2084 SELECTED SUMMARIES
The other major issues addressed by the Lowry study included lack
of correlation between TPMT activity and leukopenia, and the ab-
sence of patients homozygous-deficient for the TMPT enzyme (esti-
mated to be 1:300). This may reflect physician practice to stop purine
analogues in patients who develop early leukopenia or avoid these
drugs altogether in the TPMT-deficient patients. The authors also
showed that their previous in vitro data of the inhibitory effect of
concomitant dosing of aminosalicylates upon TPMT activity (Gastro-
enterology 1999;116:15051506) can be translated into in vivo
elevation of 6-TGN concentrations and lower WBC counts.
The take-home forecast for the clinician is partly cloudy for the
current reliance upon metabolite levels for dosing of the purine
analogues. Although some retrospective studies have shown that
target levels of 6-TGN correlate with efficacy as measured by con-
ventional disease activity indices (Gut 1996;39:401 406, Gastroen-
terology 2000;118:705713), the same does not hold true when one
applies quality of life outcomes (Gut 2001;49:665 670). One must
be aware that coadministration of an aminosalicylate may result in
increased activity of the purine analogue, and that discontinuation of
such an agent may result in an undesired decrease in efficacy of 6-MP
or AZA. I concur with the authors for the necessity of large, prospec-
tive studies of dosing protocols for these important agents, as well as
investigations into the molecular mechanisms for their actions and
metabolism.
RUSSELL D. COHEN, M.D.
Reply. I want to make 3 points regarding the Selected Summary of
our article. First, in addition to the 2 studies cited by Cohen, at least
2 other sizable studies have failed to demonstrate a relationship
between clinical efficacy and erythrocyte 6-TGN concentrations in
patients with IBD treated with azathioprine or 6-mercaptopurine
(Gastroenterology 1999;117:527535, Gastroenterology 2000;118:
A4202). Second, the assertion of Cohen that the IBDQ may not
accurately measure disease activity in patients with IBD is incorrect.
The previous studies by Cuffari (Gut 1996;39:401 406) and Dubin-
sky (Gastroenterology 2000;118:705713) that reported a relation-
ship between 6-TGN and clinical response to 6-mercaptopurine used
the Harvey Bradshaw Index (HBI) (Lancet 1980;1:514), which is in
essence a simplified version of the CDAI (Gastroenterology 1976;70:
439 444) to measure disease activity. Although the original CDAI
was rigorously developed and validated, the HBI has never been
validated, and its responsiveness to change in the context of clinical
trials has never been determined. In contrast, the IBDQ was rigor-
ously developed and validated, is sensitive to change, and correlates
well with the CDAI (Gastroenterology 1989;96:804 810, Gastroen-
terology 1994;106:287296). Thus, the IBDQ is a more valid alter-
native to the CDAI for measuring Crohns disease activity than is the
HBI. Third, in another study by Cuffari (Gut 2001;48:642 646),
nonresponding patients with IBD treated with very low doses of
azathioprine (1.1 0.1 mg/kg) who did not have leukopenia and who
had subtherapeutic 6-TGN concentrations had their azathioprine
doses gradually increased to a mean of 1.5 0.1 mg kg1 day1,
with subsequent clinical response and increase in 6-TGN concentra-
tions. It is likely that the same result could have been achieved by
simply administering the doses of azathioprine proven to be effica-
cious for Crohns disease in controlled trials (23 mg kg1 day1) enzymes that, similar to iNOS, use L-arginine as substrate
(Ann Intern Med 1995;123:132142) from the outset, without ther-
apeutic drug monitoring. Thus, the utility of routinely measuring
6-TGN concentrations in clinical practice remains unclear (Gut 2001;
48:591592). A randomized, double-blind, controlled trial compar-
GASTROENTEROLOGY Vol. 122, No.
ing standard therapy (azathioprine 2.5 mg kg1 day1 with dose
adjustment only for toxicity) to azathioprine adjusted to a target
6-TGN concentration in patients with Crohns disease is needed to
answer this question.
WILLIAM J. SANDBORN, M.D.
