RESUMO
Antigen-presenting cells (APCs) are critical cells bridging innate and adaptive immune responses by taking up, processing, and presenting antigens to naïve T cells. At steady state, APCs thus control both tissue homeostasis and the induction of tolerance. In allergies however, APCs drive a Th2-biased immune response that is directed against otherwise harmless antigens from the environment. The main types of APCs involved in the induction of allergy are dendritic cells, monocytes, and macrophages. However, these cell types can be further divided into local, tissue-specific populations that differ in their phenotype, migratory capacity, T-cell activating potential, and production of effector molecules. Understanding if distinct populations of APCs contribute to either tissue-specific immune tolerance, allergen sensitization, or allergic inflammation will allow us to better understand disease pathology and develop targeted treatment options for different stages of allergic disease. Therefore, this review describes the main characteristics, phenotypes, and effector molecules of the APCs involved in the induction of allergen-specific Th2 responses in affected barrier sites, such as the skin, nose, lung, and gastrointestinal tract. Furthermore, we highlight open questions that remain to be addressed to fully understand the contribution of different APCs to allergic disease.
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Células Apresentadoras de Antígenos , Hipersensibilidade , Humanos , Alérgenos , Linfócitos T , FenótipoRESUMO
The COVID-19 pandemic highlights the importance of efficient and safe vaccine development. Vaccine adjuvants are essential to boost and tailor the immune response to the corresponding pathogen. To allow for an educated selection, we assessed the effect of different adjuvants on human monocyte-derived dendritic cells (DCs) and their ability to polarize innate and adaptive immune responses. In contrast to commonly used adjuvants, such as aluminum hydroxide, Toll-like receptor (TLR) agonists induced robust phenotypic and functional DC maturation. In a DC-lymphocyte coculture system, we investigated the ensuing immune reactions. While monophosphoryl lipid A synthetic, a TLR4 ligand, induced checkpoint inhibitors indicative for immune exhaustion, the TLR7/8 agonist Resiquimod (R848) induced prominent type-1 interferon and interleukin 6 responses and robust CTL, B-cell, and NK-cell proliferation, which is particularly suited for antiviral immune responses. The recently licensed COVID-19 vaccines, BNT162b and mRNA-1273, are both based on single-stranded RNA. Indeed, we could confirm that the cytokine profile induced by lipid-complexed RNA was almost identical to the pattern induced by R848. Although this awaits further investigation, our results suggest that their efficacy involves the highly efficient antiviral response pattern stimulated by the RNAs' TLR7/8 activation.
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Adjuvantes Imunológicos/farmacologia , COVID-19/imunologia , Células Dendríticas/imunologia , Imunidade Celular/efeitos dos fármacos , SARS-CoV-2/imunologia , Linfócitos T/imunologia , Adolescente , Adulto , Idoso , Feminino , Humanos , Imidazóis/farmacologia , Lipídeo A/análogos & derivados , Lipídeo A/farmacologia , Masculino , Pessoa de Meia-Idade , Receptores Toll-Like/imunologiaRESUMO
BACKGROUND: The experimental fusion protein rFlaA:Betv1 was shown to efficiently suppress allergen-specific sensitization in mice. However, the detailed mechanism of rFlaA:Betv1-mediated immune modulation is not fully understood. In this study, we investigated the effect of rFlaA:Betv1 on naïve murine B cells. METHODS: Immune modulating capacity of rFlaA:Betv1 was screened in IL-10 reporter mice. B cells were isolated from spleens of naïve C57Bl/6, TLR5-/- , or MyD88-/- mice, stimulated with rFlaA:Betv1 and controls, and monitored for the expression of the regulatory B cell markers CD1d, CD24, CD38, and surface IgM by flow cytometry. Secreted cytokines, antibodies, and reactivity of the induced antibodies were investigated by ELISA and intracellular flow cytometry. Suppressive capacity of rFlaA:Betv1-stimulated B cells was tested in mDC:CD4+ T cell:B cell triple cultures. RESULTS: Upon in vivo application of rFlaA:Betv1 into IL-10-GFP reporter mice, CD19+ B cells were shown to produce anti-inflammatory IL-10, suggesting B cells to contribute to the immune-modulatory properties of rFlaA:Betv1. rFlaA:Betv1-induced IL-10 secretion was confirmed in human B cells isolated from buffy coats. In vitro stimulation of naïve murine B cells with rFlaA:Betv1 resulted in an mTOR- and MyD88-dependent production of IL-10 and rFlaA:Betv1 induced Bet v 1-reactive IgG production, which was not observed for IgA. rFlaA:Betv1-stimulated B cells formed a CD19+ CD24+ CD1d+ IgM+ CD38+ Breg subpopulation capable of suppressing Bet v 1-induced TH2 cytokine secretion in vitro. CONCLUSION: rFlaA:Betv1 can act as a thymus-independent B cell antigen, stimulating the mTOR- and MyD88-dependent differentiation of B cells displaying a regulatory phenotype, IL-10 secretion, antigen-binding antibody production, and a suppressive capacity in vitro.
