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Transplantation Biology Research Center and Department of Pathology, Massachusetts General Hospital, Harvard Medical School, Boston, MA 02129
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Successful transplantation of allogeneic or xenogeneic thymic tissue might induce donor-specific tolerance through the deletion of newly developing T cells by the same mechanism that is used in the thymus for deletion of T cells with high affinity for self. Thus, colonization of the donor thymus with bone marrow precursors would lead to negative selection to donor MHC. Migration of host APCs into the grafts would also assure negative selection to host MHC, leading to a specific state of tolerance to both self and donor-specific non-self.
Thymic tissue engraftment has been achieved in immunocompetent mice after thymectomy, followed by lethal whole body irradiation (11, 12), total lymphoid irradiation (13) or T cell and NK cell depletion with mAbs (8). In a xenogeneic pig-to-mouse model involving host thymectomy as well as T and NK cell depletion, the recovery of a tolerant recipient T cell repertoire was shown to be due in part to intrathymic deletion of donor- and host-reactive recipient thymocytes developing in the xenogeneic thymic graft (8, 14, 15). Of importance for potential clinical applications is the fact that both allo- and xenogeneic thymic grafts have been shown to support essentially normal T cell development with normal host-restricted immunocompetence (16, 17, 18).
In an attempt to transfer this technique to large animals, we have initiated a series of experiments in inbred Massachusetts General Hospital (MGH)4 miniature swine. Miniature swine provide the unique opportunity to study the effects of selective MHC matching on parameters of immunity in a reproducible fashion (19). Kidney transplantation across a class I barrier was shown to be successful in 100% of cases when high dose CyA was administered during the first 12 days after transplantation (20). We have begun these studies by using the same immunosuppressive regimen and MHC mismatch for thymic transplantation in miniature swine. We report here successful transplantation of class I-mismatched juvenile thymic tissue and the ability of such tissue to support host thymopoiesis and confer donor-specific hyporesponsiveness.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Two- to three-month-old SLAgg (class
Icc/class IIdd) donors and
4- to 5-mo-old, class I-mismatched
SLAdd
(Idd/IIdd) recipients were
selected from our herd of partially inbred miniature swine. The
immunogenetic characteristics of this herd (21, 22) and of
the intra-MHC recombinant haplotypes (23) have been
described previously and are illustrated schematically in Fig. 1
. Each experiment used one thymic tissue
donor and two recipients. One of the two recipients in each experiment
was thymectomized before thymic transplantation.
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All animals received preoperative sedation with i.m. xylazine (2 mg/kg), atropine (0.04 mg/kg), and Telazol (1.4 mg/kg; Elkins-Sinn, Cherry Hill, NJ). Isoflurane inhalation anesthesia was administered before endotracheal intubation and was continued throughout the entire operative procedure at a level permitting spontaneous respiration. Perioperatively, each animal received one prophylactic dose of cefazolin (40 mg/kg i.m.).
Thymectomy
In the thymectomized group, a complete thymectomy was performed 3–4 wk before thymic transplantation using a combined midline cervicotomy and partial sternotomy as previously described (24). Briefly, the pretracheal muscles were retracted, and the trachea, internal jugular vein, and carotid artery were exposed from the mandibular area to the ventral pericardium. The entire thymus was then carefully dissected and removed. Adherent fatty tissue was also excised when necessary to ensure completeness.
Thymic transplantation
Partial thymectomy was performed in the thymic donor on day 0
through a midline cervicotomy after exposing the major portion of the
cervical thymus. Two pieces of thymus, 
3 cm3
each, were harvested, placed in 0.9% saline solution, and immediately
transferred to one euthymic and one thymectomized animal. The thymic
tissue was minced into 10-mm3 fragments and
implanted into three prepared sites in the recipients:
Sternocephalicus muscle.
Ten small pockets, each capable of holding five pieces of tissue, were
formed in each sternocephalicus muscle using mosquito clamps. 1
cm3 of minced thymic tissue was placed into the
pockets, and the sites were closed with 4-0 Prolene (Ethicon,
Somerville, NJ), which also served to mark the implantation sites for
future biopsies.
Omentum.
After midline laparotomy, a volume of 1 cm3 of
minced thymic tissue was wrapped into an edge of the omentum and closed
with 4-0 Prolene.
