Abstract: The effects of oral RNA and intraperitoneal nucleoside-nucleotide mixture administration on methicillin-resistant Staphylococcus aureus (MRSA) strain 8985N infection were studied in mice. BALB/c mice were fed a nucleic acid-free diet or nucleic acid-free diet or nucleic acid-free diet supplemented with 0.5% or 2.5% ribonucleic acid (RNA) for 30 days. Nucleoside-nucleotide mixture or saline (control) was intraperitoneally administered daily to these rats except for the 2.5% RNA group, which received saline only. On the 10th day of this treatment, the mice were inoculated intravenously with the viable MRSA organisms. Susceptibility to the MRSA was determined by animal survival and recovery of the MRSA from the organs. The survival rates in the three groups that were administered saline were 29% for nucleic acid-free diet, 0.5% RNA, and 2.5% RNA groups, respectively, whereas in the two groups that received the nucleoside-nucleotide mixture the rates were 69% for the nucleic acid-free diet group and 55% for 0.5% RNA group. The susceptibility of the mice to the MRSA challenge was not affected by dietary RNA. The combined survival rate in the two nucleoside-nucleotide groups (64%) was statistically different (p<.01)from that in the three saline groups (34%). There was a greater reduction in viable organism recovery in the kidney and spleen of the surviving mice that had been administered the nucleoside-nucleotide mixture than in those administered saline. The study showed that intraperitoneal administration of the nucleoside-nucleotide mixture was effective in increasing the host resistance, whereas oral RNA was not adequate in offering protection to the mice against MRSA infection. Journal of Parenteral and Enteral Nutrition 17:148-152, 1993.

Abstract:
Objective: To determine if early enteral feeding, in an intensive care unit (ICU) patient population, using a formula supplemented with arginine, dietary nucleotides, and fish oil (Impact^©), results in a shorter hospital stay and a reduced frequency of infectious complications, when compared with feeding a common use enteral formula (Osmolite HN^©).
Design: A prospective, randomized, double-blind, multicenter trail.
Setting: ICUs in eight different hospitals.
Patients: Of 326 patients enrolled in the study, 296 patients  were eligible for analysis. They were admitted to the ICU after an event such as trauma, surgery, or sepsis, and met a risk assessment screen (Acute Physiology and Chronic Health Evaluation II [APACHE II] score > 10, or a Therapeutic Intervention Scoring System score of >20) and study eligibility requirements. Patients were stratified by age (<60 or > 60 yrs of age) and disease (septic or systemic inflammatory response syndrome).
Interventions: Patients were enrolled and full-strength tube feedings were initiated within 48 hrs of the study entry event. Enteral feedings were advanced to a target volume of 60 mL/hr by 96 hrs of the event. One hundred sixty-eight patients were randomized to receive the experimental formula, and 158 patients were randomized to receive the common use control formula.
Measurements and Main Results: Both groups tolerated early enteral feeding well, and the frequency of tube feeding related complications was low. There were no significant differences in nitrogen balance between groups on study days 4 and 7. Patients receiving the experimental formula had a significant ( p = .0001 ) increase in plasma arginine and ornithine concentrations by study day 7. Plasma fatty acid profiles demonstrated higher concentrations of linoleic acid (p < .01) in the patients receiving the common use formula and higher concentrations of eicosapentaenoic and docosahexaenoic acid (p<.01) in the patients the experimental formula. The mortality rate was not different between the groups and was significantly (p < .001) lower than predicted by the admission severity scores in both feeding groups. In patients who received at least 821 mL/day of the experimental formula, the hospital median length of stay was reduced by 8 days (p<.05). In patients stratified as septic, the median length of hospital stay was reduced by 10 days (p<.05), along with a major reduction in the frequency of acquired infections (p<.01) in the patients who received the experimental formula. In the septic subgroup fed at lest 821 mL/day, the median length of stay was reduced by 11.5 days, along with a major reduction in acquired infections (both p<.05) in the patients who received the experimental formula.
Conclusions: Early enteral feeding of the experimental formula was safe and well tolerated in ICU patients. In patients who received the experimental formula, particularly if they were septic on admission to the study, a substantial reduction in hospital length of stay was observed, along with a significant reduction in the frequency of acquired infections. Crit Care Med 1995; 23:436-449

Abstract:
Hypothesis: Periooperative administration of a supplemented enteral formula may reduce the rate of post-operative infections.
Designs: Prospective, randomized, double-blind clinical trial
Setting: Department of surgery at a university hospital.
Patients: Two hundred six patients with neoplasm of colorectum, stomacj and pancreas.
Intervention: Patients were randomized to drink 1L/d of either a control enteral formula (n=104) or the same formula enriched with arginine, RNA, and omega3 fatty acids (n=102) for 7 consecutive days before surgery. The 2 diets were isoenergetic and isonitrogenous. Jejunal infusion with the same formulas was started 6 hours after operation and continued until postoperative day 7.
Main Outcome Measures: Rate of postoperative infectous complications and length of hosptial stay.
Results: Both groups were comparable for age, sex, weight loss, Karnoofsky scale score, nutritional status, hemoglobin level, duration of surgery, blood loss, and rate of homologous transfusion. Intent-to-treat analysis showed a 14% (14/102) infectous complications rate in the supplemented group vs 30% (31/104) in the control group (P=.009). In the eligible population, the postoperative infection rate was 11% (9/85) in the supplemented group vs 24% (21/86) in the control group (P=.02). The mean +/- SD length of postoperative stay was 11.1+/- 4.4 days in the supplemented group and 12.9+/-4.6 in the control group (P=.01).
Conclusions: Perioperative administration of a supplemented enteral formula significantly reduced postoperative infections and length of stay in patients undergoing surgery for cancer.

The objective of this study was to determine whether dietary ribonucleotides alter immune cell phenotypes or function in the first year of life. Newborn term infants in a double-blind, 12-mo, multicenter trial were randomized to cow milk formula groups with (FN, n = 138) or without (F, n = 147) 72 mg/L supplemental ribonucleotides. A nonrandomized HMF cohort (n = 192) was concurrently enrolled. Eighty-eight immune blood cell types were characterized by flow cytometry. Data were analyzed by multivariate ANOVA (MANOVA), ANOVA, and repeated measures analysis (RMA), with adjustments made for multiple comparisons. Ribonucleotide feeding changed subpopulations of T and natural killer (NK) cells. FN had higher numbers and percentages of memory/effector (M/E) cytotoxic/suppressor (CD45R0(+)CD8(+), RMA) T, Fas(+) M/E (CD45R0(+)CD95(+)CD3(+), 6 mo) T, and CD56(+)CD16(-) NK cells (CD56(+)CD16(-)CD3(-)CD8(-), 12 mo), and higher percentages of M/E helper (CD45R0(+)CD4(+), RMA) T, Tc1 (IFN gamma(+)CD4(-)CD3(+), RMA), total interferon (IFN)gamma T (IFN gamma(+)CD4(+/-)CD3(+), RMA), Th2 (IL-4(+)CD4(+)CD3(+), 7 mo), and CD57(+) NK-T cells (CD57(+)CD56(-)CD3(+), 6 mo, 7 mo) compared with F. Percentages of naive helper T (CD45RA(+)CD4(+), 12 mo) and numbers and percentages of CD56(+) NK-T cells (CD56(+)CD16(-)CD3(+)CD8(-), 2 mo, 6 mo) were lower in FN than F. Percentages of M/E cytotoxic/suppressor, Th2, and CD56(+)CD16(-) NK cells in FN were significantly higher than F but were not different from HMF, whereas F was significantly lower than HMF. Ribonucleotide supplementation of infant formula supported increased T-cell maturation and affected immunoregulatory NK cell subsets. These FN-associated immune cell profiles either did not differ from those infants fed HMF or tended to be more like those fed HMF than those fed F.

Abstract: Protein-calorie malnutrition (PCM) has long been recognized as a cause of depressed immune function. While antibody responses and complement levels have been reported to be depressed by PCM, the most consistent suppression associated with malnutrition has been of cellular immune responses (1-3). Chandra (4) has reported that the primary focus of PCM is the phenotypic helper T lymphocyte (OKT4), with an associated reduction in the ability to generate interleukin 2 (IL-2) after immune stimulation. Although the literature has implied that provision of adequate calories and protein will improve the immunosuppression induced by PCM, few studies have focused on the optimal nutritional substrates for recovery of lost immune function. Dietary supplementation with arginine, fish oils, and nucleotides have all been reported to enhance immune response in animals compared with control animals fed isocaloric isonitrogenous diets lacking these substrates (5-10). The following studies examine the relative efficacy of fish oil, arginine, and dietary nucleotide supplements, either individually or in combination, to reverse immunosuppression induced by a protein-free diet.

Abstract: Dietary nucleotides, found in normal diets, have been recently determined to be required for normal immune defences. Rejection of cardiac transplants, graft-vs, -host disease, and delayed cutaneous hypersensitivity in animal models area all suppressed by a diet deficient in nucleotides. T lymphocytes seem to require dietary nucleotides for normal maturation and function. Host resistance to bacterial and fungal infections is decreased in mice on nucleotide free diets; addition of RNA or uracil prevents this vulnerability to infection. Dietary RNA is required to restore lost immune function after protein deprivation.  Adequate calories and protein alone do not return immune function to normal. Dietary nucleotides can restore lost immune function even during protein starvation and weight loss. Because all parenteral and most enteral nutrient solutions are nucleotide free, clinical studies were undertaken comparing a new nucleotide containing diet (Impact) to a standard high protein enteral feeding. In two separate double blind clinical studies the patients fed the enteral diet containing nucleotides had improved immune function compared with patients receiving a nucleotide free diet. In addition, infectious complications and length of hospital stay were reduced in postoperative cancer patients fed Impact compared with a control group. J.Nutr. 124: 160S-164S, 1994.

Abstract: No abstract available.

Abstract: To determine the effects of the inclusion of exogenous nucleotides in aquaculture diets on the resistance of fish to various challenge infections, trials were carried out and results presented. When added to normal fish feed formulations at a combined inclusion level of 0.03% these additional nucleotides were shown to increase resistance to challenge infections with bacterial, viral and rickettsial diseases as well as ectoparasitic infestation. When fed for 3 weeks prior to challenge, the nucleotide-supplemented diet was superior (31% mortalities; relative percent survival: RPS= 37%) to a beta-glucan-containing diet (43% mortalities; RPS=12%) in reducing mortalities due to Vibrio anguillarum infection in fish fed the control diet (49% mortalities). Mortalities resulting from infections with infectious salmon anaemia (ISA) virus and Piscirickettsia salmonis were also significantly reduced (RPS = 25.7% and 42.1%, respectively) as were the numbers of infesting sea lice (Lepeophtheirus salmonis) (37.8% reduction in the mean number of attached lice per fish). The mode of action of supplemented dietary nucleotides and advantages over recognised immunostimulants are discussed.

Abstract: Trials were carried out in Atlantic salmon to determine the effects on vaccine efficacy, vaccination and salt water transfer stress, growth performance and intestinal morphology of the inclusion in salmon diets of supplemental nucleotides. When added to normal salmon feeds at a combined inclusion level of 0.03%, these additional nucleotides were shown to significantly enhance the efficacy of vaccination as indicated by elevated antibody titres (1/144 with nucleotide diet vs. 1/160 with control diet) and lower (although non-significant) mortality following challenge. Significantly reduced blood chloride levels following salt water transfer was indicative of a greatly enhanced capacity for osmoregulation. Increases were also observed in red blood cell levels. Furthermore, the checks in growth rates normally following these particular physical stressors were negated, with fish fed nucleotide-supplemented diets having a 15-22% weight advantage after 8 weeks (5 weeks following each of the two stress events). The growth benefits described following feeding with nucleotide-supplemented diets, ever after just 3 weeks, could be due in part to an increase in the mucosal surface area of the gut due to significantly enhanced intestinal fold morphology.