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Linfócitos B Reguladores , Interleucina-10 , Camundongos , Humanos , Animais , Fator 88 de Diferenciação Mieloide/genética , Flagelina/química , Flagelina/genética , Serina-Treonina Quinases TOR , Imunoglobulina MRESUMO
PURPOSE OF REVIEW: Over the last years, we have learned that the metabolic phenotype of immune cells is closely connected to the cell's effector function. Understanding these changes will allow us to better understand allergic disease pathology and improve allergy treatment by modulating immune metabolic pathways. As part two of a two-article series, this review reports on the recent studies investigating the metabolism of the cell types involved in allergies and discusses the initial application of these discoveries in allergy treatment. RECENT FINDINGS: The cell types involved in allergic reactions display pronounced and highly specific metabolic changes (here discussed for epithelial cells, APCs, ILC2s, mast cells, eosinophils, and Th2 cells). Currently, the first drugs targeting metabolic pathways are tested for their potential to improve allergy treatment. Immune-metabolic changes observed in allergy so far are complex and depend on the investigated disease and cell type. However, our increased understanding of the underlying principles has pointed to several promising target molecules that are now being investigated to improve allergy treatment.
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Hipersensibilidade , Imunidade Inata , Humanos , Linfócitos , Hipersensibilidade/terapia , Células Th2/metabolismo , Mastócitos , Citocinas/metabolismoRESUMO
PURPOSE OF REVIEW: Recent high-level publications have shown an intricate connection between immune effector function and the metabolic state of the respective cells. In the last years, studies have begun analyzing the metabolic changes associated with allergies. As the first part of a two-article series, this review will briefly summarize the basics of immune metabolism and then focus on the recently published studies on metabolic changes observed in allergic patients. RECENT FINDINGS: In the last 3 years, immune-metabolic research in allergology had a clear focus on asthma with some studies also reporting findings in food allergy and atopic dermatitis. Current results suggest asthma to be associated with a shift in cellular metabolism towards increased aerobic glycolysis (Warburg metabolism), while also displaying substantial changes in fatty acid- and amino acid metabolism (depending on investigated patient collective, asthma phenotype, and disease severity). Understanding immune-metabolic changes in allergies will allow us to (I) better understand allergic disease pathology and (II) modulate immune-metabolic pathways to improve allergy treatment.
