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Summary Introduction : Azathioprine is effective for maintenance of remission in Crohn's disease, however, duration of efficacy and the dose response relationship has not been fully evaluated. Aims : To investigate whether patients kept in remission by azathioprine treatment for >2 years benefit from further treatment, and to explore dose–response relationship. Patients and methods : In an open 12-month trial, patients with inactive Crohn's disease after >2 years (median 37 months) of azathioprine treatment were randomized to azathioprine withdrawal or continued treatment. Primary end point was relapse defined as: (i) Crohn's disease activity index rise ≥ 75, and Crohn's disease activity index >150 or (ii) disease activity requiring intervention. Results : Of 29 patients, 28 completed the observation period or relapsed. Eleven of 13 patients (85%) continuing azathioprine remained in remission compared with seven of 15 (47%) observed without azathioprine (P = 0.043). In patients who had been treated with azathioprine >1.60 mg/kg/day the difference was even more pronounced, eight of nine (89%) vs. four of 12 (33%) respectively (P = 0.017). Conclusions : Patients with Crohn's disease in remission after >2 years of continuous azathioprine treatment will benefit from further continued treatment. Further controlled studies with azathioprine doses

There are few data on how patients on maintenance treatment of reflux oesophagitis take their medication. This study was designed to investigate the dosing patterns of patients on on-demand treatment and to compare lansoprazole with omeprazole in this regard.Patients with reflux oesophagitis, initially treated until absence of symptoms, took capsules of either lansoprazole (30 mg) or omeprazole (20 mg) for 6 months; they were instructed to take the medication only when reflux symptoms occurred. In order to document dosing patterns, the medication was dispensed in bottles supplied with a Medication Event Monitoring System recording date and time the bottles were opened. There were regular follow-up visits with assessment of symptoms.Three-hundred patients were eligible for analysis according to 'all patients treated'. A dosing pattern was found of an increased intake mornings and evenings and constant intervals between intakes. Although there was no correlation between oesophagitis grade or initial symptoms and the amount of medication consumed, the patients had significantly fewer reflux symptoms the more medication they consumed. There was no difference in the number of capsules consumed between the lansoprazole (0.73 capsules/day) and omeprazole groups (0.71 capsules/day). Nor was there any difference between the groups in reflux symptoms during the course of the study.Despite rigorous instructions to take medication on demand, the results suggest that it is patient habits more so than symptoms that determine the frequency and interval of medication intake. Symptoms are not therefore decisive for the amount of medication consumed.

Eradication of Helicobacter pylori is associated with a reduced recurrence of duodenal ulcer (DU). The relationship between H. pylori and DU has been interpreted as causal, but the evidence has been criticized for methodologic reasons. To ascertain whether an antibiotic with no effect on epithelial-cell integrity prevents DU recurrence, we conducted a randomized double-blind trial of phenoxymethylpenicillin (PEN), 2.4 twice daily, and placebo (PLA). Patients with an active DU and positive H. pylori culture from antral biopsy specimens were treated with 40 mg omeprazole daily for 4 weeks, but at week 2 they were allocated at random to PEN (85 patients) or PLA (85 patients) for up to 14 weeks. Those without recurrence during this treatment were followed up for another 6 months. Endoscopy and H. pylori culture were performed at the end of the treatment period and at the end of follow-up, and in between if ulcer symptoms recurred. During the treatment period the ulcer relapse rate was 5 of 58 (9%) in the PEN group and 34 of 68 (50%) in the PLA group (P0.0001, log-rank test), with 53% and 14%, respectively, of the patients in the two groups being H. pylori-negative. The relapse rate in the PEN group did not differ between H. pylori-negative and H. pylori-positive patients. The recurrence rate in the PEN group remained low for another 5 months but then approached the rate in the PLA group. The prevalence of H. pylori-negative patients at the end of follow-up was 20% in the PEN group and 10% in the PLA group. These data provide strong evidence that DU has a bacterial cause, with H. pylori as the likely agent.

