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Keywords:

  • Balb/c mouse;
  • cytokine modulation;
  • desensitization;
  • egg allergy;
  • hypoallergenic mutant;
  • immunoglobulin E-epitope;
  • ovomucoid;
  • Th1/Th2

Abstract

  1. Top of page
  2. Abstract
  3. Methods
  4. Results
  5. Discussion
  6. Acknowledgment
  7. References

The purpose of this study was to determine the in vivo desensitization efficacy of a hypoallergenic variant of egg white ovomucoid third domain (DIII) in Balb/c mice model. We mapped the immunodominant B-cell epitopes of ovomucoid in Balb/c mice. A hypoallergenic ovomucoid third domain (GMFA) mutant isoform having ablated allergenicity against egg allergic patient's sera was used to desensitize DIII-sensitized Balb/c mice by intraperitoneal injections. Ovomucoid DIII generated high levels of plasma histamine and specific immunoglobulin (Ig)E levels, and increased Th2 type cytokine (IL-4). On the other hand, the allergic response of mice desensitized with the GMFA was found to be significantly inhibited and abrogated by prevention of anaphylaxis reactions, low histamine levels and increased Th1-type cytokine (INF-γ). It was found that significantly higher levels of IL-10 and IL-12 were secreted in the desensitized group Desensitization with the GMFA antigen also suppressed synthesis of DIII specific-IgE levels and enhanced specific IgG2a and IgG levels compared with the group treated with the DIII antigen. The present results indicated that hyposensitization with the GMFA can desensitize or down-regulate the allergic response in Balb/c mice and this hypoallergenic variant of ovomucoid DIII can shift an ongoing allergen-specific Th2 response towards a Th1 skewed response.

Abbreviations:
DIII

third domain of ovomucoid

GMFA

modified ovomucoid third domain

PBS

phosphate buffered saline

Adverse reactions to foods cause food allergic reactions and higher incidence has been observed in the general population recently (1). Eggs are among the top eight foods that cause 90% of all allergic reactions in children (milk, peanuts, wheat, soy, fish, shellfish and tree nuts are the other seven) (2). Egg allergy is usually mild, but in rare cases can trigger anaphylaxis (3). People with an egg allergy react to the proteins in the egg white more than the egg yolk. Elimination of eggs from daily diet is the only possible curative approach available so far. Specific immunotherapy (SIT) offers an alternate therapeutic approach for the treatment of egg allergy. Egg is made up of various proteins, many of which are highly allergenic. The four major allergenic proteins of hen's egg white are ovomucoid, ovalbumin, ovotransferrin, and lysozyme. Ovomucoid is a heat-stable glycoprotein constituting about 11% of the total egg white protein and has been shown to be the major cause of allergic reactions in egg white (4). The third domain of ovomucoid has been shown to be more allergic than the first and the second domain (5). The recombinant ovomucoid third domain antigen shows molecular and immunological properties comparable with its native counterpart (6). We have recently shown that modified (engineered) isoform of ovomucoid has lower IgE-binding reactivity against egg allergic patient's sera (7). Disruption of structure via modification of the conformational B-cell epitope may have led to the complete abrogation of IgE binding reactivity against human sera.

Variants of allergens with reduced IgE binding reactivity have been produced earlier, for use in immunotherapy (8). Although the basic mechanism of desensitization is still unclear, SIT is the only curative and therapeutic approach available, for treatment of allergic symptoms because of lack of other effective treatments. Majority of the immuno-modulatory curative approaches to food allergy operate on the basis of immunoregulating the Th1/Th2 balance. Th1 and Th2 balance are primarily maintained by the regulatory cytokines such as IL-10 and IL-12 (9, 10).

In previous study, we sought to analyse the IgE-binding epitopes of ovomucoid in human and Balb/c mice, a model which is a high responder of IgE, favouring Th2 type immune response and well debicted for egg-white antigens (P. Rupa, Y. Mine, unpublished data; 11). The primary aim of this investigation was to examine whether the modified isoform of ovomucoid could desensitize the allergic response to ovomucoid third domain (DIII)-sensitized allergic Balb/c mice. Allergic immune response with regards to hypersensitivity reactions, plasma histamine levels, ovomucoid-specific antibody levels and cytokine levels in splenocytes were measured. Our experiments reveal that modified ovomucoid DIII could desensitize the DIII-induced allergic response in mice by low levels of IL-4 and high levels of IFN-γ, accompanied by reduction in IgE and IgG1 levels and elevated IgG and IgG2a levels. We have shown that modified ovomucoid DIII has the potential to prevent allergic reactions and can prevent and reverse antigen-specific immunoregulatory effects on Th1–Th2-type responses in vivo.

