Oesophageal eosinophilia accompanies food allergy to hen egg white protein in young pigs.

BACKGROUND: Esophagitis with eosinophilia, inflammation, and fibrosis represent a chronic condition in humans with food allergies. OBJECTIVE: In this investigation, we asked whether esophagitis with an eosinophilic component is observed in young pigs rendered allergic to hen egg white protein (HEWP). METHODS: Food allergy was induced in young pigs using two protocols. In one protocol, sensitized pigs were challenged by gavage with a single dose of HEWP. Clinical signs were monitored for 24 hours, and then, gastrointestinal (GI) tissues were collected for histological examination. The phenotype of circulating, ovalbumin (OVA)-specific T cells also was examined in HEWP challenged animals. In the second protocol, sensitized animals were fed HEWP for 28 days. Animals were then examined by endoscopy and gastrointestinal tissues collected for histological examination. RESULTS: In pigs challenged by gavage with HEWP, clinical signs were noted in 5/6 pigs including diarrhoea, emesis, and skin rash. Clinical signs were not seen in any control group. Histological analysis revealed significant levels of oesophageal eosinophilic infiltration (P < .05) in 4/6 of these animals, with two also displaying eosinophilic infiltration in the stomach. Eosinophils were not increased in ileum or colon samples. Increased numbers of circulating, OVA-specific CD4+ T cells also were observed in pigs that received HEWP by gavage. In the group of animals fed HEWP, endoscopy revealed clinical signs of esophagitis including oedema, granularity, white spots, and furrowing, while histology revealed oedema, immune cell infiltration, and basal zone hyperplasia. CONCLUSIONS AND CLINICAL RELEVANCE: Food allergy in the pig can be associated with esophagitis based on histological and endoscopic findings, including eosinophilic infiltration. The young pig may, therefore, be a useful large animal model for the study of eosinophilic esophagitis in humans.

Anticancer and immunomodulatory activity of egg proteins and peptides: a review.

Eggs are widely recognized as a highly nutritious food source that offer specific health benefits for humans. Eggs contain all of the proteins, lipids, vitamins, minerals, and growth factors necessary for embryonic development. In particular, egg white and yolk proteins are considered functional food substances because they possess biological activities such as antimicrobial, antioxidant, metal-chelating, antihypertensive, anticancer, and immunomodulatory activities. Peptides produced via processes such as enzymatic hydrolysis, fermentation by microorganisms, and some chemical and physical treatments of egg proteins have been shown to enhance the functional properties and solubility of these peptides. Peptide activity is strongly related to amino acid sequence, composition, and length. At present, cancer remains among the leading causes of mortality worldwide, and therefore research aimed at developing new treatments for cancer immunotherapy is of great interest. The present review focuses primarily on the anticancer and immunomodulatory activities of egg proteins and their peptides and provides some insight into their underlying mechanisms of action. A number of egg proteins and peptides have been reported to induce apoptosis in cancer cells, protect against DNA damage, decrease the invasion ability of cancer cells, and exhibit cytotoxic and antimutagenic activity in various cancer cell lines. Furthermore, egg proteins and peptides can stimulate or suppress pro- or anti-inflammatory cytokines, as well as affect the production of inflammatory mediators in a variety of cell lines. In addition, the composition of eggs and the processes of egg proteins and peptides production will be discussed.

Oral Immunotherapy with Egg Peptides Induces Innate and Adaptive Tolerogenic Responses.

