Biserka Radošević-Stašić

Dose- and time-dependent effects of luteolin on carbon tetrachloride-induced hepatotoxicity in mice

Carbon tetrachloride (CCl(4)) is a well-known model compound for producing chemical hepatic injury. This study investigated the protective effects of the flavonoid luteolin on the CCl(4)-induced hepatotoxicity in mice. Luteolin dissolved in dimethyl sulfoxide (DMSO) was administered intraperitoneally (i.p.) at 5 or 50 mg/kg as a single dose, and once daily for 2 consecutive days. Two hours after the final treatment, the mice were treated with CCl(4) (20 mg/kg, i.p.). CCl(4)-induced hepatotoxicity was reduced in a dose- and time-dependent manner, as determined by decreased serum aminotransferase activities and liver histopathology. CCl(4) intoxication resulted in an overexpression of heat shock protein gp96 in the mice liver, which was strongly attenuated by luteolin pretreatment. Luteolin has also decreased oxidative stress produced by CCl(4), as suggested by improvement in the Cu/Zn superoxide dismutase activity. The effect of luteolin on myeloperoxidase, an indicator of inflammatory cell infiltration, was also investigated. Treatment of the mice with luteolin resulted in a significant decrease in the myeloperoxidase activity. The hepatoprotective effect of luteolin against CCl(4) 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 CCl(4).

Metallothioneins and heat shock proteins 70 in marine mussels as sensors of environmental pollution in Northern Adriatic Sea

In an attempt to assess the intensity of environmental pollution in industrial zones of Kvarnerian Bay in Northern Adriatic Sea and the reactivity of Mytilus galloprovincialis to these changes, in this study we estimated the concentration of heavy metals at four locations in both sea-sediment and in the mussels. Further we tried to correlate these changes with seasonal variations in environmental temperature, pH and salinity, as well as with the expression of metallothioneins (MTs) and heat shock proteins (HSPs) in the digestive tract of the mussels. Sampling in vivo was performed monthly, during the year 2008, while under the laboratory conditions the reactivity of acclimated mussels were tested to increasing concentrations of CdCl(2) and to thermal stress. The data have shown that the induction of MTs and HSP isoforms of the 70-kDa size class were highly affected by model agents treatment including contamination of sea-sediment by Pb, Hg and Cd, implying that these stress proteins might be power biomarkers of marine pollution.

The involvement of CD14 in the activation of human monocytes by peptidoglycan monomers.

BACKGROUND: Cell-wall components of Gram-positive and Gram-negative bacteria induce the production of cytokines in human peripheral blood mononuclear cells. These cytokines are the main mediators of local or systemic inflammatory reaction that can contribute to the development of innate immunity. AIMS: This study was performed to analyze the involvement of CD14 molecule in the activation of human monocytes by peptidoglycan monomer (PGM) obtained by biosynthesis from culture fluid of penicillin-treated Brevibacterium divaricatum NRLL-2311. METHODS: Cytokine release of interleukin (IL)-1, IL-6 and tumor necrosis factor-alpha from human monocytes via soluble CD14 (sCD14) or membrane-associated (mCD14) receptor using anti-CD14 monoclonal antibody (MEM-18) or lipid A structure (compound 406) was measured in bioassays. RESULTS: The results demonstrated that PGM in the presence of human serum might induce the monokine release in a dose-dependent manner. The addition of sCD14 at physiologic concentrations enhanced the PGM-induced monokine release, while the monokine inducing capacity of PGM in the presence of sCD14 was inhibited by MEM-18. Effects of PGM were also blocked by glycolipid, compound 406, suggesting the involvement of binding structures similar to those for lipopolysaccharide. CONCLUSION: Activation of human monocytes by PGM involves both forms of CD14 molecule, sCD14 and mCD14.

Growth of allogeneic sarcoma in mice subjected to halothane anesthesia and/or surgical stress