The long-term efficacy of infliximab as rescue therapy in steroid-refractory ulcerative colitis is not well described.To examine the long-term efficacy of infliximab as a rescue therapy through a 3-year follow-up of a previous placebo-controlled trial of infliximab in acute steroid-refractory ulcerative colitis.In the original study, 45 patients were randomized to a single infusion of infliximab 5 mg/kg or placebo, and at 3 months, 7/24 patients given infliximab were operated vs. 14/21 patients given placebo. Three years or later, patients were asked to participate in a clinical follow-up.Another seven patients underwent colectomy during follow-up: five in the infliximab group and two in the placebo group. After 3 years, a total of 12/24 (50%) patients given infliximab and 16/21 (76%) given placebo (P = 0.012) had a colectomy. None of eight patients in endoscopic remission at 3 months later had a colectomy compared with 7/14 (50%) patients who were not in remission (P=0.02). There was no mortality.The benefit of rescue therapy with infliximab in steroid-refractory acute ulcerative colitis remained after 3 years. The main advantage of infliximab treatment occurred during the first 3 months, whereas subsequent colectomy rates were similar in the two groups. Mucosal healing at 3 months influenced later risk of colectomy.

The validity of using autologous leucocytes labelled with technetium -99m hexamethyl-propyleneamine-oxine (Tc-HMPAO) for scintigraphy in inflammatory bowel disease was evaluated in 12 patients with clinically active ulcerative colitis (UC) and 10 with Crohn's disease (CD). Colonoscopy and biopsy were used as reference. Scintigrams taken 1 h and 3 h after leucocyte reinjection were evaluated blindly by two independent observer groups. Full agreement was found in 11 of 12 UC patients when compared with colonoscopy but in only 3 of 10 CD patients. Segments with agreement in CD patients often showed neutrophilic granulocyte infiltration at biopsy. The judgements of clinicians and physiologists differed for only 2 of totally 70 UC segments but for 13 of 59 CD segments (kappa, 0.94 and 0.52). It is concluded that Tc-HMPAO scintigraphy might be an alternative to colonoscopy in the control of disease extent in UC. In CD patients the technique might warn about infections complications.

Trials evaluating long-term management of duodenal ulcer disease have mainly been focused on recurrence of ulcers, disregarding effects on dyspeptic and reflux symptoms. Profound acid inhibition with a proton pump inhibitor is the gold standard therapy in acid-related diseases. We aimed to compare the symptomatic effects of eradication therapy with those of long-term omeprazole treatment in a design with periods both with and without acid inhibition.Patients with active duodenal ulcer were randomized either to omeprazole, 20 mg twice daily until healing, followed by omeprazole, 20 mg/ day for 1 year, or to eradication therapy (metronidazole, amoxicillin, and omeprazole for 2 weeks) followed by placebo for 1 year. All patients were followed up passively for an additional year. Clinical controls were performed every 2 months the 1st year (maintenance phase) and every 6 months during the passive follow-up phase. The study was multicentric and double-blind. The primary end-point was discontinuation of treatment, irrespective of reason.Two hundred and seventy-six patients were randomized (139 in the eradication treatment group). In the maintenance phase there were no differences in the reporting of dyspeptic symptoms or in premature withdrawal. In the passive follow-up phase only five patients in the eradication therapy group discontinued owing to relapse of dyspeptic symptoms or ulcer, compared with 51 patients initially randomized to long-term omeprazole. There were no differences in reflux symptoms or in the development of reflux oesophagitis.Eradication therapy and long-term omeprazole are equally effective in controlling dyspeptic symptoms and reflux in duodenal ulcer patients with healed ulcers. One-quarter of the duodenal ulcer patients who start eradication therapy continue to be symptomatic or fail therapy for other reasons over a 2-year period. Eradication therapy does not increase the risk of reflux in ulcer patients.

The prolonged sedation of benzodiazepines may be inconvenient for the patient. Reversal of sedation, therefore, would be desirable. Accordingly, we assessed the efficacy of the benzodiazepine antagonist flumazenil in a placebo-controlled randomized trial including 40 adults undergoing upper gastrointestinal endoscopy under diazepam (Diazemuls®) sedation. We found no significant differences between groups with regard to either performance (two tests) or duration (within 240 min) of sedation. There were no noticeable side effects.

The aim of this study was to test the hypothesis that infection with Helicobacter pylori is essential for recurrence of duodenal ulcer. We performed a randomized controlled trial of the relapse rate of duodenal ulcer during 12 weeks treatment with penicillin V or placebo in 170 out-patients from five centres. The relapse rate was 9% during treatment with penicillin and 50% with placebo, P0.0001. It is concluded that infection with penicillin-sensitive bacteria, i.e. H. pylori, plays an important role for recurrence of duodenal ulcer disease. Penicillin V suppresses this infection but does not eradicate it.

