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Stem Cells, Vol. 15, No. 1, 43-49, January 1997
© 1997 AlphaMed Press

An Analysis of the Effects of Combined Treatment with rmGM-CSF and PEG-rHuMGDF in Murine Bone Marrow Transplant Recipients

Graham Molineux, Cynthia Hartley, Patricia McElroy, Clay McCrea, Patrick Kerzic, Ian McNiece

Amgen, Inc., Department of Developmental Hematology, Thousand Oaks, California, USA

Key Words. Megakaryocyte growth and development factor • PEG-rHuMGDF • GM-CSF • Bone marrow transplant

Correspondence: Dr. Graham Molineux, Department of Developmental Hematology, Amgen, Inc., Amgen Center, Mailstop 99-1-A, 1840 Dehavilland Drive, Thousand Oaks, CA 91320, USA.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
We have studied the potential of combination growth factor treatment with GM-CSF and megakaryocyte growth and development factor (MGDF) to stimulate hematopoietic recovery in mice following bone marrow transplantation. More rapid recovery of neutrophils occurred in mice treated with recombinant murine (rm)GM-CSF plus pegylated recombinant human (PEG-rHu)MGDF than carrier treated controls, however this recovery was equivalent to the effect of treatment with rmGM-CSF alone. PEG-rHuMGDF stimulated a more rapid recovery of platelets with no effect on neutrophil recovery. At the two tested doses of rmGM-CSF (72 and 200 µg/kg/day) the platelet recovery was inferior to that in carrier treated mice. Also, the addition of rmGM-CSF to PEG-rHuMGDF had a dose-related negative impact on platelet recovery compared to PEG-rHuMGDF alone.

These data suggest that the use of combination therapy in some clinical indications may lead to unexpected results. Furthermore, careful dosage studies may be necessary to identify the full potential of combined growth factors to obtain additive or synergistic effects on multilineage hematopoietic reconstitution in vivo.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Megakaryocyte growth and development factor (MGDF) [1], a ligand for the c-mpl encoded cytokine receptor, has been shown to increase platelet numbers in normal mice and to accelerate platelet recovery in murine models of chemotherapy-induced thrombocytopenia [2, 3], irradiation [4] and bone marrow transplantation (BMT) [5] or peripheral blood progenitor cell transplantation (PBPCT) [6]. In many transplant and chemotherapy settings recombinant human (rHu)MGDF is likely to be used not only as a single agent, but also as an adjunct to established growth factor therapy. Two colony-stimulating factors (CSFs), G-CSF and GM-CSF, have demonstrated clinical usefulness in accelerating recovery from neutropenia [7, 8]. Previous studies in mice have shown the efficacy of pegylated (PEG) rHuMGDF in promoting multilineage reconstitution in model systems of BMT [5] and PBPCT [6] in combination with rHuG-CSF. The benefits of combination PEG-rHuMGDF/rHuG-CSF therapy have therefore been established in experimental systems, but little information has been obtained on the alternative PEG-rHuMGDF/GM-CSF combination.

