Prospective study of enhanced recovery after surgery protocol in children undergoing reconstructive operations.

BACKGROUND: Enhanced recovery after surgery (ERAS) protocol is a set of peri-operative strategies to increase speed of recovery. ERAS is well established in adults but has not been well studied in children. OBJECTIVE: The purpose of the current study was to establish the safety and efficacy of an ERAS protocol in pediatric urology patients undergoing reconstructive operations. It was hypothesized that ERAS would reduce length of stay and decrease complications when compared with historical controls. STUDY DESIGN: Institutional Review Board approval was obtained to prospectively enroll patients aged <18 years if they had undergone urologic reconstruction that included a bowel anastomosis. ERAS included: no bowel preparation, administration of pre-operative oral carbohydrate liquid, avoidance of opioids, regional anesthesia, laparoscopy when feasible, no postoperative nasogastric tube, early feeding, and early removal of intravenous fluids (IVF). Recent (2009-2014) historical controls were propensity matched in a 2:1 ratio on age, sex, ventriculoperitoneal shunt status and whether the patient was undergoing bladder augmentation. Outcomes were protocol adherence, length of stay (LOS), emergency department (ED) visits, re-admission within 30 days, re-operations and adverse events occurring within 90 days of surgery. RESULTS: A total of 26 historical and 13 ERAS patients were included. Median ages were 10.4 (IQR 8.0-12.4) and 9.9 years (IQR 9.1-11), respectively (P = 0.94) (see Summary Table). There were no significant between-group differences in prior abdominal surgery (38% vs 62%), rate of augmentation (88% vs 92%) or primary diagnosis of spina bifida (both 62%). ERAS significantly improved use of pre-operative liquid load (P < 0.001), avoidance of opioids (P = 0.046), early discontinuation of IVF (P < 0.001), and early feeding (P < 0.001). Protocol adherence improved from 8/16 (IQR 4-9) historically to 12/16 (IQR 11-12) after implementation of ERAS. LOS decreased from 8 days to 5.7 days (P = 0.520). Complications of any grade per patient decreased from 2.1 to 1.3 (OR 0.71, 95% CI 0.51-0.97). There were fewer complications per patient across all grades with ERAS. No differences were seen in emergency department (ED) visits, re-admissions and re-operations. DISCUSSION: Implementation improved consistency of care delivered. Tenets of ERAS that appeared to drive improvements included maintenance of euvolemia through avoidance of excess fluids, multimodal analgesia, and early feeding. CONCLUSION: ERAS decreased length of stay and 90-day complications after pediatric reconstructive surgery without increased re-admissions, re-operations or ED visits. A multicenter study will be required to confirm the potential benefits of adopting ERAS.

Beta amino acid-modified and fluorescently labelled kisspeptin analogues with potent KISS1R activity.

Kisspeptin analogues with improved metabolic stability may represent important ligands in the study of the kisspeptin/KISS1R system and have therapeutic potential. In this paper we assess the activity of known and novel kisspeptin analogues utilising a dual luciferase reporter assay in KISS1R-transfected HEK293T cells. In general terms the results reflect the outcomes of other assay formats and a number of potent agonists were identified among the analogues, including ß(2) -hTyr-modified and fluorescently labelled forms. We also showed, by assaying kisspeptin in the presence of protease inhibitors, that proteolysis of kisspeptin activity within the reporter assay itself may diminish the agonist outputs. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.

Continuous local anesthetic infusion for children with spina bifida undergoing major reconstruction of the lower urinary tract.

