Monitoring immune modulation by nutrition in the general population: identifying and substantiating effects on human health.

Optimal functioning of the immune system is crucial to human health, and nutrition is one of the major exogenous factors modulating different aspects of immune function. Currently, no single marker is available to predict the effect of a dietary intervention on different aspects of immune function. To provide further guidance on the assessment and interpretation of the modulation of immune functions due to nutrition in the general population, International Life Sciences Institute Europe commissioned a group of experts from academia, government and the food industry to prepare a guidance document. A draft of this paper was refined at a workshop involving additional experts. First, the expert group defined criteria to evaluate the usefulness of immune function markers. Over seventy-five markers were scored within the context of three distinct immune system functions: defence against pathogens; avoidance or mitigation of allergy; control of low-grade (metabolic) inflammation. The most useful markers were subsequently classified depending on whether they by themselves signify clinical relevance and/or involvement of immune function. Next, five theoretical scenarios were drafted describing potential changes in the values of markers compared with a relevant reference range. Finally, all elements were combined, providing a framework to aid the design and interpretation of studies assessing the effects of nutrition on immune function. This stepwise approach offers a clear rationale for selecting markers for future trials and provides a framework for the interpretation of outcomes. A similar stepwise approach may also be useful to rationalise the selection and interpretation of markers for other physiological processes critical to the maintenance of health and well-being.

Dietary docosahexaenoic acid in combination with arachidonic acid ameliorates allergen-induced dermatitis in mice.

OBJECTIVE: In this study, we investigated the impact of dietary docosahexaenoic (DHA) and arachidonic acid (AA) on development and severity of allergen-induced dermatitis. STUDY DESIGN: In sensitized mice, skin inflammation was induced by ovalbumin. Mice received either a diet containing 0.015% DHA, 0.029% AA or the combination of both. The severity of dermatitis was evaluated by using a clinical skin score (CSS), followed by immunohistologic and cytokine analysis. To unravel potential mechanisms, interleukin (IL)-4 or tumor necrosis factor α-stimulated keratinocytes from the cell line Kera-308 was cultured with different DHA/AA compositions and analyzed regarding proliferation and cytokine production. RESULTS: Dietary DHA/AA significantly improved the severity of allergen-induced dermatitis as the CSS was reduced by 36 ± 23% (p=0.005). Furthermore, reduced epidermal KI67 expression, increased number of forkhead box P3(+) cells, and elevated IL-10 expression were determined in skin lesions of dietary-treated mice. Correspondingly, in vitro DHA/AA-treated keratinocytes exhibited increased IL-10 expression and produced less thymic stromal lymphopoietin. CONCLUSION: Dietary DHA/AA supplementation leads to a significant amelioration of allergen-induced dermatitis. This was accompanied with the presence of increased regulatory T cells and IL-10 expression in lesional skin. Moreover, we identify keratinocytes, which play a crucial role in the regulation of skin inflammation, as important targets of DHA/AA supplementation. Future studies are needed to clarify whether DHA/AA acts directly or whether its biologic active metabolites are responsible for these findings. This may unravel novel therapeutical compounds for allergen-induced dermatitis.

Visualization of clustered IgE epitopes on alpha-lactalbumin.

BACKGROUND: alpha-Lactalbumin (alpha-La) is a major cow's milk (CM) allergen responsible for allergic reactions in infants. OBJECTIVE: We performed molecular, structural, and immunologic characterization of alpha-La. METHODS: Recombinant alpha-lactalbumin (ralpha-La) was expressed in Escherichia coli, purified to homogeneity, and characterized by means of mass spectrometry and circular dichroism, and its allergenic activity was studied by using microarray technology, as well as in a basophil histamine release assay. IgE epitope mapping was performed with synthetic peptides. RESULTS: According to circular dichroism analysis, ralpha-La represented a folded protein with a high thermal stability and refolding capacity. ralpha-La reacted with IgE antibodies from 57.6% of patients with CM allergy (n = 66) and induced the strongest basophil degranulation with sera from patients with CM allergy who had exhibited gastrointestinal symptoms or severe systemic reactions on CM exposure. ralpha-La contained sequential and conformational IgE epitopes. Superposition of IgE-reactive peptides onto the 3-dimensional structure of alpha-La revealed a close vicinity of the N- and C-terminal peptides within a surface-exposed patch. CONCLUSIONS: ralpha-La can be used for the diagnosis of patients with severe allergic reactions to CM and serves as a paradigmatic tool for the development of therapeutic strategies for CM allergy.

