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IBS: What It‘s Like

Aug. 20, 2009 -- People with IBS suffer pain and greatly reduced quality of life -- but what hurts most is when family, friends, and doctors don't think their suffering is legitimate.

Irritable bowel syndrome greatly disrupts the lives of people who suffer from it. But unlike other gut diseases, such as inflammatory bowel disease, IBS has no known physical cause. And it presents in different ways: with unpredictable diarrhea, with extreme constipation, or both.

"IBS is a disorder with no structural abnormality like ulcers or cancers so it takes on a lower level of legitimacy for doctors and patients," IBS expert Douglas A. Drossman, MD, tells WebMD. "People say it isn't real, and patients say, 'Well then, I must be crazy.'"

That would be a lot of crazy people. An estimated 7% of the U.S. population has IBS. In the U.K., the estimate is even higher: about 10% to 20% of the population.?

Lacking a yardstick, doctors have had a hard time judging IBS severity. The FDA recently ruled that new IBS treatments must be evaluated in terms of whether patients feel better. But what do patients say IBS is like?

"Nobody was looking at how patients characterize their illness," says Drossman, co-director of the University of North Carolina Center for Functional GI and Motility Disorders.

So Drossman and colleagues designed two studies. In one, the researchers enrolled 32 IBS patients in small focus groups. In the other, Drossman's team performed an international survey of nearly 2,000 people with an IBS diagnosis.

As expected, patients reported bowel symptoms:

  • 80% experienced pain.
  • Three-fourths of patients reported bowel difficulties.
  • Half of the patients with diarrhea-predominant or mixed-type IBS reported fecal incontinence.
  • Nausea, muscle pains, and, for those with diarrhea, gas, mucus in stool, and belching were common.
  • More than two-thirds of patients reported bloating.

But just as common -- and, as the focus group members reported, at least as bothersome -- was the way IBS affected their daily function, their thoughts, and their feelings.

"The impairment in their life was far greater than you would imagine -- their own sense of degradation and the stigma they experience from others," Drossman says. "Even when they are not symptomatic, the condition still pervades their life and how they think and feel about it."

IBS: Uncertainty, Loss Add to Suffering

Stigma from friends, family, and doctors was a dominant theme, Drossman found. Patients often said that nobody understood what they were going through or truly believed they were ill. This created as great a barrier to daily function as the disease itself.

Another major theme was uncertainty, a sense of having no control over the condition. Most patients end up greatly restricting their daily activities, which results in a sense of loss: loss of freedom, loss of spontaneity, and loss of social contacts.

日期:2009年8月21日 - 来自[Health News]栏目
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Breastfeeding and risk of inflammatory bowel disease

Eyal Klement

Koret School of Veterinary Medicine
The Hebrew University of Jerusalem
Pob, 12
Rehovot
Israel
E-mail: klement@agri.huji.ac.il

Shimon Reif

Pediatric Gastroenterology Unit
Dana Children's Hospital
Tel-Aviv Sourasky Medical Center
6 Weizman Street
Tel-Aviv 64239
Israel
E-mail: shemonr{at}tasmc.health.gov.il

Dear Sir:

The recently published study by Baron et al (1) certainly sheds new light on the association between breastfeeding and inflammatory bowel disease (IBD). This study, which was conducted with the use of excellent methods, fulfills the criteria for the best-quality category in the meta-analysis we recently published (2). Incorporating the results of this study into the pooled estimate calculation would diminish the significant results of protective breastfeeding on Crohn disease (CD) [Mantel-Haenszel odds ratio (ORMH): 0.62; 95% CI: 0.27, 1.43] and would not affect significantly the summary estimate of the protective association between breastfeeding and ulcerative colitis (ORMH: 0.62; 95% CI: 0.43, 0.91). However, more important than its effect on the pooled estimate was the high heterogeneity that is implied from its inclusion in the CD studies (P < 0.001, chi-square heterogeneity test). In our study, the effects found by all of the studies had high heterogeneity, but this may have been partly attributed to the differences in studies quality, with heterogeneity in the highest-quality studies that showed no statistical significance.

Inclusion of the study by Baron et al as one of the highest-quality studies implies high heterogeneity in this group as well. Why some studies show a significant protective effect of breastfeeding while others show no effect or even suggest that breastfeeding is a risk factor for CD is an enigma that may have several possible explanations. One explanation relates to the different genetic characteristics of the studies' populations. The highest-quality studies reviewed by us were all conducted in Sweden or North America; the study conducted by Baron et al was performed in northern France. It was previously shown that the genetic background of the population has a significant influence on the effect of some risk factors. A good example is the lack of effect of smoking on the development of CD in Jewish populations as opposed to other populations (3). The second explanation relates to the fact that CD may be regarded as a cluster of diseases that have the same manifestations but that are caused by different etiologies (4). Thus, the heterogeneic effect of breastfeeding on CD may relate to its different interactions with the yet unknown various etiologies of this disease. The third explanation suggested by Jantchou et al may also account for the discrepancy between this study's findings and those of previous studies; the components of breast milk in northern France may differ significantly from the components of breast milk in less industrialized areas.

Baron et al are the first investigators to implicate breastfeeding as a risk factor for CD. This, however, is not the only new finding of this study. The observed association between some vaccinations and CD in this study is also novel. This observation and the high rate of CD in this area suggest that the population of this study is unique either in its environmental exposure or in its genetic background. Thus, we agree with Jantchou et al that breastfeeding should not be discouraged, especially on the basis of one study. On the contrary, on the basis of our meta-analysis (which showed a protective effect of breastfeeding on IBD), the biologic plausibility of this association, and the experimental evidence gathered in animal experiments (5), we still believe that breastfeeding should be encouraged. Baron et al's study does, however, emphasize the need for further high-quality studies of other population types to fully understand the association between breastfeeding and IBD.

ACKNOWLEDGMENTS

Neither author had a financial or personal conflict of interest related to any of the topics discussed in this letter.

REFERENCES

  1. Baron S, Turck D, Leplat C, et al. Environmental risk factors in pediatric inflammatory bowel diseases: a population-based case-control study. Gut 2005;54:357–63.
  2. Klement E, Cohen RV, Boxman J, Joseph A, Reif S. Breastfeeding and risk of inflammatory bowel disease: a systematic review with meta-analysis. Am J Clin Nutr 2004;80:1342–52.
  3. Reif S, Klein I, Arber N, Gilat T. Lack of association between smoking and inflammatory bowel disease in Jewish patients in Israel. Gastroenterology 1995;108:1683–7.
  4. Shanahan F. Crohn's disease. Lancet 2002;359:62–9.
  5. Madsen KL, Fedorak RN, Tavernini MM, Doyle JS. Normal breast milk limits the development of colitis in IL-10 deficient mice. Inflamm Bowel Dis 2002;8:390–8.

日期:2008年12月28日 - 来自[2005年82卷第2期]栏目

Breastfeeding and risk of inflammatory bowel disease: a systematic review with meta-analysis

Eyal Klement, Regev V Cohen, Jonathan Boxman, Aviva Joseph and Shimon Reif

1 From the Koret School of Veterinary Medicine, the Hebrew University of Jerusalem, Rehovot, Israel (EK and AJ); the Center for Vaccine Development and Evaluation, Israel Defense Forces, Ramat-Gan, Israel (EK, RVC, and JB); the Pediatric Gastroenterology Unit, Dana Children's Hospital, Tel-Aviv Sourasky Medical Center, Tel-Aviv, Israel (SR); and the Sackler Faculty of Medicine, Tel-Aviv University, Tel Aviv, Israel (SR)

2 Supported by the Tel-Aviv Sourasky Medical Center.

3 Reprints not available. Address correspondence to E Klement, Koret School of Veterinary Medicine, The Hebrew University of Jerusalem, Pob 12, Rehovot, Israel. E-mail: klement{at}agri.huji.ac.il.


ABSTRACT  
Background: It has long been believed that breastfeeding provides protection against ulcerative colitis and Crohn disease. Studies designated to test this hypothesis were conducted without reaching conclusive results.

Objective: The aim of this meta-analysis was to examine the role of breastfeeding in preventing inflammatory bowel disease and to summarize the evidence gathered about this subject.

Design: A meta-analysis was performed on 17 relevant articles that were found by using MEDLINE, EMBASE, the Internet, and articles' references. The publications were fully reviewed and divided, on the basis of their quality, into 3 groups.

Results: Studies showed heterogeneous results. The pooled odds ratios of all the 17 reviewed studies, calculated according to the random-effects model, were 0.67 (95% CI: 0.52, 0.86) for Crohn disease and 0.77 (0.61, 0.96) for ulcerative colitis. However, only 4 studies for Crohn disease and 4 for ulcerative colitis were eventually included in the highest quality group. In this group, the pooled odds ratio was 0.45 (0.26, 0.79) for Crohn disease and 0.56 (0.38, 0.81) for ulcerative colitis.

Conclusions: The results of this meta-analysis support the hypothesis that breastfeeding is associated with lower risks of Crohn disease and ulcerative colitis. However, because only a few studies were graded to be of high quality, we suggest that further research, conducted with good methodology and large sample sizes, should be carried out to strengthen the validity of these observations.

Key Words: Crohn disease • ulcerative colitis • breastfeeding • meta-analysis • epidemiology


INTRODUCTION  
Despite intensive investigation into the cause and pathogenesis of inflammatory bowel disease (IBD), its pathogenic mechanism has yet to be elucidated. Several studies indicate that a genetic basis exists for these diseases and showed a correlation between disease prevalence and the presence of specific genomic markers (1, 2). Whatever role genetic loci might play in conferring susceptibility to IBD, studies of identical twins [in which only 45% of identical twin pairs are concordant for Crohn disease (CD)] suggest that additional environmental factors are necessary for the development of this disease (3).

To determine which environmental factors contribute to the development of CD or ulcerative colitis (UC), numerous epidemiologic studies were performed (4-7). Factors such as smoking (8) and use of oral contraceptives (9) were meta-analyzed to determine their role on the risk of IBD development.

In this meta-analysis, we evaluate another factor, ie, the effect of breastfeeding, on the later development of UC and CD. The reasoning is 3-fold. First, breastfeeding protects against many immune-mediated diseases such as bronchial asthma (10), atopic dermatitis (11), allergic rhinitis (12), and type 1 diabetes mellitus (13). This effect is attributed to the immunomodulatory properties of human milk. From here we hypothesized that if the immunomodulatory effect of breastfeeding offers protection against these diseases, it is plausible to assume similar protection with regard to UC and CD. Second, the infant is exposed to human milk while developing an immune system, which seems to be important in procuring oral tolerance to specific microflora and food antigens, which can play a role in the pathogenesis of IBD (14). Third, breast-milk feeding was shown to limit the development of colitis in mice deficient for interleukin 10. This finding was explained by the change of intestinal flora of the developing mice from pathogenic bacteria to nonadherent bacteria as a result of oligosaccharides found in the milk that stimulate Bifidobacterium and Lactobacillus growth (15). A change in proinflammatory cytokine secretion can also be offered as an explanation (16).

Yet, most of the findings about the beneficial effect of breastfeeding derive from epidemiologic studies. Indeed, some studies found breastfeeding to be protective against UC or CD (17-22). However, most of the studies failed to achieve statistically significant results or found no association at all (4, 5, 23-30). Meta-analyses of observational studies present particular challenges because of inherent biases and differences in study designs (31). Thus, this meta-analysis, which is reported here according to the "proposal for reporting" published previously by Stroup et al (32), does not presume to provide a precise estimate of the association between breastfeeding and IBD but rather attempts to either support or weaken this hypothesis and to summarize the evidence that was gathered about this subject.


SUBJECTS AND METHODS  
Search strategy
A meta-analysis was performed on the basis of a computerized search of English-written epidemiologic (case-control or cohort) studies of the association between breastfeeding and UC or CD listed in the MEDLINE (National Library of Medicine, Bethesda, MD) and EMBASE (Elsevier Publishers BV, Amsterdam) data banks before November 2003. Specifically, a literature search was performed (by the investigators with the aid of a professional librarian) by using the index terms ulcerative colitis, Crohn disease, inflammatory bowel disease, bottle-feeding, breastfeeding, infant nutrition, perinatal, and milk in various combinations.

From the abstracts identified in the database search, 14 described relevant epidemiologic studies and were selected for full review (4, 5, 17-26, 28, 29). By reviewing the references of these articles, 2 additional studies were discovered (27, 30). An Internet search was conducted as well by using the same terms used in the database search to locate published studies not registered in MEDLINE or EMBASE. This search recovered one additional study (33). Thus, a total of 17 studies were fully reviewed in this meta-analysis.

We selected all studies in which the primary or secondary goal was to evaluate the association between breastfeeding and UC or CD as separate entities. These articles were independently reviewed by the authors (EK and RVC) by using a standardized report form. The articles were graded according to predefined guidelines that will be further detailed. Discrepancies were resolved in conferences.

A primary prerequisite for the inclusion of studies in the meta-analysis was the presence of a control group, which could be formed by population controls, by hospital inpatients, or by outpatients who did not suffer from IBD or other chronic diseases that might be related to lack of breastfeeding. Studies, in which the control subjects were recruited to the study by the case subjects, with no supervision of the investigators or coinvestigators, were considered to be of lower quality, because this recruitment method could inflict a serious selection bias. To further deal with the problem of selection bias, we categorized the studies according to the percentage of subjects willing to participate from the total number of subjects approached by the investigators (response rate); ie, articles in which the investigators did not detail response rates or recorded response rates of <80% in either the case or the control subjects were ranked as having a lower quality.

Studies were categorized according to the age of diagnosis, from birth to adolescence (0–18 y) or adults (>18 y). To decrease information bias, studies of adults, which did not specifically note that the classification of breastfeeding was based on information collected from parents or another older relatives of the participating subjects, were classified as low quality. This classification was not a requirement in pediatric studies (provided that data were collected during childhood), because it was assumed that the information was obtained from a parent or an older relative. No restrictions were imposed on the method in which this information was obtained (mail, interview, or clinical files).

No restrictions were imposed on the method of diagnosis of CD and UC. As long as the diagnosis was confirmed by a physician, we assumed that well-trained specialists diagnosed the cases. Otherwise, the study was assigned to the low quality group.

Breastfeeding was defined as either exclusive or nonexclusive breastfeeding for any given duration. Accordingly, no breastfeeding was defined as nonexclusive or exclusive bottle-feeding from birth. When odds ratios (ORs) were calculated for both definitions, we used the OR for "not exclusively breastfed" for any duration compared with "exclusively bottle-fed from birth" for the calculation of the pooled estimate. Duration of breastfeeding was sought and documented.

We did not exclude studies in which the investigators stated that the correlation between breastfeeding and CD or UC was insignificant and, therefore, presented neither OR nor crude data. Instead, the OR was estimated to be 1, and the CI was calculated by assuming participation of all subjects in the study, and by arbitrarily assigning them a rate of 20% bottle-feeding. In this manner we maintained a conservative attitude in which it was more difficult to spuriously reject the null hypothesis of no relation between breastfeeding and IBD.

To sum up this section, studies were graded for quality levels as follows. For grade 1 (best quality), case and control subjects were recruited by the investigators or coinvestigators. Diagnosis was always confirmed by a physician, and breastfeeding information was always confirmed by patients' mothers or other older close relative (as previously mentioned, this was not a requirement in pediatric studies). Response rate is mentioned in the article and is 80% for both case and control subjects. Grade 2 was the same as grade 1, except that the response rate is not mentioned or is <80%. For grade 3 (lowest quality), either breastfeeding information was not provided by the mother or a close relative of the patient, diagnosis was not confirmed by a physician, or control subjects were recruited to the study by the patients.

Statistical analysis
The pooled OR and its confidence limits were calculated by using the DerSimonian and Laird method (34), which is based on the random-effects model. The fixed-effects model–based OR, calculated as previously described by Greenland (35), is also presented. In both methods, the weight of each study depends on the inverse of the variance of log OR, which is estimated by the 95% CI of each study.

Heterogeneity of the studies was calculated with the following formula:

RESULTS  
Of the 17 studies that were included in the meta-analysis presented here, 11 investigated both UC and CD, 3 investigated UC alone, and 3 investigated CD alone. Together a total of 2577 patients with UC and 3551 control subjects and 3190 patients with CD and 4026 control subjects were studied. The studies are summarized for UC (Table 1) and for CD (Table 2). Four studies did not present the exact findings about breastfeeding (4, 23, 27, 30). Instead, they merely claimed that the OR was close to unity. The OR in those studies was estimated as 1, and the CI was calculated as described in "Subjects and Methods." An exception was made for the study of Gilat et al (4), because this researcher calculated the OR from the number of discordant pairs of each case subject and the matched control subject. Thus, the CI for that study was calculated with half of the control subjects mentioned in the article (302 pairs of control and case subjects for CD and 197 pairs for UC).


View this table:
TABLE 1. Case-control studies testing the association between breastfeeding and ulcerative colitis1

 

View this table:
TABLE 2. Case-control studies testing the association between breastfeeding and Crohn disease1

 
Studies were graded in accordance with the criteria mentioned in Subjects and Methods (Table 3). Overall, only 4 studies (consisting of 397 patients with UC and 766 control subjects and 583 patients with CD and 876 control subjects) were included in the highest quality groups for either CD (18, 20, 21, 24) or UC (17, 21, 24, 26). The pooled ORs and 95% CIs (calculated according to the random-effects model) for these studies were 0.56 (95% CI: 0.38, 0.81) for UC and 0.45 (95% CI: 0.26, 0.79) for CD. ORs for the 8 UC studies and the 7 CD studies graded in quality groups 1 and 2 were 0.61 (95% CI: 0.46, 0.83) and 0.55 (95% CI: 0.34, 0.87), respectively. When all studies were included in the pooled estimate, the random-effects model OR was 0.77 (95% CI: 0.61, 0.96) for UC and 0.67 (95% CI: 0.52, 0.86) for CD (Table 4, Figures 1 and 2). Thus, the protective effect of breastfeeding against both diseases remained statistically significant for all calculated pooled ORs. However, the results for both diseases appeared to be heterogeneous, primarily after adding the studies of lower quality (Pheterogeneity < 0.001 for CD and Pheterogeneity = 0.002 for UC). Heterogeneity was further explored by dividing the studies according to various characteristics related to population differences, exposure definition, and methodologic issues and by calculating summary estimates of the OR for the association of UC and CD with breastfeeding for each group (Table 5).


View this table:
TABLE 3. Quality grading of studies1

 

View this table:
TABLE 4. Pooled estimates for correlation between breastfeeding and risk of ulcerative colitis (UC) and Crohn disease (CD)1

 

View larger version (19K):
FIGURE 1.. Association between breastfeeding and ulcerative colitis. The x axis represents the odds ratio (OR) depicted on a logarithmic scale. ORs are represented by small white squares, and 95% CIs are represented by lines. Pooled ORs for group 1, groups 1 and 2, and all studies were calculated by using the random-effects model whenever possible. Otherwise, calculation was performed according to the fixed-effects model. Studies in quality groups 1 (highest) and 2 are organized according to date of publication. Studies in quality group 3 are organized according to decreasing quality.

 

View larger version (18K):
FIGURE 2.. Association between breastfeeding and Crohn disease. The x axis represents the odds ratio (OR) depicted on a logarithmic scale. ORs are represented by small white squares, and 95% CIs are represented by lines. Pooled ORs for group 1, groups 1 and 2, and all studies were calculated by using the random-effects model whenever possible. Otherwise, calculation was performed according to the fixed-effects model. Studies in quality groups 1 (highest) and 2 are organized according to date of publication. Studies in quality group 3 are organized according to decreasing quality.

