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Sensitization to major histocompatibility complex (MHC) alloantigens is critical in transplantation. The mechanism of sensitization to minor histocompatibility antigens (Mi-HAg) has not been thoroughly explored. We used a mouse model of allosensitization to Mi-HAg to study the Mi-HAg sensitization barrier in bone marrow transplantation (BMT). AKR mice were sensitized with MHC congenic Mi-HAg disparate B10.BR skin grafts. Adaptive humoral (B-cells) and cellular (T-cells) responses to Mi-HAg are elicited. In subsequent BMT, only 20% of sensitized mice engrafted, while 100% of unsensitized mice did. In vivo cytotoxicity assays showed that Mi-HAg sensitized AKR mice eliminated CFSE labeled donor splenocytes significantly more rapidly than naïve AKR mice but less rapidly than MHC-sensitized recipients. Sera from Mi-HAg sensitized mice also reacted with cells from other mouse strains, suggesting that Mi-HAg peptides were broadly shared between mouse strains. The production of anti-donor-Mi-HAg antibodies was totally prevented in mice treated with anti-CD154 during skin grafting, suggesting a critical role for the CD154:CD40 pathway in B-cell reactivity to Mi-HAg. Moreover, anti-CD154 treatment promoted BM engraftment to 100% in recipients previously sensitized to donor Mi-HAg. Taken together, Mi-HAg sensitization poses a significant barrier in BMT and can be overcome with CD154:CD40 co-stimulatory blockade.
Hypertransfusion and chelation therapy has allowed children with β-thalassemia to survive into the second decade, but it is painful, tedious, costly, and only palliative (1). Similarly, transfusion therapy has allowed children with sickle cell disease (SCD) to survive into adulthood, but infectious complications, iron overload, and end-organ failure result in premature demise (2,3). Bone marrow (BM) transplantation (BMT) is the only curative therapy for children with hemoglobinopathies (4). There is a significantly greater rate of graft rejection due to transfusion-induced sensitization (5,6). A clear definition of the mechanism, the contributing factors, and antigens for sensitization will allow the development of novel approaches for conditioning sensitized recipients in BMT. T-cell-mediated cellular immunity to major histocompatibility complex (MHC) alloantigens has been demonstrated to be the primary barrier for BM allorejection in normal recipients (7–9). The recent findings identified that B-cell responses and humoral immunity are dominant factors in sensitization to MHC alloantigens (10–13). The role of sensitization to minor antigens in MHC-matched sensitized recipients has not been fully studied until now.
Minor histocompatibility antigens (Mi-HAg) are polymorphic peptides derived from allelic cellular proteins encoded by autosomal genes or by genes of the Y-chromosome (14). The existence of minor histocompatibility loci was first hypothesized when it was found that matching of donor and recipient MHC loci is not sufficient for long-term graft survival. Recipients of human leukocyte antigen (HLA)-identical renal allografts require levels of immunosuppression similar to that for MHC-disparate recipients (15). Subsequently, the existence of Mi-HAg was further confirmed when clinical findings showed that BMT between HLA-identical siblings was still associated with graft-versus-host (GVH) reactions (16–18). Because the majority of BM transplants are currently limited to HLA-identical sibling donors, the target for rejection is likely against Mi-HAg (19). Until now, little was known about the role for Mi-HAg in transplantation sensitization and associated rejection.
In this study, we used a mouse model to explore the role of allosensitization to Mi-HAg in BM rejection and found that both humoral and cellular responses are elicited to Mi-HAg. Mi-HAg sensitization poses a significant barrier in BMT and humoral response is the major mechanism for preventing BM engraftment in Mi-HAg sensitized recipients. We also found that blockade of CD154:CD40 co-stimulatory molecule interaction during exposure to Mi-HAg prevented sensitization.
Male B6 (H-2b), B10 (H-2b), B10.BR (H-2k), AKR (H-2k), C3H (H-2k), B10.D2 (H-2d), and BALB/c (H-2d) mice were obtained from The Jackson Laboratory (Bar Harbor, ME). Animals were housed in the barrier facility and cared for according to National Institutes of Health guidelines.
B6 or AKR mice were sensitized by skin grafts from MHC-congenic B10 or B10.BR donors by a modified method described by Billingham (9,20). Grafts were scored for rejection by daily inspection for the first month and then weekly thereafter. Rejection was defined as complete when no residual viable graft could be detected.
