anti-OVA Antibody Research Articles

Cell membrane-coated mRNA nanoparticles for enhanced delivery to dendritic cells and immunotherapy

Cationic polymers such as polyethylenimine have been considered promising carriers for mRNA vaccines. However, their application is hindered by their inherent toxicity and a lack of targeted delivery capability. These issues need to be addressed to develop effective cancer vaccines. In this study, we investigated whether dendritic cell membrane-coated polyethylenimine/mRNA nanoparticles (DPN) could effectively deliver mRNA to dendritic cells and induce immune responses. For comparison, we employed red blood cell membrane-coated polyethylenimine/mRNA (RPN) and plain polyethylenimine/mRNA polyplex (PN). The dendritic cell membrane coating altered the zeta potential values and surface protein patterns of PN. DPN demonstrated significantly higher uptake in dendritic cells compared to PN and RPN, and it also showed greater mRNA expression within these cells. DPN, carrying mRNA encoding luciferase, enhanced green fluorescent protein, or ovalbumin (OVA), exhibited higher protein expression in dendritic cells than the other groups. Additionally, DPN exhibited favorable mRNA escape from lysosomes post-internalization into dendritic cells. In mice, subcutaneous administration of DPN containing ovalbumin mRNA (DPNOVA) elicited higher titers of anti-OVA IgG antibodies and a greater population of OVA-specific CD8+ T cells than the other groups. In a B16F10-OVA tumor model, DPNOVA treatment resulted in the lowest tumor growth among the treated groups. Moreover, the population of OVA-specific CD8+ T cells was the highest in the DPNOVA-treated group. While we demonstrated DPN's feasibility as an mRNA delivery system in a tumor model, the potential of DPN can be broadly extended for immunotherapeutic treatments of various diseases through mRNA delivery to antigen-presenting cells.

Immunostimulatory and immunoadjuvant capacities of soluble Rhamnan-type Ulva oligosaccharides

Algae polysaccharides were discovered to act as pathogen-associated molecular patterns (PAMPs) to trigger immune responses, while bioactivities of oligosaccharides have not been thoroughly explored. Ulva oligosaccharides from Ulva prolifera were obtained using PL25 family ulvan lyase and evaluated for cell viability and phagocytic activity. Ulva oligosaccharides with 542 Da (OUP), consisting of tetrasaccharide (∆UA-RhaS-Xyl-RhaS) and disaccharides (∆UA-RhaS), exhibited the most pronounced phagocytic activities. Besides, OUP could effectively stimulate the expressions of proinflammatory cytokines and chemokines like TNF-α, IL-1β, IL-6, IFN-γ and CXCL1 in primary macrophages and subsequently promote the proliferation and activation of T and B lymphocytes in vitro. Transcriptome analysis also gave rise to the systemic proinflammatory responses triggered by OUP in macrophages, such as activation of multiple PRRs-related signaling pathways and downstream JAK-STAT and NF-κB signaling. Furthermore, in an in vivo mouse OVA immunization model, OUP upregulated the specific anti-OVA IgG antibody production in vivo, pointing to its immunoadjuvant capacity. OUP increases cell proliferation and secretion of IL-4 and IFN-γ of splenocytes in response to OVA stimulation ex vivo, thereby enhancing both Th1 and Th2 immune responses. Collectively, all this data suggests that OUP could function as the immune stimulator to enhance host immune response to infections.

Abstract 3902: The Nanoprimer: A significant opportunity to boost the efficacy of cancer therapies

Abstract Nanomedicines are among the most promising novel delivery systems and are highly promising as therapeutic agents in oncology. However, their use is still limited by nanoparticle hepatic clearance which is responsible for low delivery to the target site. Moreover, the unintended liver distribution could cause harmful side effects. Curadigm’s innovative Nanoprimer technology aims to shift the balance of therapeutics bioavailability and toxicity. The platform is designed to decrease nanomedicines' liver trapping and increase their systemic bioavailability for optimal accumulation in target tissues. This technology can redefine the benefit/risk ratio of therapeutics, improving their clinical outcomes and treatment value. The Nanoprimer is an engineered, biocompatible liposome that transiently and specifically occupies the cells of the mononuclear phagocytic system responsible for sub-optimal therapeutics bioavailability. The Nanoprimer is administered just before the therapeutic, temporarily reducing drug clearance and elimination. Proofs-of-concept were realized by combining the Nanoprimer with different therapeutic agents such as irinotecan-loaded liposomes and nucleic acid-loaded lipid nanoparticles (LNP). One example is a study conducted in collaboration with the Langer Lab at MIT that shows that the Nanoprimer increases mRNA- and siRNA-based therapeutics efficiency by 32% and 49% respectively. Efficacy studies in a triple-negative breast cancer mouse model reveal that the Nanoprimer increases by 2-fold the anti-tumor efficacy of siRNA LNP therapy. Data demonstrate the safety of the Nanoprimer, along with its ability to maximize the inhibition of primary tumor growth and pulmonary metastasis by systemic siRNA LNP treatment. In a collaborative study with the NCL*, the Nanoprimer was shown to stimulate the immune response in a systemic vaccine model using ovalbumin-coated gold nanoparticles (GNP-OVA). Administration of the Nanoprimer before immunization of mice by intravenous injection of GNP-OVA, results in a dramatic increase in anti-OVA antibody levels with IgGs detected as early as 7 days. Furthermore, IgG and IgM antibodies remain elevated at day 28 showing a robust and sustainable immune response. Nanoprimer ability to enhance immune response shows its great potential in immunotherapy, especially for cancer vaccines. Altogether, these data demonstrate that the ubiquitous nature of the Nanoprimer mode of action allows its application to a broad spectrum of therapeutics, from nucleic acid-loaded LNP to immunotherapy. Efficacy studies conducted in mouse models highlight the potential for the Nanoprimer to empower nanotechnology for oncology treatments. *We acknowledge NCL, Frederick National Laboratory for Cancer Research sponsored by the NCI for the help with conducting these experiments. Citation Format: Julie Devallière, Laurence Poul, Maxime Bergère, Francis Mpambani, Audrey Darmon, Océane Jibault, Matthieu Germain. The Nanoprimer: A significant opportunity to boost the efficacy of cancer therapies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3902.