Guidance for substantiating the evidence for beneficial effects of probiotics: prevention and management of allergic diseases by probiotics.

Allergy is a hypersensitivity reaction mediated by specific antibody-mediated or cell-mediated immunologic mechanisms and clinically manifested as atopic eczema, allergic rhinoconjunctivitis, or asthma. During the recent decades there has been an increase in allergy prevalence, which is attributed to changes in environmental factors. The so-called "hygiene hypothesis" suggests that a lack of exposure to microbial stimulus early in childhood is a major factor involved in this trend. This provides a rationale for using probiotics to modify the gut microbiota and thereby shaping the immune response of the host, especially in infancy. Most success has been obtained in primary prevention of atopic eczema. A limited number of studies also provided evidence for a beneficial effect of different probiotics in the management of allergic diseases (atopic eczema, allergic rhinitis). However, choice of probiotic strains as well as timing of the intervention are important variables. The exact in vivo mechanism of probiotics in shaping the immune response still needs to be determined. Future studies should use uniform criteria for diagnosis and symptom scoring of atopic diseases and may identify the genes predisposing to allergic disease. There is encouraging evidence that specific probiotics can become valuable tools in the prevention and management of allergic diseases.

Guidance for substantiating the evidence for beneficial effects of probiotics: current status and recommendations for future research.

Probiotic bacteria are live microorganisms that, when administered in adequate amounts, confer a health benefit on the host. There is a growing interest in probiotics within the scientific community, with consumers, and in the food industry. The interactions between the gut and intestinal microbiota and between resident and transient microbiota define a new arena in physiology, an understanding of which would shed light on the "cross-talk" between humans and microbes. The different beneficial effects of specific probiotic strains may be translated into different health claims. However, there is a need for comprehensive and harmonized guidelines on the assessment of the characteristics and efficacy of probiotics and of foods containing them. An international expert group of ILSI has evaluated the published evidence of the functionality of different probiotics in 4 areas of (human) application: 1) metabolism, 2) chronic intestinal inflammatory and functional disorders, 3) infections, and 4) allergy. Based on the existing evidence, concrete examples of demonstration of benefits and gaps are listed, and guidelines and recommendations are defined that should help design the next generation of probiotic studies.

Cloning, expression, and mapping of allergenic determinants of alphaS1-casein, a major cow's milk allergen.

Milk is one of the first components introduced into human diet. It also represents one of the first allergen sources, which induces IgE-mediated allergies in childhood ranging from gastrointestinal, skin, and respiratory manifestations to severe life-threatening manifestations, such as anaphylaxis. Here we isolated a cDNA coding for a major cow's milk allergen, alphaS1-casein, from a bovine mammary gland cDNA library with allergic patients' IgE Abs. Recombinant alphaS1-casein was expressed in Escherichia coli, purified, and characterized by circular dichroism as a folded protein. IgE epitopes of alphaS1-casein were determined with recombinant fragments and synthetic peptides spanning the alphaS1-casein sequence using microarrayed components and sera from 66 cow's milk-sensitized patients. The allergenic activity of ralphaS1-casein and the alphaS1-casein-derived peptides was determined using rat basophil leukemia cells transfected with human FcepsilonRI, which had been loaded with the patients' serum IgE. Our results demonstrate that ralphaS1-casein as well as alphaS1-casein-derived peptides exhibit IgE reactivity, but mainly the intact ralphaS1-casein induced strong basophil degranulation. These results suggest that primarily intact alphaS1-casein or larger IgE-reactive portions thereof are responsible for IgE-mediated symptoms of food allergy. Recombinant alphaS1-casein as well as alphaS1-casein-derived peptides may be used in clinical studies to further explore pathomechanisms of food allergy as well as for the development of new diagnostic and therapeutic strategies for milk allergy.

Immunological basis and management of food allergy.

