Long-term survival of pancreatic cancer patients treated with multimodal therapy combined with WT1-targeted dendritic cell vaccines.

BACKGROUND/AIM: Pancreatic ductal adenocarcinoma (PDA) remains one of the most aggressive tumors with a dismally poor prognosis. Although surgical resection remains the only potentially curative treatment, most PDAs are not surgically resectable at diagnosis. Therefore, multimodal therapy is urgently needed to improve the long-term survival of PDA patients. METHODS: Six eligible PDA patients underwent multimodal therapy comprising dendritic cells (DCs) pulsed with Wilms' tumor 1 (WT1) peptide (DC/WT1-I) restricted by the human leukocyte antigen (HLA) class I (A*24:02 or A*02:06) allele, chemotherapy, radiation, and/or surgery. Patient laboratory data, DC/WT1-I-specific delayed-type hypersensitivity (DTH) reactions, and WT1-specific immune responses were analyzed to assess the prognostic markers of multimodal therapy. RESULTS: Compared to 2-treatment type combinations, multimodal therapy involving 3 to 4 treatment types was significantly associated with longer overall survival (p = 0.0177). Moreover, after 7 DC/WT1-I vaccinations, the progression-free survival (PFS) of PDA patients with a neutrophil to lymphocyte ratio (NLR) or C-reactive protein (CRP) level less than the median was superior to that of PDA patients with values above the median (p = 0.0246). PDA patients with an overall survival (OS)>1000 days had significantly more lymphocytes after one DC/WT1-I vaccination course than did those with an OS<1000 days. CONCLUSION: Multimodal therapy involving the DC/WT1-I vaccination may benefit patients with advanced PDA. However, comparing the limited number of PDA patients in terms of survival is difficult because the patients were at different disease stages and received different treatments. Further studies are needed to evaluate the clinical benefits of this multimodal therapy.

Possible role of nuclear ß-catenin in resistance to preoperative chemoradiotherapy in locally advanced rectal cancer.

AIMS: ß-Catenin signalling participates in the regulation of epithelial-mesenchymal transition (EMT)/cancer stem cell (CSC) properties. The aim of this study was to investigate the role of ß-catenin in resistance to neoadjuvant chemoradiotherapy in patients with rectal cancer, especially pertaining to its association with EMT/CSC features. METHODS AND RESULTS: A total of 109 cases of locally advanced rectal cancer, along with a colon cancer cell line, were investigated. Nuclear ß-catenin accumulation in pretreatment-biopsied samples was inversely associated with the therapeutic efficacy of chemoradiotherapy in resected rectal cancer. In resected tumours, nuclear ß-catenin was predominantly observed in EMT-like lesions with decreased E-cadherin and increased Snail expression, along with expression of CSC-related markers. The EMT-like lesions also showed significant decreases in both apoptosis and cell proliferation as compared with non-EMT lesions. In-vitro culture of a colon cancer cell line in STK2 was sufficient to induce EMT/CSC properties together with nuclear ß-catenin accumulation, and showed inhibition of cell proliferation and resistance to doxorubicin treatment. CONCLUSION: Nuclear ß-catenin accumulation may contribute to chemoradioresistance in locally advanced rectal cancer, probably through its regulation of EMT/CSC properties. In addition, nuclear ß-catenin in pretreatment-biopsied samples is useful in predicting the efficacy of chemoradiotherapy in patients with rectal cancer.

Distinct ß-catenin and PIK3CA mutation profiles in endometriosis-associated ovarian endometrioid and clear cell carcinomas.