The present study was designed to clarify mechanisms involved in suppression of cell-mediated immunity reported in patients undergoing major surgery with general anesthesia by determining the effects of halothane anesthesia with and without surgery on the growth of Sarcoma I (Sa I), a tumor allogeneic to BALB/c mice. Mice were given subcutaneous injections of 5 × 106 tumor cells from A/Jax mice and then immediately exposed to 0.5%-1.0% halothane for 1 hr without surgery (n = 7) or with surgery (midline laparotomy; n = 12). In control groups mice were also injected with tumor cells but were not exposed to prolonged halothane anesthesia. Some of them received only Sa 1 (n = 6), while the rest (n = 7) were also laparotomized. The rejection time of Sa I in mice exposed to halothane anesthesia was significantly longer (15.4 ± 1.25 days) than in untreated controls (12.10 ± 0.68 days) (P < 0.05). In the mice exposed to halothane tumor growth was also greater. Surgical stress per se did not significantly affect growth or rejection time of Sa I(11.0 ± 0.66 vs 12.0 ± 0.68 days). Similarly, the combination of surgical stress with halothane anesthesia did not affect the immunosuppression associated with halothane alone (12.9 ± 1.3 vs 15.4 ± 1.25; P < 0.05). The results indicate that halothane anesthesia per se may be associated with impairment of cell-mediated immunity under experimental conditions.

Effect of Olive Oil- and Corn Oil- Enriched Diets on the Tissue Mineral Content in Mice

The mineral content (zinc, iron, magnesium, and calcium) in the liver, spleen, and thymus of male Balb/C mice was analyzed. Animals were fed, over 21 d, diets enriched with corn oil (FCO diet) or olive oil (FOO diet) (5% addition to standard pellet, w/w). Olive oil with predominant oleic acid (C18:1, n-9) had a quite different composition than corn oil, in which linoleic acid (C18:2, n-6) prevails. The zinc and magnesium tissue concentrations were not changed in either group. The calcium concentration in liver as well as the calcium concentration in spleen increased in mice fed both the FCO and FOO diets. Furthermore, mice fed both the FOO and FCO diets had increased spleen iron concentration. Mice fed the FCO diet had increased thymus calcium concentration compared to controls. The results show the effect of diets with unsaturated, particularly polyunsaturated fatty acids, on the calcium and iron concentration in some organs.

Modulatory effects of octreotide on anti-CD3 and dexamethasone-induced apoptosis of murine thymocytes

In an attempt to elucidate the effects of somatostatin on two crucial processes that regulated T-cell differentiation and selection in thymus in this study, we investigated in vivo and in vitro the effects of octreotide (SMS 201-995) on dynamics of apoptosis, induced by dexamethasone (DEX) or by anti-CD3 monoclonal antibodies (mAb). The data were estimated by analysis of absolute cellularity, DNA fragmentation and maturational stage of thymocytes, detecting the CD4 and/or CD8 and T cell receptor (TCR) expression on thymocytes. The results, obtained by estimation of subdiploid peak of DNA and ladder DNA formation, have shown that SMS given in vivo, may potentiate the early phase of DEX-induced nuclear fragmentation (at 24 h), accelerating simultaneously the elimination of thymic cells with double positive (DP) CD4high CD8high phenotype (expressed both as percentage and absolute number). On the contrary, SMS, given both in vivo and in vitro, down-regulated the late process (at 72 h) of nuclear fragmentation, induced by anti-CD3 mAb, minimizing simultaneously the elimination of DP cells (expressed both as percentage and absolute number). In anti-CD3-treated cultures of thymocytes, SMS retarded also the elimination of immature thymocytes, expressing the TRC alpha/betalow or intermediate phenotype. The data emphasize that octreotide might have important regulatory effect on processes of thymic differentiation and maturation, which are crucial for T cell selection, induction of tolerance and prevention of autoimmune diseases.

Immunosuppressive and Antiproliferative Effects of Somatostatin Analog SMS 201–995

The effects of long acting somatostatin analog SMS 201-995 were examined in vivo on: 1) lymphoid morphostasis and functional reactivity of cells obtained from SMS treated donors, 2) on humoral, and 3) cellular type of immunity; and in vitro on: 1) blastic transformation of lymphocytes stimulated by activators of different transmembrane pathways (CD2 by PHA and CD3/TCR by anti-CD3 monoclonal antibody and by allogeneic cells) and 2) on growth and secretory activity of several hybridoma cell lines. The data have shown that SMS in vivo decreases the proportion of CD4+, CD5+ and Ig+ cells in spleen. The reactivity of these cells to Con A was suppressed, but their spontaneous blastic transformation was increased. SMS suppressed also the plaque forming cells generation and proliferation of cells in popliteal lymph nodes during the local host versus graft reaction. The former immunosuppression was abrogated with the use of growth hormone, while in the latter, the time dependent changes in spleen composition were also noticed. The data obtained in vitro revealed that SMS may inhibit only the CD2-induced blastogenesis (in early and late interval after the use of PHA). SMS inhibited also the spontaneous growth and/or secretion of antibodies in some hybridoma cell lines.

