Atanaska I. Kostadinova

Immunotherapy – risk/benefit in food allergy

Food allergy is a growing health concern in the westernized world with approx. 6% of children suffering from it. A lack of approved treatment has led to strict avoidance of the culprit food proteins being the only standard of care. Nowadays in‐depth research is conducted to evaluate the possible use of allergen‐specific immunotherapy (SIT) as an active therapeutic option for food allergy. Various routes of administration for the immunotherapy are investigated, including subcutaneous, oral, sublingual, and epicutaneous, and some appear to be successful in inducing a temporary tolerant state. Most research has been conducted with oral immunotherapy due to its efficacious and relatively safe profile. Increasing interest is dedicated to safer and more convenient approaches, such as sublingual and epicutaneous SIT; however, doubts exist about their possible capacity to induce temporary tolerant state and permanent oral tolerance. The high frequency of allergic adverse reactions of the various approaches and the inability to achieve permanent oral tolerance have highlighted the need of refinements in the strategies. A promising strategy for preventing IgE cross‐linking and thus enhancing safety of SIT, while still activating T cells, is the use of tolerogenic peptides. The implementation of such an immunotherapy approach has the potential of not only increasing the chance of achieving a permanent state of tolerance, but also improving the safety and tolerability of the therapy. Immunotherapy for food allergy is still not ready for the clinic, but current and upcoming studies are dedicated to collect enough evidence for the possible implementation of allergen‐SIT as a standard treatment for food allergy.

Novel immunotherapy approaches to food allergy

Purpose of reviewDespite reaching high percentages of desensitization using allergen-specific immunotherapy (SIT) in patients with food allergy, recent studies suggest only a low number of patients to reach persistent clinical tolerance. This review describes current developments in strategies to improve safety and long-term efficacy of SIT. Recent findingsModified allergens or tolerogenic peptides, ultimately optimized for human leukocyte antigen background of the patient, are explored for tolerance induction, whereas anti-IgE antibody (Omalizumab) may be used to facilitate SIT safety. Adjunct therapies to enhance efficacy may make use of TH1 polarizing agents, for example, CpG-oligodeoxynucleotides combined with modified allergen packaged in nanoparticles. Preclinical studies showed insulin-like growth factor-2, intravenous immunoglobulin, Tregitopes or allergen encased oligomannose-coated liposomes capable of inducing regulatory T-cells, recognized for their importance in clinical tolerance induction. Dietary intervention strategies utilizing herbal formula 2, VSL#3, nondigestible short-chain galacto-oligosaccharides and long-chain fructo-oligosaccharides (scGOS/lcFOS) plus Bifidobacterium breve M-16V or n-3 long-chain polyunsaturated fatty acids may facilitate safety and/or a favourable milieu for tolerance induction. SummaryCombining SIT using (adapted) allergens or tolerogenic peptides with adjunct therapy may be essential to improve safety and/or efficacy. Beyond using targeted approaches, specific dietary components may be explored to reduce side-effects and support clinical tolerance induction by SIT.

A Specific Mixture of Fructo-Oligosaccharides and Bifidobacterium breve M-16V Facilitates Partial Non-Responsiveness to Whey Protein in Mice Orally Exposed to β-Lactoglobulin-Derived Peptides

