Reflux composition influences the level of NF-κB activation and upstream kinase preference in oesophageal adenocarcinoma cells.

Oesophageal adenocarcinoma (OA) incidence is rising and prognosis is poor. Understanding the molecular basis of this malignancy is key to finding new prevention and treatment strategies. Gastroesophageal reflux disease is the primary cause of OA, usually managed with acid suppression therapy. However, this often does little to control carcinogenic bile acid reflux. The transcription factor nuclear factor kappa B (NF-κB) plays a key role in the pathogenesis of OA and its activity is associated with a poor response to chemotherapy, making it an attractive therapeutic target. We sought to decipher the role of different bile acids in NF-κB activation in oesophageal cell lines using short, physiologically relevant exposure times. The effect of an acidic or neutral extracellular pH was investigated concurrently, to mimic in vivo conditions associated with or without acid suppression. We found that some bile acids activated NF-κB to a greater extent when combined with acid, whereas others did so in its absence, at neutral pH. The precise composition of an individual's reflux, coupled with whether they are taking acid suppressants may therefore dictate the extent of NF-κB activation in the oesophagus, and hence the likelihood of histological progression and chemotherapy success. Regardless of pH, the kinase inhibitor of κB kinase was pivotal in mediating reflux induced NF-κB activation. Its importance was confirmed further as its increased activation was associated with histological progression in patient samples. We identified further kinases important in acid or bile induced NF-κB signalling in oesophageal cells, which may provide suitable targets for therapeutic intervention.

Changes in markers of oxidative stress and DNA damage in human visceral adipose tissue from subjects with obesity and type 2 diabetes.

AIMS: In the past 30 years, prevalence of obesity has almost trebled resulting in an increased incidence of type 2 diabetes mellitus and other co-morbidities. Visceral adipose tissue is believed to play a vital role, but underlying mechanisms remain unclear. Our aim was to investigate changes in markers of oxidative damage in human visceral adipose tissue to determine levels of oxidative burden that may be attributed to obesity and/or diabetes. METHODS: Visceral adipose tissue samples from 61 subjects undergoing abdominal surgery grouped as lean, obese and obese with type 2 diabetes mellitus, were examined using 3 different markers of oxidative stress. Malondialdehyde (MDA) concentration was measured as a marker of lipid peroxidation, telomere length and Comet assay as markers of oxidative DNA damage. RESULTS: No significant difference in MDA concentration, telomere length and DNA damage was observed between groups, although longer telomere lengths were seen in the obese with diabetes group compared to the obese group (P<0.05). Lower MDA concentration and longer telomere length were seen in subjects with diabetes compared to those without (P<0.05). DNA damage, analysed via Comet assay, was significantly lower in subjects with diabetes compared to those without (P<0.05). CONCLUSION: A paradoxical decrease in oxidative stress and DNA damage was observed in samples from subjects with type 2 diabetes mellitus. Further work is required to investigate this further, however this phenomenon may be due to an up regulation of antioxidant defences in adipose tissue.

Inducible nitric oxide synthase (iNOS) and nitric oxide (NO) are important mediators of reflux-induced cell signalling in esophageal cells.

Nitric oxide (NO) produced by inducible nitric oxide synthase (iNOS) has been implicated in both DNA damage induction and aberrant cell signalling in various tissue and cell backgrounds. We investigated here the role of iNOS and NO in DNA damage induction and nuclear factor-kappa B (NF-κB) signalling in esophageal cells in vitro. As esophageal adenocarcinoma develops in a background of Barrett's esophagus secondary to reflux disease, it is possible that inflammatory mediators like NO may be important in esophageal cancer development. We show that reflux components like stomach acid and bile acids [deoxycholic acid (DCA)] can induce iNOS gene and protein expression and produce NO generation in esophageal cells, using real-time PCR, western blotting and NO sensitive fluorescent probes, respectively. This up-regulation of iNOS expression was not dependent on NF-κB activity. DCA-induced DNA damage was independent of NF-κB and only partially dependent on iNOS and NO, as measured by the micronucleus assay. These same reflux constituents also activated the oncogenic transcription factor NF-κB, as measured by transcription factor enzyme-linked immunosorbent assay and gene expression studies with NF-κB linked genes (e.g. interleukin-8). Importantly, we show here for the first time that basal levels of NF-κB activity (and possibly acid and DCA-induced NF-κB) are dependent on iNOS/NO and this may lead to a positive feedback loop whereby induced iNOS is upstream of NF-κB, hence prolonging and potentially amplifying this signalling, presumably through NO activation of NF-κB. Furthermore, we confirm increased protein levels of iNOS in esophageal adenocarcinoma and, therefore, in neoplastic development in the esophagus.

