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Staphylococcus aureus is a gram-positive bacterium that may cause intestinal inflammation by secreting enterotoxins, which commonly cause food-poisoning and gastrointestinal injuries. Staphylococcal enterotoxin B (SEB) acts as a superantigen (SAg) by binding in a bivalent manner the T-cell receptor (TCR) and the costimulatory receptor CD28, thus stimulating T cells to produce large amounts of inflammatory cytokines, which may affect intestinal epithelial barrier integrity and functions. However, the role of T cell-mediated SEB inflammatory activity remains unknown. Here we show that inflammatory cytokines produced by T cells following SEB stimulation induce dysfunctions in Caco-2 intestinal epithelial cells by promoting actin cytoskeleton remodelling and epithelial cellcell junction down-regulation. We also found that SEB-activated inflammatory T cells promote the up-regulation of epithelial-mesenchymal transition transcription factors (EMT-TFs) in a nuclear factor-kB (NF-kB)- and STAT3- dependent manner. Finally, by using a structure-based design approach, we identified a SEB mimetic peptide (pSEB116-132) that, by blocking the binding of SEB to CD28, dampens inflammatory-mediated dysregulation of intestinal epithelial barrier. [ABSTRACT FROM AUTHOR]

Staphylococcus aureus is a gram-positive bacterium that may cause intestinal inflammation by secreting enterotoxins, which commonly cause food-poisoning and gastrointestinal injuries. Staphylococcal enterotoxin B (SEB) acts as a superantigen (SAg) by binding in a bivalent manner the T-cell receptor (TCR) and the costimulatory receptor CD28, thus stimulating T cells to produce large amounts of inflammatory cytokines, which may affect intestinal epithelial barrier integrity and functions. However, the role of T cell-mediated SEB inflammatory activity remains unknown. Here we show that inflammatory cytokines produced by T cells following SEB stimulation induce dysfunctions in Caco-2 intestinal epithelial cells by promoting actin cytoskeleton remodelling and epithelial cell-cell junction down-regulation. We also found that SEB-activated inflammatory T cells promote the up-regulation of epithelial-mesenchymal transition transcription factors (EMT-TFs) in a nuclear factor-κB (NF-κB)- and STAT3-dependent manner. Finally, by using a structure-based design approach, we identified a SEB mimetic peptide (pSEB116-132) that, by blocking the binding of SEB to CD28, dampens inflammatory-mediated dysregulation of intestinal epithelial barrier.

Acute Respiratory Distress Syndrome (ARDS) is triggered by a variety of insults, such as bacterial and viral infections, including SARS-CoV-2, leading to high mortality. In the murine model of ARDS induced by Staphylococcal enterotoxin-B (SEB), our previous studies showed that while SEB triggered 100% mortality, treatment with Resveratrol (RES) completely prevented such mortality by attenuating inflammation in the lungs. In the current study, we investigated the metabolic profile of SEB-activated immune cells in the lungs following treatment with RES. RES-treated mice had higher expression of miR-100 in the lung mononuclear cells (MNCs), which targeted mTOR, leading to its decreased expression. Also, Single-cell RNA-seq (scRNA seq) unveiled the decreased expression of mTOR in a variety of immune cells in the lungs. There was also an increase in glycolytic and mitochondrial respiration in the cells from SEB + VEH group in comparison with SEB + RES group. Together these data suggested that RES alters the metabolic reprogramming of SEB-activated immune cells, through suppression of mTOR activation and its down- and upstream effects on energy metabolism. Also, miR-100 could serve as novel potential therapeutic molecule in the amelioration of ARDS.

Abstract Background In Sub-Saharan Africa, herbal therapy continues to be utilized for HIV-1 disease management. However, the therapeutic benefits of these substances remain ambiguous. To date, little is known about the effects of these plant extracts on chronic CD4 + T-cell activation and exhaustion which is partly driven by HIV-1 associated microbial translocation. Methods Effects of Azadirachta indica, Momordica foetida and Moringa oleifera ethanol: water mixtures on cell viability were evaluated using the Guava PCA system. Then, an in-vitro cell culture model was developed to mimic CD4+ T cell exposures to antigens following HIV-1 microbial translocation. In this, peripheral blood mononuclear cells (PBMCs) isolated from HIV negative (n = 13), viral load 1000 copies per mL (n = 6) study participants from rural Uganda were treated with Staphylococcus enterotoxin B (SEB). Then, the candidate plant extract (A. indica) was added to test the potential to inhibit corresponding CD4+ T cell activation. Following BD Facs Canto II event acquisition, variations in %CD38, %CD69, Human Leukocyte Antigen -DR (HLA-DR), Programmed cell death protein 1 (PD-1), T-cell immunoglobulin and mucin domain-containing protein 3 (Tim-3), interferon gamma (IFN γ) and interleukin 2 (IL-2) CD4 + T cell expression were evaluated. Results Following exposure to SEB, only A. indica demonstrated a concentration-dependent ability to downregulate the levels of CD4 + T cell activation. At the final concentration of 0.500 μg/mL of A. indica, a significant downregulation of CD4 + CD38 + HLA-DR+ expression was observed in HIV negative (p

Background: In Sub-Saharan Africa, herbal therapy continues to be utilized for HIV-1 disease management. However, the therapeutic benefits of these substances remain ambiguous. To date, little is known about the effects of these plant extracts on chronic CD4 + T-cell activation and exhaustion which is partly driven by HIV-1 associated microbial translocation. Methods: Effects of Azadirachta indica, Momordica foetida and Moringa oleifera ethanol: water mixtures on cell viability were evaluated using the Guava PCA system. Then, an in-vitro cell culture model was developed to mimic CD4+ T cell exposures to antigens following HIV-1 microbial translocation. In this, peripheral blood mononuclear cells (PBMCs) isolated from HIV negative (n = 13), viral load < 1000 copies per mL (n = 10) and viral load > 1000 copies per mL (n = 6) study participants from rural Uganda were treated with Staphylococcus enterotoxin B (SEB). Then, the candidate plant extract (A. indica) was added to test the potential to inhibit corresponding CD4+ T cell activation. Following BD Facs Canto II event acquisition, variations in %CD38, %CD69, Human Leukocyte Antigen -DR (HLA-DR), Programmed cell death protein 1 (PD-1), T-cell immunoglobulin and mucin domain-containing protein 3 (Tim-3), interferon gamma (IFN γ) and interleukin 2 (IL-2) CD4 + T cell expression were evaluated. Results: Following exposure to SEB, only A. indica demonstrated a concentration-dependent ability to downregulate the levels of CD4 + T cell activation. At the final concentration of 0.500 μg/mL of A. indica, a significant downregulation of CD4 + CD38 + HLA-DR+ expression was observed in HIV negative (p < 0.0001) and both HIV infected groups (P = 0.0313). This plant extract also significantly lowered SEB induced % CD4+ T cell HLADR, PD-1 and Tim-3 levels. PD-1 and CD69 markers were only significantly downmodulated in only the HIV negative ((p = 0.0001 and p = 0.0078 respectively) and viral load< 1000 copies per ml (p = 0.0078) groups. Conclusion: A. indica exhibited the in-vitro immunomodulatory potential to inhibit the continuum of SEB induced CD4+ T-cell activation/ exhaustion without impacting general T-cell specific functions such as cytokine secretion. Additional studies are needed to confirm A. indica as a source of natural products for targeting persistent immune activation and inflammation during ART. [ABSTRACT FROM AUTHOR]

