Development of a loop-mediated isothermal amplification assay for rapid and simple detection of Erysipelothrix rhusiopathiae.

UNLABELLED: Erysipelothrix rhusiopathiae is a causative agent of swine erysipelas. We developed a novel and highly specific loop-mediated isothermal amplification (LAMP) assay for sensitive and rapid detection of E. rhusiopathiae. The LAMP assay correctly detected 39 E. rhusiopathiae strains. No LAMP products were detected from 14 non-rhusiopathiae Erysipelothrix and 16 non-Erysipelothrix strains, including E. tonsillarum serovar 10 strains, which are difficult to be discriminated from E. rhusiopathiae strains. These results were consistent with those obtained by a conventional E. rhusiopathiae-specific PCR assay. Starting with DNA extraction from a single colony, the gel-based PCR assay took 4 h to provide a result, but the LAMP assay was faster, requiring only 37-80 min. The conventional culture test required more than 3-4 days to isolate and identify E. rhusiopathiae in the enrichment cultures. In contrast, the LAMP assay required less than 22 h from the beginning of the enrichment culture to final determination. These results suggest that the LAMP assay is useful as an adjunct to facilitate early diagnosis of swine erysipelas. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first report of a loop-mediated isothermal amplification (LAMP) assay for simple and cost-effective detection of E. rhusiopathiae from swine samples. The LAMP assay provided more rapid detection of the bacterium than conventional PCR and biochemical-based assays, and it may potentially facilitate surveillance and early diagnosis of swine erysipelas in the field.

CD8+ T cells infiltrated within cancer cell nests as a prognostic factor in human colorectal cancer.

The pathophysiological significance of tumor infiltrating lymphocytes remains controversial. To clarify their role, we performed clinicopathological analysis of CD8+ T cells in 131 cases of human colorectal cancer. CD8+ T cells were classified into three groups by their localization: (a) those infiltrated within cancer cell nests; (b) those distributed in the cancer stroma; and (c) those present along the invasive margin (tumor-host interface). Of these, CD8+ T cells within cancer cell nests were most significantly associated with a better survival of patients by both mono- and multivariate analyses. The impact on survival was similar to that of Dukes' staging. Granzyme B+ cytoplasmic granules were detected in lymphocytes within cancer cell nests, confirming their activated, cytotoxic phenotype. CD8 and Ki-67 double immunohistochemistry confirmed higher proliferative activity of CD8+ T cells within cancer cell nests. Our data suggested that human colorectal cancer tissue was infiltrated by various numbers of T cells that had cytotoxic phenotype, contributing to a better survival of patients. This infiltration of colorectal cancer cell nests by CD8+ T cells could be a novel prognostic factor.

The insulin-like growth factor II receptor gene is mutated in genetically unstable cancers of the endometrium, stomach, and colorectum.

Disruption of the DNA mismatch repair system, characterized by microsatellite instability (MI), plays an important role in the course of human carcinogenesis. Repetitive sequences constitute targets for mutation in MI+ cells, and frequent mutations have indeed been reported in such regions within the transforming growth factor beta receptor II (RII) gene in genetically unstable colorectal and gastric cancers. However, other genes that are targets for mutations in MI+ cells during the course of carcinogenesis have proven elusive. Because the insulin-like growth factor II receptor (IGFIIR) gene contains several repetitive sequences within its coding region, we examined mutations of this gene in MI+ cancers occurring at various primary sites. We found frameshift mutations in the poly(G)8 tract of IGFIIR in eight tumors, all of which were MI+: 4 of 26 (15%) MI+ endometrial cancers, 3 of 12 (25%) MI+ gastric cancers, and 1 of 18 (6%) MI+ colorectal cancers. In contrast, no mutation was found in 51 pancreatic cancers, 7 of which (14%) were MI+. These results implicate abnormal IGFIIR-mediated growth control in carcinogenesis involving the endometrium, stomach, and colorectum but not the pancreas.

Functional evaluation of PTEN missense mutations using in vitro phosphoinositide phosphatase assay.

