Liver repopulation after cell transplantation in mice... : Transplantation (original) (raw)
Liver cell transplantation (LCT) involves the isolation of a hepatocyte-enriched cell suspension from a donor liver and its infusion into either the portal circulation or an ectopic site. Much data suggest LCT could provide metabolic support in the treatment of acute liver failure, chronic end-stage liver disease, and inherited, liver-based metabolic disease (1–6). The only current treatment of these conditions is orthotopic liver transplantation, which involves major surgery, is expensive and commits the patient to life-long immune suppression. More worrisome is the critical shortage of donor organs that severely limits access to needed therapy (7). LCT could help alleviate donor organ shortage and, if proven effective, would be simpler, safer, and less expensive than whole organ transplant.
LCT is hampered by inefficient repopulation of recipient livers by transplanted cells. Long-term survival and function of liver cells transplanted into mouse spleens and livers, demonstrated by Ponder et al. (8) and Gupta et al. (9) more than 10 years ago, is only 0.03% to 0.5% of the total liver mass after one cell infusion (8). A breakthrough in liver repopulation was reported by Rhim et al. (10) by using mice transgenic for urokinase-type plasminogen activator as recipients. The livers of these mice undergo progressive damage from birth, which provides a regenerative environment for transplanted cells. Transplanted cells from wild-type mice, or native cells that switch off the transgene, have a selective survival advantage. Almost complete repopulation of the recipient mouse liver can occur under these circumstances (10). These findings have been confirmed in fumarylacetoacetate hydrolase knockout mice (tyrosinemia type I phenotype), which also confers a survival advantage to congenic transplanted cells. Tyrosinemic mice exhibit significant repopulation of host livers after LCT (6). More recently, Laconi et al.(11), developed a rat model of enhanced repopulation by pretreating rats with retrorsine. Retrorsine is an alkaloid that, after exposure, exerts a strong and persistent block of native hepatocyte proliferation (12). This drug does not affect donor cells that are transplanted 2 or more weeks after drug administration. In that rat study, partial hepatectomy was performed to promote a regenerative environment. Transplanted cells proliferate, while proliferation of recipient cells is blocked, resulting in liver repopulation by transplanted cells (11). A more recent paper by the same group demonstrated in rats that retrorsine pretreatment alone was sufficient to induce repopulation of a liver by transplanted cells (13). Carbon tetrachloride (CCl4) has been used both before and after LCT to enhance repopulation by transplanted hepatocytes. When used as a sole agent in mice, however, its effect on repopulation is modest (14).
Most models of human liver-based metabolic and genetic disease are developed in mice (Wilson’s disease, alpha1-antitrypsin deficiency, ornithine carbamoyltransferase deficiency, citrullinemia, cystic fibrosis) rather than in rats. Further, clinical applications are unlikely to include hepatectomy as a means to promote engraftment. We therefore aimed to develop a high-repopulation, nonsurgical, no-genetic-advantage mouse model of LCT.
MATERIALS AND METHODS
Reagents and chemicals.
CCl4, EGTA, fetal bovine serum, and retrorsine were purchased from Sigma Chemical Co. (St. Louis, MO, U.S.A.); Earles Balanced Salt Solution (EBSS), HEPES, and penicillin/streptomycin were purchased from Gibco BRL (Grand Island, NY, U.S.A.); isoflurane was obtained from J.A. Webster Inc. (Sterling, MA, U.S.A.); liberase was from Boehringer Mannheim Co. (Indianapolis, IN, U.S.A.); Waymouth’s MB medium, minimal essential medium, and medium 199 were purchased from GIBCO BRL (Gaithersburg, MD, U.S.A.); X vivo-10 was from Biowhittaker (Walkersville, MD, U.S.A.).
Solution preparation.
(1) Retrorsine stock solution: 100 mg of retrorsine was dissolved in 5 ml of 100% ethanol at 50–60°C overnight and then kept at room temperature. (2) CCl4 solution: CCl4 was dissolved in sterile mineral oil at a 1:10 proportion and maintained in a rubber plug–sealed glass tube until used. (3) EGTA solution: EBSS without Ca2+or Mg2+, EGTA 0.5 mM, HEPES 10 mM, pH 7.5, and penicillin/streptomycin 1%. (4) Liberase solution: EBSS with Ca2+ and Mg2+ plus liberase 0.1 mg/ml, HEPES 10 mM, pH 7.5, and penicillin/streptomycin 1%. (5) Culture and wash medium: 1:3 Waymouth’s MB medium to minimum essential medium, 10% fetal bovine serum (Sigma), 1% HEPES, pH 7.4, and 1% penicillin/ streptomycin.
