Helicobacter felis Eradication Restores Normal Architecture and Inhibits Gastric Cancer Progression in C57BL/6 Mice
2005; Elsevier BV; Volume: 128; Issue: 7 Linguagem: Inglês
10.1053/j.gastro.2005.02.066
ISSN1528-0012
AutoresXun Cai, Jane E. Carlson, Calin Stoicov, Hanchen Li, Timothy C. Wang, JeanMarie Houghton,
Tópico(s)Cancer-related molecular mechanisms research
ResumoBackground & Aims: The impact of Helicobacter eradication therapy on the progression or regression of gastric lesions is poorly defined. This study examined the effects of eradication therapy on inflammation, atrophy, metaplasia, dysplasia, and cancer progression. Methods: C57BL/6 mice were infected with Helicobacter felis and received bacterial eradication therapy after 2, 6, or 12 months of infection. The gastric mucosa was examined at early, mid, and late intervals after eradication and graded for histology, expression pattern of α-catenin and β-catenin, and IQGAP1. Results: Eradication of Helicobacter infection after 2 or 6 months of infection led to a regression of inflammation, restoration of parietal cell mass, and reestablishment of normal architecture. Progression to adenocarcinoma was prevented. Bacterial eradication at 1 year was associated with the reappearance of parietal cells, partial regression of inflammation, and restoration of architecture. Hyperplasia scores significantly improved, and dysplasia did not progress. Infected mice developed antral adenocarcinoma and gastric outlet obstruction by 24 months. Only 30% of the mice receiving bacterial eradication therapy at 12 months developed antral carcinoma. Bacterial eradication at any time during the first year of infection prevented death due to gastric outlet obstruction. The expression pattern of α-catenin, β-catenin, and IQGAP1 varied with cell type and paralleled histologic changes. Conclusions: Inflammation, metaplasia, and dysplasia are reversible with early eradication therapy; progression of dysplasia was arrested with eradication therapy given as late as 1 year and prevented gastric cancer-related deaths. Background & Aims: The impact of Helicobacter eradication therapy on the progression or regression of gastric lesions is poorly defined. This study examined the effects of eradication therapy on inflammation, atrophy, metaplasia, dysplasia, and cancer progression. Methods: C57BL/6 mice were infected with Helicobacter felis and received bacterial eradication therapy after 2, 6, or 12 months of infection. The gastric mucosa was examined at early, mid, and late intervals after eradication and graded for histology, expression pattern of α-catenin and β-catenin, and IQGAP1. Results: Eradication of Helicobacter infection after 2 or 6 months of infection led to a regression of inflammation, restoration of parietal cell mass, and reestablishment of normal architecture. Progression to adenocarcinoma was prevented. Bacterial eradication at 1 year was associated with the reappearance of parietal cells, partial regression of inflammation, and restoration of architecture. Hyperplasia scores significantly improved, and dysplasia did not progress. Infected mice developed antral adenocarcinoma and gastric outlet obstruction by 24 months. Only 30% of the mice receiving bacterial eradication therapy at 12 months developed antral carcinoma. Bacterial eradication at any time during the first year of infection prevented death due to gastric outlet obstruction. The expression pattern of α-catenin, β-catenin, and IQGAP1 varied with cell type and paralleled histologic changes. Conclusions: Inflammation, metaplasia, and dysplasia are reversible with early eradication therapy; progression of dysplasia was arrested with eradication therapy given as late as 1 year and prevented gastric cancer-related deaths. The relationship between Helicobacter infection and gastric carcinoma is well known.1IARC Working Group on the evaluation of carcinogenic risks to humansSchistosomes, liver flukes and Helicobacter pylori.IARC Monogr Eval Carcinog Risks Hum. 1994; 61: 1-241PubMed Google Scholar Bacterial eradication leads to regression of mucosa-associated lymphoid tissue lymphoma2Zucca E. Cavalli F. Are antibiotics the treatment of choice for gastric lymphoma?.Curr Hematol Rep. 2004; 3: 11-16PubMed Google Scholar; however, the effects of bacterial eradication therapy on gastric adenocarcinoma are more controversial. Epidemiologically, eradication of Helicobacter pylori is linked to a decrease in the incidence of gastric adenocarcinoma3Sepulveda A.R. Coelho L.G. Helicobacter pylori and gastric malignancies.Helicobacter. 