The efficacy of human GM-CSF has been well established by continuous infusion in primates [9] and in clinical practice [10, 11]. In murine systems, however, the lack of cross-species reactivity of human material has lead to GM-CSF being relatively poorly studied, with somewhat mixed data emerging from the published reports. Early studies [12] showed an almost twofold increase in circulating neutrophils in BALB/c mice injected with up to 24 µg/kg/day rmGM-CSF. This compares with a 70-fold increase in neutrophils in mice treated with rHuG-CSF at only 10 µg/kg/day [13]. In rats injected intravenously once a day with approximately 4 µg/kg/day rmGM-CSF, Ulich et al. [14] found peak neutrophil numbers to be increased about three- to fourfold over control in comparison with a rHuG-CSF response of around 10-15 times control numbers. On longer treatment schedules, insignificant changes in hematopoietic parameters have been reported [15]. In a sublethal irradiation model of myelosuppression, GM-CSF again had relatively modest effects [16, 17] and, in models involving BMT and post-transplant rmGM-CSF treatment, significant, though modest, improvements in hematopoietic recovery rates were again reported [18]. Thus the response of normal rodents to rmGM-CSF has been studied and indicates a significant, though relatively modest, effect of the injected protein. These data are in contrast to the published results in clinical settings, where GM-CSF appears a much more useful molecule [10, 11]. From a brief review of published data there would appear to be no consensus on treatment scheduling with rmGM-CSF in mouse studies. Though this is not an uncommon feature of the disparate settings in which this and other CSFs are studied, it would appear that scheduling issues associated with the use of rmGM-CSF must be taken into account. To avoid potential scheduling problems in these experiments we have delivered rmGM-CSF by continuous s.c. infusion first to normal mice then to BMT recipients as a single agent and in combination with PEG-rHuMGDF. In normal mice more than a 100-fold increase in neutrophil counts and over 200 x 103 WBC/µl were obtained with continuous infusion. Accelerated hematopoietic reconstitution was tracked by peripheral blood cell recovery rates in BMT recipients.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Bone Marrow Transplantation
Eight- to twelve-week-old female BDF1 mice (C57Bl/6 x DBA2 F1) were irradiated at 1,200 cGy (137Cs, dose rate 106.7 cGy/min) given as a split dose (2 x 600 cGy, 4 h apart). Bone marrow cells were harvested from littermate mice by flushing the femoral contents with Hank's balanced salt solution (Life Technologies Inc.; Grand Island, NY) supplemented with 2% fetal bovine serum (Cansera International Inc.; Rexdale, Ontario). Cells were counted in a hemocytometer and dilutions made to allow the i.v. injection of 106 bone marrow cells in 500 µl into irradiated mice 4 h after completion of the irradiation. For each experiment, groups of ten mice were injected. These groups were then split into two cohorts of five mice that were alternately sampled for the duration of the study. The whole experiment was performed three times and data are presented from pooled data from all three experiments, giving 15 independent analyses at each time point.

Growth Factors
rHuMGDF was expressed in E. coli using a plasmid that encodes a truncated form of the Mpl ligand, including the erythropoietin-like amino terminus. The MGDF was purified to greater than 95% purity as previously reported [1]. The material was further derivitized with polyethylene glycol (PEG-rHuMGDF). Recombinant murine (rm)GM-CSF was prepared as in E. coli and purified to greater than 99% purity. All growth factors were prepared at appropriate dilutions to give the dose levels indicated in the Results section. Dose levels of 72 and 200 µg/kg/day of rmGM-CSF were used either alone or in combination with PEG-rHuMGDF at 50 µg/kg/day. Treatment was administered by continuous s.c. infusion via Alzet mini-osmotic pump (2002, Alza Corporation; Palo Alto, CA). Pumps were implanted the day before irradiation and bone marrow transplant. Delivery from these pumps could be expected to begin within 4 h and continue for up to 18 days. Carrier solution was phosphate buffered saline (GIBCO-BRL; Grand Island, NJ) supplemented with 0.1% bovine serum albumin (Sigma; St. Louis, MO) and was used to dilute growth factors for infusion, or infused alone to carrier treated animals.

Peripheral Blood Analysis
To determine recovery postBMT, peripheral blood was collected from the retro-orbital sinus of mice at various days up to day 21. Full blood counts were performed on a Technicon H-1E (Technicon Instruments Corp.; Tarrytown, NY) calibrated for mouse blood.

Statistical Analysis
Where indicated in the figures, differences between carrier and growth-factor treated animals were tested with Student's t-test using the Sigma Stat program (Jandel Scientific; San Rafael, CA).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Dose Finding Study in Normal Mice
Groups of 10 normal, untreated, female BDF1 mice were implanted with osmotic pumps resulting in treatment levels of rmGM-CSF of 100, 200 or 300 µg/kg/day. Data are shown in Figure 1. Continuously infused rmGM-CSF induced a significant leukocytosis as indicated by WBC counts in excess of 200 x 103/µl by day 14. The majority of recruited cells were neutrophils, which increased from less than 1 x 103/µl to greater than 100 x 103/µl, but monocytes also increased from a baseline of 0.1 x 103/µl to more than 10 x 103/µl. Accompanying the increases in myeloid elements were significant reductions in both erythrocytes and platelets. From these data a dose level of 200 µg/kg/day was selected for comparison with previous studies using rHuG-CSF at the same treatment level. A further reduction to 72 µg/kg/day was also used in an attempt to minimize the negative impact on platelet numbers.