Oral tolerance is a promising approach for allergy prevention in early life, but it strongly depends on allergen exposure and proper immune environment. Small tolerance-inducing peptides and dietary immunomodulatory components may comprise an attractive method for allergy prevention in at-risk infants. This study aimed to investigate whether early oral exposure to β-lactoglobulin-derived peptides (BLG-peptides) and a specific synbiotic mixture of short- and long- chain fructo-oligosaccharides (scFOS/lcFOS, FF) and Bifidobacterium breve (Bb) M-16V (FF/Bb) can prevent cow’s milk allergy (CMA). Three-week-old female C3H/HeOuJ mice were orally exposed to phosphate buffered saline (PBS), whey protein, or a mixture of four synthetic BLG-peptides combined with a FF/Bb-enriched diet prior to intragastric sensitization with whey protein and cholera toxin. To assess the acute allergic skin response and clinical signs of allergy, mice were challenged intradermally with whole whey protein. Serum immunoglobulins were analyzed after a whey protein oral challenge. Cytokine production by allergen-reactivated splenocytes was measured and changes in T cells subsets in the spleen, mesenteric lymph nodes, and intestinal lamina propria were investigated. Pre-exposing mice to a low dosage of BLG-peptides and a FF/Bb-enriched diet prior to whey protein sensitization resulted in a significant reduction of the acute allergic skin response to whey compared to PBS-pretreated mice fed a control diet. Serum immunoglobulins were not affected, but anaphylactic symptom scores remained low and splenocytes were non-responsive in whey-induced cytokine production. In addition, preservation of the Th1/Th2 balance in the small intestine lamina propria was a hallmark of the mechanism underlying the protective effect of the BLG-peptides–FF/Bb intervention. Prior exposure to BLG-peptides and a FF/Bb-enriched diet is a promising approach for protecting the intestinal Th1/Th2 balance and reducing the allergic response to whole whey protein. Therefore, it might have implications for developing successful nutritional strategies for CMA prevention.

Dietary Intervention with β-Lactoglobulin-Derived Peptides and a Specific Mixture of Fructo-Oligosaccharides and Bifidobacterium breve M-16V Facilitates the Prevention of Whey-Induced Allergy in Mice by Supporting a Tolerance-Prone Immune Environment

Cow’s milk allergy (CMA) prevails in infants and brings increased risk of developing other allergic diseases. Oral administration of specific β-lactoglobulin (BLG)-derived peptides (PepMix) and a specific blend of short- and long-chain fructo-oligosaccharides and Bifidobacterium breve M-16V (FF/Bb) was found to partially prevent CMA development in mice. In this study, we aimed to expand the knowledge on the preventive potential and the underlying mechanisms of this approach. Three-week-old female C3H/HeOuJ mice were orally exposed to PepMix±FF/Bb prior to a 5-week oral sensitization with whole whey and cholera toxin as an adjuvant. The acute allergic skin response was determined after an intradermal challenge with whole whey protein. Following an oral challenge with whey, regulatory T cells (Tregs) in the small intestine lamina propria (SI-LP) and mRNA expression of immune markers in the Peyer’s patches (PP) were investigated. The early impact of PepMix and FF/Bb interventions on the immune system during the oral tolerance (OT) induction phase was investigated after the last OT administration. Pre-exposing mice to PepMix+FF/Bb partially prevented the acute allergic skin response compared to PBS and increased Tregs and activated T cells in the SI-LP compared to sham-sensitized mice. It also increased the mRNA expression of Tbet over GATA3 in the PP of whey-sensitized mice. Directly upon the 6-day OT phase, FF/Bb intervention enhanced cecal content levels of propionic and butyric acid in PepMix-fed mice and the former was positively correlated with Foxp3+ cell numbers in the colon. In the PP of PepMix+FF/Bb-exposed mice, IL-22 mRNA expression increased and IL-10 followed the same tendency, while the Foxp3 expression was increased over GATA3 and RorγT. In the colon, the Tbet mRNA expression increased over GATA3, while IL-22 decreased. In addition, the Foxp3+/GATA3+ and regulatory/effector T cell ratios in the mesenteric lymph nodes and the CD11b+/CD11b− conventional dendritic cells ratio in the SI-LP were increased. In conclusion, the FF/Bb diet facilitates the capacity of the specific BLG-peptides to partially prevent the allergic response after sensitization to whole whey protein, possibly by creating a tolerance-prone environment during the OT phase. Such a dietary intervention might contribute to tailoring successful strategies for CMA prevention.

