Salivary epidermal growth factor and intestinal adaptation in male and female mice

Lawrence E. Stern, Richard A. Falcone Jr., Christopher J. Kemp, Margaret C. Braun, Christopher R. Erwin, and Brad W. Warner

Division of Pediatric Surgery, Children's Hospital Medical Center, Department of Surgery, University of Cincinnati College of Medicine, Cincinnati, Ohio 45229-3039


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Salivary epidermal growth factor (sEGF) levels are increased in male mice after small bowel resection (SBR) and may be important during intestinal adaptation. Since males have greater sEGF than females, the influence of sex on postresection adaptation was tested. Females had lower sEGF; however, sEGF substantially increased in both sexes after a massive (50%) SBR. Adaptive increases in DNA and protein content, villus height, and crypt depth, as well as crypt cell proliferation rates in the remnant ileum, were not different between males and females. Although significant postresection increases in sEGF were identified, EGF mRNA and protein did not change within the submandibular gland. Glandular kallikrein-13 and ileal EGF receptor expression were greater after SBR in female mice. Intestinal adaptation is equivalent in female and male mice after SBR. Despite lower sEGF, females demonstrated increased expression of a kallikrein responsible for sEGF precursor cleavage as well as amplified ileal EGF receptor expression. These results endorse an important differential response between sexes regarding sEGF mobilization and intestinal receptor availability during adaptation.

small bowel resection; kallikrein; salivary gland


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

FOLLOWING THE SUBSTANTIVE loss of small intestine mucosal surface area, the remnant bowel compensates by a process called adaptation. Although the exact mechanism(s) and/or mediator(s) of this important response are not well understood, there is growing evidence that implicates epidermal growth factor (EGF) and its intestinal receptor (EGF-R) as a crucial component of adaptation. After a massive (50%) small bowel resection (SBR) in mice, there is a considerable increase in the expression and activation status of EGF-R in isolated ileal enterocytes (5). Furthermore, adaptation is amplified in the presence of exogenous EGF (1, 7, 19) as well as in transgenic mice with targeted intestinal overexpression of EGF (3). Alternatively, adaptation is impaired after SBR in waved-2 mice, which have perturbed EGF-R signaling capacity (10).

In the mouse, EGF is produced primarily in the bilateral, paired submandibular glands, Brunner's glands of the duodenum, and the kidney (2). Among these sites of production, the overwhelming majority of EGF is derived from the salivary glands. It has previously been observed that pharmacological stimulation of submandibular gland secretion with systemic isoproterenol has a trophic effect on the small intestine (15). More specifically, the significance of salivary gland-derived EGF was recognized in experiments in which intestinal adaptation was substantially inhibited when the submandibular glands were surgically removed (sialoadenectomy) immediately before the intestinal resection procedure (12). Following sialoadenectomy, the impaired adaptive response was reversed following administration of EGF by either orogastric gavage or intraperitoneal injection.

During the intestinal adaptive response to massive SBR, male mice show a significant elevation in salivary EGF content and ileal EGF-R signaling (22). Male mice have significantly higher salivary EGF levels (~10-fold) than their age-matched female counterparts (8, 9, 14, 17). Although growth hormone, glucocorticoids, thyroid hormone, and androgens regulate salivary production of EGF, the sex differences in salivary EGF levels have been attributed mainly to androgens (14, 24).

To test the overall hypothesis that salivary gland-derived EGF is crucial for intestinal adaptation, a series of experiments that exploited the sex differences in murine salivary EGF content were performed. First, to establish the necessity of salivary EGF in the female intestinal adaptive process, female mice underwent SBR and were then randomized to undergo either a bilateral submandibular salivary gland manipulation (sham) or a salivary gland excision. Second, the male and female capacity for postresection intestinal adaptation was determined. Next, a mechanism for the increased salivary EGF content following SBR was sought. Finally, sex differences in ileal EGF-R expression during the adaptive period were evaluated.