Curcumin abrogates bile-induced NF-κB activity and DNA damage in vitro and suppresses NF-κB activity whilst promoting apoptosis in vivo, suggesting chemopreventative potential in Barrett's oesophagus.

INTRODUCTION: Curcumin has been suggested to possess anti-neoplastic properties. As oesophageal adenocarcinoma (OA) and Barrett's oesophagus (BO) represent a neoplastic series, we postulated that curcumin supplementation may slow neoplastic progression at this site. Our aim was to investigate the effects of curcumin in vitro and in vivo on markers of oesophageal cancer progression. METHODS: We investigated the in vitro ability of curcumin to prevent bile acid-induced DNA damage using micronucleus assay and nuclear factor-kappaB (NF-κB) activity in the oesophageal cell lines (OE33) using real-time PCR of the extracted RNA. We also analysed NF-κB p65 activation in curcumin-pre-treated OE33 cells exposed to deoxycholic acid (DCA) using ELISA. In another pilot study, BO patients took a daily 500 mg curcumin tablet for 7 days prior to their endoscopy. In biopsies collected from these patients (n=33, 16 curcumin, 17 control), we examined NF-κB-driven gene expression (interleukin (IL)-8, inhibitor- kappaB (I-κB)) using real-time PCR of the extracted RNA from the biopsy sample. The apoptotic frequency was assessed by counting the number of apoptotic bodies in the epithelial cells from the Barrett's tissue with and without curcumin. RESULTS: In vitro, curcumin (50 µM) significantly abrogated DNA damage and NF-κB activity induced by bile. Pretreating OE33 cells with curcumin (50 µM) completely abolished the ability of DCA (300 µM) to activate NF-κB. In vivo, IL-8 expression was non-significantly suppressed in the curcumin-supplemented patients compared to the squamous control tissue, whilst also showing a doubling in the apoptotic frequency compared to non-supplemented control patients. CONCLUSIONS: Curcumin abrogated bile-driven effects in vitro. The in vivo data also suggests that curcumin supplementation had beneficial effects (increased apoptosis, potentially reduced NF-κB activity) in the Barrett's tissues themselves, despite poor delivery of the curcumin to the oesophagus.

Curcumin abrogates bile-induced NF-kB activity and DNA damage in vitro and suppresses NF-kB activity whilst promoting apoptosis in vivo, suggesting chemopreventative potential in Barrett's oesophagus

INTRODUCTION: Curcumin has been suggested to possess anti-neoplastic properties. As oesophageal adenocarcinoma (OA) and Barrett's oesophagus (BO) represent a neoplastic series, we postulated that curcumin supplementation may slow neoplastic progression at this site. Our aim was to investigate the effects of curcumin in vitro and in vivo on markers of oesophageal cancer progression. METHODS: We investigated the in vitro ability of curcumin to prevent bile acid-induced DNA damage using micronucleus assay and nuclear factor-kappaB (NF-κB) activity in the oesophageal cell lines (OE33) using real-time PCR of the extracted RNA. We also analysed NF-κB p65 activation in curcumin-pre-treated OE33 cells exposed to deoxycholic acid (DCA) using ELISA. In another pilot study, BO patients took a daily 500 mg curcumin tablet for 7 days prior to their endoscopy. In biopsies collected from these patients (n=33, 16 curcumin, 17 control), we examined NF-κB-driven gene expression (interleukin (IL)-8, inhibitor- kappaB (I-κB)) using real-time PCR of the extracted RNA from the biopsy sample. The apoptotic frequency was assessed by counting the number of apoptotic bodies in the epithelial cells from the Barrett's tissue with and without curcumin. RESULTS: In vitro, curcumin (50 μM) significantly abrogated DNA damage and NF-κB activity induced by bile. Pretreating OE33 cells with curcumin (50 μM) completely abolished the ability of DCA (300 μM) to activate NF-κB. In vivo, IL-8 expression was non-significantly suppressed in the curcumin-supplemented patients compared to the squamous control tissue, whilst also showing a doubling in the apoptotic frequency compared to non-supplemented control patients. CONCLUSIONS: Curcumin abrogated bile-driven effects in vitro. The in vivo data also suggests that curcumin supplementation had beneficial effects (increased apoptosis, potentially reduced NF-κB activity) in the Barrett's tissues themselves, despite poor delivery of the curcumin to the oesophagus (AU)

