Stress up-regulates oxidative burst in juvenile Chinook salmon leukocytes.

When fish perceive stressful scenarios, their hypothalamus-pituitary-interrenal axis is activated resulting in the release of corticotropin releasing hormone, adrenocorticotropic hormone (ACTH), and finally cortisol. The physiologic stress response of fish has most often been linked to the reduced performance of the immune system, with a few exceptions where the immune system is activated. In this report, we tested the hypothesis that oxidative burst activity levels in juvenile Chinook salmon (Oncorhynchus tshawytscha) are altered when the fish is presented with a stressor. Fish were subjected to a stressor for 3 h and then allowed to recover for 20 h following the stressor. Plasma and spleens were collected from euthanized fish before the stressor, at the end of a 3 h stressor, and 23 h after the start of the experiment. Plasma was held at -80 °C until cortisol radioimmunoassay analysis was performed to confirm stress. Spleens were held in Dulbecco's Modified Eagle Medium overnight and analyzed the day following collection. Oxidative burst activity was measured in splenic leukocytes after being stimulated with phorbol 12-myristate 13-acetate. We found a significant increase in activated oxidative burst from fish subjected to the stressor as compared to unstressed fish. Speculation is given to ACTH being the leukocyte priming agent in this experiment rather than the cortisol itself.

Evaluation of seasonal and daily changes of plasma thyroxine and cortisol levels in wild masu salmon Oncorhynchus masou, sampled by a Japanese fishing method.

A new fish sampling method was developed using a Japanese bait fishing rod (8-9 m carbon rod and a nylon line with a small fine wire single hook), which is considered to catch wild salmonid juveniles with low sampling stress. Using this method, seasonal and daily changes of plasma thyroxine (T4 ) and cortisol levels were examined in wild parr, pre-smolts and smolts of masu salmon Oncorhynchus masou in contiguous locations in a coastal river (Kesen River; 44 km) in northern Honshu Island, Japan, overlapping the period of smoltification and seaward migration from August to March. Plasma T4 and cortisol were low in 0+ and 1+ year parr caught in August and September. In March, some yearling (1+ year) fish, which were judged as pre-smolts, and smolts appeared mainly in mid and lower reaches, while parr (0+ and 1+ year parr) continued to appear in the upper and mid reaches. In March, 1+ year pre-smolts and smolts showed high plasma T4 levels while the levels of 1+ year parr were low. During March 2008-2010, plasma T4 levels of 1+ year pre-smolts and smolts had high levels from early to mid-March, whereas plasma cortisol levels of 1+ year smolts were low in early March and increased towards mid-March. Based on these data, plasma cortisol increases probably occur following the increases of plasma T4 levels to lead the 1+ year O. masou to the completion of smoltification and initiation of seaward migration.

Seasonal changes in gene expression of corticoid receptors in anadromous and non-anadromous strains of rainbow trout Oncorhynchus mykiss.

To clarify the regulation of expression of corticoid receptor (CR) genes during period of parr-smolt transformation of salmonids, seasonal changes in mRNA levels of glucocorticoid receptor (GR)-1, GR-2 and mineralocorticoid receptor (MR) were examined in gill, leucocytes, spleen and brain of anadromous and non-anadromous forms of Oncorhynchus mykiss. Increases in gill Na(+) , K(+) ATPase activity, plasma thyroxine levels and hypo-osmoregulatory ability assessed by 24 h seawater challenge test represented characteristics of smoltification in anadromous O. mykiss from May to June, whereas there was no apparent increase in the values of non-anadromous O. mykiss. Plasma cortisol levels of anadromous O. mykiss were higher than levels of non-anadromous O. mykiss from April to June. In gill of non-anadromous O. mykiss, there were significant increases in mRNA levels of three types of CR in spring. Although there were significant seasonal variations of CR mRNA levels in gill of anadromous O. mykiss, they appear to be less clear than those variations in non-anadromous O. mykiss. In anadromous O. mykiss, significant elevations in mRNA levels of the three types of CR were observed especially in the spleen. In both preoptic area and basal hypothalamus of the brain, there were tendencies to increase in CR mRNA levels from spring to summer in both anadromous and non-anadromous O. mykiss. These results showed difference in regulation of CR gene expression between the two forms of O. mykiss for osmoregulatory, immune and central nervous systems.

Prevention of advanced colorectal cancer by screening using the immunochemical faecal occult blood test: a case-control study.