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Animals. A protocol for this study was approved by the Children's Hospital Research Foundation Institutional Animal Care and Use Committee (Children's Hospital Medical Center, Cincinnati OH). Male and female ICR mice (weight range 25-29 g; Harlan Laboratories, Indianapolis, IN) were housed in groups of four at 21°C on 12:12-h day-night cycles (6 AM to 6 PM). Before experimentation, the mice acclimated to their environment for at least 3 days. One day before operation, their diet was changed from regular chow to liquid rodent diet (Micro-Stabilized Rodent Liquid Diet LAD 101/101A, Purina Mills, St. Louis, MO).

SBR. An expanded description of the details of this procedure has been previously reported (13). In brief, mice were anesthetized with inhaled isoflurane anesthesia, and a midline abdominal incision was performed. An operating microscope was used for magnification. In mice undergoing sham operation, the small bowel was transected and a reanastomosis was performed 12 cm proximal to the ileocecal valve. In mice undergoing SBR, ~12.5 cm of proximal intestine was resected and an anastomosis was accomplished (50% resection). After abdominal closure, the mice were resuscitated with a 3-ml intraperitoneal injection of warmed 0.9% saline and allowed to recover in an incubator (30°C). Water was provided ad libitum for the first 24 h. Mice from each group were then pair-fed with liquid diet.

Sialoadenectomy. A more complete description of this procedure has been previously detailed (12). In brief, through a midline cervical incision, the bilateral paired submandibular salivary glands were freed of surrounding tissue and the blood supply to each gland was identified. In mice undergoing salivary gland excision, a hemostatic surgical clip was applied to each vascular pedicle and the gland was excised. The skin was closed with a 4-0 silk suture. A sham procedure was accomplished by dissection of the salivary glands but without ligation of the vascular supply or removal of any salivary tissue.

Tissue harvest. Mice were harvested on the third postoperative day for each experiment. This time point was chosen because increased postresection salivary EGF content in male mice has been demonstrated previously (22). Furthermore, the adaptive response to SBR in this model is maximal and sustained by this time (13). One hour before death, the mice received an intraperitoneal injection of 5-bromodeoxyuridine (BrdU) to allow for the subsequent determination of the crypt cell proliferation rate. At the time of death, 6 cm (roughly 1 cm from the anastomosis) of ileum was excised, the luminal contents were gently expressed with cotton swabs, and the wet weight was recorded. The proximal 1-cm section was fixed with 10% neutral buffered formalin and used for histology (see Histology); the remaining 5-cm section was utilized for the measurement of ileal protein and DNA content. Additionally, the submandibular glands were excised and immediately frozen in liquid nitrogen.

Ileal protein and DNA content. Individual samples of ileum ~2 cm distal to the anastomosis were immediately placed in liquid nitrogen at the time of harvest. The samples were subsequently homogenized in saline and used for the determination of protein and DNA content as previously detailed (26).

Histology. Formalin-fixed specimens of ileum were embedded in paraffin and oriented to provide cut sections parallel with the longitudinal axis of the bowel. Five-micrometer tissue slices were mounted and stained with hematoxylin and eosin or subjected to a biotinylated monoclonal anti-BrdU antibody system provided in a commercial kit (Zymed Laboratories, San Francisco, CA). Using the hematoxylin and eosin-stained sections, villus height and crypt depth were measured with a video-assisted integrated computer program (NIH Image; National Institutes of Health, Bethesda, MD). At least 15 villi and crypts were counted per sample. Villi were chosen if the central lymphatic channel was completely visualized, and crypts were chosen if the crypt-villus junction on both sides of the crypt was recognized. An index of the crypt cell proliferation rate was derived in BrdU-stained slides by calculating the ratio of the number of crypt cells incorporating BrdU to the total number of crypt cells. Fifteen crypts were counted per sample. All histology was performed with the investigator blinded as to the source of the ileal tissue.

Salivary EGF ELISA. Saliva was collected with a cotton swab at the time of harvest. Levels of EGF in pooled samples of saliva (n = 5 per group) were determined using an indirect antigen-inhibited ELISA corrected for total salivary protein as previously reported (23).