 

View this table:
TABLE 5. Summary estimates of the odds ratios (ORs) for the association of ulcerative colitis (UC) and Crohn disease (CD) with breastfeeding according to study characteristics1

 
Exploration of the possibility for publication bias by funnel plots (Figures 3 and 4) indicated a possible publication bias in the studies for CD. P value for a skewed funnel plot, calculated by the regression asymmetry test, was 0.23 for the UC studies and 0.003 for CD studies.


View larger version (9K):
FIGURE 3.. Funnel plot of precision estimates [calculated by using the inverse of the SE of ln(odds ratio); ie, the higher the estimate, the more precise the study] from studies that explored the association between breastfeeding and ulcerative colitis against their odds ratio. The vertical dashed line is the summary estimate of the odds ratio for all studies.

 

View larger version (9K):
FIGURE 4.. Funnel plot of precision estimates [calculated by using the inverse of the SE of ln(odds ratio); ie, the higher the estimate, the more precise the study] from studies that explored the association between breastfeeding and Crohn disease against their odds ratio. The vertical dashed line is the summary estimate of the odds ratio for all studies.

 

DISCUSSION  
The overall pooled OR of this meta-analysis demonstrates that breastfeeding has a statistically significant protective role against UC and an even greater role against CD. Because exclusion of studies is subject to criticism as a result of influence of former beliefs, we did not exclude studies but rather calculated separate pooled ORs for the best quality studies, for best and intermediate quality studies, and for all studies. The protective effect of breastfeeding against both diseases remained statistically significant for all calculated pooled ORs.

The test for heterogeneity, however, was statistically significant for both UC and CD. This finding can be partly explained by differences in the case subjects' age (children and adolescents compared with adults), control subjects characteristics (hospital based compared with population based), matching variables, and the exact definition of breastfeeding (Table 5). Heterogeneity of the studies can also be attributed to the differences in the quality of the studies, because the results become more heterogeneous when studies with lower quality are included. These differences can be due to biased results of these studies. Of special concern is the study of Thompson et al (28). That study incorporated hundreds of CD and UC case subjects in its investigation. Thus, despite methodologic problems, which made it highly prone to various biases, it had the highest influence on the pooled OR and the heterogeneity of the studies.

All but 2 studies recovered in this meta-analysis were retrospective case-control studies. That type of study constitutes a drawback because case-control studies are subject to misclassification as a result of recall bias and to selection bias. It is, however, difficult to conduct a prospective study that tests the relation between IBD and breastfeeding, because the lag between breastfeeding and the development of IBD is substantial. In 2 of the studies (24, 29), however, data about breastfeeding was collected from medical records and, thus, did not rely on the recall of mothers. Nevertheless, these data were recorded merely a few days after labor. The implications of this data collection will be further discussed later in this article.

Selection bias is potentially present in all of the reviewed studies, because no study described a comparison between subjects participating in the study and subjects excluded or not willing to participate. We set a low rank to studies in which the case subjects were instructed to obtain replies to the control subject's questionnaire by themselves, because the process of selecting and questioning the control subjects by the case subjects without the supervision of the investigators is, in our opinion, highly prone to selection bias. To further minimize the possibility of selection bias, we calculated a distinct pooled OR for the studies in which response rate was specified and was 80%.

Another potential source of bias is related to imprecise recall of breastfeeding. Thus, studies in which information about breastfeeding was not provided by the mother of the patient or an older close relative were assigned to the lowest quality group. Data provided by mothers, theoretically, could also be prone to recall bias, when one considers the prolonged lag time elapsing from infancy to development of the disease. However, we tend to think that this kind of bias was not an important problem in those studies. Our thought is supported by a study conducted by Launer et al (38) that demonstrated a high accuracy in the recall of breastfeeding duration at 18 mo after birth. Although in the reviewed studies, breastfeeding practices were inquired years after birth, the information that the mothers were asked to obtain was simple (breastfeeding, yes or no); hence, we believe it was accurate. F urthermore, our thoughts are supported by Bergstrand and Hellers (18), who mentioned in their study that "most living mothers were remarkably exact in their information regarding breastfeeding." Nevertheless, duration of breastfeeding was not documented in most of the studies. In light of the dose–response effect found for both CD and UC by Rigas et al (21) and for CD by Bergstrand and Hellers (18), we think that these missing data are probably of high importance.

As was mentioned previously in this report, most studies did not define duration and exclusivity of breastfeeding precisely, and it was not clear whether exclusive breastfeeding was being compared with nonexclusive breastfeeding or whether nonexclusive breastfeeding was being compared with exclusive bottle-feeding. Thus, it cannot be stated whether the absence of breastfeeding is the risk factor for IBD or the presence of bottle-feeding. It is also worth noting that this inadequate definition of breastfeeding duration and exclusivity can lead to nondifferential misclassification, which might obscure the protective association between breastfeeding and IBD (39). A good example for this type of nondifferential misclassification can be drawn from the study of Ekbom et al (24). That study, although conducted with almost perfect methodology, defined breastfeeding according to medical records, which merely documented breastfeeding status in the first few days after labor. It is possible that a significant portion of mothers that were assigned in this study as exclusive breastfeeders moved to partial breastfeeding or totally gave up breastfeeding a few days later. Thus, the lack of association found in that study can actually be an underestimation of an existing association that would have been discovered had breastfeeding status been recorded for a longer duration.

Confounding was not treated statistically in most of the studies. Confounding can potentially bias the results, but the few studies in which adjusted and crude OR were calculated (17, 19-21) showed little difference between the adjusted OR that controls for various confounders (diarrheal disease during infancy, sex, age, race, birthplace, sibship size, birth order, maternal age, smoking, and the use of oral contraceptives) and the crude OR. We, therefore, believe that the lack of adjustment to confounders in most of the studies probably did not lead to a significant bias of the results.

Most of the studies matched case with control subjects for sex and age. Some studies also matched other variables (region or country of birth, residential neighborhood), and in some matching was inherent in the control subject selection (neighbors, acquaintances, siblings). However, only in a few of the studies was matching statistically treated through conditional logistic regression or McNemar test. The lack of this statistical treatment in most studies can lead to bias of the OR toward unity (no relation); thus, the result presented in these studies might lead to underestimation of the protective association between breastfeeding and IBD (40).

Finally, publication bias, which results from a tendency to publish only significant data, constitutes a potential problem in every meta-analysis. The funnel plots of both CD and UC show that most of the studies have about the same precision. The funnel plot of the CD studies has an asymmetric appearance. This asymmetry is supported by the low P value (0.003) result in the test for skewed funnel plot for CD. In meta-analysis of observational studies, however, larger sample sizes do not necessarily indicate a higher validity (36). For example, it can be seen that the distorted shape of the plot is caused primarily by the study of Thompson et al (28), which, as was previously outlined, has the largest sample size but suffers from some important methodologic problems. In addition, most of the studies were performed for both CD and UC; thus, it is unlikely that publication bias exists for one but not for the other. Nevertheless, publication bias cannot be ruled out in this meta-analysis.

In conclusion, our study supports the hypothesis that breastfeeding provides protection against CD and UC development. However, it does not presume to provide an exact estimate of the OR for a certain definition of breastfeeding, but rather to provide a rough measure of the relation between breastfeeding and the risk of IBD. Our thought is that, because of a result of nondifferential misclassification, which, as we stated earlier, is inherent in many of the studies reviewed, the actual effect of breastfeeding is higher than the one estimated here. Furthermore, most of the best quality studies showed a significant protective effect. Nevertheless, because the effect found was minor and inconsistent, our study should not be regarded as final proof of this hypothesis. We think that a well-performed, documented prospective study should be held. Studies of high-risk populations that will specifically address the influence of breastfeeding (as well as its duration) are of particular importance. Because there is a clear genetic predisposition to IBD, these populations should probably be composed of families that include persons who already have IBD [such as the studies conducted by Koletzko et al (20, 26)]. That kind of study will enable the generation of breastfeeding recommendations to mothers of infants with a history of IBD in first-degree relatives.


ACKNOWLEDGMENTS  
We thank Rina Zakheim for her assistance in the database search for articles.

EK designed the study, conducted the database search, reviewed the articles, analyzed the data, and wrote the manuscript. RVC reviewed the articles, helped in designing the study and analyzing it, and reviewed the manuscript; JB reviewed the manuscript; AJ reviewed the manuscript; SR reviewed the articles and manuscript. None of the authors had any financial or personal conflicts of interest in any of the subjects discussed in this article.


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  16. Madsen KL, Fedorak RN, Tavernini MM, Doyle JS. Normal breast milk limits the development of colitis in IL-10 deficient mice. Inflamm Bowel Dis 2002;8:390-8.
  17. Acheson ED, Truelove SC. Early weaning in the aetiology of ulcerative colitis. A study of feeding in infancy in cases and controls. BMJ 1961;929-33.
  18. Bergstrand O, Hellers G. Breast-feeding during infancy in patients who later develop Crohn's disease. Scand J Gastroenterol 1983;18:903-6.
  19. Corrao G, Tragnone A, Caprilli R, et al. Risk of inflammatory bowel disease attributable to smoking, oral contraception and breastfeeding in Italy: a nationwide case-control study. Cooperative Investigators of the Italian Group for the Study of the Colon and the Rectum (GISC). Int J Epidemiol 1998;27:397-404.
  20. Koletzko S, Sherman P, Corey M, Griffiths A, Smith C. Role of infant feeding practices in development of Crohn's disease in childhood. BMJ 1989;298:1617-8.
  21. Rigas A, Rigas B, Glassman M, et al. Breast-feeding and maternal smoking in the etiology of Crohn's disease and ulcerative colitis in childhood. Ann Epidemiol. 1993;3:387-92.
  22. Whorwell PJ, Holdstock G, Whorwell GM, Wright R. Bottle feeding, early gastroenteritis, and inflammatory bowel disease. Br Med J 1979;1:382.
  23. Dietary and other risk factors of ulcerative colitis. A case-control study in Japan. Epidemiology Group of the Research Committee of Inflammatory Bowel Disease in Japan. J Clin Gastroenterol 1994;19:166-71.
  24. Ekbom A, Adami HO, Helmick CG, Jonzon A, Zack MM. Perinatal risk factors for inflammatory bowel disease: a case-control study. Am J Epidemiol 1990;132:1111-9.
  25. Gruber M, Marshall JR, Zielezny M, Lance P. A case-control study to examine the influence of maternal perinatal behaviors on the incidence of Crohn's disease. Gastroenterol Nurs 1996;19:53-9.
  26. Koletzko S, Griffiths A, Corey M, Smith C, Sherman P. Infant feeding practices and ulcerative colitis in childhood. BMJ 1991;302:1580-1.
  27. Persson PG, Leijonmarck CE, Bernell O, Hellers G, Ahlbom A. Risk indicators for inflammatory bowel disease. Int J Epidemiol 1993;22:268-72.
  28. Thompson NP, Pounder RE, Wakefield AJ. Perinatal and childhood risk factors for inflammatory bowel disease: a case-control study. Eur J Gastroenterol Hepatol 1995;7:385-90.
  29. Thompson NP, Montgomery SM, Wadsworth ME, Pounder RE, Wakefield AJ. Early determinants of inflammatory bowel disease: use of two national longitudinal birth cohorts. Eur J Gastroenterol Hepatol 2000;12:25-30.
  30. Wurzelmann JI, Lyles CM, Sandler RS. Childhood infections and the risk of inflammatory bowel disease. Dig Dis Sci 1994;39:555-60.
  31. Huston P, Naylor CD. Health services research: reporting on studies using secondary data sources. Can Med Assoc J 1996;155:1697-709.
  32. Stroup DF, Berlin JA, Morton SC, et al. Meta-analysis of observational studies in epidemiology: a proposal for reporting. Meta-analysis Of Observational Studies in Epidemiology (MOOSE) group. JAMA 2000;283:2008-12.
  33. Urashima H, Ohmori I, Shiraki K. Epidemiological survey on chronic inflammatory bowel disease developed during childhood in japan, and a case-control study on nutrition during infancy. Yonago Acta Medica 1999;42:95-102.
  34. DerSimonian R, Laird N. Meta-analysis in clinical trials. Control Clin Trials 1986;7:177-88.
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  36. Egger M, Smith GD, Schneider M, Minder C. Bias in meta-analysis detected by a simple graphical test. BMJ 1997;315:629-34.
  37. Abramson JH, Gahlinger PM. Computer programs for epidemiologists: PEPI version 4.0. Salt Lake City: Sagebrush Press, 2001.
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  39. Greenland S. Sensitivity analysis and external adjustment. In: Rothman KJ, Greenland S, eds. Modern epidemiology. 2nd ed. Philadelphia: Lippincott Williams & Wilkins, 1998:343-58.
  40. Rothman KJ, Greenland S. Matching. In: Rothman KJ, Greenland S, eds. Modern epidemiology. 2nd ed. Philadelphia: Lippincott Williams & Wilkins, 1998:147-62.
Received for publication February 27, 2004. Accepted for publication June 21, 2004.


日期:2008年12月28日 - 来自[2004年80卷第5期]栏目
循环ads

Polyunsaturated fatty acids and inflammatory bowel disease

Andrea Belluzzi, Stefano Boschi, Corrado Brignola, Alessandra Munarini, Giulio Cariani and Federico Miglio

1 From the Department of First Aid and Emergency Medicine and the Department of Clinical Pharmacology, S Orsola Hospital, Bologna, Italy, and Civil Hospital, Recanati, Italy.

2 Address reprint requests to A Belluzzi, Via Vizzani, 36, 40138 Bologna, Italy. E-mail: Belluzzi{at}altavista.net.


ABSTRACT  
The rationale for supplementation with n–3 fatty acids to promote the health of the gastrointestinal tract lies in the antiinflammatory effects of these lipid compounds. The first evidence of the importance of dietary intake of n–3 polyunsaturated fatty acids was derived from epidemiologic observations of the low incidence of inflammatory bowel disease in Eskimos. The aim of this paper was to briefly review the literature on the use of n–3 fatty acids in inflammatory bowel disease (ulcerative colitis and Crohn disease), the results of which are controversial. The discrepancies between studies may reside in the different study designs used as well as in the various formulations and dosages used, some of which may lead to a high incidence of side effects. Choosing a formulation that lowers the incidence of side effects, selecting patients carefully, and paying strict attention to experimental design are critical when investigating further the therapeutic potential of these lipids in inflammatory bowel disease.

Key Words: Polyunsaturated fatty acids • fish oil • Crohn disease • ulcerative colitis • inflammatory bowel disease • n–3 fatty acids • review


INTRODUCTION  
n–3 Fatty acids have been suggested as a treatment for various chronic inflammatory conditions, including inflammatory bowel disease, because of their antiinflammatory properties. This effect may be mediated by a lower production of the most powerful arachidonic acid metabolite, leukotriene B4 (1), which is elevated in the inflamed intestinal mucosa (2), as well as by a reduction in another arachidonic acid metabolite, thromboxane A2, which is released even in uninflamed mucosa in inflammatory bowel disease—an early abnormality of special pathogenic significance (3). It has also been shown that n–3 fatty acids can inhibit interleukin 1ß, inhibit tumor necrosis factor production (4), and possibly act as free radical scavengers (5). Moreover, multifocal gastrointestinal infarctions have been suggested as one of the first pathogenic steps in Crohn disease (CD) (6), which suggests that platelets and possibly the powerful platelet aggregator thromboxane A2 may play a pivotal role in this process (7). Treatment with n–3 fatty acids has been shown to decrease platelet responsiveness in patients with inflammatory bowel disease (8).


CLINICAL STUDIES  
In an uncontrolled study, McCall et al (9) treated 6 patients with active ulcerative colitis (UC) with 3–4 g eicosapentaenoic acid (EPA)/d (16–24 capsules of fish oil as triacylglycerol) for 12 wk and reported a significant improvement in symptoms and histologic appearance, along with a significant decrease in leukotriene B4 neutrophil production. In 1990, Salomon et al (10), in another uncontrolled study, treated 10 UC patients who were refractory to conventional treatment (steroids and salicylates) with 2.7 g EPA/d and 1.8 g docosahexaenoic acid (DHA)/d (15 capsules of fish oil as triacylglycerol) for 8 wk. All of the measures of disease activity (whether the disease was active or in remission) improved significantly in 7 patients; in 3 patients there was little or no improvement.

Lorenz et al (11) treated 29 CD patients in different stages of clinical disease activity and 10 UC patients with active disease in a 7-mo, controlled, crossover trial. Patients received 3.2 g n–3 fatty acids/d and olive oil as a placebo while conventional treatment was continued. The washout period was 1 mo. Encouraging positive results were obtained in the UC patients, whereas the activity score of the disease did not improve in the CD patients.

In 1992, Hawthorne et al (12) published the first large placebo-controlled study of n–3 fatty acids and inflammatory bowel disease. In this study, 96 UC patients in different activity stages of the disease were enrolled and assigned to receive either 4.5 g EPA/d as triacylglycerol (treatment group) or olive oil (placebo group) for 1 y. Conventional treatment was allowed to continue in both groups. In patients with active disease at entry, a significant steroid-sparing effect of fish oil was shown, but fish oil did not affect the predicted endpoint, ie, the prevention of clinical relapse in the group of patients enrolled while their disease was in remission. Remarkably, leukotriene B4 production in stimulated neutrophils was reduced by >50% in the treatment group.

Stenson et al (13) carried out a randomized, double-blind, placebo-controlled crossover study with 5.4 g n–3 fatty acids as triacylglycerol (18 capsules daily) or olive oil as the placebo in 24 patients with active UC. Patients received the treatment for 4 mo followed by a washout period lasting 1 mo. In this study, fish-oil treatment induced significant body weight gain, significantly improved the histology score, and reduced leukotriene B4 production in rectal dialysates by 60%. No significant steroid-sparing effect of fish oil compared with placebo was found, however, and the endoscopy score was not significantly improved (P = 0.06).

Aslan and Triadafilopoulos (14) carried out a similar placebo-controlled, crossover trial by giving 4.2 g n–3 fatty acids/d or corn oil as the placebo. Seventeen patients with active UC received the treatment for 3 mo followed by a washout period lasting 2 mo. A steroid-sparing effect of n–3 fatty acids was observed in 72% of patients, and in 56% of patients the activity score of the disease improved significantly. Improvements in the histology score were not significant.

Matè et al (15) reported preliminary data on a group of 38 CD patients in clinical and laboratory remission characterized by a Crohn's Disease Activity Index <150. The patients were randomly assigned to receive either a diet enriched with fish (from 100 to 250 g fish/d) or a regular diet for 2 y. Symptomatic remission of the disease was longer in those who received the enriched diet.

More recently, Loeschke et al (16) presented data from a placebo-controlled trial of prevention of UC relapse. Sixty-four patients with disease in remission were randomly assigned to receive 5.1 g n–3 fatty acids as ethyl esters or corn oil as a placebo daily for 2 y. Ongoing treatment with 5-aminosalicylic acid was allowed for 3 mo. Interestingly, after 3 mo, the fish-oil group had experienced fewer relapses than did the placebo group (P < 0.02) but this beneficial effect did not last until the end of the study (2 y). We can speculate that the fish oil and the 5-aminosalicylic acid had synergetic effects; however, we cannot rule out the possibility that compliance decreased over time in the fish-oil group, which would have affected the clinical outcome.