SCOPE: The mechanism through which peptide-based immunotherapy provides effective desensitization toward food allergy is investigated. METHODS AND RESULTS: Ex vivo experiments are conducted with intestinal epithelial cells (IECs), dendritic cells (DCs), and T cells from mice sensitized to egg white (EW) and either left untreated or tolerized by the oral administration of a hydrolysate of ovalbumin with pepsin (OP). IECs from EW-sensitized mice upregulate Il33 and Tslp to a higher extent than those from tolerized mice and induce bone marrow (BM)-DCs to express Tnfsf4 and produce pro-inflammatory cytokines. On the other hand, incubation with OP upregulates Aldh1a1 in IEC cultures and BM-DCs conditioned with supernatants of OP-pulsed IECs also overexpress Aldh1a2 and Tgfb1. DCs from tolerized mice, in co-culture with CD4+ T cells from sensitized mice, reduce the secretion of IL-5, IFN-γ, and IL-17, following stimulation with EW, to a level similar than DCs from sham-sensitized mice. Furthermore, incubation with OP of DCs and CD4+ T cells, regardless of the mouse sentitization status, promotes the secretion of TGF-ß and the generation of Foxp3+ RORγt+ cells. CONCLUSION: OP induces the expression of aldehyde dehydrogenase enzymes in cells of the innate immune system and the development of Foxp3+ RORγt+ T cells.

New methods for purification of Paralichthys olivaceus lipovitellin and immunoassay-based detection of vitellogenin.

Increasing levels of estrogenic pollution in marine environments has made the development of reliable biological detection techniques urgently needed. In this study, Japanese flounder (Paralichthys olivaceus) lipovitellin (Lv) was purified and used to establish three immunological methods for the detection of vitellogenin (Vtg), a biomarker for environmental estrogens. Firstly, five different methods were employed to purify Lv, among which water-precipitation was the fastest and easiest way to purify Lv. Japanese flounder Lv was characterized as a phospholipoglycoprotein with a molecular weight of ∼369 kDa. Using purified Lv and its specific polyclonal antibody, a sandwich enzyme-linked immunosorbent assay (ELISA) was developed. This assay had a working range from 7.8 to 250 ng/mL and a detection limit of 3.1 ng/mL. Furthermore, we developed an immunohistochemistry (IHC) and an immunofluorescence (IF) assay, both of which allowed visual detection of liver Vtg. Finally, Vtg induction in plasma and liver of juvenile Japanese flounders exposed to 17ß-ethinylestradiol (EE2) was measured using these three methods. Exposure to 10 and 50 ng/L EE2 significantly increased plasma Vtg levels, and obvious positive fluorescence signals were observed near the liver sinusoidal vessels. These results confirmed that the methods developed effectively detected estrogenic activity of exogenous chemicals. Therefore, this study provides reliable methodologies for biomonitoring of estrogenic pollution in marine environments.

IPSE, a urogenital parasite-derived immunomodulatory protein, ameliorates ifosfamide-induced hemorrhagic cystitis through downregulation of pro-inflammatory pathways.

Ifosfamide and other oxazaphosphorines can result in hemorrhagic cystitis, a constellation of complications caused by acrolein metabolites. We previously showed that a single dose of IPSE (Interleukin-4-inducing principle from Schistosoma eggs), a schistosome-derived host modulatory protein, can ameliorate ifosfamide-related cystitis; however, the mechanisms underlying this urotoxicity and its prevention are not fully understood. To provide insights into IPSE's protective mechanism, we undertook transcriptional profiling of bladders from ifosfamide-treated mice, with or without pretreatment with IPSE or IPSE-NLS (a mutant of IPSE lacking nuclear localization sequence). Ifosfamide treatment upregulated a range of proinflammatory genes. The IL-1ß-TNFα-IL-6 proinflammatory cascade via NFκB and STAT3 pathways was identified as the key driver of inflammation. The NRF2-mediated oxidative stress response pathway, which regulates heme homoeostasis and expression of antioxidant enzymes, was highly activated. Anti-inflammatory cascades, namely Wnt, Hedgehog and PPAR pathways, were downregulated. IPSE drove significant downregulation of major proinflammatory pathways including the IL-1ß-TNFα-IL-6 pathways, interferon signaling, and reduction in oxidative stress. IPSE-NLS reduced inflammation but not oxidative stress. Taken together, we have identified signatures of acute-phase inflammation and oxidative stress in ifosfamide-injured bladder, which are reversed by pretreatment with IPSE. This work revealed several pathways that could be therapeutically targeted to prevent ifosfamide-induced hemorrhagic cystitis.

Egg proteins: fractionation, bioactive peptides and allergenicity.