OBJECTIVES: We focused on the differences in molecular mechanisms in the early stages of endometriosis-associated ovarian endometrioid carcinoma (OEMCa) and ovarian clear cell carcinoma (OCCCa). METHODS: Alterations in the ß-catenin and PIK3CA genes, as well as expression of their associated markers, were investigated. RESULTS: Mutations in exon 3 of the ß-catenin gene were identified in 21 (60%) of 35 OEMCas. The mutations were also detected in the coexisting nonatypical (52.4%) and atypical (73.3%) endometriosis, and the single-nucleotide substitutions were identical in most cases. In contrast, the mutations were not identified in any of the OCCCas and their coexisting endometriosis. PIK3CA mutations were observed in 11 (31.4%) of 35 OEMCas and 10 (35.7%) of 28 OCCCas. Ten of 11 OEMCas had PIK3CA mutations in exon 9, and eight of 10 OCCCas had them in exon 20. The same mutations were also detected in the coexisting nonatypical and/or atypical endometriosis in three OEMCas and four OCCCas. In addition, significant differences in the expression of pAkt, hepatocyte nuclear factor 1ß, hypoxia-inducible factor 1α, p65, and inducible nitric oxide synthase were evident between the two types of tumors and their coexisting endometriosis. CONCLUSIONS: Distinct molecular events may occur in relatively early stages of tumorigenesis of endometriosis-associated OEMCas and OCCCas.

Transcriptional upregulation of HNF-1ß by NF-κB in ovarian clear cell carcinoma modulates susceptibility to apoptosis through alteration in bcl-2 expression.

Hepatocyte nuclear factor-1ß (HNF-1ß) is a transcriptional factor that has an important role in endometriosis-ovarian clear cell carcinoma (OCCC) sequence by modulating cell kinetics and glucose metabolism. However, little is known about the detailed molecular mechanisms that govern its regulation and function. Herein, we focus on upstream and downstream regulatory factors of HNF-1ß in OCCCs. In clinical samples, HNF-1ß expression was positively correlated with the active form of NF-κB/p65 in OCCCs, and closely linked with a low nuclear grade and non-solid architecture. In cell lines, transfection of p65 resulted in increased HNF-1ß mRNA and protein expression in TOV-21G cells (OCCC cell line with endogenous HNF-1ß expression), in line with activation of the promoter, probably through interacting with the basic transcriptional machinery. Suppression of endogenous HNF-1ß expression by siRNA increased apoptosis in TOV-21G cells, while treatment of Hec251 cells (endometrial carcinoma cell line with extremely low endogenous HNF-1ß expression) stably overexpressing exogenous HNF-1ß with doxorubicin abrogated apoptosis of the cells, along with increased ratio of bcl-2 relative to bax. Moreover, overexpression of HNF-1ß led to upregulation of bcl-2 expression at the transcriptional level in TOV-21G cells, which provided evidence for a positive correlation between HNF-1ß and bcl-2 expression in OCCCs. These data, therefore, suggest that association between HNF-1ß and NF-κB signaling may participate in cell survival by alteration of apoptotic events, particularly in mitochondria-mediated pathways, through upregulation of bcl-2 expression in OCCCs.

Acute Myeloid Leukemia Diagnosed 5 Years after Adult T-Cell Leukemia/Lymphoma.

A case of secondary acute myeloid leukemia (AML) was identified following adult T-cell leukemia/lymphoma (ATL), for which combination chemotherapy had been administered, including epipodophyllotoxin, anthracycline, and alkylating agents. AML with maturation was diagnosed by the cytological findings, cell surface markers, and chromosomal abnormalities. We previously reported two cases of AML accompanied by ATL. In this case of AML after chemotherapy for ATL, we considered that the AML was probably associated with previous chemotherapy for ATL. Although the ATL remained in remission, the therapy-related AML with complex chromosomal abnormalities proved resistant to chemotherapy, and the patient died from complications associated with AML.

A combination of nuclear ß-catenin and atypical scores as useful diagnostic markers for borderline malignancy of gastric tumours.

AIMS: Although recent advances in endoscopic technology, including submucosal dissection (ESD), contribute to significant improvement in detection and treatment for early gastric carcinomas (GCas), discrepant diagnosis between forceps biopsied and ESD sections sometimes occurs. Here, we focused on histological markers for accurate diagnosis of gastric tumours in forceps biopsied samples before ESD treatment. METHODS AND RESULTS: A total of 136 cases of gastric tumours, including 25 adenomas and 111 GCas, were investigated using a combination of forceps biopsied and ESD samples. Atypical scores based on both nuclear and branching parameters could distinguish between adenomas and GCas in both samples. The labelling indices (LIs) of nuclear ß-catenin were significantly higher in GCas than adenomas, and positively correlated with atypical scores, as well as branching parameters. In addition, nuclear ß-catenin immunoreactivity showing small cluster pattern was colocalized with immunoreactivity for aldehyde dehydrogenase 1 (ALDH1), known as a cancer stem cell (CSC) marker, which suggests the presence of some CSC population, and may be due to formation of dynamic tubular branching in GCas. CONCLUSIONS: A combination of nuclear ß-catenin and atypical scores may be useful for differential diagnosis of borderline malignancy of gastric tumours.