Concentrations of interleukin-1 beta (IL-1 beta), interleukin-2 (IL-2), and soluble IL-2 receptors (sIL-2R) were determined by enzyme linked immunosorbent assays (ELISA) in supernatants of sonicated endoscopical mucosal biopsy specimens from 31 patients with inflammatory bowel disease and 19 controls. IL-1 beta was detected in 53% of the patient supernatants (p = 0.0001), IL-2 in 35% (p = 0.0031), compared with none of the controls. Soluble IL-2R was present in 55% and 26% of the specimens, respectively (p = 0.07). The concentrations of IL-1 beta (p = 0.00015), IL-2 (p = 0.0019), and sIL-2R (p = 0.0073) were highest in the most inflamed biopsy specimens, compared with less inflamed specimens and controls. There were no significant differences in IL-1 beta, IL-2, and sIL-2R concentrations between ulcerative colitis (16) and Crohn's disease patients (15). The results suggest that enhanced cellular immunity operates in vivo at the mucosal level in active inflammatory bowel disease.

Colonoscopic screening for neoplasia was performed in a regional group of ulcerative colitis patients with a disease duration of greater than or equal to 15 years. A total of 121 patients, aged less than 80 years, were invited to participate, of whom 100 (83%) accepted colonoscopy, including biopsies in 15 standard locations of the entire colon, plus additional biopsies from all visible lesions. Unequivocal dysplasia was found in one patient with extensive colitis and a disease duration of 31 years. A polyp with highly differentiated adenocarcinoma was found in the sigmoid colon of a patient with intermittent rectum involvement, 37 years after the ulcerative colitis diagnosis had been made. Biopsy specimens from the remaining 98 patients showed no signs of dysplasia or cancer. Thus the frequency of pre-malignant or malignant changes is very low compared with the results of similar studies, and the rationale for general colonoscopic surveillance programmes for such patients is open to question.

This study tests the hypothesis that reactivation of a latent herpes simplex virus infection may be a cause of recurrent duodenal ulceration. Patients with recently healed duodenal ulcer were entered into a double blind, randomised study of maintenance treatment with the antiviral drug acyclovir (400 mg bid) versus placebo, to determine if suppression of herpes virus infection would influence the natural history of the ulcer disease. One hundred and fifteen patients entered the trial and 76 patients completed it according to the protocol. Endoscopy was performed when ulcer symptoms recurred and at the end of the 25 week trial period. In the acyclovir group the cumulated relapse rate was 63% compared with 56% in the placebo group (NS). This result suggests that reactivation of herpes simplex virus is not a cause of recurrent duodenal ulcer.

The relationship between Helicobacter pylori and duodenal ulcer has been interpreted as causal, but existing evidence has been criticized for methodologic reasons. We conducted a randomized trial of phenoxymethylpenicillin, 2.4 g twice daily, and placebo to determine whether an antibiotic, which has no effect on the integrity of the epithelial cells, prevents recurrence of duodenal ulcer. Patients with an active duodenal ulcer and positive H. pylori culture, assessed from antral biopsy specimens, were treated with omeprazole, 40 mg once daily, for a total of 4 weeks. After 2 weeks' treatment with omeprazole, these patients were randomized to receive phenoxymethylpenicillin (n = 85) or placebo (n = 85). In patients whose ulcer had healed after 4 weeks' omeprazole therapy, phenoxymethylpenicillin or placebo was continued for an additional 12 weeks. Patients without ulcer recurrence during this treatment period were followed up for a further 6 months. Endoscopy was performed and H. pylori culture assessed at...

The response to 5-aminosalicylic acid (5-ASA) in mild and moderately active Crohn's disease localized in the small bowel was studied in a randomized, double-blind, placebo-controlled trial in four centres. Sixty-seven patients were included, of whom 30 were treated with 1500 mg slow-release 5-ASA/day (Pentasa) for a scheduled period of 16 weeks. In the 5-ASA group 40% of the patients improved, versus 30% of the placebo-treated group ('intent to treat' basis; p greater than 0.1). Four of the patients treated with 5-ASA left the study owing to disease deterioration, versus 10 of the placebo-treated patients (p greater than 0.2). Seventeen patients were secondarily excluded, and the remaining 50 patients (23 receiving 5-ASA) were reevaluated in greater detail. No statistically significant differences in outcome were shown. Three patients (one given 5-ASA) were withdrawn from the study because of presumed side effects, but no serious adverse reactions were recorded. The present results indicate that 5-ASA, at least in the dosage used, is not superior to placebo. Nevertheless, trends towards a beneficial effect in Crohn's disease in the small bowel justify further clinical trials with a larger dosage of 5-ASA.