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Asma , Dermatite Atópica , Hipersensibilidade Alimentar , Humanos , Dermatite Atópica/terapiaRESUMO
PURPOSE OF REVIEW: Immunoglobulin A (IgA) mediates immune exclusion of antigens in the gut. Notably, IgA plays also a role in the prevention of IgE-mediated allergies and induction of immune tolerance. The present review addresses the role of IgA in the manifestation of IgE-mediated allergies, including allergen-specific immunotherapy (AIT), the regulation of IgA production, and the mechanism of IgA in immune cell activation. RECENT FINDINGS: The majority of studies report an association of IgA with the induction of immune tolerance in IgE-mediated allergies. However, reports on the involvement of humoral and mucosal IgA, IgA subtypes, monomeric and polymeric IgA, and the mechanism of IgA-mediated immune cell activation are confounding. Effects by IgA are likely mediated by alteration of microbiota, IgE-blocking capacity, or activation of inhibitory signaling pathways. However, the precise mechanism of IgA-regulation, the contribution of serum and/or mucosal IgA, and IgA1/2 subtypes, on the manifestation of IgE-mediated allergies, and the underlying immune modulatory mechanism are still elusive.
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Hipersensibilidade Imediata , Imunoglobulina A , Humanos , Hipersensibilidade Imediata/imunologia , Hipersensibilidade Imediata/prevenção & controle , Tolerância Imunológica , Imunidade , Imunoglobulina ERESUMO
Trained immune responses, based on metabolic and epigenetic changes in innate immune cells, are de facto innate immune memory and, therefore, are of great interest in vaccine development. In previous studies, the recombinant fusion protein rFlaA:Betv1, combining the adjuvant and toll-like receptor (TLR)5-ligand flagellin (FlaA) and the major birch pollen allergen Bet v 1 into a single molecule, significantly suppressed allergic sensitization in vivo while also changing the metabolism of myeloid dendritic cells (mDCs). Within this study, the immune-metabolic effects of rFlaA:Betv1 during mDC activation were elucidated. In line with results for other well-characterized TLR-ligands, rFlaA:Betv1 increased glycolysis while suppressing oxidative phosphorylation to different extents, making rFlaA:Betv1 a suitable model to study the immune-metabolic effects of TLR-adjuvanted vaccines. In vitro pretreatment of mDCs with cerulenin (inhibitor of fatty acid biosynthesis) led to a decrease in both rFlaA:Betv1-induced anti-inflammatory cytokine Interleukin (IL) 10 and T helper cell type (TH) 1-related cytokine IL-12p70, while the pro-inflammatory cytokine IL 1ß was unaffected. Interestingly, pretreatment with the glutaminase inhibitor BPTES resulted in an increase in IL-1ß, but decreased IL-12p70 secretion while leaving IL-10 unchanged. Inhibition of the glycolytic enzyme hexokinase-2 by 2-deoxyglucose led to a decrease in all investigated cytokines (IL-10, IL-12p70, and IL-1ß). Inhibitors of mitochondrial respiration had no effect on rFlaA:Betv1-induced IL-10 level, but either enhanced the secretion of IL-1ß (oligomycin) or decreased IL-12p70 (antimycin A). In extracellular flux measurements, mDCs showed a strongly enhanced glycolysis after rFlaA:Betv1 stimulation, which was slightly increased after respiratory shutdown using antimycin A. rFlaA:Betv1-stimulated mDCs secreted directly antimicrobial substances in a mTOR- and fatty acid metabolism-dependent manner. In co-cultures of rFlaA:Betv1-stimulated mDCs with CD4+ T cells, the suppression of Bet v 1-specific TH2 responses was shown to depend on fatty acid synthesis. The effector function of rFlaA:Betv1-activated mDCs mainly relies on glycolysis, with fatty acid synthesis also significantly contributing to rFlaA:Betv1-mediated cytokine secretion, the production of antimicrobial molecules, and the modulation of T cell responses.
