anti-OVA Antibody Research Articles

Induction of adaptive immune responses against antigens incorporated within the capsid of simian virus 40.

Simian virus 40 (SV40) is a monkey polyomavirus. The capsid structure is icosahedral and comprises VP1 units that measure 45 nm in diameter. Five SV40 VP1 molecules form one pentamer subunit, and a single icosahedral subunit comprises 72 pentamers; a single SV40 VP1 capsid comprises 360 SV40 VP1 molecules. In a previous study, we showed that an influenza A virus matrix protein 1 (M1) CTL epitope inserted within SV40 virus-like particles (VLPs) induced cytotoxic T lymphocytes (CTLs) without the need for an adjuvant. Here, to address whether SV40 VLPs induce adaptive immune responses against VLP-incorporated antigens, we prepared SV40 VLPs containing M1 or chicken ovalbumin (OVA). This was done by fusing M1 or OVA with the carboxyl terminus of SV40 VP2 and co-expressing them with SV40 VP1 in insect cells using a baculovirus vector. Intraperitoneal (i.p.) or intranasal administration of SV40 VLPs incorporating M1 induced the production of CTLs specific for the M1 epitope without the requirement for adjuvant. The production of antibodies against SV40 VLPs was also induced by i.p. administration of SV40 VLPs in the absence of adjuvant. Finally, the administration of SV40 VLPs incorporating OVA induced anti-OVA antibodies in the absence of adjuvant; in addition, the level of antibody production was comparable with that after i.p. administration of OVA plus alum adjuvant. These results suggest that the SV40 capsid incorporating foreign antigens can be used as a vaccine platform to induce adaptive immune responses without the need for adjuvant.

Development and Characterization of an Allergic Asthma Rat Model for Interventional Studies.

Allergic asthma is one of the most common chronic diseases of the airways, however it still remains underdiagnosed and hence undertreated. Therefore, an allergic asthma rat model would be useful to be applied in future therapeutic strategy studies. The aim of the present study was to develop an objective model of allergic asthma in atopic rats that allows the induction and quantification of anaphylactic shock with quantitative variables. Female Brown Norway rats were intraperitoneally sensitized with ovalbumin (OVA), alum and Bordetella pertussis toxin and boosted a week later with OVA in alum. At day 28, all rats received an intranasal challenge with OVA. Anaphylactic response was accurately assessed by changes in motor activity and body temperature. Leukotriene concentration was determined in the bronchoalveolar lavage fluid (BALF), and total and IgE anti-OVA antibodies were quantified in blood and BALF samples. The asthmatic animals’ motility and body temperature were reduced after the shock for at least 20 h. The asthmatic animals developed anti-OVA IgE antibodies both in BALF and in serum. These results show an effective and relatively rapid model of allergic asthma in female Brown Norway rats that allows the quantification of the anaphylactic response.

The impact of maternal periodontitis in the development of asthma in the offspring.

Clinical and experimental studies show an association between maternal periodontitis and adverse outcomes during gestation. On the other hand, there were no studies evaluating the impact of maternal periodontitis on the offspring. Thus, our objective was to investigate the repercussion of maternal periodontitis on the development of asthma in the offspring. Pregnant rats were submitted or not to periodontitis by ligature technique. Thirty days after birth, the puppies were sensitized and challenged with ovalbumin (OVA) in order to induce asthmatic response. Our results showed that maternal periodontitis reduced cellular infiltrate in the parenchyma of offspring, tracheal responsiveness, lung edema, and anti-OVA antibodies, without alter mucus as well as cytokines production. We concluded that periodontitis has relevant impact on the offspring's immune system, blunting the response to allergic and inflammatory stimulus. This study shows the important role of oral health during pregnancy and opens possibilities for future studies in order to explain the effects of periodontitis during pregnancy in the offspring.

