Expression pattern of the endoplasmic reticulum stress protein gp96 in monophasic and chronic relapsing form of experimental autoimmune encephalomyelitis in rats.

Gp96 is the endoplasmic reticulum (ER)-resident molecular chaperone, which is involved in the correction of unfolded proteins, in the activation of proteasome-dependent ER-associated degradation of the misfolded proteins, and in activation of the protein translation that modulates polypeptide traffic into the ER. Furthermore, owing to its peptide chaperone capacity and ability to interact with professional antigen-presenting cells, as well as with growth factors, integrins and Toll-like receptors, it is also endowed with crucial immunological functions acting as a "danger signal" to the innate and adaptive immunity. Considering these properties, in the present study the tissue expression of gp96 was examined during the monophasic and chronic relapsing form of experimental autoimmune encephalomyelitis (CR-EAE), induced in genetically susceptible DA rats by subcutaneous injection of myelin basic protein (MBP) or bovine brain homogenate in complete Freund's adjuvant (CFA). Immunohistochemical analyses were done in periods of attacks and remissions of EAE, and the results were compared with findings in intact rats and those treated only with CFA. The data revealed that the constitutive gp96 expression, found in several neurons and glial cells in the brain and spinal cord of intact animals, significantly diminished during the attacks of CR-EAE. On the contrary, the remission of disease was followed by high upregulation of gp96, mainly in the oligodendrocytes within the white matter, in the neurons of the hippocampal area, as well as in the motoneurons of lumbar spinal cord, suggesting that gp96 might be involved in proteostasis and immune-related pathways linked with the reparative processes in the CNS.

Short-term exposure of mice to gasoline vapor increases the metallothionein expression in the brain, lungs and kidney.

Environmental airborne pollution has been repeatedly shown to affect multiple aspects of brain and cardiopulmonary function, leading to cognitive and behavioral changes and to the pronounced inflammatory response in the respiratory airways. Since in the cellular defense system the important role might have stress proteins-metallothionein (MT)-I and MT-II, which are involved in sequestration and dispersal of metal ions, regulation of the biosynthesis and activities of zinc-dependent transcription factors, as well as in cellular protection from reactive oxygen species, genotoxicity and apoptosis, in this study we investigated their expression in the brain, lungs and kidney, following intermittent exposure of mice to gasoline vapor. Control groups consisted of intact mice and of those closed in the metabolic chamber and ventilated with fresh air. The data obtained by immunohistochemistry showed that gasoline inhalation markedly upregulated the MTs expression in tissues which were directly or indirectly exposed to toxic components, significantly increasing the number of MT I+II positive cells in CNS (the entorhinal cortex, ependymal cells, astroglial cells in subventricular zone and inside the brain parenchyma, subgranular and CA1-CA3 zone of the dentate gyrus in hippocampus and macrophages-like cells in perivascular spaces), in the lungs (pneumocytes type I and type II) and in the kidneys (parietal wall of Bowman capsule, proximal and distal tubules). The data point to the protective and growth-regulatory effects of MT I + II on places of injuries, induced by inhalation of gasoline vapor.

Syngeneic pregnancy induces overexpression of natural killer T cells in maternal liver.

Conditions such as stress, infection, autoimmune disease, etc. elevate the number and function of extrathymic T cells that are generated mainly in the liver. As primitive, self-reactive clones of T cells that coexpress receptors of the natural killer (NK) lineage, they mediate cytotoxicity against altered self, malignant and infected cells and have the unique potential to rapidly secrete large amount of T helper 1 (Th1) or Th2 cytokines. To elucidate whether some of these changes occur even during the syngeneic pregnancy, we made phenotypic and functional characterization of mononuclear lymphatic cells (MNLCs) isolated from the liver and spleen of pregnant C57BL/6 mice, testing their cytotoxicity against syngeneic thymocytes as well as against NK- and lymphokine-activated killer (LAK)-sensitive targets. The data have shown that on the sixteenth day of syngeneic pregnancy TCRint, NK1.1+ and IL-2Rbeta+ cells were accumulated in the liver, while the quantities of CD4+ and CD8+ T cells and total number classical NK (NK1.1+CD3- or IL-2Rbeta+CD3-) cells were increased in the spleen. Pregnancy-activated hepatic and splenic MNLCs were more cytotoxic against syngeneic thymocytes, YAC-1 and P815 targets, suggesting that the maternal liver is a main producer of autoreactive NKT clones, which subsequently augment NK- and LAK cell-mediated cytotoxicity in the liver and spleen.

