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Abstract |
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Materials and Methods
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The influences of alveolar macrophages (AM) and pulmonary surfactant on the induction of immune responses via the airways were assessed. Mice were depleted of their AM by intratracheal instillation of multilamellar vesicles containing dichloromethylene-diphosphonate followed by intratracheal instillation of a T cell-dependent antigen, trinitrophenyl-keyhole limpet hemocyanin, in vesicles of various compositions. The primary immune response was determined in the spleen of these animals using an ELI-Spot assay. The secondary immune responses in the sera of the mice were assessed using enzyme-linked immunosorbent assays. An immune response was detected in animals depleted of their AM and intratracheally instilled with antigen in small unilamellar vesicles consisting of either phosphatidylcholine cholesterol or surfactant lipids. Incorporation of surfactant protein (SP)-B in the antigen vesicles enhanced the immune response, whereas SP-A or SP-C in the antigen vesicle did not have an effect. Strikingly, intratracheal instillation of SP-B containing antigen vesicles can induce an immunoglobulin M immune response in mice without depletion of AM. These results indicate that SP-B containing vesicles can enhance the induction of immune responses via the airways and further illustrate the important roles of both AM and pulmonary surfactant in the pulmonary immune system.
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Introduction |
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Materials and Methods
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The lungs are constantly exposed to airborne particles, including pathogens and trivial antigens. To maintain normal lung function, tight control of the pulmonary defense system is required. One of the cells that plays a crucial role in the regulation of the pulmonary immune system is the alveolar macrophage. In addition to active phagocytosis of bacteria and viruses (1), alveolar macrophages are also involved in the suppression of specific immune responses in the lung (5). In line with an active suppression of the in vitro proliferation of mitogen- or antigen-stimulated T lymphocytes (5, 6), in vivo depletion of alveolar macrophages by intratracheal administration of vesicles containing dichloromethylene-diphosphonate (DMDP) led to the facilitation of immune responses in the local lung draining lymph nodes after intratracheal instillation of antigen (5).
Another important factor in the lung that regulates the immune system is pulmonary surfactant. Pulmonary surfactant lines the alveoli and is a complex mixture of lipids and proteins. One of its major functions is the prevention of alveolar collapse at end expiration. It comprises approximately 90% lipids and 10% proteins (7). Four surfactant-associated proteins have been described so far: surfactant protein (SP)-A, SP-B, SP-C, and SP-D. The hydrophobic proteins SP-B and SP-C have been implicated in the insertion of lipids into the lipid monolayer that covers the alveoli (7) and in the enhancement of the uptake of vesicles by alveolar type II cells (8). The hydrophilic proteins SP-A and SP-D are collectins and play a role in the innate defense system of the lung. SP-A was reported to enhance the phagocytosis of bacteria and viruses by alveolar macrophages (3, 4, 13), and SP-D was reported to act as a chemoattractant for neutrophils (14) and to enhance the antiviral defenses of neutrophils (15, 16). The surfactant lipids were reported to predominantly suppress the immune functions (17). Whether the hydrophobic surfactant proteins are also involved in the regulation of the pulmonary immune system is not known at present.
Particles entering the lung will come into contact with alveolar macrophages as well as with pulmonary surfactant. It seems that for a clear view on the regulation of the pulmonary immune system, both factors, i.e., alveolar macrophages and pulmonary surfactant, and their interactions need to be studied.
In the present study, the influence of alveolar macrophages and surfactant lipids and protein (SP-A, SP-B, and SP-C) containing vesicles on the induction of immune responses via the airways was assessed. A T cell-dependent antigen, trinitrophenyl-keyhole limpet hemocyanin (TNP-KLH), was entrapped in vesicles composed of lipids and various concentrations of the surfactant proteins. These vesicles were intratracheally instilled in mice that were either depleted of their alveolar macrophages or in control mice. Subsequently, the primary and secondary immune responses against the antigen in these mice were determined.
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Materials and Methods |
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Materials and Methods
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Animals
Young adult (8 to 12 wk of age, female) BALB/c mice were obtained from Harlan CPB (Zeist, The Netherlands).
