Staphylococcus Aureus Enterotoxin B Research Articles

Validation and shortcomings of the most common mouse model of chronic rhinosinusitis with nasal polyps

Chronic rhinosinusitis (CRS) is a highly prevalent airway disease worldwide. Whereas eosinophilic CRS with nasal polyps (eCRSwNP) represents its most severe phenotype, pathogenic mechanisms remain poorly understood despite a wide spectrum of in vitro and in vivo experimental models. A mouse model of experimental ovalbumin (OVA)-induced airway allergy with coadministration of Staphylococcus aureus enterotoxin B (SEB) has been widely used to study eosinophilic eCRSwNP. This study revisits the features of this model and its suitability for studying eCRS. We implemented the most used eCRSwNP mouse model based on OVA+SEB intranasal challenges. Readouts including inflammatory features by (immuno)histology of the sinonasal epithelium (NP formation, eosinophils, epithelial and basement membrane thickness, fibrosis, goblet cells, Charcot-Leyden crystals (CLC)-like, tight junctions) and IgE production by enzyme-linked immunosorbent assay (ELISA), were compared to features of the corresponding human disease. The OVA+SEB model induced eosinophilic inflammation of upper and lower airways, with epithelial and basement membrane thickening, goblet cell hyperplasia and subepithelial fibrosis in the sinuses, along increased IgE production. Except local IgE in nasal lavage (NL), which was only increased in OVA+SEB group, all other features did not differ between OVA and OVA+SEB groups. Macro- or microscopic NP were not detected. With the notable exception of local IgE production, the addition of SEB did not induce additional inflammatory or structural change in the sinuses from mice exposed to and challenged with OVA. This model might represent a model for severe upper airway allergy rather than a specific model of human eCRSwNP.

Crocin Inhibits the Type 2 Inflammatory Response Produced by ILC2s in Eosinophilic Nasal Polyps.

Chronic rhinosinusitis with nasal polyps (CRSwNP) is subdivided into type 1 and type 2 inflammatory responses according to the mucosal inflammatory patterns. Crocin can reduce the level of T-helper type 2 cell (Th2) cytokines, such as interleukin-4 (IL-4), and inhibit the nuclear factor kappa-B (NF-κB) signaling pathway. This study aimed to investigate the role of group 2 innate lymphoid cells (ILC2s) in type 2 inflammation in eosinophilic nasal polyps and the inhibitory effect of crocin on this inflammation. Immunohistochemistry and immunofluorescence were used to detect the expression of transcription factors and the infiltration of ILC2s in tissues. An ILC2 stimulation model in vitro was constructed based on IL-33 stimulation and treated with crocin. The explant models were constructed and treated with crocin to detect the expression of type 2 inflammation-related factors. Significantly more GATA-binding protein-3 (GATA3)-positive cells and chemoattractant receptor-homologous molecule expressed on T-helper type 2 cell (CRTH2)-positive cells, but fewer T-box expressed in T cell (T-bet)-positive cells, were found in eosinophilic nasal polyps (NPwEos). The expression levels of GATA3 and CRTH2 were significantly higher in NPwEos. Recombinant IL-33 stimulation increased the expression of GATA3, CRTH2, and type 2 cytokines (IL-4, IL-5, and IL-13) in ILC2s. In an IL-33-stimulated in vitro ILC2 culture model, crocin inhibited the type 2 inflammatory response, especially at lower concentrations (10 µM). The explant organoids of NPwEos were constructed in vitro, and Staphylococcus aureus enterotoxin B (SEB) was used to construct the type 2 inflammation model. Crocin at 10 µM concentration inhibited type 2 inflammation induced by SEB-stimulated explants. Crocin inhibited type 2 inflammation induced by ILC2 activation at low concentrations via inhibiting the activation of NF-κB.

A Higher Dose of Staphylococcus aureus Enterotoxin B Led to More Th1 and Lower Th2/Th1 Ratio in Th Cells.

