ovalbumin and aluminum-sulfate

Asthma is one of the most common inflammatory diseases of the lung worldwide. There has been considerable progress in recent studies to treat and prevent allergic asthma, however, various side effects are still observed in clinical practice. Six-week-old male BALB/c mice were orally administered with either sword bean pod extracts (SBP; 100 or 300 mg/kg) or dexamethasone (DEX; 5 mg/kg) once daily over 3 weeks, followed by ovalbumin sensitization (OVA/Alum.; intraperitoneal administration, 50 μg/2 mg/per mouse). Scoring of lung inflammation was performed to observe pathological changes in response to SBP treatment compared to OVA/Alum.-induced lung injury. Additionally, inflammatory cytokines were quantified in serum, bronchoalveolar lavage fluid (BALF), and lung tissue using ELISA and Western blot analyses. SBP treatment significantly reduced the infiltration of inflammatory cells, and release of histamine, immunoglobulin E, and leukotriene in serum and BALF. Moreover, the therapeutic effect of SBP was also assessed to analyze the inflammatory changes in the lung tissues. SBP markedly suppressed the activation of the MAPK signaling pathway and the expression of key inflammatory proteins (e.g., TNF-α) and Th2 type cytokines (IL-5 and IL-13). SBP was effective in ameliorating the allergic inflammation against OVA/Alum.-induced asthma by suppressing pulmonary inflammation.

Topics: Alum Compounds; Animals; Asthma; Bronchoalveolar Lavage Fluid; Canavalia; Cytokines; Dexamethasone; Disease Models, Animal; Histamine; Immunoglobulin E; Inflammation; Interleukin-13; Interleukin-5; Lung; Male; Mice; Mice, Inbred BALB C; Ovalbumin; Plant Extracts; Pneumonia; Tumor Necrosis Factor-alpha

Vaccines have played a pivotal role in improving public health, however, many infectious diseases lack an effective vaccine. Controlling the spread of infectious diseases requires continuing studies to develop new and improved vaccines. Our laboratory has been investigating the immune enhancing mechanisms of Toll-like receptor (TLR) ligand-based adjuvants, including the TLR2 ligand Neisseria meningitidis outer membrane protein, PorB. Adjuvant use of PorB increases costimulatory factors on antigen presenting cells (APC), increases antigen specific antibody production, and cytokine producing T cells. We have demonstrated that macrophage expression of MyD88 (required for TLR2 signaling) is an absolute requirement for the improved antibody response induced by PorB. Here-in, we specifically investigated the role of subcapsular CD169+ marginal zone macrophages in antibody production induced by the use of TLR-ligand based adjuvants (PorB and CpG) and non-TLR-ligand adjuvants (aluminum salts). CD169 knockout mice and mice treated with low dose clodronate treated animals (which only remove marginal zone macrophages), were used to investigate the role of these macrophages in adjuvant-dependent antibody production. In both sets of mice, total antigen specific immunoglobulins (IgGs) were diminished regardless of adjuvant used. However, the greatest reduction was seen with the use of TLR ligands as adjuvants. In addition, the effect of the absence of CD169+ macrophages on adjuvant induced antigen and antigen presenting cell trafficking to the lymph nodes was examined using immunofluorescence by determining the relative extent of antigen loading on dendritic cells (DCs) and antigen deposition on follicular dendritic cells (FDC). Interestingly, only vaccine preparations containing PorB had significant decreases in antigen deposition in lymphoid follicles and germinal centers in CD169 knockout mice or mice treated with low dose clodronate as compared to wildtype controls. Mice immunized with CpG containing preparations demonstrated decreased FDC networks in the mice treated with low dose clodronate. Conversely, alum containing preparations only demonstrated significant decreases in IgG in CD169 knockout mice. These studies stress that importance of subcapsular macrophages and their unique role in adjuvant-mediated antibody production, potentially due to an effect of these adjuvants on antigen trafficking to the lymph node and deposition on follicular dendritic cells.

Topics: Adjuvants, Immunologic; Alum Compounds; Animals; Clodronic Acid; Dendritic Cells, Follicular; Immunogenicity, Vaccine; Immunoglobulin G; Macrophages; Mice, Inbred C57BL; Mice, Knockout; Oligodeoxyribonucleotides; Ovalbumin; Porins; Sialic Acid Binding Ig-like Lectin 1; Signal Transduction; Toll-Like Receptors; Vaccination

Topics: Adjuvants, Immunologic; Allergens; Alum Compounds; Animals; Animals, Newborn; Antibodies, Anti-Idiotypic; Female; Immunoglobulin E; Immunoglobulin G; Immunosuppression Therapy; Mice, Inbred C57BL; Ovalbumin; Pregnancy

As documented in a Vietnamese traditional medical encyclopedia, Syzygium formosum (Wall.) Masam leaves have been routinely used among indigenous Vietnamese people for treatment of various allergy-like symptoms including dermatitis and rhinitis.. Anti-allergic activity of S. formosum leaves was examined with a mouse model of chicken ovalbumin (cOVA)-induced food allergy, and mechanisms underlying the anti-allergic effect were explored.. BALB/c mice were administered i.p. cOVA (20μg) plus alum (2mg) twice on day 0 and 14 for sensitization (immunization). Two weeks after the second immunization, the mice were administered cOVA (50mg) p.o. 5 times every 3 days to induce food allergy symptoms (i.e., anaphylaxis, diarrhea, and drop in the body temperature). Ethanol extract of dried leaves of S. formosum (80mg/kg or 200mg/kg body weight) was administered p.o. daily during the induction (challenge) period.. Treatment with the S. formosum leaves ethanol extract ameliorated the allergic symptoms to a significant extent and in a dose-dependent manner. The treatment also resulted in a significant improvement in the inflammatory lesion in the small intestine and reduction in the numbers of mast cells and eosinophils recruited to the lesion. The treatment also brought about a significant reduction in the levels of Th2 cytokines produced by the mesenteric lymph node cells cultured ex vivo with cOVA. The passive anaphylaxis experiment also showed that the extract treatment impaired the mast cell function.. Our study provides a scientific basis for the traditional (indigenous) use of the S. formosum leaves extract for the treatment of various allergy symptoms in Vietnam. In addition, the results show that the extract has activities to suppress antigen-specific Th2 T cell immune responses and the mast cell function, which are directly related with its anti-allergic effect.

Topics: Allergens; Alum Compounds; Animals; Anti-Allergic Agents; Chymases; Cytokines; Ethanol; Female; Flavonoids; Food Hypersensitivity; Immunoglobulin E; Immunoglobulin G; Lymph Nodes; Mice, Inbred BALB C; Ovalbumin; Phytotherapy; Plant Extracts; Plant Leaves; Solvents; Syzygium; Triterpenes

There is evidence from epidemiology studies of a negative association between infection with helminth parasites and the development of allergy and asthma. Here, we demonstrate that the excretory/secretory products of the helminth Fasciola hepatica (FHES) protected mice against ovalbumin (OVA)-induced allergic asthma when administered at time of allergen sensitization. FHES reduced the accumulation of mucus, eosinophils and lymphocytes into the airways of allergen-challenged mice. Furthermore, FHES treatment suppressed Th2 responses in the airways. Interestingly, systemic administration of FHES at allergen challenge had no effect on airway inflammation, demonstrating that alum-induced Th2 response is set following initial allergen sensitization. Our findings highlight the immunomodulatory potential of molecules secreted by F. hepatica.

Topics: Alum Compounds; Animals; Asthma; Eosinophils; Fasciola hepatica; Helminth Proteins; Immunologic Factors; Mice; Mice, Inbred BALB C; Ovalbumin; Th2 Cells

Oxidative stress is involved in various diseases, including allergies. Several studies have pointed to the preventive and therapeutic potential of antioxidants in allergic disorders. However, little is known about the immunomodulatory effects of antioxidants in type I hypersensitivity. In this study we aimed to explore the impact of a water-soluble antioxidant and α-lipoic acid derivative, sodium zinc histidine dithiooctanamide (DHL-HisZn), on mast-cell- and T-cell-mediated allergic and immune responses both in vitro and in vivo.. The therapeutic impact of DHL-HisZn on mast-cell-mediated type I hypersensitivity was evaluated by a mast-cell degranulation assay using bone marrow-derived mast cells and by a mouse model of ovalbumin (OVA)-induced allergic rhinitis. The effect of DHL-HisZn on the proportion of regulatory T cells (Tregs) was evaluated using flow cytometry.. During the course of OVA-induced allergic rhinitis in mice, serum nitrate was elevated, suggesting the involvement of oxidative stress in allergic responses. DHL-HisZn not only suppressed mast-cell degranulation but also ameliorated OVA-induced nasal hypersensitivity, with significant suppression of serum nitrate. DHL-HisZn treatment significantly suppressed OVA-specific immunoglobulin E (IgE) but enhanced OVA-specific IgG2a in OVA-sensitized and nasal-challenged mice. Furthermore, DHL-HisZn treatment suppressed interleukin-17 production in OVA-stimulated splenocytes. Finally, we demonstrated the induction of Tregs by DHL-HisZn in concanavalin A blasts.. These findings suggest that DHL-HisZn may regulate mast-cell-, T-helper 2 (Th2)-, and Th17-mediated allergic and immune responses by induction of Tregs.

Topics: Allergens; Alum Compounds; Animals; Antioxidants; Cell Degranulation; Cytokines; Disease Models, Animal; Female; Histidine; Immunoglobulin E; Immunoglobulin G; Male; Mast Cells; Mice, Inbred BALB C; Ovalbumin; Rats, Inbred Lew; Rhinitis, Allergic; Spleen; T-Lymphocytes, Regulatory; Thioctic Acid

To understand mechanisms of human olfactory dysfunction in chronic rhinosinusitis, an inducible olfactory inflammation (IOI) model has been utilized to chronically express inflammatory cytokines locally, resulting in neuronal loss, diminished odorant responses, and repressed olfactory regeneration. Knockout of the minor tumor necrosis factor α receptor 2 (TNFR2) was previously shown to partially rescue these olfactory changes. The purpose of current study was to investigate the role of the major TNF receptor, TNFR1, in chronic olfactory inflammation.. Two experimental groups of mice were studied: TNFR1 knockout in IOI background and TNFR1 knockout with allergen-induced inflammation. Olfactory function was assayed by electro-olfactogram (EOG), and olfactory tissue was processed for histology and immunohistochemical staining.. TNF-α was dramatically induced in IOI-TNFR1 knockout mice, but the olfactory epithelium did not show inflammation. EOG responses were normal after either 2 or 8 weeks of TNF-α expression. Ovalbumin-sensitized TNFR1 knockout mice developed markedly diminished eosinophilic inflammatory infiltration.. Genetic deletion of TNFR1 completely blocks TNF-α-induced inflammation and reduces allergen-induced inflammation. Preserved EOG responses suggest a TNFR1-dependent mechanism of TNF-α-induced olfactory neuron dysfunction.

Topics: Allergens; Alum Compounds; Animals; Female; Hypersensitivity; Inflammation; Male; Mice, Knockout; Nasal Lavage Fluid; Neurons; Olfaction Disorders; Olfactory Mucosa; Ovalbumin; Receptors, Tumor Necrosis Factor, Type I; Tumor Necrosis Factor-alpha

Asthmatic inflammation is dominated by accumulation of either eosinophils, neutrophils, or both in the airways. Disposal of these inflammatory cells is the key to disease control. Eosinophilic airway inflammation is responsive to corticosteroid treatment, whereas neutrophilic inflammation is resistant and increases the burden of global health care. Corticosteroid-resistant neutrophilic asthma remains mechanistically poorly understood and requires novel effective therapeutic strategies.. We sought to explore the underlying mechanisms of airway inflammation persistence, as well as corticosteroid resistance, and to investigate a new strategy of effective treatment against corticosteroid-insensitive neutrophilic asthma.. Mouse models of either eosinophil-dominated or neutrophil-dominated airway inflammation were used in this study to test corticosteroid sensitivity in vivo and in vitro. We also used vav-Bcl-2 transgenic mice to confirm the importance of granulocytes apoptosis in the clearance of airway inflammation. Finally, the Bcl-2 inhibitors ABT-737 or ABT-199 were tested for their therapeutic effects against eosinophilic or neutrophilic airway inflammation and airway hyperresponsiveness.. Overexpression of Bcl-2 protein was found to be responsible for persistence of granulocytes in bronchoalveolar lavage fluid after allergic challenge. This was important because allergen-induced airway inflammation aggravated and persisted in vav-Bcl-2 transgenic mice, in which nucleated hematopoietic cells were overexpressed with Bcl-2 and resistant to apoptosis. The Bcl-2 inhibitors ABT-737 or ABT-199 play efficient roles in alleviation of either eosinophilic or corticosteroid-resistant neutrophilic airway inflammation by inducing apoptosis of immune cells, such as eosinophils, neutrophils, T. Apoptosis of inflammatory cells is essential for clearance of allergen-induced airway inflammation. The Bcl-2 inhibitors ABT-737 or ABT-199 might be promising drugs for the treatment of airway inflammation, especially for corticosteroid-insensitive neutrophilic airway inflammation.

Topics: Adrenal Cortex Hormones; Allergens; Alum Compounds; Animals; Anti-Inflammatory Agents; Apoptosis; Asthma; Biphenyl Compounds; Bridged Bicyclo Compounds, Heterocyclic; Bronchoalveolar Lavage Fluid; Dexamethasone; Drug Resistance; Eosinophils; Freund's Adjuvant; Humans; Lung; Male; Mice, Inbred C57BL; Mice, Transgenic; Neutrophils; Nitrophenols; Ovalbumin; Piperazines; Proto-Oncogene Proteins c-bcl-2; Sulfonamides

Oral tolerance refers to the specific inhibition of immune responsiveness to T-cell-dependent antigens contacted through the oral route before parenteral immunization. Oral tolerance to one protein does not inhibit immune responses to other unrelated proteins, but parenteral injection of tolerated antigens plus adjuvant into tolerant, but not normal, mice inhibits immune responses to antigens injected concomitantly or soon thereafter. The inhibitory effect triggered by parenteral injection of tolerated proteins is known as bystander suppression or indirect effects of oral tolerance. Intraperitoneal injection of ovalbumin (OVA) plus alum adjuvant in OVA-tolerant mice soon before skin injury inhibits inflammation and improves cutaneous wound healing. However, as OVA is not a regular component of mouse chow, we tested whether indirect effects could be triggered by zein, the main protein of corn that is regularly present in mouse chow. We show that intraperitoneal injection of a single dose (10 μg) of zein plus alum adjuvant soon before skin injury in mice reduces leucocyte infiltration but increase the number of T cells and the expression of resistin-like molecule-α (a marker of alternatively activated macrophages) in the wound bed, increases the expression of transforming growth factor-β

Topics: Adjuvants, Immunologic; Alum Compounds; Animals; Bystander Effect; Cicatrix; Cytokines; Disease Models, Animal; Immune Tolerance; Immunization; Injections, Intraperitoneal; Intercellular Signaling Peptides and Proteins; Male; Mast Cells; Mice, Inbred C57BL; Myofibroblasts; Ovalbumin; Skin; T-Lymphocytes; Time Factors; Transforming Growth Factor beta3; Wound Healing; Zein

The incidence of obesity has risen to epidemic proportions in recent decades, most commonly attributed to an increasingly sedentary lifestyle, and a 'western' diet high in fat and low in fibre. Although non-allergic asthma is a well-established co-morbidity of obesity, the influence of obesity on allergic asthma is still under debate. Allergic asthma is thought to result from impaired tolerance to airborne antigens, so-called respiratory tolerance. We sought to investigate whether a diet high in fats affects the development of respiratory tolerance. Mice fed a high fat diet (HFD) for 8 weeks showed weight gain, metabolic disease, and alteration in gut microbiota, metabolites and glucose metabolism compared to age-matched mice fed normal chow diet (ND). Respiratory tolerance was induced by repeated intranasal (i.n.) administration of ovalbumin (OVA), prior to induction of allergic airway inflammation (AAI) by sensitization with OVA in alum i.p. and subsequent i.n. OVA challenge. Surprisingly, respiratory tolerance was induced equally well in HFD and ND mice, as evidenced by decreased lung eosinophilia and serum OVA-specific IgE production. However, in a pilot study, HFD mice showed a tendency for impaired activation of airway dendritic cells and regulatory T cells compared with ND mice after induction of respiratory tolerance. Moreover, the capacity of lymph node cells to produce IL-5 and IL-13 after AAI was drastically diminished in HFD mice compared to ND mice. These results indicate that HFD does not affect the inflammatory or B cell response to an allergen, but inhibits priming of Th2 cells and possibly dendritic cell and regulatory T cell activation.