Assuntos
Receptor 5 Toll-Like , Vacinas , Receptor 5 Toll-Like/metabolismo , Alérgenos , Interleucina-10/metabolismo , Flagelina/metabolismo , Hexoquinase/metabolismo , Glutaminase/metabolismo , Ligantes , Antimicina A/metabolismo , Antimicina A/farmacologia , Cerulenina/metabolismo , Cerulenina/farmacologia , Células Dendríticas , Proteínas Recombinantes/metabolismo , Citocinas/metabolismo , Adjuvantes Imunológicos/farmacologia , Vacinas/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Glicólise , Serina-Treonina Quinases TOR/metabolismo , Desoxiglucose/farmacologia , Oligomicinas/farmacologia , Ácidos Graxos/metabolismoRESUMO
Type I allergies are pathological, type 2 inflammatory immune responses against otherwise harmless environmental allergens that arise from complex interactions between different types of immune cells. Activated immune cells undergo extensive changes in phenotype and function to fulfill their effector functions. Hereby, activation, differentiation, proliferation, migration, and mounting of effector responses require metabolic reprogramming. While the metabolic changes associated with activation of dendritic cells, macrophages, and T cells are extensively studied, data about the metabolic phenotypes of the other cell types critically involved in allergic responses (epithelial cells, eosinophils, basophils, mast cells, and ILC2s) are rather limited. This review briefly covers the basics of cellular energy metabolism and its connection to immune cell function. In addition, it summarizes the current state of knowledge in terms of dendritic cell and macrophage metabolism and subsequently focuses on the metabolic changes associated with activation of epithelial cells, eosinophils, basophils, mast cells, as well as ILC2s in allergy. Interestingly, the innate key cell types in allergic inflammation were reported to change their metabolic phenotype during activation, shifting to either glycolysis (epithelial cells, M1 macrophages, DCs, eosinophils, basophils, acutely activated mast cells), oxidative phosphorylation (M2 macrophages, longer term activated mast cells), or fatty acid oxidation (ILC2s). Therefore, immune metabolism is of relevance in allergic diseases and its connection to immune cell effector function needs to be considered to better understand induction and maintenance of allergic responses. Further progress in this field will likely improve both our understanding of disease pathology and enable new treatment targets/strategies.
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Hipersensibilidade , Imunidade Inata , Basófilos , Humanos , Linfócitos , MastócitosRESUMO
PURPOSE OF REVIEW: The incidence of allergies is increasing and has been associated with several environmental factors including westernized diets. Changes in environment and nutrition can result in dysbiosis of the skin, gut, and lung microbiota altering the production of microbial metabolites, which may in turn generate epigenetic modifications. The present review addresses studies on pectin-mediated effects on allergies, including the immune modulating mechanisms by bacterial metabolites. RECENT FINDINGS: Recently, microbiota have gained attention as target for allergy intervention, especially with prebiotics, that are able to stimulate the growth and activity of certain microorganisms. Dietary fibers, which cannot be digested in the gastrointestinal tract, can alter the gut microbiota and lead to increased local and systemic concentrations of gut microbiota-derived short chain fatty acids (SCFAs). These can promote the generation of peripheral regulatory T cells (Treg) by epigenetic modulation and suppress the inflammatory function of dendritic cells (DCs) by transcriptional modulation. The dietary fiber pectin (a plant-derived polysaccharide commonly used as gelling agent and dietary supplement) can alter the ratio of Firmicutes to Bacteroidetes in gut and lung microbiota, increasing the concentrations of SCFAs in feces and sera, and reducing the development of airway inflammation by suppressing DC function. Pectin has shown immunomodulatory effects on allergies, although the underlying mechanisms still need to be elucidated. It has been suggested that the different types of pectin may exert direct and/or indirect immunomodulatory effects through different mechanisms. However, little is known about the relation of certain pectin structures to allergies.
