Regulation of the humoral type 2 immune response against allergens and helminths.

The type 2 immune response is associated with helminth infections and allergic inflammation where antibody production of the IgG1 and IgE isotypes can elicit protective or proinflammatory functions. Studies over the past few years revealed important new insights regarding the regulatory mechanisms orchestrating the humoral type 2 immune response. This includes investigations on B-cell extrinsic signals, such IL-4 and IL-21, derived from different T-helper cell subsets or discovery of new follicular helper T cells with regulatory or IgE-promoting activities. In addition, studies on B-cell intrinsic factors required for germinal center formation and class switch recombination, including the transcription factors STAT3, STAT6, and BCL-6, led to a better understanding of these processes in type 2 immune responses. Here, we review the current understanding of mechanisms controlling humoral type 2 immunity in vivo including the generation of IgE-producing plasma cells and the memory IgE response.

Modulation of the humoral immune response by constitutively active STAT6 expression in murine B cells.

The transcription factor STAT6 regulates gene expression in response to IL-4 and IL-13. To further investigate how activated STAT6 modulates B cells development and function in vivo, we characterized mice that express a constitutively active version specifically in B cells. CD19Cre_STAT6vt mice show spontaneous phosphorylation and nuclear translocation of STAT6 in B cells. About 80 genes were more than twofold up- or downregulated in splenic B cells from CD19Cre_STAT6vt as compared to control mice. B cell development, tissue localization, and populations of follicular and marginal zone B cells, B1 B cells, GC B cells, and plasma cells (PCs) appeared to be normal. However, the number of IgE+ and IgG1+ GC B cells and PCs as well as serum IgE and IgG1 levels were increased in CD19Cre_STAT6vt mice. Infection with Lymphocytic choriomeningitis virus associated with high levels of TNF and IFN-γ did not prevent the development of a significantly increased IgE and IgG1 response against the virus in CD19Cre_STAT6vt mice. These results suggest that prolonged STAT6 activation during chronic allergic inflammation may result in IgE responses during subsequent viral or bacterial infection that could further stimulate mast cell activation even in the absence of the initial allergic response.

The B-cell antigen receptor of IgE-switched plasma cells regulates memory IgE responses.

BACKGROUND: Allergic inflammation is driven by IgE-producing plasma cells (PCs), which are required for IgE-mediated activation of mast cells and basophils. Repeated antigen encounter elicits a memory IgE response with elevated serum IgE titers and accumulation of IgE-producing PCs. However, the cellular compartment and molecular signals that underlie the immunologic memory of IgE responses remain unclear. OBJECTIVE: With this study we aimed at clarifying whether inactivation of the cytoplasmic immunoglobulin tail tyrosine (ITT) motif in transmembrane IgE (mIgE) impairs the memory IgE response in mice. METHODS: We generated mice with an inactivated mIgE-ITT motif and analyzed serum IgE levels as well as the generation of IgE-producing germinal center B cells and PCs subsequent to primary and secondary infection with helminths. In vitro cultures were used to study the mIgE-ITT-controlled expression of mIgE on the surface of PCs. Systemic mast cell activation was determined by serum Mcpt1 ELISA in response to ovalbumin challenge. RESULTS: mIgE-ITT-mutant mice showed an impaired memory IgE response subsequent to helminth infection. Furthermore, sensitization and challenge of mIgE-ITT-mutant mice with ovalbumin resulted in diminished serum IgE titers and reduced mast cell activation. The mIgE-ITT motif was required for optimal cell surface expression of mIgE B-cell antigen receptors but not for intracellular IgE expression in PCs. CONCLUSION: These results indicate that the mIgE B-cell antigen receptor plays a critical role in establishing or maintaining the population of IgE-producing PCs during memory IgE responses.

Formation of melanoidins - Aldol reactions of heterocyclic and short-chain Maillard intermediates.

