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Postoperative peritonitis is characterized by a more severe clinical course than other forms of secondary peritonitis. The pathophysiological mechanisms behind this phenomenon are incompletely understood. This study used an innovative model to investigate these mechanisms, combining the models of murine Colon Ascendens Stent Peritonitis (CASP) and Surgically induced Immune Dysfunction (SID). Moreover, the influence of the previously described anti-inflammatory reflex transmitted by the vagal nerve was characterized. SID alone, or 3 days before CASP were performed in female C57BL/6 N mice. Subdiaphragmatic vagotomy was performed six days before SID with following CASP. The immune status was assessed by FACS analysis and measurement of cytokines. Local intestinal inflammatory changes were characterized by immunohistochemistry. Mortality was increased in CASP animals previously subjected to SID. Subclinical bacteremia occurred after SID, and an immunosuppressive milieu occurred secondary to SID just before the induction of CASP. Previous SID modified the pattern of intestinal inflammation induced by CASP. Subdiaphragmatic vagotomy had no influence on sepsis mortality in our model of postoperative peritonitis. Our results indicate a surgery-induced inflammation of the small intestine and the peritoneal cavity with bacterial translocation, which led to immune dysfunction and consequently to a more severe peritonitis.

Background and Purpose— The contribution of neuroinflammation and, in particular, the infiltration of the brain by lymphocytes is increasingly recognized as a substantial pathophysiological mechanism after stroke. The interaction of lymphocytes with endothelial cells and platelets, termed thromboinflammation, fosters microvascular dysfunction and secondary infarct growth. Siponimod is an S1PR (sphingosine-1-phosphate receptor) modulator, which blocks the egress of lymphocytes from lymphoid organs and has demonstrated beneficial effects in multiple sclerosis treatment. We investigated the effect of treatment with siponimod on stroke outcome in a mouse model of cerebral ischemia. Methods— Transient middle cerebral artery occlusion was induced in middle-aged wild-type mice. Animals were either treated with siponimod (3 mg/kg; intraperitoneal) or vehicle for 6 days. Stroke outcome was assessed by magnetic resonance imaging (spleen volume: prestroke, day 3, and day 7; infarct volume: days 1, 3, and 7) and behavioral tests (prestroke, day 2, and day 6). Immune cells of the peripheral blood and brain-infiltrating cells ipsilateral and contralateral were analyzed by VETScan and by flow cytometry. Results— Siponimod significantly induced lymphopenia on day 7 after transient middle cerebral artery occlusion and reduced T-lymphocyte accumulation in the central nervous system. No effect was detected for lesion size. Conclusions— For siponimod administered at 3 mg/kg in transient middle cerebral artery occlusion mouse model, our findings do not provide preclinical evidence for the use of S1PR1/5 modulators as neuroprotectant in stroke therapy.

Protection against Staphylococcus aureus is determined by the polarization of the anti-bacterial immune effector mechanisms. Virulence factors of S. aureus can modulate these and induce differently polarized immune responses in a single individual. We proposed that this may be due to intrinsic properties of the bacterial proteins. To test this idea, we selected two virulence factors, the serine protease-like protein B (SplB) and the glycerophosphoryl diester phosphodiesterase (GlpQ). In humans naturally exposed to S. aureus, SplB induces a type 2-biased adaptive immune response, whereas GlpQ elicits type 1/type 3 immunity. We injected the recombinant bacterial antigens into the peritoneum of S. aureus-naïve C57BL/6N mice and analyzed the immune response. This was skewed by SplB toward a Th2 profile including specific IgE, whereas GlpQ was weakly immunogenic. To elucidate the influence of adjuvants on the proteins’ polarization potential, we studied Montanide ISA 71 VG and Imject™Alum, which promote a Th1 and Th2 response, respectively. Alum strongly increased antibody production to the Th2-polarizing protein SplB, but did not affect the response to GlpQ. Montanide enhanced the antibody production to both S. aureus virulence factors. Montanide also augmented the inflammation in general, whereas Alum had little effect on the cellular immune response. The adjuvants did not override the polarization potential of the S. aureus proteins on the adaptive immune response.