To evaluate colorectal cancer screening with faecal occult blood testing (FOBT) in terms of prevention of advanced cancers, we conducted a case-control study in the areas where an annual screening programme with immunochemical FOBT has been offered to all inhabitants aged 40 years or over. Cases were 357 consecutive patients in the study areas clinically diagnosed as having advanced colorectal cancer or a tumour invading the muscularis propriae or deeper, that is, T(2)-T(4) in TNM classification. Three controls were selected for each case matched by gender, age, residential area and exposure status to screening within 1 year before case diagnosis. The odds ratios (ORs) of developing advanced cancer were calculated using conditional logistic regression analyses. The OR for those screened within 3 years before the diagnosis vs those not screened was 0.54 (95% confidence interval (CI) 0.29-0.99). The ORs were lower for rectum than for colon (0.32-0.73 and 0.84-1.18 for rectum and colon, respectively). For those screened within the past 3 years, OR of developing advanced cancer in the rectum was 0.32 ( 95%CI: 0.12-0.84). A screening programme with immunochemical FOBT can be effective for prevention of advanced colorectal cancer. Risk reduction appears to be larger for rectal than for colon cancer. British Journal of Cancer (2003) 89, 23-28. doi:10.1038/sj.bjc.6601002 www.bjcancer.com

The mechanism of cell death in human cultured colon adenocarcinoma cell line COLO 201 induced by beta-D-N-acetylglucosaminyl-p-nitrophenol.

COLO 201, human colon adenocarcinoma cells were incubated with artificial primers, p-nitrophenyl-glycoside derivatives at 1.0 mmol (mM) in the medium containing 10% fetal bovine serum to detect sugar chain elongation. However, when p-nitrophenyl-beta-N-acetylglucosamine (beta-GlcNAc-PNP) was added, the medium changed color to yellow and the cells were dead. To explain this finding, the cells were incubated with 1.0 mM each of beta-GlcNAc-PNP and 4-methylumbelliferyl-beta-N-acetylglucosamine, then the number of living cells was measured in a time course. In beta-GlcNAc-PNP, the living cells were decreased at 24 hours. The cells were survived with N-acetylglucosamine, whereas in the presence of p-nitrophenol (PNP) the living cells were decreased. It was suggested that PNP released from beta-GlcNAc-PNP induced the cell death. Activity of beta-D-N-acetylglucosaminidase was detected in fetal bovine serum. It was shown that PNP induced the cell death in time-and-dose dependent manner. Genomic DNA from COLO 201 analyzed by agarose gel electrophoresis was fragmentated. PNP analogues were tested for toxicity, and the results suggested that the phenolic OH-group linked to benzene ring and nitro-group linked to the structure in para-form (PNP) was the most effective.

KAI1, CAR, and Smad4 expression in the progression of colorectal tumor.

PURPOSE: The abnormal expression of several genes is involved in the development of colorectal tumors. The aim of this study was to examine the expression of KAI1, CAR, and Smad4, which has been associated with the progression of other cancers. METHODS: We examined 12 adenomas, 38 primary carcinomas, 10 liver metastases, and 7 adenocarcinoma cell lines, by fluorescent multiplex reverse transcription-polymerase chain reaction (RT-PCR). RESULTS: KAI1 expression was higher in adenomas (2.55 +/- 0.61; P < 0.05) and primary carcinomas (3.30 +/- 0.42; P < 0.005) than in normal mucosa, but it was not correlated with tumor stage. CAR expression was lower in Dukes' B (1.28 +/- 0.53) than in Dukes' A (2.38 +/- 0.48) (P < 0.05), but increased with tumor progression; Dukes' C (1.45 +/- 0.25), Dukes' D (1.53 +/- 0.14), and metastases (2.09 +/- 0.39) (P < 0.05). Smad4 expression increased in adenomas (2.30 +/- 0.46; P < 0.05), but decreased with tumor progression; Dukes' A (2.14 +/- 0.37), Dukes' B (1.65 +/- 0.41), Dukes' C (1.57 +/- 0.25), Dukes' D (1.08 +/- 0.18), and metastases (0.82 +/- 0.21) (P < 0.05). CONCLUSIONS: These results suggest that the upregulation of CAR and down-regulation of Smad4 are associated with the progression of colorectal tumors, while the upregulation of these genes and of KAI1 seems to be involved in the early stage.