Methods

  1. Top of page
  2. Abstract
  3. Methods
  4. Results
  5. Discussion
  6. Acknowledgment
  7. References

Mice and reagents

Female Balb/c mice (6–8 weeks old) were purchased from Charles River Laboratories (Missisuaga, ON, Canada) and were maintained under pathogen-free conditions at the Central Animal Facility at University of Guelph, following the standard guidelines of the Canadian Council on Animal Care. Aluminum hydroxide was used as an adjuvant for the intraperitoneal injections (Alhydrogel 2.0%, Superfos Biosector, Vedbaek, Denmark). Cytokine enzyme-linked immunosorbent assay (ELISA)s were performed according to the PharMingen standard protocols (PharMingen, San Diego, CA, USA). Opt EIA Mouse IL-10 (555252) (PharMingen) and IL-12 kits (555256) were used and for IL-4 (Cat no. 51-26611E and 51-26612E) and INF-γ paired antibodies (Cat no. 51-26571E and 51-26452E) were used as recommended by the manufacturer (PharMingen). The recombinant modified ovomucoid DIII allergen (GMFA) used for desensitization was expressed and purified as previously described (7). The GMFA antigen was run through the polymyxin B-Agarose resin (Sigma, Oakville, Canada) to remove endotoxin content from the protein before start of the experiment. The native third domain ovomucoid antigen (DIII) used for sensitization experiments was prepared by hydrolysis of ovomucoid with Staphylococcus aureus V8 (Spase V8) protease (Sigma) as described earlier (12).

Intraperitoneal ovomucoid DIII sensitization, desensitization, and challenge

Balb/c mice (four per group) were sensitized intraperitoneally with 50 μg of the native ovomucoid third domain antigen (DIII) together with 2.0 mg of alum (aluminum hydroxide) twice (day 0 and 7). Desensitization was done intraperitoneally 3 weeks after the last sensitization for three times (day 21, 28 and 35) with the modified ovomucoid third domain antigen (GMFA) and the positive control group was injected with the native DIII to maintain hypersensitivity. For negative controls mice received phosphate-buffered saline (PBS) containing alum and treatment regimen was the same as above. After the last boost (at day 42) mice were challenged intraperitoneally with a higher dose of 1 mg of ovomucoid DIII antigen (Fig. 1) The trial was performed twice.

image

Figure 1.  Experimental protocol. Balb/c mice were sensitized intraperitoneally with 50 μg of the native DIII at day 0 and 7 (n = 4/group). Group 1 (control) received PBS as a negative control. Group 2 (DIII) received intact DIII throughout. Group 3 (GMFA) received intact DIII at day 0 and 7 and desensitization with GMFA at day 21, 28 and 35. All mice were subjected to systemic anaphylaxis (ASA) with 1 mg of ovomucoid DIII i.p. injection at day 42.

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Assessment of hypersensitivity reactions and scoring

Symptoms for hypersensitivity reactions were recorded every 5 min for the first 40 min after the challenge by using the following scoring system performed by two independent investigators, no symptoms; 1, scratching and grooming of nose and head; 2, diarrhoea and puffiness around the eyes and mouth; 3,wheezing and respiratory problems; 4, tremor and convulsions; 5, death. Signs of hypersensitivity reactions became apparent in the sensitized group of mice within 15 min following i.p. injection and peaked at 20–40 min (13).

Determination of histamine levels in serum samples

Forty minutes after antigen challenge, the mice were killed and blood was collected from the heart. Sera samples from the immunized mice were analysed for histamine levels at the end point for ex vivo response to the sensitized ovomucoid DIII antigens by competitive direct ELISA using the commercial histamine ELISA kit (Neogen Corporation, Lexington, KY, USA). Standards were used and the concentration of histamine was calculated by comparison with a standard curve provided by the manufacturer to measure histamine levels in the sera samples.

