Liposome-mediated DNA immunisation via the subcutaneous route

Compared to naked DNA immunisation, entrapment of plasmid-based DNA vaccines into liposomes by the dehydration-rehydration method has shown to enhance both humoural and cell-mediated immune responses to encoded antigens administered by a variety of routes. In this paper, we have investigated the application of liposome-entrapped DNA and their cationic lipid composition on such potency after subcutaneous immunisation. Plasmid pI.18Sfi/NP containing the nucleoprotein (NP) gene of A/Sichuan/2/87 (H3N2) influenza virus in the pI.18 expression vector was incorporated by the dehydration-rehydration method into liposomes composed of 16 micromol egg phosphatidylcholine (PC), 8 micromoles dioleoyl phosphatidylethanolamine (DOPE) or cholesterol (Chol) and either the cationic lipid 1,2-diodeoyl-3-(trimethylammonium) propane (DOTAP) or cholesteryl 3-N-(dimethyl amino ethyl) carbamate (DC-Chol). This method, entailing mixing of small unilamellar vesicles (SUV) with DNA, followed by dehydration and rehydration, yielded incorporation values of 90-94% of the DNA used. Mixing or rehydration of preformed cationic liposomes with 100 microg plasmid DNA also led to similarly high complexation values (92-94%). In an attempt to establish differences in the nature of DNA association with these various liposome preparations their physico-chemical characteristics were investigated. Studies on vesicle size, zeta potential and gel electrophoresis in the presence of the anion sodium dodecyl sulphate (SDS) indicate that, under the conditions employed, formulation of liposomal DNA by the dehydration-rehydration generated submicron size liposomes incorporating most of the DNA in a manner that prevents DNA displacement through anion competition. The bilayer composition of these dehydration-rehydration vesicles (DRV(DNA)) can also further influence these physico-chemical characteristics with the presence of DOPE within the liposome bilayer resulting in a reduced vesicle zeta potential. Subcutaneous liposome-mediated DNA immunisation employing two DRV(DNA) formulations as well as naked DNA revealed that humoural responses (immunoglobulin total IgG, and subclasses IgG1 and 1gG2a) engendered by the plasmid encoded NP were substantially higher after dosing twice, 28 days apart with 10 microg liposome-entrapped DNA compared to naked DNA. At all time points measured, mice immunised with naked DNA showed no greater immune response compared to the control, non-immunised group. In contrast, as early as day 49, responses were significantly higher in mice injected with DNA entrapped in DRV liposomes containing DOTAP compared to the control group and mice immunised with naked DNA. By day 56, all total IgG responses from mice immunised with both DRV formulations were significantly higher. Comparison between the DRV formulations revealed no significant difference in immune responses elicited except at day 114, where the humoural responses of the group injected with liposomal formulation containing DC-Chol dropped to significantly lower levels that those measured in mice which received the DOTAP formulation. Similar results were found when the IgG1 and IgG2a subclass responses were determined. These results suggest that, not only can DNA be effectively entrapped within liposomes using the DRV method but that such DRV liposomes containing DNA may be a useful system for subcutaneous delivery of DNA vaccines.     