Recipient AKR mice were pretreated i.p. with anti-αβ-TCR (H57-597: 100 μg day -3) or anti-CD154 (MR-1: BioXCell, Lebanon, NH, 0.5 mg, day0 and day +3) mAb around the time of skin grafting (day0) to deplete αβ-TCR+ T-cells or block CD40/CD154 costimulatory pathway.
Mice were conditioned with 950 cGy total body irradiation (TBI, γ-cell 40; Nordion, Ontario, Canada) and transplanted with 15 × 106 untreated bone marrow cells (BMC) via lateral tail vein injection between 4–6 hours after irradiation as previously described (11).
Donor chimerism was determined by relative percentages of donor-derived peripheral blood (PB) lymphocytes with flow cytometry. In the B10.BR to AKR model, PB lymphocytes were stained with mAbs specific for antigens of donor (anti-Thy-1.2-PE B10.BR) and recipient (anti-Thy-1.1-FITC, AKR) origin. All mAbs were obtained from BD/PharMingen (San Diego, CA).
PBL were stained with anti-CD44-FITC (IM7), anti-CD62L-PE (MEL-14), anti-CD4-PerCP, and anti-CD8-APC (BD/Pharmingen). Effector/memory T-cells were identified as CD44high/CD62Llow/−.
For in vitro MLR, responder cells (1.0 × 105) were cultured 1:1 with irradiated stimulator cells (2,000 cGy) for 5 days at 37°C in 5% CO2. Each well was pulsed with 1μCi of [3H] thymidine DuPont-NEN, Boston, MA) 18-hours before harvesting. For in vivo MLR, responder cells were labeled with CFSE (Molecular Probes, Eugene, OR) at 5μM. 20 × 106 labeled cells were injected into irradiated (1,000 cGy) mice (i.v., as stimulators). At day 3, spleens were harvested and stained with anti-CD8-PerCP and anti-CD4-APC (BD PharMingen). CFSE intensities were analyzed in cells gated in either CD4+ or CD8+ cells.
Single splenocyte suspensions were prepared. Target and internal control splenocytes were then incubated with 4.0μM or 0.2μM CFSE, respectively. Cells were mixed in a 1:1 ratio, and 20 × 106 cells from each were injected intravenously. The cytotoxicity was determined by the ratio between targeting and internal control cells by flow cytometry in peripheral blood at different time points.
Data are presented as the average ± SD. The two-tailed t test (two-sample, assuming unequal variances) was used to evaluate statistical differences. The difference between groups was considered significant when P < 0.05.
Sensitization was induced by skin grafting using two MHC congenic Mi-HAg disparate strain combinations (B10 to B6 and B10.BR to AKR). B10 skin grafts survived long term in B6 recipients (>120 days), but B10.BR grafts were rejected by AKR recipients with a mean survival time (MST) of 14.9 ± 2.3 days (Figure 1A). AKR mice rejected B10.BR skin grafts with a time course similar to MHC-disparate controls (B10.BR to B10, MST: 12.3 ± 2.1 days; P > 0.05) and MHC plus Mi-HAg disparate controls (B10.BR to B6, MST: 12.5 ± 2.5 days). With flow cytometric crossmatch (FCXM) assay, anti-donor antibodies were not detected in sera of B6 mice with B10 skin grafting from 4 weeks (Figure 1C, mean fluorescence intensity [MFI]: 4.7±0.36) to up to 12 weeks. Anti-donor antibody was present in all AKR mice at 4 weeks after skin grafting from B10.BR (MFI 320.6±251). The titer directed against Mi-HAg had a MFI similar to B10 or B6 mice rejecting skin grafts with an MHC disparity (MFI: 231.3±139.0) or MHC plus Mi-HAg disparity (MFI: 301.1±141.1). The IgG subclasses of IgG1, IgG2a, and IgG2b were detected in all tested sensitized AKR mice (Figure 1D). To determine the specificity of antibody against Mi-HAg, sera from AKR mice sensitized with B10.BR skin were incubated with splenocytes from BALB/c (H-2d), B10.D2 (H-2d), B6 (H-2b), B10 (H-2b), and C3H (H-2k) donors in FCXM assay. The sensitized AKR sera showed broad cross-reactive antibody responses to all other mouse strains tested (Figure 1E). The antibody titer in sensitized sera to each strain measured by MFI was similar to the titer to donor B10.BR but significantly higher (P < 0.00001) than the titer in naïve AKR sera.
