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Due to their special metabolic pathway, medium-chain triglycerides (MCT) have been claimed to be oxidized more extensively, compared with long-chain triglycerides (LCT), when administered as a parenteral nutritional support. This enhanced lipid oxidation rate of MCT emulsions could be particularly disclosed in hyperglycemic and hyperinsulinemic conditions. In an attempt to further elucidate this question, we measured substrate oxidation rates in critically ill patients liable to experience such metabolic conditions, that is to say postoperative patients after esophageal resection receiving 1.5 times their measured energy expenditure (n = 12) or after liver transplantation (n = 8). These patients received either LCT or MCT-LCT emulsions. The metabolic measurements were performed simultaneously by two methods, namely indirect calorimetry and isotopic methods based on natural abundance of nutrients. Although both groups of patients were hyperglycemic and hyperinsulinemic, the measured carbohydrate and lipid oxidation rates were not different with whatever type of lipid was administered. The MCT-LCT emulsions did not offer clear-cut advantages over LCT emulsions in critically ill patients when lipid energetic fate was considered.

5-Aza-2'-deoxycytidine (5-AZA-CdR, Decitabine) is a nucleoside analog and an active drug for the therapy of acute leukemia. The incorporation of 5-AZA-CdR into DNA blocks DNA methylation and can result in the activation of specific genes, such as tumor suppressor genes. This novel mechanism of action of 5-AZA-CdR stimulated our interest in its potential for cancer therapy in patients with lung cancer. Using a colony assay we observed that 5-AZA-CdR showed a potent antineoplastic effect against two human lung carcinoma cell lines. The objective of this preliminary phase I-II study was to evaluate the toxicity and clinical efficacy of 5-AZA-CdR in patients with stage IV non-small cell lung carcinoma. There were 15 patients that entered the clinical study. For nine assessable patients that received 5-AZA-CdR by a single 8 h i.v. infusion of 200-660 mg/m2 for one or more cycles, the median survival duration was 6.7 months, with three patients surviving more than 15 months. The steady-state plasma concentration of 5-AZA-CdR during the infusion was estimated in some patients and was in the same range that produced activation of a tumor suppressor gene in human lung tumor cell lines as reported by other investigators. The major side effect of 5-AZA-CdR was hematopoietic toxicity which required a 5-6 week recovery period before the next cycle of therapy. This study suggests that 5-AZA-CdR may have some clinical activity against metastatic lung carcinoma using this type of dose schedule.

The induction of DNA fragmentation by cytosine arabinoside (araC) and 2',2'-difluorodeoxycytidine (dFdC, gemcitabine) was compared in human leukemic cell lines. For both araC and dFdC this process was time- and concentration-dependent and resulted in loss of clonogenic survival of HL-60 myeloid leukemic cells. There was a marked difference in the potency between these two analogs in inducing apoptosis. A 6 h exposure to 5 microM araC was required to produce DNA laddering in HL-60 cells, whereas dFdC at a concentration 100-fold less (0.05 microM) was sufficient to produce similar results. Pre-incubation of HL-60 cells with staurosporine, a non-specific protein kinase C inhibitor, increased the level of apoptosis induced by a 3 h exposure to araC or dFdC, suggesting the possible involvement of this family of enzymes in this process. Also, dFdC was able to increase the expression of both c-jun and c-fos in Molt-3 leukemic cells with a concentration known to induce apoptosis in this cell line.

To evaluate a monitor of pulmonary gas exchange (Deltatrac, Datex) in a clinical setting.After in vitro evaluation, comparison over 2 min between VO2 and VCO2 values measured by the Deltatrac and the Douglas bag technique. Comparisons were also achieved over 8 h periods between the Deltatrac and a system using a mass-spectrometer.Polyvalent intensive care unit (ICU 15 beds) in a 1200 bed general hospital.Comparison with the Douglas bag technique in 10 patients undergoing controlled ventilation. Comparison with the mass-spectrometer system in 25 other patients undergoing controlled or pressure support ventilation.Compared to the results obtained by the Douglas bag technique, the bias (+/- 2 SD) for VO2 and VCO2 was -3.5 +/- 26.6 and 6.1 +/- 12.7 ml.min-1, respectively. By comparison with the mass-spectrometer system, the bias for VO2 and RQ was -5.8 +/- 16.0 ml.min-1 and 0.018 +/- 0.048, respectively. No drift between the two systems was observed over time.The Deltatrac appears suitable for VO2 and VCO2 measurements in ventilated patients and equivalent to a mass-spectrometer system for long term measurements.

