Characterization of Lysophospholipase D Activity in Mammalian Cell Membranes.

Lysophosphatidic acid (LPA) is a lipid mediator that binds to G-protein-coupled receptors, eliciting a wide variety of responses in mammalian cells. Lyso-phospholipids generated via phospholipase A2 (PLA2) can be converted to LPA by a lysophospholipase D (lyso-PLD). Secreted lyso-PLDs have been studied in more detail than membrane-localized lyso-PLDs. This study utilized in vitro enzyme assays with fluorescent substrates to examine LPA generation in membranes from multiple mammalian cell lines (PC12, rat pheochromocytoma; A7r5, rat vascular smooth muscle; Rat-1, rat fibroblast; PC-3, human prostate carcinoma; and SKOV-3 and OVCAR-3, human ovarian carcinoma). The results show that membranes contain a lyso-PLD activity that generates LPA from a fluorescent alkyl-lyso-phosphatidylcholine, as well as from naturally occurring acyl-linked lysophospholipids. Membrane lyso-PLD and PLD activities were distinguished by multiple criteria, including lack of effect of PLD2 over-expression on lyso-PLD activity and differential sensitivities to vanadate (PLD inhibitor) and iodate (lyso-PLD inhibitor). Based on several lines of evidence, including siRNA knockdown, membrane lyso-PLD is distinct from autotaxin, a secreted lyso-PLD. PC-3 cells express GDE4 and GDE7, recently described lyso-PLDs that localize to membranes. These findings demonstrate that membrane-associated lyso-D activity, expressed by multiple mammalian cell lines, can contribute to LPA production.

A motion model-guided 4D dose reconstruction for pencil beam scanned proton therapy.

Objective.4D dose reconstruction in proton therapy with pencil beam scanning (PBS) typically relies on a single pre-treatment 4DCT (p4DCT). However, breathing motion during the fractionated treatment can vary considerably in both amplitude and frequency. We present a novel 4D dose reconstruction method combining delivery log files with patient-specific motion models, to account for the dosimetric effect of intra- and inter-fractional breathing variability.Approach.Correlation between an external breathing surrogate and anatomical deformations of the p4DCT is established using principal component analysis. Using motion trajectories of a surface marker acquired during the dose delivery by an optical tracking system, deformable motion fields are retrospectively reconstructed and used to generate time-resolved synthetic 4DCTs ('5DCTs') by warping a reference CT. For three abdominal/thoracic patients, treated with respiratory gating and rescanning, example fraction doses were reconstructed using the resulting 5DCTs and delivery log files. The motion model was validated beforehand using leave-one-out cross-validation (LOOCV) with subsequent 4D dose evaluations. Moreover, besides fractional motion, fractional anatomical changes were incorporated as proof of concept.Main results.For motion model validation, the comparison of 4D dose distributions for the original 4DCT and predicted LOOCV resulted in 3%/3 mm gamma pass rates above 96.2%. Prospective gating simulations on the p4DCT can overestimate the target dose coverage V95%by up to 2.1% compared to 4D dose reconstruction based on observed surrogate trajectories. Nevertheless, for the studied clinical cases treated with respiratory-gating and rescanning, an acceptable target coverage was maintained with V95%remaining above 98.8% for all studied fractions. For these gated treatments, larger dosimetric differences occurred due to CT changes than due to breathing variations.Significance.To gain a better estimate of the delivered dose, a retrospective 4D dose reconstruction workflow based on motion data acquired during PBS proton treatments was implemented and validated, thus considering both intra- and inter-fractional motion and anatomy changes.

Alternatives to patient specific verification measurements in proton therapy: a comparative experimental study with intentional errors.