Stable Immune Response Induced by Intradermal DNA Vaccination by a Novel Needleless Pyro-Drive Jet Injector

DNA vaccination can be applied to the treatment of various infectious diseases and cancers; however, technical difficulties have hindered the development of an effective delivery method. The efficacy of a DNA vaccine depends on optimal antigen expression by the injected plasmid DNA. The pyro-drive jet injector (PJI) is a novel system that allows for adjustment of injection depth and may, thus, provide a targeted delivery approach for various therapeutic or preventative compounds. Herein, we investigated its potential for use in delivering DNA vaccines. This study evaluated the optimal ignition powder dosage, as well as its delivery effectiveness in both rat and mouse models, while comparing the results of the PJI with that of a needle syringe delivery system. We found that the PJI effectively delivered plasmid DNA to intradermal regions in both rats and mice. Further, it efficiently transfected plasmid DNA directly into the nuclei, resulting in higher protein expression than that achieved via needle syringe injection. Moreover, results from animal ovalbumin (OVA) antigen induction models revealed that animals receiving OVA expression plasmids (pOVA) via PJI exhibited dose-dependent (10 μg, 60 μg, and 120 μg) production of anti-OVA antibodies; while only low titers (< 1/100) of OVA antibodies were detected when 120 μg of pOVA was injected via needle syringe. Thus, PJI is an effective, novel method for delivery of plasmid DNA into epidermal and dermal cells suggesting its promise as a tool for DNA vaccination.

Selective depletion of plasma cells in vivo based on the specificity of their secreted antibodies.

Antibody-mediated diseases affect more than 10% of the human population. For most, no cure is available, particularly when the pathogenic antibodies are secreted by long-lived plasma cells resistant to conventional immunosuppressive therapies. Current therapeutic approaches target not only the plasma cells that secrete pathogenic antibodies, but also those providing protective antibodies. Here, in a murine model bearing long-lived plasma cells secreting anti-OVA and -chicken gamma globulin (CGG) antibodies, we describe the first-time use of an antigen-antibody (OVA/anti-CD138 antibody) conjugate for in vivo labeling and selective ablation of plasma cells that secrete antibodies specific for the antigen OVA. The selective depletion also led to a stable reduction of the corresponding serum anti-OVA antibody levels. In contrast, CGG-specific plasma cells and circulating anti-CGG antibody levels remained unchanged. The method described here should enable the development of unique causative treatment strategies for established antibody-mediated diseases sparing humoral immunity.

Synergistic effect of IgG4 antibody and CTLs causes tissue inflammation in IgG4-related disease.

IgG4-related disease (IgG4-RD) is characterized by multi-organ irreversible damage resulting from tissue-specific infiltration of IgG4+ plasma cells and cytotoxic T lymphocytes (CTLs). However, whether IgG4 antibody contributes to the inflammation remains unclear. In this study, we established a mouse model that enabled us to evaluate the pathogenic function of IgG4 antibodies in response to a tissue-specific autoantigen using recombinant ovalbumin (OVA)-specific human IgG4 monoclonal antibody (rOVA-hIgG4 mAb) and the mice expressing OVA of the pancreatic islets (RIP-mOVA mice). We found no inflammatory effect of rOVA-hIgG4 mAb transfer alone; however, co-transfer with OVA-specific CD8 CTLs (OT-I T cells) induced tissue damage with dense lymphocytic inflammation in the pancreas of RIP-mOVA mice. rOVA-hIgG4 mAb caused accumulation of conventional DC1 cells (cDC1s) in the lymphoid tissues, and the dendritic cells (DCs) activated the OT-I T cells via cross-presentation. We also revealed that the synergistic effects of CTLs and antibodies were observed in the other subclasses including endogenous antibodies if they recognized the same antigen. The transfer of OVA-specific CD4 helper T cells (OT-II T cells) into RIP-mOVA mice induced the production of anti-OVA antibody, which had a synergistic effect, through acquisition of a T follicular helper (TFH) phenotype. Moreover, using OT-II T cells deficient in Bcl6 caused lower anti-OVA antibody production and inflammation with OT-I T cells. Our results indicated that autoreactive IgG4 antibodies play an important role of the tissue-specific CTL response in IgG4-RD.

Antibody-suppressor CD8+ T Cells Require CXCR5.