Eggs are an important source of macro and micronutrients within the diet, comprised of proteins, lipids, vitamins, and minerals. They are constituted by a shell, the white (containing 110 g kg-1 proteins: ovalbumin, ovotransferrin, ovomucoid, lysozyme and ovomucin), and the yolk (containing 150-170 g kg-1 proteins: lipovitellins, phosvitin, livetins, and low-density lipoproteins). Owing to their nutritional value and biological characteristics, both the egg white and yolk proteins are extensively fractionated using different techniques (e.g., liquid chromatography, ultrafiltration, electrophoresis, and chemical precipitation), in which liquid chromatography is the most commonly used technique to obtain individual proteins with high protein recovery and purity to develop novel food products. However, concerns over allergenic responses induced by certain egg proteins (e.g., ovomucoid, ovalbumin, ovotransferrin, lysozyme, α-livetin, and lipoprotein YGP42) limit their widespread use. As such, processing technologies (e.g., thermal processing, enzymatic hydrolysis, and high-pressure treatment) are investigated to reduce the allergenicity by conformational changes. In addition, biological activities (e.g., antioxidant, antimicrobial, antihypertensive, and anticancer activities) associated with egg peptides have received more attention, in which enzyme hydrolysis is demonstrated as a promising way to break polypeptides sequences and produce bioactive peptides to provide nutritional and therapeutic benefits for human health. © 2018 Society of Chemical Industry.

Single and multiple food allergies in infants with proctocolitis.

BACKGROUND: Food protein-induced allergic proctocolitis is a frequent cause of rectal bleeding in infants. Characteristics of infants with multiple food allergies have not been defined. OBJECTIVE: This study aimed to identify characteristics of infants with proctocolitis and compare infants with single and multiple food allergies. METHODS: A total of 132 infants with proctocolitis were evaluated retrospectively. All of the infants were diagnosed by a paediatric allergist and/or a paediatric gastroenterologist according to guidelines. Clinical features of the infants, as well as results of a complete blood count, skin prick test, specific immunoglobulin E, and stool examinations or colonoscopy were recorded. RESULTS: Cow's milk (97.7%) was the most common allergen, followed by egg (22%). Forty-five (34.1%) infants had allergies to more than one food. Infants with multiple food allergies had a higher eosinophil count (613±631.2 vs. 375±291.9) and a higher frequency of positive specific IgE and/or positive skin prick test results than that of patients with a single food allergy. Most of the patients whose symptoms persisted after two years of age had multiple food allergies. CONCLUSIONS: There is no difference in clinical presentations between infants with single and multiple food allergies. However, infants with multiple food allergies have a high blood total eosinophil count and are more likely to have a positive skin prick test and/or positive specific IgE results.

Development of an immunosensor for quantifying zebrafish vitellogenin based on the Octet system.

Vitellogenin (Vtg) is a sensitive biomarker for environmental estrogens. In this study, an immunosensor for quantifying zebrafish Vtg was developed using the Octet system. First, Protein A sensors were immobilized with purified anti-lipovitellin (Lv) antibody that demonstrated specificity to Vtg. Then, antibody-coated biosensors were immersed into zebrafish Lv standards and diluted samples. The Octet system measured and recorded kinetic parameters between antigens and captured antibody within 5 min. Sample Vtg concentrations were automatically calculated by interpolating relative binding rates observed with each sample and the immobilized anti-Lv antibody into the developed standard curve. The sensor arrays exhibited a wide linear range from 78 to 5000 ng/mL, and the inter-assay coefficient of variation was 0.66-1.97%. Furthermore, the performance of the immunosensor in detecting Vtg was evaluated by quantifying Vtg induction in juvenile zebrafish exposed to 17ß-estradiol (E2). Compared with conventional immunoassay techniques, the Vtg immunosensor developed based on the Octet system was much simpler and less time-consuming, allowing rapid Vtg quantification within 15 min. Moreover, Protein A sensors could be reused many times to ensure that the assays have high reproducibility. Therefore, we suggest that immunosensors based on the Octet system are an easily operated detection method for ecotoxicological research.