Surfactant vesicle-mediated delivery of DNA vaccines via the subcutaneous route

Compared to naked DNA immunisation, entrapment of plasmid-based DNA vaccines into liposomes by the dehydration-rehydration method has shown to enhance both humoural and cell-mediated immune responses to encoded antigens administered by a variety of routes. In this paper we have compared the potency of lipid-based and non-ionic surfactant based vesicle carrier systems for DNA vaccines after subcutaneous immunisation. Plasmid pI.18Sfi/NP containing the nucleoprotein (NP) gene of A/Sichuan/2/87 (H3N2) influenza virus in the pI.18 expression vector was incorporated by the dehydration-rehydration method into various vesicle formulations. The DRV method, entailing mixing of small unilamellar vesicles (SUV) with DNA, followed by dehydration and rehydration, yielded high DNA vaccine incorporation values (85-97% of the DNA used) in all formulations. Studies on vesicle size revealed lipid-based systems formed cationic submicron size vesicles whilst constructs containing a non-ionic surfactant had significantly large z-average diameters (>1500 nm). Subcutaneous vesicle-mediated DNA immunisation employing two DRV(DNA) formulations as well as naked DNA revealed that humoural responses (immunoglobulin total IgG, and subclasses IgG1 and 1gG2a) engendered by the plasmid encoded nucleoprotein were substantially higher after dosing twice, 28 days apart with 10 microg DRV-entrapped DNA compared to naked DNA. Comparison between the lipid and non-ionic based vesicle formulations revealed no significant difference in stimulated antibody production. These results suggest that, not only can DNA be effectively entrapped within a range of lipid and non-ionic based vesicle formulations using the DRV method but that such DRV vesicles containing DNA may be a useful system for subcutaneous delivery of DNA vaccines.     

Immunomodulatory effects of crotoxin isolated from Crotalus durissus terrificus venom in mice immunised with human serum albumin

Crotalus durissus terrificus venom and its main component, crotoxin (CTX), have the ability to down-modulate the immune system. Certain mechanisms mediated by cells and soluble factors of the immune system are responsible for the elimination of pathogenic molecules to ensure the specific protection against subsequent antigen contact. Accordingly, we evaluated the immunomodulatory effects of CTX on the immune response of mice that had been previously primed by immunisation with human serum albumin (HSA). CTX inoculation after HSA immunisation, along with complete Freund’s adjuvant (CFA) or Aluminium hydroxide (Alum) immunisation, was able to suppress anti-HSA IgG1 and IgG2a antibody production. We showed that the inhibitory effects of this toxin are not mediated by necrosis or apoptosis of any lymphoid cell population. Lower proliferation of T lymphocytes from mice immunised with HSA/CFA or HSA/Alum that received the toxin was observed in comparison to the mice that were only immunised. In conclusion, CTX is able to exert potent inhibitory effects on humoural and cellular responses induced by HSA immunisation, even when injected after an innate immune response has been initiated.     

Effect of sugars on storage stability of lyophilized liposome/DNA complexes with high transfection efficiency

Cationic lipid-based gene delivery systems have shown promise in transfecting cells in vitro and in vivo. However, liposome/DNA complexes tend to form aggregates after preparation. Lyophilization of these systems, therefore, has become of increasing interest. In this study, we investigated the feasibility of preserving complexes as a dried preparation using a modified dehydration rehydration vesicle (DRV) method as a convenient and reliable procedure. We also studied storage stability of a lyophilized novel cationic gene delivery system incorporating sucrose, isomaltose and isomaltotriose. Liposomes were composed of 3beta-[N-(N',N'-dimethylaminoethane)-carbamoyl] cholesterol (DC-Chol) and L-dioleoylphosphatidylethanolamine (DOPE), plus sucrose, isomaltose or isomaltotriose. Lyophilized liposome/DNA complexes were stored at -20, 25, 40 and 50 degrees C and their stability was followed for 50 days. Liposome/DNA complexes with sucrose could be stored even at 50 degrees C without large loss of transfection efficiency. The transfection efficiency of formulations stored at various temperatures indicated that the stabilizing effect of sugars on plasmid DNA was higher in the following order: isomaltotriose相似文献   