The tumor suppressor gene PTEN is frequently mutated in diverse human cancers and in autosomal dominant cancer predisposition disorders. Recent studies have shown that the lipid phosphatase activity of PTEN is critical for its tumor suppressor function and that PTEN negatively regulates the phosphatidylinositol 3'-kinase-protein kinase B pathway. Although more than half of PTEN mutations result in protein truncation, a significant fraction of PTEN mutations are missense mutations. To examine whether tumor-derived and germ-line-derived missense mutations inactivate PTEN lipid phosphatase function, we constructed 42 distinct types of PTEN missense mutations and expressed them in Escherichia coli. The purified (His)6-tagged PTEN proteins were tested for their ability to dephosphorylate inositol 1,3,4,5-tetrakisphosphate and phosphatidylinositol 3,4,5-triphosphate. In addition, we examined the effect of mutant PTENs on the ability of PTEN to bind to the phospholipid membrane. The results revealed that the majority of PTEN missense mutations [38 of 42 (90%)] eliminated or reduced phosphatase activity and that all of the mutations examined had no effect on the membrane binding activity of PTEN. Our study indicated that phosphoinositide phosphatase activity is important for the tumor suppressor function of PTEN and that there may be other mechanisms of PTEN inactivation that are not monitored by in vitro phosphatase assay and in vitro membrane binding assay.

Identification of thyroid hormone transporters in humans: different molecules are involved in a tissue-specific manner.

We have recently identified that rat organic anion transporters, polypeptide2 (oatp2) and oatp3, both of which transport thyroid hormones. However, in humans the molecular organization of the organic anion transporters has diverged, and the responsible molecule for thyroid hormone transport has not been clarified, except for human liver-specific transporter (LST-1) identified by us. In this study we isolated and characterized a novel human organic anion transporter, OATP-E from human brain. The isolated complementary DNA encodes a polypeptide of 722 amino acids with 12 transmembrane domains. A rat counterpart, oatp-E, was also identified. Homology analysis and the phylogenetic tree analysis revealed that OATP-E/oatp-E is a subfamily of the organic anion transporter. Human OATP-E transported 3,3',5-triiodo-L-thyronine (K(m), 0.9 microM), thyronine, and rT(3) in a Na(+)-independent manner. Although the clone was isolated from the brain, OATP-E messenger RNA was abundantly expressed in various peripheral tissues. The rat counterpart, oatp-E, also transported 3,3',5-triiodo-L-thyronine. In addition, in this study we revealed that human OATP, which is exclusively expressed in the brain, transported 3,3',5-triiodo-L-thyronine (K(m), 6.5 microM), T(4) (K(m), 8.0 microM), and rT(3). These data suggest that in humans, several different molecules are involved in transporting thyroid hormone: OATP in the brain, LST-1 in the liver, and OATP-E in peripheral tissues.

Detailed deletion mapping on chromosome 1p32-p36 in human colorectal cancer: identification of three distinct regions of common allelic loss.

Recent studies have suggested the existence of one or several tumor-suppressor genes on chromosome arm 1p in colorectal tumors. To determine the localization of the putative tumor suppressor genes, we performed LOH analysis in 1p in colorectal tumors. A total of 48 paired normal and tumor DNAs of 46 colorectal tumor patients and 21 microsatellite markers on 1p32.1-p36.3 were used for PCR-LOH analysis. Three commonly deleted regions were found: i) 1p36.3 (10-cm); ii) 1p35.1-p36.3 (2-cm); and iii) 1p34.2-p35 (1-cm). These regions overlapped with those reported in several types of tumor. No significant associations were found between LOH and clinicopathologic features. The regions identified in the present study could harbor tumor suppressor genes that would also be associated with several types of human cancer.

The somatic mutation frequency of the transforming growth factor beta receptor type II gene varies widely among different cancers with microsatellite instability.

