The candidate tumour suppressor gene, ING1, is retained in colorectal carcinomas.

ING1 plays a critical role in regulating cell cycle progression and susceptibility to apoptosis. The present study aimed to investigate allelic deletion of, and mutations within, the ING1 gene in colorectal carcinomas. Genomic DNA was extracted from 29 sporadic colorectal carcinomas and samples of adjacent normal mucosa. Losses of heterozygosity of two polymorphic dinucleotide repeat markers close to the ING1 locus at chromosome 13q32-34 were analysed. Single-stranded conformational polymorphisms of polymerase chain reaction amplified regions within the coding sequence of ING1 were examined. Microsatellite instability was noted in 5 (17%) colorectal carcinomas; this confirms selection of a subject sample representative of the population. Neither losses of heterozygosity nor changes in electrophoretic mobility of single-stranded polymerase chain reaction products were detected in any colorectal carcinoma. Thus, in common with tumour suppressor genes such as RB and BRCA2 on chromosome 13q, ING1 appears to be retained intact in colorectal carcinomas.

Characterisation and distribution of vasoactive intestinal polypeptide binding sites in human brain.

The binding of 125I-vasoactive intestinal polypeptide (VIP) to human brain membranes has been studied. The binding was saturable with a dissociation constant (KD) of 4.4 +/- 2.0 nM and maximum binding value (Bmax) of 8.6 +/- 6.2 fmoles/mg tissue (mean +/- SD, n = 4), and was displaced by unlabelled VIP with an IC50 of 4 nM; secretin was much less potent with an IC50 of approximately 8 microM. The regional distribution of 125I-VIP binding in human brain revealed highest binding values to be in frontal and temporal cortices with intermediate values in amygdala, caudate and cerebellum, with lowest values in hippocampus, substantia nigra and hypothalamus.

Co-expression in Helicobacter pylori of cagA and non-opsonic neutrophil activation enhances the association with peptic ulcer disease.

AIMS: To investigate the association of cagA positivity and non-opsonic neutrophil activation capacity in wild-type Helicobacter pylori strains with peptic ulcer disease or chronic gastritis only. METHODS: Helicobacter pylori were isolated from antral biopsies of 53 consecutive patients with chronic antral gastritis, of whom 24 had peptic ulcer disease endoscopically. The presence of cagA, a marker for the cag pathogenicity island, was determined by polymerase chain reaction with specific oligonucleotide primers, and non-opsonic neutrophil activation capacity by luminol enhanced chemiluminescence. RESULTS: The cagA gene was present in 39 of 53 (73.6%) strains, 20 of which (83.3%) were from the 24 patients with peptic ulcer disease and 19 (65.5%) from the 29 patients with chronic gastritis only. Non-opsonic neutrophil activation was found in 29 (54.7%) strains, 16 of which (66.7%) were from patients with peptic ulcer disease, and 13 (44.8%) from those with chronic gastritis. Non-opsonic neutrophil activation was found more frequently in cagA+ than cagA- strains (59% v 42.9%). Whereas four of the 14 cagA- strains and eight of the 24 non-opsonic neutrophil activation negative strains were from patients with peptic ulcer disease, only two of 24 (8.3%) peptic ulcer disease strains expressed neither cagA nor non-opsonic neutrophil activation. The cagA gene and non-opsonic neutrophil activation capacity were co-expressed in 14 of 24 (58.3%) strains from patients with peptic ulcer disease, and in nine of 29 (31%) strains from individuals with chronic gastritis. CONCLUSIONS: Positivity for cagA and non-opsonic neutrophil activation occur independently in wild-type H pylori strains. However, co-expression of the two markers enhanced the prediction of peptic ulcer disease.

CagA/cytotoxic strains of Helicobacter pylori and interleukin-8 in gastric epithelial cell lines.

