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Keywords:

  • cattle;
  • expression analysis;
  • PD-1;
  • T-cells

ABSTRACT

  1. Top of page
  2. ABSTRACT
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. ACKNOWLEDGMENTS
  7. REFERENCES

Recent work has shown that PD-1, an immune inhibitory receptor, is involved in mechanisms for down-regulating immune responses during tumor progression or chronic viral infection. However, in the case of bovine diseases, there have been no reports on this molecule due to lack of information about bovine PD-1. In this study, we performed identification and preliminary characterization of the bovine PD-1 gene in two breeds of cattle. We cloned full cDNA sequences encoding for PD-1 from both Holstein-Friesian and Japanese Black breeds, and found that both of the genes encoded a 282-amino acid protein, which had a signal sequence, transmembrane domain and an immunoreceptor tyrosine-based inhibitory motif. This bovine PD-1 showed 72.9% and 65.6% homology to human and mouse PD-1, respectively, both of which have been well characterized and documented. Quantitative real-time PCR analysis showed that bovine PD-1 is expressed predominantly in T-cells (such as CD4+ and CD8+ cells) and among PBMCs, and is strongly upregulated on T-cell stimulation via ConA. A limited number of cattle were tested yet, as expected, the degree of PD-1 mRNA expression in CD4+ and CD8+ T-cells was greater in cattle with bovine leukemia virus-induced lymphoma than in uninfected cattle. Further studies to characterize the functions of bovine PD-1 are therefore warranted, in order to elucidate the mechanism of the immunosuppression associated with progression of several diseases and therapy in cattle.

List of Abbreviations: 
AL

aleukemic

BLV

bovine leukemia virus

cDNA

complementary DNA

ConA

concanavalin A

CTL

cytotoxic T-lymphocyte

DC

dendritic cells

DNase I

deoxyribonuclease I

EBL

enzootic bovine leukemia

HTLV

human T-lymphotropic virus

ITIM

immunoreceptor tyrosine-based inhibitory motif

LCMV

lymphocytic choriomeningitis virus

MAb

monoclonal antibodies

PBMCs

peripheral blood mononuclear cells

PD-1

programmed death-1

PD-L1

programmed death-ligand 1

PL

persistent lymphocytosis

RACE

rapid amplification of cDNA ends

SD

standard deviation

SIV

simian immunodeficiency virus

VMRD

Veterinary Medical Research and Development

Cell-mediated immune reactions are essential for clearance of pathogens, including viruses, from hosts. However many pathogens, such as HIV and HTLV, are able to escape from the immune system. In chronic viral infections, although cell-mediated immune cells are present in the hosts, these immune cells appear to be anergic: they are unable to produce sufficient cytokines, undergo cell proliferation nor activate CTL activity, thus these cells cannot clear pathogens effectively (1–3).

Recent studies have shown that the immune inhibitory receptor, PD-1 and its ligand, PD-L1, are involved in the mechanisms for induction of this anergic state in immune cells. PD-1 and PD-L1 belong to the B7-CD28 super-family, and studies using PD-1 and PD-L1 blockade models have shown that the PD-1/PD-L1 pathway provides an inhibitory signal for immune cells during infectious and autoimmune diseases and in cases of tumor (4–7). PD-1 is expressed on the membrane of activated T cells (but not resting T cells) and B cells (8), while PD-L1 is constitutively expressed on the membrane of T and B cells, DC, macrophages (9), and a wide range of non-hematopoietic cells (10). In chronic viral infections, PD-1 expression is upregulated on lymphocytes, specifically virus-specific CD8+ T cells (7, 11, 12), while PD-L1 is upregulated on myeloid DC (13, 14). This expression pattern appears to disable the pathogen-specific immune cells, preventing them from eliminating the infecting virus. It has been suggested that PD-1 and/or PD-L1 expression on immune cells can be induced by the presence of viral proteins (15, 16), prolonged antigen presentation (17), and cytokine in the environment around the immune cells (18). Blockade of the PD-1/PD-L1 pathway appears to restore the immune function of immune cells which have been in the anergic state, thus making them able to inhibit viral replication (19, 20). However, no functional analysis of these molecules has been reported for cattle and bovine diseases, mainly because of the lack of sequence information.

