Nonhereditary p53 mutations in T-cell acute lymphoblastic leukemia are associated with the relapse phase

We have previously reported that greater than 60% of human leukemic T- cell lines possess mutations in the p53 tumor suppressor gene. To determine whether T-cell acute lymphoblastic leukemia (T-ALL) patient samples possess p53 mutations, we screened peripheral blood-and bone marrow-derived leukemia samples, taken at diagnosis and at relapse, for p53 mutations. Exons 4 through 9 and selected intron regions of the p53 gene were analyzed using polymerase chain reaction-single-strand conformation polymorphism and direct sequencing. p53 mutations were found in 0 of 15 T-ALL diagnosis samples, as compared with 10 of 36 (28%) T-ALL relapse samples. To determine whether p53 mutations play a role in the recurrence (relapse) of T-ALL, two special groups of T-ALL patients were studied: (1) a group of 8 relapse patients whose disease was refractory to chemotherapeutic treatment, and (2) a group of 6 "paired" T-ALL cell samples from patients for whom we possess both diagnosis and relapse samples. Three of 8 relapsed patients (37.5%) whose disease was refractory to the reinduction of remission by chemotherapy possessed missense mutations of the p53 gene. All 3 cases had mutations in exon 5. Among the paired samples, 3 of 6 patients harbored p53 mutations at disease recurrence, but possessed only wild- type p53 alleles at diagnosis. One case had mutation on exon 4, 1 case in exon 5, and 1 case in exon 8 with loss of heterozygosity. These data clearly indicate that recurrence of T-ALL is associated with missense mutations in p53. Our results indicate that (1) mutations of p53 do occur in T-ALL in vivo, and such mutations are associated with the relapse phase of the disease; and (2) p53 mutation is involved in the progression of T-ALL. This conclusion is supported by our observation that the introduction of T-ALL-derived mutant p53 expression constructs into T-ALL cell lines further increases their growth rate in culture, enhances cell cloning in methylcellulose, and increases tumor formation in nude mice.     

Evidence for an essential role of reactive oxygen species in the genesis of late preconditioning against myocardial stunning in conscious pigs.

Conscious pigs underwent a sequence of 10 2-min coronary occlusions, each separated by 2 min of reperfusion, for three consecutive days (days 1, 2, and 3). On day 1, pigs received an i.v. infusion of a combination of antioxidants (superoxide dismutase, catalase, and N-2 mercaptopropionyl glycine; group II, n = 9), nisoldipine (group III, n = 6), or vehicle (group I [controls], n = 9). In the control group, systolic wall thickening (WTh) in the ischemic-reperfused region on day 1 remained significantly depressed for 4 h after the 10th reperfusion, indicating myocardial "stunning." On days 2 and 3, however, the recovery of WTh improved markedly, so that the total deficit of WTh decreased by 53% on day 2 and 56% on day 3 compared with day 1 (P < 0.01), indicating the development of a powerful cardioprotective response (late preconditioning against stunning). In the anti-oxidant-treated group, the total deficit of WTh on day 1 was 54% less than in the control group (P < 0.01). On day 2, the total deficit of WTh was 85% greater than that observed on day 1 and similar to that observed on day 1 in the control group. On day 3, the total deficit of WTh was 58% less than that noted on day 2 (P < 0.01). In the nisoldipine-treated group, the total deficit of WTh on day 1 was 53% less than that noted in controls (P < 0.01). On days 2 and 3, the total deficit of WTh was similar to the corresponding values in the control group. These results demonstrate that: (a) in the conscious pig, antioxidant therapy completely blocks the development of late preconditioning against stunning, indicating that the production of reactive oxygen species (ROS) on day 1 is the mechanism whereby ischemia induces the protective response observed on day 2; (b) antioxidant therapy markedly attenuates myocardial stunning on day 1, indicating that ROS play an important pathogenetic role in postischemic dysfunction in the porcine heart despite the lack of xanthine oxidase; (c) although the administration of a calcium-channel antagonist (nisoldipine) is as effective as antioxidant therapy in attenuating myocardial stunning on day 1, it has no effect on late preconditioning on day 2, indicating that the ability of antioxidants to block late preconditioning is not a nonspecific result of the mitigation of postischemic dysfunction on day 1. Generation of ROS during reperfusion is generally viewed as a deleterious process. Our finding that ROS contribute to the genesis of myocardial stunning but, at the same time, trigger the development of late preconditioning against stunning supports a complex pathophysiological paradigm, in which ROS play an immediate injurious role (as mediators of stunning) followed by a useful function (as mediators of subsequent preconditioning).     

Communicating oesophageal duplication

Oesophageal duplications are rare congenital abnormalities. Most of them do not communicate with the oesophageal lumen. We present a very uncommon finding of communicating oesophageal duplication in which the connection between the oesophagus and its duplicate portion was demonstrated by CT.     

