Infiltration of CD40-positive tumor-associated macrophages indicates a favorable prognosis in colorectal cancer patients.

BACKGROUND/AIMS: Recently the role of tumor-associated macrophages (TAMs) on immunity has been variously discussed. We studied a series of cell surface antigens in TAMs in colorectal cancer tissues and their corresponding normal tissues using flow cytometry to find out prognostic indicators of these patients. METHODOLOGY: We assessed the numbers of CD14+ macrophages positive for each of the cell surface antigens (CD80, CD86, HLA-DR, CD1a, CD40 and CD83) in cancer tissues and corresponding normal tissues among 31 patients with colorectal cancer, and performed the univariate and multivariate analysis to find out prognostic indicators for overall survival among the patients. RESULTS: The numbers of CD80+, CD86+ and HLA-DR+ TAMs in the cancer tissues were higher than those in corresponding normal tissues. Inversely CD40+ and CD83+ macrophages in cancer tissues were less than those in normal tissues. With the multivariate analysis, the number of CD40+ TAMs, as well as lymph node metastasis and distant metastasis, was shown to be an independent prognostic factor of colorectal cancer patients. CONCLUSIONS: The dense infiltration of CD40+ TAM in colorectal cancer tissues indicates a favorable prognosis, which suggests that CD40 plays an important role in the tumor immunity of colorectal cancer.

Novel function of MKP-5/DUSP10, a phosphatase of stress-activated kinases, on ERK-dependent gene expression, and upregulation of its gene expression in colon carcinomas.

Mitogen-activated protein kinase phosphatase 5 (MKP-5)/DUSP10 acts as a phosphatase of stress-activated kinases (JNK and p38), but its activity towards ERK has not been demonstrated. In the present study we observed that MKP-5 interacts with ERK, retains it in the cytoplasm, suppresses its activation and downregulates ERK-dependent transcription. These data suggested a novel MKP-5 function as a scaffold protein for the ERK pathway. We analyzed MKP-5 gene expression in several tumors, and found that it is frequently upregulated in colorectal but not in lung and breast cancer, suggesting its association with the malignant phenotype of colon cancer.

Infiltration of CD14-positive macrophages at the invasive front indicates a favorable prognosis in colorectal cancer patients with lymph node metastasis.

BACKGROUND/AIMS: To clarify the distribution of CD14+ macrophages in colorectal cancer using flow cytometry and immunohistochemistry, and to elucidate the roles of CD14+ macrophages in colorectal cancer. METHODOLOGY: We studied the paired cancerous and corresponding normal tissues from 52 patients with colorectal cancer for the distribution of CD14+, CD1a+, CD83+ and CD68+ cells, and correlated the findings with the clinicopathological characteristics and with the expression of CD86 and CD80 in the CD14+ macrophages, which are co-stimulatory factors for T cell activation. RESULTS: 1) CD14+ macrophages were distributed predominantly at the invasive front of colorectal cancer tissues, rather than in the normal tissues, 2) a high percentage of the CD14+ macrophages expressed CD86 and CD80, and 3) in the colorectal cancer cases with lymph node metastasis, the 5-year overall survival rate of the high CD14 group, in which the number of CD14+ macrophages was higher than the median, was better than that of the low CD14 group. CONCLUSION: The infiltration of CD140 macrophages at the invasive front indicates a favorable prognosis in colorectal cancer patients with lymph node metastasis. In addition, the activation of CD14+ macrophages and T cells may facilitate the development of new immunotherapeutic strategies for colorectal cancer patients.

DUSP13B/TMDP inhibits stress-activated MAPKs and suppresses AP-1-dependent gene expression.

The dual-specificity phosphatase (DUSP) 13 gene encodes two atypical DUSPs, DUSP13B/TMDP and DUSP13A/MDSP using alternative exons. DUSP13B protein is most highly expressed in testis, particularly in spermatocytes and round spermatids of the seminiferous tubules, while that of DUSP13A is restricted to skeletal muscle. Here, we show that DUSP13B inactivated MAPK activation in the order of selectivity, JNK = p38>ERK in cells, while DUSP13A did not show MAPK phosphatase activity. Reporter gene analysis showed that DUSP13B had significant inhibitory effect on AP-1-dependent gene expression, but DUSP13A did not. To our knowledge, DUSP13B is the first identified testis-specific phosphatase that inhibits stress-activated MAPKs. These data suggest an important role for DUSP13B in protection from external stress during spermatogenesis.

