Mode of induction of platelet-derived extracellular vesicles is a critical determinant of their phenotype and function.

Platelet-derived extracellular vesicles (PDEVs) are the most abundant amongst all types of EVs in the circulation. However, the mechanisms leading to PDEVs release, their role in coagulation and phenotypic composition are poorly understood. PDEVs from washed platelets were generated using different stimuli and were characterised using nanoparticle tracking analysis. Procoagulant properties were evaluated by fluorescence flow cytometry and calibrated automated thrombography. EVs from plasma were isolated and concentrated using a novel protocol involving a combination of size exclusion chromatography and differential centrifugation, which produces pure and concentrated EVs. Agonist stimulation enhanced PDEV release, but did not alter the average size of EVs compared to those produced by unstimulated platelets. Agonist stimulation led to lower negatively-charged phospholipid externalization in PDEVs, which was reflected in the lower procoagulant activity compared to those generated without agonist stimulation. Circulating EVs did not have externalized negatively-charged phospholipids. None of the 4 types of EVs presented tissue factor. The mechanism by which PDEV formation is induced is a critical determinant of its phenotype and function. Importantly, we have developed methods to obtain clean, concentrated and functional EVs derived from platelet-free plasma and washed platelets, which can be used to provide novel insight into their biological functions.

Comparison of different dietary sources of n-3 polyunsaturated fatty acids on immune response in broiler chickens.

The study aims to research the effects of varied dietary sources of n-3 polyunsaturated fatty acids (PUFA) on the immune response in broiler chickens with stress on natural killer (NK) cell activity. Diets supplemented with one of the four sources of n-3 PUFA: linseed oil-, echium oil-, fish oil (FO) or algal biomass-enriched diets at levels of 18, 18, 50 and 15 g/kg fresh weight, were provided for one-d-old male Ross 308 broilers, totaling 340 in number, until they were slaughtered. The analyses included total lipid profile using gas chromatography (GC) for plasma, spleen, thymus, and blood. Additionally, NK cell activity and cell proliferation were investigated for thymocytes and splenocytes. The results indicated that the source of n-3 PUFA had a strong influence on fatty acid composition across all tissues. NK activity was highest in splenocytes and PBMCs from broilers fed linseed oil, followed by those fed algal biomass or echium oil, and lowest for those from broilers fed FO. The proliferative response of lymphocytes from algal biomass-fed chickens tended to be the highest, followed by those fed linseed oil in most cases. Lymphocytes from chickens fed fish oil showed the lowest proliferative response. These results could mean that a docosahexaenoic acid (DHA)-rich algal product might enrich chicken meat with n-3 PUFA without significant damaging effects on chicken immunity.

Effect of n-3 fatty acids on immune function in broiler chickens.

There is interest in the enrichment of poultry meat with long-chain n-3 polyunsaturated fatty acids in order to increase the consumption of these fatty acids by humans. However, there is concern that high levels of n-3 polyunsaturated fatty acids may have detrimental effects on immune function in chickens. The effect of feeding increasing levels of fish oil (FO) on immune function was investigated in broiler chickens. Three-week-old broilers were fed 1 of 4 wheat-soybean basal diets that contained 0, 30, 50, or 60 g/kg of FO until slaughter. At slaughter, samples of blood, bursa of Fabricius, spleen, and thymus were collected from each bird. A range of immune parameters, including immune tissue weight, immuno-phenotyping, phagocytosis, and cell proliferation, were assessed. The pattern of fatty acid incorporation reflected the fatty acid composition of the diet. The FO did not affect the weight of the spleen, but it did increase thymus weight when fed at 50 g/kg (P < 0.001). Fish oil also lowered bursal weights when fed at 50 or 60 g/kg (P < 0.001). There was no significant effect of FO on immune cell phenotypes in the spleen, thymus, bursa, or blood. Feeding 60 g/kg of FO significantly decreased the percentage of monocytes engaged in phagocytosis, but it increased their mean fluorescence intensity relative to that of broilers fed 50 g/kg of FO. Lymphocyte proliferation was significantly decreased after feeding broiler chickens diets rich in FO when expressed as division index or proliferation index, although there was no significant effect of FO on the percentage of divided cells. In conclusion, dietary n-3 polyunsaturated fatty acids decrease phagocytosis and lymphocyte proliferation in broiler chickens, highlighting the need for the poultry industry to consider the health status of poultry when poultry meat is being enriched with FO.

