Multidirectional Changes in Parameters related to Sulfur Metabolism in Frog Tissues exposed to Heavy Metal-related Stress.

The investigations showed changes of the cystathionine γ-lyase (CTH), 3-mercaptopyruvate sulfurtransferase (MPST) and rhodanese (TST) activity and gene expression in the brain, heart, liver, kidney, skeletal muscles and testes in frogs Pelophylaxridibundus, Xenopuslaevis and Xenopus tropicalis in response to Pb2+, Hg2+ and Cd2+ stress. The results were analyzed jointly with changes in the expression of selected antioxidant enzymes (cytoplasmic and mitochondrial superoxide dismutase, glutathione peroxidase, catalase and thioredoxin reducatase) and with the level of malondialdehyde (a product of lipid peroxidation). The obtained results allowed for confirming the role of sulfurtransferases in the antioxidant protection of tissues exposed to heavy metal ions. Our results revealed different transcriptional responses of the investigated tissues to each of the examined heavy metals. The CTH, MPST and TST genes might be regarded as heavy metal stress-responsive. The CTH gene expression up-regulation was confirmed in the liver (Pb2+, Hg2+, Cd2+) and skeletal muscle (Hg2+), MPST in the brain (Pb2+, Hg2+), kidney (Pb2+, Cd2+), skeletal muscle (Pb2+, Hg2+,Cd2+) and TST in the brain (Pb2+) and kidney (Pb2+, Hg2+, Cd2+). Lead, mercury and cadmium toxicity was demonstrated to affect the glutathione (GSH) and cysteine levels, the concentration ratio of reduced to oxidized glutathione ([GSH]/[GSSG]) and the level of sulfane sulfur-containing compounds, which in case of enhanced reactive oxygen species generation can reveal their antioxidative properties. The present report is the first to widely describe the role of the sulfane sulfur/H2S generating enzymes and the cysteine/glutathione system in Pb2+, Hg2+ and Cd2+ stress in various frog tissues, and to explore the mechanisms mediating heavy metal-related stress.

Cyclophosphamide-modified murine peritoneal macrophages induce CD4+ T contrasuppressor cells that protect contact sensitivity T effector cells from suppression.

BACKGROUND: Cyclophosphamide (CY) is one of the most widely used alkylating agents in the treatment of various cancers and some autoimmune diseases. Numerous reports suggest that CY exerts immunoregulatory effects. Animal studies have shown CY affects contact sensitivity (CS) response by depleting CD4+CD25+ T regulatory cells and CD8+ T suppressor (Ts) cells. In a mouse model of CS, we previously showed that in vivo treatment with CY shapes the immunogenic/immunoregulatory balance of peritoneal macrophages. The aim of the current study is to verify if macrophages (Mf) from CY-treated mice are indeed able to induce immunoregulatory cells that could protect from suppression. METHODS: Adoptive cell transfer of CS was used to examine immunomodulating properties of peritoneal Mf from CY-treated mice. Isolation of peritoneal Mf from animals that were (Mf-CY) or were not (Mf) treated with CY were cultured to identify cytokine repertoire. Further, we assessed spleen cell (SPLC) cytokine production following immunization with trinitrophenyl-conjugated Mf from donors treated (TNP-Mf-CY) or non-treated (TNP-Mf) with CY. RESULTS: In vitro experiments identified that Mf-CY produce more IL-6, TNF-α and TGF-ß than naïve Mf. Further, immunization with peritoneal TNP-Mf-CY induces CD4+ T contrasuppressor cells (Tcs) cells that protect CS-effector cells from suppression. Higher IL-17A secretion was observed from TNP-Mf-CY-treated mouse SPLC compared to SPLC from TNP-Mf injected mice suggesting that this cytokine might be important in mediating contrasuppression in this model. CONCLUSIONS: Our results show that in vivo treatment with CY influences mouse peritoneal Mf to induce CD4+ Tcs cells that protect CS-effector cells from suppressive signals of Ts cells.

Similar effect of sodium nitroprusside and acetylsalicylic acid on antioxidant system improvement in mouse liver but not in the brain.