ELISA for measurement of total and ovomucoid DIII specific antibody levels

Enzyme-linked immunosorbent assay was performed to determine the production of total and specific antibody levels in the anti-sera obtained from mice against the control and the desensitized groups. Whole blood was obtained by cardiac puncture at the end point and the levels of ovomucoid DIII-specific antibodies were measured. The 96-well microtitre plates were coated with the native third domain ovomucoid antigen (5 μg/ml of antigen in 50 mM sodium carbonate buffer pH 9.5) and incubated overnight at 4°C. Each well was washed three times with PBST (PBS containing 0.05% Tween 20) and blocked with 2% BSA in PBS and the plates were incubated for 2 h at 37°C. Sera samples diluted in PBS (1 : 2000 dilution) for IgG, IgG1 (1 : 1000), IgG2a (1 : 500), and IgE (1 : 20 dilution) were added and the plates were incubated 2 h at 37°C. The plates were further washed three times with PBST and tested against alkaline phosphatase-conjugated rabbit anti-mouse IgG (Sigma) (1 : 15000 dilution), for measuring IgG levels, and rat anti-mouse IgG2a and IgG1 (final concentration of 1 μg/ml) (BD Bio Science, San Jose, CA, USA) for IgG1 and IgG2a and monoclonal anti-mouse IgE conjugated to biotin (1 : 1000 dilution) (Sigma), for determining IgE levels and the bound antibodies were detected by peroxidase conjugated mouse anti-rat Ig (1 : 2000) (BD BioScience) for IgG1 and IgG2a and for IgE assay, the plate was further incubated with avidin conjugated alkaline phosphatase and specific IgE and IgG plates were developed with 100 μl of p-nitrophenol phosphate (1 mg/ml) (Sigma) in 0.1 M diethanolamine buffer (pH 9.8) for 1 h at 37°C. The reaction was terminated by adding 25 μl of 3 N sodium hydroxide. Absorbance at 405 nm was read using microplate reader (Model 550, Bio-Rad Laboratories, Hercules, CA, USA). For specific IgG1 and IgG2a the reaction was developed with 3,3′,5,5′-tetra methyl benzidine (TMB) substrate developing solution (Sigma) and stopped with 1 N H2SO4. Absorbance was measured at 450 nm. For analysis of total IgG and IgE levels the plates were coated with anti-mouse IgG (Cedarlane, Hornby, ON, Canada) and anti-mouse IgE (PharMingen) (1 μg/ml) in 50 mM sodium carbonate buffer, pH 8.5 and all other procedures were the same as mentioned above. Standard pure mouse IgG and IgE were used to quantify the levels of serum total IgG and IgE as per the manufacturer's instructions (PharMingen).

Quantitation of cytokine levels in culture supernatants of murine splenocytes

Spleen from four mice in each group were removed individually in ice cold RPMI 1640 media (Life Technologies, Gaithersberg, MD, USA) under aseptic conditions. The splenocytes were isolated after the removal of the red blood cells using red blood cell lysing buffer (RBC) (Sigma) and viability counts were performed by exclusion of trypan blue dye. More than 95% of the cells were determined viable, by trypan blue dye exclusion. Single cell suspensions of splenocytes were seeded in triplicates into 96 well flat bottom tissue culture plates (Corning Life Science, Acton, MA, USA) (1 × 106 cells/well) and cultured in RPMI 1640 media (Gibco, Grand land, NY, USA) supplemented with 8% heat-inactivated fetal calf serum (FCS) in the presence or absence of 50 μg of native ovomucoid DIII antigen for 72 h at 37°C in a humdified atmosphere of 5% CO2. Supernatants were collected after 72 h of culture and analysed for the levels of major allergy related cytokines like IL-4, IFN-γ, IL-10 and IL-12. The protocol for cytokine ELISAs were followed as per the BD Bio Science protocol instructions, with slight modifications. Paired monoclonal antibodies which are specific for cytokines, IL-4 and IFN-γ were used and for measuring IL-10 and IL-12 OptEIA mouse cytokine ELISA kits were used (PharMingen, San Diego, CA, USA) as per the standard protocol provided by the manufacturer (PharMingen). Absorbance was measured at 450 nm. Standard cytokines were used to quantify the individual cytokine levels.