We have investigated the capacity of cyclopentenyl cytosine (CPE-C), a potent inhibitor of CTP synthetase, to modulate the antineoplastic activity of 5-aza-2'-deoxycytidine (DAC) on HL-60 myeloid leukemic cells. The combination of CPE-C and DAC produced an additive effect on the growth inhibition of the cells following a treatment of 48-96 h. Cytotoxicity experiments measured by the cloning of cells in soft agar following 24 and 48 h exposures produced a more than additive effect when the drugs were used in combination. Evaluation of the effect of CPE-C and DAC on the induction of differentiation of HL-60 cells following a 48 h treatment revealed that the combination of the drugs produced a more than additive effect than when the drugs were used alone. Measurement of the intracellular pool of deoxycytidine triphosphate (dCTP) showed that a 6 h exposure to 0.05 and 0.1 microM of CPE-C reduced the pool by 60 and 88%, respectively. The decrease in the dCTP pool was correlated with a higher incorporation of radioactive DAC into DNA. The deamination of CPE-C to cyclopentenyl uridine by cytidine deaminase was investigated with the purified enzyme from human placenta. We report here that CPE-C is a very poor substrate for cytidine deaminase as compared with cytidine. These studies suggest that CPE-C could be used as a biochemical modulator to increase the antileukemic action of DAC.

Early somatosensory (SEP) and auditory (BAEP) evoked potentials, when recorded within the first seven days of the course of anoxic coma, appear to be reliable to evaluate anoxic ischemic cortical or under-cortical lesions. Prognosis depends especially on cortical SEP (N20-P25): the lack of SEP is a good outcome predictor of death (abnormal BAEP) or of vegetative status (normal BAEP); the presence of normal and bilateral cortical SEP (with normal BAEP) allows to predict awakening, without prejudging of neurologic sequelae, even if they are severe.

Measurement of the nutrient oxidation rate with 13C as a tracer requires knowledge of the value of its coefficient of fractional recovery in the expired gas (FR). We measured FR in nine intensive care patients who were mechanically ventilated and received total parenteral nutrition. NaH13CO3 was administered at a priming dose (3.75 mumol.kg-1.min-1) followed by a continuous infusion (0.05 mumol.kg-1.min-1). Metabolic rate and pulmonary carbon dioxide elimination (VCO2) were measured by using a mass-spectrometer system. The 13C-12C ratio was measured in the expired gas with an isotopic-ratio mass spectrometer and FR was calculated by using standard equations. The average value of FR was 0.899 +/- 0.026 (means +/- SE) and remained stable for each patient on 2 consecutive days. Between patients, the coefficient of variation of FR was 8.6%. Metabolic rate was the only physiological factor found to affect the FR value.

Introduction En post-operatoire precoce de transplantation hepatique (TH), le risque infectieux est majore, notamment en raison de l’immunosuppression medicamenteuse. La procalcitonine (PCT) est un biomarqueur utilise pour le diagnostic precoce d’infection bacterienne. Neanmoins, de nombreuses causes non infectieuses d’elevation de la PCT sont egalement rapportees : polytraumatisme, brulure severe, pathologies inflammatoires, causes medicamenteuses (Ig anti-thymocyte) ainsi que le syndrome de reperfusion (PRS) [1] . L’objectif de cette etude etait de mettre en evidence une association entre la survenue d’un PRS lors d’une chirurgie de TH et l’elevation de la PCT a j1 postoperatoire. Patients et methodes Il s’agit d’une etude de cohorte retrospective evaluant l’ensemble des patients ayant beneficie d’une TH entre janvier 2004 et decembre 2010. La survenue d’un syndrome de reperfusion, les valeurs de PCT a j1 ainsi que la survenue d’une infection a j1 ou pre-existante a la transplantation ont ete recueillies. Les analyses statistiques ont ete realisees avec le test de Mann-Whitney. Un syndrome de reperfusion etait defini par la survenue d’une chute de la pression arterielle moyenne de 30 % ou plus pendant au moins une minute au cours des 5 minutes suivant le declampage vasculaire [2] , ou par la necessite d’introduire des amines vasoactives, ou de doubler la dose d’amines. Resultats Deux cent quatorze patients ont ete inclus dans l’etude. Cent seize patients (54,2%) ont presente un syndrome de reperfusion, avec une valeur mediane de PCT de 13 μg/L a j1 ( Tableau 1 , Fig. 1 ). Les valeurs de PCT a j1 etaient statistiquement plus elevees (p Fig. 1 ). Quatre patients (1,9 %) ont presente une infection a j1 ou pre-existante a la transplantation. On ne retrouvait pas de difference significative des valeurs de PCT a j1, que les patients soient infectes ou non (p = 0,49). Discussion La survenue d’un PRS lors d’une TH est associee a une franche elevation des valeurs de PCT a j1, que les patients soient infectes ou non. Ces resultats suggerent que les lesions d’ischemie-reperfusion survenant lors d’une TH sont responsables d’une elevation de PCT. La physiopathologie de ce phenomene reste mal connue.