Assuntos
Microbioma Gastrointestinal , Hipersensibilidade , Fibras na Dieta , Ácidos Graxos Voláteis , Humanos , Hipersensibilidade/tratamento farmacológico , PectinasRESUMO
BACKGROUND: Fusion proteins incorporating the Toll-like receptor 5 ligand flagellin are currently undergoing clinical trials as vaccine candidates for many diseases. OBJECTIVE: We studied the mechanisms of immune modulation by a flagellin:allergen fusion protein containing the Toll-like receptor 5 ligand flagellin A from Listeria monocytogenes and the birch pollen allergen Bet v 1 (recombinant flagellin A [rFlaA]:Betv1). METHODS: BALB/c mice were vaccinated with rFlaA:Betv1 in an experimental Bet v 1 sensitization model. Myeloid dendritic cells (mDCs) were differentiated from mouse bone marrow, and PBMCs were isolated from subjects with birch pollen allergy. Cells were stimulated with equimolar amounts of rFlaA, rBet v 1, rFlaA plus rBet v 1, or the rFlaA:Betv1 conjugate and analyzed for cell activation, cytokine secretion, and metabolic state. RESULTS: rFlaA:Betv1 displayed strong immune-modulating properties both in vivo and in vitro, as characterized by secretion of both proinflammatory and anti-inflammatory cytokines from murine mDCs and PBMCs from patients with birch allergy. rFlaA:Betv1 suppressed TH2 responses from Bet v 1-specific CD4+ T cells and prevented allergic sensitization in a mouse allergy model. Aggregation of rFlaA:Betv1 resulted in stronger protein uptake accompanied by an increased resistance to microsomal digestion. Remarkably, rFlaA:Betv1 induced activation of mammalian target of rapamycin, which increased the metabolic activity of the stimulated mDCs. rFlaA:Betv1-mediated IL-10 secretion, but not proinflammatory cytokine secretion, was inhibited by rapamycin in mDCs. CONCLUSION: These results provide evidence that mammalian target of rapamycin is a key player involved in prevention of TH2 responses by flagellin A conjugate vaccines.
Assuntos
Alérgenos/imunologia , Flagelina/imunologia , Interleucina-10/imunologia , Rinite Alérgica Sazonal/imunologia , Serina-Treonina Quinases TOR/imunologia , Animais , Antígenos de Plantas/imunologia , Betula/imunologia , Medula Óssea/imunologia , Linfócitos T CD4-Positivos , Citocinas/imunologia , Células Dendríticas , Inflamação/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Pólen/imunologia , Proteínas Recombinantes/imunologia , Células Th2/imunologia , Receptor 5 Toll-Like/imunologiaRESUMO
PURPOSE OF REVIEW: Utilization of virus-like particles (VLPs) is considered to improve allergen-specific immunotherapy (AIT). AIT aims at the efficient uptake of the target allergen by antigen-presenting cells (APCs) subsequently inducing adaptive allergen-specific immune responses to induce tolerance. The purpose of this review is to describe the immune-modulating properties of VLPs per se and to summarize the application of VLPs as antigen carriers, preferably for Th2 cytokines or allergens, with and without simultaneous administration of adjuvants in order to modulate allergic immune responses. RECENT FINDINGS: Currently, a broad variety of approaches considering the origin of the VLPs, the choice of the adjuvant and antigen, and the coupling of the antigen are under preclinical investigation. The data provide evidence that VLPs used as carrier for antigens/allergens strongly increase antigen immunogenicity, and might be suitable to prevent allergies. However, systematic studies in mice showing the immunological mechanism and data from clinical studies are scarce.
