Section for Molecular Signalling, Department of Cell and Molecular Biology, Lund University, Lund, Sweden, 2Section for Tumour Immunology, Department of Cell and Molecular Biology, Lund University, Lund, Sweden, 3Department of Radiation Physics, The Jubileum Institute, Lund University, Lund, Sweden, and 4Haematology Research Laboratory, Department of Laboratory Medicine, Lund University, Lund, Sweden
Received on February 7, 2000; accepted on February 29, 2000.
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Abstract |
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Key words: 1-microglobulin/carbohydrate deletion/immune suppression/secretion/tissue distribution
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Introduction |
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The exact biological function of 1m is not known, but it has a number of in vitro immunoregulatory properties. For example, it inhibits the antigen-induced proliferation of peripheral lymphocytes, IL-2 production of T-cells and migration and chemotaxis of granulocytes (Lögdberg and Åkerström, 1981
; Méndez et al., 1986
; Wester et al., 1998
). Different antigen-models in several different species have been used, e.g., tuberculin (purified protein derivative; PPD), tetanus toxid, ovalbumin and collagen, suggesting that
1m has a general immunosuppressive role. It was also demonstrated that
1m binds to receptors on peripheral lymphocytes and T-cell hybridomas (Fernández-Luna et al., 1988
; Åkerström and Lögdberg, 1984
; Babiker-Mohamed et al., 1990a
; Wester et al., 1998
), but the connection between cell-surface binding and the immunosuppressive effects is not yet known. The antigen-induced proliferation of peripheral lymphocytes could be suppressed by a preparation of N-linked glycans from
1m as well as by the whole protein (Åkerström and Lögdberg, 1984
). The carbohydrates of
1m were then suggested to exert the immunological effects.
1m is co-synthesized in the liver together with bikunin (Kaumeyer et al., 1986
), a proteinase inhibitor which is cross-linked to one or two out of four so called heavy chains forming the various members of the inter-
-inhibitor family (Salier et al., 1996
). A precursor protein,
1m-bikunin, is formed in the hepatocytes but cleaved in the Golgi apparatus before secretion of
1m and bikunin separately (Bratt et al., 1993
). The two proteins have no known functional or structural relation after the cleavage, so the reason for the co-synthesis of the two proteins is enigmatic, but since it has been found in all species so far investigated, it is apparently of some importance.
In plasma, 1m is found in free form as well as covalently bound to other larger plasma proteins. Complexes with IgA, albumin, and prothrombin have been described in human plasma (Grubb et al., 1986
; Berggård et al., 1997
) and with fibronectin and
1-inhibitor-3, an
2-macroglobulin homologue, in rat plasma (Falkenberg, 1990
; Falkenberg et al., 1994
). Free
1m and various high-molecular weight complexes are also present in extracellular matrix of most tissues, probably originating from plasma. Its localization in placenta in areas of contact between mother and fetus, especially at sites of injury, suggests that it is involved in local immunosuppression, protecting tissues from potentially dangerous inflammatory processes (Berggård et al., 1999
).
In this work we have investigated the role of the carbohydrates of 1m for expression, secretion, structure, biochemical properties, lymphocyte binding, immunosuppressive effects and in vivo turnover of the protein. Using the baculovirus-insect cell expression system, two recombinant forms of
1m were obtained and one form of
1m was isolated from human plasma (Figure 1). Plasma
1m carries three oligosaccharides: one O-linked at pos. 5 (T5) and two N-linked at positions 17 and 96 (N17 and N96). Their exact structures have not been elucidated but were shown to contain sialic acid (Ekström et al., 1981
; Escribano et al., 1990
). Recombinant nonmutated
1m, expressed and purified from baculovirus-infected insect cells, lacked the O-linked oligosaccharide on T5 and the N-linked oligosaccharides were smaller and lacked sialic acid (Wester et al., 1997
). In this work, N17,96Q-
1m, a completely carbohydrate-free form of the protein, carrying glutamine instead of asparagine in positions 17 and 96, was made using site-directed mutagenesis and expression in an insect cell system. First, the efficiency of expression and secretion and the biochemical properties of the carbohydrate-free N17,96Q-
1m was examined and compared to nonmutated recombinant
1m. Second, all three variants were tested with respect to lymphocyte binding and immunosuppressive effects. Third, the radiolabeled proteins were injected into rats intravenously and their distribution in blood and tissues were studied. These results suggest that the carbohydrates are important for the secretion from insect cells and protein turnover in vivo, but are less important for the structure and biochemical properties of the polypeptide chain. The results also show that the lymphocyte binding and the suppressive effects of
1m on antigen-stimulation are most likely not dependent on the carbohydrates.