Selective effects of Lactobacillus casei Shirota on T cell activation, natural killer cell activity and cytokine production.

Modulation of host immunity is an important potential mechanism by which probiotics confer health benefits. This study was designed to investigate the effects of a probiotic strain, Lactobacillus casei Shirota (LcS), on immune function using human peripheral blood mononuclear cells (PBMC) in vitro. In addition, the role of monocytes in LcS-induced immunity was also explored. LcS promoted natural killer (NK) cell activity and preferentially induced expression of CD69 and CD25 on CD8(+) and CD56(+) subsets in the absence of any other stimulus. LcS also induced production of interleukin (IL)-1beta, IL-6, tumour necrosis factor (TNF)-alpha, IL-12 and IL-10 in the absence of lipopolysaccharide (LPS). In the presence of LPS, LcS enhanced IL-1beta production but inhibited LPS-induced IL-10 and IL-6 production, and had no further effect on TNF-alpha and IL-12 production. Monocyte depletion reduced significantly the impact of LcS on lymphocyte activation, cytokine production and natural killer (NK) cell activity. In conclusion, LcS activated cytotoxic lymphocytes preferentially in both the innate and specific immune systems, which suggests that LcS could potentiate the destruction of infected cells in the body. LcS also induced both proinflammatory and anti-inflammatory cytokine production in the absence of LPS, but in some cases inhibited LPS-induced cytokine production. Monocytes play an important role in LcS-induced immunological responses.

Omega-3 (n-3) fatty acids, cardiovascular disease and stability of atherosclerotic plaques.

Long-chain n-3 polyunsaturated fatty acids are found in oily fish and in fish oils and similar preparations. Substantial evidence from epidemiological and case-control studies indicates that consumption of fish, oily fish and long-chain n-3 fatty acids reduces risk of cardiovascular mortality. Secondary prevention studies using long-chain n-3 fatty acids in patients post-myocardial infarction have shown a reduction in total and cardiovascular mortality with an especially potent effect on sudden death. Long-chain n-3 fatty acids have been shown to beneficially modify a range of cardiovascular risk factors, which may result in primary cardiovascular prevention. However, reduced non-fatal and fatal events and a reduction in sudden death probably involve other mechanisms. Reduced thrombosis following long-chain n-3 fatty acids may play a role. A decrease in arrhythmias is a favoured mechanism of action of long-chain n-3 fatty acids and is supported by cell culture and animal studies. However human trials using implantable cardiac defibrillators have produced inconsistent findings and a recent meta-analysis does not support this mechanism of action. An alternative mechanism of action may be stabilisation of atherosclerotic plaques by long-chain n-3 fatty acids. This is suggested by one published human study which showed that incorporation of long-chain n-3 fatty acids into plaques collected at carotid endarterectomy resulted in fewer macrophages in the plaque and a morphology indicative of increased stability. These findings are supported from observations in an animal model and suggest that the primary effect of long-chain n-3 fatty acids might be on macrophages within the plaque.

Chemical, physical, and sensory properties of dairy products enriched with conjugated linoleic acid.

Recent studies have illustrated the effects of cis-9,trans-11 conjugated linoleic acid (CLA) on human health. Ruminant-derived meat, milk and dairy products are the predominant sources of cis-9,trans-11 CLA in the human diet. This study evaluated the processing properties, texture, storage characteristics, and organoleptic properties of UHT milk, Caerphilly cheese, and butter produced from a milk enriched to a level of cis-9,trans-11 CLA that has been shown to have biological effects in humans. Forty-nine early-lactation Holstein-British Friesian cows were fed total mixed rations containing 0 (control) or 45 g/kg (on dry matter basis) of a mixture (1:2 wt/wt) of fish oil and sunflower oil during two consecutive 7-d periods to produce a control and CLA-enhanced milk, respectively. Milk produced from cows fed the control and fish and sunflower oil diets contained 0.54 and 4.68 g of total CLA/100 g of fatty acids, respectively. Enrichment of CLA in raw milk from the fish and sunflower oil diet was also accompanied by substantial increases in trans C18:1 levels, lowered C18:0, cis-C18:1, and total saturated fatty acid concentrations, and small increases in n-3 polyunsaturated fatty acid content. The CLA-enriched milk was used for the manufacture of UHT milk, butter, and cheese. Both the CLA-enhanced butter and cheese were less firm than control products. Although the sensory profiles of the CLA-enriched milk, butter, and cheese differed from those of the control products with respect to some attributes, the overall impression and flavor did not differ. In conclusion, it is feasible to produce CLA-enriched dairy products with acceptable storage and sensory characteristics.