Abstract: Weanling mice fed chow and chow plus water supplemented with 3.5 mg of nucleotides per 100 ml of water for 6 weeks exhibited increased natural killer cell (NK) activity and lower macrophage activation compared to mice fed chow plus nonsupplemented water. In a dose-response study, NK activity, macrophage activation and spleen weight (as a percentage of body weight) were higher in mice fed up to 0.035% w/w nucleotides, however macrophage activation was decreased by feeding over 0.35% w/w compared to those receiving basal purified diet (BPD). Nucleotides in human milk may affect the immune response in breast-fed infants.

Abstract: Nucleotides (NT) and their related metabolic products play key roles in many biological processes. NT can be synthesized endogenously and thus are not considered essential nutrients. Studies have demonstrated, however, that dietary NT can have beneficial effects; the term “conditionally essential” has been used to describe their role in human nutrition. These nutrients may become essential when the endogenous supply is insufficient for normal function, even though their absence from the diet does not lead to a classic clinical deficiency syndrome. Most dietary NT are rapidly metabolized and excreted. However, some are incorporated into tissues, particularly at younger ages and with fasting. Under conditions of limited NT intake, rapid growth or certain disease states, dietary NT may spare the cost of de novo NT synthesis and optimize the function of rapidly dividing tissues such as those of the gastrointestinal and immune systems. Animals fed NT-supplemented versus non-NT supplemented diets have enhanced gastrointestinal growth and maturation, and improved recovery following small and large bowel injury. Indices of humoral and cellular immunity are enhanced, and survival rates are higher following infection with pathogens. Infants receive NT in human milk, where they are present as nucleic acids, nucleosides, nucleotides and related metabolic products. The NT content of human milk is significantly higher than most cow’s milk-based infant formulae. Dietary NT are reported to enhance the gastrointestinal and immune systems of formula-fed infants. Infants fed NT-supplemented versus non-supplemented formula have a lower incidence of diarrhea, higher antibody titers following Haemophilus influenza type b vaccination and higher natural killer cell activity. These data suggest that human milk NT may contribute to superior clinical performance of the breastfed infant.

Abstract: Nucleotide (NT) nitrogen, a component of nonprotein nitrogen, accounts for approximately 0.1% to 1.15% of the total nitrogen content of human milk. The results of studies in animals indicate that dietary NTs may be required for maintenance of normal immune function. Thirty-seven healthy term infants were either breast-fed (n=9) of fed SMA formula supplemented with 33 mg of NTs per liter (n=13, NT+) or standard SMA formula  (n=15; NT-). At 2 months of age, natural killer percent cytotoxicity was significantly higher in the breast-fed and NT+ groups compared with the NT- group (41.7 +/- 4.7, 32.2 +/- 3.4, 21.7+/- 2.2%, respectively). Interleukin-2 production by stimulated mononuclear cells was higher in the NT+ compared with the NT- group at 2 months of age(0.90 +/- 0.28 U/mL, 0.27 +/- 0.11 U/mL, respectively); neither formula-fed group differed significantly from the breast fed group. Rate of growth and incidence and severity of infections did not differ significantly among dietary groups. Nucleotides may be a component of human milk that contributes to the enhanced immunity of the breast-fed infant.

Abstract: Investigations with animals demonstrate that dietary nucleotides influence immune function. Restriction of dietary nucleotides in mice decreases several indices of cell-mediated immunity as well as resistance to challenge with Staphylococcus aureus or Candida albicans. Spleen cells of mice maintained on nucleotide free diet produce less interleukin-2 and have lower natural killer cell cytotoxicity and macrophage activation that those of animals fed nucleotide-supplemented diets. In vivo lymphoproliferative response, macrophage phagocytic activity and expression of interleukin-2 receptor and lyt1 surface marker are also lower in animals fed nucleotide-fee diets. At 2 mo of age, infants fed breast milk or nucleotide-supplemented infant formula exhibit increased natural killer cell activity compared with infants fed unsupplemented formula. Dietary nucleotide restriction in animals may also result in hepatic lipid accumulation and decreased mucosal height and gut wall thickness. Adenosine monophosphate, a mediator of hepatic and small bowel blood flow, may play a unique role among the nucleotides studied. In conclusion, de novo synthesis and salvage of nucleotides is a metabolically costly process. An exogenous source of nucleotides from diet may optimize the function of rapidly dividing tissues, particularly when growth is rapid and the diet is low in nucleotides. J. Nutr: 144S-148S, 1994.

Abstract: Nucleotides (NT) are ubiquitous intracellular compounds of crucial importance to cellular function and metabolism. Much recent interest has focused on NT as components of the non-protein nitrogen fraction of human milk. MT supplementation of infant formula has now been introduced in several countries. Biological effects of NT have been reported in several fields. Dietary NT have been shown to have important effects on several components of the immune system; they may enhance intestinal absorption of iron; they affect lipoprotein and long-chain polyunsaturated fatty acid metabolism; they may alter intestinal flora; and they have been demonstrated to have trophic effects on the intestinal mucosa and liver in several experimental situations. Clinical studies have shown NT supplementation of infant formula reduces the incidence of diarrheal episodes among socioeconomically deprived infants, and enhances catch-up growth in infants born small for gestational age. Further work will continue to try to identify other clinical situations in which NT may have a beneficial role.

Abstract: Macrophages express several P2X and P2Y nucleotide receptors and display the phenomenon of ATP-induced P2X7-dependent membrane permeabilization, which occurs through a poorly understood mechanism. Several P2 receptors are known to be coupled to the activation of mitogen-activated protein kinases (MAPKs) and Ca2+ signaling. Here, we use macrophages to investigate the phosphorylation of extracellular signal-regulated kinases 1 and 2 (ERK1/2) by nucleotides and the involvement of MAPKs and intracellular Ca2+ concentration in ATP-induced membrane permeabilization. Short-term (5 min) pre-exposure to oxidized ATP (oATP), a P2X7 antagonist that does not inhibit P2X7-associated inward currents or membrane permeabilization, inhibits the activation of ERK1/2 by ATP, ADP, the P2X7 agonist 2'-3'-O-(4-benzoylbenzoyl)-ATP (BzATP), but not by UTP and UDP. We conclude that macrophages display several P2Y receptors coupled to the ERK1/2 pathway and that oATP antagonizes the action of purine nucleotides, possibly binding to P2X7 and/or other purine-binding P2Y receptors. We also show that BzATP and ATP activate ERK1/2 by two different pathways since ERK1/2 activation by BzATP, but not by ATP, is blocked by the tryrosine kinase inhibitor, genistein, and the Src protein kinase inhibitor, tyrphostin. However, the activation of ERK1/2 by ATP is blocked by the protein kinase C (PKC) inhibitor, chelerythrine chloride. Under the same conditions, membrane permeabilization is not blocked by genistein, tyrphostin, or chelerythrine chloride, indicating that tyrosine kinase, Src protein kinase, and PKC are not required for pore opening. Membrane permeabilization is independent of ERK1/2 activation since chelerythrine, or short-term exposure to oATP or PD98059, efficiently block ERK1/2 activation without inhibiting membrane permeabilization. In addition, membrane permeabilization is not inhibited by SB203580 and SB202190, two inhibitors of p38 MAPK, nor by intracellular BAPTA, which blocks ATP-induced Ca2+ signals. These results suggest that multiple P2 receptors lead to ERK1/2 activation, that ligation of the same receptors by agonists with different affinities can lead to differential stimulation of separate pathways, and that MAPKs and intracellular Ca2+ fluxes are independent of P2X7-associated pore formation.

Abstract: No abstract available.

Abstract:
Objective: The objective of this study was to evaluate long-term enteral nutrition support in postoperative cancer patients.
Background: Multimodality therapy of surgical patients with upper gastrointestinal malignancies may improve survival, but often results in substantial malnutrition, immunosuppression, and morbidity. The benefits of combined inpatient and outpatient enteral feeding with standard diets or diets supplemented with arginine, RNA + omega-3 fatty acids are unclear.
Methods: Sixty adult patients with esophageal (22), gastric (16), and pancreatic (22) lesions were stratified by disease site and percent usual weight and randomized to receive supplemental or standard diet via jejunostomy beginning on the first postoperative day (goal = 25 kcal/kg/day) until hospital discharge. Patients also were randomized to receive (n = 37) or not receive (n = 23) enteral jejunostomy feedings (1000 kcal/day overnight) for the 12 to 16 week recovery and radiation/chemotherapy periods. Plasma and peripheral white blood cells were obtained for fatty acid levels and PGE2 production measurements.
Results: Mean plasma and cellular omega 3/omega 6 fatty acid levels (percent compostion) increased significantly (p < 0.05) in the arginine + omega-3 fatty acid group by postoperative day 7 (0.30 vs 0.13) and (0.29 vs. 014) and continued to increase over time. Mean PGE2 production decreased significantly  (p <0.05) from 2760 to 1600 ng/10(6) cells/mL at day 7 in the arganine + omega-3 fatty acid group, whereas no significant change over time was noted in the standard group. Infectious/wound complications occurred in 10% of the supplemented group compared with 43% of the standard group (p <0.05); mean length of hospital stay was 16 vs.22 (p<0.05)days, respectively. Of the patients who received postoperative chemoradiation therapy, only 1 (6%) of the 18 patients randomized to receive tube feeding did not continue, whereas 8 (61%) of the 18 patients randomized to receive tube feeding did not continue, whereas 8 (61%) of the 13 patients not randomized to tube feeding required crossover to jejunostomy nutritional support.
Conclusions: Supplemental enteral feeding significantly increased plasma and peripheral white blood cell omega 3/omega 6 ratios and significantly decreased PGE2 production and postoperative infectious/wound complications compared with standard enteral feeding. For outpatients receiving adjuvant therapy, those initially randomized to oral feedings alone required rehospitalisation more frequently, and 61% crossed over to supplemental enteral feedings.

Abstract: No abstract available.

Abstract:
Objectives: To ascertain whether a nucleotide containing nutritional supplement Nucell^® attenuates self-reported symptoms of the common cold.
Design: A randomised controlled trial.
Setting: A University.
Participants: Participants with self-reported symptoms of the common cold but otherwise healthy individuals.
Intervention: Nucell^® capsules containing a yeast-based nucleotide preparation or placebo were provided over a 28 day period.
Outcome Measures: Subjective ratings of symptoms were recorded by self-administered questionnaires using a nine-point scale.
Salivary IgA concentrations were analysed from samples collected during the first 7 days and then at days 14,21 and 28 of supplementation. Total and white blood cell counts were also measured throughout the intervention.
Results: Thirty-six participants completed the study. Nineteen received Nucell^® and 17 received the placebo. The mean age of participants was similar (29.8 + 2.5 in Nucell^® group v 30.7 + 2.7 in control group) and the time participants had been suffering from cold-related symptoms was not significantly different in each treatment group (2.5 + 0.40 days in Nucell^® v 2.9 + 0.47 days in control group).
Severity of self-reported symptoms was significantly attenuated in the Nucell® treated group in the first week of supplementation for questions asked with respect to taste, painful sinuses and earache (p< 0.05). Supplementation with Nucell^® did not adversely affect total or differential white blood counts.
Conclusion: These results suggest that Nucell® supplementation administered as a treatment for cold-related symptoms may reduce the severity of specific symptoms particularly in the early infective phase. In conclusion, Nucell^® supplementation may provide subjective relief of some cold-related symptoms and may be of significant benefit administered as a treatment in participants where sinus pain, earache and diminished taste are common symptoms

Abstract: The influence of dietary nucleotides on susceptibility to candidiasis in mice was studied using two criteria: animal survival and recovery of viable Candida ablicans organisms from the kidney and spleen. One-month-old mice were placed on one of five diets with varying nucleotide content. The results show that mice maintained on a nucleotide-free diet (NF) exhibit a significantly decreased meaned survival time and a significantly increased viable organism recovery in the spleen following intravenous injection of graded inocula of C.albicans compared to mice fed diets containing RNA or uracil as a nucleotide source.