AIMS: To investigate: (1) whether Helicobacter pylori directly induces interleukin-8 (IL-8) message expression and protein secretion in established gastric epithelial cell lines; and (2) if CagA/cytotoxin positive and negative strains of H pylori differ in their ability to induce epithelial IL-8. METHODS: Gastric epithelial cell lines were co-cultured with H pylori NCTC 11637 and 10 clinical isolates (four cytotoxic, six non-cytotoxic) and secreted IL-8 was measured by enzyme linked immunosorbent assay (ELISA). Specific induction of gastric epithelial IL-8 mRNA was examined by reverse transcription and polymerase chain reaction (RT-PCR) amplification. RESULTS: H pylori (NCTC 11637) induced IL-8 secretion from three gastric epithelial cell lines (KATO-3, ST42, AGS) but not from MKN 45 (gastric) or intestinal (SW480, HT29) cell lines. H mustelae did not stimulate IL-8 secretion from KATO-3, ST42, and AGS cells. H pylori induced IL-8 secretion was reduced by heat killing, sonication, freeze thawing or formalin fixation of the bacteria. CagA/cytotoxin positive strains of H pylori induced significantly higher IL-8 secretion than CagA/cytotoxin negative strains in the three positive gastric epithelial cell lines (KATO-3, ST42: p < 0.01; AGS: p < 0.02). A significant increase (p < 0.01) in the expression of IL-8 mRNA relative to G3PDH mRNA was observed in KATO-3 cells after three hours of co-culture with CagA/cytotoxin positive strains. CONCLUSIONS: H pylori directly increases gastric epithelial IL-8 mRNA expression and IL-8 protein secretion in a strain specific manner. Induction of epithelial IL-8 by CagA/cytotoxin positive strains is likely to result in neutrophil chemotaxis and activation and thus mucosal damage. These observations on epithelial IL-8 may explain the association between CagA/cytotoxin positive strains and gastroduodenal disease.

Helicobacter pylori induced interleukin-8 expression in gastric epithelial cells is associated with CagA positive phenotype.

AIMS: To use a range of natural phenotypically variant strains of Helicobacter pylori with disparate CagA and VacA (vacuolating cytotoxin) expression to determine which bacterial factors are more closely associated with epithelial interleukin-8 (IL-8) induction. METHODS: Gastric epithelial cells (AGS and KATO-3) were co-cultured with five H pylori strains which were variously shown to express the cagA gene/CagA protein, VacA and/or to exhibit biological cytotoxicity. Secreted IL-8 was assayed by enzyme leaked immunosorbent assay (ELISA) and IL-8 messenger RNA (mRNA) was assayed using a reverse transcription polymerase chain reaction based technique (RT-PCR). RESULTS: Strains expressing CagA, including a variant strain (D931) which is non-cytotoxic and does not express the VacA protein, were found to upregulate epithelial IL-8 secretion and gene expression. In contrast, strains with no CagA expression, even in the presence of VacA and/or biological cytotoxicity, (G104, BA142), failed to induce IL-8 protein or mRNA above control values. CONCLUSIONS: These results strongly support a role for H pylori CagA or coexpressed factors other than the cytotoxin in upregulation of gastric epithelial IL-8. Increased epithelial IL-8 secretion and concomitant neutrophil chemotaxis and activation in addition to direct cytotoxicity may be an important factor in tissue damage and ulceration.

Interleukin-10. A role in the development of postoperative immunosuppression.

