Nicole Manhart

Assessing the antioxidative status in critically ill patients.

Purpose of reviewOxidative stress is caused by a higher production of reactive oxygen and reactive nitrogen species or a decrease in endogenous protective antioxidative capacity. In all types of critical illness, such as sepsis, trauma, burn injury, acute pancreatitis, liver injury, severe diabetes, acute respiratory distress syndrome, AIDS and kidney failure, the occurrence of increased oxidative stress or a reduced antioxidative status is described. Whereas in the past, reactive oxygen and reactive nitrogen species were mainly known as harmful agents, recent investigations have given a new insight into the (patho)physiological importance of these substances as powerful messenger molecules involved in gene regulation, thereby enabling the synthesis of cytokines or adhesion molecules necessary for defending inflammatory processes. As shown in this review, there are numerous possibilities for the quantification of oxidative stress. Recent findingsSeveral investigations showed a close association of single or multiple parameters, such as total antioxidative capacity, lipid peroxidation, vitamins C and E, the activation of nuclear factor kappa B, and respiratory burst, with the patients outcome. However, no recommendation for a single parameter to be measured can be given because the assays described do not allow the definition of an overall ‘antioxidative status’ for patients. SummaryThe occurrence of oxidative stress in critically ill patients is associated with a poor prognosis. The measurement of a cluster of assays representative of the quantification of reactive species or of antioxidants may improve the usefulness of therapeutic intervention and increase knowledge of pathophysiological alterations.

Oral Feeding With Glutamine Prevents Lymphocyte and Glutathione Depletion of Peyer’s Patches in Endotoxemic Mice

ObjectiveTo determine the effect of oral glutamine feeding on lymphocyte subpopulations and glutathione metabolism in Peyer’s patches (PPs) of healthy and endotoxemic mice. Summary Background DataRecent data indicate that nutrients both maintain nitrogen and energy balances and modulate cell and organ function. In particular, glutamine has an impact on gut and immune function. This is of special importance in the perioperative phase. MethodsFemale Balb/c mice were fed a glutamine-enriched diet or a control diet for 10 days. On day 7 25 &mgr;g lipopolysaccharide (LPS) or saline was injected. On day 3 after the challenge, mice were killed, total cell yield was determined, and lymphocyte subpopulations (total T cells, CD4+, CD8+ cells, and B cells) were analyzed by flow cytometry. One experimental group was treated with buthionine sulfoximine, a specific inhibitor of glutathione synthesis. The glutathione content in PPs was measured by high-performance liquid chromatography. ResultsGlutamine administration led to a significant increase in total cell yield, including T and B cells, in PPs. The LPS-induced reduction of T cells (−45%) and of B cells (−30%) was significantly lower in glutamine-treated mice. Endotoxemia caused a 42% decrease of glutathione in control animals, but not in glutamine-treated animals. As with LPS, buthionine sulfoximine also lowered lymphocyte numbers and glutathione content of the PPs. ConclusionsAdministration of glutamine prevents LPS-stimulated lymphocyte atrophy in PPs, possibly by increasing the glutathione content in the PPs. Therefore, oral glutamine supply seems to be a suitable approach for improving intestinal immunity in immunocompromised patients.

Immunomodulatory effects of glycine on LPS-treated monocytes: reduced TNF-α production and accelerated IL-10 expression

Cytokines play a pivotal role in the pathogenesis of septic shock. Proinflammatory cytokines such as tumor necrosis factor‐α (TNF‐α) and interleukin‐1β (IL‐1β) stimulate the progression of septic shock whereas the anti‐inflammatory cytokine IL‐10 has counterregulative potency. The amino acid glycine (GLY) has been shown to protect against endotoxin shock in the rat by inhibiting TNF‐α production. In the current study we investigated the role of GLY on lipopolysaccharide (LPS) ‐induced cell surface marker expression, phagocytosis, and cytokine production on purified monocytes from healthy donors. GLY did not modulate the expression of HLA‐DR and CD64 on monocytes, whereas CD11b/CD18 expression (P<0.05) and E. coli phagocytosis (P<0.05) decreased significantly. GLY decreased LPS‐induced TNF‐α production (P<0.01) and increased IL‐10 expression of purified monocytes. Similarly, in a whole blood assay, GLY reduced TNF‐α (P< 0.0001) and IL‐1β (P<0.0001) synthesis and increased IL‐10 expression (P<0.05) in a dose‐dependent manner. The inhibitory effects of GLY were neutralized by strychnine, and the production of IL‐10 and TNF‐α was augmented by anti‐IL‐10 antibodies. Furthermore, GLY decreased the amount of IL‐1β and TNF‐α‐specific mRNA. Our data indicate that GLY has a potential to be used as an additional immunomodulatory tool in the early phase of sepsis and in different pathophysiological situations related to hypoxia and reperfusion.—Spittler, A., Reissner, C. M., Oehler, R., Gornikiewicz, A., Gruenberger, T., Manhart, N., Brodowicz, T., Mittlboeck, M., Boltz‐Nitulescu, G., Roth, E. Immunomodulatory effects of glycine on LPS‐treated monocytes: reduced TNF‐α production and accelerated IL‐10 expression. FASEB J. 13, 563–571 (1999)

Influence of fructooligosaccharides on Peyer's patch lymphocyte numbers in healthy and endotoxemic mice.