Abstract: Nucleotide-binding and oligomerization domain (NOD)-like receptors (NLRs) are a family of intracellular sensors that have key roles in innate immunity and inflammation. Whereas some NLRs - including NOD1, NOD2, NAIP (NLR family, apoptosis inhibitory protein) and NLRC4 - detect conserved bacterial molecular signatures within the host cytosol, other members of this family sense 'danger signals', that is, xenocompounds or molecules that when recognized alert the immune system of hazardous environments, perhaps independently of a microbial trigger. In the past few years, remarkable progress has been made towards deciphering the role and the biology of NLRs, which has shown that these innate immune sensors have pivotal roles in providing immunity to infection, adjuvanticity and inflammation. Furthermore, several inflammatory disorders have been associated with mutations in human NLRgenes. Here, we discuss the effect that research on NLRs will have on vaccination, treatment of chronic inflammatory disorders and acute bacterial infections.

Abstract: Dietary nucleotides have been reportedly beneficial, especially for infants, since they positively influence lipid metabolism, immunity, and tissue growth, development and repair. Rapidly proliferating tissues, such as the immune system or the intestine are not able to fulfil the needs of cell nucleotides exclusively by de novo synthesis and they preferentially utilize the salvage pathway recovering nucleosides and nucleobases from blood and diet. In the present review we describe the modulatory effect of dietary nucleotides on the immune system together with some of their effects on gut-associated lymphoid tissue. Dietary nucleotides influence lymphocyte maturation, activation and proliferation. Likewise, they affect the lymphocyte subset populations in both the small intestine and blood. Moreover, they are involved in enhancing macrophage phagocytosis and delayed hypersensitivity as well as allograft and tumour responses. In addition, they contribute to the immunoglobulin response in early life, having a positive effect on infection. In fact the incidence and duration of acute diarrhoea is lower in infants fed supplemented-nucleotide formulas. The molecular mechanisms by which dietary nucleotides modulate the immune system are practically unknown. Dietary nucleotides have been shown to enhance the production and the genetic expression of IL-6 and IL-8 by foetal small intestinal explants. Dietary nucleotides may influence protein biosynthesis as well as signal membrane transduction mediated by the interaction of exogenous nucleosides and their receptors may also contribute to modulate the expression of a number of genes, some of which can directly affect the levels of intestinal cytokines.

Abstract:
Objective: To determine whether early enteral feeding in a septic intensive care unit (ICU) population, using formula supplemented with arginine, mRNA, and omega-3 fatty acids from fish oil (Impact), improves clinical outcomes, when compared with a common use, high protein enteral feed without these nutrients.
Design: a prospective, randomized, multi-centered trial.
Setting: ICUs of six hospitals in Spain. Patients: One hundred eighty-one septic patients (122 males, 59 females) presenting for enteral nutrition in an ICU.
Interventions: Septic ICU patients with Acute Physiology and Chronic Health Evaluation (APACHE) II scores of greater than or equal to 10 received either an enteral feed enriched with arginine, mRNA, and omega-3 fatty acids from fish oil (Impact), improves clinical outcomes, when compared with a common use, high protein control (Precitene Hiperproteico).
Measurements and Main Results: One hundred and seventy-six (89 Impact patients, 87 control subjects) were eligible for intention-to-treat analysis. The mortality rate was reduced for the treatment group compared with the control group (17 of 89 vs 28 of 87; p<0.05). Bacteremias were reduced in the treatment group (7 of 89 vs 19 of 87; p = 0.01) as well as the number of patients with more than one nosocomial infection (5 of 89 vs 17 of 87; p = 0.01). The benefit in mortality rate for the treatment group was more pronounced for patients with APACHE II scores between 10 and 15 (1 of 26 vs 8 of 29; p = 0.02).
Conclusions: Immune-enhancing enteral nutrition resulted in a significant reduction in the mortality rate and infection rate in septic patients with less severe illness. (Crit Care Med 2000; 28: 643-648) Key Words: immuno-nutrition; sepsis; intensive care unit; mortality; bacteremia; Impact; enteral; arginine; nucleotides; omega-3 fatty acids

Abstract: Dietary nucleotides, like glutamine, have attracted attention as a key ingredient missing from nutritional formulae for many years. They are the building blocks of tissue RNA and DNA and of ATP and their presence in breast milk has stimulated research in babies which has indicated that supplementation of infant formula milk leads to improved growth and reduced susceptibility to infection. Animal studies have confirmed some of these data. In particular, dietary nucleotides modulate immune function, promote faster intestinal healing and have trophic effects on the intestine of parenterally-fed rats which are similar to those resulting from glutamine supplementation, but at much lower intakes. Nucleotide supplementation has also been shown to improve some aspects of tissue recovery from ischaemia/reperfusion injury or radical resection. There is, however, a fundamental paradox. The intestine and liver possess powerful homeostatic mechanisms which degrade intake of purines and pyrimidines (i.e. salvage) and replace it with de novo synthesised output. It is possible that peripheral tissues receive only small amounts of nucleotides of dietary origin. Previously, nucleotides have been proposed as being conditionally-essential nutrients that provide an adequate supply of purines and pyrimidines for nucleic acid synthesis in neonates or in the stressed patient. This review explores this puzzle in the light of recent data from nutritional studies and from research into purinergic signalling in the intestine, heart and cells of the immune system. We propose that dietary nucleotides should be considered within a pharmacological and metabolic framework.

Abstract: Human milk is recommended as the only alimentary source for the first six months of life. Additionally there is a medical and social need for safe and effective alternative forms of nutrition for infants who cannot be fed with breast milk. Recently the safety and efficacy of some ingredients in infant formulae, such as nucleotides have been discussed. This systematic review analyzed the available evidence to establish the efficacy, safety and dose-response effect of ribonucleotide-supplemented infant formulae (RSIFs). Randomised controlled clinical trials (RCTs) comparing RSIFs to formulae without nucleotides or breast milk were considered in this review. Outcome measures were: antibody titres to common paediatric vaccinations, total lymphocytes, lymphocyte subclasses and NK-cells, episodes of diarrhoea and acute respiratory infection. Publication quality was determined using Jadad and CONSORT guidelines. Results were combined using a random effects model and reported through standardised mean differences (WMD) or risk ratio (RR). Systematic review and meta-analysis showed that RSIFs were associated with a better antibody response to immunisation with Haemopillus influenzae vaccine [SMD 1.74 (99%CI 1.43-2.05), P=0.001], diphtheria toxoid [SMD 0.94 (0.75-1.12), P=0.001], oral polio vaccine [SMD 0.73 (0.51-0.95), P=0.001], and fewer episodes of diarrhoea [RR 0.67 (0.58-0.76), P=0.02]. We did not find a major risk of upper respiratory infections [RR 1.11 (0.90-1.36), P=0.50]. Available evidence suggests a positive benefit of RSFIs on infant health without any risk. These benefits begin with nucleotide addition of 1.9 mg/418.4 kJ and are maintained or increased with 10.78 mg/418.4 kJ.

Abstract: Endothelium-dependent dilation of coronary blood vessels in response to ATP and related nucleotides has been demonstrated in various animal species. The aim of the present study was to investigate a possible relaxant effect of ATP, the adenine nucleotides 2-methylthio ATP (MeSATP) and adenosine 5'-O-(2-thiodiphosphate) (ADPbetaS), and the pyrimidine nucleotide UTP in isolated human coronary artery. In endothelium-intact rings of human coronary artery precontracted with K+ (20–40 mM), the nucleotides caused relaxation. Average maximal percentage relaxations and average EC50 values (concentrations causing half-maximal relaxation) were 89% and 47.1 µM for ATP, 28% and 0.3 µM for MeSATP, 35% and 0.6 µM for ADPbetaS, and 49% and 1.6 µM for UTP. For each of the four agonists, the potency to elicit relaxation varied greatly between individual rings, so that equi-relaxing concentrations spanned several orders of magnitude. Moreover, the sensitivities to ATP and UTP, when tested in the same ring, were not correlated. Mechanical removal of the endothelium as well as NG-nitro-L-arginine methyl ester (L-NAME; 30 µM), an inhibitor of nitric oxide synthase, abolished the relaxation caused by MeSATP, ADPbetaS and UTP and greatly attenuated the response to lower concentrations of ATP (3.2–320 µM), but high concentrations of ATP (320 and 1000 µM) caused relaxation also in endothelium-denuded preparations and in the presence of L-NAME. High concentrations of ADPbetaS (32 and 100 µM) and UTP (320 and 1000 µM) caused contraction of endothelium-denuded preparations. Thus, extracellular nucleotides cause endothelium-dependent, primarily nitric oxide-mediated relaxation of human coronary artery. ATP in addition causes endothelium-independent relaxation. The receptors activated by the nucleotides appear to be unevenly distributed on the coronary endothelium.

Abstract:
Objective: To examine the effect of nucleotide (NT)-supplemented cow’s milk-based formula on growth and biochemical indices of immune function in healthy infants.
Design: Randomized controlled trial (RCT) of formula-fed term infants allocated to control formula with an innate level of NT at 10 mg/l (n=102), or formula fortified with NT at 33.5 mg/l (n=98). A parallel group 125 breastfed infants followed the same protocol as a reference.
Outcome measures: Growth was assessed at enrolment, 7 weeks, 4 months and 7 months of age. Natural killer cell activity, cytokine production and lymphocyte subpopulations were assessed at 7 weeks of age. Antibody responses to diphtheria toxoid, tetanus toxoid and Haemophilus influenza type b (Hib) immunizations were measured at 7 months of age.
Results: NT supplementation did not influence the growth of formula fed infants or any markers of immunity measured at 7 weeks of age. Antibody responses to tetanus toxoid were higher in the NT-supplemented group (n=70) at 7 months of age (median(5th, 95% percentile): 1.57(0.42, 3.43) vs 1.01(0.41, 4.66)IU/ml, P<0.03). A difference between treatments was seen in response to diphtheria toxoid but this effect disappeared when adjusted for hepatitis B Immunization at birth. There was no effect of treatment on antibody responses to Hib immunization.
Conclusions: Supplementation of formulas with NT at 33.5 mg/l resulted in a modest improvement in antibody response consistent with RCTs that used higher levels of NT supplementation. Whether this translates to clinical benefits with well-nourished infants requires further study.
Sponsorship: Supported by a grant from Wyeth Nutrition. Dr Makrides was supported by an RD Wright Fellowship from the National Health and Medical Research Council of Australia and Dr Gibson was partially supported by the MS McLeod Research Trust and a Senior Research Fellowship from the National Health and Medical Research Council of Australia.
 

Abstract: Nucleotides are low molecular weight biological molecules key to biochemical processes. Sources include de novo synthesis, recovery via salvage mechanisms, and dietary intakes. Although endogenous production serves as the main nucleotide source, evidence suggests that exogenous sources are essential to immune competence, intestinal development, and recovery. Dietary nucleotides serve a marked role in rapidly proliferating cells where they are necessary for optimal function. Accordingly, dietary nucleotides are deemed conditionally essential in the presence of various physiological stresses, including growth and development, recovery from injury, infection, and certain disease states. Clinical studies that evaluated nutrition formulations of nucleotides in combination with other specific nutrient substances demonstrated improved clinical outcomes in patients characterized as critically ill, injured, immune suppressed, or with chronic gastrointestinal conditions. However, conclusions regarding specific benefits of nucleotides are limited. Scientific substantiation of nucleotide supplementation in infant formula has been reported to improve the maturation and development of the intestinal tract as well as immune function. All medical nutrition products except for one immune-modulating formulation are devoid of nucleotides. In an effort to build on this, the current review presents the data to support potential clinical applications for nucleotides in enteral nutrition that may contribute to improved outcomes in physiologically stressed patients.

