Gene expression profiles in rat mesenteric lymph nodes upon supplementation with conjugated linoleic acid during gestation and suckling.

BACKGROUND: Diet plays a role on the development of the immune system, and polyunsaturated fatty acids can modulate the expression of a variety of genes. Human milk contains conjugated linoleic acid (CLA), a fatty acid that seems to contribute to immune development. Indeed, recent studies carried out in our group in suckling animals have shown that the immune function is enhanced after feeding them with an 80:20 isomer mix composed of c9,t11 and t10,c12 CLA. However, little work has been done on the effects of CLA on gene expression, and even less regarding immune system development in early life. RESULTS: The expression profile of mesenteric lymph nodes from animals supplemented with CLA during gestation and suckling through dam's milk (Group A) or by oral gavage (Group B), supplemented just during suckling (Group C) and control animals (Group D) was determined with the aid of the specific GeneChip(®) Rat Genome 230 2.0 (Affymettrix). Bioinformatics analyses were performed using the GeneSpring GX software package v10.0.2 and lead to the identification of 89 genes differentially expressed in all three dietary approaches. Generation of a biological association network evidenced several genes, such as connective tissue growth factor (Ctgf), tissue inhibitor of metalloproteinase 1 (Timp1), galanin (Gal), synaptotagmin 1 (Syt1), growth factor receptor bound protein 2 (Grb2), actin gamma 2 (Actg2) and smooth muscle alpha actin (Acta2), as highly interconnected nodes of the resulting network. Gene underexpression was confirmed by Real-Time RT-PCR. CONCLUSIONS: Ctgf, Timp1, Gal and Syt1, among others, are genes modulated by CLA supplementation that may have a role on mucosal immune responses in early life.

Enhancement of antibody synthesis in rats by feeding cis-9,trans-11 conjugated linoleic acid during early life.

Previous studies have demonstrated that the intake of a 1% conjugated linoleic acid (CLA) diet in an 80:20 mixture of cis-9,trans-11 and trans-10,cis-12 exerts age-specific effects on the immune system: immunoglobulin enhancement and proliferative down-modulation in neonatal and adult rats, respectively. The present study evaluates the influence of the same diet on antibody synthesis of early infant Wistar rats during suckling and/or after weaning. Dietary supplementation was performed during suckling and early infancy (4 weeks), only during suckling (3 weeks), or only in early infancy (1 week). CLA content in plasma and serum immunoglobulin (Ig) G, IgM and IgA concentration were determined. Proliferation, cytokines and Ig production were evaluated on isolated splenocytes. Cis-9,trans-11- and trans-10,cis-12-CLA isomers were detected in the plasma of all CLA-supplemented animals, and the highest content was quantified in those rats supplemented over the longest period. These rats also exhibited higher concentrations of serum IgG, IgM and IgA. Moreover, splenocytes from CLA-supplemented rats showed the highest IgM and IgG synthesis and interleukin (IL)-6 production, whereas their proliferative ability was lower. In summary, in infant rats, we observed both the enhance antibody synthesis previously reported in neonates, and the reduced lymphoproliferation previously reported in adults.

Improving the quality of infant sleep through the inclusion at supper of cereals enriched with tryptophan, adenosine-5'-phosphate, and uridine-5'-phosphate.

The present study evaluated whether the administration of cereals enriched with nutrients that are facilitators of sleep could help improve the sleep of infants who had sleep disorders at night time. Thirty infants aged 8-16 months with sleep disorders involving at least three nocturnal waking episodes took part in the study. They were given a night-time 'sleep facilitating cereal' product containing 225 mg tryptophan, 5.3 mg adenosine-5'-P, and 6.3 mg uridine-5'-P per 100 g of product. These cereals were given in a double-blind procedure lasting 5 weeks, with ingestion of the cereal between 18:00 and 06:00. In the control week, the children received a standard cereal (75 mg tryptophan/100 g product without nucleotides) dissolved in a standard formula milk (231.5 mg tryptophan, 2.6 mg adenosine-5'-P, 5 mg uridine-5'-P, per 100 g product). In one experimental week, the children received the night-time sleep facilitating cereal together with the standard formula milk. In another week, they received the sleep facilitating cereal together with a night milk specially formulated to attain the sleep rhythm (480 mg tryptophan, 8.8 mg uridine-5'-P, and 7.6 mg adenosine-5'-P per 100 g product). The three experimental weeks were separated by two wash-out weeks in which the milk and cereal administered was identical in composition to that of the control week. All the infants received a programmed writer actimeter which they wore continually, attached to their ankles, to record their motor activity. The recorded activity was used to calculate information about the time in bed, assumed sleep, actual sleep, sleep efficiency, sleep latency, immobility, and total activity. The infants receiving the enriched cereal during the time of darkness showed improvements in their sleep parameters, regardless of whether the milk they took at night was standard or enriched with tryptophan, adenosine-5'-P, and uridine-5'-P. In summary, the administration of enriched cereals led to an improvement in sleep, regardless of the type of infant milk used. These results support the concept of chrononutrition since they confirm that the sleep/wake rhythm can be influenced by diet.