PERSISTENCE AND PATHOGENESIS: A GREAT
DEFENSE FACILITATES A GOOD OFFENSE
Gobert AP, McGee DJ, Akhtar M, Mendz GL, Newton JC, Cheng
Y, Mobley HLT, Wilson KT (Departments of Medicine and
Microbiology and Immunology, University of Maryland
School of Medicine, Baltimore, Maryland; Department of Vet-
erans Affairs, Maryland Health Care System, Baltimore, Mary-
land; and School of Biochemistry and Molecular Genetics,
University of New South Wales, Sydney, Australia). Helicobac-
ter pylori arginase inhibits nitric oxide production by eukary-
otic cells: a strategy for bacterial survival. Proc Natl Acad Sci
U S A 2001;98:13844 13849.
In the nearly 2 decades since Helicobacter pylori was first
isolated, it has become increasingly clear that the true rela-
tionships between this organism and diseases of the upper
gastrointestinal tract are highly complex. Considerable efforts
have focused on delineating the precise mechanisms by which
H. pylori colonization leads to peptic ulcer disease, adenocar-
cinoma of the distal stomach, and mucosal lymphoproliferative
diseases. However, a signature feature of H. pyloriinduced
gastritis is its capacity to persist for decades without causing
serious damage in most cases. This is in marked contrast to
inflammatory reactions induced by other Gram-negative
pathogens, which either resolve over a limited time-span or
progress to eliminate the host.
Nitric oxide (NO) is an important intracellular mediator of
cell growth and apoptosis that can directly damage DNA, and
levels of this molecule are enhanced in chronic inflammatory
and autoimmune conditions. NO also possesses potent anti-
microbial activities against both intra- and extracellular patho-
gens, including H. pylori (Gut 1998;42:334 337, Infect Im-
mun 2000;68:4378 4383). NO is generated from an amino
acid substrate (L-arginine) by NO synthases, enzymes that exist
in both constitutive and inducible forms. Inducible NO syn-
thase (iNOS) is increased within foci of H. pylori gastritis,
localizing to epithelial and endothelial cells, as well as mac-
rophages within the lamina propria (Gastroenterology 1999;
116:1319 1329). Up-regulation of iNOS (and hence NO)
within H. pylori colonized tissue seems counterintuitive when
one considers the chronic and persistent lifestyle of this organ-
ism, necessitating the hypothesis that H. pylori may possess
mechanisms to counteract the effects of NO. Arginases are
(Cell Mol Life Sci 1999;55:10151028). However, instead of
generating NO, arginases catalyze the formation of urea and
L-ornithine (Cell Mol Life Sci 1999;55:10151028), thereby
diverting L-arginine away from iNOS, which results in reduced
June 2002
amounts of NO. Because H. pylori has been previously shown
to possess a gene (rocF) encoding a functional arginase ( J
Bacteriol 1999;181:7314 7322), Gobert et al. sought to de-
termine whether rocF regulates cellular production of NO, thus
contributing to the ability of H. pylori to survive within the
context of enhanced gastric mucosal levels of iNOS.
H. pylori wild-type and isogenic rocF mutants (containing an
inactivated arginase) were cocultured with macrophages in the
presence of physiologic concentrations of L-arginine substrate.
Both parental and mutant strains robustly induced iNOS
expression in macrophages, findings that are consistent
with previous observations of increased iNOS levels within
H. pylori colonized gastric tissue (Gastroenterology 1999;116:
1319 1329). However, only the rocF mutant strain induced
significantly increased levels of NO, indicating that arginase
activity in the wild-type strain was effectively siphoning L-
arginine away from iNOS, and these events occurred regardless
of macrophage activation state. L-arginine consumption studies
conducted in the absence of macrophages confirmed these
findings by demonstrating that levels of L-arginine were sig-
nificantly reduced by wild-type, but not rocF mutant, H. pylori
strains.
To delineate the bacterial components required for these
effects, Gobert et al. cocultured macrophages with live or
heat-killed H. pylori wild-type strains, crude water extracts
containing bacterial surface proteins, cytosolic extracts, or
bacteria separated from cells by a filter support. Suppression of
NO release was only found with live H. pylori, in close prox-
imity to or separated from macrophages, suggesting that viable
bacteria that are not in direct contact with macrophages con-
sume L-arginine, resulting in inhibition of iNOS-catalyzed
NO production. The investigators next complemented these
experiments with elegant functional studies in which they
measured survival rates of wild-type or rocF mutant strains
exposed to macrophages as a measure of NO-mediated killing.
As expected, macrophage coculture had no effect on survival of
the parental H. pylori strain; in contrast, the arginase-deficient
rocF mutant had a 5-log reduction in colony-forming units.