We previously reported the novel activity of alloprimed CD8 T cells that suppress posttransplant alloantibody production. The purpose of the study is to investigate the expression and role of CXCR5 on antibody-suppressor CD8 T-cell function. C57BL/6 mice were transplanted with FVB/N hepatocytes. Alloprimed CD8 T cells were retrieved on day 7 from hepatocyte transplant recipients. Unsorted or flow-sorted (CXCR5CXCR3 and CXCR3CXCR5) alloprimed CD8 T-cell subsets were analyzed for in vitro cytotoxicity and capacity to inhibit in vivo alloantibody production following adoptive transfer into C57BL/6 or high alloantibody-producing CD8 knock out (KO) hepatocyte transplant recipients. Alloantibody titer was assessed in CD8 KO mice reconstituted with naive CD8 T cells retrieved from C57BL/6, CXCR5 KO, or CXCR3 KO mice. Antibody suppression by ovalbumin (OVA)-primed monoclonal OVA-specific t-cell receptor transgenic CD8+ T cells (OT-I) CXCR5 or CXCR3 CD8 T-cell subsets was also investigated. Alloprimed CXCR5CXCR3CD8 T cells mediated in vitro cytotoxicity of alloprimed "self" B cells, while CXCR3CXCR5CD8 T cells did not. Only flow-sorted alloprimed CXCR5CXCR3CD8 T cells (not flow-sorted alloprimed CXCR3CXCR5CD8 T cells) suppressed alloantibody production and enhanced graft survival when transferred into transplant recipients. Unlike CD8 T cells from wild-type or CXCR3 KO mice, CD8 T cells from CXCR5 KO mice do not develop alloantibody-suppressor function. Similarly, only flow-sorted CXCR5CXCR3 (and not CXCR3CXCR5) OVA-primed OT-I CD8 T cells mediated in vivo suppression of anti-OVA antibody production. These data support the conclusion that expression of CXCR5 by antigen-primed CD8 T cells is critical for the function of antibody-suppressor CD8 T cells.

Engineered ovalbumin-expressing regulatory T cells protect against anaphylaxis in ovalbumin-sensitized mice

Allergy is a major public health concern, the main treatment for which is symptomatic relief with anti-inflammatory drugs. A key clinical challenge is to induce specific tolerance in order to control allergen-specific memory B and T cells, and specifically block effector cell responses. Our lab recently developed antigen-specific regulatory T-cell (Treg) therapies as a treatment for adverse responses. Recently, we created a chimeric antigen receptor (CAR) approach in which we engineered a target protein antigen, ovalbumin (OVA), linked with the transmembrane and signal transduction domains, CD28-CD3ζ to directly target B cells and sensitized mast cells in an allergy model. We named this receptor “BAR” for B-cell Antibody Receptor. Murine or human Tregs, transduced with a BAR containing OVA or control Tregs expressing an unrelated antigen, were successfully expanded in vitro and tested in the murine OVA-alum allergy model with measurable titers of anti-OVA IgE. Because BAR Tregs express the target antigen and could interact with specific IgE on sensitized mast cells, we first demonstrated that intravenously injected OVA-BAR Tregs did not directly lead to a drop in temperature or release of mediators in plasma indicative of anaphylaxis. Forty-eight hours later, mice were challenged intraperitoneally with 200 μg OVA to induce an anaphylactic reaction, and temperature immediately measured for 30 min. We found that OVA-BAR Tregs protected mice from hypothermia, whereas mice given control BARs (expressing an unrelated antigen) or PBS showed substantial temperature drops indicative of anaphylaxis when systemically challenged with OVA. Importantly, this effect was also demonstrated in a passive anaphylaxis model in which mice that received anti-OVA IgE antibody were protected from hypothermia when treated with OVA-BAR Tregs prior to systemic OVA challenge. These results provide proof of principle that engineered allergen-specific T-regulatory cells can provide clinical protection against severe allergic reactions in individuals already IgE-sensitized to an allergen.