Data analysis

Data are summarized by using statistical analysis from software package (Graphpad Instat Version 3.05, SanDiego, CA 92130, USA). The differences between groups were analysed by Tukey–Kramer 1 way analysis of variance and the level of significance was determined by P-values and P < 0.05 were considered significant.

Results

  1. Top of page
  2. Abstract
  3. Methods
  4. Results
  5. Discussion
  6. Acknowledgment
  7. References

IgE binding epitopes of ovomucoid in Balb/c mice reveals common epitope with human epitope sequence for ovomucoid

To evaluate the immunological response of the modulated derivative of the ovomucoid gene in a murine model system (Balb/c), the IgE binding epitopes of ovomucoid in mice, has been determined (P. Rupa, Y. Mine, unpublished data). The T-cell epitope mapping of ovomucoid in mice has been recently reported (14). Antigenic determinants of ovomucoid recognized by mice sera were analysed by mapping the whole ovomucoid sequence using overlapping peptides synthesized on a derivatized nitrocellulose membrane. The linear epitopes correspond to the following sequence of the second domain of ovomucoid GKVMVLCNRAFNPVC and YGNKCNFCNAVVESN of the third domain of ovomucoid (Fig. 2). The two reactivity peptides that were recognized by the sera from the Balb/c mice present in the second domain (II) region and two major epitopes in the third domain (III) region of ovomucoid were adjacent to each other and had partially overlapping sequence (P. Rupa, Y. Mine, unpublished data). Interestingly, the major epitopes containing peptide sequence, which is recognized by the Balb/c mice in the third domain, were also the major epitopes recognized by the egg allergic human patient's sera showing homologous sequence in common to both the system (P. Rupa, Y. Mine, unpublished data; 12). Amino acids critical to the IgE binding epitope of the ovomucoid allergen identified by patient's sera were targeted earlier and substitution of the critical amino acid at this epitope in the third domain of ovomucoid at position 162, where glycine was substituted to methionine (G–M) and phenylalanine at position 167 was substituted to alanine (F–A), led to complete loss of activity of IgE binding with patient's sera (7). In Balb/c mice, the relative intensity of the spot was very strong with the epitopes, in the third domain of ovomucoid, and was very faint with the epitopes in the second domain, which agrees with our previous data that the third domain is more allergenic than the first and the second domain (5).

image

Figure 2.  Sequential IgE epitopes recognized by Balb/c mice sera on the primary sequence of ovomucoid allergen. Amino acid sequence (residues) of ovomucoid are shown and amino acids in bold correspond to the IgE-binding amino acids of the entire ovomucoid molecule.

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Engineered ovomucoid DIII completely blocked hypersensitivity reactions and histamine release

As we had confirmed very weak IgE binding-activity with the GMFA against patient's sera (7), we performed hyposensitization using this modified variant of GMFA in sensitized mice and analysed various parameters. Anaphylactic scores have been reported to be associated with plasma histamine levels (15). Severe symptoms were observed in mice sensitized with native DIII inducing strong anaphylactic reactions (Fig. 3A). In contrast, mice desensitized with the GMFA antigen did not show any symptom. No anaphylactic reactions were observed in control mice. The plasma histamine levels were markedly elevated in DIII sensitized mice, whereas the histamine levels of the desensitized mice treated with GMFA antigen were the same as adjuvant treated control mice (Fig. 3B). We observed that those mice with a higher score also had the highest levels of plasma histamine indicating that histamine could be one of the mediators of anaphylaxis in this model. These results establish protection by GMFA antigen against DIII-induced hypersensitivity and blocked histamine release.

image

Figure 3.  Characterization of hypersensitivity symptoms (2A) and serum-histamine levels (2B). Blood was obtained from all immunized mice at the end point of the experiment and hypersensitivity symptoms were scored and serum-histamine levels were analysed compared with a standard histamine curve as described in Methods. Mice (n = 4 per group) were challenged intraperitoneally with native DIII at day 0 and 7. DIII group were desensitized with native DIII and GMFA group was treated by modified recombinant DIII (GMFA) at days 21, 28 and 35. Control animals were injected PBS. All mice were subjected to systemic anaphylaxis (ASA) with 1 mg/ml ovomucoid DIII i.p. injection at day 42.