To further implicate NO in H. pylori killing, peritoneal mac-
rophages isolated from wild-type or iNOS/-deficient mice
were cocultured with the H. pylori rocF mutant for 24 hours.
Compared with the profound cidal effect of wild-type macro-
phages, iNOS/ macrophages exerted no effect on the argin-
ase-deficient H. pylori mutant, indicating that iNOS-derived
NO is likely required for these antimicrobial effects.
Comment. Although chronic gastritis clearly increases the risk for
peptic ulceration and noncardia gastric adenocarcinoma, only a mi-
nority of individuals carrying H. pylori ever develop these sequelae.
Clinical complications of H. pylori colonization may instead be viewed
as imbalances in gastric homeostasis that are disadvantageous for both
the bacteria and humans, especially if death of the host occurs. In
support of this, recent data indicate that H. pylori not only induces
gastritis but also possesses means to subdue the host inflammatory
response, a requirement seemingly inherent for an organism that
persists for the lifetime of its host. H. pylori infection is associated
SELECTED SUMMARIES 2085
with increased mucosal levels of interleukin 10 (Lab Invest 1995;73:
760 770, Gastroenterology 1996;110:1744 1752, Infect Immun
1997;65:4229 4235, Gut 1997;40:739 744), an anti-inflammatory
peptide that inhibits secretion of proinflammatory cytokines from
macrophages and neutrophils. When compared with lipopolysaccha-
ride (LPS) from the Enterobacteriaceae, H. pylori LPS is 1000-fold less
active and only weakly activates macrophages (Infect Immun 1995;
63:11831187). Inactivation of a cag island gene (cag10) results in a
paradoxical increase in proinflammatory cytokine secretion compared
with levels induced by wild-type H. pylori (J Clin Pathol 1999;52:
653 657). The failure of H. pylori to invade the mucosa may con-
tribute to its long-term persistence. It is tempting to speculate that
H. pylori can also orchestrate the host response by negatively regu-
lating intracellular eukaryotic signaling pathways, as has recently
been described in Salmonella typhimurium (Science 2000;289:1560
1563). Certain nonpathogenic Salmonella attenuate interleukin-8 se-
cretion induced by pathogenic bacteria by inhibiting the ubiquitina-
tion of IB, a novel mechanism for dampening the inflammatory
response (Science 2000;289:1560 1563).
Gobert et al. have now added another component to the defensive
armamentarium of H. pylori by demonstrating that a bacterial argi-
nase can regulate iNOS activity that is induced by host-pathogen
interactions. The experiments from which these results stemmed were
rigorously controlled, and independent assays support the overall
conclusion that H. pylori arginase attenuates NO-mediated bacterial
killing. It will be interesting in the future to determine whether
complementation of rocF in an arginase-deficient H. pylori mutant
regenerates the ability to survive in the presence of macrophage-
derived NO. Arginase may not only protect the bacterium but also
may attenuate host mucosal damage, thereby contributing to long-
term persistence of H. pylori within the gastric niche. As stated by the
authors, NO is notable for its ability to induce DNA damage and
lipid peroxidation, events that are usually lethal for eukaryotic cells.
Although the experiments of Gobert et al. were performed in
isolated bacterial:macrophage systems, their results have set the stage
for important in vivo confirmations, some of which have already been
completed. Studies by this same group of investigators have revealed
that H. pylori rocF mutants have an attenuated ability to colonize
wild-type mice (J Bacteriol 1999;181:7314 7322) and that wild-
type H. pylori can infect wild-type and iNOS/ mice with no
differences in colonization efficiency or severity of inflammation,
supporting the contention that H. pylori arginase modulates NO
synthesis within inflamed tissue. A critical experiment will be to
determine if successful colonization can be restored when H. pylori
rocF mutants are infected into iNOS/ knockout mice that lack the
ability to produce NO, and such studies are ongoing at the present
time.
It is apparent that regulation of chronic gastric inflammation by H.
pylori is governed by levels of host-bacteria equilibria that are not
found during cellular interactions with acute pathogens, and clini-
cally apparent disease likely results from the cumulative effect of
multiple interactions between H. pylori and its host. Gobert et al. have
identified a novel mechanism through which H. pylori may persist for
the virtual lifetime of its cognate host. These types of studies that
focus on characterization of host-microbial interactions are critical
because they allow fundamental questions to be addressed regarding
the basis of host defense and microbial persistence.
RICHARD M. PEEK, JR.