Dietary fatty acids and chylomicron synthesis and secretion.

There is currently considerable interest in potential atherogenic and thrombogenic consequences of elevated concentrations of triacylglycerols, especially in the post-prandial state. Despite this, there is limited information on the effects of dietary fatty acids on the synthesis, secretion and metabolism of chylomicrons, the large triacylglycerol-rich lipoproteins synthesized in the enterocyte following the digestion and absorption of dietary fat. This brief review considers current approaches to the investigation of chylomicron synthesis and summarizes some of the human, cell and animal studies that have investigated effects of different fatty acids on these pathways. Potential sites for modulatory effects of dietary fatty acids on the molecular events of chylomicron synthesis are proposed in the light of the recent model that has been developed from cell and animal studies and observations based on abnormalities in chylomicron formation in human inherited autosomal recessive diseases.

A randomised clinical trial to assess the effect of total enteral and total parenteral nutritional support on metabolic, inflammatory and oxidative markers in patients with predicted severe acute pancreatitis (APACHE II > or =6).

BACKGROUND: Total enteral nutrition (TEN) within 48 h of admission has recently been shown to be safe and efficacious as part of the management of severe acute pancreatitis. Our aim was to ascertain the safety of immediate TEN in these patients and the effect of TEN on systemic inflammation, psychological state, oxidative stress, plasma glutamine levels and endotoxaemia. METHODS: Patients admitted with predicted severe acute pancreatitis(APACHE II score >5) were randomised to total enteral (TEN; n = 8) or total parenteral nutrition (TPN; n = 9). Measurements of systemic inflammation (C-reactive protein), fatigue (visual analogue scale), oxidative stress (plasma thiobarbituric acid-reactive substances), plasma glutamine and anti-endotoxin IgG and IgM antibody concentrations were made on admission and repeated on days 3 and 7 thereafter. Clinical progress was monitored using APACHE II score. Organ failure and complications were recorded. RESULTS: All patients tolerated the feeding regime well with few nutrition-related complications. Fatigue improved in both groups but more rapidly in the TEN group. Oxidative stress was high on admission and rose by similar amounts in both groups. Plasma glutamine concentrations did not change significantly in either group. In the TPN group, 3 patients developed respiratory failure and 3 developed non-respiratory single organ failure. There were no such complications in the TEN group. Hospital stay was shorter in the TEN group [7(4-14) vs. 10 (7-26) days; p = 0.05] as was time to passing flatus and time to opening bowels [1 (0-2) vs. 2 (1-5)days; p = 0.01]. The cost of TEN was considerably less than of TPN. CONCLUSION: Immediate institution of nutritional support in the form of TEN is safe in predicted severe acute pancreatitis. It is as safe and as efficacious as TPN and may be beneficial in the clinical course of this disease.

N-3 polyunsaturated fatty acids and inflammation in the arterial wall.

Atherosclerosis, leading to cardiovascular disease, is a chronic condition involving a strong inflammatory component. There is evidence that the n-3 polyunsaturated fatty acids (PUFA) present in oily fish and fish oils protect against cardiovascular disease. While these fatty acids have well-recognised effects on plasma triacylglycerol concentrations, it is likely that they exert beneficial effects through other mechanisms in addition. A large body of evidence suggests that the n-3 PUFA have anti-inflammatory properties, some of which may be manifested in the arterial wall, either directly or indirectly, to modulate the progression of atherosclerosis. This review critically evaluates the evidence for the anti-inflammatory effects of the n-3 PUFA in cells and on pathways which have a direct influence on atherogenesis in the arterial wall.

Monounsaturated fatty acids and immune function.

Animal studies generally support the idea that olive oil, rich in oleic acid, is capable of modulating functions of cells of the immune system. Importantly, several studies have demonstrated suppressive effects of oleic acid-containing diets on in vivo immune responses. There is some evidence that the effects of olive oil on immune function in animal studies are due to oleic acid rather than trace elements or antioxidants, although the evidence is not conclusive. In contrast to animal studies, consumption of a MUFA-rich diet by healthy human subjects does not appear to bring about a general suppression of immune cell functions. The lack of a clear effect of MUFA in human subjects is likely to be attributable to the much higher level of MUFA used in animal studies, which are not readily achievable or realistic in human studies. In conclusion, there is potential for MUFA to modulate immune function, but the effects in humans are likely to be far more subtle than those reported in animal studies. It remains to be seen whether MUFA will be clinically useful in immunonutrition.