Human urothelial cell lines of non-malignant and malignant origin were classified into three grades of transformation, i.e. TGr I, TGr II, and TGr III, which differ from primary and early cultures of normal bladder mucosa (TGr 0) by growth in the absence of fibroblasts, prolonged survival, increasing chromosomal abnormalities, and antigenic modifications. Non-malignant pure epithelial cell lines with a prolonged, but finite life span (18-38 passager in vitro) were classified as TGr I. These cells were characterized by chromosomal counts showing a considerable variation but with a near-diploid mode and a smaller size but otherwise normal morphology. TGr II cells differed from TGr I by infinite growth transformation and wide morphological variability. TGr III cells were above all characterized by their ability to produce tumors in nude mice and to invade and destroy fragments of embryonic heart in vitro. These immortalized cells were small with a large nucleus and a uniform polygonal shape. Chromosomal counts showed pronounced aneuploidy with an increased number of chromosomes.

The ability of 7 human cell lines to invade into cultured embryonic chick heart fragments was investigated. The 3 cell lines HU 456, HU 961 and T24 were derived from human bladder tumors of various degrees of malignancy. The 2 cell lines HCV29 and HU 609 were cultured from normal bladder and ureter epithelium, respectively. The sublines, HCV29T and HU 609T, derived from HCV29 and HU 609, showed “spontaneous” morphological transformation. The malignant and morphologically transformed cells invaded the heart fragments while the cells of normal origin apparently did not.

Cell lines from normal or malignant human urothelium, described elsewhere by morphological, cell kinetic and genotypical criteria, have been characterized further by their response to antibody or lymphocyte mediated cytotoxicity in vitro . Four groups of cell lines were included: (1) two fast proliferating cell lines from normal urothelium, (2) three fast proliferating cell lines from transitional cell carcinoma (TCC) together with two transformed sublines of normal derived cell lines, (3) six slowly proliferating TCC lines, and (4) a line from squamous cell carcinoma. HLA antigen expression was demonstrated in the cell lines of groups 1 and 3 , but not in lines from group 2 or 4 . The sensitivity to spontaneous lymphocyte mediated cytotoxicity (SLMC) of cells in group 1 and 2 exceeded that of cells from group 3 by a factor of 8 . The TCC line, HU 456 , was more susceptible to SLMC than T 24 . Specifically increased cytotoxicity of lymphocytes from patients suffering from interstitial cystitis (IC) against HU 609 from normal urothelium indicated that this line expresses tissue specific antigens, and at the same time that an immune reaction may be part of the pathogenesis of IC. By a sensitive test for antibody-dependent, cell-mediated cytotoxicity (ADCC) antibody activity against HU 456 was found in only one of nine TCC patients and none of five clinical controls. A pronounced non-disease-related blocking serum activity, frequently found, may account for some of the negative findings. The SLMC against HU 456 could be inhibited but not abolished by Fab fragments of rabbit anti-human immunoglobulin, indicating an ADCC mechanism as part of the SLMC.