Abstract: 
Background: The nucleotide-binding oligomerization domain containing 1 (NOD1) gene encodes a pattern recognition receptor that senses pathogens, leading to downstream responses characteristic of innate immunity. We investigated the role of NOD1 single nucleotide polymorphisms (SNPs) on IBD risk in a New Zealand Caucasian population, and studied Nod1 expression in response to bacterial invasion in the Caco2 cell line.
Findings: DNA samples from 388 Crohn's disease (CD), 405 ulcerative colitis (UC), 27 indeterminate colitis patients and 201 randomly selected controls, from Canterbury, New Zealand were screened for 3 common SNPs in NOD1, using the MassARRAY(R) iPLEX Gold assay. Transcriptional activation of the protein produced by NOD1 (Nod1) was studied after infection of Caco2 cells with Escherichia coli LF82. Carrying the rs2075818 G allele decreased the risk of CD (OR = 0.66, 95% CI = 0.50-0.88, p < 0.002) but not UC. There was an increased frequency of the three SNP (rs2075818, rs2075822, rs2907748) haplotype, CTG (p = 0.004) and a decreased frequency of the GTG haplotype (p = 0.02).in CD. The rs2075822 CT or TT genotypes were at an increased frequency (genotype p value = 0.02), while the rs2907748 AA or AG genotypes showed decreased frequencies in UC (p = 0.04), but not in CD. Functional assays showed that Nod1 is produced 6 hours after bacterial invasion of the Caco2 cell line.
Conclusion: The NOD1 gene is important in signalling invasion of colonic cells by pathogenic bacteria, indicative of its' key role in innate immunity. Carrying specific SNPs in this gene significantly modifies the risk of CD and/or UC in a New Zealand Caucasian population.

Abstract: Nucleotides and nucleosides have effects on improving energy metabolism and enhancing immune function. Under a surgical stress, requirement of nucleotides are increased as well as nitrogen. A well-balanced nucleoside solution (OG-VI) was developed for parenteral administration and its effect was examined in animals. The OG-VI solution contained 3.11% of nucleosides which was composed of inosine, guanosine monophosphate, cytidine, uridine and thymidine of 4:4:4:3:1 in molar ratio. The whole body protein turnover increased significantly in rats received total parenteral nutrition (TPN) with OG-VI after 70% hepatectomy and enhanced the fractional protein synthesis rates of the muscle and liver. Also myocardial contractility (%segment shortening, %SS) in dogs after occlusion of left anterior descending artery (LAD) was recovered to 70% after reperfusion in the OG-VI group. The creatine phosphate (PCr) / inorganic phosphate (Pi) was maintained the baseline level and did not decrease after hypoxia in the OG-VI group while PCr/ Pi was decreased after hypoxia in normal rat. These data suggested that the nucleoside-nucleotide (OG-VI) improved nitrogen metabolism and might stimulate synthesis of high-energy phosphate in recovery after severe surgical stress.

Abstract: Nucleotides are ubiquitous intracellular compounds of crucial importance to cellular function and metabolism. They have been demonstrated to have a wide variety of physiological effects such as antiviral, anti-proliferative, immunomodulatory and other such beneficial functions. Nucleotides can be synthesised endogenously and hence are not essential nutrients. However, their requirements go up in situations of stress like infection, surgery, tissue repair and/or when de novo synthesis may be inadequate. The need then arises for extraneous supplementation. Thus the nucleotides have come to be classified, in recent times, as ‘conditionally essential nutrient’ like the amino acid glutamine. The building blocks of nucleotides are purine and pyrimidine bases attached to a sugar (ribose or deoxyribose). When phosphate moiety is also added, it is termed a nucleotide. Table 1 gives the nucleotide nomenclature.

ABSTRACT: Previous studies indicate the importance of dietary nucleotides in maintaining optimal cellular immunity. Our studies using murine spleen cells showed that polynucleotides significantly increase in vitro antibody production in response to T-cell-dependent antigen. They seem to exert actions on T-helper cells at antigen presentation, perhaps during cognitive cell-cell interactions. They do not augment the actions of cloned, antigen-specific, activated T-helper cells, nor do they increase antibody production in response to T-cell-independent antigen or polyclonal B-cell activation. Polynucleotides increase in vitro human immunoglobulin production in response to T-cell-dependent stimuli and antigen. Humoral immune responses to T-cell-dependent antigen were depressed in mice fed a nucleotide-free diet, but were restored by a mononucleotide-nucleoside mixture. Responses to T-cell-independent antigen remained intact in these mice. The mononucleotide-nucleoside mixture had no effect on in vitro antibody production and did not further increase humoral immune responses in mice fed regular lab chow. These results suggest that the in vivo actions of polynucleotides on humoral immune responses may reflect local immune responses, perhaps at the site of inflammation. Mononucleotides and nucleosides may be incorporated into the tissue nucleotide pool fairly rapidly in a state of relative nucleotide deficiency and help restore T-cell-dependent humoral immune responses. Our findings may further support the importance of dietary nucleotides.

Abstract: Extracellular nucleotides cause neutrophil degranulation by activating the purinergic receptor subtype P2Y. However, the molecular mechanism involved in the signal pathway remains unknown. A hypothetical scheme suggesting that leukotriene(s) and leukotriene receptor(s) activation is required for extracellular nucleotide-mediated neutrophil degranulation is presented here. Subsequent to the extracellular nucleotide binding to its receptors, intracellular arachidonic acid (AA) levels are elevated. Although AA is a known substrate of the lipoxygenase pathway mediated by 5-lipoxygenase, excess AA could form a complex with S100A8/A9 for transport to the extracellular milieu. Extracellular availability of the S100A8/A9+AA complex could potentially be used for transcellular metabolism by resting and/or activated leukocytes (PMN, MN), vascular endothelium and smooth muscle cells at the inflammatory foci. Once imported into the resting and/or activated leukocytes, AA derived from the S100A8/A9+AA complex could serve as a substrate in the 5-lipoxygenase-mediated leukotriene pathway. Essentially, in addition to extracellular nucleotide-induced leukotrienes, AA derived from the S100A8/A9+AA complex could also be utilized for the synthesis of inflammatory mediators such as leukotriene B(4)(LTB(4)), which in turn could trigger leukocyte degranulation, as well as cellular damage to vascular endothelium and smooth muscle cells, thereby exacerbating inflammation.

Abstract: Microgravity and its environment have adverse effects on the immune system. Abnormal immune responses observed in microgravity may pose serious consequences, especially for the recent directions of NASA for long-term space missions to Moon, Mars and deep Space exploration. The study of space flight immunology is limited due to relative inaccessibility, difficulty of performing experiments in space, and inadequate provisions in this area in the United States and Russian space programs (Taylor 1993). Microgravity and stress experienced during space flights results in immune system aberration (Taylor 1993). In ground-based mouse models for some of the microgravity effects on the human body, hind limb unloading (HU) has been reported to cause abnormal cell proliferation and cytokine production (Armstrong et al., 1993 Chapes et al. 1993). In this report, we document that a nutritional nucleotide supplementation as studied in ground-based mirogravity analogs, has potential to serve as a countermeasure for the immune dysfunction aboserved in space travel.

Abstract:
1. Uridine triphosphate (UTP), uridine diphosphate (UDP), cytidine triphosphate (CTP),and deoxythymidine triphosphate (TTP) caused concentration-dependent increases in the release of thromboxane A2 (TXA2) form cultured glia prepared form the newborn rat cerebral cortex. Although each of the pyrimidine nucleotides displayed similar potencies, CTP and TTP were considered less effective than either UTP or UDP. The purine nucleotide ATP was equally as potent as the pyrimidine nucleotides but was marginally less effective that either UTP of UDP.
2. The ability of UTP, UDP, TTP, and CTP to promote TXA2 release from cultured glia was inhibited in a concentration-dependent manner by suramin and was markedly reduced when incubations were performed either in Ca(2+)- free medium or on cultures which had been maintained in serum-free growth medium for 4 days prior to experimentation.
3. Challenges with UTP and UDP in combination were found to elicit a response which was no different from the effects of these nucleotides alone: in addition, their effects were reversed by the phospholipase A2 inhibitor ONO-RS-082. A slight reduction in UTP- and UDP-stimulated TXA2 was observed incultures grown in the present of luecine methyl ester, a treatment reported to limit microglial survival.
4. These results suggest that glia are targets for extracellular pyrimidine nucleotides and that their ability to release eicosanoids from these cells may be important in the brain’s response to damage. 

Abstract: Extracellular nucleotides can act as important intercellular signals in diverse biological processes, including the enhanced production of factors that are key to immune response regulation. One receptor that binds extracellular adenosine triphosphate released at sites of infection and injury is P2X(7), which is an ionotrophic receptor that can also lead to the formation of a non-specific pore, activate multiple mitogen-activated protein kinases (MAPKs), and stimulate the production of immune mediators including interleukin family members and reactive oxygen species (ROS). In the present report, we have investigated the signaling mechanisms by which P2X(7) promotes monocytic cell mediator production and induces transcription factor expression/phosphorylation, as well as how receptor-associated pore activity is regulated by intracellular trafficking. We report that P2X(7) stimulates ROS production in macrophages through the MAPKs ERK1/2 and the nicotinamide adenine dinucleotide phosphate oxidase complex, activates several transcription factors including cyclic-AMP response element-binding protein and components of the activating protein-1 complex, and contains specific sequences within its intracellular C-terminus that appear critical for its activity. Altogether, these data further implicate P2X(7) activation and signaling as a fundamental modulator of macrophage immune responses.

Abstract: The influence of a nucleotide-enriched diet on the immune system, plasma cortisol levels and resistance to infectious pancreatic necrosis (IPN) was evaluated in infected juvenile rainbow trout. Non-infected fish were sampled monthly and subgroups injected with the IPN virus (IPNV) at days 60 and 120 after feeding trial onset were sampled one week after injection. Immune responsiveness was assessed by mitogen-induced lymphocyte polyclonal expansion; stress levels were measured by plasma cortisol levels using radio-immuno-assay (RIA) and pathogen presence by reverse transcription (RT) polymerase chain reaction (PCR). Non-infected trout, fed with the nucleotide diet, showed higher stimulation indexes of both "B" and "T" lymphocytes after 3 months and normal plasma cortisol levels. Results in trout infected with IPNV, indicate that the nucleotide diet was able to: a) stimulate only "B" lymphocytes and b) decrease plasma cortisol levels. All IPNV-injected and normal diet fed trout died after one week, whereas all the fish in the nucleotide enriched diet subgroup survived. All non-infected fish survived in both normal and enriched diet subgroups. It can be concluded that a nucleotide-enriched diet supplied prior to IPNV infections, is able to stimulate the humoral immune response and decrease elevated stress levels, probably caused by the disease.

Abstract: Nucleotides are compounds that play a key role in numerous intracellular biochemical processes. Synthesized de novo by the body utilizing amino acid precursors or salvaged from degraded nucleic acids and nucleotides, they cannot be considered essential nutrients. However, the term semi or conditionally essential nutrients can be applied in certain conditions where the body’s needs are greater than the amounts of nucleotides synthesized or salvaged. Rapid growth, certain disease states, limited nutrient intake or disturbed endogenous synthesis of nucleotides represent such conditions.
Nucleotides participate in several biochemical processes that are essential to the function of the living body [1].

1. As nucleic acids: being the monomeric units they carry the genetic code as DNA and RNA.
2. In biosynthesis: for example, UDP-galactose in the synthesis of lactose or UDP-glucose in the process of glycogenesis
3. As components of co-enzymes: NAD, FAD and co-enzyme A
4. As biological regulators: cyclic AMP initiates second-messenger cascades and is ubiquitous in all forms of life, playing a key role in regulating biological processes
5. As an energy source: ATP is a universal currency of energy in biological systems.