Higher immunoglobulin production in conjugated linoleic acid-supplemented rats during gestation and suckling.

Conjugated linoleic acid (CLA) has been reported to exert beneficial physiological effects on body composition and the immune system. However, little information is available on the influence of CLA on immune function during early life periods. The present study evaluates the effect of feeding an 80:20 mixture of cis-9, trans-11- and trans-10, cis-12-CLA isomers during gestation and suckling on the systemic immune response of weaned Wistar rats. Pups received dietary CLA from dams through the placental barrier and during suckling by breast milk (group A) or by oral administration (group B). Pups from group C only received CLA during suckling by oral administration. Group D constituted the reference group. Milk from dams fed the CLA diet had a high content of CLA and higher IgA and IgG concentrations than rats fed the standard diet. The plasma of pups from groups A, B and C showed six, twelve and nine times higher content of the cis-9, trans-11-CLA isomer than that of the group D pups. Rats from group A exhibited higher serum IgG concentrations than rats from the rest of the groups (22.14 (SEM 2.14) v. about 5 mg/ml; P < 0.05), whereas rats from groups A and B showed approximately 2-fold higher splenocyte IgM production than rats from groups C and D. However, CLA supplementation did not influence significantly the splenocyte proliferative response or cytokine secretion. Supplementation during gestation and suckling with an 80:20 cis-9, trans-11-trans-10, cis-12 CLA mix enhances the production of the main in vivo and in vitro Ig isotypes in Wistar rats.

Mucosal IgA increase in rats by continuous CLA feeding during suckling and early infancy.

The aim of this work was to establish the effect of the cis9,trans11 conjugated linoleic acid (CLA) isomer on mucosal immunity during early life in rats, a period when mucosal immunoglobulin production is poorly developed, as is also the case in humans. CLA supplementation was performed during three life periods: gestation, suckling, and early infancy. The immune status of supplemented animals was evaluated at two time points: at the end of the suckling period (21-day-old rats) and 1 week after weaning (28-day-old rats). Secretory IgA was quantified in intestinal washes from 28-day-old rats by ELISA technique. IgA, TGFbeta, and PPARgamma mRNA expression was measured in small intestine and colon by real time PCR, using Taqman specific probes and primers. IgA mucosal production was enhanced in animals supplemented with CLA during suckling and early infancy: in 28-day-old rats, IgA mRNA expression was increased in small intestine and colon by approximately 6- and 4-fold, respectively, and intestinal IgA protein by approximately 2-fold. TGFbeta gene expression was independent of age and type of tissue considered, and was not modified by dietary CLA. Gene expression of PPARgamma, a possible mediator of CLA's effects was also upregulated in animals receiving CLA during early life. In conclusion, dietary supplementation with CLA during suckling and extended to early infancy enhances development of the intestinal immune response in rats.

Long-term feeding of the cis-9,trans-11 isomer of conjugated linoleic acid reinforces the specific immune response in rats.

Several effects on the immune system have been ascribed to the cis9,trans11 conjugated linoleic acid (CLA) isomer. We studied whether feeding a diet enriched with an 80:20 CLA isomer mix of cis9,trans11 and trans10,cis12 CLA from gestation to adulthood affects the capacity of adult rats to achieve a specific immune response. Pregnant Wistar rats were fed a 1% CLA diet or a control diet beginning on d 7 of gestation. Weaned pups received the same diet as dams until they were 15 wk old. Rats from both groups were immunized with ovalbumin (OVA) when they were 9 wk old. Dietary CLA enhanced splenocyte OVA-specific proliferation by approximately 50% (P < 0.05) and decreased the mitogen-induced proliferative responses of these cells by approximately 10-20% (P < 0.05). The diminished splenocyte proliferative response was accompanied by a lower interleukin-2 secretion (P < 0.05). Long-term CLA supplementation did not increase serum, spleen, or mesenteric lymph node production of OVA-specific antibodies (Ab) or the number of spleen anti-OVA Ab-secreting cells. Interestingly, dietary CLA increased intestinal anti-OVA IgA production by approximately 75% (P < 0.05). In conclusion, a 1% CLA diet administered from gestation to adulthood enhanced specific systemic cell-mediated immunity as well as the mucosal IgA immune response, whereas it downregulated the polyclonal activation of the immune system. These data support the long-term effects of dietary cis9,trans11 CLA isomer on the immune system.

