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Maternal breast milk plays a key role in providing newborns with passive immunity and stimulating the maturation of an infant's immune system, protecting them from many diseases. It is known that diet can influence the immune system of lactating mothers and the composition of their breast milk. The aim of this study was to establish if a supplementation during the gestation and lactation of Lewis rats with extra virgin olive oil (EVOO), due to the high proportion of antioxidant components in its composition, has an impact on the mother's immune system and on the breast milk's immune composition. For this, 10 mL/kg of either EVOO, refined oil (control oil) or water (REF group) were orally administered once a day to rats during gestation and lactation periods. Immunoglobulin (Ig) concentrations and gene expressions of immune molecules were quantified in several compartments of the mothers. The EVOO group showed higher IgA levels in both the breast milk and the mammary glands than the REF group. In addition, the gene expression of IgA in mammary glands was also boosted by EVOO consumption. Overall, EVOO supplementation during gestation and lactation is safe and does not negatively affect the mother's immune system while improving breast milk immune composition by increasing the presence of IgA, which could be critical for an offspring's immune health.

Microbiota-host communication is primarily achieved by secreted factors that can penetrate the mucosal surface, such as extracellular membrane vesicles (EVs). The EVs released by the gut microbiota have been extensively studied in cellular and experimental models of human diseases. However, little is known about their in vivo effects in early life, specifically regarding immune and intestinal maturation. This study aimed to investigate the effects of daily administration of EVs from probiotic and commensal E. coli strains in healthy suckling rats during the first 16 days of life. On days 8 and 16, we assessed various intestinal and systemic variables in relation to animal growth, humoral and cellular immunity, epithelial barrier maturation, and intestinal architecture. On day 16, animals given probiotic/microbiota EVs exhibited higher levels of plasma IgG, IgA, and IgM and a greater proportion of Tc, NK, and NKT cells in the spleen. In the small intestine, EVs increased the villi area and modulated the expression of genes related to immune function, inflammation, and intestinal permeability, shifting towards an anti-inflammatory and barrier protective profile from day 8. In conclusion, interventions involving probiotic/microbiota EVs may represent a safe postbiotic strategy to stimulate immunity and intestinal maturation in early life.

Breastmilk contains antibodies that could protect breastfed infants from infections. In this work, we examined if antibodies in breastmilk could neutralize SARS-CoV-2 in 84 breastmilk samples from women that were either vaccinated (Comirnaty, mRNA-1273, or ChAdOx1), infected with SARS-CoV-2, or both infected and vaccinated. The neutralization capacity of these sera was tested using pseudotyped vesicular stomatitis virus carrying either the Wuhan-Hu-1, Delta, or BA.1 Omicron spike proteins. We found that natural infection resulted in higher neutralizing titers and that neutralization correlated positively with levels of immunoglobulin A in breastmilk. In addition, significant differences in the capacity to produce neutralizing antibodies were observed between both mRNA-based vaccines and the adenovirus-vectored ChAdOx1 COVID-19 vaccine. Overall, our results indicate that breastmilk from naturally infected women or those vaccinated with mRNA-based vaccines contains SARS-CoV-2 neutralizing antibodies that could potentially provide protection to breastfed infants from infection., Competing Interests: The authors declare no competing interests., (© 2023 The Author(s).)

Viral infections are described as modifying host gene expression; however, there is limited insight regarding rotavirus (RV) infections. This study aimed to assess the changes in intestinal gene expression after RV infection in a preclinical model, and the effect of 2-fucosyllactose (2'-FL) on this process. From days 2 to 8 of life, rats were supplemented with the dietary oligosaccharide 2'-FL or vehicle. In addition, an RV was inoculated on day 5 to nonsupplemented animals (RV group) and to 2'-FL-fed animals (RV+2'-FL group). Incidence and severity of diarrhea were established. A portion from the middle part of the small intestine was excised for gene expression analysis by microarray kit and qPCR. In nonsupplemented animals, RV-induced diarrhea upregulated host antiviral genes (e.g., Oas1a , Irf7 , Ifi44 , Isg15 ) and downregulated several genes involved in absorptive processes and intestinal maturation (e.g., Onecut2 , and Ccl19 ). The 2'-FL-supplemented and infected animals had less diarrhea; however, their gene expression was affected in a similar way as the control-infected animals, with the exception of some immunity/maturation markers that were differentially expressed (e.g., Ccl12 and Afp ). Overall, assessing the expression of these key genes may be useful in the evaluation of the efficacy of nutritional interventions or treatments for RV infection.

Breast milk (BM) is important for adequate infant development, and it contains bioactive compounds, such as bacteria, cytokines and some adipokines which play a role in infant microbial, metabolic, and immunological maturation. However, little is known about its impact on growth and development in early life. The objective of this study was to evaluate the impact of milk microbiota, cytokine, and adipokine profiles on the risk of overweight at 12 months of life to find the possible mechanisms of host-microbe interactions. In this study, BM samples from 100 healthy women collected during 15 d after birth were included. BM microbiota was analysed by 16S rRNA gene sequencing, and cytokine and adipokine levels were measured by the Luminex approach. In addition, infant weight and length were recorded during the first 12 months and z -scores were obtained according to the WHO databases. Infants were classified as risk of overweight (ROW) and no-risk of overweight (NOROW) based on their body mass index z -score (BMIZ) and infant weight-for-length z -score (WLZ) at 12 months. In order to study host-microbe interactions, epithelial intestinal and mammary cell lines were exposed to milk microbes to assess the host response by interleukin (IL)-6 production as a potential inflammatory marker. BM was dominated by Staphylococcus and Streptococcus genera, and the most abundant cytokines were IL-6 and IL-18. Leptin levels were positively correlated with the pregestational body mass index (BMI). Higher relative abundance of the Streptococcus genus was associated with higher IL-10 and higher relative abundance of the Bifidobacterium genus was associated with lower IL-6 concentrations in milk. Infant WLZ at 12 months could be partially predicted by Streptococcus genus proportions and IL-10 and IL-18 levels in BM. BM microbiota significantly induced cytokine responses in mammary epithelial cells. Higher levels of IL-6 production were observed in mammary cells exposed to BM microbiota from mothers with ROW offspring compared to mothers with NOROW offspring. In conclusion, BM microbiota is related to the cytokine profile. IL-10 and IL-18 levels and the abundance of the Streptococcus genus could affect early infant development. Further research is needed to clarify the specific impact of BM microbiota and cytokines on infant growth and the risk of overweight.

