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The Journal of Immunology, 1999, 162: 4496-4501.
Copyright © 1999 by The American Association of Immunologists

Modulation of Allospecific CTL Responses During Pregnancy in Equids: An Immunological Barrier to Interspecies Matings?1

Jessica M. Baker2, Anona I. Bamford3 and D. F. Antczak

James A. Baker Institute for Animal Health, College of Veterinary Medicine, Cornell University, Ithaca, NY 14853


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Maternal immune recognition of the developing conceptus in equine pregnancy is characterized by the strongest and most consistent alloantibody response described in any species, a response directed almost exclusively against paternal MHC class I Ags. This work investigated the cellular immune response to paternal MHC Ags in pregnant and nonpregnant horses and donkeys, and in horses carrying interspecies hybrid mule conceptuses. We observed profound decreases in classical, MHC-restricted, CTL activity to allogeneic paternal cells in peripheral blood lymphocytes from both horse mares and donkey jennets carrying intraspecies pregnancies, compared with cells from nonpregnant controls. This is the first evidence in a randomly bred species for a generalized systemic shift of immune reactivity away from cellular and toward humoral immunity during pregnancy. Surprisingly, mares carrying interspecies hybrid mule conceptuses did not exhibit this transient, pregnancy-associated decrease in CTL activity. The failure of interspecies pregnancy to down-regulate cellular immune responses may be a heretofore-unrecognized, subtle barrier to reproductive success between species.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Since Peter Medawar (1) first posed the problem of the fetus-as-allograft nearly 50 years ago, "Nature’s transplant" has provided a rich environment for theory and experimentation at the interfaces between mother and fetus, and between immunology and reproduction. Three recent studies in mice have provided evidence that maternal CTL responses to paternal MHC class I Ags are disrupted by pregnancy (2, 3, 4, 5). In the case of MHC differences between mother and father, there appears to be down-regulation of Ag-specific receptors and coreceptors on T cells, which is reversible after pregnancy is completed. This may account for paternal Ag-specific tolerance to MHC alloantigens during pregnancy (2, 3). However, a study using transgenic mice with TCRs specific for the H-Y minor histocompatibility Ag found evidence for two distinct types of immune regulation induced by pregnancy. Some H-Y-specific T cells were specifically deleted, while others became anergic, but without down-regulation of their receptors (4). In the case of H-Y Ag, the pregnancy-induced tolerance lasted after pregnancy was completed, and may be persistent. Together, these studies provide evidence for multiple, Ag-specific mechanisms that protect the fetal allograft from rejection. In contrast, other research has demonstrated a generalized, nonspecific shift away from cell-mediated toward humoral immunity during normal pregnancy (6, 7, 8, 9). Currently, there is active debate over the relative importance of paternal Ag-specific tolerance and/or the so-called Th1->Th2 shift in immune reactivity during pregnancy.

The horse is a good species for studies of changes in immune status during pregnancy by virtue of two striking phenomena observed after normal, histoincompatible matings. First, both primiparous and multiparous mares make strong paternal-specific cytotoxic alloantibody responses to paternal MHC class I Ags by day 60 of the 336-day horse gestation period (10, 11). Most species do not make reproducible Ab responses to paternal alloantigens, and in those species where pregnancy-induced alloantibody responses have been detected, the responses are not consistent, and the Abs do not arise as early relative to total gestation length as seen in the horse (12, 13, 14, 15, 16). A recent report indicates that there is a partial deletion of B lymphocytes specific for paternal histocompatibility Ags in pregnant mice, which may explain why alloantibody responses are rare in most species (17). The strong alloantibody responses observed in horses may be a reflection of differences in placentation between rodents and equids. Rodents have an invasive, hemochorial placenta, whereas horses have a noninvasive epitheliochorial placenta, with the exception of the endometrial cups described below (18).

A second characteristic of equine pregnancy is that there is an accumulation of CD4+ and CD8+ T lymphocytes around the invasive trophoblast of the equine endometrial cups shortly after they develop (19). The cups are formed by the migration of the equine chorionic girdle into the endometrium to form binucleate, equine chorionic gonadotrophin-secreting endometrial cup trophoblast cells (20). Interestingly, neither the alloantibody nor the cell-mediated immune response mounted by the mother to the developing horse conceptus seems to compromise pregnancy (21).

