulation of IL-6, TGF-b and MTs, reduced cellularity of granule cell layer, lower expression of gp96/granule cell, greater apoptosis and astrogliosis and elevated number of microglial and new neural cells expressing MT I/II and gp96. In SGZ the signs of an aberrant differentiation of neural precursors to doublecortin positive neuroblasts were found. Conclusions: The data show that inflammation, increased apoptosis and reduced or delayed hippocampal neurogenesis might contributed to the development of hippocampal dysfunction in chronic autoimmune CNS diseases, as well as that MT I/II and endoplasmic reticulum resident chaperons participate in the reestablishment of hippocampal integrity

Background: Metallothioneins (MTs) are small, cysteine-rich proteins, which have been implicated in variety of physiological and pathological processes, such as cell proliferation and apoptosis, detoxification of heavy metals and protection against oxidative stress and inflammation. Acting intracellularly, they regulate zinc and redox homeostasis and ensure proper functioning of metal-containing transcription factors, zinc-finger proteins and p53. Neuroprotective functions, however, might be mediated also by extracellular MTs, which signal through low-density lipoprotein family of receptors, such as lipoprotein receptor-1 and -2 (megalin). Design: In this study, we used a cuprizone-induced model of demyelination to examine the expression of MT-I+II, megalin and zinc and copper status in the brain of C57/BL6 mice at the time of massive oligodendrocyte degeneration and reactive astrogliosis (5 weeks after peroral cuprizone treatment). Results: The data have shown that MT proteins were diffusely upregulated in the white matter, as well as in the frontal cortex, hippocampus, subventricular zones and cerebellum. The majority of cells were astrocytes, but MTs were present also in some oligodendrocyte precursors. Upregulation of megalin expression was restricted to cerebral cortex. Surprisingly, qPCR analysis showed that enhanced MT protein expression was not followed by upregulation of MT-I mRNA, suggesting that progressive intoxication with cuprizone had downregulated the transcription of MT gene. The changes were associated with greater copper than zinc dyshomeostasis in the brain. Conclusion: Results show that MT I/II perform important cytoprotective and growth regulating functions in demyelination/remyelinating processes activated in the brain after toxic insults.

Background: Iron is an essential trophic factor that plays a key role in vital cell functions. It is indispensable cofactor for enzymes involved in cell proliferation, respiration, folate metabolism and DNA synthesis, having a crucial role in metabolic pathways involved in electron transfer and ATP production. The redox potential of Fe2+/Fe3+ favors its use in a number of protein complexes, but as a part of the Fenton reaction ferrous iron may also catalyze the conversion of hydrogen peroxide to highly reactive hydroxyl radicals and induce the generation of secondary lipid products that damage DNA, proteins and lipids. To analyze the mechanisms possibly involved in pathogenesis of multiple sclerosis (MS) in this study we evaluated the effects of iron overload (IO) on iron status and lipid peroxidation processes (LPO) in tissues of female and male DA rats during EAE, a well-established MS animal model. Materials and methods: Female and male DA rats were treated by iron sucrose (75 mg/kg bw/day) or with saline solution during two weeks before the sensitization with bovine brain homogenate (BBH) in complete Freund's adjuvant (CFA). Clinical symptoms of EAE were evaluated during the 30 days. Serum and tissues of CNS and liver were sampled for determination of ferritin, iron, malondialdehyde (MDA) and 4-hydroxynonenal (4-HNE) before immunization and during acute phase of EAE. The results were obtained by ELISA, ICP spectrometry and immunohistochemistry and analyzed by one way ANOVA and Kruscal Wallis tests. Results: In female EAE rats IO accelerated the onset of disease, while in male rats it accelerated second relapse and increased the mortality rate. During acute phase of EAE female IO rats sequestered more Fe in the liver, spinal cord and in the brain and produced more ferritin than male EAE rats. Male rats, however, reacted on IO by higher production of MDA or 4-HNE in the neural tissues and showed greater signs of plaque formation and gliosis in spinal cord. Conclusion: The data point to sexual dimorphism in mechanisms that regulate peripheral and brain iron homeostasis and imply that men and women during MS might be differentially vulnerable to exogenous iron overload.

Glycoprotein 96 (gp96) is an endoplasmic reticulum (ER)-resident heat shock protein. It controls the folding of nascent membrane-spanning and secretory proteins, participates in stress-induced unfolded protein response (UPR) and in pathways leading to proteolysis of damaged proteins through ER-associated degradation pathways and chaperone-mediated autophagy. In addition, gp96 controls the steroid biosynthesis and Ca2+ homeostasis and participates in insulin-IGF/signaling pathways. Besides, owing to its peptide chaperone capacity and ability to interact with antigen-presenting cells, gp96 has been implicated in priming of innate and adaptive immunity. In an attempt to visualize the intensity of ER-stress in thymus and possible participation of gp96 in generation of auto-reactive T cell clones that were detected in regenerating liver, in this study we investigated the dynamics of gp96 expression in partially hepatectomized (pHx) and sham Hx mice. Simultaneously, we detected the thymic expression of receptors responsible for endocytosis of gp96- chaperoned peptides (CD91) and intracellular activation of ER-stress pathways (TLR2), as well as the expression of TGF-β and the distribution of CD4+CD25+FoxP3+ cells. The data have shown that both pHx and sham Hx induced an accelerated apoptosis and hypoplasia in thymus. Partial Hx induced, however, a higher expression of gp96, the translocation of the CD91, TLR2 and TGF-β immunostaining from medulla to cortex and an appearance of Treg cells. The data show that pHx triggers in thymus the ERstress and UPR response and suggest that gp96 participates in the generation of natural Treg cells, which might be involved in the control of liver regeneration in the periphery.