Surfactant Proteins and Lipids
SP-A was isolated from the bronchoalveolar lavage fluid of patients with alveolar proteinosis as described (18). SP-A was dissolved in 5 mM N-2-hydroxyethylpiperazine-N'-ethane sulfonic acid (Hepes), pH 7.4 (2.2 mg/ml), and stored in small aliquots at
70°C. SP-B and SP-C were isolated from porcine lung lavage. Porcine lungs were obtained from the slaughterhouse and lavaged three to five times in a solution of 154 mM NaCl. Pulmonary surfactant was prepared from the bronchoalveolar lavage by
the method of Hawgood and coworkers (19). Lung surfactant was
extracted with 1-butanol (18). Butanol was dried by rotary evaporation, and the residue was dissolved in chloroform/methanol/
0.1 M HCl (1:1:0.05 vol/vol/vol). Insoluble material was removed
by centrifugation. SP-B and SP-C were separated from lipids and
purified to homogeneity by Sephadex LH-60 chromatography
(Pharmacia, Uppsala, Sweden) as described (20). The proteins were
stored in a mixture of chloroform/methanol (1:1 vol/vol) at 20°C.
The concentration of the proteins was determined by quantitative amino acid analysis. The LH-60 column fractions that contained only the surfactant lipids were dried by rotary evaporation. The lipids were dissolved in chloroform/methanol (1:1 vol/
vol). The phospholipid concentration was estimated by determining the phosphorus concentration as described by Bartlett (21).
Lipids
Dipalmitoylphosphatidylcholine, phosphatidylcholine (egg PC), phosphatidylglycerol (egg PG), and cholesterol were purchased from Avanti Polar Lipids (Alabaster, AL).
Alveolar Macrophage-Depleting Vesicles
A total of 86 mg egg PC and 8 mg cholesterol was dissolved in 10 ml chloroform. The chloroform was dried by rotary evaporation. The lipid film was suspended in either 4 ml phosphate-buffered saline (PBS) (control vesicles) or 10 ml DMDP in PBS (0.189 g/ml). The suspension was kept at room temperature for 2 h. Subsequently, it was sonicated for 3 min in a waterbath sonicator and kept at room temperature for 2 h. The vesicles containing DMDP were suspended in 100 ml PBS and centrifuged at 15,000 × g to remove free DMDP. The pellet was suspended in 4 ml PBS. The extent of depletion of alveolar macrophages by DMDP vesicles is 82 ± 12% (n = 6) as was determined 2 d after the instillaton of the DMDP vesicles by May-Grünwald-Giemsa staining of cells obtained by bronchoalveolar lavage.
Antigen-Containing Vesicles
The indicated concentrations of lipids were dissolved in the presence or absence of SP-B and/or SP-C in chloroform. The chloroform was dried by rotary evaporation. The lipid film was suspended in 10 ml of a solution of 0.8 mg TNP-KLH/ml PBS by vortexing. The suspension was kept at room temperature for 2 h. Subsequently, it was sonicated for 3 min in a waterbath sonicator and kept at room temperature for 2 h (multilamellar vesicles [MLV]). The small unilamellar vesicles (SUV) were prepared by additional sonification for 1 min on ice using an ultrasonic disintegrator. For the experiment depicted in Figure 2, free antigen was separated from vesicle-associated antigen. Therefore, the vesicle suspension was layered on 2 M sucrose solution in PBS and centrifuged for 4 h at 100,000 × g at 4°C. The floating lipid film that contained the antigen-associated vesicles was removed by a spatula and suspended in PBS (final volume, 10 ml). The lipid suspension was dialyzed for 2 h against PBS. Approximately 37% of the total amount of added antigen was found associated with the vesicles as was determined after solubilization of the vesicles with 1% (vol/vol) Triton X-100, using the protein assay described by Lowry and associates (22).
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To determine the amount of antigen associated with the vesicles containing the surfactant proteins, the same procedure was used, except for the measurement of the amount of antigen. The amount of antigen was determined using an enzyme-linked immunosorbent assay (ELISA) specific for TNP-KLH. No differences were observed in the amounts of antigen associated with either no surfactant proteins or vesicles containing SP-A, SP-B, or SP-C (results not shown). For the experiment with SP-A, SP-A was added to the lipid suspension together with the antigen.