Safe and efficient oral allergy immunotherapy using one-pot-prepared mannan-coated allergen nanoparticles

Allergen immunotherapy (AIT) is the only curative treatment for allergic diseases. However, AIT has many disadvantages related to efficiency, safety, long-term duration, and patient compliance. Dendritic cells (DCs) have an important role in antigen-specific tolerance induction; thus, DC-targeting strategies to treat allergies such as glutaraldehyde crosslinked antigen to mannoprotein (MAN) have been established. However, glutaraldehyde crosslinking may reduce the antigen presentation efficiency of DCs. To overcome this, we developed a MAN-coated ovalbumin (OVA) nanoparticle (MDO), which uses intermolecular disulfide bond to crosslink OVA and MAN. MDO effectively targeted DCs resulting in tolerogenic DCs, and promoted higher antigen presentation efficiency by DCs compared with OVA or glutaraldehyde crosslinked nanoparticles. In vitro and in vivo experiments showed that DCs exposed to MDO induced Treg cells. Moreover, MDO had low reactivity with anti-OVA antibodies and did not induce anaphylaxis in allergic mice, demonstrating its high safety profile. In a mouse model of allergic asthma, MDO had significant preventative and therapeutic effects when administered orally or subcutaneously. Therefore, MDO represents a promising new approach for the efficient and safe treatment of allergies.

Iron-overload affects anti-tumor immune response in pancreatic cancer.

Abstract HFE is the most frequently mutated gene in hereditary hemochromatosis, a disease in which iron accumulates in organs leading to toxicities. Worldwide, two main polymorphisms in HFE protein have been observed, namely C282Y and H63D. Even if associated to milder iron accumulations, H63D polymorphism increases the risk of cancer development and aggressiveness. As observed in pancreatic cancer patients, a great percentage of those that underwent to surgery also present H63D polymorphism but displayed a worse survival. To better characterize how the iron accumulation affects anti-tumor response in the context of the pancreatic ductal adenocarcinoma (PDA), we crossed genetically engineered mice (GEM) that spontaneously develop PDA with mice engineered to express H67D mutation (HfeH67D), the orthologue of H63D in human. Histological analysis of GEM showed that H67D polymorphism accelerated PDA progression, increased the number of metastases, and altered the composition of infiltrating immune cells. Accordingly, these mouse models also displayed a reduced overall survival compared to controls. By exploiting OVA as a well-known antigen, we injected OVA-expressing PDA cells subcutaneously in mice carrying or not the H67D mutation to assess their ability to mount a normal memory response. OVA-expressing PDA cells grew faster in the presence of H67D polymorphism, but immunized mice displayed a reduction in tumor volume similar to the WT counterpart. HfeH67Dmice displayed an increased production of anti-OVA IgG antibodies, especially IgE, but a decreased in OVA-specific IFNγ-secreting T cells. Overall, H67D polymorphism impacts the activation of anti-tumoral immune response, which consequently worsens PDA progression. Supported by grants from AIRC (ID22234; ID26341)

Fufang Shengdi mixture alleviates psoriasis-like skin inflammation via promoting Annexin-A proteins expression