Topics: Allergens; Alum Compounds; Animals; B-Lymphocytes; Dendritic Cells; Diet, High-Fat; Dietary Fats; Eosinophilia; Female; Immune Tolerance; Immunoglobulin E; Interleukin-13; Interleukin-5; Lymphocyte Activation; Mice; Mice, Inbred C57BL; Obesity; Ovalbumin; Respiratory Hypersensitivity; Respiratory System; T-Lymphocytes, Regulatory

In our previous studies, the recombinant type II macrophage migration inhibitory factor homologue (rAs-MIF) secreted from Anisakis simplex suppressed experimental inflammation mouse model through IL-10 production and CD4(+)CD25(+)Foxp3(+) T-cell recruitment. Also, TLR2 gene expression was significantly increased following rAs-MIF treatment. To know the relation between TLR2 and amelioration mechanisms of rAs-MIF, we induced allergic airway inflammation by ovalbumin and alum with or without rAs-MIF under TLR2 blocking systems [anti-TLR2-specific antibody (α-mTLR2 Ab) treatment and using TLR2 knockout mice]. As a result, the amelioration effects of rAs-MIF in allergic airway inflammation model (diminished inflammation and Th2 response in the lung, increased IL-10 secretion, CD4(+)CD25(+)Foxp3(+) T-cell recruitment) were diminished under two of the TLR2 blocking model. The expression of TLR2 on the surface of lung epithelial cell was significantly elevated by rAs-MIF treatment or Pam3CSK (TLR2-specific agonist) treatment, but they might have some competition effect on the elevation of TLR2 expression. In addition, the elevation of IL-10 gene expression by rAs-MIF treatment was significantly inhibited by α-mTLR2 Ab or Pam3CSK pretreatment. In conclusion, anti-inflammatory effects of the rAs-MIF on OVA-induced allergic airway inflammation might be closely related to TLR2.

Topics: Alum Compounds; Animals; Anisakis; Disease Models, Animal; Female; Helminth Proteins; Hypersensitivity; Inflammation; Interleukin-10; Lung; Macrophage Migration-Inhibitory Factors; Mice; Mice, Inbred C57BL; Mice, Knockout; Ovalbumin; T-Lymphocytes, Helper-Inducer; T-Lymphocytes, Regulatory; Toll-Like Receptor 2

The murine intestinal nematode Heligmosomoides polygyrus exerts multiple immunomodulatory effects in the host, including the suppression of allergic inflammation in mice sensitized to allergen presented with alum adjuvant. Similar suppression is attained by co-administration of H. polygyrus excretory/secretory products (HES) with the sensitizing dose of ovalbumin (OVA) in alum. We investigated the mechanism of suppression by HES in this model, and found it was maintained in MyD88xTRIF-deficient mice, implying no role for helminth- or host-derived TLR ligands, or IL-1 family cytokines that signal in a MyD88- or TRIF-dependent manner. We also found suppression was unchanged in µMT mice, which lack B2 B cells, and that suppression was not abrogated when regulatory T cells were depleted in Foxp3.LuciDTR-4 mice. However, reduced IL-5 production was seen in the first 12 h after injection of OVA-alum when HES was co-administered, associated with reduced activation of IL-5(+) and IL-13(+) group 2 innate lymphoid cells. Thus, the suppressive effects of HES on alum-mediated OVA sensitization are reflected in the very earliest innate response to allergen exposure in vivo.

Topics: Adaptor Proteins, Vesicular Transport; Adjuvants, Immunologic; Alum Compounds; Animals; B-Lymphocytes, Regulatory; Hypersensitivity; Immunologic Deficiency Syndromes; Lymphocytes; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Mice, Knockout; Nematospiroides dubius; Ovalbumin; Primary Immunodeficiency Diseases; Signal Transduction; T-Lymphocytes, Regulatory

Many currently licensed and commercially available human vaccines contain aluminum salts as vaccine adjuvants. A major limitation with these vaccines is that they must not be exposed to freezing temperatures during transport or storage such that the liquid vaccine freezes, because freezing causes irreversible coagulation that damages the vaccines (e.g., loss of efficacy). Therefore, vaccines that contain aluminum salts as adjuvants are formulated as liquid suspensions and are required to be kept in cold chain (2-8°C) during transport and storage. Formulating vaccines adjuvanted with aluminum salts into dry powder that can be readily reconstituted before injection may address this limitation. Spray freeze-drying of vaccines with low concentrations of aluminum salts and high concentrations of trehalose alone, or a mixture of sugars and amino acids, as excipients can convert vaccines containing aluminum salts into dry powder, but fails to preserve the particle size and/or immunogenicity of the vaccines. In the present study, using ovalbumin as a model antigen adsorbed onto aluminum hydroxide or aluminum phosphate, a commercially available tetanus toxoid vaccine adjuvanted with potassium alum, a human hepatitis B vaccine adjuvanted with aluminum hydroxide, and a human papillomavirus vaccine adjuvanted with aluminum hydroxyphosphate sulfate, it was shown that vaccines containing a relatively high concentration of aluminum salts (i.e., up to ~1%, w/v, of aluminum hydroxide) can be converted into a dry powder by thin-film freezing followed by removal of the frozen solvent by lyophilization while using low levels of trehalose (i.e., as low as 2% w/v) as an excipient. Importantly, the thin-film freeze-drying process did not cause particle aggregation, nor decreased the immunogenicity of the vaccines. Moreover, repeated freezing-and-thawing of the dry vaccine powder did not cause aggregation. Thin-film freeze-drying is a viable platform technology to produce dry powders of vaccines that contain aluminum salts.

Topics: Adjuvants, Pharmaceutic; Alum Compounds; Aluminum Hydroxide; Animals; Calorimetry, Differential Scanning; Drug Compounding; Drug Stability; Female; Freeze Drying; Hepatitis B Vaccines; Immunoglobulin G; Mice, Inbred BALB C; Microscopy, Electron, Scanning; Ovalbumin; Particle Size; Phosphates; Powders; Technology, Pharmaceutical; Tetanus Toxoid

Prostaglandin D2 (PGD2) is a major prostanoid secreted mainly by mast cells. Although PGD2 has been identified as a modulator of allergic inflammation, its precise role remains unclear. Here we investigate the role of PGD2 in food allergy. Oral administration of ovalbumin induces allergic responses in sensitized wild-type (WT) mice. Systemic gene deficiency of haematopoietic PGD synthase (H-PGDS(-/-)) exacerbates all of the manifestations accompanying severe mast cell hyperplasia in the intestine. Morphological studies show that c-kit/FcɛRI-positive WT mast cells strongly express H-PGDS. Transplantation of H-PGDS(-/-) mast cells also aggravates ovalbumin-induced mast cell hyperplasia and allergic symptoms in mast cell null mice. H-PGDS deficiency accelerates the production of SDF-1α and the activity of MMP-9 in the antigen-stimulated intestine. SDF-1α receptor blockade or MMP-9 inhibition relieves the exacerbated mast cell hyperplasia and manifestations observed in H-PGDS(-/-). Thus, PGD2 deficiency results in food antigen-induced mast cell hyperplasia.

Topics: Adjuvants, Immunologic; Alum Compounds; Animals; Chemokine CXCL12; Colon; Cytokines; Enzyme-Linked Immunosorbent Assay; Food Hypersensitivity; Hyperplasia; Intestines; Intramolecular Oxidoreductases; Lipocalins; Mast Cells; Matrix Metalloproteinase 9; Mice; Mice, Inbred C57BL; Mice, Knockout; Microscopy, Electron; Ovalbumin; Prostaglandin D2; Reverse Transcriptase Polymerase Chain Reaction

Allergic conjunctivitis from an allergen-driven Th2 response is characterized by conjunctival eosinophilic infiltration. Although CCL20-CCR6 axis has been reported to play a proinflammatory role in several murine models of autoimmune diseases including allergic diseases, their underlying mechanism needs to be investigated. We here examined whether CCL20-CCR6 axis could play a role in the development of allergic conjunctival inflammation using murine experimental allergic conjunctivitis (EAC) model induced by ovalbumin (OVA) allergen. Mice were challenged with consecutive 10days of OVA via conjunctival sac after systemic challenge with OVA and cholera toxin in alum. Several indicators for allergy were comparatively evaluated in wild-type and CCR6 KO EAC mice. Wild-type mice challenged with OVA via conjunctival sac following systemic challenge with OVA in alum had severe allergic conjunctivitis. The absence of CCR6 suppressed IgE secretion and allergic conjunctival inflammation. Reduced allergic inflammation was ascribable to reduced cytokine responses from Th-2 type in draining lymph node although Th17, regulatory T cells and dendritic cell subsets are not affected by the absence of CCR6. In addition, neutralization of CCR6 ligand, CCL20 could repress allergic conjunctival inflammation. Our findings suggested that CCR6 might be crucial for optimal development of Th2 immune responses and further allergic conjunctival inflammation in EAC model.

Topics: Allergens; Alum Compounds; Animals; Chemokine CCL20; Chemokines, CC; Cholera Toxin; Conjunctivitis, Allergic; Cytokines; Dendritic Cells; Disease Models, Animal; Female; Immunoglobulin E; Inflammation; Lymph Nodes; Mice; Mice, Inbred C57BL; Ovalbumin; Receptors, CCR6; T-Lymphocytes, Regulatory; Th17 Cells; Th2 Cells

A Cu-mediated azide-alkyne click chemistry protocol was employed for the synthesis of a focused library of novel 1,2,3-triazolyl conjugates bearing various carbohydrate-steroid/triterpenoid entities. The immunogenicity of these compounds was examined initially by ex vivo assays. The lead compound 15g was further subjected to in vivo evaluation in BALB/c mice immunized with ovalbumin. These in vivo biological studies revealed an increase in B cell-mediated proliferation, higher expression levels of IL-2, TNF-α, IL-12, and IFN-γ indicating Th1 activation, together with an enhanced OVA-specific antibody (IgG) response compared to alum, affirming adjuvanticity of these glycolipids. The primary indications of response skewed toward Th1 immunity induced by the new triazoyl analogs indicate the potential of these molecules for possible application as adjuvants.

Topics: Adjuvants, Immunologic; Alum Compounds; Animals; B-Lymphocytes; Cell Proliferation; Cells, Cultured; Drug Design; Glycolipids; Hemolysis; Immunization; Immunoglobulin G; Interferon-gamma; Interleukin-12; Lymphocyte Activation; Male; Mice, Inbred BALB C; Molecular Structure; Ovalbumin; Rats, Wistar; Structure-Activity Relationship; Th1 Cells; Triazoles; Tumor Necrosis Factor-alpha

Activated macrophages have been classified into classical (M1) and alternative (M2) macrophages. We aimed to establish a method to yield enough number of macrophages to analyze their molecular, biological and immunological functions. We used drugs; adjuvant albumin from chicken egg whites--Imject Alum (OVA-Alum) and OVA Complete Freund Adjuvant (OVA-CFA), to induce macrophages to M2 and M1 respectively. We analyzed the phenotype of purified macrophages induced under these immune conditions, using flow cytometry (FACS) to detect cell-surface molecules and the enzyme-linked immunosorbent assay (ELISA) was used to detect cytokines. The cDNA microarray was employed to measure changes in expression level of cell surface protein between M1 and M2 macrophages. Phenotype analysis of purified macrophages, induced under these immune conditions, showed macrophages induced by OVA-Alum was almost M2 while the proportion of M1 macrophages induced by OVA-CFA was significantly higher. The results also showed higher expression level of macrophage galactose N- acetyl-galactosamine specific lectin-2 protein (MGL1/2-PE), a known M2 macrophage marker, on the surface of Alum-induced macrophages. On the basis of these preliminary data, ELISA results revealed that after macrophage stimulation with lipopolysaccharides (LPS), the level of interleukin (IL)-10 produced by Alum- induced macrophages was higher than the level of IL-10 produced by CFA-induced macrophages. In contrast, the level of tumor necrosis factor-alpha (TNF-α) produced by CFA-induced macrophages was higher than Alum-induced macrophages. The cDNA microarray confirmed previous results and suggest immunoglobulin-like type 2 receptor alpha (Pilra) as a new marker for M1, macrophage galactose N-acetylgalactosamine-specific lectin 2 (Mgl2) as M2 macrophages marker.

Topics: Alum Compounds; Animals; Cell Polarity; Freund's Adjuvant; Hypersensitivity; Macrophages; Male; Mice; Oligonucleotide Array Sequence Analysis; Ovalbumin; Th1 Cells; Th2 Cells

Modulation of the immune response is an important step in the induction of protective humoral and cellular immunity against pathogens. In this study, we investigated the possibility of using a nanomaterial conjugated with the toll-like receptor (TLR) ligand CpG to modulate the immune response towards the preferred polarity. MgAl-layered double hydroxide (LDH) nanomaterial has a very similar chemical composition to Alum, an FDA approved adjuvant for human vaccination. We used a model antigen, ovalbumin (OVA) to demonstrate that MgAl-LDH had comparable adjuvant activity to Alum, but much weaker inflammation. Conjugation of TLR9 ligand CpG to LDH nanoparticles significantly enhanced the antibody response and promoted a switch from Th2 toward Th1 response, demonstrated by a change in the IgG2a:IgG1 ratio. Moreover, immunization of mice with CpG-OVA-conjugated LDH before challenge with OVA-expressing B16/F10 tumor cells retarded tumor growth. Together, these data indicate that LDH nanomaterial can be used as an immune adjuvant to promote Th1 or Th2 dominant immune responses suitable for vaccination purposes.

Topics: Adjuvants, Immunologic; Alum Compounds; Animals; Antibody Formation; CD8-Positive T-Lymphocytes; Cell Line, Tumor; Chemical Phenomena; CpG Islands; Disease Models, Animal; Drug Delivery Systems; Female; Hydroxides; Immunity, Cellular; Immunization; Immunoglobulin G; Mice; Mice, Inbred C57BL; Nanoparticles; Ovalbumin

IgG immune complexes have been shown to modify immune responses driven by APCs in either a pro- or anti-inflammatory direction depending upon the context of stimulation. However, the ability of immune complexes to modulate the inflammasome-dependent innate immune response is unknown. In this study, we show that IgG immune complexes suppress IL-1α and IL-1β secretion through inhibition of inflammasome activation. The mechanism by which this inhibition occurs is via immune complex ligation of activating FcγRs, resulting in prevention of both activation and assembly of the inflammasome complex in response to nucleotide-binding domain leucine-rich repeat (NLR) P3, NLRC4, or AIM2 agonists. In vivo, administration of Ag in the form of an immune complex during priming of the immune response inhibited resultant adaptive immune responses in an NLRP3-dependent model of allergic airway disease. Our data reveal an unexpected mechanism regulating CD4(+) T cell differentiation, by which immune complexes suppress inflammasome activation and the generation of IL-1α and IL-1β from APCs, which are critical for the Ag-driven differentiation of CD4(+) T cells.

Topics: Adjuvants, Immunologic; Alum Compounds; Animals; Antigen-Antibody Complex; CD4-Positive T-Lymphocytes; Cells, Cultured; Dendritic Cells; Gene Expression Regulation; Immunity, Innate; Inflammasomes; Interleukin-1alpha; Interleukin-1beta; Lung; Lymph Nodes; Macrophages; Mice; Mice, Inbred C57BL; Mice, Knockout; Ovalbumin; Receptors, IgG; Respiratory Hypersensitivity; Signal Transduction

There are a number of mouse models of allergic airway inflammation used to delineate various aspects of asthma. One of the hurdles with using mice is their natural resistance to developing a Th2 allergic response. This is often overcome by double priming with the allergen and an adjuvant. Here we report on an efficient 11day, single antigen/alum priming protocol that is sufficient to sensitise mice for the development of Th2-mediated inflammation in the lung following antigen challenge.

Topics: Adjuvants, Immunologic; Alum Compounds; Animals; Antigens; Asthma; Bronchoalveolar Lavage Fluid; Disease Models, Animal; Immunoglobulin E; Male; Mice; Mice, Inbred C57BL; Ovalbumin; Th2 Cells; Time Factors

Unlike other types of T helper (Th) responses, whether the development of Th2 cells requires instruction from particular subset of dendritic cells (DCs) remains unclear. By using an in vivo depletion approach, we have shown that DCs expressing CD301b were required for the generation of Th2 cells after subcutaneous immunization with ovalbumin (OVA) along with papain or alum. CD301b⁺ DCs are distinct from epidermal or CD207⁺ dermal DCs (DDCs) and were responsible for transporting antigen injected subcutaneously with Th2-type adjuvants. Transient depletion of CD301b⁺ DCs resulted in less effective accumulation and decreased expression of CD69 by polyclonal CD4⁺ T cells in the lymph node. Moreover, despite intact cell division and interferon-γ production, CD301b⁺ DC depletion led to blunted interleukin-4 production by OVA-specific OT-II transgenic CD4⁺ T cells and significantly impaired Th2 cell development upon infection with Nippostrongylus brasiliensis. These results reveal CD301b⁺ DDCs as the key mediators of Th2 immunity.