Assessment of the mutagenicity of dichloroacetic acid in lacI transgenic B6C3F1 mouse liver

Dichloroacetic acid (DCA) is a chlorination byproduct found in finished drinking water. When administered in drinking water this chemical has been shown to produce hepatocellular adenomas and carcinomas in B6C3F1 mice over the animal's lifetime. In this study, we investigated whether mutant frequencies were increased in mouse liver using treatment protocols that yielded significant tumor induction. DCA was administered continuously at either 1.0 or 3.5 g/l in drinking water to male transgenic B6C3F1 mice harboring the bacterial lacI gene. Groups of five or six animals were killed at 4, 10 or 60 weeks and livers removed. At both 4 and 10 weeks of treatment, there was no significant difference in mutant frequency between the treated and control animals at either dose level. At 60 weeks, mice treated with 1.0 g/l DCA showed a 1.3-fold increase in mutant frequency over concurrent controls (P = 0.05). Mice treated with 3.5 g/l DCA for 60 weeks had a 2.3-fold increase in mutant frequency over the concurrent controls (P = 0.002). The mutation spectrum recovered from mice treated with 3.5 g/l DCA for 60 weeks contained G:C-->A:T transitions (32.79%) and G:C-->T:A transversions (21.31%). In contrast, G:C-->A:T transitions comprised 53.19% of the recovered mutants among control animals. Although only 19.15% of mutations among the controls were at T:A sites, 32.79% of the mutations from DCA-treated animals were at T:A sites. This is consistent with the previous observation that the proportion of mutations at T:A sites in codon 61 of the H-ras gene was increased in DCA-induced liver tumors in B6C3F1 mice. The present study demonstrates DCA-associated mutagenicity in the mouse liver under conditions in which DCA produces hepatic tumors.      

Informed consent, parental awareness, and reasons for participating in a randomised controlled study

BACKGROUND: The informed consent procedure plays a central role in randomised controlled trials but has only been explored in a few studies on children. AIM: To assess the quality of the informed consent process in a paediatric setting. METHODS: A questionnaire was sent to parents who volunteered their child (230 children) for a randomised, double blind, placebo controlled trial of ibuprofen syrup to prevent recurrent febrile seizures. RESULTS: 181 (79%) parents responded. On average, 73% of parents were aware of the major study characteristics. A few had difficulty understanding the information provided. Major factors in parents granting approval were the contribution to clinical science (51%) and benefit to the child (32%). Sociodemographic status did not influence initial participation but west European origin of the father was associated with willingness to participate in future trials. 89% of participants felt positive about the informed consent procedure; however, 25% stated that they felt obliged to participate. Although their reasons for granting approval and their evaluation of the informed consent procedure did not differ, relatively more were hesitant about participating in future. Parents appreciated the investigator being on call 24 hours a day (38%) and the extra medical care and information provided (37%) as advantages of participation. Disadvantages were mainly the time consuming aspects and the work involved (23%). CONCLUSIONS: Parents' understanding of trial characteristics might be improved by designing less difficult informed consent forms and by the investigator giving extra attention and information to non-west European parents. Adequate measures should be taken to avoid parents feeling obliged to participate, rather than giving true informed consent.     

Influence of coronary collateral vessels on myocardial infarct size in humans. Results of phase I thrombolysis in myocardial infarction (TIMI) trial. The TIMI Investigators

BACKGROUND. The influence of coronary collateral vessels on infarct size in humans remains controversial, partly because no previous study has examined the impact of collaterals present at the onset of acute myocardial infarction on infarct size. METHODS AND RESULTS. The present study used the data base of the Thrombolysis in Myocardial Infarction (TIMI) Phase I trial to correlate the presence or absence of angiographically documented collaterals in the initial hours of myocardial infarct evolution with the size of the infarct as assessed by serial measurements of serum creatine kinase (CK). To avoid the confounding effects of reperfusion on enzymatic estimates of infarct size, this report is limited to those 125 patients who failed to recanalize at 90 minutes after administration of tissue plasminogen activator or streptokinase. Patients with angiographically documented collaterals (group A, n = 51) had significantly lower values of peak serum CK than patients without collaterals (group B, n = 74) (1,877 +/- 216 versus 2,661 +/- 212 IU/l, respectively [mean +/- SEM], p = 0.004). Similarly, CK-derived infarct size estimates were significantly lower in group A than in group B (20.6 +/- 2.5 versus 31.4 +/- 2.8 CK gram equivalents, p = 0.001). The infarct size observed in patients with collaterals was less for anterior infarctions as well as for infarctions of other locations; thus, the beneficial effects of collaterals were independent of the site of the infarct. In 65 of the 125 patients who failed to reperfuse, left ventricular ejection fraction (LVEF) was assessed by contrast ventriculography both at initial cardiac catheterization (before thrombolytic therapy) and at hospital discharge. Among the patients who had both studies, global LVEF tended to increase from pretreatment to hospital discharge in group A (from 50.6 +/- 1.8% to 53.4 +/- 1.8%, p = 0.10) but decreased in group B patients (from 50.3 +/- 1.8% to 47.8 +/- 1.7%, p = 0.02). At hospital discharge, global LVEF was greater in patients with coronary collaterals (53.5 +/- 1.7% versus 49.6 +/- 1.7%, p = 0.01). CONCLUSIONS. The results demonstrate that, in patients in whom thrombolytic therapy fails to induce reperfusion, the presence of coronary collateral vessels at the onset of myocardial infarction is associated with limitation of infarct size as assessed enzymatically and with improved ventricular function on discharge as assessed by LVEF.     

