Section of Digestive and Liver Diseases, Department of Medicine, University of Illinois and West Side Veterans Affairs Medical Center, Chicago, Illinois 60612
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ABSTRACT |
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The single layer of epithelial cells lining the intestinal tract is charged with a most difficult task: protecting the underlying biological compartments from both the normal commensal flora that reside within the intestinal lumen as well as the uninvited pathogens. To such an end, the intestinal epithelial cells are equipped with a panoply of defense mechanisms, both constitutive and inducible. This review focuses only on those defense mechanisms that are initiated and executed by the intestinal epithelial cell. Fitting these strict criteria are three major categories of epithelial host defense: enhanced salt and water secretion, expression of antimicrobial proteins and peptides, and production of intestinal mucins. Each of these areas is discussed in this review.
chloride secretion; enteric pathogens; antimicrobial peptides; mucins; prostaglandins
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INTRODUCTION |
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THE INTESTINAL EPITHELIUM is presented with a formidable task. In the lumen of the large intestine loom 1012 organisms/ml of contents, representing 30 genera and 500 species (71). Although the presence of intestinal microflora certainly contributes to the maintenance of human health, these organisms are beneficial only when contained within their natural residence, the intestinal lumen. The single cell layer faced with such a charge is the intestinal epithelium. Besides forming a physical barrier composed of cell membranes and interspersed tight junctions, the epithelium is armed with more active means of keeping the intestinal microflora in check. As if this singular task was not enough, the intestinal epithelium must also be able to readily discriminate between the resident flora and unwelcome pathogens. For this latter group, the epithelium maintains a separate armament.
Collectively, the mechanisms by which the intestinal epithelium controls the microflora and more actively responds to encroaching pathogens is referred to as innate intestinal epithelial defense, the subject of this review. By definition, therefore, the role of inflammatory mediators and the subsequent recruitment of inflammatory cells is not addressed, although it is well recognized that epithelia actively participate in or orchestrate this response. Similarly, although specific bacterial factors, such as cholera toxin (CT) or the heat-labile toxin of Escherichia coli (reviewed in Ref. 66) may enhance intestinal secretion of fluid and electrolytes, an accepted mechanism of host defense (see below), these mechanisms are not reviewed here since they are pathogen, not host, initiated. Only responses that originate in and are executed by the epithelium are discussed. These include mechanisms by which the epithelium promotes salt and water secretion, the production of antimicrobial peptides/proteins, and the expression of mucins.
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SECRETORY DIARRHEA |
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Numerous reviews and textbooks cite fluid secretion as a mechanism of host defense (80). The premise is that rapid and increased flow of fluid over the epithelial surface "flushes" organisms from the intestinal lumen, thus preventing their attachment to host epithelial cells or possibly dislodging adherent organisms. An alternative way to view the induction of secretory diarrhea by enteric pathogens is as a survival mechanism for the microbe. As put forth by Falkow (20), a microbial pathogen, besides necessarily possessing the ability to escape attack by the host and multiply, must create circumstances that ensure transmission to new hosts. Viewed in this manner, diarrhea may also be considered advantageous to the pathogen. Nonetheless, despite the logical and well-accepted dogma that secretory diarrhea is one mechanism of host defense, no specific studies demonstrating this phenomenon could be found. Circumstantial evidence on which such notions may be based includes the observation that infection by enteric pathogens may be prolonged by anti-motility agents (16). In addition, in models of experimental diarrhea in humans in which 1.5-2.5 liters of isotonic solution were directly infused into the small intestine over 2 h, the concentration of fecal flora was shown to decrease 30 min postpurgation (26). This reduction may have been a direct result of the flushing of organisms from the intestine and/or a disruption of the prerequisite conditions for growth in the normally "static" colon. By 18 h, the fecal flora concentrations had normalized. Neither of these studies however, specifically addressed the effect of increased intestinal secretion on microbial pathogens. No direct demonstration of beneficial effects of secretory diarrhea to the host could be found.
