All experimental procedures were carried out in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals and were approved by the Institutional Animal Care and Utilization Committee for Medical Science of Chonbuk National University (CBU2014-00052). OVA-sensitized mice model was described by Park et al. [12 (link)]. Female 8-week-old BALB/c mice were obtained from Central Lab. Animal Inc. (Seoul, Korea) and immunized intraparenterally with 20 μg of ovalbumin (OVA, grade V, Sigma, St. Louis, MO) and 2 mg aluminum hydroxide (alum, Thermo Scientific, Cramlington, UK) gel suspended in 100 μL saline. On the 14th day, the mice were given an intraperitoneal booster injection of the same antigen or alum. All mice were randomly divided into five groups (n = 7) as follows: control, OVA-sensitization, OVA + 50 mg/kg/day SHHTE, OVA + 100 mg/kg/day SHHTE, and OVA + 0.5 mg/kg/day dexamethasone. SHHTE or dexamethasone was administered orally by sonde for a period of 13 days after 2nd sensitization and then blood was collected from the retroorbital plexus. For the measurement of ear thickness, intradermal 20 μL OVA (1 mg/mL) was injected in ear tissues on the final day. Ear thickness measurements were performed by thickness gage (Mitutoyo, Japan).
Aluminum hydroxide alum
Manufactured by Thermo Fisher Scientific
Sourced in United States
Aluminum hydroxide (alum) is a laboratory chemical compound with the molecular formula Al(OH)3. It is a white, crystalline solid that is commonly used as a coagulant and flocculant in water treatment processes. Alum has the core function of facilitating the removal of suspended particles and impurities from water or other aqueous solutions.
Automatically generated - may contain errors
Lab products found in correlation
Ovalbumin (ova), by Merck Group (2 mentions)
Laboratory rodent diet 5001, by Land O'Lakes (2 mentions)
Male fvb mice, by Charles River Laboratories (2 mentions)
Chicken ovalbumin ova, by Avantor (2 mentions)
Thickness gage, by Mitutoyo (1 mentions)
Poly 1 c, by Merck Group (1 mentions)
Ova grade 6, by Merck Group (1 mentions)
Ova grade 5, by Merck Group (1 mentions)
Mouse th1 th2 staining kit, by MultiSciences Biotech (1 mentions)
Anti inducible nitric oxide synthase, by Abcam (1 mentions)
Anti lamin b1, by Santa Cruz Biotechnology (1 mentions)
Phorbol 12 myristate 13 acetate, by Merck Group (1 mentions)
Ionomycin iono, by Merck Group (1 mentions)
Iptt 300, by Avidity Science (1 mentions)
Ovalbumin (ova), by Thermo Fisher Scientific (1 mentions)
12 protocols using aluminum hydroxide alum
1
Ovalbumin-Induced Allergic Mouse Model
2
BALB/c Mice Immunization Protocol
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In total, two batches of BALB/c mice (six- to eight-weeks-old females) were purchased from the Changchun Institute of Biological Products Co., Ltd. (Changchun, China) and immunized. Poly (I:C) (Sigma, USA), aluminum hydroxide (Alum; Thermo, USA), and Montanide ISA 201VG (ISA 201VG; Seppic, France) were purchased. All research was in compliance with the Welfare and Ethics of Laboratory Animals of China (GB 14925-2001), and protocols were approved by the Animal Welfare and Ethics Committee of the Veterinary Institute at the Academy of Military Medical Sciences (JSY-DW-2018-02).
In batch I, mice were randomly divided into 6 groups and immunized as shown inTable 2 . In batch II, mice were randomly divided into 3 groups and then vaccinated with 10 μg eGP-PA-GEM alone or with ISA 201VG plus Poly (I:C) compound adjuvant. In the two batches of animal experiments, all of the mice in the control group received both the same volume of PBS at the same time points. Immunizations were performed on study days 0 and 21. Blood samples were collected at two, four, and five weeks post immunization.