End-systolic radius to thickness ratio: an echocardiographic index of regional performance during reversible myocardial ischemia in the conscious dog

Regional myocardial dysfunction induced by ischemia is associated with less thickening and a larger ventricular radius at end-systole. Thus, end-systolic radius to thickness ratio measured by echocardiography may provide an accurate index of regional left ventricular function that is totally independent of cardiac motion. To test this hypothesis, a total of 14 transient (less than or equal to 10 minutes) coronary artery occlusions (8 left anterior descending, 6 left circumflex) followed by up to 24 hours of reperfusion were performed in six chronically instrumented conscious dogs providing multiple grades of regional ventricular dysfunction. Regional myocardial thickening fraction was determined with epicardial pulsed Doppler probes and served as an independent standard for comparison with simultaneous echocardiographic measurements. End-systolic radius to thickness ratio and radial shortening fraction were derived from the two-dimensional echocardiographic short-axis view along 12 equidistant radii. In the ischemic zone, percent thickening fraction averaged 22 +/- 5% during baseline, decreased to -4 +/- 4% during occlusion with gradual return to baseline after reperfusion. End-systolic radius to thickness ratio averaged 1.39 +/- 0.25 before coronary occlusion and increased to 2.97 +/- 0.48 during occlusion with a gradual return to baseline values. A significant correlation was found between Doppler-determined thickening fraction measurements and echocardiographic end-systolic radius to thickness ratio as well as radial shortening fraction for absolute values (r = -0.83 and 0.75, respectively; n = 65) and percent change from baseline (r = -0.86 and 0.78, respectively).(ABSTRACT TRUNCATED AT 250 WORDS)     

Long-term recovery from unawareness,deficient counterregulation and lack of cognitive dysfunction during hypoglycaemia,following institution of rational,intensive insulin therapy in IDDM

Summary Hypoglycaemia unawareness, is a major risk factor for severe hypoglycaemia and a contraindication to the therapeutic goal of near-normoglycaemia in IDDM. We tested two hypotheses, first, that hypoglycaemia unawareness is reversible as long as hypoglycaemia is meticulously prevented by careful intensive insulin therapy in patients with short and long IDDM duration, and that such a result can be maintained long-term. Second, that intensive insulin therapy which strictly prevents hypoglycaemia, can maintain long-term near-normoglycaemia. We studied 21 IDDM patients with hypoglycaemia unawareness and frequent mild/severe hypoglycaemia episodes while on conventional insulin therapy, and 20 nondiabetic control subjects. Neuroendocrine and symptom responses, and deterioration in cognitive function were assessed in a stepped hypoglycaemia clamp before, and again after 2 weeks, 3 months and 1 year of either intensive insulin therapy which meticulously prevented hypoglycaemia (based on physiologic insulin replacement and continuous education, experimental group, EXP, n=16), or maintenance of the original conventional therapy (control group, CON, n=5). At entry to the study, all 21 IDDM-patients had subnormal neuroendocrine and symptom responses, and less deterioration of cognitive function during hypoglycaemia. After intensive insulin therapy in EXP, the frequency of hypoglycaemia decreased from 0.5±0.05 to 0.045±0.02 episodes/patient-day; HbA_1C increased from 5.83±0.18 to 6.94±0.13% (range in non-diabetic subjects 3.8–5.5%) over a 1-year period; all counterregulatory hormone and symptom responses to hypoglycaemia improved between 2 weeks and 3 months, with the exception of glucagon which improved at 1 year; and cognitive function deteriorated further as early as 2 weeks (p<0.05). The improvement in responses was maintained at 1 year. The improvement in plasma adrenaline and symptom responses inversely correlated with IDDM duration. In contrast, in CON, neither frequency of hypoglycaemia, nor neuroendocrine responses to hypoglycaemia improved. Thus, meticulous prevention of hypoglycaemia by intensive insulin therapy reverses hypoglycaemia unawareness even in patients with long-term IDDM, and is compatible with long-term near-normoglycaemia. Because carefully conducted intensive insulin therapy reduces, not increases the frequency of moderate/severe hypoglycaemia, intensive insulin therapy should be extended to the majority of IDDM patients in whom it is desirable to prevent/delay the onset/progression of microvascular complications.Abbreviations IDDM Insulin-dependent diabetes mellitus - IIT intensive insulin therapy - HU hypoglycaemia unawareness - EXP experimental patient group - CON control group