Nanoelectrode ensemble immunosensing for the electrochemical identification of ovalbumin in works of art

This research is aimed to the study and application of an electrochemical immunosensor for the detection of ovalbumin (OVA) from egg white (or albumen) used as a binder in some works of art, such as some historical photographic prints and tempera paintings. The immunosensor takes advantage of the interesting biodetection capabilities offered by nanoelectrode ensembles (NEEs). The NEEs used to this aim are prepared by template deposition of gold nanoelectrodes within the pores of track-etched polycarbonate (PC) membranes. The affinity of polycarbonate for proteins is exploited to capture OVA from the aqueous extract obtained by incubation in phosphate buffer of a small sample fragment (<1 mg). The captured protein is reacted selectively with anti-OVA antibody, labelled with glucose oxidase (GOx). In the case of positive response, the addition of the GOx substrate (i.e. glucose) and a suitable redox mediator (a ferrocenyl derivative) reflects in the up rise of an electrocatalytic oxidation current, which depends on the OVA amount captured on the NEE, this amount correlating with OVA concentration in the extract. After optimization, the sensor is successfully applied to identify OVA in photographic prints dating back to the late 19th century, as well as in ancient tempera paintings from the 15th and 18th centuries.

Smart pH-Responsive Nanocube-Controlled Delivery of DNA Vaccine and Chemotherapeutic Drugs for Chemoimmunotherapy.

The combination of chemotherapeutic agents with immune stimulating agents for treating degenerative diseases, called chemoimmunotherapy, has emerged as a promising cancer treatment modality. Despite the tremendous potential, chemoimmunotherapy by the combination of drugs and immune stimulators often suffers because of the lack of controlled delivery nanostructures in the microenvironment. To this end, we show that by using pH-responsive smart nanocubes (NCs), cancer cells and tumor-associated immune cells can be precisely targeted with a chemotherapeutic agent (doxorubicin, DOX) and immune stimulating agent (plasmid ovalbumin, pOVA) for enhanced chemoimmunotherapy. The pH-responsive smart NCs protect payloads from nuclease degradation and avoid renal clearance and undergo supersensitive structural change at the extracellular tumor regions that mediate efficient release. Concurrent release of pOVA vaccines encoding tumor-specific antigen laden with polyplexes were loaded on tumor-associated immune cells and produce antigen-specific humoral immune response, whereas DOX enables effective infiltration into the cancer cells and is involved in the eradication of tumor tissues. The amount of anti-OVA IgG1 antibody produced by the intravenous administration of NC formulation was similar to that of free OVA formulation. Importantly, the combined delivery of pDNA and DOX using NCs showed significantly enhanced antitumor efficacy in B16/OVA melanoma tumor xenografts, which remarkably outperforms the monotherapy counterparts. These results suggest that pH-responsive smart NCs laden with pDNA and DOX provide a promising nanostructure for chemoimmunotherapy that simultaneously involves cancer cell killing and stimulates antigen-specific immune response to prevent cancer recurrence.

Functional horseradish peroxidase−streptavidin chimeric proteins prepared using a silkworm-baculovirus expression system for diagnostic purposes

Rapid, convenient, sensitive detection methods are of the utmost importance in analytical tools. Enzyme-based signal amplification using horseradish peroxidase (HRP) is commonly implemented in clinical diagnostics kits based on enzyme-linked immunosorbent assay (ELISA), by which the limit of detection is greatly improved. Herein we report the design and preparation of recombinant fusion proteins comprising HRP and streptavidin (Stav), in which HRP was fused to either the N- or C-terminus of Stav ((HRP)4–Stav or Stav–(HRP)4, respectively) using a baculovirus-silkworm expression system. Both (HRP)4–Stav and Stav–(HRP)4 were secreted in the apo form but they were easily converted to the holo form and activated by simple incubation with hemin overnight at 4 °C. The activated (HRP)4–Stav and Stav–(HRP)4 could be combined with a commercial biotinylated anti-OVA IgG antibody to detect ovalbumin (OVA) as the antigen in ELISA. The enzymatic activity of (HRP)4–Stav was twofold higher than that of Stav–(HRP)4, and the sensitivity of (HRP)4-Stav in ELISA was higher than that of a commercial HRP–Stav chemical conjugate. The successful use of (HRP)4–Stav chimeric protein as a molecular probe in ELISA shows that the baculovirus-silkworm expression system is promising to produce enzyme–Stav conjugates to substitute for those prepared by chemical methods.