Schistosome egg antigens, including the glycoprotein IPSE/alpha-1, trigger the development of regulatory B cells.

Infection with the helminth Schistosoma (S.) mansoni drives the development of interleukin (IL)-10-producing regulatory B (Breg) cells in mice and man, which have the capacity to reduce experimental allergic airway inflammation and are thus of high therapeutic interest. However, both the involved antigen and cellular mechanisms that drive Breg cell development remain to be elucidated. Therefore, we investigated whether S. mansoni soluble egg antigens (SEA) directly interact with B cells to enhance their regulatory potential, or act indirectly on B cells via SEA-modulated macrophage subsets. Intraperitoneal injections of S. mansoni eggs or SEA significantly upregulated IL-10 and CD86 expression by marginal zone B cells. Both B cells as well as macrophages of the splenic marginal zone efficiently bound SEA in vivo, but macrophages were dispensable for Breg cell induction as shown by macrophage depletion with clodronate liposomes. SEA was internalized into acidic cell compartments of B cells and induced a 3-fold increase of IL-10, which was dependent on endosomal acidification and was further enhanced by CD40 ligation. IPSE/alpha-1, one of the major antigens in SEA, was also capable of inducing IL-10 in naïve B cells, which was reproduced by tobacco plant-derived recombinant IPSE. Other major schistosomal antigens, omega-1 and kappa-5, had no effect. SEA depleted of IPSE/alpha-1 was still able to induce Breg cells indicating that SEA contains more Breg cell-inducing components. Importantly, SEA- and IPSE-induced Breg cells triggered regulatory T cell development in vitro. SEA and recombinant IPSE/alpha-1 also induced IL-10 production in human CD1d+ B cells. In conclusion, the mechanism of S. mansoni-induced Breg cell development involves a direct targeting of B cells by SEA components such as the secretory glycoprotein IPSE/alpha-1.

Production and glyco-engineering of immunomodulatory helminth glycoproteins in plants.

Helminth parasites control host-immune responses by secreting immunomodulatory glycoproteins. Clinical trials and mouse model studies have demonstrated the potential of helminth-derived glycoproteins for the treatment of immune-related diseases, like allergies and autoimmune diseases. Studies are however hampered by the limited availability of native parasite-derived proteins. Moreover, recombinant protein production systems have thus far been unable to reconstitute helminth-like glycosylation essential for the functionality of some helminth glycoproteins. Here we exploited the flexibility of the N-glycosylation machinery of plants to reconstruct the helminth glycoproteins omega-1 and kappa-5, two major constituents of immunomodulatory Schistosoma mansoni soluble egg antigens. Fine-tuning transient co-expression of specific glycosyltransferases in Nicotiana benthamiana enabled the synthesis of Lewis X (LeX) and LDN/LDN-F glycan motifs as found on natural omega-1 and kappa-5, respectively. In vitro and in vivo evaluation of the introduction of native LeX motifs on plant-produced omega-1 confirmed that LeX on omega-1 contributes to the glycoprotein's Th2-inducing properties. These data indicate that mimicking the complex carbohydrate structures of helminths in plants is a promising strategy to allow targeted evaluation of therapeutic glycoproteins for the treatment of inflammatory disorders. In addition, our results offer perspectives for the development of effective anti-helminthic vaccines by reconstructing native parasite glycoprotein antigens.

CD4+ virtual memory: Antigen-inexperienced T cells reside in the naïve, regulatory, and memory T cell compartments at similar frequencies, implications for autoimmunity.

