This Month in GastroenterologyBy Eugene B. Chang

6-Mercaptopurine Treatment of Child-Bearing Patients With IBD Is Safe6-Mercaptopurine (6-MP) and azathioprine (the latter is rapidly converted to 6-MP in vivo) are safe and effective immunomodu-latory treatments for patients with inflammatory bowel diseases (IBD). However, they have been used sparingly in child-bearingpatients due to concerns of teratogenicity. In animal experiments, for instance, adverse outcomes such as congenital malforma-tion, prematurity, low birth weights, and genetic abnormalities have been observed at very high doses. However, at lower dosesmore in line with therapeutic human doses (1.5 mg/kg for 6-MP and 2.5 mg/kg for azathioprine), only decreased fertility and lowerbirth weights were noted. Although clinical studies have shown these agents are safe in pregnancy in patients with systemic lupusand transplant patients, little information, aside from limited retrospective studies, are available. This retrospective cohort studywas therefore undertaken to determine the safety of 6-MP in pregnant patients with IBD and their offspring. The study populationwas obtained from a private practice database of over 2800 patients with IBD, representing a high percentage of tertiary referrals.Of 462 who had received the drug, 155 had conceived at least one pregnancy after developing IBD. Patients were separated into2 groups, those who had taken 6-MP before or at the time of conception versus those that had their pregnancies before taking6-MP. There was no statistical difference between the groups with regard to failure to conceive, spontaneous abortion, abortionbecause of birth defect, major congenital abnormalities, neoplasms, or increased infections. These data therefore substantiate thesafety of 6-MP on pregnancy outcome in IBD.

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Normal Aminotransferases and Mild Liver Histology inPatients With Hepatitis C Predict a More FavorableNatural HistoryChronic hepatitis C virus (HCV) is now the major cause of chronic hepatitis in theWestern world and the single major reason for liver transplantation in the UnitedStates. While patients with aggressive disease often develop irreversible cirrhosis andend-stage liver disease, some patients remain asymptomatic and without significantliver disease for many decades, if not for life. Because current therapies are costly,have many adverse effects, and are limited in efficacy, prognostic indicators of diseaseprogression would greatly help in advising patients on their therapeutic options. Inpatients with mild disease, for instance, it may be better to advise delaying therapy,because of the continued advances in efficacy and tolerability of therapeutic regi-mens. To address this issue, this study attempted to define the natural history andprognostic factors in chronic hepatitis C. Liver histology from 123 patients of theClinical Center of the National Institutes of Health with 2 or more liver biopsies wereassessed to attempt to better define the natural history and prognostic factors inchronic hepatitis C. Hepatic fibrosis was used as the primary marker for diseaseprogression. The study showed that 39% of patients had progression of fibrosisscores, 37% exhibited no change, and 24% improved. The best predictors of progres-sion of fibrosis in chronic hepatitis C were the extent of serum aminotransferaseelevations and the degree of hepatocellular necrosis and inflammation on liver.

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Figure 1. Alanine aminotransferase at initialbiopsy (fold elevation).

Long-term Lamivudine Therapy of Hepatitis B Reduces Hepatic Necroinflammatory Activityand Reverses FibrosisChronic hepatitis B, worldwide, causes more than one million deaths per year, largely from consequences of cirrhosis and livercancer. Until recently, the progression of liver fibrosis and cirrhosis were thought to be irreversible, but there are data showingsuccessful therapeutic intervention with one year of Lamivudine, an anti-viral agent that can result in histologic improvement.Lamivudine decreases markers of hepatic fibrosis in contrast to biopsies from patients who received placebo, suppresses HBVreplication, and results in a return to normal of serum alanine aminotransferase (ALT) levels, consistent with reduced liverinflammation and reduced hepatocyte injury. However, as hepatitis B e antigen (HBeAg) seroconversion rates increase withcontinued Lamivudine treatment, YMDD-variant, Lamivudine-resistant HBV emerge with greater frequency. Nevertheless, someYMDD-variant patients continue to derive clinical benefit even after emergence of YMDD variants. In this study, the histologicalimpact of longer-term treatment Lamivudine therapy was assessed in 63 HBV patients who had previously failed to achieve HBeAgseroconversion in phase III trials and were randomly assigned to 1 year Lamivudine treatment and followed another 2 years in anopen label protocol. At the end of 1 year, 57% of patients showed histologic improvement, whereas 38% exhibited no change.After 2 additional years of lamivudine, 60% showed no further histologic progression and 19% continued to improve. After all 3years of lamivudine treatment, only 11% of patients showed worsening of histology. Not unexpectedly, patients with YMDDvariants for �2 years were least likely to improve (36%). Thus, extended lamivudine therapy appears to reduce necroinflammatoryactivity and reverse fibrosis and cirrhosis (see figure) in most patients. Because YMDD variants tend to mitigate histologicresponses, additional therapies to maintain the histological benefit of treatment may be required.

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GASTROENTEROLOGY 2003;124:1–3

Figure 2. Improvement in bridging fibrosis during lamivudine therapy. Photographs before (pre) and after (post) therapy (patient with YMDD-variant HBV).

