Pilot translational study of dietary vitamin C supplementation in Barrett's esophagus.

The transcription factor Nuclear factor kappa B (NF-kappaB) is central to the regulation of genes encoding for mediators of inflammation and carcinogenesis. In the esophagus, NF-kappaB is progressively activated from inflammation to Barrett's metaplasia and adenocarcinoma. Vitamin C, an antioxidant, can inhibit NF-kappaB in in vitro models, and the aim of this study was to prospectively assess the effect of supplemental vitamin C on NF-kappaB and associated cytokines in patients with Barrett's esophagus. Twenty-five patients with long-segment Barrett's and specialized intestinal metaplasia received dietary vitamin C (1000 mg/day) orally for four weeks, and had pre- and post-vitamin C endoscopic biopsies. NF-kappaB activity (activated p50 and p65 subunits) of nuclear extracts was assessed using the Active Motif NF-kappaB assay, and cytokines and growth factors were measured using the Evidence Investigator biochip array. NF-kappaB and related pro-inflammatory cytokines and growth factors (IL-8, VEGF, IL-10) were activated in all Barrett's tissue pre-treatment. Down-regulation in activated NF-kappaB and cytokines was observed in 8/25 (35%) patients. Dietary vitamin C supplementation may down-regulate pro-inflammatory markers in a subset of Barrett's patients. Further studies with larger numbers of endpoints will be needed to further evaluate this effect.

Vitamin C enhances chemosensitization of esophageal cancer cells in vitro.

Chemotherapy is increasingly utilised in multimodal protocols to try and improve outcomes. Cisplatin and 5-fluorouracil (5-Fu) are the mainstay of chemotherapeutic regimens, and an understanding of sensitivity and resistance of esophageal cancer to these agents is of considerable clinical importance. Antioxidants may modulate the response to chemotherapy, and in this study we examined the effect of vitamin C on 5-Fu and cisplatin cytotoxicity and related pathways in the esophageal cancer cell lines OE33 and SKGT-4. The antiproliferative effect of antitumor agents was measured by the MTT assay, and the transcription factors NF-kappaB and AP-1 pathways were assessed by electrophoretic mobility gel shift assay. 5-Fu and cisplatin demonstrated marked morphological changes and decreased cell proliferation. A combination of vitamin C with 5-Fu or cisplatin exerted a significantly enhanced cytotoxic effect compared to both drugs individually. Treatment of esophageal cancer cells with 5-Fu and cisplatin induced NF-kappaB and AP-1 activation. Pretreatment with vitamin C inhibited 5-Fu or cisplatin induced NF-kappaB nuclear translocation and DNA binding activity, but vitamin C had no effect on IkappaB-alpha protein levels. Vitamin C also inhibited 5-Fu- and cisplatin-induced AP-1 activation. Our data demonstrate that vitamin C enhances the antitumor activity of 5-Fu and cisplatin, in part by inhibiting translocation of NF-kappaB and AP-1, and sensitizes cancer cells to drug-induced cell death. The data suggest that vitamin C supplementation may improve the efficacy of chemotherapy for esophageal cancer.

Activated nuclear factor-kappa B and cytokine profiles in the esophagus parallel tumor regression following neoadjuvant chemoradiotherapy.

Esophageal adenocarcinoma is increasing in incidence; it relates to chronic gastroesophageal reflux, it is difficult to cure, and treatment modalities increasingly use chemotherapy and radiation therapy prior to resectional surgery. Nuclear factor-kappa B (NF-kappaB) is a pleiotropic transcription factor that regulates several genes for cytokines and enzymes involved in inflammation and immunity, and we have previously described sequential expression of NF-kappaB from the normal esophagus through Barrett's metaplasia to adenocarcinoma. The aim of this exploratory study was to assess the NF-kappaB status and cytokine profiles pre- and post-chemoradiotherapy for esophageal adenocarcinoma. Fresh biopsy specimens obtained from 20 patients with esophageal adenocarcinoma and normal adjacent squamous epithelium were obtained pre-, during and post-chemoradiotherapy, and NF-kappaB expression was analyzed by electrophoretic mobility shift assay. The cytokine protein content of interleukin-1 beta (IL-1beta) and interleukin-8 (IL-8) of tissue homogenates was measured using the ELISA technique. NF-kappaB was constitutively activated in tumor tissues from esophageal adenocarcinoma but was not detected in adjacent normal esophageal mucosa. Elevated levels of IL-1beta and IL-8 were significantly (P < 0.05) higher in tumor tissues compared to control tissues. Patients with a major or complete pathological response (responders) were associated with absence of activated NF-kappaB from nuclear extracts after treatment. Moreover, IL-1beta and IL-8 levels were significantly (P < 0.05) down-regulated in tumor tissues from patients who demonstrated a complete pathological response. No differences in NF-kappaB, IL-1beta and IL-8 levels were detected pre- and post-treatment in patients who did not have a major or complete pathological response (non-responders). The study suggests that monitoring of molecular and cytokine patterns in patients undergoing this neoadjuvant regimen may help subselect the cohort that derives most benefit from the multimodal approach.

Proinflammatory cytokine and nuclear factor kappa-B expression along the inflammation-metaplasia-dysplasia-adenocarcinoma sequence in the esophagus.

