Influence of intravenous vitamin E supplementation in cardiac surgery on oxidative stress: a double-blinded, randomized, controlled study.

BACKGROUND: I.V. infusions of vitamin E emulsion (all-rac-alpha-tocopherol) may reduce ischaemia-reperfusion injury after elective cardiac surgery. METHODS: Forty patients participated in a prospective, double-blind, placebo-controlled, randomized trial, receiving either placebo or four doses (270 mg each) of all-rac-alpha-tocopherol between 16 h before and 48 h after surgery. We determined plasma concentrations of vitamin E, vitamin C, malondialdehyde, creatine kinase, troponin I and interleukin 6 and other measures of clinical outcome. RESULTS: Infusion of vitamin E caused normalization of vitamin E plasma concentrations during and after surgery, but had no effect on the early increase in malondialdehyde concentration or the decreases in antioxidative capacity and the water-soluble antioxidant vitamin C. CONCLUSIONS: Normalization of plasma vitamin E concentrations with parenteral vitamin E emulsion does not affect biochemical markers of myocardial injury and does not affect clinical outcome after cardiac surgery.

Administration of proteolytic enzymes bromelain and trypsin diminish the number of CD4+ cells and the interferon-gamma response in Peyer's patches and spleen in endotoxemic balb/c mice.

Recent publications revealed that bromelain exerts a marked effect on T-cell response by inhibiting T-cell signal transduction. These experimental studies may help to explain former clinical investigations showing that Phlogenzym (PHL), a preparation consisting of the proteases bromelain and trypsin and the antioxidant rutosid, ameliorate certain diseases with an underlying inflammatory process. In this study, we showed that orally administered PHL significantly reduced lymphocyte subpopulations in Peyer's patches (PPs) of healthy and endotoxemic mice. Similarly, the number of splenic lymphocytes in endotoxin-boostered mice was significantly lowered by PHL. The effect of PHL was more pronounced on T cells than on B cells leading especially to a diminution of CD4+ cells. Moreover, PHL pretreatment decreased IFN-gamma mRNA in PPs and spleen of endotoxemic mice. These results reveal that PHL may ameliorate inflammatory process by reducing the number of CD4+ cells and by diminishing INF-gamma mRNA levels.

Oral feeding with glutamine prevents lymphocyte and glutathione depletion of Peyer's patches in endotoxemic mice.

OBJECTIVE: To 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 DATA: Recent 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. METHODS: Female Balb/c mice were fed a glutamine-enriched diet or a control diet for 10 days. On day 7 25 microg 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. RESULTS: Glutamine 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. CONCLUSIONS: Administration 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.

Postoperative glycyl-glutamine infusion reduces immunosuppression: partial prevention of the surgery induced decrease in HLA-DR expression on monocytes.

BACKGROUND AND AIMS: Surgery, trauma and inflammation reduce HLA-DR expression on monocytes, which is associated with an increased susceptibility to infection and sepsis. Furthermore, surgery decreases plasma glutamine (GLN) levels. The expression of HLA-DR on human monocytes in vitro is dependent on the concentration of GLN in the culture medium. We therefore hypothesized that postoperative infusions of glutamine-dipeptides would prevent the decreased HLA-DR expression on monocytes. METHODS: Thirty patients undergoing major abdominal surgery were randomly allocated to receive either 1500 ml Vamin (control) or an isonitrogenic formulation containing Vamin and 500 ml glycyl-glutamine (35 g GLN; 0.5g/kg BW) (GLY-GLN), or Vamin and 500 ml alanyl-glutamine (35 g GLN; 0.5 g/kg BW) (ALA-GLN) as a continuous infusion over 48 h post-operatively. Immediately and 48 h after surgery blood samples were collected to determine HLA-DR expression on monocytes by flow cytometry. RESULTS: The groups were comparable with respect to age, gender distribution and operation time. In patients receiving GLY-GLN mean HLA-DR expression on monocytes at 48 h was significantly better preserved than in controls (65.0 %+/-7 % vs 42.5 %+/-4 %;P<0.05), whereas HLA-DR expression on monocytes in patients receiving ALA-GLN was not significantly different. CONCLUSION: This is the first study comparing the dipeptides GLY-GLN and ALA-GLN in the postoperative setting. The GLY-GLN induced preservation of HLA-DR on monocytes following surgery may prevent infectious complications in these patients.

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 Peyer's 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.

Therapeutic potential of glutathione.

Reactive oxygen species, formed in various biochemical reactions, are normally scavenged by antioxidants. Glutathione in its reduced form (GSH) is the most powerful intracellular antioxidant, and the ratio of reduced to oxidised glutathione (GSH:GSSG) serves as a representative marker of the antioxidative capacity of the cell. Several clinical conditions are associated with reduced GSH levels which as a consequence can result in a lowered cellular redox potential. GSH and the redox potential of the cell are components of the cell signaling system influencing the translocation of the transcription factor NF kappa B which regulates the synthesis of cytokines and adhesion molecules. Therefore, one possibility to protect cells from damage caused by reactive oxygen species is to restore the intracellular glutathione levels. Cellular GSH concentration can be influenced by exogenous administration of GSH (as intravenous infusion or as aerosol), of glutathione esters or of GSH precursors such as glutamine or cysteine (in form of N-acetyl-L-cysteine, alpha-lipoic acid). The modulation of GSH metabolism might present a useful adjuvant therapy in many pathologies such as intoxication, diabetes, uremia, sepsis, inflammatory lung processes, coronary disease, cancer and immunodeficiency states.

