Polymerase pausing induced by sequence-specific RNA-binding protein drives heterochromatin assembly.

In Schizosaccharomyces pombe, transcripts derived from the pericentromeric dg and dh repeats promote heterochromatin formation via RNAi as well as an RNAi-independent mechanism involving the RNA polymerase II (RNAPII)-associated RNA-binding protein Seb1 and RNA processing activities. We show that Seb1 promotes long-lived RNAPII pauses at pericentromeric repeat regions and that their presence correlates with the heterochromatin-triggering activities of the corresponding dg and dh DNA fragments. Globally increasing RNAPII stalling by other means induces the formation of novel large ectopic heterochromatin domains. Such ectopic heterochromatin occurs even in cells lacking RNAi. These results uncover Seb1-mediated polymerase stalling as a signal necessary for heterochromatin nucleation.

The Nrd1-like protein Seb1 coordinates cotranscriptional 3' end processing and polyadenylation site selection.

Termination of RNA polymerase II (RNAPII) transcription is associated with RNA 3' end formation. For coding genes, termination is initiated by the cleavage/polyadenylation machinery. In contrast, a majority of noncoding transcription events in Saccharomyces cerevisiae does not rely on RNA cleavage for termination but instead terminates via a pathway that requires the Nrd1-Nab3-Sen1 (NNS) complex. Here we show that the Schizosaccharomyces pombe ortholog of Nrd1, Seb1, does not function in NNS-like termination but promotes polyadenylation site selection of coding and noncoding genes. We found that Seb1 associates with 3' end processing factors, is enriched at the 3' end of genes, and binds RNA motifs downstream from cleavage sites. Importantly, a deficiency in Seb1 resulted in widespread changes in 3' untranslated region (UTR) length as a consequence of increased alternative polyadenylation. Given that Seb1 levels affected the recruitment of conserved 3' end processing factors, our findings indicate that the conserved RNA-binding protein Seb1 cotranscriptionally controls alternative polyadenylation.

Staphylococcal enterotoxin B as DNA vaccine against breast cancer in a murine model.

Recently, many efforts have been made to treat cancer using recombinant bacterial toxins and this strategy has been used in clinical trials of various cancers. Therapeutic DNA cancer vaccines are now considered as a promising strategy to activate the immune system against cancer. Cancer vaccines could induce specific and long-lasting immune responses against tumors. This study aimed to evaluate the antitumor potency of the SEB DNA vaccine as a new antitumor candidate against breast tumors in vivo. To determine the effect of the SEB construct on inhibiting tumor cell growth in vivo, the synthetic SEB gene, subsequent codon optimization, and embedding the cleavage sites were sub-cloned to an expression vector. Then, SEB construct, SEB, and PBS were injected into the mice. After being vaccinated, 4T1 cancer cells were injected subcutaneously into the right flank of mice. Then, the cytokine levels of IL-4 and IFN-γ were estimated by the ELISA method to evaluate the antitumor activity. The spleen lymphocyte proliferation, tumor size, and survival time were assessed. The concentration of IFN-γ in the SEB-Vac group showed a significant increase compared to other groups. The production of IL-4 in the group that received the DNA vaccine did not change significantly compared to the control group. The lymphocyte proliferation increased significantly in the mice group that received SEB construct than PBS control group (p < 0.001). While there was a meaningful decrease in tumor size (p < 0.001), a significant increase in tumor tissue necrosis (p < 0.01) and also in survival time of the animal model receiving the recombinant construct was observed. The designed SEB gene construct can be a new model vaccine for breast cancer because it effectively induces necrosis and produces specific immune responses. This structure does not hurt normal cells and is a safer treatment than chemotherapy and radiation therapy. Its slow and long-term release gently stimulates the immune system and cellular memory. It could be applied as a new model for inducing apoptosis and antitumor immunity to treat cancer.

PD-1/PD-L1 Control of Antigen-Specifically Activated CD4 T-Cells of Neonates.