Ethnopharmacological relevanceTraditional Chinese medicine believes that “blood fever” is an important cause of psoriasis. Fufang Shengdi mixture (FFSD), based on the Hongban Decoction, is composed of Rehmannia glutinosa (Gaertn.) DC., Raw gypsum (Chinese: Sheng Shi Gao), and Lonicera japonica Thunb (Caprifoliaceae). FFSD has effects on nourishing Yin, clearing heat, connecting collaterals, and cooling blood. In modern medical explanation, FFSD has the effects of anti-inflammatory and immunosuppression. Our study proved that FFSD can suppress immunity and ameliorate the symptoms of imiquimod-induced psoriasis in mice. Aim of the studyThis study evaluated the efficacy and possible mechanism of FFSD in psoriasis mice. Methods and materialsFirst, the main components of FFSD were analyzed using high-performance liquid chromatography-tandem high-resolution mass spectrometry (HPLC-HRMS). An imiquimod (IMQ)-induced psoriasis mouse model was used to evaluate the efficacy of FFSD orally. Psoriasis area and severity index (PASI) scores were recorded throughout the course of the mice to reflect the severity of psoriasis. Hematoxylin-eosin staining was used to observe the pathological changes in skin lesions. Enzyme-linked immunosorbent assay (ELISA) was performed to test the level of IFN-γ and TNF-α in plasma. To further investigate the immunopharmacological effect of FFSD, we used chicken ovalbumin (OVA) to induce immunoreaction in mice. ELISA was used to detect the levels of anti-OVA antibody, IFN-γ and TNF-α in mice. Flow cytometry was performed to quantify the ratio of cell types in peripheral blood mononuclear cells (PBMCs) to evaluate the effect of FFSD on immunosuppression. Proteomics and bioinformatics analyzes were performed to find the regulation pathway of the immunosuppressive effect of FFSD. Finally, quantitative PCR (qPCR) and immunohistochemistry were used to measure the upregulation of Annexin-A proteins (ANXAs) in the skin lesion tissue of IMQ-induced mouse. ResultsOn the basis of knowing the composition of FFSD, we first proved the efficacy of FFSD in alleviating IMQ-induced psoriasis in mice. Second, we further clarified the pharmacological effect of FFSD on immunosuppression via OVA-induced mice. Subsequently, it was found that the significant up-regulation of ANXAs was caused by FFSD through proteomics analysis, and the finding was proved in the IMQ-induced psoriasis mouse model. ConclusionsThis study elucidates the immunosuppressive pharmacological effect of FFSD on improving psoriasis through up-regulating ANXAs.

Impact of Epichaperome Inhibitor PU-H71 on Anti-Tumor T Cell Responses and Its Implications for Immune and Cellular Therapy

The epichaperome is a tightly integrated network of proteins supporting critical roles in the tumor-associated microenvironment and cancer cell survival. The epichaperome inhibitor PU-H71 is under development in clinical trials. A patient with an unclassified myeloproliferative neoplasm harboring a novel PML-SYK fusion, who progressed to acute myeloid leukemia despite allogeneic stem cell transplantation was successfully treated using PU-H71 (Sugita et al. 2021, NPJ Precis Oncol). We reported changes in donor immune cells after PU-H71 treatment, suggesting that epichaperome inhibition was modulating antitumor immune responses and improving the efficacy of cellular immunotherapy. To investigate this hypothesis, we analyzed the effects of PU-H71 on antitumor immune responses, GvHD and CAR-T cells. We tested the effects of PU-H71 on C57BL/6 mice immune responses to ovalbumine (OVA) immunization. OVA-specific T- and B-cell responses were evaluated by ELISPOT and ELISA, respectively. When administered at the time of priming, PU-H71 significantly increased the number of IFNγ-secreting T cells as compared to controls (median 126 vs 42 per million of cells, p=0.0002; n=8), without an impact on T cell phenotype or anti-OVA antibody levels. In contrast, PU-H71 had no effect when administered once the OVA-specific T cell response was ongoing. Then, we tested the effect of PU-H71 on antitumor immune responses in OVA-primed mice challenged with E.G7 (OVA+) or parental EL4 lymphoma cell lines (OVAneg). PU-H71 improved in vivo anti-tumor immune responses in OVA-primed mice challenged with E.G7 but not EL4 cells (Mean survival 17.5 vs 14 days, p=0.0003, n=8 per group). Since PU-H71 augmented early but not established T cell responses, we next evaluated whether it would have an effect on a GvHD murine model. Lethally-irradiated B6C3F1 mice received bone marrow and splenocytes from C57BL/6 mice and were treated with PU-H71 or vehicle at the first signs of GvHD (D11). GvHD clinical scores and histological analysis (colon, small intestine, liver and skin) were assessed. PU-H71 did not aggravate GvHD at both clinical and histological levels. Therefore, PU-H71 augments antigen-specific mouse T cell responses by acting during priming. To better understand its effect on human T cells and its potential adjuvant effect during cellular therapy, we next evaluated in vitro and in vivo CAR-T cells phenotype and function in the presence of PU-H71. T cells were purified from healthy donor PBMCs and were transduced with a CD19.4-1Bb.3z lentiviral vector to obtain CART19 cells. First, we found that epichaperome indeed forms in CART19 cells upon activation with anti-CD3/CD28 beads vs control. In vitro cytotoxicity of CAR-T cells toward NALM6 B-ALL cells was significantly improved (55.39 vs 74.66%, p=0.0356) by PU-H71. Finally, the therapeutic effect of the combination of PU-H71 and CART19 was evaluated in a B-ALL NSG mouse model using NALM6-BLIV cells, with bioluminescence imaging and flow cytometry (CART19 and leukemic cells) monitoring. Results suggest that PU-H71 administration increases CART19 numbers in the bone marrow and improves their in vivo antitumor efficacy against NALM6. Together, our results show that PU-H71 augments antigen-specific T cell responses during priming including CAR-T cell cytotoxicity, with little effect on ongoing immune responses including GvHD. Our data suggest that PU-H71 improves the in vivo efficiency of CAR-T cells in a B-ALL mouse model. In addition to its previously demonstrated direct anti-tumor effect, PU-H71 also had an adjuvant effect on anti-tumor T cell responses while not aggravating GvHD, which supports its clinical evaluation in the post-transplant setting or in combination with immune therapies like CAR-T cells.

