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.

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.

Intraepithelial CD8+ T-cell-count becomes a prognostic factor after a longer follow-up period in human colorectal carcinoma: possible association with suppression of micrometastasis.

T-cell infiltration into human cancer tissues can be a manifestation of host immune responses to cancer cells. The present study was undertaken to explore the clinicopathological significance of intraepithelial CD8(+) T cells using 371 consecutively sampled human colorectal carcinomas. By univariate analysis, we noted that the survival curves by intraepithelial CD8(+) T cells became separated only after 1 to 2 years postoperation. Multivariate analyses revealed that the beneficial effect of this factor becomes significant only after a longer (more than 2 year), but not after a shorter (less than 2 year) follow-up period. Furthermore, the number of intraepithelial CD8(+) T cells was significantly higher in patients alive for more than 5 years than in patients who either died of cancer after a curative operation or patients who underwent a noncurative operation. Patients' cancer-specific death long after a curative operation is thought to be caused by the growth of micrometastases in other organs or near the primary sites. The effects of intraepithelial CD8(+) T cells, therefore, may be mediated by suppression of micrometastasis, rather than suppression of growth in the primary tumour. In conclusion, our data support a hypothesis on the presence of systemic immunosurveillance against micrometastasis of cancer cells.

Allelic loss of the NF1 gene in anal malignant melanoma in a patient with neurofibromatosis type 1.

A 64-year-old man with neurofibromatosis type 1 (NF1) developed a primary malignant melanoma of the anus. Genetic analysis of the resected tumor confirmed loss of heterozygosity (LOH) of the NF1 gene. Anorectal malignant melanoma in NF1 is extremely rare, and genetic studies of the NF1 gene in such patients have not been reported. The allelic loss detected in the present patient supports the previously raised idea that NF1 can function as a tumor suppressor gene in the development of malignant melanoma in patients with NF1.

LST-2, a human liver-specific organic anion transporter, determines methotrexate sensitivity in gastrointestinal cancers.

BACKGROUND & AIMS: One approach to the development of targeted cancer chemotherapy exploits increased uptake of the agent into neoplastic cells. In this scenario, higher concentrations of the agent in cancer cells are responsible for differential killing, whereas the low concentration in normal human cells decreases side effects. The aim of this study was to isolate an organic anion transporter that is weak in normal cells, but abundantly expressed in cancer cells, to deliver the anticancer drugs to the cells. METHODS: A human liver complementary DNA (cDNA) library was screened with liver-specific transporter (LST)-1 cDNA as a probe. Northern blot analyses were performed using the isolated cDNA (termed LST-2). An LST-2-specific antibody was raised, and immunohistochemical analyses including immunoelectron microscopy were performed. Xenopus oocyte expression system was used for functional analysis. We also established a permanent cell line that consistently expresses LST-2 to examine the relationship between methotrexate uptake and sensitivity. RESULTS: The isolated cDNA, LST-2, has 79.7% of overall homology with human LST-1. LST-2 exclusively expressed in the liver under normal conditions and its immunoreactivity was highest at the basolateral membrane of the hepatocytes around the central vein. Although its weak expression in the liver, LST-2 is abundantly expressed in the gastric, colon, and pancreatic cancers. On the other hand, the LST-1 was only detected in a hepatic cell line. LST-2 transports methotrexate in a saturable and dose-dependent manner. Furthermore, introduction of the LST-2 gene into mammalian cells potentiates sensitivity to methotrexate. CONCLUSIONS: LST-2 is one of the prime candidate molecules for determining methotrexate sensitivity and may be a good target to deliver anticancer drugs to the gastrointestinal cancers.

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.

Introduction of antisense CD44S CDNA down-regulates expression of overall CD44 isoforms and inhibits tumor growth and metastasis in highly metastatic colon carcinoma cells.

We created antisense CD44 transfectants using LS174T, a colon adenocarcinoma cell line and assessed the effects of overall CD44 down-regulation on colorectal tumor growth and metastasis. The expression of antisense CD44s (the standard form of CD44) cDNA markedly inhibited the overall expression of CD44 variants. In vitro studies showed a significantly reduced ability of the stable antisense transfectants (LS174TAS1 and LS174TAS2) to bind hyaluronate and osteopontin, ligands for CD44. These cells developed tumors more slowly than controls (parental LS174T and mock transfectants) when the cells were subcutaneously injected into SCID mice. However, in vitro proliferation assays demonstrated no significant difference between the antisense transfectants and the controls on a hyaluronate-coated surface, suggesting the participation of ligands other than hyaluronate in tumor growth in vivo. Intrasplenic injection of parental LS174T cells produced colonies in the liver in 10 of 11 mice, whereas mice injected with the antisense transfectants were completely free of metastasis. In peritoneal dissemination, the weight of nodules and amount of ascites were significantly reduced in LS174TAS1 and AS2 compared with the controls. In vitro adhesion assays between the transfectants or controls and human peritoneal mesothelial cells revealed that the binding of LS174T cells to mesothelial cells was partly mediated by CD44-hyaluronate interaction. These data suggest that CD44-hyaluronate interaction plays a crucial role in peritoneal dissemination in colorectal carcinoma. The results of our study demonstrate the possible application of antisense CD44s to the treatment of colorectal carcinoma.

Molecular identification of a rat novel organic anion transporter moat1, which transports prostaglandin D(2), leukotriene C(4), and taurocholate.