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Decrease in production of ovomucoid-specific IgE, IgG1 and increase in IgG2a in vivo in desensitized mice

Total and specific levels of IgG and IgE were analysed in the serum samples obtained from the control, sensitized and desensitized groups (Fig. 4). Specific IgG1 and IgG2a antibodies were also measured in serum samples using ELISA (Fig. 5). There were no discrete differences in the levels of total IgG and IgE between the sensitized control group and the desensitized group (Fig. 4). Specific IgG level was high in mice desensitized with GMFA (P < 0.01). Animals desensitized with GMFA had significantly lower levels of allergen-specific IgE (P < 0.01) compared with the mice treated with the native DIII antigen. Induction of IgG1 antibody levels were reduced strikingly in mice desensitized intraperitoneally with the modified ovomucoid antigen and was followed by marked abrogation of suppression of IgG2a response (P < 0.01) (Fig. 5). Antibody response detected in the control mice immunized with PBS was significantly very low when compared with the treatment groups.

image

Figure 4.  Serum levels of total IgG and IgE and specific IgG and IgE as determined by ELISA. Mice were intraperitoneally immunized with DIII and desensitized with GMFA antigen as described in methods section. The other set of mice received DIII throughout and control mice received PBS in aluminum hydroxide. Sera from different groups of mice (n = 4) as indicated were obtained at the end point on day 42 from both the sensitized, desensitized and control groups were analysed. Total IgG and IgE and serum-specific IgE, IgG were determined by ELISA in groups of four mice per antigen. Data are represented as means ± SEM for each group (*P < 0.001 vs control group; **P < 0.01 vs Intact DIII; ***P < 0.01 vs control group). The animal trial was carried out twice.

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image

Figure 5.  Determination of Specific IgG1 and IgG2a levels by ELISA in DIII (sensitized), GMFA (desensitized) or control (PBS) treated groups. Mice were sensitized, desensitized and challenged and sera was collected and analysed as described in Methods. Values are expressed as means ± SEM (n = 4 per group). Data are represented as means ± SEM for each group (*P < 0.001 vs control group; **P < 0.01 vs Intact DIII; ***P < 0.01 vs control group).

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Suppression of allergen specific Th2 response and augmentation of Th1 response in desensitized mice

Immunoglobulin E response is related to the balance between cytokines IL-4 and INF-γ produced by activated Th1 and Th2 cells (16). In order to determine whether the desensitization with the GMFA antigen modulates Th2 response, we investigated analysis of splenocyte cytokine profiles in both the control-sensitized group and desensitized group in vitro. Splenocytes from mice treated with the modified ovomucoid antigen showed significantly reduced levels of IL-4 production (P < 0.01; Fig. 6) and induced IFN-γ production in spleen-cell culture supernatant subjected to antigen re-stimulation with the native DIII antigen (P < 0.01). We observed a discrete shift in cytokine profile from Th2 to Th1. The immediate systemic immune response was Th1-type, with increase in IL-12 and IFN-γ levels. The levels of IL-10 and IL-12 were also considerably high in the desensitized group (P < 0.01). For control group treated with buffer alone, there were minimal levels of cytokines detected using ELISA.

image

Figure 6.  Cytokine production after in vitro restimulation with DIII of splenocyte culture supernatants from BALB/c mice sensitized and desensitized groups. Individual spleen from each mice was excised and splenocytes were isolated and levels of cytokines were checked in comparison with standard cytokines and data is represented as mean ± (SEM) of four mice (*P < 0.001 vs control group; **P < 0.01 vs intact DIII; ***P < 0.01 vs control group; ****P < 0.001 vs intact group). Non-Ag-stimulated cultures showed very low cytokine production.

