Mould allergen Alt a 1 spiked with the micronutrient retinoic acid reduces Th2 response and ameliorates Alternaria allergy in BALB/c mice.

BACKGROUND: We investigated the biological function of the mould allergen Alt a 1 as a carrier of micronutrients, such as the vitamin A metabolite retinoic acid (RA) and the influence of RA binding on its allergenicity in vitro and in vivo. METHODS: Alt a 1-RA complex formation was analyzed in silico and in vitro. PBMCs from Alternaria-allergic donors were stimulated with Alt a 1 complexed with RA (holo-Alt a 1) or empty apo-Alt a 1 and analyzed for cytokine production and CD marker expression. Serum IgE-binding and crosslinking assays to apo- and holo-protein were correlated to B-cell epitope analysis. Female BALB/c mice already sensitized to Alt a 1 were intranasally treated with apo-Alt a 1, holo-Alt a 1 or RA alone before measuring anaphylactic response, serum antibody levels, splenic cytokines and CD marker expression. RESULTS: In silico docking calculations and in vitro assays showed that the extent of RA binding depended on the higher quaternary state of Alt a 1. Holo-Alt a 1 loaded with RA reduced IL-13 released from PBMCs and CD3+CD4+CRTh2 cells. Complexing Alt a 1 to RA masked its IgE B-cell epitopes and reduced its IgE-binding capacity. In a therapeutic mouse model of Alternaria allergy nasal application of holo-Alt a 1, but not of apo-Alt a 1, significantly impeded the anaphylactic response, impaired splenic antigen-presenting cells and induced IL-10 production. CONCLUSION: Holo-Alt a 1 binding to RA was able to alleviate Th2 immunity in vitro, modulate an ongoing Th2 response and prevent anaphylactic symptoms in vivo, presenting a novel option for improving allergen-specific immunotherapy in Alternaria allergy.

AllergoOncology: Biomarkers and refined classification for research in the allergy and glioma nexus-A joint EAACI-EANO position paper.

Epidemiological studies have explored the relationship between allergic diseases and cancer risk or prognosis in AllergoOncology. Some studies suggest an inverse association, but uncertainties remain, including in IgE-mediated diseases and glioma. Allergic disease stems from a Th2-biased immune response to allergens in predisposed atopic individuals. Allergic disorders vary in phenotype, genotype and endotype, affecting their pathophysiology. Beyond clinical manifestation and commonly used clinical markers, there is ongoing research to identify novel biomarkers for allergy diagnosis, monitoring, severity assessment and treatment. Gliomas, the most common and diverse brain tumours, have in parallel undergone changes in classification over time, with specific molecular biomarkers defining glioma subtypes. Gliomas exhibit a complex tumour-immune interphase and distinct immune microenvironment features. Immunotherapy and targeted therapy hold promise for primary brain tumour treatment, but require more specific and effective approaches. Animal studies indicate allergic airway inflammation may delay glioma progression. This collaborative European Academy of Allergy and Clinical Immunology (EAACI) and European Association of Neuro-Oncology (EANO) Position Paper summarizes recent advances and emerging biomarkers for refined allergy and adult-type diffuse glioma classification to inform future epidemiological and clinical studies. Future research is needed to enhance our understanding of immune-glioma interactions to ultimately improve patient prognosis and survival.

Nutritional Provision of Iron Complexes by the Major Allergen Alt a 1 to Human Immune Cells Decreases Its Presentation.