Effects of Exercise on Stress-induced Attenuation of Vaccination Responses in Mice.

To investigate the effects of acute eccentric exercise (ECC) and voluntary wheel exercise training (VWR) on antibody and cell-mediated immune responses to vaccination in chronically stressed mice. We hypothesized that both ECC and VWR would attenuate chronic stress-induced reductions in vaccination responses. Mice were randomized into four groups: control (CON), stress (S)-ECC, S-VWR, and S-sedentary (SED). Stressed groups received chronic restraint stress for 6 h·d, 5 d·wk for 3 wk. After the first week of stress, S-ECC were exercised at 17 m·min speed at -20% grade for 45 min on a treadmill and then intramuscularly injected with 100 μg of ovalbumin (OVA) and 200 μg of alum adjuvant. All other groups were also vaccinated at this time. Stress-VWR mice voluntarily ran on a wheel for the entire experiment. Plasma was collected before, and at 1, 2, and 4 wk postvaccination. Enzyme-linked immunosorbent assay was performed to analyze anti-OVA IgG and IgM antibodies. After 3 wk of chronic stress, all mice were injected with OVA into the ear to determine the delayed-type hypersensitivity. We found that chronic restraint stress significantly reduced body weight and caused adrenal hypertrophy. We also found both S-ECC and S-VWR groups had significantly elevated anti-OVA IgG (P < 0.05), whereas no significant differences between the two exercise groups. Neither S-ECC nor S-VWR altered anti-OVA IgM or delayed-type hypersensitivity responses compared with S-SED group. Acute eccentric exercise and voluntary exercise training alleviated the chronic stress-induced anti-OVA IgG reductions in vaccination responses.

Smart vaccine delivery based on microneedle arrays decorated with ultra-pH-responsive copolymers for cancer immunotherapy

Despite the tremendous potential of DNA-based cancer vaccines, their efficacious delivery to antigen presenting cells to stimulate both humoral and cellular response remains a major challenge. Although electroporation-based transfection has improved performance, an optimal strategy for safe and pain-free vaccination technique remains elusive. Herein, we report a smart DNA vaccine delivery system in which nanoengineered DNA vaccine was laden on microneedles (MNs) assembled with layer-by-layer coating of ultra-pH-responsive OSM-(PEG-PAEU) and immunostimulatory adjuvant poly(I:C), a synthetic double stranded RNA. Transcutaneous application of MN patches onto the mice skin perforate the stratum corneum with minimal cell damage; subsequent disassembly at the immune-cell-rich epidermis/dermis allows the release of adjuvants and DNA vaccines, owing to the ultra-sharp pH-responsive nature of OSM-(PEG-PAEU). The released adjuvant and DNA vaccine can enhance dendritic cell maturation and induce type I interferons, and thereby produce antigen-specific antibody that can achieve the antibody-dependent cell-mediated cytotoxicity (ADCC) and CD8+ T cell to kill cancer cells. Strikingly, transcutaneous application of smart vaccine formulation in mice elicited 3-fold greater frequencies of Anti-OVA IgG1 serum antibody and 3-fold excess of cytotoxic CD8+ T cell than soluble DNA vaccine formulation. As a consequence, the formulation rejected the murine B16/OVA melanoma tumors in C57BL/6 mice through the synergistic activation of antigen-specific ADCC and cytotoxic CD8+ T cells. The maneuvered use of vaccine and adjuvant poly(I:C) in MNs induces humoral and cellular immunity, which provides a promising vaccine technology that shows improved efficacy, compliance, and safety.