Tumor budding, myofibroblast proliferation, and fibrosis in obstructing colon carcinoma: the roles of Hsp47 and basic fibroblast growth factor.

This study was designed to assess the mechanism of obstruction in obstructing colorectal carcinomas. Thirty-five cases of obstructing colorectal carcinoma and 34 cases of non-obstructing carcinoma were studied. The lesions were immunohistochemically analyzed using antibodies for pan-cytokeratin, α-smooth muscle actin, matrix metalloproteinase-7, 47-kDa heat shock protein (Hsp47), basic fibroblast growth factor (bFGF), myeloperoxidase, and CD68. Compared with non-obstructing cases, obstructing carcinoma cases included lesions of poorer differentiation. A higher value of tumor budding was observed in obstructing than in non-obstructing carcinoma. A higher number of α-smooth muscle actin-positive myofibroblasts, a higher expression of Hsp47 in stromal spindle cells, and a higher expression of bFGF in inflammatory cells were also significant in obstructing carcinoma. Therefore, obstructing colon carcinomas were characterized by poorer differentiation of cancer cells, a high level of tumor budding, and stromal myofibroblast proliferation resulting in fibrosis. Correlative Hsp47 expression in fibroblasts with bFGF in inflammatory cells may contribute to stromal fibrosis.

High expression of L-type amino-acid transporter 1 (LAT1) in gastric carcinomas: comparison with non-cancerous lesions.

Amino acid transporters are essential for maintenance and proliferation of both normal and transformed cells. In the present study, L-type amino-acid transporter 1 (LAT1) immunoreactive expression was investigated in gastric carcinomas, in comparison with gastric adenomas and non-neoplastic lesions, using our recently developed novel monoclonal antibody. In a total of 87 cases of advanced gastric cancer, high LAT1 expression was observed in carcinoma cells, predominantly at plasma membranes with greater intensity in non-scirrhous than scirrhous carcinomas. Gastric carcinoma cases with lymph node metastasis showed significantly higher LAT1 expression than cases without lymph node metastasis. A positive correlation with Ki-67 LI was observed and the highly expressing non-scirrhous carcinomas showed a significantly poorer prognosis than the low LAT1 group. Cox hazard test revealed that TNM stage and LAT1 expression were independent prognostic factors in non-scirrhous carcinoma group. Further, a significant poor prognosis was confirmed in high LAT1 expression group, when limited to undifferentiated carcinoma cases excluding scirrhous carcinoma. Lower levels were found in adenomas. In conclusion, LAT 1 expression may be linked with cell proliferation and prognosis of gastric carcinomas, and offers a potential target for future anticancer therapy by inhibitors.

Submucosal fibrosis and basic-fibroblast growth factor-positive neutrophils correlate with colonic stenosis in cases of ulcerative colitis.

BACKGROUND AND AIMS: The frequency of benign stenosis in ulcerative colitis (UC) is low, reported as being 3.2-11.2%, with fibrosis in the submucosa or deeper pointed out as one of the causes. The aim of the present study was to assess stenosis in UC cases using immunostaining and to analyze differences between stenotic and nonstenotic cases, focusing on basic-fibroblast growth factor (b-FGF) expression and myofibroblasts. METHODS: Totals of 9 stenotic and 17 nonstenotic UC cases were histopathologically examined and immunohistochemically stained for b-FGF, α-smooth muscle actin (α-SMA), CD34, CD68 and IL-6. To identify b-FGF-positive cells, double immunostaining for b-FGF and myeloperoxidase or CD68 was performed. RESULTS: In addition to submucosal fibrosis, a significant increase of b-FGF-positive inflammatory cells and myofibroblasts was observed in stenotic portions. Most b-FGF-positive cells were also positive for myeloperoxidase, and a correlation between b-FGF-positive and total neutrophil counts was found. CONCLUSIONS: One of the major causes of stenosis in long-standing UC is fibrosis in the bowel wall, possibly induced by infiltrating inflammatory neutrophils producing b-FGF.