BLV belongs to the family of retroviruses, and is closely related to HTLV-1(21). Infection of cattle with BLV results in an untreatable disease which causes extensive economic losses in the dairy industry (22). Most BLV-infected cattle do not express clinical symptoms (are AL), but approximately 30–40% of BLV-infected cattle show PL, characterized by polyclonal expansion of B cells. Fewer than 5% of BLV-infected cattle eventually develop EBL 5–10 years after the infection. Recent work in our laboratory has revealed that cytokine production plays a critical role in the progression of infectious diseases such as BLV-infection (23–25). Immunosuppression concomitant with significant down-regulation of mRNA for interferon-γ and interleukin-12 has been observed during disease progression in cattle with both natural and experimental infection with BLV (24). Although host immunoregulatory factors, including expression of cytokines, are clearly involved in the pathogenesis of this infection (23), the exact mechanisms of immunosuppression and disease progression from AL to PL or EBL in BLV-infected cattle are not yet known. Thus, in this study, in an attempt to identify the PD-1/PD-L1 system promoting BLV-induced immunosuppression, we cloned, sequenced and characterized the cDNA encoding for bovine PD-1, and then measured the amounts of mRNA for bovine PD-1 in BLV-infected cattle.

MATERIALS AND METHODS

  1. Top of page
  2. ABSTRACT
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. ACKNOWLEDGMENTS
  7. REFERENCES

Cloning of full length cDNA encoding for bovine PD-1

PBMCs were purified by density gradient centrifugation on Percoll (Amersham Pharmacia Biotech, Piscataway, NJ, USA) from heparinized venous blood from two breeds of cattle, Holstein-Friesian and Japanese Black. To induce PD-1 expression, PBMCs (5 × 106) were incubated at 37°C for 4 hr with 5% CO2 in the presence of ConA, 5 μg/ml (Sigma-Aldrich, St Louis, MO, USA). Total RNA was extracted from cultivated PBMCs using the Trizol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions. Residual DNA was removed from the RNA samples by treatment with DNase I (Invitrogen). 3′- and 5′- RACE was performed using the 3′-/5′- RACE system for rapid amplification of cDNA ends (Invitrogen) by using PD-1 gene-specific primers, 5′-CTG CTG GCC AGG ATG GCT CCT AGA G-3′ and 5′-GAT GCT CAT GTT AAA CT-3′. After obtaining the 5′- and 3′-end sequences, we designed new primers, 5′-ATG GGG ACC CCG CGG GCG CT-3′, and 5′- TCA GAG GGG CCA GGA GCA GT-3′, and performed PCR to clone full length bovine PD-1 cDNA. The resulting amplification products were cloned into the pGEM-T easy vector (Promega, Madison, WI, USA) and sequenced using the CEQ2000 DNA analysis system (Beckman Coulter, Fullerton, CA, USA). Sequences determined were aligned, and an unrooted neighbor-joining tree was constructed using Mega4 software (26). All primers were synthesized by Hokkaido System Science (Sapporo, Japan).

PBMCs culture for analysis of PD-1 expression

To investigate the expression of PD-1 in bovine PBMCs under conditions mimicking the physiological environment of cells, PBMCs isolated from three healthy Holstein-breed cattle were individually incubated with MAb to either bovine CD4 (CACT138T, VMRD, Pullman, WA, USA), CD8 (IL-A51, a gift from the International Livestock Research Institute, Nairobi, Kenya), CD5 (CACT105A, VMRD) or CD14 (CAM36A, VMRD) at 4°C for 30 min followed by three washes with PBS. PBMCs were then labeled with anti-Mouse IgG1 particles (BD Biosciences, Franklin Lakes, NJ, USA) in BD IMag buffer at 4°C for 30 min, after which each cell type was enriched by positive magnetic separation using the BD IMagnet cell separation magnet (BD Biosciences). The purity of each cell type prepared by this method was confirmed with the EPICS XL flow cytometry system (Beckman Coulter) using EPICS EXPO32 ADC software. Highly purified cells (>90%) were used for analysis of PD-1 expression by quantitative real-time PCR. Furthermore, to determine whether PD-1 expression is upregulated in activated T cells, PBMCs were cultured in RPMI1640 (Invitrogen) containing 10% heat-inactivated FCS (Gibco Invitrogen, Carlsbad, CA, USA), 2 mmol/l L-glutamine, 100 U/ml penicillin and 100 mg/ml streptomycin, at a cell density of 1 × 107 per well in a 6-well culture plate (Corning, New York, USA) for 18 hr in the presence or absence of anti-bovine CD3 MAb (0.2 μg/ml, MM1A, VMRD) as a T-cell specific stimulator. ConA (5 μg/ml, Sigma-Aldrich) was used as a positive control for T-cell activation. RNA was later extracted from the cultured cells, and treated with DNase I followed by cDNA synthesis as described above.