Insights Into the Genetic and Functional Significance ofNOD2 Variants Associated With Crohn’s DiseaseLast year, several groups found polymorphisms of the NOD2/CARD15 gene as beinghighly associated with the development of Crohn’s disease (CD). This gene is locatedprecisely in the IBD1 locus of chromosome 16, a site that had previously been linked toCD. However, how these mutations make individuals more susceptible to the develop-ment of CD remains a mystery, largely because of the limited understanding of what theencoded protein does. Because of a leucine rich repeat (LRR) domain located in itsC-terminal domain, a motif also found in toll-like receptors for endotoxin, investigatorshave speculated that NOD2 may function as an intracellular receptor or pattern recogni-tion molecule for bacteria-derived agents such as endotoxin or proteoglycan (PGN). Insupport of this notion, one of the mutations, L1007fsinsC, causes a frameshift andinsertion of a stop codon, resulting in truncation of the C-terminal domain of the encodedprotein. When expressed in experimental cells, a significantly decreased response toendotoxin is observed when compared with the wild type NOD2 gene. However, 2 otherNOD2 variants, R702W and G908R, do not appear to be encoded proteins that would befunctionally compromised. Moreover, all variants are found in the background of acommon P268S polymorphism. How this would contribute to disease susceptibility alsoremains undefined. This study now provides new information and insights into many ofthese questions. Genetic and case-control studies of the 3 major NOD2 variants in familialcases of CD in non-Jewish and Jewish populations were performed. In addition, thefunctional activity of the expressed NOD2 variants was assessed to better define theirroles in susceptibility for CD. The investigators found that G908R and L1007fsinsC, butnot R702W, were associated with disease susceptibility in Ashkenazi Jews. In addition,Ashkenazi Jews with CD had significantly higher allele frequency carriage of G908R, andlower carriage of R702W compared with non-Jewish whites with CD. Thus, R702W is notassociated with susceptibility to CD in Ashkenazi Jews. When the variants were expressedin experimental cell systems, G908R, R702W, and L1007fsinsC in the presence andabsence of P268S were defective in their response to bacterial LPS and PGN. This wasdetermined by measuring the activation of the downstream proinflammatory pathway,NF-�B (see figure). In contrast, P268S alone exhibited wild-type activity. The “loss offunction” observed with the aforementioned 3 variants is counter-intuitive, as this wouldseem to diminish the ability of cells to respond to proinflammatory signals. However,there is experimental evidence that supports a role for NF-�B activation in protecting thehost against invading pathogens. Diminished ability to activate this pathway wouldtherefore render the host more susceptible to not only pathogens, but also to normalcommensal bacterial flora. The latter is consistent with the observation that intestinalinflammation associated with animal models of inflammatory bowel diseases does notdevelop in the absence of bacterial flora. Much more must be learned before a clearconceptual picture can be formed. Nevertheless, the light at the end of the tunnel isclearly visible.

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Figure 3. NF-�B activation by wild-type andNOD2 variants in HEK293T cells. The differencein values between wild-type and all mutantNOD2 except P268S were significant (P �0.0005). Expression of wild-type and mutantNOD2 protein in equal amounts of cell extractsis shown on top.

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Figure 4. Livers were harvested 24 hours afterischemia-reperfusion from animals injected with(A ) Kupffer cells or (B) Ad.SOD1-infectedKupffer cells. Necrotic foci are designated byarrows.

Gene Altered Kupffer Cells for the Treatment of HepaticDiseasesThe development of gene therapy for liver disease has been intensely studied overthe past few years, but has been met with variable success. For example, severalexperimental studies have shown that adenovirus-mediated gene delivery to theliver of superoxide dismutase (SOD) can be protective in models of ischemia-reperfusion, early alcohol-induced liver injury, endotoxemia, and primary hepaticdysfunction after liver transplantation. SOD proteins detoxify superoxide radicalto hydrogen peroxide, which is rapidly degraded by catalase and glutathioneperoxidase. Overexpression of SOD may also down-regulate the proinflammatorynuclear factor kappa B (NF-�B) and activator protein-1 (AP-1) pathways. However,the problem with adenoviral gene delivery has been that the approach is indis-criminate, affecting many cell types—some not having substantial roles or impactin ameliorating reactive oxygen metabolite (ROM)-induced tissue injury. To cir-cumvent this problem, the investigators determined if repopulation or supple-ment of the liver with a specific cell type, transduced ex vivo with adenovirus,would be more effective in mitigating the injurious effects of ischemia-reperfu-sion. Hepatic Kupffer cells were selected for this purpose because they are theresident macrophages of the liver, although comprising less than 5% of the totalliver cell population. Furthermore, these cells, in contrast to hepatocytes, had notbeen extensively studied for this purpose. Kupffer cells play a major role indisease pathogenesis and in the inflammatory response through their productionof effector cytokines and ROMs. As scavenger cells, Kupffer cells also avidly takeup carbon particles, a property used by the investigators to assess the repopula-tion efficiency of injected cells in the host liver. Adenoviral-transduced Kupffercells encoding the Cu/Zn-SOD (Ad.SOD1) or the �-galactoside reporter gene(Ad.LacZ) were transplanted into recipient mice. Twenty-four hours later, a 70%hepatic ischemia-reperfusion was used to induce oxidative stress. Histologicassessments were performed 8 or 24 hours following the stress induction. Tenpercent to 20% of injected, carbon-labeled cells were found in the liver 24 hoursafter injection, representing �1% of all liver Kupffer cells. The repopulation rateof injected cells could be increased 4–10-fold by suppressing the immune systemwith a single dose of cyclosporine. The retention of injected Kupffer cells wasobserved up to 2 weeks after transplantation. As shown below, histologic injury(arrows) following ischemia-reperfusion was far less in livers repopulated withAd.SOD1-infected Kupffer cells (Figure B) than with Kupffer cells alone (FigureA). Thus, it seems that genetically altered Kupffer cells expressing SOD effectivelyprotect the liver not only by negating intracellular ROMs, but also inhibitingextracellular superoxides from other potential sources such as host Kupffer cellsand hepatocytes. These results are promising and support further investigationsand development of genetically altered Kupffer cells that can be used for thetreatment of inflammatory liver diseases.

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