Center for Surgical Research and Department of Surgery, Brown University School of Medicine and Rhode Island Hospital, Providence, 02903
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ABSTRACT |
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Previous studies have demonstrated that hemorrhagic shock
produces immunodepression in young male mice, whereas the
immunoresponsivness in young proestrus female mice is enhanced under
such conditions. This sexually dimorphic immune response to hemorrhage
appears to be related to high estrogen and testosterone levels in
females and males, respectively. Nonetheless, it is unknown what impact the age-related decline in the sex steroid levels has on the immune response after hemorrhage. To study this, young (2-3 mo) and aged (18-19 mo) male and female CBA/J NIA mice were subjected to
laparotomy (i.e., soft tissue trauma) and hemorrhage (35 ± 5 mmHg for
90 min and fluid resuscitation) or sham operation. Twenty-four hours later, splenocyte responses were assessed in vitro. Splenic T lymphocyte responses [i.e., proliferation, interleukin-2 (IL-2) and interferon- (IFN-
) release] were depressed in young
males and enhanced in young females after trauma-hemorrhage. In
contrast, in the aged male and female groups these parameters of
splenocyte function were reversed after trauma-hemorrhage (i.e.,
increased proliferation and IL-2 release in aged males compared with
suppressed proliferation and IFN-
release in aged females).
Furthermore, the release of the immunosuppressive cytokine IL-10
inversely correlated with the age- and gender-related changes in
splenocyte responses after trauma-hemorrhage. Thus the sexually
dimorphic immune response in young males and females to
trauma-hemorrhage appears to reverse as sex hormone levels decline with age.
shock; immune depression; interleukin-2; interleukin-4; interleukin-10; interferon-
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INTRODUCTION |
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HEMORRHAGIC SHOCK leads to immunodepression that
contributes to an enhanced susceptibility to sepsis under such
conditions (29, 34). Sepsis and ensuing multiple organ failure are the most common cause of death in surgical intensive care units (8). Aging
is also associated with impaired immune functions leading to a higher
incidence of bacterial sepsis, increased lipopolysaccharide (LPS)
sensitivity, and mortality due to sepsis (21, 23, 35). Studies have
demonstrated that the age-induced loss of protective immunity is
primarily related to alterations in T lymphocyte function. For
instance, aging causes a decrease in the numbers of naive T lymphocytes
and increased accumulation of memory T lymphocytes (19, 23). In
general, age-related changes in the production of T lymphocyte
cytokines [i.e., interleukin (IL) -2, IL-4, IL-10, interferon-
(IFN-
)] suggest the development of a Th-2 phenotype (i.e., IL-4, IL-10) as opposed to the predominance of a Th-1 phenotype (i.e., IL-2, IFN-
) in younger individuals (19, 23).
In addition to the effects of age on immune function, several clinical and epidemiological studies indicate that young men are more susceptible to the lethal effects of sepsis compared with women (21, 33). The importance of sex steroids in the development of immune dysfunction in a variety of disease processes has been previously reported (28). Studies indicate that estradiol leads to a more active immune system in females, resulting in the predominance of a diverse range of autoimmune diseases (28, 37). Conversely, the suppressive effects of androgens on immune functions have been observed under normal conditions (11, 36, 37) and after traumatic injury (2, 39). Experimental studies also indicate sex-related dimorphism in immune responses of young mice after hemorrhagic shock and subsequent sepsis, with females displaying enhanced immune functions compared with depressed immune functions in males (40, 41).
Recent studies have shown that with the age-related decline of
circulating sex steroids (i.e., decreased 17-estradiol in females
and decreased testosterone in males), the sexual dimorphic immune
response of macrophages after trauma-hemorrhage was reversed (16). The
findings suggest that high circulating levels of testosterone in young
males contribute to macrophage dysfunction, and high circulating levels
of estrogen in young females protect against the depression of
macrophage functions after trauma-hemorrhage. Nonetheless, it remains
unknown whether the decline of the sex hormones with age also alters
the immune response of splenic T lymphocytes after trauma-hemorrhage.
The aim of this study was, therefore, to test the hypothesis that with
aging, as circulating sex hormone levels decline, the sexually
dimorphic immune response in young males and females to
trauma-hemorrhage is reversed.
