EWS-FLI1 fusion transcripts identified in patients with typical neuroblastoma.

The t(11.22)(q24.q12) results in expression of a chimeric RNA product, EWS-FLI1. This RNA product is expressed in over 85% of tumours belonging to the Ewing's family, and is increasingly used as a definitive characteristic of these tumours. In this study, we evaluated reverse transcriptase-polymerase chain (RT-PCR) for EWS-FLI1 fusion transcripts in 18 neurally derived small round cell tumours. These included six Ewing's family tumours and 12 neuroblastomas. EWS-FLI1 fusion transcripts were identified in all six Ewing's tumours, but also in two of the 12 neuroblastomas. One neuroblastoma contained the classic type 1 fusion transcript, and the second a type 1 transcript containing a 66 bp (base pair) insert that was not derived from the EWS or FLI1 gene. The presence of EWS-FLI1 fusion products in RNA extracted from primary neuroblastoma suggests the identification of EWS-FLI1 fusion transcripts is not pathognomonic for tumours of the Ewing's family. The clinical significance of these fusion transcripts in neuroblastoma is not known.

A combined cytogenetic and molecular approach to diagnosis in a case of desmoplastic small round cell tumor with a complex translocation (11;22;21).

Desmoplastic small round cell tumor (DSRCT) has recently been described as a discrete tumor entity. It is distinguished from other small round cell tumors by its prominent desmoplastic quality, its preponderance in adolescent males, its almost exclusive intraabdominal location, a multi-immunophenotypic profile, and its aggressive nature. Diagnosis on histology alone is not always unequivocal. A recurrent t(11;22)(p13;q12) translocation has recently been described in this tumor, and a chimeric RNA fusion product formed from the WT1 and EWS genes is detectable by reverse transcriptase-polymerase chain reaction (RT-PCR). We describe the use of a multi-faceted approach using conventional G-banding, fluorescence in situ hybridization (FISH) and RT-PCR to assist the diagnosis of a case of DSRCT with a complex variant t(11;22;21)(p13;q12;q22.1) translocation and demonstrate the value of a combined approach to genetic investigation of solid tumors.

Pharmacokinetics of prednisolone in children with acute lymphoblastic leukaemia.

The pharmacokinetics of soluble oral prednisolone were studied during induction therapy in six children with acute lymphoblastic leukaemia. There was a three- to four-fold variation in the pharmacokinetics of total and free prednisolone. For total prednisolone, the mean elimination half-life was relatively short (1.37 h) and the total clearance, relatively high (15.1 ml min-1 kg-1). The mean free fraction was high (0.37).

The effect of controlled daily blood loss on the haemoglobin concentration, erythrocyte count and reticulocyte count of male rats.

0.2, 0.6 or 1 ml of blood was removed from rats once daily on 17 consecutive days. Haemoglobin concentration and erythrocyte counts fell rapidly until day 7 or 9 and were reflected after day 2 by an increase in reticulocytes. From 9 to 17 there was little further change and 14 days after the last bleed the haematological results were within the normal range. The increase in haemoglobin production correlated well with the increase in reticulocyte numbers.

The separation of unbound prednisolone in plasma by centrifugal ultrafiltration.

To study variable plasma protein binding of prednisolone in children with nephrotic syndrome we have devised a simple rapid method for measuring unbound prednisolone. The plasma was initially ultrafiltered at 37 degrees C fixed angle head at 1500 g for 30 min then the filtrate was analysed by high pressure liquid chromatography. The effects of variable ultrafiltration conditions were studied. The method was used to compare the AUC unbound prednisolone in a nephrotic child (plasma albumin concentration 18 g L-1) with a control (plasma albumin concentration 43 g L-1); their respective AUC unbound prednisolone values were 4.02 mg h L-1 and 1.07 mg h L-1.

Arterial elastic modulus for fixed and varied wall volume.

It is commonly assumed that a blood vessel maintains a constant wall volume over a wide range of intraluminal pressures. We have found that vessel wall volume decreases under increasing load. To determine the effect of decreasing wall volume on values of Elastic modulus (Em), two computer models were used. The first model held volume constant with measured values of rabbit carotid artery length and width while thickness was calculated. In the second model wall volume was allowed to vary with measured length, width and thickness. Em increased with increasing load in both models with higher values in the nonconstant volume model. Up to 30% loading, the difference was 11% or less; above 30% loading, the difference increased to over 100%.

Dynamic intra-vascular pressure harmonic analysis.

The postulate that dynamic intra vascular pressure is a function of positional wall properties has been difficult to verify due to non-linear viscoelastic influences in arteries. It was tested, in rabbit carotid artery in vitro segments using dynamic and static input. A Multifunction Pressure Generator (MPG, Millar Inc.) provide dynamic input of defined swept frequency (2-200 Hz) pressure superimposed on multiple levels of mean intraluminal pressure (IP). Bode plots were analyzed for natural frequency (omega n), gain (dB) and damping (zeta) of the IP/MPG transfer function. An increase in omega n (all zeta > 0.25) was found with increase in mean IP. An increase in segment Elastic modulus (Em) was found with increase in static force input. Relation between Em and omega n results was demonstrative of non-linear elasticity. Results supported the postulate and verified estimating vessel properties by harmonic analysis.