Kidney capsule. One cubic centimeter of minced thymic tissue was spread out under both kidney capsules as described previously (25). Briefly, a 1-cm incision was made in the kidney capsule directly opposite the hilum. The capsule was then carefully detached from the underlying cortex using an curved hemostat, and thymic tissue was inserted beneath it.
All recipients received two central venous Silastic catheters (Dow Corning, Midland, MI) placed into each external jugular vein via direct cut down. The catheters were tunneled s.c., exiting dorsally. One catheter was used for CyA administration, and the other for obtaining blood samples.
Skin grafting
Fresh split-thickness skin grafts (40 x 40 x 2.2 mm) were harvested from donors using a Zimmer dermatome and placed on graft beds, also prepared with a dermatome, on the lateral thorax as previously described (26). The day of rejection was defined as the time at which <10% of the skin graft showed signs of viability as judged by color, texture, and warmth to touch.
Immunosuppression
The only immunosuppressive treatment was a 12-day course of CyA, administered once per day to all animals beginning on day 0. The first dose on day 0 was 13 mg/kg body weight i.v. given intraoperatively. Animals were either maintained on i.v. solution or switched to an oral CyA solution (Neoral) on day 5, which was given by a gastrostomy tube inserted on the day of thymic transplantation. CyA trough levels were measured in whole blood by a mAb-based RIA. CyA dosage was adjusted daily to maintain a level between 400 and 800 ng/dl. The i.v. and oral (Neoral) preparations of CyA were provided by Novartis Pharmaceuticals (Hanover, NJ).
All procedures were performed in accordance with the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health (Publication 85-23, revised 1985) and were approved by the Massachusetts General Hospital animal research committee.
Isolation of PBL
Freshly drawn, heparinized whole blood was diluted with HBSS (Life Technologies, Gaithersburg, MD), and mononuclear cells were obtained by gradient centrifugation using lymphocyte separation medium (Organon Teknika, Durham, NC). The mononuclear cells were washed once with HBSS before the contaminating red cells were lysed with ACK lysing buffer (BioWhittaker, Walkersville, MD). Cells were then washed in HBSS again and resuspended in tissue culture medium (see below). Cell suspensions were kept at 4°C until they were used in cellular assays.
Mixed lymphocyte reaction
MLR cultures, to test for proliferative response to alloantigen, have been described previously (28). Briefly, 4 x 105 responders and an equal number of irradiated (25 Gy) stimulators were incubated in 200 µl of standard MLR medium using flat-bottom 96-well plates (Costar, Cambridge, MA). After 5 days of incubation, 1 µCi of [3H]thymidine was added to each well, followed by an additional 5-h incubation. 3H incorporation was determined in triplicate samples by liquid scintillation.
CML assays
CML assays were performed as described previously (27). Briefly, lymphocyte cultures containing 4 x 106 responder and 4 x 106 irradiated (25 Gy) stimulator PBL were incubated in 2 ml of medium for 6 days at 37°C in 7% CO2 and 100% humidity. These effector cells were harvested, and 5 x 106 cells were added to the first three wells of a 96-well round-bottom well before being serially diluted in triplicate (E:T cell ratio, 100:1, 50:1, 25:1, and 12.5:1). 51Cr (5 x 103; Amersham, Arlington Heights, IL)-labeled blasts generated from PHA (M-Form, Life Technologies) stimulation were used as specific targets and added to all wells. After 5.5 h of incubation at 37°C the supernatants were harvested with the Skatron collection system (Skatron, Sterling, VA), and 51Cr release was determined on a gamma counter (Micromedics, Huntsville, AL). Maximum lysis was obtained with a 5% solution of the nonionic detergent, Nonidet P-40 (BRL, Rockville, MD). Baseline levels were measured as the rate of spontaneous release of 51Cr from 5 x 103 targets. The results were expressed as the percentage of specific lysis: {[experimental release (cpm) - spontaneous release (cpm)]/[maximum release (cpm) - spontaneous release (cpm)]} x 100%.
Targets were matched to the stimulators, which included thymus and skin donor-matched PBL (SLAgg: class Icc/class IIdd), and skin donor-only-matched PBL (SLAhh: class Iaa/class IIdd) PBL. Targets SLA matched to the effectors were used as negative controls in all assays.