In the course of developing an assay to determine whether allospecific CTL generated from horse PBL could lyse equine trophoblast cells carrying paternal MHC class I Ags (22), we found that PBL from pregnant mares showed lower levels of CTL activity than lymphocytes from nonpregnant control mares. We have explored this phenomenon and compared it with the recent data from other species described above.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals

Horses (Equus caballus) and donkeys (Equus asinus) of the Equine Genetics Center at Cornell University College of Veterinary Medicine (Ithaca, NY) were used for these experiments, and all animal care was performed in accordance with institutional care and use guidelines. The horses were comprised of several breeds, and the animals ranged in age from 4 to 18 yr. Pregnancy was established using artificial insemination, and the day of ovulation was determined by daily transrectal ultrasound examination, from which gestational ages were deduced. The MHC class I phenotype for each animal was determined by serological typing of lymphocytes based on the equine leukocyte Ag system established by the Third International Workshop on Lymphocyte Alloantigens of the Horse (23). MHC typing of donkeys was performed using the lymphocyte microcytotoxicity assay with pregnancy sera, mixed lymphocyte culture assays, and CTL assays (J.M.B. and D.F.A., manuscript in preparation). Stallions and jack donkeys homozygous for MHC haplotypes were used as semen donors in this study.

For horses, MHC class I haplotypes are indicated by the letter A followed by a number (e.g., MHC type A2/A4, a heterozygote). For donkeys, the haplotypes are abbreviated Ag followed by a number (e.g., MHC type Ag1/Ag1, a homozygote).

CTL priming

PBMC were isolated as previously described (24) to be responder or stimulator cells. Briefly, venous whole blood was collected through jugular venepuncture into tubes or bottles containing the anticoagulant sodium heparin (Sigma, St. Louis, MO) at a final concentration of 15 IU/ml of blood. Lymphocytes were isolated by centrifugation over a Ficoll density gradient and washed twice in DAB-1-FCS: PBS containing 10% FCS, 1 IU/ml heparin, and 0.4% DAB-B salts (25 mg/ml CaCl2 · 2H2O and 25 mg/ml MgCl2 · 6H2O). Following the last wash, the pellet was resuspended in 1 ml per every 5 ml of blood originally collected, in ARM, a 1:1 (v/v) mixture of AIM V and RPMI 1640 media (Life Technologies, Grand Island, NY), containing sodium bicarbonate (Life Technologies), 10% heat-inactivated normal horse serum (pooled, Ab negative serum collected from five male horses), and supplemented with 1x penicillin-streptomycin (Life Technologies), 1x MEM nonessential amino acids (Life Technologies), 0.29 µg/ml L-glutamine (Sigma), 0.05 µg/ml sodium pyruvate (Life Technologies), and 0.025 µl/ml molecular biology grade 2-ME (Sigma). Stimulator cells were irradiated using a cesium source (Gammacell 40; Nordion International, Kanata, Ontario, Canada), at a dose of 950 rads.

One hundred million responder cells were placed into a T75 flask (Corning, Corning, NY) with 50 x 106 stimulator cells, and ARM was added for a final concentration of 3 x 106cells/ml. The flasks were incubated upright at 37°C in 5% CO2 for 7 days, and the cells were resuspended every other day. On day 7, the cultures were restimulated with freshly prepared, irradiated stimulator lymphocytes that were plated at half the density of the surviving responder cells. Additionally, half of the medium was replaced with fresh ARM. All cultures were stimulated for a total of 10 days.

Target preparation

Three days before the assay, lymphocytes (3 x 106 cells/ml in 10 ml) from each target animal were incubated with 2.5 µg/ml pokeweed mitogen (Sigma) in T25 tissue culture flasks (Corning) at 37°C in 5% CO2 for 48 h. The day before the assay, the cell concentrations were adjusted to 2.5 x 106 cells/ml and plated in one well each of a 24-well, flat-bottom tissue culture plate (Costar, Cambridge, MA). To each target well, 125 µCi of 51Cr (Na251CrO2, Dupont/NEN Research Products, Boston, MA) were added, and the plate was incubated overnight in a 37°C incubator with a CO2 concentration of 5%. The next morning, the labeled cells were transferred to tubes and washed six times in DAB-1-FCS at 1300 rpm for 5 min at 4°C.