Disruption of the DNA mismatch repair system, characterized by microsatellite instability (MSI), plays an important role in the course of human carcinogenesis. Frequent somatic mutations in a polyadenine (poly(A)) tract and two GT repeats within the coding region of the transforming growth factor beta (TGFbeta) receptor II (RII) gene were reported in colorectal cancers with MSI. We examined mutations of RII in cancers of various organs with MSI and found deletions at the poly(A) tract in eight of nine (89%) gastric cancers and four of five (80%) colorectal cancers. In contrast, no mutations were found in cancers of the pancreas, endometrium, or lungs. These results suggest that TGFbeta-mediated growth control plays a very important role in the stomach and colorectum.

Infrequent somatic mutations of the p73 gene in various human cancers.

AIMS: It has already been reported that loss of heterozygosity (LOH) on chromosome 1p is frequent in a variety of human cancers. This finding implies the presence of some important tumour suppressor genes in this region. p73, a candidate tumour suppressor gene identified recently in chromosome band 1p36.33, encodes a protein highly homologous to p53. To investigate the role of the p73 gene in human carcinogenesis, we studied genetic alterations of this gene in various human cancers. METHODS: We analysed the entire coding exons as well as their surrounding exon-intron boundaries of the p73 gene in 185 cases of various types of tumours (47 breast cancers, 43 colorectal cancers, 31 gastric cancers, 23 neuroblastomas, 21 lung cancer cell lines, and 20 pancreatic cancer cell lines); they are known as a group of tumours with frequent LOHs in the 1p region. PCR-SSCP analysis was performed and tumours in which aberrant migrating sized bands were observed were subjected to direct sequencing analyses. RESULTS: Of the 185 cases, only one somatic mis-sense mutation of glutamine from arginine at codon 269 in exon 7 was found in one breast cancer. In addition, several polymorphisms were found at codons 137, 336, 349, and 610, as well as in introns 6, 8, and 9. Monoallelic expression was also observed in pancreatic cancer cell lines. CONCLUSIONS: Our results suggest that inactivation of the p73 gene does not play a major role in the tumour types analysed in the present study.

[A case of inflammatory breast cancer treated with preoperative intra-arterial infusion chemotherapy].

A 35-year-old female with inflammatory breast cancer was treated with preoperative intra-arterial infusion chemotherapy. One cycle consisted of cyclophosphamide 700 mg, epirubicin 20 mg and 5-FU 1,750 mg. After 4-cycles of intra-arterial infusion chemotherapy, the size of tumor and regional lymph nodes were remarkably decreased. Histological examinations revealed that almost all the cancer cells had disappeared in the resected specimen. In the patient, preoperative intra-arterial infusion chemotherapy was very effective in local control, which enabled us to perform a histologically curative operation. In conclusion, multimodal treatment including intra-arterial infusion chemotherapy is a promising treatment for inflammatory breast cancer.

[A randomized trial of intrahepatic infusion chemotherapy for unresectable colorectal liver metastases. Sendai Study Group].

A prospective, controlled randomized trial of hepatic arterial infusion of 5-fluorouracil (5-FU), adriamycin (ADM) and mitomycin C (MMC) [FAM group] versus 5-FU, epirubicin (EPIR) and MMC [FEM group] in patients with unresectable liver metastasis from colorectal cancer is reported. No objective response was observed in FAM group (n = 6), while two objective responses, 1 complete and 1 partial (22.2%), were achieved in FEM group (n = 9). There was no significant difference in the 50% survival period between the two groups (468 days in FAM group (n = 8) versus 462 days in FEM group (n = 10). Long survival over 2 years was observed in FEM group, but not in FAM group. Toxicities were recorded in 50% (3/6) of FAM group, and 80% (8/10) of FEM group, but they were mild and well tolerated. In conclusion, although there was no significant difference in clinical effects between the two groups, the use of EPIR instead of ADR in combination with 5-FU and MMC might be favorable for intrahepatic infusion chemotherapy because responders and long-term survival were exclusively observed in FEM group.

[The efficacy of intra-arterial infusion chemotherapy in patients with non-resectable liver metastasis from colorectal cancer--a randomized study comparing FAM versus FEM].