Drug resistance is one of the major reasons for failure of chemotherapy of acute leukemia with cytosine arabinoside (ARA-C). In order to overcome this problem we have investigated the interaction of ARA-C with 3-deazauridine (3-DU) against HL-60 myeloid leukemic cells. 3-DU is an interesting agent to use in combination with ARA-C, since drug-resistant cells that are deficient in deoxycytidine kinase are very sensitive to this uridine analogue. We have observed that for both short and long drug exposure there was a potent synergistic interaction between ARA-C and 3-DU with respect to their cytotoxic effects on HL-60 leukemic cells. This synergy could be explained by an increased cellular uptake of ARA-C to ARA-CTP by the leukemic cells in the presence of 3-DU, due to the reduction in the pool of dCTP produced by this latter analogue. Since dCTP is a potent feedback inhibitor of the phosphorylation of ARA-C by deoxycytidine kinase, the reduction in the dCTP produced by 3-DU results in an increased rate of phosphorylation of the arabinosyl analogue. Our results suggest that ARA-C and 3-DU may be an interesting drug combination to circumvent drug resistance in the chemotherapy of acute leukemia.

2',2'-difluorodeoxycytidine (known as dFdC, Gemcitabine and LY188011) is a new analog of deoxycytidine which has demonstrated excellent antineoplastic activity against many kinds of solid tumors and leukemic cell lines. We were interested in the comparison of the antineoplastic activity of this new antimetabolite with cytosine arabinoside (ARA-C) against HL-60 myeloid, RPMI-8392 B-lymphoid and Molt-3 T-lymphoid leukemic cell lines. Our in vitro experiments showed that dFdC was a more potent cytostatic drug than ARA-C against all the leukemic lines with IC50 ranging from 3 to 10 nM for dFdC and from 26 to 52 nM for ARA-C for a 48 h exposure. The cytotoxicity of both drugs was evaluated by clonogenic assay and dFdC was found to be 100 times more potent than ARA-C against all the leukemic cell lines for both a 2 h and a 24 h exposure. The recovery of DNA synthesis after drug removal was much slower for dFdC than for ARA-C. However, in contrast to cytostatic and cytotoxicity results ARA-C was a more potent inhibitor of DNA synthesis than dFdC for all the leukemic cell lines for short exposure. Uptake and elimination of the drugs showed that dFdC accumulated to a higher degree in the leukemic cells than ARA-C and that elimination of this difluoro analog was slower than that of ARA-C. These results indicate that dFdC has more potent in vitro antileukemic activity than ARA-C.

Continuous monitoring of cardiac output during liver transplantation is essential to evaluate the patient's haemodynamic tolerance to acute volume variations. The aim of this study was to compare the cardiac output values obtained with a transoesophageal echo-Doppler and those obtained with a continuous thermodilution cardiac output pulmonary artery catheter.Twenty adult patients were prospectively studied during a 5 min hepatic vascular exclusion test performed at the end of the dissection phase. Echo-Doppler and continuous thermodilution cardiac output, mean arterial pressure and end-tidal CO2 were measured before and at the end of the test.Before the test, echo-Doppler cardiac output was 7.0 +/- 2.7 L min(-1) and thermodilution was 9.4 +/- 3.1 L min(-1), (R = 0.85, P0.001). The end test values were, respectively, 3.5 +/- 2.7 and 7.8 +/- 3.5 L min(-1) (R = 0.23, P = 0.34). Bland and Altman analysis showed a bias of -2.2 before the test, which increased to -4.4 at the end of the test. Mean arterial pressure decreased from 85.5 +/- 15 to 66.8 +/- 16 mmHg, end-tidal CO2 from 31.4 +/- 2.3 to 23.8 +/- 2.7 mmHg.Echo-Doppler cardiac output values are different from those measured by thermodilution cardiac output in these patients. Echo-Doppler cardiac output monitoring seems to detect the output changes, which can occur during acute haemodynamic changes more rapidly than thermodilution cardiac output in the course of liver transplantation.