BACKGROUND: The aim of the present study was to analyze the relative antioxidant effects of acetylsalicylic acid (ASA) and sodium nitroprusside (SNP) in mouse liver and brain. METHODS: The activity of rhodanese, 3-mercaptopyruvate sulfurtransferase (MPST) and γ-cystathionase (CSE), functioning as antioxidant proteins and capable of producing H2S, was investigated in mouse liver and brain after intraperitoneal once a day administration of sodium nitroprusside (5 mg/kg body weight) or acetylsalicylic acid (500 mg/kg body weight) continued for 5 days. The tissues were homogenized and then the obtained supernatants were used for further determinations. At the same time, the levels of sulfane sulfur, reduced and oxidized glutathione, cysteine, cystine, and cystathionine were also studied in these tissues. RESULTS: Both ASA and SNP show a statistically significant increase of sulfurtransferases activities in liver. The mechanism of action of sodium nitroprusside appears to consist in liberation of nitric oxide (NO), an important signaling molecule in the mammalian body. SNP also releases cyanide ions, which are converted in the liver to thiocyanate by the enzyme rhodanese and/or MPST and/or γ-cystathionase - the activities of all the enzymes were elevated in reaction to SNP. The action of γ-cystathionase is dependent upon converting cystathionine to cysteine, a precursor of the major cellular antioxidant, glutathione. Under oxidizing conditions, an increase in cystathionine ß-synthase activity might indirectly result in an increase in the antioxidant glutathione level; this was reflected by the increased GSH/GSSG ratio in the liver, but not in the brain, where a trace activity of γ-cystathionase is normally detected. CONCLUSION: The results of the present investigations show that ASA and SNP may stimulate the GSH-dependent antioxidant system and protect liver cells from oxidative stress. An increased activity of the H2S-producing enzymes and the increased GSH/GSSG ratio may lead to an elevated level of H2S, a molecule with antioxidant properties. A similar effect was not observed in the brain. In case of both sodium nitroprusside and aspirin administration, homeostasis of sulfane sulfur level was noted in both the liver and brain.

[Skin as a place of immunomodulation].

The skin is the largest organ of the human body which plays a key role not only in physiological processes such as thermoregulation and the maintenance of the correct water and electrolyte balance, but also forms an effective barrier against microorganisms, protecting the organism against harmful external factors. Moreover, the skin is an important organ involved in immune mechanisms. The skin is a place of drug application to treat various local and systemic diseases. Those drugs are proteins and peptides sensitive to digestion in gastrointestinal tract, or chemical molecules metabolized in the liver. Epicutaneous immunization (EC) is a new therapeutic method used to treat immunological disorders. The phenomenom of immunomodulation has already been exploited to treat allergic diseases and is called allergen specific immunotherapy. The authors describe the current state of knowledge of the immune regulatory mechanisms that allow to use the EC method to treat immunological diseases mediated by Th1, Tc1 or Th2 lymphocytes. The new aspect of this immunotherapy is immunopotentiation that involves components of the innate immune system, working via the TLRs. The first clinical trials give promising outcomes of the use of EC method in the treatment of multiple sclerosis and respiratory allergic diseases.

Exposure to lead in water and cysteine non-oxidative metabolism in Pelophylax ridibundus tissues.

Chronic, low-level exposure to metals is an increasing global problem. Lead is an environmentally persistent toxin that causes many lead-related pathologies, directly affects tissues and cellular components or exerts an effect of the generation of reactive oxygen species causing a diminished level of available sulfhydryl antioxidant reserves. Cysteine is one of substrates in the synthesis of glutathione - the most important cellular antioxidant, and it may also undergo non-oxidative desulfuration that produces compounds containing sulfane sulfur atoms. The aim of the experiment was to examine changes of the non-oxidative metabolism of cysteine and the levels of cysteine and glutathione in the kidneys, heart, brain, liver and muscle of Marsh frogs (Pelophylax ridibundus) exposed to 28mg/L Pb(NO(3))(2) for 10 days. The activities of sulfurtransferases, enzymes related to the sulfane sulfur metabolism - 3-mercaptopyruvate sulfurtransfearse, γ-cystathionase and rhodanese - were detected in tissue homogenates. The activity of sulfurtransferases was much higher in the kidneys of frogs exposed to lead in comparison to control frogs, not exposed to lead. The level of sulfane sulfur remained unchanged. Similarly, the total level of cysteine did not change significantly. The total levels of glutathione and the cysteine/cystine and GSH/GSSG ratios were elevated. Thus, it seems that the exposure to lead intensified the metabolism of sulfane sulfur and glutathione synthesis in the kidneys. The results presented in this work not only confirm the participation of GSH in the detoxification of lead ions and/or products appearing in response to their presence, such as reactive oxygen species, but also indicate the involvement of sulfane sulfur and rhodanese in this process (e.g. brain). As long as the expression of enzymatic proteins (rhodanese, MPST and CST) is not examined, no answer will be provided to the question whether changes in their activity are due to differences in the concentrations of substrates and/or compounds affecting their activity or to changes in their level in response to some parameters, e.g. associated with oxidative stress.

Effect of mercury ions on cysteine metabolism in Xenopus laevis tissues.

