Folate Deficiency Inhibits Development of the Mammary Gland and its Associated Lymphatics in FVB Mice.

BACKGROUND: Folate is essential for DNA synthesis, DNA repair, cell proliferation, development, and morphogenesis. Folic acid (FA) is a nutritional supplement used to fortify human diets. OBJECTIVES: We investigated the effects of dietary FA on early mammary gland (MG) development and hyperplasia. METHODS: Study 1: nulliparous female FVB wild-type (WT) mice were fed control (Con; 2 mg FA/kg), deficient (Def; 0 mg FA/kg), excess (Ex; 5 mg FA/kg), or super excess (S-Ex; 20 mg FA/kg) diets for 8 wk before mating to WT or heterozygous FVB/N-Tg[mouse mammary tumor virus long terminal repeat (MMTV)-polyomavirus middle T antigen (PyVT)]634Mul/J (MMTV-PyMT+/-) transgenic males. Dams were fed these diets until they weaned WT or MMTV-PyMT+/- pups, which were fed the dam's diet from postnatal day (PND) 21 to 42. Tissues were collected from female progeny at PNDs 1, 21, and 42. Study 2: Con or Def diets were fed to WT intact females and males from PND 21 to 56, or to ovariectomized females from PND 21 to 77; tissues were collected at PND 56 or 77. Growth of all offspring, development of MGs, MG hyperplasia, supramammary lymph nodes, thymus and spleen, cell proliferation, and expression of MG growth factors were measured. RESULTS: Study 1: Ex or S-Ex did not affect postnatal MG development or hyperplasia. The rate of isometric MG growth (PND 1-21) was reduced by 69% in Def female progeny (P < 0.0001). Similarly, hyperplastic growth in MGs of Def MMTV-PyMT+/- offspring was 18% of Con (P < 0.05). The Def diet reduced supramammary lymph node size by 20% (P < 0.0001) and increased MG insulin-like growth factor 2 mRNA by 200% (P < 0.05) and protein by 130%-150% (P < 0.05). Study 2: the Def diet did not affect MG growth, but it did reduce supramammary lymph node size (P < 0.05), spleen weight (P < 0.001), and thymic medulla area (P < 0.05). CONCLUSIONS: In utero and postnatal folate deficiency reduced the isometric development of the MGs and early MG hyperplasia. Postnatal folate deficiency reduced the development of lymphatic tissues.

Leptin and EGF Supplementation Enhance the Immune System Maturation in Preterm Suckling Rats.

In preterm newborns the immaturity of the immune system is remarkable, with reduced innate and adaptive immune responses. Many bioactive compounds in breast milk, such as growth factors and adipokines, contribute to the immune system's maturation in early life. However, studies on the immunoregulatory activity in preterm neonates are practically nonexistent. The aim of the present study was to determine whether a nutritional supplementation in early life with leptin or epidermal growth factor (EGF) was able to promote the maturation of the systemic and intestinal immune system in preterm conditions. For this purpose, premature rats were daily supplemented by oral gavage with leptin or EGF. Term and Preterm groups receiving vehicle were used as controls. Preterm rats showed deficiencies compared to full-term ones, such as lower body weights, erythrocyte counts, plasma IgG and IgM concentrations and B cell percentages, and higher values of Th and Tc TCRαß+ cells in mesenteric lymph nodes, and intestinal permeability, among others. However, leptin and EGF supplementation were able to revert some of these deficiencies and to improve the premature immune system's development. These results suggest that leptin and EGF are involved in enhancing the maturation of the systemic and intestinal immune system in preterm conditions.

A mixture of milk and vegetable lipids in infant formula changes gut digestion, mucosal immunity and microbiota composition in neonatal piglets.