Acute Respiratory Distress Syndrome (ARDS) is a life-threatening complication that can ensue following Staphylococcus aureus infection. The enterotoxin produced by these bacteria (SEB) acts as a superantigen thereby activating a large proportion of T cells leading to cytokine storm and severe lung injury. Δ9Tetrahydrocannabinol (THC), a psychoactive ingredient found in Cannabis sativa, has been shown to act as a potent anti-inflammatory agent. In the current study, we investigated the effect of THC treatment on SEB-induced ARDS in mice. While exposure to SEB resulted in acute mortality, treatment with THC led to 100% survival of mice. THC treatment significantly suppressed the inflammatory cytokines, IFN-γ and TNF-α. Additionally, THC elevated the induction of regulatory T cells (Tregs) and their associated cytokines, IL-10 and TGF-β. Moreover, THC caused induction of Myeloid-Derived Suppressor Cells (MDSCs). THC acted through CB2 receptor as pharmacological inhibitor of CB2 receptors blocked the anti-inflammatory effects. THC-treated mice showed significant alterations in the expression of miRNA (miRs) in the lung-infiltrated mononuclear cells (MNCs). Specifically, THC caused downregulation of let7a-5p which targeted SOCS1 and downregulation of miR-34-5p which caused increased expression of FoxP3, NOS1, and CSF1R. Together, these data suggested that THC-mediated alterations in miR expression in the lungs may play a critical role in the induction of immunosuppressive Tregs and MDSCs as well as suppression of cytokine storm leading to attenuation of SEB-mediated lung injury.

Acute Respiratory Distress Syndrome (ARDS) is a life-threatening complication that can ensue following Staphylococcus aureus infection. The enterotoxin produced by these bacteria (SEB) acts as a superantigen thereby activating a large proportion of T cells leading to cytokine storm and severe lung injury. Δ9Tetrahydrocannabinol (THC), a psychoactive ingredient found in Cannabis sativa, has been shown to act as a potent anti-inflammatory agent. In the current study, we investigated the effect of THC treatment on SEB-induced ARDS in mice. While exposure to SEB resulted in acute mortality, treatment with THC led to 100% survival of mice. THC treatment significantly suppressed the inflammatory cytokines, IFN-γ and TNF-α. Additionally, THC elevated the induction of regulatory T cells (Tregs) and their associated cytokines, IL-10 and TGF-β. Moreover, THC caused induction of Myeloid-Derived Suppressor Cells (MDSCs). THC acted through CB2 receptor as pharmacological inhibitor of CB2 receptors blocked the anti-inflammatory effects. THC-treated mice showed significant alterations in the expression of miRNA (miRs) in the lung-infiltrated mononuclear cells (MNCs). Specifically, THC caused downregulation of let7a-5p which targeted SOCS1 and downregulation of miR-34-5p which caused increased expression of FoxP3, NOS1, and CSF1R. Together, these data suggested that THC-mediated alterations in miR expression in the lungs may play a critical role in the induction of immunosuppressive Tregs and MDSCs as well as suppression of cytokine storm leading to attenuation of SEB-mediated lung injury. [ABSTRACT FROM AUTHOR]

• Leukocytes from stressed volunteers appeared to be locked in a hyporesponsive state. • Cell counting to assess protective ability seems unreliable for stressed persons. • Stressed individuals potentially may experience reduced vaccine efficacy. • Extreme stress may present difficulty for diagnosis of infection or food poisoning. Severe stress can have drastic and systemic effects with dire implications on the health and wellbeing of exposed individuals. Particularly, the effect of stress on the immune response to infection is of interest to public health because of its implications for vaccine efficacy and treatment strategies during stressful scenarios. Severe stress has previously been shown to cause an anergic state in the immune system that persists following exposure to a potent mitogen. Transcriptome and microRNA changes were characterized using blood samples collected from U.S. Army Ranger candidates immediately before and after training, followed by exposure to representative pathogenic agents: Yersinia pestis , dengue virus 2, and Staphylococcal enterotoxin B (SEB). We employed experimental and computational approaches to characterize altered gene expression, processes, pathways, and regulatory networks mediating the host's response towards severe stress; to assess the protective immunity status of the stressed host towards infection; and to identify pathogen-induced biomarkers under severe stress conditions. We observed predicted inhibition of pathways significantly associated with lymphopoiesis, wound healing, inflammatory response, lymphocyte activation, apoptosis, and predicted activation of oxidative stress. Using weighted correlation network analyses, we demonstrated preservation of these pathways across infection and stress combinations. Regulatory networks comprising a common set of upstream regulators: transcription factors, microRNAs and post-translational regulators (kinases and phosphatases) may be drivers of molecular alterations leading to compromised protective immunity. Other sets of transcripts were persistently altered in both the pre- and post-stress conditions due to the host's response to each pathogenic agent, forming specific molecular signatures with the potential to distinguish infection from that of severe stress. Our results suggest that severe stress alters molecules implicated in the development of leukopoietic stem cells, thereby leading to depletion of cellular and molecular repertoires of protective immunity. Suppressed molecules mediating membrane trafficking of recycling endosomes, membrane translocation and localization of the antigen processing mechanisms and cell adhesions indicate suboptimal antigen presentation, impaired formation of productive immunological synapses, and inhibited T-cell activations. These factors may collectively be responsible for compromised protective immunity (infection susceptibility, delayed wound healing, and poor vaccine response) observed in people under severe stress. [ABSTRACT FROM AUTHOR]