Metallothioneins (MTs) are phylogenetically old cysteine-rich proteins, which may be found in all eukaryotes and some prokaryotes. Owing to high affinity for physiological metals, such as zinc and copper, they are critical components of zinc-finger transcription factors and other regulatory proteins involved in cell growth and multiplication. Besides, providing cytoprotective action against toxic heavy metals and oxidative damages of DNA, proteins, and lipid membrane structures, they participate in anti-apoptotic and anti-inflammatory pathways, as well as in immune regulation and carcinogenesis. The aim of the study was to investigate the expression of MTs in cholecystitis induced by gallstones and to correlate it with intensity of inflammation and liberation of zinc ions. Patients and methods: Gallbladder tissue samples were obtained from 25 patients subjected to laparoscopic cholecystectomy owing to the symptomatic cholecystolithiasis. According to the standard pathohistological findings and NF-B expression the tissue changes were classified as subacute and chronic cholecystitis and the expression of MT-I/II isoforms and free zinc content were visualized by monoclonal anti-MT I+II antibodies and zinc fluorophore-Zinquin. Differences between groups were assessed by Mann-Whitney U test after cell-based staining quantification of MT and Zn expressions by Cell F v3.1 software. Results: The data have shown that chronic cholecystolithiasis is followed by overexpression MTs and accumulation of labile zinc ions in gallbladder tissue (epithelium, vascular endothelium, stromal cells). Besides, in area of inflammation were found several CD3+ cells in contact with MT+ epithelial, endothelial and monocyte-like cells, as well as the CD3+/MT+ cells. Conclusion: The data point to regulatory roles of MT/zinc network in chronic pro-inflammatory environment in gallbladder tissue induced by gallstones.

Background: Iron is an essential trophic factor that plays a key role in vital cell functions. It is indispensable cofactor for enzymes involved in cell proliferation, respiration, folate metabolism and DNA synthesis, having a crucial role in metabolic pathways involved in electron transfer and ATP production. The redox potential of Fe2+/Fe3+ favors its use in a number of protein complexes, but as a part of the Fenton reaction ferrous iron may also catalyze the conversion of hydrogen peroxide to highly reactive hydroxyl radicals that damage DNA, proteins and lipids. Therefore, the maintenance of iron homeostasis in the body and in the cells must be balanced, to ensure enough iron for the metabolism, but to avoid excessive, toxic levels of iron that provoke oxidative tissue injuries. Since the regulation of iron homeostasis is a prerequisite also for normal neurological function, in this study we analyzed the effects of iron overload on the lipid peroxidation processes (LP) in tissues and on kinetics of experimental autoimmune encephalomyelitis (EAE) in female DA rats. Material and methods: Female Dark Agouti (DA) rats, aged 2-3 months, were immunized by subcutaneous injection of bovine brain white matter homogenate (BBH) in complete Freund’s adjuvant (CFA). Animals were then subdivided into: 1) iron treated and 2) saline treated group. Experimental group consisted of iron overloaded rats, treated by intraperitoneal injections of iron sucrose (Venofer) at the dose of 75 mg/kg bw (6 times/week) for two consecutive weeks, before the immunization of rats. Control group consisted of rats treated by an equivalent volume of sterile saline. The severity of disease was clinically assessed by standard criteria (0-4). Animals were sacrificed by exsanguinations on day 13th after the active induction of disease. Tissue concentrations of Fe2+ and LP in the brain, spinal cord and liver were determined by PHILIPS PU 7000-ICP spectrometer (method ASTM D 19756-91 at a fixed wavelength of 259.940 nm) and by estimation of malondialdehyde (MDA) concentration (nmol/mg of tissue weight) based on the reaction with the thiobarbituric acid, respectively. Differences between groups were assessed by Mann-Whitney U test. Results: The data showed that iron overload markedly augmented the tissue concentration of iron in the brain and spinal cord, as well as in the liver (p