Constitutive activity of nuclear transcription factor kappaB is observed in follicular lymphoma.

Constitutive activity of nuclear transcription factor kappaB (NF-kappaB) is observed in many pathological types of lymphoma that are associated with a poor clinical course. This suggests that NF-kappaB and pathways involving NF-kappaB are possible targets for successfully treating lymphoma. We examined 28 lymph nodes from 28 patients in whom follicular lymphoma was diagnosed from 1996 to 2006 at our institution, which were formalin-fixed and paraffin-embedded. The specimens were stained with an antibody that could recognize activated NF-kappaB and p65 to determine whether they were positive or negative for NF-kappaB activation. The clinical courses of the 28 patients were then correlated with the results of the NF-kappaB staining. The 10 men and 18 women had a mean age of 57.3 years (range, 25-87 years). By follicular lymphoma grade, 10 patients had grade 1, 16 had grade 2, and 2 had grade 3a. Ten patients died due to lymphoma. NF-kappaB was positive in 6 of the 28 cases. Analysis of the positive and negative staining groups while taking into account the clinical course, sex, age, grade of follicular lymphoma, prognostic index, CD10, CD23, Bcl-2, karyotype t(14;18), and survival showed that no significant differences. Six of the 28 lymph nodes (21.4%) exhibited consistent NF-kappaB activity. Three of the eleven cases that transformed to aggressive lymphoma were positive for activated NF-kappaB. Further research to clarify the significance of constitutive NF-kappaB activity in follicular lymphoma is therefore warranted.

Dual modulation of prothrombin activation by the cyclopentapeptide plactin.

Plactin, a family of cyclopentapeptides, enhances fibrinolytic activity by elevating the activity of cellular urokinase-type plasminogen activator (u-PA), a protease involved in a variety of extracellular proteolytic events. Factor(s) in the blood plasma is an absolute requirement for this plactin activity. In this study, we found that plactin promoted plasma cofactor-dependent conversion of inactive single-chain u-PA to active two-chain u-PA on U937 cells. Using plactin-affinity chromatography, we identified prothrombin as one of the plasma cofactors. In incubations of U937 cells with prothrombin and Xa, plactin increased the formation of thrombin, which cleaved single-chain u-PA to afford the inactive two-chain form. Thrombin-cleaved two-chain u-PA was alternatively activated by cellular cystatin-sensitive peptidase activity, yielding fully active two-chain u-PA. In a purified system, plactin bound to prothrombin, altered its conformation and dually modulated factor Xa-mediated proteolytic activation of prothrombin to alpha-thrombin. Plactin inhibited the activation catalyzed by Xa in complex with Va, Ca(2+) and phospholipids (prothrombinase), whereas the activations catalyzed by nonmembrane-associated Xa were enhanced markedly by plactin. Plactin inhibited in vitro plasma coagulation, which involved prothrombinase formation. Plactin did not cause prothrombin activation or thrombosis in normal mice at doses that produced a protective effect in a thrombin-induced pulmonary embolism mouse model. Therefore, the dual modulation of prothrombin activation by plactin may be interpreted as leading to anticoagulation under physiological coagulating conditions.

Tumor budding is predictive of lymphatic involvement and lymph node metastases in submucosal invasive colorectal adenocarcinomas and in non-polypoid compared with polypoid growths.