Supplementing suckling rats with whey protein concentrate modulates the immune response and ameliorates rat rotavirus-induced diarrhea.

Group A rotaviruses (RV) are the most common causative agents of acute gastroenteritis in children <2 y. The present study was designed to establish the effect of a bovine whey protein concentrate (WPC) in a RV infection model in suckling rats. From d 3 of life, suckling Lewis rats received daily supplements of WPC, WPC plus lactoferrin (LF), standard infant formula (SIF), or water (RV-infected group and an untreated, uninfected reference group). On d 8 of life, heterologous simian RV SA-11 was inoculated orally in the WPC-RV, WPC+LF-RV, SIF-RV, and RV groups. WPC and WPC+LF reduced diarrhea incidence from approximately 90% in RV group to approximately 60% in WPC-RV and WPC+LF-RV groups (P < 0.05), whereas the area under the curve (AUC) of severity along time diminished from approximately 10 AUC in the RV group to approximately 6 AUC in both supplemented groups (P < 0.05). Serum levels of anti-RV antibodies, splenocyte proliferation, and interferon-gamma secretion after specific stimulation were significantly lower in the WPC-RV and WPC+LF-RV groups than in the SIF-RV and RV groups. In the intraepithelial intestinal compartment, RV infection increased the proportion of typical mucosal T cells (IE-T CD8alphaalpha+); however, this modification was controlled by WPC and WPC+LF supplementation. In general, for most of the parameters studied, the SIF-RV and RV groups did not differ. In summary, daily supplementation with WPC or WPC+LF in early life considerably reduces the severity of RV-induced acute gastroenteritis and modulates the immune response against the pathogen.

Bovine whey protein concentrate supplementation modulates maturation of immune system in suckling rats.

During neonatal life, challenges from breast milk and microbial flora promote immune system maturation. Immunonutrition in these stages may become an important way to increase natural defence systems. The aim of this study was to determine the effect of a daily bovine milk whey protein concentrate (WPC) supplement on the intestinal and systemic immune systems in suckling rats. The composition of intraepithelial and lamina propria lymphocytes (IEL and LPL) was analysed by flow cytometry. Systemic and intestinal humoral immune responses were determined by sera Ig levels and Ig-secreting cell quantification by ELISA and ELISPOT, respectively. From birth, suckling Wistar rats were supplemented with WPC or standard infant formula (SIF). The WPC group showed the same proportion of most of the main mucosal cell subsets as the reference animals. However, in the first days of life WPC enhanced the innate immunity by increasing the NK cell proportion in both epithelial and lamina propria (LP) compartments. A rise in intestinal CD8alphaalpha+ IEL was also induced by WPC supplementation. A time-course of sera Ig levels and spontaneous IgA, IgM and IgG production by LPL and mononuclear cells from blood and spleen, in the WPC group, exhibited a similar pattern to those pups fed only by dam's milk. In summary, the present results show the effects of WPC on enhancing mucosal innate immunity during early life.

Chrononutrition applied to formula milks to consolidate infants' sleep/wake cycle.