脂质体处方和制备方法对阿昔洛韦棕榈酸酯脂质体稳定性的影响

目的研究不同处方组成和制备方法对阿昔洛韦棕榈酸酯脂质体在4℃和25℃分别贮存90 d和180 d后的稳定性的影响。方法用卵磷脂(PC)/胆固醇(CH)/磷脂酰丝氨酸(PS),卵磷脂(PC)/胆固醇(CH)/硬脂酰胺(SA),卵磷脂(PC)/胆固醇(CH)/胆固醇硫酸酯(CS),神经酰胺(CM)/胆固醇(CH)/棕榈酸(PA)/胆固醇硫酸酯(CS),以薄膜分散法、逆向蒸发法和去水化/水化法,分别制备多室脂质体(MLV)、大单室脂质体(LUV)、去水化/水化脂质体(DRV),以平均粒径、渗漏率、pH值和Zeta电位4个指标考察不同贮存条件下脂质体的稳定性。结果脂质体稳定性顺序依次为,对不同脂质体处方:PC/CH/CS>CM/CH/PA/CS>PC/CH/PS>PC/CH/SA;对不同制备方法:LUV优于MLV和DRV;4℃时脂质体的稳定性优于25℃时的脂质体。结论脂质体的稳定性与制剂处方和制备方法密切相关。     

Effects of extrusion,lipid concentration and purity on physico-chemical and biological properties of cationic liposomes for gene vaccine applications

We developed cationic liposomes containing DNA through a conventional process involving steps of (i) preformation of liposomes, (ii) extrusion, (iii) drying and rehydration and (iv) DNA complexation. Owing to its high prophylactic potentiality against tuberculosis, which had already been demonstrated in preclinical assays, we introduced modifications into the conventional process towards getting a simpler and more economical process for further scale-up. Elimination of the extrusion step, increasing the lipid concentration (from 16 to 64?mM) of the preformed liposomes and using good manufacturing practice bulk lipids (96–98% purity) instead of analytical grade purity lipids (99.9–100%) were the modifications studied. The differences in the physico-chemical properties, such as average diameter, zeta potential, melting point and morphology of the liposomes prepared through the modified process, were not as significant for the biological properties, such as DNA loading on the cationic liposomes, and effective immune response in mice after immunisation as the control liposomes prepared through the conventional process. Beneficially, the modified process increased productivity by 22% and reduced the cost of raw material by 75%.     

Vesicular carrier constructs for topical immunisation

Topical immunisation represents a convenient and novel approach to vaccination. Skin is exploited as a route of immunisation because it shows both specific and non-specific immune responses against foreign invaders, and these responses are a result of the presence of immunocompetent cells within the skin layers. These skin-resident antigen-presenting cells are highly efficient for the initiation of humoural and cellular immune responses. Vesicular carrier systems, particularly liposomes, vesosomes, niosomes and transferosomes, have been advocated for the topical delivery of biomacromolecules. This review describes the potential and feasibility of vesicular carrier-based vaccine delivery for topical immunisation.     

Vesicular carrier constructs for topical immunisation

Topical immunisation represents a convenient and novel approach to vaccination. Skin is exploited as a route of immunisation because it shows both specific and non-specific immune responses against foreign invaders, and these responses are a result of the presence of immunocompetent cells within the skin layers. These skin-resident antigen-presenting cells are highly efficient for the initiation of humoural and cellular immune responses. Vesicular carrier systems, particularly liposomes, vesosomes, niosomes and transferosomes, have been advocated for the topical delivery of biomacromolecules. This review describes the potential and feasibility of vesicular carrier-based vaccine delivery for topical immunisation.     

脂质体介导的链霉菌染色体DNA转化原生质体的初步研究

在新霉素产生菌弗氏链霉菌中作了脂质体包埋染色体DNA转化的实验。对菌株9AS(Arg~-)作受体原生质体,9HC(His~-)作供体DNA来源。在PEG1000存在时,包有染色体DNA的脂质体转化频率较游离DNA高约1个数量级。整个转化过程是抗DNA酶的。本实验的可重复性较差,实验条件还有待进一步改进。     

Comparison of the depot effect and immunogenicity of liposomes based on dimethyldioctadecylammonium (DDA), 3β-[N-(N',N'-Dimethylaminoethane)carbomyl] cholesterol (DC-Chol), and 1,2-Dioleoyl-3-trimethylammonium propane (DOTAP): prolonged liposome retention mediates stronger Th1 responses