OBJECTIVE The purpose of this study was to determine whether fructooligosaccharides (FOS) exert an immunomodulating effect on Peyers patches (PP), the main inductive site of the intestinal immune system. We investigated the effects of FOS in healthy and endotoxemic animals. METHODS Six-week-old female Balb/c mice were fed a control diet or a diet supplemented with 10% FOS over a period of 16 d. To induce endotoxemia, mice were challenged intraperitoneally with lipopolysaccharide (LPS) on day 15. PP were excised from mice, and lymphocyte subpopulations (B lymphocytes, T lymphocytes, CD4(+) cells, and CD8(+) cells) were determined by flow cytometry. RESULTS The FOS-enriched diet increased the total cell yield in healthy and endotoxemic mice (P < 0.001). Similarly, B lymphocytes were increased in both groups (P < 0.001). In contrast, T lymphocytes were unaltered in healthy mice but increased in LPS-challenged mice after FOS enrichment (P < 0.001). In endotoxemic mice but not in control animals, the increase of CD4(+) cells (P < 0.001) was more pronounced than that of CD8(+) cells (P < 0.001), thus increasing the CD4:CD8 ratio (P < 0.01). CONCLUSION FOS showed an immunostimulating effect on PP lymphocytes under healthy and endotoxemic conditions. Thus it can be concluded that FOS administration affects not only the large intestine but also the main inductive part of the mucosal immune system in the small intestine.

Influence of short-term protein malnutrition of mice on the phenotype and costimulatory signals of lymphocytes from spleen and Peyer’s patches

The objective of this study was to investigate the impact of short-term protein malnutrition (PM) on immunoglobulin A (IgA) production and on the number and phenotype of lymphocytes in Peyers patches (PP) and in the spleen. Balb/c mice were fed for 4, 7, or 10 d with a protein-deficient diet (0.1% protein). We determined B lymphocytes (CD40(+)), T lymphocytes (CD3(+)), T-helper (CD4(+)), and T-suppressor (CD8(+)) cells and the expression of costimulatory signals B7.1 (CD80) and B7.2 (CD86) on B cells and their counter receptors CD28 and CTLA-4 on T cells by fluorescence-activated cell-sorting analysis. Luminal IgA concentration in the small intestine was determined by an enzyme-linked immunosorbent assay. Four days of PM caused a significant reduction in the number of mononuclear cells in the spleen (5.6 x 10(7) +/- 1 x 10(7) versus 2. 4 x 10(7) +/- 0.5 x 10(7), P < 0.001) and the PP (13 x 10(6) +/- 3 x 10(6) versus 8.6 x 10(6) +/- 2 x 10(6), P < 0.01). There was a relative increase of T cells in the spleen and a relative increase of B cells in the PP. Luminal IgA content of small intestine was significantly reduced after 4 d of PM (242 +/- 55 microg versus 173 +/- 39 microg, P < 0.05) and remained at about this level until day 10 of PM. Four days after PM, the costimulatory signals B7.1 and B7. 2 on B cells were upregulated in the PP but markedly downregulated in the spleen, which was inversely related to the expression of the counter receptor CD28 on T-helper cells. We conclude that short-term PM increases the activation of B cells in the PP but reduces the relative number and activation state of splenic B cells. Only 4 d of PM caused a systemic and intestinal immunodepression, as indicated by a markedly decreased content of mononuclear cells in the PP and the spleen.

Lipopolysaccharide causes atrophy of Peyer's patches and an increased expression of CD28 and B7 costimulatory ligands.

Intestinal mucosal dysfunction appears to contribute to infectious complications in critically ill patients. The current study was undertaken to investigate whether endotoxin affects lymphocyte subpopulations and the expression of costimulatory signals in Peyers patches (PP). Female Balb/c mice were given an intraperitoneal injection of 25 microg LPS and sacrified 24 h or 72 h later to determine total cell yield, lymphocyte subpopulations (B-cells, total T-cells, CD4+- and CD8+-cells), the costimulatory molecules CD28, B7.1 (CD80) and B7.2 (CD86) and the percentage of apoptotic cells in PP and in the spleen as well as small intestinal IgA concentration. Lipopolysaccharide (LPS) challenge caused a significant decrease of total cell yield in PP at both time-points (-50+/-28% and -43+/-25%, respectively; P < 0.001). This decrease was significant for all measured lymphocyte subpopulations. In contrast, total cell yield was increased (P < 0.001) in the spleen 24 h (+52+/-13%) and 72 h (+130+/-22%) after LPS. The decrease of lymphocyte numbers in the PP was accompanied by an increased percentage of lymphocytes expressing costimulatory molecules. In this respect, an increased percentage of CD40+CD80+, CD40+CD86+, and of CD4+CD28+ could be demonstrated after LPS administration. In the spleen, the percentage of CD4+CD28+ was also elevated after LPS bolus, however, the percentage of CD40+CD80+ was reduced, and that of CD40+CD86+ was unaltered. The influence of LPS on apoptosis of lymphocytes was time-dependent. The percentage of apoptotic cells 24 h after LPS was increased in PP (P < 0.01), but was unchanged in the spleen. Seventy-two hours after LPS injection, the percentage of apoptotic cells returned to normal in PP. Luminal IgA levels remained unchanged after LPS challenge. In conclusion, our data show that LPS causes atrophy of PP which seems to be counterregulated by an enhanced expression of costimulatory molecules.