Accepting, nonetheless, that the flushing action of secretory diarrhea
is a host defense mechanism, enteric pathogens have been shown to
induce the intestinal epithelium to express proteins that in an
autocrine fashion increase
Cl secretion. Using
cultured intestinal epithelial cell lines, Eckmann et al. (18) showed
that infection by invasive bacterial pathogens, such as
Salmonella, upregulated the expression
of prostaglandin (PG) E2 by
increasing PGH synthase (PGHS). PGHS catalyzes the conversion of free
arachidonic acid to PGH. Other specific synthetases then control the
subsequent conversion of PGH to other PGs (74). To demonstrate a role
for pathogen-induced PG production in ion secretion, cultured human
intestinal epithelial T84 monolayers were exposed to sterilized medium
collected from infected HT-29 monolayers (18). This resulted in active
ion transport measured as short-circuit current
(Isc).
Preincubation of T84 monolayers with
anti-PGE2 antibodies inhibited the
Isc response.
Although PGF2 was also produced in
response to infection, this particular PG did not induce ion secretion.
Interestingly, in an earlier publication, Peterson et al. (55) reported a role for PG in infectious diarrhea by demonstrating the expression of phospholipase A2 (PLA2)-activating protein in response to both CT and live Salmonella typhimurium. The rate-limiting step of PG synthesis is the availability of substrate: free arachidonate. Although free arachidonate is limited in eukaryotic cells, esterified arachidonate is found in most membrane phospholipids. PLA2 hydrolyzes esterified arachidonate from the sn-2 position of the glycerol moiety of phospholipids, thus regulating PG synthesis.
Several previous investigations have demonstrated the requirement of
active protein synthesis for CT-induced
Cl secretion (32, 48, 70).
In addition, inhibitors of protein synthesis prevented the release of
free arachidonate and subsequent PG synthesis in response to CT (54).
On the basis of these observations, Peterson et al. (55)
explored the hypothesis that CT, in the absence of live vibrios or live
S. typhimurium, increased the expression of a protein responsible for activating
PLA2,
PLA2-activating protein (PLAP).
They showed that CT induced PLAP expression in both human intestinal
epithelial cells (Caco-2) and a murine monocyte/macrophage cell line
(J774). The same response to CT and S. typhimurium was demonstrated in vivo using a mouse
intestinal loop model. Together, these studies, schematized in Fig.
1, suggest that
pathogen-induced expression of proteins that regulate the synthesis of
PGs, known secretagogues, is one mechanism of intestinal host defense.
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Continuing with the theme of host-initiated fluid and electrolyte
secretion in response to enteric pathogens, another novel mechanism has
recently been identified: upregulation of the galanin-1 receptor
(Gal1-R) by intestinal epithelial cells (7). Galanin is a neuropeptide
whose distribution lies within the central nervous system and the
gastrointestinal tract (4, 46). Although the most widely recognized
action of galanin within the gastrointestinal tract is stimulation of
intestinal motility (58), it has recently become clear that activation
of Gal1-R elicits Ca+-dependent
Cl secretion (6). On
cloning the Gal1-R gene, putative binding sites for the
inflammation-associated transcription factor nuclear factor
B
(NF-
B) were identified. Reporter gene studies demonstrated NF-
B-dependent transcription (39). Interestingly, the human galanin
gene also contains an NF-
B binding site (33), suggesting that the
expression of both receptor and ligand is coordinately regulated as a
part of the inflammatory response.
This possibility was investigated using an in vitro model of intestinal
epithelial T84 cell monolayers infected with various enteric pathogens.
Enteric bacterial pathogens, including enteropathogenic E. coli, (64) enterohemorrhagic
E. coli, enterotoxigenic
E. coli, enteroinvasive
E. coli, S. typhimurium (83), and Shigella flexneri (17), have all been shown to activate NF-B.
Furthermore, pathogen-activated NF-
B binds not only to the consensus
NF-
B probe but also to the specific NF-
B sequences with the
Gal1-R promoter (43). Correspondingly, a significant increase in Gal1-R mRNA and peptide binding was seen. Challenging of infected T84 monolayers with ligand elicited a 13-fold increase in net ion secretion, whereas galanin stimulation of uninfected monolayers enhanced Isc by
only 4-fold. Figure 2 depicts the model of
pathogen-induced expression of Gal1-R and the consequence of its
activation, Cl
secretion.