Lanthanide-Nucleotide Coordination Nanoparticles for STING Activation.

Activation of the stimulator of interferon genes (STING) is essential for blocking viral infections and eliciting antitumor immune responses. Local injection of synthetic STING agonists, such as 2'3'-cGAMP [cGAMP = cyclic 5'-guanosine monophosphate (cGMP)-adenosine monophosphate (AMP)], is a promising approach to enhance antiviral functions and cancer immunotherapy. However, the application of such agonists has been hindered by complicated synthetic procedures, high doses, and unsatisfactory systemic immune responses. Herein, we report the design and synthesis of a series of 2'3'-cGAMP surrogates in nanoparticle formulations formed by reactions of AMP, GMP, and coordinating lanthanides. These nanoparticles can stimulate the type-I interferon (IFN) response in both mouse macrophages and human monocytes. We further demonstrate that the use of europium-based nanoparticles as STING-targeted adjuvants significantly promotes the maturation of mouse bone-marrow-derived dendritic cells and major histocompatibility complex class I antigen presentation. Dynamic molecular docking analysis revealed that these nanoparticles bind with high affinity to mouse STING and human STING. Compared with soluble ovalbumin (OVA), subcutaneously immunized europium-based nanovaccines exhibit significantly increased production of primary and secondary anti-OVA antibodies (∼180-fold) in serum, as well as IL-5 (∼28-fold), IFN-γ (∼27-fold), and IFN-α/β (∼4-fold) in splenocytes ex vivo. Compared with the 2'3'-cGAMP/OVA formulation, subcutaneous administration of nanovaccines significantly inhibits B16F10-OVA tumor growth and prolongs the survival of tumor-bearing mice in both therapeutic and protective models. Given the rich supramolecular chemistry with lanthanides, this work will enable a readily accessible platform for potent humoral and cellular immunity while opening new avenues for cost-effective, highly efficient therapeutic delivery of STING agonists.

Immunomodulatory effect and histological alteration of thermally oxidized sunflower oil in male Wistar rats

Many foods are prepared by deep-frying as this provides unique flavors/textures that improve overall palatability to consumers. Sunflower oil is one of the most widely-used frying oils in the world. However, during frying, fats in the foodstuff are heated to > 150° C and concurrently come into contact with agents found in the air, water, and other foods being cooked at the same time. Since long-term consumption of oxidized/oxidation products can contribute to the development of a variety of pathologies. Used cooking oil has become of increased concern to both health administrators and consumers. Few studies have sought to examine potential immunomodulatory effects from exposures to sunflower oil or its oxidized/oxidation products. To address this gap in knowledge, here, sunflower oil was used to fry potatoes at 190° C, every 30 min; a new 100 g batch of slices was cooked. Frying was performed continuously for 5h/day for 2 and 4 days (10 and 20 h total), and then the parental uncooked oil and each of the used oils were provided to Wistar rats in their diet (at 20% level) for a total of 10 weeks. Measures of body weight\food intake indicated that diets containing the used oils led to slightly reduced food intakes and host weights. Kidney weights were not affected by consumption of the two used oils, but the liver was enlarged in rats fed with the oil treated for 20 h. Evaluation of key lipid-serum parameters, i.e., total cholesterol, HDL cholesterol, and triglycerides, revealed a diet-related decrease in each; oxidized fats reduce their concentration compared to fresh fats. Conversely, serum LDL cholesterol levels were increased. Histological analyses indicated that consumption of the oxidized oils caused both kidney (e.g., tubular/glomerular dilation, vascular congestion, inflammation, tubular necrosis) and liver (e.g., vascular congestion, inflammation, necrosis) damages. Assessments of host immune function, i.e., ability to form antibody to ovalbumin (OVA) challenge, indicated that in rats fed the used oil, there were significant decreases in serum anti-OVA antibody levels. This suggested that host immunocompetence could be compromised as a result of long-term consumption of a diet containing a relatively high (i.e., 20%) content of used sunflower oils.

Microbial metabolite butyrate promotes induction of IL-10+IgM+ plasma cells.