Recently, several infant formula manufacturers announced the addition of nucleotides to their product. This step was taken following the results of studies suggesting potential benefits to intestinal flora, immunity, iron absorption, lipid metabolism and gut development. The question of whether infants require nucleotide supplementation for optimal nutrition cannot be answered categorically until other related questions are resolved. For example: Is there sufficient clinical evidence as to their beneficial effects? Which nucleotides should be added and in what concentration and relative proportions? Are they safe? Are they stable in the formula? Should they be given on a free basis or as a more complex molecule? In an attempt to answer these questions the present review summarizes the biological effects of nucleotides as nutrients and provides an update on their known side effects.
 

Abstract: The health benefits of specific nutrients in the diet are reviewed as they pertain to the pediatric population and its unique needs. Secretory immunoglobulins, lysozyme, interferon, and growth factors, among others, are known to confer immunological advantages to breast milk. Inhibition of bacterial pathogens, as well as permissive growth of a protective colonic ecoflora occur as a result of various cellular and biochemical mechanisms at play. The immunomodulatory properties of mineral such as iron, zinc, and selenium, are presented and the newly recognized protective role of vitamin A and its importance in developing countries and in conditions of compromised nutrition are discussed. The review also covers the role of arganine, glutamine, and nucleotides in adaptive responses of the developing gut and in pathologic states such as necrotizing enterocolitis, short bowel syndrome, and inflammatory bowel disease. Probiotics (specific microbial feeds with potential benefits to the host), and prebiotics (dietary components such as complex carbohydrates able to change the colonic microenvironment fostering colonization with non-enteropathogens) are areas of current interest because they offer alternatives for the management of the growing problem of multiple antibiotic resistance and overwhelming infections in the hospitalized patient.

Abstract: Turbot (Scophthalmus maximus) fed a control or nucleotide-supplemented diet for 15 weeks were examined for the relative expression of a number of immune genes. Genes analysed included those involved in specific immune responses, such as immunoglobulin M (IgM) and recombinase activating gene 1 (RAG-1), and a number of genes important for nonspecific defences (transferrin, lysozyme, cytokines). After normalising the data to the expression of a housekeeping gene (beta-actin) in each sample analysed, it was found that IgM and RAG-1 gene expression were both significantly increased in the gill and spleen of fish fed the nucleotide-supplemented diet but decreased in the kidney of these fish. Transferrin expression was unaffected by the nucleotide supplementation, as was the expression of the cytokine gene transforming growth factor beta. Lysozyme expression was significantly decreased in the spleen and kidney of fish fed the nucleotide-supplemented diet, with no effect apparent in the gill. In contrast, a second cytokine gene examined, interleukin-1beta, showed a significant increase in expression in the kidney of the nucleotide-supplemented group. These data are discussed in relation to the differential effects seen, the function of the molecules examined and the  potential for dietary modulation of fish health.

Abstract: The objective of this study was to evaluate the influence of dietary nucleotide supplementation in preterm infants during the first month of life on the intestinal permeability to lactulose, mannitol and to beta-lactoglobulin and on the development of circulating antibodies to beta-lactoglobulin and alpha-casein. Twenty-seven preterm infants were enrolled in the study; 11 of them were fed a standard low-birth weight milk formula and 16 infants were fed the same formula supplemented with nucleotides at similar levels to those found in human milk. Blood and urine samples were obtained at 1, 7 and 30 days of age. Serum beta-lactoglobulin, serum IgG antibody to alpha-casein and serum IgG antibody to beta-lactoglobulin were measured by ELISA. The lactulose/mannitol urinary excretion rate was measured by gas liquid chromatography. Neither the intestinal permeability to saccharides nor the intestinal absorption of beta-lactoglobulin were affected by the nucleotide supplementation. However, serum concentrations of IgG antibody to beta-lactoglobulin were higher in preterm neonates fed the supplemented formula than in those fed the standard formula. According to these results, dietary nucleotides might influence the maturation of the humoral immune response in preterm newborn infants.

Abstract:
Background: Aim of this study is to evaluate the incidence of diarrhea in children fed with a nucleotide-supplemented formula (Similac FormulaPlus) in comparison with formula without nucleotide supplementation.
Methods: We investigated the effects of a nucleotide-supplemented formula on the incidence of diarrheal episodes in 3315 infants with a multicentre study conducted by 386 pediatricians since March 1998 until October 1999. The study population has been divided into 4 groups based on the type of feeding: group 1 (n=958)= exclusively nucleotide-supplemented formula, group 2 (n=824)=exclusively without nucleotide-supplementation, group 3 (n=920)=mixed breast-feeding and nucleotide-supplemented formula, group 4 (n=613)=mixed breastfeeding and formula without nucleotide supplementation. At the beginning of the study the 4 groups did not differ for body weight, length and mass index. The infants were enrolled since the first month up to the end of the third month of life and they were followed up to the end of the sixth month of life. During the period of observation the growth of length, weight and mass index was similar among the 4 groups.
Results: Monthly incidence of diarrhea was computed and the comparison between group 1 and group 2 using the summary odds-ration of Mantel-Haenszel showed that in group 1 the incidence of diarrhea was significantly lower that un group 2 (RR=0.567); CI 95%=0.440 - 0.732); similar results were obtained comparing the incidence of diarrhea between group 3 and group 4, having the former a RR=0.630 (CI 95%=0.476-0.834).
Conclusions: The conclusion drawn in that the supplementation with nucleotide of the formula milk decreases the risk of diarrhea episodes during the first six month of life in healthy infants. Such a positive effect is present both in exclusively nucleotide-supplemented formula and in mixed breast-feeding and nucleotide-supplemented formula fed infants. Interpretation of these results is that nucleotides, much more present in human milk than in formula milk, improve the immune defense of the infants stimulating particularly the cell-mediated immunity.

Abstract:
Aim: The aim of this work was to determine the ergogenic effects of a nucleotide supplement on the metabolic and immune responses to short term high intensity exercise in volunteer, trained, male subjects.
Methods: Thirty moderately trained male subjects were randomly divided into 3 equal sized groups, control (C), placebo (P) or experimental (E). Each subject undertook a 2 min maximal exercise test prior to, and after 60 days, on either a nucleotide (E) or placebo supplement. Prior to exercise testing unstimulated saliva samples and blood samples were taken. Saliva was analysed for cortisol and IgA, while blood was analysed for lactate, lactate dehydrogenase and creatine kinase.
Results: The postexercise C value was significantly higher than the pre-exercise concentration (P<0.0001; for C, P, and E). In the postsupplement C analysis, the E postexercise group was significantly lower than either the C (P<0.005) or the P group (P<0.05). In the pre- and postsupplementation periods, the pre-exercise SIgA values were significantly higher than the postexercise values (P<0.0001). However, in the postsupplementation period, the SIgA value in the E group was significantly higher than either the P (P<0.05) or C (P<0.05) groups. There were no significant changes in blood lactate, lactate dehydrogenase, or creatine kinase concentrations post supplementation.
Conclusions: We concluded that a chronically ingested nucleotide supplement blunts the response of the hormones associated with physiological stress.

Abstract:
Aim: The aim of this work was to determine the ergogenic effects of a nucleotide supplement on the salivary immunoglobulin A (SIgA) and cortisol (C) responses after prolonged endurance cycle exercise.
Methods: Fourteen moderately trained male subjects (mean body mass and VO2max) completed two 90 min cycle ergometer trials (60% VO2max) prior to and after 60 days of either a nucleotide (E group, n=7) or placebo (P group, n=7) supplement. Each of the subjects provided an unstimulated saliva sample prior to and following exercise for determination of SIgA and C. 
Results: SIgA was significantly lower after exercise trials in both E and P groups (P<0.0001) prior to as well as after the supplementation period. However, SIgA was significantly higher (P<0.01) in the E group than the P group after supplementation. There were no significant (P>0.11) differences in pre-exercise C level.  Post-exercise C concentrations were significantly (P<0.001) higher than pre-exercise levels in both groups of subjects. However, after the supplementation period, C concentration was significantly (P<0.0001) lower after exercise in E compared to P.
Conclusion: This work suggests that a nucleotide supplement, given chronically may offset the hormonal response associated with demanding endurance exercise.

Abstract: The organism constantly requires nucleotides, especially for tissues that present a high rate of turnover, such as the cells of the immune system. In certain circumstances, nucleotides may become semi-essential nutrients required by a particular organ. There is evidence of the potential role of exogenous nucleotides as regulators of the immune function. In experiments carried out with animals, studies have shown that dietary nucleotides stimulate the humoral immune response to T-dependent antigens and raise total antibody levels. The present study reveals an increase in the production of immunoglobulins and improved response to vaccines, a reduction in morbidity and increased tolerance to dietary antigens. Therefore, the addition of nucleotides to formula appears to favour the immune function.

Abstract: Our previous studies showed that dietary nucleotides fed to mice enhanced the secretion of interleukin 7 (IL-7) and transforming growth factor beta (TGF-beta) from intestinal epithelial cells (IECs). To explore whether nucleotides influence IECs directly to enhance the secretion of the cytokines or not, the effects of nucleotides added in vitro on the cytokine secretion from primary-cultured murine IECs were examined. When the mixture of nucleotide 5'-monophosphates (CMP, GMP, IMP, and UMP) or individual nucleotide 5'-monophosphates were added to the primary culture of IECs derived from BALB/c mice, the secretion of IL-7, but not that of TGF-beta, was increased significantly. Addition of nucleotides to the culture did not alter the number of the IECs. Secretion of IL-6 and granulocyte-macrophage colony-stimulating factor, which are known to be secreted from IECs, was not enhanced by the addition of nucleotides. These results demonstrate that nucleotides can affect IECs directly to enhance the secretion of IL-7, and suggest that the increased secretion of TGF-beta from IECs by dietary nucleotides was due to indirect effects of the nucleotides, which may affect intestinal microflora or cells other than IECs that in turn influence the cytokine secretion of IECs.

Abstract: We have investigated the effects of dietary nucleotides on intraepithelial lymphocytes (IEL) and intestinal epithelial cells (IEC) in weanling mice. The proportion of T-cell receptor (TCR) gammadelta+ IEL in BALB/c mice fed a diet supplemented with nucleotides (NT(+) diet) was significantly higher than that in mice fed the nucleotide-free diet, while the proportion of TCR alphabeta+ IEL in NT(+) diet-fed mice was significantly decreased. The change of the TCR alphabeta+/TCR gammadelta+ ratio was mainly observed in a CD8 alphaalpha+ subset of IEL. IEC from NT(+) diet-fed mice produced a higher level of IL-7, which is important in the development of TCR gammadelta+ IEL, than those from control diet-fed mice. The expression levels of IL-7 and IL-2 receptors on IEL were not different between the two dietary groups. Our findings suggest that the increased population of a TCR gammadelta+ IEL subset by feeding nucleotides may be caused by the increased production of IL-7 by IEC.

Abstract:
Background: It has been reported that dietary nucleotides enhance T helper cell activities. In this study, we have determined the effects of dietary nucleotides on antigen-specific. Th1 and Th2 responses and IgE responses.
Methods: Ovalbumin (OVA)-specific T cell receptor (TCR) transgenic (OVA-TCR Tg) mice, 3 weeks old, were fed a nucleotide-free diet (NT(-) diet) or the NT(-) diet supplemented with dietary nucleotides (NT(+) diet) for 4 weeks. Cytokine production by spleen cells and macrophages obtained from these mice was measured in vitro. BALB/c mice, 3 weeks old, immunized intraperitoneally with OVA adsorbed onto alum, were fed the NT(-) diet or the NT(+) diet for 4 weeks. Serum levels of antigen-specific antibodies in the BALB/c mice were determined by ELISA.
Results: The level of production of antigen-specific interferon-_ by spleen cells was significantly higher in the OVA-TCR Tg mice fed with the NT(+) diet than in control mice. The levels of secretion of bioactive IL-12 by spleen cells and peritoneal macrophages were also significantly increased in the NT(+) diet group. The serum OVA-specific IgE level was significantly decreased in BALB/c mice fed the NT(+) diet compared with those fed the NT(-) diet.
Conclusion: These results show that dietary nucleotides up-regulate the antigen-specific Th1 immune response through the enhancement of IL-12 production and suppress the antigen-specific IgE response.