The efficacy of FAM (5-FU, ADM, MMC) chemotherapy was compared with that of FEM (5-FU, EPIR, MMC) chemotherapy, in which the EPIR dose was about 1.3 times the ADM dose, by hepatic arterial infusion. Twenty-one patients with unresectable hepatic metastasis from colorectal cancer were the subjects of this multi-institutional randomized study. The response rate in evaluable 15 patients was 0% (0/6) in FAM group and 22% (2/9) in FEM group, with a 50% survival time of 15.3 months in FAM group and 15.1 months in FEM group. The major toxicities were gastrointestinal, together with hepatic dysfunction, both of which were not serious. Total incidence of > or = grade 2 toxicities was 50% (3/6) in FAM group and 80% (8/10) in FEM group. There was no statistical difference between the two groups in response rate, 50% survival time and toxicities. The clinical results of FAM and FEM were therefore considered equal, and an expected improvement in efficacy by increasing the EPIR dose was not confirmed. However, two patients in FEM group showed an objective response not observed in FAM group; one of them showed CR and survived about 3 years. These findings seem to suggest the efficacy of FEM hepatic arterial infusion chemotherapy for treatment of unresectable hepatic metastasis from colorectal cancer.

[Relationship between venous invasion and hematogenous metastasis in the gastric cancer].

We investigated the correlation between venous invasion and hematogenous metastasis in gastric cancer. In 70 patients with gastric cancer, 8 cases had hepatic metastasis at operation (group A), 26 cases were identified with hematogenous recurrence after operation (group B) and 36 cases were disease free over 5 years after operation (group C). Specimens were all step-sectioned and serial sections were stained both with HE and EM. Rates of venous invasion were calculated as following formulae; (number of cancer involving veins/total number of veins investigated) x 100 (%). Results were as follows. 1. Average rates of venous invasion (ARVI) of groups A and B were significantly higher than that of group C (A; 7.6%, B; 2.6%, C; 0.7% p < 0.05). 2. In subserosal or serosal layer, there is no significant difference between ARVI of group A and that of group B. 3. No significant difference was seen between group A and B in rates of venous invasion to larger vein (diameter > 200 microns). These observations suggest that cancer invasion to the veins located in deeper than subserosal layer or ranged in diameter over 200 microns relates to hematogenous metastasis.

[Antitumor effector cells in recombinant IL-2 activated tumor infiltrating lymphocytes from human solid cancers].

Tumor infiltrating lymphocytes (TIL) isolated from 46 human solid cancers, including 24 renal cell carcinoma (RCC), 15 cancers of digestive organs and 7 other malignancies, were cultured with 1000 units/ml of recombinant interleukin-2 (rIL-2). Induction of activated TIL was achieved in 41 cases (89.1%). Their antitumor characteristics and cell surface antigen expressions were examined using 4 hr 51Cr release assay and flow cytometric analysis in long-term culture. Cytotoxic activity of rIL-2 activated TIL derived from RCC was significantly higher than that from other tumors, but the TIL could lyse various types of cells and specificity for autologous tumor cells could not be demonstrated in any of the cultures. Though CD3+ lymphocytes were predominant in all fresh TIL preparations, antitumor activity of rIL-2 activated TIL correlated with the increase of a cell population expressing the Leu19 antigen without the CD3 antigen. Depletion of selected cell types from TIL cultures using direct complement dependent cytolysis followed by cytotoxicity tests identified the Leu19+ CD3- CD16- cells as the major effector population. CD3-, CD16+ NK cells held minor antitumor cytotoxicity and there was no detectable cytotoxic activity in the CD3+ T lymphocytes.

[Pylorus-preserving gastrectomy for early gastric cancer].

Pylorus-preserving gastrectomy (PPG) was originally proposed by Maki et al for gastric ulcer. Recently this operation with lymph node dissection has been adopted for patients with early gastric cancer locating at antrum and body of the stomach in Japan. But standard procedure of this operation has not been established yet. Grade of lymph node dissection, preservation of pyloric branch and indication criteria are fairly dependent on each surgeon when PPG is applied for gastric cancer. Although the results of this operation is not accumulated enough, it is considered that this operation may have benefits for decrease of postoperative chronic morbidity with acceptable cancer curativity as compared to conventional distal gastrectomy, Billroth-I procedure.