Administration of Vesicles
The mice were fixed in an upright position under anesthesia with 20 µl of a 4:3 (vol/vol) mixture of Aescoket (Aesculaap, Gent, Belgium) and Rompun (Bayer, Leverkussen, Germany), intramuscularly injected. Using a nylon tube connected to a 1-ml syringe, 100-µl vesicles were injected through the glottis into the trachea.
Immunization Procedures
At Day 0, mice were intratracheally instilled with 100 µl PBS or 100 µl of a suspension of either PBS or DMDP vesicles. On Day 2, mice were intratracheally injected with 100 µl of antigen-containing vesicles. To study the primary immune response, animals were killed on Day 9, and their spleen cells were isolated to determine the number of anti-TNP antibody secreting cells using an ELI-Spot assay. To study the secondary immune response, animals were injected intratracheally with the same suspensions at the same times as the mice used for the primary response and additionally injected intraperitoneally with 100 µl TNP-KLH (0.8 mg/ml PBS) on Day 23. The animals were killed on Day 30, and their sera were collected in order to determine the anti-TNP antibody titers using an ELISA.
ELI-Spot Assay
This assay was based on the method described by Sedgwick and Holt (23). Briefly, microtiter plates were coated overnight at 4°C with 5 µg/well trinitrophenylated ovalbumin (TNP-OVA). The plates were emptied and incubated with 1% (wt/vol) bovine serum albumin (BSA)/ml PBS for 1 h at room temperature. The plates were rinsed twice with PBS. A single cell suspension of spleen cells (107/ml RPMI 1640 supplemented with 1% Hepes [wt/vol], 1% BSA [wt/vol], and 10% fetal calf serum [vol/vol]) was added to the first well of each row (150 µl/well) and serially diluted 1:2 in the same medium. After incubation at 37°C, the wells were rinsed with 0.1% Tween 20 (vol/vol) in PBS (PBT) until the cells were lysed. Subsequently, goat antimouse immunoglobulin (Ig) A (1:500, in 1% BSA-PBT; Sanbio) or goat antimouse IgM (1:500, in 1% BSA-PBT; Sanbio, Uden, The Netherlands) conjugated with alkaline phosphatase was added to each well (100 µl/ well) and the plates were incubated for 2 h at 37°C. After rinsing the plates four times, the alkaline phosphatase substrate 5-bromo-4-chloro-3-indolyl-phosphate (1 mg/ml adenosine monophosphate buffer [Sigma, St. Louis, MO]) supplemented with 1% low melting agarose [wt/vol]) was added to the wells (100 µl/well) at 37°C. The plates were incubated overnight at 4°C and the cells secreting TNP antibodies were counted.
ELISA
To detect specific antibodies against TNP in the sera of mice, an ELISA was developed based on the method described by Delemarre and colleagues (24). In short, microtiter plates were coated with TNP-OVA and incubated with 1% BSA as described previously. The plates were rinsed four times with PBT. For the determination of IgM and IgG in the sera, the sera were diluted with PBT supplemented with 1% BSA (1:7). In order to measure IgA, the sera were diluted four times with the same buffer. The diluted sera were added to the first well of each row (200 µl/well) and serially diluted 1:1 in the PBT-BSA buffer. After incubation of the plates at room temperature for 2 h, the plates were rinsed four times with PBT. To each well, 100 µl of either biotinylated anti-IgM, anti-IgG, or anti-IgA was added. The plates were incubated for 1 h at room temperature and rinsed four times with PBT. The plates were incubated for 1 h at room temperature with avidin-conjugated horseradish peroxidase (1:5,000 in PBT-BSA) and washed, and the bound antibodies were visualized by incubation for 1 h at room temperature with the substrate o-phenylenediaminedihydrochloride (2 mg/ml 0.1 M phosphate-citrate buffer, pH 5.5, supplemented with 0.015% H2O2; 100 µl/well). The reaction was terminated by adding 50 µl of 2.5 M H2SO4. The absorbance at 492 nm was measured and the values were expressed as compared with positive control sera for either IgM, IgA, or IgG. Control sera were included on each plate and were raised in mice by immunization with TNP-OVA.