Expression analysis of bovine PD-1 mRNA by quantitative real-time-PCR

Quantitative real-time PCR for PD-1 mRNA was performed in the Light cycler (Roche Diagnostics, Mannheim, Germany) using SYBR Premix DimerEraser (Takara, Shiga, Japan) following the manufacturer's instructions. Briefly, less than 100 ng of cDNA was mixed with 10 μl of SYBR Premix DimerEraser (Takara) and 0.6 μl each of primers (10 pmol/μl) in a total volume of 20 μl. Primers used were 5′-AAT GAC AGC GGC GTC TAC TT-3′ and 5′-GAT GAC CAG GCT CTG CAT CT-3′ for PD-1, and 5′-CGC ACC ACT GGC ATT GTC AT-3′ and 5′-TCC AAG GCG ACG TAG CAG AG.-3′ for β-actin. The cycling conditions consists of initial template denaturing at 95°C for 30 s, followed by amplification of template for 45 cycles of 95°C for 5 s, 60°C for 30 s and 72°C for 30 s (β-actin) or 60 s (PD-1). A final melting curve analysis was performed from 65°C to 95°C at a rate of 0.1°C/s (continuous acquisition), with a final cooling to 40°C over 10 s. Each amplification procedure was done in triplicate, and the results of PD-1 mRNA expression are presented as a ratio obtained by dividing the concentration of PD-1 mRNA by that of β-actin mRNA.

PD-1 expression analysis in BLV-infected cattle

To evaluate bovine PD-1 mRNA expression in BLV-infected animals, expression analysis was conducted in three cattle diagnosed with BLV-induced leukemia at the Veterinary Teaching Hospital, Graduate School of Veterinary Medicine, Hokkaido University, between 2008 and 2009. The animals with leukemia were diagnosed clinically, and BLV infection was confirmed by nested-PCR as described previously (27). Flow-cytometric analysis and quantitative real-time PCR for PD-1 mRNA expression in freshly isolated PBMCs were conducted as above.

Statistics

Mean values were compared in Figures 2a and 3 using Student's unpaired t-test, and in Figure 2b using Student's paired t-test. Differences between groups were considered significant if probability values of P < 0.05 were obtained.

image

Figure 2. Analysis of bovine PD-1 mRNA expression by quantitative real-time PCR. (a) PD-1 mRNA expression was determined in total PBMCs from Holstein cattle, subpopulations of CD4+, CD8+, CD5+, CD14+ cells, and negative fractions of these subpopulations. The latter cells were purified from PBMCs obtained from healthy cattle. (b) PD-1 mRNA expression in PBMCs incubated with or without either anti-bovine CD3 antibody (αCD3) or ConA. The degree of PD-1 mRNA expression is shown as the ratio obtained by dividing the concentration of the PCR products from PD-1 mRNA by those from β-actin mRNA. Asterisks donate significant differences between the type of cells (*; P < 0.05 and **P < 0.01). Error bars represent the SD of the means among the four cows.

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image

Figure 3. Analysis of bovine PD-1 mRNA expression in BLV-infected cattle. (a) Flow cytometric analysis of PBMCs from BLV-negative (n= 4) and BLV-infected cattle with leukemia (n= 3). Quantification of mRNA expression for PD-1 in (b) isolated CD4+ cells, (c) CD8+ cells and (d) CD5+ cells by real-time PCR analysis. The degree of PD-1 mRNA expression is shown as the ratio obtained by dividing the concentration of PCR products from PD-1 mRNA by that from β-actin mRNA. BLV (–), BLV negative.

