A Multiscale Spatiotemporal Model Including a Switch from Aerobic to Anaerobic Metabolism Reproduces Succession in the Early Infant Gut Microbiota.

The human intestinal microbiota starts to form immediately after birth and is important for the health of the host. During the first days, facultatively anaerobic bacterial species generally dominate, such as Enterobacteriaceae. These are succeeded by strictly anaerobic species, particularly Bifidobacterium species. An early transition to Bifidobacterium species is associated with health benefits; for example, Bifidobacterium species repress growth of pathogenic competitors and modulate the immune response. Succession to Bifidobacterium is thought to be due to consumption of intracolonic oxygen present in newborns by facultative anaerobes, including Enterobacteriaceae. To study if oxygen depletion suffices for the transition to Bifidobacterium species, here we introduced a multiscale mathematical model that considers metabolism, spatial bacterial population dynamics, and cross-feeding. Using publicly available metabolic network data from the AGORA collection, the model simulates ab initio the competition of strictly and facultatively anaerobic species in a gut-like environment under the influence of lactose and oxygen. The model predicts that individual differences in intracolonic oxygen in newborn infants can explain the observed individual variation in succession to anaerobic species, in particular Bifidobacterium species. Bifidobacterium species became dominant in the model by their use of the bifid shunt, which allows Bifidobacterium to switch to suboptimal yield metabolism with fast growth at high lactose concentrations, as predicted here using flux balance analysis. The computational model thus allows us to test the internal plausibility of hypotheses for bacterial colonization and succession in the infant colon. IMPORTANCE The composition of the infant microbiota has a great impact on infant health, but its controlling factors are still incompletely understood. The frequently dominant anaerobic Bifidobacterium species benefit health, e.g., they can keep harmful competitors under control and modulate the intestinal immune response. Controlling factors could include nutritional composition and intestinal mucus composition, as well as environmental factors, such as antibiotics. We introduce a modeling framework of a metabolically realistic intestinal microbial ecology in which hypothetical scenarios can be tested and compared. We present simulations that suggest that greater levels of intraintestinal oxygenation more strongly delay the dominance of Bifidobacterium species, explaining the observed variety of microbial composition and demonstrating the use of the model for hypothesis generation. The framework allowed us to test a variety of controlling factors, including intestinal mixing and transit time. Future versions will also include detailed modeling of oligosaccharide and mucin metabolism.

Effect of bovine milk fat-based infant formulae on microbiota, metabolites and stool parameters in healthy term infants in a randomized, crossover, placebo-controlled trial.

BACKGROUND: Natural enrichment of sn-2 palmitate content of infant formulae by using bovine milk fat is known to reduce formation of faecal fatty acid soaps and to improve stool consistency, but effects on gut microbiota composition are unknown. The purpose of this study was to test the influence of milk fat-based formula high in sn-2 palmitate on the infants' gut microbiota composition and to confirm the beneficial effects of the formula on formation of faecal fatty acid soaps and stool consistency. METHODS: Twenty-two healthy term, formula-fed infants were enrolled in a single-blinded randomized, crossover, placebo-controlled trial. After a 2-week run-in period, infants received either a 50% milk fat-based formula containing 39% sn-2 palmitate (MF) or a vegetable fat-based formula (VF) containing 10% sn-2 palmitate in a 2 × 2-week crossover design. Faecal microbiota composition was the primary outcome of the study. Other outcomes included faecal fatty acid soap excretion, calcium excretion, gut comfort parameters and faecal metabolites. RESULTS: Microbiota analysis showed that bifidobacteria dominated the gut microbiota of most infants. Neither alpha- nor beta-diversity was significantly influenced by the intervention. Also, abundance of metabolic pathways was independent of the intervention. The MF formula resulted in significantly lower faecal levels of palmitic acid soap (p = 0.0002) and total fatty acid soaps (p = 0.0001) than the VF formula. Additionally, calcium excretion and palmitic acid concentration were significantly (p = 0.0335) lower in stool samples after MF intervention. Furthermore, a significant physiological effect on softer stools was observed in the MF intervention compared to the VF intervention (p = 0.02). Of the 870 measured faecal metabolites, 190 were significantly different after MF and VF intervention (FDR corrected p < 0.05). Most of these were found at higher levels after MF intervention, potentially indicative of the complex structure of milk fat. Metabolites with more than twofold change between interventions were mostly lipid-derived and included several milk fat-specific fatty acids. CONCLUSIONS: Replacing part of the vegetable fat in infant formula with bovine milk fat with high sn-2 palmitate levels did not change the microbiota composition, although a reduction in faecal palmitate soaps, total fatty acid soaps and calcium excretion while improving stool consistency in the MF intervention was confirmed. In addition, 190 faecal metabolites were significantly different, many related to the fat source. TRIAL REGISTRATION: Netherlands Trial Registry Identifier: NL7815 19/06/2019.