[Oral administration of OK-432 is an effective immunotherapy against colon cancer--a study in a murine mesenteric lymphnode metastasis model].

A simple and highly reproducible mouse colonic cancer model with mesenteric lymph node (MLN) metastasis was established by injecting COLON-26 tumor cells into lymphatic nodules in the tip of vermiform appendix of BALB/C mice. In this model, antitumor effect of orally administered OK-432 was examined. OK-432 was given orally at three different periods: before injection of COLON-26 cells (Protocol-A), early after injection of COLON-26 cells (Protocol-B), and late after injection of COLON-26 cells (Protocol-C). As a result, MLN metastasis was significantly suppressed and survival rate was significantly improved in every protocol as compared to mice without OK-432 administration. Then, in vivo anti-tumor activities of MLN cells and splenic lymphocytes were evaluated by Winn assay. Tumor neutralizing activity against syngenic COLON-26 cells was induced in the MLN cells after OK-432 administration and the effector cells were found in CD4-positive T lymphocytes because MLN cells treated with anti-L3/T4 and anti-LFA plus complement lost their anti-tumor efficacy. In conclusion, oral administration of OK-432 appears to be an effective immunotherapy against colonic cancer especially in the prevention and the treatment for MLN metastasis.

[Functional and phenotypic analysis of tumor infiltrating lymphocytes derived from solid tumors and effusion associated lymphocytes in malignant ascites cultured in recombinant interleukin 2].

Tumor infiltrating lymphocytes (TIL) and effusion associated lymphocytes (EAL) were isolated from 7 human solid tumors and 6 malignant ascitic fluids respectively, and cultured in rIL2 (700JRU/ml)-containing medium. Long-term culture (> 14 days) of separated lymphocytes with exponential increase in cell number was achieved in 5 EAL-cultures, whereas in only 2 TIL-cultures. rIL2-expanded TIL and EAL manifested significant cytotoxicity in a 4-hrs chromium release assay. The maximum NK and LAK activity were reached 21 days and 14 days after starting incubation with rIL2 respectively, followed by a rapid decrease in cytolytic potential without declining growth rate of the cells. Phenotypic analysis showed the majority of the freshly isolated TIL and EAL were CD3+ T cells, and CD16+ NK cells were rarely identified in TIL. With induction of LAK cell activity, CD8+ T cells predominantly increased in TIL-cultures, while both CD4+ and CD8+ T cells and CD16+ NK cells were increased in EAL cultures. In activated TIL most cytolytic activity was found in CD8+ T cells, in contrast CD16+ NK cells were responsible for it in activated EAL. These results indicated that EAL and TIL have similar properties in which they coexist with cancer cells at tissue level and were capable of expanding and acquiring LAK cell activity in the presence of rIL2, but apparently differ in their mechanism of rIL2-mediated activation.

Quantitative evaluation of adhesion of lactobacilli isolated from human intestinal tissues to human colonic mucin using surface plasmon resonance (BIACORE assay).

AIMS: To isolate lactobacilli from the mucus layer of the human intestine and evaluate their adhesion abilities using a BIACORE assay. METHODS AND RESULTS: Thirty strains of lactobacilli were isolated from the mucus layer of normal human intestinal tissues using conventional plate culture. The strains were identified using homology comparisons of the 16S rDNA sequence to databases as Lactobacillus salivarius (26%), Lactobacillus fermentum (13%), Lactobacillus gasseri (10%), Lactobacillus paracasei (7%), Lactobacillus casei (3%), Lactobacillus mucosae (3%) and Lactobacillus plantarum (3%). Lactobacillus plantarum LA 318 shows the highest adhesion to human colonic mucin (HCM) using the BIACORE assay at 115.30 +/- 12.37 resonance unit (RU). The adhesion of cell wall surface proteins from strain LA 318 was significantly higher to HCM than to bovine serum albumin (BSA; P < 0.05). CONCLUSIONS: We isolated 30 strains of lactobacilli. Lactobacillus salivarius was the predominant species of lactobacilli isolated in this study. The adhesion of strain LA 318 isolated from human transverse colon to its mucin was shown. The adhesion could be mediated by lectin-like components on the bacterial cell surface. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first study where lactobacilli were isolated from human intestinal tissues and shown to adhere to HCM.

