Impairment of endoplasmic reticulum in liver as an early consequence of the systemic inflammatory response in rats.

It is well known that systemic inflammatory response (SIR) often causes liver dysfunction. The aim of this study was to identify the intracellular compartment in the liver most susceptible to SIR. We analyzed morphology, ultrastructure, proteome, and expression of relevant genes in livers of rats subjected to endotoxic shock. Histological examination revealed that focal necrosis in liver was insignificant to explain liver dysfunction. Electron microscopy revealed no morphological changes in the mitochondrial structure and in the cytosol, but dilated endoplasmic reticulum (ER) cisterns were frequently observed. Apoptosis was found in white blood cells within liver tissue but not in hepatocytes. Mitochondrial, ER, and cytosolic fractions were subjected to proteome analysis by difference gel electrophoresis, and the protein spots with the highest degree of differential regulation were identified with mass spectrometry. The most pronounced proteome changes appeared in the ER, manifested as a remarkable downregulation of several proteins essential for ER functions, such as protein synthesis and transport, whereas the changes in mitochondrial and cytosolic fractions suggested a compensatory response. ER stress, as an underlying mechanism for ER impairment, was confirmed by analysis of upstream (splicing X-box-binding protein 1 mRNA) and downstream (e.g., 78-kDa glucose-regulated protein mRNA) markers, suggesting ongoing unresolved ER stress as a cause for ER dilation. Because ER is the intracellular compartment responsible for the major liver functions, our data suggest that inflammatory mediators induce unresolved ER stress, resulting in the biochemical, functional, and morphological impairment of ER that in turn causes liver dysfunction. The pathway activating ER stress in response to SIR is not known yet.

Porcine T-helper and regulatory T cells exhibit versatile mRNA expression capabilities for cytokines and co-stimulatory molecules.

T-helper (TH) and regulatory T cells (Tregs) are important modulators of immune responses. Aim of this study was to analyse their expression potential for cytokines and other immune-relevant molecules. Therefore, porcine PBMC, CD4(-) cells, CD4(+)CD25(-) resting, CD4(+)CD25(dim) activated TH cells, and CD4(+)CD25(high) Tregs were analysed on their mRNA expression potential ex vivo or after in vitro stimulation with CD3 and IL-2 by RT-qPCR. In vitro stimulation led to an increased production of pro-inflammatory (IL-6, TNFα) and TH (IL-2, IL-4, IL-17, IFN-γ) cytokines and a diverse production of immunosuppressive cytokines (IL-10 and TGF-ß) in PBMC, CD4(-), and CD4(+) cells. Resting and activated TH cells showed an increased expression of various immune-modulatory molecules indicating that porcine TH cells possess distinct immunological skills in order to react on the actual immune situation. In contrast, Tregs appear to fulfil mainly immunosuppressive functions characterized by increased production of IL-10, IL-35, CD40L, and CD25.

Transient increase of free iron in rat livers following hemorrhagic-traumatic shock and reperfusion is independent of heme oxygenase 1 upregulation.

Hemorrhagic-traumatic shock (HTS) followed by reperfusion induces heme oxygenase (HO) 1. Free iron (Fe2+) may cause oxidative stress, if not adequately sequestered. We aimed to characterize HO-1-mediated effects on Fe2+ levels in liver and transferrin-bound iron (TFBI) in plasma following HTS, including laparotomy, bleeding, and inadequate and adequate reperfusion. Anesthetized rats showed upregulated HO-1 mRNA at 40 min after HTS, which was followed by increased HO activity at 3 h after shock. Fe2+ levels were transiently increased at 40 min after shock, a time point when HO activity was not affected yet. Levels of plasma TFBI were higher in HTS animals, showing the highest levels at 40 min after shock, and decreased thereafter. In addition, we modulated HO activity 6 h before HTS by administering an inhibitor (zinc-protoporphyrin IX) or an activator (hemin) of HO. At 18 h after HTS in all shock groups, HO activity was increased, the highest being in the hemin-pretreated group. The zinc-protoporphyrin IX-treated HTS animals showed increased HO-1 mRNA and Fe2+ levels in the liver compared with the untreated HTS animals. Transferrin-bound iron levels were affected by pharmacological modulation before shock. All animals undergoing HTS displayed increased TFBI levels after reperfusion; however, in animals pretreated with hemin, TFBI levels increased less. Our data indicate that increase in Fe2+ levels in liver and plasma early after HTS is not mediated by HO-1 upregulation, but possibly reflects an increased mobilization from internal iron stores or increased cell damage. Thus, upregulation of HO activity by hemin does not increase Fe2+ levels following HTS and reperfusion.