Fatty acids and lymphocyte functions.

The immune system acts to protect the host against pathogenic invaders. However, components of the immune system can become dysregulated such that their activities are directed against host tissues, so causing damage. Lymphocytes are involved in both the beneficial and detrimental effects of the immune system. Both the level of fat and the types of fatty acid present in the diet can affect lymphocyte functions. The fatty acid composition of lymphocytes, and other immune cells, is altered according to the fatty acid composition of the diet and this alters the capacity of those cells to produce eicosanoids, such as prostaglandin E2, which are involved in immunoregulation. A high fat diet can impair lymphocyte function. Cell culture and animal feeding studies indicate that oleic, linoleic, conjugated linoleic, gamma-linolenic, dihomo-gamma-linolenic, arachidonic, alpha-linolenic, eicosapentaenoic and docosahexaenoic acids can all influence lymphocyte proliferation, the production of cytokines by lymphocytes, and natural killer cell activity. High intakes of some of these fatty acids are necessary to induce these effects. Among these fatty acids the long chain n-3 fatty acids, especially eicosapentaenoic acid, appear to be the most potent when included in the human diet. Although not all studies agree, it appears that fish oil, which contains eicosapentaenoic acid, down regulates the T-helper 1-type response which is associated with chronic inflammatory disease. There is evidence for beneficial effects of fish oil in such diseases; this evidence is strongest for rheumatoid arthritis. Since n-3 fatty acids also antagonise the production of inflammatory eicosanoid mediators from arachidonic acid, there is potential for benefit in asthma and related diseases. Recent evidence indicates that fish oil may be of benefit in some asthmatics but not others.

Dietary supplementation with gamma-linolenic acid or fish oil decreases T lymphocyte proliferation in healthy older humans.

Animal and human studies have shown that greatly increasing the amounts of flax seed oil [rich in the (n-3) polyunsaturated fatty acid (PUFA) alpha-linolenic acid (ALNA)] or fish oil [FO; rich in the long chain (n-3) PUFA eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA)] in the diet can decrease mitogen-stimulated lymphocyte proliferation. The objective of this study was to determine the effect of dietary supplementation with moderate levels of ALNA, gamma-linolenic acid (GLA), arachidonic acid (ARA), DHA or FO on the proliferation of mitogen-stimulated human peripheral blood mononuclear cells (PBMC) and on the production of cytokines by those cells. The study was randomized, placebo-controlled, double-blinded and parallel. Healthy subjects ages 55-75 y consumed nine capsules/d for 12 wk; the capsules contained placebo oil (an 80:20 mix of palm and sunflower seed oils) or blends of placebo oil with oils rich in ALNA, GLA, ARA or DHA or FO. Subjects in these groups consumed 2 g of ALNA or 770 mg of GLA or 680 mg of ARA or 720 mg of DHA or 1 g of EPA plus DHA (720 mg of EPA + 280 mg of DHA) daily from the capsules. Total fat intake from the capsules was 4 g/d. The fatty acid composition of PBMC phospholipids was significantly changed in the GLA, ARA, DHA and FO groups. Lymphocyte proliferation was not significantly affected by the placebo, ALNA, ARA or DHA treatments. GLA and FO caused a significant decrease (up to 65%) in lymphocyte proliferation. This decrease was partly reversed by 4 wk after stopping the supplementation. None of the treatments affected the production of interleukin-2 or interferon-gamma by PBMC and none of the treatments affected the number or proportion of T or B lymphocytes, helper or cytotoxic T lymphocytes or memory helper T lymphocytes in the circulation. We conclude that a moderate level GLA or EPA but not of other (n-6) or (n-3) PUFA can decrease lymphocyte proliferation but not production of interleukin-2 or interferon-gamma.

Dietary fatty acids influence the production of Th1- but not Th2-type cytokines.