The effect of mercury ions on the level of cysteine, glutathione, sulfane sulfur, and on the activity of rhodanese, 3-mercaptopyruvate sulfurtransferase (MPST) and γ-cystathionase in brain, heart muscle, liver, kidneys, testes and skeletal muscle of adult Xenopus laevis was investigated. Frogs of both sexes were exposed for 7 or 14 days to 1.353mgL(-1) (ppm) of mercury chloride (HgCl(2)) dissolved in water. The activity of the investigated enzymes participating in cysteine metabolism depends on cysteine in their active sites. Mercury ions can bind to -SH groups and, therefore, lower the activity of enzymes and change the level of sulfane sulfur, a product of l-cysteine desulfuration. The effect of mercury was found to depend on the time of exposure and the kind of tissue. In the liver, the main site of glutathione biosynthesis, the ratio of GSH to GSSG was essentially unchanged. The total glutathione level was decreased after 7 days of exposure to mercury, similarly as the activity of rhodanese. Sulfane sulfur levels were significantly increased after a shorter duration, while they decreased after a longer time of exposure. The kidney, brain and testes were able to enhance the level of GSH, probably thanks to high γ-glutamyltranspeptidase activity. These tissues showed an increased value of GSH/GSSG ratio during the shorter exposure to mercury. The activity of sulfurtransferases was decreased, especially after the longer exposure to mercury. In the heart and skeletal muscle, the level of GSH, sulfane sulfur, and the activity of the investigated sulfurtransferases was diminished after 14 days of exposure to Hg. It can be concluded that the main mechanism of toxic Hg activity is generation of reactive oxygen species in cells due to depleted GSH level, and a decreased sulfurtransferases activity either by blocking or oxidation of their -SH groups, what in consequence results in a diminished sulfane sulfur levels in tissues, especially the heart and testes.

TCRalphabeta + CD8+ and TCRgammadelta+ lymphocytes inhibit the capability of peritoneal macrophages to induce contact hypersensitivity.

Macrophages (Mf) play an important role in induction and regulation of the immune response. It was shown previously that subcutaneous injection of hapten conjugated macrophages (TNP-Mf) induces the contact hypersensitivity (CHS) response, whereas intravenous (i.v.) or intraperitioneal administration of TNP-Mf results in unresponsiveness as a result of induced T suppressor (Ts) cells. The aim of this study was to determine if different T cell populations influence macrophages to become inducers of immunological suppression. Our findings show that indeed i.v. injection of TNP labeled macrophages isolated from control mice into syngenic recipients induces unresponsiveness. However, i.v. administration of TNP substituted macrophages isolated from TCRalpha-/-, TCRdelta-/- and beta2m-/- mice induces strong CHS similar to that observed after skin painting with TNP-C1. Moreover, it was shown that TNP conjugated macrophages isolated from CD1d-/- mice were still able to promote immunosuppression when injected intravenously. This suggests that TCRalphabeta+ CD8+ and TCRgammadelta+ lymphocytes stimulate macrophages to induce immunosuppression instead of a strong CHS reaction, whereas CD1d dependent NKT cells are not involved in negative regulation of macrophage function.

The effect of aspirin on Gomori-positive glia in mouse brain.

Aspirin (acetylsalicylic acid, ASA) treatment resulted in a significant decrease in the amount of the sulfur-rich Gomori-positive material present in the cytoplasm of periventricular glia. It also caused the accumulation of the Gomori-positive neurosecretory material in the supraoptic and paraventricular nuclei and, most pronounced, in the neurosecretory axons of the paraventricular- and supraoptic-neurophypophysial tract.

The glial Gomori-positive material is sulfane sulpfur.

The Gomori-positive glia are periventricular astrocytes with abundant cytoplasmic granular material, predominantly occupying a periventricular site in the brain. These granular inclusions are strongly stained with chrome hematoxylin in the Gomori's method as well as exhibit red autofluorescence and non-enzymatic peroxidase activity. The glial Gomori-positive material (GGPM) granules are positive in the performic acid Alcian blue method indicating the presence of protein-bound sulfur, what has been shown by our previous studies. The number of cells containing glial Gomori-positive granules dropped after administration of cyanide and increased under the influence of sulfane sulfur donor (diallyl disulfide). This suggests, that sulfur of these granules is a sulfane sulfur, possibly in the form of protein-bound cysteine persulfide. Sulfane sulfur is labile, reactive sulfur atom covalently bound to another sulfur atom. In this paper we present evidence that GGPM exhibit affinity to cyanolysis and its stainability in Gomori's method is due to the presence of protein-bound sulfane sulfur. The biological role of the Gomori-positive glia connected with protective properties of sulfane sulfur has been discussed.

Epicutaneous immunization with myelin basic protein protects from the experimental autoimmune encephalomyelitis.