Newborns are highly susceptible to infections; however, the underlying mechanisms that regulate the anti-microbial T-helper cells shortly after birth remain incompletely understood. To address neonatal antigen-specific human T-cell responses against bacteria, Staphylococcus aureus (S. aureus) was used as a model pathogen and comparatively analyzed in terms of the polyclonal staphylococcal enterotoxin B (SEB) superantigen responses. Here, we report that neonatal CD4 T-cells perform activation-induced events upon S. aureus/APC-encounter including the expression of CD40L and PD-1, as well as the production of Th1 cytokines, concomitant to T-cell proliferation. The application of a multiple regression analysis revealed that the proliferation of neonatal T-helper cells was determined by sex, IL-2 receptor expression and the impact of the PD-1/PD-L1 blockade. Indeed, the treatment of S. aureus-activated neonatal T-helper cells with PD-1 and PD-L1 blocking antibodies revealed the specific regulation of the immediate neonatal T-cell responses with respect to the proliferation and frequencies of IFNγ producers, which resembled in part the response of adults' memory T-cells. Intriguingly, the generation of multifunctional T-helper cells was regulated by the PD-1/PD-L1 axis exclusively in the neonatal CD4 T-cell lineage. Together, albeit missing memory T-cells in neonates, their unexperienced CD4 T-cells are well adapted to mount immediate and strong anti-bacterial responses that are tightly controlled by the PD-1/PD-L1 axis, thereby resembling the regulation of recalled memory T-cells of adults.

The SEB1741 Aptamer Is an Efficient Tool for Blocking CD4+ T Cell Activation Induced by Staphylococcal Enterotoxin B.

Staphylococcal enterotoxin B (SEB) is a protein produced by Staphylococcus aureus, which is toxic to humans. It is well known for its ability to stimulate the exacerbated activation of proinflammatory CD4+ T cells (Th1 profile), and in vitro studies have been conducted to understand its mechanism of action and its potential use as an immune therapy. However, the efficiency of the SEB1741 aptamer in blocking SEB has not been experimentally demonstrated. METHODS: Enrichment CD4+ T cells were stimulated with SEB, and as a blocker, we used the SEB1741 aptamer, which was previously synthesised by an "in silico" analysis, showing high affinity and specificity to SEB. The efficiency of the SEB1741 aptamer in blocking CD4+ T cell activation was compared with that of an anti-SEB monoclonal antibody. Flow cytometry and Bio-Plex were used to evaluate the T-cell function. RESULTS: In vitro, SEB induced the activation of CD4+ T cells and favoured a Th1 profile; however, the SEB1741 aptamer was highly efficient in decreasing the frequency of CD4+ T cells positive to ki-67 and CD69 cells, this means that proliferation and activation of CD4+ T cells was decreased. Moreover, the production of interleukin 2 (IL-2) and interferon-gamma (IFN-γ) was affected, suggesting that the Th1 profile is not present when the SEB1441 aptamer is used. Thus, the SEB1741 function was similar to that of anti-SEB. CONCLUSIONS: The SEB1741 aptamer is a valuable tool for blocking CD4+ T cell activation and the subsequent release of proinflammatory cytokines by SEB stimulation.

Post-Translational Modification, Phase Separation, and Robust Gene Transcription.

A few recent reports reveal fundamental new insights into the intricate regulatory mechanisms that govern RNA polymerase II (Pol II)-mediated gene transcription. Whereas a histidine-rich domain (HRD) triggers phase separation, promoting transcription elongation, a phosphatase switch promotes transcription termination. A paradigm that might govern the underlying mechanisms leading to robust gene transcription is now starting to emerge.

A remote sensing-based three-source energy balance model to improve global estimations of evapotranspiration in semi-arid tree-grass ecosystems.

