Pharmacological inhibition of phosphatidylcholine biosynthesis is associated with induction of phosphatidylinositol accumulation and cytolysis of neoplastic cell lines.

De novo production of phosphatidic acid (PA) in tumor cells is required for phospholipid biosynthesis and growth of tumor cells. In addition, PA production by phospholipase D has been cited among the effects of certain oncogenes and growth factors. In this report, it has been demonstrated that enhanced phospholipid metabolism through PA in tumor cells can be exploited pharmacologically for development of anticancer agents, such as CT-2584, a cancer chemotherapeutic drug candidate currently in Phase II clinical trials. By inhibiting CTP:choline-phosphate cytidylyltransferase (CT), CT-2584 caused de novo phospholipid biosynthesis via PA to be shunted away from phosphatidylcholine (PC) and into phosphatidylinositol (PI), the latter of which was doubled in a variety of CT-2584-treated tumor cell lines. In contrast, cytotoxic concentrations of cisplatin did not induce accumulation of PI, indicating that PI elevation by CT-2584 was not a general consequence of chemotherapy-induced cell death. Consistent with this mechanism of action, propranolol, an inhibitor of PA phosphohydrolase and phosphatidylcholine biosynthesis, was also cytotoxic to tumor cell lines, induced PI accumulation, and potentiated the activity of CT-2584 in cytotoxicity assays. As expected from biophysical properties of anionic phospholipids on cellular membranes, CT-2584 cytotoxicity was associated with disruption and swelling of endoplasmic reticulum and mitochondria. We conclude that CT-2584 effects a novel mechanism of cytotoxicity to cancer cells, involving a specific modulation of phospholipid metabolism.

High dose ifosfamide in combination with etoposide and epirubicin followed by autologous stem cell transplantation in the treatment of relapsed/refractory Hodgkin's disease: a report on toxicity and efficacy.

Patients with Hodgkin's disease (HD) refractory to first line chemotherapy and those who have rapid or multiple relapses have a very poor prognosis. With the increasing use of hybrid chemotherapy these patients will have been exposed to many of the drugs active in HD so it is important to develop salvage regimens that are novel and demonstrate activity in this group of patients. We report the use of a continuous high dose infusion of ïfosfamide at a dose of 9g/m(2) over 3 days in combination with etoposide and epirubicin followed by autologous stem cell transplant with either BEAM or Melphalan/VP16 conditioning in this difficult group. Forty six patients (28M:18F) with a median age of 28 years (range 13-45) were treated. Overall 39 out of 46 (85%) patients responded to treatment, with 17 achieving complete remission and 11 a good partial remission; 28 proceeded to autologous bone marrow/stem cell transplantation. In total, 23 patients are alive and in continuous remission with a follow up of between 12 and 61 months. Median overall survival for the whole group is 36 months. Haematological toxicity, particularly neutropenia (WHO grade IV), was observed in all cases but improved over the 3 courses of treatment in all patients. Non-haematological toxicity was not a major problem; no significant cardiac, hepatic, renal, pulmonary or neuro toxicity was observed and there were no deaths on treatment. This regime shows promise in patients with difficult Hodgkin's disease and warrants further study.

Constrictive pericarditis post allogeneic bone marrow transplant for Philadelphia-positive acute lymphoblastic leukaemia.

We describe two cases of severe constrictive pericarditis arising after allogeneic BMT conditioning involving total body irradiation and melphalan to treat Philadelphia-chromosome positive ALL. Both patients required pericardectomy, resulting in marked improvement in ventricular filling. However, a degree of right-sided cardiac failure persisted in both patients secondary to restrictive cardiomyopathy. Constrictive pericarditis has not been previously described after BMT, but has been observed following thoracic radiotherapy for malignancy, usually involving a substantially higher radiation dose. Pericardial constriction and restrictive cardiomyopathy should be considered as causes of breathlessness and/or oedema occurring late after BMT. Bone Marrow Transplantation (2000) 25, 571-573.