In the course of the Maillard reaction, reducing sugars and amino compounds are converted to colorants, whose chemical structures are still mostly unknown. Active methylene compounds like norfuraneol that can initiate aldol condensation reactions are considered as key intermediates in this reaction. The aim of the present study was to characterize color formation of norfuraneol with different carbonyl compounds and to identify the underlying mechanisms of the reaction. Norfuraneol was incubated with methylglyoxal or diacetyl at elevated temperatures and the resulting reaction mixtures were analyzed by means of high-resolution mass spectrometry. It was demonstrated that aldol reactions lead to the formation of heterogeneous carbohydrate-based oligomers, which are likely to contribute to the elevated browning observed in the reaction mixtures. Furthermore, redox reactions were identified as another important part of the reaction, resulting in an increasing number of double bonds in the detected reaction products.

Structural characterization of polar melanoidins deriving from Maillard reaction intermediates - A model approach.

Chemical conversions of reducing sugars and amino compounds induce the formation of heterogenous, high-molecular-weight colorants ('melanoidins') with widely unknown chemical structures. Model experiments of reactive intermediates have proven to be suitable for unravelling the formation mechanisms of colored reaction products. Here, the active methylene norfuraneol was selected and incubated individually as well as in combination with glyoxal, glycolaldehyde, and acetaldehyde at elevated temperatures. Photometric and chromatographic methods as well as mass spectrometry were used to analyze the colored reaction products and reveal the reactivity of different carbonyls regarding the formation of heterogenous oligomers. Aqueous solutions of norfuraneol and glyoxal exceeded the color formation of all other model reaction systems and it could be shown that the initial reactants as well as their degradation products were incorporated into the colorants. The colored oligomers described herein were composed of carbohydrate-based intermediates of the Maillard reaction and defined as melanoidin precursors or pre-melanoidins.

B cell fate mapping reveals their contribution to the memory immune response against helminths.

An estimated quarter of the human world population is infected with gastrointestinal helminths causing major socioeconomic problems in endemic countries. A better understanding of humoral immune responses against helminths is urgently needed to develop effective vaccination strategies. Here, we used a fate mapping (FM) approach to mark germinal center (GC) B cells and their developmental fates by induced expression of a fluorescent protein during infection of mice with the helminth Nippostrongylus brasiliensis. We could show that FM+ cells persist weeks after clearance of the primary infection mainly as CD80+CD73+PD-L2+ memory B cells. A secondary infection elicited expansion of helminth-specific memory B cells and plasma cells (PCs). Adoptive transfers and analysis of somatic mutations in immunoglobulin genes further revealed that FM+ B cells rapidly convert to PCs rather than participating again in a GC reaction. These results provide new insights in the population dynamics of the humoral immune response against helminths.

Melanoidins Formed by Heterocyclic Maillard Reaction Intermediates via Aldol Reaction and Michael Addition.

Thermal treatment of food leads to the formation of melanoidins by reactions of carbohydrates with free amino acids or proteins in the late stage of Maillard reaction. The aim of this study was the identification of reaction mechanisms responsible for the formation of melanoidins involving active methylenes with the heterocyclic structure and the structural characterization of the resulting products. For this purpose, norfuraneol was incubated with the aldehydes pyrrole-2-aldehyde or furfural at 125 °C. With the help of high-resolution mass spectrometry, the structural composition of oligomers of up to fifteen C5-units could be described for the first time. Aldol and Michael reactions could be identified as crucial steps for the formation of new C-C-bonds. With increasing heating time, the elimination of water from the products was facilitated, and oxidation reactions of integrated reductone structures lead to the expansion of conjugated double-bond systems responsible for the color formation of the samples.

Formation of Reactive Intermediates, Color, and Antioxidant Activity in the Maillard Reaction of Maltose in Comparison to d-Glucose.

In this study, the Maillard reaction of maltose and d-glucose in the presence of l-alanine was investigated in aqueous solution at 130 °C and pH 5. The reactivity of both carbohydrates was compared in regards of their degradation, browning, and antioxidant activity. In order to identify relevant differences in the reaction pathways, the concentrations of selected intermediates such as 1,2-dicarbonyl compounds, furans, furanones, and pyranones were determined. It was found, that the degradation of maltose predominantly yields 1,2-dicarbonyls that still carry a glucosyl moiety and thus subsequent reactions to HMF, furfural, and 2-acetylfuran are favored due to the elimination of d-glucose, which is an excellent leaving group in aqueous solution. Consequently, higher amounts of these heterocycles are formed from maltose. 3-deoxyglucosone and 3-deoxygalactosone represent the only relevant C6-1,2-dicarbonyls in maltose incubations and are produced in nearly equimolar amounts during the first 60 min of heating as byproducts of the HMF formation.