Similar responses have also been demonstrated in the native intestine
employing Salmonella-infected mice
(44).
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As a result of the recent heightened interest in host-pathogen interactions, it is being acutely realized that the mechanisms of pathogen-induced diarrhea are not fully understood. Some studies, like those cited above, highlight the role of novel pathways involved in pathogen-induced intestinal secretion. Taken together, these data suggest that host intestinal epithelial cells utilize the production of secretagogues and/or specific secretagogue receptors as mechanisms of innate host defense.
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ANTIMICROBIAL PEPTIDES |
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Although the broad class of host defense effector molecules is
widespread, their existence was only recognized in the early to mid
1980s (67, 69, 75). Numerous antimicrobial peptides have now been
identified and can be separated into three general categories as
defined by Bevins (8): 1) peptides
released into internal fluids such as lymph,
2) peptides localized within
circulating phagocytic cells, and 3)
peptides released at the mucosal surface. Although arbitrarily
categorized, the mechanism of action of antimicrobial peptides is
similar, namely membrane disruption of prokaryotes. Both the charge
(cationic) and structure of these molecules are such that they interact
with anionic elements of the prokaryotic membrane, thereby inserting
themselves and creating pores. Energy and ionic gradients are
subsequently lost, and cell lysis occurs within minutes (9). In that
this review focuses on intestinal epithelial innate defense, discussion
is limited to the last category of antimicrobial peptides, those
released at the mucosal surface (Table 1).
For more extensive reviews of this topic, see Refs. 24, 30, and 36.
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-Defensins
The first intestinal defensins were identified in the Paneth cells of
mice (52). Paneth cells are located within the crypts of the small
intestine. As such, this family of mouse defensins is called cryptdins
(51, 53). Cryptdins, which are -defensins, exhibit antimicrobial
activity against a wide range of microorganisms including
E. coli, S. typhimurium, Listeria
monocytogenes, Staphylococcus aureus, Giardia
intestinalis, and Candida
albicans. The mechanisms controlling cryptdin gene
expression are not understood. Cryptdin mRNA expression does not occur
in newborn mice but gradually appears over the first 20 days after
birth. The observation that cryptdin 4 mRNA distribution is not altered
in germ-free mice compared with conventionally housed mice suggests
that expression of this gene is not responsive to microbial antigens.
This differs from the regulation of
-defensin genes (discussed
below). The contention that cryptdins are in fact
important agents of intestinal host defense is supported by the finding
that, although an avirulent mouse S. typhimurium pho
P mutant is sensitive to cryptdin 1, wild-type
S. typhimurium is completely resistant
(21, 68). This suggests that resistance of enteric pathogens to such
peptides may be crucial to the evolution of these virulent microbes.
The majority of work on intestinal defensins has been performed on the
mouse-derived cryptdins due to the paucity of fresh human tissue.
Nonetheless, two human intestinal -defensins have been identified:
human defensin 5 [HD-5 (29)] and human defensin 6 [HD-6 (28)]. Both of these genes have been cloned and found to be similar in their genomic organization. Furthermore, there is a
significant degree of sequence homology within the proximal region of
the 5' flank (41). Specifically, both possess AP-2 and NF-IL6
binding sites, suggesting functional significance. Studies to determine the regional localization of HD-5 during human
gestation revealed that its mRNA was detected within the small and
large intestines at 13.5 wk but by 17 wk was restricted to the small
intestine (only trace amounts were detected in the colon). In contrast,
HD-6 mRNA was detectable only in the small intestine over this
gestational range. The amount of message appeared to increase with the
stage of development: adult > newborn > fetus. Northern
blot analysis estimates the ratio of HD-5 to HD-6 to be ~3:1. In situ
hybridization localized both peptides to Paneth cells. Paneth cells are
identifiable by electron microscopy in the human intestine at 12 wk of
gestation, coinciding chronologically with the appearance of defensin message.