Animals.
Female C57BL/6J mice, 8–12 weeks, weighing 20–30 g were used as LCT recipients (Jackson Laboratories, Rochester, NY, U.S.A.). ROSA26 female mice (C57BL/6J-TgR(ROSA26)26Sor) (15), 8–12 weeks (Jackson Laboratories) were used as donors. All procedures were performed in accordance with the guidelines of the Institutional Animal Care and Use Committee, Children’s Memorial Institute for Education and Research at Northwestern University Medical School, and the Guide for the Care and Use of Laboratory Animals 1996 (16).
Experimental design.
An initial group of mice (n=36) was tested for toxicity and effects of a combination of retrorsine and CCl4. Because ethanol was needed to dissolve retrorsine, a nonlethal albeit moderate ethanol dose was given along with the retrorsine. Once nonlethal drug combinations were established, congenic β-galactosidase (β-gal) transgenic donor liver cells, 2×106 in 0.2 ml of medium, were transplanted into the spleens of six groups of six recipient C57BL/6J mice. Group I mice (Fig. 1) received retrorsine, 70 mg/kg, i.p. Before injection, retrorsine stock solution was diluted 1:5 in sterile phosphate-buffered saline (PBS). This dose was repeated 2 weeks later and the mice allowed to recover for 1 month. LCT was performed and 2 weeks later CCl4, 0.5 ml/kg, diluted 1:10 in sterile mineral oil, was given and repeated three times at weekly intervals. Usual i.p. injection volumes for a 20-g mouse were 350 μl of retrorsine solution and 100 μl of CCl4 solution. Brief inhaled halothane anesthesia was used before the i.p. injections. Group II received retrorsine as above, a single dose of CCl4 1 month later, and LCT 24–48 hr later. Group III received LCT and CCl4, as group I, but no retrorsine. Group IV received retrorsine and LCT, as group I, but no CCl4. Group V received a single dose of CCl4 followed by LCT, as group II, but no retrorsine. Group VI received LCT only. Some mice received LCT 2 weeks rather than 1 month after the last retrorsine dose. All recipient mice were sacrificed between 1 week and 7 months after LCT. Liver segments were flash-frozen for histology and X-gal staining; fixed for whole-segment X-gal staining; and perfused for liver cell isolation, plating, and X-gal staining for quantitation of β-gal+ donor cell engraftment and repopulation.
Experimental design. Scheme to promote and evaluate enhanced engraftment after hepatocyte transplantation (HCT).
Liver cell isolation.
Liver cells were isolated from β-gal transgenic ROSA26 donor mice or from C57/BL6 recipient mice using a method described elsewhere (17). Briefly, mice were anesthetized with isoflurane and a midline laparotomy was performed. The inferior vena cava was cannulated in the lower abdomen with a 20-gauge Teflon i.v. catheter, and perfusion was started at a rate of 2.5 ml/min. After 20–30 sec, the portal vein was sectioned, and the solution was allowed to flow through the liver, from hepatic vein to portal vein. The liver was perfused sequentially with EGTA solution (see solution preparation) for 5 min followed by liberase solution for 10 min. Hepatocytes were removed by mechanical dissociation and filtered through a sterile 150-μm nylon mesh (Scientifics, Frederick, MD, U.S.A.). The cells were washed three times in serum-containing culture medium by centrifugation at 50×g for 3 min, resuspended at a concentration of 1×107 cells/ml of medium 199 (Gibco BRL), and kept in ice until infusion. Cell preparations with high viability, as measured by >80% trypan blue exclusion (0.02% solution), were used for transplantation.
Liver cell transplantation.
Cell transplantation was performed as described in a previous publication (18). Briefly, mice were anesthetized with ketamine (Abbott Laboratory, Chicago, IL, U.S.A.), 80 mg/kg, and xylazine (Bayer Laboratories, Norwood, MA, U.S.A.), 10 mg/kg, i.p. A left lateral laparotomy was performed on a sterile field, and 2×106 β-gal transgenic liver cells in 0.2 ml of media were injected into the lower pole of the spleen for 2 min. The infusion site (spleen pole) was ligated, the abdomen was sutured, long-acting analgesia was given, and the mice were allowed to recover.