2002; 7: 37-42Crossref PubMed Scopus (58) Google Scholar, 4Ley C. Mohar A. Guarner J. Herrera-Goepfert R. Figueroa L.S. Halperin D. Johnstone I. Parsonnet J. Helicobacter pylori eradication and gastric preneoplastic conditions a randomized, double-blind, placebo-controlled trial.Cancer Epidemiol Biomarkers Prev. 2004; 13: 4-10Crossref PubMed Scopus (117) Google Scholar and appears to have the most profound effects if eradication therapy is given before the development of precancerous lesions.5Wong B.C. Lam S.K. Wong W.M. Chen J.S. Zheng T.T. Feng R.E. Lai K.C. Hu W.H. Yuen S.T. Leung S.Y. Fong D.Y. Ho J. Ching C.K. Chen J.S. China Gastric Cancer Study GroupHelicobacter pylori eradication to prevent gastric cancer in a high-risk region of China a randomized controlled trial.JAMA. 2004; 291: 187-194Crossref PubMed Scopus (1248) Google Scholar The effects of eradication therapy are less clear when infection has persisted for long periods or when mucosal changes are severe,6Leung W.K. Lin S.R. Ching J.Y. To K.F. Ng E.K. Chan F.K. Lau J.Y. Sung J.J. Factors predicting progression of gastric intestinal metaplasia results of a randomised trial on Helicobacter pylori eradication.Gut. 2004; 53: 1244-1249Crossref PubMed Scopus (351) Google Scholar suggesting there is a stage when “premalignant” changes are irreversible and progressive despite bacterial eradication. These studies raise important questions: at what point are mucosal lesions reversible, and what is the “point of no return” when they are irreversible and/or progressive? Human studies suggest improvement in gastric atrophy and intestinal metaplasia in some patients7Zhou L. Sung J.J. Lin S. Jin Z. Ding S. Huang X. Xia Z. Guo H. Liu J. Chao W. A five-year follow-up study on the pathological changes of gastric mucosa after H. pylori eradication.Chin Med J. 2003; 116: 11-14PubMed Google Scholar, 8Ito M. Haruma K. Kamada T. Mihara M. Kim S. Kitadai Y. Sumii M. Tanaka S. Yoshihara M. Chayama K. Helicobacter pylori eradication therapy improves atrophic gastritis and intestinal metaplasia a 5-year prospective study of patients with atrophic gastritis.Aliment Pharmacol Ther. 2002; 16: 1449-1456Crossref PubMed Scopus (176) Google Scholar but not in others9Ruiz B. Garay J. Correa P. Fontham E.T. Bravo J.C. Bravo L.E. Realpe J.L. Mera R. Morphometric evaluation of gastric antral atrophy improvement after cure of Helicobacter pylori infection.Am J Gastroenterol. 2001; 96: 3281-3287Crossref PubMed Google Scholar who have received successful eradication therapy. Because of limitations in human studies, investigation has turned to animal models to further address the role of bacterial eradication on reversibility of premalignant lesions and the effect on progression to gastric adenocarcinoma. Bacterial eradication in the Mongolian gerbil model appears to be effective in preventing gastric cancer if given in the early stage of inflammation10Nozaki K. Shimizu N. Ikehara Y. Inoue M. Tsukamoto T. Inada K. Tanaka H. Kumagai T. Kaminishi M. Tatematsu M. Effect of early eradication on Helicobacter pylori-related gastric carcinogenesis in Mongolian gerbils.Cancer Sci. 2003; 94: 235-239Crossref PubMed Scopus (127) Google Scholar; however, this model is limited in that infection alone does not reliably induce tumors11Watanabe T. Tada M. Nagai H. Sasaki S. Nakao M. Helicobacter pylori infection induces gastric cancer in Mongolian gerbils.Gastroenterology. 1998; 115: 642-648Abstract Full Text Full Text PDF PubMed Scopus (933) Google Scholar, 12Honda S. Fujioka T. Tokieda M. Satoh R. Nishizono A. Nasu M. Development of Helicobacter pylori-induced gastric carcinoma in Mongolian gerbils.Cancer Res. 1998; 58: 4255-4259PubMed Google Scholar, 13Ogura K. Maeda S. Nakao M. Watanabe T. Tada M. Kyutoku T. Yoshida H. Shiratori Y. Omata M. Virulence factors of Helicobacter pylori responsible for gastric diseases in Mongolian gerbil.J Exp Med. 2000; 192: 1601-1610Crossref PubMed Scopus (259) Google Scholar, 14Zhen Q. Chen Y.X. Shi Y. Xiao X.D. Development of gastric adenocarcinoma in Mongolian gerbils after long-term infection with Helicobacter pylori.J Gastroenterol Hepatol. 