OBJECTIVE: Early colorectal carcinomas (submucosal invasive adenocarcinomas) can be classified into polypoid and non-polypoid growth types, the latter progressing more rapidly to advanced malignancy. The aim of this study was to investigate the differences between invasive features of the two types of carcinoma by focusing on tumor budding (isolated single cells or small cell clusters (up to four cells) scattered at invasive tumor margins). MATERIAL AND METHODS: The number of foci in the field with the most frequent tumor budding was regarded as "activity". Tumor budding was examined using anti-cytokeratin antibodies in 98 colorectal submucosal invasive adenocarcinomas and compared with the clinicopathological findings. In addition, the relationships between tumor budding and beta-catenin and laminin-5gamma2 expression were analyzed. RESULTS: Tumor budding activity was significantly higher in non-polypoid growth carcinomas compared with polypoid growth carcinomas (p = 0.0006) and values for left-sided lesions were higher than those for right-sided lesions of the colon (p = 0.0108). Positive links with tumor budding were evident for lymphatic involvement and lymph node metastasis in non-polypoid growth carcinomas, and with laminin-5gamma2 cytoplasmic expression in polypoid growth carcinomas. Multivariate logistic analysis revealed that the activity of tumor budding was an independent risk factor for lymphatic involvement. CONCLUSIONS: The results indicate that tumor budding makes a greater contribution to progression in non-polypoid than in polypoid growth carcinomas, with possible involvement of lymph node metastasis.

L-type amino-acid transporter 1 as a novel biomarker for high-grade malignancy in prostate cancer.

To find reliable biomarkers for high-grade malignancy, the relationship between immunohistochemical L-type amino-acid transporter 1 (LAT1) expression of biopsy samples, determined with the newly developed monoclonal antibody against human LAT1, and prognosis of patients with prostate cancer, was investigated. The intensity and score of immunohistochemical LAT1 expression of first biopsy samples were assessed using the modified Sinicrope et al. method and were found to be correlated with poor survival for the study group of 114 surgically treated patients as a whole (P = 0.0002 and 0.0270, respectively). LAT1 intensity further had a significant relationship (P = 0.0057) with prognosis in pathological T3 + T4 groups. Multivariate analysis indicated that the LAT1 intensity and score were more reliable prognostic markers, compared with the Gleason score and the Ki-67 labeling index. A relationship of the LAT1 intensity and score with prognosis could also be confirmed in 63 patients with inoperable cancer (P = 0.0070 and <0.0001, respectively). Similarly, significant differences in prognosis were confirmed in clinical T3 + T4 groups (P = 0.0091 and 0.0244, respectively). Moreover, the combination of LAT1 expression and Gleason score was found to have a more reliable correlation with prognosis. Thus, elevated LAT1 expression in prostate cancers is a novel independent biomarker of high-grade malignancy, which can be utilized together with the Gleason score, which is mainly dependent on cellular and structural atypia, to assess prognosis.

Ontogeny of a new enteric peptide, neuropeptide W (NPW), in the developing rat stomach.

Neuropeptide W (NPW), a novel endogenous peptide for G protein-coupled receptors GPR7 and GPR8, is expressed in the gastric antral mucosa of rat, mouse, and human stomachs. Here, we studied the ontogeny of NPW in the developing rat stomach. Real-time RT-PCR showed that NPW gene expression was initially detectable in embryonic day 14 (E14) stomach and gradually increased during the progress of age until birth, postnatal day 1 (P1). NPW mRNA level in the stomach increased again from the weaning period (P21) until reaching adulthood. Immunohistochemistry using polyclonal antibodies raised against rat NPW revealed that NPW-positive cells were detected in the P1 antral stomach and gradually increased during the development of age. Furthermore, double immunohistochemistry demonstrated that NPW colocalized with gastrin in P1 rat stomach. These data will provide clues to physiological functions of NPW in the development of rat stomach.

Background submucosal cysts in early gastric cancer cases have unique clinicopathologic features suggestive of postgastritis and significant smoking association.

Submucosal cysts (SMCs) might result from severe gastritis and be related to gastric carcinogenesis, although direct evidence is limited. We studied clinicopathologic findings for gastric cancers arising in mucosa with SMC and the relation to gastritis. In 504 submucosal invasive cancer cases, SMC was found in 100. Comparison of degrees of gastritis using the Updated Sydney system, thickness of muscularis mucosae, and the patients' smoking and drinking habits and obesity showed significant variation between cases of cancer with and without SMC. In the stomach with SMCs, cancers were predominantly differentiated-type adenocarcinomas in men and showed a significant tendency for location in the upper gastric region. Intestinal metaplasia was significantly more severe and the muscularis mucosae were thicker in cancer cases with SMC in comparison with cases without SMC and control cases of gastrointestinal stromal tumor (GIST). Atrophy was also significantly more severe in cancer cases with and without SMC than in cases of GIST. The Brinkman index was also significantly higher. Cases of gastric cancer with SMC show characteristic clinicopathologic features, and SMC formation may be caused by gastritis and influenced by smoking.