Exploratory reference intervals on hematology and cellular immune system of multiparous Large White sows.

There is significant lack of basic hematologic and immunological data in adult sows. Therefore, aim of this study was to provide respective reference intervals. 32 clinically healthy multiparous Large White sows aged 33.5 ± 9.6 months and all of them two months postpartum were included in this study. Mean erythrocyte count was 5.5 ± 0.7 × 10(6)/µl and total leukocyte count was 12.1 ± 2.1 × 10(3)/µl. Proportion of lymphocytes was 44.7 ± 10.2% and of neutrophils 41.6 ± 11.0%. The ratio of naïve T helper (Th) cells to memory Th cells was 1:3.1 and the ratio of Th cells to cytotoxic T cells (CTLs) was 1:4.2. Proportions of regulatory T cells, NK cells, and CD21(+) B cells were lower (3.1, 2.6, and 6.0%) than those of memory Th cells ranging from 8.8 to 27.5% depending on the activation status and CTLs with 37.3%. γδ T cells were found at comparably high numbers (19.1%). Flow cytometric measurement of intracellular cytokines in PBMCs revealed marginal levels for IL-1ß, IL-2, IL-4, IL-6, IL-10, and IL-12p35, but remarkable levels for TNF-α and IFN-γ. Highest mRNA levels were found for IL-1, IL-10, and TNF-α, with TNF-α showing the least inter-individual variation.

Effect of estrogen on mitochondrial function and intracellular stress markers in rat liver and kidney following trauma-hemorrhagic shock and prolonged hypotension.

Trauma-hemorrhage (T-H) is known to impair tissue perfusion, leading to tissue hypoxia, and thus affecting mitochondria, the organelles with the highest oxygen demand. In a model of T-H and prolonged hypotension without fluid resuscitation, administration of a small volume of 17beta-estradiol (E2), but not vehicle, prolonged the survival of rats for 3 h, even in the absence of fluid resuscitation. The main finding of this study is that T-H followed by prolonged hypotension significantly affects mitochondrial function, endoplasmic reticulum (ER) stress markers and free iron levels, and that E2 ameliorated all these changes. All of these changes were observed in the liver but not in the kidney. The sensitivity of mitochondrial respiration to exogenous cytochrome c can reflect increased permeability of the outer mitochondrial membrane for cytochrome c. Increased levels of free iron are indicative of oxidative stress, but neither oxidative nor nitrosylative stress markers changed. The spliced isoform of XBP1 mRNA (an early marker of ER stress) and the expression of C/EBP homologous protein (CHOP) (a protein regulating ER stress-induced apoptosis) were elevated in T-H animals but remained unchanged if T-H rats received E2. Both the prevention of elevated sensitivity of mitochondrial respiration to cytochrome c and a decrease in ER stress by E2 maintain functional integrity of the liver and may help the organ during prolonged hypotension and following resuscitation. A decrease in free iron levels by E2 is more relevant for resuscitation, often accompanied by oxidative stress reaction. Thus, E2 appears to be a novel hormonal adjunct that prolongs permissive hypotension during lengthy transportation of the injured patient between the injury site and the hospital in both civilian and military injuries.