X-gal histochemistry.
X-gal 5-bromo-4 chloro-3-indolyl-β-d-galactoside is a chromogenic substrate for the β-galactosidase gene product expressed in the transgenic hepatocytes used. X-gal turns blue on exposure to β-galactosidase present in those cells. X-gal histochemistry was performed on liver tissues from frozen sections, whole liver segments, and isolated cells. (1) Frozen liver sections: On killing the recipient mice, liver tissue was embedded in Tissue-Tek (Sakura Finetek U.S.A, Inc., Torrance, CA, U.S.A.) and frozen on dry ice. Frozen sections, 10 μm thick, were placed on poly-l-lysine–coated glass slides, fixed at 4°C in 1.25% glutaraldehyde/PBS for 10 min, washed with PBS, and incubated in X-gal staining solution (1.3 mM MgCl2, 15.0 mM NaCl, 44.0 mM HEPES, 3 mM ferricyanide, 3 mM ferrocyanide, 0.5 mg/ml X-gal) at 37°C overnight. Slides were then rinsed and counterstained with nuclear fast red for 30 sec at room temperature, rinsed, dipped in xylene, and coverslipped with mounting medium for subsequent analysis. Standard hematoxylin and eosin staining was performed with adjacent sections. (2) Whole liver segments: Fresh samples were placed in organ fixing solution (4% paraformaldehyde), 0.1 M NaH2PO4/Na2HPO4 (pH 7.3), 2 mM MgCl2, 0.01% sodium deoxycholate, and 0.02% IGEPAL C-630 (Sigma) for 60 min at 4°C, rinsed for 30 min, three times with wash buffer (0.1 M NaH2PO4/Na2HPO4 [pH 7.3], 2 mM MgCl2, 0.01% sodium deoxycholate, and 0.02% IGEPAL C-630), then incubated in X-gal staining solution (1 mg/ml X-gal [Sigma], 5 mM ferricyanide, and 5 mM ferrocyanide at pH 7.3–7.6 in wash buffer) for 16–24 hr at 37°C, and fixed for 24–48 hr in 10% formalin. (3) Quantitation of the proportion of liver repopulation in isolated-cultured hepatocytes: Liver cells were isolated from transplantation recipient mice as already described. Cells were cultured on plastic dishes for 4–5 hr in serum containing medium, washed with PBS, fixed in 0.5% glutaraldehyde (prepared in PBS with Ca2+ and Mg2+) at 4°C for 5 min, washed twice with cold PBS, and exposed to X-gal staining solution at 37°C for 12–16 hr. Plastic dishes were examined in an inverted phase-contrast microscope, and the number of total and X-gal–staining (donor) cells counted under the high-power (×20) objective was measured. A transparent grid under the dish was used to count the same locations in all plates, and at least 500 cells were counted per dish. The percentage of X-gal–positive (donor) cells was calculated as the average obtained by two independent observers.
Statistics.
Repopulation efficiencies were compared using a one-tailed Student’s t test for two samples with equal variance.
RESULTS
Safety margin of retrorsine and CCl4 in mice.
Because it was suspected that mice might have a different response to retrorsine than that seen in other rodents (19), studies were conducted to determine the highest combined doses of retrorsine and CCl4 that could be safely administered to mice. The aim of these studies was to find the maximum nonlethal combined doses of CCl4 and retrorsine that would promote repopulation of transplanted cells. Different retrorsine dilutions in ethanol were also tested. If higher amounts of ethanol than those described in the Methods section were used, seizures and death occurred. If lower amounts of ethanol were used, retrorsine would not dissolve. After individual drug toxicity was evaluated, a total of 86 mice were studied for combined toxicity in four different groups. In the first group, retrorsine (20 mg/kg i.p.) was administered at a 2-week interval, followed by a single dose of CCl4. Doses ranging from 0.1 to 0.75 ml/kg CCl4 were administered to four mice, and no mortality was observed 2 months after treatment. In a second group (n=4), the retrorsine concentration was increased to 30 mg/kg with similar results and survival of all mice. In a third group (n=8), the CCl4 dose was maintained at 0.5 ml/kg, and retrorsine doses varied from 20 to 110 mg/kg. Only two of four mice receiving repeated retrorsine doses of 90 mg/kg and 110 mg/kg survived, whereas retrorsine doses lower than 70 mg/kg resulted in survival of all mice. In a fourth group, which included study mice, 65 of 70 mice (92%) receiving retrorsine doses of 70 mg/kg followed by CCl4 0.5 ml/kg survived. An observed side effect of retrorsine infusion was sleep for several hours, likely because of the ethanol used to dilute this drug.