PURPOSE: Although composition of infant formula has been significantly improved during the last decade, major differences with the composition and structure of breast milk still remain and might affect nutrient digestion and gut biology. We hypothesized that the incorporation of dairy fat in infant formulas could modify their physiological impacts by making their composition closer to that of human milk. The effect of milk fat and milk fat globule membrane (MFGM) fragments in infant formulas on gut digestion, mucosal immunity and microbiota composition was evaluated. METHODS: Three formulas containing either (1) vegetable lipids stabilized only by proteins (V-P), (2) vegetable lipids stabilized by a mixture of proteins and MFGM fragments (V-M) and (3) a mixture of milk and vegetable lipids stabilized by a mixture of proteins and MFGM fragments (M-M) were automatically distributed to 42 newborn piglets until slaughter at postnatal day (PND) 7 or 28, and compared to a fourth group of sow's suckling piglets (SM) used as a breast-fed reference. RESULTS: At both PND, casein and ß-lactoglobulin digestion was reduced in M-M proximal jejunum and ileum contents compared to V-P and V-M ones leading to more numerous ß-Cn peptides in M-M contents. The IFNγ cytokine secretion of ConA-stimulated MLN cells from M-M piglets tended to be higher than in V-P ones at PND 7 and PND 28 and was closer to that of SM piglets. No dietary treatment effect was observed on IL-10 MLN cell secretion. Changes in faecal microbiota in M-M piglets resulted in an increase in Proteobacteria and Bacteroidetes and a decrease in Firmicutes phyla compared to V-P ones. M-M piglets showed higher abundances of Parabacteroides, Escherichia/Shigella and Klebsiella genus. CONCLUSIONS: The incorporation of both milk fat and MFGM fragments in infant formula modifies protein digestion, the dynamic of the immune system maturation and the faecal microbiota composition.

Maresin conjugates in tissue regeneration biosynthesis enzymes in human macrophages.

Macrophages are central in coordinating immune responses, tissue repair, and regeneration, with different subtypes being associated with inflammation-initiating and proresolving actions. We recently identified a family of macrophage-derived proresolving and tissue regenerative molecules coined maresin conjugates in tissue regeneration (MCTR). Herein, using lipid mediator profiling we identified MCTR in human serum, lymph nodes, and plasma and investigated MCTR biosynthetic pathways in human macrophages. With human recombinant enzymes, primary cells, and enantiomerically pure compounds we found that the synthetic maresin epoxide intermediate 13S,14S-eMaR (13S,14S-epoxy- 4Z,7Z,9E,11E,16Z,19Z-docosahexaenoic acid) was converted to MCTR1 (13R-glutathionyl, 14S-hydroxy-4Z,7Z,9E,11E,13R,14S,16Z,19Z-docosahexaenoic acid) by LTC4S and GSTM4. Incubation of human macrophages with LTC4S inhibitors blocked LTC4 and increased resolvins and lipoxins. The conversion of MCTR1 to MCTR2 (13R-cysteinylglycinyl, 14S-hydroxy-4Z,7Z,9E,11E,13R,14S,16Z,19Z-docosahexaenoic acid) was catalyzed by γ-glutamyl transferase (GGT) in human macrophages. Biosynthesis of MCTR3 was mediated by dipeptidases that cleaved the cysteinyl-glycinyl bond of MCTR2 to give 13R-cysteinyl, 14S-hydroxy-4Z,7Z,9E,11E,13R,14S,16Z,19Z-docosahexaenoic acid. Of note, both GSTM4 and GGT enzymes displayed higher affinity to 13S,14S-eMaR and MCTR1 compared with their classic substrates in the cysteinyl leukotriene metabolome. Together these results establish the MCTR biosynthetic pathway and provide mechanisms in tissue repair and regeneration.

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.

Inhibitory effects of autoimmune disease by green tea in MRL-Faslprcg/Faslprcg mice.