Exposure to Staphylococcus aureus enterotoxin B (SEB) is one of the causes of food poisoning and is associated with several immune diseases due to its superantigen capability. This study aimed to characterize the differentiations of naïve Th cells stimulated with different doses of SEB. The expression of T-bet, GATA-3, and Foxp3 or secretion of IFN-γ, IL-4, IL-5, IL-13, and IL-10 were evaluated in wild-type (WT) or DO11.10 CD4 T cells co-cultured with bone marrow dendritic cells (BMDCs). We found that the balance of Th1/Th2 could be dominated by the doses of SEB stimulation. A higher SEB dose could induce more Th1 and a lower Th2/Th1 ratio in Th cells co-cultured with BMDCs. This different tendency of Th cell differentiation induced by the SEB complements the existing knowledge about SEB acting as a superantigen to activate Th cells. Additionally, it is also helpful in managing the colonization of S. aureus and food contamination of SEB.

Dietary supplementation with spray-dried animal plasma improves vaccine protection in aged mice.

Senescence is characterized by an aggravated inflammatory state that reduces vaccine responsiveness. Dietary supplementation with spray-dried porcine plasma (SDP) exerts anti-inflammatory effects in different mucosal areas. We aimed to determine if the anti-inflammatory properties of SDP improve the efficiency of immunization in senescent animals. Experiments were performed in 2-month-old and 6-month-old male SAMP8 mice fed control or SDP (8%) feeds for 4  months. The mice received nasal doses of 2.5  μg of Staphylococcus aureus enterotoxin B (SEB) or vehicle every 15  days (i.e., 3 times). Fifteen days after the last dose, a lethal shock was induced by intraperitoneal administration of SEB and LPS. Immunization increased anti-SEB IgA in intestinal and bronchoalveolar fluid (p < 0.05). After the lethal shock, all immunized aged mice that were supplemented with SDP survived, in contrast to only 66% of those fed the control feed (p < 0.05). Moreover, after the lethal challenge, aged mice showed higher expression levels of pro-inflammatory cytokines (Il-6, Tnf-α, Ifn-γ, and Il-1β) in jejunal and (Tnf-α, and Il-1β) in lung tissues (p < 0.05), which were reduced by SDP supplementation (p < 0.05). Furthermore, in senescent mice, SDP supplementation augmented Il-4 and Il-10 expression in both tissues (p < 0.05). SDP reduces the mucosal inflammation associated with aging, improving vaccine protection in senescent mice.

IL-17A facilitates type 2 inflammation in a modified eosinophilic chronic rhinosinusitis mouse model.

Eosinophilic chronic rhinosinusitis (ECRS) is predominantly characterized by nasal type 2 inflammation. The pathogenesis of this condition is complex. High levels of IL-17A are associated with eosinophil infiltration in some inflammatory diseases and contribute to the severity and insensitivity of corticosteroid therapy for chronic rhinosinusitis. In the first experiment, we constructed a modified ECRS mouse model using four groups of mice: phosphate-buffered saline (PBS)-sensitized and nasal instillation (control); PBS-sensitized and Staphylococcus aureus enterotoxin B (SEB) nasal instillation after nasal tamponade (SEB group); ovalbumin (OVA)-sensitized and nasal instillation (OVA group); and OVA-sensitized combined with OVA and SEB nasal instillation after nasal tamponade (OVA+SEB group). In the second experiment, we examined the role of IL-17A by dividing the mice into four groups: control group; ECRS group; ECRS+anti-IL-17A group; and ECRS + IL-17A group. The latter two groups received intraperitoneal injections of anti-IL-17A antibody or IL-17A, respectively. We constructed a modified ECRS mouse model (OVA+SEB group), where the IL-17A levels were upregulated in the nasal sinus of ECRS mice and the IL-17A levels were significantly correlated with eosinophil infiltration. We further demonstrated that IL-17A induced type 2 inflammation and eosinophil infiltration in the ECRS group of mice. In contrast, IL-17A neutralization attenuated type 2 inflammatory cytokine secretion and eosinophil infiltration. OVA sensitization and unilateral nasal tamponade, combined with SEB and OVA alternate nasal instillation (OVA+SEB group), could be used to construct a more typical ECRS mouse model in which IL-17A enhanced the expression of type 2 cytokines and eosinophil infiltration.