Whole organ X-gal staining demonstrates changed phenotype after liver cell transplantation.
Two months after LCT, animals were killed and whole liver explants were examined for phenotypic change by X-gal histochemistry. Figure 2 illustrates the change in β-gal expression induced by the transplanted transgenic hepatocytes of a representative recipient from group I. Interestingly, only 20% of cell replacement by transplanted hepatocytes were sufficient to confer light blue staining to the recipient liver. Fully transgenic β-gal–expressing liver had a stronger X-gal staining pattern. No β-gal expression was seen in livers of untreated control animals.
Whole-liver X-gal staining. A, in negative control mouse liver from C57BL/6J mice, without LCT, no blue color is seen. B, representative liver from a group I, LCT recipient mouse. Liver phenotype turns light blue owing to β-gal expression in >20% of the hepatocytes. C, mouse liver from donor β-gal transgenic mice shows strong X-gal staining.
Repopulation by transplanted liver cell clusters.
Frozen sections of LCT recipient mice livers were examined 2 months after LCT. No X-gal staining was seen in tissue sections of untransplanted control livers (Fig. 3A), whereas the majority of donor β-gal transgenic tissue turned blue on X-gal exposure (Fig. 3B). Sections from transplant recipient livers exhibited multiple clusters of X-gal+ cells, (Fig. 3C). Microscopic examination of adjacent histologic sections stained with hematoxylin and eosin showed completely normal morphology and full integration of these clusters originated from transplanted cells into the native recipient liver structure (Fig. 3, D and E). Clusters were observed in both portal and central vein areas, although initially most engraftment was periportal. Mice from group I had, on average, 20.2±2.2% of their liver replaced by transplanted cells. To determine the pattern of hepatocyte cluster repopulation, four X-gal–stained liver sections from a representative mouse from group I were analyzed morphometrically for cluster size and cell number. A total of 930 X-gal+ transplanted hepatocyte clusters were counted, containing a total of 8,792 cells. The area occupied by transplanted cells ranged from 10% to 20% of the tissue section. Although most clusters (83%) comprised 10 or fewer cells (Fig. 4A), clusters with 11 or more cells constituted the majority of cell repopulation (68%;Fig. 4B). Some clusters contained >200 cells. Transplanted cluster cell numbers were, on average, 30-fold higher in retrorsine/CCl4-treated mice than those in mice after only CCl4 treatment. These data agree with previous reports in which clusters of two to six hepatocytes were seen in mice after CCl4 and LCT (14).
Frozen sections from transplant recipient and control livers exposed to X-gal (A–D) or stained with hematoxylin and eosin (E). A, negative control. B, donor, β-gal transgenic. C, representative recipient liver from group I, 2 months after LCT, showing variable size of engrafted β-gal+ clusters. D and E, adjacent sections from transplant recipient mouse (in group I) showing normal morphology of transplanted β-gal+ cells (original magnification, ×100).
Number of cells per X-gal+ cluster was counted in a total of 930 clusters. Although clusters of <10 cells constituted 83% of the total clusters, most repopulation was effected by clusters of 21 or more cells. A, frequency distribution of transplanted versus repopulation cluster size. B, proportion of repopulation effected according to cluster size.
Significant repopulation occurs during 2 months in conditioned mice.