To investigate the cellular immune responses to Mi-HAg, recipient PB was collected 15 days after skin grafting and CD44high/CD62Llow/− effector/memory cells in CD8+ or CD4+ T-cells were enumerated (Figure 2A–B). CD8+ or CD4+ effector/memory T-cells were detected in AKR mice on day 15 at the peak of rejection. The percentage of effector/memory T-cells in CD8+ or CD4+ cell gate was 7.3±1.7% and 6.2 ± 12.5%, respectively, significantly greater than in naïve AKR mice (P <0.00001 and P = 0.018). No difference in CD8+ or CD4+ effector/memory T-cells was found in B6 mice with or without B10 skin grafts (data not shown). There was a higher percentage of effector/memory T-cells (9.9 ± 4.4% in CD8+ and 10.7 ± 4.6% in CD4+) in B10 mice that rejected MHC-disparate B10.BR skin grafts compared to Mi-HAg sensitized AKR mice but without statistic significance (P = 0.14) in CD8+ cells and with significance (P = 0.02) in CD4+ cells. CD8+/CD44high/CD62Llow/− cells have been identified as containing specific memory functions in vitro in a Mi-HAg disparity model.(21) The expression of CD71 (transferrin receptor) is upregulated after activation of T lymphocytes (22). The percentage of CD71+ cells in CD8+ or CD4+ cells in AKR mice at day 15 after skin grafting (Figure 2C, 4.2 ± 1.2% or 3.8 ± 0.9%) was similar to naïve AKR controls (P = 0.54 or P = 0.55), and significantly lower than the percentage in rejecting MHC disparate controls (B10.BR to B10, 10.3 ± 1.4%, P <0.001).
To examine the proliferative immune response to Mi-HAg, we compared in vitro and in vivo MLR assays. In in vitro MLR, splenocytes or lymph node lymphocytes from B6 or AKR were assessed for alloreactivity to Mi-HAg. B6 or AKR cells exhibited no reactivity to Mi-HAg disparate stimulators, B10 or B10.BR, respectively. Responses were similar to control autoimmune responses in B6 or AKR (Figure 3A). B6 or AKR cells were reactive to MHC plus Mi-HAg disparate stimulators, B10.BR or B6, respectively.
For in vivo MLR assays, CFSE labeled splenocytes from naïve B6 or AKR mice were injected intravenously into irradiated B10 or B10.BR mice to determine in vivo alloreactivity to Mi-HAg. B6 T-cells were not reactive to the Mi-HAg of B10 (Figure 3B). In striking contrast, T-cells from AKR mice exhibited strong alloreactivity to Mi-HAg in vivo in B10.BR mice. This alloreactivity to Mi-HAg was similar to the responses to MHC plus Mi-HAg disparate controls (AKR/B6). These results refute the previous hypothesis that proliferative T-cell responses to Mi-HAg require prior in vivo immunization (15).
We evaluated whether memory immune responses to Mi-HAg would impair engraftment. Five to seven weeks after receiving MHC congenic B10 or B10.BR skin grafts, B6 or AKR recipients were conditioned with 950 cGy TBI and reconstituted with 15 × 106 BMC from B10 or B10.BR donors. Since there is no marker to differentiate the B10 strain from B6, engraftment was followed by animal survival after ablative TBI conditioning. All B6 mice survived after being transplanted with B10 BMC. In contrast, engraftment was achieved only in 20% (n = 10) of sensitized AKR mice (Figure 4A). As expected, engraftment occurred in 100% of naïve AKR mice. None of the controls sensitized to MHC antigens or MHC plus Mi-HAg engrafted.