Inhibitory effects and mechanism of Triticum aestivum on OVA-induced anaphylaxis in mice

Objective To investigate the inhibitory effects and mechanism of Triticum aestivum (TA) on anaphylaxis in ovalbumin (OVA)-sensitized mice. Methods Twenty-five female BALB/c mice were randomly divided into five groups (n=5), including control group, OVA group, 100 mg/kg TA group, 200 mg/kg TA group and DEX (dexamethasone) group. OVA (20 μg) and aluminum hydroxide (1 mg) were dissolved in 100 μl of PBS solution and injected into the abdominal cavity of each experimental mouse, while the mice in the control group were injected with 100 μl of 0.9% normal saline. All mice were given drinking water. The above processes were repeated two weeks later, and different concentrations of experimental drugs were diluted in 1% CMC (carboxymethyl cellulose) solution to feed mice. Allergic symptoms and behaviors of each mouse were scored in the process of feeding. On the 12th day after first sensitization, mice in the control and experimental groups were subcutaneously injected in the auricle with 20 μl of 0.9% saline solution and OVA, respectively. The thickness of each mouse′s auricle was measured 6 and 24 hours later. Histopathologic changes in auricle tissues were observed by hematoxylin-eosin(HE) staining 24 hours later. The mice were euthanized by cervical dislocation in the 6th week after first sensitization. Abdominal aorta serum samples were collected and tested for specific inflammatory cytokines (IgE, IgG1, TNF-α, IL-4) and anti-OVA antibodies (IgE, IgG1) by ELISA. ELISA and RT-PCR were used to detect the expression of IFN-γ, IL-4, IL-5, IL-12 and IL-13 after spleen lymphocytes were stimulated with different concentrations of drugs. Results Both the score of allergic symptoms and behavior and the thickness of the auricle of the OVA group were the highest among all groups (P<0.05). A TA dose-dependent decrease was found in both of the two parameters, which was confirmed by histopathological analysis. ELISA results showed that the plasma levels of IgE, IgG1, IL-4 and TNF-α in the experimental groups were significantly higher than those in the control group, but all of them were decreased in a TA dose-dependent manner. TA at the high concentration of 200 mg/kg was similar to DEX in inhibiting the expression of IgE and TNF-α. RT-PCR results showed that Th1 cytokines (IFN-y and IL-12) in the experimental groups increased significantly as compared with those in the control group (P<0.05). Expression of IFN-y and IL-12 was not significantly affected by TA at various concentrations, but markedly inhibited by DEX. The levels of Th2 cytokines (IL-4, IL-5 and IL-13) in the experiment groups were significantly higher than those in the control group (P<0.05). Compared with the OVA group, the levels of IL-4, IL-5 and IL-13 were significantly reduced in TA groups, especially in the TA 100 μg/ml group in which the expression of the three cytokines was decreased by about 60%, 18% and 20%, respectively. Conclusion TA helps to maintain immune balance by selectively inhibiting the expression of Th2 cytokines (IL-4, IL-5, IL-13), IgE and IgG and not influencing Th1 cytokines (IFN-γ and IL-12). It has an anti-allergic effect as it effectively suppresses allergic responses. This research provides both theoretical and experimental basis for further development of novel anti-anaphylactic treatment with TA. Key words: Triticum aestivum; Ovalbumin; Anaphylaxis; Th1 cell; Th2 cell

Allergic airway sensitization impairs antibacterial IgG antibody responses during bacterial respiratory tract infections