One of the most common allergies in children involves cow's milk, which contains approximately 20 different proteins that can cause allergic reactions. It is well known that children exhibiting signs of cow's milk allergy early in life often go on to develop allergy-related respiratory diseases; thus, management of early sensitisations and symptoms of food allergies is crucial to preventing subsequent allergic complications. Constant allergen exposure and other environmental factors determine whether a sensitised individual will become chronically allergic and experience persistent symptoms. Management of food allergies in children focuses on minimising sensitisation and encouraging immune system maturation through the exposure of children to exogenous stimuli known to prime the immune system. Hypoallergenic molecules or allergen avoidance can also be used to induce tolerance in allergy-prone children. Available evidence suggests that the onset of the sensitisation phase and the degree of inflammation can be modulated by external factors such as nutrition, and guidelines outlining the most effective dietary regimen for the prevention of allergic disease have been published. The underlying mechanisms of tolerance induction and the potential benefits of prophylactic treatment for food allergies remain to be determined.

New visions for basic research and primary prevention of pediatric allergy: an iPAC summary and future trends.

Hydrolyzed formula feeding, delayed introduction of solid food, indoor allergen avoidance, smoke and pollutants avoidance have been applied for several decades as primary preventive measures for allergic diseases. Unfortunately, some of these strategies have had no or modest success. Therefore, resources need to be focused on better understanding of the early allergic events and on interventional studies to investigate new strategies of primary and secondary prevention. Accordingly, this review summarizes the state-of-the-art of genetic, immunological and clinical aspects of primary prevention of allergic diseases. Studies investigating gene-by-gene and gene-by-environment interactions suggest that prevention of allergic diseases must be tailored to the individual genetic susceptibilities ('gene profiling') and environmental exposures. The expanding knowledge on new T cell populations (Th17, TSLP (thymic stromal derived lymphopoietin)-dependent 'inflammatory Th2 cells') is also inspiring new concepts on the origins of allergic diseases. The old concept of 'blocking immunoglobulin G antibodies' has been re-appraised and it is likely to generate novel preventive and therapeutic strategies. The major task for future clinical research is to clearly define the timing of optimal exposure to potential allergens. In addition, the role of microbial products such as certain bacteria, or their components, and of helminths or their larvae at different times in early life, alone or with potential allergens, definitely need to be further investigated. The benefit of efficient allergy prevention, based on focusing resources on novel and promising research lines, will be of prime importance to both affluent countries and other parts of the world where allergy is only currently emerging.

Role of vitamin A elimination or supplementation diets during postnatal development on the allergic sensitisation in mice.

Vitamin A (VA) and its derivatives, the retinoids, are important factors for the development of the immune system. It has been shown in adult animals that proliferation of lymphocyte populations and antibody secretion are retinoid dependent, while little is known about the effects of retinoids during postnatal development. The aim of this study was to investigate the role of VA on allergic sensitisation during lactation and after weaning using an in vivo system for postnatal allergic sensitisation in mice. Different VA diets (basal/VA elimination/VA (as retinyl palmitate) supplemented) were fed to the dams throughout lactation and directly to the pups after weaning. Allergic sensitisation was induced with a single peritoneal ovalbumin (OVA) injection at day 28 after weaning. The phenotype of lymphocytes was analysed by flow cytometry and functional data were obtained by analysis of (IL-4/IFN-gamma) cytokine production and antibody production (OVA-specific IgG1 and IgE) in the offspring. VA/retinyl palmitate supplementation during lactation and after weaning decreased CD3+, CD4+, CD8+ and B220+ populations in splenic lymphocytes but also significantly enhanced IL-4 production and OVA-specific IgE after sensitisation. In contrast, mice fed VA-elimination diet displayed no significant alteration of lymphocyte numbers and a slightly increased IL-4 production. Our results showed that a single allergen injection during postnatal development induces allergic sensitisation whose degree is modified by the VA content of the maternal diet during lactation and the diet of the pups after weaning, indicating an important role of VA on the severity of the allergic sensitisation.

Treatment of allergic asthma by targeting transcription factors using nucleic-acid based technologies.

