Differential Biological Effects Research Articles

Glycoengineering of E-Selectin Ligands by Intracellular versus Extracellular Fucosylation Differentially Affects Osteotropism of Human Mesenchymal Stem Cells.

Human mesenchymal stem cells (MSCs) hold great promise in cellular therapeutics for skeletal diseases but lack expression of E-selectin ligands that direct homing of blood-borne cells to bone marrow. Previously, we described a method to engineer E-selectin ligands on the MSC surface by exofucosylating cells with fucosyltransferase VI (FTVI) and its donor sugar, GDP-Fucose, enforcing transient surface expression of the potent E-selectin ligand HCELL with resultant enhanced osteotropism of intravenously administered cells. Here, we sought to determine whether E-selectin ligands created via FTVI-exofucosylation are distinct in identity and function to those created by FTVI expressed intracellularly. To this end, we introduced synthetic modified mRNA encoding FTVI (FUT6-modRNA) into human MSCs. FTVI-exofucosylation (i.e., extracellular fucosylation) and FUT6-modRNA transfection (i.e., intracellular fucosylation) produced similar peak increases in cell surface E-selectin ligand levels, and shear-based functional assays showed comparable increases in tethering/rolling on human endothelial cells expressing E-selectin. However, biochemical analyses revealed that intracellular fucosylation induced expression of both intracellular and cell surface E-selectin ligands and also induced a more sustained expression of E-selectin ligands compared to extracellular fucosylation. Notably, live imaging studies to assess homing of human MSC to mouse calvarium revealed more osteotropism following intravenous administration of intracellularly-fucosylated cells compared to extracellularly-fucosylated cells. This study represents the first direct analysis of E-selectin ligand expression programmed on human MSCs by FTVI-mediated intracellular versus extracellular fucosylation. The observed differential biologic effects of FTVI activity in these two contexts may yield new strategies for improving the efficacy of human MSCs in clinical applications. Stem Cells 2016;34:2501-2511.

Abstract 2957: Generation of a novel, mono-specific, antibody against short Isoform of DCLK1 (DCLK1-S): a novel biomarker of colon carcinogenesis in patients

Abstract Double-Cortin Like Kinase I (DCLK1) marks colon cancer stem cells in mouse colons. Adenomas (Ads)/human colorectal cancers (hCRCs) express low to high levels of DCLK1. We recently reported that the canonical long isoform (DCLK1-L), transcribed from 5’(α)-promoter, is mainly expressed in normal human colons, while a novel short isoform (DCLK1-S), transcribed from IntronV (β)-promoter, is expressed in colonic tumors, with >80% hCRCs expressing significant levels of DCLK1-S (O’Connell et al., Sci Rep, 2015). We additionally reported that CRC patients, expressing high levels of DCLK1-S, had significantly lower overall survival compared to low expressers (O’Connell et al., Sci Rep, 2015), suggesting prognostic/diagnostic value of measuring DCLK1-S levels in Ads/CRCs. Since expression of DCLK1-S by CRCs was recently discovered by us, commercial antibodies (Ab) are not available for specifically identifying DCLK1-S in Ad/Carcinoma samples of patients for diagnostic/prognostic purposes. We succeeded in generating a monospecific polyclonal Ab against the few unique amino-acid sequences of DCLK1-S. By immunohistochemistry and western blot analysis, the Ab was confirmed to be specific for DCLK1-S, with no cross-reactivity for DCLK1-L, and little or no background. The DCLK1-S-Ab did not stain normal human colonic crypts, confirming absence of DCLK1-S in normal colons. In colorectal Ads, DCLK1-S was mainly located in the cytoplasm, with <10% in the nucleus, while in adenocarcinomas (AdCAs), intense staining of DCLK1-S was observed in both cytosol and nucleus, demonstrating a stage-specific increase in staining during Ad-AdCA sequence of colon carcinogenesis, with increasing localization in the nucleus. DCLK1-S-Ab and a DCLK1-L specific Ab was also used to investigate localization of the long vs short isoforms of DCLK1 in cells by immuno-electron microscopy, demonstrating important differences in their sub-cellular localization, which maybe reflective of differential biological effects. The DCLK1-S-Ab is currently being used by us to measure relative expression of DCLK1-S in Ad samples in a retrospective study, in collaboration with colleagues in the departments of pathology and biostatistics, for purposes of examining the usefulness of the Ab for prognostic purposes. Conclusion: We have generated a specific antibody against the novel short isoform of cancer stem cell marker, DCLK1, which will allow us for the first time to detect this novel cancer specific molecular biomarker in colonic tumors for prognostic purposes. Rather than lengthy and cumbersome processing, as required for most molecular markers, the Ab will allow physicians, in any setting, to make informed decisions, such as time to colonoscopy for patients, which is currently based on mostly morphological/pathological parameters, and can result in missing or over scoping patients. Citation Format: Shubhashish Sarkar, Malaney O. Connell, Vsevolod L. Popov, Gurinder Luthra, Pomila Singh. Generation of a novel, mono-specific, antibody against short Isoform of DCLK1 (DCLK1-S): a novel biomarker of colon carcinogenesis in patients. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2957.