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MATERIALS AND METHODS |
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Animals
Inbred female and male CBA/J NIA mice (Charles River Laboratories, Kingston, NY, under the contract of the National Institute of Aging, Bethesda, MD), at ages of 2-3 and 18 mo (20-26 g body wt at 2-3 mo and 27-35 g body wt at 18-19 mo) were used in this study. The animals were allowed to acclimate to the animal facility at Rhode Island Hospital for at least 1 wk before experimentation. During this period the group-housed young (2-3 mo) females were able to synchronize their estrus cycle. The stage of the female estrus cycle was determined by daily examination of the vaginal smear at the same time each day and classified according to the definitions of Rugh (30). In this study, young female mice in the proestrus stage and aged females without detectable estrus cycle [i.e., with persistent vaginal cornification or persistent anestrus (12)] were included. All procedures were carried out in accordance with the guidelines set forth in the Animal Welfare Act and the NIH Guide for the Care and Use of Laboratory Animals (7th ed. Washington, DC: Natl. Acad. Press, 1996). This project was approved by the Institutional Animal Care and Use Committee of Brown University and Rhode Island Hospital.Experimental Groups
Female and male mice, age 2-3 (young) or 18-19 (aged) mo, were randomized into one of two groups containing eight animals each. Group 1 consisted of sham-operated animals and the animals in group 2 underwent the trauma-hemorrhage procedure, previously described by Schmand et al. (31). Twenty-four hours after the end of resuscitation or sham operation, the animals were killed by methoxyflurane overdose and the spleens removed aseptically to obtain splenocytes. To avoid artifacts due to marked circadian fluctuations of plasma hormone levels, all animals were killed at 11:00 AM. To exclude the influence of such chronic diseases as hepatoma, focal leukocytic nodules of the liver, lymphoid hyperplasia of the spleen, and cancer of the liver, lung, and intestine, each mouse was inspected for visible tumors and splenomegaly after spleens were harvested. Mice were excluded if any of the above abnormalities were detected.Trauma-Hemorrhage Procedure
Mice were lightly anesthetized with methoxyflurane (Metofane, Pitman-Moore, Mundelein, IL), restrained in a supine position, and a 2-cm midline laparatomy was performed (i.e., induction of soft tissue trauma). After visual inspection of the abdominal organs to exclude iatrogen lesions, the abdominal cavity was closed in layers with 6-0 Ethilon sutures. Both femoral arteries were then cannulated aseptically with polyethylene 10 tubing (Clay-Adams, Parsippany, NJ) using a minimal dissection technique. Heparin [2 units beef lung heparin (Upjohn, Kalamazoo, MI) per 25 g body wt] was then administered and the animals allowed to awaken. Mean arterial blood pressure (MAP) was constantly monitored by attaching one of the catheters to a blood pressure analyzer (Digi-Med, Louisville, KY) coupled to a polygraph (Model 7D, Grass Instruments, Quincy, MA). On awakening, the animals were bled through the other catheter to a MAP of 35 ± 5 mmHg (MAP pre-hemorrhage was 95 ± 5 mmHg), which was maintained for 90 min. At the end of that period, the shed blood was returned and lactated Ringer solution (2 times the shed blood volume) was infused to provide adequate fluid resuscitation. The catheters were then removed, the vessels ligated, and the groin incisions closed. Sham-operated animals underwent the same surgical procedure, which included heparinization and ligation of both femoral arteries, but neither hemorrhage nor fluid resuscitation was carried out. No mortality was observed in this trauma-hemorrhage model.Preparation of Splenocyte Cultures
Twenty-four hours after trauma-hemorrhage and resuscitation or sham operation the animals were killed by an overdose of methoxyflurane. The spleens were then removed aseptically and placed in 50-ml conical tubes containing cold (4°C) PBS solution. Splenocytes were isolated as previously described, and were resuspended in RPMI 1640 (GIBCO-BRL, Grand Island, NY) containing 10% fetal bovine serum (GIBCO-BRL) (40). Splenocyte viability was greater than 95% as determined by trypan blue exclusion.An initial portion of the splenocyte suspension was used for cytokine
production. Splenocytes were cultured at a final concentration of
106 cells/ml and their ability to produce cytokines in
response to a mitogenic challenge was assessed by incubation for 48 h
(at 37°C, 5% CO2, and 90% humidity) in the presence
of 2.5 µg/ml concanavalin A (Con A). Cell-free supernatants were
harvested, aliquotted, and stored at 80°C until assayed for
cytokine levels.