Jumping translocation at 11q23 with MLL gene rearrangement and interstitial telomeric sequences.

myeloid leukemia of acute myeloid leukemia (AML) M5a showing a jumping translocation with a breakpoint at 11q23. Fluorescence in situ hybridization (FISH) demonstrated triplication of the MLL gene and the presence of interstitial telomeric sequences, supporting the role of repetitive sequences in the mechanism of jumping translocations. Southern blot analysis of the MLL breakpoint cluster region showed the presence of an MLL gene rearrangement. Jumping translocation with MLL gene rearrangement is a previously unreported phenomenon in leukemia cytogenetics.

Molecular remission in Philadelphia-positive adult acute lymphoblastic leukaemia rapidly induced by conventional-dose chemotherapy.

This report describes a patient presenting with Ph-positive ALL. RT-PCR analysis of diagnostic marrow revealed the presence of three bcr/abl transcripts; the ALL type ela2 along with the CML types, b2a2 and b3a2. After initial induction therapy, bcr/abl transcripts were only detectable after two rounds of PCR but after MIDAC consolidation, remission samples were two-round negative. The relationship between the unusual molecular biological profile of this leukaemia and the rapid attainment of molecular remission is discussed.

Endogenous natural killer enhancing factor-B increases cellular resistance to oxidative stresses.

Natural killer-enhancing factor (NKEF) was identified and cloned on the basis of its ability to increase NK cytotoxicity. Two genes, NKEF-A and -B, encode NKEF proteins and sequence analysis presented suggests that each belongs to a highly conserved family of antioxidants. To examine the antioxidant potential of NKEF, we transfected the coding region of NKEF-B cDNA into the human endothelial cell line ECV304. The stable transfectant, B/1, was found to overexpress NKEF-B gene transcript and protein. We subjected B/1 to oxidative stress by either culturing them with glucose oxidase (GO), which continuously generates hydrogen peroxide, or by direct addition of hydrogen peroxide. We found that B/1 cells were more resistant than control cell lines. Resistance to hydrogen peroxide was originally thought to be mediated mainly by catalase and the glutathione cycle. Therefore, we used inhibitors to block the two pathways and found that B/1 cells were more resistant to oxidative stress than control cells when we used inhibitors to preblock either pathway. We also examined the cellular inflammatory responses to oxidized low-density lipoprotein (LDL) and bacterial lipopolysaccharide (LPS) by measuring monocyte adhesion to endothelial cells in vitro and found that B/1 cells were resistant to such responses. Lastly, we found that B/1 cells were more resistant to a novel chemotherapeutic agent CT-2584, which appears to kill tumor cells by stimulating production of reactive oxygen intermediates in mitochondria. These results demonstrate that the NKEF-B is an antioxidant that protects cells from oxidative stress, chemotherapy agents, and inflammation-induced monocyte adhesion. Furthermore, its expression may mediate cellular responses to proinflammatory molecules.

Detection of urinary incontinence during ambulatory monitoring of bladder function by a temperature-sensitive device.

OBJECTIVES: To determine whether the difference between urinary and perineal temperatures is sufficient to allow registration of incontinent episodes by detection of temperature change alone. To design and assess the use of a diode-based temperature-sensitive device in the detection of episodes of urinary incontinence in long-term ambulatory monitoring (LTAM) studies. SUBJECTS AND METHODS: Perineal temperature recordings were made in 46 women during various activities. A temperature-sensitive device consisting of six IN4148 diodes, spanning 5 cm, and a nearby reference negative diode, was placed in a light perineal pad and attached to a portable amplifier/digitizer and recorder. The performance of the device was determined by comparison with increases in pad weight in 51 incontinent and 23 continent control subjects. RESULTS: A sufficient temperature differential existed between perineal and urinary temperature during all activities except being seated with crossed legs. Incontinence was reliably detected by the temperature-sensitive device. The device had a sensitivity of 95.2% and a specificity of 90.6% compared to a pad test. CONCLUSIONS: This temperature-sensitive device offers a new method for detecting urinary incontinence during LTAM studies. It can be fitted in an unobtrusive perineal pad and has a higher sensitivity and specificity for the detection of incontinence when compared to a pad test. It may also be used as a marker of voiding in ambulatory studies not employing an integrated voiding channel.