Alternaria alternata is a common fungus strongly related with severe allergic asthma, with 80% of affected individuals being sensitized solely to its major allergen Alt a 1. Here, we assessed the function of Alt a 1 as an innate defense protein binding to micronutrients, such as iron-quercetin complexes (FeQ2), and its impact on antigen presentation in vitro. Binding of Alt a 1 to FeQ2 was determined in docking calculations. Recombinant Alt a 1 was generated, and binding ability, as well as secondary and quaternary structure, assessed by UV-VIS, CD, and DLS spectroscopy. Proteolytic functions were determined by casein and gelatine zymography. Uptake of empty apo- or ligand-filled holoAlt a 1 were assessed in human monocytic THP1 cells under the presence of dynamin and clathrin-inhibitors, activation of the Arylhydrocarbon receptor (AhR) using the human reporter cellline AZ-AHR. Human PBMCs were stimulated and assessed for phenotypic changes in monocytes by flow cytometry. Alt a 1 bound strongly to FeQ2 as a tetramer with calculated Kd values reaching pico-molar levels and surpassing affinities to quercetin alone by a factor of 5000 for the tetramer. apoAlt a 1 but not holoAlta 1 showed low enzymatic activity against casein as a hexamer and gelatin as a trimer. Uptake of apo- and holo-Alt a 1 occurred partly clathrin-dependent, with apoAlt a 1 decreasing labile iron in THP1 cells and holoAlt a 1 facilitating quercetin-dependent AhR activation. In human PBMCs uptake of holoAlt a 1 but not apoAlt a 1 significantly decreased the surface expression of the costimulatory CD86, but also of HLADR, thereby reducing effective antigen presentation. We show here for the first time that the presence of nutritional iron complexes, such as FeQ2, significantly alters the function of Alt a 1 and dampens the human immune response, thereby supporting the notion that Alt a 1 only becomes immunogenic under nutritional deprivation.

Micronutritional supplementation with a holoBLG-based FSMP (food for special medical purposes)-lozenge alleviates allergic symptoms in BALB/c mice: Imitating the protective farm effect.

BACKGROUND: Previously, the protective farm effect was imitated using the whey protein beta-lactoglobulin (BLG) that is spiked with iron-flavonoid complexes. Here, we formulated for clinical translation a lozenge as food for special medical purposes (FSMP) using catechin-iron complexes as ligands for BLG. The lozenge was tested in vitro and in a therapeutical BALB/c mice model. METHODS: Binding of iron-catechin into BLG was confirmed by spectroscopy and docking calculations. Serum IgE binding of children allergic or tolerating milk was assessed to loaded (holo-) versus empty (apo-) BLG and for human mast cell degranulation. BLG and Bet v 1 double-sensitized mice were orally treated with the holoBLG or placebo lozenge, and immunologically analysed after systemic allergen challenge. Human PBMCs of pollen allergic subjects were flow cytometrically assessed after stimulation with apoBLG or holoBLG using catechin-iron complexes as ligands. RESULTS: One major IgE and T cell epitope were masked by catechin-iron complexes, which impaired IgE binding of milk-allergic children and degranulation of mast cells. In mice, only supplementation with the holoBLG lozenge reduced clinical reactivity to BLG and Bet v 1, promoted Tregs, and suppressed antigen presentation. In allergic subjects, stimulation of PBMCs with holoBLG led to a significant increase of intracellular iron in circulating CD14+ cells with significantly lower expression of HLADR and CD86 compared to their stimulation with apoBLG. CONCLUSION: The FSMP lozenge targeted antigen presenting cells and dampened immune activation in human immune cells and allergic mice in an antigen-non-specific manner, thereby conferring immune resilience against allergic symptoms.

AllergoOncology: Danger signals in allergology and oncology: A European Academy of Allergy and Clinical Immunology (EAACI) Position Paper.

The immune system interacts with many nominal 'danger' signals, endogenous danger-associated (DAMP), exogenous pathogen (PAMP) and allergen (AAMP)-associated molecular patterns. The immune context under which these are received can promote or prevent immune activating or inflammatory mechanisms and may orchestrate diverse immune responses in allergy and cancer. Each can act either by favouring a respective pathology or by supporting the immune response to confer protective effects, depending on acuity or chronicity. In this Position Paper under the collective term danger signals or DAMPs, PAMPs and AAMPs, we consider their diverse roles in allergy and cancer and the connection between these in AllergoOncology. We focus on their interactions with different immune cells of the innate and adaptive immune system and how these promote immune responses with juxtaposing clinical outcomes in allergy and cancer. While danger signals present potential targets to overcome inflammatory responses in allergy, these may be reconsidered in relation to a history of allergy, chronic inflammation and autoimmunity linked to the risk of developing cancer, and with regard to clinical responses to anti-cancer immune and targeted therapies. Cross-disciplinary insights in AllergoOncology derived from dissecting clinical phenotypes of common danger signal pathways may improve allergy and cancer clinical outcomes.