Cancer-associated splicing variants of the CDCA1 and MSMB genes expressed in cancer cell lines and surgically resected gastric cancer tissues.

BACKGROUND: Alternative splicing is a molecular mechanism by which different combinations of exons can be alternatively spliced to produce different mRNA isoforms. Recently, several databases have been published to predict the alternative splicing of mRNA; cancer-specific alternative splicing has also been predicted with these databases. Those variants may be potentially useful targets for cancer therapy, however, the accuracy and veracity of these databases have yet to be confirmed. METHODS: In this study, we analyzed 17 genes by reverse transcriptase-polymerase chain reaction (RT-PCR) that were predicted to have cancer-specific alternative splicing by using the splicing database, the Alternative Splicing Annotation Project (ASAP) by Lee et al, between 38 cancer cell lines from various organs and 9 corresponding normal tissues. By designing 2 types of primer sets for RT-PCR including (1) primers flanking the alternatively spliced exons and (2) primers spanning the exon/exon junctions, cancer-associated splicing variants were investigated. RESULTS: The alternatively splicing events were detected in 15 of 17 genes (88%); 35 of 43 variants (81%) were detected successfully with RT-PCR. Among these variants, M-RIP, HYAL2, CDCA1, and MSMB genes showed differential expressions between cancer cell lines and corresponding normal tissues. Furthermore, RT-PCR with surgically resected gastric cancer tissues (diffuse type, 6; intestinal type, 4) confirmed that 2 variants of CDCA1 were upregulated in cancer tissues, whereas both variants of MSMB were expressed predominantly in normal tissues. CONCLUSION: Alternative splicing variants, especially in CDCA1, were detected in this study and may be potentially useful as diagnostic markers and/or novel targets for anticancer therapy.

Successful treatment of intraoperative heart failure caused by ampulla cardiomyopathy by intra-aortic balloon pumping and percutaneous cardiopulmonary support: report of a case.

An 82-year-old woman underwent total gastrectomy for advanced gastric cancer with invasion to the lower esophagus. Her blood pressure dropped alarmingly during the operation, which was performed via the transabdominal and left-side transthoracic approach. Using echocardiography, we diagnosed intraoperative-onset reversible heart failure caused by ampulla cardiomyopathy. Because the infusion of catecholamines is associated with secondary heart failure, we gave her calcium antagonists and nicorandil, then started intra-aortic balloon pumping (IABP) and the percutaneous cardiopulmonary support system (PCPS). On postoperative day (POD) 7, the IABP and PCPS were removed and on POD 12, she was extubated successfully. The patient was discharged on POD 54 and has remained well. The factors predisposing her to ampulla cardiomyopathy were left-side thoracotomy, hypoxia caused by one-lung ventilation, and the infusion of high-dose catecholamines. Prompt diagnosis and timely treatment of the heart failure with IABP and PCPS prevented any further complications.

Outcomes after pylorus-preserving gastrectomy for early gastric cancer: a prospective multicenter trial.

The aim of the present study was to compare in a prospective, multicenter trial the results early and late after pylorus-preserving gastrectomy (PPG) versus conventional distal gastrectomy (CDG) with Billroth I anastomosis for early gastric cancer. Eighty-one patients with early gastric cancer were randomized and then underwent either PPG or CDG. Duration of operation, intraoperative blood loss, days until removal of the nasogastric tube, days until start of oral intake, and decrease in body weight were studied as parameters for outcomes early after the surgery. Late results were studied in patients followed for longer than 3 years. Change in body weight, status of oral intake, symptoms suggesting early dumping syndrome, and overall satisfaction were addressed in the questionnaire. The presence of gallstones was examined with ultrasonography. There were no differences in early results between PPG and CDG. The incidence of early dumping syndrome was lower in PPG (8%) than in CDG (33%). Other late results including the incidence of gallstones were not different between the 2 groups. These results indicate that PPG is as safe as CDG and has an advantage in terms of early dumping syndrome.