Background: Inflammatory demyelinating diseases, such as MS and experimental autoimmune encephalomyelitis (EAE) are often followed by cognitive deficits associated with the neuronal injury and synaptic loss within the hippocampal CA1 pyramidal layer and an altered neurogenesis in the subgranular zone (SGZ) in the dentate gyrus (DG). Changes have been related with an imbalance in cellular and environmental homeostasis that is normally created by the continuous interaction of immune competent cells, glial cells and resident neural cells. Since, in the maintenance of CNS homeostasis participate metallothioneins (MTs) and heat shock proteins (HSPs), which protect the neurons from metal, free radical, cytokine-induced and proteotoxic injuries and contribute to the repair of neural tissues, in this study we analyzed the expression of MTs and endoplasmic reticulum (ER) resident HSP-gp96 in the hippocampus of Dark Agouti (DA) and Albino Oxford (AO) rats that differ in the susceptibility to induction of EAE. Methods: AO and DA rats were immunized with bovine brain homogenate (BBH) emulsified in complete Freund adjuvant (CFA) or only with CFA. The expression patterns of hippocampal MT I+II proteins, gp96, IL-6, NF-B and transforming growth factor (TGF)-ß were estimated on day 12 after immunization (when EAE-prone DA rats develop first attack of clinical symptoms), as well as in intact AO and DA rats, by immunohistochemistry, cell-based staining quantification with Cell F v3.1 software and by Western blot analysis in the lysates of the whole hippocampal tissue. Differences between groups were assessed by Mann-Whitney U test. Results: The data showed the hippocampal content of induced MTs, Il-6 and TGF-ß proteins was significantly greater in EAE-prone DA rats than in EAE-resistant AO rats, but that AO rats had greater constitutive expression of these proteins in intact hippocampus. Immunohistochemical data revealed that these changes were distributed mostly in the SGZ in DG, as well as that in DA rats immunized with encephalitogen increased also the nuclear expression of NF-B in some granule cells and the cytoplasmic expression of TGF-ß on some cells in the hilus. Furthermore, the data obtained by immunohistochemistry suggested that the expression of gp96 in granular layer of DA rats was downregulated in comparison with AO rats, but the total protein content of gp96 in the whole hippocampal tissue was not significantly different. Conclusions: The data show that autoimmune attack during EAE is followed by marked changes in the expressions MTs, ER-resident HSPs, IL-6 and TGF-ß in the hippocampal regions responsible for the adult neurogenesis, suggesting also that the interplay of these factors contribute to the different susceptibility of DA and AO rats to the induction of EAE.

Background: Albino Oxford (AO) rats in comparison to the Dark Agouti (DA) strain exhibit lower susceptibility to the induction of EAE. Mechanisms include the differences in peripheral response to immunization and those linked with the CNS milieu, which contribute to initiation and limitation of the injury. In a search for peripheral factors related to these differences, herein we estimated the role of the liver in the pathogenesis of EAE, analyzing the expression pattern of cysteine rich proteins-metallothioneins (MT) I+II that maintain the metal ion homeostasis and of endoplasmic reticulum (ER)-resident heat shock protein (HSP)-gp96, which have marked anti-inflammatory, immunoregulatory and cytoprotective properties. Besides, since the differentiation of naive T cells into the regulatory T or into the pathogenic Th17 cells may be regulated particularly by the transforming growth factor (TGF)-ß and its combination with IL-6, in this study we compared also the hepatic levels of these two cytokines in EAE prone and EAE-resistant rats. Methods: AO and DA rats were immunized with bovine brain homogenate (BBH) emulsified in complete Freund adjuvant (CFA) or only with CFA. The expression pattern of hepatic MT-I mRNA and MT I+II proteins, gp96 and cytokines were estimated on day 12 after immunization, as well as in intact AO and DA rats, by immunohistochemistry, real time PCR and Western blot analysis. Results: AO rats, injected with BBH+CFA have not shown any clinical sign of EAE, in contrast to DA rats, which developed a typical chronic-relapsing form of disease, characterized by two peaks of clinical symptoms (on the 12th and the 22nd postimmunization day). Herein, we show that these rats significantly differ also in the constitutive and inducible expression of MTs, gp96 and cytokines in the liver. Thus, during the first attack of EAE the significant upregulation of MTs and gp96 was found only in EAE-prone DA rats, where the levels of MT I+II and Gp96 proteins were three times greater that those in the intact liver (p

Metallothioneins (MTs) are evolutionary old, low molecular weight proteins rich in cysteine residues. Owing to a large number of thiolate groups within their primary structure, they can rapidly bind and release metal ions, acting thus as important regulators of numerous metaloenzymes and transcription factors involved in cell proliferation, differentiation and apoptosis. Additionally, thiolate groups can be easily oxidised, what provide to MTs the ability to act as one of the most potent endogenous antioxidant with strong anti-inflammatory effects. Induced by variety of stressful events, MTs therefore represent an importnat protective response to etiologically heterogeneous forms of tissue injuries, promoting at the same time regenerative mechanisms. Since the aging process is accompanied with the accumulation of reactive oxygen species-induced damages, as well as with decreased ability of regulated cell proliferation, the aim of this study was to compare metallothioneins expression in the tissues of young (aged 2 months) and old (aged 2 years) mice. Immunohistochemical staining showed increased expression of MTs in almost all analyzed tissues of old mice in relation to the tissues of young animals. The exceptions were testicular tissue, in which the higher expression was found in young mice, and the skeletal muscle, with no notable differences. Data suggest that aging-induced MTs, given to their well-known anti-oxidative and anti-inflammatory properties, can be considered as an endogenous, adaptive and protective response during physiological aging process.