Background Gemcitabine, a deoxycytidine nucleoside analog, is the current standard chemotherapy used as first-line treatment for patients with locally advanced or metastatic cancer of the pancreas, and extends life survival by 5.7 months. Advanced pancreatic cancer thus remains a highly unmet medical need and new therapeutic agents are required for this patient population. Troxacitabine (Troxatyl™) is the first unnatural L-nucleoside analog to show potent preclinical antitumor activity and is currently under clinical investigation. Troxacitabine was recently evaluated as a first-line therapy in 54 patients with advanced adenocarcinoma of the pancreas and gave comparable overall results to those reported with gemcitabine in recently published randomized trials. Methods The human pancreatic adenocarcinoma cell lines, AsPC-1, Capan-2, MIA PaCa-2 and Panc-1, were exposed to troxacitabine or gemcitabine alone or in combination, for 72 h, and the effects on cell growth were determined by electronic particle counting. Synergistic efficacy was determined by the isobologram and combination-index methods of Chou and Talalay. Mechanistic studies addressed incorporation of troxacitabine into DNA and intracellular levels of troxacitabine and gemcitabine metabolites. For in vivo studies, we evaluated the effect of both drugs, alone and in combination, on the growth of established human pancreatic (AsPC-1) tumors implanted subcutaneously in nude mice. Statistical analysis was calculated by a one-way ANOVA with Dunnett as a post-test and the two-tailed unpaired t test using GraphPad prism software. Results Synergy, evaluated using the CalcuSyn Software, was observed in all four cell-lines at multiple drug concentrations resulting in combination indices under 0.7 at Fa of 0.5 (50% reduction of cell growth). The effects of drug exposures on troxacitabine and gemcitabine nucleotide pools were analyzed, and although gemcitabine reduced phosphorylation of troxacitabine when cells were exposed at equal drug concentrations, there was no effect on phosphorylated pools at drug combinations that were synergistic. The amount of troxacitabine incorporated into DNA was also not affected by the presence of gemcitabine. In vivo testing against a human pancreatic (AsPC-1) xenograft mouse tumor model indicated that both drugs were more than additive at well-tolerated doses and schedule. The biological basis for this synergy is unclear as we did not observe changes in apoptosis, DNA repair, troxacitabine incorporation into DNA or troxacitabine metabolism in the presence of gemcitabine. Conclusion These data, together with phase I clinical data showing tolerability of both agents when combined, suggest combination therapy with troxacitabine and gemcitabine warrants further evaluation in advanced pancreatic cancer patients.

By applying a novel cell- and caspase-based HTS assay, 2-amino-3-cyano-7-(dimethylamino)-4-(3-methoxy-4,5-methylenedioxyphenyl)-4H-chromene (1a) has been identified as a potent apoptosis inducer. Compound 1a was found to induce nuclear fragmentation and PARP cleavage, as well as to arrest cells at the G(2)/M stage and to induce apoptosis as determined by the flow cytometry analysis assay in multiple human cell lines (e.g. Jurkat, T47D). Through structure-activity relationship (SAR) studies of the 4-aryl group, a 4- and 7-fold increase in potency was obtained from the screening hit 1a to the lead compounds 2-amino-4-(3-bromo-4,5-dimethoxyphenyl)-3-cyano-7-(dimethylamino)-4H-chromene (1c) and 2-amino-3-cyano-7-(dimethylamino)-4-(5-methyl-3-pyridyl)-4H-chromene (4e), with an EC(50) of 19 and 11 nM in the caspase activation assay in T47D breast cancer cells, respectively. The 2-amino-4-aryl-3-cyano-7-(dimethylamino)-4H-chromenes also were found to be highly active in the growth inhibition MTT assay, with GI(50) values in the low nanomolar range for compound 1c. Significantly, compound 1c was found to have a GI(50) value of 2 nM in the paclitaxel resistant, p-glycoprotein overexpressed, MES-SA/DX5 tumor cells. Functionally, compound 1c was found to be a potent inhibitor of tubulin polymerization and to effectively inhibit the binding of colchicine to tubulin. These results confirm that the cell-based caspase activation assay is a powerful tool for the discovery of potent apoptosis inducers and suggest that the 4-aryl-4H-chromenes have the potential to be developed into future anticancer agents.