Immune modulation via T regulatory cell enhancement: Disease-modifying therapies for autoimmunity and their potential for chronic allergic and inflammatory diseases-An EAACI position paper of the Task Force on Immunopharmacology (TIPCO).

Therapeutic advances using targeted biologicals and small-molecule drugs have achieved significant success in the treatment of chronic allergic, autoimmune, and inflammatory diseases particularly for some patients with severe, treatment-resistant forms. This has been aided by improved identification of disease phenotypes. Despite these achievements, not all severe forms of chronic inflammatory and autoimmune diseases are successfully targeted, and current treatment options, besides allergen immunotherapy for selected allergic diseases, fail to change the disease course. T cell-based therapies aim to cure diseases through the selective induction of appropriate immune responses following the delivery of engineered, specific cytotoxic, or regulatory T cells (Tregs). Adoptive cell therapies (ACT) with genetically engineered T cells have revolutionized the oncology field, bringing curative treatment for leukemia and lymphoma, while therapies exploiting the suppressive functions of Tregs have been developed in nononcological settings, such as in transplantation and autoimmune diseases. ACT with Tregs are also being considered in nononcological settings such as cardiovascular disease, obesity, and chronic inflammatory disorders. After describing the general features of T cell-based approaches and current applications in autoimmune diseases, this position paper reviews the experimental models testing or supporting T cell-based approaches, especially Treg-based approaches, in severe IgE-mediated responses and chronic respiratory airway diseases, such as severe asthma and COPD. Along with an assessment of challenges and unmet needs facing the application of ACT in these settings, this article underscores the potential of ACT to offer curative options for patients with severe or treatment-resistant forms of these immune-driven disorders.

The Role of IgG4 in the Fine Tuning of Tolerance in IgE-Mediated Allergy and Cancer.

Among the four immunoglobulin G (IgG) subclasses, IgG4 is the least represented in serum of a healthy human and it is considered an "odd" antibody. The IgG4 antibody has unique structural features that affect its biological function. These include the ability to undergo antigen-binding fragment (Fab)-arm exchange, to create fragment crystallizable (Fc) - Fc binding with other IgG4 and other IgG subclass antibodies, have a unique affinity profile for Fc gamma receptors (FcγRs) and no binding to complement component C1q. Altogether, these characteristics support anti-inflammatory roles of IgG4 leading to immune tolerance. Under conditions of chronic antigenic stimulation and Th2-type inflammation, both tissue and serum IgG4 levels are increased. This review seeks to highlight how in allergen immunotherapy IgG4 can confer a protective role as a "blocking" antibody and safeguard from subsequent allergen exposure, while IgG4 can confer immunomodulatory functions to support malignancy. While Th2 conditions drive polarization of macrophages to the M2a subtype, chronic antigen stimulation drives B cell class switching to IgG4 to further support phenotypical macrophage changes towards an M2b-like state. M2b-like macrophages can secrete chemokine (C-C motif) ligand 1 (CCL1) and interleukin-10 (IL-10) to support regulatory cell recruitment and to further shape a tolerogenic microenvironment. Thereby, IgG4 have a Janus-faced role, favorable in allergy but detrimental in cancer.

Filling the Antibody Pipeline in Allergy: PIPE Cloning of IgE, IgG1 and IgG4 against the Major Birch Pollen Allergen Bet v 1.

Birch pollen allergy is among the most prevalent pollen allergies in Northern and Central Europe. This IgE-mediated disease can be treated with allergen immunotherapy (AIT), which typically gives rise to IgG antibodies inducing tolerance. Although the main mechanisms of allergen immunotherapy (AIT) are known, questions regarding possible Fc-mediated effects of IgG antibodies remain unanswered. This can mainly be attributed to the unavailability of appropriate tools, i.e., well-characterised recombinant antibodies (rAbs). We hereby aimed at providing human rAbs of several classes for mechanistic studies and as possible candidates for passive immunotherapy. We engineered IgE, IgG1, and IgG4 sharing the same variable region against the major birch pollen allergen Bet v 1 using Polymerase Incomplete Primer Extension (PIPE) cloning. We tested IgE functionality and IgG blocking capabilities using appropriate model cell lines. In vitro studies showed IgE engagement with FcεRI and CD23 and Bet v 1-dependent degranulation. Overall, we hereby present fully functional, human IgE, IgG1, and IgG4 sharing the same variable region against Bet v 1 and showcase possible applications in first mechanistic studies. Furthermore, our IgG antibodies might be useful candidates for passive immunotherapy of birch pollen allergy.