It is widely accepted that central and effector memory CD4+ T cells originate from naïve T cells after they have encountered their cognate antigen in the setting of appropriate co-stimulation. However, if this were true the diversity of T cell receptor (TCR) sequences within the naïve T cell compartment should be far greater than that of the memory T cell compartment, which is not supported by TCR sequencing data. Here we demonstrate that aged mice with far fewer naïve T cells, respond to the model antigen, hen eggwhite lysozyme (HEL), by utilizing the same TCR sequence as their younger counterparts. CD4+ T cell repertoire analysis of highly purified T cell populations from naive animals revealed that the HEL-specific clones displayed effector and central "memory" cell surface phenotypes even prior to having encountered their cognate antigen. Furthermore, HEL-inexperienced CD4+ T cells were found to reside within the naïve, regulatory, central memory, and effector memory T cell populations at similar frequencies and the majority of the CD4+ T cells within the regulatory and memory populations were unexpanded. These findings support a new paradigm for CD4+ T cell maturation in which a specific clone can undergo a differentiation process to exhibit a "memory" or regulatory phenotype without having undergone a clonal expansion event. It also demonstrates that a foreign-specific T cell is just as likely to reside within the regulatory T cell compartment as it would the naïve compartment, arguing against the specificity of the regulatory T cell compartment being skewed towards self-reactive T cell clones. Finally, we demonstrate that the same set of foreign and autoreactive CD4+ T cell clones are repetitively generated throughout adulthood. The latter observation argues against T cell-depleting strategies or autologous stem cell transplantation as therapies for autoimmunity-as the immune system has the ability to regenerate pathogenic clones.

A novel enzyme-linked immunosorbent assay based on anti-lipovitellin monoclonal antibodies for quantification of zebrafish (Danio rerio) vitellogenin.

Vitellogenin (Vtg) in zebrafish (Danio rerio) is a recommended biomarker endpoint for detecting estrogenic activity of chemicals under the OECD test guidelines. The present paper reports the development of a sensitive and rapid enzyme-linked immunosorbent assay (ELISA) for the quantification of zebrafish Vtg based on monoclonal antibodies (MAbs) against lipovitellin (Lv), the major yolk protein derived from Vtg. The purified Lv was used to immunize mice and the spleen cells of mice were fused with myeloma cells. Two high-affinity MAbs (H3A8 and H4D9) were screened from hybridoma cells. Western blot analysis revealed that two MAbs were highly specific to zebrafish Vtg and recognized different antigenic epitopes because MAb H3A8 detected a main band of 143kDa in purified Vtg, while MAb H4D9 reacted with two clear bands of purified Vtg at 117 and 102kDa. Using MAb H3A8 as the coating antibody, HRP-labeled MAb H4D9 or HRP-labeled PAbs as the detecting antibody, two sandwich ELISAs for Vtg quantification were developed. The sandwich ELISA developed using HRP-labeled MAb H4D9 had a working range of 1.95-250ng/mL, with a detection limit of 0.78ng/mL, which was lower than that of the assay based on HRP-labeled PAbs. Parallelism between Lv standard curves and dilution curves of whole-body homogenates (WBH) from E2-treated male zebrafish confirmed the validity of the ELISAs for quantifying zebrafish Vtg. Finally, the usefulness of two assays for detecting estrogenic activity was verified by quantifying Vtg inductions in zebrafish exposed to 17ß-estradiol.

Lymphocyte Activation Dynamics Is Shaped by Hereditary Components at Chromosome Region 17q12-q21.

Single nucleotide polymorphisms (SNPs) located in the chromosome region 17q12-q21 are risk factors for asthma. Particularly, there are cis-regulatory haplotypes within this region that regulate differentially the expression levels of ORMDL3, GSDMB and ZPBP2 genes. Remarkably, ORMDL3 has been shown to modulate lymphocyte activation parameters in a heterologous expression system. In this context, it has been shown that Th2 and Th17 cytokine production is affected by SNPs in this region. Therefore, we aim to assess the impact of hereditary components within region 17q12-q21 on the activation profile of human T lymphocytes, focusing on the haplotype formed by allelic variants of SNPs rs7216389 and rs12936231. We measured calcium influx and activation markers, as well as the proliferation rate upon T cell activation. Haplotype-dependent differences in mRNA expression levels of IL-2 and INF-γ were observed at early times after activation. In addition, the allelic variants of these SNPs impacted on the extent of calcium influx in resting lymphocytes and altered proliferation rates in a dose dependent manner. As a result, the asthma risk haplotype carriers showed a lower threshold of saturation during activation. Finally, we confirmed differences in activation marker expression by flow cytometry using phytohemagglutinin, a strong polyclonal stimulus. Altogether, our data suggest that the genetic component of pro-inflammatory pathologies present in this chromosome region could be explained by different T lymphocyte activation dynamics depending on individual allelic heredity.