Evaluation of the association between serum anti-Lewis X antibody and inflammatory infiltration into the gastric mucosa infected with Helicobacter pylori.

BACKGROUND: Lipopolysaccharides of Helicobacter pylori have an antigenic structure that mimics Lewis X occurring in gastric mucosa. The pathogenic role of antigenic mimicry in Helicobacter pylori-induced gastritis has been of recent interest. AIM: To examine whether this molecular mimicry affects gastric mucosal inflammation in patients infected with Helicobacter pylori. PATIENTS: A total of 59 patients (mean age 58.0 years, 35 males, 24 females) were studied. METHODS: Serum samples were collected to measure IgG antibodies to Helicobacter pylori and Lewis X. Biopsy specimens were obtained from the antrum and corpus for the grading of gastritis. Correlation coefficients between serum Lewis X antibody titre and histological grades of inflammatory infiltration were determined. RESULTS: There was no significant correlation between anti-Lewis X antibody titre and the grade of mononuclear or polymorphonuclear cell infiltration. High titre of anti-Lewis X antibody was seen only in patients who had increased inflammatory infiltration in the corpus. CONCLUSIONS: Serum anti-Lewis X antibody, possibly induced by Helicobacter pylori infection, does not seem to play a major role in gastric mucosal inflammation in patients with Helicobacter pylori infection.

The involvement of sex steroid hormones in downstream and upstream migratory behavior of masu salmon.

From May through July when masu salmon, Oncorhynchus masou, commence downstream migration under natural conditions, yearling precocious male masu salmon (resident form) showed higher GSI and plasma levels of testosterone (T) and 11-ketotestosterone (11-KT) in contrast to immature smolts (migratory form). From March through September coinciding with the upstream migration period, 2-year-old male and female adults also showed higher GSI and plasma levels of T, estradiol-17beta (E(2)) 11-KT, 17alpha-hydroxyprogesterone and 17alpha,20beta-dihydroxy-4-pregnene-3-one (DHP). In order to test the effects of steroid hormones on migratory behaviors, silascone tube capsules containing 500 microg of T, E(2), 11-KT, DHP, or a vehicle was implanted into smolts, castrated precocious males, or immature parr, and downstream and upstream behavior were observed in artificial raceways in spring and autumn. Downstream behavior of smolts was inhibited significantly by T, E(2) and 11-KT. Upstream behavior was stimulated by T and 11-KT in castrated precocious males and stimulated by T, E(2) and 11-KT in immature parr. These results indicate that T, E(2) and 11-KT are the factors regulating downstream and upstream migratory behavior. In particular, because of its changing patterns in plasma and significant effects, T, the common precursor hormone of E(2) (female) and 11-KT (male), is considered to play central roles in both types of behavior.

Changes in intraluminal pressure in rat large intestines with aging and effects of dietary fiber.

Changes in intraluminal pressure in rat colon with aging and with the effects of dietary fiber were measured. A pressure sensor was inserted into the rat large intestine under endoscopic guidance. The intraluminal pressure curve in the colon was recorded, and the motility index was calculated by this curve. The rats were divided into three groups with a fiber-free diet, a cellulose diet (10% w/w), or a pectin diet (10% w/w). Intraluminal pressure was measured in the proximal, middle, and distal colon at 2, 4, 8, 12, and 16 months after birth. Intraluminal pressure in three sites increased with age and decreased in the latter half of the study. The motility index was lower during the course in the fiber groups, especially the pectin group more than the nonfiber group. This result suggests that long-term ingestion of dietary fiber might have a prophylactic effect on the development of diverticula.

The effects of testosterone on upstream migratory behavior in masu salmon, Oncorhynchus masou.