IgG4 drives M2a macrophages to a regulatory M2b-like phenotype: potential implication in immune tolerance.

BACKGROUND: Macrophages can be converted in vitro into immunoregulatory M2b macrophages in the presence of immune complexes (ICs), but the role of the specific subclasses IgG1 or IgG4 in this phenotypic and functional change is not known. OBJECTIVE: We aimed to refine the original method by applying precisely defined ICs of the subclasses IgG4 or IgG1 constructed by two independent methods. METHODS: Monocyte-derived macrophages (MDMs) were treated with M-CSF, followed by IL-4/IL-13 to induce the M2a allergic phenotype. To mimic unspecific or allergen-specific ICs, plates were coated with myeloma IgG1 or IgG4, or with grass pollen allergen Phl p 5 followed by recombinant human Phl p 5-specific IgG1 or IgG4. M2a polarized macrophages were then added, cultured, and examined for cellular markers and cytokines by flow cytometry, ELISA, and rtPCR. Alternatively, immune complexes with IgG1 or IgG4 were formed using protein L. RESULTS: IgG4 ICs down regulated CD163 and CD206 on M2a cells, and significantly increased IL-10, IL-6, TNFα, and CCL1 secretion, indicating a shift to an M2b-like phenotype. Treatment with IgG4 ICs resulted in expression of FcγRII and down modulation of FcγRII compared with IgG1 treated cells (P = 0.0335) or untreated cells (P < 0.00001). CONCLUSION: Immune complexes with subclasses IgG1 and IgG4 can in vitro be generated by plate absorption, and in fluid form by protein L. Cross-linking of FcγRIIb by the IgG4 subclass redirects pro-allergic M2a macrophages to an M2b-like immunosuppressive phenotype. This suggests an interplay of macrophages with IgG4 in immune tolerance, likely relevant in allergen immunotherapy.

AllergoOncology: Microbiota in allergy and cancer-A European Academy for Allergy and Clinical Immunology position paper.

The microbiota can play important roles in the development of human immunity and the establishment of immune homeostasis. Lifestyle factors including diet, hygiene, and exposure to viruses or bacteria, and medical interventions with antibiotics or anti-ulcer medications, regulate phylogenetic variability and the quality of cross talk between innate and adaptive immune cells via mucosal and skin epithelia. More recently, microbiota and their composition have been linked to protective effects for health. Imbalance, however, has been linked to immune-related diseases such as allergy and cancer, characterized by impaired, or exaggerated immune tolerance, respectively. In this AllergoOncology position paper, we focus on the increasing evidence defining the microbiota composition as a key determinant of immunity and immune tolerance, linked to the risk for the development of allergic and malignant diseases. We discuss novel insights into the role of microbiota in disease and patient responses to treatments in cancer and in allergy. These may highlight opportunities to improve patient outcomes with medical interventions supported through a restored microbiome.

Comparing biologicals and small molecule drug therapies for chronic respiratory diseases: An EAACI Taskforce on Immunopharmacology position paper.

Chronic airway diseases such as asthma and chronic obstructive pulmonary disease (COPD), together with their comorbidities, bear a significant burden on public health. Increased appreciation of molecular networks underlying inflammatory airway disease needs to be translated into new therapies for distinct phenotypes not controlled by current treatment regimens. On the other hand, development of new safe and effective therapies for such respiratory diseases is an arduous and expensive process. Antibody-based (biological) therapies are successful in treating certain respiratory conditions not controlled by standard therapies such as severe allergic and refractory eosinophilic severe asthma, while in other inflammatory respiratory diseases, such as COPD, biologicals are having a more limited impact. Small molecule drug (SMD)-based therapies represent an active field in pharmaceutical research and development. SMDs expand biologicals' therapeutic targets by reaching the intracellular compartment by delivery as either an oral or topically based formulation, offering both convenience and lower costs. Aim of this review was to compare and contrast the distinct pharmacological properties and clinical applications of SMDs- and antibody-based treatment strategies, their limitations and challenges, in order to highlight how they should be integrated for their optimal utilization and to fill the critical gaps in current treatment for these chronic inflammatory respiratory diseases.