Lorenz-Meyer et al (17) published data from a large, placebo-controlled trial in which 204 CD patients recovering from an acute relapse were randomly assigned to receive n–3 fatty acids (n = 70; 5.1 g/d as ethyl esters), a diet poor in carbohydrate (n = 69), or a placebo (n = 65) for 1 y. Low doses of prednisolone were allowed for 8 wk. In an intent-to-treat analysis, none of the treatments prevented clinical flare-ups, but in the per-protocol analysis the diet poor in carbohydrate (20 dropouts) seemed to be effective (P < 0.05). It is important to stress that >60% of patients treated with steroids after an acute relapse have further relapses at 6 mo, after steroid treatment is suspended (18).

The crossover design of most of these studies, with short washout periods between the 2 treatments, did not allow for a complete displacement of the extra n–3 fatty acids from the membrane, which may have interfered with the final results. Endres et al (4) showed that the inhibition of cytokine production lasts for >10 wk after the suspension of supplementation with n–3 fatty acids.


PLACEBO EFFECTS  
The choice of placebo is another crucial consideration in studies of n–3 fatty acids and inflammatory bowel disease because it has been shown that olive oil exerts some important beneficial effects, such as free radical scavenging (19) and inhibition of eicosanoid formation (20). Even corn oil is a rich source of linoleic acid, an essential n–6 fatty that is desaturated and elongated to dihomo--linolenic acid, a precursor of the 1-series of prostanoids that may have antiinflammatory properties in many chronic inflammatory disorders (21).

Moreover, in many of the studies in which fish oil was used, patient compliance was poor (22–24). This poor compliance was caused by the poor palatability of the diets and by minor but annoying side effects such as halitosis, belching, and diarrhea resulting from the high daily intake of fish-oil preparations, which is necessary for satisfactory intestinal absorption and incorporation of n–3 fatty acids into membranes.

We carried out a study of patient tolerance in a group of CD patients with a new n–3 fatty acid preparation that consisted of 500 mg enteric-coated (gastric resistant) capsules of EPA (40%) and DHA (20%) as a free fatty acid mixture. This was compared with a traditional preparation of n–3 fatty acids as triacylglycerol. In addition to patient tolerance, we evaluated the incorporation of the n–3 fatty acids into phospholipids in plasma and red blood cell membranes. The new preparation showed the best incorporation of EPA and DHA in red blood cell phospholipid membranes and had no associated side effects (25).

We also performed a 1-y, double-blind, placebo-controlled study to investigate the effect of this new formulation in 78 CD patients who had a high risk of relapse (26). Patients with a well-established diagnosis of CD in clinical remission were evaluated for eligibility for this study according to the Crohn's Disease Activity Index. This index incorporates 8 items—the daily number of liquid or very soft stools, abdominal pain, general well-being, extraintestinal manifestations of CD, use of opiates to treat diarrhea, abdominal mass, hematocrit, and body weight—to yield a composite score ranging from 0 to 600. Higher scores indicate more disease activity. Patients with scores of 150 are considered to have inactive disease. The main eligibility criteria for our study were a Crohn's Disease Activity Index <150 for 3 mo but <2 y and 1 of the following: serum 1 acid glycoprotein >1.3 g/L (reference range: <1.2 mg/L), serum 2 globulin >9 g/L (reference range: <8 g/L), erythrocyte sedimentation rate >40 mm/h (reference range: <20 mm/h). Patients received either 9 capsules containing a total of 2.7 g n–3 fatty acids or 9 placebo capsules (a mixture of capric and caprylic acids) daily. A special coating protected the capsules against acidity for 30 min. Of the 39 patients in the n–3 fatty acid group, 11 (28%) had relapses, 4 dropped out because of diarrhea, and 1 withdrew from the study. In contrast, of the 39 patients in the placebo group, 27 (69%) had relapses, 1 dropped out because of diarrhea, and 1 withdrew from the study (P < 0.001). After 1 y, 23 patients (59%) in the n–3 fatty acid group remained in remission compared with 10 (26%) in the placebo group (P = 0.003). The Kaplan-Meier life-table curves for patients remaining in clinical remission are shown in Figure 1. Logistic regression analysis indicated that only n–3 fatty acids and not sex, age, previous surgery, duration of disease, or smoking status affected the likelihood of relapse (26).


View larger version (18K):
FIGURE 1. . Kaplan-Meier life-table curves showing the percentage of patients remaining in clinical remission during the treatment period. Reprinted from reference 26. Copyright 1996 Massachusetts Medical Society. All rights reserved.

 
Only one placebo-controlled study of the use of n–6 fatty acids in patients with UC has been performed. Greenfield et al (27) compared the effectiveness of evening primrose oil, which is rich in -linolenic acid and linoleic acid, with that of n–3 fatty acids and olive oil in 43 patients with UC in different phases of activity. The patients' normal treatments were continued. After 6 mo, no beneficial effects were shown in the patients taking evening primrose oil except for an increase in stool consistency.


CONCLUSION  
In conclusion, this overview indicates a potential effectiveness of n–3 fatty acids in the therapy of CD and UC. The conflicting results reported can be ascribed to differences in study design (such as patients selection, influence of concomitant therapy, and choice of placebo) as well as to different formulations of the fatty acids, which have been shown to influence not only the delivery and absorption of the fatty acids but also patient compliance. The administration of lower doses of new formulations of free fatty acids may reduce side effects and improve the therapeutic potential of n–3 fatty acids, offering a new perspective in the management of inflammatory bowel disease.


REFERENCES  

  1. Lee TH, Hoover RL, Williams D, et al. Effect of a dietary enrichment with eicosapentaenoic acid and docosahexaenoic acids on in vitro polymorphonuclear and monocyte leukotriene generation and polymorphonuclear leukocyte function. N Engl J Med 1985;312:1217–24.
  2. Sharon P, Stenson WF. Enhanced synthesis of leukotriene B4 by colonic mucosa in inflammatory bowel disease. Gastroenterology 1984;86:453–60.
  3. Rampton DS, Collins CE. Review article: thromboxanes in inflammatory bowel disease: pathogenic and therapeutic implications. Aliment Pharmacol Ther 1993;7:357–67.
  4. Endres S, Ghorbani R, Kelly VE, et al. The effect of dietary supplementation with n–3 fatty acids on the synthesis of interleukin-1 and tumor necrosis factor by mononuclear cells. N Engl J Med 1989;320:265–70.
  5. Fisher M, Upchurch KS, Levine PH, et al. Effect of dietary fish oil supplementation on polymorphonuclear leucocyte inflammatory potential. Inflammation 1986;10:387–91.
  6. Wakefield AJ, Sawyerr AM, Dhillon AP, et al. Pathogenesis of Crohn's disease: multifocal gastrointestinal infarction. Lancet 1989;1:1057–62.
  7. Webberley MJ, Hart MT, Melikian V. Thromboembolism in inflammatory bowel disease: role of platelets. Gut 1993;34:247–51.
  8. Jaschoenek K, Clemens MR, Scheurlen M. Decreased responsiveness of platelets to a stable prostacyclin analogue in patients with Crohn's disease. Reversal by n–3 polyunsaturated fatty acids. Thromb Res 1991;63:667–72.
  9. McCall TB, O'Leary D, Bloomfield J, et al. Therapeutic potential of fish oil in the treatment of ulcerative colitis. Aliment Pharmacol Ther 1989;3:415–24.
  10. Salomon P, Asher A, Kornbluth, Janowitz HD. Treatment of ulcerative colitis with fish oil n–3 fatty acid: an open trial. J Clin Gastroenterol 1990;12:157–61.
  11. Lorenz R, Weber PC, Szimnau P, Heldwein W, Strasser T, Loeschke K. Supplementation with n–3 fatty acids from fish oil in chronic inflammatory bowel disease: a randomized, placebo-controlled, double-blind cross-over trial. J Intern Med 1989;225(suppl):225–32.
  12. Hawthorne AB, Daneshmend TK, Hawkey CJ, et al. Treatment of ulcerative colitis with fish oil supplementation: a prospective 12 month randomised controlled trial. Gut 1992;33:922–8.
  13. Stenson WF, Cort D, Rodgers J, et al. Dietary supplementation with fish oil in ulcerative colitis. Ann Intern Med 1992;116:609–14.
  14. Aslan A, Triadafilopoulos G. Fish oil fatty acid supplementation in active ulcerative colitis: a double-blind, placebo-controlled, crossover study. Am J Gastroenterol 1992;87:432–7.
  15. Matè J, Castanos J, Garcia-Samaniego J, Pajares JM. Does dietary fish oil maintain the remission of Crohn's disease (CD): a study case control. Gastroenterology 1993;100:A–228(abstr).
  16. Loeschke K, Ueberschaer B, Pietsch A, et al. n–3 Fatty acids retard early relapse in ulcerative colitis in remission. Dig Dis Sci 1996;41:2087–94.
  17. Lorenz-Meyer H, Bauer P, Nicolay C, et al. Omega-3 fatty acids and carbohydrate diet for maintenance of remission in Crohn's disease. Scand J Gastroenterol 1996;31:778–85.
  18. Brignola C, De Simone G, Belloli C, et al. Steroid treatment in active Crohn's disease: a comparison between two regimens of different duration. Aliment Pharmacol Ther 1994;8:465–8.
  19. Budiarso IT. Fish oil versus olive oil. Lancet 1990;336:1313–4 (letter).
  20. Petroni A, Blasevich M, Salami M, et al. Inhibition of platelet aggregation and eicosanoid production by phenolic components of olive oil. Thromb Res 1995;78:151–60.
  21. Leventhal L, Boyce EG, Zurier RB. Treatment of rheumatoid arthritis with gammalinolenic acid. Ann Intern Med 1993;119:867–73.
  22. Appel LJ, Miller ER, Seidler AJ, Whelton PK. Does supplementation of diet with "fish oil" reduce blood pressure? A meta-analysis of controlled clinical trials. Arch Intern Med 1993;153:1429–38.
  23. O'Connor GT, Malenka DJ, Olmstead EM, Johnson PS, Hennekens CH. A meta-analysis of randomized trials of fish oil in prevention of restenosis following coronary angioplasty. Am J Prev Med 1992;8:186–92.
  24. Kunzel U, Bertsch S. Klinsche erfahrungen mit einem standardiserten fischolkonzenntrat. Feldstudie mit 3.958 hyperlipamischen patienten in der praxis des niedergelassenen arztes. (Clinical results on the treatment of 3,958 patients with hyperlipidemia by using a concentrated fish oil preparation.) Fortschr Med 1990;108:437–42 (in German).
  25. Belluzzi A, Brignola C, Campieri M, et al. Effects of a new fish oil derivative on fatty acid phospholipid-membrane pattern in a group of Crohn's disease patients. Dig Dis Sci 1994;39:2589–94.
  26. Belluzzi A, Brignola C, Campieri M, et al. Effect of an enteric-coated fish oil preparation on relapses in Crohn's disease. N Engl J Med 1996;334:1557–616.
  27. Greenfield SM, Green AT, Teare JP, et al. A randomized controlled study of evening primrose oil and fish oil in ulcerative colitis. Aliment Pharmacol Ther 1993;7:159–66.

日期:2008年12月28日 - 来自[2000年71卷第1期]栏目

Black Box Warning for Bowel Prep Drugs

Dec. 11, 2008 -- The FDA today ordered a "black box" warning, the FDA's sternest warning, for the prescription oral sodium phosphate products Visicol and OsmoPrep, which are used to cleanse the bowel before a colonoscopy and other medical procedures.

The warning pertains to the risk of acute phosphate nephropathy, which is a type of acute kidney injury.

The FDA also recommends that consumers not use over-the-counter oral sodium phosphate products, such as Fleet Phospho-soda, for bowel cleansing.

According to the FDA, over-the-counter oral sodium phosphate products, when used for bowel cleansing, pose the same kidney risk as the prescription oral sodium phosphate products. But that risk doesn't apply to those over-the-counter products used as laxatives.

Kidney Injury Reports

The FDA has received 20 reports of kidney injury associated with OsmoPrep since OsmoPrep's approval in 2006. Three of those cases were confirmed by biopsy. Some cases happened within hours of use; others were reported days or weeks later.

The FDA has also received reports of 19 cases of acute renal (kidney) failure, seven of which were confirmed by biopsy.

Korvik notes that one reason for the risk might be that some people may be dehydrated and not drinking enough fluid when they use oral sodium phosphate products for bowel cleansing, despite the instructions on the products.

"We don't believe that all of the patients are at risk," Korvik said at a news conference.

Charles Ganley, MD, who directs the FDA's Office of Nonprescription Products, agrees, noting that "the majority" of patients who use those products "don't run into problems."

The FDA has asked Salix Pharmaceuticals, which makes Visicol and OsmoPrep, to do a study to learn more about the kidney risk and how to manage it.

Spokespeople for Salix Pharmaceuticals and for C.B. Fleet Company, which makes Fleet Phospho-soda, were not immediately available for comment.

No Reason to Postpone Colonoscopy

Patients 18 and older can still use Visicol and OsmoPrep for bowel cleansing before colonoscopy or other procedures.

But the FDA recommends that people use caution with those products if they are in the following risk groups:

  • People older than 55
  • People with dehydration, kidney disease, acute colitis, or delayed bowel emptying
  • People taking certain medicines that affect kidney function, such as diuretics, ACE inhibitors, angiotensin receptor blockers, and possibly nonsteroidal anti-inflammatory drugs (NSAIDs).

The FDA's warning doesn't apply to Golytely, Colyte, Nulytely, Trilyte, and Halflytely, which are polyethylene glycol preparations that can be used for bowel cleansing.

日期:2008年12月13日 - 来自[Health News]栏目
循环ads

Treatment of murine Th1- and Th2-mediated inflammatory bowel disease with NF-B decoy oligonucleotides

Mucosal Immunity Section, Laboratory of Host Defenses, National Institute of Allergy and Infectious Diseases, NIH, Bethesda, Maryland, USA.

    Abstract

The Th1 and Th2 T cell responses that underlie inflammatory bowel diseases (IBDs) are likely to depend on NF-B transcriptional activity. We explored this possibility in studies in which we determined the capacity of NF-B decoy oligodeoxynucleotides (decoy ODNs) to treat various murine models of IBD. In initial studies, we showed that i.r. (intrarectal) or i.p. administration of decoy ODNs encapsulated in a viral envelope prevented and treated a model of acute trinitrobenzene sulfonic acid–induced (TNBS-induced) colitis, as assessed by clinical course and effect on Th1 cytokine production. In further studies, we showed that NF-B decoy ODNs were also an effective treatment of a model of chronic TNBS-colitis, inhibiting both the production of IL-23/IL-17 and the development of fibrosis that characterizes this model. Treatment of TNBS-induced inflammation by i.r. administration of NF-B decoy ODNs did not inhibit NF-B in extraintestinal organs and resulted in CD4+ T cell apoptosis, suggesting that such treatment is highly focused and durable. Finally, we showed that NF-B decoy ODNs also prevented and treated oxazolone-colitis and thus affect a Th2-mediated inflammatory process. In each case, decoy administration led to inflammation-clearing effects, suggesting a therapeutic potency applicable to human IBD.

    Introduction

Results of recent studies of the idiopathic inflammatory bowel diseases (IBDs; Crohn disease and ulcerative colitis) strongly suggest that these diseases are due to inappropriate and/or excessive responses to antigens present in the normal bacterial microflora (1-6). Crohn disease is characterized by a transmural, granulomatous inflammation occurring anywhere in the alimentary canal but is usually centered in the terminal ileum and ascending colon; ulcerative colitis, in contrast, is marked by a superficial inflammation causing epithelial cell destruction (ulceration) that is centered in the rectum and colon (1, 2). Despite having a common basis in overresponsiveness to mucosal antigens, the 2 diseases have considerably different pathophysiologies. Crohn disease is associated with a Th1 T cell–mediated response induced by IL-12 and possibly IL-23, whereas ulcerative colitis is associated with an atypical Th2-mediated response characterized by NKT cell secretion of IL-13 (6-10). In recent years, a great number of murine models of mucosal inflammation mimicking these diseases have been described, and their study has led to a profound increase in our understanding of their immunologic bases. One such model, hapten-induced colitis in mice caused by intrarectal (i.r.) instillation of trinitrobenzene sulfonic acid (TNBS-colitis) is a Th1 T cell–mediated colitis that captures many of the features of Crohn disease (11-14). On the other hand, a second hapten-induced colitis in mice (oxazolone-colitis), caused by i.r. instillation of oxazolone, reproduces many of the features of ulcerative colitis (5, 15).

Both in human IBD and in murine models of IBD, the inflammation is likely to depend, at least in part, on the activation and nuclear translocation of NF-B family members (16-19). This would certainly be the case in Th1-mediated inflammations dependent on IL-12 and/or IL-23, since the synthesis of these cytokines is regulated by NF-B transcription factors (20-23). In addition, it would also be true of Th2-mediated inflammations if these inflammations depend on IL-4 or IL-13, since the synthesis of these cytokines is also dependent on NF-B transcription factors, albeit more indirectly than IL-12/IL-23 (24-26). This suggests that one method of treating the inflammation of IBD is to administer agents that inhibit NF-B activity, and indeed, in previous studies of hapten-induced murine models of IBD, inflammation has been successfully prevented with the administration of antisense oligodeoxynucleotides (ODNs) specific for the p65 component of NF-B (16, 27). If this approach is to be applied to the treatment of IBD in humans, however, it must be shown that such agents can very effectively reverse established disease when cytokines not directly dependent on NF-B may be operative. In addition, it must be shown that such agents can have major therapeutic effects in Th2 as well as Th1 inflammations. Finally, methods of delivering anti–NF-B agents to intracellular sites within the inflamed tissue must be devised to achieve an effective level of therapy with concentrations of inhibitors that do not have widespread toxic effects.

The major family of NF-B transcription factors consists of 5 members, c-Rel, p65, RelB, p50, and p52, all of which contain domains that bind to a similar binding site in the promoters of genes encoding key inflammatory proteins (such as IL-12 and IL-23) (28). This creates the possibility of blocking most forms of NF-B activity with a single NF-B decoy ODN sequence that is identical to the consensus sequence of the NF-B binding site. In this study, we utilized a decoy with such a consensus binding site sequence in association with a novel way of delivering the NF-B decoy ODNs to the inside of cells. The latter involves the delivery of decoy ODNs in a viral envelope derived from the hemagglutinating virus of Japan (HVJ-E) (Sendai virus) that has a high capacity to fuse with mammalian cells (29).

In the present study, we show that either locally (i.r.) or systemically (i.p.) administered NF-B decoy ODNs represent a highly effective means of preventing and treating both acute TNBS-colitis and oxazolone-colitis as well as chronic TNBS-colitis. In addition, even when given late in the course of a chronic model of TNBS-colitis, this form of treatment prevents the development of fibrosis. Thus, NF-B decoy ODNs targeting the NF-B consensus binding sequence emerge as an effective tool for the treatment of both major forms of IBD and their complications.

    Results

NF-B decoy ODNs block DNA-binding activity of NF-B family members.