Statistics
Data were analyzed by an analysis of variance followed by the Student-Newman-Keuls test.
Ethical Guidelines
All the animal studies were approved by the local welfare committee for animal experiments.
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Results |
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Materials and Methods
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Effect of Alveolar Macrophage Depletion and Incorporation of Antigen in Vesicles on the Immune Response
Previously we showed that depletion of alveolar macrophages using MLV containing DMDP followed by intratracheal instillation of antigen leads to an immune response in the lymph nodes draining the lung but not the spleen (5). In the present experiment, we investigated whether stronger systemic immune responses could be evoked via the airways by entrapment of antigen in vesicles. Using alveolar macrophage-depleting vesicles, we tested whether administration in the lungs of vesicle-entrapped antigen (TNP-KLH) would lead to a systemic immune response. Indeed, a systemic immune response can be induced when antigen is incorporated in SUV as was determined by the splenic immune response using an ELI-Spot assay (Figure 1). No substantial serum responses can be observed using this protocol; however, if the mice are additionally boosted with antigen, IgA, IgG, and IgM antibodies are detected in the sera (Table 1).
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Effect of Association of Antigen with Vesicles versus Incorporation of Antigen in Vesicles on the Immune Response
Next, we wished to determine whether it was important to have the antigen entrapped in the vesicles used for immunization or whether mere association of antigen with the vesicles was sufficient. From Figure 2 it is clear that only vesicles that contained the antigen were effective in the induction of a substantial immune response as measured by IgM. Similar results were obtained determing the IgG and IgA antibody titers (results not shown).
Effects of the Lipid Composition as Well as the Size of the Antigen Vesicle on the Immune Response
The importance of the lipid composition of the antigen vesicle was tested using antigen vesicles of different composition: PC/cholesterol (9:1 wt/wt), PC/PG (9:1 wt/wt), or surfactant lipids (devoid of the surfactant proteins). The PC/cholesterol as well as the surfactant vesicles induced good secondary immune responses after instillation in the trachea of mice and depletion of the alveolar macrophages (Figure 3). No immune response was observed in mice that received antigen associated with vesicles that were composed of PC/PG. In addition, the size of the antigen vesicle, SUV versus MLV, is also an important factor that determines the immune response. No immune response was detected when the antigen was entrapped in MLV, only antigen entrapped in SUV could induce immune responses (Table 1).
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Entrapment of Antigen in Surfactant Vesicles
The physiologic properties of pulmonary surfactant can be improved if surfactant lipids and hydrophobic surfactant proteins are included in the surfactant. This, and the efficiency of surfactant vesicles in inducing immune responses, prompted us to study the effect on the immune response of incorporation of the hydrophobic proteins SP-B and SP-C in the antigen-containing SUV.
A maximal IgM immune response in the spleen cells is obtained at a lipid concentration of 5 mg/ml when TNP-KLH is incorporated in SUV that contained the surfactant lipids and also the surfactant proteins SP-B and SP-C. To obtain a similar immune response using antigen vesicles that consist of only the surfactant lipids, approximately four times as much lipid is required (Figure 4). The half maximal immune response for the surfactant SUV that contained SP-B and SP-C was observed at a phospholipid concentration of 3 mg/ml. This concentration was used in the following experiments. No other classes of immunoglobulins were produced and secreted by the spleen cells (results not shown).