Assuntos
Dessensibilização Imunológica , Hipersensibilidade/terapia , Vacinas de Partículas Semelhantes a Vírus , Alérgenos/administração & dosagem , Animais , HumanosRESUMO
UNLABELLED: To induce and trigger innate and adaptive immune responses, antigen-presenting cells (APCs) take up and process antigens. Retroviral particles are capable of transferring not only genetic information but also foreign cargo proteins when they are genetically fused to viral structural proteins. Here, we demonstrate the capacity of lentiviral protein transfer vectors (PTVs) for targeted antigen transfer directly into APCs and thereby induction of cytotoxic T cell responses. Targeting of lentiviral PTVs to APCs can be achieved analogously to gene transfer vectors by pseudotyping the particles with truncated wild-type measles virus (MV) glycoproteins (GPs), which use human SLAM (signaling lymphocyte activation molecule) as a main entry receptor. SLAM is expressed on stimulated lymphocytes and APCs, including dendritic cells. SLAM-targeted PTVs transferred the reporter protein green fluorescent protein (GFP) or Cre recombinase with strict receptor specificity into SLAM-expressing CHO and B cell lines, in contrast to broadly transducing vesicular stomatitis virus G protein (VSV-G) pseudotyped PTVs. Primary myeloid dendritic cells (mDCs) incubated with targeted or nontargeted ovalbumin (Ova)-transferring PTVs stimulated Ova-specific T lymphocytes, especially CD8(+) T cells. Administration of Ova-PTVs into SLAM-transgenic and control mice confirmed the observed predominant induction of antigen-specific CD8(+) T cells and demonstrated the capacity of protein transfer vectors as suitable vaccines for the induction of antigen-specific immune responses. IMPORTANCE: This study demonstrates the specificity and efficacy of antigen transfer by SLAM-targeted and nontargeted lentiviral protein transfer vectors into antigen-presenting cells to trigger antigen-specific immune responses in vitro and in vivo. The observed predominant activation of antigen-specific CD8(+) T cells indicates the suitability of SLAM-targeted and also nontargeted PTVs as a vaccine for the induction of cytotoxic immune responses. Since cytotoxic CD8(+) T lymphocytes are a mainstay of antitumoral immune responses, PTVs could be engineered for the transfer of specific tumor antigens provoking tailored antitumoral immunity. Therefore, PTVs can be used as safe and efficient alternatives to gene transfer vectors or live attenuated replicating vector platforms, avoiding genotoxicity or general toxicity in highly immunocompromised patients, respectively. Thereby, the potential for easy envelope exchange allows the circumventing of neutralizing antibodies, e.g., during repeated boost immunizations.
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Antígenos CD/imunologia , Vetores Genéticos/genética , Ovalbumina/imunologia , Receptores de Superfície Celular/imunologia , Linfócitos T Citotóxicos/imunologia , Proteínas Virais de Fusão/imunologia , Animais , Antígenos CD/biossíntese , Células CHO , Cricetulus , Células Dendríticas/imunologia , Proteínas de Fluorescência Verde/biossíntese , Células HEK293 , Humanos , Integrases/biossíntese , Integrases/genética , Lentivirus/genética , Ativação Linfocitária/imunologia , Vírus do Sarampo/genética , Glicoproteínas de Membrana/biossíntese , Glicoproteínas de Membrana/genética , Camundongos , Camundongos Knockout , Transporte Proteico , Receptores de Superfície Celular/biossíntese , Membro 1 da Família de Moléculas de Sinalização da Ativação Linfocitária , Transfecção , Vacinas de Subunidades Antigênicas/imunologia , Proteínas do Envelope Viral/biossíntese , Proteínas do Envelope Viral/genética , Proteínas Virais de Fusão/genéticaRESUMO
Recent advances in immunology have greatly improved our understanding of the pathomechanisms of food allergies. Food allergies are caused and maintained by complex interactions of the innate and adaptive immune system involving antigen-presenting cells (APC), T cells, group 2 innate lymphoid cells (ILC2), epithelial cells (EC) and effectors cells. Additionally, epigenetic factors, the intestinal microbiome and nutritional factors modulating the gastrointestinal lymphatic tissue probably have a significant impact on allergy development. However, why certain individuals develop tolerance while others mount allergic responses, the factors defining the allergenicity of food proteins, as well as the immunological mechanisms triggering allergy development have yet to be analyzed in detail.