Cloning of the thaumatin I cDNA and characterization of recombinant thaumatin I secreted by Pichia pastoris.

Thaumatin is a sweet-tasting protein comprising a mixture of some variants. The major variants are thaumatins I and II. Although the amino acid sequence of thaumatin I was known and the nucleotide sequence of cDNA of thaumatin II was elucidated, the nucleotide sequence of thaumatin I has been controversial. We have cloned two thaumatin cDNAs from the fruit of Thaumatococcus daniellii Benth. One is the same nucleotide sequence as that of thaumatin II already reported, and the other is a novel nucleotide sequence. The amino acid sequence deduced from the novel cDNA was the same amino acid sequence as that of thaumatin I, the only exception being the residue at position 113 (Asp instead of Asn), indicating that the novel thaumatin cDNA is that for thaumatin I. This thaumatin I cDNA was transformed into Pichia pastoris X-33, and the recombinant thaumatin I expressed was purified and characterized. The threshold value of sweetness of the recombinant thaumatin I was the same as that of the plant thaumatin I, although several unexpected amino acid residues were attached to the N-terminal of the recombinant thaumatin I. These indicate that the N-terminal portion of thaumatin is not critical for the elicitation of sweetness.

Genetic instability on chromosome 17 in the epithelium of non-polypoid colorectal carcinomas compared to polypoid lesions.

Early colorectal carcinomas (submucosal invasive adenocarcinomas) can be classified into polypoid growth carcinoma (PG-Ca) and non-polypoid growth carcinoma (NPG-Ca) types, the latter transforming more rapidly to advanced carcinoma. Previously, we indicated that stromal genetic instability might contribute to tumorigenesis of both sporadic and ulcerative colitis-associated colorectal adenocarcinomas. In the present study, we analyzed genetic instability of both epithelial and surrounding stromal components in PG-Ca and NPG-Ca. In 99 colorectal submucosal invasive adenocarcinomas, epithelial and stromal genetic instability was analyzed with National Cancer Institute standard microsatellite markers, chromosome 17 (Chr.17) markers and tumor suppressor gene-related markers, using a combination of the laser-captured microdissection and GeneScan approaches. Immunohistochemical analysis was carried out for hMLH1, hMSH2, MGMT and p53. In addition, we investigated methylation of the hMLH1 and MGMT promoters. The frequencies of epithelial microsatellite instability (MSI) with Chr.17 markers were significantly higher in NPG-Ca (33.3%) compared to PG-Ca (10.4%), particularly with D17S579 and D17S796. For loss of heterozygosity, only D17S786 showed a significant difference. The frequencies of stromal MSI with all markers were 31.7% and 25.9% in NPG-Ca and PG-Ca, respectively, but D17S579 and TP53 showed higher MSI in NPG-Ca than PG-Ca. Immunohistochemically, p53 protein expression in PG-Ca was significantly higher in loss of heterozygosity-positive cases with altered Chr.17 markers overall, especially the D17S796 marker, compared to cases without genetic instability. These results suggest that epithelial and stromal MSI of Chr.17 markers contributes more to carcinogenesis in NPG-Ca, whereas stromal genetic instability might be necessary for the development of both types of colorectal carcinoma.

Significant increase of colonic mutated crypts correlates with age in sporadic cancer and diverticulosis cases, with higher frequency in the left- than right-side colorectum.