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Effect of the Different Surfactant Proteins on the Immune Response
To determine which surfactant protein(s) entrapped in antigen surfactant vesicles can enhance the immune response after depletion of the alveolar macrophages, the following experiment was performed. Antigen-containing vesicles were prepared that were composed of the surfactant lipids and the various surfactant proteins. The lipid-to-protein ratio was approximately the same as the physiologic ratio, i.e., for SP-A, phospholipid/SP-A (15:1 [wt/wt]), for SP-B, phospholipid/SP-B (100:0.5 to 1 [wt/wt]) and for SP-C, phospholipid/SP-C (100:0.5 to 1) (20, 25, 26). When the primary immune responses were determined, it was found that the antigen vesicles that contained SP-B yielded a higher immune response than did the other vesicles (Figure 5). In addition, incorporation of SP-C in SP-B-containing antigen vesicles did not influence the SP-B-mediated enhancement of the immune response. In this experiment, SP-A was encapsulated in the antigen vesicles together with the antigen. When SP-A was added to the antigen vesicles and was not incorporated in the vesicles, similar results were obtained (results not shown). The concentration dependency of the SP-B-induced enhancement of the immune response was tested, incorporating various concentrations of SP-B in the antigen-containing vesicles, while keeping the lipid concentration constant (3 mg/ml). A plateau in the immune response was observed at a phospholipid-to-protein ratio of 100:0.5 (3 mg phospholipid:15 µg SP-B) (Figure 6).
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Effect of Alveolar Macrophage Depletion on the SP-B-Mediated Stimulation of the Immune Response
Because of the high efficiency of SP-B-containing surfactant vesicles in inducing an immune response, we wished to investigate whether such vesicles in themselves would be sufficient for immunologic priming without the need for prior depletion. Indeed, antigen encapsulated in vesicles containing SP-B can induce a substantial increase in anti-TNP IgM, in both a primary (data not shown) and secondary response (Figure 7) without prior depletion of alveolar macrophages. The extent of the response is lower than the IgM response with depletion of alveolar macrophages (Figure 7A). Interestingly, antigen vesicles containing SP-B were not able to induce anti-TNP IgG titers without depletion of alveolar macrophages (Figure 7B).
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This study demonstrates that antigen entrapped in vesicles can induce an immune response in mice after intratracheal instillation and depletion of their alveolar macrophages using vesicles containing DMDP.
In addition, memory cells are formed, which lead to a secondary immune response comprising IgG and IgM antibodies, after an intraperitoneal booster in these mice. For the induction of the immune response via the airways, the antigen has to be entrapped in vesicles. Besides the localization of the antigen in the vesicles (inside), the lipid composition as well as the size of the antigen vesicles determines the induction of the immune response. No responses are observed when the antigen is not encapsulated in SUV. The lipid composition of the SUV has to be either PC/cholesterol (9:1 wt/wt) or the lipids present in pulmonary surfactant in order to observe an immune response.
The half maximal response can be further increased if the antigen vesicle contains SP-B and the lipids of pulmonary surfactant, suggesting that entrapment of antigen in these vesicles leads to a more effective immune response. The SP-B effect is concentration-dependent, reaching a maximum at the physiologic phospholipid:SP-B ratio of 100:0.5 to 1 (20, 25, 26). Inclusion of antigen in vesicles is required to induce an immune response via the airways. One possible explanation is that due to encapsulation of antigen in vesicles, the antigen is slowly released from the vesicle and the overall time that the antigen is present in the lung is lengthened, thereby increasing the chance to induce an immune response.
However, entrapment of antigen in MLV does not lead to the induction of an immune response after intratracheal instillation because the antigen in MLV is even more slowly released from the vesicle than from the immune response inducing SUV. It seems more likely that the incorporation of antigen in SUV is needed to target the antigen to a particular cell in the lung.
In the lungs, only two cell types were reported to readily internalize vesicles: alveolar macrophages and alveolar type II cells. It seems very likely that the alveolar type II cells may in some way be connected to the induction of an immune response when the antigen is packed in vesicles because the alveolar macrophages are known to suppress immune responses and are not present due to intratracheal instillation of the DMDP-containing vesicles. How can alveolar type II cells contribute to the induction of an immune response? After internalization of antigen vesicles by the alveolar type II cells, the antigen may become processed and/or passed to antigen-presenting cells such as dendritic cells in the lung interstitium (27). These cells may transport the antigen-derived peptides to the spleen and present it to local T cells, thereby inducing an immune response.