Multiple sclerosis (MS) is a chronic inflammatory autoimmune disease of the central nervous system (CNS) with limited treatment modalities. One of the experimental methods that protect from autoimmune diseases is oral tolerance. However, this method failed to show therapeutic efficacy in clinical trials. In our previous work, we found that epicutaneous (ec) immunization with a protein antigen induces a state of profound immunosuppression that inhibits inflammatory response in contact sensitivity (CS), experimental autoimmune encephalomyelitis (EAE) in B10.PL mice that develop chronic form of disease, and also delayed allogeneic skin graft rejection. In the current work, we showed that ec immunization with MBP protects from relapsing and remitting EAE. Protection from the disease correlated with decreased number of mononuclear cells isolated from CNS. Additionally, histological examination showed only a slight mononuclear cell infiltration in spinal cords of mice ec immunized with MBP when compared to positive control where animals were ec treated with PBS before disease induction.

Cadmium toxicity related to cysteine metabolism and glutathione levels in frog Rana ridibunda tissues.

The level of glutathione and sulfane sulfur and sulfurtransferases activity in adult frogs Rana ridibunda were investigated after the exposure to 40 mg or 80 mg CdCl(2) L(-1) for 96 h or 240 h. Cd accumulation in the liver, kidneys and testes was confirmed, and the highest Cd level was found in the testes. In the liver, the exposure to Cd resulted in an increase of GSH level and the activity of rhodanese, while the activity of 3-mercaptopyruvate sulfurtransferase and cystathionase decreased. The kidneys and brain showed the elevated level of GSH and the activity of all investigated sulfurtransferases, as well as sulfane sulfur especially in brain. In such tissues as the testes, muscles and heart, the level of GSH and the activity of 3-mercaptopyruvate sulfurtransferase were significantly diminished. The increased level of sulfane sulfur was determined in the testes and muscles and the increased activity of rhodanese in the testes and the heart. These findings suggest the possible role of sulfane sulfur and/or sulfurtransferases in the antioxidation processes, which can be generated in cells by cadmium.

Aspirin augments the concentration of endogenous hydrogen sulfide in mouse brain and liver.

Intraperitoneal injections of lysine acetylsalicylate (L-ASA, aspirin) in a dose of 10 mg during 5 consecutive days to BALB/c and B10.PL mice increased the concentration of endogenous hydrogen sulfide in their livers. The rise of hydrogen sulfur levels was shown also in brains of BALB/c females and B10.PL males, however in BALB/c male brains there is no statistically significant difference. The mechanism of aspirin action on H2S concentration is discussed.

Lipopolysaccharide aggravates cerebral pathology in B10.PL-derived CD1-/-, beta2m-/-, TCRalpha-/-, and TCRdelta-/- knockout mice.

Adult B10.PL-derived immunological genes knockout mice injected with 100 microg lipopolysaccharide (LPS) showed severe hydrocephalus and meningitis. A consequence of the hydrocephalus is pineal hyperplasia, sponginess of periventricular parenchyma, gliosis and, at the last stage of hydrocephalus formation, disappearance of the ependymal layer and the Gomori-positive subependymal astrocytes. Possible mechanisms for the aggravation of cerebral pathology induced by LPS are discussed.

Season dependent response of the Marsh frog (Rana ridibunda) to cadmium exposure.

Cadmium toxicity related to cysteine metabolism and glutathione levels in several tissues of the Marsh frog (Rana ridibunda) collected in late spring were investigated after exposure to 80 mg CdCl2 L(-1) for 168 h. The results were compared to those obtained in a previous experiment carried out in autumn. The most striking changes involved the brain which could not maintain a proper glutathione level and the testes in which neither GSH nor sulfane sulfur levels recovered. Substantial damage is expected in the presence of Cd which decreases the antioxidant status of these tissues. It seems that in spring, frogs had lesser tolerance for Cd in comparison with frogs in autumn. This may be caused by the transition to aerobic respiration after hibernation.

Cerebral pathology in immunodeficient gnotobiotic laboratory mice.

Gnotobiotic autoimmunity prone, antigen presentation, T lymphocyte receptor gene knockout mice show cerebral pathology in the form of meningitis, venous blood statis with subarachnoid hemorrhages and massive hemosiderin deposits. A more or less severe hydrocephalus was present in all animals examined. In all cases except the CD 1(-/-) animals, the pineal gland was considerably reduced in mass. Only in the latter strain was a strong pineal hypertrophy in the form of a benign tumor present in ca. 50% of the animals. A possible sequence of events leading to hydrocephalus is discussed. Endogenous vertically transmitted facultative pathogens, active in the immunocompromised animals, probably play a primary etiological role. The results show that caution is needed in planning immunobiological studies on the B10.PL and B10.PL-derived mice, and possibly other strains not examined for possible neuropathological changes.

[Regeneration in the central nervous system and neuro-transplantation]. / Regeneracja w osrodkowym ukladzie nerwowym i neurotransplantacja.

A review article on recent literature data on regeneration and transplantation in central nervous system is presented. The role of embryonal and adult stem cells is emphasized and possible therapeutic implications are indicated in injuries of the spinal cord and brain and in neurodegenerative diseases.