OBJECTIVE: While many options for postoperative analgesia are available to the general patient population, choices are limited for individuals with spinal dysraphism. We hypothesized that the use of continuous local anesthetic infusion following major reconstruction of the lower urinary tract in children with spina bifida would significantly decrease need for opiate use, while maintaining adequate pain control. MATERIALS AND METHODS: Children with spina bifida who underwent major reconstruction of the lower urinary tract at Children's Hospital Colorado were identified from January, 2003 through January, 2013 were identified. In addition to enterocycstoplasty, procedures included Mitrofanoff or Monti creation, bladder neck reconstruction, and Malone antegrade continence enema. Patients who had local anesthetic infusion catheters placed in the incision were compared to patients without catheters. Opioid consumption was calculated by conversion of any opiates into IV morphine (mg/kg) on postoperative days (POD) 0-3. Pain was assessed by mean and maximum FLACC scores on POD 0-2. Use of antiemetic medications and wound related complications were recorded as secondary metrics. Patients with other etiologies for neurogenic bladder and bowel were excluded. Patients whose pain was assessed by other assessment scales were excluded. Chi-squared analysis was used for nominal variables, students t-test was used for analysis of continuous variables. P values <0.05 were considered significant. RESULTS: 36 myelomeningocele patients who underwent primary enterocystoplasty met the inclusion criteria. All surgeries were open procedures. 24 patients in the infusion catheter group were compared to 12 patients who received primary analgesia by PCA or IV narcotics. There were no significant differences in age, sex, weight or spinal defect level between the two groups. Opioid use, as defined by IV morphine equivalents, was significantly less in the wound soaker group on all PODs. The total opioid use after POD #0-3 was 0.55 mg/kg in the wound soaker group vs 1.66 mg/kg in the IV/PCA group (p = 0.03). FLACC scores were uniformly lower in the wound soaker group, but were not significantly different. There was a significant decrease in need for postoperative antiemetic use in the wound soaker group (36.5% vs 83.3%, p = 0.014). Complications and hospital stay were similar between both groups. DISCUSSION: The advantage of local anesthesia is the reduction of systemic opioids and their subsequent adverse side effects. Our results suggest that in children with spina bifida undergoing major reconstruction of the lower urinary tract narcotic consumption is approximately 1/3 when continuous local anesthetic catheters are placed into the incision. The need for antiemetic medication is also significantly less. While this technique has been validated in a variety of other settings, it may be most beneficial in patients with myelomeningocele or other spinal dysraphism where epidural placement is generally contraindicated and narcotic use may have a particularly deleterious effect on preexisting neurogenic bowel function. The primary limitation of our study is that it is a retrospective review of a limited number of patients. Patients were not randomized and subject to other management differences that could have influenced our results in unknown ways. CONCLUSIONS: Continuous local anesthetic catheters are a simple, effective alternative strategy to provide postoperative analgesia while reducing systemic opiate use and associated adverse effects.

How do I do it: Continuous local anesthetic infusion for children with spinal dysraphism undergoing major reconstruction of the lower urinary tract.

Postoperative pain control is a fundamental aspect of contemporary pediatric surgery. While many options for analgesia are available to the general patient population, choices are limited for individuals with spinal dysraphism who undergo major urologic procedures. Continuous infusion of local anesthetics has been shown to improve postoperative pain scores and decreases the need for systemic analgesia. We present our technique for continuous local anesthetic infusion utilizing readily available equipment with limited additional cost.

Mycophenolate mofetil therapy for refractory inflammatory bowel disease.

BACKGROUND: Mycophenolate mofetil (MMF) is an immunomodulatory drug, and its use in inflammatory bowel disease has previously been reported. The aim of this study was to review the Leeds Colitis Clinic experience of the safety and efficacy of MMF in treating patients with refractory Crohn's disease (CD) and ulcerative colitis (UC). This is an extension of a previously published study from our center with a longer follow-up period and approximately twice the number of patients. METHODS: A retrospective analysis was performed of the records of all patients treated with MMF for inflammatory bowel disease over a 5-year period. RESULTS: Of 70 patients identified, 67 had previously been treated with azathioprine unsuccessfully. Seventeen of the 70 patients had been successfully maintained in remission with MMF for an average duration of 33 months. Treatment with MMF was discontinued for 53 patients, 17 because of side effects and 36 because they had not responded to the treatment. CONCLUSIONS: In our series, 17 patients (24.3%) had a sustained steroid-free remission with MMF therapy. Nineteen patients (27%) experienced side effects, of which 17 (24.3% of the total group) had to discontinue therapy. An additional 36 (51.4%) required an escalation in medical therapy or surgery because of failure of the MMF therapy. MMF may have a role in the treatment of refractory inflammatory bowel disease, especially in patients who have previously failed standard therapies such as azathioprine.