Vitamin B6 deficiency during pregnancy translates into a severe vitamin B6 deficiency (plasma levels decreased by 97%) in new-born rats. Further, hallmarks are increased (+89%) concentrations of homocysteine, gross changes in gene methylation and expression, and metabolic alterations including lipid metabolism. This study focuses on determining the effects of vitamin B6-deficiency on cardiolipin composition and oxidative phosphorylation in liver. For this purpose, hepatic cardiolipin composition was analyzed by means of LC/MS/MS, and mitochondrial oxygen consumption was determined by using a Clark-type electrode in a rat model of vitamin B6 deficiency. Liver mitochondria from new-born rats with pre-term vitamin B6 deficiency responded with substantial alterations in cardiolipin composition that include the following changes in the amounts of cardiolipin incorporated fatty acids: increase in C16, decrease in C18, decrease in saturated fatty acid, as well as increase in amount of oxidized cardiolipin species. These changes were accompanied by significantly decreased capacity of oxidative phosphorylation. In conclusion, vitamin B6 deficiency in new born rats induces massive alterations of cardiolipin composition and function of liver mitochondria. These findings support the importance of sufficient periconceptional supply of vitamin B6 to prevent vitamin B6 deficiency.Impact statementVitamin B6 (VitB6) is an active co-enzyme for more than 150 enzymes and is required for a great diversity of biosynthesis and metabolic reactions. There is an increased need for VitB6 during pregnancy and sufficient supply of VitB6 is crucial for the prevention of cleft palate and neural tube defects. We show that liver mitochondria from new-born rats with pre-term VitB6 deficiency respond with substantial alterations in cardiolipin (CL) composition and in the amount of oxidized CL species. These changes are associated with a decrease in the efficiency of oxidative phosphorylation. The results of this study support the significance of sufficient supply of VitB6 during pregnancy (and periconceptional) for diminishing the number of early abortions and minimizing malformation. The established link between VitB6 deficiency, CL composition, and mitochondrial respiration/energy production provides mechanistic insight as to how the VitB6 deficiency translates into the known pathophysiological and clinically relevant conditions.

sBackgroundObesity has become a severe problem among the world’s population with clearly increasing prevalence over the last decades. Because obesity is associated with several comorbidities (e.g. hypertension or cancer) it constitutes an increasing burden for the health care system. Correlations between obesity and cognition have been studied in humans with ambivalent results. Here, we studied the effects of obesity on hippocampus dependent learning and memory and cell morphology in a mouse model of obesity.MethodsThe body mass of male and female Lep+/+(wt) and Lepob/ob(ob/ob) animals with access to food and water ad libitum was measured between postnatal day 60-200 and animals with clear adiposity (4-6 months) were further analyzed. Adult hippocampal neurogenesis in the dentate gyrus was examined using phosphohistone H3 as a marker for proliferation, doublecortin as a marker for differentiation and caspase3 as a marker for apoptosis. Moreover, the density of dendritic spines on apical and basal dendrites of pyramidal neurons of the cornu ammonis 1 (CA1) were analyzed using Golgi impregnation. In addition, mice were subjected to the open field and Morris water maze test in order to analyze locomotor activity and spatial learning.ResultsThe body weight of ob/ob mice nearly doubled during the first 120 postnatal days. Adult hippocampal neurogenesis was reduced in ob/ob mice due to reduced cell proliferation. Dendritic spine densities in the hippocampal area CA1 were not altered in ob/ob mice. Four to six months old ob/ob mice showed reduced locomotor activity in the open field test but similar performance in the Morris water maze compared to control mice.ConclusionOur data show that alterations in adult neurogenesis in leptin-deficient mice are not associated with an impairment in spatial learning abilities. Moreover, ob/ob mice are inconspicuous in the Morris water maze and do not display altered spine densities in the hippocampus, suggesting that obesity does not have a severe impact upon hippocampal neuronal plasticity and spatial learning.

Using a combination of transcriptional profiling and Ingenuity Pathway Analysis (IPA, www.ingenuity.com) we investigated acute and chronic psychological stress induced alterations of hepatic gene expression of BALB/c mice. Already after a 2-h single stress session, up-regulation of several LPS and glucocorticoid-sensitive immune response genes and markers related to oxidative stress and apoptotic processes were observed. Support for the existence of oxidative stress was gained by measuring increased protein carbonylation, but no alterations of immune responsiveness or cell death were measured in mice after acute stress compared to the control group. When animals were repeatedly stressed during 4.5-days, we found reduced transcription of antigen presentation molecules, altered mRNA levels of immune cell signaling mediators and persisting high expression of apoptosis-related genes. These alterations were associated with a measurable immune suppression characterized by a reduced ability to clear experimental Salmonella typhimurium infection from the liver and a heightened hepatocyte apoptosis. Moreover, genes associated with anti-oxidative functions and regenerative processes were induced in the hepatic tissue of chronically stressed mice. These findings indicate that modulation of the immune response and of apoptosis-related genes is initiated already during a single acute stress exposure. However, immune suppression will only manifest in repeatedly stressed mice which additionally show induction of protective and liver regenerative genes to prevent further hepatocyte damage.