To investigate the expression pattern of staphylococcal enterotoxin B (SEB) in food and the genotypic diversity of SEB-encoding gene in association with molecular characteristics and antimicrobial resistance of S. aureus , 498 isolates from retail food were screened for seb gene and detected for SEB production in S. aureus. In addition, the seb nucleotide sequences, virulence genes, resistance genes, antimicrobial susceptibility and molecular characteristics of S. aureus were examined. A total of 45 (9.0 %) seb -positive S. aureus strains were identified, all of which expressed SEB. The detection rate of SEB-production strains was significantly higher from dairy-related sources than those from other sources (P < 0.05). In vitro simulations showed that S. aureus could grow and express SEB in both milk and pork, with SEB expression exceeding 20 ng/g after 1 day of storage at room temperature. There were 2 distinct SEB genotyping (SEB1 and SEB2) in the SEB amino acid sequences of the 45 isolates, including 4 amino acid differences (Ala-13Val, Ser14Ala, Asn192Ser, and Met222Leu). There was no significant difference (P > 0.05) in SEB production between SEB1 and SEB2 genotyping strains. Based on MLST clustering analysis, the same molecular type strains were found to have the same SEB genotyping, virulence gene profile, resistance gene profile and drug resistance profile. Among them, the dominant molecular types of SEB1 and SEB2 strains were CC1-ST188-t189 and CC59-ST59-t437, respectively. Compared to the CC1-ST188-t189 clonal strain, the CC59-ST59-t437 clonal strain carried a higher number of virulence and resistance genes and exhibited a broader resistance profile. Therefore, understanding the characteristics of the strains and their expression patterns in food can be effective in preventing food poisoning incidents. • Higher level SEB-producing S. aureus contamination existed in dairy-related samples than in pork, chicken, and egg. • Two different SEB genotypes were identified, with 4 different amino acids between SEB1 and SEB2. • CC1-ST188-t189 and CC59-ST59-t437 were the dominant clone SEB1 and SEB2 strains, respectively. • The CC59-ST59-t437 clone strain carries multiple virulence and resistance genes and exhibits a broad resistance profile. [ABSTRACT FROM AUTHOR]

Acute Respiratory Distress Syndrome (ARDS) is triggered by a variety of insults, such as bacterial and viral infections, including SARS-CoV-2, leading to high mortality. In the murine model of ARDS induced by Staphylococcal enterotoxin-B (SEB), our previous studies showed that while SEB triggered 100% mortality, treatment with Resveratrol (RES) completely prevented such mortality by attenuating inflammation in the lungs. In the current study, we investigated the metabolic profile of SEB-activated immune cells in the lungs following treatment with RES. RES-treated mice had higher expression of miR-100 in the lung mononuclear cells (MNCs), which targeted mTOR, leading to its decreased expression. Also, Single-cell RNA-seq (scRNA seq) unveiled the decreased expression of mTOR in a variety of immune cells in the lungs. There was also an increase in glycolytic and mitochondrial respiration in the cells from SEB + VEH group in comparison with SEB + RES group. Together these data suggested that RES alters the metabolic reprogramming of SEB-activated immune cells, through suppression of mTOR activation and its down- and upstream effects on energy metabolism. Also, miR-100 could serve as novel potential therapeutic molecule in the amelioration of ARDS., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Alghetaa, Mohammed, Singh, Wilson, Cai, Putluri, Nagarkatti and Nagarkatti.)

Protein-imprinted polyacrylamide gel beads (IPGB) were synthesized via inverse suspension polymerization, using staphylococcal enterotoxin B (SEB) as template. The adsorption capacity of SEB-IPGB was almost three times as much as that of non-imprinted gel beads. The Langmuir adsorption models were applied to describe the equilibrium isotherms. The results showed that an equal class of adsorption was formed in the SEB-IPGB with the maximum adsorption capacity of 8.40 mg SEB/g imprinted beads. The selectivity test of imprinted beads shows that they exhibited good recognition for SEB as compared with the other proteins. The formation of multiple hydrogen bonds and complementary shape between the imprinting cavities and the template proteins would be the two factors that led to the imprinting effect. The obtained SEB-IPGB would be used as a potential material for protein toxin separation, extraction, and purification. Copyright © 2014 John Wiley & Sons, Ltd. [ABSTRACT FROM AUTHOR]

Food allergy is a rapidly growing public health concern because of its increasing prevalence and life-threatening potential. Animal models of food allergy have emerged as a tool for identifying mechanisms involved in the development of sensitization to normally harmless food allergens, as well as delineating the critical immune components of the effector phase of allergic reactions to food. However, the role animal models might play in understanding human diseases remains contentious. This review summarizes how animal models have provided insights into the etiology of human food allergy, experimental corroboration for epidemiologic findings that might facilitate prevention strategies, and validation for the utility of new therapies for food allergy. Improved understanding of food allergy from the study of animal models together with human studies is likely to contribute to the development of novel strategies to prevent and treat food allergy. [Copyright &y& Elsevier]

Abstract: Staphylococcal enterotoxin B (SEB) is a potent exotoxin produced by the Staphylococcus aureus. This toxin is classified as a superantigen because of its ability to directly bind with MHC-II class molecules followed by activation of a large proportion of T cells bearing specific Vβ-T cell receptors. Commonly associated with classic food poisoning, SEB has also been shown to induce toxic shock syndrome, and is also considered to be a potential biological warfare agent because it is easily aerosolized. In the present study, we assessed the ability of indole-3-carbinol (I3C) and one of its byproducts, 3,3′-diindolylmethane (DIM), found in cruciferous vegetables, to counteract the effects of SEB-induced activation of T cells in mice. Both I3C and DIM were found to decrease the activation, proliferation, and cytokine production by SEB-activated Vβ8 + T cells in vitro and in vivo. Interestingly, inhibitors of histone deacetylase class I (HDAC-I), but not class II (HDAC-II), showed significant decrease in SEB-induced T cell activation and cytokine production, thereby suggesting that epigenetic modulation plays a critical role in the regulation of SEB-induced inflammation. In addition, I3C and DIM caused a decrease in HDAC-I but not HDAC-II in SEB-activated T cells, thereby suggesting that I3C and DIM may inhibit SEB-mediated T cell activation by acting as HDAC-I inhibitors. These studies not only suggest for the first time that plant-derived indoles are potent suppressors of SEB-induced T cell activation and cytokine storm but also that they may mediate these effects by acting as HDAC inhibitors. [Copyright &y& Elsevier]