It is well documented that energy balance and other remote sensing-based evapotranspiration (ET) models face greater uncertainty over water-limited tree-grass ecosystems (TGEs), representing nearly 1/6th of the global land surface. Their dual vegetation strata, the grass-dominated understory and tree-dominated overstory, make for distinct structural, physiological and phenological characteristics, which challenge models compared to more homogeneous and energy-limited ecosystems. Along with this, the contribution of grasses and trees to total transpiration (T), along with their different climatic drivers, is still largely unknown nor quantified in TGEs. This study proposes a thermal-based three-source energy balance (3SEB) model, accommodating an additional vegetation source within the well-known two-source energy balance (TSEB) model. The model was implemented at both tower and continental scales using eddy-covariance (EC) TGE sites, with variable tree canopy cover and rainfall (P) regimes and Meteosat Second Generation (MSG) images. 3SEB robustly simulated latent heat (LE) and related energy fluxes in all sites (Tower: LE RMSD ~60 W/m2 ; MSG: LE RMSD ~90 W/m2 ), improving over both TSEB and seasonally changing TSEB (TSEB-2S) models. In addition, 3SEB inherently partitions water fluxes between the tree, grass and soil sources. The modelled T correlated well with EC T estimates (r > .76), derived from a machine learning ET partitioning method. The T/ET was found positively related to both P and leaf area index, especially compared to the decomposed grass understory T/ET. However, trees and grasses had contrasting relations with respect to monthly P. These results demonstrate the importance in decomposing total ET into the different vegetation sources, as they have distinct climatic drivers, and hence, different relations to seasonal water availability. These promising results improved ET and energy flux estimations over complex TGEs, which may contribute to enhance global drought monitoring and understanding, and their responses to climate change feedbacks.

Resveratrol-mediated attenuation of superantigen-driven acute respiratory distress syndrome is mediated by microbiota in the lungs and gut.

Acute Respiratory Distress Syndrome (ARDS) is triggered by a variety of agents, including Staphylococcal Enterotoxin B (SEB). Interestingly, a significant proportion of patients with COVID-19, also develop ARDS. In the absence of effective treatments, ARDS results in almost 40% mortality. Previous studies from our laboratory demonstrated that resveratrol (RES), a stilbenoid, with potent anti-inflammatory properties can attenuate SEB-induced ARDS. In the current study, we investigated the role of RES-induced alterations in the gut and lung microbiota in the regulation of ARDS. Our studies revealed that SEB administration induced inflammatory cytokines, ARDS, and 100% mortality in C3H/HeJ mice. Additionally, SEB caused a significant increase in pathogenic Proteobacteria phylum and Propionibacterium acnes species in the lungs. In contrast, RES treatment attenuated SEB-mediated ARDS and mortality in mice, and significantly increased probiotic Actinobacteria phylum, Tenericutes phylum, and Lactobacillus reuteri species in both the colon and lungs. Colonic Microbiota Transplantation (CMT) from SEB-injected mice that were treated with RES as well as the transfer of L. reuteri into recipient mice inhibited the production of SEB-mediated induction of pro-inflammatory cytokines such as IFN-γ and IL-17 but increased that of anti-inflammatory IL-10. Additionally, such CMT and L. reuteri recipient mice exposed to SEB, showed a decrease in lung-infiltrating mononuclear cells, cytotoxic CD8+ T cells, NKT cells, Th1 cells, and Th17 cells, but an increase in the population of regulatory T cells (Tregs) and Th3 cells, and increase in the survival of mice from SEB-mediated ARDS. Together, the current study demonstrates that ARDS induced by SEB triggers dysbiosis in the lungs and gut and that attenuation of ARDS by RES may be mediated, at least in part, by alterations in microbiota in the lungs and the gut, especially through the induction of beneficial bacteria such as L. reuteri.

Recent advances in sensitive surface-enhanced Raman scattering-based lateral flow assay platforms for point-of-care diagnostics of infectious diseases.

This review reports the recent advances in surface-enhanced Raman scattering (SERS)-based lateral flow assay (LFA) platforms for the diagnosis of infectious diseases. As observed through the recent infection outbreaks of COVID-19 worldwide, a timely diagnosis of the disease is critical for preventing the spread of a disease and to ensure epidemic preparedness. In this regard, an innovative point-of-care diagnostic method is essential. Recently, SERS-based assay platforms have received increasing attention in medical communities owing to their high sensitivity and multiplex detection capability. In contrast, LFAs provide a user-friendly and easily accessible sensing platform. Thus, the combination of LFAs with a SERS detection system provides a new diagnostic modality for accurate and rapid diagnoses of infectious diseases. In this context, we briefly discuss the recent application of LFA platforms for the POC diagnosis of SARS-CoV-2. Thereafter, we focus on the recent advances in SERS-based LFA platforms for the early diagnosis of infectious diseases and their applicability for the rapid diagnosis of SARS-CoV-2. Finally, the key issues that need to be addressed to accelerate the clinical translation of SERS-based LFA platforms from the research laboratory to the bedside are discussed.

