Genome complexity in acute lymphoblastic leukemia is revealed by array-based comparative genomic hybridization.

Chromosomal abnormalities are important for the classification and risk stratification of patients with acute lymphoblastic leukemia (ALL). However, approximately 30% of childhood and 50% of adult patients lack abnormalities with clinical relevance. Here, we describe the use of array-based comparative genomic hybridization (aCGH) to identify copy number alterations (CNA) in 58 ALL patients. CNA were identified in 83% of cases, and most frequently involved chromosomes 21 (n=42), 9 (n=21), 6 (n=16), 12 (n=11), 15 (n=11), 8 (n=10) and 17 (n=10). Deletions of 6q (del(6q)) were heterogeneous in size, in agreement with previous data, demonstrating the sensitivity of aCGH to measure CNA. Although 9p deletions showed considerable variability in both the extent and location, all encompassed the CDKN2A locus. Six patients showed del(12p), with a common region encompassing the ETV6 gene. Complex CNA were observed involving chromosomes 6 (n=2), 15 (n=2) and 21 (n=11) with multiple regions of loss and gain along each chromosome. Chromosome 21 CNA shared a common region of gain, with associated subtelomeric deletions. Other recurrent findings included dim(13q), dim(16q) and enh(17q). This is the first report of genome-wide detection of CNA in ALL patients using aCGH, and it has demonstrated a higher level of karyotype complexity than anticipated from conventional cytogenetic analysis.

Chk1-dependent slowing of S-phase progression protects DT40 B-lymphoma cells against killing by the nucleoside analogue 5-fluorouracil.

Chk1 plays a crucial role in the DNA damage and replication checkpoints in vertebrates and may therefore be an important determinant of tumour cell responses to genotoxic anticancer drugs. To evaluate this concept we compared the effects of the nucleoside analogue 5-fluorouracil (5FU) on cell cycle progression and clonogenic survival in DT40 B-lymphoma cells with an isogenic mutant derivative in which Chk1 function was ablated by gene targeting. We show that 5FU activates Chk1 in wild-type DT40 cells and that 5FU-treated cells accumulate in the S phase of the cell cycle due to slowing of the overall rate of DNA replication. In marked contrast, Chk1-deficient DT40 cells fail to slow DNA replication upon initial exposure to 5FU, despite equivalent inhibition of the target enzyme thymidylate synthase, and instead accumulate progressively in the G1 phase of the following cell cycle. This G1 accumulation cannot be reversed rapidly by exogenous thymidine or removal of 5FU, and is associated with increased incorporation of 5FU into genomic DNA and severely diminished clonogenic survival. Taken together, these results demonstrate that a Chk1-dependent replication checkpoint which slows S phase progression can protect tumour cells against the cytotoxic effects of 5FU.

Derivative chromosome 9 deletions are a significant feature of childhood Philadelphia chromosome positive acute lymphoblastic leukaemia.

Deletions from the derivative chromosome 9, der(9), of the translocation, t(9;22)(q34;q11), at the site of the ABL/BCR fusion gene, have been demonstrated by fluorescence in situ hybridisation (FISH), in both Philadelphia chromosome (Ph)-positive chronic myeloid leukaemia (CML) and acute lymphoblastic leukaemia (ALL). In CML they occur in 10-15% of cases and appear to indicate a worse prognosis, whereas in ALL, the situation is unclear. This study presents the findings of dual fusion FISH used to detect such deletions in a series of 27 BCR/ ABL-positive childhood ALL patients. Metaphase FISH was essential for the accurate interpretation of interphase FISH signal patterns. Three cases (11%) had a single fusion signal, resulting from deletions of the der(9). Three other patients with variant translocations and one with an insertion, also had a single fusion, but with no evidence of deletions. Gain of a fusion in approximately one-third of patients indicated a second Ph, which appears to be a diagnostic marker of Ph-positive ALL. This study shows that the incidence of deletions from the der(9) in childhood ALL is at least as high as that reported for CML.

Duty-related stressors and PTSD symptoms in suburban police officers.

This study examined the effects of duty-related stress on police officers. Using a sample of 100 suburban police officers, an anonymous questionnaire requested demographic information and included a measure of duty-related stressors, SCL-90-R, the Posttraumatic Stress Disorder scale of the Impact of Events Scale-Revised, and a locus of control scale. Also assessed was whether Critical Incident Stress Debriefing was experienced. The results showed significant correlations between scores on duty-related stress, somatization, and symptoms of PTSD. 13% of the sample met the DSM-IV (1994) diagnostic criteria for PTSD. Results of the regression analysis showed the best predictors for the diagnosis of PTSD were associated with the factor of Exposure to Death and Life Threat, which corresponds to the DSM-IV AI criteria. Finally, 63% of the respondents stated that a critical incident debriefing would be beneficial following an extremely stressful event related to duty.