Dietary Vitamin D Supplementation Is Ineffective in Preventing Murine Cow's Milk Allergy, Irrespective of the Presence of Nondigestible Oligosaccharides.

INTRODUCTION: Cow's milk allergy (CMA) is one of the most common food allergies especially early in life. A mixture of nondigestible short-chain galacto-oligosaccharides, long-chain fructo-oligosaccharides, and pectin-derived acidic-oligosaccharides (GFA) may reduce allergy development and allergic symptoms in murine CMA. Recently, vitamin D (VitD) has been suggested to have beneficial effects in reducing allergy as well. OBJECTIVE: In this study, the immune modulatory effect on allergy prevention using the combination of GFA and VitD was investigated. METHODS: Female C3H/HeOuJ mice were fed a control or GFA-containing diet with depleted, standard (1,000 IU/kg), or supplemented (5,000 IU/kg) VitD content for 2 weeks before and during whey sensitization (n = 10-15). Mice were sensitized 5 times intragastrically with PBS as a control, whey as cow's milk allergen, and/or cholera toxin as adjuvant on a weekly interval. One week after the last sensitization, mice were intradermally challenged in both ear pinnae and orally with whey, subsequently the acute allergic skin response and shock symptoms were measured. After 18 h, terminal blood samples, mesenteric lymph nodes, and spleens were collected. Whey-specific immunoglobulin (Ig) E and IgG1 levels were measured by means of ELISA. T cell subsets and dendritic cells (DCs) were studied using flow cytometry. RESULTS: Additional VitD supplementation did not lower the allergic symptoms compared to the standard VitD diet. CMA mice fed the GFA diet supplemented with VitD (GFA VitD+) significantly decreased the acute allergic skin response of whey sensitized mice when compared to the CMA mice fed VitD (VitD+) group (p < 0.05). The effect of GFA was not improved by extra VitD supplementation even though the CMA mice fed the GFA VitD+ diet had a significantly increased percentage of CD103+ DCs compared to the VitD+ group (p < 0.05). The VitD-deprived mice showed a high percentage of severe shock and many reached the humane endpoint; therefore, these groups were not further analyzed. CONCLUSIONS: High-dose VitD supplementation in mice does not protect against CMA development in the presence or absence of GFA.

Development and validation of bioengineered intestinal tubules for translational research aimed at safety and efficacy testing of drugs and nutrients.

Currently used intestinal cell models have limited translational value, therefore, development of novel in vitro intestinal models that recapitulate the human in vivo setting more closely are of interest. Here, an advanced intestinal model was developed by the incorporation of physiological parameters, such as extracellular matrix (ECM) elements and shear stress, to cultured Caco-2 cells in a 3-dimensional environment. Caco-2 cells grown on ECM-coated hollow fiber membranes (HFM) under physiological shear stress show an improved phenotype, as demonstrated by the presence of enterocytes, goblet, Paneth, enteroendocrine and stem cells. Additionally, this model showed signs of an improved morphology due to the appearance of villi-like structures. Similar to epithelial cells grown on Transwells™, the current model remains easy to use, cost efficient and allows apical and basolateral access. The bioengineered intestinal tubule was validated by exposure to Clostridium difficile toxin A, the leading cause of healthcare-associated diarrhea. The loss of the tight junction network was supported by an increase in inulin-FITC leakage and the number of goblet cells increased, in agreement with clinical findings. In addition to toxicity screening, the bioengineered intestinal tubules are considered useful for drug and nutrient safety and efficacy testing.

The Combination Therapy of Dietary Galacto-Oligosaccharides With Budesonide Reduces Pulmonary Th2 Driving Mediators and Mast Cell Degranulation in a Murine Model of House Dust Mite Induced Asthma.