Highlights: [•] CD4+ Treg develop in transgenic mice expressing mutant MHC II incapable of binding to CD4. [•] Relative numbers of CD25+CD4+ Treg are increased. [•] Activated Treg suppress IL-2 secretion in normally selected CD4+ T cells. [•] Treg suppression is contact-dependent. [ABSTRACT FROM AUTHOR]

Abstract: In this paper, a novel electrochemical immunosensor to detect staphylococcal enterotoxin B based on bio-magnetosomes, polyaniline nano-gold composite and 1,2-dimethyl-3-butylimidazolium hexafluorophosphate ionic liquid, was developed, and found to exhibit high sensitivity and stability. The specific antibody to staphylococcal enterotoxin B conjugated with the magnetosomes showed rapid immunoreactions and good dispersion, which contributed to the formation of a nanostructurally smooth and dense film on the surface of a gold electrode. Polyaniline nano-gold composite and 1,2-dimethyl-3-butylimidazolium hexafluorophosphate ionic liquid were used to modify the electrode as mediators to improve the electron transfer and offer an excellent biocompatible microenvironment for the antibody to retain its activity to enhance the response of the electrochemical sensor. Under optimal conditions, the developed immunosensor showed a good linear response in the range from 0.05 to 5ng/mL (R 2=0.9957) with a detection limit as low as 0.017ng/mL, compared with the one without magnetosomes (0.05–5ng/mL, 0.033ng/mL), this developed immunosensor showed a wider response range and a reduced detection limit. And a good specificity with little adsorption to staphylococcal enterotoxin A, C and Na+, K+, Ca2+ was obtained. Moreover, the immunosensor exhibited a good long-time stability at 4°C reaching up to 60 days, which showed a relatively long working life. Meanwhile the immunosensor could be regenerated four times using NaOH elution. The sensor also displayed a good repeatability with a relative standard deviation of 5.02% for staphylococcal enterotoxin B detection (1ng/mL, n=9). Furthermore, high recoveries in milk samples from 81% to 118% were achieved and successfully applied to milk sample detection. The obtained results demonstrate that the developed electrochemical immunosensor is a promising tool for the detection of staphylococcal enterotoxin B in food. [Copyright &y& Elsevier]

Superantigens (SAgs) are a class of immunostimulatory exotoxins that activate large numbers of T cells, leading to overproduction of cytokines and subsequent inflammatory reactions and systemic toxicity. Staphylococcal enterotoxin C (SEC), a SAg secreted by Staphylococcus aureus, has been implicated in various illnesses including non-menstrual toxic shock syndrome (TSS) and necrotizing pneumonia. SEC has been shown to cause TSS illness in rabbits and the toxin contributes to lethality associated with methicillin-resistant S.aureus (MRSA) in a rabbit model of pneumonia. With the goal of reducing morbidity and mortality associated with SEC, a high-affinity variant of the extracellular variable domain of the T-cell receptor beta-chain for SEC (∼14 kDa) was generated by directed evolution using yeast display. This protein was characterized biochemically and shown to cross-react with the homologous (65% identical) SAg staphylococcal enterotoxin B (SEB). The soluble, high-affinity T-cell receptor protein neutralized SEC and SEB in vitro and also significantly reduced the bacterial burden of an SEC-positive strain of MRSA (USA400 MW2) in an infective endocarditis model. The neutralizing agent also prevented lethality due to MW2 in a necrotizing pneumonia rabbit model. These studies characterize a soluble high-affinity neutralizing agent against SEC, which is cross-reactive with SEB, and that has potential to be used intravenously with antibiotics to manage staphylococcal diseases that involve these SAgs. [ABSTRACT FROM AUTHOR]

Abstract: We investigated the role of the extracellular matrix component, hyaluronic acid (HA) in SEB-induced ALI/ARDS. Intranasal exposure of mice to SEB led to a significant increase in the level of soluble hyaluronic acid in the lungs. Similarly, in an endothelial cell/spleen cell co-culture, SEB exposure led to significant increases in soluble levels of hyaluronic acid, cellular proliferation, and cytokine production compared with SEB-exposed spleen cells or endothelial cells alone. Exposure of SEB-activated spleen cells to hyaluronic acid led to increased cellular proliferation and increased cytokine production. SEB-induced cytokine production and proliferation in vitro were significantly reduced by the hyaluronic acid blocking peptide, Pep-1. Finally, treatment of SEB-exposed mice with Pep-1 significantly reduced SEB-induced ALI/ARDS, through reduction of cytokine production and numbers of lung inflammatory cells, compared to mice treated with a control peptide. Together, these results suggest the possibility of targeting HA for the treatment of SEB-induced ALI/ARDS. [Copyright &y& Elsevier]

We fabricated a two-dimensional (2D) molecularly imprinted sol-gel thin film-coated quartz crystal microbalance (QCM) for the rapid detection of staphylococcal enterotoxin B (SEB) by combining organosilanes and the template protein SEB on the surface of piezoelectric quartz crystal (PQC) Au-electrode by in-situ immobilization. The detection process was monitored by the QCM's frequency shift (Δf). The working range of this method was 1.0 × 10−1–1.0 × 103 µg/mL. The detection limit was 6.1 ng/mL, which was lower than that of the PQC immunosensor, and the detection period was within 0.5 h. The reproducibility of the imprinted film-coated QCM was satisfactory due to no significant statistical difference (P > 0.05) in the rapid detection of SEB between intra- and inter-batch. The selectivity of the imprinted sol-gel film showed that it could discriminate the template molecule from its analogues and other guest molecules. Compared with immunochip, the imprinted film-coated QCM is more advantageous in terms of simplicity, rapidity, low cost, and sensitivity. Moreover, in real sample analysis, the recoveries of this method were 89.4–106.63%, which can be considered a favorable and applicable method for the rapid determination of SEB in real samples. [ABSTRACT FROM AUTHOR]

n enzyme-linked immunosorbent assay, a horseradish peroxidase-catalyzed fluorogenic reaction, and chemiluminescence (CL) analysis have been combined to develop a sandwich ELISA for Staphylococcal enterotoxin B (SEB) using monoclonal antibodies for different epitopes of SEB. The enzyme catalyzed reaction of 3-(4-hydroxyphenyl propionate) with the urea complex of hydrogen peroxide produced a fluorescent dimer which was detected by chemiluminescence analysis. The CL response to SEB is linear in the range from 6.0 to 564 pg mL ( r = 0.9993), and the detection limit is 3.3 pg mL (S/N = 3). Intra- and interassay coefficients of variation are <7.0% at three concentrations (24, 96 and 384 pg mL). The method was applied to the analysis of SEB in serum, lake water and milk samples. The results compared well with those obtained by conventional ELISAs. [Figure not available: see fulltext.] [ABSTRACT FROM AUTHOR]