A conserved ncRNA-binding protein recruits silencing factors to heterochromatin through an RNAi-independent mechanism.

Long noncoding RNAs (lncRNAs) can trigger repressive chromatin, but how they recruit silencing factors remains unclear. In Schizosaccharomyces pombe, heterochromatin assembly on transcribed noncoding pericentromeric repeats requires both RNAi and RNAi-independent mechanisms. In Saccharomyces cerevisiae, which lacks a repressive chromatin mark (H3K9me [methylated Lys9 on histone H3]), unstable ncRNAs are recognized by the RNA-binding protein Nrd1. We show that the S. pombe ortholog Seb1 is associated with pericentromeric lncRNAs. Individual mutation of dcr1+ (Dicer) or seb1+ results in equivalent partial reductions of pericentromeric H3K9me levels, but a double mutation eliminates this mark. Seb1 functions independently of RNAi by recruiting the NuRD (nucleosome remodeling and deacetylase)-related chromatin-modifying complex SHREC (Snf2-HDAC [histone deacetylase] repressor complex).

Staphylococcal enterotoxin B increased severity of experimental model of multiple sclerosis.

BACKGROUND: Superantigens can be absorbed trans-mucosal and trans-cutaneous in individuals colonized with superantigen producing Staphylococcus aureus. Ability of superantigens to activate a large numbers of T cells suggests that they may play a role in the course of autoimmune diseases including human multiple sclerosis and experimental autoimmune encephalomyelitis (EAE). In this study we investigated the role of staphylococcal enterotoxin B in immunologic and pathologic changes in experimental animal model of multiple sclerosis. METHODS: C57BL/6 female mice were treatment with SEB protein prior or post immunization with MOG33-35 peptide. Mice were monitored daily and scored for clinical symptoms following EAE induction. Spleen and spinal cord of mice were removed and used for ELISA and histological studies, respectively. RESULTS: Treatment with SEB prior induction of EAE, increased clinical score, the concentration of IL-17A, IFN-γ and histological changes compared to control group. Treatment with SEB after induction of EAE caused these changes, but less severe. DISCUSSION: Since SEB causes demyelination of spinal cord and increases the level of pro-inflammatory cytokine response, infiltration of T-lymphocytes and macrophages to CNS, it may exacerbate the clinical signs of EAE in mice and multiple sclerosis in human.

Molecular alterations induced by Yersinia pestis, dengue virus and Staphylococcal enterotoxin B under severe stress.

BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

Polyadenylation site selection: linking transcription and RNA processing via a conserved carboxy-terminal domain (CTD)-interacting protein.

Despite the fact that the process of mRNA polyadenylation has been known for more than 40 years, a detailed understating of the mechanism underlying polyadenylation site selection is still far from complete. As 3' end processing is intimately associated with RNA polymerase II (RNAPII) transcription, factors that can successively interact with the transcription machinery and recognize cis-acting sequences on the nascent pre-mRNA would be well suited to contribute to poly(A) site selection. Studies using the fission yeast Schizosaccharomyces pombe have recently identified Seb1, a protein that shares homology with Saccharomyces cerevisiae Nrd1 and human SCAF4/8, and that is critical for poly(A) site selection. Seb1 binds to the C-terminal domain (CTD) of RNAPII via a conserved CTD-interaction domain and recognizes specific sequence motifs clustered downstream of the polyadenylation site on the uncleaved pre-mRNA. In this short review, we summarize insights into Seb1-dependent poly(A) site selection and discuss some unanswered questions regarding its molecular mechanism and conservation.

Mechanisms mediating enhanced neutralization efficacy of staphylococcal enterotoxin B by combinations of monoclonal antibodies.

