Inactivation of p21WAF1/cip1 enhances intestinal tumor formation in Muc2-/- mice

In the Apc1638(+/-) mouse model of intestinal tumorigenesis, targeted inactivation of the cyclin-dependent kinase inhibitor p21(WAF1/cip1) is highly effective in enhancing Apc-initiated tumor formation in the intestine. Because p21(WAF1/cip1) plays a critical role in regulating intestinal cell proliferation, maturation, and tumorigenesis, we examined whether its inactivation would enhance tumor formation in a different mouse model of colon cancer. Therefore, we mated p21(-/-) mice with mice carrying a genetic deficiency of the Muc2 gene, which encodes the major gastrointestinal mucin. Muc2(-/-) mice develop tumors in the small and large intestine and the rectum, but in contrast to tumors in Apc1638(+/-) mice, this does not involve increased expression or nuclear localization of beta-catenin. We found that inactivation of p21(WAF1/cip1) significantly increased the frequency and size of intestinal tumors in Muc2 knockout mice and also led to development of more invasive adenocarcinomas. This enhanced tumorigenesis significantly decreased mouse life span. Further, inactivation of p21(WAF1/cip1) increased cell proliferation, decreased apoptosis, and decreased intestinal trefoil factor expression in the mucosa of both the small and large intestine. Surprisingly, reduced expression of p27(kip1) was also observed in the Muc2(-/-), p21(+/-), and p21(-/-) mice. In contrast, the expression of c-myc was significantly elevated. Thus, p21 modulates the formation of tumors whose initiation does (Apc) or does not (Muc2) involve altered beta-catenin-Tcf4 signaling, but which may converge on common elements downstream of this signaling pathway.     

Persistent hepatitis and enterocolitis in germfree mice infected with Helicobacter hepaticus.

Helicobacter hepaticus has been associated with naturally occurring hepatitis in certain inbred strains of mice, and in A/JCr mice it has been linked to the development of hepatic adenomas and adenocarcinomas. H. hepaticus was orally inoculated into 30 axenic, outbred female mice, and the mice were studied longitudinally to fulfill Koch's postulates and to ascertain the pathogenic potential of the organism under defined germfree conditions. Ten cage contact mice were also housed in the same germfree isolator to study transmission patterns, and 10 germfree mice were maintained in separate isolators as controls. Mice serially euthanized from 3 weeks through 24 months postinoculation (p.i.) were surveyed by culture and PCR for H. hepaticus in liver and intestinal tissues. Tissues were analyzed for histopathological changes, and sera were assayed for the presence of immunoglobulin G antibody to H. hepaticus and changes in the liver enzyme alanine aminotransferase. Inoculated mice and cage contact mice were persistently infected with H. hepaticus as identified by culture and PCR, in both the intestine and, less frequently, the liver, for the duration of the 2-year study. Animals developed persistent chronic hepatitis, and in some animals enterocolitis was noted. Hepatocellular carcinoma was diagnosed in one H. hepaticus-infected mouse. The level of H. hepaticus serum antibody was highest in experimentally infected mice at 12 to 18 months p.i.; this corresponded in general to the time interval when the highest levels of alanine aminotransferase were recorded. Although cage contact mice became persistently infected with H. hepaticus, lesions were less severe and the levels of serological biomarkers utilized in the study were lower. The H. hepaticus-infected mouse will provide an ideal model to study putative bacterial virulence determinants and how they interact with the host to induce chronic inflammation and tumorigenesis.     