Non-covalent pomegranate (Punica granatum) hydrolyzable tannin-protein complexes modulate antigen uptake, processing and presentation by a T-cell hybridoma line co-cultured with murine peritoneal macrophages.

In this work we characterize the interaction of pomegranate hydrolyzable tannins (HT) with hen egg-white lysozyme (HEL) and determine the effects of non-covalent tannin-protein complexes on macrophage endocytosis, processing and presentation of antigen. We isolated HT from pomegranate and complex to HEL, the resulting non-covalent tannin-protein complex was characterized by gel electrophoresis and MALDI-TOF MS. Finally, cell culture studies and confocal microscopy imaging were conducted on the non-covalent pomegranate HT-HEL protein complexes to evaluate its effect on macrophage antigen uptake, processing and presentation to T-cell hybridomas. Our results indicate that non-covalent pomegranate HT-HEL protein complexes modulate uptake, processing and antigen presentation by mouse peritoneal macrophages. After 4 h of pre-incubation, only trace amounts of IL-2 were detected in the co-cultures treated with HEL alone, whereas a non-covalent pomegranate HT-HEL complex had already reached maximum IL-2 expression. Pomegranate HT may increase rate of endocytose of HEL and subsequent expression of IL-2 by the T-cell hybridomas.

Lipovitellin as an antigen to improve the precision of sandwich ELISA for quantifying zebrafish (Danio rerio) vitellogenin.

Vitellogenin (Vtg) in zebrafish (Danio rerio) is a core biomarker for screening environmental estrogens in test guidelines of the Organization for Economic Cooperation and Development. To accurately quantify zebrafish Vtg, lipovitellin (Lv), the main Vtg-derived yolk protein, was used as the antigen to establish a sandwich enzyme-linked immunosorbent assay (ELISA). The purified Lv was a phospholipoglycoprotein with apparent molecular weight of ~445kDa, and separated into three polypeptides corresponding to ~117, ~102, and ~23.8kDa by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Immunological analysis confirmed the specificity of the anti-Lv antibody for Vtg and the immunological similarity between Vtg and Lv. Using the purified Lv and anti-Lv antibody, a sandwich ELISA with a detection limit of 4.3ng/mL and a detection range from 7.8 to 250ng/mL was developed. The intra- and inter-assay coefficients of variation were both below 10%. Moreover, the Lv standard curve was nearly identical to the Vtg standard curve, and paralleled serial whole-body homogenate dilutions of male zebrafish exposed to 17ß-estradiol, demonstrating that the Lv-based ELISA could be used for quantification of zebrafish Vtg. Zebrafish Lv showed high stability during purification process, heat treatment, -80°C storage, and repeated freeze/thaw cycles. Additionally, the standard curve of Lv stored at -80°C for 3months exhibited higher robustness than that of Vtg stored under the same conditions. Finally, the usefulness of the ELISA for detecting estrogenic activity was verified by quantifying Vtg inductions in zebrafish exposed to monocrotophos.

Pro-Cognitive Properties of the Immunomodulatory Polypeptide Complex, Yolkin, from Chicken Egg Yolk and Colostrum-Derived Substances: Analyses Based on Animal Model of Age-Related Cognitive Deficits.