To investigate the effects of air pollution related with gasoline/petrochemical industry the expression of metallothionein I (MT-I) mRNA and tissue metals were analyzed in organs of mice, exposed to the gasoline (G) vapor in laboratory conditions. Control groups consisted of intact mice and of those exposed in the metabolic chamber to fresh air. The data obtained by RT-PCR and inductively coupled plasma spectrometry have shown that exposure to G vapor leads to upregulation of MT-I mRNA in organs that receive a strong respiratory and olfactory input or participate in gasoline degradation and elimination (lungs, brain, kidney and liver). Simultaneously, the tissue content of Zn2+ decreased in the brain and the concentration of Cu2+ and Mg2+ in the brain, lungs, kidney and liver (p

Background: Liver regeneration that follows partial hepatectomy (pHx) is a well-known model for investigating mechanisms related with the control of normal growth and maintenance of morphostasis. The process is characterized by 1) initial priming phase, linked with cytokine induction, 2) proliferative phase, related with growth-factor-induced progression of cells through the G1 restriction point and 3) growth termination phase, induced by inhibitory factors TGF and IL1. Activating and inhibiting signals come from the parenchymal and nonparenchymal cells in the liver, but also from the peripheral organs and the thymus, showing that regeneration of the liver is regulated by cooperative signals from various sources. Moreover, in the control of reparatory processes participate the autoreactive lymphocyte clones with regulatory and protective effects, such as CD1-restricted NKT cells that express a semi-invariant T-cell receptor specific for conserved self ligands and CD4+CD25+Foxp3+ regulatory T cells (TReg) cells, which may have a strong influence on reestablishment of self-tolerance after disturbance of morphostasis. Material and methods: In an attempt to enlarge our previous findings, pointing to the involvement of endoplasmic reticulum (ER)-resident heat shock proteins (HSP) in these events, in the thymus of mice subjected to pHx we estimated the expression of gp96, CD91, TLR2 and TGF-, and the distribution of TReg cells and cells reactive to Dolichos biflorus agglutinin (DBA) lectin. The data, obtained by immunohistochemistry and real-time PCR (for the detection of gp96 mRNA) were compared with findings in intact and sham operated mice. Results: The data have shown that 1/3 pHx in C57/BL6 mice leads to high upregulation of gp96 in the thymic cortex (on stromal and epithelial cells and some lymphatic cells), and to the enhanced expression of its receptors - CD91 and TLR2 that are responsible for endocytosis of gp96-chaperoned peptides and for the intracellular activation of ER-stress pathways, respectively. The highest increase in the expression of gp96 protein was observed 48h after pHx, i.e. at the time when the high apoptosis of thymocytes was also seen. At the transcriptional level, two peaks of gp96 mRNA were detected (at 2h and at 48h after pHx). Additionally, it has been found that pHx induces a marked upregulation of TGF- immunoreactivity in cortical areas. Simultaneously, in thymic cortex increased the number of DBA+ cells that are specific for terminal alpha-linked N-acetyl-D-galactosamine residues. Several of these cells were in direct contact with cells expressing gp96 or exhibited the cytoplasmic gp96 immunopositivity. Furthermore, 48h after pHx, in the cortex of thymus, numerous CD4+/Foxp3+ and CD25+/Foxp3+ T cells were found, indicating that pHx has stimulated also the generation of regulatory T cells. Conclusion: The data imply that liver regeneration is followed by breakdown of central tolerance to self-antigens and show that ER-resident HSP-gp96 may participate in ER-quality control and in the interactions that determine the positive and negative selection events during normal thymocyte differentiation. Mechanisms are unclear, but there is a high possibility that gp96 participated in the maintenance of immunologic self-tolerance, acting as a chaperon of self-peptides and/or as a factor responsible for the delivery of co-stimulatory signals by classical and non-classical APC in thymic epithelial network. The data point also to the importance of glycosylation in various steps of T-cell development and suggest that upregulated TGF- in the thymic cortex may contribute to the positive selection of TReg cells, which during liver regeneration might be involved in the restrain the fast liver growth and in the suppression of transient autoimmune events activated by pHx.

Albino Oxford (AO) rats, compared to the Dark Agouti (DA) strain, exhibit lower susceptibility to the induction of experimental autoimmune encephalomyelitis (EAE). Mechanisms include the differences in peripheral response to immunization and those linked with the CNS milieu, which contribute to limit the injury. In the search for factors related to these differences, previously we found that these strains significantly differ in the constitutive gene and protein expression of the cysteine rich proteins-metallothioneins I and II (MTs) that maintain the metal ion homeostasis and have marked anti-inflammatory and neuroprotective properties. In attempt to enlarge these findings, in this study we correlated the protein expression of MTs with tissue concentrations of Zn+2, Cu+2 and Fe+2 in the brain and its regions, which were highly sensitive to autoimmune attack - i.e. in hippocampus, cerebellum and lumbar spinal cord. Rats were immunized with bovine brain homogenate (BBH) emulsified in Freund's Complete Adjuvant (CFA) or with CFA only. On days 7 and 12 after immunization the tissue concentrations of Zn2+, Cu2+ and Fe2+ were estimated in the whole brain and its regions, and in the spinal cord by inductively coupled plasma spectrometry. AO rats did not show any clinical signs of EAE after immunization with BBH+CFA, but MTs protein expression were upregulated in some regions of CNS. This strain of rats had, however, greater basal levels of Zn+2 and Cu+2 in hippocampus and lower concentrations of Zn+2, Cu+2 and Fe+2 in spinal cord. Besides, in comparison to EAE-susceptible DA rats they were able to better maintain the metal homeostasis in the brain and spinal cord tissue after immunization. The data point to genetic differences in the mechanisms that during the autoimmune attack through activation of MTs and metal uptake or release ensure protection of the most sensitive brain regions against oxidative and nitrosative injuries.