2004; 19: 1192Crossref PubMed Scopus (49) Google Scholar and use of the cocarcinogen N-methyl-N-nitrosourea does not produce tumors in all infected animals. Mouse models of infection recapitulate human disease where infection causes gastric adenocarcinoma without the need for a chemical carcinogen, making the murine model very attractive for further study. Infection of the C57BL/6 strain of mice with SS1, a mouse-adapted H pylori strain, results in chronic colonization and hypertrophy, but these mice do not appear to consistently develop dysplasia and carcinoma.15Thompson L.J. Dannon S.J. Wilson J.E. O’Rourke J.L. Salama N.R. Falkow S. Mitchell H.J. Lee A. Chronic Helicobacter pylori infection with Sydney strain 1 and a newly identified mouse-adapted strain (Sydney strain 2000) in C57BL/6 and BALB/c mice.Infect Immun. 2004; 723: 4668-4679Crossref Scopus (74) Google Scholar On the other hand, Helicobacter felis infection in the C57BL/6 mouse model reproducibly results in the classic sequence of histologic changes seen in human infection: chronic gastritis, atrophy, metaplasia, dysplasia, and adenocarcinoma, with adenocarcinoma occurring in 100% of mice by 15 months of infection.16Wang T.C. Goldenring J.R. Dangler C. Ito S. Mueller A. Jeon W.K. Koh T.J. Fox J.G. Mice lacking secretory phospholipase A2 show altered apoptosis and differentiation with Helicobacter felis infection.Gastroenterology. 1998; 114: 675-689Abstract Full Text Full Text PDF PubMed Scopus (206) Google Scholar, 17Rogers A.B. Fox J.G. Inflammation and cancer. I. Rodent models of infectious gastrointestinal and liver cancer.Am J Physiol Gastrointest Liver Physiol. 2004; 286: G361-G366Crossref PubMed Scopus (116) Google Scholar, 18Houghton J. Stoicov C. Nomura S. Rogers A.B. Carlson J. Li H. Cai X. Fox J.G. Goldenring Jr., Wang T.C. Gastric cancer originating from bone marrow derived cells.Science. 2004; 306: 1568-1571Crossref PubMed Scopus (1049) Google Scholar However, longer-term studies in this model and the effects of eradication on histologic regression or progression of lesions are lacking. The purpose of this study was to establish the long-term natural history of H felis infection (beyond 15 months) and determine the effects of successful bacterial eradication on the progression and/or resolution of histologic changes and to define the expression pattern of 3 potential markers of neoplastic transformation during natural progression of H felis infection and after successful bacterial eradication. All work was performed at the University of Massachusetts Medical School. Approval was obtained from the Institution Animal Care and Use Committee of the University of Massachusetts Medical School before initiation of the study. Six-week-old male C57BL/6 mice that were viral antibody-free, parasite-free, and bacterial pathogen-free inclusive of Helicobacter species were purchased from Jackson Laboratories (Bar Harbor, ME), housed in microisolator cages, fed standard chow, allowed free access to water, and acclimated for 2 weeks before entry into the study. H felis (strain 49179) was grown as recommended; bacteria were enumerated,19Houghton J. Macera-Block L.S. Harrison L. Kim K.H. Korah R.M. Tumor necrosis factor alpha and interleukin 1β up-regulate gastric mucosal Fas antigen expression in Helicobacter pylori infection.Infect Immun. 2000; 68: 1189-1195Crossref PubMed Scopus (78) Google Scholar diluted with culture medium to 1 × 107 colony-forming units/mL, and used immediately in animal experiments. Eight-week-old C57BL/6 mice were infected with H felis (1 × 107 colony-forming units) by oral gavage 3 times at 2-day intervals. Bacterial eradication was performed at 2, 6, or 12 months with tetracycline HCl (0.5 mg/30-g mouse/day), metronidazole (0.675 mg per 30-g mouse/day), and bismuth subsalicylate (0.185 mg per 30-g mouse/day) dissolved or suspended in a total volume of 500 μL sterile water and given by oral gavage daily for 14 days or sham eradicated with 500 μL sterile water daily for 14 days. Mice were killed 2, 6, or 12 months after eradication therapy. Uneradicated mice and control/sham-infected mice served as controls for each time point (Figure 1). Mice received 1 mg/kg bromodeoxyuridine (BrdU) exactly 1 hour