Stressful situations such as a high-intensity exercise or exhausting training programs can act as immune disruptors leading to transitory immunodepression status, which can be accompanied by alterations of the gastrointestinal functions. Hesperidin intake has demonstrated ergogenic activity and is able to influence the intestinal ecosystem and immunity. We aimed to investigate the effect of hesperidin consumption in rats submitted to an intense training and a final exhaustion test, focusing on the functionality of the intestinal immune system and on the cecal microbiota. Rats, supplemented or not with hesperidin, were intensively trained on a treadmill for 5 weeks. Samples were obtained 24 h after a regular training session, and immediately and 24 h after a final exhaustion test. Cecal microbiota and composition and function of mesenteric lymph node (MLN) lymphocytes and mucosal immunoglobulin A (IgA) were determined. Results showed that chronic intense exercise followed by an exhausting test induced changes in the intestinal immune compartment such as the distribution and function of MLN lymphocytes. Although the hesperidin supplementation did not prevent these alterations, it was able to enhance IgA synthesis in the intestinal compartment. This could be important in enhancing the immune intestinal barrier in this stressful situation.

The aim of this study was to establish the influence of flavonoid-enriched diets on the immune alterations induced by an intensive training and a final exhaustion test in rats. A flavanol-enriched diet (with 10% cocoa, C10 diet) and a flavanol and flavanone-enriched diet (C10 plus 0.5% hesperidin, CH diet) were used. Lewis rats were fed either a standard diet, C10 diet or CH diet while they were submitted to an intensive running training on a treadmill. After 6 weeks, samples were obtained 24 h after performing a regular training (T groups) and after carrying out a final exhaustion test (TE groups). The C10 diet attenuated the increase in plasma cortisol induced by exhaustion, while both the C10 and the CH diets prevented the alterations in the spleen Th cell proportion. The experimental diets also induced an increase in serum immunoglobulin concentration and an enhancement of spleen natural killer cytotoxicity, which may be beneficial in situations with a weakened immunity. Most of the effects observed in the CH groups seem to be due to the cocoa content. Overall, a dietary intervention with flavonoids enhances immune function, partially attenuating the alterations in systemic immunity induced by intensive training or exhausting exercise.

Galectins (Gal) are a family of conserved soluble proteins with high affinity for β-galactoside structures. They have been recognized as important proteins for successful pregnancy. However, little is known about their presence in breast milk and their role in early infancy. Gal-1, -3 and -9 concentrations were evaluated by Multiplex immunoassays in mother-infant pairs from the MAMI cohort in maternal plasma (MP) ( n = 15) and umbilical cord plasma (UCP) ( n = 15) at birth and in breast milk samples ( n = 23) at days 7 and 15 postpartum. Data regarding mother and infant characteristics were collected. Gal-9 was present in a lower concentration range than Gal-1 and Gal-3 in plasma, specifically in UCP. A major finding in the current study is that Gal-1, -3 and -9 were detected for the first time in all the transitional breast milk samples and no differences were found when comparing the two breastfeeding time points. Finally, Gal levels were associated with some maternal and infant characteristics, such as gestational age, pregnancy weight gain, maternal diet, the gender, infant growth and infant infections. In conclusion, Gal levels seem to be involved in certain developmental aspects of early life.

Scope: Lack of information about the impact of maternal severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection on the elemental and metabolomic profile of human milk (HM)., Methods and Results: An observational study on HM from mothers with COVID-19 is conducted including a prepandemic control group. Maternal-infant clinical records and symptomatology are recorded. The absolute quantification of elements and untargeted relative metabolomic profiles are determined by inductively coupled plasma mass spectrometry and gas chromatography coupled to mass spectrometry, respectively. Associations of HM SARS-CoV-2 antibodies with elemental and metabolomic profiles are studied. COVID-19 has a significant impact on HM composition. COVID-19 reduces the concentrations of Fe, Cu, Se, Ni, V, and Aluminium (Al) and increases Zn compared to prepandemic control samples. A total of 18 individual metabolites including amino acids, peptides, fatty acids and conjugates, purines and derivatives, alcohols, and polyols are significantly different in HM from SARS-CoV-2 positive mothers. Aminoacyl-tRNA biosynthesis, phenylalanine, tyrosine and tryptophan biosynthesis, phenylalanine, and linoleic acid pathways are significantly altered. Differences are obtained depending on COVID-19 symptomatic and asymptomatic status., Conclusions: This study provides unique insights about the impact of maternal SARS-CoV-2 infection on the elemental and metabolomic profiles of HM that warrants further research due the potential implications for infant health., (© 2022 The Authors. Molecular Nutrition & Food Research published by Wiley-VCH GmbH.)