UNLABELLED: Some 30% of pre-weaning infants present problems of sleep during the night, especially those who are bottle-fed. The solution is for them to be breast-fed for as long as possible, or, if this is not possible, for the formula milk to reproduce breast-milk's natural circadian variations in the concentrations of tryptophan and those nucleotides which have a beneficial effect in consolidating the circadian sleep-wake cycle. OBJECTIVE: To study in pre-weaning infants the effect on nocturnal sleep of the administration of formula milk dissociated into its day/night components. MATERIALS AND METHODS: A prospective study was carried out on 30 pre-weaning infants of 4-20 weeks in age who preferentially showed sleep problems. The day dissociated formula, administered from 06:00-18:00, had lower levels of tryptophan and carbohydrates, and higher levels of proteins together with cytosine-5P, guanosine-5P, and inosine-5P. The night dissociated formula, administered from 18:00-06:00, had lower levels of proteins and medium-chain triglycerides, higher levels of tryptophan and carbohydrates, together with adenosine-5P and uridine-5P. In a random, double-blind, design, three one-week diets were administered: Diet A (Control): normal initiation milk; Diet B: 06:00-18:00 normal initiation milk, 18:00-06:00 dissociated night formula; and Diet C: day/night formulas with the schedule given above. The sleep patterns were analyzed by means of actimeters (Actiwatch). Statistical analysis consisted of an ANOVA with a Scheffe F-test, taking a value of p<0.05 to be statistically significant. RESULTS: The children receiving the week of Diet C (with the day/night formulas in synchrony with the environment) showed increased hours of actual sleep (7.68 +/- 0.54 h vs. 6.77 +/- 0.12 h for the Diet A control) and improved sleep latency (0.44 +/- 0.04 h vs. 0.60 +/- 0.08 h for the Diet A control). The same children receiving the Diet B in another different week showed an improvement in sleep efficiency (76.43 +/- 3.4% vs. the Diet A control 69.86 +/- 0.94%) and sleep latency (0.45 +/- 0.04 h vs. the Diet A control 0.60 +/- 0.08h) The parents also reported, in response to follow-up questions, an improvement in the sleep of their infants during the Diet C week. CONCLUSION: Day/night infant formula milks designed according to the principles of chrononutrition help to consolidate the sleep/wake rhythm in bottle-fed infants.

Dietary glutamine affects mucosal functions in rats with mild DSS-induced colitis.

The development of inflammatory bowel disease may involve immune dysfunction. Because enteral glutamine is the main source of amino acids for the intestinal mucosa and is metabolized at high rates by both enterocytes and immunocytes, the aim of this study was to ascertain the protective role of glutamine supplementation in a DSS-induced model of mild experimental colitis on metabolic, immune, and intestinal variables. Lewis rats were fed diets supplemented with glutamine (glutamine diet, G group) or an isoenergetic isonitrogenous control diet (C group) from postnatal d 21 (weaning) and continuing to d 35. On d 30, half of the rats from both groups were given 0.5% DSS in drinking water (G-DSS and C-DSS groups). Glutamine supplementation increased the plasma concentrations of Thr, Gln, Cit, His, and Arg and enhanced the ratio of essential to nonessential amino acids irrespective of DSS treatment. DSS administration increased the plasma Gln concentration, indicating a reduced utilization of this amino acid by the intestinal tissue. Regarding the gut-associated lymphoid tissue lymphocyte populations, DSS increased the percentages of CD3(+) T lymphocytes from Peyer's patches, NK and B lymphocytes from mesenteric lymph nodes, and NK CD8(-) cells from intraepithelial lymphocytes. The administration of glutamine did not affect the inductive populations nor did it modify T-cell subtypes or the percentage of intraepithelial lymphocytes of gut-associated lymphoid tissue. However, glutamine supplementation reduced the feces water contents in the DSS-treated but not in the untreated rats. These results indicate that glutamine supplementation can improve barrier function in rats with colitis.

Effects of fish-oil and folate supplementation of pregnant women on maternal and fetal plasma concentrations of docosahexaenoic acid and eicosapentaenoic acid: a European randomized multicenter trial.

BACKGROUND: Pregnant women usually meet their increased energy needs but do not always meet their increased micronutrient requirements. The supply of both folic acid and docosahexaenoic acid (DHA) has been related to positive pregnancy and infant outcomes. OBJECTIVE: We aimed to assess whether fish-oil (FO) supplementation with or without folate from gestation week 22 to birth improves maternal and fetal n-3 long-chain polyunsaturated fatty acid (n-3 LC-PUFA) status. DESIGN: We conducted a multicenter (Germany, Hungary, and Spain), randomized, double-blind, 2 x 2 factorial, placebo-controlled trial. From gestation week 22 until delivery, 311 pregnant women received daily a preparation with FO [0.5 g DHA and 0.15 g eicosapentaenoic acid (EPA)], 400 microg methyltetrahydrofolic acid (MTHF), FO with MTHF, or placebo. Outcome measures included maternal and cord plasma DHA and EPA contents at gestation weeks 20 and 30 and at delivery, indicators of pregnancy outcome, and fetal development. RESULTS: FO significantly (P<0.001) increased maternal DHA and EPA (% by wt), as shown by 3-factor repeated-measures ANOVA (ie, MTHF, FO, and time) with adjustment for maternal baseline DHA and EPA. In addition, FO significantly (P<0.001) increased cord blood DHA (% by wt; 2-factor ANOVA). MTHF was significantly (P=0.046) associated with increased maternal DHA (% by wt). There was no FO x MTHF interaction for the time course of DHA or EPA (P=0.927 and 0.893). Pregnancy outcomes and fetal development did not differ significantly among the intervention groups. CONCLUSIONS: FO supplementation from gestation week 22 until delivery improves fetal n-3 LC-PUFA status and attenuates depletion of maternal stores. MTHF may further enhance maternal n-3 LC-PUFA proportions.