Mild periodic acid-Schiff (mPAS) staining can discriminate non-O-acetylated (mPAS-positive) from O-acetylated (mPAS-negative) epithelial sialoglycoproteins in human colonic mucosa, allowing the three haplotypes expressed from a single polymorphic autosomal gene (oat) to be distinguished. In heterozygotes, we previously demonstrated wholly mPAS-positive (stem cell mutated) crypts and clusters of two or more mPAS-positive crypts to be significantly increased with duration of ulcerative colitis. To establish whether such an increase in the number of mutated crypts with age also occurs in normal individuals or in cases with diverticulosis, the O-acetylation phenotype in the non-cancerous colonic mucosa of 47 sporadic colorectal cancer patients who were heterozygotes for oat was tested with mild-PAS staining. PAS-positive crypts were assessed histologically in relation to age and compared between the left (sigmoid colon and rectum) and right (cecum and ascending colon) sides of the colorectum. Wholly mPAS-positive (stem cell mutated) crypts and foci in heterozygotes were found to be increased significantly (P < 0.0001) in the left side with aging (r = 0.598 and 0.643, respectively). Such a positive correlation with aging was also confirmed in 19 diverticulosis cases without cancer (r = 0.797 and 0.793, respectively). The frequency of mutated crypts and foci on the right side was significantly lower than on the left side in both spontaneous colorectal cancer and diverticulosis cases. The results provide support for an intimate relationship between accumulation of mutated crypts with aging, possibly with significance for colorectal cancer development. Furthermore, the environment in the right side of the colon may be different from that in the left side in this regard.

Peripheral interaction of ghrelin with cholecystokinin on feeding regulation.

Ghrelin and cholecystokinin (CCK) are gastrointestinal hormones regulating feeding. Both transmitted via the vagal afferent, ghrelin elicits starvation signals, whereas CCK induces satiety signals. We investigated the interaction between ghrelin and CCK functioning in short-term regulation of feeding in Otsuka Long-Evans Tokushima fatty (OLETF) rats, which have a disrupted CCK type A receptor (CCK-AR), and their lean littermates, Long-Evans Tokushima Otsuka (LETO) rats. Intravenous administration of ghrelin increased 2-h food intake in both OLETF and LETO rats. Because OLETF rats are CCK insensitive, iv-administered CCK decreased 2-h food intake in LETO, but not in OLETF, rats. Although preadministration of CCK to LETO rats blocked food intake induced by ghrelin, CCK preadministration to OLETF rats did not affect ghrelin-induced food intake. Conversely, preadministration of ghrelin to LETO rats blocked feeding reductions induced by CCK. In electrophysiological studies, once gastric vagal afferent discharges were altered by ghrelin or CCK administration, they could not be additionally affected by serial administrations of either CCK or ghrelin, respectively. The induction of Fos expression in the hypothalamic arcuate nucleus by ghrelin was also attenuated by CCK preadministration. Using immunohistochemistry, we also demonstrated the colocalization of GH secretagogue receptor (GHS-R), the cellular receptor for ghrelin, with CCK-AR in vagal afferent neurons. These results indicate that the vagus nerve plays a crucial role in determining peripheral energy balance. The efficiency of ghrelin and CCK signal transduction may depend on the balance of their respective plasma concentration and/or on interactions between GHS-R and CCK-AR.

Identification of ghrelin and its receptor in neurons of the rat arcuate nucleus.

Ghrelin, an acylated peptide originally identified in rat stomach as the endogenous ligand for the growth hormone secretagogue receptor (GHS-R), stimulates both food intake and growth hormone (GH) secretion. Ghrelin is predominantly synthesized by a subset of endocrine cells in the oxyntic gland of human and rat stomach. Previous studies using immunohistochemistry have shown that ghrelin is also present in the hypothalamic arcuate nucleus, a region critical for the control of feeding and GH secretion, but its expression pattern in this region and the details of its molecular form has yet to be clarified. In this report, we examined the presence of ghrelin in the arcuate nucleus using reverse-phase liquid chromatography combined with radioimmunoassay (RIA) and immunohistochemistry. Neurons in the arcuate nucleus were observed to react positively to ghrelin antibodies. In addition, we confirmed the existence of ghrelin mRNA expression using the reverse-transcription polymerase chain reaction (RT-PCR). We also observed the colocalization of GHS-R with neuropeptide Y (NPY) and growth-hormone-releasing hormone (GHRH) in the arcuate nucleus. The present study clearly indicates that ghrelin is synthesized in the arcuate nucleus, which will further our understanding of ghrelin's actions in the central nervous system, including feeding behavior and GH secretion.