In batch I, mice were randomly divided into 6 groups and immunized as shown in
3
Evaluation of Nanoparticle Immunomodulatory Effects
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To evaluate the immunomodulatory effect of NPs, a protocol of mild allergic inflammation in the lung was used, as previously described [21 ,36 (link)]. Briefly, mice were sensitized by repetitive intraperitoneal injections of 1 μg ovalbumin (OVA) (grade VI; Sigma-Aldrich, St Louis, MO, USA) in PBS adsorbed to 2.5 mg aluminum hydroxide (alum) (Thermo Fisher Scientific, Waltham, MA, USA) on days 0, 7, 14, 28, and 42. Blood samples were taken before and after sensitization. OVA/alum-sensitized mice (S mice), compared to non-sensitized mice (NS mice), were characterized by high titers of OVA-specific immunoglobulin E (8.05 ± 1.64 vs. 0.1 ± 0.03 μg/mL). At day 52, mice were intratracheally instilled with 50 μg (1 mg/mL) of SiO2 NPs, or with their correspondent supernatant control (CTL). NP instillation was followed by OVA aerosol challenge for 20 min with 1% OVA in PBS that was delivered by a PARI BOY nebulizer (PARI GmbH, Starnberg, Germany). Five days after OVA challenge, lungs were snap frozen in liquid nitrogen and stored at −80 °C until use.
4
Evaluating Nanoparticle Immunomodulation in Allergic Lung Inflammation
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To evaluate the immunomodulatory effect of NPs, a protocol of mild allergic inflammation in the lung was used, as previously described (Figure 1A ).23 Briefly, mice were sensitized by repetitive intraperitoneal injections of 1 μg ovalbumin (OVA) (grade VI; Sigma-Aldrich, St Louis, MO, USA) in PBS adsorbed to 2.5 mg aluminum hydroxide (alum) (Thermo Fisher Scientific, Waltham, MA, USA) on days 0, 7, 14, 28, and 42. Blood samples were taken before and after sensitization. OVA/alum-sensitized mice (S mice), compared to non-sensitized mice (NS mice), were characterized by high titers of OVA-specific immuno-globulin E (8.05±1.64 versus 0.1±0.03 μg/mL). At day 52, mice were intratracheally instilled with 50 μg (1 mg/mL) of SiO2 NPs, or with their correspondent SUP (Figure 1B ). For dose-response studies, S mice were instilled with 12.5, 25, or 50 μg of SiO2 and SiO2-PEG NPs. NP instillation was followed by OVA aerosol challenge for 20 minutes with 1% OVA in PBS delivered by a PARI BOY nebulizer (PARI GmbH, Starnberg, Germany). Lung function, flow cytometry, and confocal analysis were performed on day 53. Bronchoalveolar lavage (BAL), histology, scanning electron microscopy (SEM), and messenger (m)RNA expression analysis were performed 5 days after OVA challenge (day 57).
5
Isolation of Eosinophil-Rich Splenocytes
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To collect activated splenocytes including high proportions of eosinophils (SPLhEos), donor mice (IL-5 Tg) were sensitized with three intraperitoneal injections of PBS or antigen: 50 µg OVA (grade V; Sigma, St. Louis, Missouri, USA), 10 µg Dermatophagoides farinae (Der f) house dust mite (HDM) allergen (Institute of Tokyo Environmental Allergy, Tokyo, Japan), or 50 µg Aspergillus (Institute of Tokyo Environmental Allergy, Tokyo, Japan) in 2 mg of aluminum hydroxide (Alum) (Thermo Fisher Scientific, Waltham, Massachusetts, USA) on Days 0, 7, and 14 (Figure 1 A). Donor mice were sacrificed at Day 15, and splenocytes were prepared. Cells were counted in a hemocytometer and using Diff Quik (Dade Behring AG, Dudingen, Switzerland) following cytospin (Thermo Shandon, Pittsburgh, PA, USA). The eosinophil percentage among splenocytes was greater than 50%. The character of splenocytes from IL-5 Tg mouse as determined by flow cytometric analysis is shown in Figure S1 .
6
Ovalbumin-Induced Allergic Response
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Ovalbumin (OVA, Grade V) was bought from Yuanye Bio-Technology Co., Ltd., Shanghai, China. Aluminum hydroxide (alum) was obtained from Thermo Fisher Scientific, Waltham, MA. All ELISA kits (OVA-specific IgE and IgG1; IL-4, IL-10; TNF-α) were purchased from Wuhan Fine Biotech Co., Ltd., Wuhan, China. The mouse Th1/Th2 staining kit was bought from Multi Sciences Biotech Co., Ltd., Hangzhou, China. The lotus-seed resistant starch was prepared as described in previous studies [11 (link)].