Topics: Allergens; Alum Compounds; Animals; Cell Differentiation; Dendritic Cells; Gene Expression Regulation; Immunity, Cellular; Interleukin-4; Lectins, C-Type; Mice; Mice, Transgenic; Nippostrongylus; Ovalbumin; Signal Transduction; Skin; Strongylida Infections; Th2 Cells

Adjuvant efficiency is critical for inducing a protective and long-lasting immune response against weak immunogenic antigens. Discovered more than 70 years ago, aluminum salts remain the most widely used adjuvant in human vaccine. Prone to induce a strong humoral response, alum fails to drive a cell-mediated immunity, which is essential to fight against intracellular pathogens. Adjuvant systems that contain more than one component may represent an excellent alternative for completing the lack of T cell immunity associated with the injection of alum-based vaccine. In this work, we demonstrated that the adjuvant effects of alum strongly benefited from combining with a cationic lipid, the diC14 amidine. Indeed, we measured a significant improvement of alum-driven IL-1β release when human macrophages were co-cultured with a mixed suspension of alum and the diC14 amidine. Morphological analysis suggested that diC14 amidine improved the alum uptake by phagocytes. Furthermore, the addition of diC14 amidine to alum efficiently enhanced antigen processing and cross-presentation by antigen presenting cells. The biological relevance of these in vitro data was assessed by measuring the in vivo development of a cytotoxic activity and the enhanced synthesis of antigen-specific immunoglobulins after immunization with alum combined to diC14 amidine. Mechanistically, we demonstrated that diC14 amidine supported the alum adjuvanticity independently of the TLR-4 and caspase-1 agonist activities of the cationic lipid. Based on our findings, we conclude that diC14 amidine works synergistically with alum to achieve higher immune protection after vaccination.

Topics: Adjuvants, Immunologic; Alum Compounds; Amidines; Animals; Caspase 1; Cells, Cultured; Female; Immunity, Cellular; Immunity, Humoral; Interleukin-1beta; Macrophages; Male; Mice; Mice, Inbred C57BL; Models, Animal; Ovalbumin; Toll-Like Receptor 4; Vaccines, Subunit

Alum adjuvants have been in continuous clinical use for more than 80 yr. While the prevailing theory has been that depot formation and the associated slow release of antigen and/or inflammation are responsible for alum enhancement of antigen presentation and subsequent T- and B-cell responses, this has never been formally proven. To examine antigen persistence, we used the chimeric fluorescent protein EαGFP, which allows assessment of antigen presentation in situ, using the Y-Ae antibody. We demonstrate that alum and/or CpG adjuvants induced similar uptake of antigen, and in all cases, GFP signal did not persist beyond 24 h in draining lymph node antigen-presenting cells. Antigen presentation was first detectable on B cells within 6-12 h of antigen administration, followed by conventional dendritic cells (DCs) at 12-24 h, then finally plasmacytoid DCs at 48 h or later. Again, alum and/or CpG adjuvants did not have an effect on the magnitude or sequence of this response; furthermore, they induced similar antigen-specific T-cell activation in vivo. Notably, removal of the injection site and associated alum depot, as early as 2 h after administration, had no appreciable effect on antigen-specific T- and B-cell responses. This study clearly rules out a role for depot formation in alum adjuvant activity.

Topics: Adjuvants, Immunologic; Alum Compounds; Animals; Antigen Presentation; Antigen-Presenting Cells; Antigens; B-Lymphocytes; Dendritic Cells; Female; Flow Cytometry; Lymph Nodes; Lymphocyte Activation; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Mice, Transgenic; Oligodeoxyribonucleotides; Ovalbumin; T-Lymphocytes; Time Factors

Previous studies in young rats reported the impact of cocoa intake on healthy immune status and allow suggesting it may have a role in the prevention of some immune-mediated diseases. The aim of this study was to ascertain the effect of a cocoa diet in a model of allergy in young rats. Three-week-old Brown Norway rats were immunized by i.p. injection of ovalbumin (OVA) with alum as adjuvant and Bordetella pertussis toxin. During the next 4 weeks rats received either a cocoa diet (containing 0.2% polyphenols, w/w) or a standard diet. Animals fed a standard diet showed high concentrations of anti-OVA IgG1, IgG2a, IgG2b and high anti-OVA IgE titres, which is the antibody involved in allergic response. In contrast, animals fed a cocoa diet showed significantly lower concentrations of anti-OVA IgG1 and IgG2a antibodies. Interestingly, the cocoa diet prevented anti-OVA IgE synthesis and decreased total serum IgE concentration. Analysis of cytokine production in lymph node cells at the end of the study revealed that, in this compartment, the cocoa diet decreased the tumor necrosis factor (TNF)-α and the interleukin (IL)-10 secretion but not IL-4 production. In conclusion, a cocoa-enriched diet in young rats produces an immunomodulatory effect that prevents anti-allergen IgE synthesis, suggesting a potential role for cocoa flavonoids in the prevention or treatment of allergic diseases.

Topics: Alum Compounds; Animals; Anti-Allergic Agents; Body Weight; Cacao; Diet; Disease Models, Animal; Hypersensitivity; Immunoglobulin E; Interleukin-10; Interleukin-4; Lymph Nodes; Ovalbumin; Pertussis Toxin; Polyphenols; Rats; Rats, Inbred BN; Time Factors; Tumor Necrosis Factor-alpha

The efficacy of a vaccine is generally dependent on an adjuvant, which enhances the immune functions and alum has been widely used in human immunization. Alum activates the intracellular stress sensors inflammasomes, but whether these are responsible for the adjuvanticity is controversial. The objectives of this investigation were to examine the hypothesis that alum-mediated adjuvanticity is a function of stress and conversely that stress agents will elicit adjuvanticity. The investigation was carried out in BALB/c mice by SC immunization with ovalbumin (OVA) mixed with alum. This elicited inflammasomes, with significant activation of caspase 1, production of IL-1β, and adjuvanticity, demonstrated by enhancing OVA-specific serum IgG antibodies, CD4(+) T cells, and proliferation. The novel finding that alum induced HSP70 suggests that stress is involved in the mechanism of adjuvanticity. This was confirmed by inhibition studies with PES (phenylethynesulfonamide), which disrupts inducible HSP70 function, and inhibited both inflammasomes and the adjuvant function. Parallel studies were pursued with an oxidative agent (sodium arsenite), K-releasing agent (Gramicidin) and a metal ionophore (dithiocarbamate). All 3 stress agents induced HSP70, inflammasomes, and the adjuvant functions. Furthermore, up-regulation of membrane associated IL-15 on DC and CD40L on T cells in the animals treated with alum or the stress agents mediate the interactions between splenic CD11c DC and CD4(+) or CD8(+) T cells. The results suggest that the three stress agents elicit HSP70, a hallmark of stress, as well as inflammasomes and adjuvanticity, commensurate with those of alum, which may provide an alternative strategy in developing novel adjuvants.

Topics: Adjuvants, Immunologic; Alum Compounds; Animals; Anti-Bacterial Agents; Arsenites; Caspase 1; CD4-Positive T-Lymphocytes; CD40 Ligand; CD8-Positive T-Lymphocytes; Cell Proliferation; Chelating Agents; Dendritic Cells; Ditiocarb; Enzyme Activation; Enzyme Inhibitors; Gramicidin; HSP70 Heat-Shock Proteins; Humans; Immunization; Immunoglobulin G; Inflammasomes; Interleukin-15; Interleukin-1beta; Mice; Mice, Inbred BALB C; Ovalbumin; Oxidants; Sodium Compounds; Stress, Physiological

Alum is thought to induce inflammation resulting in the release of danger signals such as uric acid (UA) which in turn enhances the immune response to an antigen. Hydrogen peroxide (H(2)O(2)) is produced as a byproduct in the purine catabolic pathway that leads to the production of UA. In addition, serum nitric oxide (NO) levels are increased in inflammation.. To further explore the mechanism of action of alum, this study was designed to determine the effects of catalase and 1400W on the number of interleukin-4 (IL-4) and interferon-γ (IFN-γ) secreting spleen cells in mice given ovalbumin (OVA) with alum.. Groups of BALB/c mice were injected intraperitoneally with alum + OVA, alum, OVA, catalase, or 1400W. Other groups were treated with catalase or 1400W and given alum + OVA. The number of IL-4 and IFN-γ secreting spleen cells were determined at days 4 and 7 postinjection by enzyme-linked immunosorbent spot (ELISPOT).. Catalase and 1400W caused a decrease in the number of IL-4 secreting spleen cells induced by alum + OVA. 1400W caused a decline in the IFN-γ secreting spleen cells induced by alum + OVA. Catalase caused an increase in IFN-γ secreting spleen cells.. It appears that H(2)O(2) and NO are needed for alum-induced production of a T-helper 2 cytokine. NO also appears to be needed, whereas H(2)O(2) appeared to inhibit an alum-induced production of a T-helper 1 cytokine. These results might explain why alum is mainly a promoter of a T-helper 2 response.

Topics: Adjuvants, Immunologic; Alum Compounds; Amidines; Animals; Benzylamines; Catalase; Cattle; Cell Count; Enzyme Inhibitors; Female; Hydrogen Peroxide; Interferon-gamma; Interleukin-4; Mice; Mice, Inbred BALB C; Nitric Oxide; Ovalbumin; Spleen

Inflammatory mediators from peripheral tissues may control dendritic cell (DC) development in the bone marrow. In this study, DCs (CD11c(+) cells) differentiated from the bone marrow of mice with inflammation of the airways, or the peritoneal cavity had poor priming ability resulting in reduced, long-lived responses to that antigen in vivo. This indicates enhancement of regulatory mechanisms of immune responses through a peripheral tissue-bone marrow axis. If CD11c(+) cells, expanded from the bone marrow of mice with tissue inflammation were antigen pre-loaded and injected into mice already sensitized to that antigen, then subsequent contact hypersensitivity responses were significantly reduced. The effects of inflammation were imprinted in vivo and were independent of in vitro culture conditions for DC differentiation. The effect of tissue inflammation on the bone marrow DC precursors was not detected in mice treated subcutaneously with slow-release indomethacin pellets, suggesting a role for prostanoids, including prostaglandin E(2), in differentiation of regulatory CD11c(+) cells from bone marrow. Our study represents an important homeostatic process with potential for therapeutic use in the future.

Topics: Alum Compounds; Animals; B7-2 Antigen; Bone Marrow Cells; CD11c Antigen; Cell Differentiation; Cell Movement; Cross-Priming; Dendritic Cells; Granulocyte-Macrophage Colony-Stimulating Factor; Haptens; Homeostasis; Hypersensitivity; Immunization; Indomethacin; Inflammation; Interleukin-4; Lung; Lung Diseases; Lymph Nodes; Membrane Proteins; Mice; Mice, Inbred BALB C; Myelopoiesis; Ovalbumin; Peritoneal Cavity

CCR4 is highly expressed on Th2 cells. These cells play an important role in acute inflammatory responses, including those involved in allergic rhinitis. We determined whether disrupting the CCR4 ligand interaction with CCR4 antagonist could alleviate allergic rhinitis in a mouse model.. BALB/c mice were sensitized with ovalbumin and alum by intraperitoneal injection and challenged with intranasally administered ovalbumin. Compound 22, which has been reported as a novel small-molecule antagonist of CCR4, was also administered intranasally. In addition, budesonide, an efficient glucocorticoid, was used as a positive control. The effects of compound 22 were quantified by multiple parameters of allergic responses in both nasal and pulmonary tissues.. Compound 22 significantly improved symptoms of allergic rhinitis and suppressed levels of total IgE of serum. It dramatically reduced the levels of IL-4 in bronchoalveolar lavage fluid and also decreased the number of inflammatory cells in the fluid. The infiltration of inflammatory cells, especially eosinophils, was markedly reduced in the nasal and pulmonary tissues. The number of IL-4+ cells was also significantly reduced in these tissues. Moreover, the numbers of Foxp3+ cells and IL-17+ cells were reduced, though not to a statistically significant degree.. In our research, CCR4 antagonists such as compound 22 were proven for the first time to alleviate murine allergic rhinitis when administered nasally. CCR4 antagonists may have therapeutic potential for the treatment of allergic rhinitis.

Topics: Administration, Intranasal; Alum Compounds; Animals; Bronchoalveolar Lavage Fluid; Budesonide; Chemotaxis; Disease Models, Animal; Female; Glucocorticoids; HEK293 Cells; Humans; Immunization; Immunoglobulin E; Immunologic Factors; Interleukin-4; Mice; Mice, Inbred BALB C; Neutrophils; Ovalbumin; Receptors, CCR4; Rhinitis, Allergic, Perennial; Th2 Cells

TNFR superfamily (TNFRSF)4 (OX40, CD134) and TNFRSF25 are costimulatory receptors that influence CD4(+) and CD8(+) T cell responses to cognate Ag. Independently, these receptors have been described to stimulate overlapping functions, including enhanced proliferation and activation for both regulatory T cells (CD4(+)Foxp3(+); Tregs) and conventional T cells (CD4(+)Foxp3(-) or CD8(+)Foxp3(-); Tconvs). To determine the relative functionality of TNFRSF4 and TNFRSF25 in T cell immunity, the activity of TNFRSF4 and TNFRS25 agonistic Abs was compared in the context of both traditional protein/adjuvant (OVA/aluminum hydroxide) and CD8(+)-specific heat shock protein-based (gp96-Ig) vaccine approaches. These studies demonstrate that both TNFRSF4 and TNFRSF25 independently and additively costimulate vaccine-induced CD8(+) T cell proliferation following both primary and secondary Ag challenge. In contrast, the activities of TNFRSF4 and TNFRSF25 were observed to be divergent in the costimulation of CD4(+) T cell immunity. TNFRSF4 agonists were potent costimulators of OVA/aluminum hydroxide-induced CD4(+) Tconv proliferation, but they only weakly costimulated Treg proliferation and IgG2a production, whereas TNFRSF25 agonists were strong costimulators of Treg proliferation, producers of IgG1, IgG2a, and IgG2b, and weak costimulators of CD4(+) Tconv proliferation. Interestingly, Ag-specific cellular and humoral responses were uncoupled upon secondary immunization, which was dramatically affected by the presence of TNFRSF4 or TNFRSF25 costimulation. These studies highlight the overlapping but nonredundant activities of TNFRSF4 and TNFRSF25 in T cell immunity, which may guide the application of receptor agonistic agents as vaccine adjuvants for infectious disease and tumor immunity.

Topics: Adjuvants, Immunologic; Alum Compounds; Animals; CD4-Positive T-Lymphocytes; CD8-Positive T-Lymphocytes; Cell Proliferation; Cells, Cultured; Cross-Priming; Immunization, Secondary; Lymphocyte Activation; Mice; Mice, Inbred C57BL; Mice, Transgenic; NIH 3T3 Cells; Ovalbumin; Receptors, OX40; Receptors, Tumor Necrosis Factor, Member 25; Vaccines

NF-κB1-dependent signaling directs the development of CD4(+) Th2 cells during allergic airway inflammation and protective responses to helminth infection. Here, we show that IL-4 and IL-13 production is NF-κB1-dependent in mouse OVA-specific CD4(+) (OTII) T cells responding to alum-precipitated OVA (alumOVA) immunization. More surprisingly, we found that NF-κB1 deficiency in OTII cells also selectively impairs their CXCR5 induction by alumOVA without affecting upregulation of BCL6, IL-21, OX40 and CXCR4 mRNA and PD-1 protein. This results in functional impairment of follicular helper T cells. Thus, fewer germinal center B cells develop in LN responses to alumOVA in T-cell-deficient mice reconstituted with NF-κB1(-/-) OTII cells as opposed to NF-κB1(+/+) OTII cells, while plasma cell numbers are comparable. Unlike CXCR5 induction in CD4(+) T cells, NF-κB1-deficient recirculating follicular B cells are shown to express normal levels of CXCR5. The selective effects of NF-κB1-deficiency on Th2 and follicular helper T cell induction do not appear to be due to altered expression of the Th2-associated transcription factors - GATA-3, c-Maf and Ikaros. Altogether, these results suggest that NF-κB1 regulates the expression of CXCR5 on CD4(+) T cells primed in vivo, and thus selectively controls the T-cell-dependent germinal center component of B-cell response to alumOVA.

Topics: Adoptive Transfer; Alum Compounds; Animals; B-Lymphocytes; CD4-Positive T-Lymphocytes; Cells, Cultured; Germinal Center; Immunization; Lymphocyte Activation; Mice; Mice, Inbred C57BL; Mice, Knockout; Mice, Transgenic; NF-kappa B; Ovalbumin; Receptors, CXCR5; Th2 Cells; Up-Regulation

Vaccines can greatly reduce the spread of and deaths from many infectious diseases. However, many infections have no successful vaccines. Better understanding of the generation of protective CD8 memory T cells by vaccination is essential for the rational design of new vaccines that aim to prime cellular immune responses. Here we demonstrate that the combination of two adjuvants that are currently licensed for use in humans can be used to prime long-lived memory CD8 T cells that protect mice from viral challenge. The universally used adjuvant, aluminum salts, primed long-lived memory CD8 T cells; however, effective cytotoxic T-cell differentiation occurred only in the presence of an additional adjuvant, monophosphoryl lipid A (MPL). MPL-induced IL-6 was required for cytotoxic differentiation. The IL-6 acted by inducing granzyme B production and reducing expression of inhibitory molecule PD1 on the surface of the primed CD8 T cells. CD8 memory T cells generated by antigen delivered with both aluminum salts and MPL provided significant protection from influenza A challenge. These adjuvants could be used in human vaccines to prime protective memory CD8 T cells.

