Mira Ćuk

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.

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.

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.

TISSUE ZINC DYNAMICS DURING THE IMMUNE REACTION IN MICE

Owing to the importance of zinc for the functioning of the immune system, the role of endogenous Zn, located both in lymphoid and nonlymphoid organs, was investigated during the standard humoral and cellular types of immune response. For this purpose, the dynamics of hepatic, thymic, splenic, and renal Zn content was determined in mice sensitized with (a) sheep red blood cells and (b) semiallogeneic lymphocytes during the local host vs graft reaction (HVGR). The data obtained by ion-coupled plasma spectrometry revealed that the humoral type of immunity is characterized by a significant increase of Zn concentration in the liver and in the thymus. Simultaneously, linear regression analysis showed that the generation of plaque-forming cells in the individual mouse was highly positively correlated with Zn concentration in the liver (r = 0.897), and spleen (r = 0.833), and negatively with Zn concentration in the thymus (r = -0.624). Similar relationships between the intensity of local immune reaction and tissue Zn levels were found in local HVGR at the fifth day in the liver and spleen (r = 0.861 andr = 0.695, respectively), at the seventh day in the thymus (r = -0.797), and at the tenth day in the liver (r = -0.859). The data emphasize the necessity of Zn for the development of normal immune response and point to the existence of a Zndependent hepato-thymic axis during the humoral and cellular types of immune reactivity.

On the Role of T Lymphocytes in Stimulation of Humoral Immunity Induced by Peptidoglycan–Monomer Linked with Zinc

Effects of peptidoglycan linked with zinc (PGM–Zn) were investigated on plaque–forming cell (PFC) generation to sheep red blood cell (SRBC) and SRBC–unrelated antibody production in primary and secondary immune response in mice depleted in vivo of CD4+ and/or CD8+ T lymphocytes. PGM–Zn in nondepleted mice stimulated the PFC generation and IgM or IgG and IgG1 production in primary and secondary reaction. Single depletion of CD4 or CD8+ T cells did not change this ability. The effects of PGM–Zn after CD8+ depletion were even greater than those in nondepleted mice. Depletion of both T cell subsets, however, completely abrogated immunostimulatory effects of PGM on PFC generation (primary and secondary response), as well as on primary SRBC–unrelated antibody production, leaving only the increase of IgG in secondary response unchanged. Immunostimulatory effects and isotype switching to IgG1 and IgG2a correlated with the changes in splenic CD4+, CD8+, CD5+ cells, pointing to the regulatory role of these cells and/or their cytokines in PGM–Zn–induced immunostimulation. Altogether the data suggest that PGM–Zn may potentiate the costimulatory signals coming from activated T cells and act on B cells without the T cell help.

Immunoregulating effects of peptidoglycan monomer linked with zinc in adult mice.

Since it is well known that both zinc ions and bacterial immunostimulants influence the function of the immune system, in the present study we investigated the immunomodulating activity of a new analog of peptidoglycan monomer (PGM), in which the basic molecule was linked to zinc (PGM-Zn). Its effects in BALB/c mice, aged 10-12 months, were compared with the effects of equimolar doses of PGM and ZnCl2. The treatment lasted 26 days (one i.p. injection every fifth day). The results showed that PGM-Zn may markedly enhance antibody production to sheep red blood cells, as well as spontaneous and concanavalin A (ConA)-induced blastogenesis. The generation of plaque-forming cells in individual mouse was positively correlated with the expression of class II antigens in the liver and negatively correlated with the total quantity of hepatic proteins. PGM-Zn also induced the appearance of peritoneal macrophages, which in cocultures with syngeneic splenocytes were less able to enhance the spontaneous, and particularly the ConA-induced blastic transformation. The enhancing activities of PGM-Zn were in some respects more closely correlated with the action of PGM, whereas the induction of suppressive macrophages resembled more the activity of ZnCl2. The data emphasize that PGM-Zn may both stimulate and inhibit immunoregulative pathways by mechanisms which are not identical to those of PGM or ZnCl2.

Immunological consequences of lesions of nucleus basalis in rats

In this study we followed up the effects of acute (ten days) and chronic (two months) stereotaxic lesions of NB in rats on: 1.) cellularity of lymphatic organs, 2.) humoral and 3.) cellular immunity and 4.) in vitro functional reactivity of splenocytes to polyclonal mitogens. Experiments were done on (AoxDA) F1 male rats, aged 4 months and maintained in standard laboratory conditions. The coordinates for bilateral stereotaxic lesions were taken from atlas of Pellegrino. Electrolysis was performed unipolarly at the anode with 10 mA for 10s. Histological verification of the lesions was performed by the method of Wolf. For the evaluation of humoral or cell mediated immunity standard plaque forming cell assay to sheep red blood cells (SRBC) or transplantation of allogeneic skin were used. The data have shown that bilateral electrolytic lesions of NB cause the prolongation of rejection time to allogeneic skin, transplanted on tenth p.o. (15.8+-0.4 days versus 12.8+-0.5 in intact control, p<.001). Some prolongation of allograft survival was, however, seen also in group of rats in which electrodes were lowered in NB without turning off the electric current, indicating the consequences of mechanical lesions of NB on cell mediated immunity. Humoral immunity tested after sensitization of rats with SRBC, on tenth p.o. was found also to be slightly depressed and followed by depletion of thymus, but the values were on the border of statistical significance. Chronic electrolytic lesions of NB in rats which were not otherwise sensitized, revealed that in comparison to appropriate controls, the cellularity of thymus of NB lesioned rats is higher, while the cellularity of spleen is decreased. Although the tendency of hypoplasia of bone marrow seems to exist, the results are not statistically significant. The data imply that NB is not important only for several behavioural functions but also for mechanisms involved in the maintenance of normal immunological response and composition of lymphoid organs. It is obvious that NB might play an important role in the reestablishment of homeostasis after the initiation of an immune response.

THE ROLE OF LYMPHATIC TISSUE IN THE COMPENSATORY RENAL GROWTH

Compensatory renal growth (CRG) has been analyzed in three types of experiments: a) in situations when normal lymphoid homeostasis had been disturbed by thymectomy (Tx) or splenectomy (Sx), b) under influence of cellular /lymphoid cells/ and humoral /serum/ factors from unilaterally nephrectomized donors, c/ in situations when simultaneously with compensatory growth immunological functions were induced by allogeneic skin transplantation. The data have shown the following: Tx performed one month before UNx inhibited CRG (41, 6 % of normal, p<0.01) ; syngeneic splenocytes obtained from UNx donors had renothropic features ; while the splenocytes from intact donors produced a marked inhibition of the nephrocompensation. When allogeneic cells combination was used the renothropic capabilities of lymphatic cells were lost. Furthermore, the inhibition of CRG was induced by transplantation of allogeneic skin.

Thymic alterations induced by partial hepatectomy: upregulation of glycoprotein 96, CD91 and TLR2 and generation of regulatory T cells

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-ß 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-ß immunostaining from medulla to cortex and an appearance of Treg cells. The data show that pHx triggers in thymus the ER-stress 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.