Mycophenolate mofetil in refractory inflammatory bowel disease.

BACKGROUND: Mycophenolate mofetil has been claimed to be effective and well tolerated in refractory inflammatory bowel disease although there is little information regarding its use in clinical practice. AIM: To review our experience in achieving and maintaining remission in refractory inflammatory bowel disease and to document tolerability, major toxicity and efficacy. METHODS: A retrospective audit was performed of the records of all patients with inflammatory bowel disease treated with mycophenolate mofetil (1-2 g/day) over a 3-year period. RESULTS: Thirty-nine patients were identified. Almost all had been intolerant of, or had not responded to azathioprine, and 38 were steroid-dependent. mycophenolate mofetil was discontinued in 22 patients, 11 due to intolerance and 10 because of lack of efficacy. Of the 17 on treatment at the end of the study period 16 were in remission and off all steroid therapy, but one needed infliximab to maintain remission. No major toxicity was noted and there was no major sepsis. CONCLUSIONS: Approximately 40% of patients with severe refractory inflammatory bowel disease achieved remission and complete steroid withdrawal on mycophenolate mofetil therapy, almost 30% could not tolerate the drug, and a further 30% did not respond. Mycophenolate mofetil therapy may have a role for steroid-dependent patients refractory to azathioprine.

DNA damage levels are raised in Barrett's oesophageal mucosa relative to the squamous epithelium of the oesophagus.

Barrett's oesophagus (BE) is a pre-malignant metaplastic tissue predisposing to oesophageal adenocarcinoma (EC), and gastro-oesophageal reflux is a risk factor for both conditions. Reflux of acid and bile can cause mucosal injury and initiate chronic inflammation. These processes can induce DNA damage, possibly via an oxidative stress mechanism, thus increasing the likelihood of progression from Barrett's metaplasia to dysplasia and finally carcinoma. The comet assay was optimized for the detection of DNA damage (strand breaks and alkali-labile sites) in oesophageal biopsies, including incorporation of the DNA repair enzyme Fapy-DNA glycosylase (Fpg). Fpg allows the detection of 8-hydroxy-2-deoxyguanosine (8-OHdG) sites, a known pro-mutagenic DNA lesion. BE patients were recruited from BE surveillance clinics and oesophageal biopsies collected at endoscopy. Comet analysis revealed significantly increased (p < 0.001) DNA damage in Barrett's epithelium compared with matched squamous epithelium, with median % tail DNA values of 25.1% (first to third quartile 21.7-29.6%) and 18.6% (first to third quartile 16.9-21.4%), respectively. The median % tail DNA was up to 70% higher in the matched BE tissue compared with squamous epithelium from the same patient. Fpg sensitive sites were demonstrated in both tissue types at similar levels. The raised level of DNA damage in the premalignant BE may contribute to the accumulation of genetic alterations occurring during progression to EC. Understanding these underlying mechanisms provides a basis for cancer prevention strategies in BE patients.

A novel 'sort-suicide' fusion gene vector for T cell manipulation.