The effects of lactate and acid on articular chondrocytes function: Implications for polymeric cartilage scaffold design

Poly (lactic-co-glycolic acid) (PLGA) and poly-l-lactate acid (PLLA) are biodegradable polymers widely utilized as scaffold materials for cartilage tissue engineering. Their acid degradation products have been widely recognized as being detrimental to cell function. However, the biological effects of lactate, rather than lactic acid, on chondrocytes have never been investigated. This is the major focus of this study. The amounts of lactate and the pH value (acid) of the PLGA and PLLA degradation medium were measured. The effects of PLGA and PLLA degradation medium, as well as different lactate concentrations and timing of exposure on chondrocytes proliferation and cartilage-specific matrix synthesis were investigated by various techniques including global gene expression profiling and gene knockdown experiments. It was shown that PLGA and PLLA degradation medium differentially regulated chondrocyte proliferation and matrix synthesis. Acidic pH caused by lactate inhibited chondrocyte proliferation and matrix synthesis. The effect of lactate on chondrocyte matrix synthesis was both time and dose dependent. A lactate concentration of 100mM and exposure duration of 8h significantly enhanced matrix synthesis. Lactate could also inhibit expression of cartilage matrix degradation genes in osteoarthritic chondrocytes, such as the major aggrecanase ADAMTS5, whilst promoting matrix synthesis simultaneously. Pulsed addition of lactate was shown to be more efficient in promoting COL2A1 expression. Global gene expression data and gene knock down experiments demonstrated that lactate promote matrix synthesis through up-regulation of HIF1A. These observed differential biological effects of lactate on chondrocytes would have implications for the future design of polymeric cartilage scaffolds. Statement of SignificanceLactic acid is a widely used substrate for polymers synthesis, PLGA and PLLA in particular. Although physical and biological modifications have been made on these polymers to make them be better cartilage scaffolds, little concern has been given on the biological effect of lactic acid, the main degradation product of these polymers, on chondrocytes. Our finding illustrates the differential biological function of lactate and acid on chondrocytes matrix synthesis. These results can facilitate future design of lactate polymers-based cartilage scaffolds

TH‐CD‐209‐12: Spatial Mapping of Scanned Proton Biologic Effect Using the High‐Throughput Technique, Continued

Purpose:To investigate the biologic effects of scanned protons by evenly sampling dose‐averaged LET (LETd) values.Methods:Our previous high‐throughput clonogenic study demonstrated a distinct relationship between RBE and LETd. However, our initial experimental design resulted in over‐sampling the low LETd values in the plateau region of the Bragg curve while under‐sampling in the region proximal to the Bragg peak as well as the high LETd values in the distal edge of the Bragg curve. To further examine the relationship between RBE and LETd, we refined the experimental design to more evenly sample proton LETd values from 1 to 20 keV/µm by optimizing the thicknesses of the irradiation jig steps. We used the clonogenic survival as the biological endpoint for the H460 lung cancer cell line cultured in 96‐well plates (12 columns by 8 rows). In the irradiation, the 8 wells in each column received a uniform dose‐LETd pair. The dose‐LETd pairs of the 12 different columns were sampled along the Bragg curve of 81.4 MeV scanned protons. Five peak dose levels from 1.5 Gy to 7.5 Gy were delivered with an increment of 1.5 Gy in the preliminary test. Two 96‐well plates were irradiated simultaneously to decrease the statistical uncertainties.Results:In the proximal region, for LETd = 5 keV/µm and 8 keV/µm, we did not observe any distinct differential biologic effects between the survival curves. At the Bragg peak (LETd = 9.5 keV/µm) and in the distal edge, irradiation with increasing LET values resulted in decreasing cell survival.Conclusion:The survival curves from the new experimental design support our previous findings that below 10 keV/µm, the LET effect in cell kill is obscured, but above 10 keV/µm, the biologic effects increase with LETd.Funding Support: U19 CA021239‐35 and R21 CA187484‐01