Ultrastructure of the islets of Langerhans in protein-energy malnutrition.

Significant hormonal changes have been reported in childhood malnutrition, including high serum levels of growth hormone and cortisol, and low levels of circulating insulin. The ultrastructure of the endocrine pancreas in such patients has hitherto not been reported. A light microscopy survey of the pancreatic islets was carried out on 69 malnourished children dying from protein-energy malnutrition. In seven of these cases, a rapid autopsy protocol allowed tissues to be fixed for electron microscopy within 75 minutes of death. This paper presents the first ultrastructural observations on the Islets of Langerhans in childhood protein-energy malnutrition. In all cases, there was a variable degree of degeneration of all cell types with membrane damage, loss of ribosomes, vesiculation and mitochondrial swelling. In addition, the B-cells showed a high proportion of precursor granules compared to crystal forms, possibly accounting for low insulin serum levels reported by other workers. It is suggested that islet cell changes may be related to free radical damage secondary to depletion of glutathione and other antioxidants, as well as relative deficiencies of cysteine and zinc. In addition, the effects of agonal anoxia, and a short fixation delay after death must be considered.

The effect of malnutrition on insulin binding to rat erythrocytes.

Insulin binding to erythrocyte receptors was compared in malnourished and control rats. Percentage specific insulin binding to malnourished rat erythrocytes was significantly lower than to control erythrocytes (P less than 0.001). The low insulin binding in the malnourished rat erythrocytes was accompanied by low insulin receptor affinity (P = 0.035).

The effect of DPT inoculation on the hormonal control of glucose homeostasis in children recovered from malnutrition.

The effect of a controlled stress (DPT inoculation) on the hormonal control of glucose homeostasis was investigated in children nutritionally rehabilitated from severe malnutrition. The age range of the 15 children studied was 6-26 months. Plasma insulin (INS), growth hormone (GH) and interleukin-1 (IL-1) were measured by radioimmunoassay; plasma glucose (GLU) by a glucose oxidase method; and red cell insulin binding (%SB) was determined, using A-14 monoiodinated insulin. Measurements were made on two occasions: (T-0) at 10 a.m., 12 hr before DPT inoculation, and (T-36) 36 hr. after inoculation. On both occasions, 4 hr post-prandial blood samples were used, and the mean body temperature (T) on the day of the test was determined. Red cell insulin binding (%SB) was significantly higher at T-36 than at T-0 (16.8 +/- 1.7 vs 12.1 +/- 1.2 (14), p = 0.005). (Results were expressed as mean +/- SEM, numbers of paired observations in parentheses). The higher %SB after DPT was accompanied by an increase in the number of receptor sites (S) (29.05 +/- 6.5 vs 15.6 +/- 2.5 (14), p = 0.025). However, insulin receptor affinity (K x 10(9) M-1) was decreased (0.7 +/- 0.1 vs 1.5 +/- 0.3 (14), p = 0.008). There were no significant differences in the plasma levels of insulin, glucose and interleukin-1, but plasma growth hormone (microU/ml) was increased after DPT, (18.0 +/- 3.0 vs. 11.5 +/- 1.2 (13), p = 0.04). Body temperature (degree C) was also significantly increased after DPT, (99.6 +/- 0.4 vs. 98.3 +/- 0.2 (14), p = 0.006).(ABSTRACT TRUNCATED AT 250 WORDS)

The effect of DPT inoculation on the hormonal control of glucose homeostatis in children recovered from malnutrition