Aims: Methicillin Resistant Staphylococcus aureus is among common pathogens that its prevalence has increased all around the world during past three decades. Staphylococcal Enterotoxin B (SEB) is one of the most important Staphylococcal enterotoxins, which is considered as an important pathogenic factor. The aim of this study was investigating the correlation between SEB and resistance to Methicillin. Material & Methods: This cross-sectional study was performed on inpatients of one of the hospitals of Tehran in year 2008 who were selected by accessible sampling method. Correlation between SEB gene in Methicillin Resistant strains was investigated in 100 samples isolated from wounds. Antibiogram was done by disc diffusion and presence of SEB gene was confirmed by PCR. Data was analyzed by SPSS 17 software using descriptive statistical methods and Chi square test. Results: Only 8% of strains had SEB gene. 75% of Staphylococcus aureus strains that had SEB gene were Methicillin resistant and only 15% of them were Methicillin sensitive. A significant correlation was observed between the SEB gene and Methicillin resistance (p<0.05). Conclusion: There is e significant correlation between presence of SEB gene and Methicillin resistance and this issue can reinforce the hypothesis that SEB gene has role in Methicillin resistance. [ABSTRACT FROM AUTHOR]

A previously developed fluorescence sensing platform, combining spatial illumination using electroluminescence (EL) semiconductor strips with charge coupled device (CCD)-based detection (EL-CCD), was adapted to a new 96-well chip for colorimetric immunological assays, enhancing the capabilities of the EL-CCD platform. The modified system was demonstrated using a colorimetric-based enzyme linked immunosorbent assay (ELISA) for detection of staphylococcal enterotoxin B (SEB). Limits of detection (LODs) of 3.9 ng/mL (±2.4 ng/mL) SEB were determined with the ELISA chip measured using the EL-CCD platform, following a standard 4-h ELISA protocol. The LODs were comparable to those obtained using standard 96-well ELISA plates measured using a standard laboratory 96-well plate reader. The miniature 96-well ELISA chip however required as little as 5-µL samples, representing a tenfold reduction in sample volume compared to a standard 96-well ELISA plates. The ELISA chip also demonstrated detection of SEB spiked into various food matrices (milk, mushrooms, and mayonnaise) using limited-to-no sample preparation, with LODs ranging from 3.9 to 18.5 ng/mL depending on the matrix. The EL-CCD platform is versatile, capable of multi-mode detection (e.g., fluorescent and colorimetric along with solution and solid phase assays), and could readily be applied to other field portable or point-of-care applications. [Figure not available: see fulltext.] [ABSTRACT FROM AUTHOR]

Abstract: Murine models for bacterial superantigens like staphylococcal enterotoxin B (SEB) have to date been rather cumbersome. The reasons include: (1) necessary use of potentiating agents such as actinomycin D, d-galactosamine, lipopolysaccharide (LPS), or viruses; (2) high toxin amounts required to elicit effects; and/or (3) generation of phenotypic-stable transgenic animals. Our study employed readily available C3H/HeJ (TLR4 negative, LPS-nonresponsive) mice with intranasal and intraperitoneal administration of low microgram quantities of SEB. These animals responded to SEB with severe lung inflammation and hypothermia, culminating in death. A survey of cytokines/chemokines in sera and lungs after lethal intoxication revealed that monocyte chemoattractant protein-1 and interleukin-2 were associated with effects in this model. In contrast, SEB had minimal effects upon congenic (TLR4 positive, LPS-responsive) C3H/OuJ mice. Lethality of SEB in C3H/HeJ mice was neutralized with SEB-specific antibodies, suggesting potential utility of this model for future therapeutic studies. [Copyright &y& Elsevier]

The bacterial superantigen staphylococcal enterotoxin B (SEB) is a potent inducer of cytotoxic T-cell activity and cytokine production in vivo. We investigated the possibility of the therapeutic application of SEB in patients with fibrosarcoma. The anti-tumor effect of SEB in mice with inoculated fibrosarcoma (WEHI-164) was examined by intravenous (IV) and intratumoral (IT) injection and the sizes of the inoculated tumors, IFN-γ production, and CD4+/CD8+ T cell infiltration were determined. The inoculated tumors were also examined histologically. In the mice in the IV-injected group, a significant reduction ( P < 0.02) of tumor size was observed in comparison with mice in the IT-injected and control groups. Furthermore, the mice in the IV-injected group showed significantly higher levels of IFN-γ ( P < 0.009) and CD4+/CD8+ T cell infiltration when compared with the other groups ( P < 0.02). A significantly higher frequency of necrosis in tumor tissues was also observed in mice in the IV-injected group ( P < 0.05). Our present findings suggest that tumor cell death is caused by increased cytotoxic T-cell activity and cytokine levels in response to the IV injection of SEB and that SEB may be a good option for use as a novel therapy in patients with fibrosarcoma. [ABSTRACT FROM AUTHOR]

Abstract: The high-density attachment of active antibodies or other recognition molecules to the capture surface is one of the fundamental processes in route to developing effective biosensors. One method applied frequently to enzymatic sensor systems has been the layer-by-layer assembly of the bioactive surface. Cui et al. [Cui, X., Pei, R., Wang, Z., Yang, F., Ma, Y., Dong, S., Yang, X., 2003. Biosens. Bioelectron. 18, 59–67] extended this concept to immunosensors, where they formed multilayers composed of avidin and biotinylated antibody and reported this construct to be a potent way to form an effective surface for surface plasmon resonance biodetection. We reexamined this concept in an effort to establish a simple method to improve the activity of polystyrene capture surfaces used in sandwich fluoroimmunoassays for the detection of the target, staphylococcal enterotoxin B (SEB). Using multilayers prepared by alternating between NeutrAvidin and either biotinylated mono or polyclonal anti-SEB antibody, we found that virtually all the SEB-binding activity was derived from the final layer added; and that additional layers provided no observable enhancement in fluoroimmunoassay signal strength. [Copyright &y& Elsevier]