There is considerable evidence that T-helper 2 (Th2) cells play a central role in the pathogenesis of allergic diseases such as bronchial asthma, hay fever or food allergy. The differentiation of naïve T cells into Th2 cells producing a specific pattern of cytokines is tightly controlled and regulated by transcription factors. Thus down-regulation of mRNA-levels of a single transcription factor leads to a "knock-down" of several mediators simultaneously, representing an advantage compared to earlier approaches involving down-regulation of one intercellular inflammatory mediator, which is unlikely to influence all pathophysiological aspects of the disease. We review the impact of specific and master transcription factors involved in Th2 cell commitment and evaluate approaches for the down-regulation of these proteins by degradation of their mRNA using nucleic-acid based technologies including antisense oligonucleotides, ribozymes, DNAzymes, decoys oligonucleotides and RNA interference.

Suppression of IL-2 and IFN-gamma production in women with spontaneous preterm labor.

AIMS: To determine the TH-1/TH-2 cytokine pattern in peripheral blood leukocytes (PBL) in late second- and third trimester in normal pregnancies, in comparison to patients with spontaneous preterm delivery (PTD; < 37 completed weeks' gestation). METHODS: A cross-sectional retrospective study was performed in a tertiary care obstetric unit with healthy non-pregnant women (n=7); healthy pregnant women (n=25); patients (n=25) with preterm labor (defined as uterine contractions or changes in cervical length). The phenotypic analysis of TH-1/TH-2 immune deviation was examined in PBL. RESULTS: 26% PTD (n=13) were recorded. Patients delivering at term (n=37, 74%) were characterized by an upregulation of IL-2, IFN-gamma and IL-4 production during weeks 20-25, followed by a strong suppression in cytokine production, except for TGF-beta. Towards the end of pregnancy cytokine levels returned to normal as observed in non-pregnant women. In contrast, PTD showed an inverse pattern for IL-2 and IFN-gamma with marked suppression in IL-2 and IFN-gamma production between weeks 20-25, followed by a strong stimulation of these cytokines. No differences were observed in IL-4 and TGF-beta production. CONCLUSION: An inverse pattern in IL-2 and IFN-gamma production in PBLs between weeks 20-30 is seen in PTD as compared to patients delivering at term.

Perinatal events affecting the onset of allergic diseases.

The prevalence of asthma and related allergic disorders has increased considerably over the last several decades. Since the genetic makeup of humans has not changed during this time, it is likely that environmental factors may have influenced this rise in allergic diseases. Furthermore, there is increasing evidence to suggest that many aspects of health and disease are determined during the perinatal period and that alterations in lifestyle and diet later in life are secondary to the effects of the immunological programming that occurs during pregnancy and early infancy. This is directly applicable to allergic disease where immune responses at birth implicate intrauterine exposure as a primary sensitization event. Moreover, infants who experience allergy early in life already have an altered immune response at birth and most therapeutic approaches focus on altering the expression of the disease. Therefore, a better understanding of the underlying mechanisms that shapes the immune response towards allergy development is fundamental to strengthening "natural" protective stimuli or developing preventative rather than treatment therapies.

NO2-induced airway inflammation is associated with progressive airflow limitation and development of emphysema-like lesions in C57bl/6 mice.

The major features of chronic obstructive pulmonary disease (COPD) comprise a not fully reversible airflow limitation associated with an abnormal inflammatory response, increased mucus production and development of emphysema-like lesions. Animal models that closely mimic these alterations represent an important issue for the investigation of pathophysiological mechanisms. Since most animal models in this area have focused on specific aspects of the disease, we aimed to investigate whether exposure of C57BL/6 mice to nitrogen dioxide (NO2) may cause a more complex phenotype covering several of the characteristics of the human disease. Therefore, mice were exposed to NO2 for 14h each day for up to 25 days. Initial dose response experiments revealed the induction of a significant inflammatory response at a dose of 20 ppm NO2. Mice developed progressive airway inflammation together with a focal inflammation of the lung parenchyma characterized by a predominant influx of neutrophils and macrophages. In addition, goblet cell hyperplasia was detected in the central airways and increased collagen deposition was found in the lung parenchyma. NO2-exposed mice developed emphysema-like lesions as indicated by a significantly increased mean linear intercept as compared to control mice. Finally, the assessment of lung functional parameters revealed the development of progressive airway obstruction over time. In conclusion, our data provide evidence that the inflammatory response to NO2 exposure is associated with increased mucus production, development of airspace enlargement and progressive airway obstruction. Thus, NO2-exposed mice may serve as a model to investigate pathophysiological mechanisms that contribute to the development of human COPD.