To elucidate the role of metallothioneins (MTs) in the pathomechanisms of autoimmune CNS disorders we estimated the expression of MTs I+II and the tissue concentrations of Zn2+ and Cu2+ in the brain, spinal cord (SC) and in the liver during the periods of attacks and remissions in chronic relapsing experimental autoimmune encephalomyelitis (CR-EAE). Disease was induced in the genetically susceptible Dark Agouti (DA) rats by subcutaneous injection of bovine brain homogenate in CFA. Control rats were treated with CFA or with saline solution. The data, obtained by clinical assessment, immunohistochemistry and inductivity coupled plasma spectrometry, have shown that during the first attack (12th day) MTs I+II were markedly upregulated in subarachnoid regions and perivascular space on astrocytes, microglia and spinal neurons. Simultaneously, increased the concentration of zinc in the SC and zinc and copper in the liver. During the second attack (22nd day) a new overexpression of MTs was found in the cerebellum, in sulcus hippocampi, in spinal neurons and particularly on hepatocytes around the central vein. Concomitantly, in the brain and SC increased the concentration of copper. The data point to neuroprotective role of MTs and to important regulatory role of essential metals and hepatic MTs in the pathogenesis of CR-EAE

Osteopontin (Opn) is an O-glycosylated phosphoprotein that is synthesized in a variety of tissues and cells and has pleiotropic functions including roles in inflammation and in immunity. Secreted form of Opn acts as a Th1 cytokine and as a chemoattractant for many types of cells through integrin receptors and CD44. Recently, it has been shown that intracellular form of Opn is a critical regulator for TLR-9, TLR-7-dependent interferon-α expression by plasmacytoid DCs and Th17 development. Furthermore, OPN expression is frequently up-regulated in response to various stressors, when it promotes cell survival. Additionally, in inflammatory situations OPN stimulates both pro- and anti-inflammatory processes, what can be either beneficial or harmful depending on other inputs on the cells. In this study we estimated the expression of OPN in the brain, spinal cord, liver and the thymus early in the course of chronic relapsing experimental autoimmune encephalomyelitis (CR-EAE) pathogenesis, i.e. before the onset of any clinical symptoms. Disease was induced in the genetically susceptible Dark Agouti (DA) rats by subcutaneous injection of bovine brain homogenate in complete Freund’s adjuvant (CFA). Control animals were treated with CFA alone. The data obtained by immunofluorescence have shown that in the presymptomatic phase of CR-EAE (on the seventh post-immunization day) OPN was markedly upregulated in the brain parenchyma and blood vessels, as well as in the spinal cord neurons. Furthermore, we found that shortly after immunization OPN was also induced in the liver and the thymus. The data point to the role of OPN early during the CR-EAE development, in the CNS as a target tissue, but also in the liver, as the site of acute phase response and in the thymus, as a place of autoreactive clones generation.

To elucidate the role of iron in the pathomechanisms of autoimmune CNS disorders we estimated the tissue concentrations of Fe2+ in the brain, spinal cord (SC) and liver in monophasic and chronic relapsing (CR) forms of experimental autoimmune encephalomyelitis (EAE), induced in Dark Agouti (DA) strain of rats, by subcutaneous injection of myelin basic protein (MBP) in complete Freund’s adjuvant (CFA) or by bovine brain homogenate (BBH) in CFA, respectively. Control rats were treated with CFA or with saline solution. The data obtained by clinical assessment and by inductively coupled plasma spectrometry have shown that in both monophasic EAE and in CR-EAE the attacks of disease were followed by high accumulation of iron in the liver (on the 12th post-immunization day, and on the 12th and the 22nd day). Simultaneously, in monophasic EAE decreased the concentration of Fe2+ in the brain (during the remission phase) and in the cervical spinal cord (during attacks and remission). Similarly, in CR-EAE the lower iron content was found in cervical spinal cord during the second attack of disease. The data point to regulatory effects of iron and hepatic trace elements in the pathogenesis of EAE.