Retroviral suicide gene vectors have successfully been used in clinical studies to improve the safety of adoptive immunotherapy with allogeneic T lymphocytes in the treatment of malignant and viral diseases. At the same time these studies have revealed several problems that are yet to be resolved including impaired T cell function due to long ex vivo culture. Here we present new retroviral vectors co-expressing truncated CD34, a gene transfer marker which ensures rapid enrichment of transduced cells using commercially available GMP-approved devices, and a splice-corrected variant of Herpes simplex virus thymidine kinase (scHSVtk) which confers high sensitivity to the prodrug ganciclovir. We show that a retroviral hybrid vector, MP71, based on the myeloproliferative sarcoma virus (MPSV) and the murine embryonic stem cell virus (MESV), encoding a tCD34/scHSVtk fusion protein mediates high expression of the 'sort-suicide' selection marker, thereby allowing for highly efficient purification and selective elimination of transduced cells.

The effect of Helicobacter pylori infection on levels of DNA damage in gastric epithelial cells.

BACKGROUND: Helicobacter pylori infection leads to an increased risk of developing gastric cancer. The mechanism through which this occurs is not known. We aimed to determine the effect of H. pylori and gastritis on levels of DNA damage in gastric epithelial cells. METHODS: Epithelial cells were isolated from antral biopsies from 111 patients. DNA damage was determined using single cell gel electrophoresis and the proportion of cells with damage calculated before and 6 weeks after eradication of H. pylori. Cell suspensions generated by sequential digestions of the same biopsies were assayed to determine the effect of cell position within the gastric pit on DNA damage. RESULTS: DNA damage was significantly higher in normal gastric mucosa than in H. pylori gastritis [median (interquartile range) 65% (58.5-75.8), n = 18 and 21% (11.9-29.8), n = 65, respectively, p <.001]. Intermediate levels were found in reactive gastritis [55.5% (41.3-71.7), n = 13] and H. pylori negative chronic gastritis [50.5% (36.3-60.0), n = 15]. DNA damage rose 6 weeks after successful eradication of H. pylori[to 39.5% (26.3-51.0), p =.007] but was still lower than in normal mucosa. Chronic inflammation was the most important histological factor that determined DNA damage. DNA damage fell with increasing digestion times (r = -.92 and -.88 for normal mucosa and H. pylori gastritis, respectively). CONCLUSIONS: Lower levels of DNA damage in cells isolated from H. pylori infected gastric biopsies may be a reflection of increased cell turnover in H. pylori gastritis. The investigation of mature gastric epithelial cells for DNA damage is unlikely to elucidate the mechanisms underlying gastric carcinogenesis.

Dietary antioxidants and DNA damage in patients on long-term acid-suppression therapy: a randomized controlled study.

Free radicals and reactive species produced in vivo can trigger cell damage and DNA modifications resulting in carcinogenesis. Dietary antioxidants trap these species limiting their damage. The present study evaluated the role of vitamins C and E in the prevention of potentially premalignant modifications to DNA in the human stomach by supplementing patients who, because of hypochlorhydria and possible depletion of gastric antioxidants, could be at increased risk of gastric cancer. Patients undergoing surveillance for Barrett's oesophagus (n 100), on long-term proton pump inhibitors were randomized into two groups: vitamin C (500 mg twice/d) and vitamin E (100 mg twice/d) for 12 weeks (the supplemented group) or placebo. Those attending for subsequent endoscopy had gastric juice, plasma and mucosal measurements of vitamin levels and markers of DNA damage. Seventy-two patients completed the study. Plasma ascorbic acid, total vitamin C and vitamin E were elevated in the supplemented group consistent with compliance. Gastric juice ascorbic acid and total vitamin C levels were raised significantly in the supplemented group (P=0.01) but supplementation had no effect on the mucosal level of this vitamin. However, gastric juice ascorbic acid and total vitamin C were within normal ranges in the unsupplemented group. Mucosal malondialdehyde, chemiluminescence and DNA damage levels in the comet assay were unaffected by vitamin supplementation. In conclusion, supplementation does not affect DNA damage in this group of patients. This is probably because long-term inhibition of the gastric proton pump alone does not affect gastric juice ascorbate and therefore does not increase the theoretical risk of gastric cancer because of antioxidant depletion.