Iridescent polymeric film with tunable color responses to ultra-trace Staphylococcus aureus enterotoxin B

Colorimetric sensing materials have a rising demand in detection applications, especially in the field of naked-eye detection. Herein, a novel iridescent polymeric film that can respond to different Staphylococcus aureus enterotoxin B (SEB) concentrations in the visible range is reported. This film is fabricated by polymerization of methacrylic acid (MAA) and ethylene dimethacrylate (EGDMA) owing an inverse opal array (IOA) structure, and gold nanoparticles (AuNPs) are enchased on the polymer skeleton. This composite structure facilitates the film to respond to SEB at a LOD of 4.29 fg mL−1, making it the best performing colorimetric material to date. Cryo-Scanning Electron Microscopy and 3D AFM were used to observe the morphology change of the iridescent polymeric film after SEB binding. This film possesses great advantages for ultrasensitive visual detection of real samples. In addition, the iridescent polymeric film has the potential to become a universal sensing material, realizing the ultrasensitive visual detection of hazardous substances without equipment in the field by replacing the recognition element.

Oral exposure to Staphylococcus aureus enterotoxin B could promote the Ovalbumin-induced food allergy by enhancing the activation of DCs and T cells.

Recent work highlighted the importance of environmental contaminants in the development of allergic diseases. The intestinal mucosal barrier, Th (helper T) cells, DCs (dendritic cells), and intestinal flora were analyzed with flow cytometry, RNA-seq, and 16s sequencing in the present study to demonstrate whether the exposure of enterotoxins like Staphylococcus aureus enterotoxin B (SEB) in allergens could promote the development of food allergy. We found that co-exposure to SEB and Ovalbumin (OVA) could impair the intestinal barrier, imbalance the intestinal Th immune, and cause the decline of intestinal flora diversity in OVA-sensitized mice. Moreover, with the co-stimulation of SEB, the transport of OVA was enhanced in the Caco-2 cell monolayer, the uptake and presentation of OVA were promoted in the bone marrow dendritic cells (BMDCs), and Th cell differentiation was also enhanced. In summary, co-exposure to SEB in allergens should be considered a food allergy risk factor.

Novel immunological tools to investigate CD4+ T cell activation in the canine species

Abstract Opportunities to identify T cell epitopes in the dog have been thwarted by a lack of immunological tools specific for the species. As a result, antigen-specific triggers in diseases that affect dogs like autoimmune disorders and allergies remain unknown. Our objective was to develop immunological tools such as: IFN-γ enzyme-linked immunosorbent spot (ELISpot) and activation induced marker (AIM) assays, and an MHC class II tetramer to help overcome these hurdles. We collected splenocytes from dogs that underwent a routine splenectomy. We used these splenocytes to develop: an ELISpot assay detecting canine IFN-γ production by stimulated T cells, an AIM assay to assess early activation by CD40L expression on CD4+CD5+ canine splenocytes using a cross-reactive anti-human monoclonal antibody, and finally, we produced the first-ever canine MHC II tetramer specific for a common canine DR allele. We confirmed the efficacy of these tools in the presence of positive and negative controls, Staphylococcus aureus enterotoxin B (SEB) and dimethyl sulfoxide (DMSO), respectively. SEB induces a robust canine IFN-γ ELISpot readout quantifiable in canine splenocytes. Similarly, we discovered that canine CD40L expression peaks at 3 hours post-stimulation with SEB. Finally, we confirmed the development of the first canine pMHCII tetramer through size discriminatory SDS-PAGE analysis and peptide exchange assays. Our novel canine-specific reagents will help identify and characterize antigen-specific T cells and their cognate antigens driving canine MHC class II-restricted disease, thus guiding research towards the advancement of antigen-specific immunotherapies. Supported by a grant from the NIH (K01 OD027058)

A real-time immunocapture PCR (RT-IPCR) without interference of protein A for convenient detection of staphylococcal enterotoxin B from food and environmental samples