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Results |
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Size, shape, charge, and color
Some biochemical and physicochemical properties of nonmutated and N17,96Q-1m were analyzed and are displayed in Table I. The N-terminal sequences were identical. As described above, the molecular mass was lower for nonglycosylated, N17,96Q-
1m, 24,000 Da as compared to 29,000 Da for the glycosylated, nonmutated
1m. The Stokes radius and frictional ratio were similar for the two proteins. The secondary structure of the proteins was studied by far UV CD-analysis. The shape of the CD spectra obtained (Figure 4C) were similar to previously reported spectra for urinary, recombinant and plasma
1m (Gavilanes et al., 1984
; Wester et al., 1997
), indicating no major shift in the overall secondary structure of nonglycosylated
1m.
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Antibody binding
Surface epitopes on 1m were investigated by RIA, a competitive binding assay, and SPRIA, a direct-binding assay (Figure 5, A and B, respectively). The binding of polyclonal and monoclonal antibodies to recombinant nonmutated and N17,96Q-
1m was compared to human plasma and urinary
1m. All antibodies were prepared against human urinary
1m, and the monoclonal antibodies have been demonstrated to recognize non-carbohydrate structures only (Babiker-Mohamed et al., 1991
). As shown in Figure 5, similar results were obtained with the four
1m variants in RIA and the three
1m variants tested in SPRIA, suggesting that the surface epitopes are not structurally altered on N17,96Q-
1m.
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Discussion |
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The secretion of carbohydrate-free N17,96Q-1m from the recombinant insect cells was severely reduced as compared to nonmutated
1m. Most of the mutated protein was found inside the cells. Nonmutated
1m, which is normally secreted in high amounts, also accumulated intracellularly when it was expressed in the presence of tunicamycin. This indicates that the lack of carbohydrates rather than the mutation, an exchange of asparagine to glutamine, cause the reduced secretion. A defect secretion has been reported for other carbohydrate-deleted proteins expressed in the baculovirus-insect cell system, either as a result of point mutations (Sareneva et al., 1994
) or culturing in the presence of tunicamycin (Chawla and Owen, 1995
). It is generally believed that N-linked oligosaccharides are important for secretion of proteins (for a review, see Helenius et al., 1992
). The mechanism behind this could be that the N-linked oligosaccharides are needed for a correct folding in the endoplasmatic reticulum and that the absence of the carbohydrates leads to misfolding and aggregation (Helenius et al., 1992
). Another possibility is that a structure on the N-linked oligosaccharides is necessary for secretion. Our data support the former alternative since polyclonal antibodies showed a much weaker binding to intracellular
1m, indicating misfolding of the protein. The fact that the carbohydrate-deleted protein was partly secreted argues against the N-glycans as a necessary signal for secretion. The secreted protein was recognized by polyclonal antibodies and presumably correctly folded. This can be explained by the activity of chaperone proteins, a system only capable of handling a part of the overexpressed protein.