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  		Figure 1. Blood cell responses in normal mice infused with rmGM-CSF at 100, 200, or 300 µg/kg/day delivered by continuous s.c. infusion. Mean ± 1 SD, n = 10.

 
Platelet Recovery after BMT
Platelet numbers fell to a nadir of around 100 x 103 platelets/µl of blood at or around day 7 after ablation and transplant (Figs. 2A and 3A). This compares with a pretreatment level of around 1342 x 103/µl. Platelet counts in untreated recipients then recover along a predictable curve to attain around 1,000 x 103 by day 16, a level which remains stable beyond day 21.



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  		Figure 2A. Leukocyte and platelet recovery in transplanted mice treated with innocuous carrier, PEG-rHuMGDF (50 µg/kg/day) and/or rmGM-CSF at 72 µg/kg/day. Each data point is the mean ± SD of 15 independent animals, pooled from three complete repeats of the experiment. *p < 0.05, **p < 0.01.

 


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  		Figure 3A. Leukocyte and platelet recovery in transplanted mice treated with innocuous carrier, PEG-rHuMGDF (50 µg/kg/day) and/or rmGM-CSF at 200 µg/kg/day. Each data point is the mean ± SD of 15 independent animals, pooled from three complete repeats of the experiment. *p < 0.05, **p < 0.01.

 
PEG-rHuMGDF Effect
As has been previously shown, the administration of PEG-rHuMGDF during recovery from BMT accelerates platelet recovery [5]. After attaining a nadir comparable with carrier-treated recipients at the same time point, platelet numbers were consistently and significantly higher in the PEG-rHuMGDF-treated animals than in carrier or rmGM-CSF-treated animals (Figs. 2A and 3A). The arbitrary point of 1,000 x 103 platelets per microliter was passed on day 13-14, four days earlier in the PEG-rHuMGDF group than in controls, and final numbers attained were around 3,000 x 103/µl on day 16, around three times higher than carrier-treated recipients and more than twofold higher than pretreatment numbers (Figs. 2A and 3A).

GM-CSF Effect
Two doses of rmGM-CSF were given to BMT recipients (Figs. 2 and 3). The lower of the two doses, 72 µg/kg/day, resulted in platelet counts significantly lower than counts in carrier-treated animals. Platelet nadir coincided with carrier-treated animals at day 7, but the platelet count at this point was around 40-50 x 103/µl compared to around 100 x 103 in carrier-treated recipients (significantly lower, p < 0.05). Platelet recovery was delayed compared to carrier-treated mice until day 21 of the study (Fig. 2A)

The higher dose (200 µg/kg/day) of rmGM-CSF depressed platelet counts more seriously than the lower dose (Fig. 3A). A nadir of 50 x 103/µl, attained at day 7 after BMT, preceded delayed recovery and numbers lower than control at every time point. Five days after rmGM-CSF infusion stopped, platelet numbers were around 400 x 103/µl when counts in carrier treated recipients were approximately 1,000 x 103/µl (significantly lower than carrier, p < 0.05), and PEG-rHuMGDF recipients had around 2,000 x 103 platelets per microliter of blood (significantly higher than carrier, p < 0.01).

GM-CSF/PEG-rHuMGDF Combination Treatment
As outlined above, the effects of the two growth factors on platelet recovery were in contrast to one another. It was not perhaps surprising, therefore, that the effects of the combination treatment were intermediate between the effect of either factor alone. Thus, the suppression of platelet recovery resulting from rmGM-CSF treatment was reversed by the inclusion of PEG-rHuMGDF in the treatment schedule. On the other hand, the effect of PEG-rHuMGDF on accelerating platelet recovery was minimized in the presence of rmGM-CSF. Indeed the nadir of around 50 x 103 platelets per microliter in rmGM-CSF + PEG-rHuMGDF recipients on day 7 was similar in terms of time after ablation, depth of thrombocytopenia and duration to that noted in BMT recipients of only rmGM-CSF treatment. Following the nadir with platelet numbers about half of the number in carrier or PEG-rHuMGDF-treated recipients, the recovery in combination-treated mice was indistinguishable from control. This resulted in peak platelet numbers at slightly below 1000 x 103/µl (Fig. 3A). The advantage of around three days in recovery of platelet numbers to 300, 500 or 1,000 x 103 platelets per microliter noted in PEG-rHuMGDF-treated BMT recipients was also lost in mice treated with the combinations of rmGM-CSF plus PEG-rHuMGDF.