PurposeA real-time immunocapture PCR (RT-IPCR) has been fabricated for the detection of Staphylococcus aureus enterotoxin B (SEB) from food and environmental samples.MethodsConsidering the fact, anti-SEB immunoglobulin G (IgG) has affinity towards protein A, produced by nearly all S. aureus, and generates false-positive read out in all immuno-based assay. We have employed avian anti-SEB antibody (SEB-IgY) as capture probe, since IgY interact less efficiently to protein A and biotinylated SEB-specific monoclonal antibody (SEB -MAb) conjugated with reporter DNA as revealing probe for real-time PCR amplification and signal generation. Sensitivity and selectivity of the assay were evaluated employing closely related enterotoxins and other toxins.ResultsThe RT-IPCR is highly specific and sensitive (100 fg/mL). The practical applicability of the assay was tested using spiked food sample as well as naturally contaminated food samples. The sensitivity and specificity of RT-IPCR were not compromised by the foods tested and was able to detect SEB conveniently. Further, the assay was validated comparing with the in-house developed PCR, and plausible result was obtained.ConclusionThe developed assay can be utilized as a low-cost detection system of SEB in routine food testing laboratories.

Altered Mitochondrial Functions and Morphologies in Epithelial Cells Are Associated With Pathogenesis of Chronic Rhinosinusitis With Nasal Polyps.

PurposeChronic rhinosinusitis with nasal polyps (CRSwNP) is a complex inflammatory disease of the nasal and paranasal sinus mucosa. The disease is associated with mitochondrial dysfunction, structural changes in the mitochondria, and reactive oxygen species (ROS) generation. This study investigated whether there are functional and morphological changes in the mitochondria in the epithelial cells of nasal polyps (NPs) and Staphylococcus aureus enterotoxin B (SEB)-stimulated nasal epithelial cells.MethodsIn all, 30 patients with CRSwNP and 15 healthy subjects were enrolled. Mitochondrial ROS (mtROS) and changes in mitochondrial functions and structures were investigated in the uncinate tissue (UT) of healthy controls, the UT or NPs of CRSwNP patients, and human nasal epithelial cells with or without SEB stimulation.ResultsOxidative phosphorylation complexes showed various responses following SEB stimulation in the nasal epithelial cells, and their expressions were significantly higher in the NPs of patients with CRSwNP than in the UT of controls. Generation of mtROS was increased following SEB exposure in nasal epithelial cells and was reduced by pretreatment with MitoTEMPO, which is used as an mtROS scavenger. In the tissues, mtROS was significantly increased in the NPs of CRSwNP patients compared to the UT of controls or CRSwNP patients. The expressions of fusion- and fission-related molecules were also significantly higher in SEB-exposed nasal epithelial cells than in non-exposed cells. In tissues, the expression of fission (fission mediator protein 1)- and fusion (membrane and mitofusin-1, and optic atrophy protein 1)-related molecules was significantly higher in the NPs of CRSwNP patients than in UT of controls or CRSwNP patients. Transmission electron microscopy revealed elongated mitochondria in SEB-exposed nasal epithelial cells and epithelial cells of NPs.ConclusionsProduction of mtROS, disrupted mitochondrial function, and structural changes in nasal epithelial cells might be involved in the pathogenesis of CRSwNP.

Immunological profiling of key inflammatory drivers of nasal polyp formation and growth in chronic rhinosinusitis.

Chronic rhinosinusitis (CRS) is a chronic inflammatory condition of the upper airways, often associated with the formation of nasal polyps (CRSwNP). It is well established that macroscopically normal (non-polypoidal) sinonasal mucosa in CRSwNP patients can undergo polypoidal change over time, turning into frank polyps. However, little is known about what drives this process. This study aimed to investigate potential drivers of nasal polyp formation or growth through comparison of the immunological profiles of nasal polyps with contiguous non-polypoidal sinonasal mucosa, from the same patients. The immune profiles of three types of tissue were compared; nasal polyps and adjacent non-polypoidal sinonasal mucosa from 10 CRSwNP patients, and sinonasal mucosa from 10 control patients undergoing trans-sphenoidal pituitary surgery. Nasal polyp and control samples were also stimulated with Staphylococcus aureus enterotoxin B (SEB) using a nasal explant model, prior to cytokine analysis. Real time quantitative polymerase chain reaction (IL-5, T-bet, IL-17A, FoxP3, TLR-4, IL-8, IL-1beta and IL-6) and Luminex (IFNgamma, IL-5 and IL-17A) were used to quantify pro-inflammatory responses. Nasal polyps and contiguous non-polypoidal sinonasal mucosa from CRSwNP patients displayed a very similar pro-inflammatory profile. When stimulated with SEB, nasal polyps displayed a Th2/Th17 mediated response when compared to controls. In CRSwNP, nasal polyps and non-polypoidal sinonasal mucosa from the same patient displayed a similar pro-inflammatory profile skewed towards the Th2/Th17 pathway in nasal polyps following SEB stimulation, with evidence of disordered bacterial clearance. These factors may contribute to enhanced survival of bacteria and development of a chronic inflammatory milieu, potentially driving new polyp formation and recurrence following surgical removal.