Elimination of the truncated message from the herpes simplex virus thymidine kinase suicide gene.

Introduction of the Herpes simplex virus thymidine kinase (HSV-tk) gene into target cells renders them susceptible to killing by ganciclovir (GCV). We are studying the use of HSV-tk-transduced T lymphocytes in the context of hematopoietic stem cell transplantation. We have previously shown, in vitro and in vivo, the occurrence of transduced cells resistant to GCV due to a deletion within HSV-tk. This deletion, a consequence of the presence of cryptic splice donor and acceptor sites, originates in the retroviral producer cell. Here we adopt two different methods that introduce third-base degenerate changes at the cryptic splice sites and so prevent splicing. Consequently, the HSV-tk protein is unaltered and the sensitivity of the target cells to GCV is preserved. The use of this mutated HSV-tk should reduce the likelihood of the development of resistant genetically modified cells during clinical trials.

Distinct hematopoietic support by two human stromal cell lines.

OBJECTIVE: The hematopoietic microenvironment is complex, and the role of myofibroblast in its function is crucial. In order to obtain a stable model reflecting this particular cell type, we have previously established human bone marrow cell lines from primary myofibroblastic Stro1(+) population (pStro1(+)). We placed HPV16 E6 and E7 expression under the control of different promoters. Here, we have characterized and studied the hematopoietic support for two cell lines corresponding to the promoters alpha-SM (alphaSM-56 line) and SV40 (SV40-56 line). MATERIALS AND METHODS: The expression profile was analyzed at the RNA level by gene array and at the protein level by Western blot, flow cytometry, and ELISA. Hematopoietic support determined using colony-forming unit (CFU) and stroma-adherent colony-forming cell (SA-CFC) assays. RESULTS: The phenotype of cell lines was not significantly modified compared with primary myofibroblastic cells. They secreted a broad spectrum of hematopoietic cytokines and nonspecific mediators. The two lines allowed the growth of hematopoietic precursors and had different support capabilities. CONCLUSIONS: We have extensively characterized two novel human bone marrow stromal cell lines. They retained a myofibroblastic phenotype and have substantial but different hematopoietic support capabilities. These lines provided a basis for determining stromal factors involved in stem-cell regulation.

Preservation of the myofibroblastic phenotype of human papilloma virus 16 E6/E7 immortalized human bone marrow cells using the lineage limited alpha-smooth muscle actin promoter.

The in vitro study of mammalian hematopoiesis is hindered by the lack of immortalized human stromal cell lines that support hematopoiesis. We have immortalized human stromal vascular smooth muscle cells characterized by the expression of the alpha-smooth muscle (alpha-SM) actin. This marker is usually down-regulated as a result of oncogenic transformation. To correct this dedifferentiation, we placed the expression of human papilloma virus 16 E6/E7 oncogenes under the control of the tissue-specific alpha-SM actin promoter. The immortalization event is rare and requires polyclonal culture, but the corresponding established line retains alpha-SM actin expression. Moreover, when compared with other lines derived from the same cells from vectors made with the same oncogenes but driven by either an internal SV40 promoter or the viral long terminal repeat, this line is less transformed as shown by anchorage-independent growth assay. We show therefore that the use of a physiological promoter allows the production of human cell lines with a conserved phenotype.

The New Zealand rugby injury and performance project. VI. A prospective cohort study of risk factors for injury in rugby union football.