Salivary gland EGF Western blot. Individual submandibular gland samples were homogenized in a 5× volume of homogenization buffer (10 mM Tris · HCl, pH 8.0, 0.1 M EGTA, pH 8.0, 1.0 M dithiothreitol, 0.1 M Na3VO4, 5 mM Na2MoO4, 1.0 M beta -glycerol-phosphate, 0.1 M Na4P2O7, 1 mg/ml aprotinin, 1 mg/ml leupeptin, and 100 mM phenylmethylsulfonyl fluoride). Total protein was quantified by using a modified Lowry assay (16). One hundred micrograms of protein was added to an equal volume of 2× protein sample buffer (250 mM Tris · HCl, pH 6.8, 4% SDS, 10% glycerol, 0.003% bromophenol blue, and 2% beta -mercaptoethanol) and then resolved on a 4-20% gradient polyacrylamide gel (Owl Separation Systems; Woburn, MA) at 4°C with standard protein running buffer (0.192 M glycine, 0.025 M Tris base, and 0.10% SDS). The protein was transferred to a PVDF-Plus membrane (Micron Separations; Westboro, MA) and, after blocking in 5% nonfat milk, exposed for 1 h at room temperature to a 1:1,000 dilution of mouse anti-EGF antibody (Harlan, Indianapolis, IN). After five washings, antibody detection was accomplished by the incubation of the membrane for 1 h at room temperature in a 1:15,000 dilution of horseradish peroxidase-avidin anti-mouse IgG (Transduction Laboratories, Lexington, KY) followed by the use of a chemiluminescence system (Renaissance; NEN Life Science Products, Boston, MA) and exposure to X-ray film (Biomax ML; Eastman Kodak, Rochester, NY). Band intensity was quantified using Image Quant 5.0 software (Molecular Dynamics, Sunnyvale, CA).

Salivary gland EGF and kallikrein RT-PCR. Submandibular glands were homogenized and total RNA isolated using TRIzol reagent (GIBCO BRL, Gaithersburg, MD) following the instructions of the manufacturer. The concentration of total RNA was determined spectrophotometrically at A260. A RT reaction was performed using 2.5 µg of total RNA and reverse transcriptase (GIBCO BRL) according to the manufacturer's instructions. The product of the RT reaction then was utilized for a RT-PCR. The primers used were as follows: EGF 156-bp fragment, 5'-AATAGTTATCCAGGATGCCC, 3'-ACGCAGCTCCCACCATCGTA; kallikrein-9 (mGK-9) 230-bp fragment, 5'-GCAAGCCTGCTGACATCAC, 3'-CCATCTGTCTC- TCCTGCACAC; kallikrein-13 (mGK-13) 300-bp fragment, 5'-CTCAGCACCGATTGGTCAGC, 3'-CTTTGGCACAGTTCTCATTGGG; kallikrein-22 (mGK-22) 199-bp fragment, 5'-CAAGCCTGCTGACATCAC, 3'-CTGTCACCTTCAGTATATGGG. All PCR reactions were run using 18S rRNA (Ambion, Austin, TX) as an internal control. The intensities of the bands were measured using Image Quant 5.0 software (Molecular Dynamics) and normalized to 18S rRNA intensity.

Ileal EGF receptor protein. Remnant intestine samples were pooled (n = 3-5 per group) and homogenized, and protein was extracted as previously described (26). Total protein concentration was determined using a modified Lowry assay (16). One hundred micrograms of protein was serially diluted with sample buffer and added to the wells of a dot blot apparatus with a nitrocellulose membrane. Immunoblotting was done as previously described (26), except that a mouse anti-EGF receptor antibody (Transduction Laboratories) was utilized for EGF receptor identification. Blot intensity was quantified using Image Quant 5.0 software.