Effects of testosterone (T) on upstream migratory behavior in masu salmon, Oncorhynchus masou, were studied by use of artificial raceways. In Experiment 1, yearling precocious males castrated in August were implanted with a capsule of medical silicone tube containing 500 microg of T in September. Their upstream migration was recorded in an artificial raceway with castrated and sham-operated precocious males implanted with a capsule containing vehicle for 2 months. In Experiment 2, upstream migratory behavior of yearling immature parr implanted with a capsule containing T 500 microg or vehicle was observed from September through November. In Experiment 3, upstream migratory behavior of castrated, castrated + T 50 microg, castrated + T 500 microg, and sham-operated precocious males was observed from September through October. In Experiment 4, upstream migratory behavior of the control, T 50 microg-, T 500 microg-, and T 1000 microg-treated immature parr was observed from September through October. In each experiment, plasma and pituitary samples were taken from the fish that moved upstream and remained to measure levels of T, gonadotropin (GTH) II, thyroxine (T(4)), and triiodothyronine (T(3)) by radioimmunoassay. Administration of T caused increases in plasma T levels within the physiological range. In Experiment 1, the frequency of migration upstream was 0, 19.2, and 35.7% in the castrated, castrated + T 500 microg, and sham-operated groups, respectively. The frequency was higher in the castrated + T 500 microg and sham-operated groups than in the castrated group. In Experiment 2, the frequency was higher in T 500 microg-treated parr (22%) than in the control (2.6%). In Experiments 1 and 2, pituitary contents of GTH II in the T-treated groups and precocious males were higher than those in the castrated precocious males and immature parr. In Experiment 3, castrated + T 50 microg, castrated + T 500 microg, and sham-operated fish showed upstream migratory behavior, whereas castrated fish without T did not. In Experiment 4, the frequency was 5.7, 22.9, 17.1, and 28.6% in the control, T 50 microg-, T 500 microg-, and T 1000 microg-treated groups, respectively. In each experiment, plasma levels of T(4) in migrants were lower than those in nonmigrants, whereas plasma levels of T(3) did not show such changes. From these results, it is inferred that T is a factor influencing upstream migration in masu salmon.

Multiple accumulation of Vdelta2+ gammadelta T-cell clonotypes in intestinal mucosa from patients with Crohn's disease.

The gammadeltaT cells have been known to play an important role in the regulation of the mucosal immune system, but the relationship between these cells and the pathogenesis of Crohn's disease (CD) has remained obscure. We now demonstrate the T-cell receptor (TCR) Vdelta2 gene transcripts characterize antigenic immune response in the intestinal mucosa from patients with CD. TCR Vdelta2 gene transcripts of six patients with CD and six controls were subcloned and 20 randomly selected clones from each sample were subjected to nucleotide sequencing. Sequence analysis demonstrated that the different clones in the intestinal mucosa of CD were significantly increased (11.833 +/- 0.946) compared to controls (7.167 +/- 1.470) (P = 0.0374). The presence of intraindividual dominance and oligoclonality of TCR Vdelta2 gene transcripts in normal controls appears reflect positive selection and expansion of specific gammadelta T cells in normal controls. By contrast TCR Vdelta2 gene transcripts in the intestinal mucosa of CD can express different clonotypes. We conclude that accumulation of multiple Vdelta2+ gammadelta T-cell clonotypes are involved in the pathogenesis of CD.

Safety and efficacy of granulocyte and monocyte adsorption apheresis in patients with active ulcerative colitis: a multicenter study.

Active ulcerative colitis (UC) is characterized by activation and infiltration of granulocytes and monocytes/macrophages into the colonic mucosa. The infiltrated leukocytes can cause mucosal damage by releasing degradative proteases, reactive oxygen derivatives, and proinflammatory cytokines. The aim of this trial (conducted in 14 specialist centers) was to assess safety and efficacy of granulocyte and monocyte adsorption apheresis in patients with active UC most of whom were refractory to conventional drug therapy. We used a new adsorptive type extracorporeal column (G-1 Adacolumn) filled with cellulose acetate beads (carriers) of 2 mm in diameter, which selectively adsorb granulocytes and monocytes/macrophages. Patients (n = 53) received five apheresis sessions, each of 60 minutes duration, flow rate 30 ml per minute for 5 consecutive weeks in combination with 24.4 +/- 3.60 mg prednisolone (mean +/- SE per patient per day, baseline dose). During 60 minutes apheresis, 26% of granulocytes, 19.5% of monocytes and 2% of lymphocytes adsorbed to the carriers. At week 7, 58.5% of patients had remission or improved, the dose of prednisolone was reduced to 14.2 +/- 2.25 mg (n = 37). The apheresis treatment was fairly safe, only eight non-severe side effects (in 5 patients) were reported. Based on our results, we believe that in patients with active severe UC, patients who are refractory to conventional drugs, granulocyte and monocyte adsorption apheresis is a useful adjunct to conventional therapy. This procedure should have the potential to allow tapering the dose of corticosteroids, shorten the time to remission and delay relapse.