OBJECTIVES: Although the nature of rugby injury has been well documented, little is known about key risk factors. A prospective cohort study was undertaken to examine the association between potential risk factors and injury risk, measured both as an injury incidence rate and as a proportion of the playing season missed. The latter measure incorporates a measure of injury severity. METHODS: A cohort of 258 male players (mean (SD) age 20.6 (3.7) years) were followed through a full competitive season. At a preseason assessment, basic characteristics, health and lifestyle patterns, playing experience, injury experience, training patterns, and anthropometric characteristics were recorded, and then a battery of fitness tests were carried out. RESULTS: A multiple regression model identified grade and previous injury experience as risk factors for in season injury, measured as an injury incidence rate. A second model identified previous injury experience, hours of strenuous physical activity a week, playing position, cigarette smoking status, body mass index, years of rugby participation, stress, aerobic and anaerobic performance, and number of push ups as risk factors for in season injury, measured as proportion of season missed. CONCLUSIONS: The findings emphasise the importance of previous injury as a predictor of injury incidence and of missing play. They also show the importance of considering both the incidence rate and severity of injury when identifying risk factors for injury in sport.

Molecular mechanism for ganciclovir resistance in human T lymphocytes transduced with retroviral vectors carrying the herpes simplex virus thymidine kinase gene.

The herpes simplex virus thymidine kinase gene type 1 (HSV-Tk) ganciclovir (GCV) system is a novel therapeutic strategy for the modulation of graft-versus-host disease (GVHD), a major complication of allogeneic stem cell transplantation (allo-SCT). Retroviral-mediated gene transfer of the HSV-Tk gene into donor T lymphocytes before allo-SCT may allow their in vivo selective depletion after treatment with GCV. The expression of the HSV-Tk gene was analyzed in vitro in CEM cells, a human lymphoblastoid cell line, transduced with 2 different vectors, each containing the HSV-Tk gene and a selectable marker gene. GCV-resistant clones were identified within the clones expressing the marker gene. Characterization of the molecular events leading to this resistance revealed a 227-bp deletion in the HSV-Tk gene due to the presence of cryptic splice donor and acceptor sites within the HSV-Tk gene sequence. Furthermore, it was confirmed that this deletion was present in human primary T cells transduced with either vector and in 12 patients who received transduced donor T cells, together with a T-cell-depleted allo-SCT. In vivo circulating transduced T cells containing the truncated HSV-Tk gene were identified in all patients immediately after infusion and up to 800 days after transplantation. In patients who received GCV as treatment for GVHD, a progressive increase in the proportion of transduced donor T cells carrying the deleted HSV-Tk gene was observed. These results suggest that the limitations within the HSV-Tk/GCV system can be improved by developing optimized retroviral vectors to ensure maximal killing of HSV-Tk-transduced cells.

Health-related quality of life in survivors of Wilms' tumor and advanced neuroblastoma: aA cross-sectional study.

PURPOSE: In pediatric oncology, Wilms' tumor and advanced neuroblastoma represent opposite ends of the spectra of survival probability and therapeutic intensity. Consequently, it was envisaged that survivors of Wilms' tumor would enjoy better health status and health-related quality of life (HRQL) than survivors of advanced neuroblastoma. PATIENTS AND METHODS: Health status questionnaires were sent to the parents of all eligible children and to the children themselves if they were > or = 8 years of age. Responses were received from 84% of 93 eligible families. Responses were converted by established algorithms into levels of two multiattribute health status classification systems known as Health Utilities Index Mark 2 and Mark 3. These systems are linked to measures of preference, in the form of multiattribute utility functions, which provide scores of morbidity for single-attribute levels and of global HRQL for comprehensive health states. RESULTS: A greater burden of morbidity was identified in the survivors of advanced neuroblastoma than in survivors of Wilms' tumor based on the assessments of the parents of these children. In particular, survivors of advanced neuroblastoma exhibited deficits in hearing and speech. It is possible that this morbidity burden reflects the prevalent use of platinum compounds (causing ototoxicity) in this group. Within parent-child dyads there was a high level of percentage agreement on responses in all attributes except cognition. CONCLUSION: Extension of this study to a larger sample size of patients will provide clarification of these observations.

Development of a competitive PCR method for in vitro and in vivo quantification of herpes simplex virus thymidine kinase and neomycin resistance-expressing cells used in a clinical trial.