A second portion of the splenocyte suspension was cultured in a 96-well microtiter plate (Corning Glass, Corning, NY) at a concentration of 2 × 105 cells/200 µl/well. The ability of the cells to proliferate in response to mitogenic stimulation with 0 (negative control) or 2.5 µg/ml Con A was determined by incubation for 48 h at 37°C in a 5% CO2 atmosphere with 90% humidity. Proliferation was determined by measuring the incorporation of [3H]thymidine as previously described by Stephan et al. (34).
Cell-Line Maintenance
The macrophage cell line RAW 264.7, used for the IFN-Assessment of Cytokine Production
IFN-The capacity of the splenocyte cultures to produce IL-2 was assessed by
determining the amount of IL-2 in the culture supernatant. Serial
dilutions of the supernatants were added to CTLL-2 cells (1 × 105 cells/ml) and incubated for 48 h at 37°C and 5%
CO2. At the end of this period, 1 µCi of
[3H]thymidine (sp. act. 6.7 Ci/mmol, New
England Nuclear, Wilmington, DE) was added to each well and the
cultures incubated for an additional 16 h. The cells were then
harvested onto glass-fiber mats and the -decay detected by liquid
scintillation radiography as previously described (20).
IL-3 activity in the splenocyte supernatants was detected by adding
serial dilutions of the supernatants to FDC-P1 cells (2.5 × 105 cells/ml). After an incubation period of 24 h at
37°C and 5% CO2, similar to the IL-2 assay, 1 µCi of
[3H]thymidine (sp. act. 6.7 Ci/mmol, New
England Nuclear) was added to each well and the cultures incubated for
an additional 16 h. The cells were then harvested onto glass-fiber mats
and the -decay detected by liquid scintillation radiography as
previously described (20). Relative units per milliliter of cytokine
activity present in the unknown samples were determined by
comparison of the curves produced by dilutions of the unknown
samples to those generated by a dilution of recombinant murine
cytokine standard (IL-2 and IL-3 standard: 100 U/ml, Genzyme,
Cambridge, MA) as previously described (20).
IL-4 and IL-10 levels in the splenocyte supernatants were determined using the sandwich ELISA technique previously described (4, 25). Matched monoclonal antibodies were obtained from PharMingen (San Diego, CA). Sandwich ELISA was performed according to the manufacturer's instructions.
Statistical Analysis
The results are presented as means ± SE. One-way ANOVA and Student-Newman-Keuls method were employed to determine the significance of the differences between experimental means. P < 0.05 was considered significant. ![]() |
RESULTS |
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Splenocyte Proliferative Responses
The proliferative response of splenocytes from young female mice was significantly increased (P < 0.05) after trauma-hemorrhage compared with sham-operated animals (Fig. 1). In contrast, splenocyte proliferation in young male mice was significantly suppressed (P < 0.05) after trauma-hemorrhage (Fig. 1). It is interesting that this gender-dependent pattern was partially reversed in aged mice. Aged female mice showed no change in splenocyte proliferation after trauma-hemorrhage compared with sham-operated animals, whereas aged male mice had a significantly increased (P < 0.05) proliferative response after trauma hemorrhage (Fig. 1). Aging in itself did not significantly alter the proliferative response of splenocytes from sham-operated females; however, proliferation was significantly less (P < 0.05) in splenocytes from aged male sham-operated mice compared with their younger counterparts. Transformation of the splenocyte proliferation data to log base 2 did not alter the results of the statistical analysis (data not shown).
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Cytokine Productive Capacity
IL-3.
Splenocytes harvested from young female mice produced significantly
greater (P < 0.05) amounts of IL-3 after trauma-hemorrhage compared with shams, whereas IL-3 release by splenocytes from young
male mice was significantly reduced under such conditions (P < 0.05; Fig. 2). The IL-3 release by
splenocytes from aged female mice was reduced after trauma-hemorrhage
(P < 0.05). In contrast, no change in IL-3 release was
evident in the aged male group after trauma hemorrhage. The basal
(sham) IL-3 release in the aged female group was significantly
increased (P < 0.05) compared with the young female group. In
contrast, the basal (sham) IL-3 release from the aged male group was
significantly reduced compared with the young male group (Fig. 2).