Circular dichroism analysis of secreted recombinant nonmutated and N17,96Q-1m indicated a similar overall structure. Human
1m has previously been shown to consist of mainly ß-sheet structure with minor contributions of
-helix (Ekström and Berggård, 1977
; Gavilanes et al., 1984
; Wester et al., 1997
). The CD-spectrum analysis confirmed this also for the N17,96Q-mutated protein. Moreover, a panel of antibodies recognized the recombinant nonmutated and N17,96Q-mutated proteins to the same degree, indicating that the same epitopes are present on the surface of the two proteins. Both proteins were found to be heterogeneously charged and carrying yellow-brown chromophores. The structure and identity of the chromophores are still unknown, but they are most likely covalently bound to the protein core. There is a correlation between the degree of yellow-brown color and the net charge of
1m (Calero et al., 1996
; Wester et al., 1997
), which is further supported in this work: N17,96Q-
1m displayed somewhat higher net charge and was more intensely colored. It was previously shown that the chromophore is not associated with the carbohydrate part of
1m, since a complete removal of the carbohydrate by glycosidase treatment did not alter the optical properties (Åkerström et al., 1995
). The results presented here show that the carbohydrates are not needed at all for the formation of the chromophore of
1m. Finally, both recombinant forms of
1m were significantly more colored than plasma
1m (Wester et al., 1997
), suggesting that the insect cell expression system somehow promotes the formation of the chromophore.
Based on the immunosuppressive properties of 1m, several authors have suggested a regulatory role of
1m on the immune system (Méndez et al., 1986
; Åkerström, 1992
; Santin and Cannas, 1999
). Carbohydrates have been shown to be involved in many important processes in the immune system, as exemplified by selectins and orosomucoid (Lasky, 1992
; Shiyan and Bovin, 1997
). Glycopeptides isolated from human
1m showed an inhibitory effect on the antigen-stimulated proliferation of human peripheral leukocytes (Åkerström and Lögdberg, 1984
). The effect was also shown to be independent of the polypeptide backbone, and it was suggested that the N-linked oligosaccharides of
1m are responsible for the immunosuppressive effect. The hypothesis was tested in this paper, on antigen-induced stimulation of mouse T cell hybridomas, a system in which plasma and recombinant
1m previously have been shown to exert an inhibitory effect (Wester et al., 1998
), and on PPD-induced stimulation of human peripheral lymphocytes, one of the most commonly used antigen model systems and identical to the one previously used to show the effect of isolated N-linked oligosaccharides of
1m (Åkerström and Lögdberg, 1984
). It was unexpectedly found that carbohydrate-free
1m had the same immunosuppressive effects and bound to lymphocytes to the same degree as fully glycosylated
1m. We can therefore conclude that the oligosaccharides are not responsible for the immunosuppressive effects or binding to lymphocytes. A possible explanation for the immunosuppressive effects of the glycopeptide preparation is that a co-purified low molecular weight substance exerts the effect. From the results in this paper it must be considered unlikely that a contaminating substance, i.e. not related to
1m, causes the effect, since
1m isolated from two completely different sources like human plasma and serum-free cell culture medium exert the same effect. Instead, the hypothetical lipocalin ligand could be a possible candidate for such a substance.
1m has been shown to bind to receptors on PBL and T cell hybridomas from mouse, and to the human histiocytic cell-line U937 (Fernández-Luna et al., 1988
; Babiker-Mohamed et al., 1990a
; Wester et al., 1998
). We have shown here that the protein also binds to human peripheral T cells, B cells and NK cells. The binding is weak, similar to the previous results. The binding is consistent with immunohistochemical findings, which have shown a staining of lymphocytes, monocytes and macrophages with antibodies against
1m ((Berggård et al., 1999
; Bouic et al., 1984
). Thus, it can be concluded that
1m binds weakly to a wide variety of blood cells. This is logical considering that the protein has been shown to regulate many different functions of several blood cell populations (Lögdberg and Åkerström, 1981
; Lögdberg et al., 1986
; Méndez et al., 1986
; Babiker-Mohamed et al., 1990a
,b; Wester et al., 1998
).