Decoy ODNs consist of short chains of double-stranded DNA containing the consensus binding sequences of specific transcription factors. Thus, transfected decoy ODNs can bind to a specific transcription factor and inhibit its interaction with its target gene promoter (30-32). In the following studies utilizing NF-B decoy ODNs to inhibit NF-B–mediated transcription, transfection of decoy ODNs was performed with the use of HVJ-E vesicles, which have been shown to achieve high transfection efficiencies both in vitro and in vivo (see Discussion) (30, 33, 34).

In initial studies to determine the inhibitory effect of NF-B decoy ODNs on each of the family members of NF-B, we measured the binding activity of all major subunits of NF-B to a plate-bound consensus binding sequence in the presence and absence of decoy ODNs using the TransFactor assay (see Methods). Nuclear extracts derived from HeLa cells subjected to TNF- stimulation were the source of the activated NF-B family members, p65, c-Rel, and p50, whereas unstimulated Raji cells (cells in which NF-B family members are constitutively activated) were the source of RelB and p52. The inhibitory effect of NF-B decoy ODNs was compared with that of scrambled ODNs, a 22-bp double-stranded DNA sequence not containing any known binding sites for a transcription factor. As shown in Figure 1A, the ability of all measured NF-B subunits to bind to the plate-bound consensus sequence was decreased to baseline levels by NF-B decoy ODNs, whereas scrambled ODNs showed no inhibitory effects. These findings indicate that NF-B decoy ODNs are a potent inhibitor of all subunits of the NF-B transcription factor family that might thus have similar inhibitory effects in vivo.

   Figure 1

Basic properties of NF-B decoy ODNs. (A) Effect of NF-B decoy ODNs on NF-B DNA-binding activity. HeLa cells activated by TNF- (20 ng/ml) or Raji cells (constitutively activated) were transfected with NF-B decoy ODNs or scrambled ODNs encapsulated in a HVJ-E; 30 minutes after stimulation, the binding activity of p65, c-Rel, and p50 was determined in nuclear extracts of HeLa cells, whereas binding activity of Rel B and p52 was directly determined in nuclear extracts of Raji cells using the TransFactor assay. Data shown are mean values ± SD obtained from 2 independent experiments. Results are presented as absorbance at 450 nm (A450) wave length. (B) In vivo transfection of NF-B decoy ODNs into CD4+ T cells and non-CD4+ T cells in the colonic lamina propria. Mice were administered FITC-conjugated NF-B decoy ODNs (or unconjugated NF-B decoy ODNs) i.r. 4 hours after TNBS administration or i.p. 4, 24, and 48 hours after TNBS administration; then, 5 days after TNBS-colitis induction, colonic lamina propria cells were isolated, stained with PE–anti-CD4, and analyzed by flow cytometry.

NF-B decoy ODNs encapsulated in the HVJ-E are effectively transfected into both CD4+ T cells and non-T cells of the lamina propria.

In further studies mapping the basic characteristics of NF-B decoy ODN activity, we determined the types of cells undergoing in vivo transfection with decoy ODNs following both i.r. and i.p. administration of decoy ODNs. To this end, we administered FITC-conjugated NF-B decoy ODNs encapsulated in HVJ-E after TNBS-colitis induction in C57BL/10 mice via an i.r. or i.p. route. The decoy ODNs were administered once by the i.r. route, at 4 hours after TNBS induction, whereas it was administered 3 times by the i.p. route, at 4, 24, and 48 hours after TNBS induction. Then, on day 5 after induction, colonic lamina propria mononuclear cells (LPMCs) were isolated and subjected to flow cytometry after staining with PE-labeled anti-CD4 Ab. As shown in Figure 1B, cells from mice administered unlabeled NF-B decoy ODNs exhibited background FITC fluorescence (except for minimal autofluorescence most probably arising from epithelial cells). In contrast, cells from mice administered labeled decoy ODNs exhibited very considerable positive fluorescence in both CD4+ T cells and non-CD4+ cells (a cell population containing APCs, epithelial cells, and possibly CD8+ T cells). FITC-positive cells were seen in cell populations obtained from mice administered decoy ODNs by both the i.r. and i.p. routes, but was somewhat higher in the populations from mice given i.r. decoy ODNs. These studies establish that NF-B decoy ODNs encapsulated in HVJ-E transfect both CD4+ T cells and non-CD4+ cells following both i.r. and i.p. administration. It should be noted, however, that because the decoy ODNs could not be labeled with high intensity, the data shown offers a qualitative rather than quantitative estimate of cell transfection.

Administration of NF-B decoy ODNs encapsulated in HVJ-E prevents nascent TNBS-colitis and reverses established TNBS-colitis.

TNBS-colitis induced in SJL/J or C57BL/10 mice is a rapidly evolving transmural colitis that, like Crohn disease, is a Th1-mediated inflammation dependent on the production of IL-12 (and presumably IL-23). To determine whether NF-B decoy ODNs could prevent the development of this colitis in C57BL/10 mice, we induced colitis in these mice by i.r. instillation of TNBS in ethanol (see Methods) and then, 4 hours later, instilled 75 μg NF-B decoy ODNs or scrambled ODNs (encapsulated in HVJ-E) (see Methods); alternatively, we administered these ODNs (again 75 μg packaged in HVJ-E) by i.p. injection at 4 hours after TNBS administration and again on days 1 and 2 after TNBS administration. The mice were then monitored by weight loss (or gain), mortality, colon histology, and cytokine secretion by cells extracted from tissues and stimulated in vitro. As shown in the weight curves depicted in Figure 2A and the mortality graph depicted in Figure 2B, whereas mice administered TNBS/ethanol alone and treated with scrambled ODNs exhibited progressive weight loss and high mortality, those treated with NF-B decoy ODNs (either by i.r. instillation or i.p. injections) had a course similar to that observed in control mice treated with ethanol alone or, indeed, HVJ-E alone (without ODNs). Similarly, the macroscopic appearance of the colons and, as shown in Figure 2, C and D, histological examination of the colons of the NF-B decoy ODN–treated mice showed no evidence of inflammation, whereas the colons of the scrambled ODN–treated mice showed severe inflammation.

   Figure 2

Treatment of TNBS-colitis by administration of NF-B decoy ODNs. (A–D) TNBS-colitis was induced by i.r. administration of TNBS in ethanol. Mice were treated with NF-B decoy ODNs (or scrambled ODNs) via an i.r. route (at 4 hours) or via an i.p. route (at 4, 24, and 48 hours). Data shown are representative of 3 independent experiments. (A) Body weight as a percent of starting weight. Data shown are mean values ± SD and derived from at least 7 mice per group. (B) Animal survival during the first 5 days after TNBS administration. (C) H&E staining of representative colon cross-sections on day 5 after TNBS administration. Magnification, x5. (D) Histological scores shown are mean values ± SD from at least 7 mice per group. (E–H) TNBS-colitis was induced by i.r. administration of TNBS in ethanol. On day 5, mice with at least 20% body weight loss and not in recovery phase were pooled and divided into treatment groups. Mice were treated with NF-B decoy ODNs (or scrambled ODNs) via an i.r. route (day 5) or via an i.p. route (days 5–7). Data shown are representative of 2 independent experiments. (E) Body weight as a percent of starting weight. Data shown are mean values ± SD from at least 7 mice per group. (F) Animal survival in percent until day 9 after TNBS administration. (G) H&E staining of representative colon cross-sections on day 9 after TNBS administration. Magnification, x5. (H) Histological scores are shown as mean values ± SD from at least 7 mice per group. *P < 0.05.

A more stringent test of the efficacy of NF-B decoy ODNs as a treatment of TNBS-colitis is whether or not they can reverse already established colitis. To explore this question, we again induced TNBS-colitis in C57BL/10 mice and then monitored the mice for weight loss, ultimately only selecting those for treatment with NF-B decoy ODNs (or scrambled ODNs) that had lost 20% of body weight by the fifth day after colitis induction. Then, on day 5 after colitis induction, we administered 1 dose of NF-B decoy or scrambled ODNs (75 μg) by the i.r. route or 3 doses of these ODNs by the i.p. route on consecutive days (in both cases encapsulated in HVJ-E) as in the prevention study described above. Care was taken to ensure that the various experimental groups to be compared had lost equivalent amounts of weight. As shown in Figure 2, E and F, untreated mice and mice treated with scrambled ODNs continued to lose weight and exhibited a mortality rate at day 9 as high as 50%. In contrast, mice that were treated with NF-B decoy ODNs (by either the i.r. or i.p. route) exhibited a reversal in weight loss and a mortality rate of only 15% at day 9. Furthermore, as shown in Figure 2, G and H, these weight loss and mortality data correlated with histological evaluation of colonic sections. In a similar experiment, mice were treated on day 4 and sacrificed on day 7 after TNBS administration, with comparable positive treatment effects by NF-B decoy ODNs.

Finally, culture and stimulation of isolated colonic LPMCs from NF-B decoy ODN–treated mice, obtained either from the mice administered NF-B decoy ODNs at the time of TNBS-colitis induction to prevent disease or 5 days after induction to treat established disease led to secretion of baseline levels of Th1 cytokines. In contrast, culture and stimulation of cells from untreated or scrambled ODN–treated mice led to secretion of high levels of these cytokines. Thus, as shown in Figure 3A, which depicts the cytokine response of cells isolated from mice with established TNBS-colitis, cells extracted from untreated mice or scrambled ODN–treated mice produced higher levels of IL-12 p70, TNF-, and IFN- than did cells from mice treated with NF-B decoy ODNs when appropriately stimulated in vitro (see Methods), whereas cells extracted from the lamina propria of NF-B decoy ODN–treated mice exhibited levels of cytokine secretion similar to those observed in control ethanol-treated mice. However, as also shown in Figure 3A, secretion of IL-10, a cytokine with antiinflammatory properties, was greatly enhanced in mice treated with NF-B decoy ODNs as compared with scrambled ODN–treated or untreated mice. Entirely similar cytokine responses were obtained with cells isolated from mice treated with NF-B decoy ODNs at the time of TNBS-colitis induction (data not shown). Taken together, these data indicate that NF-B decoy ODNs administered i.r. or i.p. are highly effective both in the prevention of nascent TNBS-colitis and in the treatment of established TNBS-colitis. It should be noted that mice treated with "naked" NF-B decoy ODNs not packaged in HVJ-E (at the time of TNBS-colitis induction), then monitored by all of the parameters mentioned above, exhibited no amelioration of colitis (data not shown). In addition, treatment of mice (at the time of TNBS-colitis induction) with only 1 i.p. injection of NF-B decoy ODNs had virtually no effect on the development of TNBS-colitis.

   Figure 3

Treatment of established TNBS-colitis with NF-B decoy ODNs — effect on cytokine production, NF-B activity, and T cell apoptosis. (A–D) TNBS-colitis was induced by i.r. administration of TNBS in ethanol. On day 5, mice with at least 20% weight loss and not in a recovery phase were pooled and divided into treatment groups. Mice were treated with NF-B decoy ODNs (or scrambled ODNs) via an i.r. route (day 5) or via an i.p. route (days 5–7). Data shown are representative of 2 independent experiments. (A) Cytokine production of colonic lamina propria cells on day 9 after TNBS administration. Cells were extracted from the lamina propria and cultured for 48 hours in the presence of stimulants (see Methods). Cytokine concentration was determined in the supernatants by ELISA. (B) DNA-binding activity of p65 and c-Rel in nuclear extracts derived from colonic lamina propria cells on day 9 after TNBS administration and measured by TransFactor assay. (C) Apoptosis of CD4+ cells in colonic lamina propria 1 day after i.r. treatment of established TNBS-colitis. Mice were treated with NF-B decoy ODNs (or scrambled ODNs) on day 5, and colonic lamina propria cells were isolated on day 6 by flow cytometry. Apoptotic cells were determined by annexin V staining. (D) TUNEL staining of representative colon cross-sections on day 7 after TNBS administration. Magnification, x40. Mice were treated with NF-B decoy ODNs (or scrambled ODNs) on day 5, and colonic cross-sections were stained on day 7.

The results of 2 subsequent studies correlated with and expanded on these results. First, we conducted studies to determine whether the administration of NF-B decoy ODNs (encapsulated in HVJ-E) results in a persistent inhibition of NF-B components (p65 and c-Rel) in the context of an existent inflammatory state. Accordingly, we obtained nuclear extracts of mononuclear cells isolated from the lamina propria of mice 4 days after i.r. administration of NF-B decoy ODN or scrambled ODN treatment (9 days after induction of TNBS-colitis) and then estimated the DNA-binding activity of p65 and c-Rel in the extracts by the extent of binding of these components to plate-bound consensus NF-B sequences in the TransFactor assay. As shown in Figure 3B, extracts of cells from NF-B decoy ODN–treated mice exhibited greatly decreased p65 and c-Rel binding to the consensus sequence, whereas the extracts of scrambled ODN–treated mice exhibited high levels of binding. These data indicate that suppression of binding of NF-B components occurs during inflammation and, indeed, is present during the period when the inflammation is subsiding. The latter finding, together with the data described above relating to the persistence of FITC-labeled NF-B decoy ODNs, indicates that the inflammation-clearing effects of NF-B decoy ODNs are surprisingly long lasting.

Second, we investigated whether or not NF-B decoy ODN treatment is associated with apoptosis of effector cells in TNBS-colitis, as previously shown (13) in treatment of TNBS-induced colitis with anti–IL-12. To this end, on day 5 after induction of TNBS-colitis (or on day 5 after administration of ethanol alone), mice were i.r. treated with NF-B decoy ODNs or scrambled ODNs; then, 24 hours later, colonic LPMCs were isolated and stained with annexin V (see Methods) for detection of apoptotic cells by flow cytometric analysis. As shown in Figure 3C, whereas in the untreated or scrambled ODN–treated TNBS-colitis mouse groups, or the ethanol-treated mouse group, less than 4% of the CD4+ cells were annexin V positive, in the i.r. NF-B decoy ODN–treated TNBS-colitis mouse group, 23% of CD4+ cells were annexin V positive. To visualize this effect in situ, we performed TUNEL staining of colonic cross sections of mice with TNBS-colitis 2 days after treatment with NF-B decoy ODNs. As shown in Figure 3D, only colonic lamina propria of mice with TNBS-colitis and treated with NF-B decoy ODNs showed increased numbers of TUNEL-positive inflammatory cells. These data show clearly that NF-B decoy ODN administration causes apoptosis of CD4+ T cells in the inflamed gut, suggesting that this form of therapy might have durable effects.

In further studies to identify the cells subject to apoptosis following treatment with NF-B decoy ODNs, we isolated colonic LPMCs and separated the latter into CD4+ and CD11b+ subsets using Ab-coated magnetic beads. After separation, we subjected a portion of the 2 cell populations to transfection with NF-B decoy ODNs (or scrambled ODNs) and then cultured both transfected and untransfected cells with TNF- for 24 hours. Finally, we determined the extent of apoptosis occurring in each cell population by flow cytometric analysis of annexin V and propidium iodide staining. As shown in Supplemental Figure 1 (supplemental material available online with this article; doi:10.1172/JCI24792DS1 http://dx.doi.org/10.1172/JCI24792DS1), after in vitro culture and transfection of CD4+ and CD11b+ cells, we found that both CD4+ and CD11b+ cells exhibited greatly enhanced apoptosis after transfection with NF-B decoy ODNs when cultured with TNF-. The relatively high background apoptosis observed in control cultures was due to the fact that even after cell purification, the cell populations still contain substantial numbers of colonic epithelial cells and/or granulocytes that undergo cell death during the culture period. These data suggest that NF-B decoy ODN treatment induces apoptosis in both T cell and APC populations and thus undermines the inflammation-causing immune response at 2 levels.

Administration of NF-B decoy ODNs packaged in HVJ-E prevents the development of colonic fibrosis in chronic TNBS-colitis.

In the studies described so far, the effects of NF-B decoy ODNs were studied in an acute model of TNBS-colitis. Recently, a more chronic form of this type of experimental colitis has been reported in which the colitis is induced in BALB/c mice by weekly i.r. administration of increasing doses of TNBS (see Methods) (27). This form of TNBS-colitis in BALB/c mice differs from the more acute form in SJL/J or C57BL/10 mice by the fact that it has both a Th1 and Th2 component. In addition, it leads to the development of fibrosis after the sixth week of TNBS treatment. This model therefore allowed us to again determine the effects of NF-B decoy ODNs on an established colitis (in this case with a somewhat different pathophysiology) and at the same time to determine the effect of such treatment on the development of cytokine-mediated fibrosis.

In these studies we administered i.r. TNBS to mice each week for 7 weeks. On day 35 after initiation of TNBS administration, mice were assembled into weight-matched subgroups for various types of treatment. We treated one group of mice with i.r. NF-B decoy ODNs encapsulated in HVJ-E on days 37 and 44 and a second group with i.p. NF-B decoy ODNs daily on days 37–39 and days 44–46. A similar regimen was followed for mice treated with scrambled ODNs. It should be noted that the TNBS-colitis in these mice did not cause death after week 3, suggesting that at this point the remaining mice had achieved a steady state of inflammation compatible with continued survival.

As shown in Figure 4A, BALB/c mice administered TNBS as described above lost weight during the first 7 days following the initial dose of i.r. TNBS but thereafter gained weight and reached their starting weight by day 28 despite readministration of TNBS. In the following weeks, untreated mice and scrambled ODN–treated mice with chronic TNBS-colitis did not gain additional weight, whereas NF-B decoy ODN–treated mice gained additional weight after their first treatment on day 37. As shown in Figure 4B, these weight changes correlated with the histological evaluation of colonic tissues of the various mouse groups. Untreated and scrambled ODN–treated mice with chronic TNBS-colitis exhibited inflammation of the colonic lamina propria as well as marked thickening of the colon wall, whereas NF-B decoy ODN–treated mice showed comparatively little inflammation of the colonic lamina propria associated with reduced thickness of the colon wall. In addition, as shown in Figure 4C, while colon tissues stained with the Masson trichrome technique revealed increased amounts of collagen in the subepithelial and in deeper layers of the colonic lamina propria of untreated mice or scrambled ODN–treated mice, no increase in collagen deposition was observed in NF-B decoy ODN–treated mice. As shown in Figure 4D, this reduction in collagen deposition was corroborated by the Sircol collagen assay: the amount of collagen after NF-B decoy ODN treatment was significantly reduced to almost basal levels.

   Figure 4

Treatment of chronic TNBS-colitis by NF-B decoy ODNs. (A–D) Chronic TNBS-colitis was induced by 7 weekly i.r. administrations of TNBS in ethanol. Mice were treated with NF-B decoy ODNs (or scrambled ODNs) via an i.r. route (day 37 and day 44) or via an i.p. route (days 37–39 and days 44–46). (A) Body weight as a percent of starting weight. Data are shown as mean values ± SD and are representative of 2 independent experiments. (B) H&E staining of representative colon cross-sections on day 49 after TNBS administration. Magnification, x5. (C) Masson trichrome staining of representative colon cross-sections on day 49 after TNBS administration. Magnification, x5. (D) Collagen content of the colon. Collagen content was determined on day 49 by a Sircol assay. Data shown are mean values ± SD and are derived from at least 4 mice per group. *P < 0.05.