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RESULTS

  1. Top of page
  2. ABSTRACT
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. ACKNOWLEDGMENTS
  7. REFERENCES

Sequence analysis of bovine PD-1

To clone the bovine PD-1 gene, total RNA isolated from bovine PBMCs was used to synthesize total cDNA from which the full gene sequence was generated by 5′- and 3′-RACE. The full-length bovine PD-1 cDNA sequence and its putative amino acid sequence obtained from Holstein cattle is shown in Figure 1a (GenBank accession number AB510901). The complete nucleotide sequence is 1708 bp in length, and contains 44 nucleotides upstream of the initial ATG codon and 815 nucleotides downstream of the stop-codon. The Kozak consensus sequence and the polyadenylation site, ATTAAA, are located at the translation start site and 17 bases upstream of the poly (A) tail, respectively. The Japanese Black breed cattle-derived PD-1 cDNA sequence was found to be identical to that obtained from Holstein breed cattle (Table 1). These sequences were also identical to that of the cDNA from Hereford breed cattle on the NCBI database (BC123854). In comparison to other artiodactyls species, the predicted amino acid sequence of bovine PD-1 was found to be most similar (98.6%) to that of water buffalo. The bovine PD-1 has amino acid identities of 76.6%, 73.6%, 72.9%, 68.3%, 65.6% and 50.4% to cat, monkey, human, rat, mouse and chicken PD-1, respectively (Table 1). Sequence analysis of bovine PD-1 showed that the amino acid residues 1 to 26 represent a putative signal peptide, and that the residues 172 to 194 represent the transmembrane domain (Fig. 1b). Its extracellular domain, corresponding to residues 27 to 171, contains the immunoglobulin domain. The immunoreceptor tyrosine-based inhibitory motif (ITIM: I/L/V/S/TxYxxL/V/I) pair corresponding to residues 223 to 228 and 248 to 253, is conserved in bovine PD-1 (Fig. 1b). Phylogenetic analysis revealed that vertebrate PD-1 is divided into two groups, mammalian (Primate, Rodentia, Carnivora and Artiodactyla) and avian, with bovine PD-1 clustering in the artiodactyl species group (Fig. 1c).

image

Figure 1. Cloning of bovine PD-1. (a) Nucleotide and deduced amino acid sequences of cDNA encoding for bovine PD-1. A bold line indicates a poly(A) addition signal. (b) Alignment of deduced amino acid sequences of PD-1 from several animal species. Amino acid residues identical to bovine PD-1 (cattle) are boxed in black, and conservative substitutions are shown in gray letters. Bold lines indicate predicted signal (1–26) and transmembrane domain (172–194) of bovine PD-1. The proposed immunoreceptor tyrosine-based inhibitory motif (ITIM: I/L/V/S/TxYxxL/V/I) pair is boxed. (c) A phylogenetic tree constructed based on the nucleotide sequences of the PD-1 genes of animal species. The tree was built using the neighbor-joining method by the MEGA 4.0 software. Numbers indicate the bootstrap percentage (100 replicates). The scale indicates the divergence time.

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Table 1.  The similarities in nucleic and amino acid sequences (%) of PD-1 among animal species
Species (GenBank accession number)Cattle (Holstein)Cattle (Japanese Black)Cattle (Hereford)Water BuffaloCatMonkeyHumanRatMouseChicken
  1. Upper section shows percent homologies in nucleic acid level, and lower section shows those in deduced amino acid level. GenBank accession numbers are in parentheses after species names.