The effect of a controlled stress (DPT inoculation) on the hormonal control of glucose homeostasis was investigated in children nutritionally rehabilitated from severe malnutrition. The age range of the 15 children studied was 6-26 months. Plasma insulin (INS), growth hormone (GH) and interleukin-1 (IL-1) were measured by radioimmunoassay; plasma glucose (GLU) by a glucoseoxidase method; and red cell insulin binding (%SB) was determined, using A-14 monoiodinated insulin. Measurements were made on two occasions: (T-O) at 10 a.m.,12 hr before DPT inoculation, and (T-36) 36 hr. after inoculation. On both occasions, 4 hr post-prandial blood samples were used, and the mean body temperature(T) on the day of the test was determined. Red cell insulin binding (%SB) was significantly higher at T-36 than at T-O (16.8 ñ 1.7 vs 12.1 ñ 1.2 (14), p=0.005). (Results were expressed as mean ñ SEM, numbers of paired observations in parentheses). The higher %SB after DPT was accompanied by an increase in the number of receptor sites (S) (29.05 ñ 6.5 vs 15.6 ñ 2.5 (14),p=0.025). However, insulin receptor affinity (K x 10(9)M(-1)) was decreased 0.7 ñ 0.1 vs 1.5 ñ 0.3(14), p=0.008). There were no significant differences in the plasma levels of insulin, glucose and interleukin-1, but plasma growth hormone (*U/ml) was increased after DPT, (18.0 ñ 3.0 vs 11.5 ñ 1.2 (13), p=0.04). Body temperature (-C) was also significantly increased after DPT,(99.9 ñ 0.4 vs 98.3 ñ 0.2(14), p=0.006). The change in plasma glucose from T-O to T-36 tended to be associated with both a change in plasma insulin (p=0.06) and plasma growth hormone (p=0.07). Increased insulin binding, as one index of increased insulin sensitivity during fever, can contribute to a reductionin blood glucose. However, the elevation in plasma growth hormone cold buffer the hypoglycaemic effect of insulin, and help to maintain glucose homeostasis

Changes in red cell insulin receptors during recovery from severe malnutrition.

Red cell insulin binding was studied in 13 Jamaican children (age range 4-24 months), while malnourished (MAL), during early recovery (GI), late recovery (GII), and after anthropometric recovery (REC). The rate of weight gain (RW), the energy intake (EN), and the protein intake (PR) were monitored at each phase of the study. Four-hour fasting blood samples were used, and the insulin binding characteristics were investigated in the physiological range of insulin concentrations (16.7-1670 pM). Analyses of variance were used to examine differences in the variables measured at the four phases. Red cell-specific insulin binding (SB) was lower in MAL than in GI (P less than 0.001) and in (GII) (P = 0.026). SB in REC and MAL were not significantly different. Insulin receptor affinity (K) was also lower in MAL than in GI (P less than 0.001). GII (P = 0.001), and REC (P = 0.012). The insulin receptor number (S) appeared to be high in malnutrition and to decrease as recovery progressed; however the decrease was not significant. Children with fever demonstrated high insulin binding. Plasma insulin (IN) rose during recovery, and was significantly higher in GII than in MAL (P = 0.01). There was no difference in plasma glucose (G) at any phase of the study. The interrelationships among the variables measured were investigated longitudinally using multiple regression analyses, SB was positively associated with S (P = 0.032), EN (P = 0.029), and PR (P = 0.0076). S was negatively associated with K (P less than 0.001). The associations of S and K with PR were positive and approached significance (P = 0.09 and P = 0.07 respectively). RW was positively associated with PR (P less than 0.001), and with EN (P = 0.001). There were no significant relationships between G and any of the other variables longitudinally. However, correlations of the variables within phases demonstrated that in MAL, G was negatively associated with SB (P less than 0.05) and with K (P less than 0.05); but in REC, G was positively associated with SB (P less than 0.05). These results demonstrated that in severe malnutrition, the red cell insulin receptor affinity was low. During catch-up growth when protein and energy intakes were increased, both insulin receptor affinity and specific insulin binding were also increased. The negative relationship between insulin binding and plasma glucose during malnutrition may be related to carbohydrate intolerance.

Insulin receptor studies of erythrocytes and mononuclear leucocytes in phasic insulin diabetes mellitus.

This study was designed to investigate any differences in cellular binding of insulin between phasic insulin-dependent (malnutrition-related) diabetes mellitus (PIDDM) and insulin-dependent, non-insulin-dependent, and normal controls. Isolated, washed red and white blood cells obtained after 12-14 hr fast, were separately incubated with varying concentrations of non-radioactive insulin, and a fixed quantity of radioactively labelled insulin. After the 3-hr incubation, cells were washed with buffer, and radioactivity determined on an autogamma counter. Percentage binding, receptor sites number and affinity were all determined by linear regression of the Scatchard plot. Fasting plasma insulin and glucose levels were also assayed. The results obtained show decreased binding of insulin in red blood cells [11.3 +/- 1.3%) and white blood cells 2.9 +/- 0.5%) in PIDDM. This was due to decreased receptor sites (red blood cells 39 +/- 11; white blood cells 0.5 +/- 0.11 x 10(4] as well as decreased affinity (red blood cells 0.14 +/- 0.03 x 10(9) M-1; white blood cells 0.17 +/- 0.04 x 10(9) M-1) when compared to the normal and diabetic (both insulin and non-insulin-dependent) controls. Phasic insulin-dependent diabetes (malnutrition-related diabetes mellitus) is characterized by decreased red and white cellular binding to insulin, in addition to decreased production of insulin.