Abstract: Candidate HIV-1 vaccines currently being evaluated in clinical trials are designed to elicit HIV-1-specific cellular immunity. Intracellular cytokine staining (ICS) assays allow sensitive, quantitative ex vivo assessments of antigen-specific T cells including immunophenotyping of responding cells and measurement of multiple effector functions. Additionally, the use of banked cryopreserved PBMC samples makes this assay attractive in the setting of large efficacy trials where it is less feasible to perform immunoassays on freshly isolated samples. Here we describe extensive studies to optimize and quantitatively validate the 8-color ICS assay for use in clinical trials of candidate vaccines, which includes measurement of viable IFN-γ, IL-2, TNF-α and IL-4 producing CD4+ and CD8+ T cells. We show that omission of viability dye staining results in an over-estimate of the true antigen-specific T cell response by up to two-fold. After optimization, the 8-color assay was validated for specificity, precision, linearity, limit of quantitation and robustness. The assay has a lower quantitation limit generally below 0.04%, depending on the cytokine subset. Additionally, with appropriate gating, the 8-color assay gives comparable cytokine-positive responses to those observed with the conventional 4-color assay. In conclusion, we provide the first description of a quantitatively validated ICS assay, which permits quantitative and qualitative evaluation of vaccine-induced immunogenicity and analysis of immune correlates of protection. [Copyright &y& Elsevier]

Abstract: Administration of bacterial superantigen, such as staphylococcal enterotoxin B (SEB), induces in vivo stimulation of T cell proliferation and cytokine production such as interleukin-2 (IL-2). It has been previously reported that SEB administration induces fever, c-Fos expression in the brain, and hypothalamus–pituitary–adrenal axis activation, demonstrating that the brain is able to sense and respond to SEB. Previously it had been shown that immune functions can be behaviourally conditioned pairing a novel gustatory stimulus together with an immunomodulatory drug or an antigen. We designed an experimental protocol using Dark Agouti rats in which saccharin taste, as conditioned stimulus, was paired with an i.p. injection of SEB (2 mg/kg), as unconditioned stimulus. Six days later, when conditioned animals were re-exposed to the conditioned stimulus they displayed strong conditioned taste avoidance to the saccharin. More importantly, re-exposure to the conditioned stimulus significantly increased IL-2, interferon-γ and corticosterone plasma levels, in comparison with conditioned animals which had not been re-exposed to saccharin taste. These results demonstrate a behavioural-immune-endocrine conditioned response using a superantigen as unconditioned stimulus. In addition, they illustrate the brain abilities to mimic the unconditioned effects of a superantigen by yet unknown mechanisms. [Copyright &y& Elsevier]

In general non-human primates are highly susceptible to infections with Mycobacterium tuberculosis which therefore presents an explosive health threat to colonies. To screen for M. tuberculosis infections in non-human primates, the skin test is routinely used. However, the reliability of this test in primates is debatable. The aim of this study was to compare relatively easy in vitro diagnostic tests for TB with the skin test for detection of a tuberculosis (TB) infection. Two in vitro assays, a whole blood interferon-γ (WB IFN-γ) assay and in vitro stimulation of isolated lymphocytes (PBMC IFN-γ) were evaluated during both experimental TB infections in macaques as well as during an outbreak of TB in a macaque quarantine facility. The WB IFN-γ assay was also evaluated on healthy old and new world monkeys. Our results show that both in vitro assays detected TB infection in macaques. All experimentally infected animals showed TB-specific responses in both assays. In contrast, several TB animals were not diagnosed TB positive using the skin test. In addition, during the outbreak in the quarantine facility one animal was not detected using the routinely used skin test, but it showed strong positive responses in the WB assay. In conclusion, the in vitro assays are a valuable tool for screening non-human primates for TB infection, especially because the assays cause relatively less stress for the animals compared to the skin test and give reproducible and reliable results. [Copyright &y& Elsevier]

The enzyme-linked immunospot (ELISPOT) assay is a highly sensitive and reproducible method for quantifying T cell-mediated immune responses, and has been used to measure antigen-specific responses post-vaccination. While there are several advantages of the ELISPOT assay for use in field settings for large-scale vaccination trials, blood draw volumes are often limited, and the number of antigen-specific responses that can be measured is constrained by the limited cell number. We reasoned that it should be possible to salvage and rescue viable cells from a completed ELISPOT assay post-incubation, to use for further experimentation. Here, we show that cells rescued from an ELISPOT plate after assay are viable, and may be used in a second cytokine-producing assay, in a proliferation assay, or to provide a source of DNA for genetic studies such as human leukocyte antigen (HLA) typing. Rescue of cells after an ELISPOT assay will be particularly useful for increasing sample utility and maximizing data collection from T cell assays in vaccine trials. [Copyright &y& Elsevier]

The enzyme-linked immune spot (ELISPOT) assay is receiving increased attention as a means for quantifying antigen-specific CD8 T-cell responses in rhesus macaques. Further improving the sensitivity of this assay could aid in the evaluation of vaccine candidates and/or immune therapeutic candidates. Interleukin (IL)-15 has been demonstrated to stimulate expansion of human immunodeficiency virus (HIV)-specific cytotoxic T lymphocytes (CTL) and to regulate homeostatic proliferation of CD8+ memory cells. We evaluated the in vitro effect of IL-15 to increase the detection of interferon-γ (IFN-γ) production by antigen-specific stimulated lymphocytes from a group of rhesus macaques exposed to simian-human immunodeficiency virus (SHIV) and a second group infected with SIVmac251, before and after antiretroviral treatment (ART). Results from these studies demonstrate that the presence of IL-15 during stimulation in a peptide-based ELISPOT assay greatly enhanced IFN-γ production in both SHIV and simian immunodeficiency virus (SIV)-infected macaques. IFN-γ production was mainly mediated by CD8 lymphocytes. The optimal concentrations of IL-15 that give enhancement of IFN-γ production to specific antigen, without a significant increase in the spontaneous IFN-γ release, ranged from 0.5 to 2.5 ng/ml. The mean number of IFN-γ spots was increased 3.1- to 3.6-fold in response to SIV gag or HIV env peptide pools, respectively, in peripheral blood mononuclear cells (PBMC) from SHIV-infected macaques. Similarly, in SIV-infected macaques, IL-15 increased the mean number of IFN-γ spots 2.7-fold in response to both SIV gag and env peptide pools. In samples obtained after ART in the same macaques, the increase factor was 2.5 for SIV gag and 1.8 for the env peptide pools. Thus, the sensitivity of the ELISPOT assay can be enhanced by addition of IL-15. This modified assay will be useful for detection of low frequencies of IFN-γ producing cells in rhesus macaques. [Copyright &y& Elsevier]