Troxacitabine (BCH-4556, l-(-)-OddC, Troxatyl) is a novel beta- l-nucleoside analogue with potent antineoplastic activity both in vitro and in several tumor models in vivo, and is presently in phase II clinical trials. The combination of the cytosine analogues troxacitabine and araC (1-beta- d-arabinofuranosylcytosine, cytarabine) has shown promising activity in patients with acute myelogenous leukemia. To further examine the interactions between these two analogues, we investigated the in vitro and in vivo effects of their combination against a human leukemia cell line, CCRF-CEM.. The in vitro cytotoxic effect of the combination of troxacitabine and araC on the survival of CCRF-CEM cells was measured using a standard MTT assay and combination indices were generated with the CalcuSyn software. For in vivo studies, we evaluated the effect of both drugs, alone and in combination, on survival of CCRF-CEM tumor-bearing animals. Mechanistic studies addressed recovery of DNA synthesis, intracellular levels of araC metabolites, feedback inhibition by triphosphate species and pharmacokinetics of both drugs.The combination of troxacitabine and araC in vitro was synergistic with combination indices between 0.1 and 0.7. This appeared to be related to the impact of the combination on DNA synthesis recovery, which was significantly delayed following exposure to the combination of troxacitabine and araC compared to either agent alone. Analysis of the effect of troxacitabine on the intracellular metabolites of araC revealed that troxacitabine did not inhibit araC deamination and caused a slight decrease in the overall intracellular accumulation of araCTP. The lower accumulation of araCTP could not be attributed to feedback inhibition caused by troxacitabine triphosphate on dCK. Furthermore, our in vivo experiments demonstrated that the combination of araC and troxacitabine was better at slowing down the progression of leukemia in SCID mice than either agent used alone without additive toxicities. Injections of 10 mg/kg troxacitabine i.p. daily for 5 days in combination with araC at 10 mg/kg led to an increase in median survival time of 58 days compared to 49.5 and 53.5 days for araC and troxacitabine, respectively, given as single agents. This represents an increase in life span of 17%, respectively when compared to araC alone. A pharmacokinetic study revealed that troxacitabine did not influence the disposition of araC when coadministered.Overall, our results show that the antileukemic activity of troxacitabine and araC is complementary when the two nucleoside analogues are combined in vivo. These effects appear to be related to their interaction at the level of DNA repair rather than to pharmacokinetic interactions. These results encourage the use of troxacitabine and araC in combination in patients with acute leukemia.

The use of a two-arm robot for assembling two objects, with each being held by one arm, is presented. More precisely, the authors propose some solutions for determining how and where such an assembly task should be performed. For the approach phase (before contact), they define the motion of one end-effector with respect to the other, without reference to any fixed frame. This allows them to take advantage of the two-arm robot redundancy. For the insertion phase, they use a symmetric hybrid control scheme. Real experiments with two PUMA robots have shown that the success of the assembly is highly dependent on the location where the assembly is performed. This is due to the control of contact forces between the objects, which can be done more or less properly. This problem is treated by using the concept of manipulability, represented by ellipsoids.

Nucleoside phosphonates are widely used therapeutic agents with a broad spectrum of antiviral activity. However, only a few of them are reported to have antitumor activity. In this study, we show that a tetrahydrofuran phosphonate analogue of guanosine, (-)-2-R-dihydroxyphosphinoyl-5-(S)-(guanin-9'-ylmethyl) tetrahydrofuran (BCH-1868), previously reported as having antiviral activity, also displays antitumor activity. In vitro, BCH-1868 inhibited the proliferation of several murine and human cancer cell lines with IC50s in the microM range independently of the tissue type or the presence of multidrug resistance protein MRP/gp190. In vivo, BCH-1868 was active against a variety of human tumor xenograft models (Caki-1, HT-29, DU 145, COLO 205, and CCRF-CEM). In all tumors tested, a significant tumor growth inhibition was noted at 40-50 mg/kg (daily x 5), but no tumor regression was observed in the settings used. To better understand these results, we partially characterized, at the cellular level, the mechanism of action of this new cyclic nucleoside phosphonate and investigated its pharmacokinetic characteristics in mice. We showed that BCH-1868 exerts its antitumor activity by an inhibitory mechanism at the level of DNA polymerase a, resulting in arrest of DNA synthesis and a block of cell division at the S phase of the cell cycle. Low-circulating plasma concentration (Cmax = 87 microM; area under the curve = 1138 micromol x min/liters; after a bolus i.v. injection of 10 mg/kg) and rapid clearance of the drug (terminal half-life, t1/2 = 16 min) may contribute to the modest antitumor efficacy observed in vivo.

The authors present solutions for manipulating objects with two robotic arms, when force control is necessary. The applications of interest are the transport and assembly of rigid objects, the deformation, transport, and assembly of flexible objects, and the assembly in space of two objects, each being held by one arm. The authors present a theoretical solution for modeling the tasks and practical solutions implemented for controlling two six-axis arms. >