51Cr release assay

The 51Cr release assay we used in this study was adapted from previous studies (25, 26). In most assays, killer cells were plated at four concentrations: 1 x 106, 5 x 105, 2.5 x 105, and 1.25 x 105 cells/well, in a volume of 100 µl/well in round-bottom tissue culture plates (Fisher Scientific, Pittsburgh, PA). Targets were always plated at 1 x 104 cells/well in 25 µl, giving killer:target ratios of 100:1, 50:1, 25:1, and 12.5:1.

Spontaneous release wells contained only target cells and medium. Total release was obtained by adding 50 µl of 10% Triton X-100 (Sigma) to wells that contained only targets and medium. The average spontaneous release did not exceed 30% in any assay. All test dilutions and controls were done in triplicate.

The plates were incubated at 37°C in 5% CO2 for 4 h. Afterwards, the supernatants from each well were harvested using Skatron (Sterling, VA) harvesting frames and the 51Cr activity was measured in a gamma counter. Counts generally ranged from 500 to 12,000 cpm. The percent cytotoxicity was normalized to the spontaneous and total release controls using the following formula: % cytotoxicity = [mean sample release - mean spontaneous release]/[mean total release - mean spontaneous release].

Monoclonal Abs

The mAb WS#57 (MHC class I) (27) was created in our laboratory. Monoclonal Abs WS#72 (equine CD4) (28) and WS#12 (equine CD8) (28) were obtained from Dr. William Davis of Washington State University (Pullman, WA). The specificity of these Abs were verified in two International equine leukocyte Ag workshops (27, 28).

When Ab blocking was attempted, 100 µl of the mAbs were preincubated with either the targets ({alpha}MHC class I, neat supernatant) or the effectors ({alpha}CD4 or {alpha}CD8, ascites product diluted to 1:80) for 1 h. The cells were washed and then plated as described above.

Statistical analyses

Student’s t tests were used to determine significance of pregnant vs nonpregnant data (see Fig. 3Go); a value of p <= 0.05 was considered significant.



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  		FIGURE 3. The ability to make cytotoxic T cells against paternal histocompatibility Ags is decreased in horse mares and jennet donkeys carrying intraspecies pregnancies at the time of blood collection. A, Horse mare 2470 (MHC type A6/A7). B, Donkey jennet 2175 (Ag2/Ag4). Each point for pregnancy data is representative of at least five experiments and at least two different pregnancies. The mare was mated with stallion 0834 (A2/A2), and the jennet was mated with jack 1992 (Ag1/Ag1). Each point for the nonpregnant data is representative of at least nine experiments, with data collected both before and after pregnancy. The letters above symbols indicate values are significantly different, p < 0.05.

 
For mule vs horse pregnancy comparisons, ANOVA ruled out an effect of the species, the mating stallion or jack, the day of pregnancy, histocompatibility Ags expressed, and the mare. With the exception of one mare who showed significance based on her histocompatibility type (and who was subsequently removed from the analysis so that the model could be generalized, and thus independent of MHC effects), none of these variables was significant (all with p <= 0.1). Testing the data by the general linear model ensured that the day of pregnancy did not have an effect (p <= 0.4), and nested ANOVA results showed that there was no effect of parity (p <= 0.09) on the outcome. Because none of the other tests resulted in significance, the data from each treatment group (intraspecies pregnancy or interspecies pregnancy) were pooled and subjected to a Student’s t test. A value of p <= 0.05 was considered statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
MHC specificity of equine CTL activity

PBL from nonpregnant female horses and ponies were used as responder cells and stimulated with irradiated lymphocytes from an MHC homozygous stallion. The CTL activity in this system was specific for polymorphic class I Ags of the equine MHC (Fig. 1Go). The in vitro primed CTL lysed targets from the stimulator of the 10-day culture and target lymphocytes from a genetically unrelated animal that carried the same MHC class I type as the stimulator, but did not lyse the autologous control lymphocytes or target cells from an animal carrying a MHC type different from that of the stimulator. Fig. 1Go shows data representative of that obtained in >85 similar experiments, in which we tested animals of various ages and genetic backgrounds. A sample of the MHC specificities of different responder and stimulator combinations tested is shown in Table IGo.