Staphylococcal enterotoxin B (SEB) is a superantigen that cross-links the major histocompatibility complex class II and specific V-ß chains of the T-cell receptor, thus forming a ternary complex. Developing neutralizing mAb to disrupt the ternary complex and abrogate the resulting toxicity is a major therapeutic challenge because SEB is effective at very low concentrations. We show that combining two SEB-specific mAbs enhances their efficacy, even though one of the two mAbs by itself has no effect on neutralization. Crystallography was employed for fine-mapping conformational epitopes in binary and ternary complexes between SEB and Fab fragments. NMR spectroscopy was used to validate and identify subtle allosteric changes induced by mAbs binding to SEB. The mapping of epitopes established that a combination of different mAbs can enhance efficacy of mAb-mediated protection from SEB induced lethal shock by two different mechanisms: one mAb mixture promoted clearance of the toxin both in vitro and in vivo by FcR-mediated cross-linking and clearance, whereas the other mAb mixture induced subtle allosteric conformational changes in SEB that perturbed formation of the SEB·T-cell receptor·major histocompatibility complex class II trimer. Finally structural information accurately predicted mAb binding to other superantigens that share conformational epitopes with SEB. Fine mapping of conformational epitopes is a powerful tool to establish the mechanism and optimize the action of synergistic mAb combinations.

sCD48 is anti-inflammatory in Staphylococcus aureus Enterotoxin B-induced eosinophilic inflammation.

BACKGROUND: Staphylococcus aureus, one of the most important pathogens, is heavily associated with allergy. S. aureus and its toxins interact with eosinophils through CD48, a GPI-anchored receptor important in allergy mainly as expressed by the eosinophils (mCD48). CD48 can exist in a soluble form (sCD48). Our aim was to investigate SEB-induced regulation of eosinophil CD48 and the possible formation and role of sCD48 in SEB-mediated eosinophil activation in vitro and in vivo. METHODS: Human peripheral blood eosinophils were activated by SEB with or without inhibitors for phospholipases (PL) (-C or -D), or cycloheximide, or brefeldin A. We evaluated eosinophil activation (CD11b expression or EPO/IL-8 release), mCD48 (flow cytometry), sCD48 (ELISA), SEB binding to sCD48 (ELISA), and chemotaxis toward SEB. C57BL/6 mice were pre-injected (ip.) with sCD48, and then, peritonitis was induced by SEB injection; peritoneal lavages were collected after 48 h and analyzed by flow cytometry and ELISA. RESULTS: SEB-activated human eosinophils formed sCD48, directly correlating with CD11b expression, through cell-associated PL-C and -D. mCD48 remained stable due to up-regulation in CD48 transcription and cellular trafficking. sCD48 bound to SEB and down-regulated SEB stimulatory effects on eosinophils as assessed by EPO and IL-8 release and eosinophil chemotaxis toward SEB. sCD48 showed anti-inflammatory activity in a SEB-induced mouse peritonitis model. CONCLUSIONS: SEB regulates CD48 dynamics on eosinophils. Our data indicate sCD48 as a SEB-induced 'decoy' receptor derived from eosinophil and therefore as a potential anti-inflammatory tool in S. aureus-induced eosinophil inflammation often associated with allergy.

Combined multiplex loop-mediated isothermal amplification with lateral flow assay to detect sea and seb genes of enterotoxic Staphylococcus aureus.

UNLABELLED: Staphylococcal enterotoxins (SEs) are the most common cause of food poisoning worldwide and can induce symptoms, such as diarrhoea, vomiting and abdominal cramping. Thus, the aim of this study is to develop a multiplex loop-mediated isothermal amplification combined with a lateral flow assay (m-LAMP/LFA) to simultaneously detect the sea and seb genes of Staphylococcus aureus. The amplicons of the sea gene were labelled with digoxigenin (Dig) and biotin while those of seb gene were labelled with fluorescein isothiocyanate (FITC) and biotin. These amplicons were detected using a multiplex LFA with NeutrAvidin-tagged gold nanoparticles as the detection reagent. After optimization, the detection limit of this assay was 10(2)  CFU ml(-1) Staph. aureus, which was one tenth that of a multiplex PCR. This assay did not exhibit any cross-reactivity in detecting other enterotoxic Staph. aureus strains or other food pathogens. After 6 h of enrichment, this developed assay detected 1 CFU ml(-1) of Staph. aureus in milk, apple juice, cheese and rice. The developed m-LAMP/LFA method does not require expensive equipment and can be completely implemented within an 8-h workday. Therefore, this method can provide an effective means of quickly screening staphylococcal enterotoxin A- and/or staphylococcal enterotoxin B-producing Staph. aureus in food samples. SIGNIFICANCE AND IMPACT OF THE STUDY: Staphylococcus aureus is one of the major foodborne pathogens worldwide, and its staphylococcal enterotoxin A and B are strongly associated with food poisoning. This work developed a multiplex loop-mediated isothermal amplification combined with a lateral flow assay (m-LAMP/LFA) to simultaneously detect the sea and seb genes of Staph. aureus in food samples. The assay has good specificity and sensitivity with ease-of-use features, making it ideal for on-site detection.