Background: Dietary non-digestible galacto-oligosaccharides (GOS) suppress allergic responses in mice sensitized and challenged with house dust mite (HDM). Budesonide is the standard therapy for allergic asthma in humans but is not always completely effective. Aim: To compare the efficacy of budesonide or different doses of GOS alone or with a combination therapy of budesonide and GOS on HDM-allergic responses in mice. Methods:BALB/c mice were sensitized and challenged with HDM, while fed a control diet or a diet supplemented with 1 or 2.5 w/w% GOS, and either or not oropharyngeally instilled with budesonide. Systemic and local inflammatory markers, such as mucosal mast cell protease-1 (mMCP-1) in serum, pulmonary CCL17, CCL22, and IL-33 concentrations and inflammatory cell influx in the bronchoalveolar lavage fluid (BALF) were determined. Results: Budesonide or GOS alone suppressed the number of eosinophils in the BALF of HDM allergic mice whereas budesonide either or not combined with GOS lowered both eosinophil and lymphocyte numbers in the BALF of HDM-allergic mice. Both 1 w/w% and 2.5 w/w% GOS and/or budesonide suppressed serum mMCP-1 concentrations. However, budesonide nor GOS alone was capable of reducing Th2 driving chemokines CCL17, CCL22 and IL-33 protein levels in supernatants of lung homogenates of HDM allergic mice, whereas the combination therapy did. Moreover, IL-13 concentrations were only significantly suppressed in mice treated with budesonide while fed GOS. A similar tendency was observed for the frequency of GATA3+CD4+ Th2 and CD4+RORγt+ Th17 cells in the lungs of the allergic mice. Conclusion: Dietary intervention using GOS may be a novel way to further improve the efficacy of anti-inflammatory drug therapy in allergic asthma by lowering Th2 driving mediators and mast cell degranulation.

IL-10 Receptor or TGF-ß Neutralization Abrogates the Protective Effect of a Specific Nondigestible Oligosaccharide Mixture in Cow-Milk-Allergic Mice.

Background: Dietary nondigestible, short-chain galacto-, long-chain fructo-, and pectin-derived acidic oligosaccharides (GFAs) lower the effector response in cow-milk-allergic (CMA) mice; and forkhead box P3 (Foxp3)-positive regulatory T cells (Tregs) were shown to contribute to this. Objective: The aim of this study was to assess the contribution of interleukin 10 (IL-10) and transforming growth factor ß (TGF-ß) to the protective effect of the GFA diet in CMA mice. Methods: Female C3H/HeOuJ mice, 3-4 wk old, were orally sensitized with cholera toxin (Sham) or whey and cholera toxin (Whey) 1 time/wk for 5 consecutive weeks and challenged with whey 1 wk later. The mice were fed a control or 1% GFA (9:2:1) (Whey+GFA) diet starting 2 wk before the first sensitization. In a second experiment, the mice were also injected with αIL-10 receptor (αIL-10r), αTGF-ß, or isotype control antibodies 24 h before each sensitization. The acute allergic skin response, anaphylaxis score, whey-specific IgE, mucosal mast cell protease 1 (mMCP-1), and Treg frequency in the mesenteric lymph nodes (MLNs) and intestinal Foxp3, Il10, and Tgfb mRNA expression were determined. Results: In Whey+GFA mice, intestinal Il10, Tgfb, or Foxp3 mRNA expression was 2-10 times higher (P < 0.05) and the MLN Treg frequency was 25% higher compared with Whey mice (P < 0.05). The acute allergic skin response was 50% lower in Whey+GFA mice compared with Whey mice (P < 0.01), and IL-10 receptor (IL-10r) or TGF-ß neutralizing antibodies prevented this protective effect (P < 0.001). The Whey mice had higher serum mMCP-1 concentrations and whey-immunoglobulin E (-IgE) levels than Sham mice (P < 0.01), whereas these were not higher in Whey+GFA mice, and neutralizing antibodies partially interfered with these responses. Conclusions: Dietary GFAs enhance the Treg frequency in the MLNs and mucosal IL-10 and TGF-ß transcription while suppressing the allergic effector response. Neutralizing antibodies showed that the allergy-protective effect of the GFA diet was mediated by IL-10 and TGF-ß in CMA mice.

Identification of peptides with tolerogenic potential in a hydrolysed whey-based infant formula.

