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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.

Ciljevi: Osteopontin (OPN) je fosforilirani glikoprotein koji posjeduje različita djelovanja uključujući nastanak i metastaziranje tumora. Kočenje apoptoze jedan je od mehanizama kojim OPN doprinosi napredovanju tumora, a jezgreni čimbenik kappa B (NF-κB) je uključen u signalne putove kočenja apoptoze. Cilj ovog istraživanja je proučavanje izražaja OPN-a u normalnom tkivu bubrega i svijetlostaničnom karcinomu bubrežnih stanica (SKBS) i određivanje njegovog prognostičkog značenja te ujedno određivanje odnosa između NF-κB i izražaja OPN-a na proteinskoj i genskoj razini kao i njihovog odnosa prema apoptozi u uzorcima SKBS-a. Materijali i postupci: Izražaj proteina OPN-a istražen je metodom imunohistokemije na standardnim tkivnim rezovima 171 SKBS-a i uspoređen s uobičajenim kliničkopatološkim čimbenicima. Korištenjem imunohistokemijske metode i tehnike tkivnih mikroareja (TMA) na 94 SKBS-a određen je izražaj proteina OPN-a te p50 i p65 podjedinice NF-κB i međusobno uspoređen kao i s apoptotičkom aktivnosti. Korištenjem kvantitativne lančane reakcije polimerazom (QPCR) određena je količina OPN glasničke RNA (mRNA) iz svježe smrznutog tumorskog tkiva i uspoređena s izražajem proteina OPN-a i NF-κB u 22 SKBS-a. Rezultati: Izražaj OPN-a je u tumorskom tkivu bio veći u odnosu na normalno tkivo bubrega i primijećen je u 61 SKBS-u (35.7 %) kao citoplazmatsko bojanje različite jačine. Utvrđena je povezanost povećanog izražaja OPN-a s pokazateljima loše prognoze kao što su veličina tumora (p, Objectives: Osteopontin (OPN) is a phosphorylated glycoprotein with diverse functions including tumorigenesis and tumor cell metastasis. Apoptosis inhibition is one of the OPN’s mechanisms that contribute cancer progression and nuclear factor-kappa B (NF-κB) is involved in antiapoptotic signaling pathway. The aim of this study was to analyze the expression of OPN in normal renal tissue and clear cell renal cell carcinomas (CRCC), and to assess its prognostic significance together with determining relations between NF-κB and OPN expression at protein and gene level as well as their relation to apoptosis in specimens of CCRCC. Material and Methods: The expression of OPN protein was analyzed using immunohistochemistry in whole-tissue sections of 171 CRCC and compared to usual clinicopathological parameters. Using tissue microarray (TMA) and immunohistochemical technique the expression of OPN protein and p50 and p65 NF-κB subunits was analyzed in 94 CCRCC and compared mutually and to apoptotic index. Using real-time PCR OPN mRNA was quantified from snap frozen tumor tissue and compared to OPN and NF-κB protein expression in 22 CCRCC. Results: Compared to normal renal parenchyma OPN was upregulated in tumor tissue and was observed in 61 CRCC (35.7%) in the form of cytoplasmic staining of various intensities. The association of OPN expression and poor prognostic indicators as tumor size (p