before they were killed. Strips of gastric tissue along the lesser curvature from the squamocolumnar junction through the pylorus were fixed in 10% neutral buffered formalin or Prefer fixative (Anatech Ltd, Battle Creek, MI) for 4 hours, processed by standard methods, embedded in paraffin, cut into 5-μm sections, and stained with H&E and Giemsa stain or processed for immunohistochemistry (IHC). Histology was scored by 2 blinded reviewers using a numeric scale of 1–4 using the MIT scoring criteria as described previously.20Stoicov C. Whary M. Rogers A.B. Lee F.S. Klucecsek K. Li H. Cai X. Reza S. Ge Z. Khan I. Luster A. Fox J.G. Houghton J. Co-infection modulates inflammatory response and clinical outcomes of Helicobacter felis and Toxoplasma gondii infection.J Immunol. 2004; 173: 3329-3336PubMed Google Scholar Mice found moribund or who had lost >15% of their greatest achieved weight were killed; the tissues were evaluated and mice were counted as mortalities. A 2 × 2-mm piece of gastric mucosa taken at the fundus/antral border was snap frozen at the time of necropsy. All samples were processed together as described previously.20Stoicov C. Whary M. Rogers A.B. Lee F.S. Klucecsek K. Li H. Cai X. Reza S. Ge Z. Khan I. Luster A. Fox J.G. Houghton J. Co-infection modulates inflammatory response and clinical outcomes of Helicobacter felis and Toxoplasma gondii infection.J Immunol. 2004; 173: 3329-3336PubMed Google Scholar Each sample was analyzed in triplicate. Mice were scored as infected if the 225–base pair flaB fragment was amplified with ≤40 cycles and scored as negative if >45 cycles fail to produce a band of appropriate size in the eradicated mouse while producing an appropriate size band in a known infected control. Proliferation was assayed using the BrdU in situ detection kit (BD Biosciences Pharmingen, San Diego, CA) according to the manufacturer’s protocol. Well-oriented sections were visualized and photographed using the Olympus BX51 microscope and analyzed using Olympus Microsuite 835V (Melville, NY). Sections were scored and reported as BrdU-positive staining cells per 20 pit/glands (labeling index [LI]). Three sections per mouse were scored, and the average number for each animal was used in calculations. Paraffin-embedded tissue was cut into 5-μm sections, rehydrated, washed, and endogenous peroxidase inhibited with 3% hydrogen peroxide in methanol for 20 minutes. Subsequently, slides were heated to 89°C for 20 minutes in citrate buffer (0.1 mol/L sodium citrate, pH 6.0) for antigen retrieval. After blocking with 5% normal goat serum, slides were incubated with primary rabbit antibody H+,K+–adenosine triphosphatase (H+,K+-ATPase) 1:800 dilution (EMD Biosciences Inc, San Diego, CA), IQGAP1, and α-catenin or β-catenin 1:200 dilution (Santa Cruz Biotechnology, Santa Cruz, CA) at 4°C overnight. Slides were washed with phosphate-buffered saline and incubated with biotinylated anti-rabbit secondary antibody (Vectastain ABC kit; Vector Laboratories, Burlingame, CA) for 30 minutes at room temperature followed by an additional 30-minute incubation with ABC reagent (Vectastain ABC kit) at room temperature. Color was developed with 3,3′-diaminobenzidine tetrahydrochloride using the DAKO Liquid DAB Substrate-Chromogen System (DAKO Corp, Carpinteria, CA), counterstained with hematoxylin (Fisher Chemicals, Springfield, NJ), and analyzed. Pathologic data are compared using the Mann-Whitney analysis of nonparametric data and considered significant at a value of P < .05. Survival data are shown using the Kaplan-Meier curve. Data for the BrdU LI are reported as the mean ± SD and considered significant if P < .05. The infection and eradication protocols are outlined in Figure 1A. H felis infection was eradicated according to schedule using triple therapy for 14 days. Infection status was assessed in all mice at the time of necropsy by a combination of H&E staining, Giemsa staining, and flaB polymerase chain reaction (Figure 1B). Uninfected control mice ranging in age from 8 weeks to 24 months did not differ in stomach/body weight ratios or any histologic parameter evaluated and are therefore discussed together. Under control, noninfected conditions, the mucosa of the fundus (Figure 2A) had rare submucosal and mucosal mononuclear leukocytes (Figure 2A, thick arrow). Chief