Leukocyte Recovery after BMT
Our previous experience (data not shown) indicated that the nadir in leukocyte numbers after BMT does not necessarily coincide with the nadir in platelet counts. Our first post-BMT study point in these experiments was designed to examine the nadir in platelet numbers and therefore probably does not correspond to the nadir in WBC. We were, however, able to show that leukocyte numbers on day 5 were around 0.1 x 103 WBC/µl in all treatment groups. A steady recovery was then sustained through day 14 when numbers approached 5 x 103/µl, a stable level that persisted beyond day 21. Control (pretreatment) numbers were 12.8 ± 3.3 x 106/ml (SD, n = 30), and this level had not been attained by the end of the study period in mice that did not receive growth factor support (Figs. 2A and 3A).

PEG-rHuMGDF Effect
No effect of PEG-rHuMGDF was noted on leukocyte recovery after BMT. At no point between 5 and 21 days did the recovery of WBC in PEG-rHuMGDF treated recipients differ significantly from the recovery in carrier-treated recipients (Figs. 2A and 3A).

rmGM-CSF Effect
From day 7 onwards leukocyte numbers were higher in both rmGM-CSF treatment groups than in mice receiving only carrier solution. Both doses were similarly effective, doubling WBC counts at day 7 (from 0.17 x 103/µl in carrier, to 0.33 x 103/µl) in rmGM-CSF recipients. The advantage in rmGM-CSF treatment was maintained throughout the study period, resulting in peak leukocyte numbers of over 100 x 103/µl (between days 12 and 16) and maintaining numbers of 20 x 103 at 72 µg/kg/day and over 60 x 103/µl at 200 µg/kg/day through the end of the study at day 21 (Figs. 2A and 3A).

rmGM-CSF/PEG-rHuMGDF Combination Treatment
Leukocyte recovery was accelerated in recipients of the rmGM-CSF/PEG-rHuMGDF combination over that in carrier recipients. At no point on the recovery curves was there a significant difference between leukocyte counts in rmGM-CSF and rmGM-CSF + PEG-rHuMGDF-treated mice. The numbers were, however, consistently and significantly higher than in PEG-rHuMGDF or carrier-treated BMT mice (p varies, Figs. 2A and 3A).

Leukocyte Differential Analysis
The analysis of various leukocyte populations in the blood of mice recovering from BMT did not reveal any qualitative effects of PEG-rHuMGDF, substantiating the absence of any quantitative change as noted above. Neutrophils accounted for the majority of leukocytes at most time points studied. Detailed data are presented in Figure 4 including data from normal, nontransplanted mice. Carrier-treated BMT mice had 0.04 x 103 neutrophils/µl at day 5, the low point, and stabilized at between 3 and 4 x 103/µl from day 14 onwards. Beyond day 14 lymphocyte numbers were noted above 1 x 103/µl. The recovery curves of PEG-rHuMGDF-treated mice were very similar to control with the same numbers of neutrophils, lymphocytes, monocytes, eosinophils and basophils noted at every time point. During accelerated leukocyte recovery under the influence of infused rmGM-CSF, the majority of recruited cells were neutrophils — over 60 x 103/µl on day 12 — the peak level attained. Later than this, lymphocytes represented an increasing proportion of leukocytes, reaching over 55 x 103/µl at the peak on day 16. Accompanying this excess of lymphocytes was a significant number of monocytes — over 34 x 103/µl on day 16, compared with a maximum number of less than 0.5 x 103/µl at any time point in the recovery of carrier- or PEG-rHuMGDF-treated BMT recipients. No notable differences were seen between the various leukocyte populations in mice treated with rmGM-CSF and rmGM-CSF/PEG-rHuMGDF combination recipients.



