Hop-derived fraction rich in beta acids and prenylflavonoids regulates the inflammatory response in dendritic cells differently from quercetin: unveiling metabolic changes by mass spectrometry-based metabolomics.

Dendritic cells (DCs) represent a heterogeneous family of immune cells that link innate and adaptive immunity and their activation is linked to metabolic changes that are essential to support their activity and function. Hence, targeting the metabolism of DCs represents an opportunity to modify the inflammatory and immune response. Among the natural matrices, Humulus lupulus (Hop) compounds have recently been shown to exhibit immunomodulatory and anti-inflammatory activity. This study aimed to evaluate the ability of specific Hop fractions to modulate DCs metabolism after stimulation with lipopolysaccharide (LPS) by an untargeted metabolomics approach and compare their effect with flavonol quercetin. Following liquid chromatography-based fractionation, three fractions (A, B, and C) were obtained and tested. Cytokine and gene expression were evaluated using ELISA and qPCR, respectively, while the untargeted metabolomics analysis was performed using a combined HILIC-HRMS and DI-FT-ICR approach. The HOP C fraction and quercetin could both reduce the production of several inflammatory cytokines such as IL-6, IL-1α, IL-1ß, and TNF, but differently from quercetin, the HOP C mechanism is independent of extracellular iron-sequestration and showed significant upregulation of the Nrf2/Nqo1 pathway and Ap-1 compared to quercetin. The untargeted analysis revealed the modulation of several key pathways linked to pro-inflammatory and glycolytic phenotypes. In particular, HOP C treatment could modulate the oxidative step of the pentose phosphate pathway (PPP) and reduce the inflammatory mediator succinate, citrulline, and purine-pyrimidine metabolism, differently from quercetin. These results highlight the potential anti-inflammatory mechanism of specific Hop-derived compounds in restoring the dysregulated metabolism in DCs, which can be used in preventive or adjuvant therapies to suppress the undesirable inflammatory response.

Effect of perennial dust mites allergy on symptom severity of autumn allergic rhinitis in adults.

Background: Multiple immunoglobulin E (IgE) mediated sensitizations and/or allergies often coexist in patients with allergic rhinitis (AR). Several simultaneous allergen exposures in multiple IgE-mediated sensitizations and/or allergies may increase the allergen load and be related to disease severity. No study has verified whether positive allergen serum IgE levels and allergen categories together are associated with AR severity in adults. Objective: To investigate the effects of perennial dust mites (DMs) allergy and multiple serum sIgE-mediated autumn pollen allergy coexistence on symptom severity in adult patients with AR in autumn. Methods: In total, 153 patients with AR and with autumn pollen allergy (Artemisia argyi, ragweed, and hop) with or without DMs allergy were recruited in the autumn pollen season. Symptom severity was assessed by using the Chinese version of the visual analog scale (VAS): four rhinitis symptoms (sneezing, rhinorrhea, nasal pruritus, and nasal congestion) and two ocular symptoms (ocular itching and/or grittiness and/or redness, and ocular tearing) were scored at approximately the same period. We measured allergen serum sIgE levels for the inhaled allergens. The effects of DMs allergy and multiple autumn pollen allergy coexistence on symptom severity were analyzed. Results: Neither the sum of the autumn pollen allergens categories (total number of positive autumn pollen allergens, i.e., Artemisia argyi or ragweed or hop positive: 1; Artemisia argyi and ragweed positive: 2; Artemisia argyi, ragweed, and hop positive: 3) nor serum sIgE levels( total sIgE levels of positive autumn pollen allergens) exerted any influence on the severity of nasal and ocular symptoms (p > 0.05). When the concomitant DMs allergy status was considered, the sum of the positive autumn pollen allergen categories and accumulated positive autumn pollen and DMs serum sIgE levels (total levels of serum sIgE of positive autumn pollen allergens plus the levels of serum sIgE of DMs) had no influence on patients' symptom severity (p > 0.05). Conclusion: The coexistence of perennial DMs allergy and multiple autumn pollen allergy did not affect the severity of symptoms among adult patients with AR and with autumn pollen allergy in autumn.

Studies on relationships between air pollutants and allergenicity of Humulus Scandens pollen collected from different areas of Shanghai.