Abstract: We examined the effects of dietary nucleotides on the immune response balance in two subtypes of T helper cells, type 1 (Th1 ) and type 2 (Th2) cells. In experiment 1, BALB/c mice were maintained on a nucleotide-free diet (NT(-) diet) or the NT(-) diet supplemented with dietary nucleotides (NT(+) diet) from 4 weeks prior to mating and throughout the experiments. The second generations of these mice (F1) were maintained on the same diet as the respective dams. Male F1 -generation mice were used to determine the serum immunoglobulin levels. The serum IgE levels were significantly decreased in mice fed the NT(+) diet (p 0.05) compared with ones fed the NT(-) diet. The serum IgG1 and IgG2a levels were not significantly different between the two dietary groups. However, the IgG1:IgG2a level ratio was significantly decreased in mice fed the NT(+) diet (p 0.05). In experiment 2, BALB/c weanling mice were placed on the NT(-) diet or NT(+) diet for 10 weeks. The production of splenic interleukin-4, a cytokine produced by Th2, was decreased in mice-fed the NT(+) diet compared with ones fed the NT(-) diet. On the other hand, the production of splenic interferon-gamma, a cytokine produced by Th1, was increased in mice fed the NT(+) diet. These results suggest that dietary nucleotides may up-regulate the Th1 immune response in systemic immunity

Abstract: We have investigated the influence of dietary nucleotides on the intestinal immune system in ovalbumin (OVA)-specific T-cell receptor (TCR) transgenic mice (OVA-TCR Tg mice). When mice were supplied with water supplemented with 2% OVA ad libitum, the faecal OVA-specific immunoglobulin A (IgA) level significantly increased in those fed a nucleotide-supplemented diet (NT(+) diet) compared with those fed a nucleotide-free control diet (NT(-) diet). In the NT(+) diet-fed mice, secretion of transforming growth factor beta (TGF-beta), which is an isotype-specific switch factor for IgA, from intestinal epithelial cells (IECs) was significantly increased. Furthermore, an increased proportion of intestinal intraepithelial lymphocytes (IELs) bearing gammadelta TCR (TCRgammadelta(+) IELs) and increased secretion from IECs of interleukin 7 (IL-7), which is essential for the development of TCRgammadelta(+) IELs, were also observed in OVA-TCR-Tg mice fed the NT(+) diet, as we previously demonstrated using BALB/c mice (Nagafuchi et al., Biosci. Biotechnol. Biochem. 64: 1459-65 (2000)). Considering that TCRgammadelta(+) T cells and TGF-beta are important for an induction of the mucosal IgA response, our results suggest that dietary nucleotides augment the mucosal OVA-specific IgA response by increasing the secretion of TGF-beta from IECs and the proportion of TCRgammadelta(+) IELs.

Abstract: We examined the effects of nucleotide supplementation to a preterm adapted milk formula on the lymphocyte subsets and plasma IgG, IgM and IgA levels in preterm infants for the first three months of life.  Two groups of preterm infants received a milk formula or the same formula supplemented with CMP, AMP, UMP, GMP and IMP to mimic the concentration of acid soluble nucleotides found in human milk. Blood samples were obtained at birth, 10 days, 20-30 days and 3 months of age. Preterm infants fed the nucleotide formula exhibited higher plasma levels of IgM in all postnatal study periods than neonates fed the standard formula; moreover, IgA was also higher at 3 months of age in nucleotide formula fed infants. No major differences were seen between groups for IgG levels and lymphocyte subsets. Thus, dietary nucleotides appear to exert actions on immature human neonate lymphocytes enhancing the in vivo production of Ig which may have a role in the defence capacity of neonates.

Abstract: Evidence exists regarding the potential role of exogenous nucleotides as regulators of the immune function in physically active humans, yet the potential use of nucleotides has been hindered by their low bioavailability after oral administration. We conducted a double-blind, placebo-controlled, randomized trial to assess the effect of sublingual nucleotides (50 mg/day) on salivary and serum immunity indicators as compared to placebo, both administered to healthy males aged 20 to 25 years for 14 days. Sublingual administration of nucleotides for 14  days increased serum immunoglobulin A, natural killer cells count and cytotoxic activity, and offset the post-exercise drop of salivary immunoglobulins and lactoferrin (P < 0.05), with no adverse effects reported. No significant differences in fasting salivary antimicrobial proteins (alpha-amylase, lysozyme and lactoferrin) were found before or after the treatment (P > 0.05). It seems that sublingual administration of nucleotides for two weeks considerably affected immune function in healthy males.

Abstract: Human milk is the gold standard of nutrition for the infant. Some epidemiological studies indicate that enteric and respiratory infections are less common among breast-fed infants. This protective effect has been attributed to a variety of immunologic factors such as lysozyme, lactoferin, leukocytes, and immunoglobulins. Human milk contains nucleic acids, nucleotides, and related metabolites which have been theorized to improve immunity. Because of this and other potential beneficial effects, two manufacturers of infant formula have recently added nucleotides to their milks. This discussion will review some studies that support the concept that dietary nucleotides may have a positive effect on immunity.

Extracellular nucleotides regulate macrophage function via P2X nucleotide receptors that form ligand-gated ion channels. In particular, P2X7 activation is characterized by pore formation, membrane blebbing, and cytokine release. P2X7 is also linked to mitogen-activated protein kinases (MAPK) and Rho-dependent pathways, which are known to affect cytoskeletal structure in other systems. As cytoskeletal function is critical for macrophage behavior, we have tested the importance of these pathways in actin filament reorganization during P2X7 stimulation in RAW 264.7 macrophages. We observed that the P2X7 agonists adenosine 5'-triphosphate (ATP) and 3'-O-(4-benzoylbenzoyl) ATP (BzATP) stimulated actin reorganization and concomitant membrane blebbing within 5 min. Disruption of actin filaments with cytochalasin D attenuated membrane blebbing but not P2X7-dependent pore formation or extracellular-regulated kinase (ERK)1/ERK2 and p38 activation, suggesting that these latter processes do not require intact actin filaments. However, we provide evidence that p38 MAPK and Rho activation but not ERK1/ERK2 activation is important for P2X7-mediated actin reorganization and membrane blebbing. First, activation of p38 and Rho was detected within 5 min of BzATP treatment, which is coincident with membrane blebbing. Second, the p38 inhibitors SB202190 and SB203580 reduced nucleotide-induced blebbing and actin reorganization, whereas the MAPK kinase-1/2 inhibitor U0126, which blocks ERK1/ERK2 activation, had no discernable effect. Third, the Rho-selective inhibitor C3 exoenzyme and the Rho effector kinase, Rho-associated coiled-coil kinase, inhibitor Y-27632, markedly attenuated BzATP-stimulated actin reorganization and membrane blebbing. These data support a model wherein p38- and Rho-dependent pathways are critical for P2X7-dependent actin reorganization and membrane blebbing, thereby facilitating P2X7 involvement in macrophage inflammatory responses.

Abstract: Macrophage activation is critical in the innate immune response and can be regulated by the nucleotide receptor P2X7. In this regard, P2X7 signaling is not well understood but has been implicated in controlling reactive oxygen species (ROS) generation by various leukocytes. Although ROS can contribute to microbial killing, the role of ROS in nucleotide-mediated cell signaling is unclear. In this study, we report that the P2X7 agonists ATP and 3'-O-(4-benzoyl) benzoic ATP (BzATP) stimulate ROS production by RAW 264.7 murine macrophages. These effects are potentiated in lipopolysaccharide-primed cells, demonstrating an important interaction between extracellular nucleotides and microbial products in ROS generation. In terms of nucleotide receptor specificity, RAW 264.7 macrophages that are deficient in P2X7 are greatly reduced in their capacity to generate ROS in response to BzATP treatment (both with and without LPS priming), thus supporting a role for P2X7 in this process. Because MAP kinase activation is key for nucleotide regulation of macrophage function, we also tested the hypothesis that P2X7-mediated MAP kinase activation is dependent on ROS production. We observed that BzATP stimulates MAP kinase (ERK1/ERK2, p38, and JNK1/JNK2) phosphorylation and that the antioxidants N-acetylcysteine and ascorbic acid strongly attenuate BzATP-mediated JNK1/JNK2 and p38 phosphorylation but only slightly reduce BzATP-induced ERK1/ERK2 phosphorylation. These studies reveal that P2X7 can contribute to macrophage ROS production, that this effect is potentiated upon lipopolysaccharide exposure, and that ROS are important participants in the extracellular nucleotide-mediated activation of several MAP kinase systems.

Abstract:
Objective: To determine whether human milk and nucleotides added to infant formula at levels present in human milk enhance development of the immune system during infancy.
Methods: A 12-month, controlled, randomized and blinded, multisite feeding trial was conducted on two infant formulas: Iron-fortified, milk-based control formula (Control) or the same formula fortified with nucleotides (Nucleotide). The level (72 mg/L) and ratio of individual nucleotides selected were patterned after those available in human milk. A third group fed human milk exclusively for 2 months and then human milk or Similac with iron until 12 months of age also was studied. Response to immunizations was chosen to assess development of the immune system. Infants followed the immunization schedule recommended by the American Academy of Pediatrics in 1991.
Outcome Variables: Antibody responses were determined at 6, 7, and 12 months of age to Haemophilus influenzae type b polysaccharide (Hib), to diphtheria and tetanus toxoides, and to oral polio virus (OPV) immunizations.
Results: Of 370 full-term, healthy infants enrolled, 311 completed the study (107 Control, 101 Nucleotide, 103 human milk/Similac with iron). Intake, tolerance, and growth of infants were similar in all three groups. Compared with the Control group 1 month after the third immunization (7 months of age), the Nucleotide group had a significantly higher Hib antibody concentration (geometric mean concentrations of 7.24 vs 4.05 micrograms/mL, respectively), and a significantly higher diphtheria antibody concentration (geometric mean of 1.77 vs 1.38 U/mL). The significantly higher Hib antibody response in the Nucleotide group persisted at 12 months. The antibody responses to tetanus and OPV were not enhanced by nucleotide fortification. There also was an effect of breastfeeding on immune response. Infants who breastfed had significantly higher neutralizing antibody titers to polio virus than either formula-fed group (1:346 vs 1:169 and 1:192 in the Control and Nucleotide groups, respectively) at 6 months of age.
Conclusion: Infant formula fortified with nucleotides enhanced H influenza type b and diphtheria humoral antibody responses. Feeding human milk enhanced antibody responses to OPV. Dietary factors play a role in the antibody response of infants to immunization. Pediatrics 1998:101:242-249.