Dietary unsaturated long-chain Fatty acids modify D-glucose absorption in weaning rats.

OBJECTIVE: The authors evaluated the effects of dietary long-chain polyunsaturated fatty acids on D-glucose absorption in weaning rats. METHODS: Pups were born from control mothers fed a diet containing (per kg of total fatty acids) 280 g of saturated fatty acids, 496 g of monounsaturated fatty acids and 222 g of polyunsaturated fatty acids or from mothers fed a diet containing a high proportion of saturated fatty acids (920 g/kg) and a low proportion of unsaturated fatty acids (low-unsaturated fatty acid, 80 g/kg), initiated 2 weeks before mating and continued throughout pregnancy. When pups from low-unsaturated fatty acid mothers were 15 days old, they were subdivided into two groups: one control (low-unsaturated fatty acid-C) and one fed a long-chain polyunsaturated fatty acid supplement rich in arachidonic acid and docosahexaenoic acid (low-unsaturated fatty acid-S) until weaning. At day 21, the kinetics of D-glucose absorption was studied in brush-border membrane vesicles from the jejunoileal segment. RESULTS: The maximal transport rate (V(max)) of glucose in the low-unsaturated fatty acid-C and low-unsaturated fatty acid-S groups was higher than in control rats: 160 and 130 versus 98 pmol/(mg protein.s), respectively (P < 0.05). Rats fed the low-unsaturated fatty acid diet had a lower diffusion constant (K(d)) than control rats did: 21.6 and 29.2 nL/(mg protein.s), respectively (P < 0.05). However, rats receiving the long-chain polyunsaturated fatty acid supplement and control rats had similar Kd values. CONCLUSION: These results indicate that dietary long-chain polyunsaturated fatty acid supplementation can restore, in part, the kinetic characteristics of intestinal D-glucose absorption in pups from mothers maintained on a low-unsaturated fatty acid diet.

The source of long-chain PUFA in formula supplements does not affect the fatty acid composition of plasma lipids in full-term infants.

Supplementation of formulas for full-term infants with long-chain (LC) PUFA [arachidonic acid (AA) and docosahexaenoic acid (DHA)] at levels resembling human milk is recommended because they provide biochemical and functional benefits to the neonate. The objective of this work was to determine whether the source of dietary LC-PUFA affects the bioavailability in full-term infants. Treatment groups were as follows: full-term infants were fed from birth to 3 mo breast-milk (n = 11, 0.4 and 0.3 g/100 g total fatty acids as AA and DHA, respectively), formula containing LC-PUFA in the form of egg phospholipids (n = 12), or a formula supplemented with LC-PUFA in the form of triglycerides synthesized by single cells of algal and fungal microorganisms (n = 12). Both formulas provided 0.4 and 0.1 g/100 g total fatty acids as AA and DHA, respectively. We compared the fatty acid compositions of the main plasma lipid fractions (phospholipids, triglycerides, and cholesteryl esters) at birth and 3 mo. At 3 mo, lower levels of nervonic acid (NA), docosapentaenoic (DPA) acid, and DHA were found in all plasma lipid fractions from infants fed formula compared with those in the human milk-fed infants, irrespective of the source of the formula supplement (P < 0.02). These data demonstrate that the form of dietary LC-PUFA (triglycerides or phospholipids) does not influence their bioavailability. Similarly, absorption of LC-PUFA depends mainly on the lipid composition of the diet fed. These results suggest that the levels of NA, DPA, and DHA in formulas for full-term infants should be increased.