Pollen pollution and allergy are becoming prominent issues in China. However, few studies on pollinosis have been reported. As an allergen in the atmosphere, allergenic Humulus scandens pollen was collected from four districts of Shanghai, including Wusong (WS), Jiading (JD), Xujiahui (XJH) and Songjiang (SJ). The mass concentrations of SO2, NO2, O3, PM10, and PM2.5 (particulate matter with air dynamic diameter less than 10 and 2.5 µm, respectively) near the four sampling sites were also recorded during Humulus scandens pollen season. The allergenicity of the Humulus scandens pollen was assessed by using of a rat model and enzyme linked immunosorbent assay (ELISA). Relationships between the allergenicity and air pollutants were correlated. Our results demonstrated that the biological viability of the pollens collected from the four districts exhibited no significant differences. ELISA and dot blotting results further demonstrated that the serum of sensitized rats exhibited much higher immune-reactive response than that of control groups. Western blotting showed that the 15 KD (1KD = 1000 dalton) proteins of Humulus pollen led to the allergic response. The allergenic intensity of Humulus pollen protein from different samples followed the pattern: WS > JD > XJ > SJ. There was a negative relationship between the allergenicity of Humulus pollens and PM10 (R = -0.99) / PM2.5 (R = -0.73), and a positive relationship with O3 (R = 0.92). These data clearly showed that PM10 and PM2.5 could enhance Humulus pollen protein release, and O3 could aggravate the allergenicity of the Humulus pollen.

ISO-alpha-acids improve the hematoma resolution and prevent peri-hematoma inflammations by transforming microglia via PPARgamma-CD36 axis in ICH rats.

OBJECTIVE: To elucidate the effects of ISO-α-acids (IAAs), a PPAR-γ agonist, on ICH rats and its potential mechanism. MATERIAL AND METHODS: The Sprague Dawley rats ICH model was induced by stereotactic injecting of 100 µl autologous artery blood. Ninety male rats were randomly allocated to five groups: autologous blood and IAAs (IAA); received autologous blood, IAAs and PPAR-γ inhibitor (IAA + GW9662); autologous blood and normal Saline (Saline); only autologous blood (Mock); and only needle injection (Sham). Neurological functions were assessed by mNSS. Hematoma volume, brain water content, surface proteins and inflammatory factors were detected. The microglia anti-inflammatory abilities were also evaluated. RESULTS: IAAs were able to significantly decrease ICH rat's mNSS scores, alleviate brain water content, improve hematoma resolution than Saline, Mock (p < 0.05). More "M2" microglial/macrophage can be induced by IAAs. The expression of CD 36 was statistically higher in IAA than other groups (p < 0.05). Injection of IAAs led to a greatly increasing in CD 11b and CD 206 double-positive anti-inflammatory type microglial/macrophage, moreover, a reduction of inflammatory cytokines expression (p < 0.05). Such protective effects can be relieved by GW9662. CONCLUSIONS: This is the first study to elucidate the relationship between IAAs and ICH. IAAs were able to accelerate hematoma absorption, alleviate brain edema, suppress peri-hematoma inflammations and finally improved the outcome of ICH rats. The phenotype was due to the IAAs induction of "M2" microglial/macrophage via activating of PPAR-γ and increasing CD 36 expression.

Allergenicity of recombinant Humulus japonicus pollen allergen 1 after combined exposure to ozone and nitrogen dioxide.

Ozone (O3) and nitrogen dioxide (NO2) are thought to play primary roles in aggravating air pollution-induced health problems. However, the effects of joint O3/NO2 on the allergenicity of pollen allergens are unclear. Humulus japonicus pollen allergen 1 (Hum j1) is a profilin protein that causes widespread pollinosis in eastern Asia. In order to study the effects of combined O3/NO2 on the allergenicity of Hum j1, tandem six-histidine peptide tag (His6)-fused recombinant Hum j1 (rHum j1) was expressed in a prokaryotic system and purified through His6 affinity chromatography. The purified rHum j1 was used to immunize SD rats. Rat sera with high titers of IgG and IgE antibodies against rHum j1 were used for allergenicity quantification. The rHum j1 was exposed to O3/NO2, and changes in allergenicity of the exposed rHum j1 were assayed using the immunized rat antibodies. Tandem LC-MS/LC (liquid chromatography-mass spectrometer/liquid chromatography spectrometer) chromatography and UV and circular dichroism (CD) spectroscopy were used to study the structural changes in rHum j1. Our data demonstrated that a novel disulfide bond between the sulfhydryl groups of two neighboring cysteine molecules was formed after the rHum j1 exposure to joint O3/NO2, and therefore IgE-binding affinity was increased and the allergenicity was reinforced. Our results provided clues to elucidate the mechanism behind air pollution-induced increase in pollinosis prevalence.