The immunostimulatory capacities of cationic liposomes are well-documented and are attributed both to inherent immunogenicity of the cationic lipid and more physical capacities such as the formation of antigen depots and antigen delivery. Very few studies have however been conducted comparing the immunostimulatory capacities of different cationic lipids. In the present study we therefore chose to investigate three of the most well-known cationic liposome-forming lipids as potential adjuvants for protein subunit vaccines. The ability of 3β-[N-(N',N'-dimethylaminoethane)carbomyl] cholesterol (DC-Chol), 1,2-dioleoyl-3-trimethylammonium propane (DOTAP), and dimethyldioctadecylammonium (DDA) liposomes incorporating immunomodulating trehalose dibehenate (TDB) to form an antigen depot at the site of injection (SOI) and to induce immunological recall responses against coadministered tuberculosis vaccine antigen Ag85B-ESAT-6 are reported. Furthermore, physical characterization of the liposomes is presented. Our results suggest that liposome composition plays an important role in vaccine retention at the SOI and the ability to enable the immune system to induce a vaccine specific recall response. While all three cationic liposomes facilitated increased antigen presentation by antigen presenting cells, the monocyte infiltration to the SOI and the production of IFN-γ upon antigen recall was markedly higher for DDA and DC-Chol based liposomes which exhibited a longer retention profile at the SOI. A long-term retention and slow release of liposome and vaccine antigen from the injection site hence appears to favor a stronger Th1 immune response.     

DC-Chol/DOPE cationic liposomes: A comparative study of the influence factors on plasmid pDNA and siRNA gene delivery

Cationic liposomes (CLs) composed of 3β-[N-(N′,N′-dimethylaminoethane) carbamoyl] cholesterol (DC-Chol) and dioleoylphosphatidylethanolamine (DOPE) (DC-Chol/DOPE liposomes) have been classified as one of the most efficient gene delivery systems. Our study aims to examine the effect of the molar ratio of DC-Chol/DOPE, PEGylation and serum on the pDNA (plasmid pDNA) and siRNA (small interfering RNA) transfection of DC-Chol/DOPE liposomes. The results showed that the most efficient DC-Chol/DOPE liposomes for pDNA or siRNA delivery were at a 1:2 or 1:1 molar ratio of DC-Chol/DOPE, respectively. The transfection efficiency of DC-Chol/DOPE liposomes increased along with increased weight ratio of DC-Chol/siRNA. However, the pDNA transfection efficiency decreased along with increased weight ratio of DC-Chol/pDNA from 3/1. As expected, PEGylation decreased siRNA and pDNA transfection efficiency of DC-Chol/DOPE liposomes. In PEGylated DC-Chol/DOPE liposomes, increased weight ratio of DC-Chol/pDNA from 3/1 did not lead to higher pDNA transfection efficiency, whereas increased weight ratio of DC-Chol/siRNA resulted in increased siRNA transfection efficiency. Furthermore, the serum did not significantly inhibit the pDNA and siRNA transfection efficiency of DC-Chol/DOPE liposomes. In conclusion, our results elucidated the influence factors of DC-Chol/DOPE liposome transfection and would reveal that siRNA and pDNA transfection mechanisms were different in DC-Chol/DOPE liposomes.     

The influence of lipid composition and lamellarity of liposomes on the physical stability of liposomes upon storage

The physical stability of the four liposomal systems was determined on storage at 4 and 25°C over a 6-month period. A correlation of the mean volume diameter, zeta potential and pH lead to the conclusion that stability follows the order of egg lecithin (PC)/cholesterol (CH)/stearylamine (SA) < PC/CH/phosphatidylserine (PS) < bovine brain ceramides (CM)/CH/palmitic acid (PA)/CS < PC/CH/cholesteryl sulphate (CS) at 4°C, as well as at 25°C, after a 6-month storage period. Large unilamellar vesicles (REV) proved to be superior to multilamellar liposomes (MLV) and dehydration/rehydration liposomes (DRV) systems as far as physical stability was concerned. Instability was exaggerated in the systems stored at 25°C as compared to storage at 4°C.     