Dietary, nondigestible oligosaccharides and Bifidobacterium breve M-16V suppress allergic inflammation in intestine via targeting dendritic cell maturation

Dietary intervention with short‐chain galacto‐oligosaccharides (scGOS), long‐chain fructo‐oligosaccharides (lcFOS) and Bifidobacterium breve M‐16V (Bb) (GF/Bb) suppresses food allergic symptoms in mice, potentially via intestinal epithelial cell (IEC)–derived galectin‐9. Furthermore, in vitro studies showed galacto‐ and fructo‐oligosaccharides (GF) to enhance the immunomodulatory capacity of a TLR9 ligand representing bacterial CpG DNA when exposed to IEC. In this study, we investigated whether GF/Bb modulates dendritic cells (DCs) and subsequent Th2 and regulatory T cell (Treg) frequency in the small intestinal lamina propria (SI‐LP). BALB/c mice were fed GF/Bb during oral OVA sensitization. DC and T cell phenotype were determined in SI‐LP mononuclear cells using flow cytometry. Murine bone marrow‐derived DCs (BMDCs) were exposed to recombinant galectin‐9 or human monocyte‐derived DCs (moDCs) and were cultured in IEC‐conditioned medium from GF and TLR9 ligand‐exposed HT‐29 cells. GF/Bb reduced allergic symptoms and enhanced serum galectin‐9 levels, while suppressing activation, restoring phagocytic capacity, and normalizing CD103 expression of SI‐LP DCs of OVA‐allergic mice. In vitro, galectin‐9 suppressed LPS‐induced activation markers and cytokine secretion by BMDCs, and IEC‐conditioned medium suppressed moDC activation in a galectin‐9–dependent manner. Besides suppression of SI‐LP DC activation, dietary GF/Bb also lowered the frequency of activated Th2 cells, while enhancing Treg in the SI‐LP of OVA‐allergic mice compared to the control diet. Dietary intervention with GF/Bb enhances galectin‐9 and suppresses allergic symptoms of OVA‐allergic mice in association with reduced intestinal DC and Th2 activation and increased Treg frequency in these mice.

Galectin-9 Produced by Intestinal Epithelial Cells Enhances Aldehyde Dehydrogenase Activity in Dendritic Cells in a PI3K- and p38-Dependent Manner

Intestinal epithelial cells (IEC) drive regulatory T cell (Treg) responses by promoting the differentiation of aldehyde dehydrogenase (ALDH)-expressing CD103+ dendritic cells (DC). Apical stimulation of TLR9 by CpG DNA on IEC supports galectin-9 expression by IEC, which is promoted by short-chain galacto-oligosaccharides and long-chain fructo-oligosaccharides (GF). While galectin-9 can induce the maturation of monocyte-derived DC (moDC), the contribution of galectin-9 on the induction of ALDH activity in DC is not known. To this end, DC were stimulated with galectin-9, and ALDH activity and the expression of CD103 were assessed. ALDH activity was increased by moDC exposed to galectin-9, while the expression of CD103 remained unaltered. Galectin-9 secreted by IEC apically exposed to CpG DNA and GF enhanced ALDH activity, but not CD103 expression by moDC, which was abrogated upon galectin-9 neutralization. Similar observations were found in murine GM-CSF-cultured bone marrow-derived DC (BMDC). Using Flt3L-cultured BMDC and ex vivo murine splenic DC, it was observed that galectin-9 only enhanced ALDH activity in the presence of GM-CSF in CD103- cells. The induction of ALDH activity in BMDC was dependent on p38 and PI3K signaling. These data indicate a novel role for galectin-9 in modulating innate immunity by inducing ALDH activity in DC.

PLGA nanoparticles loaded with beta-lactoglobulin-derived peptides modulate mucosal immunity and may facilitate cow's milk allergy prevention