C57B16 mice were fed for 6 weeks on a low-fat diet or on high-fat diets containing coconut oil (rich in saturated fatty acids), safflower oil [rich in n-6 polyunsaturated fatty acids (PUFAs)], or fish oil (rich in n-3 PUFAs) as the main fat sources. The fatty acid composition of the spleen lymphocytes was influenced by that of the diet fed. Thymidine incorporation into concanavalin A-stimulated spleen lymphocytes and interleukin (IL)-2 production were highest after feeding the coconut oil diet. Interferon (IFN)-gamma production was decreased by safflower oil or fish oil feeding. IL-4 production was not significantly affected by diet, although production was lowest by lymphocytes from fish oil-fed mice. The ratio of production of Th1- to Th2-type cytokines (determined as the IFN-gamma/IL-4 ratio) was lower for lymphocytes from mice fed the safflower oil or fish oil diets. After 4 h of culture, IL-2 mRNA levels were higher in cells from mice fed coconut oil, and IFN-gamma mRNA levels were higher in cells from mice fed coconut oil or safflower oil. After 8 h of culture, IL-2, IFN-gamma, and IL-4 mRNA levels were lowest in cells from mice fed fish oil. The ratio of the relative levels of IFN-gamma mRNA to IL-4 mRNA was highest in cells from mice fed coconut oil and was lowest in cells of mice fed fish oil. The influence of individual fatty acids on IL-2 production by murine spleen lymphocytes was examined in vitro. Although all fatty acids decreased IL-2 production in a concentration-dependent manner, saturated fatty acids were the least potent and n-3 PUFAs the most potent inhibitors, with n-6 PUFAs falling in between in terms of potency. It is concluded that saturated fatty acids have minimal effects on cytokine production. In contrast, PUFAs act to inhibit production of Th1-type cytokines with little effect on Th2-type cytokines; n-3 PUFAs are particularly potent. The effects of fatty acids on cytokine production appear to be exerted at the level of gene expression.

The effects of phenolic components of tea on the production of pro- and anti-inflammatory cytokines by human leukocytes in vitro.

Epidemiological evidence suggests protective effects of dietary flavonoids against cardiovascular disease. Tea provides a major source of dietary flavonoids in many countries and its polyphenolic components have well-recognised antioxidant properties. However, scavenging of free radicals may not be the sole mechanism by which tea-derived polyphenols exert their protective effects. This study investigates the effects of four major tea-derived catechins and a black tea extract on the production of pro- and anti-inflammatory cytokines by human leukocytes in vitro. Epicatechin gallate, epigallocatechin and epigallocatechin gallate decreased the production of interleukin 1beta and enhanced the production of interleukin 10, but had no effect on the production of interleukin 6 or tumour necrosis factor-alpha. Although these effects suggest anti-inflammatory properties of the tea-derived catechins, they were observed at concentrations which were unlikely to be achievable in plasma in vivo and are therefore unlikely to contribute to the protective effects of tea-derived flavonoids in inflammatory diseases.

Dietary supplementation with eicosapentaenoic acid, but not with other long-chain n-3 or n-6 polyunsaturated fatty acids, decreases natural killer cell activity in healthy subjects aged >55 y.

BACKGROUND: Animal studies showed that dietary flaxseed oil [rich in the n-3 polyunsaturated fatty acid alpha-linolenic acid (ALA)], evening primrose oil [rich in the n-6 polyunsaturated fatty acid gamma-linolenic acid (GLA)], and fish oil [rich in the long-chain n-3 polyunsaturated fatty acids eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA)] can decrease natural killer (NK) cell activity. There have been no studies of the effect on NK cell activity of adding these oils to the diet of humans. OBJECTIVE: Our objective was to determine the effect of dietary supplementation with oil blends rich in ALA, GLA, arachidonic acid (AA), DHA, or EPA plus DHA (fish oil) on the NK cell activity of human peripheral blood mononuclear cells. DESIGN: A randomized, placebo-controlled, double-blind, parallel study was conducted. Healthy subjects aged 55-75 y consumed 9 capsules/d for 12 wk; the capsules contained placebo oil (an 80:20 mix of palm and sunflower seed oils) or blends of placebo oil and oils rich in ALA, GLA, AA, DHA, or EPA plus DHA. Subjects in these groups consumed 2 g ALA, 770 mg GLA, 680 mg AA, 720 mg DHA, or 1 g EPA plus DHA (720 mg EPA + 280 mg DHA) daily, respectively. Total fat intake from the capsules was 4 g/d. RESULTS: The fatty acid composition of plasma phospholipids changed significantly in the GLA, AA, DHA, and fish oil groups. NK cell activity was not significantly affected by the placebo, ALA, GLA, AA, or DHA treatment. Fish oil caused a significant reduction (mean decline: 48%) in NK cell activity that was fully reversed by 4 wk after supplementation had ceased. CONCLUSION: A moderate amount of EPA but not of other n-6 or n-3 polyunsaturated fatty acids can decrease NK cell activity in healthy subjects.