Colorectal cancers in a new mouse model of familial adenomatous polyposis: influence of genetic and environmental modifiers

Murine models of familial adenomatous polyposis harbor a germinal heterozygous mutation on Apc tumor suppressor gene. They are valuable tools for studying intestinal carcinogenesis, as most human sporadic cancers contain inactivating mutations of APC. However, Apc(+/-) mice, such as the well-characterized Apc(Min/+) model, develop cancers principally in the small intestine, while humans develop mainly colorectal cancers. We used a Cre-loxP strategy to achieve a new model of germline Apc invalidation in which exon 14 is deleted. We compared the phenotype of these Apc(Delta14/+) mice to that of the classical Apc(Min/+). The main phenotypic difference is the shift of the tumors in the distal colon and rectum, often associated with a rectal prolapse. Thus, the severity of the colorectal phenotype is partly due to the particular mutation Delta14, but also to environmental parameters, as mice raised in conventional conditions developed more colon cancers than those raised in pathogen-free conditions. All lesions, including early lesions, revealed Apc LOH and loss of Apc gene expression. They accumulated beta-catenin, overexpressed the beta-catenin target genes cyclin D1 and c-Myc, and the distribution pattern of glutamine synthetase, a beta-catenin target gene recently identified in the liver, was mosaic in intestinal adenomas. The Apc(Delta14/+) model is thus a useful new tool for studies on the molecular mechanisms of colorectal tumorigenesis.     

Intestinal epithelial cell-specific CD98 expression regulates tumorigenesis in Apc(Min/+) mice

The transmembrane glycoprotein CD98 regulates integrin signaling that in turn controls cell proliferation and survival. CD98 expression is upregulated in various carcinomas, including colorectal cancer. Recently, by generating gain- and loss-of-function mouse models featuring genetic manipulation of CD98 expression specifically in intestinal epithelial cells (IECs), we have explored the crucial role of CD98 in the regulation of intestinal homeostasis and inflammation-associated tumorigenesis. In the present study, we investigated the contribution of CD98 to intestinal tumorigenesis in Apc(Min/+) mice and the underlying mechanism of action. Mice featuring IEC-specific CD98 overexpression (Tg animals) were crossed with Apc(Min/+) mice, and the characteristics of intestinal adenoma formation were assessed. Compared with Apc(Min/+) mice, Tg/Apc(Min/+) animals exhibited increases in both intestinal tumor incidence and tumor size; these parameters correlated with enhanced proliferation and decreased apoptosis of IECs. IEC-specific CD98 overexpression resulted in increased synthesis of the oncogenic proteins c-myc and cyclin-D1 in Apc(Min/+) mice, independently of the Wnt-APC-β-catenin pathway, suggesting the implication of CD98 overexpression-mediated Erk activation. IEC-specific CD98 overexpression enhanced the production of proinflammatory cytokines and chemokines that are crucial for tumorigenesis. We validated our results in mice exhibiting IEC-specific CD98 downregulation (CD98(flox/+)VillinCre animals). IEC-specific CD98 downregulation efficiently attenuated tumor incidence and growth in Apc(Min/+) mice. The reduction of intestinal tumorigenesis upon IEC-specific CD98 downregulation was caused by the attenuation of IEC proliferation and cytokine/chemokine production. In conclusion, we show that CD98 exerts an oncogenic activity in terms of intestinal tumorigenesis, via an ability to regulate tumor growth and survival.     

Identification of the modifier of Min 2 (Mom2) locus, a new mutation that influences Apc-induced intestinal neoplasia.

Min (Multiple intestinal neoplasia) mice carry a dominant mutation in the adenomatous polyposis coli (Apc) gene and develop multiple adenomas throughout their intestinal tract (Moser et al. 1990; Su et al 1992). Polyp multiplicity in Min mice is greatly influenced by genetic background. A modifier locus, Mom1 (Modifier of Min 1), was identified and localized to distal mouse chromosome 4 (Moser et al. 1992; Dietrich et al. 1993), and accounts for some of the genetic variance in polyp multiplicity. Mom1 is a semidominant modifier of polyp size and multiplicity in Min mice (Gould and Dove 1997), and encodes the secretory type II nonpancreatic phospholipase A2 (Pla2g2a) gene (MacPhee et al. 1995; Cornier et al. 1997, 2000). We now report the identification of a second Modifier of Min 2 (Mom2) locus that is the result of a spontaneous mutation. One resistant Mom2 allele can suppress 88%-95% of polyps detected in Apc(Min)/+ mice, indicating that Mom2 acts in a dominant fashion. Linkage analysis has localized Mom2 to distal mouse chromosome 18. The effects of the Mom2 locus on reducing polyp multiplicity are stronger than the effects of the Mom1 locus, in both the small and large intestines. Some Apc(Min)/+ mice that carried one resistant Mom2 allele were tumor-free at 21 weeks of age, even in the absence of a resistant Mom1 allele. Thus, the resistant Mom2 allele can, in some cases, completely suppress the penetrance of the Apc(Min) mutation.     