Abstract:
Background: Strenuous exercise has been classically associated to immune-suppression and consequently to an increased risk of infections, especially at the upper respiratory tract. The administration of dietary nucleotides has been demonstrated useful to maintain the immune function in situations of stress and thus could be an appropriate strategy to counteract the decline of the immune function associated to strenuous exercise. The aim of the present study was to asses the impact of a specific nucleotide formulation (Inmunactive^®) on the markers of immune function of athletes after a heavy exercise bout under cold conditions.
Methods: Twenty elite male taekwondo athletes were randomly divided into two groups of 10 subjects that were supplemented with placebo (P) or Inmunactive (I) at 480 mg/day during 30 days. At baseline (day 0) and after 4 wk of supplementation (day 30) each subject undertook an exhaustion exercise test using a cycloergometer. Skin temperature, core temperature, heart rate, lactate concentration and rating of perceived exertion (RPE) were recorded during the test. Blood and saliva samples were obtained before and after each exercise test for determination of blood cell concentrations, PHA-stimulated lymphocyte proliferation (PHA-LP) and salivary immunoglobulin A (SIgA).
Results: Exercise tests induced neutrophilia and reduction in lymphocyte blood counts on day 0 and on day 30 in both groups. However, the I group exhibited a faster recovery from the lymphopenic response than the P group, so that lymphocyte levels were higher after 150 min (P < 0.0028). Furthermore, the lymphoproliferative response was modulated by nucleotide supplementation, since it was higher in the I group on day 30 despite an almost significant (P < 0.06) exercise-evoked decrease at baseline.
Conclusion: Our results suggest that exogenous nucleotides may have a protective effect on the on the markers immune response of athletes after strenuous exercise. According to the recent findings, it could be hypothesized that this protection could be mediated by a preventive effect against apoptosis induced by different stress stimuli. However further studies are required to elucidate the mechanisms of action of dietary nucleotides, as well as to evaluate their potential in prevention of immune disturbances.

Abstract: The interplay between pro- and anti-inflammatory mechanisms during inflammatory and immune responses is critical for avoiding excessive tissue damage. Extracellular nucleotides (e.g., adenosine 5'-triphosphate) may represent constitutive signals that can alert the immune system of abnormal cell death. Relatively high doses of nucleotides induce rapid release of proinflammatory mediators and favor pathogen killing. However, recent findings on antigen presenting cells, particularly dendritic cells, revealed a more complex role for these molecules. Chronic exposure to low-dose nucleotides can redirect cellular responses to prototypic activation stimuli, leading to suppressed inflammation and immune deviation.

Abstract: It remains a goal of pediatric nutrition to provide optimal nourishment for infants who are not fed human milk. Investigators have attempted to emulate the composition and functionality of human milk, the gold standard for infant nutrition. These efforts began with the analysis of milk components and continued with assessments of biological effects that culminated in clinical studies in infants. This chapter summarizes the path that researchers followed to study ribonucleotides and their role in infant nutrition. Based on analytical methods for the quantification of ribonucleotides in human milk, investigators assessed their potential impact on the immune systems of infants and looked for concomitant mechanistic explanations. These inquiries evolved into clinical trials in which ribonucleotide-supplemented formula performance was compared with that of non-supplemented formulas and with human milk. This chapter intends to summarize an area of pediatric nutrition that has yielded both enlightening evidence and seemingly contradictory data.

Abstract: The objective of this study was to further explore previously identified defects of supplemental ribonucleotides on infant immune status as measured by antibody responses to routine infant immunizations. Infants were randomized to a milk-based formula with (FN, n=138) or without (F, n=147) 72 mg ribonucleotides/L. A cohort of human milk-fed (HMF, n = 192) infants was also followed. Subjects were given Haemophilus influenza type b (Hib), diphtheria tetanus acellular pertussis, and oral poliovirus vaccinations at 2, 4, and 6 mo of age, and specific antibody responses were assessed at 2, 6,7, and 12 mo. Growth and safety data were also monitored. Using a two-group repeated measures analysis (RMA), FN-fed infants had significantly higher poliovirus type 1 neutralizing antibody (PV-VN1) responses than F-fed infants (p = 0.045). Using three-group RMA, PV-VN1 responses in HMF infants were not different from FN-fed infants, while HMF-fed infant PV-VN1 responses were significantly higher than F-fed infants at 6 (p = 0.0004) and 12 mo (p = 0.0001). FN-fed infants had responses to Hib Farr, diphtheria, tetanus toxoid, oral poliovirus-specific IgA, and PV-VN3 not significantly different from those of F and HMF infants. Growth, gastrointestinal tolerance, and adverse events were equivalent among the three groups. The FN-associated increase in PV-VN1 response and nonstatistically significant trends toward increased Hib and diphtheria antibody responses were consistent with observations from earlier studies, indicating immune benefits of nucleotide supplementation of infant formula.

Abstract:
Hypothesis: Perioperatively administered enteral immunonutrition will improve early postoperative morbidity and cost-effectiveness after gastrointestinal tract surgery.
Design: A prospective, randomized, double-blind, multicenter clinical trial.
Setting: Surgical departments in German university and teaching hospitals.
Patients: One hundred fifty-four patients with upper gastrointestinal tract malignant neoplasms who were eligible for analysis.
Intervention: Preoperatively, patients received 5 days of oral immunonutrition (an arginine-, RNA-, and omega3 fatty acid-supplemented diet) or an isoenergetic control diet (1 L/d). Early postoperative enteral feeding with immunonutrition or an isoenergetic, isonitrogenous control diet using a catheter jejunostomy was performed for 10 days.
Main outcome measures: Postoperative infectious complications, their treatment costs, and cost-effectiveness of immunonutrition were analyzed. Plasma levels of the fatty acids eicosapentaenoic acid and docosahexaenoic acid were measured.
Results: In the immunonutrition group, significantly fewer infectious complication events occurred (14 vs 27; P = .05). The number of patients with complications was significantly lower in the supplemented diet group after postoperative day 3 (7 vs 16; P = .04). The treatment costs of complications in the supplemented diet group were suggestively lower than in the control diet group (DM 75172 vs DM 204273). Cost-effectiveness was DM 1503 in the experimental group vs DM 3587 in the control group, where DM denotes deutsche mark (German currency).
Conclusion: The perioperative administration of an enteral immunonutrition significantly (P = .05) decreased the early occurrence of postoperative infections and reduced substantially the treatment costs of the complications after major upper gastrointestinal tract surgery.

Abstract: It remains a goal of pediatric nutrition to provide optimal nourishment for infants who are not fed human milk. Investigators have attempted to emulate the composition and functionality of human milk, the gold standard for infant nutrition. These efforts began with the analysis of milk components and continued with assessments of biological effects that culminated in clinical studies in infants. This chapter summarizes the path that researchers followed to study ribonucleotides and their role in infant nutrition. Based on analytical methods for the quantification of ribonucleotides in human milk, investigators assessed their potential impact on the immune systems of infants and looked for concomitant mechanistic explanations. These inquiries evolved into clinical trials in which ribonucleotide-supplemented formula performance was compared with that of non-supplemented formulas and with human milk. This chapter intends to summarize an area of pediatric nutrition that has yielded both enlightening evidence and seemingly contradictory data.

Abstract: Nucleotides, nucleosides and nucleobases belong to the non-protein-nitrogen (NPN) fraction of milk. The largest amounts of ribonucleosides and ribonucleotides--ribose forms only were considered in this review--were measured directly after parturition in bovine milk and other ruminants as well as in the milk of humans. Generally, concentrations of most of the nucleos(t)ides tend to decrease gradually with advancing lactation period or nursing time. The species-specific pattern of these minor constituents in milk from different mammals is a remarkable property and confirms, at least, the specific physiological impact of these minor compounds in early life. The physiological capacity of these compounds in milk is given by the total potentially available nucleosides. The main dietary sources of nucleos(t)ides are nucleoproteins and nucleic acids which are converted in the course of intestinal digestion into nucleosides and nucleobases the preferred forms for absorption in the intestine. Thus, nucleosides and nucleobases are suggested to be the acting components of dietary and/or supplemented nucleic acid-related compounds in the gut. They are used by the body as exogenous trophochemical sources and can be important for optimal metabolic functions. Up to 15 % of the total daily need for a breast-fed infant was calculated to come from this dietary source. Concerning their biological role they not only act as metabolites but are also involved as bioactive substances in the regulation of body functions. Dietary nucleotides affect immune modulation, e.g. they enhance antibody responses of infants as shown by a study with more than 300 full-term healthy infants. Dietary nucleos(t)ides are found to contribute to iron absorption in the gut and to influence desaturation and elongation rates in fatty acid synthesis, in particular long-chain polyunsaturated fatty acids in early stages of life. The in vitro modulation of cell proliferation and apoptosis has been described by ribonucleosides, in particular by modified components using human cell culture models. Due to the bio- and trophochemical properties of dietary nucleos(t)ides, the European Commission has allowed the use of supplementation with specific ribonucleotides in the manufacture of infant and follow-on formula. From the technochemical point of view, the ribonucleoside pattern is influenced by thermal treatment of milk. In addition ribonucleosides are useful indicators for quantifying adulterations of milk and milk products.

Abstract: Nucleotide supplementation can reduce postexercise immunosuppression and hypothalamic-pituitary axis (HPA) axis activation in endurance exercise models. Nucleotide supplementation may aid recovery from other exercise modalities, such as heavy resistance exercise. Thus, the purpose of this investigation was to investigate the effects of nucleotide supplementation on the acute cortisol and immune responses to heavy resistance exercise and its effects on recovery. A double-blinded, crossover, mixed methods design with 10 men and 10 women was used. Each performed an acute heavy resistance exercise protocol (AHREP) after a loading period with a nucleotide or placebo supplement. Before and after the AHREP, and at 24, 48, and 72 hours post, blood samples were analyzed for cortisol, myeloperoxidase (MPO), and absolute neutrophil, lymphocyte, and monocyte counts. Creatine kinase (CK) was analyzed before and 24, 48, and 72 hours after the AHREP. Performance measures, including peak back squat isometric force and peak countermovement jump power were also analyzed. Nucleotide supplementation resulted in significant (p < 0.05) decreases in cortisol and MPO immediately after the AHREP, and significantly lower CK values 24 hours later. The AHREP significantly affected leukocyte counts; however, no treatment effects were observed. Greater isometric force was observed immediately after AHREP and at 24 hours and 48 hours with nucleotide supplementation. Nucleotide supplementation seems to attenuate muscle damage, HPA axis and immune system activation, and performance decrements after heavy resistance exercise.

Abstract:
Background: Adequate nutritional support of severe primary protein energy malnutrition (PEM) is indispensable for achieving normal immune response. However, an ideal dietetic criterion has not yet been established or accepted by the majority of health professionals caring for malnourished infants. Our objective was to demonstrate improvement in immune response in infants with PEM who received starting infant formula with nucleotides.
Methods: Twelve malnourished infants 3-18 months of age were include in our study and were fed through a nasogastric tube with infant formula (3.35kJ/mL) for 2  weeks and ad libitum for an additional 2 weeks. Anthropometric measurements and immunologic indicators such as phagocytosis, microbicidal activity, chemotaxis, and cell proliferation index were determined. The sample was divided into two groups; group 1 (n = 6) was fed formula and nucleotides added, while group 2 (n=6) was fed a formula with no nucleotides. Paired t test was used to determine differences between initial and final results for each group for comparison between 1 vs. group 2; a nonparametric Mann-Whitney test was used for immunologic indicators.
Results: Comparison of immunologic indicators showed no significant difference between groups. Groups 1 and 2 showed improvement in phagocytosis and microbicidal activity (p<0.05) and group 2 additionally showed improvement improvement in cell proliferation index.
Conclusions: Infant formula with intake of 837 kJ/kg/d (200 kcal/kg/d) and proteins of 4 g/kg/d in infants with PEM had a favourable impact on immunologic indicators regardless of additional of nucleotides
 

Abstract: Nucleotides are well known for being the universal currency of intracellular energy transactions, but over the past decade it has become clear that they are also ubiquitous extracellular messengers. In the immune system there is increasing awareness that nucleotides serve multiple roles as stimulants of lymphocyte proliferation, ROS generation, cytokine and chemokine secretion: in one word as pro-inflammatory mediators. However, although often neglected, extracellular nucleotides exert an additional more subtle function as negative modulators of immunity, or as immunodepressants. The more we understand the peculiar biochemical composition of the microenvironment generated at inflammatory sites, the more we appreciate how chronic exposure to low extracellular nucleotide levels affect immunity and inflammation. A deeper understanding of this complex network will no doubt help design more effective therapies for cancer and chronic inflammatory diseases.