Abstract Beta‐lactoglobulin (BLG)‐derived peptides may facilitate oral tolerance to whey and prevent cows milk allergy (CMA). Loading of BLG‐peptides in poly(lactic‐co‐glycolic acid) (PLGA) nanoparticles (Pep‐NP) may improve this. Here we studied the uptake of NP and the capacity of NP and Pep‐NP to activate bone marrow dendritic cells (BMDC). Furthermore, CMA prevention was evaluated by orally exposing three‐week‐old female C3H/HeOuJ mice to Pep‐NP, NP or free peptides (PepMix) for 6 days before oral sensitization with whole whey protein and effects on the spleen and small intestine lamina propria (SI‐LP) were studied. In BMDC, NP and Pep‐NP enhanced CD40 expression and IL‐6 and TNF‐&agr; secretion, while tended to decrease CD80 expression and prevented PepMix‐induced IL‐12 secretion. In vivo, oral exposure to Pep‐NP, but not NP or PepMix, prior to whey sensitization tended to partially prevent the acute allergic skin response to whole whey protein. Splenocytes of NP‐pre‐exposed mice secreted increased levels of whey‐specific IL‐6, but this was silenced in Pep‐NP‐pre‐exposed mice which also showed reduced TNF‐&agr; and IFN‐&ggr; secretion. In the SI‐LP, Pep‐NP pre‐exposure reduced the CD4+ T cell frequency in CMA mice compared to PBS pre‐exposure. In addition, while NP increased whey‐specific IL‐6 secretion in the SI‐LP, Pep‐NP did not and maintained regulatory TGF‐&bgr; secretion. This study presents a proof‐of‐concept that PLGA nanoparticles facilitate the capacity of BLG peptides to suppress the allergic response to whole whey protein. Hence, PLGA nanoparticles may be further developed as an adjunct strategy for BLG‐peptide‐based oral tolerance induction and CMA prevention.

Dietary Interventions in Infancy

The prevalence of food allergies in infants has increased, indicating that maturation of the infants immune system and the development of oral tolerance to food antigens are hampered. Exposure to an altered Western diet and hygienic living conditions appear to skew the infant’s immune response toward an atopic phenotype. Allergen avoidance strategies are challenged nowadays and active tolerance induction via immunomodulatory food components and/or modified allergens is of key interest. Dietary interventions with probiotics, prebiotics, synbiotics, n-3 polyunsaturated fatty acids, or antioxidants show potential in supporting tolerance induction. Clinical trials have provided promising results. However, variation in the timing, dose, duration, and type of dietary intervention used delay its implementation in early-life strategies for allergy prevention. Modulation of the infants immune response might benefit from applying dietary interventions prenatally as well as postnatally.

Combined exposure to betalactoglobulin-derived tolerogenic peptides and synbiotics alleviates food allergy response in vivo

Background: Although pretreatment is not routinely used before immunotherapy, it has been shown to reduce the reactions in patients using rush, cluster and conventional schedules. Our aim is to determine the possible preventive effects of various premedication usage on systemic and local reactions during cluster immunotherapy. Method: One hundred and eighteen patients receiving a total of 137 cluster immunotherapy protocols to mites, pollens, bee and wasp venoms were recruited in the study. Patients were randomized into 5 different groups according to premedication status as shown here below: Group 1: daily levosetirizine, Group 2: levosetirizine only 2 h prior to injections, Group 3: daily montelukast, Group 4: combinations of montelukast and levosetirizine and, Group 5: no premedication Patients were followed during build up and maintenance phase of immunotherapy, systemic and local reactions were reported. Reactions are reported according to World Allergy Organization Subcutaneous Systemic Reaction Grading System. Results: Most of the patients were female (%64.2), the most frequent allergen was house dust mites (50.4%). Of 69.3% of patients took premedication and 19.7% of patients had reactions during the build up phase. Reactions were more frequent in patients who have pollen allergy and have received pollen immunotherapy [P = 0.034; OR = 2.65 CI:95%, 1.05–6.63)]. The total frequency of the hypersensitivity reaction was significantly higher in patients not receiving premedication [14 (14.7%) vs 13 (31.0%), P = 0.028 ]. However no difference was detected between groups taking various drugs for premedication. The patients in the premedication group significantly experienced less local reactions (P = 0.033) although no difference in the frequency of systemic reactions was seen between the groups. Conclusion: These preliminary results suggest that the reaction risk is increased in pollen immunotherapy and premedication does not seem to prevent the frequency or severity of systemic reactions although it decreases the frequency of local reactions. As the number of patients recruited in the study increases, the effect of premedication on reactions may be clearer.A three year follow-up of asthma, respiratory symptoms and self-reported allergy, among pilots and cabin attendants