Effect of ileo-jejunal transposition on ileal longitudinal smooth muscle contractility in vitro in rats.

The aim of the present paper was to study the effects of ileo-jejunal transposition (IJT) on ileal contractile activity in vitro in rats. Male Sprague-Dawley rats were divided into three groups: control, IJT, and sham. In rats with IJT, the distal ileum was interposed isoperistaltically into the proximal jejunum. The jejunoileum was transected and anastomosed at three sites in the sham group. Rats were sacrificed 17-20 weeks postoperatively and the ileal segment was removed. Isometric contractile activity of the isolated ileal longitudinal muscle was measured in tissue chambers. Spontaneous contractile activity was decreased in the IJT group (0.16 +/- 0.03 g/min per mg tissue) as compared with the control group (0.25 +/- 0.02 g/min per mg tissue, p < 0.05). The motor response to cholinergic agonist bethanechol in the IJT group was greater than in the control group above 10(-6) M dosage. The dose-response curves to adrenergic agonist norepinephrine did not differ between groups. A nitric oxide synthase inhibitor reversed electrical field stimulation-induced inhibition of spontaneous activity in all groups. These results indicate that the response to bethanechol in the IJT group was enhanced in rat ileal longitudinal smooth muscle and this may be an adaptive response to compensate for decreased spontaneous contractile activity.

[Thymidine phosphorylase expressed in monocyte-macrophages enhanced anticancer effect of 5'-deoxy-5-fluorouridine on colorectal carcinoma cells].

OBJECTIVE: To detect the thymidine phosphorylase (dThdPase) expression in colorectal carcinoma tissue, and clarify whether dThdPase expressed in macrophage-like cell lines, and monocytes from human peripheral blood can modulate the anticancer effect of 5'-deoxy-5-fluorouridine (5'-DFUR) on colorectal carcinoma cells. METHODS: Forty specimens resected from 40 patients with colorectal carcinoma were immunohistochemically stained by the monoclonal antibodies 654-1 (anti-dThdPase) and PG-M1 (anti-macrophage marker CD68). Then morphometrical analysis and positive cell counting were performed. In 27 of 40 specimens, dThdPase activity analysis was assayed by HPLC. The dThdPase level were also measured by ELISA in 4 colorectal cancer cell lines, LS174T, Clone A, Colo320, MIP101, and 2 macrophage-like cell lines, THP-1, U937. After estimated the drug sensitivities of each colorectal carcinoma cell both to 5'-DFUR and 5-Fu by MTT assay, THP-1, U937, or monocytes isolated from human blood were incubated in the medium containing 5-Fu or 5'-DFUR for 24 hours, respectively. Then the supernatant was collected and 2-fold serially diluted with the medium, in which the macrophage-like cells, or monocytes were also cultivated for 24 h without anticancer agents. Using the serially diluted medium, MTT assay were also carried out on 4 colorectal carcinoma cell lines. Each experiment was repeated six times and the means of IC50 value +/- standard errors (mean +/- S.E.M.) were calculated. Finally, THP-1 or U937 cells were cultured in medium containing 400 micro mol/L of 5'-DFUR for 24 hours, then the supernatants were collected and the amount of generated 5-Fu was measured by HPLC. RESULTS: dThdPase activities was significantly increased (139.7 micro g x 5-Fu x h(-1) x ml(-1) +/- 61.5 micro g x 5-Fu x h(-1) x ml(-1)) in colorectal carcinoma tissues compared with adjacent normal tissues (42.2 micro g x 5-Fu x h(-1) x ml(-1) +/- 21.4 micro g x 5-Fu x h(-1) x ml(-1)), P < 0.001. In immunohistochemical analysis, it was confirmed that most cells expressed dThdPase-positive cells were the stromal cells, especially macrophages, which surrounding cancer nests, or along the invasive margin of cancer. The distribution patterns of dThdPase-positive stromal cells are similar to that of the CD68-positive cells. The number of dThdPase positive cells was correlated with the number of macrophages in the cancerous tissues, r = 0.76. The dThdPase protein were detected at the levels of 18.2 unit/mg in THP-1, 19.3 unit/mg in U937, and 0.5 unit/mg in LS174T, however, not detected in other 3 colorectal carcinoma cells. The values of IC50 of 5'-DFUR on the 4 colorectal carcinoma cell lines were 11.5 approximately 84.8 times higher than those of 5-Fu (all P < 0.01). It was showed that no inhibiting effect for 5'-DFUR on the growth rates of THP-1, U937, and monocyte cells. After incubated with THP-1 or U937, 5'-DFUR expressed a significant enhanced antitumor effect (P < 0.0001), while almost no change is observed to 5-Fu (P > 0.05). Same conclusion was also demonstrated in using the monocytes instead of THP-1 or U937. 40.2 micro mol/L and 29.5 micro mol/L of 5-Fu were detected in the medium containing 400 micro mol/L of 5'-DFUR treated with THP-1 and U937 cells, respectively. CONCLUSION: 5'-DFUR cannot be converted into 5-Fu in colorectal carcinoma cells in vitro because no dThdPase is expressed in those cells. After being incubated with macrophage-like cell, THP-1, U937, or human monocytes, the anticancer effect of 5'-DFUR is significantly increased due to the activation by dThdPase expressed in above cells.