Blood lymphocytes from small groups of patients with transitional-cell carcinoma of the urinary bladder (TCC), clinical controls (CC) or healthy donors (HD) were tested for cytotoxicity in vitro by a 51Cr-release assay. The target cells were either from TCC or control tissue (long-term cultures) or were from short-term TCC cultures, kept in vitro for 10-20 transfer generations. When tested with allogeneic target cells from long-term cultures, TCC patients' lymphocytes tended to be more cytotoxic to TCC targets than to control targets. For the control lymphocytes this was not seen. A large proportion but not all of the cytotoxicity to these target cells was due to immunoglobulin-dependent cellular reactions, probably mediated by natural and disease-related antibodies of the lymphocyte donors, since it was significantly inhibited by Fab-fragments of rabbit antibodies to human immunoglobulin. Moreover, it was, to a large extent, mediated by lymphocytes with Fc-receptors for IgG. For seven of the TCC target cell cultures (two long-term and five short-term) autologous lymphocytes were also available for testing. While two patients were non-reactive to their own tumor cells, five reacted strongly in the autologous combinations. These autologous reactions were immunoglobulin-independent and were mediated by Fc-receptor-negative effector cells. In some instances, autologous cytotoxicity was accompanied by similar reactions to some of the allogeneic TCC targets but not to the allogeneic non-TCC control targets. On the basis of available information on HLA antigens in this material, the pattern of cross-reactions suggests that the cytotoxicity encountered in the autologous and in some of the allogeneic TCC-combinations may be the expression of antibody-independnet but specific CTL-mediated reactions, regulated by HLA. However, at the present stage of the investigation, other mechanisms must also be considered since the target cells from short-term TCC cultures were sometimes lysed by control lymphocytes in immunoglobulin-independent reactions. Whatever the explanation, the results show that the cytotoxicity observed in the in vitro systems is usually the net result of several different types of reaction. Which effector cell types and which mechanism of recognition will predominate in a given lymphocyte/target cell combination is greatly influenced by the nature and origin of the target cells used.

Titrations of spontaneous lymphocyte-mediated cytotoxicity (SLMC) in a 44-hour microcytotoxicity assay (MA) by serial dilutions of effector lymphocytes were performed to characterize the lymphocyte-dose response relationship, and to investigate the methodological basis for comparative cytotoxicity studies. The following lymphocyte fractions were used: unseparated, E rosetting, non-E rosetting, EA rosetting, and non-EA rosetting. Assays were performed against target cell lines from normal and malignant urothelium with high or low sensitivity to SLMC in allogeneic and autologous combinations, and against an osteosarcoma cell line. Analysis of SLMC titration curves showed that two different mathematical models could be used to describe the relationship between lymphocyte concentration and SLMC, I: linear relationship (log lymphocytes, CI) with exclusion of both high and low CI values, and II: sigmoid relationship (log lymphocytes, CI) which was transformed to linear relationship (log lymphocytes, log CI/100 - CI), including also high and low values. It is pointed out that perfect quantitative comparisons of SLMC in different combinations were complicated by statistically significant differences between the curve slopes. However, estimations based on experimental results in a range close to 50% cytotoxicity index (CI) may be acceptable, as the differences between slopes were small.

Cytotoxicity of lymphocytes from T1 or T2 bladder cancer patients and patients with other diseases was tested in 44-hour microcytotoxicity assay against 3 different target cell lines: 1) HU 456, derived from human bladder carcinoma and established in our laboratory, 2) HU 609, a line derived from normal human bladder tissue and established in our laboratory and 3) SAOS-2, a human osteosarcoma cell line from the Memorial Sloan-Kettering Cancer Institute. Lymphocyte concentrations ranging from 7.8 time 10(4) to 2.5 times 10(6) lymphocytes per ml. were tested against each cell line. Lymphocytes from both groups of patients demonstrated a cytotoxicity against all 3 target cell lines, proportional to the lymphocyte concentration used. There was no difference in reactivity to HU 609 or to SAOS-2 between bladder cancer and control patients but the lymphocytes of bladder cancer patients showed a statistically greater cytotoxicity for HU 456, demonstrating a tumor type-specific cellular immune reaction superimposed on a background of non-specific cytotoxicity.

Thirty-five subjects treated with a gluten-free diet for a chronic gastrointestinal disorder (no biopsy performed) in early childhood were re-examined 14-38 (median, 28) years later. Twenty-one accepted jejunal biopsy at re-examination. None of those studied were on a gluten-free diet at the time of the study. Ten of 21 biopsy specimens were abnormal--6 with flat mucosa and very low disaccharidase levels, indicating coeliac disease, and 4 with low disaccharidase activity but morphologically normal mucosa. Three of the six subjects with flat mucosa showed a slight fat and lactose malabsorption, but their clinical condition did not suggest serious intestinal disease. Ingestion of gluten throughout most of childhood did not appear to affect growth, as evaluated by final height. Jejunal biopsy is recommended in all patients with gastrointestinal symptoms that are not otherwise explainable if their medical history shows a period of gluten-free diet for a chronic gastrointestinal disorder in childhood.