Epidermal cell culture model with tight stratum corneum as a tool for dermal gene delivery studies

The purpose of this study was to evaluate the feasibility of organotypic cultures of rat epidermal cells as a tool to study non-invasive dermal gene delivery. Also, a novel transfection method employing liposomal pre-treatment of stratum corneum (SC) was evaluated. Rat epidermal cells were cultured on Transwell tissue culture inserts and formation of stratum corneum barrier was evaluated in permeability studies with two model compounds. Transfections were performed with naked pCMV-SEAP2 plasmid and 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP)/dioleyl-phosphatidylethanolamine (DOPE)/DNA lipoplexes. Naked DNA was administered on the stratum corneum of the cell culture model with or without prior treatment of the stratum corneum with DOTAP/DOPE liposomes. Transfection was evaluated non-invasively by monitoring concentrations of secreted alkaline phosphatase (SEAP) in the culture medium of the basolateral compartment at 24-h intervals. Transfection with lipoplexes produced significant gene expression in rat epidermal keratinocyte (REK) epidermal culture model. Likewise, delivery of naked DNA on stratum corneum after DOTAP/DOPE liposome pre-treatment produced gene expression. Naked DNA alone did not result in detectable gene expression. In dermal gene delivery studies REK epidermal culture model is a suitable tool that includes tight stratum corneum and allows transgene expression in viable epidermis and non-invasive sampling of secreted gene product in the basolateral compartment. Liposomal pre-treatment of the stratum corneum augments transfection of viable epidermis.     

Predicting the influence of liposomal lipid composition on liposome size,zeta potential and liposome-induced dendritic cell maturation using a design of experiments approach

In this study, the effect of liposomal lipid composition on the physicochemical characteristics and adjuvanticity of liposomes was investigated. Using a design of experiments (DoE) approach, peptide-containing liposomes containing various lipids (EPC, DOPE, DOTAP and DC-Chol) and peptide concentrations were formulated. Liposome size and zeta potential were determined for each formulation. Moreover, the adjuvanticity of the liposomes was assessed in an in vitro dendritic cell (DC) model, by quantifying the expression of DC maturation markers CD40, CD80, CD83 and CD86. The acquired data of these liposome characteristics were successfully fitted with regression models, and response contour plots were generated for each response factor. These models were applied to predict a lipid composition that resulted in a liposome with a target zeta potential. Subsequently, the expression of the DC maturation factors for this lipid composition was predicted and tested in vitro; the acquired maturation responses corresponded well with the predicted ones. These results show that a DoE approach can be used to screen various lipids and lipid compositions, and to predict their impact on liposome size, charge and adjuvanticity. Using such an approach may accelerate the formulation development of liposomal vaccine adjuvants.     

靶向肿瘤新生血管的阿霉素阳离子脂质体的体外研究

本研究采用3β-［N-(N′,N′-二甲基胺乙基)胺基甲酰胺基］胆固醇(DC-Chol)和二棕榈酰磷脂酰胆碱为脂材制备了各种DC-Chol含量不同的阿霉素阳离子脂质体，考察了阿霉素阳离子脂质体的体外性质，同时以大鼠主动脉内皮细胞为模型，考察它们对不同阳离子脂质体的摄取情况，并采用静脉注射FITC-Dextran(M_r 500 000)标记体内肿瘤新生血管，为体内靶向肿瘤血管提供依据。结果表明阿霉素阳离子脂质体包封率均在90%以上，粒径在100~200 nm。随着DC-Chol含量的增加，zeta电位升高，但PEG的加入会降低zeta电位。DC-Chol含量的增加会增大阿霉素的释放量，同时也促进脂质体被内皮细胞的摄取，加快摄取速度。因此在进行体内靶向肿瘤血管考察时应充分关注这些体外实验结果。FITC-Dextran标记法可以显影体内新生血管，为体内肿瘤血管靶向实验提供直观的观察方法。     