1m is synthesized almost exclusively by the liver (see, for instance, Berggård et al., 1998
). It is secreted to the blood and, due to its relatively small size, filtrated and degraded in the kidneys. However, it is also found in the interstitial tissue of all examined organs except the brain (Bouic et al., 1984
; Ødum and Nielsen, 1994
; Berggård et al., 1998
). The transcription of the
1m-gene was increased during inflammation but the levels of
1m were unchanged in blood and urine (Falkenberg et al., 1997
). It has therefore been speculated that, after synthesis in the liver and secretion to blood,
1m is transported to tissues where its immunosuppressive properties help protect these tissues from unwanted immune and inflammatory reactions (Ødum and Nielsen, 1994
; Falkenberg et al., 1997
; Berggård et al., 1999
). This view is supported by the results in this work. In our rat model, the protein was cleared from blood with a half-life of 3.1 min, but more than half of the protein was found in other compartments than blood, liver, and kidneys after 45 min. The results also show that the distribution of
1m is dependent on the carbohydrate part of the molecule. Carbohydrate-free
1m was metabolized by the kidneys faster than wild-type
1m, and the sialic acid apparently protected the protein from uptake, and possibly degradation (Ashwell and Morell, 1974
) by the liver.
The results in this paper have implications for future applications of 1m in immunotherapy. We have shown that immunologically functional
1m can be expressed in large amounts. Furthermore, the structure of the carbohydrates is probably important for the delivery of
1m to adequate body compartments. The carbohydrates apparently prolong the half-life of the protein in the blood and sialic acid prevents it from uptake in the liver. In line with this, it should be possible to use other expression systems to obtain a functional
1m in which the structure of the carbohydrate part and the number of oligosaccharide units have been optimized for drug delivery purposes.
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Materials and methods |
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Analysis of 1-microglobulin secretion
Nonmutated 1m and N17,96Q-mutated
1m were expressed in the presence or absence of tunicamycin. Insect cells, 1.5 x 106, were grown in 6-well plates (Corning Costar, Bodenheim, Germany) and inoculated with 10 pfu/cell of baculovirus. The cells were left for incubation in 1.5 ml medium at 27°C. In some wells tunicamycin (Calbiochem, La Jolla, CA) was added to the medium at a final concentration of 10 µg/ml. Each day between day 1 and 6 after inoculation, infected cells cultured in presence or absence of tunicamycin, were harvested. Cells and media were separated by low-speed centrifugation. Protease inhibitors were added to the media (leupeptin 10 µg/ml, antipain 5 µg/ml, and pepstatin 1 µg/ml). The cell-pellets were suspended in 100 µl of PBS and lysed by incubation with 25 µl of lysis-buffer (50 mM TrisHCl, pH 6.9, 25% glycerol, 10% SDS, 25% ß-mercaptoethanol and 0.25% bromphenolblue) at 100°C for 5 min. 10 µl of cell media and 2.5 µl of the cell lysates were analyzed by SDSPAGE and Western blotting.
Purification of 1-microglobulin
All 1m-variants were purified by anti-
1m affinity chromatography and gel chromatography. Nonmutated and N17,96Q-mutated recombinant
1m were purified as described by Wester et al. (1997)
. Plasma and urinary
1m were isolated as described by Wester et al. (1997)
and Åkerström et al. (1995)
, respectively.
Radiolabeling
Proteins were labeled with 125I using the chloramine T method (Greenwood et al., 1963). Labeled proteins were separated from free iodide by gel-filtration on Sephadex G-25 columns (Pharmacia). The specific radioactivity achieved was around 1 MBq/µg.
Electrophoresis
SDSPAGE under reducing conditions was done according to the procedure described by Laemmli (1970) and detailed previously (Wester et al., 1997
). Agarose electrophoresis was done according to (Johansson, 1972
) with a 0.8% agarose gel (SeaKem ME, FMC BioProducts, Rockland, ME) as outlined (Wester et al., 1997
).
Immunochemical methods
Proteins were separated by SDSPAGE and transferred to polyvinylidene difluoride (PVDF) membranes (Immobilon, Millipore, Bedford, MA) as described previously (Madisudaira, 1987).
1m was then detected by Western blotting as described previously (Wester et al., 1997
). Briefly, the membranes were blocked and incubated with polyclonal rabbit anti-human
1m antibodies, K: 324 (prepared in our laboratory) and finally with 125I-goat anti-rabbit IgG (prepared in our laboratory) (0.5 x 106 c.p.m./ml).