Adrenal insufficiency following bariatric surgery.

A 35-year-old woman with morbid obesity and amenorrhoea underwent a bilo-pancreatic diversion (BPD). Surgery was successful with good weight loss, restoration of menstruation and almost immediately she conceived for the first time. She was commenced on routine vitamin supplements after surgery but failed to attend follow-up clinic. Five years later, she presented with limb girdle pains, lethargy, night blindness, skin pigmentation, amenorrhoea and dizziness. She had stopped taking supplements prescribed after the surgery. Investigations showed severe vitamin A and D deficiency along with iron and calcium deficiency. Her cholesterol was low at 3.5 mmol L⁻¹. Despite aggressive vitamin replacement, she continued to complain of lethargy and dizziness. Subsequently, three short adrenocorticotropic hormone-stimulation tests were suboptimal (basal cortisol: 196, 185 and 223 nmol L⁻¹; 30 min cortisol: 421, 453 and 435 nmol L⁻¹). She was subsequently commenced on adrenal replacement and her symptoms resolved and she conceived. We describe for the first time in the literature the unexpected finding of adrenal insufficiency following a BPD.

Temporal changes in glucose and insulin homeostasis after biliopancreatic diversion and laparoscopic adjustable gastric banding.

BACKGROUND: Obesity surgery is associated with improvement in type 2 diabetes mellitus. Our aim was to examine the effects of biliopancreatic diversion (BPD) and laparoscopic adjustable gastric banding (LAGB) on the body mass index, fasting insulin level, glucose level, and insulin resistance in morbidly obese subjects with type 2 diabetes mellitus. The setting was the Department of Surgery, Morriston Hospital (Swansea, Wales, United Kingdom). METHODS: A total of 13 morbidly obese patients (7 BPD, 6 LAGB) underwent serial measurements of fasting glucose and insulin at baseline, immediately after surgery (days 1-7), and 1, 6, and 12 months postoperatively. The homeostasis model of assessment-insulin resistance was calculated. RESULTS: In the BPD group, the glucose levels had normalized by day 3 (5.6 ± 1 mmol/L) and the difference was statistically significant at 6 and 12 months postoperatively (5 ± .7 and 4.4 ± .5 mmol/L, respectively). The insulin levels had improved from day 1, and the difference was statistically significant at days 2, 5, 6, and 7 (19 ± 9, 14.2 ± 7, 15.2 ± 8, and 17.4 ± 8 mU/L, respectively). All diabetes medications were stopped on the fourth postoperative day. In the LAGB group, no statistically significant changes were seen in the glucose levels. Statistically significant changes in insulin were seen on days 1 and 2 (19 ± 13 and 13 ± 6.5 mU/L, respectively). The homeostatic model of assessment-insulin resistance had improved in both groups (BPD, 1.6 ± 1.2, P < .01; and LAGB, 4.3 ± 1.4, P < .05). CONCLUSION: BPD causes immediate remission of type 2 diabetes mellitus. Leptin might play an important role in the early improvement of insulin resistance in fasting states after BPD. In the LAGB group, glucose homeostasis improved, but the patients still required diabetes medications, although the dosages were reduced.