The tubular maximum for calcium reabsorption: a normal range in children.

OBJECTIVE: We aimed to establish a normal range for the tubular maximum rate of reabsorption of calcium corrected for glomerular filtration rate. DESIGN: A prospective survey was used. PATIENTS: One hundred and ten normal children aged 2-14 years were studied. MEASUREMENTS: Total plasma calcium, ultrafiltrable calcium and parathyroid hormone. Urinary calcium, sodium and creatinine excretion. RESULTS: The normal range was found to be 1.87-3.39 mmol/l of glomerular filtrate with a geometric mean value of 2.40 mmol/l. There was a significant inverse relationship between the tubular maximum for calcium and the urinary sodium excretion. No significant relationship was found between the tubular maximum for calcium and the level of parathyroid hormone in this group of normal children. CONCLUSIONS: The normal range seen in children is higher than that seen in adults with a higher mean value. This normal range should be useful in the assessment of renal calcium handling in children with disorders of calcium homeostasis.

Intra-arterial pressure wave parameters modeled using electrical analogs.

An Electrical Model was developed to help identify parameters obtained from dynamic pressure data on the in vitro rat aortic artery. The data was obtained using a Multifunction Pressure Generator (MPG) and recording MPG Input Pressure (Pi) and Intraarterial Pressure (Po). Transfer functions of the form Po/Pi = (A1S+Ao)/(B2S2 + B1S+Bo) were obtained and it is necessary to link A1, Ao, B2, B1 and Bo to the Biological Parameters of Inertance (M), Vascular Resistance (R) and Compliance (C). Using the Electrical Analogs to P, M, R and C which are Voltage (V), Inductance (L), Resistance (Re), and Capacitance (Ce), an Electrical Model was built. The Electrical Model has the form Vo/Vi = (Re1S + 1/Ce)/[LS2 + (Re1 + Re2)S + 1/Ce]. Since Ao = Bo = 1 from our experimental data we multiplied the denominator and numerator by Ce to obtain Vo/Vi = (CeRe1S + 1)/[CeLS2 + Ce(Re1 + Re2)S + 1]. We then transformed our Electrical Model to its Pressure Equivalent and obtained Po/Pi = (CR2S + 1)/[CMS2 + C(R1 + R2)S + 1]. Since R2 is less than R1 + R2 we theorize that total R is composed of two viscoelastic or resistive elements R1 and R2. Using measured values of compliance it should be possible to obtain reasonable values for R1, R2 and Inertance.

Indices of intact serum parathyroid hormone and renal excretion of calcium, phosphate, and magnesium.

Up to date reference ranges were established for fasting renal excretion of calcium, phosphorus, and magnesium on 101 healthy children aged 2-15 years. A normal range for intact parathyroid hormone was also measured. The indices of calcium and magnesium excretion showed no correlation with age or sex so that a common range for all children could be established. The 97th centile values for urinary calcium:creatinine and magnesium:creatinine ratios were 0.69 mmol:mmol and 1.05 mmol:mmol respectively. The calculated tubular maximum for phosphate/litre of glomerular filtrate (TmPO4/GFR) showed no correlation with age with a geometric mean value of 1.67 mmol/l. The normal range for intact serum parathyroid hormone for the age group was 11-35 ng/l, which is lower than the adult normal range using the same assay. There was an inverse correlation between TmPO4/GFR and intact parathyroid hormone in this group of normal children.

Modular data acquisition system updated using LabWindows/CVI Graphical User Interface.

The Biomechanics Laboratory of the Neuroscience Department of the Medical College of Wisconsin is currently engaged in research involving trauma biomechanics. For some experiments, 24 channels of analog data must be sampled at 10,000 Hertz. The Modular Data Acquisition System (MDAS) is able to acquire up to 60 channels of analog data at sampling intervals as low as 6 microseconds. This excellent hardware system has only menu-driven utility software that is no longer supported and required updating to a Graphical User Interface (GUI). Using National Instruments LabWindows/CVI software a GUI was developed. The GUI consists of 9 Graphical User Interface panels controlled by a 3000 line C program "MDAS3SAM". "MDAS3SAM" controls a driver program "MDAS_SAM" which communicates with the MDAS unit via a National Instruments GPIB interface. The 9 GUI panels allow the user to configure the MDAS system (selecting channels, sampling interval, triggering levels etc.), start sampling the data, writing the data to hard disk, graphing the data and printing the graphs. The new system allows the user to quickly reconfigure the MDAS unit and obtain accurate results.

Progressive renal toxicity due to ifosfamide.