P-选择素对ApcMin/+小鼠肠道肿瘤的作用

目的: 研究P-选择素(P-selectin)在Apc^Min/+小鼠肠道肿瘤中的作用。方法: 采用P-selectin 基因缺失的基因工程小鼠和肠道肿瘤模型Apc^Min/+小鼠杂交,计数Apc^Min/+小鼠与Apc^Min/+ P-selectin ^-/-杂交小鼠小肠及大肠肿瘤的数目,并测量其肿瘤体积,研究P-selectin对Apc^Min/+小鼠肠道肿瘤的作用。结果: 与Apc^Min/+小鼠相比,Apc^Min/+P-selectin^-/-杂交小鼠在9周龄时肠道肿瘤数目与总负荷明显减少。结论: P-selectin 缺失能够显著抑制Apc^Min/+小鼠肠道肿瘤的生长。     

Developmental abnormalities in multiple proliferative tissues of Apc(Min/+) mice

Germ-line mutation of the Apc gene has been linked to familial adenomatous polyposis (FAP) that predisposes to colon cancer. Apc(Min/+) mice, heterozygous for the Apc gene mutation, progressively develop small intestinal tumours in a manner that is analogous to that observed in the colon of patients with FAP (Su et al. 1992; Fodde et al. 1994; Moser et al. 1995). We have studied the effects of Apc gene mutation on murine intestinal and extra-intestinal, proliferatively active tissues. We have contrasted the histology to that of the age- and sex-matched wild-type C57BL/6 mice. Histological assessment of the normal appearing intestinal mucosa demonstrates minimal change in size of crypts. In contrast, villi are longer in the ileum of Apc(Min/+) mice relative to C57BL/6 mice at 12 and 15 weeks of age. Vigorous splenic haematopoiesis in Apc(Min/+) mice was seen at 12 and 15 weeks of age, as reflected by marked splenomegaly, increased splenic haematopoietic cells and megakaryocytes. Peripheral blood counts, however, did not differ between C57BL/6 and Apc(Min/+) mice at 15 weeks of age. Lymphoid depletion in Apc(Min/+) mice was characterized by diminished numbers of splenic lymphoid follicles and small intestinal Peyer's patches. The ovaries of 12- and 15-week-old Apc(Min/+) mice exhibited increased numbers of atretic follicles, and estrous cycling by serial vaginal smears showed tendency of elongation in the mutant mice during these age ranges. The testicles of 10-week-old Apc(Min/+) mice showed increased numbers of underdeveloped seminiferous tubules. Collectively, these data suggest that, in addition to its obvious effects upon intestinal adenoma formation, Apc gene mutation causes impairment of developmental and apparent differentiation blockade in proliferative tissues, including those of the haematopoietic system, lymphoid and reproductive tract.     

Apc mice: models, modifiers and mutants

The mouse provides an excellent in vivo system with which to model human diseases and to test therapies. Mutations in the Adenomatous polyposis coli (APC) gene are required to initiate familial adenomatous polyposis (FAP) and are also important in sporadic colorectal cancer tumorigenesis. The (multiple intestinal neoplasia Min) mouse contains a point mutation in the Apc gene, develops numerous adenomas and was the first model used to study the involvement of the Apc gene in intestinal tumorigenesis. The model has provided examples of modifying loci (called Modifiers of Min: Mom) in mice, demonstrating the principle of genetic modulation of disease severity. A spectrum of Apc mutant mice has since been developed, each with defining characteristics, some more able to accurately model human polyposis and colon cancer. We will focus our review on Apc mutant mouse models, the advent of models with concurrent or compound mutations and the importance of genetic background when modeling polyposis and cancer. Brief consideration will be given to the use of these models in drug testing.     