Chromosome aberrations in peripheral blood lymphocytes in subjects occupationally exposed to ionizing radiation or chemical clastogens.

To get an insight into genotoxic risk in some occupations, in this study the chromosome aberration analysis of peripheral blood lymphocytes was made in 20 physicians and nurses exposed to a low dose of ionizing radiation in a hospital, 12 individuals working with X-rays in a cement factory and 19 technicians working with some chemical toxic agents in the laboratories of a medical school. The control group consisted of 14 sex- and age-matched unexposed persons living in the same district area. The data showed that the total number of chromosome aberrations in 200 scored metaphases in all examined groups were almost the same and inside the low-permitted values. In hospital workers, however, the percentage of acentric and dicentric fragments (1.63 +/- 0.28 vs 0.31 +/- 0.21 and 0.47 +/- 0.18 vs 0.0, respectively) increased predominantly in contrast to cement-factory employees and laboratory workers, where a higher incidence of minutes (0.58 +/- 0.19 vs 0.31 +/- 0.2) or gaps (2.21 +/- 0.37 vs 1.15 +/- 1.15) was noticed. Moreover, in groups exposed to low doses of ionizing radiation (hospital and factory), a positive correlation was found between the total number of chromosome aberrations and the 6-year absorption dose or working period, suggesting an effect of cumulative dosage. The data emphasize that the continuous chromosomal aberration analysis should be obligatory for individuals exposed to various genotoxic substances, even in occupational conditions where according to dosimetric analysis they are exposed to permitted levels of radiation.

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.

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.

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.

High incidence of chromosome aberrations after radiochemotherapy for Hodgkin's disease: a report of a case and a review of the literature.

To emphasize the importance of information obtained by conventional cytogenetic tests, in this study we will present a case report showing the development of HD in a young engineer of medical radiology, who had been professionally exposed to ionizing radiation during 4 years. Bio-dosimeter data and chromosomal aberration analysis made in a group of colleagues working in the same ionizing conditions excluded the possibility that she was overexposed to ionizing radiation, but retroactive analysis showed that at the time of employment she had a moderately increased level of chromosomal aberrations in peripheral blood lymphocytes (4.6%), with higher than normal incidence of chromatid breaks (22 x 10(-3) per cell) and dicentric fragments (5 x 10(-3) per cell). After the treatment of lymphoma by chemotherapy in combination with radiotherapy she also demonstrated a very high post-therapeutic level of chromosomal aberrations (21.5%), which consisted mostly of increased dicentric fragments (65 x 10(-3) per cell). Although her illness is now clinically cured, the observed genotoxic changes point to a greater risk for delayed complications after HD, emphasizing the necessity for further continuous survey of this patient.

Comparison of chromosome aberrations in peripheral blood lymphocytes from people occupationally exposed to ionizing and radiofrequency radiation.

The genotoxic effects of occupational exposure to ionizing and non-ionizing radiation were investigated in 25 physicians and nurses working in hospitals and in 20 individuals working at radio-relay stations. Examination was conducted by chromosome aberration analysis of peripheral blood lymphocytes. The data showed that total number of chromosome aberrations in people exposed to ionizing and radio-frequency radiation (4.08 +/- 0.37 and 4.35 +/- 0.5 on 200 scored metaphases, respectively) were almost equally higher than those of non-irradiated subjects. The increase was in proportion to the number of individuals having more that 5-aberration/200 metaphases. Acentric fragments comprised the most frequently seen type of aberration. The average numbers in examined groups (11.8 x 10(-3) and 14.8 x 10(-3) per cell, respectively), were significantly higher than 4.2 x 10(-3), which was observed in controls, unexposed individuals. Dicentric fragments were also frequent (4.8 x 10(-3) and 6.25 x 10(-3), respectively, vs. 0.52 x 10(-3) in control). In contrast, the frequency of chromatid breaks increased only after ionizing radiation (3.8 x 10(-3) vs. 0.26 x 10(-3) in control). A positive correlation between the total number of chromosome aberrations and cumulative 6-years dosage was also found. The data emphasized the dangerous effects of prolonged exposure to both types of radiation and indicated that chromosomal aberration analysis should be obligatory for individuals working at radio-relay stations.

Immunoprotective properties of peptidoglycan monomer linked with zinc in cholestatic jaundice.