Purpose. We have recently identified a deoxycytidine nucleoside analogue, troxacitabine (β-L-dioxolane cytidine, Troxatyl; Shire BioChem), which has potent antitumor activity against both leukemia and solid tumors. In contrast to the cytidine nucleoside analogues currently in clinical use (cytarabine and gemcitabine), troxacitabine is a poor substrate of nucleoside transporters and enters cells primarily by passive diffusion. This unusual property led us to evaluate the efficacy of troxacitabine in multidrug resistant (MDR) and multidrug resistance-associated protein (MRP) tumors. Methods. The in vitro antiproliferative activity of troxacitabine was investigated in the human nasopharyngeal epidermoid carcinoma cell line, KB, and its vincristine-resistant derivative (KBV), as well as in human leukemia cell lines of myeloid and lymphoblastoid origin, HL60 and CCRF-CEM, respectively, and their MDR (HL60/R10 and CCRF-CEM/VLB) and MRP (HL60/ADR) derivatives, using the thymidine incorporation assay. For in vivo studies, we compared the antitumor efficacy of troxacitabine with that of doxorubicin and vinblastine in xenograft models of these solid and hematological human anthracycline-resistant tumor xenografts. Results. Troxacitabine demonstrated potent antiproliferative activity against both P-glycoprotein-positive (KBV, HL60/R10, CCRF-CEM/VLB) and P-glycoprotein-negative (HL60/ADR) multidrug-resistant cell lines with IC50 values ranging from 7 to 171 nM. Tumor regression was observed in the KBV xenograft following a 5-day treatment with 20, 50 and 100 mg/kg of troxacitabine, with percent total growth inhibition (TGI) of 81, 96 and 97, respectively, and some cures at the two highest dose levels. In the HL60, HL60/R10, HL60/ADR and CCRF-CEM/VLB xenografts, the effect of troxacitabine was evaluated on survival time. In the HL60 promyelocytic human xenograft models, troxacitabine treatment (25, 50 and 100 mg/kg per day for 5 days) was initiated 10 days after tumor cell inoculation, once animals had developed disseminated tumors. In all three promyelocytic leukemia xenografts, troxacitabine was quite potent, producing T/C values of 162% to 315% as well as complete cures at the higher dose levels. In the CCRF-CEM/VLB T-lymphoblastoid leukemia xenograft, troxacitabine treatment (10, 30 or 250 mg/kg total doses using different schedules) was initiated 20 days after tumor cell inoculation. Troxacitabine was not as potent in this model but did result in significant antileukemic activity (T/C of 131%) when administered at 10 mg/kg on days 20, 27 and 34. Conclusions. These results indicate that troxacitabine has a potent in vivo antitumor activity associated with tumor regressions and complete cures in animals with tumors refractory to current chemotherapeutic agents.

Publisher Summary This chapter focuses on a general overview of ribozymes and their applications for cancer gene therapy. Ribozymes are small RNA molecules that possess specific catalytic activities and are being actively investigated for their therapeutic applications in the field of gene therapy. Ribozymes have the ability to modulate specific gene expression because of their site-specific cleavage activity. Ribozymes can be designed for any disease in which a specific protein has been linked to its etiology, and may offer some advantages over antisense oligonucleotide strategies. Since the discovery of the protooncogene, cancer has been defined as a genetic disease. Therefore, gene therapy could be a rational and promising strategy for the treatment of specific cancers. So, oncogenes are obvious targets for the therapeutic application of anti-oncogene ribozymes. One of the problems for successful gene therapy is to define the role of specific oncogenes in specific tumors. Ribozyme technology can be used to help define and delineate the role of oncogenes in cancer and can be used as a therapeutic agent as well. Extensive studies have investigated the efficacy of antioncogene ribozymes, and have shown successful alteration of the human malignant phenotype in vivo. The appearance of gene therapy as an alternative treatment for cancer and other diseases has led researchers toward the development of efficient delivery systems. Among the first methods developed for gene delivery, viral systems have been potentially the most promising. Although viral transfer shows huge potential for clinical settings, nonviral delivery systems are receiving increasing attention. Thus, effective delivery systems with minimal toxicity may advance ribozymes as important therapeutic modalities in the clinical field. Ribozymes could have an important impact on the field of gene therapy in the near future.

The delineation of gene function has always been an intensive subject of investigations. Recent advances in the synthesis and chemistry of oligonucleotides have now made these molecules important tools to study and identify gene function and regulation. Modulation of gene expression using oligonucleotides has been targeted at different levels of the cellular machinery. Triplex forming oligonucleotides, as well as peptide nucleic acids, have been used to inhibit gene expression at the level of transcription; after binding of these specific oligonucleotides, conformational change of the DNA's helical structure prevents any further DNA/protein interactions necessary for efficient transcription. Gene regulation can also be achieved by targeting the translation of mRNAs. Antisense oligonucleotides have been used to down-regulate mRNA expression by annealing to specific and determined region of an mRNA, thus inhibiting its translation by the cellular machinery. The exact mechanism of this type of inhibition is still under intense investigation and is thought to be related to the activation of RNase H, a ribonuclease that is widely available that can cleave the RNA/DNA duplex, thus making it inactive. Another well-characterized means of interfering with the translation of mRNAs is the use of ribozymes. Ribozymes are small catalytic RNAs that possess both site specificity and cleavage capability for an mRNA substrate, inhibiting any further protein formation. This review describes how these different oligonucleotides can be used to define gene function and discusses in detail their chemical structure, mechanism of action, advantages and disadvantages, and their applications.