Mice that lack the guanine nucleotide exchange factor (GEF) Vav1 exhibit particular defects in antigen-triggered T cell activation but may have an autoreactive T cell repertoire due to impaired intra-thymic negative selection. MOG35–55-induced experimental autoimmune encephalomyelitis (EAE) was used to test the susceptibility of Vav1−/− mice to organ-specific autoimmunity. Vav1−/− animals were found to be resistant to MOG35–55–EAE since the priming and in vivo expansion of myelin oligodendrocyte glycoprotein (MOG)-specific T cells was inefficient despite fully functional antigen presentation. Protection from cell-mediated autoimmunity was not due to a Th2 bias, to the lack of IL-2 or a failure of Vav1−/− T cells in terms of chemotactic mobility. [Copyright &y& Elsevier]

Endotoxin (lipopolysaccharide, LPS) inducible cytokine release by human whole blood is increasingly used to model inflammatory responses in vitro, to detect the presence of pyrogenic contaminations as well as to monitor disease states or immunomodulatory treatments ex vivo. However, the LPS-stimulated blood model primarily allows the assessment of monocyte responses. Here, a whole blood model was established which allows assessment of lymphocyte responses. Four different superantigens, namely staphylococcal enterotoxin A and B (SEA, SEB), toxic shock syndrome toxin-1 (TSST-1) or streptococcal exotoxin A (SPEA) were tested with respect to the induction of lymphokine release. All superantigens were capable of inducing significant amounts of the lymphokines interferon-γ (IFNγ), interleukin 2 (IL-2), IL-4, IL-5, IL-13 and tumor necrosis factor β (TNFβ) after 72 h of incubation. Concentration-dependencies and kinetics were determined. Blood from 160 healthy donors was used to assess the variability of SEB-inducible lymphokine release. Interindividual differences were more pronounced compared to LPS-inducible monokine release. However, the individual response was maintained when blood from six donors was tested once a week for 8 weeks, suggesting that the individual response represents a donor characteristic. The model appears to be suitable for the evaluation of immunomodulatory agents in vitro as well as ex vivo. [Copyright &y& Elsevier]

The importance of the enzyme-linked immunosorbent spot (ELISPOT) assay as a tool for studying immune responses in vitro is becoming increasingly apparent. However, there remains a need for enhanced sensitivity for the detection of low frequency antigen-specific T cell responses. We reasoned that the addition of a combination of the cytokines interleukin (IL)-7 and IL-15 would selectively increase interferon-gamma (IFN-γ) production from antigen-stimulated CD4+ and CD8+ effector memory T cells. Freshly isolated or cryopreserved peripheral blood mononuclear cells (PBMC) from four healthy donors were analysed by ELISPOT for the frequency of purified protein derivative (PPD)-specific CD4+ T cells or cytomegalovirus (CMV) peptide-specific CD8+ T cells. Addition of IL-7 and IL-15 increased the number of PPD-specific CD4+ T cells up to 2.4-fold in fresh PBMC and up to 18-fold in cryopreserved PBMC. The cytokines also increased the number of CMV peptide-specific CD8+ T cells in fresh PBMC up to 7.5-fold. No additional increases were seen when antibodies to co-stimulatory molecules CD28 and CD49d were applied together with the cytokine combination. These data demonstrate that the sensitivity of the ELISPOT assay may be significantly augmented by addition of the cytokines IL-7 and IL-15 to antigen-stimulated cells. This method will be particularly useful for the assessment of antigen-stimulated cytokine production by T cells in cryopreserved biological specimens. [Copyright &y& Elsevier]

We have achieved sensitive, rapid and reproducible detection of three biological threat agents in a variety of biological and environmental matrices using the DELFIA time-resolved fluorometry (TRF) assay system (Perkin-Elmer Life Sciences, Akron, OH). Existing ELISA assays for the detection of Francisella tularensis, Clostridium botulinum A/B neurotoxin (BotNT A/B), and Staphylococcus aureus enterotoxin B (SEB) were converted to TRF assays. They use 100 μl of positive control or unknown per test well and require just over 2 h to run. Fluorescent signal read time is a fraction of a second per well. The assay format consists of a capture ELISA utilizing a biotinylated capture antibody, prebound to a streptavidin-coated 96-well plate and a lanthanide (Europium, Eu3+)-labeled detector antibody. The bound Eu-labeled detector antibody produces a fluorescent signal upon the addition of an enhancement solution. The signal results from the dissociation of the Europium from the antibody, creating a micelle, thus amplifying the signal nearly one million-fold. Sensitivities achieved by these assays were between 4 and 20 pg/ml in buffer. Additionally, we have tested this system in different matrices such as serum, urine, dirt, and sewage. Concentration curves generated from standard solutions produced a wide linear range making serial dilutions of unknown samples unnecessary. DELFIA TRF assays are significantly better in terms of sensitivity, linear range, and run time than standard capture ELISAs and should facilitate early detection of potential biological warfare agents in clinical and environmental samples. [Copyright &y& Elsevier]