Induction of protective and specific antibodies against cocaine by intranasal immunisation using a glyceride adjuvant

The goal of this study was to investigate an intranasal cocaine vaccine containing the mucosal adjuvant macrogol-6-glycerol capylocaprate (RhinoVax). Cocaine-KLH conjugate was prepared and administered in two formulations. Ten mice were immunised intranasally using RhinoVax as adjuvant and ten subcutaneously using aluminium hydroxide as an adjuvant. A negative control group (n=10) received unconjugated KLH with RhinoVax intranasally. Specific cocaine antibodies in serum were measured following primary and booster immunisation. Relative antibody responses in serum indicated that the immunisation was successful. Animals were then challenged with cocaine either intranasally or intraperitoneally with subsequent measurement of drug distribution into the serum, brain and olfactory bulb. The cocaine-immunised groups revealed significantly lower cocaine levels in the brain compared to the negative control group. The inhibition of cocaine distribution to the brain in the intranasal immunised group was comparable to that of the subcutaneous immunised group. This was unexpected because the cocaine specific antibody levels in serum were fivefold lower in the intranasal immunised group. However, the presence of mucosal cocaine specific antibodies after intranasal immunisation could play an important role in hindering direct access of cocaine into the brain via the olfactory bulb.     

Physicochemical and pharmacokinetic characteristics of cationic liposomes

After intravenous administration of plasmid DNA (pDNA)/cationic liposome complexes, the gene expression is predominantly observed in the lung due to the physicochemical properties of the liposome complexes and the physiology of the lung. To determine the physicochemical properties and distribution behavior of cationic liposomes for lung-selective drug and/or gene delivery systems, N-[1-(2,3-dioleyloxy)propyl]-n,n,n-trimethylammonium chloride (DOTMA)/cholesterol and 1,2-dioleoyl-3-trimethyl-ammoniopropane (DOTAP)/cholesterol liposomes were studied. The particle sizes of DOTMA/cholesterol and DOTAP/cholesterol liposomes were very similar: 126 and 128 nm, respectively. Furthermore, the zeta potentials of these two liposomes were 51 and 66 mV, respectively. After intravenous injection into mice, both cationic liposomes were rapidly eliminated from the blood circulation and preferentially recovered in the lung. Interestingly, the highest lung accumulation was observed at 1 min, and then, decreased gradually. The distribution characteristics of DOTMA/cholesterol and DOTAP/cholesterol liposomes were almost identical due to the similarities in their physicochemical properties. These results demonstrated that DOTMA/cholesterol and DOTAP/cholesterol liposomes, which possess a positive charge, are promising carriers for lung-selective drug and/or gene delivery systems.     

Non-ionic surfactant based vesicles (niosomes) for non-invasive topical genetic immunization against hepatitis B

DNA vaccines are capable of eliciting both humoral as well as cellular immune responses. Liposomes have been widely employed for DNA delivery through topical route; however, they suffer from certain drawbacks like higher cost and instability. In present study, non-ionic surfactant based vesicles (niosomes) for topical DNA delivery have been developed. DNA encoding hepatitis B surface antigen (HBsAg) was encapsulated in niosomes. Niosomes composed of span 85 and cholesterol as constitutive lipids were prepared by reverse phase evaporation method. Prepared niosomes were characterized for their size, shape and entrapment efficiency. The immune stimulating activity was studied by measuring serum anti-HBsAg titer and cyokines level (IL-2 and IFN-gamma) following topical application of niosomes in Balb/c mice and results were compared with naked DNA and liposomes encapsulated DNA applied topically as well as naked DNA and pure recombinant HBsAg administered intramuscularly. It was observed that topical niosomes elicited a comparable serum antibody titer and endogenous cytokines levels as compared to intramuscular recombinant HBsAg and topical liposomes. The study signifies the potential of niosomes as DNA vaccine carriers for effective topical immunization. The proposed system is simple, stable and cost effective compared to liposomes.     