Topics: Adjuvants, Immunologic; Alum Compounds; Animals; Antigen Presentation; Antigens, Surface; Apoptosis Regulatory Proteins; Cattle; Cytokines; Female; Humans; Immunologic Memory; Influenza A virus; Interleukin-6; Lipid A; Mice; Mice, Knockout; Mice, Transgenic; Nucleocapsid Proteins; Orthomyxoviridae Infections; Ovalbumin; Programmed Cell Death 1 Receptor; RNA-Binding Proteins; Serum Albumin, Bovine; T-Lymphocytes, Cytotoxic; Vaccines, Subunit; Viral Core Proteins

Oligonucleotides containing CpG motifs (cytosine-phosphate-guanosine oligodeoxynucleotide [CpG ODN]) display strong immunostimulatory effects, and polycations have been previously reported as cellular delivery system. In the present study, we investigated the adjuvant properties of combinations of a CpG ODN with various polycations (poly-arginine, poly-lysine, poly-histidine, or chitosan) in an ovalbumin vaccination model. We showed that, when combined to CpG ODN, poly-arginine and poly-histidine, but not poly-lysine or chitosan, enhanced efficiently both the IgG antibody production and the number of splenocytes secreting interferon-gamma after stimulation with a CD8+ T cell-restricted peptide. Interestingly, CpG ODN-poly-arginine, which was the most efficient, compared favorably to the complete Freund's adjuvant and aluminium salts and induced no local toxicity, making this combination a very attractive adjuvant for vaccines.

Topics: Adjuvants, Immunologic; Alum Compounds; Animals; Cells, Cultured; Chitosan; Drug Stability; Electrophoretic Mobility Shift Assay; Freund's Adjuvant; Histidine; Immune Sera; Immunity, Cellular; Immunity, Humoral; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Oligodeoxyribonucleotides; Ovalbumin; Particle Size; Peptides; Polyamines; Polyelectrolytes; Polylysine; Vaccination

Mouse mast cells (MCs) express a large number of serine proteases including tryptases, mouse mast cell protease (mMCP)-6 and -7; chymases, mMCP-1, -2, and -4; and an elastase, mMCP-5; along with carboxypeptidase-A3 (CPA3). In helminth-infected mouse intestine, distinct protease phenotypes are observed for connective tissue MCs (CTMCs) (mMCP-4(+)-7(+), and CPA3(+)) and mucosal MCs (MMCs) (mMCP-1(+) and 2(+)). To determine whether the protease phenotype was regulated by the tissue, we compared the phenotype of constitutive CTMCs and induced MMCs in trachea and large airways in antigen-sensitized unchallenged and challenged mice to MCs in skin and helminthic-infected intestine. We found that in the trachea, unlike in skin and intestine, CTMCs and MMCs both express all six serine proteases and CPA3 (mMCP-1(+), -2(+), 4(+)-7(+), CPA3(+)). This phenotype also holds for the lung CTMCs in the proximal bronchi, whereas the induced MMCs express only four proteases, mMCP-1, -2, -6, and -7. Thus, the T-cell-dependent induction of MMCs in trachea, large bronchi, and small intestine provides numbers but does not determine the protease phenotype. Furthermore, the CTMCs, which are constitutive, also show striking differences at these tissue sites, supporting the view that the differences in expression are tissue directed and not dependent on inflammation.

Topics: Aerosols; Alum Compounds; Animals; Antibody Specificity; Antigens; Bronchi; Cell Count; Cell Differentiation; Connective Tissue Cells; Immunization; Mast Cells; Mice; Mice, Inbred BALB C; Mucous Membrane; Organ Specificity; Ovalbumin; Peptide Hydrolases; Phenotype; Stem Cells; Time Factors; Trachea

Using a T helper (Th)1/Th2 disease model, we previously showed that genetically determined Th development depends on dendritic cell-derived interleukin (IL)-1alpha. In Leishmania major infections, Th1 immunity develops if IL-1alpha is present during T cell priming, whereas at later time points, IL-1alpha worsens disease outcome. In the present study, we determined the role of IL-1alpha in other Th2-mediated diseases.. BALB/c mice were subjected to delayed-type hypersensitivity (DTH) or ovalbumin (OVA)/alum-induced allergic asthma in the presence or absence of IL-1alpha.. In DTH, mice treated with IL-1alpha during sensitization with keyhole limpet hemocyanin (KLH)/alum developed decreased footpad swelling associated with elevated KLH-specific interferon-gamma levels. In asthma, significantly decreased airway hypersensitivity responses (AHRs) were detected upon treatment with IL-1alpha during T cell priming. In contrast to control mice, IL-1alpha-treated mice showed reduced peribronchial inflammatory infiltrates. The bronchoalveolar lavage (BAL) fluid contained significantly decreased eosinophil numbers (approximately 50%), but 4 times more neutrophils. The BAL fluid of IL-1alpha-treated BALB/c exhibited reduced amounts of IL-5 and OVA-specific IgE serum levels. In contrast, IL-1alpha treatment at later time points after sensitization or during allergen challenge worsened AHR, had no effect on lung inflammation and BAL fluid cell composition. Furthermore, cytokine levels (IL-5, IL-13) and antigen-specific IgE were increased or unaltered under these conditions.. Similarly to leishmaniasis, IL-1alpha administration during sensitization of Th2-mediated allergic reactions suppresses the course of disease by shifting the immune response towards Th1, whereas later treatments worsen disease outcome. Future studies will elucidate the therapeutic value of IL-1alpha in asthmatic patients.

Topics: Alum Compounds; Animals; Asthma; Bronchoalveolar Lavage Fluid; Cell Count; Eosinophils; Female; Hemocyanins; Hypersensitivity, Delayed; Immunoglobulin E; Interferon-gamma; Interleukin-1alpha; Interleukin-5; Mice; Mice, Inbred BALB C; Neutrophils; Ovalbumin; Th2 Cells

The formylpeptide receptor-like 1, now officially termed FPR2, in human and its mouse homolog mFPR2 mediate leukocyte migration in response to agonists associated with inflammation and immune responses. To clarify the in vivo role of the receptor, we generated mice deficient in mFPR2. mFPR2(-/-) mice showed markedly reduced severity in OVA/alum-induced allergic airway inflammation. This was associated with diminished recruitment of CD11c(+) dendritic cells into the airway mucosa and secondary lymphoid organs, as well as reduced production of Type 2 cytokines and Igs. We also found that the bronchoalveolar lavage fluid from wild type mice with airway inflammation contained mFPR2 agonist activity. This study reveals a critical role for mFPR2 in the progression of allergic airway inflammation and immune responses.

Topics: Allergens; Alum Compounds; Animals; Bronchoalveolar Lavage Fluid; Cell Separation; Chemotaxis, Leukocyte; Cytokines; Enzyme-Linked Immunosorbent Assay; Flow Cytometry; Fluorescent Antibody Technique; Hypersensitivity; Immunohistochemistry; Inflammation; Lung; Mice; Mice, Inbred C57BL; Mice, Knockout; Ovalbumin; Receptors, Formyl Peptide; Respiratory Hypersensitivity; Reverse Transcriptase Polymerase Chain Reaction

Aluminium (ALUM) is used as experimental and clinical adjuvant for parenteral vaccine formulation. It is also contained in anti-acid drugs like sucralfate (SUC). These anti-acids have been shown to cause sensitization to food proteins via elevation of the gastric pH. The aim of this study was to assess the oral adjuvant properties of ALUM, alone or contained in SUC, in a BALB/c mouse model.. Mice were fed SUC plus ovalbumin (OVA) and compared with groups where ALUM or proton pump inhibitors (PPI) were applied as adjuvants. The humoral and cellular immune responses were assessed on antigen-specific antibody and cytokine levels. The in vivo relevance was investigated in skin tests.. The highest OVA-specific immunoglobulin G1 (IgG1) and IgE antibody levels were found in mice fed with OVA/SUC, followed by OVA/ALUM-treated animals, indicating a T helper 2 (Th2) shift in both groups. Antibody levels in other groups revealed lower (OVA/PPI-group) or baseline levels (control groups). Positive skin tests confirmed an allergic response in anti-acid or adjuvant-treated animals.. Our data show for the first time that ALUM acts as a Th2-adjuvant via the oral route. This suggests that orally applied SUC leads to an enhanced risk for food allergy, not only by inhibiting peptic digestion but also by acting as a Th2-adjuvant by its ALUM content.

Topics: Adjuvants, Immunologic; Administration, Oral; Alum Compounds; Animals; Antacids; Female; Food Hypersensitivity; Gastric Acidity Determination; Immunoglobulin E; Immunoglobulin G; Interferon-gamma; Interleukin-5; Mice; Mice, Inbred BALB C; Ovalbumin; Skin Tests; Sucralfate; Th2 Cells

Recent clinical trials, epidemiological studies and animal experiments have suggested that probiotics may help suppress the development of allergic responses.. To investigate whether the application of the probiotic Escherichia coli strain Nissle 1917 (EcN) protects mice from developing ovalbumin (OVA)-specific T helper-2 responses in the airways.. OVA-specific Th2 responses were induced by 2 intraperitoneal (i.p.) injections with OVA/alum followed by 1 intranasal (i.n.) challenge with OVA. EcN was given orally during the entire sensitization and challenge period, together with OVA/alum during the i.p. sensitizations, or i.n. before or during the airway challenge with OVA.. We found that when the bacteria were given together with OVA/alum airway eosinophilia, airway hyper-reactivity, goblet cell metaplasia and IL-5 levels in the bronchoalveolar lavage and mediastinal lymph node cell cultures were reduced. This effect was associated with increased numbers of IFN-gamma producing T helper-1 cells and IFN-gamma levels in the airways and strongly increased OVA-specific IgG(2a) titers in the serum. The suppressive effect on airway eosinophilia was dependent on IFN-gamma but not TLR-4. Applying EcN i.n. or orally did not reduce the development of allergen-specific Th2 responses.. Our results suggest that EcN can inhibit the development of allergic responses when the bacteria are present at the site of Th2 cell priming and that this immunomodulatory effect is due to a shift from Th2 to Th1 response. The data support the hypothesis that probiotics may help reduce allergic responses and that EcN may also be used as adjuvant therapy to induce allergen-specific Th1 responses.

Topics: Adjuvants, Immunologic; Administration, Intranasal; Administration, Oral; Allergens; Alum Compounds; Animals; Bronchial Hyperreactivity; Bronchoalveolar Lavage Fluid; Dendritic Cells; Eosinophilia; Escherichia coli; Female; Goblet Cells; Hypersensitivity; Interferon-gamma; Interleukin-5; Lipopolysaccharides; Mice; Mice, Inbred C57BL; Ovalbumin; Probiotics; Th1 Cells; Th2 Cells

In our previous work, a new water-soluble polysaccharide (PAP) from the mycelium of Polyporus albicans was identified for the first time. Preliminary tests in vitro showed PAP have potent stimulating effects on murine lymphocyte proliferation induced by mitogen. So in this study, the immunomodulatory effect and the adjuvant potential of PAP on the cellular and humoral immune response of ICR mice against ovalbumin were investigated. In vivo toxicity assays of PAP showed not to be lethal for mice in doses ranging from 0.5 to 4 mg. ICR mice were immunized subcutaneously with OVA 0.1 mg alone or with OVA 0.1 mg dissolved in saline containing alum (0.2 mg) or PAP (0.5, 1 and 2 mg) on days 1 and 15. Two weeks later (day 28), concanavalin A (Con A)-, lipopolysaccharide (LPS)- and OVA-stimulated splenocyte proliferation and OVA-specific serum antibodies were measured. PAP (0.5, 1 and 2 mg) other than alum significantly enhanced the Con A-, LPS- or OVA-induced splenocyte proliferation in the OVA-immunized mice (P<0.05 or <0.01). The OVA-specific IgG, IgG1 and IgG2b antibody levels in serum were also significantly enhanced by PAP (0.5, 1 and 2 mg) compared with OVA control group (P<0.05 or <0.01). Moreover, alum (0.2 mg) only induces IgG and IgG1 antibody responses to OVA in mice. In conclusion, the results suggest that PAP could be a safe efficacious adjuvant capable of boosting cellular and humoral immunity without unacceptable toxicity. Thus adjuvants based on PAP have enormous potential for use in vaccines against both pathogens and cancer.

Topics: Adjuvants, Immunologic; Alum Compounds; Animals; Antibodies; Cell Proliferation; Immunoglobulin G; Lymphocytes; Male; Mice; Mice, Inbred ICR; Mycelium; Ovalbumin; Polyporaceae; Polysaccharides; Spleen

CD4 T helper (Th) cell differentiation defined by in vitro cytokine-directed culture systems leaves major gaps in our knowledge of the mechanisms driving divergent Th differentiation. This is evident from our analysis of the response of mouse ovalbumin-specific CD4 T cells to different forms of ovalbumin that induce markedly distinct responses in vivo. We show that live attenuated ovalbumin-expressing Salmonella (SalOVA) induce Th1-associated T-bet and IFN-gamma. Conversely, alum-precipitated ovalbumin (alumOVA) induces the Th2-associated GATA-3 and IL-4. The early diversity occurring within these CD4 T cells isolated 3 days after immunization was assessed using real-time RT-PCR microfluidic cards designed with 384 selected genes. The technique was validated both at the population and single cell levels at different stages of the responses, showing beta2-microglobulin to be a more stably expressed reference mRNA than either beta-actin or 18S RNA. SalOVA was then shown selectively to induce the OVA-specific CD4 T cells to produce many chemokines and pro-inflammatory cytokines, contrasting with alumOVA-induced cells that only produced a few Th2-associated cytokines. Several cytokines and features associated with follicular helper functions were induced in the OVA-specific CD4 T cells by both antigens. Finally, IL-17RB is strongly associated with OVA-specific CD4 T cells responding to alumOVA, suggesting that alum may promote Th2 immune response through a role for the IL-25/IL-17RB pathway.

Topics: Alum Compounds; Animals; CD4-Positive T-Lymphocytes; Cell Differentiation; Gene Expression Profiling; Gene Expression Regulation; Immunization; Immunoprecipitation; Interferon-gamma; Interleukin-4; Mice; Mice, Inbred C57BL; Mice, Transgenic; Oligonucleotide Array Sequence Analysis; Ovalbumin; Salmonella; Salmonella Infections; Th1 Cells; Th2 Cells

Alum, the only adjuvant approved for clinical applications, can induce strong humoral (Th2) but weak cellular (Th1) immune responses. It is necessary to develop safe and effective adjuvants capable of inducing both humoral and cellular immune responses. We previously showed that activation-associated protein-1 (ASP-1) derived from Onchocerca volvulus has potent adjuvant activity. In this study, we have further evaluated the adjuvanticity of recombinant ASP-1 using a panel of recombinant proteins or synthetic peptide-based antigens, including ovalbumin (OVA), synthetic HIV peptide (HIV-p), recombinant HIV gp41 (rgp41) and HBV HBsAg, as well as three commercially available inactivated vaccines against haemorrhagic fever with renal syndrome (HFRS), Influenza and Rabies. Our results indicate that ASP-1 induced significantly higher IgG1 (Th2-associated) and IgG2a (Th1-associated) responses than alum adjuvant against OVA antigen, HIV-p, and rgp41. Consistently, it induced similar level of IgG1 responses as alum but higher level of IgG2a and IFN-gamma-producing T cell responses than alum adjuvant against HBsAg. Further, ASP-1 improved both IgG1 and IgG2a responses to three commercial inactivated vaccines when used separately or in combination. In conclusion, the recombinant ASP-1, unlike alum adjuvant, is able to induce both Th1 and Th2-associated humoral responses and Th1 cellular responses, suggesting that it can be further developed as a promising adjuvant for subunit-based and inactivated vaccines.

Topics: Adjuvants, Immunologic; Alum Compounds; Animals; Antibodies, Viral; Helminth Proteins; Hepatitis B Surface Antigens; Hepatitis B virus; HIV; HIV Envelope Protein gp41; Immunoglobulin G; Male; Mice; Mice, Inbred BALB C; Onchocerca volvulus; Ovalbumin; Recombinant Proteins; Th1 Cells; Vaccines, Inactivated; Vaccines, Synthetic; Viral Vaccines

Biocompatible lipid implants which promote the sustained release of antigen have potential as novel vaccine delivery systems for subunit antigen as they may reduce or remove the requirement for multiple administrations. Of particular interest are sustained release systems that release antigen incorporated into particles. Previous work has demonstrated that lipid implants prepared from phosphatidylcholine, cholesterol, the adjuvant Quil-A, and ovalbumin as the model antigen could stimulate an immune response equivalent to that induced by a prime and boost with a comparable injectable vaccine. However, entrapment of antigen into particles released from the implant was low. Therefore the aim of this study was to firstly determine if the inclusion of a cationic derivative of cholesterol, DC-cholesterol, into the implants increased antigen entrapment and immunogenicity, and secondly, if a cationic implant could induce at least a comparable immune response as compared to a prime and boost with an injectable vaccine. The inclusion of DC-cholesterol had only a minor effect on antigen entrapment into particles released from the implants and the implants did not stimulate cellular responses as effectively as the comparable injectable vaccine or the unmodified implant containing Quil-A and cholesterol, although the vaccine did induce stronger responses than either soluble protein alone, or protein co-delivered in alum.