BACKGROUND: Failure to induce oral tolerance may result in food allergy. Hydrolysed cow's milk-based infant formulas are recommended in subjects with a high risk of developing allergic disease. Presentation of T cell epitopes is a prerequisite to generate regulatory T cells that could contribute to oral tolerance. OBJECTIVE: To investigate whether a specific hydrolysed whey-based infant formula contains peptides that function as T cell epitopes to support the development of oral tolerance to whey. METHODS: First, a novel liquid chromatography-mass spectrometry (LC-MS) method was developed to characterize ß-lactoglobulin-derived peptides present in a specific infant formula with a focus on region AA#13-48 of ß-lactoglobulin, which has previously been described to contain T cell epitopes with tolerogenic potential. Second, the formula was subjected to the ProImmune ProPresent® antigen presentation assay and MHC class II binding algorithm to identify relevant HLA-DRB1-restricted peptides. Third, identified peptides were tested on human cow's milk protein-specific T cell lines to determine T cell recognition. RESULTS: Thirteen peptides of minimal 9AAs long that overlap with AA#13-48 of ß-lactoglobulin were identified. Six of them were found across all batches analysed. It was further confirmed that these peptides were processed and presented by human dendritic cells. The identified HLA-DRB1-restricted peptides were correlated to AA#11-30 and AA#23-39 of ß-lactoglobulin. Importantly, the proliferation assay showed that the synthetic peptides were recognized by cow's milk protein-specific T cell lines and induced T cell proliferation. CONCLUSION AND CLINICAL RELEVANCE: This study demonstrates that the tested hydrolysed infant formula contains functional HLA-DRB1-restricted T cell epitopes, which can potentially support the development of oral tolerance to whey.

Evaluating Human Intestinal Cell Lines for Studying Dietary Protein Absorption.

With the global population rising, the need for sustainable and resource-efficiently produced proteins with nutritional and health promoting qualities has become urgent. Proteins are important macronutrients and are involved in most, if not all, biological processes in the human body. This review discusses these absorption mechanisms in the small intestine. To study intestinal transport and predict bioavailability, cell lines are widely applied as screening models and often concern Caco-2, HT-29, HT-29/MTX and T84 cells. Here, we provide an overview of the presence and activities of peptide- and amino acid transporters in these cell models. Further, inter-laboratory differences are discussed as well as the culture micro-environment, both of which may influence cell culture phenotype and performance. Finally, the value of new developments in the field, including culturing cells in 3-dimensional systems under shear stress (i.e., gut-on-chips), is highlighted. In particular, their suitability in screening novel food proteins and prediction of the nutritional quality needed for inclusion in the human diet of the future is addressed.

Oligosaccharides in Urine, Blood, and Feces of Piglets Fed Milk Replacer Containing Galacto-oligosaccharides.

Human milk oligosaccharides (HMOs) are absorbed into the blood (about 1% of the HMO intake) and subsequently excreted in urine, where they may protect the infant from pathogen infection. As dietary galacto-oligosaccharides (GOS) have partial structural similarities with HMOs, this study investigated the presence of GOS and oligosaccharides originating from milk replacer in blood serum, urine, and cecal and fecal samples of piglets, as a model for human infants. Using liquid chromatography-mass spectrometry and capillary electrophoresis with fluorescence detection, oligosaccharides originating from piglet diet including 3'-sialyllactose and specific GOS ranging from degree of polymerization 3 to 6 were detected in blood serum and in urine of piglets. In blood serum, GOS levels ranged from 16 to 23 µg/mL, representing about 0.1% of the GOS daily intake. In urine, approximately 0.85 g of GOS/g of creatinine was found. Cecum digesta and feces contained low amounts of oligosaccharides, suggesting an extensive GOS intestinal fermentation in piglets.

Human Milk Blocks DC-SIGN-Pathogen Interaction via MUC1.

Beneficial effects of breastfeeding are well-recognized and include both immediate neonatal protection against pathogens and long-term protection against allergies and autoimmune diseases. Although several proteins have been identified to have anti-viral or anti-bacterial effects like secretory IgA or lactoferrin, the mechanisms of immune modulation are not fully understood. Recent studies identified important beneficial effects of glycans in human milk, such as those expressed in oligosaccharides or on glycoproteins. Glycans are recognized by the carbohydrate receptors C-type lectins on dendritic cell (DC) and specific tissue macrophages, which exert important functions in immune modulation and immune homeostasis. A well-characterized C-type lectin is dendritic cell-specific intercellular adhesion molecule-3-grabbing non-integrin (DC-SIGN), which binds terminal fucose. The present study shows that in human milk, MUC1 is the major milk glycoprotein that binds to the lectin domain of DC-SIGN and prevents pathogen interaction through the presence of Lewis x-type oligosaccharides. Surprisingly, this was specific for human milk, as formula, bovine or camel milk did not show any presence of proteins that interacted with DC-SIGN. The expression of DC-SIGN is found in young infants along the entire gastrointestinal tract. Our data thus suggest the importance of human milk glycoproteins for blocking pathogen interaction to DC in young children. Moreover, a potential benefit of human milk later in life in shaping the infants immune system through DC-SIGN cannot be ruled out.