The microbially-derived short-chain fatty acid butyrate is a central inhibitor of inflammatory innate and adaptive immune responses. Emerging evidence suggests that butyrate induces differentiation of IL-10-producing (IL-10+) regulatory B cells. However, the underlying mechanisms of butyrate-driven modulation of B cell differentiation are not fully defined. Given the dominant role of regulatory plasma cells (PCs) as the main source of anti-inflammatory cytokines including IL-10 and the observation that butyrate also induces the differentiation of PCs, we here investigated the effect of the microbial metabolite butyrate on the induction of regulatory IL-10+ PCs and underlying mechanisms. Here we show that butyrate induces the differentiation of IL-10+IgM+ PCs. Ex vivo, butyrate, but hardly propionate, another microbially-derived short-chain fatty acid, induced the differentiation of IL-10+IgM+ CD138high PCs from isolated splenic murine B cells. In vivo, administration of butyrate via drinking water or by daily intraperitoneal injection increased the number of IL-10+IgM+ CD138high PCs in the spleens of Ovalbumin (Ova)/complete Freund’s adjuvant-immunized mice. The induction of these regulatory PCs was associated with an increase of anti-Ova IgM, but a reduction of anti-Ova class-switched pathogenic IgG2b serum antibodies. Based on the knowledge that butyrate inhibits histone deacetylases (HDACs) thereby increasing histone acetylation, we identified here that HDAC3 inhibition was sufficient to induce PC differentiation and IL-10+ expression. Furthermore, reduced mitochondrial superoxide levels following butyrate treatment and HDAC3 inhibition were necessary for PC differentiation, but not IL-10 expression. In summary, the microbial metabolite butyrate promotes the differentiation of IgM+ PCs and their expression of IL-10. HDAC3 inhibition may be involved as an underlying pathway for both PC differentiation and IL-10 expression, while reduced mitochondrial superoxide levels are crucial only for PC differentiation. The induction of regulatory IL-10+IgM+ PCs and the inhibition of class switching to antigen-specific pathogenic IgG subclasses might represent important pathways of butyrate to limit inflammation.

Double qualitative immunochromatographic test for simultaneous control of chicken muscles and eggs in food

This study presents the development of a qualitative double immunochromatographic analysis (ICA) for the rapid simultaneous determination of ovalbumin (OVA) and immunoglobulins of class Y (IgY) of chicken origin. The latter serve as biomarkers to control the presence of chicken meat and eggs in food products. Polyclonal antibodies against OVA were produced in rabbits. The test system was based on the two-stage sandwich ICA with gold nanoparticles (AuNPs) as a label for anti-OVA and anti-IgY antibodies. Limits of detection (LODs)/cutoffs of IgY and OVA were 24.8/20 ng/mL and 3.6/2 ng/mL, respectively; the assay was performed within 18 min. The analysis allowed for the determination of OVA and IgY in raw meat mixtures containing chicken meat and dry egg white with visual cutoffs of 0.125 % and 0.0001 % (w/w) for chicken and egg white additives. The analysis in foodstuffs—ravioli with known composition—demonstrated that the developed double ICA can reveal IgY in poultry meat. The test system has a great potential for food product analysis when reliable determination and differentiation of chicken egg white and chicken meat as separate components are required.

Endangered Lymphocytes: The Effects of Alloxan and Streptozotocin on Immune Cells in Type 1 Induced Diabetes.

Alloxan (ALX) and streptozotocin (STZ) are extensively used to induce type 1 diabetes (T1D) in animal models. This study is aimed at evaluating the differences in immune parameters caused by ALX and STZ. T1D was induced either with ALX or with STZ, and the animals were followed for up to 180 days. Both ALX and STZ induced a decrease in the total number of circulating leukocytes and lymphocytes, with an increase in granulocytes when compared to control mice (CT). STZ-treated mice also exhibited an increase in neutrophils and a reduction in the lymphocyte percentage in the bone marrow. In addition, while the STZ-treated group showed a decrease in total CD3+, CD4−CD8+, and CD4+CD8+ T lymphocytes in the thymus and CD19+ B lymphocytes in the pancreas and spleen, the ALX group showed an increase in CD4−CD8+ and CD19+ only in the thymus. Basal levels of splenic interleukin- (IL-) 1β and pancreatic IL-6 in the STZ group were decreased. Both diabetic groups showed atrophy of the thymic medulla and degeneration of pancreatic islets of Langerhans composed of inflammatory infiltration and hyperemia with vasodilation. ALX-treated mice showed a decrease in reticuloendothelial cells, enhanced lymphocyte/thymocyte cell death, and increased number of Hassall's corpuscles. Reduced in vitro activation of splenic lymphocytes was found in the STZ-treated group. Furthermore, mice immunized with ovalbumin (OVA) showed a more intense antigen-specific paw edema response in the STZ-treated group, while production of anti-OVA IgG1 antibodies was similar in both groups. Thereby, important changes in immune cell parameters in vivo and in vitro were found at an early stage of T1D in the STZ-treated group, whereas alterations in the ALX-treated group were mostly found in the chronic phase of T1D, including increased mortality rates. These findings suggest that the effects of ALX and STZ influenced, at different times, lymphoid organs and their cell populations.

S100A4 Is Critical for a Mouse Model of Allergic Asthma by Impacting Mast Cell Activation.