Blocking IL-15 prevents the induction of allergen-specific T cells and allergic inflammation in vivo.

IL-15 has been shown to accelerate and boost allergic sensitization in mice. Using a murine model of allergic sensitization to OVA, we present evidence that blocking endogenous IL-15 during the sensitization phase using a soluble IL-15Ralpha (sIL-15Ralpha) suppresses the induction of Ag-specific, Th2-differentiated T cells. This significantly reduces the production of OVA-specific IgE and IgG and prevents the induction of a pulmonary inflammation. Release of proinflammatory TNF-alpha, IL-1beta, IL-6, and IL-12 as well as that of Th2 cytokines IL-4, IL-5, and IL-13 into the bronchi are significantly reduced, resulting in suppressed recruitment of eosinophils and lymphocytes after allergen challenge. It is of clinical relevance that the airway hyper-responsiveness, a major symptom of human asthma bronchiale, is significantly reduced by sIL-15Ralpha treatment. Ex vivo analysis of the draining lymph nodes revealed reduced numbers of CD8, but not CD4, memory cells and the inability of T cells of sIL-15Ralpha-treated mice to proliferate and to produce Th2 cytokines after in vitro OVA restimulation. This phenomenon is not mediated by enhanced numbers of CD4(+)/CD25(+) T cells. These results show that IL-15 is important for the induction of allergen-specific, Th2-differentiated T cells and induction of allergic inflammation in vivo.

The IL-6R alpha chain controls lung CD4+CD25+ Treg development and function during allergic airway inflammation in vivo.

The cytokine IL-6 acts via a specific receptor complex that consists of the membrane-bound IL-6 receptor (mIL-6R) or the soluble IL-6 receptor (sIL-6R) and glycoprotein 130 (gp130). In this study, we investigated the role of IL-6R components in asthma. We observed increased levels of sIL-6R in the airways of patients with allergic asthma as compared to those in controls. In addition, local blockade of the sIL-6R in a murine model of late-phase asthma after OVA sensitization by gp130-fraction constant led to suppression of Th2 cells in the lung. By contrast, blockade of mIL-6R induced local expansion of Foxp3-positive CD4+CD25+ Tregs with increased immunosuppressive capacities. CD4+CD25+ but not CD4+CD25- lung T cells selectively expressed the IL-6R alpha chain and showed IL-6-dependent STAT-3 phosphorylation. Finally, in an in vivo transfer model of asthma in immunodeficient Rag1 mice, CD4+CD25+ T cells isolated from anti-IL-6R antibody-treated mice exhibited marked immunosuppressive and antiinflammatory functions. IL-6 signaling therefore controls the balance between effector cells and Tregs in the lung by means of different receptor components. Furthermore, inhibition of IL-6 signaling emerges as a novel molecular approach for the treatment of allergic asthma.

Volumetric analysis of mice lungs in a clinical magnetic resonance imaging scanner.

Small animal models are widely used to study various pathologies. Magnetic resonance imaging (MRI) allows investigation of these animals in a non-invasive way. Therefore, the aim of our study was to develop and evaluate a low-cost approach to measure lung volumes in small animal MRI using a clinical scanner and a specially designed RF coil. Five mice (three of an established emphysema model and two controls) were investigated in a 1.0-T clinical scanner using a specially built small animal saddle coil and three different three-dimensional sequences; overall imaging time was approximately 16 min. Lung volumes were calculated from these images using an interactive watershed transform algorithm for semi-automatic image segmentation. The gold standard for the volume measurement was water displacement after surgical explantation. MRI measured volumes correlated significantly with ex vivo measurements on the explanted lungs (r = 0.99 to 0.89; p < 0.05). Mean lung volume in emphysema model mice was larger than in controls. High-resolution, small animal MRI using a clinical scanner is feasible for volumetric analysis and provides an alternative to a dedicated small animal scanner.

Induction of autoallergy with an environmental allergen mimicking a self protein in a murine model of experimental allergic asthma.

