Koji Nozaki

Effect of early eradication on Helicobacter pylori-related gastric carcinogenesis in Mongolian gerbils

Helicobacter pylori (Hp) infection and gastric carcinogenesis are known to have a close relationship, but the effect of eradication of Hp on Hp‐related gastric carcinogenesis has not been fully studied experimentally. To evaluate the effect of eradication in gastric carcinogenesis, an experimental model with eradication in the early, middle or late period was studied using Hp‐infected and N‐methyl‐N‐nitrosourea (MNU)‐treated Mongolian gerbils. The animals were divided into seven groups: group A (MNU+Hp+ eradication at 15 w), group B (MNU+Hp+eradication at 35 w), group C (MNU+Hp+eradication at 55 w), group D (MNU+Hp), group E (MNU), group F (Hp) and group G (control). The tumor incidences at week 75 in the early (group A), middle (group B) and late (group C) eradicated groups were 6.7%, 27.3% and 38.2%, respectively. The incidence of 56.3% in the non‐eradicated group was the highest (group D). Incidences in group E, group F and group G were 6.3%, 0.0% and 0.0%, respectively. The tumor incidences were related to the period of inflammatory status induced by Hp infection. Hp infection strongly enhanced gastric carcinogenesis initiated with a chemical carcinogen, and following eradication at an early period this enhancing effect was effectively reduced. Eradication at an early stage of inflammation might be effective in preventing Hp‐related gastric carcinogenesis. (Cancer Sci 2003; 94: 235–239)

Reversibility of Heterotopic Proliferative Glands in Glandular Stomach of Helicobacter pylori-infected Mongolian Gerbils on Eradication

Helicobacter pylori (Hp) infection is an important factor in human gastric disorders. Mongolian gerbils can be easily infected with Hp and represent excellent experimental models to clarify the role of Hp in chronic active gastritis, peptic ulcers, intestinal metaplasia, and gastric carcinoma. We have proved the enhancing effects of Hp infection on all histological types of gastric cancers in Mongolian gerbils exposed to chemical carcinogens. Heterotopic proliferative glands (HPGs) also frequently develop with Hp infection in the glandular stomach of infected gerbils, with a slightly dysplastic change of constituent cells. Distinguishing reversible inflammatory lesions from true neoplasms upon eradication is necessary for further biological or histochemical investigations using this model. We employed an experimental model of long‐term Hp infection and eradication in gerbils. HPGs finally developed with a phenotypic shift of intestinalization with Paneth cells. After eradication, HPGs were obviously reduced, and gastric lesions in mucosa also improved with few remnants of the former injury. This shows that reversible HPGs are frequently induced solely by Hp infection in this annual species, and are related to severe gastritis, rather than being malignant in character. Thus, distinguishing reversible lesions from true neoplasms is necessary to investigate the relationship of Hp infection and gastric carcinogenesis in this animal model.

Expression of the intestine-specific transcription factors, Cdx1 and Cdx2, correlates shift to an intestinal phenotype in gastric cancer cells

Purpose It is well known that gastric cancers (GCs) at early stages, independent of the histological type, mainly consist of gastric phenotype malignant cells, while those at advanced stage tend to have a more intestinal phenotype with progression. However, the connection between this shift and expression of homeobox genes, which are important factors to maintain tissue character, has remained unclear. We therefore evaluated the expression of Cdx1/2 in relation to the phenotype of GCs.Methods We analyzed the expression of Cdx1/2 mRNAs by Northern blotting and Cdx2 protein by immunohistochemistry in seventy advanced GCs, and evaluated phenotypically using mucin- and immunohistochemistry.Results Seventy GCs were divided phenotypically into 16 gastric (G type), 18 gastric and intestinal mixed (GI type), 18 intestinal (I type), and 18 null (N type) phenotypes, independent of the histological classification. Cdx1 and Cdx2 mRNAs statistically demonstrated an increase with shift from G to I (P=0.042 and P=0.0082, respectively). Cdx2 nuclear staining was observed immunohistochemically in the intestinal phenotypic cancer cells, but could not be detected in those with only the gastric phenotype.Conclusions These results show that Cdx1 and Cdx2 might be indispensable for intestinal phenotypic expression even in gastric cancer cells.

Earlier Helicobacter pylori infection increases the risk for the N-methyl-N-nitrosourea-induced stomach carcinogenesis in Mongolian gerbils

Helicobacter pylori (H. pylori) is now well known to be associated with stomach cancer, with infection during childhood rather than as an adult considered to be more important for carcinogenesis. To evaluate the difference in susceptibility to stomach carcinogenesis in relation to age of acquisition of H. pylori infection, we designed an experiment involving inoculation of H. pylori ATCC43504 followed by N‐methyl‐N‐nitrosourea (MNU) treatment at different ages. Four‐week‐ old male Mongolian gerbils (MGs) were divided into twelve groups. H. pylori was inoculated at 4, 18 and 32 weeks of age, as representatives of early, middle and late infection, respectively. Two weeks later, the animals were treated with MNU. Groups without H. pylori and/or MNU were included as controls. The incidences of adenocarcinomas at 52 weeks after the inoculation in the early (H. pylori+MNU), middle (H. pylori+MNU), and late (H. pylori+MNU) group were 60% (12/ 20), 18.4% (2/11), and 10% (2/20), respectively. The corresponding figures were 14.8% (4/27), 0% (0/11), and 0% (0/21) in the MNU‐alone groups. A higher titer of serum IgG for H. pylori and higher gastrin level were seen in the early‐infected compared to the middle and the late groups (P<0.01). The results clearly demonstrated that early acquisition of H. pylori significantly increases gastric chemical carcinogenesis with MNU, as compared to the case with later infection, possibly because of differences in host gastric mucosal factors and immunologic responses.