Immediately after LCT and for a few weeks, all groups had similar engraftment and repopulation efficiencies. However, preferential proliferation and repopulation of the recipient livers by transplanted cells occurred only in those groups receiving both retrorsine and CCl4. The time course of this repopulation is illustrated in Figure 5. This graph suggests that there was no significant repopulation in mice with the use of retrorsine alone even a long time after LCT as was seen in rats (13). Species differences might account for these observations. Table 1 compares the hepatocyte engraftment efficiencies in the different treatment groups after counting isolated and plated X-gal–stained liver cells as described in the Methods section. The differences between groups I and II as well as with the control group VI were significant with a P <0.001. Hepatocytes were isolated from recipient mice, allowed to attach to plastic, cultured for 4 hr, fixed, and exposed to X-gal. At least 500 cells were counted, and the number of X-gal+ and X-gal− cells recorded. The percentage of positive cells was calculated as the average obtained by two independent observers. Although CCl4 or retrorsine alone increased efficacy of repopulation several fold above that of the control nonconditioned mice, final repopulation was low and approximately 1% of the total liver (Table 1). However, when both a cell cycle inhibitor and a stimulus for proliferation were given, significant repopulation occurred. At least a 20-fold enhancement of repopulation over control values was observed in mice from group I receiving two doses of retrorsine before LCT and three doses of CCl4 after LCT. The 1-month period between LCT and the last retrorsine dose was chosen based on rat models (11). In that model, the interval was used to prevent surgical mortality and retrorsine effect on transplanted cells. Because we did not use hepatectomy, we reduced the interval between the last retrorsine dose and LCT to 2 weeks with no increase in mortality and similar results in liver cell repopulation (data not shown).
Time course of hepatocyte engraftment and repopulation. Efficiency of repopulation was evaluated by cell isolation, culture, and X-gal staining from livers of groups I through VI at different time points (one mouse per time point). The percentage of positive cells increased with time in conditioned livers from mice in groups I and II. RET, retrorsine.
Repopulation efficiency of conditioned and control liversa
DISCUSSION
This study used a no-genetic-advantage, nonsurgical approach to enhance the repopulation of transplanted liver cells in vivo, the first time that such approach has been used in mice. We combined the use of a recipient liver cell cycle inhibitor, retrorsine, with a liver-cell-injury agent, CCl4, to stimulate a regenerative environment that, in the proliferation-inhibited recipient livers, would promote preferential proliferation of transplanted cells. The resulting efficiency of liver repopulation, 20%, was sufficient to attempt cure of mouse models of liver-based metabolic disease and could be useful in other cell transplant research applications. Human applications would be more complex as they will involve allogeneic cell transplantation and the need for clinically usable drugs.
The strategy to use the CCl4 after LCT was based on previous studies that showed hepatocyte engraftment in periportal locations after intrasplenic infusion (20). We allowed cells to become integrated into the liver parenchyma for a period of 2 weeks before the first CCl4 dose. Migration of transplanted periportal hepatocytes toward the perivenous areas of the liver, after CCl4-induced perivenous injury, was observed (21). CCl4 treatment can induce a favorable environment for significant proliferation and engraftment of transplanted liver cells in rats (21). In our setting, proliferating cell nuclear antigen (PCNA) expression was used as a marker of hepatocyte entry into the cell cycle and to demonstrate the induction of a proliferative environment in the liver. PCNA immunohistochemistry of mouse liver sections revealed >60% of hepatocytes were proliferated by 2 days after CCl4 treatment. Untreated control mouse livers exhibited no proliferation (data not shown). Intraperitoneal injections of CCl4 were also used in the past to promote repopulation of mouse livers after LCT (14). That work in mice demonstrated transplanted cell clusters of only two to six hepatocytes, suggesting at most three rounds of cell division after CCl4 and LCT. In the current study, the combination of retrorsine followed by LCT and CCl4 enhanced repopulation of the recipient liver by transplanted congenic β-gal transgenic hepatocytes by 20-fold to 200-fold as compared with that of control groups. A much more modest enhancement in repopulation was seen in mice after a single dose of CCl4.
β-gal transgenic mouse models have been used in the past to follow engraftment of transplanted liver cells (10). The donor mice used in our study, Rosa 26, express the transgene in the liver and other tissues under a constitutional promoter (trap) that does not require activation and facilitated quantitation. Indeed, our initial efforts using β-gal transgenic donor hepatocytes dependent on the activation of an inducible metalloprotease promoter were hampered by variability in the results owing to variability in transgene expression (data not shown). An additional advantage of using donor Rosa 26 mice was a low rate of transgene extinction of 10–20%, such that most of the hepatocytes remain transgenic even in older mice and, as we observed, in proliferative cell clusters after transplantation. Of note, whole liver X-gal staining is useful to suggest phenotypic change after liver repopulation with β-gal–expressing cells but could mislead if used as a method for quantitation of cell engraftment. In this study, the use of X-gal histochemistry of liver tissue sections and of recipient liver cell isolation and plating for X-gal cell counts allowed for more accurate quantitation of cell engraftment and liver repopulation.