Preparation of a new arabinoxylooligosaccharide from wheat bran hemicellulose and its structure.

A novel arabinoxylooligosaccharide was prepared from wheat bran and its structure was analyzed. Wheat bran hemicellulose was digested with a commercial Aspergillus japonicus hemicellulase preparation and an oligosaccharide was purified by carbon column and gel filtration chromatographies to be a single component. The oligosaccharide has D.P. of 6 and contains arabinose and xylose with a molar ratio of 2:1. Xylotetraose was formed as an intermediate hydrolyzate on acid hydrolysis of this oligosaccharide. Methylation analysis indicated that both reducing and non-reducing end xylose residues were free from arabinose residues and that there is only one xylose residue attached by two arabinose residues. The structure of this saccharide was finally identified by 13C NMR as beta-D-Xylp-(1-->4)[alpha-L-Araf-(1-->2)][alpha-L-Araf -(1-->3)]-beta-D- Xylp-(1-->4)-beta-D-Xylp-(1-->4)-D-Xylp.

Cell adhesion molecule expression by vascular endothelial cells as an immune/inflammatory reaction in human colon carcinoma.

The cell adhesion of inflammatory cells to vascular endothelial cells is an important process in the recruitment of inflammatory cells to the site. In cancer tissue, infiltration of inflammatory cells has been suggested to be a mechanism of host resistance. To clarify this infiltration mechanism, we investigated cell adhesion molecule expression (E-selectin, P-selectin, and ICAM-1) in vascular endothelial cells by immunohistochemistry in colon carcinoma. Venules distributed along the invasive margin expressed E- and P-selectins and ICAM-1. These phenotypical features are identical to those of endothelial cells observed in active inflammatory lesions, and the vessels can, therefore, be designated as immunologically activated vessels. Nevertheless, the majority of blood vessels within the tumor lacked immunoreactivity for all these adhesion molecules and, therefore, could be designated as immunologically inactive vessels. Granulocytes, lymphocytes and macrophages, bearing the counter-receptors of these adhesion molecules, were more densely distributed along the invasive margin. In contrast, few inflammatory cells were present within the tumor. In conclusion, the present study has demonstrated the phenotypical heterogeneity of tumor vessels; those for inflammatory cell infiltration to the tumor and those for the nutrient supply to the tumor.

Enhanced expression of lipocortin-1 as a new immunosuppressive protein in cancer patients and its influence on reduced in vitro peripheral blood lymphocyte response to mitogens.

To clarify the mechanism of immunosuppression in cancer-bearing hosts, the expression of lipocortin-1 (LC1), a new immunosuppressant, and its effects on the in vitro mitogen responsiveness of peripheral blood mononuclear cells (PBMC) were investigated in cancer patients. Immunohistochemical studies showed LC1 expression in the cytoplasm of inflammatory cells morphologically recognized as a macrophage lineage, infiltrating the tumor interstices of gastric cancer. LC1 protein was detected in the ascitic fluid from gastric cancer patients using Western blot analysis. LC1 expression in PBMC was studied using a fluorescence-activated cell sorter (FACScan), which revealed that the percentage of CD14 and LC1 double positive cells was much greater in cancer patients than in healthy individuals. The proliferative response of PBMC by concanavalin A (ConA) stimulation was significantly suppressed in patients with advanced cancer, while the intact mitogen responsiveness in healthy individuals was inhibited when recombinant LC1 was added to the cultures. A similar inhibitory effect was induced by adding the supernatant of cancerous ascites or spleen cell cultures derived from advanced cancer patients. These inhibitory effects were eliminated, and the suppressed mitogen responsiveness in cancer patients recovered to the control level of healthy individuals when anti-LC1 antibody was added to the cultures. These findings indicate that LC1 is produced and expressed in cancer patients, and deeply involved in the immunosuppressive mechanism of tumor-bearing hosts.