Qualitative and Quantitative Analysis of the Immune Synapse in the Human System Using Imaging Flow Cytometry.

The immune synapse is the area of communication between T cells and antigen-presenting cells (APCs). T cells polarize surface receptors and proteins towards the immune synapse to assure a stable binding and signal exchange. Classical confocal, TIRF, or super-resolution microscopy have been used to study the immune synapse. Since these methods require manual image acquisition and time-consuming quantification, the imaging of rare events is challenging. Here, we describe a workflow that enables the morphological analysis of tens of thousands of cells. Immune synapses are induced between primary human T cells in pan-leukocyte preparations and Staphylococcus aureus enterotoxin B (SEB)-loaded Raji cells as APCs. Image acquisition is performed with imaging flow cytometry, also called In-Flow microscopy, which combines features of a flow cytometer and a fluorescence microscope. A complete gating strategy for identifying T cell/APC couples and analyzing the immune synapses is provided. As this workflow allows the analysis of immune synapses in unpurified pan-leukocyte preparations and hence requires only a small volume of blood (i.e., 1 mL), it can be applied to samples from patients. Importantly, several samples can be prepared, measured, and analyzed in parallel.

Type 2/Th2-driven inflammation impairs olfactory sensory neurogenesis in mouse chronic rhinosinusitis model.

BackgroundChronic rhinosinusitis (CRS) with nasal polyps (CRSwNP) is a chronic inflammatory disease often accompanied by impairment of sense of smell. This symptom has been somewhat overlooked, and its relationship to inflammatory cytokines, tissue compression, neuronal loss, and neurogenesis is still unclear.MethodsIn order to elucidate potential mechanisms leading to CRS in humans, we have established a type 2/T helper type 2 cell (Th2)‐mediated allergic CRS mouse model, based on house dust mite (HDM) and Staphylococcus aureus enterotoxin B (SEB) sensitization. The inflammatory status of the olfactory epithelium (OE) was assessed using histology, biochemistry, and transcriptomics. The sense of smell was evaluated by studying olfactory behavior and recording electro‐olfactograms (EOGs).ResultsAfter 22 weeks, a typical type 2/Th2‐mediated inflammatory profile was obtained, as demonstrated by increased interleukin (IL)‐4, IL‐5, and IL‐13 in the OE. The number of mast cells and eosinophils was increased, and infiltration of these cells into the olfactory mucosa was also observed. In parallel, transcriptomic and histology analyses indicated a decreased number of immature olfactory neurons, possibly due to decreased renewal. However, the number of mature sensory neurons was not affected and neither the EOG nor olfactory behavior was impaired.ConclusionOur mouse model of CRS displayed an allergic response to HDM + SEB administration, including the type 2/Th2 inflammatory profile characteristic of human eosinophilic CRSwNP. Although the sense of smell did not appear to be altered in these conditions, the data reveal the influence of chronic inflammation on olfactory neurogenesis, suggesting that factors unique to humans may be involved in CRSwNP‐associated anosmia.

1020 Assessment of the anti-inflammatory effects of cannabidiol and its fluorinated derivative in in vitro and in vivo models of atopic dermatitis