Background: Heat shock protein gp96, has been reported to play important role in the endoplasmic reticulum stress response, acting as a molecular chaperone in the unfolded protein response. Additionally, owing to it’s ability to bind protein and nonprotein molecules with exposed hydrophobic residua, it’s function in antigen presentation and cross-presentation, activation of macrophages and lymphocytes, and activation and maturation of dendritic cells has been also proposed Material and methods: In this study we analyzed the expression of gp96 in the regenerating liver, thymus and spleen after 1/3 partial hepatectomy (pHx) in C57/Bl6 mice. The data, obtained by immunohistochemistry and quantitative real-time PCR, were correlated with phenotype and cytotoxicity of hepatic and splenic mononuclear lymphatic cells against NKT and NK-sensitive targets, as well as with the intensity of apoptosis in the liver and thymus. Results: High time-dependent changes were found in the expression of gp96 protein and mRNA in regenerating liver, thymus, and spleen. Simultaneously, in the liver and thymus augmented the proportion of autoreactive NKT and regulatory T cells, increased the cytotoxicity against the syngeneic thymocytes and augmented the apoptosis in the liver and thymus. Additionally, in thymus and spleen a marked upregulation of TLR2 was noticed. Conclusions: The data suggest that after tissue damages and disturbance of morphostasis gp96 may serve as a natural adjuvant for chaperoning antigenic self peptides and nonprotein molecules into the immune surveillance pathways, resulting in activation of autoreactive NKT and Treg cells that might participate in reestablishment of self tolerance.

Metallothioneins (MTs) are small, cysteine-rich proteins that have been implicated in various forms of stress providing cytoprotective action against oxidative injury, DNA damage and apoptosis. Owing to their high affinity for physiological metals, such as zinc and copper MTs are also critical components of multiple regulatory proteins involved in cell growth and multiplication, as well as in the maintenance of immune homeostasis, affecting the functions of immune cells, TLR signaling, upregulation of co-stimulatory molecules on antigen-presenting cells (APC) and the production of pro-inflammatory cytokines. Therefore, their expression may be induced through several response elements in the MT gene promoter: 1) metal response elements, which are activated by the metal-responsive transcription factor-1 after zinc occupancy, 2) glucocorticoid responsive elements activated by stress, 3) elements activated by signal transducers and activators of transcription proteins through cytokine signaling and 4) the antioxidant (or electrophile) response element (ARE) activated in response to redox status. The extensive data show that these proteins confer a protective effect also within the mammalian CNS, where the MT I+II expression dramatically increases in response to many types of CNS and spinal cord (SC) injury or ischemia, as well as in senescence and in several neurodegenerative and autoimmune diseases. To obtain an insight into the changes of MT-trace metal circuit during the development of an autoimmune disease, in this study we correlated the expression of MT I+II in the brain, SC and liver with total zinc and copper content in these organs, during clinically different stages of chronic relapsing (CR) experimental autoimmune encephalomyelitis (EAE): 1) in early, asymptomatic phase (on the 7th post-immunization day ; 2) during periods of attacks (on the 12th and the 22nd day), and 3) during the period of remissions of EAE (on the 18th and the 28th day). Disease was induced in the genetically susceptible DA rats by subcutaneous injection of encephalitogen (bovine brain homogenate in CFA). Rats in the control groups were treated only with CFA. MTs I+II and tissue metals were evaluated by immunocytochemistry and by inductively coupled plasma spectrometry, respectively. Additionally, in the asymptomatic stage of EAE the free intracellular Zn2+ content in the brain and SC were visualized by Zinquin (a selective Zn 2+ fluorophore). The data revealed an early upregulation of MTs and free Zn2+ content in the brain and SC in preclinical phase of disease and their marked overexpression during both attacks at sites of invasion of myelin-reactive T cells into CNS and their reactivation on local APC (on endothelial cells, ventricular subependyma, microglia/macrophages and astroglial cells around the ventricle walls and blood vessels), as well as in the molecular layer of cerebellum, in subgranular layer of gyrus dentatus, and on several neurons and oligodendrocytes in SC. Simultaneously, increased the content of free Zn2+ at these sites and the total Zn2+ content in the cervical SC. Furthermore, during attacks in the liver markedly arose the expression of MT I+II and tissue concentrations of total zinc and copper. The data point to neuroprotective role of MTs and to important regulatory role hepatic MTs in pathogenesis of EAE (Supported by grant 0621341-1337 from Croatian Ministry of Science).

Background: CR-EAE, which may be induced in genetically susceptible Dark Agouti rats, mimics many of the clinical and immunopathological features of multiple sclerosis in humans. The triggering event is invasion of peripherally activated myelin-specific immune cells in CNS, where they interact with APC and microglial cells that drive the inflammatory cascade leading to tissue damage and an amplification of the initial immune reaction. The final outcome may, however, depend on the presence of metallothioneins (MTs)-cysteine-rich heavy metal binding proteins, which have marked anti-inflammatory and neuroprotective properties. Material and methods: Since, previously, we found that attacks of CR-EAE were followed by enhanced expression of MT-I+II in both CNS and in the liver, in this study we estimated their expression before the appearance of clinical symptoms, i.e. on the 7th day after immunization of rats with bovine brain homogenate+CFA or only with CFA. Simultaneously the tissue concentrations of Zn2+ and Cu2+ were evaluated by inductively coupled plasma spectrometry. Results: Prior to any clinical manifestation of disease in the liver markedly arose the expression of MT I+II and tissue concentrations of zinc and copper. Simultaneously, in the brain increased MTs immunoreactivity, particularly on cells that form blood-brain and the blood-cerebrospinal fluid barrier and provide stimulatory signals for T cells invasion (endothelial cells, ventricular subependyma, microglia/macrophages and astroglial cells around the ventricle walls). Conclusions: The data imply that hepatic MT I+II play pivotal roles in early pathogenesis of EAE, owing to their involvement in Zn and Cu homeostasis and in mechanisms that maintain tolerance to self antigens.