Transfer of CD45RBhi CD4+ naı¨ve T cells into severe combined immunodeficient (SCID) mice induces colitis and skin lesions. Recipients treated with cyclosporin A (CsA), CTLA4-Ig, or vehicle were evaluated for weight loss, skin lesions, and cutaneous blood flow. Necropsy, histological, hematological and cytokine analyses were performed at the conclusion of the experiment to confirm the clinical findings. Vehicle-treated mice lost weight and had 100% incidence of skin lesions by 46-days. CsA-treated mice also lost weight, but only 3/8 mice developed mild, clinically evident skin lesions. In contrast, all CTLA4-Ig-treated mice gained weight and did not develop skin lesions. Increase in cutaneous blood flow correlated with the development of skin lesions. Granulocyte numbers, which were high or moderately high in the vehicle- or CsA-treated mice, respectively, remained as low in the CTLA4-Ig-treated group as in untreated mice. IFN-γ, IL-1β, and TNF-α levels in the gut and skin correlated with the extent of inflammation in both organs. Histology revealed that CTLA4-Ig but not CsA effectively prevented both autoimmune disorders. The ability of CTLA4-Ig to prevent both colitis and skin lesions suggests that CD28-dependent co-stimulation of T cells is critical for generation of pro-inflammatory cytokines and induction of clinical disease in such autoimmune disorders. [ABSTRACT FROM AUTHOR]

Background: In Sub-Saharan Africa, herbal therapy continues to be utilized for HIV-1 disease management. However, the therapeutic benefits of these substances remain ambiguous. To date, little is known about the effects of these plant extracts on chronic CD4 + T-cell activation and exhaustion which is partly driven by HIV-1 associated microbial translocation., Methods: Effects of Azadirachta indica, Momordica foetida and Moringa oleifera ethanol: water mixtures on cell viability were evaluated using the Guava PCA system. Then, an in-vitro cell culture model was developed to mimic CD4+ T cell exposures to antigens following HIV-1 microbial translocation. In this, peripheral blood mononuclear cells (PBMCs) isolated from HIV negative (n = 13), viral load < 1000 copies per mL (n = 10) and viral load > 1000 copies per mL (n = 6) study participants from rural Uganda were treated with Staphylococcus enterotoxin B (SEB). Then, the candidate plant extract (A. indica) was added to test the potential to inhibit corresponding CD4+ T cell activation. Following BD Facs Canto II event acquisition, variations in %CD38, %CD69, Human Leukocyte Antigen -DR (HLA-DR), Programmed cell death protein 1 (PD-1), T-cell immunoglobulin and mucin domain-containing protein 3 (Tim-3), interferon gamma (IFN γ) and interleukin 2 (IL-2) CD4 + T cell expression were evaluated., Results: Following exposure to SEB, only A. indica demonstrated a concentration-dependent ability to downregulate the levels of CD4 + T cell activation. At the final concentration of 0.500 μg/mL of A. indica, a significant downregulation of CD4 + CD38 + HLA-DR+ expression was observed in HIV negative (p < 0.0001) and both HIV infected groups (P = 0.0313). This plant extract also significantly lowered SEB induced % CD4+ T cell HLADR, PD-1 and Tim-3 levels. PD-1 and CD69 markers were only significantly downmodulated in only the HIV negative ((p = 0.0001 and p = 0.0078 respectively) and viral load< 1000 copies per ml (p = 0.0078) groups., Conclusion: A. indica exhibited the in-vitro immunomodulatory potential to inhibit the continuum of SEB induced CD4+ T-cell activation/ exhaustion without impacting general T-cell specific functions such as cytokine secretion. Additional studies are needed to confirm A. indica as a source of natural products for targeting persistent immune activation and inflammation during ART.

There has been considerable investigation of host-microbial interactions in patients with chronic rhinosinusitis (CRS) in hopes of elucidating mechanisms of disease and better treatment. Most attention has been paid to bacterial infection and potential underlying defects in innate immunity. Bacterial biofilm is present in most patients with CRS undergoing surgical intervention, and its presence is associated with more severe disease and worse surgical outcomes. A role for viral or fungal infection in patients with CRS is less clear. There is no evidence for a primary defect in mucociliary clearance in most patients with CRS. Decreased levels of certain antimicrobial proteins, most notably lactoferrin, have been found in sinus secretions, whereas levels of other antimicrobial proteins have been found to be normal. No primary defects in Toll-like receptors have been found in patients with CRS, although a 50% reduced expression of Toll-like receptor 9 was reported in patients with recalcitrant nasal polyps. A polymorphism in a bitter taste receptor was recently associated with refractory CRS and persistent Pseudomonas aeruginosa infection. A downregulation of innate immunity by maladaptive TH2 tissue inflammation has also been described in patients with recalcitrant nasal polyps, suggesting a link to persistent infection. To date, an effective means of restoring host-microbial balance and mitigating disease in patients with CRS remains elusive. [Copyright &y& Elsevier]

Background: Although the cytokine IL-31 has been implicated in inflammatory and lymphoma-associated itch, the cellular basis for its pruritic action is yet unclear. Objective: We sought to determine whether immune cell–derived IL-31 directly stimulates sensory neurons and to identify the molecular basis of IL-31–induced itch. Methods: We used immunohistochemistry and quantitative real-time PCR to determine IL-31 expression levels in mice and human subjects. Immunohistochemistry, immunofluorescence, quantitative real-time PCR, in vivo pharmacology, Western blotting, single-cell calcium imaging, and electrophysiology were used to examine the distribution, functionality, and cellular basis of the neuronal IL-31 receptor α in mice and human subjects. Results: Among all immune and resident skin cells examined, IL-31 was predominantly produced by TH2 and, to a significantly lesser extent, mature dendritic cells. Cutaneous and intrathecal injections of IL-31 evoked intense itch, and its concentrations increased significantly in murine atopy-like dermatitis skin. Both human and mouse dorsal root ganglia neurons express IL-31RA, largely in neurons that coexpress transient receptor potential cation channel vanilloid subtype 1 (TRPV1). IL-31–induced itch was significantly reduced in TRPV1-deficient and transient receptor channel potential cation channel ankyrin subtype 1 (TRPA1)–deficient mice but not in c-kit or proteinase-activated receptor 2 mice. In cultured primary sensory neurons IL-31 triggered Ca2+ release and extracellular signal-regulated kinase 1/2 phosphorylation, inhibition of which blocked IL-31 signaling in vitro and reduced IL-31–induced scratching in vivo. Conclusion: IL-31RA is a functional receptor expressed by a small subpopulation of IL-31RA+/TRPV1+/TRPA1+ neurons and is a critical neuroimmune link between TH2 cells and sensory nerves for the generation of T cell–mediated itch. Thus targeting neuronal IL-31RA might be effective in the management of TH2-mediated itch, including atopic dermatitis and cutaneous T-cell lymphoma. [Copyright &y& Elsevier]