To further study HD-5, Porter et al. (56) biosynthesized this peptide using a baculovirus-insect cell system. In this way, they were able to produce antibodies, which allowed immunolocalization studies to be performed, and characterize the antibacterial activity (57). These investigators were able to demonstrate that HD-5 was selectively localized to secretory granules within Paneth cells of the human adult small intestine. In contrast to the previously reviewed findings that mouse cryptdin exhibited antimicrobial activity against avirulent S. typhimurium but not wild type, recombinant HD-5 (rHD-5) was active against both (57). Even after exposure to concentrations of trypsin that are normally present in the intestinal lumen, which partially cleaved rHD-5, essentially full antimicrobial activity was observed. Such observations support the belief that these naturally occurring human intestinal peptides most certainly play an active role in both controlling the concentration of normal flora as well as providing protection against pathogens.
-Defensins
In humans, only two -defensins have been identified: hBD-1 and
hBD-2. hBD-1 was originally isolated from hemodialysate (5). Later,
hBD-1 was found to be expressed in a number of epithelia, including
submaxillary gland, trachea, prostate, placenta, thymus, small
intestine, and testis (82). In contrast to the inducible nature of the
bovine
-defensins, TAP and LAP, hBD-1 appears not to be upregulated
in response to lipopolysaccharide, TNF-
, interleukin-6 (IL-6), and
interferon-
(IFN-
) alone or in combination (82). The upstream regulatory region of the hBD-1 gene lacks an NF-
B binding site; however, it does contain consensus sites for NF-IL6 and
IFN-
, suggesting that there is potential for upregulation to
specific inflammatory mediators (34).
The second human -defensin, hBD-2, was only recently identified (2)
by homology to hBD-1. Like hBD-1, hBD-2 is expressed in many epithelial
tissues including, most prominently, lung, but it also is expressed
throughout the gastrointestinal tract. Studies regarding regulation of
hBD-2 have not been performed; therefore, it is not known whether its
expression is induced in response to bacterial infection. In a more
recent publication, however, Singh et al. (73) provided evidence that
hBD-2, but not hBD-1, expression is induced by inflammation. They found
that exposure of primary airway epithelial cultures to IL-1
stimulated the production of hBD-2 but not hBD-1. Furthermore, hBD-2
was demonstrated in bronchoalveolar lavage fluid only from cystic fibrosis patients or those with inflammatory conditions
of the lung. In contrast, hBD-1 was isolated from bronchoalveolar
lavage fluid from all patients. Such findings suggest that hBD-1 is
constitutively expressed, whereas hBD-2 is induced as a part of the
inflammatory response. Whether a similar paradigm extends to intestinal
regulation has yet to be determined. It is known, however, that there
is antimicrobial synergy with hBD-2 and the antimicrobial proteins lysozyme and lactoferrin (2). In fact, minimum inhibitory
concentrations (MICs) against E. coli and S. aureus
dropped two- to fourfold for hBD-2 in the presence of either
lactoferrin or lysozyme.
The most recently recognized family of antimicrobial peptides is the cathelicidins (81). The prosequence from which these peptides originate is highly homologous to that of a protein isolated from porcine leukocytes called cathelin (59), hence the name. The first human cathelicidin, LL37/hCAP18 (18-kDa cationic antimicrobial protein), was cloned from bone marrow (35). LL37/hCAP18, although primarily expressed in myeloid cells, has also been identified in inflamed skin (23) and testis (1). Most recently, LL37/hCAP18 was also shown to be expressed diffusely in human epithelial tissues including the gastrointestinal tract (3). This peptide was also demonstrated to be secreted into the surface fluid of the airway. Synergy with the antimicrobial protein lactoferrin, but not lysozyme, occurred as the MICs for numerous bacteria tested dropped two- to fourfold (2). Whether expression of this peptide is upregulated in response to infection has yet to be determined.