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Discussion

  1. Top of page
  2. Abstract
  3. Methods
  4. Results
  5. Discussion
  6. Acknowledgment
  7. References

Hyposensitization or desensitization is a reduction in sensitivity after challenge with the relevant allergen. Allergen-SIT can prevent the progression of allergic diseases (17). It has now become possible to analyse the immunological effects of immunotherapy using recombinant allergen molecules and for the controlled reduction of allergen activity (18). Modulation of allergen-specific IgE responses is key targets for successive immunotherapy, which could be achieved by targeting the critical IgE reactivity site in an allergen. Mutation of the recombinant allergen to modify the IgE binding epitope results in change in surface topography and folding pattern of the allergen and disruption of the native conformation of the allergen. Although two-third of egg allergy is outgrown with age in children, in some, it could be life threatening. We have earlier demonstrated that double mutation of IgE binding epitope of ovomucoid completely abrogates specific IgE binding against egg allergic patient's sera (7). It is of interest to note that the immunodominant epitope binding sequence against Balb/c mice sera were homologous to human IgE binding sequence and this prompted us to go with the murine model system, as a model for egg allergy to validate the efficacy of the modified ovomucoid DIII antigen.

Desensitization experiments with homologous sequences have been attempted recently in mice model and have been shown to down-regulate peanut-specific immune response (19). Although variations could occur in response to the modified ovomucoid DIII antigen (GMFA) in mice and human, it is noteworthy to try if the mutated antigen would suppress and down-regulate ovomucoid DIII-specific immune response. Elimination of all anaphylaxis symptoms were observed in mice desensitized with the modified ovomucoid DIII antigen and significant reduction in plasma histamine levels indicated that the GMFA antigen could obliterate hypersensitivity reactions in ovomucoid DIII-sensitized mice.

Complete protection of the allergic symptoms by the GMFA antigen was observed using several parameters like anaphylaxis scoring, serum-histamine levels, specific antibody levels and with the modulation in the cytokine profile. In mice allergic disease model, it is confirmed that IgG1, IgG2a and IgE antibody isotypes levels are reflections of cytokine secreting Th1/Th2 response (16). Th1 cells secrete IFN-γ and promote cellular immunity and the production of IgG2a. Th2 cells in contrast are known to secrete IL-4 and induce production of IgE antibodies, thereby the Th1 and Th2 responses are reciprocally regulated. We found that GMFA antigen inhibited antigen-specific IgE response and enhanced specific IgG and IgG2a production in desensitized mice in contrast to the control-sensitized group, which showed significant levels of specific IgE levels. The reduction in IgE binding of the desensitized mice was further confirmed in results obtained by standard histamine release assays. The control groups sensitized with the DIII did not reduce histamine release. Desensitization with the GMFA may have induced IgG blocking antibodies which inhibit allergen-IgE interaction by binding to the same epitopes, on other hand they may bind to surface regions which are not affected by the mutations and cause significant suppression in IgE binding to the natural allergen by sterical hindrance, thereby no longer binds IgE from allergic serum and instead induces a strong Th1 (IFN-γ) response. Various immunological approaches have been discussed recently for the treatment of food allergic disorders (20). GMFA antigen effectively prevented development of a Th2 immune response by lowering the levels of IL-4 production and allowed a strong Th1 response in the desensitized group by enhancing the levels of INF-γ. While Th2 cytokines trigger an Ig isotype switch towards production of IgE and IgG1, the Th1 cytokine IFN-γ induces production of IgG2a by B-lymphocytes (21). It was evident from our results that there is an existence of a close link between Th1 cytokine induction and immunoglobulin isotype switch in mice that received the modified antigen. It was also evident that production of IL-10 is associated with improvement of allergic symptoms (21–24). We found elevated levels of IL-10 and IL-12, which are again a Th1 inducing cytokine. Therefore increase in IL-12 drives production of INF-γ to more of a Th1 skewed response thereby suppressing allergic symptoms by reduction in specific IgE, which is important for Th2 response. In conclusion we have shown that desensitization with the hypoallergenic variant of ovomucoid DIII could completely protect allergic symptoms and successfully abrogate allergic mechanisms in mice.

Acknowledgment

  1. Top of page
  2. Abstract
  3. Methods
  4. Results
  5. Discussion
  6. Acknowledgment
  7. References

This work was supported by a grant from Natural Sciences and Research Council of Canada (NSERC) under a discovery grant program.

References

  1. Top of page
  2. Abstract
  3. Methods
  4. Results
  5. Discussion
  6. Acknowledgment
  7. References
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