BACKGROUND: Previously it was shown that a new immunostimulator, peptidoglycan monomer linked with zinc (PGM-Zn), might have immunocorrective and hepatotropic effects. Owing to this in the present study we investigated its effects on jaundice-induced immunodysfunction, which might be responsible for serious peri- and postoperative complications in biliary obstruction. METHODS: In vivo effects of PGM-Zn were analyzed in mice subjected to common bile duct ligation (CBDL), where we estimated phenotypic profile and cell cycle of thymocytes, splenocytes and phagocytic function of peritoneal macrophages. In vitro effects of PGM-Zn were evaluated on blastogenesis of human peripheral blood mononuclear cells (PBMNC), obtained from healthy donors and stimulated with anti-CD3 monoclonal antibody and/or PMA, in the presence or absence of jaundice serum obtained from patients with biliary calculosis. RESULTS AND DISCUSSION: Jaundice induced marked disarrangement of lymphatic homeostasis, which at several points might be blocked by PGM-Zn. In mice it delayed the CBDL-induced decline of CD4+ CD8+ thymocytes, decreased the proportion of CD8+ T cells, and increased the percentage of CD4- CD8- thymocytes, augmenting simultaneously the proportion of thymic cells in S and G2 + M phase of cycle. Similar hyperplastic reaction with increased percentage of CD4+, Ig+ and CD5+ cells was noticed in the spleen, together with the enhanced phagocytic ability of peritoneal macrophages. In human PBMNC jaundice reduced the percentages of CD3, CD5, CD4, CD8 and HLA-DR-expressing cells and increased the proportion of CD25 and perforin-positive lymphocytes. PGM-Zn given in vitro was able to abrogate the antiproliferative activity of jaundice serum on PMA and anti-CD3 + PMA-induced blastogenesis.

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.

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 and r=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 Zn-dependent hepato-thymic axis during the humoral and cellular types of immune reactivity.

Zinc and metallothioneins on cellular immune effectiveness during liver regeneration in young and old mice.

Partial hepatectomy in young mice (pHx) induces thymic atrophy, disregulation of thymocytes subsets and a strong accumulation of zinc in thymic tissue after 1-2 days of liver regeneration. Zinc is relevant for good immune functioning. Restoration of zinc into both the thymus and thymocytes subsets in the late period of liver regeneration is observed in young pHx mice. These findings have suggested a link between the thymus and the liver influencing T-cell functions and involving zinc. This kind of link could be relevant in aging because thymic involution, negative crude zinc balance and crippled immune functions are constant events. The preminence of a liver extrathymic T-cell pathway after pHx or during aging has been suggested. Thus the study of pHx in young and old mice may offer a good model to better understand the role played both by thymic involution and by liver extrathymic T-cell pathway and the role of zinc in these physiological processes during aging. Young pHx mice after 1-2 days of liver regeneration show: reduced thymic endocrine activity, increment of double negative (DN) thymocytes subsets, impairment of peripheral immune efficiency (PHA, NK activity and IL-2) and negative crude zinc balance, which are all restored in the late period of liver regeneration. By contrast the thymic and peripheral immune defects and the negative crude zinc balance, already present in old sham mice, are not modified during liver regeneration in old pHx mice. Circulating leukocytes and lymphocytes are not significantly modified both in young and old pHx mice as compared to respective sham controls. Zinc may also be crucial for extrathymic T-cells pathway, being preminent in aging, rather than in young age, due to its metallothioneins (MT) binding capacity. MT are significantly increased in young pHx and in aging inducing a low zinc-free quota for thymic and peripheral immune efficiency in young pHx mice, and for extrathymic T-cell pathway, in old age. Thus low zinc bioavailability, due to MT, may play a pivotal role, not only for thymocytes but also for liver extrathymic T-cell pathway.

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.

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.

Somatostatin promotes accumulation of phospholipids in regenerating liver tissue of rats.

Effects of somatostatin (SOM) on tissue contents of proteins, total lipids and phospholipids were investigated in regenerating and intact liver tissue of Y-59 rats. Whereas SOM inhibited protein accumulation in regenerating liver, the hormone evoked an increase in total lipids, and specially in phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine (PS) and phosphatidylinositol (PI). Since the same effects were not seen when intact liver was analyzed, it is assumed that SOM acts primarily on tissue stimulated to rapid growth. The increase of PS + PI fractions indicates a specific effect of SOM on the metabolism of phosphatidylinositides. Such an effect might result from the interference of the hormone with the action of growth factors that accelerate phosphatidylinositol breakdown.

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 X 10(6) 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 I (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 (12.0 +/- 0.68 days) (P less than 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 less than 0.05). The results indicate that halothane anesthesia per se may be associated with impairment of cell-mediated immunity under experimental conditions.