Statistical analysis. Results are presented as means ± SE. When experiments included only two groups, unpaired Student's t-test was used. When the experiments included more than two groups, statistical differences were determined by ANOVA followed by a post hoc pairwise multiple comparison using the Student-Newman-Keuls method. The SigmaStat statistical package (Jandel Scientific, San Rafael, CA) was utilized for all statistical analysis A P value of <0.05 was considered significant.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Salivary gland removal impairs intestinal adaptation in female mice. To ascertain whether or not salivary gland-derived EGF is a necessary component in the female adaptive process, female mice were subjected to a SBR, followed by either a salivary gland excision or a sham neck operation. Similar to male mice (12), the remnant ileal wet weight and protein content were substantially reduced after combined SBR and salivary gland excision in female mice (remnant ileal wet weight: 36.8 ± 1.1 vs. 27.6 ± 1.9 mg/cm, sham vs. sialoadenectomy, P = 0.0006; remnant ileal total protein: 2,933.6 ± 509.7 vs. 1,475.1 ± 176.2 mg/cm, sham vs. sialoadenectomy, P = 0.005).

Adaptation after intestinal resection is similar in male and female mice. Mice from all groups tolerated the surgical procedure well, with an overall survival of >80%. As shown in Table 1, the cellular parameters of adaptation, which included ileal protein and DNA content, were not different after SBR between male and female mice. The morphological parameters demonstrated similar increases in villus height and crypt depth between the sexes. Finally, the rate of crypt cell proliferation in response to massive SBR was indistinguishable between the sexes.

                              
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Table 1.   Parameters of intestinal adaptation

Intestinal resection increases salivary EGF in both male and female mice. Consistent with previous observations, male sham mice had an ~10-fold higher level of EGF in the saliva than their female counterparts (213 vs. 22.6 pg EGF/µg total protein). However, SBR resulted in an ~10-fold increase in salivary EGF content in both male and female mice (Fig. 1).


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Fig. 1.   Concentration of epidermal growth factor (EGF) in the saliva of male and female mice following either a sham operation (transection and reanastomosis only) or 50% proximal small bowel resection (SBR) as determined by ELISA. Samples were pooled (n = 4-10 per group).

To resolve whether the increased EGF in the saliva occurred at the transcriptional level, EGF mRNA levels in the submandibular gland were evaluated. There was no appreciable difference between sham and SBR EGF mRNA levels in either sex (data not shown). To determine if increased salivary EGF content occurred at the translational level, EGF protein levels in submandibular gland homogenates were analyzed. Concordant with the salivary EGF content measured by ELISA, an ~10-fold difference between males and females was identified; however, no measurable differences between sham and SBR mice were observed (Fig. 2).


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Fig. 2.   Salivary gland EGF levels in male and female mice following either a sham operation (transection and reanastomosis only) or 50% proximal SBR as determined by Western blot analysis. No significant differences were appreciated between sham and SBR. * P < 0.05, females vs. males by ANOVA.

Submandibular gland kallikrein isoforms are differentially expressed between sexes and following intestinal resection. Although no appreciable changes in submandibular gland EGF mRNA and protein were identified after intestinal resection, increased salivary EGF content may have been due to accelerated cleavage of preformed, precursor EGF. To test this hypothesis, the expression of three mouse glandular kallikrein genes (mGK) was examined. Kallikrein and its associated isoforms account for the majority of EGF precursor cleavage within the salivary glands (2). As seen in Fig. 3, A and B, all three mGK genes were significantly higher in male mice when compared with the female mice. Following SBR, there were no significant changes in the male mice (Fig. 4A). On the other hand, a 260% increase in mGK-13 was observed after SBR in the female mice (Fig. 4B). The expression of mGK-9 and -22 was unchanged after SBR in these mice.


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Fig. 3.   A: submandibular gland RT-PCR for the mouse glandular kallikrein genes (mGK) -9, -13, and -22 of female and male mice that had undergone a sham (transection and reanastomosis only) operation. * P < 0.05, females vs. males by t-test. B: submandibular gland RT-PCR for the mGK-9, -13, and -22 of female and male mice that had received a 50% proximal SBR. * P < 0.005, females vs. males by t-test.



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Fig. 4.   A: submandibular gland RT-PCR for mGK-9, -13, and -22 of male mice that had undergone either a sham (transection and reanastomosis only) operation or a 50% proximal SBR. B: submandibular gland RT-PCR for mGK-9, -13, and -22 of female mice that had undergone either a sham (transection and reanastomosis only) operation or a 50% proximal SBR. * P < 0.005, sham vs. SBR by t-test.