On the other hand, the type II cells may present antigen themselves to circulating T cells because type II cells were shown to possess major histocompatibility complex class II molecules and the costimulatory signals intercellular adhesion molecule-1 and B7 needed for effective antigen presentation on their outer membrane (28). The involvement of alveolar type II cells in the induction of an immune response using antigen-entrapped vesicles may also explain the observed effects of SP-B incorporation in these antigen vesicles. SP-B was shown to enhance the uptake of vesicles by alveolar type II cells (9), leading to increased uptake of antigen entrapped in vesicles. However, simply stimulating the internalization of antigen incorporated in vesicles by type II cells may not be the only requirement for an induction of an immune response. SP-A as well as SP-C was also reported to enhance the uptake of vesicles by type II cells (8, 33) and do not further enhance the immune response induced by antigen-containing vesicles when entrapped or present on the outside of antigen-containing vesicles after intratracheal instillation.
Apparently, the route of the antigen in the type II cell may also be an essential factor because the SP-A- and SP-C-mediated uptake by type II cells was suggested to differ from the SP-B-mediated uptake of vesicles (8). In addition, SP-A may inhibit the SP-C-mediated uptake of vesicles by type II cells (11, 12).
Targeting antigen to alveolar type II cells is not the only requirement to induce a strong immune response. Alveolar macrophages also need to be absent or the antigen packed in SP-B-containing vesicles. Alveolar macrophages were shown to inhibit immune responses probably via the suppression of T cells and dendritic cells (5, 34). It seems likely that alveolar macrophages may completely inhibit either the uptake of antigen encapsulated in ordinary, i.e., not SP-B-containing, vesicles or the processing/antigen presentation by the alveolar type II cells. Surprisingly, without depletion of the alveolar macrophages an immune response can be induced if antigen vesicles are used with incorporated SP-B, but the response is significantly lower than in alveolar macrophage-depleted animals. A simple explanation for this observation may be that depletion of alveolar macrophages leads to the destruction of cells that compete with the alveolar type II cells for the uptake of antigen and SP-B-containing vesicles. Alternatively, alveolar macrophages may suppress the uptake of SP-B-containing antigen vesicles by type II cells and/or alveolar macrophages may influence the route of the SP-B-containing antigen vesicles in the type II cells. Besides a higher immune response in animals that were depleted of their alveolar macrophages and intratracheally instilled with SP-B-containing antigen vesicles compared with mice that were intratracheally injected with only the SP-B-containing antigen vesicles, the secondary IgG immune response in the latter animals is very low after an intraperitoneal boost. This result suggests that alveolar macrophages may influence the shift of an IgM to an IgG response in these animals. Briefly, this is the first report that suggests that SP-B may be involved in the induction of an immune response via the airways. No depletion of alveolar macrophages is necessary when SP-B-containing antigen vesicles are instilled in the trachea to induce a systemic immune response. Although the concentration of lipids used in the present study is high, 300 µg per mouse, compared with the amount of lipids present in surfactant, approximately 250 µg per mouse (35), the observation that an immune response can be induced after intratracheal instillation of SP-B-containing antigen vesicles warrants further studies. In addition, this study also suggests that the lipid metabolism by type II cells is closely linked to the immune system of the lung. Therefore, determination of the exact mechanism(s) by which the immune response is induced after intratracheal instillation of SP-B-containing vesicles may not only advance our knowledge of the pulmonary immune system but also our understanding of the factors that may govern the metabolism of the surfactant system.
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Address correspondence to: J. Freek van Iwaarden, Laboratory of Pediatrics, Erasmus University Rotterdam, Dr. Molewaterplein 50, 3015 GE Rotterdam, The Netherlands. E-mail: vaniwaarden{at}kgk.fgg.eur.nl
(Received in original form May 5, 2000 and in revised form November 2, 2000).
Abbreviations: bovine serum albumin, BSA; dichloromethylene-diphosphonate, DMDP; enzyme-linked immunosorbent assay, ELISA; immunoglobulin, Ig; multilamellar vesicles, MLV; phosphate-buffered saline, PBS; Tween-20 in PBS, PBT; phosphatidylcholine, PC; phosphatidylglycerol, PG; surfactant protein, SP; small unilamellar vesicles, SUV; trinitrophenyl-keyhole limpet hemocyanin, TNP-KLH; trinitrophenyl-ovalbumin, TNP-OVA.Acknowledgments: This study was supported by a grant from the Stichting Technische Wetenschappen.
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References |
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References
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