BACKGROUND: The cause of diminished monocyte major histocompatibility complex class II antigen expression after surgery or trauma is unclear. Interleukin-10 (IL-10) regulates inflammatory cytokine production and major histocompatibility complex class II (HLA-DR) expression in vitro. OBJECTIVES: To quantify in vivo IL-10 messenger RNA (mRNA) and protein and monocyte HLA-DR expression after major surgery and to investigate the effects of IL-10 neutralizing blockade on monocyte HLA-DR expression in vitro. DESIGN: Inception cohort study of 48 surgical patients from preoperative status to postoperative day 7 and 9 healthy volunteers (controls). SETTING: Large teaching hospital, Northern England. PATIENTS: Monocyte HLA-DR and cytokine mRNA expression was determined in 32 of 48 consecutive patients undergoing elective major resectional surgery. Mononuclear cells for in vitro studies and serum samples for IL-10 measurement were obtained from the remaining 16 patients. MAIN OUTCOME MEASURES: Monocyte HLA-DR expression determined by flow cytometry, IL-10, and tumor necrosis factor mRNA in peripheral blood mononuclear cells assayed by multiplex reverse transcriptase polymerase chain reaction, and serum IL-10 determined by enzyme-linked immunosorbent assay. RESULTS: Monocyte HLA-DR expression (in mean channel fluorescence units [MCF]) was significantly reduced 24 hours after surgery (MCF [+/- SEM], 32.6 +/- 2.3 vs 16.3 +/- 1.2; P < .001) and remained low during the first postoperative week. A relative increase in IL-10 to G3PDH mRNA ratio (mean [+/- SEM], 0.95 +/- 0.08 vs 0.59 +/- 0.06; P < .01) and serum IL-10 (mean [+/- SEM], 18.1 +/- 4.1 vs 5.4 +/- 0.8 pg/mL; P < .01) was noted on the first postoperative day. A significant correlation existed between HLA-DR antigen expression and the presence of IL-10 mRNA transcript on the first postoperative day (P < .01). Lipopolysaccharide-induced up-regulation of monocyte HLA-DR expression was significantly impaired on the first postoperative day (mean [+/- SEM], 151% +/- 24.4% vs 60% +/- 10.1%; P < .01), but this was partially reversed by IL-10 neutralizing antibody (mean [+/- SEM], 60% +/- 10.1% vs 115% +/- 11.6%; P < .01). CONCLUSIONS: Interleukin-10 gene expression correlates with the fall in monocyte HLA-DR antigen expression in patients undergoing major abdominal surgery and may account for the immunosuppression associated with surgical injury.

Dyskinesia in the primate following injection of an excitatory amino acid antagonist into the medial segment of the globus pallidus.

Injection of an excitatory amino acid antagonist, kynurenic acid, into the medial segment of the globus pallidus of the conscious monkey elicited dyskinesia of the contralateral limbs. In most respects the dyskinesia was indistinguishable from the disorder that is produced by ablation of the subthalamic nucleus, or injection of a GABA antagonist into the subthalamic nucleus. Injections of kynurenic acid into the lateral segment of the globus pallidus, by contrast, did not provoke dyskinesia. The effect of kynurenic acid is attributed to the blockade of neurotransmission from the subthalamic nucleus to the medial pallidal segment, and the results suggest that the neurotransmitter utilised by this pathway is an excitatory amino acid.

The effects of oxytocin and vasotocin analogues on the responses of rat brainstem neurones to oxytocin.

The effects of microiontophoretically applied oxytocin on the firing rates of neurones in the rat caudal brainstem have been examined. Of the sample of 156 units recorded the firing rates of 33.3% were reduced and those of 21.2% were raised by microiontophoretically applied oxytocin, whilst the remainder were unaffected. The distribution of units facilitated by oxytocin tended to overlap with the distribution of oxytocin demonstrated immunocytochemically, whereas many units inhibited by oxytocin were in regions where no oxytocin was observed. Overall the excitatory but not the inhibitory effects of oxytocin could be blocked by oxytocin and vasotocin analogues with antagonist properties.

Reduced high affinity cholecystokinin binding in hippocampus and frontal cortex of schizophrenic patients.

Cholecystokinin (CCK) binding sites were assessed in post-mortem brain membrane preparations from controls and schizophrenic patients. 125I-BH CCK33 specific binding was reduced by 40% (p less than 0.02) in the hippocampus and by 20% (p less than 0.01) in the frontal cortex of schizophrenic patients compared with controls. There were no differences in 125I-BH CCK33 binding between the two groups in the amygdala, temporal cortex or caudate nucleus.

Role of epidermal growth factor and transforming growth factor alpha in the developing stomach.