Increased activity of the tryptophan-metabolizing enzyme indoleamine 2,3-dioxygenase (IDO) is associated with immunological and neurological disorders, and inhibition of its enzyme activity could be a therapeutic approach for treatment of these disorders. The aim of the present study was to establish a large animal model to study the accumulation of the potential IDO inhibitor 1-methyltryptophan (1-MT) in blood and different organs of domestic pigs (Sus scrofa domestica). Because 1-MT has not been previously evaluated in pigs, the pharmacokinetics of a single subcutaneous 1-MT application was investigated. Based on this kinetic study, a profile for repeated 1-MT applications over a period of five days was simulated and tested. The results show that a single administration of 1-MT increases its concentrations in blood, with the maximum concentration being obtained at 12 h. Repeated daily injections of 1‑MT generated increasing plasma concentrations followed by a steady-state after two days. Twelve hours after the final application, accumulation of 1-MT was observed in the brain and other organs, with a substantial variability among various tissues. The concentrations of 1-MT measured in plasma and tissues were similar to, or even higher, than those of tryptophan. Our data indicate that repeated subcutaneous injections of 1-MT provide a suitable model for accumulation of 1-MT in plasma and tissues of domestic pigs. These findings provide a basis for further research on the immunoregulatory functions of IDO in a large animal model.

We analyzed the putative association between abdominal obesity (measured in waist circumference) and gray matter volume (Study of Health in Pomerania: SHIP-2, N=758) adjusted for age and gender by applying volumetric analysis and voxel-based morphometry (VBM) with VBM8 to brain magnetic resonance (MR) imaging. We sought replication in a second, independent population sample (SHIP-TREND, N=1586). In a combined analysis (SHIP-2 and SHIP-TREND) we investigated the impact of hypertension, type II diabetes and blood lipids on the association between waist circumference and gray matter. Volumetric analysis revealed a significant inverse association between waist circumference and gray matter volume. VBM in SHIP-2 indicated distinct inverse associations in the following structures for both hemispheres: frontal lobe, temporal lobes, pre- and postcentral gyrus, supplementary motor area, supramarginal gyrus, insula, cingulate gyrus, caudate nucleus, olfactory sulcus, para-/hippocampus, gyrus rectus, amygdala, globus pallidus, putamen, cerebellum, fusiform and lingual gyrus, (pre-) cuneus and thalamus. These areas were replicated in SHIP-TREND. More than 76% of the voxels with significant gray matter volume reduction in SHIP-2 were also distinct in TREND. These brain areas are involved in cognition, attention to interoceptive signals as satiety or reward and control food intake. Due to our cross-sectional design we cannot clarify the causal direction of the association. However, previous studies described an association between subjects with higher waist circumference and future cognitive decline suggesting a progressive brain alteration in obese subjects. Pathomechanisms may involve chronic inflammation, increased oxidative stress or cellular autophagy associated with obesity.

Indoleamine 2,3-dioxygenase (IDO) is a rate-limiting enzyme for the degradation of tryptophan (Trp) along the kynurenine (Kyn) pathway, and its increased activation is associated with immunologic disorders. Because the specific role of IDO activation is not yet completely clear, the aim of the present study was to establish a pig model of IDO activation for further research. The activation of IDO in pigs was induced experimentally by LPS stimulation in vivo and ex vivo. IDO activation was characterized by measuring Trp, Trp metabolites and IDO protein expression in blood, liver, lung, muscle and different brain areas. The results show that the in vivo LPS administration induced increased plasma concentrations of TNF-α and IL-10, a depletion of Trp and an increase of Kyn, indicating an elevated enzymatic activity of IDO. This was supported by an LPS-induced IDO protein expression in blood, liver and lung. The ex vivo LPS stimulation also resulted in increased TNF-α concentrations and an IDO activation, characterized by an increase of Trp metabolites and IDO protein expression. In conclusion, our data emphasize that the LPS stimulation is a suitable model for IDO activation in the domestic pig, which provides a basis for further research on immunoregulatory IDO functions.

It has recently been shown that an increased plasma level of the tryptophan catabolite kynurenine is an early indicator for the development of sepsis in major trauma patients. We examined the predictive value of kynurenine pathway activity for ongoing sepsis in patients being admitted to a surgical intensive care unit for different reasons. In addition, we asked whether an accumulation of kynurenines in patients’ plasma depends on reduced renal clearance. We conducted a prospective observational study including 100 consecutive patients and monitored laboratory variables, physiological and adverse events, sepsis and outcome. Using tandem mass spectrometry, we quantified the five indoleamines tryptophan, serotonin (5-HT), kynurenine, quinolinic acid and kynurenic acid at baseline and twice a week during the intensive care unit stay. Among the patients enrolled, 50 did not develop sepsis in the intensive care unit (non-septic), 18 patients did not have sepsis at baseline but developed sepsis later on (preseptic) and 32 patients already fulfilled the criteria of severe sepsis and septic shock at baseline (septic). In general, non-septic critically ill patients showed activation of the kynurenine pathway, but septic shock coincided with an exacerbation of kynurenine pathway activity even in the absence of renal failure. Importantly, plasma concentrations of quinolinic acid (area under the curve 0.832 [95% confidence interval 0.710 to 0.954]) and the Quin/Trp ratio (area under the curve 0.835 [95% confidence interval; 0.719 to 0.952]) showed the best discrimination between non-septic and pre-septic patients at baseline. These findings open new avenues for further investigations on the pathophysiology of sepsis.