The digestion of gluten generates toxic peptides, among which a highly immunogenic proline-rich 33-mer from wheat α-gliadin, that trigger coeliac disease. Neprosin from the pitcher plant is a reported prolyl endopeptidase. Here, we produce recombinant neprosin and its mutants, and find that full-length neprosin is a zymogen, which is self-activated at gastric pH by the release of an all-β pro-domain via a pH-switch mechanism featuring a lysine plug. The catalytic domain is an atypical 7+8-stranded β-sandwich with an extended active-site cleft containing an unprecedented pair of catalytic glutamates. Neprosin efficiently degrades both gliadin and the 33-mer in vitro under gastric conditions and is reversibly inactivated at pH > 5. Moreover, co-administration of gliadin and the neprosin zymogen at the ratio 500:1 reduces the abundance of the 33-mer in the small intestine of mice by up to 90%. Neprosin therefore founds a family of eukaryotic glutamate endopeptidases that fulfils requisites for a therapeutic glutenase., (© 2022. The Author(s).)

Mastitis is an inflammation of the mammary gland occurring in 3-33% of the breastfeeding mothers. The majority of mastitis cases have an infectious etiology. More than 75% of infectious mastitis are caused by Staphylococcus epidermidis and Staphylococcus aureus and involves breast milk microbiota alteration, which, may have an impact in lactating infant. The aim of this study was to analyze in rats during the suckling period and later in life the impact of a high and a low overload of Staphylococcus epidermidis , similarly as it occurs during the clinical and the subclinical mastitis, respectively. From days 2 to 21 of life, suckling rats were daily supplemented with low (Ls group) or high (Hs group) dose of S. epidermidis . Body weight and fecal humidity were periodically recorded. On days 21 and 42 of life, morphometry, hematological variables, intestinal gene expression, immunoglobulin (Ig) and cytokine profile and spleen cells' phenotype were measured. Although no differences were found in body weight, Ls and Hs groups showed higher body length and lower fecal humidity. Both doses induced small changes in lymphocytes subpopulations, reduced the plasma levels of Ig and delayed the Th1/Th2 balance causing a bias toward the Th2 response. No changes were found in cytokine concentration. The low dose affected the Tc cells intestinal homing pattern whereas the high dose had an impact on the hematological variables causing leukocytosis and lymphocytosis and also influenced the intestinal barrier maturation. In conclusion, both interventions with Staphylococcus epidermidis overload during suckling, affects the immune system development in short and long term., Competing Interests: MO was employed by Biosearch Life SA. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Morales-Ferré, Franch, Castell, Olivares, Rodríguez-Lagunas and Pérez-Cano.)

Background: Following intensive sports events, a higher rate of upper respiratory tract infections and the appearance of gastrointestinal symptomatology have been reported. We aimed to evaluate the effect of a cocoa-enriched diet on the cecal microbiota and mucosal immune system of rats submitted to high-intensity acute exercise, as well as to elucidate the involvement of cocoa fiber in such effects., Methods: Wistar rats were fed either a standard diet, a diet containing 10% cocoa providing 5% fiber and a diet containing only 5% cocoa fiber. After 25 days, half of the rats of each diet performed an exhaustion running test. Sixteen hours later, samples were obtained to assess, among others, the cecal microbiota and short chain fatty acids (SCFAs) composition, mesenteric lymph nodes (MLNs) and Peyer's patches (PPs) lymphocyte composition, and immunoglobulin (Ig) content in salivary glands., Results: The intake of cocoa, partially due to its fiber content, improved the SCFA production, prevented some changes in PPs and in MLNs lymphocyte composition and also decreased the production of proinflammatory cytokines. Cocoa diet, contrary to cocoa fiber, did not prevent the lower salivary IgM induced by exercise., Conclusion: A cocoa dietary intake can partially attenuate the alterations in microbiota and mucosal immunity induced by a single session of intensive exercise., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Ruiz-Iglesias, Massot-Cladera, Rodríguez-Lagunas, Franch, Camps-Bossacoma, Castell and Pérez-Cano.)

Intensive acute exercise can induce oxidative stress, leading to muscle damage and immune function impairment. Cocoa diet could prevent this oxidative stress and its consequences on immunity. Our aim was to assess the effect of a cocoa-enriched diet on the reactive oxygen species (ROS) production by peritoneal macrophages, blood immunoglobulin (Ig) levels, leukocyte counts, and the physical performance of rats submitted to an intensive acute exercise, as well as to elucidate the involvement of cocoa fiber in such effects. For this purpose, Wistar rats were fed either a standard diet, i.e., a diet containing 10% cocoa (C10), or a diet containing 5% cocoa fiber (CF) for 25 days. Then, half of the rats of each diet ran on a treadmill until exhaustion, and 16 h later, the samples were obtained. Both C10 and CF diets significantly prevented the increase in ROS production. However, neither the cocoa diet or the cocoa fiber-enriched diet prevented the decrease in serum IgG induced by acute exercise. Therefore, although the cocoa-enriched diet was able to prevent the excessive oxidative stress induced by intensive exercise, this was not enough to avoid the immune function impairment due to exercise.

Rotavirus (RV) is the main cause of gastroenteritis in children. Prebiotics and, more recently, postbiotics are used for preventing and treating gastrointestinal infections. The aim of this study was to analyze the effects of a Lactofidus TM , short-chain galacto-oligosaccharides (scGOS) and long-chain fructo-oligosaccharides (lcFOS) mixture, and their combination on RV infection, in a rat model, for early life diarrhea. Fifteen litters of suckling rats were intragastrically administered daily with the vehicle, the prebiotic mixture, the postbiotic or the combination. The RV was inoculated on day 5 and then fecal samples were clinically evaluated daily. Viral shedding, intestinal permeability assay, in vitro blocking assay, immunoglobulin profiles, and anti-RV response were assessed at day 8 and 16 of life. Cecal microbiota composition, intestinal gene expression, and short chain fatty acids (SCFAs) were analyzed at day 16. The incidence and severity of diarrhea were significantly reduced by all the supplementations. Moreover, they showed blocking activity, changes in the immunoglobulin profiles, in gut microbiota, and in the intestinal gene expression. The prebiotic mixture reduced gut permeability and changed the SCFA profile, whereas the postbiotic enhanced the expression of Toll-like receptors (TLRs). The combination preserved most of the individual observed effects, and furthermore, complementary effects, such as an increase in white blood cells and lymphocytes recruitment, as well as upregulation of TLR7 and TLR9 gene expression.