The study aimed to assess the effect of the polypeptide Y complex (Yolkin), isolated from chicken egg yolk, on behavioural and cognitive functions. It also aimed to compare this activity with colostrum-derived substances (Colostrinin, Coloco), which have a confirmed impact on learning and memory. In the study, the effect of Yolkin, administered to rats of different ages, who performed various tasks involving spatial and episodic memory, motor functions and exploratory behavior, was assessed. The experiment was carried out in rats which were 6 and 12 months old. Two different doses of the studied specimens based on previous comparative studies and two different routes of administration (oral and retroperitoneal) were used. A series of behavioural tests were carried out, including an open field test, a novel object recognition test and a Morris water maze. They were used to evaluate the impact of the studied specimen on improving locomotor function and exploratory behaviour, preventing their decline and assess the functioning of episodic and spatial memory in aging rats. The administration of Yolkin gave distinct effects compared to colostrum-derived substances, although confirmed its suggested pro-cognitive action. Therefore, it may be used to enhance cognitive functions and inhibit the progression of dementia in the course of neurodegenerative disorders.

The helminth T2 RNase ω1 promotes metabolic homeostasis in an IL-33- and group 2 innate lymphoid cell-dependent mechanism.

Induction of a type 2 cellular response in the white adipose tissue leads to weight loss and improves glucose homeostasis in obese animals. Injection of obese mice with recombinant helminth-derived Schistosoma mansoni egg-derived ω1 (ω1), a potent inducer of type 2 activation, improves metabolic status involving a mechanism reliant upon release of the type 2 initiator cytokine IL-33. IL-33 initiates the accumulation of group 2 innate lymphoid cells (ILC2s), eosinophils, and alternatively activated macrophages in the adipose tissue. IL-33 release from cells in the adipose tissue is mediated by the RNase activity of ω1; however, the ability of ω1 to improve metabolic status is reliant upon effective binding of ω1 to CD206. We demonstrate a novel mechanism for RNase-mediated release of IL-33 inducing ILC2-dependent improvements in the metabolic status of obese animals.

The HIV-1 envelope protein gp120 is captured and displayed for B cell recognition by SIGN-R1(+) lymph node macrophages.

The HIV-1 envelope protein gp120 is both the target of neutralizing antibodies and a major focus of vaccine efforts; however how it is delivered to B cells to elicit an antibody response is unknown. Here, we show that following local gp120 injection lymph node (LN) SIGN-R1(+) sinus macrophages located in interfollicular pockets and underlying SIGN-R1(+) macrophages form a cellular network that rapidly captures gp120 from the afferent lymph. In contrast, two other antigens, phycoerythrin and hen egg lysozyme, were not captured by these cells. Intravital imaging of mouse LNs revealed persistent, but transient interactions between gp120 bearing interfollicular network cells and both trafficking and LN follicle resident gp120 specific B cells. The gp120 specific, but not the control B cells repetitively extracted gp120 from the network cells. Our findings reveal a specialized LN antigen delivery system poised to deliver gp120 and likely other pathogen derived glycoproteins to B cells.

Development of a lipovitellin-based sandwich ELISA for quantification of vitellogenin in surface mucus and plasma of goldfish (Carassius auratus).

Goldfish (Carassius auratus) vitellogenin (Vtg) is an efficient biomarker for estrogen contamination in aquatic environments. In this study, Vtg and lipovitellin (Lv) were purified from the plasma of 17ß-estradiol (E2)-induced male goldfish and unfertilized eggs of females, and were used to generate polyclonal antibodies against Vtg (anti-Vtg) and Lv (anti-Lv), respectively. SDS-PAGE and Western blot were performed to confirm the specificity of the two antibodies and the immunological similarity between Vtg and Lv. As anti-Lv recognized more antigen epitopes than anti-Vtg, it was used to develop a sandwich enzyme-linked immunosorbent assay (ELISA) for goldfish Vtg with purified Lv as the standard. The detection limit of the assay was 1.82ng/mL, and the working range was 3.9-250ng/mL. The use of Lv instead of Vtg as the standard provided greater precision and strengthened the robustness of the sandwich ELISA. Western blot and the Lv-based ELISA were used to detect Vtg inductions in surface mucus and plasma of E2-induced goldfish. The surface mucus Vtg level in E2-induced males was significantly higher than that in the control males and E2-induced females, and was much closer to the plasma Vtg level in E2-induced males than that in E2-induced females. Therefore, the surface mucus Vtg level of male goldfish may be a reliable indicator of estrogenic activity in the aquatic environment.