Lactobacillus reuteri promotes Helicobacter hepaticus-associated typhlocolitis in gnotobiotic B6.129P2-IL-10(tm1Cgn) (IL-10(-/-) ) mice

To model inflammatory bowel disease, we assessed infection with Helicobacter hepaticus 3B1 (ATCC 51449) and a potential probiotic Lactobacillus reuteri (ATCC PTA-6475) in gnotobiotic B6.129P2-IL-10(tm1Cgn) (IL-10(-/-) ) mice. No typhlocolitis developed in germ-free controls (n=21) or in L. reuteri (n=8) or H. hepaticus (n=18) mono-associated mice for 20 weeks post-infection. As positive controls, three specific pathogen-free IL-10(-/-) mice dosed with H. hepaticus developed severe typhlocolitis within 11 weeks. Because L. reuteri PTA-6475 has anti-inflammatory properties in vitro, it was unexpected to observe significant typhlocolitis (P<0·0001) in mice that had been infected with L. reuteri followed in 1 week by H. hepaticus (n=16). The H. hepaticus colonization was not affected through 20 weeks post-infection but L. reuteri colonization was lower in co-infected compared with L. reuteri mono-associated mice at 8-11 weeks post-infection (P<0·05). Typhlocolitis was associated with an increased T helper type 1 serum IgG2c response to H. hepaticus in co-infected mice compared with H. hepaticus mono-associated mice (P<0·005) and similarly, mRNA expression in caecal-colonic tissue was elevated at least twofold for chemokine ligands and pro-inflammatory interleukin-1α (IL-1α), IL-1β, IL-12 receptor, tumour necrosis factor-α and inducible nitric oxide synthase. Anti-inflammatory transforming growth factor-β, lactotransferrin, peptidoglycan recognition proteins, Toll-like receptors 4, 6, 8 and particularly 9 gene expression, were also elevated only in co-infected mice (P<0·05). These data support that the development of typhlocolitis in H. hepaticus-infected IL-10(-/-) mice required co-colonization with other microbiota and in this study, required only L. reuteri. Although the effects other microbiota may have on H. hepaticus virulence properties remain speculative, further investigations using this gnotobiotic model are now possible.     

Intestinal tumours induced by the food carcinogen 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine in multiple intestinal neoplasia mice have truncation mutations as well as loss of the wild-type Apc(+) allele.

C57BL/6J-Min/+ (multiple intestinal neoplasia) is a murine model for familial adenomatous polyposis (FAP), where the mice are heterozygous for a nonsense Apc(Min) (adenomatous polyposis coli) mutation, and therefore develop numerous spontaneous adenomas in the small intestine and colon. Neonatal exposure of Min/+ mice to the food carcinogen 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) (eight subcutaneous injections of 25 or 50 mg/kg PhIP to pups or 50 mg/kg PhIP to lactating dams) markedly increased (2--9-fold) the number of intestinal tumours, especially in the small intestine. We examined whether the Apc gene was affected in small intestinal and colonic tumours induced by PhIP. In spontaneous tumours formed in these mice, the main mechanism for tumour induction is loss of the wild-type Apc(+) allele, i.e. loss of heterozygosity (LOH). Also in the PhIP-induced tumours, this is a major mechanism, since large fractions of PhIP-induced tumours had LOH in APC: However, mechanisms other than LOH must also prevail, since a lower frequency of LOH was found in the small intestinal tumours from male mice exposed to PhIP either via breast milk (65%) or by direct injection (68%), compared with the untreated controls (92%). Tumours that had retained the wild-type Apc(+) allele were further analysed for presence of truncated Apc proteins with in vitro synthesized protein (IVSP) assay. Truncated Apc proteins, indicating truncation mutations in exon 15 of the Apc gene, were detected in 20% (8 of 40) of the tumours not showing LOH from the small intestine after PhIP exposure, all in segment 2 (codons 686--1217). Seventeen percent (2 of 12) of the colonic tumours had a truncated Apc protein in segment 3 (codons 1099--1693). Importantly, no truncated proteins were detected in tumours from unexposed mice with apparently retained wild-type Apc(+) allele. These results show that PhIP induces intestinal tumours in the Min/+ mice both by causing LOH and truncation mutations in the wild-type Apc(+) allele.     