Humoral responses
Animals were tested for the presence of anti-donor cytotoxic Abs using a two-stage, complement-dependent lymphocytotoxicity assay as described previously (26). The medium used consisted of medium 199 (Life Technologies) with 1% FCS added as a protein source. Targets that were of the same SLA haplotype as the donor thymus were used to test for the presence of cytotoxic Abs. Specific lysis was measured using the same formula as that used in the CML assays (see above). All targets were tested against a positive control (alloantiserum) and a baseline serum sample from the experimental animal to control for nonspecific complement cytotoxicity. To determine whether cytotoxic activity was due to IgM or IgG, sera were incubated with 0.2 M DTT (Sigma, St. Louis, MO) to eliminate IgM activity before the assay. The cytotoxicity index was calculated by multiplying the maximum percent lysis by the reciprocal dilution of half-maximum lysis.
Flow cytometry
Flow cytometry was used to follow the course of serum Abs
sampled serially before and after thymic transplantation. Polyclonal,
fluoresceinated, goat anti-swine IgG (
-chain) or IgM (µ-chain)
Abs (Kirkegraard & Perry Laboratories, Gaithersburg, MD) were used as
second reagents after incubation for 30 min with the specific test
sera. All steps were performed at 4°C using flow cytometry buffer
(HBSS containing 0.5% BSA and 0.5% sodium azide). The total bound Abs
were measured as the median fluorescence.
Macrochimerism was determined by one-color indirect Ab staining. Mouse anti-pig mAbs reactive with SLA class I (2.27.3a: IgG1), class Ic (16.7.E4.2: IgM), and class Id (2.12.3a; IgM) as previously described (29) were added in separate tubes and incubated for 30 min at 4°C. Non-cross-reacting anti-mouse class I mAbs (36-7-5, IgG2a and 12-2-2, IgM) were used as negative control Abs. All Abs were FITC conjugated. After washing twice, cells were resuspended in 0.5 ml of flow cytometry buffer. Analysis was performed using a Becton Dickinson FACScan II (San Jose, CA).
Histology
Sequential biopsies were performed on the day of transplantation; wk 2, 4, 8, 12, 16, 20, and 24; and every 6 wk thereafter, as well as postmortum, and staining was carried out using hematoxylin and eosin. Every scheduled biopsy of the sternocephalicus muscle was performed by completely removing one of the 20 grafts.
Immunoperoxidase staining
Frozen sections of sequential thymic graft biopsies were analyzed using the avidin-biotin-HRP complex technique reported previously (30). Donor and recipient cells were stained with murine anti-pig mAbs. An SLA class Ic-specific mAb (16.7.E4.2) was used to identify donor tissue, whereas an SLA class Id mAb (2.12.3a) served as a marker for graft-infiltrating cells. Immature thymocytes were stained with anti-CD1 mAb (76-7-4: IgG2a) (31), whereas mature thymocytes were stained with anti-CD3 mAb (2-6-15: IgG2a) (24). Briefly, 2- to 4-µm sections were incubated with 1% normal horse serum to inhibit nonspecific binding of horse IgG and with avidin to block endogenous biotin. After 20 min, the tissue was covered with optimally diluted primary Ab (mouse anti-pig mAb) and incubated for 60 min at room temperature. Sections were rinsed with PBS and incubated in a solution of biotin (1 mg/100 ml in PBS) with 0.3% hydrogen peroxide for 30 min to block endogenous peroxidase. The biotinylated secondary Ab (horse anti-mouse) was added and incubated for 30–45 min. After a PBS wash, sections were incubated in an optimal dilution of avidin-biotin-peroxidase complex for 60 min, rinsed in PBS, and stained in a solution containing 3-amino-9-ethyl carbazole for 2–5 min. Staining was stopped by dipping slides into distilled water. Sections were then fixed in 4% paraformaldehyde and counterstained with Gill’s single-strength hematoxylin. Controls included omission of primary Ab or horse anti-mouse Ab.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To evaluate the optimal conditions required for thymic tissue engraftment and development, four animals received class I-mismatched minced thymic tissue placed into three distinct sites: sternocephalicus muscle, kidney capsule, and omentum. Two animals were thymectomized, and two were left euthymic before thymic transplantation to investigate the role of the recipient thymus in allogeneic thymic engraftment. CyA, given during the first 12 postoperative days, was the only immunosuppression used in these animals. The same immunosuppressive regimen has previously been shown to induce long term tolerance to SLA class I-mismatched kidney grafts in euthymic recipients, whereas untreated recipients uniformly rejected (20).