Abstract: Extracellular ATP (eATP), the most abundant among nucleotides, can act as a mediator during inflammatory responses by binding to plasmamembrane P2 purinergic receptors, which are widely expressed on cells of the immune system. Extracellular ATP is generally considered as a classical danger signal, which stimulates immune responses in the presence of tissue damage. Converging evidence from several studies employing murine models of chronic inflammation have supported this hypothesis; however, the role of eATP in the regulation of human immune function appears to be more complex. Chronic stimulation with micromolar eATP concentrations inhibits the proliferation of T and natural killer (NK) lymphocytes and enhances the capacity of dendritic cells to promote tolerance. The effect of eATP depends on multiple factors, such as the extent of stimulation, eATP concentration, presence/absence of other mediators in the microenvironment, and pattern of P2 receptor engagement. Small but significant differences in the pattern of P2 receptor expression in mice and humans confer the diverse capacities of ATP in regulating the immune response. Such diversity, which is often overlooked, should therefore be carefully considered when evaluating the role of eATP in human inflammatory and autoimmune diseases.

Abstract: It is apparent from the number of symposia published in the last few years that the role of nucleotides in the diet is an extremely important issue. This roundtable, entitled Nucleotides and Nutrition, helped update our knowledge on the use of nucleotides as a supplement in newborns and in older children and adults. We were very fortunate to have many of the world’s authorities in the field available to discuss the salient issues surrounding this topic.
In session 1: Frederick B. Rudolph and Lewis A. Barness reviewed the current knowledge on the chemistry, physiology and composition of nucleotides in breast milk from parturition until weaning. Dr. Rudolph described the metabolism of nucleotides, underscoring their importance in cellular function and the consequences of dysfunction vis-à-vis various human disease states. He reviewed his studies with Charles T. Van Buren over the last several years examining the role of nucleotides in optimal host defense against infection and as a substrate for a normal immune response to noxious foreign stimuli. He pointed out, based on his research, that the absence of nucleotides tends to cause an arrest in the cell cycle between the G1-G2 and the S phases of T helper cells. It was apparent from this review that additional basic research is necessary to further define the importance of nucleotides within various metabolic pathways of specific cell types and under various conditions in which there may be higher requirements for them as substrates for normal host defense function. Dr. Barness reviewed data on the composition of the nonprotein nitrogen pool in breast milk and the contribution made by nucleotides. He also indicated that the total nucleotide pool, including the nucleotides in the soluble fraction and those released by the breakdown of cells in the breast milk, may be grossly underestimated and that any consideration of nucleotide supplementation of infant formula must include a re-examination of these fluids using such modern quantitative techniques as HPLC. Very little is now known about the capacity of the enterocytes in premature and full-term human infants to use nucleotides through the salvage pathway or the de nove synthesis pathway. Again, this presentation underscored the need for further molecular and cellular studies in human development, as well as for a reassessment of our knowledge of the composition and quantity of nucleotides, before recommendations for meaningful supplementation in premature infants and in posttraumatic stress can be made.
In session 2: Ian R.Sanderson and Harumi Jyonouchi reviewed molecular studies involving nucleotide uptake and metabolism in human intestinal cell lines and organ cultures from human foetuses, as well as in vivo and in vitro studies of specific mechanisms of lymphocyte function. Dr. Sanderson reviewed the data obtained in his laboratory using Caco-2 cells, a human cancer cell line that differentiates in a manner similar to that of the crypt villus differentiation pathway, and IEC-6 cells, a noncancer rodent crypt cell line that requires growth factors from an extracellular matrix for maturation to occur. In the neoplastic cells (Caco-2) the de novo synthetic pathway for nucleotides was active and, under conditions of normal growth, exogenous nucleotides were not required However, under conditions of nutritional stress, they became essential. With the non-neoplastic crypt cells (IEC-6) in the presence of an extracellular matrix (Matrigel), nucleotides provided an additive stimulus for the differentiation.

This study investigated the influence of an enteral diet supplemeted with arginine, omega-3 fatty acids, and nucleotides (Impact, Sandoz Nutrition, Berne, Switzerland) on the incidence of systemic inflammatory response syndrome (SIRS) and multiple organ failure (MOF) in patients after severe trauma. Thirty two patients with an injury severity score >20 were included in this prospective, randomized, double blind, controlled study. Primary endpoints were the incidence of SIRS and MOF. Secondary endpoints were parameters of acute phase and immune response as well as infection rate, morbidity, and gospital stay. For statistical analysis 29 patients (test group n=16, control n=13) were eligible. In the test group, significantly fewer SIRS days per patient were found during 28d. The difference was higly significant between d 8-14 (P<0.001). MOF score was significantly lower in the test group on d 3 and d 8-11 (P<0.05). Acute phase parameters showed lower C-reactive protein serum levels (significant on D day 4) and fibrinogen plasma levels (significant on d 12 and 14; P<0.05). HLA-DR expression on monocytes showed significantly higher fluorescence activity on d 7. No significant difference was found for T-lymphocyte CD4/CD8 ratio, interleukin-2 receptor expression, infection rate, mortality (2/16 vs 4/13), and hospital stay. The results of the study provide further support for beenficial effects of arginine, omega-3-fatty acids and nucleotide-supplemented enteral diet in critically ill patients.

In the intestine innate recognition of microbes is achieved through pattern recognition receptor (PRR) families expressed in immune cells and different cell lineages of the intestinal epithelium. Toll-like receptor (TLR) and nucleotide-binding and oligomerization domain-like receptor (NLR) families are emerging as key mediators of immunity through their role as maturation factors of immune cells and triggers for the production of cytokines and chemokines and antimicrobial factors. At the mucosal surface chronic activation of the immune system is avoided through the epithelial production of a glycocalyx, steady-state production of antimicrobial factors as well as the selective expression and localization of PRRs. Additionally, the polarization of epithelial TLR signaling and suppression of NF-kappaB activation by luminal commensals appears to contribute to the homeostasis of tolerance and immunity. Several studies have demonstrated that TLR signaling in epithelial cells contributes to a range of homeostatic mechanisms including proliferation, wound healing, epithelial integrity, and regulation of mucosal immune functions. The intestinal epithelium appears to have uniquely evolved to maintain mucosal tolerance and immunity, and future efforts to further understand the molecular mechanisms of intestinal homeostasis may have a major impact on human health.

Abstract: Emerging evidence indicates the importance of nucleosides and nucleotides in the maintenance of functions of the bone marrow hematopoietic cells, intestinal mucosa, and the brain, which have limited de novo synthesis of purine and pyrimidine bases. We have found that nucleosides and nucleotides stimulate hemopoieses and increase peripheral neutrophil counts in mice treated with cyclophosphoamide. Intraperitoneal administration of nucleosides and nucleotides decreased bacterial translocation, the number of colony-forming units, and increased survival against methicillin-resistant Staphylococcus aureus. In vitro immune studies in mice showed that nucleosides and nucleotides increase the delayed-type cutaneous hypersensitivity and the popliteal lymph node blastogenic response to antigens, allogens, and mitogens. Both intraperitoneal and oral administration of nucleosides and nucleotides reduced endotoxin-induced bacterial translocation and improved injury to the gut in protein-deficient mice. However, oral administration of nucleosides and nucleotides in experimental colitis resulted in a worsening of colitic conditions and increased interleukin-8 and tumor necrosis factor-alpha concentrations in inflamed colonic portions, indicating the pro-inflammatory activities of nucleosides and nucleotides. Memory-deficient senescence-accelerated mice and mice with dementia showed improved memory with dietary nucleosides and nucleotides supplementation. These results indicate that supplementation with nucleosides and nucleotides is beneficial to the functions of the system and the brain. However, beneficial effects to the gut appear to depend on the type of damage sustained by the gut.

Abstract: Microgravity and stress of spaceflights result in immune dysfunction. The role of nutrition, especially nucleotide supplementation, has become an area of intensive research and significant interest in immunomodulation for maintenance of cellular immune responses. The studies presented here evaluate the plausibility of administering nucleotides to obviate immune dysfunction in an Earth-based in vivo analog of microgravity as studied in anti-orthostatic tail suspension (AOS) of mice. Mice were divided into three housing groups: group, isolation, and AOS. Mice were fed either control chow diet (CD), or RNA-, adenine-, or uracil-supplemented CD for the 1-wk duration of the experiments. In AOS mice, supplemental nucleotides significantly increased in vivo lymph node proliferation and ex vivo lymphoproliferation response to alloantigen and mitogens, respectively, and interleukin-2 and interferon-gamma production. A lower corticosterone level was observed in uracil-supplemented CD compared with CD. These results suggest that exogenous nucleotide supplementation, especially uracil, of normal diet is beneficial in the maintenance and restoration of the immune response during the microgravity analog conditions. 

Abstract: Human milk has a higher concentration of nucleotides than bovine milk which is the source of most infant formulas. As the composition of human milk is considered the 'gold standard,' an increasing number of infant formulas are supplemented with nucleotides. This review summarises the biology of human milk nucleotides and evaluates the studies which investigated the clinical benefits of feeding infants with nucleotide-supplemented formulas. Although dietary nucleotides have been suggested to have beneficial gastrointestinal and immunological effects, nucleotide-supplemented formula feeding has not been shown to confer the same benefits as breast feeding, and randomised controlled trials have yet to prove that healthy term infants fed nucleotide-supplemented formulas compared to those fed nonsupplemented formulas, have accelerated physical growth and neurological development, better growth and development of their gastrointestinal tract resulting in improved digestive and absorptive functions, enhanced development of their immune system resulting in increased resistance to infection and lower bacterial and viral infection rates during infancy, and a more favourable intestinal microflora associated with a lower rate of infectious diarrhoea. However, a randomised controlled trial has reported that term infants with severe intrauterine growth retardation do have better catch-up growth with nucleotide supplementation. The hypothesis that nucleotides are semi-essential nutrients needs to be further studied, in particular in the presence of prematurity, fetal growth retardation, intestinal injury and limited nutrient intake. As no deleterious effects have been reported with the use of nucleotide-supplemented formulas, the first of which was introduced over 30 years ago, such products are considered safe when nucleotides are supplemented to an amount equivalent to the free nucleotide concentration of human milk. More basic and clinical research studies are awaited to further define the biology and role of human milk nucleotides, and to critically assess the potential benefits and appropriate level of nucleotide supplementation of infant formula.

Abstract: Human milk has a higher concentration of nucleotides than bovine milk which is the source of most infant formulas. As the composition of human milk is considered the ‘gold standard’ an increasing number of infant formulas are supplemented with nucleotides. This review summarises the biology of human milk nucleotides and evaluates the studies which investigated the clinical benefits of feeding infants with nucleotide-supplemented formulas. Although dietary nucleotides have been suggested to have beneficial gastrointestinal and immunological effects, nucleotide-supplemented formula feeding has not been shown to confer the same benefits as breast feeding, and randomised controlled trials have yet to prove that healthy term infants fed nucleotide supplemented formulas compared to those fed nonsupplemented formulas, have accelerated physical growth and neurological development, better growth and development of their gastrointestinal tract resulting in improved digestive and absorptive functions, enhanced development of their immune system resulting in increased resistance to infection and lower bacterial and viral infection rates during infancy, and a more favourable intestinal microflora associated with a lower rate of infectious diarrhoea. However, a randomised controlled trial has reported that term infants with sever intrauterine growth retardation do have better catch-up growth with nucleotide supplementation. The hypothesis that nucleotides are semi-essential nutrients needs to be further studied, in particular in the presence of prematurity, fetal growth retardation, intestinal injury and limited nutrient intake. As no deleterious effects have been reported with the use of nucleotide supplemented formulas, the first of which was introduced over 30 years ago, such products are considered safe when nucleotides are supplemented to an amount equivalent to the free nucleotide concentration of human milk. More basic and clinical research studies are awaited to further define the biology and role of human milk nucleotides, and to critically assess the potential benefits and appropriate level of nucleotide supplementation of infant formula.