Topics: Adjuvants, Immunologic; Alum Compounds; Animals; Antibodies; Antibody Formation; CD4-Positive T-Lymphocytes; CD8-Positive T-Lymphocytes; Cholesterol; Drug Compounding; Drug Implants; Immunity, Cellular; Injections; Kinetics; Liposomes; Lymphocyte Activation; Male; Mice; Mice, Inbred C57BL; Ovalbumin; Phosphatidylcholines; Quillaja Saponins; Saponins; Solubility; Vaccines

The efficiency of cross-presentation of exogenous antigens by dendritic cells (DCs) would seem to be related to the level of antigen escape from massive degradation mediated by lysosomal proteases in an acidic environment. Here, we demonstrate that a short course of treatment with chloroquine in mice during primary immunization with soluble antigens improved the cross-priming of naïve CD8(+) T lymphocytes in vivo. More specifically, priming of chloroquine-treated mice with soluble ovalbumin (OVA), OVA associated with alum, or OVA pulsed on DCs was more effective in inducing OVA-specific CD8(+) T lymphocytes than was priming of untreated mice. We conclude that chloroquine treatment improves the cross-presentation capacity of DCs and thus the size of effector and memory CD8(+) T cells during vaccination.

Topics: Adjuvants, Immunologic; Alum Compounds; Animals; CD8-Positive T-Lymphocytes; Cells, Cultured; Chloroquine; Cytotoxicity Tests, Immunologic; Dendritic Cells; Female; Mice; Mice, Inbred C57BL; Ovalbumin; Vaccination

It has been suggested that one possible contributor to the increasing prevalence of IgE-mediated allergic diseases in Europe and the US is exposure to chemicals that may act as adjuvants. It has been reported previously that certain commonly used phthalate plasticizers, such as di-(2-ethylhexyl) phthalate (DEHP), are able to modify immune responses induced in mice by the common hens' egg allergen ovalbumin (OVA). However, the significance of these observations for human health is unclear, not least because the relevant studies have been conducted exclusively using subcutaneous administration of phthalates. We have therefore investigated the ability of DEHP when applied topically to affect anti-OVA antibody responses induced by subcutaneous exposure to OVA in BALB/c strain mice. Doses of DEHP (50mg) were used that resulted in a marked (approximately 30%) increase in liver weight. Dose-responses were conducted in order to identify doses of OVA that were sub-optimal for both anti-OVA IgG1 and IgE antibody responses: 1microg and 0.05microg, respectively. Under these conditions of exposure, topical administration of DEHP was without impact on antibody responses, regardless of whether DEHP was applied local or distant to the site of OVA immunization. Topical application of concentrations of DEHP that provoked marked systemic effects was without effect on the induction of immune responses.

Topics: Adjuvants, Immunologic; Administration, Topical; Allergens; Alum Compounds; Animals; Diethylhexyl Phthalate; Enzyme-Linked Immunosorbent Assay; Female; Immunoglobulin E; Immunoglobulin G; Injections, Intraperitoneal; Mice; Mice, Inbred BALB C; Ovalbumin; Passive Cutaneous Anaphylaxis

Alum is used as a vaccine adjuvant and induces T(h)2 responses and T(h)2-driven antibody isotype production against co-injected antigens. Alum also promotes the appearance in the spleen of Gr1+IL-4+ innate cells that, via IL-4 production, induce MHC II-mediated signaling in B cells. To investigate whether these Gr1+ cells accumulate in the spleen in response to other T(h)2-inducing stimuli and to understand some of their functions, the effects of injection of alum and eggs from the helminth, Schistosoma mansoni, were compared. Like alum, schistosome eggs induced the appearance of Gr1+IL-4+ cells in spleen and promoted MHC II-mediated signaling in B cells. Unlike alum, however, schistosome eggs did not promote CD4 T cell responses against co-injected antigens, suggesting that the effects of alum or schistosome eggs on splenic B cells cannot by themselves explain the T cell adjuvant properties of alum. Accordingly, depletion of IL-4 or Gr1+ cells in alum-injected mice had no effect on the ability of alum to improve expansion of primary CD4 T cells. However, Gr1+ cells and IL-4 played some role in the effects of alum, since depletion of either resulted in antibody responses to antigen that included not only the normal T(h)2-driven isotypes, like IgG1, but also a T(h)1-driven isotype, IgG2c. These data suggest that alum affects the immune response in at least two ways: one, independent of Gr1+ cells and IL-4, that promotes CD4 T cell proliferation and another, via Gr1+IL-4+ cells, that participates in the polarization of the response.

Topics: Adjuvants, Immunologic; Alum Compounds; Animals; Antigens, Helminth; B-Lymphocytes; CD4-Positive T-Lymphocytes; Female; Genes, MHC Class II; Immunity, Innate; Immunoglobulin Isotypes; Interleukin-4; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Mice, Mutant Strains; Ovalbumin; Receptors, Chemokine; Schistosoma mansoni; Spleen; Th1 Cells; Th2 Cells

Alum (aluminum hydroxide) is the most widely used adjuvant in human vaccines, but the mechanism of its adjuvanticity remains unknown. In vitro studies showed no stimulatory effects on dendritic cells (DCs). In the absence of adjuvant, Ag was taken up by lymph node (LN)-resident DCs that acquired soluble Ag via afferent lymphatics, whereas after injection of alum, Ag was taken up, processed, and presented by inflammatory monocytes that migrated from the peritoneum, thus becoming inflammatory DCs that induced a persistent Th2 response. The enhancing effects of alum on both cellular and humoral immunity were completely abolished when CD11c(+) monocytes and DCs were conditionally depleted during immunization. Mechanistically, DC-driven responses were abolished in MyD88-deficient mice and after uricase treatment, implying the induction of uric acid. These findings suggest that alum adjuvant is immunogenic by exploiting "nature's adjuvant," the inflammatory DC through induction of the endogenous danger signal uric acid.

Topics: Adjuvants, Immunologic; Alum Compounds; Animals; Antibody Formation; Antigen Presentation; Antigens; CD11c Antigen; Cell Movement; Cross-Priming; Dendritic Cells; Immunity; Immunity, Innate; Inflammation; Injections, Intramuscular; Injections, Intraperitoneal; Lymph Nodes; Mice; Monocytes; Myeloid Differentiation Factor 88; Ovalbumin; Signal Transduction; Uric Acid

Vaccine efficacy largely depends upon DC targeting and activation. The most potent TLR soluble ligands induce diffuse DC activation, which may be associated with marked pro-inflammatory responses and possibly adverse effects. This raises the concern that effective vaccine adjuvants may similarly rely on widespread DC activation. Using a promising candidate vaccine against tuberculosis (fusion protein of Ag85B and 6-kDa early secretory antigenic target (ESAT-6)) formulated in the potent IC31 adjuvant, DC targeting and activation was studied in vivo, following the fate of antigen and adjuvant in the draining lymph nodes, to define the magnitude of DC targeting/activation required in vivo to induce protective vaccine responses. Unexpectedly, protective IFN-gamma-mediated Ag85B-ESAT-6/IC31 responses were associated to the activation of a minute population (less than 0.3%) of CD11c(+) lymph node DC, without detectable systemic pro-inflammatory responses. This activated peripheral tissue-derived DC population, characterized by enhanced CD80, CD86, CD40 and IL-12p40 expression, was only identified when focusing on adjuvant- or antigen-labeled CD11c(+) DC, which were found to support T cell proliferation. Immunization with aluminum hydroxide adjuvant (Alum) resulted in a similar proportion of antigen-associated DC but without detectable enhancement of CD80, CD86, CD40 or IL-12p40 expression. Thus, potent protective IFN-gamma-producing responses may be elicited by the exquisite activation of a minute number of in vivo targeted DC.

Topics: Adjuvants, Immunologic; Alum Compounds; Animals; Antigen Presentation; Antigens, Bacterial; Antigens, CD; Bacterial Proteins; CD11c Antigen; CD4-Positive T-Lymphocytes; Cell Proliferation; Dendritic Cells; Interferon-gamma; Interleukin-12 Subunit p40; Lymph Nodes; Lymphocyte Activation; Mice; Mice, Inbred C57BL; Mycobacterium bovis; Oligodeoxyribonucleotides; Ovalbumin; Recombinant Fusion Proteins; Spleen; T-Lymphocytes; Tuberculosis; Vaccination

Eosinophils play a major role in the development and severity of asthma. Robust and rapid preclinical animal models are desirable to profile novel therapeutics inhibiting the influx of eosinophils into the airways. To develop a rapid, airway eosinophil recruitment model in the rat, Brown-Norway (BN) rats were immunised with ovalbumin (OVA)/alum on day 0, 1 and 2 and challenged with OVA aerosol on day 5 and 6. On day 7 bronchoalveolar lavage fluid (BALF) was analysed for eosinophil numbers, eosinophil peroxidase (EPO) activity and cytokines. Lung sections were also examined. The immunised animals showed a strong selective influx of eosinophils into the airways correlating with enhanced EPO activity, Interleukin (IL-4), IL-5 and monocytes chemo attractant protein levels in the BALF in comparison to sham-sensitised rats. In addition the immunised rats developed goblet cell metaplasia in the lung and showed OVA specific IgG1 and IgE levels in the serum but no airway hyperreactivity after metacholine challenge. Airway inflammation was suppressed by applying the steroids Budesonide (intra tracheally) and Prednisolone (per orally), Roflumilast a phosphodiesterase-4 inhibitor, and the H1 receptor antagonists Epinastine and Ketotifen. Montelukast, a Leukotriene receptor antagonist and Chromoglycate, a mast cell stabiliser, had no effect in this model. In summary, in this novel preclinical rat model therapeutics expected to inhibit the development of airway eosinophilia can rapidly be tested.

Topics: Alum Compounds; Aminopyridines; Animals; Anti-Inflammatory Agents; Asthma; Benzamides; Bronchoalveolar Lavage Fluid; Budesonide; Cyclopropanes; Dibenzazepines; Disease Models, Animal; Eosinophils; Histamine H1 Antagonists; Imidazoles; Ketotifen; Lung; Mice; Mice, Inbred BALB C; Ovalbumin; Phosphodiesterase Inhibitors; Prednisolone; Rats; Rats, Inbred BN

The mechanisms underlying exacerbation of asthma induced by respiratory syncytial virus (RSV) infection have been extensively studied in human and animal models. However, most of these studies focused on acute inflammation and little is known of its long-term consequences on remodelling of the airway tissue.. The aim of the study was to use a murine model of prolonged allergen-induced airway inflammation to investigate the effect of RSV infection on allergic airway inflammation and tissue remodelling.. We subjected mice to RSV infection before or during the chronic phase of airway challenges with OVA and compared parameters of airway inflammation and remodelling at the end-point of the prolonged allergen-induced airway inflammation protocol.. RSV infection did not affect the severity of airway inflammation in any of the groups studied. However, RSV infection provoked airway remodelling in non-sensitized, allergen-challenged mice that did not otherwise develop any of the features of allergic airways disease. Increased collagen synthesis in the lung and thickening of the bronchial basal membrane was observed in non-sensitized allergen-challenged mice only after prior RSV infection. In addition, fibroblast growth factor (FGF)-2 but not TGF-beta(1) was increased in this group following RSV infection.. Our data show for the first time that RSV infection can prime the lung of mice that are not previously systemically sensitized, to develop airway remodelling in response to allergen upon sole exposure via the airways. Moreover, our results implicate RSV-induced FGF-2 in the remodelling process in vivo.

Topics: Alum Compounds; Analysis of Variance; Animals; Asthma; Bronchoalveolar Lavage Fluid; Collagen; Cytokines; Extracellular Matrix; Female; Fibroblast Growth Factor 2; Immunohistochemistry; Lung; Mice; Mice, Inbred BALB C; Ovalbumin; Respiratory Syncytial Virus Infections; Respiratory Syncytial Viruses; Statistics, Nonparametric; Transforming Growth Factor beta

Since liposomes are known as strong adjuvants, we attempted to use liposomes in immunotherapy as adjuvants, and to achieve desensitization in pre-sensitized mice. At first, we sensitized mice with intraperitoneal injection of model antigen, 100 microg ovalbumin (OVA), with Alum and treated them with liposome composed of distearoylphosphatidylcholine (DSPC) and cholesterol (2:1 as a molar ratio), which was coupled with a small amount of OVA (10 microg OVA in 400 nmol DSPC and 200 nmol cholesterol-liposome was injected into 20 g mouse). It is well known that antigen-specific immunotherapy increases IgG blocking antibodies and decreases in IgE antibodies. The treatment with i.v. injection of OVA-liposome at days 8, 10, and 12 after sensitization strongly suppressed OVA-specific IgE production without affecting IgG level after the boost (100 microg OVA with Alum). Moreover, the treatment with high-density OVA-liposome (10 microg OVA in 80 nmol DSPC and 40 nmol cholesterol-liposome/20 g mouse) not only strongly suppressed IgE levels but also reduced IgG production after the boost of OVA-sensitized mice suggesting the importance of liposomal characteristic in desensitization immunotherapy. Next we reduced the dose of OVA-liposome and the desensitization effect was also observed at the dose of as low as 1 microg OVA on OVA-liposome/mouse. On the contrary, free OVA did not affect the production of both IgG and IgE levels. Biodistribution study indicated that OVA-liposome was highly accumulated in spleen of OVA-sensitized mice compared to control liposome at 3 h after i.v. injection. These results suggest that the liposomal OVA effectively interacts with and desensitizes immune cells, therefore, liposomes coupling with a certain antigen may be effective in allergy immunotherapy.

Topics: Adjuvants, Immunologic; Alum Compounds; Animals; Antigens; Cholesterol; Desensitization, Immunologic; Dose-Response Relationship, Immunologic; Drug Administration Schedule; Enzyme-Linked Immunosorbent Assay; Female; Hypersensitivity; Immunoglobulin E; Immunoglobulin G; Liposomes; Mice; Mice, Inbred BALB C; Ovalbumin; Phosphatidylcholines; Spleen; Tissue Distribution

Toluene diisocyanate (TDI), a highly reactive industrial chemical, is one of the leading causes of occupation-related asthma in industrialized countries. The pathogenesis of TDI-induced asthma, however, remains not fully understood, in part due to lack of appropriate animal models. Twenty five female BALB/c mice (age: 8 weeks) were randomly divided into 5 groups: Ovabumin (OVA); OVA peptide amino acid residues No. 323-339 (Pep); TDI; alum and physiological saline. Mice were intraperitoneally injected with 25 microg OVA or pep absorbed on 300 microg alum, 300 microg alum or saline on days 0, 7 and 14. For the TDI group, mice were sensitized subcutaneously with 20 microl neat TDI on day 0; 20 microl of TDI in olive oil (1:10) on days 7 and 14; on days 21-23. Then each group was challenged intranasally with 20 microl of 1% OVA, 1% Pep, 1% TDI, 10% alum and saline respectively. On day 28, mice were killed under pentothal anesthesia. The results demonstrated that neutrophil-dominant inflammation with a few eosinophil infiltration occurred in the peri-bronchial and peri-vascular regions of the lungs. This was accompanied by hyperplasia/hypertrophy of cells lining the airways and mucus production as shown by HE staining. Positive immunohistochemical MBP staining in parenchyma was also shown. Th2 cytokine IL-4 and IgE production were significant increased 5 days after last challenge while IFN-gamma level was below the detection limit.. the clear elevation of IL-4 and IgE could allow to conclude a possible Th2-like dominated allergic response in TDI-exposed BALB/c mouse model.

Topics: Alum Compounds; Animals; Asthma; Bronchi; Bronchoalveolar Lavage Fluid; Cytokines; Disease Models, Animal; Eosinophils; Female; Immunoglobulin E; Inflammation; Interleukin-4; Mice; Mice, Inbred BALB C; Neutrophils; Ovalbumin; Th2 Cells; Toluene 2,4-Diisocyanate

CpG ODN are toll-like receptor 9 (TLR9) agonists that can enhance antigen presentation by antigen presenting cells (APCs) such as dendritic cells (DCs). The most potent antigen-specific responses are seen when CpG ODN and the antigen are co-localized in the same APC. CpG ODN-antigen fusion molecules and biodegradable microparticles entrapping CpG ODN and antigen can ensure both components are delivered to the same APC. In this study, we compared the efficacy of the CpG-ODN fusion molecules against biodegradable microparticles entrapping antigen and CpG ODN. Microparticles were prepared using a double emulsion solvent evaporation methodology. CpG ODN-OVA fusion molecules were prepared by mixing maleimide-activated protein with thiolated CpG ODN. Both CpG ODN-OVA fusion molecules and microparticles co-entrapping CpG ODN and OVA generated stronger IgG2a and interferon-gamma (IFN-gamma) responses than delivery of soluble CpG ODN and OVA. The microparticles generated stronger IgG2a and IFN-gamma immune responses than did CpG ODN-antigen fusion molecules.

