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Premature delivery influences the immunological composition of colostrum and transitional and mature human milk

Human breast milk is the ideal nutrition for the newborn, and in addition to its nutritional contribution, necessary for infant growth and development, it contains various immune bioactive factors that confer some of the numerous beneficial effects of breastfeeding. The current study analyzed the concentrations of IgA, growth factors such as epidermal growth factor (EGF), TGFβ1, and TGFβ2, cytokines IL-6, IL-8, IL-10, IL-13, and TNFα, and TNF-receptor I (TNF-RI) in colostrum and transitional and mature milk from mothers with mature, premature, and very premature infants. Human milk samples were collected from mothers delivering at term (T), preterm (PT), and very preterm (VPT). Milk from all the mothers was collected at 3 different time points after delivery corresponding to colostrum and transitional and mature milk. After obtaining milk whey, IgA, EGF, TGFβ1, and TGFβ2 were determined by ELISA and IL-6, IL-8, IL-10, IL-13, TNFα and TNF-RI by cytometric bead array immunoassay. The colostrum of the PT group was extremely rich in most of the factors studied, but higher concentrations than in the T group were only found for IL-6 (P = 0.051), TGFβ1, and TGFβ2 (P < 0.05). Conversely, the colostrum of the VPT group had lower concentrations of IgA, IL-8, IL-10, and TNFα than those in the T group (P < 0.05). Results suggest that maternal lactogenic compensatory mechanisms accelerating the development of immature breast-fed preterm infants may take effect only after wk 30 of gestation.

Cocoa modulatory effect on rat faecal microbiota and colonic crosstalk

Previous studies have reported the effect of a cocoa-enriched diet on the intestinal immune system in rats. Cocoa contains fibre and polyphenols that can directly influence the intestinal ecosystem and its relationship with the immune system. The aim of this study was to evaluate the effects of a cocoa-enriched diet on gut microbiota, toll-like receptor (TLR) expression and immunoglobulin (Ig) A (IgA) intestinal secretion in rats. Four-week-old Wistar rats were fed a standard or cocoa diet for 6 weeks. Faecal samples were collected before the beginning of the diet and at the end of the study. After the nutritional intervention, colon samples were obtained to quantify TLR and IgA gene expression and IgA protein. Microbiota composition was characterized by fluorescent in situ hybridization (FISH) coupled to flow cytometry (FCM) analysis using specific probes directed to 16S rRNA of the main bacteria genus present in rat intestine. The cocoa dietary intervention resulted in a differential TLR pattern and a decrease in the intestinal IgA secretion and IgA-coating bacteria. Moreover there was a significant decrease in the proportion of Bacteroides, Clostridium and Staphylococcus genera in the faeces of cocoa-fed animals. In conclusion, cocoa intake affects the growth of certain species of gut microbiota in rats and is associated with changes in the TLR pattern which could be responsible for the changes observed in the intestinal immune system.

Spleen lymphocyte function modulated by a cocoa-enriched diet

Previous studies have shown the down‐regulating in vitro effect of cocoa flavonoids on lymphocyte and macrophage activation. In the present paper, we report the capacity of a long‐term rich cocoa diet to modulate macrophage cytokine secretion and lymphocyte function in young rats. Weaned rats received natural cocoa (4% or 10% food intake), containing 32 mg flavonoids/g, for 3 weeks. Spleen immune function was then evaluated through the analysis of lymphocyte composition, their proliferative response and their ability to secrete cytokines and Ig. In addition, the status of activated peritoneal macrophages was established through tumour necrosis factor (TNF)‐α secretion. The richest cocoa diet (10%) caused a reduction of TNF‐α secretion by peritoneal macrophages showing anti‐inflammatory activity. Similarly, although a 10% cocoa diet increased lymphocyte proliferation rate, it down‐regulated T helper 2 (Th2)‐related cytokines and decreased Ig secretion. These changes were accompanied by an increase in spleen B cell proportion and a decrease in Th cell percentage. In summary, these results demonstrate the functional activity of a cocoa‐high dosage in down‐regulating the immune response that might be beneficial in hypersensitivity and autoimmunity.