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Th-1 cytokines (IL-2, IFN-).
IL-2 release by splenocytes from young female mice was significantly
(P < 0.05) enhanced after trauma-hemorrhage compared with
sham animals. Conversely, splenocytes harvested from young male mice
showed significantly decreased IL-2 release after trauma-hemorrhage (Fig. 3A). In aged mice,
trauma-hemorrhage did not significantly alter IL-2 release compared
with age- and gender-matched sham mice. IL-2 release by splenocytes
from aged mice was significantly (P < 0.05) less than that
observed in the corresponding young group (Fig. 3A).
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Th-2 cytokines (IL-4, IL-10).
Trauma-hemorrhage had no effect on splenocyte IL-4 release,
irrespective of gender or age (Fig.
4A). IL-4 release by splenocytes harvested from aged sham-operated female mice, however, was increased compared with their respective young group, whereas the IL-4 release in
the sham-operated aged male group showed no difference compared with
their equivalent young group (Fig. 4A).
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DISCUSSION |
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The data presented here indicate that with aging, which is associated
with decreased levels of circulating sex steroids (16), profound
changes occur in T lymphocyte-dependent immune function after
trauma-hemorrhage. The sexual dimorphism in the T lymphocyte immune
response that exists in young mice after trauma-hemorrhage (40, 41) is
lost with age. Splenocyte immune function after trauma-hemorrhage was
maintained, or enhanced, in young females, whereas young males
experienced immune depression. Moreover, decreased susceptibility to
the lethal effects of sepsis, a major complication following
trauma-hemorrhage, was found in females (41). The data presented here
are consistent with these findings, indicating that splenocytes from
young proestrus females display enhanced proliferative responses, IL-2,
IL-3, and IFN- release after trauma-hemorrhage as opposed to
depressed responses in age-matched males. Moreover, the release of
IL-10, an immunosuppressive Th-2 cytokine, by splenocytes from young
females was not increased after trauma-hemorrhage as it was in the
young male group. In contrast, in the aged female group cytokine
release and proliferative responses were suppressed or unchanged after
trauma-hemorrhage, whereas splenocytes from aged males displayed no
change in proliferation or IL-2, IL-3, and IFN-
release after
trauma-hemorrhage, and IL-10 release was not increased under such conditions.
Aging is associated with impaired immune functions mainly due to
changes in the T cell compartment. The sham splenocyte immune responses
in both aged male and female mice were markedly altered compared with
young mice in this study. Previous studies have demonstrated that age
leads to an accumulation of memory T cells and a decline of naive T
cells (14, 19, 23). This change in the T lymphocyte population is
associated with decreased release of IL-2 by naive T cells (19, 23). An
increase in sham IFN- release by splenocytes from aged mice was
observed in both gender groups, which is likely to be due to increased
numbers of memory T cells (19, 23). Memory T cell numbers appear to
increase with aging and they produce higher levels of IFN-
than
naive T cells (19). Thus an increase in memory T cells, with a
proportional decrease in naive T cells, in the splenocyte population
might in part explain the increase in splenocyte IFN-
production in the aged group.
IL-3 is a growth factor that affects the proliferation of early T cell precursors and enhances the growth of specific T cell subsets (26, 32). It is interesting that basal IL-3 release displayed age-dependent changes that were gender specific. Splenocytes from aged male mice exhibited significantly reduced IL-3 release, whereas aged females displayed enhanced IL-3 levels. These findings are in accordance with the findings of other investigators who demonstrated diminished IL-3 release by splenocytes from aged male mice (14, 19, 22). Furthermore, Kubo and Cinader (18) also demonstrated that T lymphocytes from aged female mice produce significantly higher IL-3 levels compared with their younger counterparts. A potential functional significance of changes in IL-3 release by splenocytes after trauma-hemorrhage is subsequent parallel changes in T lymphocyte lymphopoiesis and cell-mediated immune responses (i.e., depressed IL-3 release causes depression in T lymphocyte lymphopoiesis and cell-mediated immunity).