A Randomised, Cross-Over, Placebo-Controlled Study of Aloe vera in Patients with Irritable Bowel Syndrome: Effects on Patient Quality of Life.

Background. Irritable bowel syndrome (IBS) is a chronic, difficult to treat condition. The efficacy of Aloe vera in treating IBS symptoms is not yet proven. The purpose of this study was to determine if Aloe vera is effective in improving quality of life. Methods. A multicentre, randomised, double-blind, cross-over placebo controlled study design. Patients were randomised to Aloe vera, wash-out, placebo or placebo, washout, Aloe vera. Each preparation (60 mL) was taken orally twice a day. Patient quality of life was measured using the Gastrointestinal Symptoms Rating Score, Irritable Bowel Syndrome Quality of Life, EuroQol and the Short-Form-12 at baseline and treatment periods 1 and 2. Results. A total of 110 patients were randomised, but only 47 completed all questionnaires and both study arms. Statistical analysis showed no difference between the placebo and Aloe vera treatment in quality of life. Discussion. This study was unable to show that Aloe vera was superior to placebo in improving quality of life. Drop outs and other confounding factors may have impacted on the power of the study to detect a clinically important difference. Conclusion. This study failed to find Aloe vera superior to placebo in improving quality of life proven Irritable Bowel Syndrome patients.

In vitro and ex vivo models of extended reflux exposure demonstrate that weakly acidic mixed reflux heightens NF-kB-mediated gene expression.

The development of Barrett's esophagus and its progression to adenocarcinoma are clearly linked to reflux of acid and bile. Our objective in this study was to develop an optimized ex vivo biopsy culture technique to study the molecular signaling events induced after insult with individual refluxate constituents. We illustrate the utility of this method by showing results for NF-kB centered cell signaling, and compare the results with those obtained from esophageal cell lines. We show that upregulation of the two NF-kB target genes show differences in pH preference, with IL-8 being preferentially upregulated by DCA at neutral pH, and IkB being upregulated by neutral DCA, acidic DCA, and acid alone. This was found to be true in both cell lines and biopsy cultures. The maximum responses were noted in both models when mixed reflux (DCA at pH 6) was utilized, perhaps reflecting the pH preference of DCA (pKa 6.2). Both the optimized ex vivo models, and the in vitro cell lines show that bile and acid are capable of inducing NF-kB dependent gene expression, with some interesting differences in preferred transcriptional target. In conclusion, in both cells and cultured biopsies, similar reflux driven gene expression changes were noted, with maximum effects noted with DCA exposures at pH 6.

Influence of bariatric surgery on indices of cardiac autonomic control.

BACKGROUND: Obesity is associated with reduced heart rate variability (HRV), reflecting detrimental changes in cardiac regulation by the autonomic nervous system (ANS). Weight loss reverses this change and ANS dysfunction is thought to have a role in obesity-related cardiac pathology. Few studies have examined the influence of weight-reduction (bariatric) surgery on cardiac autonomic control. This study therefore sought to assess longitudinal changes in indices of cardiac autonomic control following two types of bariatric procedure, laparascopic gastric banding (LGB) and biliopancreatic diversion (BPD). METHODS: Eleven morbidly obese subjects aged 47.8 +/- 7.9 years (mean+/-SD) with BMI 48.2 +/- 6.9 kg x m(-2) underwent weight-reduction surgery: five received BPD and six received LGB. Holter ECG was recorded and HRV was quantified together with a QT variability index (QTVI), a complexity index (SampEn), and a fractal (scaling) index (DFAalpha). Repeated measures ANOVA compared the indices for the two groups as a function of time (1, 6 and 12 months follow-up). RESULTS: BMI was reduced by up to 24% (p=0.008) post-surgery despite patients remaining obese at one-year follow-up. Several indices showed prompt and persistent improvement with progressive weight loss, QTVI being the most sensitive discriminator of recovery time (F(3,216)=16.86; p<0.0005; eta(2)=0.190). Autonomic responsiveness was functionally normal throughout. The bariatric procedures induced similar changes in cardiac autonomic control, despite their differing mechanisms of action. CONCLUSIONS: This pilot study suggests that the mechanism responsible for improving cardiac regulation following bariatric surgery might be the weight loss itself. Furthermore, post-surgery improvement in QTVI implies that weight loss reduces the risk of ventricular arrhythmic events.