Mycoplasma pneumoniae, an atypical human pathogen, has been associated with asthma initiation and exacerbation. Asthmatic patients have been reported to have higher carriage rates of M pneumoniae compared with nonasthmatic subjects and are at greater risk for invasive respiratory infections. We sought to study whether prior allergen sensitization affects the host response to chronic bacterial infection. BALB/cJ and IL-4 receptor α-/- mice were sensitized with ovalbumin (OVA) and then infected with M pneumoniae or Streptococcus pneumoniae. Immune parameters were analyzed at 30days postinfection and included cellular profiles in bronchoalveolar lavage fluid (BALF) and serum IgG and IgE antibody levels to whole bacterial lysate, recombinant P1 adhesin, and OVA. Total lung RNA was examined for transcript levels, and BALF was examined for cytokine protein profiles. Anti-M pneumoniae antibody responses were decreased in allergen-sensitized, M pneumoniae-infected animals compared with control animals, but OVA-specific IgG responses were unaffected. Similar decreases in anti-S pneumoniae antibody levels were found in OVA-sensitized animals. However, M pneumoniae, but not S pneumoniae, infection augmented anti-OVA IgE antibody responses. Loss of IL-4 receptor signaling partially restored anti-M pneumoniae antibody responses in IgG2a and IgG2b subclasses. Inflammatory cytokine levels in BALF from OVA-sensitized, M pneumoniae-infected or S pneumoniae-infected animals were reduced compared with those in uninfected OVA-sensitized control animals. Unexpectedly, airway hyperreactivity to methacholine was essentially ablated in M pneumoniae-infected, OVA-sensitized animals. An established type 2-biased host immune response impairs the host immune response to respiratory bacterial infection in a largely pathogen-independent manner. Some pathogens, such as M pneumoniae, can augment ongoing allergic responses and inhibit pulmonary type 2 cytokine responses and allergic airway hyperreactivity.

Polymeric Immunoglobulin Receptor Mediates Immune Excretion of Mucosal IgM-Antigen Complexes Across Intestinal Epithelium in Flounder (Paralichthys olivaceus).

Polymeric immunoglobulin receptor (pIgR) is one important player of mucosal defenses, but very little is known on pIgR-mediated immune excretion of the antigens that penetrate mucosal surface in fish. Previously, we cloned the pIgR of flounder (Paralichthys olivaceus) and developed anti-pIgR antibody. In this study, the flounders were immunized intraperitoneally with the chicken ovalbumin (OVA) and the control protein bovine serum albumin (BSA) to elicit mucosal IgM antibody and pIgR response, and then challenged with OVA via caudal vein injection after the immunized OVA was absent from fish body at the fourth week after immunization. After OVA challenge, strong OVA-positive fluorescence signals were observed in lamina propria (LP) submucosa and epithelial cells of the hindgut at 30 min, increased proceeding toward the distal portion of intestinal folds, reached a peak at 2–3 h, and then weakened and disappeared at 12 h, indicating that the OVA rapidly diffused from bloodstream into LP submucosa and excreted across intestinal epithelium. Whereas in BSA-immunized and OVA-challenged control fish, the OVA was detected in LP submucosa but not in intestinal epithelium due to the lack of OVA-specific antibody. Accordingly, in intestinal epithelium, the transepithelial transport of OVA was confirmed by immunogold electron microscopy, and co-localization of OVA, IgM, and pIgR was illuminated by multiple-label immunofluorescence confocal microscopy and analyzed using Image J software. Furthermore, in gut mucus but not in serum, an ~800-kDa protein band showed IgM-positive, OVA-positive, and pIgR-positive simultaneously, and the OVA, together with IgM and secretory component (SC) of pIgR, could be immunoprecipitated by anti-OVA antibody, demonstrating the existence of SC–polymeric IgM–OVA complexes. All these results collectively revealed that the pIgR could transport mucosal IgM–OVA complexes from LP across intestinal epithelium into gut mucus via the transcytosis in flounder. These new findings provided direct evidences for pIgR-mediated immune excretion of IgM–antigen complexes, and better understanding the role of pIgR in mucosal immunity in teleost fish.

Engineered ovalbumin-expressing regulatory T cells protect from chicken egg white-induced allergy in vivo