Objectives: To develop and validate a specific protocol for SARS-CoV-2 detection in breast milk matrix and to determine the impact of maternal SARS-CoV-2 infection on the presence, concentration and persistence of specific SARS-CoV-2 antibodies., Design and Patients: This is a prospective, multicentre longitudinal study (April-December 2020) in 60 mothers with SARS-CoV-2 infection and/or who have recovered from COVID-19. A control group of 13 women before the pandemic were also included., Setting: Seven health centres from different provinces in Spain., Main Outcome Measures: Presence of SARS-CoV-2 RNA in breast milk, targeting the N1 region of the nucleocapsid gene and the envelope (E) gene; presence and levels of SARS-CoV-2-specific immunoglobulins (Igs)-IgA, IgG and IgM-in breast milk samples from patients with COVID-19., Results: All breast milk samples showed negative results for presence of SARS-CoV-2 RNA. We observed high intraindividual and interindividual variability in the antibody response to the receptor-binding domain of the SARS-CoV-2 spike protein for each of the three isotypes IgA, IgM and IgG. Main Protease (MPro) domain antibodies were also detected in milk. 82.9% (58 of 70) of milk samples were positive for at least one of the three antibody isotypes, with 52.9% of these positive for all three Igs. Positivity rate for IgA was relatively stable over time (65.2%-87.5%), whereas it raised continuously for IgG (from 47.8% for the first 10 days to 87.5% from day 41 up to day 206 post-PCR confirmation)., Conclusions: Our study confirms the safety of breast feeding and highlights the relevance of virus-specific SARS-CoV-2 antibody transfer. This study provides crucial data to support official breastfeeding recommendations based on scientific evidence. Trial registration number NCT04768244., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2022. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Different cocoa populations have demonstrated a protective role in a rat model of allergic asthma by attenuating the immunoglobulin (Ig) E synthesis and partially protecting against anaphylactic response. The aim of this study was to ascertain the effect of diets containing two native Peruvian cocoa populations ("Amazonas Peru" or APC, and "Criollo de Montaña" or CMC) and an ordinary cocoa (OC) on the bronchial compartment and the systemic and mucosal immune system in the same rat model of allergic asthma. Among other variables, cells and IgA content in the bronchoalveolar lavage fluid (BALF) and serum anti-allergen antibody response were analyzed. The three cocoa populations prevented the increase of the serum specific IgG1 (T helper 2 isotype). The three cocoa diets decreased asthma-induced granulocyte increase in the BALF, which was mainly due to the reduction in the proportion of eosinophils. Moreover, both the OC and CMC diets were able to prevent the leukocyte infiltration caused by asthma induction in both the trachea and nasal cavity and decreased the IgA in both fecal and BALF samples. Overall, these results highlight the potential of different cocoa populations in the prevention of allergic asthma.

Human milk serves as a model for infant formula providing nutritional solutions for infants not able to receive enough mother's milk. Infant formulas aim to mimic the composition and functionality of human milk by providing ingredients reflecting those of the latest human milk insights, such as prebiotics, probiotics and postbiotics. The aim of this study was to examine the effects of the supplementation with a postbiotic (Lactofidus TM ) and its combination with the prebiotics short-chain galactooligosaccharides (scGOS) and long-chain fructooligosaccharides (lcFOS) in a preclinical model of healthy suckling rats. Pups were supplemented daily with Lactofidus TM (POST group) and/or scGOS/lcFOS (P+P and PRE groups, respectively). Body weight and fecal consistency were analyzed. At the end of the study, immunoglobulin (Ig) profile, intestinal gene expression, microbiota composition and short chain fatty acid (SCFA) proportion were quantified. The supplementation with all nutritional interventions modulated the Ig profile, but the prebiotic mixture and the postbiotic induced differential effects: whereas scGOS/lcFOS induced softer feces and modulated microbiota composition and SCFA profile, Lactofidus™ upregulated Toll-like receptors gene expression. The use of the combination of scGOS/lcFOS and Lactofidus™ showed the effects observed for the oligosaccharides separately, as well as showing a synergistic impact on animal growth. Thus, the combined use of both products seems to be a good strategy to modulate immune and microbial features in early life.

Breast milk components contribute to the infant's immune development and protection, and among other immune factors, immunoglobulins (Igs) are the most studied. The presence of IgA in milk has been known for a long time; however, less information is available about the presence of other Igs such as IgM, IgG, and their subtypes (IgG1, IgG2, IgG3, and IgG4) or even IgE or IgD. The total Ig concentration and profile will change during the course of lactation; however, there is a great variability among studies due to several variables that limit establishing a clear pattern. In this context, the aim of this review was firstly to shed light on the Ig concentration in breast milk based on scientific evidence and secondly to study the main factors contributing to such variability. A search strategy provided only 75 studies with the prespecified eligibility criteria. The concentrations and proportions found have been established based on the intrinsic factors of the study-such as the sampling time and quantification technique-as well as participant-dependent factors, such as lifestyle and environment. All these factors contribute to the variability of the immunoglobulinome described in the literature and should be carefully addressed for further well-designed studies and data interpretation.