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  		Figure 4. Recovery of different blood cell populations through day 21 after BMT and growth factor treatment as indicated. NEUT = neutrophils, LYMPH = lymphocytes, MONO = monocytes, EOS = eosinophils, BASO = basophils. Each portion of each stacked bar is a mean from 15 individual analyses; errors are not shown. Normal data from nontransplanted, untreated animals are shown at right. Scales vary between panels. Absolute normal values were: NEUT = 1.29 ± 0.76 x 103/µl; LYMPH = 7.21 ± 2.09 x 103/µl; MONO = 0.23 ± 0.09 x 103/µl; EOS = 0.15 ± 0.09 x 103/µl; BASO = 0.06 ± 0.04 x 103/µl (mean ± 1 SD, n = 30).

 
Erythroid and Monocyte Recovery
Monocytes recovered much more quickly in the mice supported with rmGM-CSF (Figs. 2B and 3B). Peak numbers were comparable at either dose of rmGM-CSF. Red cell parameters were also generally comparable at both tested rmGM-CSF doses. There was a marked reduction in red cell count, hemoglobin and hematocrit particularly towards the end of the treatment period up to day 21 (the end of the study). The degree of impairment of erythroid recovery was generally worse at the higher dose of GM-CSF. Red cell distribution width, a measure of anisocytosis, was much higher in the GM-CSF recipient mice, indicating a greater degree of variation in red cell size (Figs. 2B and 3B). PEG-rHuMGDF did not affect red cell recovery one way or the other.



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  		Figure 2B. Erythrocyte and monocyte parameters in the recovery phase after BMT in mice supported with rmGM-CSF (72 µg/kg/day), PEG-rHuMGDF, or both. RBC = red blood cell count (x 106/µl); RDW = relative distribution width; HCT = hematrocrit. Indications of significance are omitted for clarity; data points represent the mean of 15 individual counts ± 1 SD.

 


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  		Figure 3B. Erythrocyte and monocyte parameters in the recovery phase after BMT in mice supported with rmGM-CSF (200 µg/kg/day), PEG-rHuMGDF, or both. RBC = red blood cell count (x 106/µl); RDW = relative distribution width; HCT = hematrocrit. Indications of significance are omitted for clarity; data points represent the mean of 15 individual counts ± 1 SD.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Murine GM-CSF has proven difficult to exploit in mouse models of bone marrow transplantation due to problems in defining protocols in which the material shows readily documented effects. It is possible that these difficulties were due, at least in part, to scheduling issues. In these studies we have delivered the growth factors by continuous s.c. infusion. In normal mice treated with rmGM-CSF we have noted two log increases in neutrophils and monocytes—effects more in keeping with the responses obtained in patients than the previously published data from mice. Having thus defined a delivery system which reflects more fully the potential of GM-CSF as a hematopoietic modulator, we have been able to document some of the effects of rmGM-CSF in a murine model of BMT.

PEG-rHuMGDF has been shown previously to be highly effective in sustaining accelerated platelet recovery in models of BMT [5], and recent preliminary Phase I clinical data indicate that enhanced thrombopoiesis is also a feature of PEG-rHuMGDF use in patients [19]. As a logical extension to our previous work with combinations of rHuG-CSF and PEG-rHuMGDF in murine models of both PBPCT and BMT, we have now studied the effects of rmGM-CSF in combination with PEG-rHuMGDF in murine BMT recipients. It has been previously reported with thrombopoietin (TPO) that beneficial effects on platelet and erythrocyte recovery can be obtained with bone marrow donor TPO treatment after cells are subsequently transplanted to ablated mice which received no further growth factor support [20]. This is in contrast to our experience with TPO-treated PBPC donors where post-transplant treatment was many fold more effective than donor treatment in respect to platelet recovery time [6]. For this reason, in these experiments we have chosen to restrict our observations to the effects of post-transplant recipient treatment.