Patient specific verification (PSV) measurements for pencil beam scanning (PBS) proton therapy are resource-consuming and necessitate substantial beam time outside of clinical hours. As such, efforts to safely reduce the PSV-bottleneck in the clinical work-flow are of great interest. Here, capabilities of current PSV methods to ensure the treatment integrity were investigated and compared to an alternative approach of reconstructing the dose distribution directly from the machine control- or delivery log files with the help of an independent dose calculation (IDC). Scenarios representing a wide range of delivery or work-flow failures were identified (e.g. error in spot position, air gap or pre-absorber setting) and machine files were altered accordingly. This yielded 21 corrupted treatment files, which were delivered and measured with our clinical PSV protocol. IDC machine- and log file checks were also conducted and their sensitivity at detecting the errors compared to the measurements. Although some of the failure scenarios induced clinically relevant dose deviations in the patient geometry, the PSV measurement protocol only detected one out of 21 error scenarios. However, 11 and all 21 error scenarios were detected using dose reconstructions based on the log and machine files respectively. Our data suggests that, although commonly used in particle therapy centers, PSV measurements do a poor job detecting data transfer failures and imperfect delivery machine performance. Machine- and log-file IDCs have been shown to successfully detect erroneous work-flows and to represent a reliable addition to the QA procedure, with the potential to replace PSV.

Validating a Monte Carlo approach to absolute dose quality assurance for proton pencil beam scanning.

For radiotherapy, it is crucial to guarantee that the delivered dose matches the planned dose. Therefore, patient specific quality assurance (QA) of absolute dose distributions is necessary. Here, we investigate the potential of replacing patient specific QA for pencil beam scanned proton therapy with Monte Carlo simulations. First, the set-up of the automated Monte Carlo model is presented with an emphasis on the absolute dose validation. Second, the absolute dose results obtained from the Monte Carlo simulation for a comprehensive set of patient fields are compared to patient specific QA measurements. Absolute doses measured with the Farmer chamber are shown to be 1.4% higher than the doses measured with the Semiflex chamber. For single energy layers, Monte Carlo simulated doses are 2.1% ± 0.4% lower than the ones measured with the ionization chamber and 1.1% ± 1.0% lower than measurements compared to patient field verification measurements. After rescaling to account for this 1.1% discrepancy, 98 fields (94.2%) agree within 2% to measurements, the maximum difference being 2.3%. In conclusion, an automated, easy-to-use Monte Carlo calculation system has been set up. This system reproduced patient specific QA results over a wide range of cases, showing that the time consuming measurements could be reduced or even replaced using Monte Carlo simulations without jeopardizing treatment quality.

Contour scanning for penumbra improvement in pencil beam scanned proton therapy.

Proton therapy, especially in the form of pencil beam scanning (PBS), allows for the delivery of highly conformal dose distributions for complex tumor geometries. However, due to scattering of protons inside the patient, lateral dose gradients cannot be arbitrarily steep, which is of importance in cases with organs at risk (OARs) in close proximity to, or overlapping with, planning target volumes (PTVs). In the PBS approach, physical pencil beams are planned using a regular grid orthogonal to the beam direction. In this work, we propose an alternative to this commonly used approach where pencil beams are placed on an irregular grid along concentric paths based on the target contour. Contour driven pencil beam placement is expected to improve dose confirmation by allowing the optimizer to best enhance the penumbra of irregularly shaped targets using edge enhancement. Its effectiveness has been shown to improve dose confirmation to the target volume and reduce doses to OARs in head-and-neck planning studies. Furthermore, the deliverability of such plans, as well as the dosimetric improvements over conventional grid-based plans, have been confirmed in first phantom based verifications.

Assessing the quality of proton PBS treatment delivery using machine log files: comprehensive analysis of clinical treatments delivered at PSI Gantry 2.

Pencil beam scanning (PBS) proton therapy requires the delivery of many thousand proton beams, each modulated for position, energy and monitor units, to provide a highly conformal patient treatment. The quality of the treatment is dependent on the delivery accuracy of each beam and at each fraction. In this work we describe the use of treatment log files, which are a record of the machine parameters for a given field delivery on a given fraction, to investigate the integrity of treatment delivery compared to the nominal planned dose. The dosimetry-relevant log file parameters are used to reconstruct the 3D dose distribution on the patient anatomy, using a TPS-independent dose calculation system. The analysis was performed for patients treated at Paul Scherrer Institute on Gantry 2, both for individual fields and per series (or plan), and delivery quality was assessed by determining the percentage of voxels in the log file dose distribution within +/- 1% of the nominal dose. It was seen that, for all series delivered, the mean pass rate is 96.4%. Furthermore, this work establishes a correlation between the delivery quality of a field and the beam position accuracy. This correlation is evident for all delivered fields regardless of individual patient or plan characteristics. We have also detailed further usefulness of log file analysis within our clinical workflow. In summary, we have highlighted that the integrity of PBS treatment delivery is dependent on daily machine performance and is specifically highly correlated with the accuracy of beam position. We believe this information will be useful for driving machine performance improvements in the PBS field.