Routine laboratory procedures, such as handling or transporting animals or carrying out injections on animals, are stressful for animals but are necessary in many pre-clinical studies. Here, the authors show that multiple injections of the non-toxic vehicle cyclodextrin moderately increased plasma corticosterone concentrations in female BALB/c mice. Additionally, male BALB/c mice that had received a single intraperitoneal injection of harmless saline had an increased glucocorticoid response to a second saline injection. The authors found that female mice that had been exposed to an acute psychological stress session had a decreased glucocorticoid response to a second homotypic stressor. In contrast, multiple psychological stress sessions led to increased glucocorticoid release in female mice. Acute injection(s) of saline in male mice and of cyclodextrin in female mice led to transient lymphocytopenia. Further analysis showed that repeated stress-induced lymphocytopenia is glucocorticoid-dependent. The authors conclude that laboratory stress can affect physiological parameters in mice, potentially altering study results.

Background Laboratory routine procedures such as handling, injection, gavage or transportation are stressful events which may influence physiological parameters of laboratory animals and may interfere with the interpretation of the experimental results. Here, we investigated if female BALB/c mice derived from in-house breeding and BALB/c mice from a vendor which were shipped during their juvenile life differ in their HPA axis activity and stress responsiveness in adulthood. Results We show that already transferring the home cage to another room is a stressful event which causes an increased HPA axis activation for at least 24 hours as well as a loss of circulating lymphocytes which normalizes during a few days after transportation. However and important for the interpretation of experimental data, commercially available strain-, age- and gender-matched animals that were shipped over-night showed elevated glucocorticoid levels for up to three weeks after shipment, indicating a heightened HPA axis activation and they gained less body weight during adolescence. Four weeks after shipment, these vendor-derived mice showed increased corticosterone levels at 45-min after intraperitoneal ACTH challenge but, unexpectedly, no acute stress-induced glucocorticoid release. Surprisingly, activation of monoaminergic pathways were identified to inhibit the central nervous HPA axis activation in the vendor-derived, shipped animals since depletion of monoamines by reserpine treatment could restore the stress-induced HPA axis response during acute stress. Conclusions In-house bred and vendor-derived BALB/c mice show a different stress-induced HPA axis response in adulthood which seems to be associated with different central monoaminergic pathway activity. The stress of shipment itself and/or differences in raising conditions, therefore, can cause the development of different stress response phenotypes which needs to be taken into account when interpreting experimental data.

Stress is a powerful modulator of neuroendocrine, behavioral, and immunological functions. After 4.5-d repeated combined acoustic and restraint stress as a murine model of chronic psychological stress, severe metabolic dysregulations became detectable in female BALB/c mice. Stress-induced alterations of metabolic processes that were found in a hepatic mRNA expression profiling were verified by in vivo analyses. Repeatedly stressed mice developed a hypermetabolic syndrome with the severe loss of lean body mass, hyperglycemia, dyslipidemia, increased amino acid turnover, and acidosis. This was associated with hypercortisolism, hyperleptinemia, insulin resistance, and hypothyroidism. In contrast, after a single acute stress exposure, changes in expression of metabolic genes were much less pronounced and predominantly confined to gluconeogenesis, probably indicating that metabolic disturbances might be initiated already early but will only manifest in repeatedly stressed mice. Thus, in our murine model, repeated stress caused severe metabolic dysregulations, leading to a drastic reduction of the individual’s energy reserves. Under such circumstances stress may further reduce the ability to cope with new stressors such as infection or cancer.

Most studies on the biogenesis of mitochondrial proteins have been carried out using fungal mitochondria as a model system. In particular, baker's yeast, Saccharomyces cerevisiae, combines several experimental advantages, allowing both genetic and biochemical approaches and thus a combination of investigations in vivo and in vitro. However, the red bread mold Neurospora crassa has also been an important research tool. Isolated mitochondria can be used from both organisms for import experiments in a reconstituted system, using radiolabeled precursor proteins synthesized in reticulocyte lysate or purified preproteins. Assays are available for studies on the import pathways and localization of mitochondrial proteins and for the characterization of the components of the protein import machinery.