Impact of the α-Synuclein Initial Ensemble Structure on Fibrillation Pathways and Kinetics.

The presence of intracellular filamentous α-synuclein (αS) aggregates is a common feature in Parkinson's disease. Recombinant expressed and purified human αS is also capable of forming fibrils in vitro. Many studies have shown that solution conditions heavily influence αS fibrillation kinetics, fibril structure, and morphology that exhibit differential biological effects. Nevertheless, the αS ensemble structure in various solution conditions is not well characterized; furthermore, how the initial solution ensemble structures impact αS assembly kinetics and pathways that result in diverse fibril structure and morphology remains elusive. Here, we mainly employed NMR spectroscopy to characterize the initial ensemble structure of αS in the presence or absence of a 150 mM sodium chloride (NaCl) solution, where two polymorphs of αS were demonstrated in previous studies. Our data show that αS exhibits distinct conformations and fibrillation kinetics in these two solutions. αS adopts a more compact and rigid ensemble structure that has faster fibrillation kinetics in the absence of NaCl. On the basis of the ensemble structure and dynamics, we proposed a possible molecular mechanism in which αS forms different polymorphs under these two conditions. Our results provide novel insights into how the initial conformation impacts fibrillation pathways and kinetics, suggesting that a microenvironment can be used to regulate the intrinsically disordered proteins assembly.

The osteogenic capacity of biomimetic hierarchical micropore/nanorod-patterned Sr-HA coatings with different interrod spacings

Advanced titanium based bone implant with fast established, rigid and stable osseointegration is stringently needed in clinic. Here the hierarchical micropore/nanorod-patterned strontium doped hydroxyapatite (Ca9Sr1(PO4)6(OH)2, Sr1-HA) coatings (MNRs) with different interrod spacings varying from about 300 to 33nm were developed. MNRs showed dramatically differential biological performance closely related to the interrod spacing. Compared to micropore/nanogranule-patterned Sr1-HA coating (MNG), MNRs with an interrod spacing of larger than 137nm resulted in inhibited in vitro mesenchymal stem cell functions and in vivo osseointegration, while those of smaller than 96nm gave rise to dramatically enhanced the biological effect, especially those of mean 67nm displayed the best effect. The differential biological effect of MNRs was related to their modulation on the focal adhesion mediated mechanotransduction. These results suggest that MNRs with a mean interrod spacing of 67nm may give rise to an advanced implant of improved clinical performance.

Foetal bovine serum-derived exosomes affect yield and phenotype of human cardiac progenitor cell culture.

Introduction: Cardiac progenitor cells (CPCs) represent a powerful tool in cardiac regenerative medicine. Pre-clinical studies suggest that most of the beneficial effects promoted by the injected cells are due to their paracrine activity exerted on endogenous cells and tissue. Exosomes are candidate mediators of this paracrine effects. According to their potential, many researchers have focused on characterizing exosomes derived from specific cell types, but, up until now, only few studies have analyzed the possible in vitro effects of bovine serum-derived exosomes on cell proliferation or differentiation. Methods: The aim of this study was to analyse, from a qualitative and quantitative point of view, the in vitro effects of bovine serum exosomes on human CPCs cultured either as cardiospheres or as monolayers of cardiosphere-forming cells. Results: Effects on proliferation, yield and molecular patterning were detected. We show, for the first time, that exogenous bovine exosomes support the proliferation and migration of human cardiosphere-forming cells, and that their depletion affects cardiospheres formation, in terms of size, yield and extra-cellular matrix production. Conclusion: These results stress the importance of considering differential biological effects of exogenous cell culture supplements on the final phenotype of primary human cell cultures.