The data have shown that prior to any clinical manifestation of disease in the liver markedly arose the expression of metallothioneins I+II and tissue concentration of zinc and copper. Simultaneously, in the brain increased MTs immunoreactivity on cells that form blood-brain and blood cerebrospinal fluid barrier.

Multiple sclerosis is a chronic, autoimmune disease of the central nervous system, which mainly affects young women during a reproductive period of life. Since, its symptoms might be significantly affected by pregnancy, in this study we investigated the development and kinetics of disease in the model of chronic relapsing experimental autoimmune encephalomyelitis (CR-EAE), induced in genetically susceptible Dark Agouti (DA) strain of rats. They were sensitized with bovine brain white matter homogenate (BBH) in complete Freund's adjuvant during the first, second or third week of pregnancy, and the disease scores were compared between treatment groups, and identically treated nongravid females. Additionally, the susceptibility to the induction of EAE was tested in offspring of mothers that during the pregnancy were sensitized with BBH. The data have shown that pregnancy does not block the induction of EAE, but that it significantly changes the course of diseases, depending on time of immunization. In rats sensitized during the first week of gestation the onset of the clinical signs was delayed, but after the delivery the intensity of disease significantly increased. Similar aggravation, with appearance of monophasic form of disease was observed in the group of rats sensitized during the third week of gestation. On the contrary, in rats sensitized during the second week of gestation the beneficial effects were observed, with later onset of attacks, and lower disease score. Furthermore, offspring of these rats after immunization with BBH developed a monophasic form of EAE of lower intensity, suggesting that some protective factors might be transferred across the placenta.

Heat shock proteins (HSPs) are phylogenetically conserved proteins, present in all prokaryotes and eukaryotes that are involved in various physiological and pathological processes. According to their function and size, or cellular localization, they are classified into major classes (small HSPs, HSP40, 60, 70, 90, and 110 families) and several members of a family. Gp96 (known also as glucose-regulated protein 94) is the endoplasmic reticulum (ER)-resident protein belonging to the HSP90 family. It is upregulated in response to glucose starvation and other stressful stimuli that disrupt protein synthesis in the ER, acting as a molecular chaperon involved in the correction of unfolded proteins, in the activation of proteasome-dependent ER-associated degradation of the misfolded proteins and in activation of protein translation that modulate the polypeptide traffic into the ER. In addition, it has been implicated in antigen presentation and MHC class I and II upregulation, in the activation and maturation of dendritic cells and proinflammatory cytokine secretion, as well as in chaperoning of Toll-like receptors, acting as a "danger signal" to the innate and adaptive immunity. Moreover, owing to its specific function in Ca2+ homeostasis and in the insulin-IGF/signaling pathways it was proposed that gp96 participates in mechanisms that are critical for cell growth, differentiation and responses to ER stress. To underline some aspects of these functions, in this survey we will present the data showing the expression of gp96 in the conditions of: 1) normal growth (liver regeneration after partial hepatectomy, syngeneic pregnancy, fetal organogenesis), 2) stress and ageing and 3) autoimmunity (chronic relapsing experimental autoimmune encephalomyelitis ; CR-EAE). Tissue expression of gp96 protein and mRNA was estimated in the liver, thymus and spleen and the data were correlated with phenotype and cytotoxicity of hepatic and splenic mononuclear lymphatic cells against the NKT and NK-sensitive targets. In the model of CR-EAE gp96 expression in the brain was correlated with the intensity of clinical symptoms. The data have shown that gp96 is highly upregulated in fastly proliferating and remodeling tissues (in regenerating liver, at the fetoplacental barrier, during fetal organogenesis), as well as in healing tissues after autoimmune attack (in the brain and spinal cord during the remission phases of EAE). Enhanced level of gp96 expression was also found in the thymus of aged and stress-exposed mice. Moreover, the kinetic studies made in the model of liver regeneration showed that gp96 upregulation was followed by maturation of dendritic cells, accumulation of CD3intermediate/NK1.1+/CD69+cells in the liver and CD4+CD25+Fox3+ cells in the liver and thymus, as well as by augmentation of NKT and NK-mediated cytotoxicity in the liver and in the spleen. The data imply that during the disturbance of morphostasis gp96 may serve as a natural adjuvant for chaperoning antigenic self peptides into the immune surveillance pathways, resulting in activation of autoreactive NKT, which in cooperation with Treg cells participate in more or less successful reestablishment of self tolerance (Supported by grant 0621341-1337 from Croatian Ministry of Science).

The data revealed that prior during attack of CR-EAE significantly diminished the gp96 constitutive expression, found in several neurons and gill cells in the brain and spinal cord of control animals. On the contrary, the remission of disease was followed by upregulation of gp96 expression in the brain.