Expression of a mutant prohibitin from the aP2 gene promoter leads to obesity-linked tumor development in insulin resistance-dependent manner.

A critical unmet need for the study of obesity-linked cancer is the lack of preclinical models that spontaneously develop obesity and cancer sequentially. Prohibitin (PHB) is a pleiotropic protein that has a role in adipose and immune functions. We capitalized on this attribute of PHB to develop a mouse model for obesity-linked tumor. We achieved this by expressing Y114F-PHB (m-PHB) from the aP2 gene promoter for simultaneous manipulation of adipogenic and immune signaling functions. The m-PHB mice develop obesity in a sex-neutral manner, but only male mice develop impaired glucose homeostasis and hyperinsulinemia similar to transgenic mice expressing PHB. Interestingly, only male m-PHB mice develop histiocytosis with lymphadenopathy, suggesting that metabolic dysregulation or m-PHB alone is not sufficient for the tumor development and that both are required for tumorigenesis. Moreover, ovariectomy in female m-PHB mice resulted in impaired glucose homeostasis, hyperinsulinemia and consequently tumor development similar to male m-PHB mice. These changes were not observed in sham-operated control m-Mito-Ob mice, further confirming the role of obesity-related metabolic dysregulation in tumor development in m-PHB mice. Our data provide a proof-of-concept that obesity-associated hyperinsulinemia promotes tumor development by facilitating dormant mutant to manifest and reveals a sex-dimorphic role of PHB in adipose-immune interaction or immunometabolism. Targeting PHB may provide a unique opportunity for the modulation of immunometabolism in obesity, cancer and in immune diseases.

Independent dose calculations for commissioning, quality assurance and dose reconstruction of PBS proton therapy.

Pencil beam scanning proton therapy allows the delivery of highly conformal dose distributions by delivering several thousand pencil beams. These beams have to be individually optimised and accurately delivered requiring a significant quality assurance workload. In this work we describe a toolkit for independent dose calculations developed at Paul Scherrer Institut which allows for dose reconstructions at several points in the treatment workflow. Quality assurance based on reconstructed dose distributions was shown to be favourable to pencil beam by pencil beam comparisons for the detection of delivery uncertainties and estimation of their effects. Furthermore the dose reconstructions were shown to have a sensitivity of the order of or higher than the measurements currently employed in the clinical verification procedures. The design of the independent dose calculation tool allows for a high modifiability of the dose calculation parameters (e.g. depth dose profiles, angular spatial distributions) allowing for a safe environment outside of the clinical treatment planning system for investigating the effect of such parameters on the resulting dose distributions and thus distinguishing between different contributions to measured dose deviations. The presented system could potentially reduce the amount of patient-specific quality assurance measurements which currently constitute a bottleneck in the clinical workflow.

Assessment of posttranslational modification of mitochondrial proteins.

Mitochondria play vital roles in the maintenance of cellular homeostasis. They are a storehouse of cellular energy and antioxidative enzymes. Because of its immense role and function in the development of an organism, this organelle is required for the survival. Defects in mitochondrial proteins lead to complex mitochondrial disorders and heterogeneous diseases such as cancer, type 2 diabetes, and cardiovascular and neurodegenerative diseases. It is widely known in the literature that some of the mitochondrial proteins are regulated by posttranslational modifications. Hence, designing methods to assess these modifications in mitochondria will be an important way to study the regulatory roles of mitochondrial proteins in greater detail. In this chapter, we outlined procedures to isolate mitochondria from cells and separate the mitochondrial proteins by two-dimensional gel electrophoresis and identify the different posttranslational modifications in them by using antibodies specific to each posttranslational modification.

Synthesis and anticancer activity of new flavonoid analogs and inconsistencies in assays related to proliferation and viability measurements.