Aneuploidy involving chromosome 1 may be an early predictive marker of intestinal type gastric cancer.

Intestinal type gastric cancer is a significant cause of mortality, therefore a better understanding of its molecular basis is required. We assessed if either aneuploidy or activity of the oncogenic transcription factor nuclear factor kappa B (NF-kappaB), increased incrementally during pre-malignant gastric histological progression and also if they correlated with each other in patient samples, as they are both induced by oxygen free radicals. In a prospective study of 54 (aneuploidy) and 59 (NF-kappaB) consecutive patients, aneuploidy was assessed by interphase fluorescent in situ hybridisation (FISH) for chromosome 1. NF-kappaB was assessed by expression of interleukin-8 (IL-8), and in a subset, by immunohistochemistry (IHC) for active p65. Aneuploidy levels increased incrementally across the histological series. 2.76% of cells with normal histology (95% CI, 2.14-3.38%) showed background levels of aneuploidy, this increased to averages of 3.78% (95% CI, 3.21-4.35%), 5.89% (95% CI, 3.72-8.06%) and 7.29% (95% CI, 4.73-9.85%) of cells from patients with gastritis, Helicobacter pylori positive gastritis and atrophy/intestinal metaplasia (IM) respectively. IL-8 expression was only increased in patients with current H. pylori infection. NF-kappaB analysis showed some increased p65 activity in inflamed tissues. IL-8 expression and aneuploidy level were not linked in individual patients. Aneuploidy levels increased incrementally during histological progression; were significantly elevated at very early stages of neoplastic progression and could well be linked to cancer development and used to assess cancer risk. Reactive oxygen species (ROS) induced in early gastric cancer are presumably responsible for the stepwise accumulation of this particular mutation, i.e. aneuploidy. Hence, aneuploidy measured by fluorescent in situ hybridisation (FISH) coupled to brush cytology, would be worthy of consideration as a predictive marker in gastric cancer and could be clinically useful in pre-malignant disease to stratify patients by their cancer risk.

The bile acid deoxycholic acid has a non-linear dose response for DNA damage and possibly NF-kappaB activation in oesophageal cells, with a mechanism of action involving ROS.

Deoxycholic acid (DCA) is a secondary bile acid implicated in various cancers of the gastrointestinal (GI) tract. In oesophageal adenocarcinoma, DCA is believed to contribute to carcinogenesis during reflux where stomach contents enter the lower oesophagus. It is imperative that we understand the mechanisms whereby oesophageal carcinogens function in order that therapeutic options may be developed. We have previously shown that DCA can damage chromosomes and does so through its generation of reactive oxygen species (ROS). We show here, after detailed experiments, that DCA appears to have a non-linear dose response for DNA damage. DCA induces DNA damage (as measured by the micronucleus assay) at doses of 100 microM and higher in oesophageal OE33 cells, but fails to induce such DNA damage below this cut-off dose. We also show that in terms of NF-kappaB activation (as measured by up-regulation of two NF-kappaB target genes) by DCA, a similar dose response is observed. This dose-response data may be important clinically as DCA exposure to the oesophagus may be used as a way to identify the 10% of Barrett's oesophagus patients currently progressing to cancer from the 90% of patients who do not progress. Only quantitative studies measuring DCA concentrations in refluxates correlated with histological progression will answer this question. We further show here that ROS are behind DCAs ability to activate NF-kappaB as antioxidants (epigallocatechin gallate, resveratrol and vitamin C) abrogate DCAs ability to up-regulate NF-kappaB-controlled genes. In conclusion, low doses of DCA appear to be less biologically significant in vitro. If this were to be confirmed in vivo, it might suggest that reflux patients with low DCA concentrations may be at a lower risk of cancer progression compared to patients with high levels of DCA in their refluxate. Either way, antioxidant supplementation may possibly help prevent the deleterious effects of DCA in the whole GI tract.