Topics: Adjuvants, Immunologic; Alum Compounds; Animals; Cells, Cultured; Dendritic Cells; Drug Carriers; Drug Compounding; Female; Immunoconjugates; Immunoglobulin G; Interferon-gamma; Lactic Acid; Mice; Mice, Inbred C57BL; Mice, Transgenic; Oligodeoxyribonucleotides; Ovalbumin; Particle Size; Polyglycolic Acid; Polylactic Acid-Polyglycolic Acid Copolymer; Polymers; Receptors, Antigen, T-Cell; Technology, Pharmaceutical; Vaccines, Synthetic

In this study, we examined the effects of dietary lactosucrose (LS, a non-digestible oligosaccharide) on the IgE response in mice immunized with ovalbumin (OVA)/alum. In addition to IgG1 and IgG2a responses, the anti-OVA IgE response in mice fed LS diets was dose-dependently suppressed, as compared with the control mice, while the serum total IgG levels were comparable. Moreover, dietary LS feeding inhibited antigen-specific IgE and IgG1 productions even after a second immunization. Regarding with cytokine production, when stimulated in vitro with OVA, splenocytes obtained from LS-fed mice produced a similar level of IFN-gamma, and lower levels of IL-4 and IL-5, as compared with the control mice. But IL-10 production by OVA-stimulated splenocytes was augmented in LS-fed mice, suggesting that IL-10 producing cells are responsible for the immunoregulatory effect of LS. Our findings indicate the further possibility that dietary LS supplementation can be used to prevent IgE-mediated allergic diseases.

Topics: Adjuvants, Immunologic; Allergens; Alum Compounds; Animals; Antibody Formation; Cytokines; Dietary Carbohydrates; Hypersensitivity; Immunization; Immunoglobulin E; Immunoglobulin G; Immunosuppression Therapy; Mice; Ovalbumin; Spleen; Trisaccharides

In this study, the haemolytic activities of Glycyrrhiza uralensis saponins (GLS) and its adjuvant potentials on the cellular and humoral immune responses of ICR mice against ovalbumin (OVA) were evaluated. We determined the haemolytic activity of GLS using 0.5% rabbit red blood cell. Haemolytic percents of GLS-treated red blood cell were 11.20 and 5.54% at the concentrations of 500 and 250 microg/ml, respectively. ICR mice were immunized subcutaneously with OVA 100 microg alone or with OVA 100 microg dissolved in saline containing Alum (200 microg), QuilA (10 and 20 microg), or GLS (50, 100, or 200 microg) on Days 1 and 15. Two weeks later (Day 28), concanavalin A (Con A)-, lipopolysaccharide (LPS)-, and OVA-stimulated splenocyte proliferation and OVA-specific serum antibodies were measured. GLS significantly enhanced the Con A-, LPS-, and OVA-induced splenocyte proliferation in the OVA-immunized mice at a dose of 100 microg (P<0.025). OVA-specific IgG, IgG1, and IgG2b antibody titers in serum were also significantly enhanced by GLS compared with OVA control group (P<0.025). Moreover, no significant differences (P>0.05) were observed between enhancing effect of GLS and QuilA on the OVA-specific IgG, IgG1, and IgG2b antibody responses to OVA in mice. The results suggest that GLS showed a slight haemolytic effect and enhanced significantly a specific antibody and cellular response against OVA in mice, and deserved further researches to be developed as immunological adjuvant.

Topics: Adjuvants, Immunologic; Alum Compounds; Animals; Antibodies; Antibody Formation; Erythrocytes; Glycyrrhiza uralensis; Hemolysis; Immunoglobulin G; Injections, Subcutaneous; Lymphocyte Activation; Lymphocytes; Male; Mice; Mice, Inbred ICR; Ovalbumin; Quillaja Saponins; Rabbits; Saponins

Previous studies show that the generation of maximal T cell responses requires B cell antigen presentation and the differential expression of costimulatory molecules by B cells may affect polarization of naïve T cells to Th1 or Th2 phenotypes. We have therefore characterized the expression of activation and costimulatory molecules on antigen-specific T and B cells following immunisation with Alum or Alum/LPS to induce Th2 or Th1 responses in vivo. While antigen-specific B cells show similar levels of activation with respect to MHCII upregulation following Th1 or Th2 induction, they differentially express costimulatory molecules. Although ICOS-B7RP-1 interactions were originally implicated in Th2 generation, surprisingly this receptor-ligand pair was only upregulated on antigen-specific T and B cells following Th1 induction. In conclusion, these studies indicate that during the generation of antigen-specific Th1 or Th2 responses, adjuvants induce differential costimulation in antigen-specific B cells that may subsequently influence T cell polarization.

Topics: Adjuvants, Immunologic; Alum Compounds; Animals; Antigens, Differentiation, B-Lymphocyte; Antigens, Differentiation, T-Lymphocyte; B-Lymphocytes; B7-1 Antigen; Histocompatibility Antigens Class II; Inducible T-Cell Co-Stimulator Ligand; Inducible T-Cell Co-Stimulator Protein; Lipopolysaccharides; Lymphocyte Activation; Membrane Glycoproteins; Mice; Ovalbumin; OX40 Ligand; T-Lymphocytes; Th1 Cells; Th2 Cells; Tumor Necrosis Factors; Up-Regulation

Resistance to several prevalent infectious diseases requires both cellular and humoral immune responses. T cell immunity is initiated by mature dendritic cells (DCs) in lymphoid organs, whereas humoral responses to most antigens require further collaboration between primed, antigen-specific helper T cells and naive or memory B cells. To determine whether antigens delivered to DCs in lymphoid organs induce T cell help for antibody responses, we targeted a carrier protein, ovalbumin (OVA), to DCs in the presence of a maturation stimulus and assayed for antibodies to a hapten, (4-hydroxy-3-nitrophenyl) acetyl (NP), after boosting with OVA-NP. A single DC-targeted immunization elicited long-lived T cell helper responses to the carrier protein, leading to large numbers of antibody-secreting cells and high titers of high-affinity antihapten immunoglobulin Gs. Small doses of DC-targeted OVA induced higher titers and a broader spectrum of anti-NP antibody isotypes than large doses of OVA in alum adjuvant. Similar results were obtained when the circumsporozoite protein of Plasmodium yoelii was delivered to DCs. We conclude that antigen targeting to DCs combined with a maturation stimulus produces broad-based and long-lived T cell help for humoral immune responses.

Topics: Adjuvants, Immunologic; Alum Compounds; Animals; Antibody Formation; Antigen Presentation; B-Lymphocytes; Chickens; Dendritic Cells; Haptens; Humans; Immunization; Immunoglobulin G; Immunologic Memory; Mice; Mice, Inbred BALB C; Ovalbumin; Plasmodium yoelii; Protozoan Proteins; T-Lymphocytes

It is well known that the immunoregulatory cytokine interleukin (IL)-10 inhibits the accessory function of human dendritic cells (DC) in vitro. Recently, we have shown that these IL-10 DC inhibit the production of T helper cell 1 (Th1) and T helper cell 2 (Th2) cytokines by T cells from atopic individuals in vitro. The current study was set out to analyze whether IL-10 DC also exert inhibitory effects in vivo in a murine model of allergy to ovalbumin adsorbed to the adjuvant aluminium hydroxide (OVA/alum).. OVA-pulsed or unpulsed bone marrow-derived DC, treated with IL-10 or left untreated during generation, were injected intravenously into BALB/c mice prior to and during OVA/alum sensitization, and sera and immune responses of mesenterial lymph node cells were analyzed. Additionally, bronchoalveolar lavage was performed after intranasal challenge with OVA.. Treatment of BALB/c mice with OVA-pulsed DC led to a significantly enhanced proliferation as well as Th2 (IL-4, IL-5), Th1 (interferon-gamma) and IL-10 cytokine production after restimulation of lymph node cells with OVA in vitro compared with OVA immunization alone. In contrast, using OVA-pulsed IL-10 DC for transfer, proliferation and cytokine production by lymph node cells were not enhanced. OVA-specific immunoglobulin G1 (IgG1) and IgG2a production were significantly increased after transfer of OVA-pulsed DC and OVA-pulsed IL-10 DC, respectively, whereas anti-OVA IgE production and airway eosinophilia remained unchanged.. Our data indicate that IL-10 treatment of DC decreases the Th1 and Th2 stimulatory capacity of DC but does not actually inhibit systemic (IgE) and local (airway inflammation) allergen-specific immune responses in a murine model of allergy.

Topics: Adjuvants, Immunologic; Alum Compounds; Animals; Cell Proliferation; Cell Transplantation; Cytokines; Dendritic Cells; Disease Models, Animal; Female; Flow Cytometry; Hypersensitivity; Immune Tolerance; Immunoglobulin E; Interleukin-10; Lymphocyte Activation; Mice; Mice, Inbred BALB C; Ovalbumin; Th2 Cells

Adjuvants in vaccines are immune stimulants that play an important role in the induction of effective and appropriate immune responses to vaccine component(s). Diphtheria-tetanus-pertussis (DPT) vaccine contains not only aluminum hydrate (alum) to enhance the immune response to the vaccine ingredients, but also, both for that purpose and as a principal ingredient, pertussis toxin (PT). However, both adjuvants strongly promote T helper (Th) 2 type immune responses. Th1 and Th2 type immune responses are counterbalanced in vivo, and a Th2-prone immune response is not effective against intracellular infections but promotes IgE production, which is related to allergic disease. In this study, we used the CpG motif contained in oligodeoxynucleotide (CpG-ODN), which has an adjuvant effect and also induces the Th1 response, as an adjuvant to this vaccine, and we investigated its adjuvanticity and its potential to modulate immune responses to DPT vaccine. Administration of DPT vaccine with CpG-ODN (DPT-alum/ODN) to mice significantly reduced the total IgE levels and increased the anti-PT specific IgG2a titer in serum, in comparison with ordinary DPT vaccine (DPT-alum). Moreover, we investigated the antibody response to orally administrated ovalbumin (OVA) after vaccine administration. In the DPT-alum/ODN-administered group, the OVA specific IgE production in serum greatly decreased in comparison with that in the DPT-alum-administered group. These data indicate that CpG-ODN was not useful only as an efficient vaccine adjuvant but also shifted the immune responses substantially toward Th1 and modulated the Th1/Th2 immune response in DPT vaccine. These data suggested new applications of CpG-ODN as adjuvants in DPT vaccine.

Topics: Adjuvants, Immunologic; Alum Compounds; Animals; Antigens; CpG Islands; Diphtheria-Tetanus-Pertussis Vaccine; Enzyme-Linked Immunosorbent Assay; Female; Hypersensitivity; Immunity, Cellular; Immunoglobulin G; Intubation, Gastrointestinal; Mice; Mice, Inbred BALB C; Oligonucleotides; Ovalbumin; Th1 Cells; Th2 Cells

A viable hypomorphic allele of mouse retinoid X receptor alpha (Rxralpha) was created by random germline mutagenesis. The mutation (I273N) alters the ligand binding and heterodimerization domain, and causes a 90% decline in ligand-inducible transactivation. Homozygotes develop progressive alopecia and dermal cysts, and progressive exaggeration of Th1 and loss of Th2 responses to antigen. Th1 skewing is directly caused by aberrant function of both antigen-presenting cells and naïve CD4 T cells; the predominant Th1 response to antigen is attributable to decreased suppression of regulatory T cells in mutant mouse. Dietary depletion of vitamin A in Th2-prone wild-type mice mimics the immune phenotype caused by the mutation. Hence, RXRalpha plays an important post-developmental role in the regulation of adaptive immune responses, and provides a plausible link between nutritional environment and the type of adaptive response that results from immunization.

Topics: Aging; Alum Compounds; Animals; Cell Proliferation; Female; Immunity, Cellular; Immunization, Passive; Male; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Mutagenesis, Site-Directed; Ovalbumin; Retinoid X Receptor alpha; Th1 Cells; Th2 Cells

Most studies investigating the induction of oral tolerance (OT) use purified proteins such as ovalbumin (OVA), bovine serum albumin (BSA) and beta-lactoglobulin (beta-LG). Little information is available regarding the induction of OT to a protein mixture, e.g. cow's milk. In this study we compared the regulatory mechanisms induced after the oral administration of a whey protein concentrate (WP) derived from cow's milk following immunization with two different adjuvants, complete Freund's adjuvant (CFA) and alum. OVA was used as a control antigen. Animals were given a single feed of these proteins at an equivalent dose of 1 mg/g body weight before they were immunized seven days later with the antigen in Freund's adjuvant or alum. Delayed type hypersensitivity (DTH) responses were suppressed by both a feed of WP and OVA after immunization with CFA. However, only OVA feeding suppressed antigen specific IgG responses. In an attempt to investigate whether WP would tolerize the more susceptible IgE responses, alum immunization replaced CFA as the adjuvant used for systemic immunizations. WP, after a single feed, significantly primed for DTH and IgE responses indicating oral sensitization to WP. In contrast, OVA suppressed DTH, IgE and IgG responses. Antigen specific proliferation of mononuclear cells was suppressed in mice fed OVA, but primed in those fed with WP. In addition cells taken from sensitized mice fed WP up-regulated levels of specific interleukin (IL) -4, -10 and -12 in vitro whereas these cytokines were suppressed in cultures from tolerant WP fed mice. Global suppression was obtained in cultures from tolerant OVA fed mice. TGF-beta was not detected in draining PLN cell cultures of either tolerant or sensitized mice. These data suggest that a whey protein mixture induces divergent responses following immunization with either CFA or alum despite being fed at an identical dose. We suggest that that the choice of the adjuvant may determine the immunoregulatory outcome and this is also reflected by the systemic cytokine profile.

Topics: Administration, Oral; Alum Compounds; Animals; Cattle; Cells, Cultured; Cytokines; Female; Freund's Adjuvant; Hypersensitivity, Delayed; Immune Tolerance; Immunization; Immunoglobulin E; Immunoglobulin G; Lactoglobulins; Lymph Nodes; Mice; Mice, Inbred BALB C; Milk Proteins; Ovalbumin; Whey Proteins

Cationic antimicrobial peptides (CAMPs) are active defence components of the innate immune system. Several artificial CAMPs have been designed as antibiotic peptide therapeutics, but none have been reported to exert adjuvant activity in animal models. Here we show for the first time that an artificial CAMP, KLKLLLLLKLK (KLKL5KLK), is a potent inducer of adaptive immunity to co-injected antigens in vivo. High levels of antigen-specific antibodies were obtained after co-injection of KLKL5KLK with the model antigen ovalbumin (OVA) or a commercially available influenza vaccine. We show that KLKL5KLK induces a sustained immune response with a prevalent TH2 profile when co-injected with proteinaceous and peptide-based antigens. Furthermore, the immuno-enhancing activity of peptide KLKL5KLK was retained when C-terminally amidated or synthesised as retro-all-D-peptide. We provide evidence that KLKL5KLK enhances the association of antigen to antigen-presenting cells and forms a depot of antigen at the site of injection, making it an interesting adjuvant for novel vaccine design.

Topics: Adjuvants, Immunologic; Alum Compounds; Animals; Anti-Infective Agents; Antibodies, Viral; Antigen-Presenting Cells; Antigens; Enzyme-Linked Immunosorbent Assay; Epitopes; Flow Cytometry; Fluorescent Dyes; Genes, MHC Class I; Hemagglutination Inhibition Tests; Immunity, Cellular; Immunoglobulin G; Influenza Vaccines; Interleukin-4; Interleukin-5; Mice; Mice, Inbred C57BL; Oligopeptides; Ovalbumin; Spleen; Th2 Cells

IgE is critical in the pathogenesis of allergic disorders. In this report, we investigated the differential regulation of antigen-specific and by-stander IgE. Ovalbumin (OVA) immunization did not increase IgE producing cells in the spleen, but significantly enhanced the intracellular IgE content of all IgE+ cells. In contrast, OVA induced a significant increase of IgE+ cells in the draining lymph nodes (LN). Furthermore, OVA-specific IgE was detected only in the ex vivo cultures of the draining LN but not the spleen cells, while total IgE was increased in both cultures. These results indicated that antigen-specific IgE was mainly produced in the draining LN, while the spleen was a major source for by-stander IgE. Anti-IL-4, but not anti-IL-13, antibody blocked the expansion of IgE producing cells in the draining LN as well as systemic OVA-specific and total IgE levels, indicating IL-4 was important in both antigen-specific IgE generation and total IgE upregulation.

Topics: Adjuvants, Immunologic; Alum Compounds; Animals; Enzyme-Linked Immunosorbent Assay; Epitopes; Female; Flow Cytometry; Hypersensitivity; Immunoglobulin E; Immunoglobulin G; Interleukin-13; Interleukin-4; Lymph Nodes; Mice; Mice, Inbred BALB C; Ovalbumin; Specific Pathogen-Free Organisms; Spleen; Up-Regulation

Allergic asthma is characterized by airway hyperreactivity, inflammation, and a Th2-type cytokine profile favoring IgE production. Beneficial effects of TGF-beta and conflicting results regarding the role of Th1 cytokines have been reported from murine asthma models. In this study, we examined the T cell as a target cell of TGF-beta-mediated immune regulation in a mouse model of asthma. We demonstrate that impairment of TGF-beta signaling in T cells of transgenic mice expressing a dominant-negative TGF-beta type II receptor leads to a decrease in airway reactivity in a non-Ag-dependent model. Increased serum levels of IFN-gamma can be detected in these animals. In contrast, after injection of OVA adsorbed to alum and challenge with OVA aerosol, transgenic animals show an increased airway reactivity and inflammation compared with those of wild-type animals. IL-13 levels in bronchoalveolar lavage fluid and serum as well as the number of inducible NO synthase-expressing cells in lung infiltrates were increased in transgenic animals. These results demonstrate an important role for TGF-beta signaling in T cells in the regulation of airway responses and suggest that the beneficial effects observed for TGF-beta in airway hyperreactivity and inflammation may be due to its regulatory effects on T cells.