BackgroundThe calcium-binding protein S100A4 demonstrates important regulatory roles in many biological processes including tumorigenesis and inflammatory disorders such as allergy. However, the specific mechanism of the contribution of S100A4 to allergic diseases awaits further clarification.ObjectiveTo address the effect of S100A4 on the regulation of mast cell activation and its impact on allergy.MethodsBone marrow-derived cultured mast cells (BMMCs) were derived from wild-type (WT) or S100A4-/- mice for in vitro investigation. WT and S100A4-/- mice were induced to develop a passive cutaneous anaphylaxis (PCA) model, a passive systemic anaphylaxis (PSA) model, and an ovalbumin (OVA)-mediated mouse asthma model.ResultsFollowing OVA/alum-based sensitization and provocation, S100A4-/- mice demonstrated overall suppressed levels of serum anti-OVA IgE and IgG antibodies and proinflammatory cytokines in serum, bronchoalveolar lavage fluid (BALF), and lung exudates. S100A4-/- mice exhibited less severe asthma signs which included inflammatory cell infiltration in the lung tissue and BALF, and suppressed mast cell recruitment in the lungs. Reduced levels of antigen reencounter-induced splenocyte proliferation in vitro were recorded in splenocytes from OVA-sensitized and challenged mice that lacked S100A4-/-. Furthermore, deficiency in the S100A4 gene could dampen mast cell activation both in vitro and in vivo, evidenced by reduced β-hexosaminidase release and compromised PCA and PSA reaction. We also provided evidence supporting the expression of S100A4 by mast cells.ConclusionS100A4 is required for mast cell functional activation, and S100A4 may participate in the regulation of allergic responses at least partly through regulating the activation of mast cells.

Platelet-Targeted Gene Transfer Induces Immune Tolerance in a Primed Model

Our previous studies have demonstrated that platelet-specific gene transfer under control of the αIIb promoter (2b) is effective in inducing immune tolerance not only to the coagulation factor VIII and IX, but also to the non-coagulant protein ovalbumin (OVA) in a model without pre-existing immunity (the non-primed model). In the present study, we explored whether platelet-specific gene transfer could induce immune tolerance in the primed model. To address this question, we first used C57BL6 wild type (WT) mouse models. Both donors and recipients were immunized with OVA to induce anti-OVA antibody development. OVA expression was introduced by 2bOVA lentivirus (LV) transduction of hematopoietic stem cells (HSCs) from WT/CD45.2 donors followed by transplantation into OVA-primed WT/CD45.1 recipients preconditioned with a sub-lethal 6.6Gy total body irradiation (TBI). 2bGFP was used as a control vector in parallel. Animals were analyzed after at least 4 weeks of bone marrow (BM) reconstitution. The viability of OVA genetically modified HSCs was confirmed by PCR in all 2bOVA-transduced recipients. After full bone marrow constitution, which takes about 12 weeks, the engraftment in the 2bOVA group (87.4±4.4%, n=5) was similar to that in the 2bGFP group (87.8±3.9%, n=5) as determined by flow cytometry. The OVA protein level was 6.7±2.8 ng/108platelets (n=5) in 2bOVA transduced recipients. Anti-OVA antibody titers declined with time in both groups. Six months after transplantation, recipients were rechallenged with OVA. All of the anti-OVA titers in 2bGFP-transduced recipients (n=5) increased dramatically, while only one in the 2bOVA group slightly increased. Normalized anti-OVA antibody titers in the 2bOVA group (90.8±152.7) were significantly lower than those in the 2bGFP group (1278±2101) after OVA rechallenge, demonstrating that platelet-specific OVA gene delivery to HSCs can suppress anti-OVA immune responses in OVA-primed WT mice.To explore how immune suppression is established after platelet-specific gene transfer in the OVA-primed model, we transduced HSCs from OVA-specific TCR transgenic mice (OTII/CD45.2, in which greater than 97% CD4 T+ cells are OVA-specific) with 2bOVALV and transplanted into OVA-primed WT/CD45.1 recipients preconditioned with 6.6Gy TBI. Untransduced transplanted mice were used as a control. After BM reconstitution, the OVA protein level was 32.3±6.3 ng/108 platelets (n=5) in the 2bOVA-transduced group. Anti-OVA antibody titers in both groups declined with time. When all the recipients were rechallenged with OVA, 2 of 3 anti-OVA titers in the untransduced group increased, while none of the titers in the 2bOVA group increased. Normalized anti-OVA antibody titers in the 2bOVA group (4.1±4.9) were significantly lower than those in the un-transduced group (152.3±214.5), demonstrating that platelet-specific OVA gene delivery to HSCs can also suppress the anti-OVA immune response in the OVA-primed WT mice that received 2bOVA-transduced HSCs from OTII mice (the OTII/WT model). After BM reconstitution, the engraftments in the 2 groups were similar (81.4±2.6%, and 80.9±2.5%, respectively), but donor-derived CD45.2+CD4+ (OTII/CD4) T cells in the 2bOVA (0.2±0.1%) group were consistently significantly lower than in the untransduced group (4.2±1.1%) in peripheral blood during the entire study course. Also, donor-derived CD45.2+CD4+ T cells in both spleen and lymph nodes were significantly lower in the 2bOVA group compared to the untransduced group. Interestingly, there were no differences in either percentage or total cell number of CD45.2+CD4+ T cells in the thymus between the 2 groups, indicating that central tolerance may not play a role in the immune tolerance induced after platelet-targeted gene therapy. Meanwhile, the frequency and total number of endogenous CD4+ T cells were similar in the 2 groups. Of note, the percentages of donor-derived regulatory T (Treg) cells in the 2bOVA group were significantly higher than those in the untransduced group in peripheral blood, spleen, and lymph nodes, while there were no significant differences in the total numbers of donor-derived Treg cells between the 2 groups.Taken together, our studies demonstrate that platelet-specific gene therapy can induce immune tolerance in a primed system through two distinct pathways, peripheral antigen-specific CD4+ T cell clone deletion and regulatory T cell induction. DisclosuresNo relevant conflicts of interest to declare.