Nutritional deficiencies after bariatric surgery.

A current review of nutritional complications following bariatric procedures is presented, focusing on the most common and clinically important deficiencies. A brief outline of nutritional supplementation protocol is presented, highlighting the need for a standardized, national or international set of guidelines for pre- and postoperative nutritional screening and appropriate supplementation.

Obesity, metabolic syndrome and sleep apnoea: all pro-inflammatory states.

Obesity is associated with significant morbidity and mortality and is increasing in prevalence worldwide. Associated conditions include insulin resistance (IR), diabetes, hypertension and dyslipidaemia; a clustering of these has recently been termed as metabolic syndrome. Weight gain is a major predictor of the metabolic syndrome with waist circumference being a more sensitive indicator than body mass index as it reflects both abdominal subcutaneous adipose tissue and visceral adipose tissue (VAT). VAT has more metabolic activity and secretes a number of hormones and pro-inflammatory cytokines which are linked with the metabolic abnormalities listed above. Central obesity also increases the risk of obstructive sleep apnoea syndrome (OSAS), where the sleep disordered breathing may also independently lead to/or exacerbate IR, diabetes and cardiovascular risk. The contribution of OSAS to the metabolic syndrome has been under-recognized. The putative mechanisms by which OSAS causes or exacerbates these other abnormalities are discussed. We propose that activation of nuclear factor kappa B by stress hypoxia and/or by increased adipokines and free fatty acids released by excess adipose tissue is the final common inflammatory pathway linking obesity, OSAS and the metabolic syndrome both individually and, in many cases, synergistically.

Immunohistochemical study of nuclear factor-kappaB activity and interleukin-8 abundance in oesophageal adenocarcinoma; a useful strategy for monitoring these biomarkers.

AIMS: To determine if immunohistochemistry (IHC) could be used to monitor nuclear factor-kappaB (NF-kappaB) activity in oesophageal adenocarcinoma and pre-malignant (Barrett's) oesophageal tissues, relative to normal oesophageal mucosa. The pro-inflammatory cytokine interleukin-8 (IL-8), a transcriptional target of NF-kappaB, was also studied to better understand NF-kappaB functionality; its RNA and protein levels were assessed in oesophageal tissues. METHODS: IHC was employed using an antibody against the nuclear localisation sequence (NLS) of the p65 subunit as well as an antibody against IL-8. To assess NF-kappaB function, changes in gene expression of NF-kappaB controlled genes (IL-8 and I-kappaB) were also assessed in the histological sequence using real-time PCR. More global expression changes were also studied using membrane arrays. RESULTS: IHC was effective at monitoring overall NF-kappaB activity and IL-8 abundance. This method also allowed NF-kappaB activity and IL-8 abundance to be pinpointed in specific cell types. There were significant increases in nuclear NF-kappaB activity and IL-8 abundance across the histological series. Gene expression analysis also showed consistent up-regulation of IL-8, confirming the IHC data and showing enhanced transcriptional NF-kappaB activity. I-kappaB (another NF-kappaB target) showed down-regulation in dysplastic and adenocarcinoma tissues. Down-regulation of I-kappaB gene expression may partly explain increased NF-kappaB activity. CONCLUSION: IHC, using antibodies against the NLS of p65, may be useful in monitoring overall NF-kappaB activity in oesophageal tissues. As IHC is amenable to high-throughput screening (whereas traditional electrophoretic mobility shift assay methods are not), this may lead to the development of a better screening tool for early cancer risk.

Severe proctitis, perforation, and fatal rectal bleeding secondary to cytomegalovirus in an immunocompetent patient: report of a case.

Cytomegalovirus (CMV) infection is associated with significant morbidity and mortality in immunocompromised patients. In immunocompetent individuals, the infection is usually subclinical but it can sometimes be life threatening. We describe a case of fatal CMV proctitis in a 71-year-old man following an Ivor-Lewis esophagectomy. After surgery he developed renal failure, methicillin-resistant Staphylococcus aureus pneumonia, and acute respiratory distress syndrome. He recovered but developed melena and massive fresh rectal bleeding. Sigmoidoscopy revealed severe proctitis and a biopsy was consistent with ischemia. Despite undergoing a proctectomy he continued to bleed and died despite every effort. The final histological examination of the rectum revealed a CMV infection.