Ileal EGF receptor expression is increased after SBR and greater in female mice. Because the EGF content in the saliva was lower in female mice, but both sexes increased salivary EGF content in response to SBR, determination of whether there were sex differences in the expression of ileal EGF-R was undertaken. Consistent with prior work on male mice (11), SBR resulted in augmented ileal EGF-R expression in both sexes (Fig. 5A and B). Although both male and female mice were able to increase ileal EGF-R levels after SBR, the expression of this receptor was notably greater in the female mice in both the sham and SBR groups.


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Fig. 5.   Top: immunoblots for the ileal EGF receptor in male and female mice following either a sham operation (transection and reanastomosis only) or 50% proximal SBR. Samples were pooled (n = 3-5 per group). Bottom: ileal remnant EGF receptor protein quantification in male and female mice following either a sham operation (transection and reanastomosis only) or 50% proximal SBR as determined by immunoblot. Samples were pooled (n = 3-5 per group).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

This study has demonstrated for the first time that salivary gland-derived EGF is a necessary component in the female adaptive response to massive SBR; however, in spite of lower salivary EGF levels, female mice have the capacity to adapt to massive SBR to the same degree as male mice. Furthermore, despite intrinsic sex differences in salivary EGF content, intestinal resection resulted in increased salivary EGF in both male and female mice, a response that did not appear to be transcriptionally or translationally evident within the submandibular gland. An increase in mGK-13 in females without such a change in males may represent sex-specific regulation of EGF precursor cleavage and may help explain the ability of female mice to achieve equivalent adaptation despite significantly lower baseline salivary EGF. Finally, the expression of EGF-R within the ileum was greater in female mice and increased after SBR in both sexes. Together, these findings support an important role for salivary EGF as a critical mediator of the intestinal adaptive response to massive SBR.

The premise of this study, to determine whether a specific response in female mice is different from that in their male counterparts because of lower salivary EGF levels, is not novel. In another study focusing on the ability of mice to heal tongue wounds, female mice were able to heal their injuries as well as males (18). In another study, it was observed that acid-induced gastric ulcers in female rats healed more slowly than similar lesions in their male counterparts. They attributed this observation to the lower EGF content in female orogastric secretions (27). Neither of these studies reported the actual level of salivary EGF or whether there was a change following injury. The current study is the first to demonstrate that, despite a large difference in basal salivary EGF concentration between sexes, both males and females respond to SBR with a 10-fold increase in salivary EGF content. It appears that the change in EGF concentration, and not the absolute level, is crucial during adaptation.

At present, the mechanism for increased salivary EGF output in response to murine SBR is unknown. Although androgens likely account for the sex difference observed in basal salivary EGF levels, they do not play a role in the actual stimulus for EGF secretion (8, 17). The regulation of salivary EGF synthesis and concentration appears to be multifactorial and controlled by separate mechanisms. Glandular protein synthesis is regulated by growth hormone, prolactin, androgens, thyroid hormones, and corticosteroids (20). Glandular secretion is influenced by alpha - and beta -adrenergic stimuli, muscarinic stimuli, substance P, and vasoactive intestinal polypeptide (20). It is possible that SBR results in a greater physiological stress and associated adrenergic stimulation to the mice compared with sham operation. The mice in both groups were healthy-appearing, anabolic, and gaining weight at the time of death. This argues against significant, ongoing catabolism in the SBR group. Future studies designed to both stimulate and inhibit alpha - and beta -adrenergic receptors may help elucidate the mechanism for enhanced EGF levels following SBR.