Peritoneal inflammation in pigs is associated with early mitochondrial dysfunction in liver and kidney.

The objective of this study was to investigate early effects of peritoneal inflammation on the mitochondrial function in the vital organs, liver and kidney, and their relation to inflammatory and oxidative stress mediators. The study was performed on 14 domestic pigs. Peritoneal inflammation was induced in anesthetized pigs after a midline laparotomy by autologous feces. Fluid resuscitation maintained a MAP above 60 mmHg. Animals were sacrificed 12 h later, and tissue samples were obtained to determine mitochondrial function, mRNA levels of relevant genes [inducible NO synthase (iNOS), inducible HO (HO-1), tumor necrosis factor-alpha (TNF-alpha)], generation of reactive oxygen species (ROS), and HO-1 activity. We found impaired mitochondrial function in both liver and kidney, based on decreased state 3 respiration in the liver and increased states 2 and 4 respiration in the kidney at 12 h. This was accompanied by increased TNF-alpha protein in the blood and up-regulation of TNF-alpha mRNA in the liver. Free iron was elevated in the liver but not in the kidney. In the kidney, mitochondrial ROS production was increased. Nitric oxide levels in blood remained unchanged, corresponding to unchanged levels of iNOS mRNA expression in liver and kidney. Similarly, HO-1 mRNA and heme oxygenase (HO)-activity were unchanged. The inflammatory response in the absence of characteristic septic symptoms was not associated with morphological organ damage at this early time point. Peritoneal inflammation in pigs caused mitochondrial dysfunction in liver and kidney, preceding signs of organ damage. We did not find proof that mitochondrial dysfunction was due to increased levels of either nitric oxide (NO) or products of HO, but it was accompanied by increased levels of oxidative stress markers.

Reperfusion does not induce oxidative stress but sustained endoplasmic reticulum stress in livers of rats subjected to traumatic-hemorrhagic shock.

Oxidative stress is believed to accompany reperfusion and to mediate dysfunction of the liver after traumatic-hemorrhagic shock (THS). Recently, endoplasmic reticulum (ER) stress has been suggested as an additional factor. This study investigated whether reperfusion after THS leads to increased oxidative and/or ER stress in the liver. In a rat model, including laparotomy, bleeding until decompensation, followed by inadequate or adequate reperfusion phase, three time points were investigated: 40 min, 3 h, and 18 h after shock. The reactive oxygen and nitrogen species and its scavenging capacity (superoxide dismutase 2), the nitrotyrosine formation in proteins, and the lipid peroxidation together with the status of endogenous antioxidants (alpha-tocopherylquinone-alpha-tocopherol ratio) were investigated as markers for oxidative or nitrosylative stress. Mitochondrial function and cytochrome P450 isoform 1A1 activity were analyzed as representatives for hepatocyte function. Activation of the inositol-requiring enzyme 1/X-box binding protein pathway and up-regulation of the 78-kDa glucose-regulated protein were recorded as ER stress markers. Plasma levels of alanine aminotransferase and Bax/Bcl-XL messenger RNA (mRNA) ratio were used as indicators for hepatocyte damage and apoptosis induction. Oxidative or nitrosylative stress markers or representatives of hepatocyte function were unchanged during and short after reperfusion (40 min, 3 h after shock). In contrast, ER stress markers were elevated and paralleled those of hepatocyte damage. Incidence for sustained ER stress and subsequent apoptosis induction were found at 18 h after shock. Thus, THS or reperfusion induces early and persistent ER stress of the liver, independent of oxidative or nitrosylative stress. Although ER stress was not associated with depressed hepatocyte function, it may act as an early trigger of protracted cell death, thereby contributing to delayed organ failure after THS.

Endotoxin causes functional endoplasmic reticulum failure, possibly mediated by mitochondria.