It is common wisdom in pharmacology that fluorination can significantly increase the efficacy of the active components in pharmaceuticals – actually, ca. 30% of the best-selling drugs worldwide contain fluorinated compounds. The aim of the current study was to assess the potential anti-inflammatory effects of cannabidiol (CBD), the major non-psychoactive component of the pant Cannabis sativa, and its fluorinated derivative (HUF-101) in various experimental systems modeling atopic dermatitis (AD). For the in vitro AD model, human epidermal keratinocytes were challenged with the combination of Staphylococcus aureus enterotoxin B (SEB) and thymic stromal lymphopoietin (TSLP), and expressions of certain marker molecules were assessed by RT-qPCR and ELISA. For the in vivo model, mice were sensitized with 2% oxazolone (OXA) before elicitation. Test compounds were applied topically (1 and 10 μM) after inducing skin inflammation and edema formation (in the ears) was measured with an engineer’s micrometer. In the in vitro model, expressions of certain pro-inflammatory cytokines (e.g. interleukin [IL]-1α, IL-1β, IL-6 and IL-8) were significantly down-regulated upon the administration of CBD and HUF-101. Of great importance, however, HUF-101 exhibited significantly higher potency in comparison to CBD. In the in vivo model, topical application of 1 μM CBD significantly reduced the OXA-induced ear edema; however, 10 μM CBD exerted insignificant effect. In contrast, HUF-101 attenuated OXA-induced edema formation at both concentrations. Intriguingly, similar to the in vitro conditions, the anti-inflammatory potency of HUF-101 was significantly greater than that of CBD. Our study provides the first evidence that CBD and its fluorinated derivative exert significant anti-inflammatory actions in models of AD. These intriguing data invite further pre-clinical and clinical studies to exploit the therapeutic potential of certain CBD derivatives in cutaneous inflammatory conditions.

Association between impaired IL-10 production following exposure to Staphylococcus aureus enterotoxin B and disease severity in eosinophilic chronic rhinosinusitis

BackgroundIL-10 is a major anti-inflammatory cytokine that prevents inflammation-mediated tissue damage. We characterized the production of IL-10 by sinonasal tissue cells following exposure to Staphylococcus aureus enterotoxin B (SEB), which elicits cellular responses and is associated with the pathogenesis of eosinophilic chronic rhinosinusitis (ECRS). MethodsDispersed nasal polyp (NP) cells and uncinate tissue (UT) cells were prepared from patients with CRS with and without NP, respectively. Cells were incubated with SEB, and then the levels of IL-10 in the cell supernatants were determined. The effect of neutralizing IL-10 on SEB-induced IL-5, IL-13, IFN-γ, and IL-17A production was examined. Expression of IL-10 in NPs was also determined. ResultsIL-10 was expressed in infiltrating inflammatory cells in NPs. NP cells, especially non-adherent NP cells, produced substantial amounts of IL-10 in response to SEB. Although baseline production of IL-10 was significantly higher in NP cells than UT cells, the degree of IL-10 response to SEB was not significantly different between the cell types. The degree of IL-10 production was negatively correlated with the degree of eosinophilia both in tissues and peripheral blood whereas positively correlated with the 1-s forced expiratory volume/forced vital capacity ratio. Patients with severe ECRS displayed a significant decrease in IL-10 production compared with those with non-ECRS. IL-10 neutralization significantly augmented SEB-induced IL-13 and IFN-γ production by NP cells. ConclusionsImpaired IL-10 production in response to SEB in NP may exacerbate the pathophysiology of ECRS including eosinophilia and lower airway obstruction.

Anti-inflammatory effect of bee venom in an allergic chronic rhinosinusitis mouse model.

Bee venom (BV) has long been used as anti-inflammatory agent in traditional oriental medicine; however, the effect of BV on chronic rhinosinusitis (CRS) is not commonly studied. The aim of the present study was to determine the anti-inflammatory effect of BV on an allergic CRS mouse model. An allergic CRS mouse model was established following the administration of ovalbumin with Staphylococcus aureus enterotoxin B (SEB) into the nose. A total of 0.5 or 5ng/ml of BV were intranasally applied 3times a week for 8weeks. Histopathological alterations were observed using hematoxylin and eosin, and Periodic acid Schiff staining. The levels of inflammatory cell infiltration, interleukin (IL)‑4, IL‑10 and interferon (INF)‑γ in nasal lavage fluid (NLF) were measured. Nuclear factor (NF)‑κB and activator protein (AP)‑1 expressions were also determined by immunohistochemical staining. The group treated with BV had significantly decreased inflammatory cell infiltration and PAS‑positive cells. The levels of INF‑γ, and neutrophil and eosinophil counts in NLF were significantly decreased, and the SEB‑induced NF‑κB and AP‑1 expressions in mouse nasal mucosa were significantly suppressed by 0.5 and 5ng/ml BV. Thus, BV exerted significant anti‑inflammatory effects in an allergic CRS mouse model and may have potential value for the treatment of CRS.