Effect of Theobroma cacao flavonoids on immune activation of a lymphoid cell line

We analysed the effect of (-)-epicatechin and cocoa extract on the activation of a lymphoid cell line. Particularly the expression of IL-2 receptor alpha (IL-2Ralpha or CD25) and, the secretion of IL-2 and IL-4 were established after flavonoid treatment. Two media culture conditions (1 and 10 % of fetal calf serum supplementation) and the different moments of flavonoid addition (simultaneously or 2 h before cell-activation) were compared. IL-2Ralpha (CD25) expression on activated cells was significantly reduced by epicatechin and cocoa extract in a dose-dependent manner, achieving the highest inhibition of about 50 % when flavonoids were added 2 h before stimulation. IL-2 secretion was also inhibited by the presence of both epicatechin and cocoa extract, displaying 60 and 75 % of inhibition, respectively. Cocoa flavonoids were also able to enhance 3-4.5-fold IL-4 release. In summary, cocoa extract down-modulated T lymphocyte activation and therefore the acquired immune response. This fact could be important in some states of the immune system hyperactivity such as autoimmune or chronic inflammatory diseases.

Maintenance of breast milk immunoglobulin A after high-pressure processing

Human milk is considered the optimal nutritional source for infants. Banked human milk is processed using low-temperature, long-time pasteurization, which assures microbial safety but involves heat denaturation of some desirable milk components such as IgA. High-pressure processing technology, the subject of the current research, has shown minimal destruction of food macromolecules. The objective of this study was to investigate the influence of pressure treatments on IgA content. Moreover, bacterial load was evaluated after pressure treatments. The effects of high-pressure processing on milk IgA content were compared with those of low-temperature, long-time pasteurization. Mature human milk samples were heat treated at 62.5 degrees C for 30min or pressure processed at 400, 500, or 600MPa for 5min at 12 degrees C. An indirect ELISA was used to measure IgA in human milk whey obtained after centrifugation at 800xg for 10min at 4 degrees C. All 3 high-pressure treatments were as effective as low-temperature, long-time pasteurization in reducing the bacterial population of the human milk samples studied. After human milk pressure processing at 400MPa, 100% of IgA content was preserved in milk whey, whereas only 72% was retained in pasteurized milk whey. The higher pressure conditions of 500 and 600MPa produced IgA retention of 87.9 and 69.3%, respectively. These results indicate that high-pressure processing at 400MPa for 5min at 12 degrees C maintains the immunological protective capacity associated with IgA antibodies. This preliminary study suggests that high-pressure processing may be a promising alternative to pasteurization in human milk banking.

Liposomal encapsulation enhances and prolongs the anti-inflammatory effects of water-soluble dexamethasone phosphate in experimental adjuvant arthritis

IntroductionThe objective of this study was to evaluate the efficacy of intravenous (i.v.) injection of liposomally encapsulated dexamethasone phosphate (DxM-P) in comparison to free DxM-P in rats with established adjuvant arthritis (AA). This study focused on polyethylene glycol (PEG)-free liposomes, to minimize known allergic reactions caused by neutral PEG-modified (PEG-ylated) liposomes.MethodsEfficacy was assessed clinically and histologically using standard scores. Non-specific and specific immune parameters were monitored. Activation of peritoneal macrophages was analyzed via cytokine profiling. Pharmacokinetics/biodistribution of DxM in plasma, synovial membrane, spleen and liver were assessed via mass spectrometry.ResultsLiposomal DxM-P (3 × 1 mg/kg body weight; administered intravenously (i.v.) on Days 14, 15 and 16 of AA) suppressed established AA, including histological signs, erythrocyte sedimentation rate, white blood cell count, circulating anti-mycobacterial IgG, and production of interleukin-1beta (IL-1β) and IL-6 by peritoneal macrophages. The suppression was strong and long-lasting. The clinical effects of liposomal DxM-P were dose-dependent for dosages between 0.01 and 1.0 mg/kg. Single administration of 1 mg/kg liposomal DxM-P and 3 × 1 mg/kg of free DxM-P showed comparable effects consisting of a partial and transient suppression. Moreover, the effects of medium-dose liposomal DxM-P (3 × 0.1 mg/kg) were equal (in the short term) or superior (in the long term) to those of high-dose free DxM-P (3 × 1 mg/kg), suggesting a potential dose reduction by a factor between 3 and 10 by liposomal encapsulation. For at least 48 hours after the last injection, the liposomal drug achieved significantly higher levels in plasma, synovial membrane, spleen and liver than the free drug.ConclusionsThis new PEG-free formulation of macrophage-targeting liposomal DxM-P considerably reduces the dose and/or frequency required to treat AA, with a potential to enhance or prolong therapeutic efficacy and limit side-effects also in the therapy of rheumatoid arthritis. Depot and/or recirculation effects in plasma, inflamed joint, liver, and spleen may contribute to this superiority of liposomally encapsulated DxM-P.