Browning Potential of C6-α-Dicarbonyl Compounds under Maillard Conditions.

In this work, the three major C6-α-dicarbonyl compounds glucosone (GLUC), 1-deoxyglucosone (1-DG), and 3-deoxyglucosone (3-DG) were synthesized and examined under Maillard conditions (aqueous solutions with the addition of l-alanine at 130 °C and pH 5/8). For the first time, the resulting color formation, antioxidant activity, and generation of short-chained α-dicarbonyls were investigated and compared to incubations of d-glucose and d-fructose. An additive effect on the formation of color, an antagonistic effect on the generation of α-dicarbonyl compounds, and a synergistic effect on the antioxidant activity could be observed for the 1-DG/GLUC combination. Despite their common degradation products, different extinctions could be measured, with 3-DG showing the strongest color formation, followed by GLUC and 1-DG. The analyzed α-dicarbonyl compounds have no direct impact on the formation of color but are precursors for most of the colored compounds. The main difference between the three substances is their ability to form different heterocyclic degradation products, such as pyranones (1-DG), furanones (1-DG), furans (GLUC and 3-DG), and the corresponding N-heterocycles in the presence of amino components. This seems to be the main reason for their varying browning potential and antioxidant activity.

Antioxidant Properties of Heterocyclic Intermediates of the Maillard Reaction and Structurally Related Compounds.

It is well established that a wide range of reductones is formed in the course of the Maillard reaction and that these substances contribute to the oxidative stability of food. The aim of this study was to analyze 12 important heterocyclic intermediates with and without reductone structure as well as structurally related substances under equal conditions to compare their antioxidant properties in detail. For this purpose, five methods were selected including photometrical methods such as the trolox equivalent antioxidant capacity assay and an electron paramagnetic resonance spectroscopic method. Reductones with furan-3-one structure and 2,3-dihydro-3,5-dihydroxy-6-methyl-4H-pyran-4-one were reducing in all assays, whereas isomaltol and maltol did not react in assays based on the reduction of metal ions because of their complexing abilities. The introduction of protecting groups to the free hydroxyl functions of selected reductones could nearly eliminate their reducing abilities. In addition, the oxidation products of the different reductive heterocycles were compared after treatment with iodine. Mainly short-chained organic acids such as lactic, glycolic, and glyceric acid are formed as result of the degradation, which indicates 1,3-dicarbonyl cleavage reactions of corresponding tricarbonyl compounds as intermediates of the oxidation.

Antioxidant capacity of 1-deoxy-D-erythro-hexo-2,3-diulose and D-arabino-hexo-2-ulose.

The antioxidant capacity of two 1,2-dicarbonyl compounds, 1-deoxy-d-erythro-hexo-2,3-diulose (1-deoxyglucosone) and d-arabino-hexo-2-ulose (d-glucosone), was investigated. Both compounds are key intermediates of the Maillard reaction, and both possess a reductone-like structure. The reductive potential of the reductones was measured with the trolox equivalent antioxidant capacity (TEAC) assay and the Folin-Ciocalteu reagent (FCR) assay. Their antioxidant capacity set them apart from their precursors and other typical Maillard reaction products. Using electron paramagnetic resonance (EPR) spectroscopy, the special radical scavenging behavior of 1-deoxyglucosone and d-glucosone was measured. Both exhibited a slow, but constant, scavenging ability over the course of several hours, even days. It was postulated that this characteristic behavior is caused by the isomeric composition and the transformation to the particular antioxidant form. Reaction mixtures of 1-deoxyglucosone showed a correlation between the decrease of antioxidant properties and the decomposition of 1-deoxyglucosone.

Reactivity of thermally treated α-dicarbonyl compounds.