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  		FIGURE 1. MHC specificity of equine CTL activity. PBL were collected from a nonpregnant mare (MHC type A6/A7) and cultured in vitro for 10 days with irradiated lymphocytes from a genetically unrelated horse (A2/A2) to prime CTL. On day 10, these CTL effectors were incubated for 4 h with 51Cr-labeled lymphocyte targets, and cell lysis was determined by the formula shown in Materials and Methods. Lysis was specific for the MHC type of the priming lymphocytes (A2/A2), as seen by the strong killing against the primary lymphocytes and MHC-matched lymphocytes (A2/A10). Lysis was negligible against the autologous control (A6/A7) and MHC-mismatched (A10/?) targets. Data are shown as mean of triplicate wells ± SD. See Table IGo for a list of other genetic combinations tested.

 

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  		Table I. MHC specificity of equine CTL assays MHC types1

 
For Ab blocking experiments, CTL were primed in vitro as described above, but only lymphocytes from the stimulating stallion were used as targets. Where Ab blocking was attempted, mAbs were added to the effector or target cells as described in Materials and Methods. The E:T ratio for these assays was 50:1. Cytolysis of the primary target was blocked by mAbs to equine CD8 (added to the effector cells) and equine MHC class I molecules (added to the targets), but not by a mAb specific for equine CD4 (also added to the effector cells) (Fig. 2Go).



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  		FIGURE 2. Monoclonal Ab blocking of equine CTL killing. Ab blocking was performed just before the start of the 4-h 51Cr release assay. Monoclonal Abs directed against MHC class I, CD8, or CD4 (26, 27) were added to the target wells (anti-MHC class I) or to effector cells (anti-CD8 or anti-CD4) and incubated for 1 h. The data demonstrate percent change in cytotoxicity from control (no Ab blocking). Data are shown as mean ± SD of five observations. *, p < 0.001 by a Student’s t test; significant inhibition of cytotoxicity compared with control or anti-CD4 cytotoxicity levels.

 
Effect of intraspecies pregnancy on CTL activity

Normal, intraspecies horse and donkey pregnancies were established using artificial insemination. In vitro mixed lymphocyte cultures were set up between days 7 and 325 of gestation to prime CTL from the pregnant mares to MHC class I Ags of the sire of the mating. The CTL were tested in the 4-h, 51Cr release assay. After 10 days of culture, CTL activity was dramatically decreased when the responder female from which lymphocytes were isolated was pregnant at the time of blood collection (Fig. 3Go). This effect was observed in lymphocyte cultures from both horse mares and donkey jennets. There was a significant depression in the CTL responses to the mating males by the females while they were pregnant, when compared with the cytotoxicity generated against the same male while the animals were nonpregnant. The CTL activity to autologous and third party targets was very low in these assays (data not shown in Fig. 3Go for clarity; see Fig. 1Go and Table IIGo for examples). CTL activity returned to normal levels after pregnancy was terminated.


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  		Table II. Antipaternal CTL activity of mares carrying either intraspecies horse or interspecies hybrid mule pregnancies1

 
Interspecies hybrid pregnancy did not cause a shift in maternal antipaternal CTL activity

Four mares were inseminated using semen from either a jack donkey (for interspecies hybrid mule pregnancy) or a horse stallion (for intraspecies horse pregnancy) and tested for their ability to mount CTL against PBL from the respective sire (Table IIGo). For mares 2446 and 1406, the order was interspecies followed by intraspecies pregnancy. For mares 2996 and 2470, intraspecies pregnancy was established first, followed by the interspecies pregnancy. PBL were isolated from each mare and the breeding male between days 14 and 30 after the establishment of pregnancy, and in vitro cultures were established as described. Each mare was tested twice in each pregnancy. Between days 33 and 35 of pregnancy, conceptuses were removed by means of nonsurgical uterine lavage (29), and mares were allowed to remain nonpregnant for one or more estrous cycles. The mares were then inseminated with semen from either a jack donkey or horse stallion for the reciprocal mating. Mares pregnant with horse conceptuses (intraspecies pregnancy) had decreased ability to make CTL responses against paternal alloantigens (mean [test-control] values approached zero). In contrast, the same mares, when carrying interspecies mule conceptuses, made strong CTL responses against paternal alloantigens.