The composition of bioactive factors with immune activity in human breast milk is widely studied. However, the knowledge on rat milk immune factors during the whole lactation period is still scarce. This study aimed to analyze rat breast milk's immunoglobulin (Ig) content and some critical adipokines and growth factors throughout the lactation period, and to assess relationships with corresponding plasma levels. During lactation, milk concentration of the transforming growth factor (TGF)-β2 and -β3 showed a punctual increase in the first week, whereas adiponectin and leptin remained stable. In the second period of lactation (d14-21), despite the increase in the milk epidermal growth factor (EGF), a decrease in fibroblast growth factor 21 (FGF21) was detected at day 21. Milk IgA concentration had a progressive increase during lactation, while no significant changes were found in IgM and IgG. Regarding plasma levels, a decrease in all studied adipokines was observed in the second period of lactation, with the exception of IgA and TGF-β1, which reached their highest values at the end of the study. A positive correlation in IgM, IgG, and adipokine concentration was detected between milk and plasma compartments. In summary, the changes in the pattern of these bioactive compounds in rat milk and plasma and their relationships during lactation are established.

Flavonoids are attracting increasing attention due to their antioxidant, cardioprotective, and immunomodulatory properties. Nevertheless, little is known about their role in exercise performance in association with immune function. This systematic review firstly aimed to shed light on the ergogenic potential of flavonoids. A search strategy was run using SCOPUS database. The returned studies were screened by prespecified eligibility criteria, including intervention lasting at least one week and performance objectively quantified, among others. Fifty-one studies (54 articles) met the inclusion criteria, involving 1288 human subjects, either physically untrained or trained. Secondly, we aimed to associate these studies with the immune system status. Seventeen of the selected studies (18 articles) assessed changes in the immune system. The overall percentage of studies reporting an improved exercise performance following flavonoid supplementation was 37%, the proportion being 25% when considering quercetin, 28% for flavanol-enriched extracts, and 54% for anthocyanins-enriched extracts. From the studies reporting an enhanced performance, only two, using anthocyanin supplements, focused on the immune system and found certain anti-inflammatory effects of these flavonoids. These results suggest that flavonoids, especially anthocyanins, may exert beneficial effects for athletes' performances, although further studies are encouraged to establish the optimal dosage and to clarify their impact on immune status.

Exhausting exercise can disturb immune and gastrointestinal functions. Nevertheless, the impact of it on mucosal-associated lymphoid tissue has not been studied in depth. Here, we aim to establish the effects of an intensive training and exhausting exercise on the mucosal immunity of rats and to approach the mechanisms involved. Rats were submitted to a high-intensity training consisting of running in a treadmill 5 days per week for 5 weeks, involving 2 weekly exhaustion tests. At the end, samples were obtained before (T), immediately after (TE) and 24 h after (TE24) an additional final exhaustion test. The training programme reduced the salivary production of immunoglobulin A, impaired the tight junction proteins' gene expression and modified the mesenteric lymph node lymphocyte composition and function, increasing the ratio between Tαβ+ and B lymphocytes, reducing their proliferation capacity and enhancing their interferon-γ secretion. As a consequence of the final exhaustion test, the caecal IgA content increased, while it impaired the gut zonula occludens expression and enhanced the interleukin-2 and interferon-γ secretion. Our results indicate that intensive training for 5 weeks followed or not by an additional exhaustion disrupts the mucosal-associated lymphoid tissue and the intestinal epithelial barrier integrity in rats.

Cocoa contains bioactive components, which vary according to genetic and environmental factors. The present study aimed to ascertain the anti-allergic properties of native Peruvian cocoa populations ("Blanco de Piura" or BPC, "Amazonas Peru" or APC, "Criollo de Montaña" or CMC, "Chuncho" or CCC, and an ordinary cocoa or OC). To do so, after an initial in vitro approach, an in vivo study focused on the induction of an anaphylactic response associated with allergic asthma in Brown Norway rats was carried out. Based on their polyphenol content, antioxidant activity and in vitro effects, the APC and CMC were selected to be included in the in vivo study. Cocoa diets were tested in a model of allergic asthma in which anaphylactic response was assessed by changes in body temperature, motor activity and body weight. The concentration of specific immunoglobulin E (IgE), mast cell protease and leukotrienes was also quantified in serum and/or bronchoalveolar lavage fluid. CMC and OC populations exhibited a protective effect on the allergic asthma rat model as evidenced by means of a partial protection against anaphylactic response and, above all, in the synthesis of IgE and the release of mast cell protease.

The study's objective was to ascertain whether a nutritional multivitamin and mineral supplement enriched with two different dietary fibers influences microbiota composition, mineral absorption, and some immune and metabolic biomarkers in adult rats. Nine-week-old Wistar rats were randomly assigned into four groups: the reference group; the group receiving a daily supplement based on a food matrix with proteins, vitamins, and minerals; and two other groups receiving this supplement enriched with inulin (V + I) or acacia (V + A) fiber for four weeks. Microbiota composition was determined in cecal content and mineral content in fecal, blood, and femur samples. Intestinal IgA concentration, hematological, and biochemical variables were evaluated. Both V + I and V + A supplementations increased Firmicutes and Actinobacteria phyla, which were associated with a higher presence of Lactobacillus and Bifidobacterium spp. V + A supplementation increased calcium, magnesium, phosphorus, and zinc concentrations in femur. V + I supplementation increased the fecal IgA content and reduced plasma total cholesterol and uric acid concentration. Both fiber-enriched supplements tested herein seem to be beneficial to gut-health, although differently.