Cattle (Holstein)-10010097.578.875.174.967.866.658
Cattle (Japanese Black)100-10097.578.875.174.967.866.658
Cattle (Hereford, BC123854)100100-97.578.875.174.967.866.658
Water Buffalo (FJ827144)98.698.698.6-78.875.474.768.567.558
Cat (NM 001145510)76.676.676.678.4-73.773.967.266.355.1
Monkey (NM 001114358)73.673.673.67473.6-95.871.971.353.6
Human (NM 005018)72.972.972.973.373.398.3-7271.455.5
Rat (NM 001106927)68.368.368.369.365.972.271.2-91.353.8
Mouse (NM 008798)65.665.665.66764.272.671.590.3-54.9
Chicken (XM 422723)50.450.450.452.148.849.348.347.751-

Comparative expression analysis of PD-1 in various bovine PBMCs-derived cell types

In order to investigate the degree of expression of bovine PD-1 mRNA in PBMCs, we used real-time PCR to quantify the expression of bovine PD-1 mRNA in fresh and cultured cells. Firstly, we evaluated bovine PD-1 mRNA expression kinetics in several cell types among PBMCs (mean ± SD: 7.288 ± 3.678). As expected, expression in CD5+ cells (12.882 ± 7.098), a pan lymphocyte fraction, was greater than that in CD14+ cells (0.864 ± 1.196), which are representative of monocytes (Fig. 2a). More specifically, the mean degree of expression of PD-1 in CD4+ T cells (15.096 ± 4.770, P < 0.05) and CD8+ T cells (17.656 ± 7.736, P < 0.05) was significantly greater than that in CD14+ cells (Fig. 2a). PD-1 mRNA expression was also observed in the negative fraction (7.987 ± 1.082), which was considered to contain natural killer cells and B cells among others. To determine the effect of T-cell stimulation on bovine PD-1 mRNA expression, PBMCs from healthy animals were cultured in the presence of anti-CD3 MAb. As shown in Figure 2b, the addition of this MAb enhanced bovine PD-1 mRNA expression in PBMCs (13.895 ± 1.247) to a magnitude similar to that induced by ConA (16.61 ± 2.911, P < 0.05), a lectin which induces T-cell proliferation by specific interaction with the T-cell receptor complex. There were no notable differences in the degree of expression of bovine PD-1 mRNA before and after cell cultivation without stimulation (9.551 ± 4.448).

PD-1 expression analysis in BLV-infected cattle with leukemia

To evaluate the degree of PD-1 mRNA expression in BLV-infected animals, we examined PD-1 mRNA expression in PBMCs freshly isolated from BLV-infected and uninfected cattle, although a limited number of cattle were tested (Fig. 3). In BLV-infected cattle with leukemia, the mean degree of expression of PD-1 in CD4+ T cells (50.05 ± 51.10) and CD8+ T cells (25.52 ± 23.30) was greater than that of control cattle (CD4+ T cells: 15.10 ± 4.770, CD8+ T cells: 17.66 ± 7.736), although the difference was not statistically significant (Fig. 3b and c). On the other hand, expression in unusual expanding CD5+ cells (2.884 ± 3.454), as BLV-target cells, was less than that in control cattle (12.88 ± 7.098).

DISCUSSION

  1. Top of page
  2. ABSTRACT
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. ACKNOWLEDGMENTS
  7. REFERENCES

PD-1 plays a crucial role in immunomodulation. Binding of PD-1 to PD-L1 downregulates immune responses, and many recent reports suggest that this immune modulation is exploited in chronic viral infections in human and mice in which pathogens evade immune surveillance (28). In cattle, however, there has been no genetic information on PD-1 nor information on the role of PD-1/PD-L1 molecules. The present study provides genetic information about bovine PD-1 and sets the stage for elucidation of the functions of PD-1/PD-L1 molecules in cattle. Our findings with regard to the genetic characteristics of bovine PD-1 underscore earlier reports which suggest that there is structural conservation of PD-1 among several vertebrate species. By comparative analysis, the hydrophobic N-terminal (signal sequence) and the transmembrane domains are almost conserved in bovine PD-1. In addition, the immunoreceptor tyrosine-based inhibitory motif, a characteristic feature in inhibitory receptors, was also found to be conserved in bovine PD-1. In the human and mouse, several immunomodulatory activities of PD-1 have been reported: PD-1 has been shown to be particularly expressed on CD4+ and CD8+ T cells and on activated lymphocyte cells. Our findings show that the bovine PD-1 sequence is highly similar to human and murine PD-1 sequences and that, like human and murine PD-1, bovine PD-1 is also predominantly expressed in T-cells, including CD4+ and CD8+ cells, and its expression is up-regulated on specific stimulation of T-cells. These observations raise the possibility that bovine PD-1, similarly to human PD-1, can act as an immune inhibitory receptor. However, detailed analyses will be needed to elucidate its exact role in modulation of the immune system in cattle.