The degradation reaction of thermally treated 3-deoxy-d-erythro-hexos-2-ulose and methylglyoxal, both key intermediates in Maillard chemistry, was investigated. Different analytical strategies were accomplished to cover the broad range of formed products and their different chemical behavior. These involved HPLC-DAD and accordingly LC/MS analysis of the quinoxaline derivates, GC/MS analysis of the acetylated quinoxalines, and GC-FID analysis of the decyl ester of acetic acid. As a main degradation product of 3-deoxy-d-erythro-hexos-2-ulose, 5-(hydroxymethyl)furfural could be identified. At alkaline pH values, 3-deoxy-d-erythro-hexos-2-ulose generated various acids but no colored products. In contrast, thermal treatment of methylglyoxal yielded high molecular weight, brownish products. A dimer of methylglyoxal, first precursor for aldol-based polymerization of methylglyoxal, could be clearly identified by GC/MS.

Milieu dependence of isomeric composition of D-arabino-hexo-2-ulose in aqueous solution determined by high-resolution NMR spectroscopy.

In this study, high-resolution (1)H NMR spectroscopy (600.03 MHz) and (13)C NMR spectroscopy (150.89 MHz) were used to elucidate the structures of equilibrating d-arabino-hexo-2-ulose (GLUC) (1) isomers in aqueous solution. Four isomers were formed from the investigated ketohexose, and their equilibrium is dependent on the pH value and temperature. Only hydrated GLUC (1) isomers were identified. The (2)C5-ß-2,6-pyranoid and the ß-2,5-furanoid GLUC (1) isomer were exclusively formed in aqueous solution. Thus, (4)C1-1,5-pyranoid isomers are predominating in the crystalline state. An increase in solution pH or temperature led to a pairwise conversion of configurative information. Thus, changing the measurement conditions permits control over the equilibrium's characteristic. Furthermore, all GLUC (1) isomers showed comparable reaction behavior regarding pH- and temperature-dependent degradation reactions.

Milieu dependence of isomeric composition of 1-deoxy-D-erythro-hexo-2,3-diulose in aqueous solution determined by high-resolution NMR spectroscopy.

In this study, high-resolution (1)H (600.03 MHz) and (13)C NMR spectroscopy (150.89 MHz) were used to elucidate the structures of the identifiable isomers of a complex mixture of equilibrating 1-deoxy-D-erythro-hexo-2,3-diulose (1-DG) (1) isomers in aqueous solution. Approximately 15 isomers were formed from the investigated deoxy-ketohexose, and the equilibrium of the isomers is dependent on the pH values and temperature. In contrast to non-hydrated isomers, hydrated 1-DG (1) isomers were exclusively formed in aqueous solution. Their relative concentration increased when the pH was increased to more basic conditions. An increase in solution temperature led to the elimination of water and an increase in the relative concentration of the non-hydrated 1-DG (1) isomers. The observed influence of different conditions on the isomeric composition was explained for all isomers. Also, these observations provided information for structure elucidation. Furthermore, the reaction behaviour of 1-DG (1) under basic conditions at high temperatures (up to 347.5K) was interpreted and described.

Efficient preparation of photoswitchable dithienylethene-linker-conjugates by palladium-catalyzed coupling reactions of terminal alkynes with thienyl chlorides and other aryl halides.

Three photochromic dithienylethene-linker-conjugates with an adamantane core containing different spacer lengths and footprint areas with carboxylic anchoring groups are synthesized. The synthetic routes start either from the ethynylene-linker 5 or the iodo-substituted linker 8. Reaction conditions for the final Sonogashira coupling step between ethynylene-linker 5 with the chloro-substituted dithienylethene 4 in the presence of [PdCl(2)(CH(3)CN)(2)]/X-Phos and Cs(2)CO(3) or K(3)PO(4) are optimized using 2-chloro-5-methylthiophene (9) and triethylsilylacetylene or triisopropylsilylacetylene (10a,b) as model compounds. Experimental conditions are found to suppress the activation of the C(sp)-Si bond in TIPS-acetylene 10b, a reaction leading to a subsequent cross-coupling reaction to form by-product 12. Furthermore, activation of the C(sp)-Si bond in the presence of the fluorinated backbone of the chloro-substituted dithienylethene 4 can also be prevented. The photochromic properties of the conjugate 3 and its precursor dithienylethene 7b are also investigated.