Topics: Administration, Inhalation; Aerosols; Alum Compounds; Animals; Antibody Specificity; Bronchial Hyperreactivity; Bronchoalveolar Lavage Fluid; CD2 Antigens; Cell Movement; Epitopes, T-Lymphocyte; Humans; Immunoglobulin E; Immunohistochemistry; Inflammation; Interferon-gamma; Interleukin-13; Lung; Mice; Mice, Inbred Strains; Mice, Transgenic; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Ovalbumin; Protein Serine-Threonine Kinases; Receptor, Transforming Growth Factor-beta Type II; Receptors, Transforming Growth Factor beta; T-Lymphocyte Subsets; Th1 Cells; Transforming Growth Factor beta

The IgE production was compared in the presence and absence of aluminum hydroxide gel (alum). Without alum, the IgE production was induced within a suitable range of the antigen dosage; however, alum enhanced it. Alum did not affect the minimum requirement for the antigen dosage, indicating that alum may not take part in the efficiency of antigen presentation.

Topics: Adjuvants, Immunologic; Alum Compounds; Animals; Antibody Specificity; Enzyme-Linked Immunosorbent Assay; Immunoglobulin E; Immunoglobulin G; Mice; Mice, Inbred BALB C; Ovalbumin; Passive Cutaneous Anaphylaxis

Recently, we demonstrated that prostaglandin (PG)I2 has a regulatory role in allergic responses through the receptor, IP; however, the role of PGI2 in airway remodeling associated with chronic airway inflammation has not been elucidated. In the present study, we examined the role of PGI2 in allergen-induced airway remodeling using IP gene-deficient mice. Mice were sensitized to ovalbumin (OVA) with alum, and exposed daily for 3 wk to aerosolized OVA. Twenty-four hours after the final antigen inhalation, bronchoalveolar lavage, biochemical, and histopathologic examinations were performed. In wild-type mice, prolonged allergen exposure in sensitized animals induced the increases in the numbers of inflammatory leukocytes (including eosinophils and lymphocytes), levels of T helper type 2 (Th2) cytokines (interleukin [IL]-4, IL-5, and IL-13), levels of OVA-specific immunoglobulin (Ig)E and IgG1 in serum, and amount of hydroxyproline in the right lungs associated with transforming growth factor-beta1 levels in bronchoalveolar lavage fluid. Moreover, goblet cell hyperplasia and subepithelial fibrosis were also appreciated after repeated allergen challenge. In contrast, the disruption of IP gene significantly augmented all these parameters. These findings suggest that PGI2 has a regulatory role in allergen-induced airway remodeling as well as airway eosinophilic inflammation, Th2 cytokine production and IgE production, and that a PGI2 agonist is a therapeutic approach for the treatment of airway remodeling in allergic asthma.

Topics: Allergens; Alum Compounds; Animals; Antigens; Asthma; Bronchoalveolar Lavage; Bronchoalveolar Lavage Fluid; Cytokines; Eosinophils; Epoprostenol; Female; Genotype; Hydroxyproline; Hyperplasia; Immunoglobulin E; Interferon-gamma; Interleukin-13; Interleukin-4; Interleukin-5; Leukocytes; Lymphocytes; Mice; Mice, Inbred C57BL; Mice, Knockout; Ovalbumin; Th2 Cells; Time Factors

There is great potential for novel vaccines based on recombinant proteins and synthetic peptides. Unfortunately these antigens often lack the immunogenicity of whole, killed pathogens used in traditional vaccines. Thus there is strong interest in the identification of immunological adjuvants with low reactogenicity, but high potency, to enhance immune responses and realize the potential of these new vaccine strategies. CD40 antibodies have been shown to have adjuvant effects when administered at very high doses. These large doses are impractical and induce a cascade of cytokine release giving rise to septic shock-like symptoms, as well as splenomegaly and polyclonal antibody production. We show here that a very small amount of CD40 antibody can exhibit potent adjuvant effects when attached to soluble antigen. The lack of detectable systemic effects indicates that this method may be a powerful and practical means of enhancing the efficacy of recombinant vaccines.

Topics: Adjuvants, Immunologic; Alum Compounds; Animals; Antibodies, Monoclonal; Antigen-Antibody Complex; CD40 Antigens; Dose-Response Relationship, Immunologic; Female; Immunization; Mice; Mice, Inbred BALB C; Ovalbumin; Vaccines, Synthetic

Unfractionated GPI-0100 (UFGPI-0100) containing semi-synthetic derivatives of deacylated Quillaja saponins (DS saponins) modified at the glucuronic acid residue was resolved by reverse phase low-pressure liquid chromatography (RP-LPLC) into two fractions, RP18-1 and RP18-2, with different compositions and adjuvanticity. The fraction RP18-1 contained DS saponin adducts of N-dicyclohexylurea, and stimulated Th2 immunity with production of IgG1, while the RP18-2 fraction contained the dodecylamide derivatives of DS saponins and stimulated Th1 immunity with production of IgG2a, IFN-gamma, IL-2, and CTL. The strong immune stimulatory properties of RP18-2, relative to RP18-1, and the formation of RP18-1/RP18-2 mixed micelles may account for the effective stimulation of Th1 immunity by UFGPI-0100. UFGPI-0100 was free of acylated quillaja saponin components, including the more stable QS-7.

Topics: Adjuvants, Immunologic; Alum Compounds; Animals; Antigens; Carbohydrate Sequence; Cell Line; Chromatography, Liquid; Enzyme-Linked Immunosorbent Assay; Female; Immunity, Cellular; Immunoglobulin G; Indicators and Reagents; Interferon-gamma; Interleukin-2; Mass Spectrometry; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Molecular Sequence Data; Ovalbumin; Quillaja; Saponins; Th1 Cells

Control of IgE Ab production is important for the prevention of IgE-related diseases. However, in contrast to the existing information on the induction of IgE production, little is known about the regulation of the production of this isotype, with the exception of the well-documented mechanism involving T cell subsets and their cytokine products. In this study, we demonstrate an alternative approach to interfere with the production of IgE, independent of the activity of T cells, which was discovered during the course of an investigation intended to clarify the mechanism of IgE-selective unresponsiveness induced by surface-coupled liposomal Ags. Immunization of mice with OVA-liposome conjugates induced IgE-selective unresponsiveness without apparent Th1 polarization. Neither IL-12, IL-10, nor CD8(+) T cells participated in the regulation. Furthermore, CD4(+) T cells of mice immunized with OVA-liposome were capable of inducing Ag-specific IgE synthesis in athymic nude mice immunized with alum-adsorbed OVA. In contrast, immunization of the recipient mice with OVA-liposome did not induce anti-OVA IgE production, even when CD4(+) T cells of mice immunized with alum-adsorbed OVA were transferred. In the secondary immune response, OVA-liposome enhanced anti-OVA IgG Ab production, but it did not enhance ongoing IgE production, suggesting that the IgE-selective unresponsiveness induced by the liposomal Ag involved direct effects on IgE, but not IgG switching in vivo. These results suggest the existence of an alternative mechanism not involving T cells in the regulation of IgE synthesis.

Topics: Adjuvants, Immunologic; Adoptive Transfer; Alum Compounds; Animals; Antibodies, Monoclonal; Antigens, Surface; CD4-Positive T-Lymphocytes; Cells, Cultured; Cytokines; Female; Freund's Adjuvant; Immunization, Secondary; Immunoglobulin E; Injections, Intraperitoneal; Interleukin-12; Liposomes; Mice; Mice, Inbred BALB C; Mice, Knockout; Mice, Nude; Ovalbumin; Spleen; T-Lymphocyte Subsets

We have previously reported that intraperitoneal injection with OVA-liposome conjugates induces OVA-specific and IgE-selective unresponsiveness in mice.. In the present study, the effects of oral pre-treatment with OVA-liposome conjugates or with plain OVA solution on anti-OVA IgG antibody production were investigated in mice after subsequent immunization with alum-adsorbed OVA. Control mice received only the immunization.. The levels of serum anti-OVA IgG antibody in mice receiving oral administration of OVA-liposome were comparable to those in the control mice. However, in mice receiving oral administration of the same dose of plain OVA, levels of serum anti-OVA IgG antibody were significantly lower than those in control mice. Surprisingly, anti-OVA IgE antibody production was completely inhibited in mice receiving oral administration of OVA-liposome conjugates. Splenic CD4(+) T cells of mice receiving oral administration of OVA-liposome and those of control mice produced comparable levels of cytokines, while those of mice receiving oral administration of plain OVA solution produced significantly lower levels of cytokines than those in the other two groups.. Orally administered OVA-liposome did not affect anti-OVA IgG production but did inhibit anti-OVA IgE antibody production, while orally administered OVA solution inhibited production of both IgG and IgE antibodies. These results suggest that antigen-liposome conjugates can possibly be orally administered in order to control antigen-specific IgE antibody production, without affecting IgG antibody production.

Topics: Administration, Oral; Allergens; Alum Compounds; Animals; Antibody Specificity; Cytokines; Female; Immunization Schedule; Immunoglobulin E; Immunoglobulin G; Liposomes; Mice; Mice, Inbred BALB C; Ovalbumin

Exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) induces immunosuppression in humans and animals. However, the effect of TCDD on Th2-type immune responses such as allergic reactions has been unclear. Using NC/Nga mice that developed atopic dermatitis-like skin lesions with marked elevation in plasma of total IgE when bred under conventional conditions, we investigated the effects of a single oral dose of TCDD on immune responses. NC/Nga mice received a single oral dose (0 or 20 microg/kg body weight) of TCDD. On day 7, treatment with TCDD alone decreased the cellularity of thymus. However, treatment with TCDD modified the cellularity of spleens and mesenteric lymph nodes (MLNs) but not of the thymus on day 28. When NC/Nga mice received ip immunization with OVA and alum on the same day as the TCDD treatment (0, 5, or 20 microg/kg body weight), TCDD markedly suppressed the concentrations of Th2-type cytokines (e.g., IL-4 and IL-5) in culture supernatants of spleen cells, whereas IFN-gamma production significantly increased. TCDD exposure reduced anti-OVA and total IgE antibody titers in plasma and did not induce the development of atopic dermatitis-like lesions in the pinnae or dorsal skin of NC/Nga mice. These results suggest that in NC/Nga mice, exposure to TCDD may impair the induction of Th2-type immune responses.

Topics: Administration, Oral; Alum Compounds; Animals; Cell Division; Cytokines; Environmental Pollutants; Flow Cytometry; Immunization; Immunoglobulin E; Immunoglobulin G; Lymph Nodes; Male; Mice; Mice, Inbred Strains; Organ Size; Ovalbumin; Polychlorinated Dibenzodioxins; Skin; Spleen; Th1 Cells; Th2 Cells; Thymus Gland

Adjuvants are compounds that, when combined with an antigen, potentiate an immune response in an immunized species. There are numerous pathogens for which there are no protective vaccines and since alum is the only adjuvant licensed for use in humans, there is a clear need for more effective adjuvant preparations. In this study we describe the immunopotentiating properties of three novel molecular aggregate formulations based on tomatine (RAM1), a glycosylamide lipid (RAM2) and a fifth generation dendrimeric polymer (RAM3) respectively. These formulations were evaluated for their ability to augment antigen-specific antibody responses when administered with a soluble protein antigen. All three adjuvants were shown to be nontoxic to mice and elicited antigen-specific antibody responses. Of the three formulations, RAM1 was found to induce the highest titers of antibody; these were substantially higher than those induced by reference control adjuvants. RAM1 elicited antibodies of the IgG1 and IgG2a subclasses indicating, indirectly, that this adjuvant can stimulate Th2 and Th1 type immunity.

Topics: Adjuvants, Immunologic; Alum Compounds; Animals; Antibody Formation; Female; Freund's Adjuvant; Glycolipids; Immunoglobulin G; Immunoglobulin Isotypes; Immunoglobulin M; ISCOMs; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Ovalbumin; Tomatine

A previous study has shown that Sm37-5 is a major B cell epitope of Sm37-GAPDH. This epitope is highly antigenic in human infections and IgG antibody reactivity toward this determinant is associated with adolescent resistance to reinfection. This led us to test a synthetic peptide corresponding to Sm37-5, coupled to ovalbumin, as an anti-schistosome vaccine. Although mice injected with Sm37-5-OVA in Freund's adjuvant showed significant protection, immunization in aluminium hydroxide failed to induce protection. The adjuvant effect of cytokines (GM-CSF or IL-12) associated with the antigen on alum was investigated. With each of these two cytokines, significant reductions in the worm burden were obtained (32-38% with GM-CSF and 27% with IL-12, respectively). In addition, a reduction of the egg number trapped in the liver of immunized mice was also observed. Thus, protections were obtained with formulations that could potentially be used in humans.

Topics: Adjuvants, Immunologic; Adsorption; Alum Compounds; Animals; Antibodies, Helminth; Antigens, Helminth; Epitopes, B-Lymphocyte; Female; Glyceraldehyde-3-Phosphate Dehydrogenases; Granulocyte-Macrophage Colony-Stimulating Factor; Immunity, Innate; Immunoglobulin G; Injections, Intramuscular; Interleukin-12; Mice; Mice, Inbred CBA; Molecular Weight; Ovalbumin; Schistosoma mansoni; Schistosomiasis mansoni

In the following study, we demonstrate that medium responder PVG rats immunized i.p. with OVA complexed to the adjuvant aluminum hydroxide exhibit a moderate IgE response (400-1000 ng/ml). In these rats, we demonstrate that underlying the MHC class II-restricted CD4+ T cell response, there is an MHC class I-restricted CD8+ T cell component that plays an important role in restricting the magnitude and duration of the IgE response. We show that in vivo depletion of CD8+ T cells effects a massive increase in IgE (20-fold), and that they are MHC class I-restricted, OVA-specific, cytolytic cells that universally produce IFN-gamma (25-69 ng/ml) and IL-2 (7.6-22 U/ml), and occasionally secrete IL-4 (68-81 U/ml IL-4), and when adoptively transferred into CD8-depleted recipients, can effect a significant reduction in IgE (3- to 50-fold). We also demonstrate that this in vivo inhibition of IgE is dependent on the Ag-specific activation of the CD8+ T cells, and that the activated CD8+ T cells will suppress total/bystander IgE in an Ag-nonspecific manner. These data are consistent with a growing literature demonstrating sensitization of MHC class I-restricted CD8+ T cells by exogenous protein Ags delivered to mucosal sites, and may represent a mechanism whereby a selective pressure can be applied on the functional outcome of an immunoglobulin response to environmental allergens.

Topics: Adoptive Transfer; Alum Compounds; Animals; Antibodies, Blocking; Antibodies, Monoclonal; Antigen-Presenting Cells; CD3 Complex; CD4 Antigens; CD8 Antigens; Cells, Cultured; Clone Cells; Cytotoxicity, Immunologic; Drug Combinations; Epitopes; Fatty Acids, Monounsaturated; Female; Fluorescent Dyes; Hemocyanins; Histocompatibility Antigens Class I; Histocompatibility Antigens Class II; Immunoglobulin E; Interferon-gamma; Interleukin-2; Interleukin-4; Lymph Nodes; Lymphocyte Activation; Lymphocyte Count; Lymphocyte Depletion; Mollusca; Ovalbumin; Quaternary Ammonium Compounds; Rats; Rats, Inbred Strains; Receptors, Antigen, T-Cell, alpha-beta; Receptors, Antigen, T-Cell, gamma-delta; Stem Cells; T-Lymphocyte Subsets; T-Lymphocytes, Regulatory; Trypsin

Ovalbumin (OVA) was coupled with murine red blood cells (MRBC) using glutaraldehyde. The OVA-MRBC conjugate induced anti-OVA IgG antibody in mice at almost the same level as OVA in alum. However, no IgE antibody production specific for OVA was observed in OVA-MRBC-injected mice. A significant increase in IGG2a production was obtained with OVA-MRBC immunization, whereas the production of IgG1 predominated in OVA in alum immunization. Am OVA-liposome conjugate induced IgE-specific unresponsiveness in mice in the same manner as OVA-MRBC. Similar results were obtained when antigens other than OVA, such as tetanus toxoid or diphtheria toxoid, were coupled to liposome. These results show the potential of antigen-liposome conjugates for the development of vaccine that induces sufficient IgG antibody production without IgE synthesis.