In keeping with previous data, PEG-rHuMGDF stimulated accelerated recovery of platelets after BMT but had little if any impact on the recovery of leukocytes. Treatment with PEG-rHuMGDF confers an advantage of between two and three days in the time of recovery to several specific levels (200, 500, 1000 x 103 platelets/µl), reflecting a reduced duration of serious thrombocytopenia. The treatment of mice post-transplant with rmGM-CSF delayed the recovery of platelets and erythrocytes. In fact, recovery of both populations was slower than we had noted in mice receiving only carrier in the post-transplant phase. Recovery of platelets to any of the above levels was delayed by a day or more. This suppression of platelet recovery was dependent on the dose of rmGM-CSF. Later in the treatment period the 200 µg/kg/day dose had kept platelet numbers down to less than 500 x 103/µl when carrier-treated mice had recovered to 1000 x 103/µl and PEG-rHuMGDF-supported mice had around 2,000 x 103 platelets/µl. By combining PEG-rHuMGDF with infused rmGM-CSF it proved possible to retain the beneficial effect upon leukocyte recovery obtained with rmGM-CSF and to minimize the suppression of platelet recovery. However, the fastest recovery obtained in mice treated with rmGM-CSF, even with the support of PEG-rHuMGDF, was comparable only to that in carrier-treated mice.

Recovery of erythroid parameters is also clearly delayed by GM-CSF administration (Figs. 2B and 3B). The delay is manifested as a falling red cell count and corresponding decreases in hematocrit and hemoglobin concentrations. Accompanying the reducing red cell parameters, an increasing anisocytosis was noted, corresponding to the inclusion of a population of cells of higher volume (>200 fl) in the erythrocyte window. The nature of this anisocytosis could not be determined, but it is possibly associated with the emergence of macrocytes, the relative lack of smaller erythroid cells, or inclusion of inappropriate cells in the counting window. At this stage these possibilities cannot be distinguished from each other.

The degree of leukocytosis in rmGM-CSF-treated BMT animals was greater than would have been expected based on our preliminary dose-finding studies in normal mice (Fig. 1). Indeed leukocyte numbers noted in BMT mice in response to rmGM-CSF were higher than had been seen in normal mice treated with the same dose. Normal mice, as well as BMT mice treated with rmGM-CSF, showed a marked reduction in circulating platelet numbers. It is possible that the reduction in platelet recovery is associated with the stimulation of excessive leukocyte counts. Though the dampening effect on platelet counts in rHuG-CSF-treated BMT recipients is generally less severe than that associated with rmGM-CSF treatment, a similar dose relationship was noted, i.e., higher doses, not necessarily associated with yet further increased WBC counts, had a greater impact on platelet numbers. The mechanism of this effect is not understood, but it may be a more general phenomenon associated not only with platelet numbers and CSF treatment, but also involving erythropoiesis (Figs. 1, 2B and 3B, [13, 21-23]).

Data from in vitro studies do not indicate a suppressive effect of GM-CSF on megakaryocyte or erythroid precursor cell growth, indeed, in some systems [24] GM-CSF has actually been suggested to be an erythroid burst-promoting molecule. G-CSF, which also dampens platelet response in some settings [5], lacks any negative impact on in vitro precursors of these lineages. There would appear, therefore, to be no in vitro correlate to these observations, which makes the definition of a mechanism particularly difficult. It is interesting that accelerated platelet recovery under the influence of MGDF is readily documented in murine recipients of PBPC grafts and, in this setting, G-CSF has a minimal impact on the degree of this acceleration [6]. In BMT recipients, the dampening is much more marked and, with GM-CSF at least, it also appears to extend to the erythroid lineage.

Clinical data on platelet recovery during GM-CSF therapy are mixed. Standard-dose GM-CSF has been associated with delayed or reduced platelet counts [25-28] but also "no increase in platelet transfusion requirements" [29] and even a reduction in platelet transfusion requirements at low treatment levels [30]. Therefore, lower doses given to patients do not appear to negatively impact platelet numbers; higher doses may produce delayed platelet recovery. Our studies in mice argue that high doses of rmGM-CSF suppress platelet recovery and also that lower doses may have a smaller impact on platelet numbers. The implication is also that the degree of leukocytosis is not a good predictor of the effect on platelets.

These data demonstrate that an overlap of effects between platelets and neutrophils occurs with combined treatment with rmGM-CSF and PEG-rHuMGDF. It is possible that with careful clinical studies evaluating various dosage combinations it will be possible to maximize the combined effects of both cytokines. However, these studies should proceed with caution, especially in patients who have limited numbers of progenitor cells at transplantation.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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Received on May 15, 1996; accepted for publication on August 13, 1996.




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