Glucose regulated proteins, such as Grp94 (gp96), Grp78 (BiP), Grp58, calnexin and calreticulin are located primarily in the rough endoplasmic reticulum, where they interact with many secretory and membrane proteins during course of their maturation, functioning as chaperones that monitor the folding and assembly of glycoproteins. In addition, when released in forms of complexes with self peptides from damaged, infected, stressed or transformed cells, they may act as endogenous danger-associated signal that participates in stimulation of innate immune system and in generation of both-antigen specific and non-specific T cell response. Hypothesizing that gp96, as natural carrier of self-peptides may participate in the activation of self-reactive CD1-dependent NKT cells and specialized CD25+CD4+ T regulatory cells, in this report we investigated the tissue expression of gp96 in the thymus, liver and spleen: 1) after breakdown of morphostasis induced by partial hepatectomy and 2) in psychosocial stress induced by closure of mice in metabolic chamber. The data, evaluated by immunohistochemistry and real time PCR, were correlated with phenotype and cytotoxicity of hepatic and splenic mononuclear lymphatic cells (MNLC) against the NKT- and NK-cells sensitive targets. In both experimental models high overexpression of gp96 was found in the liver, as well as in the thymus (in the cortex and at cortico-medullary junction). Simultaneously, in the liver accumulated CD3intermediate/NK1.1+/CD69+cells and CD4+CD25+FoxP3+ cells, while hepatic and splenic MNLC became highly cytotoxic against syngeneic thymocytes and YAC-1 targets, implying that during the disturbance of morphostasis gp96 probably acts as a natural adjuvant for chaperoning of self peptides into the immune surveillance pathway, leading to activation of autoreactive clones NKT and Treg cells that participate in reestablishment of self tolerance.

Aim: In the genetically susceptible Dark Agouti rats (DA) experimental autoimmune encephalomyelitis may be induced in chronic relapsing form (CR-EAE) that resembles to multiple sclerosis, which is an inflammatory, demyelinating disease the central nervous system. It is caused by erroneous activation of self-reactive T cells specific for myelin antigen, but the severity of symptoms depends on the balance between the aggressive and the localized, or systemic protective tissue reaction to infection or injury. Methods: Since in the pathogenesis of disease a significant role may have the metallothioneins (MTs), which are the antioxidant proteins with high metal-binding properties, in this study we estimated the expression of MTs I+II, as well as the tissue concentrations of Zn2+ and Cu2+ in the brain, spinal cord and liver during the first and second attack (on the 12th and 22nd post-immunization day, respectively) and during the remission phases (on the 18th and 28th day) of CR-EAE, induced in DA rats by subcutaneous injection of encephalitogen (bovine brain homogenate in complete Freund’ s adjuvant). Controls consisted of rats treated only with CFA. Clinical assessment was performed according to the standard criteria, while MTs and metal ions were analyzed by immunocytochemistry and inductivity coupled plasma spectrometry, respectively. Results: The data showed that during the first attack MTs I+II were upregulated in the brain (subpial region, perivascular space and parenchymal astrocytes) and in the spinal cord (glial cells and neurons). Simultaneously, increased the concentration of Zn2+ in the spinal cord and Zn2+ and Cu2+ in the liver. During the second attack a very high, new overexpression was found in the molecular layer of cerebellum, in sulcus hippocampi, on several neurons and oligodendrocytes in spinal corn, and particularly on hepatocytes around the central vein. Simultaneously, in the brain and spinal cord increased the concentration of Cu2+. Conclusions: The data point to neuroprotective role of MTs and to important regulatory role of essential metals and hepatic MTs for the recovery from acute attacks of EAE.

Objectives. The hepatoprotective effect of luteolin was poorly studied. Literature overview shows that most of the studies on luteolin actions have been performed in vitro. Carbon tetrachloride (CCl4) is a well-known model compound for producing chemical hepatic injury. This study investigated the protective effects of the flavonoid luteolin on the CCl4-induced hepatotoxicity in mice. Methods. Luteolin dissolved in DMSO was administered intraperitoneally (i.p.) at 5 or 50 mg/kg as a single dose, and once daily for 2 consecutive days, respectively. Two hours after the final treatment, the mice were treated with CCl4 (20 mg/kg, i.p.). The mice were killed 24 hours after administrating CCl4. Results. CCl4-induced hepatotoxicity was reduced in a dose- and time-dependent manner, as determined by decreased serum aminotransferase activities and liver histopathology. CCl4 administration caused extensive centrilobular necrosis with loss of hepatocyte structure, vacuolar fatty change and marked inflammatory cell infiltration. Luteolin pretreatment greatly reduced necrosis, inflammatory infiltration and fatty changes. CCl4 intoxication resulted in an overexpression of heat shock protein gp96 in the mice liver, which was almost completely attenuated by the luteolin pretreatment. Conclusions. The data suggest that the overexpression of gp96 in liver tissue is an early molecular response in acute, chemically induced liver damage in mice, which correlates with the degree of tissue damage. The hepatoprotective effect of luteolin against CCl4 hepatotoxicity was higher in animals pretreated with luteolin for 2 consecutive days. This suggests that the protection might be due to induction of some adaptive mechanisms. The data indicate that luteolin could be effective in protecting mice from the hepatotoxicity produced by CCl4.