Encapsulated fish oil enriched in alpha-tocopherol alters plasma phospholipid and mononuclear cell fatty acid compositions but not mononuclear cell functions.

BACKGROUND: Several studies have reported that dietary fish oil (FO) supplementation alters cytokine production and other functional activities of peripheral blood mononuclear cells (PBMC). However, few of these studies have been placebo controlled and few have related the functional changes to alterations in PBMC fatty acid composition PATIENTS AND METHODS: Healthy subjects supplemented their diets with 9 g day-1 of encapsulated placebo oil (3 : 1 mix of coconut and soybean oils), olive oil (OO), safflower oil (SO), evening primrose oil (EPO) or FO [providing 2.1 g eicosapentaenoic acid (EPA) plus 1.1 g docosahexaenoic acid (DHA) per day] for 12 weeks; the capsules also provided 205 mg alpha-tocopherol per day. Blood was sampled at 4-weekly intervals and plasma and PBMC prepared. Plasma phospholipid and PBMC fatty acid composition, plasma alpha-tocopherol and thiobarbituric acid-reactive substance concentrations, plasma total antioxidant capacity, the proportions of different PBMC subsets, the proportions of PBMC expressing the adhesion molecules CD2, CD11b and CD54, and PBMC functions (lymphocyte proliferation, natural killer cell activity, cytokine production) were measured. All measurements were repeated after a 'washout' period of 8 weeks. RESULTS: The placebo, OO and SO capsules had no effect on plasma phospholipid or PBMC fatty acid composition. The proportion of dihomo-gamma-linolenic acid in plasma phospholipids was elevated in subjects taking EPO and was decreased in subjects taking FO. There was no appearance of gamma-linolenic acid in the plasma phospholipids or PBMC in subjects taking EPO. There was a marked increase in the proportion of EPA in the plasma phospholipids (10-fold) and PBMC (four-fold) of subjects taking FO supplements; this increase was maximal after 4 weeks of supplementation. There was an increase in the proportion of DHA in plasma phospholipids and PBMC, and an approximately 20% decrease in the proportion of arachidonic acid in plasma phospholipids and PBMC, during FO supplementation. Plasma concentrations of alpha-tocopherol were significantly elevated during supplementation in all subjects and returned to baseline values after the washout period. There were no effects of supplementation with any of the capsules on total plasma antioxidant activity or plasma thiobarbituric acid-reactive substances or on the proportion of different PBMC subsets, on the proportion of PBMC expressing adhesion molecules, on natural killer cell activity, on the proliferation of mitogen-stimulated whole blood cultures or PBMC, or on the ex vivo production of a range of cytokines by whole blood cultures or PBMC cultures stimulated by either concanavalin A or lipopolysaccharide. CONCLUSION: Supplementation of the diet with 3.2 g EPA plus DHA per day markedly alters plasma phospholipid and PBMC fatty acid compositions. The lack of effect of FO upon PBMC functions may relate to the level of alpha-tocopherol included in the supplements.

Glutamine and the immune system.

Glutamine is utilised at a high rate by cells of the immune system in culture and is required to support optimal lymphocyte proliferation and production of cytokines by lymphocytes and macrophages. Macrophage-mediated phagocytosis is influenced by glutamine availability. Hydrolysable glutamine dipeptides can substitute for glutamine to support in vitro lymphocyte and macrophage functions. In man plasma and skeletal muscle glutamine levels are lowered by sepsis, injury, burns, surgery and endurance exercise and in the overtrained athlete. The lowered plasma glutamine concentrations are most likely the result of demand for glutamine (by the liver, kidney, gut and immune system) exceeding the supply (from the diet and from muscle). It has been suggested that the lowered plasma glutamine concentration contributes, at least in part, to the immunosuppression which accompanies such situations. Animal studies have shown that inclusion of glutamine in the diet increases survival to a bacterial challenge. Glutamine or its precursors has been provided, usually by the parenteral route, to patients following surgery, radiation treatment or bone marrow transplantation or suffering from injury. In most cases the intention was not to stimulate the immune system but rather to maintain nitrogen balance, muscle mass and/or gut integrity. Nevertheless, the maintenance of plasma glutamine concentrations in such a group of patients very much at risk of immunosuppression has the added benefit of maintaining immune function. Indeed, the provision of glutamine to patients following bone marrow transplantation resulted in a lower level of infection and a shorter stay in hospital than for patients receiving glutamine-free parenteral nutrition.