To investigate whether green tea has inhibitory effects on the development of autoimmune disease (AID), one-month-old MRL-Faslprcg/Faslprcg mice were fed diets containing 2% green tea powder (GTP) for 3 months. At the end of GTP feeding, the weights of body, subcutaneous (s.c.) and intraperitoneal (i.p.) lymph nodes (LN), kidneys, spleen and intraperitoneal adipose tissue (IPAT), serological abnormalities and renal lesions were compared between GTP-fed and control mice. SCLN, IPLN, kidneys and IPAT weights in both sexes, spleen weight in males and body weight increase in males were significantly lower in GTP-fed mice. Particularly, LN hyperplasia and fatty accumulation were markedly reduced by GTP. Serum levels of anti-DNA antibodies and immune complexes (IC) were significantly lowered and proteinuria and blood urea nitrogen tended to be improved by GTP. The incidence of serious glomerulonephritis was significantly lower and nephric vasculitis was almost completely prevented in GTP-fed mice. Moreover, the survival of mice was significantly prolonged by GTP feeding for 6 months. These results indicate that the progression of lupus-like syndrome including glomerulonephritis was significantly delayed by reduced production of autoantibodies and IC in GTP-fed MRL-Faslprcg/Faslprcg mice, which led to the prolonged survival.

Molecular control of bone remodeling and osteoporosis.

Osteoprotegerin ligand (OPGL, TNFS11) and its receptor RANK (TNFRS11A) are essential for the development and activation of osteoclasts and critical regulators of physiological bone remodeling and osteoporosis. Production of OPGL by activated T cells can directly regulate osteoclastogenesis and bone remodeling. This may explain why autoimmune diseases, cancers, leukemias, asthma and chronic viral infections such as hepatitis and HIV result in systemic and local bone loss. OPGL is also the pathogenetic factor that causes bone and cartilage destruction and clinical crippling in arthritis. Inhibition of OPGL binding to RANK via the natural decoy receptor osteoprotegerin (OPG) prevents bone loss in postmenopausal osteoporosis and cancer metastases and completely blocks crippling in a rat model of arthritis. Moreover, OPG expression is induced by estrogen which provides a molecular explanation of postmenopausal osteoporosis in women.

An immunomodulating protein, Ling Zhi-8 (LZ-8) prevents insulitis in non-obese diabetic mice.

Ling Zhi-8 (LZ-8), a novel and recently discovered immunomodulatory protein having in vivo immuno-suppressive activity, was tested for in vivo effect against Type 1 (insulin-dependent) diabetes mellitus in the nonobese diabetic mouse, the disease having immunologically mediated aetiology in this animal. LZ-8 had mitogenic activity in vitro towards spleen cells of the non-obese diabetic mice as previously shown towards those of DBA/2 mice. Intraperitoneal administration of LZ-8 twice weekly into the mice (10.3-12.6 mg/kg body weight) from 4 weeks of age prevented insulitis and an almost normal number of insulin producing cells were observed. Extreme insulitis and reduction of the number of insulin producing cells were observed in the pancreata of the untreated non-obese diabetic mouse. No cumulative incidence of diabetes mellitus was observed in the LZ-8 treated group, while cumulative incidences of 70% and 60% were observed in an untreated group followed up to 42 weeks of age when the incidence of diabetes was defined as a plasma glucose level of greater than 11 mmol/l and as a urine glucose level of greater than 2+, respectively. T cell subset population analysis was performed to further investigate the action of LZ-8 on the non-obese diabetic mouse which revealed that LZ-8 treatment increased in L3T4'/Lyt-2+ ratio.

Ontogenetic development of lipopolysaccharide-reactive B cells against bromelain-treated mouse erythrocytes in mouse lymphoid tissues.

In lymphoid tissues of mice, there exist LPS-reactive B cells which can differentiate to IgM-secreting plaque-forming cells (IgM-PFC) and PFC secreting antibodies against bromelain-treated mouse erythrocytes (BrMRBC) by LPS activation. In this study, four groups of LPS-reactive B cells in spleen, peritoneal cavity and mesenteric lymph nodes from 2- and 10-week-old mice were compared and enumerated as precursors of IgM-PFC and anti-BrMRBC PFC on days 1 and 2 after LPS activation in quantitative culture conditions. The induction of each of four PFC responses in peritoneal cells was sensitive to LPS and anti-mouse IgM antibodies as much as the induction of the respective PFC response in spleen cells. The ratios of four groups of PFC to each other were different among three tissues and between two ages. These findings support the view that the four groups of LPS-reactive B cells in each tissue are mostly in distinct subpopulations of B cells from each other, and the respective groups of different lymphoid tissues at different ages belong to the same subpopulation.