Flavonoids have been studied intensely for their ability to act as anti-carcinogenic, anti-inflammatory, anti-viral and anti-aging agents and are often marketed as supplements related to their anti-inflammatory activity. Previous studies have primarily focused on the effects of polar natural flavonoids. We examined the activity of novel hydrophobic and lipophilic flavonols against human DU-145 and PC-3 prostate cancer cell lines. All flavonol analogs were more active than the naturally occurring flavonols quercetin, kaempferol, kaempferide and galangin. The most potent analogs were 6.5-fold more active against DU-145 and PC-3 cells than quercetin and fell within the biologically relevant concentration range (low micromolar). We also evaluated the potential toxic effects of flavonol analogs on normal cells, an assessment that has frequently been ignored when studying the anticancer effects of flavonoids. During these analyses, we discovered that various metabolic and DNA staining assays were unreliable methods for assessing cell viability of flavonoids. Flavonoids reduce colorimetric dyes such as MTT and Alamar Blue in the absence of cells. We showed that flavonol-treated prostate cancer cells were stained less intensely with crystal violet than untreated cells at non-toxic concentrations. The trypan blue exclusion assay was selected as a reliable alternative for measuring cell viability.

Structure-activity relationship studies of flavonol analogues on pollen germination.

Flavonoids are polyphenolic compounds required in the fertilization process in many, if not all, plants. However, the exact biological mechanism(s) and the interacting proteins are unknown. To determine the characteristics important in activating or inhibiting the pollination sequence, a structure-activity relationship analysis of natural and synthetic flavonols was conducted. Flavonol analogues were synthesized through a modified "one-pot" procedure that utilized a Baker-Venkataraman type rearrangement and a Suzuki-Miyaura cross-coupling of a halo-flavonol with an organotrifluoroborate. Of the flavonols tested, kaempferol was the only compound to act as a full agonist. The other smaller, less sterically hindered flavonols (galangin, kaempferide, and 4'-methyl flavonol) acted as partial agonists. Larger more hydrophobic flavonol analogues (3'- and 4'-benzoyl, 3'- and 4'-phenyl, and 3'- and 4'-iodo flavonols) had minimal or no agonist activity. Competition assays between kaempferol and these minimally activating flavonols showed that these analogues inhibited the action of kaempferol in a manner consistent with noncompetitive antagonism. The results suggest that steric hindrance is the most important factor in determining a good agonist. Hydrogen bonding also had a positive effect as long as the substituent did not cause any steric hindrance.

Mutually exclusive acetylation and ubiquitylation among enzymes involved in glucose metabolism.

The posttranslational modification (PTM) in protein occurs in a regiospecific manner. In addition, the most commonly occurring PTMs involve similar residues in proteins such as acetylation, ubiquitylation, methylation and sumoylation at the lysine residue and phosphorylation and O-GlcNAc modification at serine/threonine residues. Thus, the possibility of modification sites where two such PTMs may occur in a mutually exclusive manner (ME-PTM) is much higher than known. A recent surge in the identification and the mapping of the commonly occurring PTMs in proteins has revealed that this is indeed the case. However, in what way such ME-PTM sites are regulated and what could be their relevance in the coordinated network of protein function remains to be known. To gain such potential insights in a biological context, we analyzed two most prevalent PTMs on the lysine residue by acetylation and ubiquitylation along with the most abundant PTM in proteins by phosphorylation among enzymes involved in glucose metabolism, a fundamental process in biology. The analysis of the PTM data sets has revealed two important clues that may be intrinsically associated with their regulation and function. First, the most commonly occurring PTMs by phosphorylation, acetylation and ubiquitylation are widespread and clustered in most of the enzymes involved in glucose metabolism; and the prevalence of phosphorylation sites correlates with the number of acetylation and ubiquitylation sites including the ME-modification sites. Second, the prevalence of ME-acetylation/ubiquitylation sites is exceptionally high among enzymes involved in glucose metabolism and have distinct pattern among the subset of enzymes of glucose metabolism such as glycolysis, tricarboxylic acid (TCA) cycle, glycogen synthesis, and the irreversible steps of gluconeogenesis. We hypothesize that phosphorylation including tyrosine phosphorylation plays an important role in the regulation of ME-acetylation/ubiquitylation sites and their similar pattern among the subset of functionally related proteins allows their coordinated regulation in the normal physiology. Similarly their coordinated dysregulation may underlie the disease processes such as reprogrammed metabolism in cancer, obesity, type 2 diabetes, and cardiovascular diseases. Our hypothesis provides an opportunity to understand the regulation of ME-PTMs in proteins and their relevance at the network level and is open for experimental validation.