Dietary glutamine enhances cytokine production by murine macrophages.

To examine the effects of dietary glutamine on cytokine production by macrophages, mice were fed for 2 wk on a control diet that included 200.0 g casein/kg providing 19.6 g glutamine/kg or a glutamine-enriched diet that provided 54.8 g glutamine/kg partly at the expense of casein. There were no differences in weight gain between animals fed the two diets. The plasma concentrations of a number of amino acids differed according to the diet fed; this variation largely reflected the variation in the levels of the different amino acids in the diets. Plasma glutamine concentration was not significantly affected by dietary glutamine level. The production of three cytokines, tumor necrosis factor-alpha, interleukin-1 beta, and interleukin-6, was greater for lipopolysaccharide-stimulated macrophages from mice fed the glutamine-enriched diet. Thus, increasing the amount of glutamine in the murine diet enhances the ability of macrophages to respond to stimulation, at least in terms of cytokine production. These observations suggest that increasing the availability of glutamine orally could promote immune responses involving macrophage-derived cytokines.

Comparison of cytokine production in cultures of whole human blood and purified mononuclear cells.

In order to examine inter- and intra-individual variations in cytokine production, blood was collected from 48 healthy subjects on each of 4 occasions separated by 4 weeks. Whole blood (diluted 1:10) and mononuclear cell (MNC) cultures were stimulated for 24 h with either concanavalin A (Con A) or bacterial lipopolysaccharide (LPS) and the concentrations of IL-1alpha, IL-1beta, IL-2, TNF-alpha, IL-10 and IFN-gamma in the culture medium measured. There were highly significant inter-individual variations in the production of each of the cytokines measured. However, the level of the production of each cytokine appeared to be characteristic of an individual. There were significant correlations between production of each cytokine in whole blood and MNC cultures. It is concluded that there is significant inter-individual variation in cytokine production which is unaffected by time or by the stimulus used to elicit cytokine production, and that whole blood cultures can be used instead of MNC cultures to measure cytokine production.

Dietary glutamine enhances murine T-lymphocyte responsiveness.

To examine the effects of dietary glutamine on lymphocyte function, male mice aged 6 wk were fed for 2 wk one of three isonitrogenous, isocaloric diets, which varied in glutamine concentration. The control diet included 200 g casein/kg, providing 19.6 g glutamine/kg; the glutamine-enriched diet provided 54.8 g glutamine/kg partly at the expense of casein; and the alanine + glycine-enriched diet provided 13.3 g glutamine/kg. The plasma concentrations of a number of amino acids varied because of the diet fed. The plasma glycine concentration was greater in mice fed the alanine + glycine-enriched diet (380 +/- 22 micromol/L) than in mice fed the control (177 +/- 17 micromol/L) or the glutamine-enriched (115 +/- 18 micromol/L) diets. The plasma glutamine concentration was greater in mice fed the glutamine-enriched diet (945 +/- 117 micromol/L) than in those fed the diet enriched with alanine + glycine (561 +/- 127 micromol/L), but was not different from that in mice fed the control diet (791 +/- 35 micromol/L). There was a significant linear relationship between the amount of glutamine in the diet and plasma glutamine concentration (r = 0.655, P = 0.015). Plasma alanine concentration was unaffected by diet. The reason for the lack of effect of increasing the amount of alanine in the diet upon its concentration in the circulation may relate to its use by the liver. Thymidine incorporation (56 +/- 18 kBq/well versus <10 kBq/well), expression of the alpha-subunit of the interleukin-2 receptor (62 versus 30% receptor positive cells) and interleukin-2 production [189 +/- 28 versus 106 +/- 5 (control) or 61 +/- 13 (alanine + glycine enriched) ng/L] were greater for concanavalin A-stimulated spleen lymphocytes from mice fed the glutamine-enriched diet compared to those from mice fed the other two diets. Thus, increasing the amount of glutamine in the murine diet enhances the ability of T lymphocytes to respond to mitogenic stimulation. Taken together, these observations suggest that increasing the oral availability of glutamine could promote the T-cell driven, cell-mediated immune response.