The animals in the first experiment (12311 Thx and 12364 EuThy) received a 12-day course of i.v. CyA. Their CyA trough levels were within the desired range between 400 and 800 ng/dl throughout this period. In the second experiment, the two animals (12578 Thx and 12579 EuThy) were switched to Neoral on day 5. Both animals had supratherapeutic trough levels and suffered from neurologic side effects (tremor and somnolence), but suffered no permanent sequelae.
Thymectomy before allogeneic thymic transplantation (12311 Thx and
12578 Thx) did not support engraftment in this model. In contrast, both
euthymic animals showed thymic graft development with evidence of host
thymopoiesis (12364 EuThy and 12579 EuThy). Initial graft biopsies
performed on these animals in all three sites at 2 and 4 wk showed
complete loss of thymic architecture (Fig. 2, a and b), with
clusters of mature recipient-type T cells, as indicated by positive CD3
and class Id staining (data not shown). These
cells were most likely infiltrating T cells rather than locally
maturing T cells, although the latter cannot be ruled out. As judged by
immunohistochemistry, stromal cells expressed mainly donor-type SLA
class I (Fig. 3, d and
f), whereas the vascular endothelial cells expressed either
donor or host-type SLA class I (Fig. 3, c–f), indicating
partial revascularization by the host. Immature, CD1-positive
thymocytes were decreased at wk 2 (compared with the normal thymus) and
were undetectable at wk 4. These immature T cells were probably
remaining donor cells that disappeared over the first 4 wk. The thymic
grafts were then progressively repopulated with CD1-positive T cells in
both euthymic animals. The CD1 positive cells (Fig. 3b)
expressed class Id weakly (Fig. 3, c
and e), consistent with thymopoiesis involving host T cell
progenitors. The percentages of CD1- and class
Id-positive host thymocytes were used as
measurements of thymic graft development. These levels continuously
increased compared with the levels of mature T cells observed in the
thymic grafts in both animals until wk 16 (Table I). The immature T cells were distributed
around the mature donor-type T cells, forming a thymic neocortex and
medulla. Within the medulla, new Hassall’s corpuscles developed (Figs. 2c and 3). By wk 12, the grafts could not be distinguished
histologically from native thymus except by size. Macroscopically, the
size of the i.m. grafts increased to 3–4 times that of the original
implants. The omentum grafts also increased in size, but the kidney
capsule grafts did not. Histology obtained on grafts from the omentum
and kidney capsule revealed the same findings as those from the
sternocephalicus muscle (data not shown). However, because the
procedure for taking biopsies from the muscle was technically easier
and less invasive, muscle grafts were biopsied preferentially at later
time points. The histologic appearance of the thymic grafts was
evaluated at 4-wk intervals, and with each biopsy performed, at least
one graft was excised. Therefore, the total amount of thymic grafts
continuously decreased. The euthymic animals were followed over a
period of 12 mo after transplantation, until the majority of the grafts
were removed. In one of the animals (12579 EuThy), the remaining grafts
were structurally intact (Fig. 2d). The remaining grafts of
the other animal (12364 EuThy) may have been rejected after the
recipient was sensitized with donor skin grafts (see below).
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Decrease in donor-specific T cell response in thymic-grafted animals
MLR responses in naive animals were mainly directed against SLA class II, whereas the SLA class I proliferative responses were generally low (SI = 2–10). In contrast, CML responses in naive animals directed against SLA class I were always vigorous (32).
Rejection of the thymic grafts in the thymectomized animals led to a
marked increase in anti-donor MLR (12311 Thx: SI = 35; 13578
Thx: SI = 43; measured at wk 8), consistent with sensitization
(Fig. 4). In contrast, euthymic animals
that accepted their thymic grafts continued to have low anti-donor
responses, suggesting that sensitization did not occur.