Topics: Alum Compounds; Animals; Erythrocytes; Female; Immunization; Immunoglobulin E; Immunoglobulin G; Liposomes; Mice; Mice, Inbred BALB C; Ovalbumin; Rats; Rats, Sprague-Dawley

Allergic reactions to food are characterized by enhanced allergen-specific IgE serum levels and the activation of intestinal mast cells. Here we describe a murine model for the onset of food allergy and the role of cytokines in the regulation of food-induced IgE responses.. Mice were primed systemically with low doses of alum-precipitated ovalbumin. Subsequent intragastric challenge led to enhanced sensitization.. Compared with baseline ovalbumin-specific IgE levels before challenge (0.23 +/- 0.06 optical density [OD] units), ovalbumin-challenged mice showed significantly elevated IgE levels (0.86 +/- 0.23 OD units) after intragastric challenge, which were not observed in control animals (0.29 +/- 0.06 OD units). IgE levels mirrored intestinal mast cell activation, measured by decreased histamine levels in duodenal specimens, in ovalbumin-challenged mice (92.6 +/- 7.9 ng/0.1 gm tissue weight) but not in saline-challenged mice (135.4 +/- 18.3 ng/0.1 gm tissue weight), compared with baseline levels (141.1 +/- 4.1 ng/0.1 gm tissue weight). Changes in IgE and histamine levels after intragastric challenge could be blocked by treating the animals with neutralizing antibodies against IL-4 or IL-10. Although it is generally accepted that ingestion of food allergens leads to a state of immunologic unresponsiveness (i.e., oral tolerance), it is shown here that low-dose systemic priming followed by intragastric challenge leads to sensitization instead of unresponsiveness.. Our murine model shows an important correlation between TH2 cytokines, IgE production, and histamine release. Hence, this in vivo model provides a useful tool with which the complex mechanism underlying sensitization to food allergens can be studied.

Topics: Allergens; Alum Compounds; Animals; Antibody Specificity; Cytokines; Disease Models, Animal; Female; Food Hypersensitivity; Histamine Release; Immunization; Immunoglobulin E; Interleukin-10; Interleukin-4; Intestine, Small; Mast Cells; Mice; Mice, Inbred BALB C; Neutralization Tests; Ovalbumin

The role of interleukin (IL)-4 in the activity of two frequently used vaccine adjuvants, Freund's complete adjuvant (FCA) and the aluminum hydroxide gels (alum), was studied using the standard antigen ovalbumin (OVA) in IL-4 genedisrupted mice (IL-4 -/-). In the absence of adjuvant, there was an overall reduction in antibody production to OVA in IL-4 -/- mice and significantly greater amounts of interferon (IFN)-gamma were produced following restimulation of splenocytes with antigen in vitro compared with immunocompetent controls (IL-4 +/+). FCA and alum boosted the immune response to OVA in both IL-4 -/- and IL-4 +/+ mice. In IL-4 +/+ mice, while FCA stimulated a wide-spectrum immunoglobulin response, including both Th1-associated IgG2a and Th2-associated IgG1, alum enhanced only Th2 antibody production and no OVA-specific IgG2a could be detected. In IL-4-deficient mice, however, not only was IgG2a production increased in all adjuvant-treated groups, but alum was as potent at stimulating this antibody subclass as FCA. Similarly, increased production in vitro by splenocytes of the Th1 cytokine IFN-gamma, equivalent to that produced after inoculation with FCA/OVA, was only detected in IL-4 -/- mice inoculated with alum/OVA. There was no IgE production in IL-4 -/- mice and OVA-specific IgG1 production, although still at significant levels, was reduced compared with wild-type mice irrespective of the adjuvant used. However, although production of the Th2 cytokine IL-5 was totally inhibited in IL-4-deficient mice inoculated with FCA/OVA, there was no significant difference in IL-5 production between the two strains when alum was used as adjuvant.

Topics: Adjuvants, Immunologic; Alum Compounds; Animals; Cytokines; Female; Freund's Adjuvant; Immunoglobulin G; Interleukin-4; Mice; Mice, Inbred C57BL; Ovalbumin; Th1 Cells; Th2 Cells

Ovalbumin-loaded poly (D,L-lactide co-glycolide) [OVA-loaded PLG] microparticles, produced by emulsion/solvent evaporation stimulated the production of high serum IgG antibody levels after a single subcutaneous (s.c.) administration in mice and the duration of the immune response paralleled the degradation rate of the carrier. Formulations based on slow resorbing PLG maintained relatively constant peak antibody levels for 26 weeks and high titres for over 1 year at a level approximating the peak response to the faster resorbing, OVA-loaded particles which was of lower duration. Vaccine formulations prepared by simple mixing of blank PLG microparticles and OVA exhibited low primary immune responses which were only elevated by boosting. OVA-loaded PLG microparticles exhibited a substantial surface protein component amounting to ca 40% and 60% of the total protein loading for slow resorbing and fast resorbing PLG, respectively. These findings suggest that sustained presentation of surface protein to the immune system was a major factor in the induction and long-term maintenance of high antibody titres following a single s.c. administration of OVA-loaded microparticles.

Topics: Alum Compounds; Animals; Antibodies; Biocompatible Materials; Drug Carriers; Immunoglobulin G; Lactic Acid; Mice; Microspheres; Ovalbumin; Particle Size; Polyglycolic Acid; Polylactic Acid-Polyglycolic Acid Copolymer; Polymers; Vaccines

Six different immunisation regimes were used in an attempt to elevate the concentration of ovine IgE. The response was measured by a passive cutaneous anaphylaxis (PCA) test. These regimes included infection with Ascaris suum and the combination of infection and parenteral administration of Ascaris antigens and ovalbumin in adjuvants. The regime producing the highest titre of IgE was the combination of Ascaris infection with parenteral administration of Ascaris extract in Bordetella pertussis. This regime consistently produced a transient high titre (1:1280-1:2560) of serum IgE 9-12 days after immunisation. The response to ovalbumin was transient and the maximum PCA titre obtained was 1:10.

Topics: Administration, Oral; Alum Compounds; Animals; Antigens, Helminth; Ascariasis; Ascaris suum; Bacterial Vaccines; Body Fluids; Female; Immunization; Immunization Schedule; Immunoglobulin E; Ovalbumin; Ovum; Pertussis Vaccine; Sheep

The primary objectives of the present study were to produce poly(lactide-co-glycolide) (PLGA) microspheres with different diameters, to characterize these microspheres which were loaded with a model antigen, ovalbumin and to evaluate the effect of microsphere particle size on the serum antibody levels following administration to mice. Four kinds of ovalbumin-loaded PLGA microspheres with different diameters (1.2, 3.5, 7.0 and 14.3 microns as mean volume diameter) were manufactured by a w/o/w emulsion/solvent evaporation method. Low loading percent (0.08%-0.25% w/w) and efficiencies (8-25% w/w) were observed. Examination using scanning electron photomicrographs showed smooth spherical particles. The in-vitro release of ovalbumin from microspheres showed an expected burst release with all batches and the extent of the burst release increased with decreasing diameters of spheres; PLGA microspheres with the smallest diameter (1.2 microns) showed an 80% burst release within one day. Approximately 10-60% of ovalbumin remained unreleased 30 days later. The single subcutaneous administrations of ovalbumin-loaded PLGA microspheres with different diameters to mice induced good antibody responses above ovalbumin saline negative controls at 3, 6, 9, and 12 weeks after inoculation. Especially, 0.16% ovalbumin-loaded PLGA microspheres having mean volume diameter of 3.5 microns exhibited the best immune responses with values greater than those obtained after inoculation with adjuvants such as complete Freund's adjuvant or alum as positive control. The strong adjuvant activity of PLGA microspheres as vaccine formulation was suggested.

Topics: Alum Compounds; Animals; Antibody Formation; Drug Delivery Systems; Enzyme-Linked Immunosorbent Assay; Female; Freund's Adjuvant; In Vitro Techniques; Injections, Subcutaneous; Lactic Acid; Mice; Mice, Inbred BALB C; Microscopy, Electron, Scanning; Microspheres; Ovalbumin; Particle Size; Polyglycolic Acid; Polylactic Acid-Polyglycolic Acid Copolymer; Polymers; Vaccines, Synthetic

Adjuvant effect of liposomes was compared to that of aluminium hydroxide (Alum) using ovalbumin (OVA) as a model antigen, and the difference in adjuvanticity of liposomes from Alum has been evaluated on the basis of cytokine production. Both adjuvants enhanced the IgG levels, but a remarkable difference was observed in the production of IgG subclass; Alum enhanced IgG1 levels, and liposomes enhanced IgG2a, IG2b, and IG3 levels. Further, Alum enhanced antigen-specific IgE levels, whereas liposomes did not. To clarify the difference in adjuvant effect of these adjuvants, secretion of cytokines, especially interferon-gamma (IFN-gamma) and interleukin-4 (IL-4) which regulate IgE production, was estimated ex vivo. Overnight culture of splenic cells obtained from mice immunized with liposomes encapsulating OVA elicits IFN-gamma secretion, but not IL-4 secretion. On the other hand, the production of both cytokines was elevated by the immunization with OVA-Alum complex. The results indicate that the difference of adjuvant activity between liposomes and Alum may come from the difference in the secretion of IL-4 and that, consequently, a different class of antibody response is developed. More importantly, negatively charged liposomes containing phosphatidylserine remarkably promoted IFN-gamma secretion compared to neutral liposomes.

Topics: Adjuvants, Immunologic; Alum Compounds; Animals; Electrochemistry; Immunoglobulin E; Immunoglobulin G; Interferon Inducers; Interferon-gamma; Interleukin-4; Liposomes; Male; Mice; Mice, Inbred BALB C; Ovalbumin

Ovalbumin (OVA) was coupled with murine red blood cells (MRBC) using glutaraldehyde. OVA-MRBC conjugate induced anti-OVA IgG antibody production in mice at almost the same level as OVA in alum. However, no IgE antibody production specific for OVA was observed in OVA-MRBC-injected mice. IgE-specific unresponsiveness was also observed in mice injected with OVA coupled with sheep red blood cells (OVA-SRBC), suggesting that our present observation was not restricted to the property of MRBC. These results show the potential ability of antigen-RBC conjugate for the development of vaccine that induces sufficient IgG antibody production without IgE synthesis.

Topics: Alum Compounds; Animals; Erythrocytes; Female; Immunization; Immunoglobulin E; Immunoglobulin G; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Ovalbumin; Rats; Rats, Sprague-Dawley

Groups of BALB/c mice were sham infected or inoculated intranasally (IN) with live RSV. From Day 4 to 8 after infection, the animals were exposed IN to ovalbumin (OVA) with or without alum adjuvant. At different intervals, levels of OVA concentration in serum, IgG-anti-OVA antibody activity in serum, and IgA-anti-OVA antibody activity in bronchial washings were determined, employing the ELISA technique. IgE-anti-OVA antibody titers in serum and bronchial washings were assessed by PCA. OVA concentrations in serum were significantly higher in RSV-infected animals compared to uninfected controls. The use of alum adjuvant also increased OVA uptake in uninfected animals but to a lesser extent than RSV infection. RSV-infected animals developed significantly higher OVA-specific antibody titers of IgG isotype in serum and IgA isotype in bronchial washings than the uninfected controls, while alum enhanced the immune response less markedly but still significantly in uninfected mice. An IgE antibody response to OVA in serum was demonstrable in 50% of RSV-infected mice immunized IN with OVA and alum, while all uninfected animals and RSV-infected animals immunized with OVA alone (without adjuvant) failed to develop a detectable IgE response. These findings suggest that infections with viral agents such as RSV may function as adjuvants for other antigens inhaled during acute respiratory infection. These observations may explain the alterations in the immune response to other antigens in patients with acute viral-induced bronchopulmonary diseases.

Topics: Adjuvants, Immunologic; Administration, Inhalation; Alum Compounds; Aluminum; Animals; Antigens; Bronchoalveolar Lavage Fluid; Immunoglobulin A; Immunoglobulin E; Immunoglobulin G; Lung; Male; Mice; Mice, Inbred BALB C; Ovalbumin; Respiratory Syncytial Viruses; Respiratory Tract Infections; Respirovirus Infections; Sulfates

High IgE responder rats were sensitized intraperitoneally with alum-adsorbed ovalbumin and Bordetella pertussis organisms. At day 0, 7, 14, 21 and 28 following sensitization the peritoneal cells were harvested and further processed for light and immunoelectron microscopical investigations using a specific anti-rat IgE immunogold sandwich method. The results obtained show that the number of peritoneal mast cells increase significantly during the course of sensitization. There is a time-dependent increase in the amount of immunogold particles on mast cell surfaces together with a shift of particle distribution towards the surface folds. Sensitized mast cells exhibit altered releasability as is indicated by slightly degranulated cells at day 21 and day 28 postsensitization. Additionally, about 25% of small lymphocytes which had entered the peritoneal cavity between day 7 and day 14 are heavily stained with the immunogold complex between day 14 and day 28 postsensitization. This is not so for macrophages (less than or equal to 0.2% positive cells) nor for eosinophils which do not show any staining activity at all despite they are present in high numbers.

Topics: Adsorption; Alum Compounds; Aluminum; Animals; Bordetella pertussis; Immunization; Immunoglobulin E; Inflammation; Leukocytes; Ovalbumin; Peritoneal Cavity; Rats; Rats, Inbred BN; Sulfates; Time Factors

We examined the effects of glucocorticosteroids (GCS) on antigen-induced bronchial anaphylactic reactions (BAR) in SD rats immunized with ovalbumin (OA) and alum. The animals were treated with vehicle, budesonide (BUD), dexamethasone (DEX), or hydrocortisone (HC) at various times before intravenous (i.v.) antigen challenge. The drugs were administered either intraperitoneally (i.p.) or intratracheally (i.t.); the BAR was elicited by a low or by a high challenge dose of antigen. A BAR elicited by a low challenge dose of antigen was reduced in a dose-dependent way by all GCS after i.p. administration; at 1 mg/kg, BUD and DEX significantly reduced BAR and at 50 mg/kg all three of the examined compounds inhibited the BAR by 50% or more. For BUD, maximum effect was recorded when it was given 12 h before test. There was only a slight variation in the inhibitory effects of the GCS with immunization conditions of test animals. I.t. instillation of the drugs did not markedly increase their inhibitory capacity as compared to i.p. administration. BAR elicited by a high antigen dose was at best marginally affected by the GCS when given either i.p. or i.t. Thus, antigen-induced airway reactivity in rats can be reduced by GCS treatment provided that this is performed sufficiently long before the test and that the challenge dose of antigen is not too high.

Topics: Acetophenones; Administration, Topical; Alum Compounds; Aluminum; Anaphylaxis; Animals; Antigens; Bronchial Diseases; Budesonide; Cromolyn Sodium; Dexamethasone; Dose-Response Relationship, Drug; Drug Interactions; Glucocorticoids; Immunization; Injections, Intraperitoneal; Injections, Intravenous; Male; Ovalbumin; Pregnenediones; Quinacrine; Rats; Rats, Inbred Strains; Respiration; Sulfates; Time Factors

The adjuvant effects of aluminum silicate on IgE and IgG1 antibody production were investigated. BALB/c mice were immunized intraperitoneally with 10 micrograms ovalbumin (OA) adsorbed on 0.2, 2, or 20 mg aluminum silicate. The enhancement of anti-OA IgE antibody production was observed in the mice injected with aluminum silicate and antigen compared with the mice injected with antigen alone. Anti-OA IgE antibody production with 2 and 20 mg aluminum silicate was greater than that with aluminum hydroxide (alum) as an adjuvant. Similar adjuvant effects with 2 mg aluminum silicate or alum were observed in AKR and C57BL/6 mice using 10 micrograms OA, and in BALB/c mice using 2 micrograms DNP-KLH (dinitrophenyl keyhole limpet hemocyanin): IgE antibody production induced by aluminum silicate adjuvant persisted for weeks in these experiments. The enhancement of IgG1 antibody production to OA mixed with aluminum silicate was also demonstrated. However, no difference between aluminum silicate and alum was observed on the IgG1 antibody production.

Topics: Adjuvants, Immunologic; Adsorption; Alum Compounds; Aluminum; Aluminum Silicates; Animals; Antibody Formation; Dinitrobenzenes; Hemocyanins; Immunoglobulin E; Immunoglobulin G; Mice; Mice, Inbred AKR; Mice, Inbred BALB C; Mice, Inbred C57BL; Ovalbumin; Rats; Rats, Inbred Strains; Sulfates

Topics: Alum Compounds; Animals; Cattle; Injections, Intramuscular; Ovalbumin; Serum Albumin; Serum Albumin, Bovine

After subcutaneous injection of hen's ovalbumin or diphtheria toxoid precipitated with aluminum phosphate, the production of antibody, as judged by the presence in the tissues of antibody-containing cells, proceeds partly within the regional lymphatic glands and partly in the granulation tissue surrounding the nodule which develops at the site of injection. The first production of antibody takes place in the regional lymphatic gland and antibody production in the local granuloma becomes apparent only from 14 days onwards (rabbit). Antibody-containing plasma cells were demonstrated in the local granuloma up to 7 weeks. Antibody-containing cells in the regional lymphatic glands reach maximum numbers at 2 weeks following injection and decrease thereafter to few cells at 5 weeks. The adjuvant effect of the aluminum phosphate is interpreted as due partly to the delay in absorption of antigen from the local site of its injection which results in prolongation of stimulation of cells within the regional lymphatic glands, and partly to the production of a local granuloma which contains antibody-producing plasma cells.

Topics: Adjuvants, Immunologic; Alum Compounds; Animals; Antibodies; Antibody Formation; Antigens; Granuloma; Lymph Nodes; Ovalbumin; Rabbits