BACKGROUND: Allergy and autoimmunity are traditionally considered as 2 exclusive entities related to the development of either TH2-dominated or TH1-dominated immune responses. OBJECTIVE: This study investigated whether allergic sensitization to a foreign antigen mimicking a self protein can induce allergy accompanied by an autoimmune response. METHODS: BALB/c mice were sensitized to human alpha-NAC, an evolutionary conserved component of the nascent polypeptide-associated complex, recently identified as an IgE-reactive autoantigen in patients with severe forms of atopy. By using nitrocellulose-blotted murine lung and skin extracts, purified recombinant human as well as murine alpha-NAC and murine alpha-NAC-derived synthetic peptides, the IgE, IgG1, and IgG2a antibody responses were measured, and their epitope specificity was mapped. RESULTS: Cross-reactivity of IgE and IgG antibodies with murine alpha-NAC was found in mice sensitized with human alpha-NAC. The biological relevance of the antibody response was demonstrated by the induction of immediate skin reactions in sensitized mice and by the fact that skin sensitivity could be passively transferred with serum to naive mice. Antigen challenge of sensitized mice resulted in airway inflammation accompanied by eosinophil and neutrophil accumulation, airway hyperresponsiveness to methacholine and perivasculitis of lung veins. CONCLUSION: Our data demonstrate that sensitization with a foreign antigen mimicking self can induce an allergic immune response of a mixed TH2 and TH1 profile that is associated with autoreactivity. Cross-sensitization to self may represent an important pathomechanism involved in the maintenance of severe and chronic forms of allergy.

Animal models of type I allergy using recombinant allergens.

Various animal models including guinea pigs, monkeys, dogs, rats, and mice have been established in an attempt to provide insights into the complex immunological and pathophysiological mechanisms of human type I allergic diseases. The detailed knowledge of the murine genome, the various components of the murine immune system, and the generation of engineered mice has made the murine system the most attractive among all animal models. The availability of multitude technologies and reagents to characterize and manipulate immunological pathways and mediators adds to the outstanding opportunities to assess the pathology of allergic diseases and to develop novel therapeutic strategies in mice. Numerous sensitization protocols with food and aero-allergens are used to establish an allergic/asthma-like phenotype in mice. Requirements for an appropriate murine model include a close resemblance to the pathology of the disease in humans, the objective measurement of the physiologic parameters, as well as reliability and reproducibility of the experimental data. With respect to reproducible experimental conditions, it has been recognized that extract preparations from natural allergen sources can vary in their allergen-content and -composition. This might influence the degree of sensitization or the outcome of treatment strategies in dependence of the applied extract preparation. The use of recombinant allergens in experimental in vivo and in vitro systems can overcome these problems. Another aspect, that has become obvious from the experimental studies, is that allergens can differ in their immunogenicity as well as in their capacity to act as tolerogens. Therefore, it seems important that the efficacy of the different allergen-molecules to act as therapeutic agents is individually examined. In this review, examples of animal models are described, in which recombinant allergens have been used for sensitization and/or treatment of allergic responses and how they have been used to enhance our understanding of the pathology of allergic diseases.

Inhibition of IgE-production by peroxisome proliferator-activated receptor ligands.

In this study we analyzed the effect of peroxisome proliferator-activated receptor-alpha and -gamma ligands on immunoglobulin synthesis and cytokine production in non-allergic and atopic dermatitis donors in vitro, but also in vivo ovalbumin-sensitized mice. A significant inhibition in CD40+ interleukin-4-mediated, but also basal IgE synthesis from peripheral blood mononuclear cells of atopic dermatitis donors was observed in the presence of the peroxisome proliferator-activated receptor-alpha ligand (up to 47+/-12%) and peroxisome proliferator-activated receptor-gamma ligand (69+/-5%). By contrast, the production of other isotypes such as IgA, IgG, and IgM was only modest inhibited. Analysis of cytokine production from the peripheral blood mononuclear cells shows inhibition of several cytokines by both peroxisome proliferator-activated receptor ligands. The most inhibitory effect on cytokine production was observed by the peroxisome proliferator-activated receptor-gamma ligand in peripheral blood mononuclear cells from atopic dermatitis donors with high IgE baseline production. Coculture experiments show that the decrease of IgE production by ciglitazone was monocyte dependent (up to 63+/-7%). Finally, in vivo experiments from ovalbumin-sensitized mice confirmed the in vitro findings showing that the interleukin-4-mediated immune response was inhibited in ciglitazone-treated mice.