Macrophage induced gelsolin in response to Group B Streptococcus (GBS) infection.

Group B Streptococcus (GBS) has evolved several strategies to avoid host defences. We have shown that interaction of macrophages with GBS causes macrophage calpain activation, cytoskeletal disruption and apoptosis, consequences of intracellular calcium increase induced by membrane permeability alterations provoked by GBS-ß-haemolysin. Open question remains about what effect calcium influx has on other calcium-sensing proteins such as gelsolin, involved in cytoskeleton modulation and apoptosis. Therefore we analysed the effect of GBS-III-COH31:macrophage interaction on gelsolin expression. Here we demonstrate that an early macrophage response to GBS-III-COH31 is a very strong gelsolin increase, which occurs in a time- and infection-ratio-dependent manner. This is not due to transcriptional events, translation events, protein turnover alterations, or protein-kinase activation, but to calcium influx, calpain activation and caspase-3 degradation. In fact, EGTA and PD150606 (calpain inhibitor) prevented gelsolin increase while BAF (caspase inhibitor) enhanced it. Since gelsolin increase is induced by highly ß-haemolytic GBS-III-NEM316 and GBS-V-10/84, but not by weakly ß-haemolytic GBS, or GBS-III-COH31 in conditions suppressing ß-haemolysin expression/activity and the presence of dipalmitoylphosphatidylcholine (ß-haemolysin inhibitor), GBS-ß-haemolysin is solely responsible for gelsolin increase causing, through membrane permeability defects, calcium influx and calpain activation. Early gelsolin increase could represent a macrophage response to antagonize apoptosis since gelsolin knockdown increases macrophage susceptibility to GBS-induced apoptosis. This response seems to be GBS specific because macrophage apoptosis by Staurosporine or Cycloeximide does not induce gelsolin.

Glucocorticoid-induced tumor necrosis factor receptor family-related ligand triggering upregulates vascular cell adhesion molecule-1 and intercellular adhesion molecule-1 and promotes leukocyte adhesion.

The interaction of glucocorticoid-induced tumor necrosis factor receptor-family related (GITR) protein with its ligand (GITRL) modulates different functions, including immune/inflammatory response. These effects are consequent to intracellular signals activated by both GITR and GITRL. Previous results have suggested that lack of GITR expression in GITR(-/-) mice decreases the number of leukocytes within inflamed tissues. We performed experiments to analyze whether the GITRL/GITR system modulates leukocyte adhesion and extravasation. For that purpose, we first evaluated the capability of murine splenocytes to adhere to endothelial cells (EC). Our results indicated that adhesion of GITR(-/-) splenocytes to EC was reduced as compared with wild-type cells, suggesting that GITR plays a role in adhesion and that this effect may be due to GITRL-GITR interaction. Moreover, adhesion was increased when EC were pretreated with an agonist GITR-Fc fusion protein, thus indicating that triggering of GITRL plays a role in adhesion by EC regulation. In a human in vitro model, the adhesion to human EC of HL-60 cells differentiated toward the monocytic lineage was increased by EC pretreatment with agonist GITR-Fc. Conversely, antagonistic anti-GITR and anti-GITRL Ab decreased adhesion, thus further indicating that GITRL triggering increases the EC capability to support leukocyte adhesion. EC treatment with GITR-Fc favored extravasation, as demonstrated by a transmigration assay. Notably, GITRL triggering increased intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) expression and anti-ICAM-1 and anti-VCAM-1 Abs reversed GITR-Fc effects. Our study demonstrates that GITRL triggering in EC increases leukocyte adhesion and transmigration, suggesting new anti-inflammatory therapeutic approaches based on inhibition of GITRL-GITR interaction.

Characterization of a new regulatory CD4+ T cell subset in primary Sjögren's syndrome.