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). Anti-third-party CML responses were
generally normal, but anti-SLAhh responses
were diminished, suggesting possible cross-tolerance to determinants
shared by class I of SLAc and
SLAa (Fig. 1
). Stimulators from fully allogeneic
outbred (Yucatan) animals produced vigorous responses in nearly all
experiments. Representative data for one of the euthymic
animals (12579 EuThy) at wk 0, 4, and 8, demonstrating the development
of donor specific nonresponsiveness, are illustrated in Fig. 5.
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Thymic grafted animals did not show humoral tolerance
Anti-donor cytotoxic Abs were detected 4 wk after thymic
transplantation in both thymectomized and euthymic animals, as measured
on donor-type PBL. Serum treatment with DTT did not diminish the
cytotoxicity, suggesting that Abs of IgG class were responsible for at
least part of the complement-mediated cytotoxicity. Representative data
for one of the two engrafted animals (12579 EuThy) are shown in Fig. 6.
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Flow cytometry was performed to assess macrochimerism in a thymectomized and a euthymic animal (12311 Thx and 12364 EuThy). PBL, gated on lymphocytes, were stained for donor-type SLA class I. Samples were obtained from both animals on days 0, 4, 7, 10, 16, and 30 and again on day 126 in the euthymic pig only (12364 EuThy). In addition, the host thymus was analyzed in the same animal on days 0, 16, and 126. No SLA class Ic-positive cells could be detected at a detection level of 0.05% (data not shown).
Influence of skin graft rejection on thymic graft survival
In an attempt to determine whether tolerance to the thymic donor
might have been induced, skin grafts obtained from the original thymic
donor were transplanted onto each thymic graft recipient, 8 wk after
thymic transplantation. Naive animals from our inbred herd of miniature
swine generally reject class I-mismatched skin grafts within 8–10 days
(33). Both animals that were thymectomized before thymic
transplantation rejected the skin grafts between 9 and 10 days (12311
Thx and 12578 Thx, respectively). One of the euthymic animals (12364
EuThy) also rejected in 10 days, while the other (12579 EuThy) showed a
prolonged skin survival of 18 days. After skin graft rejection,
subsequent thymic graft development was impaired in both of these
animals, as measured by the relative levels of immature CD1-positive
thymocytes compared with the mature T cells in the grafts (Table I). In
addition, both euthymic animals experienced an immediate increase in
their levels of cytotoxic Abs (Fig. 6), and an increase several weeks
later in their anti-donor CML responses (Table II). This apparent
rejection process was seen only transiently in the animal with
prolonged skin graft survival (12579 EuThy). The other animal (12364
EuThy) may have rejected its remaining thymic grafts between 28 and 40
wk after skin grafting (i.e., 36 and 48 wk after thymic
transplantation).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Class I-mismatched kidney grafts are uniformly accepted in euthymic pigs when transplantation is followed by a 12-day course of CyA. In contrast, such kidney grafts are rejected when transplanted without immunosuppression (20). Therefore, we adopted the same CyA treatment regimen and SLA disparity for development of a thymic transplantation model. We compared animals who underwent thymectomy before thymic transplantation to those who remained euthymic. Because previous studies in rodent models have demonstrated host thymectomy to be an absolute requirement for allogeneic or xenogeneic thymic tissue engraftment, it seemed possible that the same might be true in miniature swine (13, 35). In theory, the host thymus might be expected to continuously produce allo-reactive T cells and cause rejection of the thymic graft, while removal of the host thymus in conjunction with T cell depletion might allow the transplanted thymus to survive and sustain T cell development after engraftment.
Contrary to this expectation, not only was removal of the host thymus found to be unnecessary to permit engraftment of class I-mismatched thymic tissue, but such thymectomy was also found to be detrimental. Thus, after host thymectomy and class I-mismatched thymic transplantation followed by a 12-day course of CyA, sequential graft biopsies revealed that as early as 2 wk and continuing throughout the entire observation period, there was no evidence for viable thymic graft tissue. In contrast, the nonthymectomized animals successfully engrafted, and by 8 wk post-transplant the grafts had differentiated into tissue that fulfilled all the morphologic criteria of a functional thymus. The grafts were repopulated with immature, host-type thymocytes and showed normal thymic architecture, including a neocortex and medulla. In subsequent weeks, the relative number of immature thymocytes continuously increased, and Hassall’s corpuscles developed.