Loss of heterozygosity in spontaneous and X-ray-induced intestinal tumors arising in F1 hybrid min mice: evidence for sequential loss of APC(+) and Dpc4 in tumor development

Min (multiple intestinal neoplasia) mice carry a mutant allele of the murine Apc (adenomatous polyposis coli) locus and are predisposed to intestinal adenoma formation in the intestinal tract. Early studies have shown complete loss of function of Apc by whole chromosome loss on the tumor-sensitive C57BL/6J genetic background and in AKR x B6 F1 hybrids. Gamma-radiation-induced chromosomal losses focus the critical region on wt Apc, but because of the limited number of polymorphic markers used, no other critical regions of loss on chromosome 18 were identified. Using intestinal tumors arising spontaneously and induced by X-rays in CBA/H x C57BL/6J F1 hybrid mice and high-resolution microsatellite loss of heterozygosity (LOH) techniques, we provide mapping data for wt Apc loss, which confirms and extends earlier observations. In addition, high-frequency loss events at the Dpc4 locus were found in both spontaneous and radiation-induced tumors. These data identified LOH of Dpc4 as a critical secondary event following complete functional loss of Apc. LOH across the Trp53 genomic region of chromosome 11 was not observed. No LOH was recorded for the Mom1 candidate gene Pla2g2a or for 9 out of 10 polymorphic markers from the Mom1 genomic region on murine chromosome 4. One marker mapping distal to Pla2g2a showed LOH in a small minority of spontaneous tumors. These data support the contention that Mom1 does not act as a classical tumor suppressor. Overall, our data indicates a significant role for Dpc4 mutation in intestinal tumor progression in the mouse and provides further evidence for the importance of interstitial chromosome losses in radiation tumorigenesis.     

A urokinase-type plasminogen activator deficiency diminishes the frequency of intestinal adenomas in ApcMin/+ mice

The interaction of urokinase-type plasminogen activator (uPA) and its receptor, uPAR, on cell surfaces facilitates the generation of cell-bound plasmin, thus allowing cells to establish a proteolytic front that enables their migration through protein barriers. This complex also activates cell signalling pathways that influence cell functions. Clinical studies have identified uPA as an indicator of poor overall survival in patients with colorectal cancer. In the current study, a mouse model of colon cancer, Apc(Min/+), with an additional deficiency of uPA (Apc(Min/+)/Plau-/-) was used to determine the effects of uPA on tumour initiation and growth. Utilizing this model, it was found that the number of tumours was diminished in these mice relative to Apc(Min/+) mice, which correlated with the decreased leukocyte infiltration in the tumours. However, tumour growth was not impeded in Apc(Min/+)/Plau-/- mice, and proliferation and tumour vascularization were, in fact, enhanced in Apc(Min/+)/Plau-/- mice. These latter effects are consistent with a mechanism involving up-regulation of COX-2 expression and Akt pathway activation in Apc(Min/+)/Plau-/- mice. The results from this study suggest that uPA plays dual and opposing roles in regulating lesion development: one early, during the transition from normal epithelia to dysplastic lesions, and another later during tumour growth.     

Cytolethal distending toxin is essential for Helicobacter hepaticus colonization in outbred Swiss Webster mice