OBJECTIVE: CD4(+)CD25(low)GITR(+) T lymphocytes expressing FoxP3 and showing regulatory function have been recently described in healthy donors (HD). The objective of the study was to investigate their presence and role in patients with primary SS (pSS). METHODS: CD4(+)CD25(low)GITR(+) cells circulating in peripheral blood (PB) of patients with pSS were isolated by MACS technique, their phenotype was studied by flow cytometry and real-time PCR, and their function was studied by in vitro co-culture. CD4(+)CD25(low)GITR(+) cells infiltrating salivary glands (SGs) were revealed by immunohistochemistry. RESULTS: Results indicated that conventional CD4(+)CD25(high) regulatory T cells (Tregs) are decreased, whereas CD4(+)CD25(low)GITR(+) cells are expanded in the PB of pSS as compared with HD. Phenotypic analysis demonstrated that CD4(+)CD25(low)GITR(+) cells display Treg markers, including FoxP3, TGF-ß and IL-10, and functional experiments demonstrated that they exert a strong inhibitory activity against autologous effector cells. CD4(+)CD25(low)GITR(+) cells were detectable in great number in the SG inflammatory infiltrate. Interestingly, PB CD4(+)CD25(low)GITR(+) cell expansion was evident only in patients with inactive disease, while conventional CD4(+)CD25(high) Treg number was not associated with disease activity. CONCLUSION: The present data demonstrate that circulating CD4(+) cells expressing GITR, but with low levels of CD25 (CD4(+)CD25(low)GITR(+)), are detectable in pSS patients. These cells, displaying Treg phenotype and function, are present in SG inflamed tissues and are expanded in the PB of subjects with inactive disease. Data suggest that the expansion of CD4(+)CD25(low)GITR(+) cells in pSS may represent a counter-regulatory attempt against autoimmune-driven inflammation and may provide a new target for future treatment strategies.

CD4(+) CD25(low) GITR(+) cells: a novel human CD4(+) T-cell population with regulatory activity.

Treg subsets play a role in sustaining peripheral tolerance, are characterized by markers such as forkhead winged-helix transcription factor (FOXP3) and CD25, and produce suppressive cytokines, such as IL-10 and TGF-ß. Glucocorticoid-induced TNF receptor family-related (GITR) protein has been suggested to regulate Treg activity in mice. The aim of our study was to investigate GITR expression in human CD4(+) T lymphocytes and its possible role in Treg function. Results indicate that a subset of CD4(+) T cells in the peripheral blood expresses GITR and low levels of CD25 (CD4(+) CD25(low) GITR(+) ). These cells show Treg features as they express FOXP3, IL-10, TGF-ß and are anergic but, as opposed to natural Tregs, express low levels of CTLA-4 and are CD127(high) . CD4(+) CD25(low) GITR(+) cells represent a low percentage of the CD4(+) T-cell population (0.32-1.74%) and are mostly memory cells. Functional experiments demonstrated that CD4(+) CD25(low) GITR(+) cells have relevant suppressive activity that depends on TGF-ß. Moreover, an anti-GITR Ab inhibited their suppressive activity, as observed in CD4(+) CD25(+) murine Tregs. Taken together, these data indicate that human CD4(+) CD25(low) GITR(+) cells represent a distinct Treg subpopulation.

Ameliorating Atopy by Compensating Micronutritional Deficiencies in Immune Cells: A Double-Blind Placebo-Controlled Pilot Study.

BACKGROUND: Functional iron deficiency facilitates allergy development and amplifies the symptom burden in people experiencing allergies. Previously we selectively delivered micronutrients to immune cells with ß-lactoglobulin as carrier (holoBLG), resulting in immune resilience and allergy prevention. OBJECTIVE: The clinical efficacy of a food for special medical purposes-lozenge containing ß-lactoglobulin with iron, polyphenols, retinoic acid, and zinc (holoBLG lozenge) was assessed in allergic women. METHODS: In a randomized, double-blind, placebo-controlled pilot study, grass- and/or birch pollen-allergic women (n = 51) were given holoBLG or placebo lozenges over 6 months. Before and after dietary supplementation, participants were nasally challenged and the blood was analyzed for immune and iron parameters. Daily symptoms, medications, pollen concentrations, and well-being were recorded by an electronic health application. RESULTS: Total nasal symptom score after nasal provocations improved by 42% in the holoBLG group versus 13% in the placebo group. The combined symptom medication score during the birch peak and entire season as well as the entire grass pollen season improved in allergic subjects supplemented with the holoBLG lozenge by 45%, 31%, and 40%, respectively, compared with the placebo arm. Participants ingesting the holoBLG lozenge had improved iron status with increased hematocrit values, decreased red cell distribution width, and higher iron levels in circulating CD14+ cells compared with the placebo group. CONCLUSIONS: Targeted micronutrition with the holoBLG lozenge seemed to be effective in elevating the labile iron levels in immune cells and reducing the symptom burden in allergic women in this pilot study. The underlying allergen-independent mechanism provides evidence that dietary nutritional supplementation of the immune system is one of the ways to combat atopy.