The aim of this study was to set up a sensitive and specific method to quantify the number of gene-modified cells in a gene therapy clinical trial currently underway at our institution. This trial involves the use of retrovirally transduced allogeneic T cells expressing the herpes simplex-1 thymidine kinase (HSV-TK) and neomycin-phosphotransferase (NeoR) resistance gene. Quantification by competitive PCR was performed, with two homologous internal standards (deltaTK, deltaNeoR), 30 bp shorter than the target sequences (TK, NeoR), coupled to fluorescent laser-based detection. Assessment of the amplification systems procedures was carried out for each sequence. The 30-bp deletion did not affect the amplification efficiency significantly. Determination of the plateau phase of both amplified sequences demonstrated that each sample must be quantified during the predetermined exponential phase. Finally, a blinded study of a transduced cell dilutions panel validated the overall methodology. The competitive PCR was applied to quantification of the retroviral transduction process by quantifying the NeoR gene in transduced PBMC samples (prior to G418 selection) from 18 donors in our clinical trial. A mean transduction efficiency of 9.78% +/- 1.37% was observed. We also quantified TK-expressing donor transgenic T cells in a murine GvHD model. Results demonstrated on initial expansion of donor HSV-TK- expression T cells as well as a significant ganciclovir (GCV)-induced decrease correlated with the number of circulating gene-modified T cells. Therefore, we have developed an efficient gene quantification tool that should be useful for in vivo monitoring of gene-modified cells.

Flat and depressed colonic neoplasms: a prospective study of 1000 colonoscopies in the UK.

BACKGROUND: Flat and depressed colorectal tumours were originally thought to be unique to the Japanese population. Recently there have been reports of flat and depressed lesions in western countries but they have been thought to be uncommon. METHODS: In this prospective study, 1000 consecutive patients attending for routine colonoscopy were examined for flat or depressed lesions. The examinations were done by one European colonoscopist using methods developed in Japan. FINDINGS: 321 adenomas were found: 202 (63%) were polypoid, 36% (117) were flat and 2 (0.6%) appeared depressed. Most adenomas contained areas of mild or moderate dysplasia but 10% (31) were severely dysplastic. Six Dukes' A adenocarcinomas were identified together with 25 more advanced adenocarcinomas. The likelihood of Dukes' A cancer or severe dysplasia increased from 4% (3/70) in small flat lesions, to 6% (9/154) in small polyps, 16% (8/50) in larger polyps, 29% (14/49) in large flat lesions, and 75% (3/4) in depressed lesions. 54% (20/37) lesions containing severe dysplasia or Dukes' A carcinoma were flat or depressed. INTERPRETATION: The polyp-carcinoma hypothesis prompts colonoscopists to search only for polypoid lesions when screening for cancer, and many early colorectal neoplasms may therefore be missed. Colonoscopists require training in the recognition of flat and depressed lesions to detect colorectal tumours in the early stages.

Loss of Fas (CD95/APO-1) expression by antigen-specific cytotoxic T cells is reversed by inhibiting DNA methylation.

Elimination of clonally expanded peripheral CD8 T cells was thought to involve apoptosis induction mediated principally by TNF, but recently Fas (CD95/APO-1) has been shown to play a role in certain responses. Here we study Fas expression and sensitivity to its ligation on murine CD8 cells specific for the CW3 antigen expressed by transfected P815 cells. Fas was progressively downregulated after successive in vitro restimulations of antigen-specific CD8 cells, until clones became Fas negative and totally resistant to the effects of recombinant Fas ligand. In contrast, Fas expression by in vivo restimulated antigen-specific cells did not diminish. Loss of Fas expression in vitro was not totally irreversible, since it could be reinduced by inhibition of DNA methylation. Understanding how Fas may be differentially regulated in vivo and in vitro is an important issue for the optimal manipulation of T cells for adoptive immunotherapy protocols.