This requirement for the presence of the host thymus is probably peculiar to the use of our CyA treatment regimen for induction of tolerance across an MHC class I barrier (20). Under these circumstances, mature T cells that would otherwise react with the graft are apparently inactivated by a peripheral mechanism, currently under investigation in our renal transplantation studies. In those class I-mismatched renal transplantation studies, prior thymectomy was also found to be detrimental to the induction of tolerance (24). On the other hand, when T cell depletion is used to permit engraftment, it is likely that host thymectomy may not be detrimental and may even be required, as was the case in murine studies (8). We are now attempting to extend our thymic transplantation studies to more extensive histocompatibility barriers in our miniature swine, where, on the basis of our previous studies of renal transplantation, we anticipate that CyA alone will not be sufficient to induce long term graft acceptance (26). We therefore plan to use T cell depletion in these studies and will again examine the effects of thymectomy (G. W. Haller et al., work in progress).
There are additional reasons why thymectomy may have been detrimental to successful engraftment in these studies. These include recent evidence that the host thymus may be involved in the development of donor-specific regulatory cells (36, 37). In addition, in murine models, CyA treatment has been shown to lead to thymic medullary involution with destruction of the medullary dendritic cells. After CyA treatment is stopped, the thymus rapidly repopulates with dendritic cells (38, 39). It is therefore possible that in the present studies either donor dendritic cells or host cells presenting donor peptides may repopulate the thymus early after CyA treatment. This repopulation, which would of course require the presence of the host thymus, could be responsible for subsequent deletion of developing donor-reactive thymocytes.
In vitro cellular assays revealed that the thymic graft recipients had developed donor-specific hyporesponsiveness. Antidonor proliferative responses as well as cell-mediated cytotoxicity were markedly reduced or absent as early as 4 wk after transplantation. Surprisingly, however, the animals not only produced an anti-donor IgM response, but also demonstrated a class switch to IgG 4 wk after thymic transplantation. Because such class switching is thought to require T cell help, we speculate that T cell responsiveness may have been present transiently after cessation of CyA treatment on day 11. This T cell response may have disappeared or been suppressed by the time at which the MLR was first measured (4 wk after transplantation). However, the thymic grafts continued to develop, and endothelial cells remained positive for donor SLA class I despite the presence of donor-specific Abs. This finding suggests either a local suppressive phenomenon or the general absence of effector mechanism leading to destruction of Ab-coated cells in the thymus. Consistent with the latter possibility, failure of mAbs to deplete mature T cells in the thymus has previously been reported in murine studies (40).
Despite successful engraftment, we speculate that the immunosuppressive treatment selected for these experiments was insufficient to prevent sensitization. However, it is remarkable that these grafts continued to develop and remained viable as long as 1 yr after transplantation in the presence of alloantibodies. Even after sensitization with donor skin, which was originally performed with the intention of assessing donor-specific tolerance, one of the animals was able to recover donor-specific hyporesponsiveness in CML. These results demonstrate the potential of allogeneic thymus to induce tolerance in large animals and suggest that thymus may be resistant to both cellular and humoral rejection. These findings may be of particular importance in xenotransplantation, where preformed natural Abs are present (reviewed in 41).
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Acknowledgments |
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Footnotes |
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2 Current address: Department of Surgery, Brigham and Women’s Hospital, 75 Francis Street, Boston, MA 02115.
3 Address correspondence and reprint requests to Dr. David H. Sachs, Transplantation Biology Research Center, Massachusetts General Hospital, Harvard Medical School, MGH-East, Building 149-9019, 13th Street, Boston, MA 02129. E-mail address:
4 Abbreviations used in this paper: CyA, cyclosporin A; CML, cell-mediated lympholysis; EuThy, euthymic pig; MGH, Massachusetts General Hospital; POD, postoperative day; SI, stimulation index; SLA, swine leukocyte Ag; Thx, thymectomized pig.
Received for publication April 13, 1999. Accepted for publication July 14, 1999.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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