Evidence for existence of oligoclonal tumor-infiltrating lymphocytes and predominant production of T helper 1/T cytotoxic 1 type cytokines in gastric and colorectal tumors.

Tumor-infiltrating lymphocytes (TIL) play a central role in cellular immunity against tumor. We have revealed the characteristics of TILs in terms of T-cell receptor (TCR) repertoire, T-cell clonality, and cytokine production. TCR repertoire analyses and CDR3 size spectratyping were performed using peripheral blood mononuclear cells (PBMCs) and tissue specimens of gastric or colorectal cancers surgically resected from 11 patients. The cytokine expression was measured by real-time quantitative polymerase chain reaction. TCR repertoires were similar among multiple tissue specimens from different sites of the same tumor. Similar peak patterns of CDR3 size spectratyping were observed among these tumor specimens, but not in normal tissues or PBMCs. In addition, identical peaks were detected in multiple specimens of the same tumor. The ratio of the levels of IFN-gamma to that of IL-4 is significantly higher for tumor lesions compared with PBMCs. These results suggested that a limited number of TILs locally expand in response to tumor antigens exiting within gastric or colorectal cancers and local predominant production of the T helper 1/T cytotoxic 1 type cytokine may affect the anti-tumor immune response of TILs.

New reconstructive procedure after intestinal resection for Crohn's disease: modified side-to-side isoperistaltic anastomosis with double Heineke-Mikulicz procedure.

Although side-to-side isoperistaltic anastomosis is a useful strictureplasty technique when long segments of intestinal stenosis complicate Crohn's disease, concerns have been raised regarding disease recurrence adjacent to the anastomosis. We performed side-to-side isoperistaltic anastomosis without spatulated intestinal ends as a method of reconstruction after intestinal resection for Crohn's disease; both intestinal ends were transversely closed like a Heineke-Mikulicz-type strictureplasty. With this procedure, the luminal diameter proximal and distal to the anastomosis became wider than the original diameter of the intestine. This new procedure, which we refer to as the "modified side-to-side isoperistaltic anastomosis with double Heineke-Mikulicz procedure" could become an alternative operation after intestinal resection in persons with Crohn's disease, although long-term outcome analysis is necessary.

Expression of thymidine phosphorylase by macrophages in colorectal cancer tissues.