In vivo cytotoxicity assays were performed to determine the effect of Mi-HAg sensitization on rejection of donor cells (Figure 4B). B10 mice sensitized to MHC alloantigens eliminated the majority of CFSE-labeled B10.BR splenocytes within 1 hour (93.7 ± 5.3%) and the remainder (99.4 ± 1.0%) by day 1. This hyperacute cytotoxicity indicates antibody-mediated killing that represents the predominant barrier for MHC alloreactivity (12). Naïve AKR mice eliminated only 5.4 ± 4.7% Mi-HAg disparate donor cells 1 hour after cell infusion and gradually eradicated donor cells to almost 100% at day 7. AKR mice sensitized to Mi-HAg eliminated 38.1 ± 4.4% and 56.4±6.3% CFSE labeled BALB/c cells at 1 and 3 hrs, respectively, and almost 100% by day 3. The cytotoxicity in mice sensitized to Mi-HAg was significantly higher than in unsensitized recipients (P <0.0001 through all the time points tested at 1 hr, 3 hr and 1, 2 and 3 days except day 7), but significantly less than in MHC sensitized recipients (P < 0.05 to 0.0001 through all the time points tested).
The engagement of CD40, a co-stimulatory molecule on B-cells, with its ligand CD154 on T-cells, is critical to initiate effective T and B-cell activation (23). Here, we examined effector factors that affect the humoral immune response at the time of exposure to Mi-HAg. Two commonly used conditioning regimens were tested: 1) T-cell lymphodepletion using anti-αβ-TCR mAb; or 2) co-stimulatory blockade with anti-CD154 mAb. Recipients pretreated with anti-CD154 alone rejected their skin grafts (17.2 ± 2.4 days) with a kinetic similar to untreated controls (14.9 ± 2.3 days, P > 0.1; Figure 5A). Skin grafts were slightly prolonged in mice treated with anti-αβ-TCR mAb but without significance (20.5 ± 5.3 days; P > 0.1). Antibody titers at 4 weeks after skin grafting in mice treated with anti-CD154 mAb (MFI: 6.2 ± 3.9) were only slightly higher than in naïve mice (Figure 5B, MFI: 4.2 ± 0.47; P > 0.05). In contrast, mice treated with anti-αβ-TCR mAb alone produced anti-donor antibody at significantly higher levels (MFI: 76.93 ± 33.1; P < 0.05) compared with naïve mice, but significantly lower levels (P < 0.05) compared to controls that received skin grafts only.
To evaluate the influence of the conditioning regimens used to prevent sensitization to Mi-HAg on subsequent transplant, BMT was performed on AKR mice 5–7 weeks after B10.BR skin grafting with 950 cGy TBI and 15 × 106 BMC from B10.BR (Figure 5C). Engraftment occurred in only 25% of mice treated with anti-αβ-TCR mAb (n = 4) at skin grafting and. In contrast, 100% of mice treated with anti-CD154 engrafted. Donor chimerism averaged 86.5±8.8% at 1 month. Engraftment was stable ≥6 months. The percentage of engraftment and the level of donor chimerism in mice treated with anti-CD154 at skin grafting were similar to unsensitized AKR.
We further evaluated the effect of anti-CD154 mAb on T-cells. Anti-CD154 treatment resulted in a significant reduced alloreactive T-cells (Figure 5D): the percentage of CD4+/CD44+ in spleen lymphocytes was significantly lower compared with control mice that received skin grafts without treatment (P < 0.001), and the percentage of CD8+/CD44+ was decreased compared with untreated controls (P = 0.062). Moreover, the IFN-γ expression in CD4+/CD44+ activated T-cells was significantly inhibited by anti-CD154 treatment (Figure 5F; P < 0.001) at day 15 compared with the control group. However, even the IFN-γ expression in CD8+/CD44+ T-cells was similar to naïve mice but not significantly lower (Figure 5E; P = 0.082) compared with the control group that received skin grafting without treatment.
The existence of histocompatibility antigens in addition to MHC was indicated first in inbred mice and then in humans when skin grafts exchanged between HLA-identical siblings were rejected (15,24–28). Recently, the molecular characterization of Mi-HAg has resulted in the observation that although large numbers of Mi-HAg exist, not all Mi-HAg are equal in immunogenicity (29). Only one or a few are immunodominant (15). Our present findings are consistent with these observations. The Mi-HAg disparity between B6 and B10 is not immunogenic and does not elicit any immune responses as evidenced by the acceptance of skin grafts and failure to elicit Ab generation. In contrast, AKR mice generated strong alloimmune responses to antigens from Mi-HAg-disparate mice. Our two models further suggest that the immunogenicity of Mi-HAg in MHC-congenic pairs is different. A worldwide phenotype frequency analysis of Mi-HAg found that Mi-HAg frequencies were significantly different between ethnic populations, suggesting that some populations might display more phenotypic diversity than others (30). The clinical significance of these Mi-HAg is relatively unknown and may provide valuable information for tissue typing to identify the optimal donor for BM and solid organ transplantation.