In additional studies of separate groups of mice, we evaluated the effect of NF-B ODN treatment of BALB/c mice with chronic TNBS-colitis on in vitro cytokine production by lamina propria cells isolated on day 49. As shown in Figure 5A, cells from untreated and scrambled ODN–treated mice with chronic TNBS-colitis produced elevated levels of Th1 cytokines, such as IL-12p70, IFN-, TNF-, IL-23, and IL-17. In addition, these cells produced markedly increased amounts of TGF-?1 as well as increased amounts of Th2 cytokines, including modestly elevated amounts of IL-4 and markedly elevated amounts of IL-13. In contrast, cells from mice treated with NF-B decoy ODNs produced only the basal amounts of these cytokines produced by control mice.

   Figure 5

Treatment of chronic TNBS-colitis by NF-B decoy ODNs — effect on cytokine production and NF-B binding activity. (A and B) Chronic TNBS-colitis was induced by 7 weekly i.r. administrations of TNBS in ethanol. Mice were treated with NF-B decoy ODNs (or scrambled ODNs) via an i.r. route (day 37 and day 44) or via an i.p. route (days 37–39 and days 44–46). (A) Cytokine production of colonic lamina propria cells on day 49 after the initial TNBS administration. Cells were extracted from the lamina propria and cultured for 48 hours in the presence of stimulants (see Methods). Cytokine concentrations were determined in culture supernatants by ELISA. Data shown are mean values ± SD and are representative of 2 independent experiments. (B) DNA-binding activity of p65 on day 49 after initial TNBS administration in nuclear extracts from colonic lamina propria cells, measured by TransFactor assay. *P < 0.01.

Finally, we evaluated the DNA-binding activity of the NF-B subunit p65 in nuclear extracts of day 49 LPMCs from mice treated with scrambled ODNs or i.r. NF-B decoy ODNs. As shown in Figure 5B, extracts of cells from NF-B decoy ODN–treated mice once again exhibited low levels of binding to plate-bound NF-B consensus sequences in the TransFactor assay.

Taken together, these results show that NF-B decoy ODNs can block the development of colitis as well as the development of fibrosis in a chronic model of TNBS-colitis. Given the fact that the development of fibrosis in this model is probably secondary to IL-13 secretion and induction of TGF-?1 (see Discussion), this study shows that such treatment also blocks aspects of this inflammation mediated by Th2 cytokines.

Administration of NF-B decoy ODNs packaged in HVJ-E prevents oxazolone-colitis and treats established oxazolone-colitis.

In a further series of studies, we sought to determine the capacity of NF-B decoy ODNs to treat the Th2 cytokine–mediated model of hapten-induced colitis that resembles ulcerative colitis, namely oxazolone-colitis. Accordingly, we induced acute oxazolone-colitis in C57BL/10 mice by i.r. administration of oxazolone in ethanol (see Methods) and then determined the course of colitis in untreated mice or mice treated on the day of oxazolone administration with either i.r. or i.p. NF-B decoy ODNs or scrambled ODNs packaged in HVJ-E. As shown in Figure 6, A and B, untreated mice and mice treated with scrambled ODNs exhibited severe weight loss and a 50% mortality rate by day 3 after induction of oxazolone-colitis, whereas mice treated with NF-B decoy ODNs by either route exhibited a weight equivalent to that of mice administered ethanol alone and a greatly reduced mortality. Moreover, as shown in Figure 6, C and D, these weight changes and survival figures correlated with histological examination of colons from mice in the various groups of acute oxazolone-colitis: untreated and scrambled ODN–treated mice exhibited high levels of inflammation associated with extensive epithelial cell ulceration, whereas NF-B decoy ODN–treated mice exhibited virtually no inflammation.

   Figure 6

Prevention and treatment of oxazolone-colitis by administration of NF-B decoy ODNs. (A–D) Oxazolone-colitis was induced by i.r. administration of oxazolone (Oxa) in ethanol. Mice were treated with NF-B decoy ODNs (or scrambled ODNs) via an i.r. route (4 hours) or via an i.p. route (4 hours, 24 hours). Data shown are representative of 2 independent experiments. (A) Body weight as a percent of starting weight. Data shown are mean values ± SD derived from at least 4 mice per group and are representative of 2 independent experiments. (B) Animal survival in percent until day 3 after oxazolone administration. (C) H&E staining of representative colon cross-sections on day 3 after oxazolone administration. Magnification, x5. (D) Histological scores shown are mean values ± SD derived from at least 4 mice per group. (E–H) Treatment of established oxazolone-colitis by administration of NF-B decoy ODNs. Oxazolone-colitis was induced by i.r. administration of oxazolone in ethanol 4 days after skin presensitization with oxazolone. Mice were treated with NF-B decoy ODNs (or scrambled ODNs) via an i.r. route (day 4) or via an i.p. route (days 4–6). (E) Body weight as a percent of starting weight. Data shown are mean values ± SD derived from at least 3 mice per group. (F) Animal survival in percent until day 7 after oxazolone administration. (G) H&E staining of representative colon longitudinal sections on day 7 after oxazolone administration. Magnification, x5. (H) Histological scores shown are mean values ± SD derived from at least 3 mice per group. *P < 0.01.

In additional studies to explore the question of whether NF-B decoy ODNs are able to reverse an established Th2 cytokine–mediated inflammation, we determined whether NF-B decoy ODNs administration can treat a more prolonged form of oxazolone-colitis obtained by subjecting mice to skin presensitization with oxazolone prior to administration of i.r. oxazolone (5). To induce this form of oxazolone-colitis, we presensitized male C57BL/10 mice by painting the skin with 3% oxazolone in 100% ethanol and 4 days later administered 1.75% oxazolone in 47.5% ethanol i.r. Then, on day 4 after colitis induction, we administered 1 dose of NF-B decoy or scrambled ODNs (75 μg) by the i.r. route or 3 doses of these ODNs by the i.p. route on consecutive days (in both cases encapsulated in HVJ-E). Care was taken to ensure that the various experimental groups to be compared had lost equivalent amounts of weight. As shown in Figure 6, E and F, weight loss and mortality were severe in untreated presensitized oxazolone-colitis and in groups of presensitized oxazolone-colitis–administered scrambled ODNs. However, administration of NF-B decoy ODNs by either the i.p. or i.r. routes was effective in reversing this weight loss and mortality. In addition, as shown in Figure 6, G and H, this positive clinical effect was also evident in the histological examination and scoring of H&E-stained colonic tissues.

Importantly, the effects of NF-B decoy ODN administration were also reflected in the measurements of local cytokine secretion by cells extracted from the lamina propria and then stimulated ex vivo. Thus, as shown in Figure 7A, colonic lamina propria cells extracted on day 3 after induction of the acute oxazolone-colitis from untreated and scrambled ODN–treated mice produced high levels of both IL-4 and IL-13 upon stimulation, whereas cells from NF-B decoy ODN–treated mice produced basal amounts of these cytokines. Similarly, as shown in Figure 7B, secretion of a Th2-associated chemokine, monocyte-derived chemokine/CCL22 (MDC/CCL22), was increased in colon cultures from untreated and scrambled ODN–treated mice but was undetectable in colon cultures of cells from NF-B decoy ODN–treated mice. In contrast, as also shown in Figure 7A, whereas colonic LPMCs isolated from mice with ongoing acute inflammation (day 3 after instillation of oxazolone) secreted low levels of the antiinflammatory cytokine IL-10, colonic LPMCs from mice treated with NF-B decoy ODNs exhibited greatly enhanced IL-10 secretion. This, plus the fact that IL-10 secretion is also increased in TNBS-colitis (Figure 3A) following treatment with NF-B decoy ODNs, indicates that such treatment may have a positive effect on the secretion of antiinflammatory cytokines, and this may be an additional mechanism of its antiinflammatory effect.

   Figure 7

Prevention and treatment of oxazolone-colitis with NF-B decoy ODNs — effect on cytokine production and intracellular proteins. (A) Cytokine production in cultures of colonic LPMCs isolated on day 3 after oxazolone administration. Cytokine concentrations were determined in culture supernatant by ELISA. Data shown are mean values ± SD and are representative of 2 independent experiments. (B) Monocyte-derived chemokine/CCL22 (MDC/CCL22) production in ex vivo colon cultures isolated on day 3 after oxazolone administration; chemokine concentrations in the culture supernatant were determined by ELISA. Data shown are mean values ± SD and are representative of 2 independent experiments. (C) Cytokine production in cultures of colonic LPMCs on day 7 after oxazolone administration and day 3 after oligonucleotide administration; each culture contained cells pooled from at least 3 mice; cytokine concentrations were determined in culture supernatant by ELISA. (D) DNA-binding activity of p65 in nuclear extracts of cells derived from lamina propria on day 3 after oxazolone administration measured in nuclear extracts from colonic lamina propria cells by TransFactor assay. (E) IRF4 protein expression is reduced in colonic LPMCs. Total colonic LPMC lysates were analyzed by Western blotting on day 3 after oxazolone administration. *P < 0.01. n.d., not detectable.

Similar results were obtained upon analysis of cytokine responses of cells isolated from the lamina propria of mice treated with NF-B decoy ODNs after oxazolone-colitis had been established. Thus, as shown in Figure 7C, cells isolated on day 7, i.e., 3 days after initiation of treatment regimens on day 4, cells from untreated mice or mice treated with scrambled ODNs produced large amounts of IL-13, whereas cells from mice administered NF-B decoy ODNs produced baseline levels of this effector cytokine. In addition, cells from untreated mice or scrambled ODN–treated mice displayed low levels of IL-10, whereas cells from NF-B decoy ODN–treated mice exhibited high levels of IL-10 secretion.

In further studies we determined the effect of NF-B decoy ODN administration on NF-B function in oxazolone-colitis using the TransFactor assay. As shown in Figure 7D, nuclear extracts of colonic LPMCs obtained on day 3 after instillation of oxazolone from untreated or scrambled ODN–treated mice exhibited high levels of binding of p65 to plate-bound NF-B consensus sequences, whereas equivalent extracts of LPMCs from NF-B decoy ODN–treated mice exhibited low levels of binding. Thus, as in the case of the TNBS-induced inflammatory states, NF-B decoy ODN administration to mice with oxazolone-induced inflammation inhibits binding of NF-B components to NF-B consensus sequences.

Finally, to establish a theoretical basis for the mechanism by which NF-B decoy ODN administration inhibits Th2 inflammation, we determined whether such administration downregulates the expression of a NF-B–dependent protein necessary for the induction of Th2 cytokines. In particular, we determined whether NF-B decoy ODN administration affects the expression of IFN regulatory factor 4 (IRF4), an NF-B–dependent intracellular protein that has been shown to be necessary for the induction of Th2 cytokines (35, 36). Accordingly, we obtained colonic LPMCs from mice 3 days after i.r. oxazolone administration and then subjected lysates from these cells to Western blot analysis for the detection of IRF4. As shown in Figure 7E, LPMCs from mice treated with NF-B decoy ODNs exhibited an IRF4 protein band with greatly reduced intensity when compared with LPMCs cells from control mice. These studies strongly suggest that at least 1 mechanism of action of NF-B decoy ODNs in the inhibition of Th2 inflammation is the inhibition of NF-B–dependent proteins necessary for Th2 differentiation.

Taken together, these studies show that NF-B decoy ODN administration has profound effects on Th2 models of colonic inflammation as well as on Th1 models.

Administration of NF-B decoy ODNs via the i.r. route suppresses local (mucosal) NF-B activity but not NF-B activity in a distant organ.

In a final series of studies, we determined the effects of i.r. and i.p. NF-B decoy ODN administration on NF-B activation outside of the colon. Accordingly, mice administered i.r. TNBS to induce TNBS-colitis were treated with NF-B decoy ODNs by i.r. (4 hours after TNBS administration) or i.p. (4 hours, 1 day, and 2 days after TNBS administration) routes. Then, on day 5 after TNBS induction, nuclear extracts from mononuclear cells isolated from the colonic lamina propria, spleen, and liver were obtained. The extracts were then subjected to a TransFactor assay to determine p65 DNA-binding activity. As shown in Figure 8, whereas i.p. administration of NF-B decoy ODNs led to decreased p65 activity in cells from all 3 organs, i.r. administration led to greatly decreased activity in colonic lamina propria cells but not in splenic or hepatic cells. These studies thus show that local administration of NF-B decoy ODNs has little effect on extraintestinal mononuclear cells.

   Figure 8

Effect of NF-B decoy ODNs administered via an i.r. route on NF-B binding activity in extraintestinal mononuclear cells. TNBS-colitis was induced by i.r. instillation of TNBS in ethanol. Mice were treated with NF-B decoy ODNs (or scrambled ODNs) via an i.r. route (4 hours) or via an i.p. route (4, 24, and 48 hours). DNA-binding activity of p65 on day 5 after TNBS administration was measured in nuclear extracts derived from colonic LPMCs, liver mononuclear cells, and splenocytes by TransFactor assay. Data shown are representative of 2 independent experiments involving at least 3 mice in each group. *P < 0.01.

    Discussion

In this study, we determined whether blockade of NF-B transcriptional activity by administration of decoy ODNs corresponding to the consensus binding site of the major NF-B components and encapsulated in a viral envelope derived from HVJ could prevent and/or treat experimental models of inflammatory bowel diseases. Indeed, we found that such treatment was also very effective in preventing both the development of a Th1-mediated colitis (TNBS-colitis in C57BL/10 mice) as well as a Th2-mediated colitis (oxazolone-colitis in C57BL/10 mice) and thus appear to be comparable to the effects of administration of Abs (as shown in previous studies) capable of neutralizing the cytokines that drive these inflammations: anti–IL-12p40 in the case of TNBS-colitis and anti–IL-4 or soluble IL-13R2–Fc in the case of oxazolone-colitis (5, 13, 15). In addition, such treatment was equally effective in reversing the course of an already-established TNBS-colitis both in an acute model induced in C57BL/10 mice and a chronic model induced in BALB/c mice. Since anti–IL-12p40 treatment of TNBS-colitis (and other experimental colitides) has now been at least partially replicated in patients with Crohn disease (8), the high efficacy of treatment of TNBS-colitis with NF-B decoy ODNs must now be seriously considered as a possible treatment of some forms of human Crohn disease and as a possible treatment of human ulcerative colitis.

The favorable treatment outcomes we obtained in these studies can be attributed to several factors. First, we took advantage of the fact that exogenous DNA such as NF-B decoy ODNs can be encapsulated in a viral vesicle formed by inactivated HVJ, a Sendai virus, and that these vesicles can bind to and fuse with cell membranes, thus releasing the encapsulated DNA into the cell cytoplasm (30, 33, 34). The efficacy of this cell transfection method was shown in in vitro studies in which we observed that primary mouse T cells can be transfected with plasmid constructs packaged in HVJ-E at an efficiency of greater than 90% (data not shown). Perhaps more importantly, the efficacy of this method was also shown in vivo, where it was observed that administration of the NF-B decoy ODNs to mice with the various types of TNBS-colitis studied here resolved the inflammation and led to virtually complete disappearance of NF-B binding activity for all 5 major NF-B family members in nuclear extracts of cells obtained from colonic tissue. Of interest, we were able to deliver sufficient amounts of decoy ODNs to cells with unmodified HVJ-E, rather than cationic liposome HVJ complexes that have been shown to be highly efficient delivery vehicles in previous in vitro transfection studies (37). However, such complexes are tedious to assemble, inherently unstable, and do not readily target many types of cells in vivo unless fine tuned vis-à-vis phospholipid content. Finally, it should be noted that the NF-B decoy ODNs had more potent effects when delivered directly to the site of inflammation by i.r. administration, since a greater amount of decoy was needed for effective systemic administration. Such i.r. application of decoy ODNs was also advantageous in that it led to less effect of decoy ODNs on mononuclear cells in extraintestinal organs (liver and spleen) than was the case with i.p. administration of decoy. This strongly suggests that administration of decoy ODNs by the i.r. route would not give rise to systemic side effects consequent to NF-B blockade in the immune system as a whole.

Second, we found that, as in the case of treatment with anti–IL-12p40, treatment with NF-B decoy ODNs led to increased apoptosis of CD4+ T cells, as detected by flow cytometric enumeration of annexin V–stained cells in populations of T cells extracted from the lamina propria (13). The mechanism of apoptosis induction following inhibition of NF-B signaling is probably multifactored. One possible mechanism involves the fact that activation of NF-B leads to the synthesis of proteins that block Jun N-terminal kinase, an enzyme utilized by TNF- to induce apoptosis (38, 39). Thus, the inhibition of NF-B signaling by NF-B decoy ODNs leads to relief from this block and facilitation of TNF-–induced apoptosis. Another possible mechanism is related to the fact that NF-B signaling leads to the synthesis of inhibitor of apoptosis proteins, a family of as-yet poorly understood antiapoptotic proteins (40). Finally, it is important to recall that NF-B is necessary for the production of IL-12, a cytokine that has significant antiapoptotic effects on Th1 T cells; thus, by shutting down IL-12 production, NF-B decoy ODNs lead to apoptosis of Th1 cells. Regardless of the mechanism by which decoy ODNs induce apoptosis, the very fact that such apoptosis occurs and leads to the loss of T effector cells suggests that treatment with NF-B decoy ODNs is likely to have a durable therapeutic effect.

Third, the design of the decoy ODNs used in these studies allowed the best possible chance of inhibiting the inflammation-inducing potential of NF-B. The NF-B transcription factor family consists of 5 different subunits, c-Rel, p65, RelB, p50, and p52, each capable of forming homodimers and heterodimers and each having in common an amino-terminal region containing dimerization, nuclear-localization, and DNA-binding domains. Importantly, the latter DNA-binding domains bind to a consensus binding site on target promoters; thus, a decoy ODN that mimics the sequence of the consensus binding site, such as that used in these studies, inhibits binding and transcriptional activity by all major components of the NF-B family. The importance of such broad-spectrum inhibition in an inflammatory process like that occurring in inflammatory bowel disease is inherent in the fact that despite the existence of a consensus binding sequence, the role of NF-B transactivation cannot be assumed to be due to any specific NF-B family member. Thus, while antisense ODNs that are specific for p65 have been shown to be effective in the treatment of TNBS-colitis, IL-12 production has been shown to be highly dependent on c-Rel (16, 41). Finally, it is important to mention that certain NF-B components, especially p50 homodimers, can act as transcriptional repressors because they bind to consensus binding sequences but lack carboxyterminal domains that bind to nonhomologous transactivation sites (42). Thus, the fact that blocking NF-B activity with the NF-B decoy ODN also inhibits p50 DNA-binding activity could conceivable have a proinflammatory effect. However, since the decoy ODNs used here inhibit all major components, it would theoretically inhibit both the positive and negative effects of NF-B components and have a net antiinflammatory effect. This view is strongly supported by the effects of NF-B decoy ODNs studied here as well as in previous studies of experimental inflammations of various types (43-45).

A fourth and final reason for the success of the NF-B decoy ODN treatment is that the main cytokine drivers of the T cell–mediated response in the inflammations are, as already implied above, dependent on NF-B transcriptional activation of the relevant cytokine genes. Thus, in the case of the Th1-mediated inflammation studied, it is now well established that not only the p35 component of IL-12, but also the p40 component of IL-12/IL-23 as well as IL-17 are dependent on NF-B transcriptional activity (20-23, 46). Likewise, in the case of the Th2-mediated inflammation studied, NF-B is needed for the activation of a number of genes that are necessary for IL-4 and IL-13 production, including IL-2 and IRF4, the latter a factor induced by TLR stimulation and the subsequent production of type I interferons (35, 36).