7
Influenza Virus Propagation and Antigen Preparation
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Influenza virus strains, including A/Korea/01/2009 (H1N1) and A/Philippines/2/1982 (H3N2) viruses, were grown in 10-day-old embryonic chicken eggs at 37ºC or in Madin-Darby Canine Kidney (MDCK) cells. After 48 h or after showing cytopathic effects, the allantoic fluid or cell culture supernatant was harvested and stored at −80ºC until use. Aluminum hydroxide (alum) and AddaVax® were purchased from Thermo Fisher Scientific (Waltham, MA, USA) and InvivoGen (San Diego, CA, USA), respectively.
8
Immune Regulation by PMA, Ionomycin, and OVA
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Phorbol 12-myristate 13-acetate (PMA; #P1585), ionomycin (Iono; #I0634), and OVA (#A5503) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Aluminum hydroxide (alum, #77161) was purchased from Thermo Fisher Scientific (Waltham, MA, USA) and C20 (#S6577) from Selleckchem (Houston, TX, USA). Antibodies against phospho(p)-PKCθ (#9377), p-STAT6 (#5654), STAT6 (#5397), p-NF-κB (#3033), and NFAT (#4389) were obtained from Cell Signaling Technology (Danvers, MA, USA), and anti-PKCθ (#sc-212), anti-β-actin (#sc-47778), and anti-lamin B1 (#sc-374015) antibodies from Santa Cruz Biotechnology (Dallas, TX, USA). Anti-GATA3 (#ab61052). Anti-inducible nitric oxide synthase (iNOS; #ab136918) antibodies were purchased from Abcam (Cambridge, UK).
9
OVA-Induced Anaphylaxis in Mice
10
Hydrogel-Delivered Allergen Immunotherapy in Mice
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Female C57BL6/J mice were divided into five groups (n = 10). The non-allergic and non-allergic–hydrogel groups represented non-allergic, non-sensitized control groups. The non-allergic–hydrogel group was injected with unloaded hydrogel to exclude any effects of hydrogel itself. The allergic group represented allergic, untreated individuals, whereas the AIT group included allergic mice that underwent the standard AIT treatment. Mice in the AIT–hydrogel group were allergic, and received AIT treatment delivered in hydrogel.
Allergic mice and mice of the AIT groups were sensitized via intraperitoneal (i.p.) injections on days 1, 7, and 21 with 30 µg of ovalbumin grade V (OVA) (Merck) and 2 mg of aluminum hydroxide (alum) (Thermo Fisher Scientific, Waltham, MA, USA) in 200 µL PBS. Non-allergic mice received injections of 2 mg alum in PBS (i.p.). After sensitization, the AIT group without hydrogel was treated via subcutaneous (s.c.) injections of 500 µg OVA in 200 µL PBS on days 35, 38, 41, and 44; the AIT group with hydrogel received 500 µg OVA in 200 µL hydrogel. Non-allergic mice were treated with 200 µL PBS or 200 µL hydrogel (s.c.), whereas allergic mice were treated with 200 µL PBS only. All mice were challenged with 1% nebulized OVA for 15 min on days 57, 60, and 63, and euthanized on day 64.
Allergic mice and mice of the AIT groups were sensitized via intraperitoneal (i.p.) injections on days 1, 7, and 21 with 30 µg of ovalbumin grade V (OVA) (Merck) and 2 mg of aluminum hydroxide (alum) (Thermo Fisher Scientific, Waltham, MA, USA) in 200 µL PBS. Non-allergic mice received injections of 2 mg alum in PBS (i.p.). After sensitization, the AIT group without hydrogel was treated via subcutaneous (s.c.) injections of 500 µg OVA in 200 µL PBS on days 35, 38, 41, and 44; the AIT group with hydrogel received 500 µg OVA in 200 µL hydrogel. Non-allergic mice were treated with 200 µL PBS or 200 µL hydrogel (s.c.), whereas allergic mice were treated with 200 µL PBS only. All mice were challenged with 1% nebulized OVA for 15 min on days 57, 60, and 63, and euthanized on day 64.
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