Interestingly, the activity of antimicrobial peptides is not limited to the killing of microorganisms. Instead, several other actions have been identified. The human neutrophil defensin HNP-1 has been shown to be a chemotaxin for monocytes (77). Furthermore, defensins may serve to potentiate the opsonizing ability of macrophages (22). Antimicrobial peptides may stimulate cell proliferation (50) and enhance the production of extracellular matrix proteins (25), suggesting their participation in wound healing. Other activities attributed to defensins include the inhibition of ACTH-stimulated production of steroids (72) and inhibition of protein kinase C (11). The biological significance of these activities, however, is not apparent.
To this point, discussion has focused on two broad categories of host
defense, namely fluid and electrolyte secretion and antimicrobial
peptides. Interestingly, the molecules discussed so far are not solely
restricted to the specific defense mechanism with which they were
initially associated. For example, although cryptdins possess potent
antimicrobial activity, an elegant study by Lencer et al. (37)
demonstrated that the exposure of cryptdins 2 and 3 (but not 1, 4, 5, or 6) to the apical surface of cultured intestinal epithelial (T84)
monolayers stimulated the secretion of
Cl. Cryptdin-induced
Cl
secretion could not be
ascribed to any of the classical signaling pathways, i.e., increased
cAMP or cGMP, but instead was attributed to the formation of membrane
pores likely related to those that are responsible for the killing of microorganisms.
In a similar vein, an enzyme (PLA2), discussed above as being crucial to the production of secretagogues (namely PGs) in response to bacteria and/or their toxins, has itself been shown to have antimicrobial activity (27). In an attempt to purify cryptdins from murine Paneth cells, Harwig et al. (27) noted the presence of a larger polypeptide that exhibited potent antimicrobial activity. A series of studies identified this molecule as type 2 (intestinal/splenic) PLA2. Although the precise bactericidal mechanism of PLA2 has not been established, intestinal PLA2 has an unusual affinity for bacterial phospholipids compared with other phospholipase substrates (27, 42). It is interesting, therefore, that molecules such as defensins and PLA2 seem to possess overlapping strategies for providing host defense.
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HISTONES |
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As early as 1942, Miller et al. (47) reported that histone possessed antibiotic action. Most recently however, Rose et al. (60) demonstrated that histone H1 may provide antimicrobial protection within the human intestinal tract. Using immunohistochemical techniques, these investigators were able to demonstrate the presence of histone H1 not only within the nuclei but also within the cytoplasm of villus, but not crypt, cells. Furthermore, detached villus cells were shown to release histone H1 while undergoing apoptosis. Both histone H1 and fragments of histone H1 extracted from human ileal mucosa possess antimicrobial properties. These findings suggest, therefore, that, in addition to the protection afforded by the classical defensins localized primarily to the intestinal crypt, specifically Paneth cells, villus cells are also armed with antimicrobial molecules. The presence of histone H1 within the cell cytoplasm may help protect against invading pathogens, whereas histone H1 released from exfoliated apoptotic villus cells may provide a broader level of protection against luminal microorganisms. A number of pathogens have been shown to induce apoptosis of host cells (12, 31). It is widely speculated that the induction of apoptosis by pathogenic organisms may itself be a mechanism of host defense (for review of this issue see Refs. 38 and 84).
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MUCINS |
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Mucins, or mucus glycoproteins, are highly complex glycoproteins ranging in size from 200 to 2,000 kDa. They are comprised of a peptide backbone to which O-glycosidic bonds link carbohydrates to serine or threonine residues. Other posttranslational modifications complete the structure. The mucosal surface of the gastrointestinal tract is coated by mucins in such a way that a gellike layer interfaces the apical intestinal epithelial membrane and the intestinal lumen. Strategically positioned, the intestinal mucus layer has long been viewed as the first line of intestinal defense against infectious agents. The carbohydrate structures found on mucin macromolecules are extraordinarily diverse. In this way, a vast array of potential binding sites for both commensal and pathogenic organisms is provided. The intestinal mucus layer, therefore, may serve as a niche for microbial colonization. Such colonization can lead to two different scenarios. First, competition of mucin binding sites with those on the underlying host epithelial cells may impede microbial and epithelial interactions that can trigger injurious host cell responses or even a full-blown inflammatory reaction. Second, restriction of microbes to the mucus layer may enhance their removal by peristaltic flow. Alternatively, by providing a temporary foothold for microorganisms, mucins may allow motile microbes to track to the epithelial surface where they directly exert their deleterious effects. Whether a harmful or beneficial outcome results from the attachment of microbes to intestinal mucins may depend on other factors, including the composition and quantity of mucins, intestinal motility, and the rate of intestinal fluid flow. If the rate of microbial replication exceeds that of removal, infection is likely to result.