Insulin receptor studies of erythrocytes and mononuclear leucocytes in phasic insulin diabetes mellitus

This study was designed to investigate any diferences in cellular binding of insulin between phasic insulin-dependent (malnutrition-related) diabetes mellitus (PIDDM) and insulin-dependent, non-insulin-dependent, and normal controls. Isolated, washed red and white blood cells obtained after 12 - 14hr fast, were separately incubated with varying concentrations of non-radioactive insulin, and a fixed quantity of radioactively labellede insulin. After the 3hr incubation, cells were washed with buffer, and radioactivity determined on an autogamma counter. Percentage binding, receptor sites number and affinity were all determined by linear regression of the Scathard plot. Fasting plasma insulin and glucose levels were were also assayed. The results obtained showed decreased binding of insulin in red blood cells (11.3+or -1.3%) and white blood cells 2.9 + or -o.5%) in PIDDM. This was due to decreased receptor sites (red blood cells 39+ or -11; white blood cells 0.5+ or -0.11x 10 to the 4th) as well as decreased affinity (red blood cells 0.14+ or -0.03 x 10 to the 9th M-1; white blood cells 0.17 + or -0.04 x10 to the 9th M-1) when compared to the normal and diabetic (both insulin and non-insulin-dependent) controls. Phasic insulin-dependent diabetes (malnutition-related diabetes mellitus) is characterized by decreased red and white cellular binding to insiulin, in addition to decreased production of insulin

Plasma somatomedin-C in Nigerian malnourished children fed a vegetable protein rehabilitation diet.

Plasma somatomedin-C (pSm-C) was measured by immunoassay in Nigerian malnourished children treated with a mainly vegetable diet. In oedematous children, the mean intake was 4.31 +/- 0.23 g protein and 611 +/- 46 kJ per kg body weight per day, and in marasmic children 5.22 +/- 0.62 g protein and 795 +/- 131 kJ/kg body weight/d. PSm-C concentration (U/ml) was measured at weekly intervals to determine the response to this rehabilitation diet. By our assay the value for 39 normal children (age range 6-36 months) was 0.315 +/- 0.035 U/ml. The average initial level of pSm-C in the malnourished children was 0.19 +/- 0.03 (n = 24). The values were higher (P less than 0.05) in the 7 marasmic children (0.26 +/- 0.1) than in the 11 with oedema (0.15 +/- 0.02). Eight days after admission pSm-C had risen to 0.20 +/- 0.02 (n = 24) and at discharge after approximately 19 d, pSm-C concentration was normal, 0.30 +/- 0.05. In oedematous malnutrition, pSm-C level at discharge was lower than in marasmus, 0.27 +/- 0.06 (n = 17) compared with 0.37 +/- 0.06 (n = 7) (P less than 0.05). Because the childrens' stay in hospital was short (average 19 d), they were far from attaining normal weight for height by the time of discharge. However, they had gained on average 0.9 kg and their clinical condition was satisfactory. It is concluded that the vegetable-based diet produced satisfactory recovery, at least in the initial stages. Increases in pSm-C compared well with those found in an earlier study with milk-based diets.

Free and total triiodothyronine and thyroxine in malnourished Jamaican infants. The effect of diet on plasma levels of thyroid hormones, insulin and glucose during recovery.

Plasma concentrations of free triiodothyronine (FT3), total triiodothyronine (TT3) and total thyroxine (TT4) were reduced to 63.0, 37.7, 61.7 per cent of controls respectively in protein-energy malnutrition (PEM), while free thyroxine (FT4) was elevated by 23 per cent. There was a gradual increase of both TT4 and TT3 during recovery. The ratio of free to bound hormones was high in malnutrition and declined with recovery, indicating a deficiency of thyroid-hormone binding in malnutrition. The observation of a significant reduction (P less than 0.05) in T3/T4 ratios, which occurred in malnutrition and was induced during recovery after 3 d on a low energy maintenance diet, suggested depressed conversion of T4 to T3 due to energy restriction. Energy restriction also significantly (P less than 0.001) depressed plasma insulin concentrations in the presence of nearly constant plasma glucose levels.