AIMS: To determine whether epidermal growth factor (EGF) or the related transforming growth factor alpha (TGF alpha) may have a role in the developing human stomach; to substantiate the presence of EGF in human liquor in the non-stressed infant and whether EGF in amniotic fluid is maternally or fetally derived. METHODS: The temporal expression and localisation of EGF, TGF alpha, and their receptors during fetal and neonatal life were examined in 20 fetal and five infant stomachs. Simultaneously, samples of amniotic fluid and fetal urine from 10 newborn infants were collected and assayed for EGF by radioimmunoassay. RESULTS: EGF immunoreactivity was not noted in any of the specimens examined. In contrast, TGF alpha immunoreactivity was shown in mucous cells from 18 weeks of gestation onwards. EGF receptor immunoreactivity was seen on superficial mucous cells in gastric mucosa from 18 weeks of gestation onwards. The median concentration of EGF was 30 and 8.5 pg/ml in amniotic fluid and fetal urine, respectively, suggesting that EGF is not produced by the fetus. CONCLUSIONS: This study adds weight to the hypothesis that swallowed EGF, probably produced by the amniotic membranes, and locally produced TGF alpha, may have a role in the growth and maturation of the human stomach.

Reversible impairment in monocyte major histocompatibility complex class II expression in malnourished surgical patients.

BACKGROUND: Upregulation of major histocompatibility complex (MHC) class II antigen in response to the T-cell lymphokine interferon-gamma (IFN-gamma) is central to T cell-macrophage cooperation and immune homeostasis. We evaluated this property in malnourished surgical patients and assessed the impact of nutrition repletion with total parenteral nutrition (TPN). METHODS: Sixty-two patients were studied: 37 malnourished and 25 controls. Whole blood was cultured with or without IFN-gamma (100 U mL-1), dual-labeled with anti-CD14 (monocyte) and anti-human leukocyte antigen-DR antibodies and analyzed by flow cytometry. Phagocytosis was measured by flow cytometry. In a second study, 10 severely malnourished patients received 5 days of TPN and MHC class II expression was measured at the end of this period. RESULTS: The magnitude of the increase in monocyte MHC class II expression in response to IFN-gamma was significantly increased in the control group compared with the malnourished group (107% vs 53%; p < .05). This impairment directly correlated with severity of malnutrition, but did not correlate with age or disease type. The number of bacteria phagocytozed per cell was significantly decreased (p < .05) in the malnourished group. In study 2, there was a significant increase in MHC class II induction with IFN-gamma after short-term TPN (58% before vs 173% after, p < .001). CONCLUSIONS: MHC class II induction in response to IFN-gamma is significantly impaired in malnourished patients, correlating with the severity of malnutrition. This defect is reversed by short-term TPN. These data identify the reversible loss of a key mechanism, fundamental to host defense, that may enhance the risk of infection in malnourished patients.

Host Responses to H. pylori : Molecular Analysis of Cytokine Gene Expression.

The interactions between Helicobacter pylori and the human gastric epithelium have been modeled in an in vitro cell culture system. The model permits investigation of the interplay between the bacteria and epithelial cells in a controled environment. Following coculture, the cells, bacteria, and culture supernatants are available for analysis.

Activated mesothelial cells produce heparin-binding growth factors: implications for tumour metastases.

Curative surgery for gastrointestinal malignancy is commonly thwarted by local tumour recurrence. The heparin-binding growth factors, basic fibroblast growth factor (bFGF), heparin-binding epidermal growth factor-like growth factor (HB-EGF) and vascular epidermal growth factor (VEGF) are all implicated in the metastatic process, but whether or not these essential growth factors are produced by the activated peritoneum is unknown. This study reveals that peritoneal mesothelial cells constitutively express mRNA for bFGF, HB-EGF and two VEGF spliced variants, VEGF121 and VEGF165. Mesothelial activation with interleukin (IL)-1b or tumour necrosis factor (TNF)-a produced an up-regulation of mRNA for HB-EGF and VEGF, but not bFGF expression. IL-6 failed to stimulate growth factor expression, whereas IL-2 produced a marked suppression in HB-EGF and bFGF, but not VEGF expression. Mesothelial cells were shown to predominantly express mRNA for the intermediate affinity (bg(c)) IL-2 receptor. Cytokine-induced growth factor up-regulation was confirmed at the protein level using Western blotting of mesothelial cell lysates for HB-EGF and culture supernatant enzyme-linked immunosorbent assay for VEGF. The production of these growth factors by human mesothelial cells may play a significant role in post-operative peritoneal tumour recurrence. Their common heparin-binding property offers a potential therapeutic target for manipulating the growth factor environment of the human peritoneum.