Gastro-ileal stenosis and gastroparesis after a biliopancreatic diversion.

Biliopancreatic diversion (BPD) is a very effective bariatric operation particularly for super-obese patients (BMI > or = 50 kg/m(2)). We present the development of a stricture at the gastro-ileal anastomotic site, with subsequent dilatation and aperistalsis of the stomach in a female patient who had undergone a standard open Scopinaro BPD. The patient remained symptomatic and persisted in losing weight, despite endoscopic balloon dilatations of the stricture and surgical revision of the anastomosis. She finally underwent conversion to a standard Roux-en-Y proximal gastric bypass. We describe the development of the stricture after the use of the stapling gun, subsequent gastric dilatation and dysmotility.

Laparoscopic gastric bypass in a patient with malrotation of the intestine.

The increased prevalence of morbid obesity is associated with an increased prevalence of obesity co-morbidities. Bariatric surgery is generally the only effective treatment. Gastric bypasses are the most common bariatric operation in many countries, and more than half are performed laparoscopically. We discuss the challenges encountered in performing laparoscopic gastric bypass and cholecystectomy in a morbidly obese patient who was found to have malrotated small and large bowel when the procedure started. In the absence of past gastrointestinal symptoms and investigations, there is no way of diagnosing this anomaly preoperatively. However, when such a problem is posed at the time of surgery, it is safe to perform the planned operation if the surgeon has experience and skills in advanced laparoscopic techniques.

Parathyroid carcinoma.

BACKGROUND: Parathyroid carcinoma is a rare malignancy affecting 0.5-5 per cent of all patients with primary hyperparathyroidism. This article reviews the literature on the pathogenesis, pathology, clinical features, diagnosis and management of parathyroid carcinoma. METHODS: A Medline search was performed and all relevant English language articles published between 1970 and 2005 were retrieved. The search words included 'parathyroid carcinoma', 'pathology', 'genetics', 'management' and 'radiotherapy'. Secondary references were obtained from key articles. RESULTS AND CONCLUSION: The exact aetiology of parathyroid carcinoma remains obscure. Recently, the HRPT2 gene has been implicated in its pathogenesis and may prove to be a genetic target in future. Surgical resection is the accepted 'gold standard'. There is now a growing consensus on the use of adjuvant radiotherapy as it has been shown to provide a survival benefit.

Generation of locus-specific probes for interphase fluorescence in situ hybridisation--application in Barrett's esophagus.

Despite the wide range of probes commercially available for interphase fluorescence in situ hybridisation (FISH), the supply of locus-specific probes is limited to genes or chromosomal regions commonly altered in genetic diseases or during carcinogenesis. Generation of these probes is therefore desirable to accommodate individual research requirements. Hence, we detail the methodology required to design and produce custom locus-specific interphase FISH probes for any human genomic region of interest and their application was illustrated in cytogenetic investigations of Barrett's tumourigenesis. Previously utilising FISH, we observed that Barrett's tissues demonstrated chromosome 4 hyperploidy [Gut 52 (2003) 623], but as centromeric probes were used in this analysis, it was not known if the whole chromosome was amplified. We consequently generated single-copy sequence probes for the 4p16.3 and 4q35.1 subtelomeric loci. Multicolour FISH was subsequently performed on interphase preparations originating from patients with Barrett's esophagus at varying histological grades, thus demonstrating the whole region of chromosome 4 was amplified within the tissues. Additionally, probes for the DNA methyltransferase genes were produced to determine if gene dosage alterations were responsible for increasing methylation activity during Barrett's neoplastic progression. No significant alterations at the DNMT1 and DNMT3a loci were detected. An increased copy number of these genes is therefore not the basis for the hypermethylation commonly observed in this premalignant lesion.