SU-E-T-461: Fractionation Schedule Optimization for Lung Cancer Treatments Using Radiobiological and Dose Distribution Characteristics.

PURPOSE: Lung cancer radiotherapy treatments employ a wide variety of fractionation protocols. The choice among protocols mostly depends on the size of the target volume (GTV or ITV) and the volume of normal tissue receiving a critical dose. Rigorous mathematical criteria for normal tissue (NT) dose distributions were derived to determine the type of dose per fraction schedule that maximizes linear-quadratic tumor effect. METHODS: Selecting the individual doses per fraction that maximize a linear-quadratic effect in the tumor while constraining the normal tissue complication probability according to the Lyman-Kutcher-Burman model leads to an optimization problem. For time-independent parameters, the solution is always an equal dose per fraction schedule; depending on parameter values, two different class solutions are suggested: minimal number of fractions clinically realized with hypo-fractionation, or minimizing dose per fraction clinically realized with standard- or hyper-fractionation. The value of a single scale-free "bifurcation" number, derived from the DVH of the NT dose distribution suggests which solution is preferred for a given plan with respect to a given normal tissue. The clinical relevance of the bifurcation number in selecting fractionation schemes was tested for 30 patients previously treated for non-small-cell lung cancer according to various fractionation protocols. RESULTS: The bifurcation numbers for both lung and esophagus were a good classifier for the hypofractionated and the conventional fractionation groups. The variability of the numbers within patients of the conventional fractionation group was much smaller than the variability of the treated ITV volumes or the ITV to lung volume ratios. The prescribed fractionations were also consisted with the currently accepted alpha-beta values for tumor (10) and radiation-induced pneumonities in the lung (4). CONCLUSIONS: Model-based criteria such as the bifurcation number may replace the more empirical volume criteria to decide the optimal fractionation protocol once the dose distribution has been optimized.

Conformational changes in bacteriophage P22 scaffolding protein induced by interaction with coat protein.

Many prokaryotic and eukaryotic double-stranded DNA viruses use a scaffolding protein to assemble their capsid. Assembly of the double-stranded DNA bacteriophage P22 procapsids requires the interaction of 415 molecules of coat protein and 60-300 molecules of scaffolding protein. Although the 303-amino-acid scaffolding protein is essential for proper assembly of procapsids, little is known about its structure beyond an NMR structure of the extreme C-terminus, which is known to interact with coat protein. Deletion mutagenesis indicates that other regions of scaffolding protein are involved in interactions with coat protein and other capsid proteins. Single-cysteine and double-cysteine variants of scaffolding protein were generated for use in fluorescence resonance energy transfer and cross-linking experiments designed to probe the conformation of scaffolding protein in solution and within procapsids. We showed that the N-terminus and the C-terminus are proximate in solution, and that the middle of the protein is near the N-terminus but not accessible to the C-terminus. In procapsids, the N-terminus was no longer accessible to the C-terminus, indicating that there is a conformational change in scaffolding protein upon assembly. In addition, our data are consistent with a model where scaffolding protein dimers are positioned parallel with one another with the associated C-termini.

Is above knee bypass defunct?

As endovascular treatments improve, the inevitable progress will result in the abandonment of conventional bypasses. First and foremost in this regard is the use of above knee bypass, particularly with prosthetic graft material. Already, endovascular success approaches or exceeds the patency seen with this bypass technique. As a result, in centers with endovascular expertise in infrainguinal intervention, bypass surgery is increasingly being replaced by these techniques and conventional bypass surgery is disappearing. Over the next few years, above knee bypass will be replaced by endovascular techniques in most centers as our results with these techniques improve.

Is ultrasound more accurate than axial computed tomography for determination of maximal abdominal aortic aneurysm diameter?