BACKGROUND: The incidence of esophageal adenocarcinoma has increased significantly in the western world over the last 20 yr. Most cases arise in a background of chronic gastroesophageal reflux, and specialized intestinal metaplasia in Barrett's esophagus is frequently an antecedent phenotype or evident in association with adenocarcinoma. The molecular events that characterize the pathway from inflammation to metaplasia to dysplasia and adenocarcinoma are poorly understood. AIMS: To examine the expression of the proinflammatory cytokines IL-8 and IL-1beta along the esophagitis, metaplasia, dysplasia, and adenocarcinoma pathway, and to correlate this with histological changes and expression of the transcription factor NF-kappaB. PATIENTS AND METHODS: Fresh biopsy specimens were collected from patients with reflux esophagitis (n=15), Barrett's esophagus (n=35), Barrett's adjacent to adenocarcinoma (n=8), and esophageal adenocarcinoma (n=35). IL-8 and IL-1beta expression were measured using enzyme-linked immunosorbent assay. NF-kappaB expression was measured by electrophoretic mobility shift assay. RESULTS: Elevated expression of NF-kappaB was found in 2 (13%) out of 15 patients with reflux esophagitis, 21 (60%) out of 35 patients with Barrett's esophagus, and 28 (80%) out of 35 patients with esophageal adenocarcinoma. All 5 patients with Barrett's esophagus and high-grade dysplasia showed elevated expression of NF-kappaB. IL-8 and IL-1beta were significantly increased in esophagitis, Barrett's, and adenocarcinoma compared with squamous epithelium, and in adenocarcinoma compared with all other groups. There was a stepwise increase in the expression of IL-8, IL-1beta, and NF-kappaB from normal through Barrett's epithelium to adenocarcinoma in eight cases of esophageal adenocarcinoma. The levels of both IL-8 and IL-1beta in adenocarcinoma patients correlated with stage of disease. Patients with adenocarcinoma who were NF-kappaB positive had significantly higher levels of both IL-8 (p=0.04) and IL-1beta (p=0.03) compared to adenocarcinoma patients who were NF-kappaB negative. CONCLUSIONS: The proinflammatory cytokines IL-8 and IL-1beta are elevated in esophagitis and Barrett's epithelium, and markedly elevated in adenocarcinoma. NF-kappaB activation is infrequent in esophagitis, but is increased in Barrett's epithelium and adenocarcinoma. The association of NF-kappaB activation with cytokine upregulation was only evident in patients with adenocarcinoma. These patterns may play an important role in Barrett's inflammation and tumourigenesis, and inhibition of the NF-kappaB/proinflammatory cytokine pathway may be an important target for future chemoprevention strategies.

Helicobacter pylori activates the early growth response 1 protein in gastric epithelial cells.

The early growth response 1 (Egr-1) transcription factor is rapidly induced by various stimuli and is implicated in the regulation of cell growth, differentiation, and gene expression. The aim of this study was to examine the effect of Helicobacter pylori on the expression of Egr-1 and Egr-1-regulated genes in gastric epithelial AGS cells. Egr-1 expression was assayed by immunoblotting and electrophoretic mobility shift assays using H. pylori-stimulated AGS cells. Transient transfection experiments with promoter-reporter constructs of CD44, ICAM-1, and CD95L were used for expression studies. H. pylori induced the expression of Egr-1 in gastric epithelial cell lines in a dose-dependent manner, with the rapid kinetics that are typical of this class of transcription factors. Immunohistochemical studies of biopsies revealed that Egr-1 expression is more abundant in H. pylori-positive patients than in uninfected individuals. Reporter-promoter transfection studies indicated that Egr-1 binding is required for the H. pylori-induced transcriptional promoter activity of the CD44, ICAM-1, and CD95L (APO-1/Fas) constructs. The blocking of egr-1 with an antisense sequence prevented H. pylori-induced Egr-1 and CD44 protein expression. The MEK1/2 signaling cascade participates in H. pylori-mediated Egr-1 expression, but the p38 pathway does not. The data indicate that H. pylori induces Egr-1 expression in AGS cells in vitro and that the Egr-1 protein is readily detectable in biopsies from H. pylori-positive subjects. These observations suggest that H. pylori-associated Egr-1 expression may play a role, in part, in H. pylori-induced pathology.

Diagnosis of toxoplasmosis in children with malignancy.

The study aimed at the diagnosis of toxoplasmosis in 73 children with malignancy; 31 with lymphoma (22 with Hodgkin's and 9 with non-Hodgkin's lymphoma) and 42 with leukemia (34 with acute lymphoblastic leukemia and 8 with acute myelogenic leukemia). In positive cases toxoplasmosis was manifested by any of the following; fever, lymph node enlargement, neurological manifestations and/or hepatosplenomegaly. The indirect hemagglutination test (IHA) for toxoplasmosis detected 4 (5.4%) positive cases with malignancy, 2 with Hodgkin's lymphoma, one with non-Hodgkin's lymphoma and one with acute lymphoblastic leukemia. The immunoglobulin M enzyme-linked immunosorbent assay (IgM ELISA) detected only one (1.4%) case with Hodgkin's lymphoma. Immunoglobulin G (IgG) ELISA detected 6 (8.2%) positive cases, 3 with Hodgkin's lymphoma, one with non-Hodgkin's lymphoma and 2 cases with acute lymphoblastic leukemia. Polymerase chain reaction for detection of parasite DNA in blood (PCR) was the most useful in diagnosing toxoplasmosis with malignancy, as it was able to detect 9 (12.3%) positive cases; 5 (6.8%) with Hodgkin's lymphoma, one (1.4%) with non-Hodgkin's lymphoma and 3 (4.1%) with acute lymphoblastic leukemia. No positive toxoplasmosis cases were detected with acute myelogenic leukemia by any of the above methods.

A proposed computer programme for determining infant survival status in demographic surveys.

"Determining infant survival status in demographic surveys is of great importance in analysis of infant mortality. Missing data representing age at death in complete months and complete years, if any, will affect the accuracy of any related measurement. A computer programme to reduce the percentage of such missing data is proposed."