Secretory protein beta-lactoglobulin in cattle stable dust may contribute to the allergy-protective farm effect.

BACKGROUND: Growing up on a cattle farm and consuming raw cow's milk protects against asthma and allergies. We expect a cattle-specific protein as active component in this farm effect. METHODS: Dust was collected from cattle and poultry stables and from mattresses of households. Urine was obtained from cattle, and ambient aerosols were sampled. Samples were analysed for BLG by SDS PAGE/immunoblot and mass spectrometry, and for association with metals by SEC-ICP-MS. PBMC of healthy donors were incubated with BLG +/- zinc, and proliferation and cytokines determined. BALB/c mice were pre-treated intranasally with stable dust extract containing BLG or depleted of BLG, and subsequent allergy response after sensitization was evaluated on antibody and symptom level. RESULTS: A major protein in dust from cattle farms and ambient air was identified as BLG. Urine from female and male cattle is a major source of BLG. In dust samples, BLG was associated with zinc. In vitro, zinc-BLG provoked significantly lower proliferation of CD4+ and CD8+ cells while inducing significantly higher levels of IFN-γ and IL-6 than the apo-BLG devoid of zinc. In vivo, pre-treatment of mice with dust extract containing BLG resulted in lower allergy symptom scores to BLG and unrelated Bet v 1 than pre-treatment with extract depleted of BLG. These in vitro and in vivo effects were independent of endotoxin. CONCLUSION: The lipocalin BLG is found in large amounts in cattle urine, accumulates in bovine dust samples and is aerosolized around farms. Its association with zinc favorably shapes the human cellular immune response towards Th1-cytokines in vitro. BLG together with zinc in stable dust protects mice from allergic sensitization. BLG with its associated ligands may in an innate manner contribute to the allergy-protective farm effect.

IgG4 induces tolerogenic M2-like macrophages and correlates with disease progression in colon cancer.

IgG4 subclass antibodies are expressed in alternative Th2 environments featuring high IL-10 expression, including several solid tumors such as melanoma. To induce tolerance, allergen immunotherapy mediates antibody class switching from pro-inflammatory IgE to anti-inflammatory IgG4. We previously reported that IgG4 drives allergic M2 macrophages toward tolerogenic states. Here we assessed the roles of IgG4 and macrophage activation in colorectal cancer (CRC). In this observer-blinded, case-control study, we analyzed total circulating serum IgE, IgG1 and IgG4 levels in CRC (n = 38) patients with (n = 13, TxNxM1) or without (n = 25, TxNxM0) metastasis, and in healthy donors (n = 21). Primary cultures of circulating monocyte-derived macrophages from healthy controls and CRC patients were further evaluated in their responses to stimulation with IgG1 or IgG4. We found higher absolute serum levels of IgG4 in patients with CRC. IgG4 enabled polarization of macrophages derived from CRC patients and healthy controls into alternatively-activated tolerogenic M2b phenotypes. IgG4-stimulated M2 macrophages were characterized by lower surface CD206, CD163, CD14, and CD11b expression and higher CCL-1, IL-10, and IL-6 production. IgG4 was less potent that IgG1 in triggering antibody-dependent cell-mediated phagocytosis (ADCP) of cancer cells. Further, higher z-normalized IgG4/-IgE sera level ratios correlated with the presence of metastasis (p = .0247 and p = .0009, respectively) in CRC patients. High IgG4 in CRC synergizes with macrophages in shaping an immunosuppressive microenvironment and impairs anti-cancer effector cell functions. The shift of serum IgG4/IgE ratios toward enhanced tolerance induction in metastatic disease indicates a role for high IgG4 in disease progression and poor prognostic outcome.