Role of mast cells in Staphylococcus aureus enterotoxin Binduced atopic dermatitis-like skin inflammation in mice

Objective To investigate the role of mast cells in Staphylococcus aureus enterotoxin B (SEB) -induced atopic dermatitis (AD) -like skin inflammation in BALB/c mice. Methods A total of 24 BALB/c mice were randomly and equally divided into 4 groups to be topically treated with ovalbumin (OVA group) , SEB (SEB group) , OVA+ SEB (OVA+ SEB group) and sodium chloride physiological solution (control group) respectively, so as to establish mouse models of epicutaneously induced AD-like skin inflammation. The AD-like skin lesions were evaluated by clinical observation and eczema area and severity index (EASI) . Biopsy specimens were obtained from lesional skin of mice and then subjected to toluidine blue staining and immunohistochemical staining to count the mast cells, observe the morphology and distribution of mast cells, and calculate the percentage of degranulated mast cells. Results After 7-week treatment, the OVA group, SEB group and OVA+ SEB group all showed severer local skin inflammation, higher EASI scores and denser infiltration of inflammatory cells compared with the control group. Moreover, the OVA+ SEB group showed significantly severer local skin inflammation, skin lesions and degree of infiltration of inflammatory cells compared with the OVA group and SEB group (all P < 0.05) . The number of mast cells in the dermis of AD-like skin lesions per high-power field (× 400) was significantly higher in the OVA group (median[quartile range]: 10.625[3.675]) , SEB group (11.000[4.163]) and OVA+ SEB group (13.875[8.813]) than that in the control group (5.925[2.088], all P < 0.05) . The SEB group (71.083% ± 14.519%) and OVA+ SEB group (58.767% ± 16.978%) both showed significantly higher percentage of degranulated mast cells compared with the OVA group (24.050% ± 11.161%, both P < 0.05) and control group (23.617% ± 8.132%, both P < 0.05) . Bivariate correlation analysis showed that the number of mast cells in the skin lesions was positively linearly correlated with the EASI scores (P < 0.05) . Conclusions Epicutaneous application of SEB can induce AD-like skin lesions in mice, and can exacerbate the severity of OVA-induced AD-like skin lesions. Mast cell proliferation, activation/degranulation and tryptase release may participate in the inflammation. Key words: Dermatitis, atopic; Mast cells; Staphylococcus aureus; Enterotoxins; Tryptases; Ovalbumin

Role of Staphylococcus aureus enterotoxin B in activation of LAD2 mast cells

Objective To investigate the role of Staphylococcus aureus enterotoxin B (SEB) in non-IgE mediated activation of mast cells (MCs) by in vitro co-culture of laboratory of allergic disease 2 (LAD2) cells and SEB. Methods The LAD2 cells were incubated with SEB at different concentrations of 0.01, 0.1, 1, 10 and 100 μg/ml, A23187 positive control and negative control separately for 30 minutes. Then, effects of SEB on the morphology of MCs were observed by using a light microscope, and culture supernatants of the above incubation systems were collected. The concentration of tryptase released from MCs was analyzed by enzymatic activity assay, and the level of histamine was detected by enzyme-linked immunosorbent assay (ELISA) . Results After 30-minute co-culture of LAD2 cells and SEB, MCs showed larger size, obscure boundaries, increased number of protuberances on the cell surface and decreased refractivity, with a radial burr fin-like appearance. After 30-minute co-culture of LAD2 cells and SEB at different concentrations of 0.01, 0.1, 1, 10 and 100 μg/ml, the concentrations of tryptase in the culture supernatants were 4.116 ± 0.651, 5.344 ± 0.874, 3.806 ± 0.459, 1.309 ± 0.247, 0.310 ± 0.199 ng/ml respectively. Additionally, the tryptase levels were significantly higher in the 0.01-, 0.1-, 1-μg/ml SEB groups than in the negative control group (1.538 ± 0.490, all P < 0.05) , and gradually decreased along with the increase of SEB concentrations. The histamine levels in the 0.01-, 0.1-, 1-, 10-and 100-μg/ml SEB groups were 242.409 ± 63.915, 522.491 ± 73.466, 550.926 ± 84.466, 334.397 ± 33.640, 226.527 ± 5.678 ng/ml respectively. In the 0.01-, 0.1-, 1-μg/ml SEB groups, the levels of histamine released from MCs were gradually increased along with the increase of SEB concentrations, and were significantly higher than those in the negative control group (146.436 ± 3.100, all P < 0.05) . However, with the continued increase of SEB concentrations, the histamine levels gradually decreased. Conclusion SEB can directly activate MCs by a non-IgE mediated mechanism, followed by morphologic changes of MCs and release of tryptase and histamine. Key words: Mast cells; Staphylococcus aureus; Enterotoxins; Tryptases; Histamine; Dermatitis, atopic