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Hipersensibilidade Alimentar/genética , Hipersensibilidade Alimentar/imunologia , Imunidade Inata/genética , Imunidade Inata/imunologia , Fenômenos Imunogenéticos/genética , Epigênese Genética/genética , Epigênese Genética/imunologia , Predisposição Genética para Doença/genética , Humanos , Modelos Genéticos , Modelos ImunológicosRESUMO
Type I hypersensitivity, or so-called type I allergy, is caused by Th2-mediated immune responses directed against otherwise harmless environmental antigens. Currently, allergen-specific immunotherapy (AIT) is the only disease-modifying treatment with the potential to re-establish clinical tolerance towards the corresponding allergen(s). However, conventional AIT has certain drawbacks, including long treatment durations, the risk of inducing allergic side effects, and the fact that allergens by themselves have a rather low immunogenicity. To improve AIT, adjuvants can be a powerful tool not only to increase the immunogenicity of co-applied allergens but also to induce the desired immune activation, such as promoting allergen-specific Th1- or regulatory responses. This review summarizes the knowledge on adjuvants currently approved for use in human AIT: aluminum hydroxide, calcium phosphate, microcrystalline tyrosine, and MPLA, as well as novel adjuvants that have been studied in recent years: oil-in-water emulsions, virus-like particles, viral components, carbohydrate-based adjuvants (QS-21, glucans, and mannan) and TLR-ligands (flagellin and CpG-ODN). The investigated adjuvants show distinct properties, such as prolonging allergen release at the injection site, inducing allergen-specific IgG production while also reducing IgE levels, as well as promoting differentiation and activation of different immune cells. In the future, better understanding of the immunological mechanisms underlying the effects of these adjuvants in clinical settings may help us to improve AIT.
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Dessensibilização Imunológica , Hipersensibilidade , Humanos , Adjuvantes Imunológicos/uso terapêutico , Alérgenos , Hidróxido de Alumínio , Adjuvantes FarmacêuticosRESUMO
In order to ensure valid diagnostics for occupational test allergen solutions despite the ongoing reduction in the availability of commercial test extracts, a plan B was initiated for the possible production of skin prick test (SPT) solutions in public pharmacies. For important occupational allergen sources (wheat and rye, storage mites, animal epithelia, mold material) laboratory extraction methods were analyzed in comparison to pharmacy compatible extraction methods regarding protein quantity and quality in SDS-PAGE combined with silver staining. Subsequently, using the example of bovine epithelia, adapted extraction procedures as well as in-process and final product controls were transferred to a public pharmacy. Allergen sources with a high protein content, such as wheat and rye grains as well as storage mites, showed good comparability of the extractable protein quantity and protein pattern, regardless of the applied extraction method. In contrast, allergen source materials with a low total protein content, such as animal epithelia and molds, can benefit from laboratory extraction conditions such as mechanical disruption and specific buffer additives. In the qualitative protein silver staining, characteristic protein patterns were identified for each allergen source. Depending on the extraction method, only minor differences in total protein patterns were observed in animal epithelia and molds. Using source materials from two suppliers, the resulting allergen extracts displayed clear differences in protein content in storage mites and quantitative and qualitative differences in molds. A practical preparation attempt of SPT solutions in a public pharmacy was successful. SPT solutions prepared with adapted pharmacy extraction methods showed a comparable protein and Bos d 2 allergen content and equivalent qualities in the protein pattern compared to a previously available commercial SPT solution. Accordingly, it can be assumed that standardized SPT solutions with sufficient allergen quality for occupational allergen sources can be prepared in public pharmacies if certified allergen sources with appropriate protein content are available.
RESUMO
The availability of high-quality skin test allergens is a prerequisite for the reliable diagnosis of occupational type I allergies. Due to the withdrawal of existing marketing authorizations (MAs) by pharmaceutical companies and the lack of new MAs for commercial test allergens, there is an increasing diagnostic gap in Germany and other EU member states, which makes it necessary to investigate alternative ways of providing in vivo diagnostics. The German Medicinal Products Act (Arzneimittelgesetz = AMG) allows for the possibility of preparing medicinal products in pharmacies without the need for an MA or a manufacturing authorization pursuant to Section 13 (2) No. 1 in conjunction with Section 13 (2a) Sentence 2 No. 3 AMG. This also includes test allergens. In addition to the AMG, the requirements of the German Ordinance on the Operation of Pharmacies (Apothekenbetriebsordnung - ApBetrO) and the European Pharmacopoeia apply in particular. Medicolegal and practical challenges, as well as potentials of manufacturing skin prick test solutions in public pharmacies are presented based on examples of different allergen source materials.