Metallothionein expression and tissue metal kinetics after partial hepatectomy in mice

To better elucidate previous results showing that partial hepatectomy noticeably changes the tissue content of zinc, calcium, magnesium, and iron(II) ions in regenerating the liver, thymus, and spleen, we report on the correlation of these metal tissue kinetics in these organs with the expression of metallothionein-I+II (MT-I+II) proteins and MT-I mRNA in early postoperative period (1, 2, 6, 12, and 24 h) after one-third hepatectomy (pHx). The results showed that 2 h after pHx the regenerating liver accumulated Zn2+, Ca2+, Mg2+, and Fe2+ ions while decreasing the concentration of all these metals in the spleen and of Zn2+ in the thymus. On the 24th h, a new high accumulation of Zn2+ and Ca2+ was seen in the regenerating liver and of Zn2+, Ca2+, and Fe2+ in the spleen. Simultaneously, MT-I mRNA increased in the liver and spleen. In hepatocytes and on several spleen and thymus mononuclear lymphatic cells, the increased expression of MT proteins was found mainly in the cytoplasm and nuclei. The areas expressing MTs in regenerating liver inversely correlated with those containing apoptotic cells, suggesting that these proteins participate in tissue restoration through reduction or increase of metal ions after injury to the liver.

Metal tissue kinetics in regenerating liver, thymus, spleen and submandibular gland after partial hepatectomy in mice

Liver regeneration after partial hepatectomy (pHx) is a well-defined process, which involves the concerted action of extra- and intracellular factors resulting in induction of cell replication and its inhibition at the time when the entire liver mass is restored. Concomitantly, the breakdown of previously maintained tolerance and the exposure of self-antigens lead to the activation of preimmune and immune repertoires, which participate in surveillance against aberrant cells and the re-establishment of previous morphostasis. Because, in these events, important biological function might have tissue minerals that are affecting the structural integrity and enzyme activities, transduction signals, transcription and replication factors during cell proliferation and apoptosis, as well as the development and maintenance of immune functions and cytokine production, in this study we analyzed tissue dynamics of zinc, ioron magnesium, and calcium in the liver, thymus, spleen, and submandibular gland in intact and pHx mice on the 1st, 2nd, 7th, and 15th d after one-third pHx, using microwave digestion and inductivity coupled plasma spectrometry. The data showed that pHx induces significant and interconnected changes in all of the estimated metals not only in the regenerating liver but also in the lymphatic tissues and submandibular gland, indicating their importance for the control of growth processes.

Lymphoid System as a Regulator of Morphostasis and Hormonal Modulation of These Functions

It is generally accepted that the primary function of the immune system is the elimination of alien macromolecules and the monitoring of self components. The immunologic attack occurs only when major histocompatibility complex (MHC)-restricted T cells recognize antigen on the surface of cells bearing the appropriate MHC gene, product. The antigen:Ia complexes promote receptor aggregation on the cloned Thelper cell, which leads to the release of many non-antigen-specific mediator molecules, which then affect other lymphatic and nonlymphatic cells.’ In the clonal induction approach to immunology, Burch and Burwell’ anticipate that similar events occur during the maintenance of morphostasis, which may be the final outcome of the physiological contact of lymphoid cells with their own tissue, performing the function of growth control. It seems that any disturbance of morphostasis (by damage of tissue or surgical resection, for example) leads to the activation of self-reactive clones of lymphocytes because Ia-bearing cells, in these situations, probably expose to the lymphatic system some new antigens connected with the dedifferentiation and changes in the cytoskeletal elements of the regenerating cells? Thus, Ia-antigen-bearing Kupffer cells from regenerating murine liver stimulate T lymphocytes in vivo and in ~ i t r o . ~ These lymphocytes “primed” by partial hepatectomy or unilateral nephrectomy initiate and then stop the growth of liver or kidney until the morphostasis reappears. Transfer experiments show that these lymphocytes affect the growth of liver and kidney in the recipients as well.’s6 Because an increasing amount of data today supports the hypothesis that bidirectional circuits exist between the immune and neuroendocrine systems’ we attempted in this study to see if similar mechanisms operate when growth-regulatory capacities of the lymphatic system are viewed. For this purpose we examined: (1) the consequences of partial hepatectomy on the cellularity of various lymphatic organs; (2) the capacity of splenocytes primed by hepatectomy to change the intensity of compensatory liver growth of recipients and (3) the possibilities of hormonal modulations of these functions.