Rotaviruses (RVs) are the leading pathogens causing severe and acute diarrhea in children and animals. It is well known that sex contributes to shaping immune responses, thus it could also influence the incidence and severity of the RV infection. The aim of this study was to analyze the influence of sexual dimorphism on RV infection and its antibody (Ab) immune response in a suckling rat model. Neonatal suckling rats were intragastrically RV-inoculated and clinical indexes derived from fecal samples, as well as immune variables were evaluated. Higher severity of diarrhea, fecal weight and viral elimination were observed in males compared to females ( p < 0.05). Regarding the adaptative immunity, the RV shaped the immune response to lower IgG1 levels and an increased Th1/Th2-associated Ab response ( p < 0.05). Although females had lower IgG2a levels than males ( p < 0.05), the specific anti-RV antibody levels were not sex influenced. In fact, at this age the passive transfer of anti-RV antibodies through breast milk was the critical factor for clustering animals, independently of their sex. It can be concluded that male and female diarrhea severity in RV infection is slightly influenced by sexual dimorphism and is not associated with the specific immune response against the virus.

Breast milk is a rich fluid containing bioactive compounds such as specific growth factors (GF) that contribute to maturation of the immune system in early life. The aim of this study was to determine whether transforming growth factor-β2 (TGF-β2), epidermal growth factor (EGF) and fibroblast growth factor 21 (FGF21), compounds present in breast milk, could promote systemic immune maturation. For this purpose, newborn Wistar rats were daily supplemented with these GF by oral gavage during the suckling period (21 days of life). At day 14 and 21 of life, plasma for immunoglobulin (Ig) quantification was obtained and spleen lymphocytes were isolated, immunophenotyped and cultured to evaluate their ability to proliferate and release cytokines. The main result was obtained at day 14, when supplementation with EGF increased B cell proportion to reach levels observed at day 21. At the end of the suckling period, all GF increased the plasma levels of IgG1 and IgG2a isotypes, FGF21 balanced the Th1/Th2 cytokine response and both EGF and FGF21 modified splenic lymphocyte composition. These results suggested that the studied milk bioactive factors, mainly EGF and FGF21, may have modulatory roles in the systemic immune responses in early life, although their physiological roles remain to be established.

Allergic asthma is one of the most common chronic diseases of the airways, however it still remains underdiagnosed and hence undertreated. Therefore, an allergic asthma rat model would be useful to be applied in future therapeutic strategy studies. The aim of the present study was to develop an objective model of allergic asthma in atopic rats that allows the induction and quantification of anaphylactic shock with quantitative variables. Female Brown Norway rats were intraperitoneally sensitized with ovalbumin (OVA), alum and Bordetella pertussis toxin and boosted a week later with OVA in alum. At day 28, all rats received an intranasal challenge with OVA. Anaphylactic response was accurately assessed by changes in motor activity and body temperature. Leukotriene concentration was determined in the bronchoalveolar lavage fluid (BALF), and total and IgE anti-OVA antibodies were quantified in blood and BALF samples. The asthmatic animals' motility and body temperature were reduced after the shock for at least 20 h. The asthmatic animals developed anti-OVA IgE antibodies both in BALF and in serum. These results show an effective and relatively rapid model of allergic asthma in female Brown Norway rats that allows the quantification of the anaphylactic response.

Intensive training and exhausting exercise can disrupt innate and acquired immunity. The flavanone hesperidin has shown immunomodulatory properties in physiological and some pathological conditions, and positive effects on exercise-induced oxidative stress. Nevertheless, it remains uncertain whether it also prevents exhausting exercise-induced immune alterations. The aim of this study was to establish the effect of oral hesperidin supplementation on the systemic immune system in rats following an intensive training and exhausting exercise. For this purpose, female Wistar rats were randomized into an intensive training group or a sedentary group. Intensive training was induced by running in a treadmill 5 days per week (including two exhausting tests) for five weeks. Throughout the training period, 200 mg/kg of hesperidin or vehicle was administered by oral gavage three times per week. At the end, blood, thymus, spleen and macrophages were collected before, immediately after and 24 h after an additional final exhaustion test. Hesperidin supplementation enhanced natural killer cell cytotoxicity and the proportion of phagocytic monocytes, attenuated the secretion of cytokines by stimulated macrophages, prevented the leukocytosis induced by exhaustion and increased the proportion of T helper cells in the thymus, blood and spleen. These results suggest that hesperidin can prevent exhausting exercise-induced immune alterations.

It is known that intensive physical activity alters the immune system's functionality. However, the influence of the intensity and duration of exercise needs to be studied in more depth. We aimed to establish the changes in the innate immune response induced by two programmes of intensive training in rats compared to sedentary rats. A short training programme included 2 weeks of intensive training, ending with an exhaustion test (short training with exhaustion, S-TE). A second training programme comprised 5-week training including two exhaustion tests and three trainings per week. In this case, immune status was assessed before (T), immediately after (TE) and 24 h after (TE24) an additional final exhaustion test. Biomarkers such as phagocytic activity, macrophage cytokine and reactive oxygen species (ROS) production, and natural killer (NK) cell activity were quantified. S-TE was not enough to induce changes in the assessed innate immunity biomarkers. However, the second training was accompanied by a decrease in the phagocytic activity, changes in the pattern of cytokine secretion and ROS production by macrophages and reduced NK cell proportion but increased NK cytotoxic activity. In conclusion, a 5-week intense training programme, but not a shorter training, induced alterations in the innate immune system functionality.