The pathways in which PD-1 and PD-L1 are involved have been recently identified and shown to contribute significantly to the development of aberrant immunoregulatory states in chronic viral infections in the mouse and human (20, 29). According to established reports, the negative regulatory PD-1/PD-L1 pathway has been implicated in the induction of effecter lymphocyte exhaustion during chronic viral infection. Indeed, expression of PD-1/PD-L1 on lymphocytes has been closely correlated with disease progression in hosts infected with HIV, SIV (20, 29), LCMV (30) and HTLV-1 (11, 12). In particular, an intimate relationship between PD-1/PD-L1-induced CTL exhaustion and disease progression has been well documented in retroviral infections such as HIV and HTLV-I which are structurally and functionally related to BLV. Thus, to gain preliminary insights into how the expression of bovine PD-1 influences disease outcome in cattle, we firstly determined the expression profile of PD-1 in cattle naturally infected with BLV, albeit with a limited number of experimental animals. Contrary to the prediction that PD-1 mRNA expression in whole PBMCs would be augmented in cattle infected with BLV, the magnitude of PD-1 mRNA expression in infected cattle was not statistically different when compared to control uninfected animals, though notable upregulation of PD-1 mRNA expression was observed in some BLV-infected animals (data not shown). This discrepancy can be attributed to the fact that the progression pattern of BLV infection in these animals was not established. Upregulation of PD-1 expression has been reported in T-cells derived from HTLV-1 infected adults with symptoms of leukemia/lymphoma, but not from asymptomatic HTLV-1 carriers (11). Similarly, the observations in the present study may be due to the animals used in the experiment having been in the aleukemic stage, though infected. Therefore, in order to investigate the differences in degree of expression of PD-1 in BLV-infected cattle in the late stages of disease, we used real-time PCR to quantify the expression of PD-1 mRNA in fresh PBMCs-derived cell types isolated from cattle with leukemia (Fig. 3). A limited number of cattle were tested (three Holstein) yet, as expected, the degree of PD-1 mRNA expression in CD4+ and CD8+ T-cells was higher in cattle with leukemia than in uninfected cattle, although the difference was not statistically significant. The PD-1mRNA in CD5+ T-cells was less expressed than in control cattle. Thus, these results suggest that BLV can influence the degree of expression of PD-1 in effector cells, such as CD4+ and CD8+ T-cells, and subsequent elimination of BLV as HTLV-1 infection (11). However, to clearly elucidate the correlation of PD-1 dynamics with disease progression in cattle, a detailed study with a large number of animals is required. Furthermore, prior to that study complete determination of the health status, or disease stage if infected, of the experimental animals would be necessary.

This is the first report on genetic characterization and expression of PD-1 in various sub-populations of T-cells in cattle. As has been reported in mouse and human PD-1, bovine PD-1 may also have important immunomodulatory roles in progression of various diseases in cattle. Further elucidation of the role of PD-1 in immunomodulation in cattle is necessary to fully understand the cell signaling pathways involved in the modulation of host immune responses. Studies are now in progress to evaluate the possible involvement of PD-1 in immunomodulation during disease progression in BLV-infected cattle.

ACKNOWLEDGMENTS

  1. Top of page
  2. ABSTRACT
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. ACKNOWLEDGMENTS
  7. REFERENCES

We are indebted to Dr. William H. Witola for help in preparing the manuscript. This work was supported by grants-in-Aid for Scientific Research from the Japan Society for the Promotion of Science (JSPS), and by a special grant for the Promotion of Basic Research Activities for Innovative Biosciences from the Bio-oriented Technology Research Advancement Institution (BRAIN).

REFERENCES

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  2. ABSTRACT
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. ACKNOWLEDGMENTS
  7. REFERENCES
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