Retrorsine is one of the main alkaloids present in the Senecio plant species and it induces cell cycle arrest of hepatocytes, which leads to an accumulation of cells in late S or G2 phases (12,22). It can also cause liver cell damage by inducing apoptotic pathways, dependent on relative levels and localization of Bax and Bcl-xl proteins (23). The use of retrorsine on recipient livers to inhibit proliferation of native hepatocytes and promote repopulation by transplanted cells has been well established in rats (11,13). A different species-specific response to this drug has delayed this model’s application in mice. Early toxicity studies revealed that the lethal dose for 50% of mice was 65 mg/kg (19). Mice died 1–4 days after an i.p. dose of retrorsine with a hemorrhagic liver and often ascites (19). Contrary to these reports our study found that two doses of retrorsine, 70 mg/kg, were tolerated by >90% of the animals. One possible explanation to these discrepant findings could be the different solvents used to infuse retrorsine. Whereas early toxicity studies (19) and those in rats (23) use 1 N HCl to dissolve the drug, followed by NaOH and saline solution, our study used ethanol followed by dilution in PBS. Different solubility and absorption pharmacokinetics, or different strain responses, could explain the observed 50% lethal dose differences. It was originally believed that retrorsine acted preferentially in male recipient rats (24). However, recent studies (13) and our current data in female mice show that retrorsine can promote hepatocyte repopulation in both male and female rodents.
The retrorsine and partial hepatectomy model in rats is characterized by proliferation of oval cells and appearance of foci of small endogenous hepatocytes (25). In the present study, multicellular clusters of transplanted cells were seen, suggesting clonal expansion of a small proportion of donor cells. Although colony size and distribution throughout the recipient liver was not uniform, β-gal–stained colonies were more prominent in the periphery of each mouse liver lobe. Repopulating clusters had normal histology as examined in adjacent X-gal– and hematoxylin and eosin–stained histologic sections. The β-gal+ hepatocytes were indistinguishable from resident hepatocytes on hematoxylin and eosin–stained sections. Transplanted repopulating liver cells arise both from mature donor hepatocytes and from small hepatocytes or nonparenchymal cells (26). Data using the retrorsine model in rats suggest that small hepatocytes have three times higher proliferation potential than parenchymal hepatocytes (27). Subpopulations of small cells also have varying proliferative potential. For example, those having less cytoplasmic granularity and autofluorescence form clusters nearly four times larger after transplantation (27). These characteristics were not examined in our study. Our study showed a large range of cell cluster size from 1 to >200 cells, consistent with variable proliferation after transplantation of a nonpurified hepatocyte mixture containing both small and parenchymal hepatocytes. Notably, control group mice exhibited an average engraftment of 0.05% in mice receiving 2×106 cells. Assuming approximately 4×107 hepatocytes per mouse liver, the 2×106 cells constitute 5% of the total hepatocytes, which implies that up to 99% of transplanted cells do not engraft after one intrasplenic cell infusion. Such low overall engraftment is consistent with prior observations (8,9) and is suggestive of a high selective pressure and death of transplanted cells. Further, most engrafted liver cells, 83%, did not exhibit significant proliferation, remaining in clusters of <10 cells. Taken together, these findings support the hypothesis that liver repopulation results from the active proliferation of a small percentage of engrafted cells.
In summary, the data showed that >20% of total cells in the recipient liver were reconstituted by donor cells 2–7 months after LCT in experimental mice conditioned with retrorsine and treated with CCl4. These levels of liver repopulation in wild-type mice using freshly isolated hepatocytes were obtained without the need for partial hepatectomy or intrinsic selective advantage of the transplanted cells. The described LCT protocol could be used to study the treatment of mouse models of liver-based metabolic disease and in further research into the mechanisms involved in liver cell engraftment and repopulation.
Acknowledgments.
The authors thank Dr. Bernard L. Mirkin and Dr. Peter F. Whitington for their editorial assistance, Dr. William H. Schnaper, Dr. Thomas P. Green, and Mr. Patrick Magoon for their encouragement and support, Dr. Jose Hernandez for his veterinary expertise, and Ms. Adrienne Woodworth for preparing the manuscript.
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