Abstract Allergy is a major public health concern, the main treatment for which is symptomatic relief with anti-inflammatory drugs. A key clinical challenge is to induce specific tolerance in order to control allergen-specific memory B and T cells. Our lab recently developed antigen-specific regulatory T cell therapies as a treatment for adverse responses in hemophilia (Kim et al. BLOOD 125: 1107, 2015; Yoon et al. BLOOD 129: 238–245, 2017). In this report, we created a chimeric antigen receptor (CAR) approach in which we engineered the target protein antigen, ovalbumin (OVA), linked with the transmembrane and signal transduction domains, CD28-CD3ζ to directly target B cells to induce tolerance in an allergy model. We named this receptor “BAR” for B-cell Antibody Receptor. Murine Tregs, retrovirally transduced with a BAR containing OVA or control cells, were successfully expanded in vitro and tested in a standard murine OVA-alum allergy model with high titered circulating anti-OVA IgE antibodies. We first confirmed that intravenously injected OVA-BAR Tregs did not directly lead to temperature drops indicative of anaphylaxis. Forty eight hours later, mice were challenged intraperitoneally with 200μg of OVA to induce an anaphylactic reaction, and temperature taken immediately again after challenge for 30 minutes. Our results demonstrated that OVA-BAR Tregs protected mice from hypothermia, whereas the control BARs or PBS-treated mice showed temperature drops indicative of anaphylaxis. These results were repeated and confirmed using human Tregs (CD4+CD25hiCD127low) transduced and expanded and injected into mice. These data provide proof of principle that engineered OVA-BAR Tregs may be useful to treat IgE based allergies.

Sublingual administration of 2-hydroxyethyl methacrylate enhances antibody responses to co-administered ovalbumin and Streptococcus mutans

Objective: The oral mucosa of patients undergoing dental procedures is often exposed to residual monomers leaking from incompletely cured acrylic resins. We investigated whether 2-hydroxyethyl methacrylate (HEMA) monomers applied to the sublingual mucosa in mice modulate the antibody responses towards co-administered ovalbumin (OVA) or live oral bacteria.Material and methods: OVA, live mouse oral commensal Lactobacillus murinus or live human oral commensal Streptococcus mutans were administered sublingually with or without HEMA to BALB/c mice on four weekly occasions. One week after the last administration, the experiment was terminated and serum antibody levels were analyzed using ELISA.Results: Significantly increased IgG and IgE anti-OVA antibody activity was found in the sera from mice immunized with OVA together with HEMA, as compared to mice immunized with OVA alone. Likewise, S. mutans together with HEMA induced an IgG anti-S. mutans antibody response that was significantly higher than the antibody response detected after application of S. mutans alone. No IgG anti-L. murinus antibody response was detected in mice immunized with L. murinus together with HEMA, as compared to the background activity.Conclusions: We report that HEMA monomers have adjuvant properties when sublingually administered in combination with OVA or S. mutans.

Isotype-specific agglutination-PCR (ISAP): A sensitive and multiplex method for measuring allergen-specific IgE

Isotype-specific agglutination-PCR (ISAP): A sensitive and multiplex method for measuring allergen-specific IgE

Triethylene glycol dimethacrylate: adjuvant properties and effect on cytokine production

Objective: Leakage of monomers from dental fillings due to incomplete curing is very common. The objective of the present study was to examine the cytokine profile in cells exposed to triethyleneglycol dimethacrylate (TEGDMA) and the adjuvant properties of TEGDMA.Materials and methods: Human peripheral blood mononuclear cells were exposed to TEGDMA (500 and 1000 μM) for 24 h in vitro. Bio-Plex Pro™ assays were used for analysis and detection of cytokines. In vivo, BALB/c mice were immunized subcutaneously in the base of the tail with TEGDMA in combination with ovalbumin (OVA).Results: The cytokine levels of IL-8, IL-18, GRO-α and MCP-1 were significantly increased for both concentrations. IL-1β, IL-6 and TNF-α was only significantly increased in cultures exposed to 500 μM TEGDMA. The concentration of TNF-α was significantly decreased in cultures exposed to 1000 μM TEGDMA. Animals immunized with OVA co-administrated with TEGDMA had a significantly higher IgE and IgG anti-OVA antibody levels in blood than animals immunized with OVA only.Conclusions: TEGDMA affects production of proinflammatory cytokines IL-1β, IL-6, IL-8, IL-18 and TNF-α. This inflammatogenic capacity renders TEGDMAs adjuvant properties, which may interfere with the homeostasis between the immune system and the indigenous microflora in the oral cavity.