cells (Figure 2A, thin arrow, darker cell) populate the lower third of the fundic mucosa. Parietal cells, located in the lower two thirds of the mucosa, are larger than chief cells and are seen wedged between the chief cells (Figure 2A, thin arrow, pink cells). Parietal cells are shown at higher power using IHC directed toward the H+,K+-ATPase (Figure 2C, brown staining). The antrum (Figure 2B) consists of well-ordered glands extending down the entire thickness of the mucosa to abut the muscularis mucosae. There is a lack of inflammatory cells. At 4 months of infection (Figure 2D), inflammation was prominent and seen as both submucosal (thin arrow) and intramucosal lymphoplasmacytic infiltrates (thick arrow). There was antralization of glands, with loss of parietal and chief cells (Figure 2D [bar] and F [IHC]). Hypertrophy was prominent, with infected mucosa 50% thicker than the uninfected specimen (compare Figure 2D and A). The antrum shows hypertrophy and metaplasia of glands (Figure 2E [bar]) and mild inflammation in both the mucosa and submucosa (arrow). Neither metaplasia nor dysplasia were prominent at this early time point of infection in either the fundus or the antrum. After 8 months of infection, there was moderate to severe inflammation and hyperplasia, with near complete parietal and chief cell loss (Figure 2G and I). Metaplastic glands are seen as corkscrew-shaped and/or dilated glands, extending through the mucosa, with basally located nuclei and abundant cytoplasm (Figure 2G, arrow), resembling Brunner’s glands.21Kushima R. Manabe R. Hattori T. Borchard F. Histogenesis of gastric foveolar metaplasia following duodenal ulcer a definite reparative lineage of Brunner’s gland.Histopathology. 1999; 35: 38-43Crossref PubMed Scopus (26) Google Scholar We defined the group of long-term infected mice as those infected for 12–22 months. In these mice, inflammation remained severe (Figure 2J [12 months], M [18 months], and P [22 months]). Architectural distortion became increasingly severe, with marked hypertrophy, widespread metaplasia, and a significant increase in dysplasia. From 12 months onward, there was a complete absence of parietal cells in the sections studied by histology (Figure 2J, M, and P) or IHC (Figure 2L and O). Dysplasia progressed from large intramucosal cystic glands lined with uniform cuboidal cells (low grade)22Boivin G.P. Washington K. Yang K. Ward J.M. Pretlow T.P. Russell R. Besselsen D.G. Godfrey V.L. Doetschman T. Dove W.F. Pitot H.C. Halberg R.B. Itzkowitz S.H. Groden J. Coffey R.J. Pathology of mouse models of intestinal cancer consensus report and recommendations.Gastroenterology. 2003; 124: 762-777Abstract Full Text Full Text PDF PubMed Scopus (418) Google Scholar to invasive cystic glands penetrating beneath the muscularis mucosa with marked cellular pleomorphism and piling up of nuclei (high grade)22Boivin G.P. Washington K. Yang K. Ward J.M. Pretlow T.P. Russell R. Besselsen D.G. Godfrey V.L. Doetschman T. Dove W.F. Pitot H.C. Halberg R.B. Itzkowitz S.H. Groden J. Coffey R.J. Pathology of mouse models of intestinal cancer consensus report and recommendations.Gastroenterology. 2003; 124: 762-777Abstract Full Text Full Text PDF PubMed Scopus (418) Google Scholar (Figure 2J, M [thick arrow], and P). Fifty percent of mice examined at 12 months had adenocarcinoma of the fundus. At 18 and 22 months, 100% of mice had adenocarcinoma in the fundus with invasion through the muscle layer to the serosa (Figure 2M). Changes within the antrum lagged behind those of the fundus, became prominent at 12 months, and progressed as time of infection lengthened. There was marked antral hypertrophy, a mucous cell metaplasia similar to that seen in the fundus along with the formation of exophytic polypoid lesions consisting of severely dysplastic glands with loss of epithelial cell nuclear polarity and pronounced nuclear atypia. These lesions progressed in architectural complexity to include cribriform corkscrew glands, followed in time by bizarre glands embedded in a desmoplastic stroma with extension of the lesions below the muscularis mucosa (Figure 2K, N, and Q). These antral masses became large (Figure 2R), were found in all mice examined from the 18-month and later infection groups, and caused gastric outlet obstruction. A total of 75% fit the criteria of polypoid adenocarcinomas, defined as polypoid lesions composed of tubular and/or tubulovillous glands penetrating below the muscularis mucosa into the stalk of the polyp.22Boivin G.P. Washington K. Yang K. Ward J.M. Pretlow T.P. Russell R. Besselsen D.G. Godfrey V.L. Doetschman T. Dove W.F. Pitot H.C. Halberg R.B. Itzkowitz S.H. Groden J. Coffey R.J. Pathology of mouse models of intestinal cancer consensus report and recommendations.Gastroenterology. 