Influence of enteral diets supplemented with key nutrients on lymphocyte subpopulations in Peyer's patches of endotoxin-boostered mice.

BACKGROUND AND AIMS: This study was undertaken to compare the effect of different key nutrients on lymphocyte subsets of Peyer's patches (PP) and spleen in endotoxemic mice. METHODS: Female Balb/c mice were fed over a period of 10 days either with an isocaloric and isonitrogenous control diet (Control), a glutamine enriched diet (Diet I) or a diet containing glutamine, arginine, glycine, and n-3 fatty acids (Diet II). On day 7 the mice were challenged intraperitoneally with 25 microg LPS. The lymphocyte subpopulations (B cells, T cells, CD4+ and CD8+) of PP and spleen were analysed by flow cytometry. Glutathione content of small intestinal mucosa and spleen was determined by HPLC and luminal small intestinal IgA by ELISA. RESULTS: Both experimental diets increased the number of B and T cells in the PP and that of T cells in the spleen (P<0.01). Glutathione content in PP and spleen was higher under administration of key nutrients (P<0.05). Diet II reduced luminal small intestinal IgA content in comparison to the two other groups. CONCLUSION: The addition of arginine, glycine and n-3 fatty acids to a glutamine supplemented diet does not enhance lymphocyte numbers in PP and spleen, but reduces intestinal IgA content.

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 Peyer's 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.

Polytrauma induces increased expression of pyruvate kinase in neutrophils.

Polytrauma (PT) leads to systemic activation of polymorphonuclear neutrophils (PMNs). Organ damage commonly found in these patients is ascribed to respiratory bursts of activated PMNs. With the use of sodium dodecyl sulfate-polyacrylamide gel electrophoresis, PMN extracts from PT patients were found to contain a clear protein band not seen in control PMNs from healthy volunteers. This band was identified by amino acid sequencing and Western blotting as pyruvate kinase (PK). Enzymatic assays revealed a 600-fold increase in PK activity in PMNs of PT patients, with the highest levels occurring between the fifth and seventh posttraumatic day. In lymphocytes, no such increase was detectable. As PK is a major regulatory enzyme in glycolysis, glucose-dependent lactate production in PMNs from PT patients was assayed. These cells showed a higher glycolytic lactate production than controls. It was additionally demonstrated that acute activation of respiratory burst activity depends mainly on breakdown of glucose to lactate via the pentose-phosphate pathway and glycolysis. In PMNs from PT patients, this glucose-dependent respiratory burst activity was more than twofold higher than in controls. The increase in expression and activity of PK in PMNs from PT patients may contribute to the high glucose-dependent respiratory burst activity seen in these cells.

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

Cytokines play a pivotal role in the pathogenesis of septic shock. Proinflammatory cytokines such as tumor necrosis factor-alpha (TNF-alpha) and interleukin-1beta (IL-1beta) 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-alpha 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-alpha production (P<0.01) and increased IL-10 expression of purified monocytes. Similarly, in a whole blood assay, GLY reduced TNF-alpha (P<0.0001) and IL-1beta (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-alpha was augmented by anti-IL-10 antibodies. Furthermore, GLY decreased the amount of IL-1beta and TNF-alpha-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.

Receptor and non-receptor mediated formation of superoxide anion and hydrogen peroxide in neutrophils of intensive care patients.

Generation of reactive oxygen intermediates (ROI) has been implicated in tissue damage in a variety of disease states including sepsis and trauma. On the other hand, generation of ROI in polymorphonuclear granulocytes (PMN) presents a crucial element in the defence of the host against invading microorganisms. In the present study we investigated the generation of superoxide anions (O2-) and hydrogen peroxide (H2O2) by neutrophils (PMN)5 of 17 critically ill patients treated at a intensive care unit (ICU) after polytrauma (n = 6), heart operation (n = 6) or during septic shock (n = 5) using flow cytometry. O2- production of PMN from ICU patients was significantly lower (p < 0.01) than that in healthy volunteers (HV) during non-receptor mediated stimulation with phorbol-myristate-acetate (PMA) but higher (p < 0.001) during receptor mediated stimulation with formylmethionine-leucine-phenylalanine (FMLP). H2O2 generation of PMN from ICU patients was increased after stimulation with FMLP (p < 0.01) and remained unchanged after stimulation with PMA. Patients in septic shock had lower O2(-)-generation of PMN than did injured patients and patients after heart operations. We conclude that receptor mediated formation of O2- and H2O2 is stimulated in ICU patients. However, in patients in septic shock O2(-)-generation decreases, which potentially might contribute to the immunoparalysis present in septic shock.