Purpose: Cocoa intake has been associated with health benefits, improving cardiovascular function and metabolism, as well as modulating intestinal immune function. The aim of this study was to take an in-depth look into the mechanisms affected by the cocoa intake by evaluating the colonic gene expression after nutritional intervention, and to ascertain the role of the fiber of cocoa in these effects., Methods: To achieve this, Wistar rats were fed for 3 weeks with either a reference diet, a diet containing 10 % cocoa (C10), a diet based on cocoa fiber (CF) or a diet containing inulin (I). At the end of the study, colon was excised to obtain the RNA to evaluate the differential gene expression by microarray. Results were validated by RT-PCR., Results: The C10 group was the group with most changes in colonic gene expression, most of them down-regulated but a few in common with the CF diet. The C10 diet significantly up-regulated the expression of Scgb1a1 and Scnn1 g and down-regulated Tac4, Mcpt2, Fcer1a and Fabp1 by twofold, most of them related to lipid metabolism and immune function. The CF and I diets down-regulated the expression of Serpina10 and Apoa4 by twofold. Similar patterns of expression were found by PCR., Conclusion: Most of the effects attributed to cocoa consumption on genes related to the immune system (B cell and mast cell functionality) and lipid metabolism in the colon tissue were due not only to its fiber content, but also to the possible contribution of polyphenols and other compounds.

Objective: Cocoa has been highlighted as a food with potential benefits to human health because of its polyphenol content. However, few studies show the contribution of cocoa and chocolate products in polyphenol intake. The aim of this work was to develop a food frequency questionnaire (FFQ) for evaluating the intake of food products containing cocoa (C-FFQ)., Methods: A sample of 50 university students was recruited to complete the 90-item questionnaire, a validated questionnaire (called here European Food Safety Authority [EFSA]-Q) as well as a 24-hour dietary recall (24 HDR). Spearman correlation test, Bland-Altman plots, and quintile classification analysis were conducted together with the Wilcoxon test and descriptive statistics., Results: Significant correlations between the C-FFQ and the EFSA-Q for the most common cocoa/chocolate products were observed (P < 0.05), as well as between data from the C-FFQ and 24 HDR (P < 0.05). However, a number of cocoa/chocolate products frequently consumed by the participants were detected by the C-FFQ and 24 HDR which were not included in the EFSA-Q. According to the C-FFQ, chocolate bars were the main source of cocoa in university students, but dairy products also provided an important amount of cocoa., Conclusion: The developed C-FFQ questionnaire can be considered as a valid option for assessing the consumption frequency of cocoa/chocolate-derived products, thereby allowing the evaluation of cocoa polyphenol intake in further studies., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Interaction between host cells and microbes is known as crosstalk. Among other mechanisms, this takes place when certain molecules of the micro-organisms are recognized by the toll-like receptors (TLRs) in the body cells, mainly in the intestinal epithelial cells and in the immune cells. TLRs belong to the pattern-recognition receptors and represent the first line of defense against pathogens, playing a pivotal role in both innate and adaptive immunity. Dysregulation in the activity of such receptors can lead to the development of chronic and severe inflammation as well as immunological disorders. Among components present in the diet, flavonoids have been suggested as antioxidant dietary factors able to modulate TLR-mediated signaling pathways. This review focuses on the molecular targets involved in the modulatory action of flavonoids on TLR-mediated signaling pathways, providing an overview of the mechanisms involved in such action. Particular flavonoids have been able to modify the composition of the microbiota, to modulate TLR gene and protein expression, and to regulate the downstream signaling molecules involved in the TLR pathway. These synergistic mechanisms suggest the role of some flavonoids in the preventive effect on certain chronic diseases.

Flavonoids are secondary products of plants that include thousands of compounds classified in several classes. Preclinical studies mainly carried out in rodents suggest that they may have a role in the prevention of immunoglobulin E (IgE) synthesis and mast cell degranulation. Interestingly, using animal models with allergic asthma, it can be concluded that preventive treatment with particular flavonoid classes can reduce airway hyperresponsiveness, which is accompanied by lowered inflammatory mediators such as histamine and cytokines, and cell infiltration. In addition, there are some clinical trials in patients with allergic asthma or rhinitis that offer promising results with regard to these natural compounds. On the other hand, the dissection of cellular mechanisms that have interacted with flavonoids allow their effectiveness to be understood. Among these mechanisms there is a lower expression of IgE receptor or other membrane receptors, the modulation of calcium influx, and the downregulation of particular signaling pathways that eventually produces lower primary and secondary mediator release. In conclusion, some particular flavonoids could be an alternative or complementary therapy in the prevention and treatment of some allergies. Nevertheless, an increased number of clinical trials is required in order to confirm the therapeutic role of flavonoids.

Previous studies have reported that a diet containing 10% cocoa, a rich source of flavonoids, has immunomodulatory effects on rats and, among others effects, is able to attenuate the immunoglobulin (Ig) synthesis in both systemic and intestinal compartments. The purpose of the present study was focused on investigating whether these effects were attributed exclusively to the flavonoid content or to other compounds present in cocoa. To this end, eight-week-old Lewis rats were fed, for two weeks, either a standard diet or three isoenergetic diets containing increasing proportions of cocoa flavonoids from different sources: one with 0.2% polyphenols from conventional defatted cocoa, and two others with 0.4% and 0.8% polyphenols, respectively, from non-fermented cocoa. Diet intake and body weight were monitored and fecal samples were obtained throughout the study to determine fecal pH, IgA, bacteria proportions, and IgA-coated bacteria. Moreover, IgG and IgM concentrations in serum samples collected during the study were quantified. At the end of the dietary intervention no clear changes of serum IgG or IgM concentrations were quantified, showing few effects of cocoa polyphenol diets at the systemic level. However, in the intestine, all cocoa polyphenol-enriched diets attenuated the age-related increase of both fecal IgA and IgA-coated bacteria, as well as the proportion of bacteria in feces. As these effects were not dependent on the dose of polyphenol present in the diets, other compounds and/or the precise polyphenol composition present in cocoa raw material used for the diets could be key factors in this effect.