Root replacement for all allograft aortic valves: preferred technique or too radical?

From November 1985 to January 1994, 146 patients have received a viable cryopreserved allograft for aortic root replacement. The follow-up was complete, with all events included to March 1st, 1994. The median age of patients was 49 years; 83.6% were male. Valve dysfunction (91 patients), primary aortic wall disease (45 patients), and a combination of both (10 patients) were the indications for aortic root replacement. The current operative mortality is 1.7% (three deaths in 172 patients to July 1st, 1994). Four late deaths have occurred, with an 8-year actuarial survival of 85% +/- 8% (95% confidence limits). Endocarditis (two events) and thromboembolism (four events) had a low incidence. Structural deterioration (three events) and reoperation for all causes (nine events) have constituted low morbidity and are compared with the results after non-root allograft implantation techniques. The clinical and echocardiographic evidence indicates that the immediate results of valve function with root replacement are superior. But no statistical difference between aortic root replacement and non-root procedures is apparent at 8 years, indicating that a longer follow-up is required before the answer to the question "preferred technique or too radical" can be answered.

Estimation of sieving coefficients of convective absorption of drugs in perfused rat jejunum.

Intestinal absorption of many hydrophilic drugs cannot be explained solely in terms of pH-partition and solvent-drag effects have been described in a number of cases. However, quantitative estimates of sieving coefficient (phi) for drug molecules have tended to be variable. In the present work an in situ perfused intestinal loop preparation in the rat has been used to measure the disappearance of five hydrophilic drugs from the intestinal lumen and a mathematical model of drug absorption in the presence of net and unidirectional fluid fluxes has been developed. The model allows separate estimation of the convective (solvent drag) and nonconvective (partition) components of drug absorption from the experimental data. The five drugs studied were found to have phi values ranging from 0.1-0.9; this was highly dependent on molecular size. Analysis of the data shows that three of the drugs are absorbed almost exclusively by the convective process (caffeine, cimetidine, hydrochlorthiazide) while the other two are absorbed by both convective and nonconvective processes (salicylate, oxprenolol). We conclude that the methodology is a useful and reliable means of deriving separate estimates of these two components of drug absorption.

Association of pRas and pRaf-1 in a complex correlates with activation of a signal transduction pathway.

BACKGROUND: A key pathway for transduction of proliferative, developmental and oncogenic stimuli from receptors at the cell surface to transcription factors located in the nucleus involves the activation of pRas and pRaf-1. Recent publications have described a physical interaction between pRas and pRaf-1, either as ectopic proteins in yeast or as recombinant proteins added to cellular extracts. Until now, however, physical complexes that include pRas and pRaf-1 have not been identified as native structures in mammalian cells. RESULTS: We have directly identified a pRas-pRaf-1 complex in extracts of mammalian cells. Formation of the complex is augmented in neoplastically transformed cells expressing constitutively activated pRas. Moreover, the complexes form in concert with the activation of pRas during intracellular signalling through the T-cell receptor in T-leukemia cells. CONCLUSIONS: We propose that, pRas signals to pRaf-1 in vivo by means of a direct physical interaction that results in activation of the pRaf-1 protein kinase.

Predisposition to neoplastic transformation caused by gene replacement of H-ras1.

Homologous recombination was used to introduce a nominally transforming mutation into an endogenous H-ras1 gene in Rat1 fibroblasts. Although both the mutant and the remaining normal allele were expressed equally, the heterozygous cells were not neoplastically transformed. Instead, spontaneously transformed cells arose from the heterozygotes at a low frequency, and the majority of these cells had amplified the mutant allele. Thus, the activated H-ras1 allele was not by itself dominant over the normal allele but predisposed cells to transformation by independent events, such as amplification of the mutant allele.

A cost-effective peripheral venous port system placed at the bedside.