It is interesting that the immunoenhancing effect of trauma-hemorrhage on splenocyte proliferation in the young female group is lost with aging, whereas the suppression of proliferation in the young male group is not evident in the aged group compared with sham controls. Aging decreased the proliferative response of T lymphocytes from males; however, sham proliferation was unaffected by age in the females. The reason for this is unclear, but may be in part related to an age-dependent accumulation of memory T cells (28).
The finding that trauma-hemorrhage leads to increased IL-10 and unchanged IL-4 release by splenocytes of young male mice after hemorrhage is in accordance with Coimbra et al. (7) and previous work from our group (4). IL-10 is a potent inhibitor of cell-mediated immune responses in macrophages and Th-1 T lymphocytes (6, 9). Ayala et al. (5) have suggested that IL-10-induced suppression of T lymphocyte function in young males after hemorrhage is in part mediated by IL-4, since anti-IL-4 antibodies suppressed IL-10 production by purified splenic T cells after hemorrhage. The observation that splenocyte IL-10 production was not elevated in the young female group after trauma-hemorrhage further suggests that increased IL-10 in young males may be a causative factor for the immunodepressed state; however, IL-4 production was not elevated in this group after trauma-hemorrhage. The apparent difference between these findings and those of Ayala et al. (5) may be related to the fact that their conclusions are based on the effect of antibodies against IL-4 on IL-10 production, whereas in our study we measured immunoreactive IL-4 levels in cell supernatants. Furthermore, Ayala et al. (5) used purified splenic T cells, whereas the present study used unfractionated splenocytes containing B cells and macrophages (which also produce IL-10).
Other studies also have shown that cells (e.g., monocytes and Th-2
cells) from aged donors produce higher amounts of Th-2 cytokines
compared with those harvested from young individuals (19, 22, 23, 35).
IL-4 production was not altered after trauma-hemorrhage in either
gender group, suggesting that this anti-inflammatory cytokine may not
play an important role in trauma-hemorrhage-induced immune depression
in the young male group. However, gender-related differences in IL-4
release, independent of trauma-hemorrhage were observed. Aged females
produced elevated levels of IL-4 compared with aged males and young
females. This gender-related IL-4 release has also been observed in
other studies. Kubo and Cinader (18), using female mice, also showed
elevated IL-4 response to a mitogenic stimulus in aged individuals,
whereas Engwerda et al. (10), using male mice, demonstrated unchanged
IL-4 levels in aged mice. In contrast, Hobbs et al. (14) observed that
CD4+ splenic T lymphocytes from aged male mice produce
increased levels of IL-4 compared with their younger counterparts.
Because IL-4 and IL-10 are potent inhibitors of the Th-1 cytokine
synthesis (5, 18, 22), the altered IL-4 and IL-10 release after
trauma-hemorrhage and with age may contribute to the reciprocal changes
in Th-1 cytokine (i.e., IL-2, IFN-) release in the present study.
Trauma-hemorrhage represents an example of the problems with
oversimplification of the Th-1-Th-2 paradigm, since in young males
IL-4 production was not enhanced in parallel with IL-10. Numerous other
exceptions to Th-1-Th-2 paradigm have been documented (1).
Nonetheless, a reciprocal decrease in Th-1 cytokines (i.e., IL-2,
IFN-) was observed in this study and normalization of IL-10 production in aged males after trauma-hemorrhage to sham levels was
paralleled by normalization of IFN-
and IL-2 production.
These reciprocal changes in IL-10 and IFN- release by splenocytes
after trauma-hemorrhage may also play an important role in the sexually
dimorphic macrophage response to trauma-hemorrhage observed recently
(16). In this regard, young males have enhanced IL-10 and suppressed
IFN-
production after trauma-hemorrhage and suppressed
proinflammatory cytokine production (i.e., IL-1
, IL-6) by
macrophages (16). In contrast, aged mice have normalized (i.e., sham
levels) macrophage proinflammatory cytokine production, as well as
splenocyte IFN-
production. It does not appear that the absolute
levels of IL-10 and IFN-
are necessarily the critical factors, since
splenocyte IL-10 levels were higher in aged mice than in young mice.