T-cells are thought to be the major effector cells in response to Mi-HAg (15,31). T-cells are capable of recognizing and responding to immunogenic Mi-HAg loci (32,33). They are believed to be responsible for the rejection of MHC-matched BMC via responses directed at Mi-HAg (34). It was previously thought that prior in vivo immunization was required for in vitro T-cell responses to Mi-HAg (15). Studies from mice and humans clearly show that both CD4+ and CD8+ T-cells are involved in the generation of in vivo responses to minor-histocompatibility peptide epitopes (35). In our current study, we observed disparate immune responses to Mi-HAg between in vitro and in vivo MLR in AKR mice responses to B10.BR, where the in vivo proliferative response was robust while the in vitro response was not. The reason for this difference is not clear. The in vitro culture environment may lack some factors present in vivo that are critical for generation of efficient T-cell immune responses to Mi-HAg. Nonetheless, our present findings show the value of defining the mechanism of reactivity to Mi-HAg using in vivo models.
The humoral immune response to Mi-HAg has not been appreciated until now. In fact, a recent editorial in Blood echoed the clinical paradigm that cellular responses are dominant in MHC-identical transplants (31). Our findings not only confirm the previous studies in T-cell responses against Mi-HAg (33), but additionally provide new insight into the previously unappreciated role of humoral immune responses to Mi-HAg. In cellular adaptive immune responses, AKR mice rejected B10.BR skin grafts with a similar time course as controls with MHC disparity pairs and generated a higher percentage of effector/memory phenotypes in T-cells. Interestingly, we found that the upregulation of CD71 on T-cells was substantially different in immune responses to MHC versus Mi-HAg, which may suggest a difference in T-cell response to MHC antigen vs. Mi-HAg. The more interesting finding in the current study is that significantly high levels of anti-donor antibody are generated in response to Mi-HAg. It is of note that the antibody produced by AKR mice sensitized to B10.BR Mi-HAg showed strong cross-reactivity against numerous other strains with different MHC and/or Mi-HAg backgrounds in FCXM assay. The broad cross-reactive antibody responses suggest that Mi-HAg peptides are shared broadly between mouse strains. Our findings suggest that in approaches to treat allosensitization in MHC-matched BMT one must target both humoral and cellular immunity.
Sensitization to MHC antigens is considered to be one of the most critical challenges in clinical transplantation (36,37). Allorejection mediated by preformed antibodies has recently been recognized as a major cause of graft loss in sensitized recipients (11,13). In a sensitized mouse model, we found that as little as 25μL serum adoptively transferred from sensitized recipients abrogated allogeneic donor marrow engraftment in naïve recipients, reflecting the dominance of the humoral immune barrier in sensitized recipients (11). The existence of sensitization to Mi-HAg has been suggested clinically because HLA matched BMT has a higher rate of failure in patients who have undergone chronic transfusion therapy, such as in SCD and thalassemia (19). Chronic transfusion immunizes the recipients to multiple donor antigens, including Mi-HAg, which mediates subsequent rejection of BMC in HLA-matched BMT. In our current model using MHC-congenic, Mi-HAg-disparate donor/recipient pair (B10.BR to AKR), we found that the recipients generated high levels of anti-donor antibody. This posed a strong barrier to subsequent BMT from that same donor, as only 20% of sensitized AKR mice engrafted. Taken together, our findings suggest that BMC are the target in memory immune responses to Mi-HAg. This could have a profound impact in immunotherapeutic applications of BMT and identifying the optimal donors for transplantation in sensitized recipients.