[Immunological characteristics of the recombinant major pollen allergen pTSX2 of Humulus scandens].

OBJECTIVE: To identify the immunological characteristics of the recombinant major pollen allergen pTSX2 of Humulus scandens and evaluate its safety in immunotherapy of allergic asthma in mice. METHODS: Western blotting was used to characterize the immunological properties of pTSX2, and its immunogenicity in normal mice was evaluated by detecting sIgG and sIgE levels. The mouse models of allergic asthma were immunized with pTSX2 and examined for sIgE and sIgG levels, total cells and eosinophils percentage in BALF, interleukin-4 (IL-4) and interferon-γ (IFN-γ) levels in BALF and spleen homogenate, and changes in lung pathologies. RESULTS: Western blotting showed that pTSX2 reacted with the majority (about 70%) of sera from patients allergic to Humulus pollen. In normal mice, pTSX2 mainly induced the production of sIgG. In mouse models of allergic asthma, intervention with pTSX2 caused a significant reduction of sIgE and an increase of sIgG (P<0.05), significantly decreased the total cells and eosinophils in BALF (P<0.05), obviously lowered IL-4 but increased IFN-γ in BALF and spleen homogenate (P<0.05), and diminished inflammatory cell infiltration and percentage of eosinophils in the lung tissues. CONCLUSIONS: pTSX2 shows a definite therapeutic effect and safety in the treatment of allergic asthma in mice possibly by inhibiting sIgE and inducing sIgG production, suppressing airway allergic inflammation and regulating the balance between Thl and Th2.

[Purification and analysis of the immunoactivity of humulus pollen allergen].

OBJECTIVE: To purify the humulus pollen allergen and study the allergenicity and immunogenicity of it. METHODS: Crude humulus pollen extracts were purified by gel filtration with Sephadex G-75 and Sephacryl S-200HR. Various fractions of the allergen protein were collected respectively. The molecular weights of protein were confirmed by SDS-PAGE. The inhibition rate and reaction rate with sIgG and sIgE of patient's serum were determined by ELISA inhibition test and western blotting. RESULTS: Two peaks were obtained from crude humulus pollen extracts by gel filtration of Sephadex G-75. The first peak contained most of protein while the second peak contained little protein and lots of pigments. So the second peak was thrown away. P solution which contained the first peak and valley fraction were purified by gel filtration of Sephacryl S-200HR and four components that included the 1st peak, the valley, the 2nd peak and the end fraction were obtained. The results of electrophoresis demonstrated that purified humulus pollen contained more than 20 kinds of protein with the molecular weights ranged from 5.0 x 10(3) to 97.4 x 10(3). The fraction of the 1st peak contained protein with the molecular weights ranged from 43 x 10(3) to 97.4 x 10(3), and the fraction of the valley and the 2nd peak contained protein with the molecular weights ranged from 5.0 x 10(3) to 43 x 10(3). The fraction of the end contained protein with the molecular weights lower than 5.0 x 10(3). The results of ELISA inhibition test showed that the inhibition rate of the 1st peak, the valley, the 2nd peak and the end fraction to sIgG were 68%, 70%, 95%, 5% respectively, and those to sIgE were 25%, 64%, 71%, 11% respectively. The results of western blotting demonstrated that the reaction rate of the 1st peak, the valley, the 2nd peak and the end fraction with sIgG of patients' serum were 65.63%, 78.13%, 87.50%, 6.25% respectively, and those with patients' sIgE were 25.00%, 71. 88%, 84.38%, 15.63% respectively. CONCLUSION: Humulus pollen contained more than 20 kinds of protein. The proteins with molecular weights ranged from 5.0 x 10(3) to 43 x 10(3) were the major allergen with strong allergenicity and immunogenicity. The proteins with molecular weights ranged from 43 x 10(3) to 97.4 x 10(3) were subordinated allergen with strong immunogenicity and weak allergenicity.