2003; 124: 762-777Abstract Full Text Full Text PDF PubMed Scopus (418) Google Scholar The remaining 25% were deemed adenomas, because they had all of the above criteria except extension through the muscularis mucosa.22Boivin G.P. Washington K. Yang K. Ward J.M. Pretlow T.P. Russell R. Besselsen D.G. Godfrey V.L. Doetschman T. Dove W.F. Pitot H.C. Halberg R.B. Itzkowitz S.H. Groden J. Coffey R.J. Pathology of mouse models of intestinal cancer consensus report and recommendations.Gastroenterology. 2003; 124: 762-777Abstract Full Text Full Text PDF PubMed Scopus (418) Google Scholar Histologic changes are summarized in Figure 3. We next examined the impact of eradication on the natural progression of mucosal changes. All infected mice tested positive for H felis by a combination of polymerase chain reaction, detection of organisms by routine histology, and Giemsa staining. There was no evidence of infection in the mice that received eradication therapy. Mice were infected for 2 months, received sham or bacterial eradication therapy, and were examined at early, mid, and late time points after eradication. Within 2 months after successful bacterial eradication, inflammatory infiltrates disappeared completely, parietal cell mass was restored to control levels, and mucosal architecture was reestablished (Figure 4B). These changes were durable, because the mice examined 12 months after successful eradication had architecture indistinguishable from control mice of the same age (Figure 4C and D). There was no progression to carcinoma in situ or invasive carcinoma and no increase in mortality when compared with uninfected control mice. Histology scores are shown in Figure 3B. Mice were infected for 6 months (midpoint infection), received sham or bacterial eradication therapy, and were examined at early, mid, and late time points after eradication. Infection in this group resulted in architectural distortion with metaplasia, antralization of glands, and complete loss of parietal and chief cells (Figure 4E). Inflammation, hyperplasia, and dysplasia scores were substantially elevated (Figure 3C) relative to control values. As early as 2 months posteradication, inflammatory infiltrates markedly improved (Figure 4F); the inflammatory scores decreased to less than half of peak levels, returned to uninfected control levels at 6 months, and remained there for the duration of the study (Figure 3C). Hyperplasia and dysplasia scores decreased rapidly, achieved control levels at 2 months posteradication, and remained there for the duration of the study (Figure 3C). Additionally, within 2 months of treatment, parietal cell mass was reestablished (Figure 4F). This reversion to normal architecture, although slightly delayed compared with the earlier group (described previously), was durable to 1 year posteradication (Figure 4G) when parietal cell repopulation was confirmed by IHC (Figure 4H). There was no progression to carcinoma in situ or invasive carcinoma and no increase in mortality when compared with uninfected control mice. Mice infected for 12 months developed severe inflammation, with marked architectural distortion, metaplasia, severe dysplasia, and cystic dilation of fundic glands extending into the submucosa (Figure 4I), with 50% meeting the criteria for gastrointestinal neoplasia.22Boivin G.P. Washington K. Yang K. Ward J.M. Pretlow T.P. Russell R. Besselsen D.G. Godfrey V.L. Doetschman T. Dove W.F. Pitot H.C. Halberg R.B. Itzkowitz S.H. Groden J. Coffey R.J. Pathology of mouse models of intestinal cancer consensus report and recommendations.Gastroenterology. 