Eradication of Helicobacter pylori induces apoptosis and inhibits proliferation of heterotopic proliferative glands in infected Mongolian gerbils

Mongolian gerbils infected with Helicobacter pylori (H. pylori) develop heterotopic proliferative glands (HPGs) in the glandular stomach submucosa. To investigate the effects of H. pylori eradication on cell turnover in HPGs, three antibiotics, lansoprazole, amoxicillin and clarithromycin, were administered at 50 or 75 weeks after inoculation of H. pylori, and the stomachs were excised for histological examination at 1, 2, 4, 8 or 25 weeks thereafter. The HPGs were classified into gastric type (G‐type) and others (Gl+I‐type), which included both pure intestinal (I‐type) and gastric‐and‐intestinal mixed type (Gl‐type). Apoptosis and cell proliferation were evaluated by means of TUNEL assay and BrdU labeling, respectively. At 8 weeks post‐eradication, apoptotic indices were significantly increased in the eradication group (G‐type: 2.5%; Gl+I‐type: 7.2%) compared to the non‐eradication group (G‐type: 0.6%; Gl+I‐type: 2.1%: P<0.01), while BrdU labeling indices were significantly decreased (G‐type: 1.9%; Gl+I‐type: 6.8% as compared with 4.3% and 13.2%, respectively, P<0.01 for both). At 25 weeks, the apoptotic indices were similarly higher [G‐type: 0.4 (eradication group) vs. 0.2% (non‐eradication group); Gl+I‐type: 5.8 vs. 1.1%, both P<0.01], and the BrdU labeling indices (G‐type: 0.8 vs. 2.2%, P<0.01; Gl+I‐type: 5.1 vs. 11%, P<0.05) continued to be lower in HPGs. Furthermore, there were highly significant reductions in the areas of HPGs at 8 and 25 weeks post‐eradication. These findings demonstrated that eradication results in apoptosis and reduction of proliferation of HPGs in H. pylori‐infected gerbils, these lesions thus being apparently reversible through regulation of cell kinetics.

Helicobacter pylori infection and gastric carcinogenesis in animal models.

Abstract.The effects of Helicobacter pylori infection on gastric disorders have been proven by many epidemiological and experimental studies. To explore the relationships between H. pylori infection and gastric carcinogenesis, many factors, including host responses, environmental status, and the virulence factors of the bacteria should be taken into account. Mongolian gerbils (Meriones unguiculatus) can be easily infected with H. pylori, and provide an excellent in-vivo experimental model to clarify the role of H. pylori in active gastritis, peptic ulcers, intestinal metaplasia, and gastric carcinoma. Studies have revealed that H. pylori infection markedly enhances all histological types of gastric cancers in gerbils treated with a chemical carcinogen. Eradication reduced the enhancing effect of H. pylori on gastric carcinogenesis, whereas a high-salt diet synergistically enhanced the effect of H. pylori. Various factors involving inflammation, cell proliferation, and cell differentiation could be examined with this experimental model to help elucidate this mechanisms of gastric carcinogenesis.

β-Catenin mutations and nuclear accumulation during progression of rat stomach adenocarcinomas

Aberrant Wnt/β‐catenin signaling caused by mutations in exon 3 of the β‐catenin gene has been identified in a number of human malignancies, including stomach cancer. However, studies of mutation frequency have yielded conflicting results, and timing during progression remains largely unknown. In this study, we utilized an animal model to address this question. A total of 20 ACI male rats were treated with N‐methyl‐N′‐nitro‐N‐nitrosoguanidine (MNNG) in the drinking water and 22 induced differentiated adenocarcinomas were histopathologically and immunohistochemically evaluated for β‐catenin localization. Fourteen tumors (63.6%) that showed homogeneous low‐grade morphology, preserving cell polarity, were found to harbor β‐catenin protein on the cell membranes (M). Eight tumors exhibited regions of high‐grade morphology among areas with low‐grade morphology, and they were characterized by denser cell growth and loss of cell polarity. Among these 8 tumors, 4 (18.2%) showed cytoplasmic localization (C) of β‐catenin in small regions. The remaining 4 tumors (18.2%) contained more dysplastic regions that displayed nuclear (N) β‐catenin staining. Analysis of DNA obtained by microdissection demonstrated that all of 4 regions with C staining and 20 with M staining, as well as 17 samples of surrounding normal mucosa (S) had wild‐type β‐catenin. In contrast all of 3 regions with N staining featured mutations (3 of 3=100%; N vs. C, P<0.05; N vs. M and N vs. S, P<0.001, Fishers exact test) in exon 3, at glycine 34, threonine 41, and serine 45, which affected phosphorylation sites. In conclusion, β‐catenin mutations appear to be associated with the late progression stage of adenocarcinoma development in rat stomach carcinogenesis, in contrast to the case of colorectal cancers, in which mutations appear to occur in the early stages.