No change in the salivary gland EGF mRNA or protein concentration was identified postresection. The absence of measurable differences within the gland has several potential explanations. First, the amount of EGF mRNA does not necessarily correlate directly with the amount of EGF protein within a specific tissue. It has been previously observed that there is a severalfold greater content of EGF protein in the salivary gland and the kidney despite only modest differences in absolute mRNA levels between these tissues (17). It appears that the regulation of EGF availability occurs post-translationally. Therefore, a change in mRNA expression before or coincident with a change in the amount of active EGF may not be appreciated (21). Second, because the salivary gland has nearly a 1,000-fold higher concentration of EGF compared with the saliva (17), it is conceivable that a 10-fold increase in salivary EGF protein concentration can occur without a measurable change in the vast amount of EGF protein within the gland itself. This notion is supported by a prior study in which exogenous EGF administration to mice resulted in a 66% reduction in salivary EGF concentration without a significant change in the EGF content within the salivary gland (25).

Because EGF is produced as a precursor molecule and requires subsequent cleavage to an active form, a change in the proportion of precursors to active molecules may explain the equivalent adaptive response in females despite lower total salivary EGF concentration. Because the ELISA utilized in this study contained an antibody that recognized both precursor and active EGF, it was not possible to distinguish between the different forms of EGF. Glandular kallikreins are arginine bond-specific serine esteropeptidases that process prohormones and precursor peptides into their active form (6). Mouse glandular kallikreins (mGK) are a multigene family located on chromosome 7 (4). Three of the twenty-four identified mGK genes (9, 13, and 22) have been implicated to be important for the generation of biologically active EGF (2). These kallikreins have been referred to as EGF binding proteins (EGFBP) A, B, and C, although a more recent study has questioned the role of mGK-22 as an EGFBP (4). The increase in mGK-13 that was observed in the female mice following SBR without such a change in the male SBR group supports the notion that EGF bioavailability in the saliva is increased in females, a factor that may compensate for the lower absolute quantity of EGF.

This study has shown that, despite significant differences in the absolute levels of salivary EGF, no differences existed in the morphological structure of the intestine between male and female mice. Although EGF is known to be an important factor for both the development and maintenance of the gastrointestinal epithelium, most physiological systems exhibit a homeostasis between ligand and receptor availability. The finding of greater EGF-R expression in the ileum of female mice that have lower salivary EGF concentrations supports this concept. It is intuitive that female mice should have a higher basal level of EGF-R expression to compensate for the diminished levels of salivary EGF available to the intestine. In a previous study, upregulation of EGF-R expression in male mice that had undergone massive SBR was identified (26). In the current study, female mice increased EGF-R expression by 39% after SBR and reached a level that was 55% greater than their male counterparts. Through various second-messenger pathways, homo- and heterodimerization of EGF-R, and increased absolute number of EGF receptors, the effect of increased salivary EGF levels can be significantly amplified. This data may also explain why females achieve equivalent adaptation despite lower absolute levels of salivary EGF.

By taking advantage of naturally occurring sex differences in salivary EGF content, this study has attempted to elucidate a mechanism for increased levels of salivary EGF during adaptation. The results in this series of experiments suggest a unique mechanism for the intestinal adaptive response to massive SBR and demonstrate that SBR triggers an increase in salivary EGF in both male and female mice. Additionally, changes in EGF bioavailability in female mice and upregulation of ileal EGF-R expression in both sexes may serve to initiate and/or sustain the postresection adaptive response. The lack of change in salivary gland EGF protein may indicate greater secretion of preformed EGF rather than increased production, thus focusing further work on the critical signal(s) for salivary EGF secretion. A more complete understanding of the physiological and hormonal changes that take place during the adaptive process will permit a comprehensive understanding of intestinal adaptation and provide a method for enhancing this important response.


    ACKNOWLEDGEMENTS

This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grants F-32-DK-09882 (L. Stern) and RO-1-DK-53234 (B. Warner), and a grant from the Children's Hospital Campaign for Children Fund, Children's Hospital Medical Center, Cincinnati, OH


    FOOTNOTES

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Address for reprint requests and other correspondence: B. W. Warner, Division of Pediatric Surgery, Children's Hospital Medical Center, 3333 Burnet Ave., Cincinnati, OH 45229-3039 (E-mail: warnb1{at}chmcc.org).

Received 5 October 1999; accepted in final form 26 January 2000.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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