Supply of R-α-lipoic acid and glutamine to casein-fed mice influences the number of B lymphocytes and tissue glutathione levels during endotoxemia

ZusammenfassungHINTERGRUND: Reaktive Sauerstoffspezies und eine verringerte antioxidative Kapazität spielen eine wichtige Rolle in der Modulierung des Immunsystems kritisch Kranker. Ziel dieser Studie war es, den Einfluss einer gemeinsamen oralen Zufuhr der Antioxidantien R-α-Liponsäure (LA) und Glutamin (GLN) auf das Immunsystem und den Glutathion-Stoffwechsel bei einer Endotoxinämie an der Maus zu untersuchen. METHODIK: Weibliche Balb/c Mäuse erhielten über einen Zeitraum von 10 Tagen Nahrungen, die mit GLN (3 g/100 kcal), LA (0.74 mg/100 kcal) oder einer Kombination aus GLN und LA angereichert waren, wobei eine isokalorische und isonitrogene Kontrolle als Vergleichsnahrung diente. Zweiundsiebzig Stunden nach der intraperitonealen Verabreichung von 25 μg Lipopolysaccharid am Tag 7 wurden Anzahl und Phänotyp der Lymphozyten aus den Peyerschen Plaques und der Milz ermittelt. Zusätzlich wurde der Glutathion-Gehalt im Dünndarm, in der Milz und in der Leber gemessen. RESULTATE: Nur eine kombinierte Zufuhr von GLN und LA war in der Lage, die Gesamtzellzahl in den Peyerschen Plaques (+19%) zu erhöhen, was hauptsächlich auf einen Anstieg der B-Lymphozyten zurückzuführen war. In der Milz steigerten sowohl die LA (+17%) als auch die gemeinsame Gabe von GLN und LA (+22%) die Gesamtzellzahl. Der Glutathion-Gehalt des Dünndarms wurde durch LA erhöht, wohingegen GLN plus LA in der Milz am effektivsten war. SCHLUSSFOLGERUNG: Die gemeinsame Gabe von GLN und LA ist in der Lage bei experimenteller Endotoxinämie die Zahl sowohl systemischer als auch intestinaler Blymphozyten selektiv zu erhöhen. Weiters führte LA im Dünndarm zu einem Anstieg des GSH-Gehaltes, dem mengenmäßig am häufigsten vorkommenden intrazellulären Antioxidans. Auf Grundlage dieser Daten ist die Untersuchung einer gemeinsamen Gabe von LA und GLN in septischen Patienten zu empfehlen.SummaryBACKGROUND: An overwhelming production of reactive oxygen species concomitant with a decrease in antioxidative capacity plays an important role in modulation of the immune system in critically ill patients. The purpose of this study was to assess the influence of a combined oral supply of the antioxidants R-α-lipoic acid (LA) and glutamine (GLN) on the immunity of endotoxemic mice, with a special focus on tissue glutathione levels. METHODS: Female Balb/c mice were fed diets enriched with GLN (3 g/100 kcal), LA (0.74 mg/100 kcal), a combination of GLN and LA, or an isocaloric and isonitrogenous control diet for 10 days. On day 7, the mice were challenged intraperitoneally with 25 μg lipopolysaccharide. Seventy-two hours later, the number and phenotype of lymphocytes in Peyers patches (PP) and spleen of the endotoxemic mice were measured. In addition, glutathione levels were determined in the small intestine, spleen and liver. RESULTS: In PP only the combined supply of GLN and LA significantly increased the total cell yield (+19%), which was predominantly due to an increased number of B cells. In the spleen, both LA (+17%) and the combination of GLN and LA (+22%) were able to enhance total cell yield. The glutathione content of the small intestine was increased by feeding LA alone, whereas in the spleen GLN plus LA was most effective. CONCLUSION: Supplying combined GLN and LA to endotoxemic mice is effective in selectively increasing the number of systemic and intestinal B lymphocytes. Furthermore, LA augmented the level of the main intracellular antioxidant glutathione in the small intestine. On the basis of these data we recommend investigation of the effects of LA and GLN supplementation in patients with sepsis.