These results were robust, and they were detected in animals of diverse genetic backgrounds (Fig. 4Go). When mares were pregnant with interspecies mule conceptuses, their antipaternal CTL activity was greater than the CTL activity generated by nonpregnant control females challenged in vitro with the same histocompatibility Ag barrier (p < 0.05). In contrast, when mares or jennets were pregnant with intraspecies horse or donkey conceptuses, respectively, the antipaternal CTL activity was less than that generated in lymphocyte cultures from nonpregnant control females tested in the same experiments (p < 0.001).



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  		FIGURE 4. Differences in CTL activity during intraspecies (horse and donkey) vs interspecies (mule) pregnancy. For horse pregnancies, mares were inseminated with semen from either stallion 0834 (MHC type A2/A2) or stallion 2505 (A3/A3). In some cases, data are from two or more pregnancies in one mare, with both stallions used as sires. Mule and donkey pregnancies were established by inseminating mares and jennets with semen from either jack 1989 (Ag3/Ag3) or jack 1992 (Ag1/Ag1). Again, in some cases, data are representative of two or more pregnancies. The MHC types of each female are shown in the . "?" indicates that alloantibodies were not available for the MHC type of that animal, so its haplotype was undetermined. Each bar indicates the difference between the mean CTL activity of lymphocytes from a pregnant animal and a control, nonpregnant animal tested at the same time. Open bars represent interspecies hybrid mule pregnancy. Hatched bars represent intraspecies horse or donkey pregnancies. Data represent varying numbers of tests and pregnancies. CTL activity from mares or jennets carrying horse or donkey intraspecies conceptuses, respectively, was always less than the control (p < 0.001), and CTL activity from mares carrying interspecies hybrid mule conceptuses was always greater than or equal to the control values (p < 0.05).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
These data demonstrate a dramatic decrease in antipaternal alloantigenic CTL activity in lymphocytes from mares or jennets carrying intraspecies pregnancies (Figs. 3Go and 4Go). This effect appears similar to the hyporesponsiveness of T cells to paternal alloantigens described in mice (2, 3). Furthermore, to our knowledge, it is the first demonstration of systemic decreases in antipaternal T cell reactivity during pregnancy in any randomly bred species. It is important that these alterations in maternal immune reactivity occur in species with very different placental structure. The mare has an epitheliochorial (noninvasive) placenta, and the mouse has a hemochorial (invasive) placenta (18). Moreover, the type of immune cells that are present in the uterus do not appear to affect the described effects. In mice, the predominant immune cell in the uterus is the NK-like granulated metrial gland cell (30), while the uterus of the mare primarily contains classical CD4+ and CD8+ T cells (19).

Contrary to the finding by Jiang and Vacchio (4) for the H-Y minor histocompatibility Ag, the described hyporesponsiveness in our experiments is transient rather than permanent, and full responsiveness to paternal alloantigens returned postpartum (Fig. 3Go). Thus, the horse results correspond to research in both transgenic and nontransgenic mice that demonstrated that T cell responses to paternal MHC alloantigens were restored shortly after parturition (2, 3). There may be many reasons for the discrepancies between the different studies; one simple explanation is that there are multiple, overlapping mechanisms of fetal survival, and not all mechanisms are utilized by every species. However, Jiang and Vacchio (4) argue that their results differ from those previously described because H-Y is present at more physiological levels than the MHC Ags used in other experiments (5). Our work, and that done by Robertson et al. (3), also demonstrate physiological systems, since the antigenic stimuli are fetal and paternal alloantigens, and the strong alloreactions permit the use of nontransgenic T cells for the experiments. Although it appears that the hyporesponsiveness in our model is reversible postpartum, it is possible that there is permanent deletion of some T cells clones that is impossible to measure in our system.

Other evidence from equids indicates that pregnancy may also induce a generalized shift away from cell-mediated immunity, and toward Ab-mediated responses, as Wegmann and his colleagues have proposed for mice (6, 8, 9) and humans (7). Our conclusion was drawn from a comparison of immunological aspects of horse and mule pregnancy.