Helicobacter hepaticus, which induces chronic hepatitis and typhlocolitis in susceptible mouse strains, produces a cytolethal distending toxin (CDT) consisting of CdtA, CdtB, and CdtC. A cdtB-deficient H. hepaticus isogenic mutant (HhcdtBm7) was generated and characterized for colonization parameters in four intestinal regions (jejunum, ileum, cecum, and colon) of outbred Swiss Webster (SW) mice. Inactivation of the cdtB gene abolished the ability of HhcdtBm7 to colonize female mice at both 8 and 16 weeks postinfection (wpi), whereas HhcdtBm7 colonized all of four intestinal regions of three of five males at 8 wpi and then was eliminated by 16 wpi. Wild-type (WT) H. hepaticus was detected in the corresponding intestinal regions of both male and female mice at 8 and 16 wpi; however, colonization levels of WT H. hepaticus in the cecum and colon of male mice were approximately 1,000-fold higher than in females (P < 0.0079) at 16 wpi. Infection with WT H. hepaticus, but not HhcdtBm7, at 8 wpi was associated with significantly increased mRNA level of ileal and cecal gamma interferon (IFN-gamma) in females (P < 0.016 and 0.031 between WT H. hepaticus-infected and sham-dosed females, respectively). In contrast, the mRNA levels of IFN-gamma were significantly higher in the colon (P < 0.0079) and trended to be higher in the cecum (P < 0.15) in the HhcdtBm7-colonized male mice versus the sham-dosed controls at 8 wpi. In addition, mRNA levels of ileal IFN-gamma were significantly higher in the control females than males at 8 wpi (P < 0.016). There were significantly higher Th1-associated immunoglobulin G2a (IgG2a), Th2-associated IgG1 and mucosal IgA (P < 0.002, 0.002, 0.002, respectively) responses in the mice infected with WT H. hepaticus when compared to HhcdtBm7 at 16 wpi. Colonic interleukin-10 (IL-10) expressions at 16 wpi were significantly lower in both female and male mice colonized by WT H. hepaticus or in males transiently colonized through 8 wpi by HhcdtBm7 versus control mice (P < 0.0159). These lines of evidence indicate that (i) H. hepaticus CDT plays a crucial role in the persistent colonization of H. hepaticus in SW mice; (ii) SW female mice are more resistant to H. hepaticus colonization than male mice; (iii) there was persistent colonization of WT H. hepaticus in cecum, colon, and jejunum but only transient colonization of H. hepaticus in the ileum of female mice; (iv) H. hepaticus colonization was associated with down-regulation of colonic IL-10 production.     

Natural history of Helicobacter hepaticus infection in conventional A/J mice,with special reference to liver involvement

It has been reported that Helicobacter hepaticus infection of mice leads to chronic hepatitis and hepatocarcinoma. Our aim was to monitor a cohort of 80 conventional A/J mice in which half of the mice were infected by H. hepaticus in order to study the evolution of the infection and the pathological changes in comparison to uninfected mice. H. hepaticus was detected by culture only in some colon and cecum specimens after 17 months of age, while PCR detected H. hepaticus in the intestines of all inoculated mice after only 5 months of infection. The percentage of mice in which H. hepaticus was detected in the gallbladder, bile ducts, and liver by PCR, as well as the number of bacteria present in the liver, tended to increase with increasing age and longer infection time. Anti-H. hepaticus immunoglobulin G antibodies were positive by enzyme-linked immunosorbent assay only in inoculated mice. Pathological findings were also more frequent as the mice grew older: fibrosis was present (especially in the peripheral part of the liver), and significant portal inflammation including lymphoid nodules was present in almost all infected animals. Biliary lesions of neutrophilic acute cholangitis or lymphocytic cholangitis were noted. However, lesions were also observed in uninfected animals, although at a significantly lower level, and the only hepatocellular carcinoma occurred in an uninfected mouse. The evolution towards hepatocarcinoma is not always the endpoint and may depend on the bacterial strain and on the environmental conditions.     

The genetic background modifies the spontaneous and X-ray-induced tumor spectrum in the Apc1638N mouse model.