In recent years a number of quite disparate agents that inhibit NF-B have been shown to have positive effects on experimental mucosal inflammation. These include agents derived from plants (catalposide, fucoidan, or curcumin) (47-49), low-molecular-weight molecules such as the MAPKs and RICK inhibitor (SB203580), the rho kinase inhibitor (Y27632), the PPAR ligand (pioglitazone), and gliotoxin (50-53). For the most part, these inhibitors have been tested in studies of either dextran sulphate sodium–induced colitis or hapten-induced colitis, in which their capacities to prevent mucosal inflammatory disease were assessed rather than their capacities to reverse established inflammatory disease, a more stringent test. In addition, these inhibitors were administered via a systemic route, and the specificity of their inhibitory effects for NF-B was not assessed; thus they could have significant side effects. For example, SB203580 and curcumin not only inhibit NF-B activation but also activation of the MAPK pathway. In addition, the rho kinase inhibitor Y27632, in that it has activity in a number of rather different disorders, such as hypertension, Alzheimer disease, bronchial asthma, and coronary heart disease, is likely to affect several signaling pathways (54-57). Finally, gliotoxin is a known inducer of cellular apoptosis and can cause free radical damage (58, 59). In contrast to these more-or-less nonspecific NF-B inhibitors, the previously studied p65 antisense phosphorothioate ODN NF-B inhibitor is as specific for NF-B as the NF-B decoy ODNs studied here (16). In initial studies that were in fact the first studies of an NF-B inhibitor in experimental colitis (16), this agent was shown to be an effective preventative treatment of an acute model of TNBS-colitis. In later, very recent studies, the same agent was again shown to be an effective preventive agent in a chronic model of TNBS-colitis, the TNBS-colitis induced in BALB/c mice, but in this case, it was only partially effective as a treatment agent (27). The reason for this partial effect may be 2-fold. First, since it inhibits p65 mRNA synthesis and not other NF-B components, it only partially blocks NF-B signaling. Second, since it was delivered as naked ODNs rather than a nucleotide encapsulated in a viral envelop that has a high capacity to penetrate mammalian cells, it may not have been efficiently delivered to key inflammatory cells.

One of the important features of the antiinflammatory effects of NF-B decoy ODNs packaged in HVJ-E was its capacity to prevent the development of and to treat an already established inflammation occurring in a Th2-mediated mucosal inflammation. This was first shown in the chronic TNBS-colitis established in BALB/c mice that begins as a Th1 inflammation and then gradually evolves into a mixed Th1/Th2 inflammation in which Th2 cytokines, especially IL-13, are secreted along with Th1 cytokines. Thus, the fact that administration of NF-B decoy ODNs packaged in HVJ-E led to resolution of inflammation after the colitis had evolved into a mixed Th1/Th2 inflammation provided initial evidence that this treatment was also relevant to a Th2-driven inflammatory process.

A second and perhaps more definitive body of evidence relevant to the efficacy of NF-B decoy ODN treatment of a Th2 colitis was obtained with its use in oxazolone-colitis. In previous studies it has been shown that this model of colitis is due to the induction of NKT cells that produce IL-13 and that administration of agents that eliminate NKT cells and/or block IL-13 function ameliorate the disease (5). Thus, the demonstration in this study that NF-B decoy ODN administration can both prevent nascent oxazolone-colitis and reverse established oxazolone-colitis provides strong evidence that decoy administration is in fact an effective therapeutic agent in Th2 inflammation. In this context, it should be noted that oxazolone-colitis bears a histologic resemblance to ulcerative colitis, and, indeed, the human disease is also characterized by the increased production of IL-13 by NKT cells. Thus, NF-B decoy ODN treatment may prove useful in the treatment of the ulcerative colitis, particularly in light of the fact that this disease is limited to the colon and thus can be effectively treated with an agent administered by the i.r. route.

The mechanism of action of the NF-B decoy ODNs in oxazolone-colitis (and in other forms of Th2 colitis) is probably multifactorial, but it is reasonable to assume that several cytokines, chemokines, or intracellular factors necessary for the generation of the Th2 response are dependent on NF-B signaling and thus susceptible to blockade by NF-B decoy ODNs. The possible cytokine/chemokine targets of the decoy include IL-2, a cytokine that has recently been shown to play a role in the initiation of Th2 T cell differentiation, as well as MDC/CCL22, a TNF-–dependent chemokine that attracts Th2 T cells to inflammatory sites (60-62). In the same vein, the possible intracellular signaling factors that are decoy targets include IRF4 and IRF5, factors induced by TLR signaling that have been shown to be necessary for Th2 T cell differentiation and Th2-mediated inflammation (35, 36). In partial confirmation of this latter supposition, we were able to show that NF-B decoy ODN treatment exerts a downregulatory effect on the expression of IRF4. Finally, it should be noted that while the development of NKT cells is known to be critically dependent on the NF-B component RelB, it is unlikely that NF-B decoy ODNs prevented acute oxazolone-colitis through the inhibition NKT cell development, since the decoy ODN effect was too rapid (63). It is possible, however, that human ulcerative colitis would be ameliorated by blockade of RelB-dependent NKT cell development through repeated administration of NF-B decoy ODNs.

Yet another important feature of NF-B decoy ODN treatment is its capacity to prevent the development of fibrosis that invariably occurs in the chronic TNBS model induced in BALB/c mice. Such fibrosis is also a constant complication of long-standing IBD and one that can lead to some of the direst IBD symptoms, such as loss of gut motility and obstruction. In the chronic TNBS model studied here, fibrosis becomes evident after 6 weeks of TNBS administration and is marked by increasing collagen deposition in the subepithelial layer as well as in the lamina propria associated with colonic wall thickening and rigidity. Interestingly, NF-B decoy ODN treatment, even when begun well after the establishment of chronic colitis, could effectively minimize collagen deposition, suggesting that decoy ODN treatment could block fibrosis in IBD even after a long period of inflammation. A similar result was noted in the aforementioned treatment of mice with chronic TNBS-colitis with p65 antisense ODNs. As to the mechanism of this antifibrotic effect, it is now known that TGF-?1, an important fibrogenic factor, is induced by IL-13, and we have found in unpublished studies that such induction depends on NF-B–dependent IL-13 signaling (64). Thus, it is likely that the antifibrotic effect of the decoy resulted from its ability to indirectly downregulate TGF-?1 through its ability to prevent IL-13 secretion.

In summary, these studies strongly suggest that NF-B decoy ODNs targeting the consensus NF-B binding site and encapsulated in an HVJ-E represent a very potent approach to the treatment of experimental mucosal inflammation. They thus indicate that not only the cytokines responsible for initiation of mucosal inflammation but also those responsible for the persistence of inflammation are dependent on NF-B. These mouse studies set the stage for clinical studies of the efficacy of NF-B decoy ODNs in patients with IBD, particularly those with colonic inflammation that can readily be treated with i.r. decoy ODNs.

    Methods

Mice.

Male C57BL/10 mice (6–8 weeks old) were used in studies of both the prevention and treatment of TNBS-colitis and oxazolone-colitis. Female BALB/c mice (8–10 weeks old) were used in studies of a chronic form of TNBS-colitis. All mice were obtained from Jackson Laboratory and were maintained in the National Institute of Allergy and Infectious Diseases (NIAID) animal holding facilities. Animal use adhered to NIH Laboratory Animal Care Guidelines, and all animal experiments were approved by the NIAID Animal Care and Use Committee review board.

Induction of colitis.

Mice were lightly anesthetized with isoflurane and then administered a haptenating agent (either TNBS or oxazolone dissolved in ethanol) i.r. via a 3.5 French (F) catheter equipped with a 1-ml syringe; the catheter was advanced into the rectum until the tip was 4 cm proximal to the anal verge, at which time the haptenating agent was administered in a total volume of 150 μl. To ensure distribution of the haptenating agent within the entire colon and cecum, mice were held in a vertical position for 30 seconds after the i.r. injection. Control mice were administered an ethanol solution without haptenating agent using the same technique. 3.75 mg TNBS (Sigma-Aldrich) in 50% ethanol was administered for studies of prevention of acute TNBS-colitis, 3 mg TNBS in 45% ethanol for studies of treatment of established acute TNBS-induced colitis, and 1.5–2.5 mg TNBS (in increasing doses) in 45% ethanol was administered each week for studies of treatment of chronic TNBS-induced colitis. Six milligrams oxazolone (Sigma-Aldrich) in 47.5% ethanol was administered for studies of prevention of acute oxazolone-colitis. To induce a longer-lasting form of oxazolone-colitis, we presensitized mice by applying a 3% solution of oxazolone in 100% ethanol to a shaved 2 x 2 cm field of the abdominal skin 4 days prior to i.r. administration of 1.75% oxazolone in 47.5% ethanol.

Preparation of HVJ-E vector and its loading with ODNs.

HVJ-E vector was prepared as previously described (41). In brief, suspended Sendai virus (25,600 hemagglutinating units; AnGes MG) was inactivated by ?-propiolactone followed by ultraviolet irradiation and purified by column chromatography. The HVJ-E thus obtained was mixed with 37.5 μl of protamine sulfate (1 mg/ml) and then incubated for 10 minutes on ice. Insertion of ODNs into the vector was accomplished using a packaging technique that allowed direct insertion of ODNs into the viral envelope. This involved mixing DNA (1 mg/100 μl) and 13.8 μl of 3% Triton X-100 with the HVJ-E and then incubating the resultant mixture for 15 minutes on ice. Finally, the HVJ-E–ODNs were centrifuged at 15,000 g for 15 minutes and resuspended in 500 μl of PBS containing 72 μg of protamine sulfate.

Decoy ODNs.

Double-stranded DNA ODNs with sequences corresponding to the consensus NF-B combining site or scrambled were provided by AnGes MG. Seventy-five micrograms of NF-B decoy or scrambled ODNs were administered during each in vivo treatment. The following sequences were used: NF-B decoy ODNs, 5'-CCTTGAAGGGATTTCCCTCC-3' and 3'-GGAACTTCCCTAAAGGGAGG-5'; scrambled decoy ODNs, 5-CATGTCGTCACTGCGCTCAT-3' and 3'-GTACAGCAGTGACGCGAGTA-5'.

ELISA.

Cytokine protein concentrations in culture supernatants were measured by ELISA kits according to the manufacturer’s instructions. Isolated colonic LPMCs were stimulated for 48 hours. To determine IFN-, IL-4, and IL-13 protein concentrations, cells were stimulated with plate-bound anti-CD3 Ab and soluble anti-CD28 Ab (BD Biosciences — Pharmingen). For measurement of IL-12p70, TNF-, and TGF-?1 colonic LPMCs were stimulated 48 hours with Staphylococcus aureus Cowan I (EMD Biosciences) and IFN- (R&D Systems). IL-23 was determined after 48 hours stimulation of colonic LPMCs with peptidoglycan (Sigma-Aldrich). ELISA kits for IFN-, IL-4, IL-12p70, TNF-, and IL-10 were purchased from BD Biosciences — Pharmingen and for TGF-?1 from BioSource International and IL-23 from eBioscience.

Flow cytometry.

Colonic LPMCs were stained with annexin V, propidium iodide, anti-CD4 Ab (BD Biosciences — Pharmingen). Nonspecific binding of Abs was blocked by preincubation with Fc Block (BD Biosciences — Pharmingen). Cells were acquired using a BD FACScan and analyzed utilizing FlowJo software.

Collagen assay.

Colons of TNBS-treated mice were harvested on day 49 and homogenized in 0.5 M acetic acid containing 1 mg pepsin (at a concentration of 10 mg tissue/10 ml of acetic acid solution). The resulting mixture was then incubated for 24 hours at 4°C with stirring. Colon collagen content was determined by assaying total soluble collagen using the Sircol Collagen Assay kit (Biocolor Ltd.) (65). Acid soluble type I collagen supplied with the kit was used to generate a standard curve.

Assay of activated NF-B components.

Nuclear extracts from colonic LPMCs were obtained using the TransFactor Extract Kit (Active Motif). The extracts were then tested for DNA-binding activity using the NF-B TransFactor Kit (BD Biosciences — Clontech) according to the manufacturer’s instructions. In brief, nuclear extract (15–30 μg) was applied to each well coated with NF-B consensus ODNs, and then wells were incubated with specific Abs for each of the NF-B subunits followed by HRP-labeled secondary Abs (41). After color development with TMB substrate was stopped by adding H2SO4, absorbance was measured at 450 nm wavelength.

Western blot analysis.

Total cell lysates from colonic LPMCs were subjected to SDS-PAGE, and the separated proteins thus obtained were transferred to a nitrocellulose membrane. A protein band for mouse IRF4 was detected by incubation with a polyclonal goat anti-IRF4 Ab (Santa Cruz Biotechnology Inc.) followed by incubation with HRP-conjugated anti-goat IgG (Zymed Laboratories Inc.). Membranes were developed with SuperSignal West Pico Chemiluminescent Substrate (Pierce Biotechnology Inc.) and exposed to x-ray film.

Histological examination.

Colons were fixed in 10% buffered formalin and embedded in paraffin. Paraffin-embedded colon sections were cut and then stained with H&E or by the Masson’s trichrome method. To visualize apoptotic cells, in situ paraffin sections of colonic tissue were subjected to TUNEL staining (66). TUNEL-positive cells were visualized by fuchsin red, and sections were counterstained with hematoxylin (Sigma-Aldrich). For calculation of inflammation indices in treated and control group of mice, the H&E sections were read by investigators blinded to the experimental protocol and evaluated according to formerly published scoring systems for TNBS-colitis and oxazolone-colitis (13, 67).

Statistics.

Statistical differences were assessed using the 2-tailed Student’s t test. P values less than 0.05 were considered statistically significant.

    Acknowledgments

This research was supported by the Intramural Research Program of the NIH, NIAID, and by the Material Cooperative Research and Development Agreement (MCRADA) with AnGes, MG Inc. and GenomIdea Inc.

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日期:2007年5月11日 - 来自[2005年第115卷第11期]栏目

Induction of mucosal tolerance in Peyer‘s patch‘deficient, ligated small bowel loops

1Immunobiology Center,

2Department of Liver Transplant, and

3Department of Microbiology, Mount Sinai School of Medicine, New York, New York, USA.

4Division of Nutritional Sciences and Department of Food Science and Human Nutrition, University of Illinois at Urbana-Champaign, Urbana, Illinois, USA.

    Abstract

To explore the requirement for M cells and the Peyer’s patch (PP) in induction of oral tolerance and address the potential in vivo role of intestinal epithelial cells as nonprofessional APCs, we have attempted to induce tolerance in mice with ligated small bowel loops without M cells and Peyer’s patches. A 2-centimeter section of vascularized small bowel was spliced away from the gut without disruption of the mesenteric attachments. We introduced OVA directly into the lumen of the loop prior to footpad immunization. By excising segments of bowel that contain PPs in some mice and segments without patches in others, we could study the necessity of the M cell and the underlying patch versus epithelial cells in induction of mucosal tolerance. We show that OVA-specific T cell proliferation and serum antibody responses are reduced in mice that have previously been given OVA both in PP-containing loops and in loops without patches. Furthermore, both high- and low-dose tolerance could be induced in the absence of PPs. Low-dose tolerance is associated with bystander suppression and requires IL-10, which indicates active suppression and the induction of regulatory cells. These data suggest that there is a critical role for components of the mucosal immune system other than PPs in antigen sampling and induction of oral tolerance.

    Introduction

The mucosal immune system utilizes a very complicated and tightly regulated system of suppression and controlled inflammation to distinguish between non-self that is harmless (dietary antigens  and normal enteric flora) and non-self that is harmful (pathogenic organisms) in the intestinal lumen. Although the exact mechanisms governing this regulation remain to be elucidated, evidence exists for the involvement of a variety of cell types, including CD8+ suppressor T cells (1), CD4+ regulatory T cells (2), B cells (3, 4), and DCs (5).

One phenomenon linked to this regulation is termed oral tolerance. Large quantities of non-self proteins are taken up daily from the intestinal lumen, yet they are presented to T cells in such a way as to promote tolerance. The systemic immune system is not ignorant of these foreign proteins, though, as in vitro stimulation with experimentally fed Ags can elicit Th2 cytokine responses (6, 7). Tolerance to soluble Ags that have been introduced at mucosal sites has been observed in many animal models as well as in human trials (8-10). It has been shown that low doses of fed Ag can suppress Th1 responses by swaying the response to a Th2 (IL-4, IL-5, IL-10), Th3 (TGF-?), or Treg 1 (Tr1) (IL-10) cytokine profile (11-13). High doses of fed Ag can induce tolerance by anergizing Ag-specific T cells or by clonal deletion (14).

Mucosal tolerance is thought to be contingent upon the very specific environment of the mucosa-associated lymphoid tissue. Regulation is most likely the result of the cytokine microenvironment as well as the immunoregulatory properties of the specialized cells that line the intestinal surface, the M cell and the absorptive epithelial cell.

The M cell resides only in the follicle-associated epithelium overlying a Peyer’s patch (PP). M cells arise from undifferentiated enterocytes that overlie PPs (15), and differentiation is induced by B cells in the patch (16). M cells are very efficient at taking up particulate Ags such as virus particles, bacteria, and other macromolecules in the lumen and passing them through to the underlying patch, where they will be taken up, processed, and presented by the resident macrophages and DCs. Conversely, the M cell is inefficient at taking up soluble proteins. The absorptive intestinal epithelial cells (IECs), however, have been shown to take up soluble Ags and even process and present these Ags on their basolateral surface (17, 18). In humans, they express MHC class I, class II, and nonclassical class I molecules (19), as well as a unique costimulatory molecule, gp180, which acts to induce proliferation of CD8+ T cells in culture (20). CD8+ T cells activated in this manner could either play a role in local suppression (controlled physiologic inflammation) or systemic tolerance (21).

In attempting to understand the roles that the M cell and PP play in induction of oral tolerance, investigators have relied on gene-targeted knockouts, such as the lymphotoxin  knockout (LT–/–) and LT?–/–, in which PPs are absent or aberrantly developed (22, 23), or else have treated mice in utero with LT? receptor Ig fusion protein to generate mice lacking PPs (24). These mice fail to develop tolerance to orally administered Ags. However, in addition to undeveloped PP, these mice also have aberrantly developed splenic architecture that may result in altered immune responses. The LT–/– mice also have no mesenteric lymph nodes, making interpretation of the results difficult. Others have suggested that PPs are not relevant to tolerance induction, although the findings in these studies were not absolute (25).

To understand the role of the M cell and the underlying PP, as well as the IEC in mucosal tolerance, we have surgically isolated a segment of small intestine and repositioned it in the abdomen so as to have access to the lumen. By choosing segments of small bowel that contain PPs and segments that do not, we were able study the necessity for M cells and that of the PP in mucosal tolerance in an immunologically intact animal. We administered multiple doses of either whole OVA, OVA fragments, or OVA peptides via the loops followed by immunization in the footpad. Our results show that mice can be tolerized by Ag administration through both M cell and PP–containing and M cell and PP–deficient loops. We show that administration of peptide into either loop leads to the induction of bystander suppression, which provides evidence for the generation of regulatory cells. In addition, we show that IL-10 is a critical cytokine for low-dose tolerance in both PP-independent and -dependent tolerance induction. Moreover, oral tolerance was not achieved using whole OVA as the tolerogen in the loops, which supports the concept that gastric digestion is needed in induction of oral tolerance.

    Results

Oral tolerance in normal mice.