Mechanisms underlying immune effects of dietary oligosaccharides.

The WHO refers to human milk as the nutritional gold standard for term infants. Human milk contains many immunomodulatory compounds, including oligosaccharides. Human-milk oligosaccharides can serve as prebiotics because the nondigestible oligosaccharides present in human milk show a clear bifidogenic effect on the gut microbiota. Dietary oligosaccharide structures that have prebiotic effects similar to human-milk oligosaccharides include galacto-oligosaccharides, fructo-oligosaccharides, and pectin-derived acidic oligosaccharides. Both animal studies and human clinical trials showed that dietary intervention with these dietary oligosaccharides in early life could lead to the prevention of atopic dermatitis, food allergy, and allergic asthma. The immune-modulating effects of these oligosaccharides are likely assisted via alteration of the intestinal microbiota or in a microbiota-independent manner by direct interaction on immune cells or both. In this review, an overview of the prebiotic role of dietary oligosaccharides on the microbiota and the microbiota-independent immune modulation by these prebiotics is provided. In addition, recent publications that report on the pathways by which the oligosaccharides might exert their direct immunomodulatory effect are summarized.

Difficulties in describing allergic disease modulation by pre-, pro- and synbiotics.

The so-called hygiene hypothesis is, at least in part, accountable for the increase in allergic diseases in the developed countries. Although there is support for one of its primary predictions that host-microbe interactions in early life have longterm effects on the development of disease across populations, the theory has already proven to be imperfect as many more recent increases in certain diseases cannot be explained by the hygiene hypothesis. Nevertheless, many research groups are interested in the host-microbe interactions and are exploring the use of "live micro-organisms which, when administered in adequate amounts, confer a health benefit to the host" (probiotics) and "selectively fermented ingredients that result in specific changes, in the composition and/or activity of the gastrointestinal microbiota, thus conferring benefit(s) upon host health" (prebiotics) to reduce the allergic disease onset or clinical outcomes. As the definitions of pre- and probiotics by itself were already adapted after their original dictation, it is not surprising that producing generalistic conclusions on the effectiveness of pre-, pro and synbiotic intervention in allergic diseases is very challenging as large differences exist in used species, methodologies, prebiotic(s) (mixtures) and probiotic strains. In this review we elucidate on the hurdles in describing prebiotics, probiotics and the combination being synbiotics in allergic manifestations.

Food-derived oligosaccharides exhibit pharmaceutical properties.

Breast feeding is considered as the best nutrition for growth and development of an infant. Human milk consists of a unique combination of nutritional components each with different characteristics. Oligosaccharides or non-digestible carbohydrates as one of these components, are generally accepted to have a beneficial effect by selectively stimulating the growth and/or activity of one or a limited number of bacterial species. Recently more evidence is rising for direct effects of oligosaccharides on the immune system. Oligosaccharides often used as dietary supplements for their beneficial effects on the host and its immune system, are derived from nutritional sources. In this review we aim to summarize the pharmaceutical properties of these food-borne oligosaccharides early in life.

In vivo and in vitro evaluation of the residual allergenicity of partially hydrolysed infant formulas.

Hypoallergenic infant formulas are commonly used for genetically predisposed children and infants diagnosed with cow's milk allergy. This study describes both in vitro and in vivo approaches to assess residual allergenicity of partially hydrolysed infant formulas. Electrophoretic patterns indicated that ß-lactoglobulin and other whey proteins were largely degraded. For safety reasons, according to the European commission-guidelines, it is required that the sensitizing capacity of hypoallergenic formulas is tested in an animal model. In contrast to whey sensitization, no elevated levels of whey-specific IgE, anaphylactic reactions or drop in body temperature were observed in sensitized mice exposed to whey hydrolysates. This indicates that the whey hydrolysates lost their putative sensitizing capacity in a mouse model using oral sensitization, which is highly relevant in relation to the human situation. In combination with the lost capacity of hydrolysed infant formulas to cross-link human IgE antibodies on RBL-huFcɛRI in vitro, both the sensitization and the challenge phase of the allergic response were studied. This combination of assays is proposed as a strategy for the screening of new hypoallergenic formulas aimed at preventing sensitization in atopic children and avoiding clinical symptoms in infants suffering from cow's milk allergy.