Isolation of Helicobacter pylori from feces of patients with dyspepsia in the United Kingdom.

BACKGROUND/AIMS: Epidemiological studies suggest that fecal-oral spread of Helicobacter pylori potentially represents an important route of infection. However, the bacterium has never been isolated from feces of adults in the developed world. This study attempted to isolate H. pylori from stool specimens donated by 36 adults from the United Kingdom who had dyspepsia. METHODS: Fresh fecal samples were obtained and, after centrifugation to harvest bacteria, cultured onto H. pylori selective growth media. Pure colonies of H. pylori were obtained by subculture and were then analyzed using polymerase chain reaction to confirm genotypic identity. RESULTS: H. pylori was isolated from feces in 12 of 25 subjects with dyspepsia proven to be H. pylori-positive at endoscopy and/or 14C urea breath test. Initial bacterial identification was made on the basis of various phenotypic methods. Genotypic confirmation that the bacterium was indeed H. pylori was also made. CONCLUSIONS: This study is the first to conclusively show that H. pylori can be isolated from feces obtained from adults in the United Kingdom. The implication of this finding is that transmission of H. pylori infection by the fecal-oral route is feasible.

Somatostatin binding in normal and malignant human gastrointestinal mucosa.

Somatostatin is a regulatory peptide implicated in the control of cellular proliferation in epithelial tissues and this regulation may occur directly via membrane bound receptor activation. The aim of this study was to investigate somatostatin binding in human gastrointestinal cancer and normal mucosa. Plasma membranes were prepared from specimens of tumour and normal mucosa from 51 patients undergoing surgical resection for malignancy (28 gastric, 23 colorectal). Using a competitive displacement assay, specific 125I-tyrosine-11-somatostatin-14 binding to plasma membranes was assessed and and characterised in terms of receptor affinity (Kd) and maximum binding capacity (Bmax) as determined by Scatchard analysis. Specific low affinity (Kd = 166 nM), high capacity (Bmax = 1.2 pmol mg-1 protein) somatostatin binding was demonstrated in 22 of the gastric cancers and 17 of the colorectal cancers (Kd = 140 nM, Bmax = 1.8 pmol mg-1 protein). Similar affinity and binding capacity was demonstrable in normal mucosal samples. High affinity receptors for somatostatin were expressed by one gastric carcinoma (Kd = 0.9 nM; Bmax = 0.23 pmol mg-1 protein). Thus, low affinity, high capacity binding is a common feature of gastrointestinal tumours and normal mucosa, and high affinity receptors may occasionally be demonstrated. The functional significance of these low affinity binding sites requires elucidation to determine whether long-acting somatostatin analogues may have therapeutic benefit in gastrointestinal malignancy.

Expression of the antiapoptosis gene, survivin, predicts death from recurrent colorectal carcinoma.