First, using the protocol developed by Liu et al. (26), we tested the low-dose tolerance of mice with unligated loops. One group of mice was fed 1 mg OVA by gastric intubation every day for 5 days, then immunized in both footpads 3 days after the last feeding. Two weeks later, mice were bled and sacrificed; the lymphocytes from the popliteal lymph nodes (PLNs) were restimulated in vitro with 10–1,000 μg/ml OVA, and the response was compared with that of mice that were immunized without OVA feeding. The results (Figure 1A) show that T cell proliferative responses were reduced 52–64% in mice that were fed OVA compared with nonfed mice. Serum was analyzed for the presence of anti-OVA IgG, and as shown in Figure 1B, the levels of OVA-specific IgG were reduced by 45–61% in mice fed OVA compared with nonfed mice. Cytokine production by T cells from OVA-fed and nonfed mice upon in vitro stimulation was also measured. As can be seen in Figure 1C, OVA-fed mice displayed a decreased secretion of IFN- and IL-2 and enhanced secretion of IL-4 and IL-10 compared with nonfed mice. The above results are consistent with those of previously published reports (26) and confirm the effectiveness of the tolerance protocol in our study.

   Figure 1

Induction of low-dose oral tolerance in normal mice. (A) Antigen-specific T cell proliferation in nonfed and OVA-fed mice. The tolerance protocol was performed as described in Methods. *P < 0.035. (B) Serum anti-OVA antibody levels in nonfed and OVA-fed mice. Serum from mice that were (open circles) or were not (filled circles) fed OVA prior to OVA immunization was diluted (1.25 x 10–5 to 5 x 10–5) and analyzed for the presence of anti-OVA IgG by ELISA. Serum from a nonimmunized animal (normal mouse serum ; open squares) was used as a negative control. The titers of OVA-specific IgG were significantly lower in mice fed OVA compared with nonfed mice. #P < 0.007. (C) Cytokine profile of cells from OVA-fed or nonfed mice. Cells from the PLN of OVA-fed (black bars) and nonfed (white bars) OVA-immunized mice were cultured with 100 μg/ml OVA for 24–96 hours, and cytokine secretion was measured by ELISA. The results indicate that the OVA-fed mice had increased secretion of IL-4 (72 hours) and IL-10 (72 hours) and a decreased secretion of IFN- (24 hours) and IL-2 (24 hours) compared with nonfed mice.

Isolated loops remain intact, without evidence of inflammation.

Before initiating studies involving Ag administration in the loops, we needed to ensure that the surgical procedure itself would not induce local inflammation or disruption of mucosal architecture, as this could prevent tolerance induction by altering Ag trafficking patterns. Furthermore, we needed to confirm the presence or absence of PPs. Therefore, 10 days after surgery, we excised loops for histologic analysis. As shown in Figure 2, H&E staining of a cross-section of a representative loop without PPs (E loop; Figure 2, A and B) and a representative M loop (containing PPs and M cells; Figure 2, C and D) revealed normal architecture and no evidence of active inflammation.

   Figure 2

Microscopic evaluation of intestinal loops. E and M loops were excised 10 days after surgery and fixed in 10% formalin for H&E staining or immediately fixed in glutaraldehyde for electron microscopy. Cross-sections were cut and stained with hematoxylin and counterstained with eosin. (A and B) Representative sections from the E loop (no PP). (C and D) Representative sections from the M loop. The PP is indicated by an arrow in C. Original magnification, x10 (A and C) and x40 (B and D). (E and F) Electron micrographs of a loop 10 days after surgery (E) compared with normal bowel (F) at x40,000 magnification. Desmosomes (arrows) indicate the presence of tight junctions. As shown, cross-sections of the loops revealed normal architecture as well as an intact epithelium and no evidence of active inflammation. (G and H) Deconvolution micrograph demonstrating the presence of DCs in the lamina propria of distal jejunal segments in CD11c-GFP mice (G) and in controls (H). Note that the dendrites failed to reach the epithelium in these segments (in contrast to the distal ileum). A 3D reconstruction (supplemental data) further demonstrates the failure of DCs to invade the epithelium in this part of the small intestine.

It was also important to assess the possibility that the surgical procedure had damaged the mucosal barrier, allowing for paracellular transport of the OVA. Therefore, we analyzed the loops for the presence of tight junctions by electron microscopy. Figure 2E shows a representative section of the loop 10 days after surgery. The arrows indicate the presence of desmosomes. The cell-cell contacts between epithelial cells in the loop appear normal when compared with normal small bowel from mice that had not undergone surgery (Figure 2F). Importantly, no subepithelial DCs were noted on any of the sections.

Previous studies have suggested that intestinal DCs express tight junction proteins and can intercalate their dendrites between absorptive epithelial cells (27). These findings suggest that subepithelial/intraepithelial DCs might sample Ag from the lumen and carry these Ags to local draining lymph nodes. More recent studies suggest that these DCs are present predominantly in the distal small bowel (ileum). The loops generated in our model are from the jejunoileal junction. In order to determine whether intraepithelial DCs were present in the loops, we used 2 different approaches. We used CD11c-GFP transgenic mice, originally generated by the Littman laboratory (28). DCs from these mice fluoresce spontaneously. Using deconvolution microscopy, we were able to demonstrate DCs within the lamina propria of the jejunum. Fluorescent dendrites did not extend into the epithelium in any section (Figure 2G and supplemental data; supplemental material available online with this article; doi:10.1172/JCI19102DS1). Direct staining with a FITC-conjugated anti-CD11c mAb also failed to demonstrate the presence of intraepithelial DCs (data not shown). In summary, we analyzed loops using fluorescence microscopy and electron microscopy and could not document intraepithelial dendrites using either of these approaches.

Permeability in loops remains unaltered.

To eliminate the possibility that the surgical procedure could have influenced the permeability of the loops to Ag, we performed electrical resistance studies, analyzing the tissues in Ussing chambers. Epithelial permeability has been associated with a loss of tolerance, and this would have prevented us from drawing valid conclusions from this study. Mice were sacrificed within 2 weeks after surgery, and the permeability of the isolated loops was compared with that seen in intact bowel segments from either the operated mice (bowel in continuity) or nonoperated control mice. As shown in Table 1, the resistance across the barrier within the loops was comparable to that seen across the intact intestine. These findings as well as the failure to generate an immune response to intact OVA in the loops (see below) provided further evidence that the epithelium of the loops exhibited normal barrier function during the interval of the experiment.

   Table 1

Permeability of bowel segments is unaltered after surgery

Loops are functional and respond as well as intact small bowel to cholera toxin and OVA.

We next assessed whether the loops, 10 days after surgery, were immunologically intact. Cholera toxin (CT) is a known mucosal adjuvant that stimulates both local and systemic immune responses when given orally, alone or in association with soluble Ag. CT and OVA were coadministered into the loop on days 1, 21, and 28. Two weeks later, the mice were bled, sacrificed, and tested for serum anti-OVA IgG levels and anti-OVA splenic T cell proliferation. As shown in Figure 3, mice immunized with CT/OVA in the loops responded by OVA-specific T cell proliferation (Figure 3A) and production of anti-OVA antibodies (Figure 3B). The responses were similar in E and M loops, which suggests that repositioned bowel loops both with and without PP responded normally to Ag in the presence of the mucosal adjuvant CT.

   Figure 3

Response to immunization with OVA and CT in E and M loops. Ten micrograms CT and 5 mg OVA were coadministered in the loop on days 1, 21, and 28. Mice were bled and sacrificed 2 weeks later. (A) OVA-specific T cell proliferation. Splenocytes from mice immunized with OVA and CT in either E loops (white bars) or M loops (black bars) were cultured with or without 10–1,000 μg/ml OVA for 72 hours, and this was followed by addition of thymidine for 16 hours. Cells were harvested, and incorporated thymidine was read on a MicroBeta counter. The results show that splenocytes from mice immunized with CT plus OVA in an E loop or an M loop proliferated similarly in response to OVA. (B) Serum anti-OVA levels. Serum was diluted to the indicated titers and analyzed for the presence of OVA-specific IgG by ELISA. Normal mouse serum (indicated by an X) and serum from mice immunized i.p. with OVA in complete Freund’s adjuvant (triangles) were used as negative and positive controls, respectively. OVA-specific antibody titers from E loop (open circles) or M loop (filled circles) mice were statistically similar and not different from those from normal mice at the indicated dilutions.

Tolerance could be achieved with OVA fragments but not whole OVA in both E and M loops.

We next attempted to induce mucosal tolerance by administering OVA (without CT) directly into the ligated loops. As shown in Figure 4, A and B, neither tolerance nor immunization was seen in either the M loops or the E loops when whole OVA was used as the tolerogen. T cell proliferative responses were variable but not significantly decreased in the "loop-fed" mice (Figure 4A), and the serum anti-OVA IgG levels were unchanged (Figure 4B). These data suggest that the epithelial barrier is intact in the surgically repositioned intestinal loops, as described above, and that alteration of the native Ag may be required for tolerance induction. Since early reports have suggested that gastric digestion might be critical for achieving mucosal tolerance (29-31), we repeated the above experiment using pepsin-treated fragments of OVA as the tolerogen. Interestingly, administration of these fragments into the loop prior to systemic immunization did cause a significant reduction in the OVA-specific T cell proliferation (Figure 4C) as well as a reduction in the levels of serum anti-OVA antibodies (Figure 4D). This could be seen both in E and M loop mice, but it appears that pepsin-digested Ag introduced into M loops induced greater tolerance (40% of control) compared with that achieved with fragments introduced into loops without PPs (72% of control).

   Figure 4

The effect of administration of whole OVA or OVA fragments in E or M loops. (A) OVA-specific proliferation of PLN cells was measured as in Figure 2. As shown, T cell proliferation was not significantly suppressed in the mice with intact OVA administered through the E or M loop compared with OVA-nonfed animals. (B) Serum anti-OVA IgG levels. Serum from loop-fed and control mice was analyzed for OVA-specific IgG by ELISA. As shown, OVA-specific IgG levels were not reduced in mice that had received OVA either in M loops (triangles) or in E loops (squares) compared with nonfed animals (diamonds). These results indicate that neither mucosal tolerance nor immunization could be achieved by administration of whole OVA directly into either E or M loops. (C and D) OVA was digested by pepsin, and purified OVA fragments (3–30 kDa) were administered via either E loops or M loops prior to immunization and in vitro stimulation as described in Methods. (C) OVA-specific proliferative responses were reduced in T cells from the PLN of mice administered OVA through the E loop by 28% (P = 0.0016 compared with nonfed controls). Mice fed through the M loop had T cell proliferative responses reduced by 60% (P < 0.001 compared with nonfed controls). (D) Reduced serum anti-OVA antibody levels in E and M loop mice. Serum from mice that were fed OVA fragments per os (triangles) or in E loops (filled circles) or M loops (open circles) was diluted and analyzed for anti-OVA IgG and compared with serum from nonfed mice (diamonds). Serum anti-OVA antibody levels were reduced in E loops, and the reduction was enhanced in M loops. *P < 0.03, E loop compared with nonfed controls; #P < 0.001, M loop compared with nonfed controls.

High- and low-dose oral tolerance are induced in small bowel loops.

In order to study the induction of high- and low-dose tolerance in the small bowel loops, we switched to a more controllable system using OVA peptide 323–339 (OVA-pep). We explored the induction of high-dose oral tolerance by feeding 0.5 mg OVA-pep into the lumen of either M loops or E loops. This dose was chosen following a series of dose-ranging studies (data not shown). OVA-specific in vitro proliferation of LN cells from both E and M loop mice that were fed OVA-pep into the loop was decreased more than 50% (P < 0.01) compared with that of LN cells from nonfed mice (Figure 5A). Furthermore, we observed a reduction of the IFN- and IL-2 secretion by LN cells from both E and M loop mice compared with nonfed mice, whereas the levels of IL-4 and IL-10 secretion were unchanged (Figure 5, B–E).

   Figure 5

High-dose tolerance to OVA-pep can be achieved in both E and M loops. Loop mice were either not fed or fed 0.5 mg OVA-pep into E or M loops and then immunized with the same antigen in the footpads. Twelve days later, PLN cells were harvested and restimulated with OVA-pep in vitro. Proliferation (A) and cytokine production (B–E) were measured after 72 hours of incubation. Each data point represents an individual mouse, while each column represents the mean value of 4 mice, and error bars indicate SEM. **P < 0.01, #P < 0.001 vs. nonfed.

In order to investigate the induction of low-dose tolerance in the presence and absence of PP, we reduced the dose 50-fold and administered 0.01 mg OVA-pep into the loops. Upon restimulation with OVA-pep in vitro, the proliferation of LN cells both from OVA-fed E loop and M loop mice was reduced by more than 50% compared with loop that did not receive OVA-pep (P < 0.01) (Figure 6A). The levels of IFN- and IL-2 were significantly suppressed, whereas the IL-4 levels remained unchanged, and the IL-10 levels were increased in mice fed OVA-pep (Figure 6, B–E).

   Figure 6

Low-dose tolerance to OVA-pep can be achieved in both E loops and M loops. Mice with small bowel loops were either not fed or fed 0.01 mg OVA-pep into E or M loops and then immunized with the same antigen in the footpads. Twelve days later, PLN cells were harvested and restimulated in vitro. Proliferation of PLN cells upon in vitro restimulation with OVA-pep (A) and cytokine production after 72 hours of incubation (B–E). Each data point represents an individual mouse, while each column represents the mean value of 4 mice, and error bars indicate SEM. *P < 0.05, **P < 0.01 vs. nonfed.

These results indicate that both high- and low-dose tolerance to OVA peptide can be induced equally well in the presence and absence of PP.

Bystander suppression in low-dose tolerance.

Low-dose tolerance is thought to be dependent upon the generation of regulatory T cells. In order to investigate whether the generation of Th3/Tr1 cells is dependent on the presence of PP, we examined the induction of bystander suppression in loop mice. This was performed by administering OVA-pep (0.01 mg) into the loops as before, then immunizing with either a mixture of OVA-pep and another immunogen, keyhole-limpet hemocyanin (KLH), or immunizing with KLH alone. We then restimulated cells from the draining LN in vitro with KLH to assess whether there was bystander suppression. The results showed a marked decrease in KLH responsiveness in T cells in the animals that were immunized with both OVA and KLH (Figure 7A), whereas no tolerance to KLH was induced in mice fed OVA-pep and immunized with KLH alone (Figure 7B). The results were similar in M and E loop mice, which suggests that bystander suppression does occur in both PP-independent and PP-dependent systems.

   Figure 7

Bystander tolerance. Mice with small bowel loops were either fed or not fed 0.01 mg OVA-pep into loops with or without PP. The mice were then immunized in the footpads either with a mixture of OVA-pep and KLH (A) or with KLH alone (B). PLN cells were isolated 12 days later and restimulated in vitro with KLH. (A) KLH-specific proliferation of LN cells from mice immunized with OVA-pep and KLH together. (B) KLH-specific proliferation of LN cells from OVA-pep fed mice immunized with KLH alone. Proliferation was measured after 72 hours of incubation. Each data point represents an individual mouse. **P < 0.01 vs. nonfed.

IL-10 but not TGF- ? is critical for low-dose tolerance through M and E loops.

Since regulatory cells were generated by feeding through both PP- and non–PP-containing loops, we wanted to investigate the necessity for TGF-? and IL-10 for induction of tolerance in the loop mice. We first asked whether TGF-? secreting Th3 cells were critical to M loop or E loop tolerance. We treated mice with neutralizing anti–TGF-? mAbs prior to and during Ag administration (1 mg every other day for 10 days starting 1 day prior to Ag administration). This protocol has previously been shown to block TGF-? activity (26). As seen in Figure 8, when compared with treatment with an isotype-matched control mAb, this treatment failed to inhibit tolerance induction in either loop mice or intact mice. Thus TGF-? may not be critical for tolerance induction.

   Figure 8

Low-dose tolerance is independent of TGF-?. Intact or loop mice were treated with either an anti–TGF-? mAb or an isotype-matched control for 1 day prior and every other day during OVA-pep administration (total of 5 mg over 10 days). This dosing regimen had previously been shown to neutralize TGF-? activity in vivo (24). Mice were immunized on day 11 as described above, and T cell proliferation (data not shown) and IFN- secretion by draining LN T cells were assessed 5 days later (as described for Figures 4). Neutralization of TGF-? failed to inhibit tolerance induction in either intact or loop (results for E loop are shown here) mice.

We then performed tolerance studies in IL-10–/– mice and wild-type littermates (C57BL/6 mice). In contrast to the results of the studies using anti–TGF-? mAbs, there was a failure to generate low-dose tolerance in the IL-10–/– mice, whether mice were fed OVA-pep orally (data not shown) or via the loops (Figure 9, A and B), which demonstrates that low-dose tolerance generated both in the presence and absence of PPs is dependent on IL-10. Neither T cell proliferation nor IFN- secretion was decreased upon OVA administration (Figure 9, A and B). These data are consistent with those of previous studies that reported that low-dose tolerance could not be achieved in IL-10–/– mice (32) and support a role for this cytokine in induction of oral tolerance. In contrast, high-dose tolerance (0.5 mg/d for 5 days) was independent of IL-10, regardless of whether it was induced in the presence or absence of PP. T cell proliferation and IFN- secretion were significantly decreased upon OVA administration in both E and M loops (Figure 9, C and D).

   Figure 9

IL-10 is necessary for the induction of low-dose tolerance both in the presence and absence of PP. IL-10–/– mice with small bowel loops were either not fed or fed a low dose (0.01 mg) (A and B) or high dose (0.5 mg) (C and D) of OVA-pep into E or M loops and then immunized in the footpads with OVA-pep in Freund’s complete adjuvant. Twelve days after immunization, PLN cells were restimulated by OVA-pep in vitro, and proliferation and IFN- production were measured after 72 hours of incubation. (A and C) Proliferation of LN cells from IL-10–/– mice. (B and D) IFN- production from IL-10–/– mice. Each data point represents an individual mouse, while each column represents the mean of 4 mice. Error bars indicate SEM. As previously reported, low-dose tolerance cannot be achieved in intact IL10–/– mice fed orally (data not shown). **P < 0.01.

In summary, IL-10, but not TGF-?, appears to be a critical component of oral tolerance in a PP-independent model as well as a PP-dependent model.

    Discussion

M cells are quite efficient at taking up lumenal bacteria and specific viruses and transporting them into the PP for presentation by the resident APCs to T cells. Because of this capacity, the M cell has been proposed as the obvious candidate for transport of proteins in mucosal tolerance induction.

Previous studies from our laboratory have focused on the potential role of the IEC as an APC (17, 33). Another study suggests that normal IEC–T cell interactions result in the activation of regulatory CD8+ T cells controlled by the class Ib molecule CD1d and a novel CD8 ligand, gp180 (34). These studies were performed in vitro, in human systems, and there have been no in vivo studies to suggest a role for the IEC in mucosal Ag presentation, regulation of physiological inflammation in the gut, or oral tolerance.

In the present study, we took a different approach in order to study the role of PPs and M cells versus epithelial cells in the genesis of oral tolerance. By administering Ag directly into isolated noncontiguous segments of the small intestine, in which a PP was either present or absent, we could study the necessity of PPs for mucosal tolerance in an otherwise normal mouse.