Solid-phase radioimmunoassay was performed following mainly the description of Nilson et al. (1986). Briefly, serial dilutions of the proteins were coated on microtiter plates overnight, at room temperature. The plates were then incubated with either one of five monoclonal mouse antibodies (BN 11.1, BN 11.2, BN 11.3, BN 11.5 or BN 11.10), described in Babiker-Mohamed et al. (1991)
at a concentration of 10 µg/ml, or with polyclonal rabbit anti human-
1m antiserum, K:324, (diluted 500 times). They were then incubated with either radiolabeled rabbit anti-mouse Ig (DAKO A/S, Denmark) or goat anti-rabbit Ig (5 kBq/ml). Finally, the plates were washed, cut and counted in a
-counter (Packard, Meriden, CT). Background level was set by excluding the primary antibody step.
Radioimmunoassay (RIA) was done by mixing fixed amounts of 125I-labeled 1m and polyclonal goat anti-human urinary-
1m antibodies (prepared in our laboratory) with samples of unknown
1m concentration (Åkerström, 1985
). The mixture was left overnight at 4°C, and then precipitated by 10% polyethylene glycol 6000. The pellets were analyzed for radioactivity in a
-counter.
Stokess radius and frictional ratio
Stokess radius, rs, and frictional ratio (f/f0) were determined by gel chromatography (Laurent and Killander, 1964) on a Sephacryl S-200 column as described previously (Bratt and Åkerström, 1995
) Bovine serum albumin (BSA) (Boehringer-Mannheim, GmbH, Mannheim, Germany) was used as a standard with known Stokess radius.
Carbohydrate analysis
1m variants were digested sequentially with neuraminidase, O-Glycosidase and N-Glycosidase F as described previously (Wester et al., 1997
). Agarose-insolubilized neuraminidase (from Clostridium perfringens) was purchased from Sigma Chemical Co. O-Glycosidase (from Diplococcus pneumoniae), and N-Glycosidase F (from Flavobacterium meningosepticum) were purchased from Boehringer-Mannheim, Mannheim, Germany. The binding of the lectins Concanavalin A (ConA) and peanut agglutinin (PNA) was tested by incubation of proteins, separated by SDSPAGE and transferred to PVDF-membranes, with digoxigenin-labeled lectins (DIG Glycan Differentation Kit, Boehringer-Mannheim) using the protocol supplied with the kit.
Circular dichroism studies
CD spectra were recorded at 37°C using a JASCO J-720 spectropolarimeter with a thermostated cell holder. Ten scans were performed on each protein sample using a scan speed of 20 nm/min, a sampling interval of 1 nm, and 4 s response time. Measurements were performed on protein samples dissolved in 20 mM sodium-phosphate buffer, pH 7.0 in 0.1 cm quartz cuvettes. The contribution from the buffer was subtracted and the spectra were moderately noise reduced. The protein concentration of the samples was determined by absorbance at 280 nm.
Flow cytometric analysis
The binding of 1m to different cells was analyzed by flow cytometry. The mouse CD4+ T hybridoma cell line, HCQ.4, which is specific for rat collagen II, was cultivated as described (Michaëlsson et al., 1994
). Human peripheral lymphocytes were prepared from blood from normal healthy donors by centrifugation on lymphoprep (Nycomed Amersham plc) at 800 x g for 20 min and washed once by PBS at 400 x g for 10 min. The contaminating red blood cells were lysed by treating the cells with ortholyse (Ortho-mune Lysing reagent, Ortho Diagnostic Systems Inc., Johnson & Johnson, Raritan, NJ) for 7 min. Cells were centrifuged and resuspended in PBS + 1 mg/ml BSA. Flow cytometric analysis was made by incubating the cells with 1 mg/ml plasma
1m, recombinant nonmutated or N17,96Q-
1m or BSA (control), in the PBS-BSA buffer. After 15 min , the cells were washed and incubated for 10 min with 10 µg/ml monoclonal mouse anti-
1m antibodies (BN 11.3), washed and incubated 10 min with FITC-conjugated goat anti-mouse immunoglobulin (GAM-FITC, DAKO A/S, Denmark). PBL were then washed carefully to remove excess of GAM-FITC and finally incubated with phycoerythrin-conjugated mouse monoclonal anti-human CD8, CD19 (DAKO) or CD4, CD56 (BD immunocytometry systems, San Jose, CA) diluted 1:10 in PBS+1 mg/ml BSA. The cells were washed, fixed with 1% paraformaldehyde and analyzed on a FACScan flow cytometer (Becton Dickinson AB, Sweden). At least 10,000 cells were registered and a lymphocyte region combined with a region including the CD4, CD8, CD19, and CD56 positive cells, respectively, was analyzed to evaluate the binding of
1m to each lymphocyte subset.