The effect of the genetic background on the tumor spectrum of Apc1638N, a mouse model for attenuated familial adenomatous polyposis (FAP), has been investigated in X-irradiated and untreated F1 hybrids between C57BL/6JIco-Apc1638N (B6) and A/JCrIBR (A/J), BALB/cByJIco (C) or C3H/HeOuJIco (C3). Similar to the ApcMin model, the Apc1638N intestinal tumor multiplicity seems to be modulated by Mom1. Moreover, several additional (X-ray-responsive) modifier loci appear also to affect the Apc1638N intestinal tumor number. The genetic background did not significantly influence the number of spontaneous desmoids and cutaneous cysts in Apc1638N. In general, X-irradiation increased the desmoid multiplicity in Apc1638N females but had no effect in males. The opposite was noted for the cyst multiplicity after X-rays. Surprisingly, X-irradiated CB6F1-Apc1638N females were highly susceptible to the development of ovarian tumors, which displayed clear loss of the wild-type Apc allele.     

CD4+ CD25+ regulatory T lymphocytes inhibit microbially induced colon cancer in Rag2-deficient mice

Inflammatory bowel diseases, including ulcerative colitis and Crohn's disease, increase the risk of colorectal cancer in humans. It has been recently shown in humans and animal models that intestinal microbiota and host immunity are integral in the progression of large bowel diseases. Lymphocytes are widely believed to prevent bacterially induced inflammation in the bowel, and lymphocytes are also critical in protecting against primary tumors of intestinal epithelia in mice. Taken together, this raises the possibility that lymphocytes may inhibit colon carcinogenesis by reducing bacterially driven inflammation. To examine the role of bacteria, lymphocytes, and inflammatory bowel disease in the development of colon cancer, 129/SvEv Rag-2-deficient and congenic wild-type mice were orally inoculated with a widespread enteric mouse bacterial pathogen, Helicobacter hepaticus, or sham-dosed with media only. H. hepaticus-infected Rag2-/-, but not sham-dosed Rag2-/- mice, rapidly developed colitis and large bowel carcinoma. This demonstrated a link between microbially driven inflammation and cancer in the lower bowel and suggested that innate immune dysregulation may have an important role in inflammatory bowel disease and progression to cancer. H. hepaticus-infected wild-type mice did not develop inflammation or carcinoma showing that lymphocytes were required to prevent bacterially induced cancer at this site. Adoptive transfer with CD4+ CD45RBlo CD25+ regulatory T cells into Rag-deficient hosts significantly inhibited H. hepaticus-induced inflammation and development of cancer. These results suggested that the ability of CD4+ T cells to protect against intestinal cancer was correlated with their ability to reduce bacterially induced inflammatory bowel disease. Further, regulatory T cells may act directly on the innate immune system to reduce or prevent disease. These roles for T cells in protection against colon carcinoma may have implications for new modes of prevention and treatment of cancer in humans.     

Fluorogenic PCR-Based Quantitative Detection of a Murine Pathogen, Helicobacter hepaticus

Helicobacter hepaticus infection in mice is being used as an animal model for elucidating the pathogenesis of gastrointestinal and biliary diseases in humans. H. hepaticus, which forms a spreading film on selective agar, is not amenable to routine quantitative counts of organisms in tissues using a CFU method. In this study, a fluorogenic PCR-based assay was developed to quantitatively detect H. hepaticus in mouse ceca and feces using the ABI Prism 7700 sequence detection system. A pair of primers and a probe for this assay were generated from the H. hepaticus cdtB gene (encoding subunit B of the H. hepaticus cytolethal distending toxin). Using this assay, the sensitivity for detection of H. hepaticus chromosomal DNA prepared from pure culture was 20 fg, which is equivalent to approximately 14 copies of the H. hepaticus genome based on an estimated genome size of approximately 1.3 Mb determined by pulsed-field gel electrophoresis. H. hepaticus present in feces and cecal samples from H. hepaticus-infected mice was readily quantified. The selected PCR primers and probe did not generate fluorescent signals from eight other helicobacters (H. canis, H. cineadi, H. felis, H. mustelae, H. nemestrinae, H. pullorum, H. pylori, and H. rodentium). A fluorescent signal was detected from 20 ng of H. bilis DNA but with much lower sensitivity (10(6)-fold) than from H. hepaticus DNA. Therefore, this assay can be used with high sensitivity and specificity to quantify H. hepaticus in experimentally infected mouse models as well as in naturally infected mice.