Disposable electrochemical microfluidic device for ultrasensitive detection of egg allergen in wine samples

Food allergies have been increasing all over the world. Egg is an important component in the food industries and the second most common cause of food allergy, shortly after milk. In the wine industry, egg white is applied as a fining agent for tannin removal. In this study, a sandwich-based immunoassay for ultrasensitive detection of ovalbumin (OVA) in wine samples was developed. The assay involves the use of magnetic beads (MBs) decorated with a polyclonal anti-OVA antibody (Ab2) and horseradish peroxidase (HRP), used as label for the quantification in a disposable electrochemical microfluidic device (DEμD) here developed. The Ab2-MB-HRP prepared was applied to capture, separate, and pre-concentrate OVA from wine samples. In the DEμD, OVA was immune-magnetically captured (OVA-Ab2-MB-HRP), producing a sandwich structure (GO-Ab1-OVA-Ab2-MB-HRP) on the electrode's surface. This arrangement results in an ultrasensitive device, achieving the ultralow limit of detection of 0.2 fg mL−1 OVA. Five samples of wines were analyzed by using the immuno-magneto-assay which presents excellent accuracy compared with enzyme-linked immunosorbent assay (ELISA).

Induction of food tolerance is dependent on intestinal inflammatory state

Food allergies are usually managed by food avoidance. Hidden allergens in food, due to cross-contamination and/or allergenic additives added during production, place an important concern in today’s increasing food allergy cases worldwide. Previous studies showed that the introduction of unacquainted food components, in an inflamed intestine, results in sensitization to this food. Thus, our aim was to evaluate the kinetics of multiple food allergy induction. Adult male C57BL/6 mice were divided into five groups, four of which were submitted to an intestinal inflammation induction protocol to peanuts. Egg white (OVA) diluted 1:5 v/v in distilled water was instilled by gavage 6h-before (PRIOR), concomitant (AT) and 6h-after (DURING) the onset of the peanut challenge diet. Positive control (POS CONT) and NEG CONT received saline per gavage. Finally, animals were challenged with subcutaneous injections of OVA. Results showed no changes in diet intake were observed. Anti-OVA polyisotypic IgG antibody titers significantly increased in AT. Flow cytometry revealed significant decrease in CD4+CD25+Foxp3+ and significant increase in TCD8+ in AT. Histomorphometrically, AT and DURING were classified as Infiltrative and Partial Destruction stages. PRIOR was classified as Infiltrative, while POS CONT was classified as Partial Destruction. NEG CONT was classified as Normal. Together, our results confirm that the introduction of unfamiliar food only a few hours before the initiation of a gut inflammation process is able to induce oral tolerance, however the introduction of a dietary protein concomitant to the onset or during an ongoing gut inflammation may induce multiple allergies.

Mannose-Functionalized Biodegradable Nanoparticles Efficiently Deliver DNA Vaccine and Promote Anti-tumor Immunity.

Cancer vaccines have attracted increasing attention for their application in tumor immunotherapy. DNA vaccines are one of them that have been proven very promising with the advantages of safety, rapid design, and low cost. However, the low stability, ineffective cell internalization, and low immunostimulation hinder their wide application. Thus, developing targeted and safe systems to effectively deliver DNA vaccines becomes a vital step. In this study, we report the development of mannose- and bisphosphonate (BP)-modified calcium phosphate (CP) nanoparticles (NPs) as efficient vaccine delivery vehicles by targeting C-type lectin receptors (CLRs) on antigen-presenting cells (APCs). Using a model antigen ovalbumin (OVA)-encoded plasmid DNA (pOVA) as a model vaccine, we demonstrate that mannose-modified and BP-stabilized CP (MBCP) nanoparticles are mono-dispersed for enhanced uptake by APCs and subsequently induce OVA antigen presentation and immunostimulation. Mice immunized with MBCP-pOVA nanovaccines show a significantly stronger anti-OVA antibody response with a quicker IgG1 and IgG2a antibody production than unmodified NPs. Moreover, MBCP-pOVA immunization significantly inhibits the growth of OVA-expressing E.G7 tumor cells in C57BL/6J mice. Our data collectively suggest that the modifications to enhance the stability and targeting ability of MBCP NPs are essential for effective delivery of DNA vaccines and promote robust anti-tumor immunity.