Point mutations in the ras gene have been found in approximately 90% of human pancreatic carcinomas. These alterations can be used as potential targets for specific ribozyme-mediated reversal of the malignant phenotype. We have evaluated the efficacy of a hammerhead ribozyme directed against codon 12 (GUU) of the activated K-ras gene in a Capan-1 human pancreatic carcinoma cell line using different delivery systems. Our results have demonstrated that the anti-Kras ribozyme cloned into the pHs plasmid was able to efficiently suppress K-ras gene expression and to inhibit the proliferation of transfected Capan-1 cells. In contrast, the anti-K-ras ribozyme was less efficient against the Capan-1 cells when cloned into a pLNCX retroviral plasmid. In addition, our results showed that adenoviral-mediated expression of the ribozyme RNA was more effective than the two other plasmid vectors. Our studies have characterized different viral and non-viral delivery systems for the therapeutic application of an anti-K-ras ribozyme against a human pancreatic carcinoma cell line. In the near future, ribozymes could emerge as important therapeutic agents against human malignancies, and optimal delivery systems are necessary to achieve maximal gene therapy benefit.

Phosphorylation of cytosine analogs by deoxycytidine kinase (dCK) and deamination by cytidine deaminase (CDA) are two important processes in the activation and elimination of these drugs. We have investigated the kinetic parameters of 2',2'-difluorodeoxycytidine (dFdC) using purified enzymes from human cells. Deoxycytidine (CdR) and dFdC had Km values of 1.5 and 4.6 microM for dCK, respectively. Feedback inhibition of dCK by deoxycytidine 5'-triphosphate (dCTP) was also studied. Our results show that dCTP produced a greater inhibition of the phosphorylation of dFdC than CdR with concentrations of dCTP ranging from 1 to 25 microM. dFdC was a good substrate for CDA. Kinetic studies with this enzyme gave Km values for CdR and dFdC of 46.3 and 95.7 microM, respectively. The effect of competitive inhibitors of CDA on the deamination of dFdC was also investigated. Diazepinone riboside was a more potent inhibitor than tetrahydrouridine using either CdR or dFdC as the substrate. Inhibitors of CDA could be useful in clinical trials in patients with cancer to increase the chemotherapeutic effectiveness of dFdC.

Early somatosensory (SEP) and auditory (BAEP) evoked potentials, when recorded within the first seven days of the course of anoxic coma, appear to be reliable to evaluate anoxic ischemic cortical or under-cortical lesions. Prognosis depends especially on cortical SEP (N20-P25): the lack of SEP is a good outcome predictor of death (abnormal BAEP) or of vegetative status (normal BAEP); the presence of normal and bilateral cortical SEP (with normal BAEP) allows to predict awakening, without prejudging of neurologic sequelae, even if they are severe.

Forty-eight hours after a women was poisoned by ingesting Amanita phalloides mushrooms, she developed fulminant hepatic failure with collapse, pH 7.24, lactic acidosis 7.6 mmol/l, hypoglycaemia 3.5 mmol/l, anuria and stage IV coma requiring tracheal intubation and mechanical ventilation. Transaminase level was up to 8,000 UI/l. Prothrombin and factor V levels were below 10 percent, with an APT time of 86 s versus a 29 s control time. Twenty-four hours after her admission, the patient underwent orthotopic liver transplantation. The postoperative period was uneventful, with return to consciousness and rapid normalization of hepatic biochemistry values, without signs of acute rejection. This 10th published case of orthotopic liver transplantation for Amanita phalloides poisoning with acute hepatic necrosis confirms that this type of treatment must be systematically envisaged in all such cases.

We report a case with focal neurological deficits suggesting vertebro-basilar system ischemia, in the course of pre-eclampsia. An early CT scan showed a large hypodensity throughout the midbrain. Brainstem auditory evoked potentials initially showed an abolition of III and V pikes suggesting brainstem injury. Two days later both neurological examination and brain stem auditory evoked potentials returned to normal. A CT scan performed three weeks after the onset was normal. These findings suggest a vasospasm which may have been due to sympathomimetic agents given two weeks before the onset of toxemia for preterm labor.

For both clinical and research purposes, the monitoring of pulmonary gas exchange in mechanically ventilated patients is of great interest.

Early somatosensory (ESEP) and auditory (EAEP) evoked potentials were recorded in 27 patients with severe coma (Glasgow score less than 5) following cardiorespiratory arrest, within the first 7 days of its course. Somatosensory responses were elicited by stimulation of the median nerve. ESEP were abolished in 17 patients due to a parietal thalamo-cortical lesion. Among these, 6 patients died within one month and 11 presented with a persistent vegetative state. In all patients EAEP were obtained, showing functional brainstem activity. Low-voltage EAEP, especially for peak V (inferior colliculus or upper part of the brainstem), was sometimes observed. One patient, in whom ESEP and EAEP were initially abolished, died rapidly. In 9 other patients scalp-recorded ESEP and EAEP were normal; all emerged from coma including 5 with good neurological recovery and 4 with neurological sequelae. Clinical, electroencephalographic and computerized tomographic data appeared to be devoid of predictive value at the same initial period. In view of their sensitivity to anoxia and to cerebral oedema, even with neurosedative drugs, ESEP seemed to be reliable in predicting outcomes and in evaluating central nervous system lesions at cortical and subcortical levels (basal ganglia and brainstem) after cardiorespiratory arrest.