Identification of the Mhc region as an asthma susceptibility locus in recombinant congenic mice.

Mouse models of allergic asthma are characterized by airway hyperreactivity (AHR), Th2-driven eosinophilic airway inflammation, high allergen-specific IgE (anti-OVA IgE) levels in serum, and airway remodeling. Because asthma susceptibility has a strong genetic component, we aimed to identify new asthma susceptibility genes in the mouse by analyzing the asthma phenotypes of the Leishmania major resistant (lmr) recombinant congenic (RC) strains. The lmr RC strains are derived from C57BL/6 and BALB/c intercrosses and carry congenic loci on chromosome 17 (lmr1) and 9 (lmr2) in both backgrounds. Whereas the lmr2 locus on chromosome 9 contributes to a small background-specific effect on anti-OVA IgE and AHR, the lmr1 locus on chromosome 17 mediates a strong effect on Th2-driven eosinophilic airway inflammation and background-specific effects on anti-OVA IgE and AHR. The lmr1 locus contains almost 600 polymorphic genes. To narrow down this number of candidate genes, we performed genome-wide transcriptional profiling on lung tissue from C.lmr1 RC mice and BALB/c control mice. We identified a small number of differentially expressed genes located within the congenic fragment, including a number of Mhc genes, polymorphic between BALB/c and C57Bl/6. The analysis of asthma phenotypes in the C.B10-H2b RC strain, carrying the C57Bl/6 haplotype of the Mhc locus in a BALB/c genetic background, reveals a strikingly similar asthma phenotype compared with C.lmr1, indicating that the differentially expressed genes located within the C.B10-H2b congenic fragment are the most likely candidate genes to contribute to the reduced asthma phenotypes associated with the C57Bl/6 allele of lmr1.

Effect of nutrient deficiencies on in vitro Th1 and Th2 cytokine response of peripheral blood mononuclear cells to Plasmodium falciparum infection.

BACKGROUND: An appropriate balance between pro-inflammatory and anti-inflammatory cytokines that mediate innate and adaptive immune responses is required for effective protection against human malaria and to avoid immunopathology. In malaria endemic countries, this immunological balance may be influenced by micronutrient deficiencies. METHODS: Peripheral blood mononuclear cells from Tanzanian preschool children were stimulated in vitro with Plasmodium falciparum-parasitized red blood cells to determine T-cell responses to malaria under different conditions of nutrient deficiencies and malaria status. RESULTS: The data obtained indicate that zinc deficiency is associated with an increase in TNF response by 37%; 95% CI: 14% to 118% and IFN-gamma response by 74%; 95% CI: 24% to 297%. Magnesium deficiency, on the other hand, was associated with an increase in production of IL-13 by 80%; 95% CI: 31% to 371% and a reduction in IFN-gamma production. These results reflect a shift in cytokine profile to a more type I cytokine profile and cell-cell mediated responses in zinc deficiency and a type II response in magnesium deficiency. The data also reveal a non-specific decrease in cytokine production in children due to iron deficiency anaemia that is largely associated with malaria infection status. CONCLUSIONS: The pathological sequels of malaria potentially depend more on the balance between type I and type II cytokine responses than on absolute suppression of these cytokines and this balance may be influenced by a combination of micronutrient deficiencies and malaria status.

Alterations in early cytokine-mediated immune responses to Plasmodium falciparum infection in Tanzanian children with mineral element deficiencies: a cross-sectional survey.