A controlled investigation of lymphocyte-mediated cytotoxicity has been carried out in patients with transitional cell carcinoma (TCC) of the urinary bladder, three long-term established cell lines with comparable sensitivities to the natural cytotoxicity (NC) being used as targets, namely HU 456 derived from human TCC, HU 609 from normal human urothelium, and SAOS 2 from a human osteosarcoma. The 44-h incubation microcytotoxicity assay (MA) was used with ‘blinded’ semiautomatic visual counting of target cells. The cytotoxicity was determined by titration with a serial dilution of lymphocyte suspensions 1:2, five different concentrations being used in each experiment. Fifty tests were performed with lymphocytes from 48 TCC patients. As controls, 65 patients with diseases other than TCC were tested simultaneously. The average cytotoxicity of lymphocytes from TCC patients against HU 456 was only slightly and insignificantly higher than that of the control patients. However, a marked decrease of the NC against the control cells HU 609 and SAOS 2 was noted with TCC patient lymphocytes. After correction of the data for the depression of background NC, increased tumor-specific cytotoxicity (TSC) and tumor type-specific cytotoxicity (TTSC) were demonstrated in TCC patients with noninvasive malignant tumors (grade 2 or 3), whereas no increased specific cytotoxicity was found in TCC patients with invasive grade 2 or 3 tumors. Neither was any increased specific cytotoxicity found in patients with very well-differentiated grade tumors. The presence of specific reactivity in patients with noninvasive TCC in contrast to invasive TCC is supposed to indicate growth-controlling function of the cellular immune reaction. Tests were also performed with lymphocytes from 35 patients in the postoperative phase without any history of TCC. Surprisingly, this group revealed a significantly elevated TSC but not increased TTSC when compared with the 65 untreated control patients, thus indicating that serious reservations must be made in the interpretation of the cytotoxocity assay.

Publisher Summary This chapter reviews the relation between effector mechanisms to target cell origin. Cell-mediated immune reactions in vitro to a variety of human and animal tumors have been studied extensively during recent years. The most commonly used in vitro approaches have been to assess lymphocyte cytotoxicity against a multiplicity of cultured cell lines. However, the issue of specificity of these reactions and of their biological significance remain controversial. The cytotoxic effector cells active in many of these systems are not well characterized. The chapter discusses studies to establish the occurrence of a disease-related lymphocyte cytotoxicity in patients with transitional cell-carcinoma (TCC) of the urinary bladder in both allogeneic and autochtonous lymphocyte-target cell combinations and to characterize the effector cell(s) operative in these systems. Cytotoxicity to allogeneic target cells from established cell lines is frequently, but not invariably due to effector lymphocytes of the K-type lysing target cells through an immunoglobulin-dependent mechanism. This holds for both the disease-related reactions of cells from TCC-bladder patients and for the selective cytotoxicity of lymphocytes from clinical controls or healthy donors.

A 44-hour incubation microcytotoxicity assay (MA) was used to titrate the lymphocyte-mediated cytotoxicity in 47 transitional cell carcinoma (TCC) bladder cancer patients and 65 clinical control patients. All titrations included three target cell lines: HU 456 (TCC), HU 609 (normal urothelium), and SAOS 2 (osteosarcoma). Tumor-specific cytotoxicity (TSC) was calculated as the difference between cytotoxicity to HU 456 and HU 609, and tumor type-specific cytotoxicity (TTSC) as the difference between cytotoxicity to HU 456 and SAOS 2. On the basis of TSC and TTSC values obtained before treatment TCC patients were divided into one group with high-grade specific cytotoxicity (HSC) and another with low-grade specific cytotoxicity (LSC). A prospective follow-up study of these patients revealed a significantly lower survival rate for patients with LSC compared with patients with HSC, even when the groups were corrected for differences in distributions according to clinical and histological tumor gradation. This indicates a growth-controlling function of the cellular immune reaction.

In numerous in vitro studies of cell-mediated cytotoxicity directed against transitional cell carcinomas (TCC) urothelial cells of normal origin have served as control material. In the present work, which includes 13 human bladder cell lines of non-malignant or malignant origin, a comparison was made of morphology, chromosomal mode, growth fraction and tumorigenicity in nude mice. Signs of apparently spontaneous transformation were seen in two cell lines of non-malignant origin. The possibilities for cross-contamination as an explanation of these signs of ‘spontaneous’ transformation are discussed. During these studies radiation-dependent cytotoxicity of disposable microtissue culture plates was revealed as another important source of error.