High costs and a paucity of available operating time have led us to seek alternatives to operatively placed vascular access systems. This prospective study is the initial report of a peripheral port system (P.A.S. PORT System, Pharmacia Deltec, Inc.) placed at the bedside. Seventy-nine patients (52 male, 27 female), ages 3-92 years, had ports implanted by surgical residents with attending supervision. Sixty-eight (86%) received the P.A.S. PORT for long-term antibiotics, antifungal, or antiviral therapy; four (5%) for TPN infusion; three (4%) for blood products; two (3%) for chemotherapy; and two (3%) for iv narcotics. Ports were placed in 10 (13%) HIV(+) patients, three (4%) who were fully anticoagulated, and one who was a hemophiliac with a platelet count of zero. Eight patients (10%) developed superficial phlebitis, all of which resolved with nonsteroidal anti-inflammatory agents within 48 hr without port removal. Seven patients (9%) had their port removed due to infection. The average hospital charge to place the P.A.S. PORT System was $1488.00 vs $2811.00 for a tunneled external chest catheter and $3729.00 for the placement of a chest port. Bedside insertion of vascular access devices can be safely performed with acceptable infection rates allowing more efficient use of hospital operating rooms and with substantial cost savings.

The classwide peer tutoring program: implementation factors moderating students' achievement.

We conducted a study designed to assess implementation of the classwide peer tutoring program and the relationship between implementation variation and student outcome. A clinical replication design was used. Five volunteer elementary teachers were trained to implement the program; their implementation was monitored for 19 consecutive weeks during 1 school year. Overall, the results indicated that specific variations in program implementation were associated with students' responses to treatment. It was also demonstrated that different teachers' applications of the program produced differential levels of student outcome. Implementation factors related to lower spelling achievement were (a) reduced opportunities to receive program sessions, (b) reduced probabilities of students' participation in program opportunities, (c) too many students assigned unchallenging spelling words, and (d) reduced rates of daily point earning reflecting lower levels of spelling practice during tutoring sessions. The implications of these findings and methods of preventing these implementation problems are discussed in the context of quality assurance and social validity.

Measurement of lithotripsy pulses through biological media.

Lithotripsy is now the method of choice for the treatment of renal calculi. The mechanism of destruction is not clearly understood, and detailed knowledge of the shock-wave characteristics at the calculus would aid understanding of the phenomenon. Current methods of measuring the pressure pulse by observing it through a water path are not well characterized, and the results may not represent the actual pressure fluctuations produced in vivo. In order to determine the actual pressure pulse experienced at the site of the calculus, measurements have been made through a variety of biological media. The results show that there are considerable differences between measurements taken through a water path and through biological media. This paper describes the pressure fluctuations in the time domain. The implications of the results for lithotripsy are discussed.

Volatile anesthetic effects on left ventricular relaxation in swine.

The effects of halothane (0.5, 1.0, and 1.5%; n = 10), enflurane (1.0, 2.0, and 3.0%; n = 8), and isoflurane (0.75, 1.5, and 2.25%; n = 8) on isovolumic relaxation were studied in open-chest swine. The time constant for isovolumic left ventricular pressure decline, T, was determined at each anesthetic concentration at the intrinsic heart rate and during atrial pacing to 150 beats per min. The effect of increased left ventricular afterload on T was investigated by partial occlusion of the thoracic aorta to raise the left ventricular systolic pressure to baseline in the presence of volatile anesthetics, and 20% above baseline in the absence of volatile anesthetics. Heart rate and left ventricular systolic pressure decreased substantially with all three anesthetics, whereas left ventricular end-diastolic pressure increased (by 3-4 mmHg). Relaxation time constants increased with all three anesthetics at the intrinsic heart rate; when the heart rate was controlled by pacing, T increased in the halothane and enflurane, but not in the isoflurane, experiments. T was significantly prolonged (by 30-100%) by partial aortic occlusion in the presence of anesthetic, but not in the control measurements. T did not change significantly in the isoflurane experiments when atrial pacing was employed with partial aortic occlusion. The volatile anesthetics, particularly halothane, seem to impair the relaxation process of the left ventricle; further investigation of the mechanisms of this interference, such as anesthetic effects on intracellular calcium movement and total left ventricular load, is warranted.