BACKGROUND: Deficiencies in vitamins and mineral elements are important causes of morbidity in developing countries, possibly because they lead to defective immune responses to infection. The aim of the study was to assess the effects of mineral element deficiencies on early innate cytokine responses to Plasmodium falciparum malaria. METHODS: Peripheral blood mononuclear cells from 304 Tanzanian children aged 6-72 months were stimulated with P. falciparum-parasitized erythrocytes obtained from in vitro cultures. RESULTS: The results showed a significant increase by 74% in geometric mean of TNF production in malaria-infected individuals with zinc deficiency (11% to 240%; 95% CI). Iron deficiency anaemia was associated with increased TNF production in infected individuals and overall with increased IL-10 production, while magnesium deficiency induced increased production of IL-10 by 46% (13% to 144%) in uninfected donors. All donors showed a response towards IL-1beta production, drawing special attention for its possible protective role in early innate immune responses to malaria. CONCLUSIONS: In view of these results, the findings show plasticity in cytokine profiles of mononuclear cells reacting to malaria infection under conditions of different micronutrient deficiencies. These findings lay the foundations for future inclusion of a combination of precisely selected set of micronutrients rather than single nutrients as part of malaria vaccine intervention programmes in endemic countries.

T cell responses in fresh and cryopreserved peripheral blood mononuclear cells: kinetics of cell viability, cellular subsets, proliferation, and cytokine production.

Polyclonal stimuli like phorbol myristate acetate (PMA) plus calcium ionophore (Ca-I), concanavalin A (ConA) or anti-CD3 plus anti-CD28 (alphaCD3/alphaCD28) are widely used T cell stimuli. All three stimuli act at different sites and in different ways to activate the T cell receptor pathway and are widely used in different concentrations, stimulation durations and read-out systems. This study was designed to establish the most suitable polyclonal stimulus in human peripheral blood mononuclear cells (PBMC) experiments by assessing the kinetics of cell viability, present immunophenotypes, proliferation, and cytokine production of the PBMC. In addition, changes in these read-out parameters due to cryopreservation have been investigated by comparing fresh and cryopreserved PBMC cultures at days 1, 3, 5, and 7. This study showed a reduction in the cytokine levels after cryopreservation of PMA/Ca-I stimulated PBMC, whereas no significant differences due to the cryopreservation were observed in ConA or alphaCD3/alphaCD28 stimulated PBMC. Cryopreservation did not alter the maximal proliferation capacity of ConA or alphaCD3/alphaCD28 stimulated PBMC, whereas it did delay the proliferation. Although cryopreservation had no effect on the CD3+CD4+ or CD3+CD8+ T cell subsets, PMA/Ca-I significantly reduced the amount of both T cell subsets over time. In conclusion, PMA/Ca-I is suitable as a positive control in experiments where high cytokine production is expected and only fresh PBMC are used. Proliferation and effects on the T cell subsets in long-term PBMC cultures should use ConA or alphaCD3/alphaCD28 as positive control.

Immunomodulatory capacity of fungal proteins on the cytokine production of human peripheral blood mononuclear cells.

Immunomodulation by fungal compounds can be determined by the capacity of the compounds to influence the cytokine production by human peripheral blood mononuclear cells (hPBMC). These activities include mitogenicity, stimulation and activation of immune effector cells. Eight mushroom strains (Agaricus blazei, Coprinus comatus, Flammulina velutipes, Ganoderma lucidum, Grifola frondosa, Volvariella volvacea, Lentinus edodes, and Pleurotus ostreatus) were tested for the immunomodulating activity of the isolated protein fractions and polysaccharides fractions present in mycelia and culture liquid. The fungal proteins and polysaccharides have been investigated for their in vitro effect on the cytokine profile (IFN-gamma, IL-4, IL-10, IL-12 and TNF-alpha) of unstimulated or hPBMC stimulated with the polyclonal stimulations PMA/Ca-I, ConA or LPS. In addition to their influence on the cytokine profile, the hemagglutination activity of the fungal proteins on rabbit red blood cells was determined. Proteins from V. volvacea and G. lucidum showed immunomodulating activity without the presence of any mitogen, however, neither of them decreased the production of IL-4 and IFN-gamma in combination with a stimulus. All used stimuli resulted in an induction of IL-12 in the presence of the protein extracts, suggesting a direct effect on monocytes. This effect might lead to the indirect immunomodulation of T cell activation and cytokine production. In addition, both protein extracts showed more hemagglutination activity after trypsin treatment of the rabbit red blood cells, indicating the presence of carbohydrate-binding proteins, like lectins and FIPs. In conclusion, the protein extracts of V. volvacea and G. lucidum contain immunomodulating activity by acting directly on monocytes and thereby modulating T cell activation. Further purification of the fungal extracts is needed to clarify whether there are FIPs or lectins present that are responsible for this immunomodulating activity.