The antibody titer to anti-Mi-HAg in the AKR/B10.BR strain combination was similar to that in response to MHC antigen. However, the antibody titer in AKR response to Mi-HAg did not correlate with the robustness in rejection of donor cells compared with the donor-specific antibody against MHC. We found that sensitization to Mi-HAg was somewhat less effective in mediating rejection compared with sensitization to MHC antigen. Mi-HAg sensitized recipients had significantly less cytotoxicity in vivo and exhibited better engraftment in subsequent BMT irrespective of the titers. In Mi-HAg sensitized recipients, the cytotoxicity in vivo is significantly more rapid compared to naïve mice. The rapidity of the cytotoxicity in MHC-sensitized recipients suggests that the mechanism was antibody-mediated. The kinetics for cytotoxicity observed in the naïve mice is mediated by cellular mechanism as no antibodies against donor were generated. The intermediate kinetic of cytotoxicity in Mi-HAg sensitized recipients suggests that both humoral and cellular mechanisms may be involved. Our finding confirmed that Mi-HAg disparity is an immune obstacle in transplantation, but less potent than that for MHC alloantigens.
We found that blocking the interaction of CD154:CD40 with anti-CD154 prevented allosensitization to Mi-HAg and promoted engraftment following BMT. This is similar to the outcomes we observed in preventing sensitization to MHC alloantigens where we recently demonstrated that CD154:CD40 costimulatory pathway plays a critical and specific role in B-cell activation to MHC antigens through a T-cell-dependent mechanism (12). In our present Mi-HAg sensitization mouse model, we found that 100% of mice preconditioned with anti-CD154 mAb at the time of skin transplantation engrafted. The efficient engraftment suggests that preventing generation of humoral responses to Mi-HAg in vivo with co-stimulatory blockade of CD154 prevents humoral allosensitization to Mi-HAg and promotes subsequent BMT. CD44 is a marker of T-cell activation and a property of memory cells and implicated in cell migration, activation, and differentiation (38–40). IFN-γ is a cytokine produced mostly by activated T-cells and IFN-γ-producing phenotype corresponds to effector function in graft rejection (41–44). Notably, blockade of CD154 significantly decreased the generation of CD4+/CD44+ activated cells to the level of naïve mice, but not CD8+/CD44+ cells. We have also found that anti-CD154-treated recipients had a significant reduction of IFN-γ–producing cells. These findings may help explain the dissociation between T-cell tolerance (skin graft rejection) and B-cell tolerance (inhibition of Ab generation) observed in recipients with anti-CD154 treatment. The inhibition of CD4 T-cell activation results in effective abrogation of Th2 response and subsequent Ab generation. The partial inhibition of CD8 T-cell activation suggests their effector function in mediating skin graft rejection.
Here we show Mi-HAg sensitization poses a significant barrier in BMT and antibodies against donor Mi-HAg is the most likely mechanism for preventing BM engraftment. This is consistent with BMC rejection in MHC sensitized recipients: the BMC rejection is dominantly mediated by humoral immune responses (11,45), although the sensitization to Mi-HAg was less effective in BMC rejection compared with sensitization to MHC antigen. One practical justification for gaining knowledge of the nature and identity of Mi-HAg is the potential to use it to manipulate immune responses to such epitopes in clinical protocols. Current tissue typing for transplantation focuses primarily on MHC matching between donor and recipient. Thus, the combination of a tissue typing for immunodominant minor antigens with routine typing for MHC of stem cell and solid organ transplant donor and recipient may be clinically relevant to manage allosensitization not only to MHC but also to Mi-HAg.
The authors thank Haval Shirwan for review of the manuscript and helpful comments; Carolyn DeLautre for manuscript preparation; and the staff of the animal facility for outstanding animal care.
This work was supported in part by the following: NIH R01 DK069766 and NIH 5RO1 HL063442; JDRF 1-2005-1037 and JDRF 1-2006-1466; and The Department of the Navy, Office of Naval Research. This publication was also made possible by Award No.W81XWH-07-1-0185 and W81XWH-09-2-0124 from the U.S. Army Medical Research Acquisition Activity, 820 Chandler Street, Fort Detrick, MD,21702-5014 (Any opinions, findings, and conclusions or recommendations expressed in this material are those of the author(s) and do not necessarily reflect the views of the Office of Army Research); the National Foundation to Support Cell Transplant Research; the Commonwealth of Kentucky Research Challenge Trust Fund; the W. M. Keck Foundation; and The Jewish Hospital Foundation. Research was conducted in compliance with the Animal Welfare Act Regulations and other Federal statues relating to animals and experiments involving animals and adheres to the principles set forth in the Guide for Care and Use of Laboratory Animals, National Research Council, 1996.
The authors have no conflicts of interest to declare.