Cationic liposome (DC-Chol/DOPE=1:2) and a modified ethanol injection method to prepare liposomes, increased gene expression

Cationic liposomes composed of 3beta-[N-(N',N'-dimethylaminoethane)-carbamoyl] cholesterol (DC-Chol) and dioleoylphosphatidylethanolamine (DOPE) (DC-Chol/DOPE liposome, molar ratio, 1:1 or 3:2) prepared by the dry-film method have been often used as non-viral gene delivery vectors. The formulation and preparation of DC-Chol/DOPE liposomes, as well as the formation of their lipoplexes were investigated in an attempt to improve transfection efficiency in vitro. A more efficient transfection in medium with serum was achieved using DC-Chol/DOPE liposomes (molar ratio, 1:2) than those (3:2), and preparation method by a modified ethanol injection than the dry-film. The most efficient DC-Chol/DOPE liposome for gene transfer was molar ratio (1:2) and prepared by a modified ethanol injection method. The enhanced transfection might be related to an increase in the release of DNA in the cytoplasm by the large lipoplex during incubation in optiMEM, not to an increased cellular association with the lipoplex. The use of a modified ethanol injection method might enhance the role of DOPE that is aid in destabilization of the plasma membrane and/or endosome. These findings suggested that cationic liposomes rich in DOPE prepared by a modified ethanol injection method will help to improve the efficacy of liposome vector systems for gene delivery.     

Effects of simulated downwind coal combustion emissions on pre-existing allergic airway responses in mice

Context: Coal-fired power plant emissions can contribute a significant portion of the ambient air pollution in many parts of the world.

Objective: We hypothesized that exposure to simulated downwind coal combustion emissions (SDCCE) may exacerbate pre-existing allergic airway responses.

Methods: Mice were sensitized and challenged with ovalbumin (OVA). Parallel groups were sham-sensitized with saline. Mice were exposed 6?h/day for 3 days to air (control, C) or SDCCE containing particulate matter (PM) at low (L; 100 μg/m³), medium (M; 300 μg/m³), or high (H; 1000 μg/m³) concentrations, or to the H level with PM removed by filtration (high-filtered, HF). Immediately after SDCCE exposure, mice received another OVA challenge (pre-OVA protocol). In a second (post-OVA) protocol, mice were similarly sensitized but only challenged to OVA before air/SDCCE. Measurement of airway hyperresponsiveness (AHR), bronchoalveolar lavage (BAL), and blood collection were performed ~24?h after the last exposure.

Results: SDCCE significantly increased BAL macrophages and eosinophils in OVA-sensitized mice from the post-OVA protocol. However, there was no effect of SDCCE on BAL macrophages or eosinophils in OVA-sensitized mice from the pre-OVA protocol. BAL neutrophils were elevated following SDCCE in both protocols in nonsensitized mice. These changes were not altered by filtering out the PM. In the post-OVA protocol, SDCCE decreased OVA-specific IgG₁ in OVA-sensitized mice but increased levels of total IgE, OVA-specific IgE and OVA-specific IgG₁ and IgG_2a in non-sensitized animals. In the pre-OVA protocol, SDCCE increased OVA-specific IgE in both sensitized and non-sensitized animals. Additionally, BAL IL-4, IL-13, and IFN-γ levels were elevated in sensitized mice.

Conclusion: These results suggest that acute exposure to either the particulate or gaseous phase of SDCCE can exacerbate various features of allergic airway responses depending on the timing of exposure in relation to allergen challenge.