Using this loop model to study oral tolerance induction, we demonstrated that OVA-specific antibody production as well as T cell proliferative responses are reduced in mice that received the tolerogen (pepsin-digested OVA fragments) in loops devoid of M cells and PP, although greater suppression was seen after administration of Ag into loops with M cells and PP. However, E and M loop mice that received OVA-pep tolerized equally well and to a greater extent (up to 80% inhibition) than those receiving pepsin-digested fragments. This suggests that tolerance to mucosally administered Ag can occur without the presence of M cells to absorb the soluble protein or PPs. Most investigators have noted that M cells do not develop in the absence of PPs or lymphoid follicles, although the existence of M cells in the villi (in the absence of PPs) has been suggested (35).

Several lines of evidence support the fact that the mucosal barrier was intact in both E and M loops. First, by electron microscopy, we demonstrated that the tight junctions were intact. Second, the administration of intact OVA into the loops failed to elicit any form of immune response. Moreover, H&E staining showed normal mucosa free of inflammation. Last, barrier integrity of the loops, as assayed by Ussing chamber studies, was comparable to that seen in intact bowel. Thus, taken together, these data support, but do not unequivocally prove, a direct role for the IEC in the sampling of Ag involved in the regulation of mucosal immune responses.

Lymphoid aggregates are occasionally seen in the small intestine mucosa. Although their function is unknown, it is possible that they are sites of lumenal Ag presentation to T cells. Recent studies by Lorenz and colleagues have suggested that there is a compensatory increase in lymphoid nodules in LT?R-Ig fusion protein–treated mice (36). These nodules are predominantly located in the terminal ileum and not in the more proximal segments of the small intestine (R. Lorenz, personal communication). The loops in the mice described in this study were derived from the distal jejunum/proximal ileum and therefore would be less likely to contain such aggregates/nodules. However, to exclude the possibility that lymphoid aggregates were in our PP-deficient loops, we performed multiple (serial) sectioning of both E and M loops. In no section did we find evidence for lymphocyte clusters.

We also found no evidence for subepithelial DCs in any of these loops. Rescigno et al. have reported that DCs can reside below the basement membrane and extend processes between the epithelium (expressing tight junction proteins) to "sample" lumenal Ags (27). Subepithelial DCs typically reside in the distal small bowel, and the loops in the present study were generated from the distal jejunum/proximal ileum. However, we still looked for the presence of such DCs using 2 different approaches. In neither case could we identify dendrites from DCs extending into the epithelium. These findings provide further support for a more primary role of IECs or M cells in Ag sampling. Indeed, previous studies by our laboratory as well as others (37, 38) have demonstrated that IECs could take up soluble Ag either in intact mice or in surgical loops via an endolysosomal pathway.

Other laboratories have also shown that some form of tolerance can be induced at mucosal sites free of M cells and PPs. Nasal tolerance (39), as well as vaginally induced tolerance (40), have been demonstrated in a murine system. In our model, the enhancement of tolerance to pepsin-digested OVA fragments in the M loops suggests that there might be multiple mechanisms, both M cell/PP–dependent and –independent, involved in oral tolerance.

Interestingly, we were not able to induce tolerance by administering whole OVA into the loops. This is in agreement with previously published data (30, 31) that report that direct administration of whole OVA into the jejunum or ileum cannot elicit tolerance, while OVA fragments or peptide administered similarly can tolerize.

In the present study, the cytokines analyzed that were significantly different in both E and M loop mice compared with nonfed mice were IFN- and IL-2. The levels of IL-10 were only increased in mice fed low-dose OVA peptide via the E loop. The decreased levels of IFN- and the moderately increased levels of IL-10 suggest that regulatory cells might be generated during tolerance induction. In support of this was the finding that low-dose tolerance could not be achieved in either E or M loops in IL-10–/– mice, while tolerance was achieved in IL-10–/– mice with high-dose OVA-pep. In contrast, neutralization of TGF-? had no effect in this system. Thus, IL-10 appears to be a more critical cytokine in our model of induction of oral tolerance. Attempts to define the nature of the cells generated by administration of Ag into the loops were thwarted by the low yield of cells isolated from the loops. Further studies using in situ hybridization and immunohistochemistry should shed some light on the cytokines induced following Ag administration into the loops.

The fact that the absorptive area in the loop is much smaller than that in the intact small bowel (by approximately 10%) could influence the amount of Ag that is absorbed from the lumen. On the other hand, as the gastric digestion processes are bypassed, it is very likely that much more immunologically intact Ag is in contact with the mucosa after loop administration than after gastric feeding. Importantly, we were able to demonstrate that OVA-pep administered into either E or M loops induced tolerance as well. This allowed for specific dose-ranging studies and the demonstration of both low- and high-dose tolerance in the loops. Although E and M loops are of the same size and both act as internal controls for absorption, it will be important for us to estimate the amount of protein in the circulation immediately following administration and compare it with that following gastric feeding.

Thus far we have not identified differences in tolerance induction generated via either E or M loops (other than an increase in IL-10 in E loop–tolerized mice). If in fact there are distinct mechanisms involved (e.g., direct presentation of Ag by IEC to lamina propria lymphocytes in E loops), we should be able to identify these using different transgenic and knockout mice (e.g., CD28–/–, CD2–/–, CD8–/–, CD4–/–, OVA TCR–transgenic mice, etc.). Such studies are currently in progress.

    Methods

Surgical procedure.

The studies were approved by the Mount Sinai Medical Center Institutional Animal Care and Use Committee. Eight week-old BALB/c mice (Jackson Laboratory) were used in this study. The mice were taken off solid food 18 hours before surgery. Under anesthesia, a 2-cm section of small intestine from approximately midway between the ileum and jejunum either with a PP (M loop) or without (E loop) was clamped and excised from the intestine without disruption of the mesenteric attachments or vascularization. The remaining intestine was religated and released from clamps. The bypassed loop was closed at one end with surgical sutures; the other end was sutured to the abdominal wall, and a small opening (ostomy) was brought to the skin surface, which allowed access to the lumen of the loop. Oral feeding with mouse chow was resumed 2 days after surgery. Histological analysis of the loops confirmed the presence or absence of PPs. No inflammation or disruption of mucosal architecture was noted. The survival rate was 50–60% over the 3-week study and 90% after the first 3 days following surgery. Mice that failed to survive died of intestinal obstruction. The isolated loops appeared viable and well vascularized. The surviving mice were not malnourished nor did they show signs of inflammation or infection.

Similar studies were performed in IL-10–/– mice (Jackson Laboratories) and wild-type littermates (C57BL/6). These mice were operated on at 8 weeks of age. Since IL-10–/– mice housed at the Mount Sinai Animal Facility have been shown to develop colitis by 14 weeks of age, the studies were completed by 11 weeks of age. These mice showed no macroscopic signs of colitis (i.e., weight loss, diarrhea, ruffled fur) at the time of sacrifice, and histological examination of the colon and small intestine did not reveal any inflammation.

Mucosal tolerance protocol.

All mice were rested for 10 days after surgery to allow for recovery. Tolerance induction was attempted with repeated administrations of OVA (Sigma-Aldrich). The Ag administration schedule was 1 mg whole OVA or 0.25 mg of 3–30 kDa fragments of pepsin-treated OVA either by gastric intubation or by administration of Ag using a flexible tube directly into the lumen of the loop for 5 consecutive days (days 1–5). On day 8, mice were immunized with 2 footpad injections of 100 μg OVA emulsified in complete Freund’s adjuvant (Sigma-Aldrich). On day 18, blood was collected from the retro-orbital plexus or tail vein, and the mice were sacrificed. Spleens and the PLNs were aseptically removed and teased into single-cell suspensions. Each mouse was analyzed separately.

In some experiments, OVA-pep was used as the Ag. OVA-pep was purchased from the peptide synthesis core at the Mount Sinai Medical Center. The purity was greater than 90%. Oral tolerance was induced as described above using 0.01–0.5 mg of peptide in 100 μl of PBS.

In a separate series of experiments, mice with E or M loops or without loops were treated with neutralizing anti–TGF-? mAbs (1 mg i.p. every other day for 10 days just prior to and during Ag administration; a kind gift of Ivan Fuss, NIH, Bethesda, Maryland, USA). This regimen had previously been shown to block TGF-? in vivo (41).

Pepsin digestion of OVA.

OVA was resuspended in deionized water and adjusted to pH 2.0 with HCl. OVA was incubated with insoluble pepsin-agarose beads (Sigma-Aldrich) overnight at 37°C, and an aliquot was run on SDS-PAGE to determine efficiency of digestion. A smear centered at 8 kDa was seen after digestion. This is in agreement with previous studies that resolved pepsin-treated OVA fragments at 8 kDa and less than 2 kDa. The 8-kDa fragment, previously found to be tolerogenic if administered orally and immunogenic if injected with adjuvant (42), was purified by consecutive centrifugations through Centricon columns (Millipore) with membrane cutoffs of 3 kDa and 30 kDa. The purified fragment was dialyzed against PBS and sterile filtered, and the concentration was determined by optical absorbance (Spectronic 601; Milton Roy) at 280 nm.

Serum anti-OVA antibody measurements.

Serum anti-OVA antibody levels were measured by ELISA. Serum was separated from blood collected from the tail vein or from the retro-orbital plexus at the time of sacrifice. Diluted serum samples were incubated for 1 hour on ELISA plates (Nalge Nunc) previously coated overnight at 4°C with 5 μg/ml OVA in 0.01 M carbonate buffer, pH 9.5. Plates were washed and incubated for 1 hour with 100 μl/well HRP-conjugated goat anti-mouse IgG (Roche Diagnostics Corp.) diluted to 0.2 μg/ml. A/B substrate (BD Biosciences — Pharmingen) was added, and colormetric analysis was performed on an ELISA reader (Bio-Tek Instruments Inc.) at a wavelength of 650 nm. As controls, sera from nonimmunized mice (normal mouse sera) and OVA-immunized (by i.p. injection) mice were used at concentrations identical to those of the samples.

In vitro analysis of T cell anti-OVA response.

PLNs were teased into single-cell suspensions and washed twice with PBS. Cells were then cultured at 2 x 106 cells/ml in RPMI 1640 supplemented with 10% FCS (Atlantic Biologicals), 2-mercapto-ethanol (5 x 10–5 M), and 1% penicillin–1% streptomycin–glutamine (2 mM) (Invitrogen Corp.) and analyzed for Ag-specific cytokine release and T cell proliferation.

Cytokine measurements.

Cells (2 x 106/ml) were cultured in the presence or absence of OVA (10–1,000 μg/ml) for 96 hours, and supernatants were collected at 24-hour intervals. Culture supernatant was analyzed for the presence of IL-10, IL-4, IL-2, and IFN- by OptEIA ELISA kits (BD Biosciences — Pharmingen) following the manufacturer’s protocol. A standard curve was generated using recombinant cytokines, and concentrations of samples were determined by a polynomial curve fit analysis.

T cell proliferation.

Cells from the PLN were cultured in the presence or absence of OVA (10–1000 μg/ml) for 72 hours, followed by a 16-hour pulse with 1 μCi thymidine (ICN Pharmaceuticals). Incorporated radioactivity was measured on a flatbed MicroBeta counter (Wallac). As a positive control for T cell proliferative capacity, cells were stimulated with Concanavalin A (Sigma-Aldrich) at 1 μg/ml.

Bystander suppression.

In order to assess induction of bystander suppression in loop mice, we administered 0.01 mg OVA-pep into the loops as described above and immunized mice in the footpads and the flank with 50 μl of either KLH (Sigma-Aldrich) alone or a mixture of KLH and OVA-pep, both at a protein concentration of 4 mg/ml and emulsified 1:1 in complete Freund’s adjuvant. KLH and OVA-pep–specific T cell proliferation was measured by culturing cells from the PLN in the presence or absence of KLH or OVA (10–100 μg/ml), respectively, for 72 hours, followed by a 16-hour pulse with 1 μCi thymidine (ICN). Incorporated radioactivity was measured as described above.

Immunohistochemistry.

Upon sacrifice, bowel loops were removed and immediately fixed in 10% formalin or snap-frozen for immunohistochemical analysis. For analysis of loop architecture and inflammatory infiltrates, sections were stained and counterstained with H&E.

Electron microscopy.

Small (1 mm3) blocks of tissue were fixed in 3% formaldehyde plus 2% glutaraldehyde in PBS, pH 7.4, for 2 hours at room temperature. After fixation, the tissue was washed 3 times in PBS and postfixed in 1% OsO4 in PBS, pH 7.4, for 60 minutes. The tissue was then washed 3 times in PBS, en bloc stained in 1% aqueous uranyl acetate for 2.5 hours, and subsequently dehydrated through a graded ethanol series (50%, 70%, 95%, 100%, and 100%; 10 minutes for each step). After dehydration, the samples were placed in a 2:5 mixture of ethanol/propylene oxide followed by a change through propylene oxide. The tissue was infiltrated overnight (on a rotator) with EMbed 812 (Electron Microscopy Sciences). The tissue was embedded in EMbed 812 and polymerized for 24 hours at 68°C. Eighty-nanometer sections were cut (using a Reichert Ultracut E ultramicrotome), collected on 200 mesh copper grids, and stained with uranyl acetate (15% saturated in 30% ethanol) for 10 minutes followed by Reynolds lead citrate for 1.5 minutes. Sections were viewed and photographed with a Hitachi H7000 transmission electron microscope operated at 75 kV.

Deconvolution microscopy.

CD11c-GFP transgenic mice (28) and were the kind gift of Akiko Iwasaka (Yale University, New Haven, Connecticut, USA). Jejunal segments were isolated, fixed in formalin, sectioned following embedding in paraffin (6-μM sections), and stained with DAPI. In some experiments, isolated loops were snap-frozen in Tissuetek OCT (Bayer Corp.), sectioned (4-μM sections), and stained with anti-CD11c FITC. The images were captured using an Olympus BX61WI work station with a motorized XY stage allowing lateral movement between the X and Y positions and a Z focusing drive to allow the focal plane to be rapidly changed. The microscope was equipped with a Sutter Lambda DG-4 high-speed wavelength changer (Sutter Instrument Co.) and 175-W xenon light source with excitation filters (360 nm, 480 nm, and 590 nm) matched to a triple-band filter (DAPI, FITC, and Cy5) and individual filter sets for DAPI, FITC, Cy3, TexasRed, and Cy5 in the body of the microscope. The image was collected using a x60 objective (NA1.4) and Coolsnap camera (Roper Inc.) (1,392 x 1,040 pixels) and analyzed by deconvolution microscopy using Slidebook 4.0 (Intelligent Imaging Innovations Inc.).

Assessment of intestinal permeability (barrier function) in loops.

Techniques assessing gastrointestinal function through measurement of ion flux have been previously described (43). The intestine was mounted in modified Ussing chambers (Physiologic Instruments Inc.) with 2.0 mm (0.031/cm2) of the mucosal and serosal surfaces exposed to 4 ml oxygenated (95% O2, 5% CO2) modified Krebs buffer maintained at 37°C with a circulating water bath (Fischer Scientific International). The buffer contained 140 mM Na, 119.8 mM Cl, 25 mM HCO3, 12 mM Mg, 1.2 mM Ca, 4.8 mM K, 2.4 mM HPO4, and 0.4 mM H2PO4, pH 7.4. Mannitol (10 mM) was added to the mucosal side, and glucose (10 mM) was added to the serosal side of the tissue for osmotic balance. Following tissue equilibration (20–30 minutes), passive ion transport, or transmural resistance ( x cm2), was measured as an indicator of barrier function.

Statistical analyses.

In all experiments, each mouse was analyzed separately. In the figures showing results for oral tolerance/T cell proliferation analyses, data points represent values for individual mice; for cytokine and serum dilution analyses, values from mice within the same group were averaged, and error bars represent the SEM. P values represent the probability associated with the 2-tailed Student’s t test. P < 0.05 was considered statistically significant.

    Acknowledgments

This work was supported by NIH grants AI23504, AI24671, and AI44236 (to L. Mayer). Electron microscopy was performed at the Mount Sinai School of Medicine–Microscopy Shared Resource Facility, supported, in part, with funding from NIH–National Cancer Institute shared resources grant (1 R24 CA095823-01). A portion of this work appears as part of the doctoral thesis of T.A. Kraus.

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日期:2007年5月11日 - 来自[2005年第115卷第8期]栏目
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Irritable Bowel, Pain Syndromes Linked

Sept. 28, 2006 -- Doctors have long suspected a link between irritable bowel syndrome, pain syndromes, and depression. New data now strongly support this theory.

The findings come from data on 97,593 people with irritable bowel syndrome enrolled in a large U.S. health plan from 1996 to 2002. J. Alexander Cole, DSc, MPH, and colleagues at Boston University compared these patients with 27,402 people seeking routine health care.

Their results show that people with irritable bowel syndrome are:

  • 80% more likely to suffer fibromyalgiafibromyalgia
  • 60% more likely to suffer migraine
  • 40% more likely to suffer depression
  • Overall, 60% more likely to suffer fibromyalgia, migraine, or depression

"Perhaps what is driving the relation between irritable bowel syndrome and these other conditions is some underlying biological disorder," Cole tells WebMD. "Nobody is sure what this could be. But people suggest that there is this constellation of symptoms among people with irritable bowel syndrome, fibromyalgia, migraine, and depression that might present in different ways."

Cole and colleagues report their findings in the Sept. 28 issue of the online journal BMC Gastroenterology.

Common Cause of Pain Syndromes?

Cole, now an epidemiologist with i3 Drug Safety, is not an expert on irritable bowel syndrome. Reza Shaker, MD, is. Shaker, chief of gastroenterology and hepatology at the Medical College of Wisconsin, was not involved in the Cole study.

"Clinical observations of patients with pain syndromes indicate that we are dealing with a syndrome bigger than a single organ," Shaker tells WebMD. "These findings confirm these previous observations."

Shaker says people with irritable bowel syndrome and people with pain syndromes such as fibromyalgia and migraine have something in common. They all have nerve pathways which somehow have become vastly oversensitive to pain signals -- a process doctors call sensitization.

Perhaps, Shaker suggests, there's a common problem at the crossroads where these nerve pathways intersect.

"Is it possible that there is an event -- possibly an early life event -- that affects the crossroads of all these nerve pathways?" he asks. "In areas where these nerves cross, it could be that there is sensitization occurring, affecting different neural circuits."

Cole suggests that different doctors looking at the same underlying illness might make different diagnoses. A gastroenterologist, for example, might diagnose irritable bowel syndrome, while a rheumatologist might diagnose fibromyalgia.

This sounds a lot like the blind men who, on first encountering an elephant, declare it to be like a snake or a tree depending on whether they are touching the elephant's trunk or its leg. Shaker says this analogy is apt. But most doctors, he says, will examine the whole elephant, not just its parts.

"A professional doesn't just focus on one symptom. If we see irritable bowel syndrome along with noncardiac chest pain or fibromyalgia, then we tackle this," he says. "But we doctors need to have a more global picture of this, instead of pigeonholing our diagnosis according to our own specialty or subspecialty."


SOURCES: Cole, J.A. BMC Gastroenterology, Sept. 28, 2006; vol 6: pp 26. J. Alexander Cole, DSc, MPH, epidemiologist, i3 Drug Safety. Reza Shaker, MD, chief, division of gastroenterology and hepatology, Medical College of Wisconsin, Milwaukee.

日期:2006年9月29日 - 来自[General Health]栏目
共 2 页,当前第 1 页 9 1 2 :

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