Scope: Previous studies have shown that a highly enriched cocoa diet affects both intestinal and systemic immune function in young rats. The aim of this study was to elucidate whether diets containing lower amounts of cocoa could also influence the systemic and intestinal humoral immune response., Methods and Results: Fecal and serum samples were collected during the study and, at the end, intestinal washes were obtained and mesenteric lymph nodes and small-intestine walls were excised for gene expression assessment. IgA, IgM, IgG1, IgG2a, IgG2b and IgG2c concentrations were quantified in serum whereas S-IgA and S-IgM were determined in feces and intestinal washes. Animals receiving 5 and 10% cocoa for 3 wk showed no age-related increase in serum IgG1 and IgG2a concentrations, and IgG2a values were significantly lower than those in reference animals. Serum IgM was also decreased by the 10% cocoa diet. The 5 and 10% cocoa diets dramatically reduced intestinal S-IgA concentration and modified the expression of several genes involved in IgA synthesis. A diet containing 2% cocoa had no effect on most of the studied variables., Conclusion: The results demonstrate the downregulatory effect of a 5% or higher cocoa diet on the systemic and intestinal humoral immune response in adult rats., (Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Commensal bacteria, including some species of lactobacilli commonly present in human breast milk, appear to colonize the neonatal gut and contribute to protection against infant infections, suggesting that lactobacilli could potentially modulate immunity. In this study, we evaluated the potential of two Lactobacillus strains isolated from human milk to modulate the activation and cytokine profile of peripheral blood mononuclear cell (PBMC) subsets in vitro. Moreover, these effects were compared to the same probiotic species of non-milk origin. Lactobacillus salivarius CECT5713 and Lactobacillus fermentum CECT5716 at 10⁵, 10⁶ and 10⁷ bacteria/mL were co-cultured with PBMC (10⁶/mL) from 8 healthy donors for 24 h. Activation status (CD69 and CD25 expressions) of natural killer (NK) cells (CD56+), total T cells (CD3+), cytotoxic T cells (CD8+) and CD4+ T cells was determined by flow cytometry. Regulatory T cells (Treg) were also quantified by intracellular Foxp3 evaluation. Regarding innate immunity, NK cells were activated by addition of both Lactobacillus strains, and in particular, the CD8+ NK subset was preferentially induced to highly express CD69 (~90%, p<0.05). With respect to acquired immunity, approximately 9% of CD8+ T cells became activated after co-cultivation with L. fermentum or L salivarius. Although CD4+ T cells demonstrated a weaker response, there was a preferential activation of Treg cells (CD4+CD25+Foxp3+) after exposure to both milk probiotic bacteria (p<0.05). Both strains significantly induced the production of a number of cytokines and chemokines, including TNFα, IL-1β, IL-8, MIP-1α, MIP-1β, and GM-CSF, but some strain-specific effects were apparent. This work demonstrates that L salivarius CECT5713 and L. fermentum CECT5716 enhanced both natural and acquired immune responses, as evidenced by the activation of NK and T cell subsets and the expansion of Treg cells, as well as the induction of a broad array of cytokines., (Copyright © 2010 Elsevier GmbH. All rights reserved.)

Background: Cocoa is a rich source of flavonoids that, among other functions, can act as antioxidants. In living systems, the production of reactive oxygen species (ROS) activate an array of intracellular cascades, including mitogen-activated protein kinases (MAPK), that are closely associated with cell death or survival pathways., Aim of the Study: To ascertain the role of a cocoa extract and its main flavonoid, (-)-epicatechin, in an in vitro model of oxidative stress induced in a neuronal cell line., Methods: We analyzed ROS production by fluorometry (dichlorofluorescein assay), and activation of MAPK pathways including extracellular signal-regulated kinases 1/2 (ERK 1/2), c-Jun N-terminal kinase (JNK), and p-38, by Western blot analysis., Results: Cells incubated with cocoa extract or (-)-epicatechin, reduced ROS production in a dose-dependent manner, reaching 35% inhibition. pJNK and p38, involved in apoptosis, were down-modulated by cocoa extract and (-)-epicatechin with p38 inhibition reaching up to 70%., Conclusions: Our results show that cocoa extract and (-)-epicatechin may exert a neuroprotective action by reducing ROS production and modulating MAPK activation.

Objective: To determine the immunomodulatory effects of the anti-rat CD28 monoclonal antibody (Mab) JJ316 on the onset of rat adjuvant arthritis (AA). JJ316 is a superagonistic Mab that induces polyclonal T cell proliferation in the absence of T cell receptor (TCR) ligation and promotes the expansion of regulatory T cells., Methods: Female Wistar rats in which AA was induced were treated with JJ316 on Day 0 and Day 9 postinduction. A parallel treatment with JJ319, a "conventional" CD28-specific Mab that costimulates anti-TCR triggered proliferation, was performed. Severity of arthritis was monitored by means of an arthritic score, and by recording hindpaw volume and body weight increases. Serum antibodies against the AA-inducing mycobacteria were also determined by ELISA. To ascertain the effect of JJ316 on T lymphocytes in vivo, blood CD4+CD45RChigh (Th1-like) and CD4+CD45RClow (Th2-like) cells were analyzed by flow cytometry, and the relative levels of interleukin 2 (IL-2), IL-10, and interferon-g (IFN-g) mRNA in synovial tissue were measured by real-time reverse transcription-polymerase chain reaction., Results: JJ316 efficiently prevented the inflammatory process of AA. This effect was associated with a specific decrease in the blood CD4+CD45RChigh/CD4+CD45RClow T cell ratio and high IL-10 mRNA expression in the synovia. In addition, anti-mycobacteria antibody levels decreased in JJ316 treated animals. In contrast, administration of the conventional anti-CD28 Mab JJ319 did not improve inflammation., Conclusion: JJ316, a stimulatory CD28-specific Mab known to promote Th2 function and the expansion of regulatory T cells, provides effective protection from AA.