Several specific mucin domains have been identified as interacting with specific bacterial adhesins. Although a complete discussion of these data is beyond the scope of this review, some pertinent examples follow: oligomannosides of mucin N-glycans bind type 1 pili of certain E. coli 0157:H7 (62); saccharides are recognized by several pili on enterotoxigenic E. coli (49); fucose-containing receptors provide recognition sites for S. typhimurium (19) and Campylobacter jejuni (45); and Gal-GalNAc are receptors for Entamoeba histolytica trophozoites (10).
The role of intestinal mucins in innate intestinal defense is highlighted by recent publications demonstrating that the protective effects of probiotic organisms, such as Lactobacillus species, may lie in their ability to stimulate mucin production (40). Equally important is the observation that an invertebrate intestinal mucin is the primary substrate for a baculovirus-encoded metalloprotease called enhancin (78). The in vivo degradation of this intestinal mucin by enhancin correlated strongly with enhancement of infection as well as mortality. That viruses have evolved a mechanism by which to penetrate the intestinal mucus layer lends further credence to the concept that these complex molecules do indeed provide protection to the host.
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SPECULATED MECHANISMS OF DEFENSE |
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Apoptosis
It has been demonstrated that infection of cultured human intestinal epithelial cells in vitro with invasive bacterial pathogens, specifically Salmonella and Shigella, induces apoptosis of a limited percentage of cells (31). It has been hypothesized that this may afford the infected epithelium a mechanism for shedding infected cells, thereby prompting the clearance of these unwanted microbes. Whether pathogen-induced apoptosis occurs in vivo is not known. As an extension, therefore, it is impossible to conclude whether this response might offer an advantage to the host except to possibly expedite mucosa repair. Complex studies in animal models of pathogen-specific infection are needed to address this question.Nitric Oxide
A role for nitric oxide (NO) production by epithelial cells in response to microbial pathogens as a mechanism of host defense has also been suggested but not proven. The effect of infection by enteric pathogens on NO production by intestinal epithelial cells has been controversial. Initial studies by Salzman et al. (63) demonstrated that NO production by cultured human intestinal epithelial cells was stimulated by a bacterial enteric pathogen S. dublin, but only in the presence of IFN- ![]() |
SUMMARY |
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In summary, I have tried to provide an overview of the "built-in" mechanisms by which the intestinal epithelium can provide defense against a potentially hostile environment. Although I have focused on three specific areas, active secretion of water and electrolytes, antimicrobial peptides/proteins, and mucins, I realize that I have either not addressed or only lightly touched upon other potential mechanisms of defense including apoptosis, the production of NO, the generation of free radicals, and others. This is an exciting and rapidly expanding area. Coupled with the rate at which new information regarding the direct effects of pathogenic organisms on eukaryotic host cells is being generated, we can be assured that the recognition of many more novel mechanisms will further expand our knowledge of the panoply of intestinal epithelial defenses realized today.
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ACKNOWLEDGEMENTS |
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A special acknowledgment is extended to my colleague and collaborator Dr. Richard V. Benya, without whom the galanin receptor studies would not have been conceived. Thanks also to Lisa Mosher for her tremendous assistance with the preparation of this manuscript.
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FOOTNOTES |
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The author is supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-50694 and grants from the Department of Veterans Affairs (Merit Award and Research Enhancement Awards Program).
Address for reprint requests and other correspondence: G. Hecht, Univ. of Illinois, Dept. of Medicine, Digestive and Liver Disease (M/C 787), 840 South Wood St., CSB Rm. 704, Chicago, IL 60612 (E-mail: gahecht{at}uic.edu).
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