Pulmonary gas exchange measurements can be performed in ICU with commercially available devices. During open-circuit anaesthesia, measurement of VO2 and VCO2 requires the acquisition of fractional concentration of inspired and expired nitrogen, the appropriate calibration of sensors according to the use of anaesthetic gases and to take into account the unsteady state of nitrogen body stores after a change in FiN2. This technique can be readily used to measure energetic expenditure or to specific applications as oxygen cost of breathing, respiratory effects of parenteral nutrition or the metabolic effects of various anaesthetic procedures in man.

The in vitro inhibitory action and metabolism of 1-beta-D-arabinofuranosylcytosine (ara-C) on human myeloid (HL-60), B-lymphoid (RPMI-8392), and T-lymphoid (Molt-3) leukemic cells was compared. Ara-C produced greater inhibitory effects in Molt-3 cells than in either HL-60 or RPMI-8392 cells. At a 48 h exposure, ara-C was 7 and 10 times more cytotoxic to Molt-3 cells than to HL-60 and RPMI-8392 cells, respectively. The total ara-C uptake to nucleotides and the formation of 1-beta-D-arabinofuranosylcytosine 5'-triphosphate (ara-CTP) was about 5 times greater in Molt-3 cells than in either HL-60 or RPMI-8392 cells. The incorporation of ara-C into DNA was also higher in Molt-3 cells than in either HL-60 or RPMI-8392 cells. The mean intracellular half-life of ara-CTP was 31.7, 59.4, and 155 min for RPMI-8392, HL-60, and Molt-3 leukemic cells, respectively. The Km and Vmax values of ara-C for deoxycytidine kinase and the feedback inhibition of this enzyme by ara-CTP in the different leukemic cell lines could not explain the differences in metabolism of this analogue in these cells. These data indicate the increased sensitivity of T-lymphoid leukemic cells to ara-C than as compared with B-lymphoid and myeloid leukemic cells was due to an increased rate of formation and a longer half-life of ara-CTP in the T-cells.

CONFUSIONAL STATE IN ICU PATIENTS AFTER. WEANING FROM MECHANICAL VENTILATION AND SEDATION. F Pochard, JJ Lanore, M Belghith, JP Mira, B Renaud, F Bellivier, I Hamy, JF Vax; laire, F Brunet, JF Dhainaut. Confusional state (CS) is known to be frequent in ICU pts. It can result in a poorer prognosis at time of weaning from mechanical ventilation (MV). The aim of this study was to prospectively assess the incidence of CS in pts requiring MV and sedation during more than 24 hours, at time of weaning. Thirty-three survivor pts, Age 48.3, SAPS 15.4, OSF 1.7, OMEGA score 34a, were consecutively included. Reason for admission to ICU was acute lung injury (20), cardiac failure (5), septic shock (3), other (5). Length of MV was 17 days. Pharmacological agents used were flunitrazepam for narcose (33 pts, 35 mg/d, 11 d) and phenoperidine for analgesia (30 pts, 39 mg/d, 12 d). Neither pt had psychosis previous history, nor neurological disease. Fourteen pts suffered CS, defined as the onset of at least 2 of the following signs: disorientation, anxiety, auditory or visual hallucinations, agitation, clouding of consciousness, incoherence, perceptive disturbances, sleep disorders, after weaning from MV. No CS appeared in the 10 pts who had sedation during less than 5 days; in the other23 pts, incidence of CS was 64 % (p < .001). No metabolic etiology was found. The incidence of CS is high in ICU patients when weaning from MV and sedation; this incidence increases with the duration of MV. It should be treated with neuroleptics drugs. Further studies should be performed, in order to consider prevention of CS.

A 65-year-old man developed postsurgical septic shock, unresponsive to plasma volume expansion and administration of dopamine and dobutamine. A continuous norepinephrine infusion was then started and the dose increased to 0.62 micrograms.kg-1.min-1 until the mean arterial pressure was 70 mmHg. Prior to and during the norepinephrine infusion, oxygen consumption was continuously measured with a mass spectrometer system. There was a parallel increase in mean arterial pressure and oxygen consumption (+ 35%). There was also an increase in cardiac index and oxygen delivery. Systemic vascular resistance was only transiently increased. In this case with septic shock, norepinephrine infusion improved hemodynamic variables with an associated increase in oxygen consumption.