Staphylococcal α-toxin induces a functional upregulation of TLR-2 on human peripheral blood monocytes.

Resistance to bacterial skin infections, for example with Staphylococcus aureus (S. aureus), is based on the function of intact innate immune mechanisms. Toll-like receptor (TLR)-2 recognizes components of S. aureus and is known to be expressed on monocytes. Staphylococcal exotoxins such as staphylococcal enterotoxin B (SEB) or α-toxin are produced by many S. aureus strains. To investigate TLR-2 regulation and function on human monocytes upon stimulation with staphylococcal exotoxins to elucidate a putative feedback loop between different staphylococcal components. Monocytes were stimulated with α-toxin or SEB, respectively. TLR-2 expression and regulation as well as functional effects of TLR-2 stimulation with Pam3Cys (TLR-2/TLR-1), lipoteichoic acid (LTA) (TLR-2/TLR-6) and peptidoglycan (PGN) (TLR-2 and Nod) were then investigated both at the mRNA and protein level and compared to monocytes from patients with psoriasis. α-toxin significantly upregulated TLR-2 expression. TLR-2 mediated IL-1ß, IL-6 and IL-8 secretion was significantly augmented after upregulation with staphylococcal exotoxins. CD36 expression was significantly more downregulated after TLR-2 upregulation with SEB and consecutive LTA stimulation and TLR-2 upregulation with α-toxin following LTA and PGN stimulation, respectively. PGN enhanced CD54 expression after upregulation of the receptor with α-toxin. Expression of HLA-DR was unaffected. However, no differences were observed in monocytes from psoriasis patients compared to healthy controls. Together, our findings provide a new link between staphylococcal α-toxin and TLR-2 signalling in monocytes which may have implications for skin diseases where skin colonization with S. aureus and dysregulation of TLR-2 have been described.

Food allergy: Insights into etiology, prevention, and treatment provided by murine models.

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.

A sensory neuron-expressed IL-31 receptor mediates T helper cell-dependent itch: Involvement of TRPV1 and TRPA1.

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 Ca(2+) 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.

Screening and characterization of anti-SEB peptides using a bacterial display library and microfluidic magnetic sorting.

Bacterial peptide display libraries enable the rapid and efficient selection of peptides that have high affinity and selectivity toward their targets. Using a 15-mer random library on the outer surface of Escherichia coli (E.coli), high-affinity peptides were selected against a staphylococcal enterotoxin B (SEB) protein after four rounds of biopanning. On-cell screening analysis of affinity and specificity were measured by flow cytometry and directly compared to the synthetic peptide, off-cell, using peptide-ELISA. DNA sequencing of the positive clones after four rounds of microfluidic magnetic sorting (MMS) revealed a common consensus sequence of (S/T)CH(Y/F)W for the SEB-binding peptides R338, R418, and R445. The consensus sequence in these bacterial display peptides has similar amino acid characteristics with SEB peptide sequences isolated from phage display. The Kd measured by peptide-ELISA off-cell was 2.4 nM for R418 and 3.0 nM for R445. The bacterial peptide display methodology using the semiautomated MMS resulted in the discovery of selective peptides with affinity for a food safety and defense threat. Published 2014. This article is a U.S. Government work and is in the public domain in the USA.