BACKGROUND/AIMS: Inhibition of programmed cell death (apoptosis) is associated with increased tumour aggressiveness, and expression of Survivin, an antiapoptosis gene, in colorectal carcinomas may provide important prognostic information. PATIENTS/METHODS: Expression of Survivin messenger RNA was evaluated by reverse transcription-polymerase chain reaction in 144 colorectal carcinomas and 86 adjacent histologically normal mucosa samples from patients for whom long term follow up data were available. RESULTS: Survivin transcripts were detected in a significantly greater proportion of carcinomas (63.5%) than normal mucosa samples (29.1%; p<0.001). The prevalence of Survivin expression was independent of advancing pathological stage. Death due to recurrent cancer following curative resection was predicted independently by tumour expression of Survivin (hazard ratio (HR) 2.60; 95% confidence interval (95% CI) 1. 17-5.75) and lymph node metastases (HR 2.38; 95% CI 1.21-4.70). On stage wise analysis, the predictive value of Survivin expression was limited to patients with stage II colorectal carcinomas; those with Survivin negative tumours had a five year survival rate of 94.4% compared with 44.8% for patients with Survivin positive tumours (p=0. 004, log rank test). CONCLUSION: In patients with stage II colorectal carcinomas, Survivin expression provides prognostic information that may have important therapeutic implications.

Risk of neoplasia in renal transplant patients.

Renal allograft recipients are at an increased risk of neoplasia, although the extent of the problem has not been established in a typical European transplant population. To assess this risk we did a comprehensive, retrospective study of 918 patients transplanted at one centre over 24 years. The centre (Leeds) serves Yorkshire and Humberside, a region in northern England with a population of 3.6 million. The search, which made use of six sources of information, revealed 70 patients (7.6%) who had developed a neoplastic lesion, 10 patients having more than one type. More than half (42) were cutaneous lesions (mostly squamous cell carcinomas). The risk of developing neoplasia in the first 10 years after transplantation was calculated to be 14%. By 20 years this had risen to 40% compared with a 6% cumulative risk of neoplasia in an age-matched control population (p < 0.005). The full extent of this problem in the European transplant population has been underestimated and, now that recipients are surviving longer, there is a clear need for both lifelong surveillance and closer investigation of these patients.

Somatostatin binding in human gastrointestinal tissues: effect of cations and somatostatin analogues.

This study characterises the somatostatin binding site in human gastrointestinal cancer and mucosa in terms of cationic specificity and relative affinity for three somatostatin analogues. Competitive displacement assays were performed on plasma membranes from human gastric and colonic tissues using radiolabelled somatostatin-14 as ligand. Comparison was made with the somatostatin binding site in rat cerebral cortex. In gastrointestinal tissue, magnesium decreased and sodium increased specific binding. By contrast, in rat cerebral cortex, the converse cationic effect was seen. These changes resulted from alterations in receptor density, with no change in receptor affinity. Displacement studies were then performed with somatostatin-14 and somatostatin analogues RC-160, somatuline, and octreotide. RC-160 and somatuline displaced radiolabel from binding sites in gastric and colonic cancer and mucosa with 10-fold lower affinity than the native peptide. Octreotide did not displace radioligand in gastric or colonic cancer at any concentration tested. By contrast, in rat cortex, although all three analogues displaced with a lower affinity than the native peptide, there was no difference between analogues. These data suggest a distinct somatostatin receptor subtype in gastrointestinal tissues.

Death from early colorectal cancer is predicted by the presence of transcripts of the REG gene family.

An intrinsic component of colorectal carcinogenesis may be the capacity to activate regenerative responses simultaneously with inhibition of apoptosis. Since apoptosis is known to be inhibited in colorectal cancer, this study sought evidence for the activation of the REG family of genes which are considered to be activated during regeneration of intestinal mucosa. Transcripts for the REG gene were found in 53% of colorectal cancers and for the PAP gene in 60% of colorectal cancers, by RT-PCR. Using in situ hybridization, the REG transcripts were found to be present in the tumour cells themselves rather than inflammatory or stromal cells. There were no significant correlations between the expression of these two genes and tumour stage, age or sex of the patient population or tumour site. However, in patients with non-metastatic disease who underwent ostensibly curative surgery, the expression of REG alone and co-expression of REG with PAP had a highly significantly adverse effect on survival. These data provide support for the concept that, in some tumours, carcinogenesis involves a regenerative process which co-exists with apoptotic inhibition and may provide a valuable selective indicator of the need for adjuvant therapy in those patients with early-stage colorectal cancer whose disease is destined to recur after curative surgery.