OBJECTIVE(S): Clinical assessment of maximal abdominal aortic aneurysm (AAA) diameter assumes clinical equivalency between ultrasound (US) and axial computed tomography (CT). Three-dimensional (3D) CT reconstruction allows for the assessment of AAA in the orthogonal plane and avoids oblique cuts due to AAA angulation. This study was undertaken to compare maximal AAA diameter by US, axial CT, and orthogonal CT, and to assess the effect that AAA angulation has on each measurement. METHODS: Maximal AAA diameter by US (US(max)), axial CT (axial(max)), and orthogonal CT (orthogonal(max)) along with aortic angulation and minor axis diameters were measured prospectively. Spiral CT data was processed by Medical Media Systems (West Lebanon, NH) to produce computerized axial CT and reformatted orthogonal CT images. The US technologists were blinded to all CT results and vice versa. RESULTS: Thirty-eight patients were analyzed. Mean axial(max) (58.0 mm) was significantly larger (P<0.05) than US(max) (53.9 mm) or orthogonal(max) (54.7 mm). The difference between US(max) and orthogonal(max) (0.8 mm) was insignificant (P>0.05). When aortic angulation was <==25 degrees, axial(max) (55.3 mm), US(max) (54.3 mm), and orthogonal(max) (54.1 mm) were similar (P>0.05); however, when aortic angulation was >25 degrees, axial(max) (60.1 mm) was significantly larger (P<0.001) than US(max) (53.8 mm) and orthogonal(max) (55.0 mm). The limits of agreement (LOA) between axial(max) and both US(max) and orthogonal(max) was poor and exceeded clinical acceptability (+/-5 mm). The variation between US(max) and orthogonal(max) was minimal with an acceptable LOA of -2.7 to 4.5 mm. CONCLUSION: Compared to axial CT, US is a better approximation of true perpendicular AAA diameter as determined by orthogonal CT. When aortic angulation is greater than 25 degrees axial CT becomes unreliable. However, US measurements are not affected by angulation and agree strongly with orthogonal CT measurements.

Successful targeted radiotherapy with 90Y-DOTATOC in a patient with Merkel cell carcinoma. A Case Report.

Merkel cell carcinomas (MCC) belong to the family of neuroendocrine tumors. In addition to other markers, they express somatostatin receptors. They are uncommon, highly malignant skin tumors with an aggressive clinical course. They develop in sun-exposed areas of the skin, mostly in elderly patients. In addition to frequent locoregional recurrences, there is a high incidence of distant metastases. Treatment is stage dependent and consists of operation and chemo- and/or radiotherapy, respectively. The advanced age of patients often impedes adequate therapy. (90)Y-DOTATOC is a novel radiolabeled somatostatin analogue containing the active octapeptide of somatostatin. It is very well tolerated and offers the option of treating somatostatin receptor-positive tumors by targeted radiotherapy. We report the case of an 83-year-old woman with recurrent MCC of the left cheek. The primary tumor and several relapses were treated with surgery and locoregional radiotherapy. After the 3rd relapse, she was treated 4 times with (90)Y-DOTATOC and two complete remissions were achieved. The fourth administration after the 2nd relapse was ineffective and conventional chemotherapy was started. There were no side effects of the (90)Y-DOTATOC. We conclude that due to its good tolerability, (90)Y-DOTATOC therapy should be evaluated further as a new therapy for somatostatin receptor-positive MCC.

Prodrug modification increases potassium tricyclo[5.2.1.0(2,6)]-decan-8-yl dithiocarbonate (D609) chemical stability and cytotoxicity against U937 leukemia cells.

Potassium tricyclo[5.2.1.0(2,6)]-decan-8-yl dithiocarbonate (D609) is a selective antitumor agent, potent antioxidant, and cytoprotectant. It has the potential to be developed as a unique chemotherapeutic agent that may provide dual therapeutic benefits against cancer, e.g., enhancing tumor cell death while protecting normal tissues from damage. However, D609 contains a dithiocarbonate (xanthate) group [O-C(=S)S(-)/O-C(=S)SH], which is chemically unstable, being readily oxidized to form a disulfide bond with subsequent loss of all biological activities. Therefore, we developed the synthesis of a series of S-(alkoxyacyl) D609 prodrugs by connecting the xanthate group of D609 to an ester via a self-immolative methyleneoxyl group. These S-(alkoxylacyl)-D609 prodrugs are designed to release D609 in two steps: esterase-catalyzed hydrolysis of the acyl ester bond followed by conversion of the resulting hydroxymethyl D609 to formaldehyde and D609. Three S-(alkoxyacyl) D609 prodrugs were synthesized by varying the steric bulkiness of the acyl group. These prodrugs are stable to ambient conditions, but readily hydrolyzed by esterases to liberate D609 in a controlled manner. More importantly, the lead prodrug methyleneoxybutyryl D609 is biologically more effective than D609 in inhibiting sphingomyelin synthase, thereby increasing the level of ceramide and inducing apoptosis in U937 leukemia cells. The prodrug has a significantly lower LD(50) value than that of D609 (56.6 versus 117 microM) against U937 cells. These findings demonstrate that prodrug modification of the xanthate moiety with an alkoxyacyl group can improve D609 oxidative stability and enhance its antitumor activity.