AIM: To detect the thymidine phosphorylase (dThdPase) expression in human colorectal cancer tissues and cells. METHODS: Forty specimens resected from patients with colorectal cancer were immunohistochemically stained by 654-1, anti-dThdPase monoclonal antibody, PG-M1, anti-macrophage marker CD68 monoclonal antibody. Morphometrical analysis and positive cell counting were performed. In 27 of 40 specimens, dThdPase activity was also assayed by HPLC. Otherwise, the dThdPase level was measured by ELISA in 6 colorectal cancer cell lines, LS174T, Clone A, Colo320, CX-1, Lovo, and MIP101, as well as in 2 macrophage-like cell lines, THP-1 and U937. RESULTS: dThdPase activity was significantly increased in cancer tissues compared with adjacent normal tissue (P<0.01). In immunohistochemical analysis, it was confirmed that most cells expressed dThdPase were the stromal cells surrounding cancer nests or along the invasive margin of cancer. Based on their morphometrical characteristics, we found that most of them were tumor-associated macrophages (TAMs). The number of dThdPase-positive stromal cells was significantly correlated with the number of CD68-positive macrophages (r=0.76, P<0.0001). By ELISA, 18.2 unit/mg and 19.3 unit/mg of dThdPase protein were detected in THP-1 and U937, but only little was detected in 6 colorectal cancer cell lines. CONCLUSION: The present data suggest that dThdPase expression is seldom detected in colorectal carcinoma cells. TAM is the most important source of dThdPase in colorectal cancer tissues.

Thymidine phosphorylase expressed in macrophages enhances antitumor effect of 5'-deoxy-5-fluorouridine on human colorectal carcinoma cells.

BACKGROUND: Thymidine phosphorylase (dThdPase) is a key enzyme in the activation of the pro-drugs of 5-fluorouracil (5-FU), 5-deoxy-5-fluorouridine (5'-DFUR) and capecitabine. In colorectal carcinoma (CRC), the major cells expressing dThdPase have been shown to be stromal cells, particularly macrophages. MATERIALS AND METHODS: The present study was designed to clarify whether dThdPase expressed in macrophage-like cell lines, THP-1 and U937, and monocyte-rich mono-nuclear cells (MoMNCs) from human peripheral blood can modulate the antitumor effect of 5'-DFUR on CRC cells. RESULTS: dThdPase protein was found in THP-1 and U937 by ELISA, while little or no dThdPase could be detected in the CRC cell lines tested. Incubation of 5'-DFUR with the macrophage-like cells significantly enhanced the antitumor effect of 5'-DFUR in a 5-DFUR sensitivity assay compared with untreated 5'-DFUR. MoMNCs also showed a similar effect. When the media containing 5'-DFUR was treated with either THP-1 or U937 cells, detectable levels of 5-FU could be measured in the treated media. CONCLUSION: These data suggest that macrophages convert 5'-DFUR to 5-FU and release the converted 5-FU, resulting in an enhancement of the antitumor effect of 5'-DFUR.

Interleukin-12 administration is more effective for preventing metastasis than for inhibiting primary established tumors in a murine model of spontaneous hepatic metastasis.

PURPOSE: The effect of interleukin-12 (IL-12) on tumor growth at the primary site and its therapeutic efficacy against metastasis were examined using a model of spontaneous hepatic metastasis. METHODS: IL-12 was peritumorally injected into RL male1 tumor-bearing BALB/c male mice at the different tumor stage. RESULTS: Striking inhibition of hepatic metastasis in both athymic and euthymic mice was induced by the administration of IL-12 irrespective of the stage of tumor progression. In contrast, IL-12 failed to produce any antitumor effect in athymic mice which lack conventional T cells. These results suggest that the antitumor effect of IL-12 is mediated mainly by T cells, and that the antimetastatic effect is mediated mainly by natural killer (NK) and/or NKT cells. Next we examined the direct effect of IL-12 on these cells. IL-12-induced T-cell proliferation was remarkably augmented in the early stage, then decreased dramatically in the advanced stage, while IL-12-induced cytotoxic activity, mediated by NK and NKT cells, was not attenuated even in the advanced stage. This dissociation in IL-12 responsiveness appeared to be the reason for the remarkable antimetastatic effect but insufficient antitumor effect of IL-12 in the advanced stage. CONCLUSION: The findings of this study support the clinical use of IL-12 for immunotherapy against either occult or evident liver metastasis.