Silicone Oil-Based Nanoadjuvants as Candidates for a New Formulation of Intranasal Vaccines.

Many conventional vaccines are administered via a needle injection, while most pathogens primarily invade the host via mucosal surfaces. Moreover, protective IgA antibodies are insufficiently induced by parenteral vaccines. Mucosal immunity induces both local and systemic response to pathogens and typically lasts for long periods of time. Therefore, vaccination via mucosal routes has been increasingly explored. However, mucosal vaccines require potent adjuvants to become efficacious. Despite many efforts to develop safe and robust adjuvants for mucosal vaccines, only a few have been approved for use in human formulations. The aim of our study was to design, develop and characterize new silicone oil-based nanoadjuvant candidates for intranasal vaccines with potential to become mucosal adjuvants. We have developed an array of nanoadjuvant candidates (NACs), based on well-defined ingredients. NAC1, 2 and 3 are based on silicone oil, but differ in the used detergents and organic solvents, which results in variations in their droplet size and zeta potential. NACs’ cytotoxicity, Tumor Necrosis Factor α (TNF-α) induction and their effect on antigen engulfment by immune cells were tested in vitro. Adjuvant properties of NACs were verified by intranasal vaccination of mice together with ovalbumin (OVA). NACs show remarkable stability and do not require any special storage conditions. They exhibit bio-adhesiveness and influence the degree of model protein engulfment by epithelial cells. Moreover, they induce high specific anti-OVA IgG antibody titers after two intranasal administrations. Nanoadjuvant candidates composed of silicone oil and cationic detergents are stable, exhibit remarkable adjuvant properties and can be used as adjuvants for intranasal immunization.

Biodegradable Cationic Polycarbonates as Vaccine Adjuvants.

In this study, biodegradable cationic polycarbonate and polylactide block copolymers were synthesized and successfully used as novel vaccine adjuvants to provide enhanced anticancer immunity. The polymers formed nanoparticles with the model vaccine, ovalbumin (OVA), and the immunostimulant toll-like receptor 3 agonist poly(I:C) (a synthetic analog of the double-stranded RNA). Higher uptake of poly(I:C) by the bone marrow-derived dendritic cells and macrophages and OVA by dendritic cells was observed when delivered using the polymer adjuvant. In vivo experiments showed that these nanoparticles remained longer in the subcutaneous injection site as compared to OVA alone and led to higher production of anti-OVA specific antibodies with prolonged immunostimulation. When OVA was combined with poly(I:C) that was either co-entrapped in the same particles or as separate particles, a comparable level of anti-OVA IgG1 antibodies and interleukin-6 (IL-6) was produced in mouse blood plasma, and a similar level of cytotoxic T lymphocyte (CTL) response in mice was stimulated as compared to OVA/Alum particles. Furthermore, tumor rejection in the mice that were vaccinated for 9 months with the formulations containing the polymer adjuvant was stronger than the other treatment groups without the polymer. Notably, the cationic polycarbonates were not associated with any adverse in vivo effects. Thus, these biodegradable polymers may be promising substitutes for aluminum-based adjuvants in vaccine formulations.

Chlorogenic Acid Alleviates Allergic Inflammatory Responses Through Regulating Th1/Th2 Balance in Ovalbumin-Induced Allergic Rhinitis Mice.

BackgroundAllergic rhinitis (AR) is a prevalent atopic disorder caused by immune imbalance. Chlorogenic acid (CGA) has antibacterial, antiviral, antioxidative and immunoregulatory effects, but its role in anaphylactic disease remains unclear. The current study aimed to investigate the function of CGA in AR.Material/MethodsAR mice models were induced with ovalbumin (OVA) by orally administrating the mice with 50 mg/kg (L-CGA), 100 mg/kg (M-CGA), or 200 mg/kg (H-CGA) of CGA. The number of nasal rubbings and sneezes was recorded. Afterward, the mice were sacrificed for the collection of blood, nasal lavage fluid (NALF), and nasal tissues. The cells in NALF were counted by hemocytometer and stained by Diff-Quick. Nasal mucosa was observed by H&E staining. ELISA testing was conducted for detecting the levels of anti-OVA antibodies and Th1/Th2-related cytokine. Quantitative real-time polymerase chain reaction experiments were conducted to determine mRNA expressions of Th1/Th2-related cytokines.ResultsIn the OVA-induced AR mice, CGA treatment reduced nasal rubbing and sneezing, and also suppressed the number of total cells, eosinophils, neutrophils, lymphocytes, macrophages, and epithelial cells in NALF. OVA-induced up-regulation of nasal mucosa thickness was inhibited by CGA, and the effects of OVA on IgE, IgG1, and IgG2a were reversed by CGA. The regulatory effects of OVA on mRNA expressions and levels of Th1/Th2-related cytokines were abolished by CGA treatment in AR mice.ConclusionsCGA can alleviate allergic inflammatory responses through regulating Th1/Th2 balance in OVA-induced allergic rhinitis mice.