Inhibition of wild-type human immunodeficiency virus and reverse transcriptase inhibitor-resistant variants by Phyllanthus amarus.

Substantial progress has been made in research on natural products which effectively inhibit HIV-1 replication. Many active compounds were isolated from traditionally used medicinal plants including Phyllanthus species. This study shows that aqueous as well as alcohol-based Phyllanthus amarus extracts potently inhibit HIV-1 replication in HeLa CD4(+) cells with 50% effective concentration (EC(50)) values ranging from 0.9 to 7.6 microg/ml. A gallotannin enriched fraction showed enhanced activity (0.4 microg/ml), and the purified gallotannins geraniin and corilagin were most active (0.24 microg/ml). HIV-1 replication was also blocked in CD4(+) lymphoid cells with comparable EC(50) values. Applying a cell-based internalization assay, we could demonstrate 70-75% inhibition of virus uptake at concentrations of 2.5 microg/ml for the water/alcohol extract and geraniin. In addition, a concentration-dependent inhibition of HIV-1 reverse transcriptase (RT) could be demonstrated in vitro. The 50% inhibitory concentration (IC(50)) values varied from 1.8 to 14.6 microg/ml. The ability to inhibit replication of a variety of RT inhibitor-resistant HIV-1 strains points to the potential of P. amarus extracts, as natural products, in the chemotherapy of HIV infections.

Endotension after endovascular aneurysm repair: the Ancure experience.

PURPOSE: The expansion of aneurysms after endovascular repair is a consequence of persistent sac pressure, usually resulting from an endoleak. Several authors have suggested that sac expansion can occur even in the absence of endoleak, referring to this phenomenon as endotension. This study undertakes a review of the largest US endograft trial data to better define the significance of aneurysm expansion in the absence of endoleak. METHODS: The core laboratory imaging database from the Ancure (Guidant) endovascular graft Phase I and Phase II trials approved by the Food and Drug Administration was reviewed with attention to aneurysm size and endoleak. Aneurysm size was measured with standardized two-dimensional computed tomography (CT) scan at the area of largest initial aneurysm diameter. Endoleak was detected with CT scans, color duplex ultrasound scans, and angiography in selected cases. Patients were evaluated at baseline, 3 months, 6 months, 12 months, and every 12 months thereafter. An endograft was classified as leaking if any endoleak was detected with any modality at any time point. RESULTS: A total of 658 patients were entered into these protocols and the data submitted to the core laboratory. A control group of 120 conventional aortic patients and a group of 62 without baseline CT data were excluded from further analysis. Of the remaining 476 patients, 144 (60 tube, 60 bifurcated, and 24 mono-iliac) were free of endoleak at all intervals and had baseline CT measurements to allow comparison. Overall, the average size decrease in this nonleaking group was 9.9 +/- 9.4 mm (range, -50.6-11.1 mm) at a mean follow-up of 23.3 months. Evaluation for overall aneurysm expansion revealed 17 patients who had an increase of 2.3 +/- 2.9 mm (range, 0.3-11.1 mm) at a mean follow-up of 14.1 months. Only two patients without evidence of endoleak exhibited growth of more than 5 mm at maximum follow-up (7.6 mm at 12 months and 11.1 mm at 36 months). Additional analysis of sealed endoleaks and late endoleaks failed to demonstrate any group with expansion in the absence of detectable endoleak. CONCLUSIONS: Endotension appears to be rare in this large series of prospectively evaluated endografts. From this review, endotension seems more likely to represent missed endoleak than true aneurysm expansion in the absence of perigraft flow.