Influence of a cocoa‐enriched diet on specific immune response in ovalbumin‐sensitized rats

Previous studies in young rats have reported the impact of 3 weeks of high cocoa intake on healthy immune status. The present article describes the effects of a longer-term cocoa-enriched diet (9 weeks) on the specific immune response to ovalbumin (OVA) in adult Wistar rats. At 4 weeks after immunization, control rats produced anti-OVA antibodies, which, according their amount and isotype, were arranged as follows: IgG1 > IgG2a > IgM > IgG2b > IgG2c. Both cocoa diets studied (4% and 10%) down-modulated OVA-specific antibody levels of IgG1 (main subclass associated with the Th2 immune response in rats), IgG2a, IgG2c and IgM isotypes. Conversely, cocoa-fed rats presented equal or higher levels of anti-OVA IgG2b antibodies (subclass linked to the Th1 response). Spleen and lymph node cells from OVA-immunized control and cocoa-fed animals proliferated similarly under OVA stimulation. However, spleen cells from cocoa-fed animals showed decreased interleukin-4 secretion (main Th2 cytokine), and lymph node cells from the same rats displayed higher interferon-gamma secretion (main Th1 cytokine). These changes were accompanied by a reduction in the number of anti-OVA IgG-secreting cells in spleen. In conclusion, cocoa diets induced attenuation of antibody synthesis that may be attributable to specific down-regulation of the Th2 immune response.

The Suckling Rat as a Model for Immunonutrition Studies in Early Life

Diet plays a crucial role in maintaining optimal immune function. Research demonstrates the immunomodulatory properties and mechanisms of particular nutrients; however, these aspects are studied less in early life, when diet may exert an important role in the immune development of the neonate. Besides the limited data from epidemiological and human interventional trials in early life, animal models hold the key to increase the current knowledge about this interaction in this particular period. This paper reports the potential of the suckling rat as a model for immunonutrition studies in early life. In particular, it describes the main changes in the systemic and mucosal immune system development during rat suckling and allows some of these elements to be established as target biomarkers for studying the influence of particular nutrients. Different approaches to evaluate these immune effects, including the manipulation of the maternal diet during gestation and/or lactation or feeding the nutrient directly to the pups, are also described in detail. In summary, this paper provides investigators with useful tools for better designing experimental approaches focused on nutrition in early life for programming and immune development by using the suckling rat as a model.

A diet enriched with cocoa prevents IgE synthesis in a rat allergy model

Previous studies in young rats reported the impact of cocoa intake on healthy immune status and allow suggesting it may have a role in the prevention of some immune-mediated diseases. The aim of this study was to ascertain the effect of a cocoa diet in a model of allergy in young rats. Three-week-old Brown Norway rats were immunized by i.p. injection of ovalbumin (OVA) with alum as adjuvant and Bordetella pertussis toxin. During the next 4 weeks rats received either a cocoa diet (containing 0.2% polyphenols, w/w) or a standard diet. Animals fed a standard diet showed high concentrations of anti-OVA IgG1, IgG2a, IgG2b and high anti-OVA IgE titres, which is the antibody involved in allergic response. In contrast, animals fed a cocoa diet showed significantly lower concentrations of anti-OVA IgG1 and IgG2a antibodies. Interestingly, the cocoa diet prevented anti-OVA IgE synthesis and decreased total serum IgE concentration. Analysis of cytokine production in lymph node cells at the end of the study revealed that, in this compartment, the cocoa diet decreased the tumor necrosis factor (TNF)-α and the interleukin (IL)-10 secretion but not IL-4 production. In conclusion, a cocoa-enriched diet in young rats produces an immunomodulatory effect that prevents anti-allergen IgE synthesis, suggesting a potential role for cocoa flavonoids in the prevention or treatment of allergic diseases.

Influence of breast milk polyamines on suckling rat immune system maturation.

The aim of this study was to ascertain whether the supplementation of polyamines present in breast milk, i.e. spermine (SPM) and spermidine (SPD), influenced the post-natal maturation of the systemic and intestinal immune system in rats. From birth, pups daily received SPM or SPD. At 5, 11 and 18 days old, small intestine intraepithelial lymphocytes (IEL), lamina propria lymphocytes (LPL) and splenocytes were phenotypically characterized. SPM and, less evidently, SPD accelerated the maturation of CD8+ IEL, and enhanced the presence of intraepithelial NK cells and IEL related with specific immune responses on the proximal and distal small intestine, respectively. Polyamines increased the percentage of more mature CD4+ LPL and enhanced the early presence of splenic B cells and, later, that of NK cells. However, no effect on Ig-secretory function was detected. These results suggest that breast milk polyamines improve the maturation of the rat intestinal and systemic immune system.