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Pectins are dietary fibers that are attributed with several beneficial immunomodulatory effects. Depending on the degree of esterification (DE), pectins can be classified as high methoxyl pectin (HMP) or low methoxyl pectin (LMP). The aim of this study was to investigate the effects of pectin methyl-esterification on intestinal microbiota and its immunomodulatory properties in naive mice. Supplementation of the diet with LMP or HMP induced changes in the composition of the intestinal microbiota in mice toward Bacteroides, which was mainly promoted by HMP. Metabolome analysis of stool samples from pectin-fed mice showed a different effect of the two types of pectin on the levels of short-chain fatty acids and bile acids, which was consistent with highly efficient in vivo fermentation of LMP. Analysis of serum antibody levels showed a significant increase in IgG and IgA levels by both pectins, while FACS analysis revealed a decrease of infiltrating inflammatory cells in the intestinal lamina propria by HMP. Our study revealed that the structural properties of the investigated pectins determine fermentability, effects on microbial composition, metabolite production, and modulation of immune responses. Consumption of HMP preferentially altered the gut microbiota and suppressed pro-inflammatory immune responses, suggesting a beneficial role in inflammatory diseases.
Assuntos
Microbioma Gastrointestinal , Pectinas , Camundongos , Animais , Pectinas/química , Esterificação , Fibras na Dieta/farmacologia , Fibras na Dieta/metabolismo , FermentaçãoRESUMO
Background: A recombinant fusion protein combining the adjuvant and TLR5-ligand flagellin with the major birch pollen allergen Bet v 1 (rFlaA:Betv1) has been suggested to prevent the manifestation of birch allergy. Noteworthy, rFlaA:Betv1 induced both pro- and anti-inflammatory responses which were differentially regulated. However, the mechanism by which flagellin fusion proteins modulate allergen-specific immune responses, especially the mechanisms underlying IL-1ß secretion and their contribution to the overall immune responses remains elusive. Objective: To investigate the mechanisms underlying the production of IL-1ß from rFlaA:Betv1 stimulated macrophages. Methods: Macrophages were derived from mouse peritoneal-, human buffy-coat-, and PMA-differentiated THP-1 (wild type or lacking either ASC, NLRP3, or NLRC4) cells. Macrophages were stimulated with non-modified rFlaA:Betv1, mutant variants lacking either the flagellin DC0 domain or a sequence motif formerly described to mediate TLR5-activation, and respective controls in the presence or absence of inhibitors interfering with MAPK- and NFκB-signaling. Cytokine secretion was analyzed by ELISA and intracellular signaling by Western Blot. To study the contribution of IL-1ß to the overall immune responses, IL1R-deficient mouse peritoneal macrophages were used. Results: rFlaA:Betv1 consistently activated all types of investigated macrophages, inducing higher IL-1ß secretion compared with the equimolar mixture of both proteins. rFlaA:Betv1-induced activation of THP-1 macrophages was shown to be independent of either the TLR5-activating sequence motif or the flagellin DC0 domain but depended on both NLRP3- and NLRC4-inflammasomes. In addition, NFκB and SAP/JNK MAP kinases regulated rFlaA:Betv1-induced inflammasome activation and cytokine secretion by modulating pro-Caspase-1- and pro-IL-1ß-expression in THP-1 macrophages. Finally, lack of IL-1ß positive feedback via the IL1R strongly diminished the rFlaA:Betv1-induced secretion of IL-1ß, IL-6, and TNF-α from peritoneal macrophages. Conclusion: The mechanisms contributing to rFlaA:Betv1-induced IL-1ß secretion from macrophages were shown to be complex, involving both NLRC4- and NLRP3-inflammsomes, as well as NFκB- and SAP/JNK MAP kinase-signaling. Better understanding the mechanisms regulating the activation of immune cells by novel therapeutic candidates like the rFlaA:Betv1 fusion protein will allow us to further improve and develop new treatment strategies when using flagellin as an adjuvant.