Serum anti-Lewis X antibody is associated with VacA seropositivity but not atrophic gastritis in patients with Helicobacter pylori infection.

OBJECTIVES: Lipopolysaccharides of Helicobacter pylori have an antigenic structure that mimics Lewis X occurring in gastric mucosa. The pathogenic role of antigenic mimicry in H. pylori-induced gastritis has been of recent interest. The aim of this study was to examine the relevance of anti-Lewis X antibody in the development of atrophic gastritis in H. pylori infection. METHODS: A total of 72 patients were studied. Serum samples were collected to measure IgG antibodies to H. pylori, CagA, VacA and Lewis X. Biopsy specimens were obtained from the antrum and the corpus to examine the grade and the type of atrophic gastritis. RESULTS: Mean anti-Lewis X antibody titres were higher in 38 VacA-seropositive patients than in 13 seronegative patients (P < 0.05). The difference was not significant between patients with diffuse-type atrophic gastritis and those with multi-focal type. No significant correlation was observed between the titre of anti-Lewis X antibody and the grade of glandular atrophy, whereas CagA seropositivity was associated with glandular atrophy. CONCLUSIONS: Anti-Lewis X antibody may play a role in persistent gastric inflammation, particularly in VacA-seropositive H. pylori infection. However, anti-Lewis X antibody does not seem itself to be associated with atrophic gastritis in patients with H. pylori infection.

Ecabet sodium inhibits the ability of Helicobacter pylori to induce neutrophil production of reactive oxygen species and interleukin-8.

The pathogenic role of human neutrophils has been implicated in Helicobacter pylori-associated gastritis. Ecabet sodium, a locally acting antiulcer drug, has anti-H. pylori actions. The aim of this study was to examine the effects of ecabet on the ability of H. pylori to stimulate human neutrophils. H. pylori were added to 1 x 10(5) neutrophils and incubated for 30 min in the presence of ecabet. Bacterial suspensions which had been incubated with ecabet for 30 min were also used to stimulate neutrophils. The intracellular production of reactive oxygen species was measured with a FACScan. Bacterial suspensions were also added to neutrophils in the presence of ecabet and incubated at 37 degrees C for 12 h to measure interleukin (IL)-8 production by enzyme-linked immunosorbent assay. The mean fluorescence intensity was found to be attenuated dose-dependently by ecabet (P < 0.01). Ecabet also inhibited IL-8 production by neutrophils in a dose-dependent manner (P < 0.001). Bacteria with prior incubation with ecabet induced significantly lower IL-8 production than those without this incubation (P < 0.05). Ecabet sodium has preventive effects on the ability of H. pylori to stimulate human neutrophils. It may lead to reduced gastritis activity and decreased oxidative damage of the gastric mucosa in H. pylori-associated gastritis.

Existence of variant gamma delta T cells in Crohn's disease.

BACKGROUND AND OBJECTIVES: gamma delta T cell populations are well-known for their unique distribution (e.g. intra-epithelial lymphocytes). Though their ligands play a major role in the immune response they have remained largely obscure. To shed light on this issue, we have analyzed in this study the complementarity determining region (CDR) 3 of the T cell receptor (TCR) V delta 2 chains. This provides an insight into the antigenic immune response in the intestinal mucosa in sickness and health. METHODOLOGY: Total RNA was extracted from surgically resected intestinal mucosa of patients with Crohn's disease (CD) and controls. TCR V delta 2 cDNA was PCR-amplified using V delta 2 sense primer and C delta antisense primer. The PCR products were then subcloned in pUC18 plasmid. From each sample, 20 subclones were randomly selected and subjected to nucleotide sequence analysis. RESULTS: Sequence analysis revealed that the CDR3 sequences of the TCR V delta 2 chains were unique to each individual. The evidence also showed a significant restriction of the junctional diversity of the TCR V delta 2 chains while no such restriction was found for CD. CONCLUSIONS: The marked complexity of the TCR V delta 2 junctional sequences and the oligoclonality of the TCR V delta 2 genes in the control subjects are indicative of a positive selection and expansion of specific T cells in the normal, healthy condition. In CD patients, however, the expression of distinct, non-overlapping TCR V delta 2 clonotypes can be found, suggesting polyclonal activation of gamma delta T cells in the diseased colon of CD patients. These findings have led us to conclude that accumulation of multiple gamma delta T cell clonotypes may be involved in the pathogenesis of CD.