Severity of gastritis determines glandular stomach carcinogenesis in Helicobacter pylori-infected Mongolian gerbils

Helicobacter pylori (H. pylori) infection causes chronic gastritis and is also related to gastric carcinoma. The present study focused on severity of H. pylori‐induced gastritis as a determinant of carcinogenesis. Seven‐week‐old male Mongolian gerbils were inoculated with H. pylori at experimental weeks 0, 12, or 18, then given N‐methyl‐N‐nitorosourea (MNU) from weeks 20–40. At week 70, stomachs were then excised for histological examination 70, 58, or 52 weeks after H. pylori inoculation, respectively (Groups A, B, and C for long‐, middle‐, and short‐term). The respective incidences of glandular stomach adenocarcinomas were 65.0% (13/20), 20.0% (2/10), and 23.0% (3/13) (P < 0.05). Higher scores of infiltration of inflammatory cells, hyperplasia, intestinal metaplasia and mucosal bromodeoxyuridine (BrdU) labeling index in antrum and corpus mucosa, were seen in group A than B or C (P < 0.05) and serum anti‐H. pylori IgG titer and gastrin levels were also significantly higher, along with mRNA levels for mucosal interleukin‐1β (IL‐1β), tumor necrosis factor‐α (TNF‐α), cyclooxygenase‐2 (COX‐2), and inducible nitric oxide synthase (iNOS). The results demonstrated the term and severity of H. pylori infection to play important roles in gastric carcinogenesis, with essential involvement of chronic inflammation, especially increased rates of cell proliferation, in H. pylori‐associated carcinogenesis. (Cancer Sci 2007; 98: 478–483)

Altered gastric chief cell lineage differentiation in histamine-deficient mice

The orderly differentiation of cell lineages within gastric glands is regulated by a complicated interplay of local mucosal growth factors and hormones. Histamine secreted from enterochromaffin-like cells plays an important role in not only stimulated gastric acid secretion but also coordination of intramucosal growth and lineage differentiation. We have examined histidine-decarboxylase (HDC)-deficient mice, which lack endogenous histamine synthesis, to evaluate the influence of histamine on differentiation of fundic mucosal lineages and the development of metaplasia following induction of acute oxyntic atrophy. Stomachs from HDC-deficient mice and wild-type mice were evaluated at 8 wk and 12 mo of age. DMP-777 was administrated orally to 6-wk-old mice for 1 to 14 days. Sections of gastric mucosa were stained with antibodies against Mist1, intrinsic factor, H/K-ATPase, trefoil factor 2 (TFF2), chromogranin A, and Ext1 and for the cell cycle marker phospho-histone H3. HDC-deficient mice at 8 wk of age demonstrated a prominent increase in chief cells expressing Mist1 and intrinsic factor. Importantly Mist1-positive mature chief cells were present in the midgland region as well as at the bases of fundic glands, indicating a premature differentiation of chief cells. Mice dually deficient for both HDC and gastrin showed a normal distribution of chief cells in fundic glands. Treatment of HDC-deficient mice with DMP-777 led to loss of parietal cells and an accelerated and exaggerated emergence of mucous cell metaplasia with the presence of dual intrinsic factor and TFF2-expressing cells throughout the gland length, indicative of the emergence of spasmolytic polypeptide-expressing metaplasia (SPEM) from chief cells. These findings indicate that histamine, in concert with gastrin, regulates the appropriate differentiation of chief cells from mucous neck cells as they migrate toward the bases of fundic glands. Nevertheless, histamine is not required for emergence of SPEM following acute oxyntic atrophy.

Guidelines for the management of cutaneous lymphomas (2011): A consensus statement by the Japanese Skin Cancer Society - Lymphoma Study Group

In 2010, the first Japanese edition of guidelines for the management of cutaneous lymphoma was published jointly by the Japanese Dermatological Association (JDA) and the Japanese Skin Cancer Society (JSCS) – Lymphoma Study Group. Because the guidelines were revised in 2011 based on the most recent data, we summarized the revised guidelines in English for two reasons: (i) to inform overseas clinicians about our way of managing common types of cutaneous lymphomas such as mycosis fungoides/Sézary syndrome; and (ii) to introduce Japanese guidelines for lymphomas peculiar to Asia, such as adult T‐cell leukemia/lymphoma and extranodal natural killer/T‐cell lymphoma, nasal type. References that provide scientific evidence for these guidelines have been selected by the JSCS – Lymphoma Study Group. These guidelines, together with the degrees of recommendation, have been made in the context of limited medical treatment resources, and standard medical practice within the framework of the Japanese National Health Insurance system.