Assay for inhibition of IL-2 production
The 1m inhibition of IL-2 production of the HCQ.4 T cell line stimulated by antigen (a rat collagen II peptide) in the presence of mouse (B10Q x DBA/1)F1 splenocytes was studied as described (Wester et al., 1998
). Briefly, serial dilutions of
1m variants were mixed with mouse T hybridoma cells, mouse splenocytes, and antigen. After 24 h at 37°C, the supernatants were analyzed for IL-2 content with an ELISA Development system for mouse IL-2 (Duoset, Genzyme Diagnostics, USA).
Assay for inhibition of proliferation
The procedure followed mainly the description of Åkerström and Lögdberg (1984). I principle, human PBL were incubated with an antigen-mixture from tubercle bacilli, PPD (purified protein derivative, also called tuberculin), which is used for testing of immunity against tuberculosis, and the resulting cell proliferation was inhibited by various concentrations of added
1m. PBL were isolated from buffy coats obtained from normal healthy donors on Ficoll-Paque (Amersham-Pharmacia biotech, Uppsala, Sweden). The cells were then washed once in RPMI 1640 medium (Gibco, Paisley, UK) supplemented with 10 mM HEPES, 4 mM L-glutamine, 1 mM pyruvat, 0.1% NaHCO3, and 10% fetal calf serum and then resuspended in this medium. The cells were cultured in flat-bottomed 96-well microtiter plates (Corning Costar) at 37°C in a CO2- incubator. Each well contained 2 x 105 cells in 0.2 ml medium. PPD, 1 mg/ml in PBS (Statens seruminstitut, Copenhagen, Denmark) was added to 50 µg/ml, and plasma
1m, recombinant nonmutated or N17,96Q-
1m were added to final concentrations of 250, 125, 62.5, 31.3, 15.6, 7.81, 3.90, and 0 µg/ml. The cultivation time was 4 days. Four hours before termination the cells were pulsed with 0.5 µCi methyl-[3H]thymidine and then harvested onto filter paper and the thymidine incorporation was measured in a ß-scintillation counter. The inhibition studies were carried out in triplicate. The maximal proliferation was set by the mean value of triplicate of cells cultured in the presence of PPD and absence of
1m and a minimal proliferation was set by the mean value of triplicate of cells cultured in the absence of PPD and
1m.
Biokinetic studies
The biokinetic distribution of 125I-labeled plasma 1m, neuraminidase-digested plasma
1m, recombinant nonmutated
1m and N17,96Q-
1m was performed in female Sprague-Dawley rats. The animals were anaesthetized first firmly with ether and then intraperitoneally with chloral hydrate (5%) using a volume of 0.6 ml/100 g body weight. Labeled protein containing an activity of 4.25 ± 0.27MBq 125I in a volume of 0.6 ml was injected intravenously in the right femoral vein. Dynamic studies were carried out during 45 min using a scintillation camera (Maxi Camera I, General Electric), equipped with a Low Energy High Resolution Parallel collimator. Immediately after the end of the study the animals were killed and dissected. Blood sample and whole organs (heart, kidneys, liver, spleen, lungs, muscle, stomach, intestine, appendix, and thymus) were removed and taken to analysis for static image collection. In order to generate time-curves for the blood clearance and the activity uptake in organs, regions of interest were selected over the heart, liver, kidneys and bladder in the dynamic images. The T-values for the blood clearance were obtained by regression analysis (least-square curve fitting to exponential polynoms) of the values over the heart regions. To estimate the activity in the dissected organs a scintillation camera correction factor for 125I was used.
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Acknowledgments |
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Footnotes |
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References |
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