Inflammatory response has recently been shown to induce endoplasmic reticulum (ER) stress and the unfolded protein response (UPR), which either recovers proper ER function or activates apoptosis. Here we show that endotoxin (lipopolysaccharide = LPS) can lead to functional ER failure tentatively via a mitochondrion-dependent pathway in livers of rats. Histological examination did not reveal significant damage to liver in form of necroses. Electron microscopy displayed transparent rings appearing around morphologically unchanged mitochondria, which were identified as dilated ER. The spliced mRNA variant of X-box protein-1 (XBP1) and also the mRNA of 78 kDa glucose-regulated protein (GRP78) were up-regulated, both typical markers of ER stress. However, GRP78 was down-regulated at the protein level. A pro-apoptotic shift in the bax/bcl-XL mRNA ratio was not accompanied by translocation of apoptosis inducing factor (AIF) to the nucleus, suggesting that the cells entered a pre-apoptotic state, but apoptosis was not executed. Monooxygenase activity of p450, representing the detoxification system in ER, was decreased after administration of endotoxin. Biochemical analysis of proteins important for ER function revealed the impairment of protein folding, transport, and detoxification suggesting functional ER failure. We suggest that functional ER failure may be a reason for organ dysfunction upon excessive inflammatory response mediated by endotoxin.

A novel endotoxin-induced pathway: upregulation of heme oxygenase 1, accumulation of free iron, and free iron-mediated mitochondrial dysfunction.

Mitochondria are involved in the development of organ failure in critical care diseases. However, the mechanisms underlying mitochondrial dysfunction are not clear yet. Inducible hemoxygenase (HO-1), a member of the heat shock protein family, is upregulated in critical care diseases and considered to confer cytoprotection against oxidative stress. However, one of the products of HO-1 is Fe2+ which multiplies the damaging potential of reactive oxygen species catalyzing Fenton reaction. The aim of this study was to clarify the relevance of free iron metabolism to the oxidative damage of the liver in endotoxic shock and its impact on mitochondrial function. Endotoxic shock in rats was induced by injection of lipopolysaccharide (LPS) at a dose of 8 mg/kg (i.v.). We observed that the pro-inflammatory cytokine TNF-alpha and the liver necrosis marker aspartate aminotransferase were increased in blood, confirming inflammatory response to LPS and damage to liver tissue, respectively. The levels of free iron in the liver were significantly increased at 4 and 8 h after onset of endotoxic shock, which did not coincide with the decrease of transferrin iron levels in the blood, but rather with expression of the inducible form of heme oxygenase (HO-1). The proteins important for sequestering free iron (ferritin) and the export of iron out of the cells (ferroportin) were downregulated facilitating the accumulation of free iron in cells. The temporarily increased concentration of free iron in the liver correlated with the temporary impairment of both mitochondrial function and tissue ATP levels. Addition of exogenous iron ions to mitochondria isolated from control animals resulted in an impairment of mitochondrial respiration similar to that observed in endotoxic shock in vivo. Our data suggest that free iron released by HO-1 causes mitochondrial dysfunction in pathological situations accompanied by endotoxic shock.

Increased mRNA levels of the mitochondrial complex I 75-kDa subunit. A potential peripheral marker of early onset schizophrenia?

Recently, the dopamine D3-receptor mRNA on blood lymphocytes and platelet mitochondrial complex I were suggested as biological markers of schizophrenia in adults. We investigated the mRNA level of the dopamine D3-receptor and complex I subunits in whole blood cells of early-onset schizophrenic patients compared to healthy controls using quantitative real-time PCR. We found an increased mRNA expression of the complex I 75-kDa subunit (referred to beta-actin in schizophrenic patients (0.57 +/- 0.24 versus 0.23 +/- 0.18 in controls, P < 0.01)), but were unable to analyse the dopamine D3-mRNA expression. This increase appears to be inherent to schizophrenia, because it was found in neuroleptic-naive patients and it was not affected by neuroleptic treatment. Our preliminary findings suggest the mitochondrial complex I as a potential peripheral marker of schizophrenia and its involvement in the pathophysiology of this illness.