Effects of a hop water extract on the compound 48/80-stimulated vascular permeability in ICR mice and histamine release from OVA-sensitized BALB/c mice.

The antiallergic properties of a hop water extract (HWE) were studied by evaluating the Evans blue leakage from ICR mice caused by compound 48/80 stimulation, and the histamine release from ovalbumin (OVA)-sensitized BALB/c mice. An oral administration of HWE significantly inhibited the vascular permeability and histamine release. HWE itself did not have any influence on the total and antigen-specific immunoglobulin E (IgE) production in OVA-sensitized mice. These results indicate that HWE exerted an antiallergic effect by inhibiting the release of chemical mediators from mast cells and basophiles.

Cloning, expression, and characterization of pollen allergens from Humulus scandens (Lour) Merr and Ambrosia artemisiifolia L.

AIM: To clone the pollen allergen genes in Humulus scandens (Lour) Merr (LvCao in Chinese) and short ragweed (Ambrosia artemisiifolia L) for recombinant allergen production and immunotherapy. METHODS: The allergen genes were selectively amplified in the weed pollen cDNA pool by using a special PCR profile, with the primers designed by a modeling procedure. Following truncated gene cloning and confirmation of the pollen source, unknown 3'cDNA ends were identified by using the 3'-RACE method. The gene function conferred by the full-length coding region was evaluated by a homologue search in the GenBank database. Recombinant proteins expressed in Escherichia coli pET-44 RosettaBlue cells were subsequently characterized by N-terminal end sequencing, IgE binding, and cross-reactivity. RESULTS: Three full-length cDNAs were obtained in each weed. Multiple alignment analysis revealed that the deduced amino acid sequences were 83% identical to each other and 56%-90% identical to panallergen profilins from other species. Five recombinant proteins were abundantly expressed in non-fusion forms and were confirmed by using the N-terminal end sequence identity. Sera from patients who were allergic to A artemisiifolia reacted not only with rAmb a 8(D03) derived from A artemisiifolia, but also with recombinant protein rHum s 1(LCM9) derived from H scandens, which confirmed the allergenicity and cross-reactivity of the recombinant proteins from the 2 sources. Comparison of the degenerate primers used for truncated gene cloning with the full-length cDNA demonstrated that alternative nucleotide degeneracy occurred. CONCLUSION: This study demonstrates a useful method for cloning homologous allergen genes across different species, particularly for little-studied species. The recombinant allergens obtained might be useful for the immunotherapeutic treatment of H scandens and/or A artemisiifolia pollen allergies.

[Cloning full-length homologous cDNAs of pollen allergens in Humulus Scandens(Lour.) Merr by degenerate primer].

AIM: To establish a stable and reliable method for fast cloning homologous genes of pollen allergens in allergen-containing plants. METHODS: Degenerate primers were designed based on the bioinformatic analysis of numerous allergens available from the database. Subsequent amplification of the allergen genes was conducted in the weed pollen cDNA pool by a selective PCR profile. Following the truncated gene cloning, RACE method was used to isolate full-length cDNA. Gene function was deduced by sequence alignment in GenBank database. The degenerate ability of the primer was compared with the full-length cDNA sequences. RESULTS: Three full-length cDNAs were obtained. Sequence analysis showed that these new genes shared as high as 79%-85% homology with a large amount of known allergen profilins and were hence regarded as members of panallergen profilin family. Comparing these genes with the degenerate primers that were initially used in truncated gene cloning revealed that alternative nucleotide degeneracy occurred beyond the degenerate site predesigned, suggesting that further degeneracy was expanded by Touchdown-gradient PCR. CONCLUSION: Cloning of homologous genes or allergen genes can be efficiently achieved by using the combination of degenerate primer with Touchdown-gradient RT-PCR in the species such as Humulus scandens that has not yet been investigated.