A prospective and follow up study of renal tubular and glomerular function in 11 children receiving ifosfamide treatment was conducted. Each child received between four and 14 courses of ifosfamide, given as a continuous infusion of 3 g/m2 over 24 hours for two or three days. Evidence of renal toxicity was seen in all patients. There was a treatment related rise in urinary tubular markers (N-acetyl-glucosaminidase and alpha 1 microglobulin). Recovery was limited, so that by the fourth course of treatment all values remained abnormal. There was an associated treatment related reduction in plasma phosphate concentration. Urinary albumin also showed a treatment related rise, but with fewer abnormal values. Electrophoresis was used to confirm tubular or glomerular patterns. Glomerular toxicity was less severe and occurred in fewer patients. The follow up study showed persistence of tubular damage in all seven patients examined, and there was evidence of glomerular damage in five of the seven children. Children receiving ifosfamide need to be carefully monitored for renal toxicity both during treatment and at follow up.

Finite element model of human cervical spinal column.

The purpose of this study was to develop a detailed anatomically accurate finite element model (FEM) of the whole cervical column (C2-T1) using sagittal and coronal computed tomography (CT) scan images and cryomicrotome anatomical sections. The bony vertebrae were defined using CT scan images. The geometrical details of intervertebral discs, uncovertebral joints and ligaments were obtained from cryomicrotome sections. The following steps were used to develop the FEM: wire mesh, surface model and solid model. The wire mesh of each vertebra and disc was generated from the CT and cryomicrotome sections, respectively. The surface model was developed by connecting the wire mesh lines. The solid model was obtained by filling the volume enclosed by the surface model. Finally, the FEM was constructed by discretizing the solid model using the mapped-mesh option. Appropriate finite element types were assigned to each component. For example, the isoparametric eight-noded solid elements were used to define the cancellous bone of a vertebra. The material properties reported in literature were adopted in the model. Commercially available software (IDEAS and ABAQUS) was used to develop the FEM.

Pharmacokinetics of prednisolone in children with nephrosis.

The pharmacokinetics of prednisolone given intravenously were studied in 11 children with relapsed steroid responsive nephrotic syndrome, and four control subjects. The clearance of both total and unbound drug was decreased in these children and the unbound fraction of the drug in plasma was significantly correlated with the degree of hypoalbuminaemia. We conclude that changes in the clearance of prednisolone and altered protein binding might account for some of the variability in both therapeutic responses and the incidence of toxicity in patients treated with standard dosage regimens.

Thalidomide treatment for chronic graft-versus-host disease.

The treatment of chronic graft-versus-host disease (GVHD) may present a difficult therapeutic problem. We used thalidomide to treat six patients with severe chronic GVHD who failed to respond to standard immunosuppressive agents. Four of the six patients showed a clear response to thalidomide, with the fifth patient showing a partial response. The best results were seen in patients with chronic cutaneous GVHD. Two of the patients developed neurophysiological evidence of a peripheral neuropathy, associated with clinical signs in one patient. Measurement of thalidomide plasma levels and pharmacokinetic curves showed a significant inter-patient variation. Peak plasma levels varied from 0.47 to 1.46 micrograms/ml. Thalidomide has a role to play in the management of chronic GVHD and further studies are needed.

Effect of timing of cefuroxime dosage on its protection of rats against gentamicin nephrotoxicity.

The neophrotoxic effect on rats of once daily treatment with 40 or 45 mg gentamicin/kg/day for ten days was substantially reduced by administration of 4 g cefuroxime/kg/day, either at the same time or eight hours later. This dosage of cefuroxime was protective when given for only two consecutive days starting on the first to third days of gentamicin treatment, but enhanced gentamicin nephrotoxocity when started on the sixth or subsequent days.

Detection of colorectal cancer cells in peripheral blood by reverse-transcriptase polymerase chain reaction for cytokeratin 20.

The staging of colorectal cancer currently depends on pathological examination of the surgical specimen and regional lymph nodes, accompanied by imaging tests such as computed tomography (CT) scanning. However, alternative molecular methods to detect circulating tumour cells in blood or bone marrow may provide additional information about the extent of disease and prognosis. We have previously reported the development of a reverse-transcriptase polymerase chain reaction (RT-PCR) for cytokeratin 20 (CK 20) mRNA to detect circulating epithelial tumour cells. In this study, we report on the application of this method for detecting circulating tumour cells in patients with colorectal cancer. Using this method, CK 20 mRNA was detected in 8/8 human colorectal cancer cell lines, in 8/9 biopsies from primary colorectal tumours and in 9/10 biopsies of liver metastasis in patients with metastatic colorectal cancer, suggesting that CK 20 may be a useful target for the detection of circulating tumour cells in this patient group. In spiking experiments, 10 cells were consistently identified in 2 ml of whole blood (1 x 10(6)-1 x 10(7) mononuclear cells). In 12/25 (48%) peripheral blood samples from patients with known metastatic colorectal cancer, CK 20 mRNA was detected. However, there was no correlation between the detection of CK 20 mRNA in the peripheral blood and disease progression and survival in this group of patients. CK 20 mRNA was detected in 1/12 normal blood samples, which raises questions about the absolute specificity of CK 20 expression.