We have isolated a rat novel multispecific organic anion transporter, moat1. The isolated clones were originated by alternative splicing of the moat1 mRNA. The nucleotide sequences predict a protein of 682 amino acids with moderate sequence similarity to LST-1, the oatp family, and the prostaglandin transporter. Northern blot analysis of rat moat1 identified a predominant transcript of 4.4 kilonucleotides in all tissues. Northern blot and in situ hybridization analyses of rat brain further indicated that moat1 mRNA is widely distributed in neuronal cells of the central nervous system, especially in the hippocampus and cerebellum. moat1 transports prostaglandin D(2) (K(m); 35.5 nM), leukotriene C(4) (K(m); 3.2 microM) and taurocholate (K(m); 17.6 microM) in a sodium-independent manner. moat1 also transports prostaglandin E(1), E(2), thromboxane B(2), and iloprost but not dehydroepiandrosterone sulfate and digoxin, of which the substrate specificity is similar, but definitively different from those of any other organic anion transporters.

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.

Chromosome band 16q24 is frequently deleted in human gastric cancer.

We have analysed the loss of heterozygosity (LOH) on chromosome bands 16q22-q24 in 24 primary gastric cancer tissues and found three regions of frequent allelic loss (16q22, 16q24.1-q24.3 and 16q24.3). The region for the most frequent allelic loss (63%) was in 16q24.1-q24.3. LOH of this region had no relationship with histological subtype, but a significant association between LOH and microscopic lymphangial invasion was observed. Although not significant, vascular and gastric wall invasions are also associated with LOH. The region includes the locus for the H-cadherin gene. Therefore we examined the genetic and epigenetic alterations of this gene. Markedly reduced expression was observed in gastric cancer cell lines compared with that of normal gastric mucosa. However, no mutation was found in this gene in any of the gastric cancer tissues or the gastric cancer cell lines. Furthermore, we analysed the methylation status of the 5'-flanking region of the gene, but no significant association was found. We suggest that some other tumour suppressor gene(s) in 16q24.1-q24.3 may be responsible for gastric carcinogenesis.

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.

Differing expression of MMPs-1 and -9 and urokinase receptor between diffuse- and intestinal-type gastric carcinoma.

Gastric cancer is classified into intestinal and diffuse types, which exhibit different biological behavior. Urokinase-type plasminogen activator (uPA), its receptor (uPAR) and matrix metalloproteinases (MMPs)-1 and -9 are considered to play important roles in cancer invasion and metastasis. We have already suggested a functional duality of these matrix-degrading enzymes/factors; they may also be involved in the matrix turnover (remodeling) or host immune/inflammatory reactions as far as they are expressed by host cells. We performed a retrospective study on the immuno-histochemical expression of these enzymes/factors in surgical specimens from patients with gastric cancer, including 26 with the diffuse and 78 with the intestinal type. We also evaluated macrophages since they are major sources of uPAR. The positivity rate for uPA in cancer cells was significantly lower in diffuse-type than in intestinal-type. Stromal expression was seen mainly along the invasive margin (tumor-host interface). The degree of stromal expression of uPAR and MMP-9 and the macrophage number were markedly decreased in diffuse-type compared with intestinal-type. Stromal expression of uPAR and macrophage number in intestinal-type were higher in patients without liver metastasis than in patients with liver metastasis, while uPA expression in cancer cells was more pronounced in patients with liver metastasis. Studies using frozen sections revealed that the expression of MMP-1, restricted to the stromal area, was more decreased in diffuse-type (18 patients) than in intestinal-type (21 patients). Our results show that the in situ expression of matrix-degrading enzymes/factors in gastric cancer is significantly more diminished in diffuse-type than in intestinal-type, suggesting a multifunctional aspect of the matrix-degradation process in cancer tissue.

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.

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.

CD44H participates in the intrahepatic growth of murine colon 26 adenocarcinoma cells.

The purpose of this study was to determine if CD44, a metastasis-associated cell adhesion molecule, is involved in the hepatic colonization by murine colon 26 adenocarcinoma cells. Indirect membrane immunofluorescence and FACS analysis showed strong expressions of CD44 and integrin beta 1 on colon 26 cells. Injection of 1 x 10(5) colon 26 cells into the superior mesenteric vein of syngeneic BALB/c mice produced macroscopic hepatic nodules in 92% (22/24) of the mice 14 days after inoculation. When colon 26 cells were pretreated with an anti-CD44 monoclonal antibody (mAb), IM7, only 30% (3/10) of the mice produced minute nodules in the liver on day 14 (P < 0.001), though IM7 did not inhibit growth of the cells in vitro. Pretreatment of colon 26 cells with an anti-integrin beta 1 mAb did not significantly block the hepatic metastasis. Histologically, microcolonies of tumor cells were detected in all of the livers on day 14 including the IM7-pretreatment mice that were free of gross nodules. However, percentages of tumor-occupied areas in the liver were consistently lower in IM7-pretreatment mice than in control mice (0.82% vs. 5.0% on day 14; P < 0.005). Reverse transcription-polymerase chain reaction (RT-PCR) amplification of mRNA revealed that colon 26 cells and splenocytes only expressed the hematopoietic isoform of CD44 (CD44H), which had no insertion of variant exons, while normal colonocytes expressed possible variant isoforms. These data suggest that malignant transformation of murine colonic epithelium altered the expression pattern of CD44 isoforms and that CD44H participates in the intrahepatic growth of colon 26 cells.

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.

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.

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.

[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.