Mares make high-titered Ab responses to paternal MHC class I Ags that are detected early in gestation in normal horse pregnancy (10). By contrast, in mares carrying hybrid mule conceptuses, the antipaternal Ab responses arise later and are of lower titer (11, 31). These responses are directed against allelic epitopes of paternal MHC Ags of the mating donkey (J.M.B. and D.F.A., unpublished observations).

A second difference between horse and mule pregnancy is seen in the leukocyte infiltration around the invasive trophoblast of the endometrial cups. In pregnant horse mares, there is a triphasic leukocytic response to the endometrial cup cells (19). The first phase occurs immediately following the invasion of the chorionic girdle trophoblast cells to form the endometrial cups and is characterized by a striking infiltration of CD4+ and CD8+ T lymphocytes around the cups at about day 40 of gestation. The second phase occurs in the midlife of the cups (around day 60), where the lymphocytes are greatly diminished in number. In the third phase, a more complex leukocytic infiltration is observed that may bring about the demise of the cups between days 80 and 120. In mule pregnancy, the leukocyte response to the endometrial cups seems to progress uniformly, with no reduction in leukocyte numbers around day 60 (31, 32). In fact, the mule endometrial cups are usually destroyed by this stage of pregnancy.

Considered together with the differences in maternal antipaternal CTL activity between intraspecies and interspecies pregnancy described in this paper, these observations suggest a unifying hypothesis: in normal intraspecies horse pregnancy there may be a shift away from cell-mediated immunity (Figs. 3Go and 4Go), toward humoral immunity (or from Th1 to Th2 type immune responses) that does not occur in interspecies mule pregnancy (Table IIGo, Fig. 4Go). These differences may reflect a subtle immunological barrier to interspecies mating that in itself does not prevent interspecies pregnancy, but which might compromise the ability of females to carry to term hybrids made between closely related species.

In 1949, Coombs (33) reviewed data obtained by Caroli and Bessis (34) demonstrating an 8% mortality in newborn mules caused by hemolytic disease of the newborn resulting from isoimmunization during pregnancy. Moreover, an immunological basis for pregnancy failure has been proposed in interspecies hybrid (sheep x goat) pregnancies, based on the discovery of antifetal hemolytic activity in serum from does after hybrid pregnancy and a faster rejection of second hybrid pregnancies than first pregnancies (35, 36).

Thus, there is strong evidence from both pre- and postnatal studies indicating pathological consequences of maternal immune reactivity to hybrid interspecies conceptuses. We propose that such untoward consequences result in part from abnormal signaling between mother and hybrid fetus early in pregnancy. This fails to establish an immunological relationship emphasizing recognition (as opposed to destruction) of the developing fetus by the maternal immune system. In both the horse and donkey, two very closely related species, similar mechanisms appear to operate to ensure intraspecies pregnancy success (a shift away from cell-mediated immunity), yet this protective mechanism seems to break down when the two species are mated to create an interspecies hybrid conceptus. Perhaps it is correct, as hypothesized by Mourant (33) of hemolytic disease of the newborn, that its effect "will thus lead to the development of new species" by perpetuating immunogenetic differences arising in reproductively separate populations. The subtle differences between intraspecies horse and interspecies mule pregnancy described here may be a newly recognized manifestation of mechanisms with similar outcome.


   Acknowledgments
 
We thank James Hardy for assistance with animal breeding; Jane Miller, Melissa Carlson, Laurie Lantagne, and Sarah Deacon for their technical help; and Dr. Charles McCulloch from the Department of Biometrics at Cornell University for statistical consultation.


   Footnotes
 
1 This work was supported by research Grants NICHD-15799 and NICHD-80436 from the National Institutes of Health and by the Dorothy Russell Havemeyer Foundation. J.M.B. was supported by a U.S. Department of Agriculture Training Grant in Biotechnology, No. 96-38420-3061. Back

2 Address correspondence and reprint requests to Dr. Jessica M. Baker, James A. Baker Institute for Animal Health, College of Veterinary Medicine, Cornell University, Ithaca, NY 14853. E-mail address: Back

3 Current address: Bio-Rad Laboratories, Hertfordshire, United Kingdom. Back

Received for publication October 9, 1998. Accepted for publication January 28, 1999.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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