2003; 124: 762-777Abstract Full Text Full Text PDF PubMed Scopus (418) Google Scholar Eradication therapy given at this point substantially decreased the level of inflammation (Figures 3D, 4J, and 4K). However, even 12 months after eradication therapy, residual inflammatory infiltrates remained and inflammation scores had not returned to basal levels. Indeed, the mucosa retained a substantial number of inflammatory cells within both the mucosa and the submucosal space. Hyperplasia decreased but not to a significant degree until 6 and 12 months after eradication and never achieved uninfected levels. Dysplasia scores remained high despite successful eradication therapy and did not decrease significantly even at 12 months after eradication (Figure 3D). Interestingly, though, dysplasia scores did not progress further in the eradicated group compared with a continual increase in the untreated infected mice (Figure 3A), suggesting that eradication therapy halted the progression of dysplasia. At 12 months of infection, the incidence of severe gastrointestinal intraepithelial neoplasia was 50%; 2 months after eradication, the rate remained 50% (3 of 6). The rate of severe GIN declined to 40% (2 of 5) 6 months after eradication and to only 30% (3 of 10) 10 months after eradication, suggesting a regression of established lesions with successful eradication therapy. In mice that did develop gastrointestinal intraepithelial neoplasia, lesions were fewer in number and of lower histologic grade when compared with lesions in mice actively infected. The lesions remained as large dilated cystic glands with less nuclear stratification and fewer bizarre mitoses. Gland structure of the gastrointestinal intraepithelial neoplasia was more preserved, and there was no extension to the serosa. In addition, eradication therapy restored parietal cell mass to near-normal levels at late stages of infection, as shown by both histology (Figure 4K) and IHC (Figure 4L), despite the persistence of dysplasia. Parietal cells are seen intercalating between dysplastic cells and were found abundantly in areas of dilated cystic glands and gastrointestinal intraepithelial neoplasia (Figure 4K and L). All mice infected for 12 months developed moderate to severe dysplasia; by 16 months, all developed invasive lesions (dysplasia grade 3.5–4) in both the fundus and the antrum. Fundus lesions, while becoming more aggressive histologically, remained grossly small and did not metastasize or become locally invasive beyond the stomach wall. In contrast to this were lesions in the antrum. Antral dysplasia was present in all mice infected for 12 months, and adenocarcinoma was microscopically detected in all mice infected for 16 months or more. Antral lesions rapidly increased in size, becoming obstructing in 70% at 22 months and in 100% at 24 months. Eradication therapy given at 12 months markedly decreased the progression from antral dysplasia to carcinoma, with only 30% of the eradicated mice developing invasive antral carcinoma by 24 months. The lesions in these eradicated mice were not grossly apparent and were only seen microscopically with meticulous examination of serial section. There were no mortalities in this group, and there was no incidence of gastric outlet obstruction (Figure 5). It is not clear if lesions would eventually progress, albeit at a slower rate than those in the actively infected group, because mice were not followed up beyond 24 months posteradication. Increased proliferation and widening of the proliferative zone is a hallmark of Helicobacter infection and inflammation and is believed to underlie the increased cancer risk associated with Helicobacter infection. We next addressed the effect of eradication therapy on gastric mucosal proliferation at early, mid, and late time points of infection. These alterations in proliferation were measured 2 months after effective therapy was completed to determine short-term growth effects and after 1 year to ascertain if these effects were durable. Under normal conditions, proliferating cells in the fundus are confined to the isthmus region of the gland, where the presumed multipotent gastric stem cells reside.23Karam S.M. Straiton T. Hassan W.M. Leblond C.P. Defining epithelial cell progenitors in the human oxyntic mucosa.Stem Cells. 2003; 21: 322-336Crossref PubMed Scopus (119) Google Scholar Helicobacter infection caused a marked increase in proliferation, as measured by BrdU incorporation. Mice receiving eradication therapy early in infection (Figure 6A) h
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