The Anti-Inflammatory Effect of Spray-Dried Plasma Is Mediated by a Reduction in Mucosal Lymphocyte Activation and Infiltration in a Mouse Model of Intestinal Inflammation.

Spray-dried preparations from porcine and bovine plasma can alleviate mucosal inflammation in experimental models and improve symptoms in patients with enteropathy. In rodents, dietary supplementation with porcine spray-dried plasma (SDP) attenuates intestinal inflammation and improves the epithelial barrier function during intestinal inflammation induced by Staphylococcus aureus enterotoxin B (SEB). The aim of this study was to discern the molecular mechanisms involved in the anti-inflammatory effects of SDP. Male C57BL/6 mice were fed with 8% SDP or control diet (based on milk proteins) for two weeks, from weaning until day 33. On day 32, the mice were given a SEB dose (i.p., 25 µg/mouse) or vehicle. SEB administration increased cell recruitment to mesenteric lymph nodes and the percentage of activated Th lymphocytes and SDP prevented these effects). SDP supplementation increased the expression of interleukin 10 (IL-10) or transforming growth factor- β (TGF-β) compared to the SEB group. The SEB challenge increased six-fold the expression of mucosal addressin cell adhesion molecule 1 (MAdCAM-1) and intercellular adhesion molecule 1 (ICAM-1); and these effects were attenuated by SDP supplementation. SEB also augmented NF-κB phosphorylation, an effect that was prevented by dietary SDP. Our results indicate that the anti-inflammatory effects of SDP involve the regulation of transcription factors and adhesion molecules that reduce intestinal cell infiltration and the degree of the inflammatory response.

Staphylococcus aureus Enterotoxin B Down-Regulates the Expression of Transforming Growth Factor-Beta (TGF-β) Signaling Transducers in Human Glioblastoma

BackgroundIt has been revealed that Staphylococcus aureus enterotoxin B (SEB) may feature anti-cancer and anti-metastatic advantages due to its ability to modify cell immunity processes and signaling pathways. Glioblastoma is one of the most aggressive human cancers; it has a high mortality nature, which makes it an attractive area for the development of novel therapies.ObjectivesWe examined whether the SEB could exert its growth inhibitory effects on glioblastoma cells partially through the manipulation of a key tumor growth factor termed transforming growth factor-beta (TGF-β).Materials and MethodsA human primary glioblastoma cell line, U87, was treated with different concentrations of SEB. The cell quantity was measured by the MTT assay at different exposure times. For molecular assessments, total ribonucleic acid (RNA) was extracted from either non-treated or SEB-treated cells. Subsequently, the gene expression of TGF-β transducers, smad2/3, at the messenger RNA (mRNA) level, was analyzed via a quantitative real-time polymerase chain reaction (qPCR) using the SYBR Green method. Significant differences between cell viability and gene expression levels were determined (Prism 5.0 software) using one-way analyses of variance (ANOVA) test.ResultsWe reported that SEB could effectively down-regulate smad2/3 expression in glioblastoma cells at concentrations as quantity as 1 μg/mL and 2 μg/mL (P < 0.05 and P < 0.01, respectively). The SEB concentrations effective at regulating smad2/3 expression were correlated with those used to inhibit the proliferation of glioblastoma cells. Our results also showed that SEB was able to decrease smad2/3 expression at the mRNA level in a concentration- and time-dependent manner.ConclusionsWe suggested that SEB could represent an agent that can significantly decrease smad2/3 expression in glioblastoma cells, leading to moderate TGF-β growth signaling and the reduction of tumor cell proliferation.