Therefore, this suggests that the relative balance of T lymphocyte
IL-10 to IFN-
may determine the degree of macrophage activation
(i.e., production of proinflammatory cytokines) after
trauma-hemorrhage. For example, a high ratio of IL-10 to IFN-
suppresses macrophage activity, whereas a high ration of IFN-
to
IL-10 leads to normalized or enhanced macrophage activity. Further
studies are required to determine the influence of the IL-10/IFN-
ratio on macrophage activity, as well as cell-mediated immune responses
in general.
In this study only acyclic aged female mice and young female mice in proestrus state were included, thus it is unknown exactly at which time point during female maturation (aging) that the transition (initial hormonal decline) in estrus females took place. Nonetheless, the proestrus state of female mice, like the follicular phase of women, is correlated to the surge of estradiol, which is followed by the lutenizing hormone and prolactin peak leading to ovulation (6, 38). With regard to the murine model we have utilized in this study, the estrus cycle of females does not stop with age, but the frequency of the cycle decreases. Although the aged CBA/J NIA mice may not have become truly postmenopausal, we have previously observed a significant decrease in plasma estradiol levels (~33%) in the aged females (16). In this regard, plasma levels have been reported to decrease by 38% in postmenopausal women (38). Moreover, the average lifespan of mice is 24 mo, therefore, 18-19 mo of age in the mouse is comparable to 60 years of age in humans. Taking into consideration that the age of menopause in women is ~50 years, the murine model we have utilized is consistent with the postmenopausal state in humans with regard to circulating estrogen levels and relative age. Nonetheless, caution should exerted in applying results in aged female mice to those of postmenopausal women, since other factors such as metabolic rate are not similar.
Although several studies have implicated the immunomodulatory effects of sex steroids in the immune depression seen in response to shock, trauma, or sepsis, the exact mechanism of these steroids' actions still remains unclear (2, 27, 28, 39-41). Estrogen and androgen receptors have been found on thymocytes as well as on peripheral T lymphocytes with direct effects on the cytokine release (13, 28). Therefore, sex hormones could act directly on T lymphocytes and lead to the changes of the immune response observed in this study, although this was not determined in this study. Further studies on sex hormone and hormone receptor interactions after trauma-hemorrhage are required to determine the mechanism(s) by which a sexually dimorphic immune response is mediated and how aging influences it.
The data presented here indicate that the sexual dimorphism in the
immune response after trauma-hemorrhage observed in young mice is lost
with age. The changes in the gender-related immune functions seen with
age appear to be associated with the decreased levels of
17-estradiol in females and free testosterone in males (16). The
loss of the immune protective effects in aged females suggests that the
associated survival advantage against sepsis evident in these younger
mice may be lost in aged females. Therefore, an overlooked potential
benefit of hormone (estrogen) replacement therapy is that it may act
directly or indirectly to provide postmenopausal females with an
enhanced immune system. Thus improvement in the ability of the patients
to ward off infectious challenges encountered after traumatic injury
and shock might be expected in females on estrogen replacement therapy.
Alternatively, the administration of female sex hormones may also
provide a comparative benefit in both young and aged males. In fact,
recent studies have shown that the administration of 17
-estradiol to
males after trauma-hemorrhage restored immune responses to sham levels
(3). Moreover, recent observations from our laboratory indicate that
surgical ovariectomy produces immunodepression in young females after
trauma-hemorrhage that is reversible by the administration of
17
-estradiol (17, and unpublished observations). In conclusion,
these findings suggest that further evaluation of such therapeutic
approaches, both experimentally and clinically, are warranted.
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ACKNOWLEDGEMENTS |
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This work was supported by National Institutes of Health Grant R37 GM-37127.
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FOOTNOTES |
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Present addresses: V. Kahlke, Department of General Surgery, University of Kiel, 24105 Kiel, Germany; M. K. Angele, Department of Surgery, Ludwig-Maximilians University, 81377 Munich, Germany.
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: I. H. Chaudry, Center for Surgical Research, Rhode Island Hospital and Brown Univ. School of Medicine, Middle House II, 593 Eddy St., Providence, RI 02903.
Received 14 July 1999; accepted in final form 18 October 1999.
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