Exosomes from bulk and stem cells from human prostate cancer have a differential microRNA content that contributes cooperatively over local and pre-metastatic niche.

The different prostate cancer (PCa) cell populations (bulk and cancer stem cells, CSCs) release exosomes that contain miRNAs that could modify the local or premetastatic niche. The analysis of the differential expression of miRNAs in exosomes allows evaluating the differential biological effect of both populations on the niche, and the identification of potential biomarkers and therapeutic targets. Five PCa primary cell cultures were established to originate bulk and CSCs cultures. From them, exosomes were purified by precipitation for miRNAs extraction to perform a comparative profile of miRNAs by next generation sequencing in an Illumina platform. 1839 miRNAs were identified in the exosomes. Of these 990 were known miRNAs, from which only 19 were significantly differentially expressed: 6 were overexpressed in CSCs and 13 in bulk cells exosomes. miR-100-5p and miR-21-5p were the most abundant miRNAs. Bioinformatics analysis indicated that differentially expressed miRNAs are highly related with PCa carcinogenesis, fibroblast proliferation, differentiation and migration, and angiogenesis. Besides, miRNAs from bulk cells affects osteoblast differentiation. Later, their effect was evaluated in normal prostate fibroblasts (WPMY-1) where transfection with miR-100-5p, miR-21-5p and miR-139-5p increased the expression of metalloproteinases (MMPs) -2, -9 and -13 and RANKL and fibroblast migration. The higher effect was achieved with miR21 transfection. As conclusion, miRNAs have a differential pattern between PCa bulk and CSCs exosomes that act collaboratively in PCa progression and metastasis. The most abundant miRNAs in PCa exosomes are interesting potential biomarkers and therapeutic targets.

SU‐E‐T‐526: Irradiation of Human Cell Lines Using Carbon Ions: Real Time Dosimetry Using Gaf‐Chromic Film

Purpose:The purpose of this study is to investigate and quantify several factors affecting biological effects of carbon ions such as cell type, dose, energy and position where the cells are irradiated along the pristine Bragg curve.Methods:Experiments to quantify clonogenic cell survival in three human lung cancer cell lines and a fibroblast cell line were performed at the NASA Space Radiation Laboratory, BNL, Upton, USA. A system of water or media‐filled T25 flasks lined up along the beam axis was designed so that the cell‐containing surfaces of the flasks were placed at specific depths along the Bragg curve. Gaf‐chromic films were placed between the flasks to monitor the dose distribution in the sample as soon as the irradiation was finished. Additional studies were conducted at four selected depths along the Bragg curve to obtain full cell survival dose response curves for the four cell lines.Results:The percent depth dose of the beams was determined using an ionization chamber and showed that the physical Bragg peak is at 22.5 cm water depth. However, the clonogenic cell survival data indicated that the maximum cell killing occurred at 21.5 cm. Gaf‐chromic films revealed some inhomogeneity in the dose distribution on the flasks near the peak, presumably due to lack of scattering from the sides of the flasks, which might account for the differences. Depending on the cell line and radiation dose, the maximum cell killing (i.e., the greatest RBE) is at the 21.5 (the peak) or 24 cm (distal fall off) depth.Conclusion:There is a difference in biological effect along the Bragg curve of a carbon ion beam, indicating an elevated RBE at or beyond the end of the range. Gaf‐chromic films are proven to be effective in monitoring the 2D irradiation pattern to the flasks.Research supported by NIH/NCI through grant no. R21 CA182259.

The trans-octadecenoic fatty acid profile of beef: Implications for global food and nutrition security

In addition to being an important source of macro- and micronutrients, beef can also be a source of trans (t)-18:1 fatty acids (FA). Overall, trans-FA have been associated with an increased incidence of cardiovascular disease in humans. Recent research has, however, shown that t-18:1 FA are a group of distinct isomers which elicit differential biological effects. The current review examines the potential of different nutritional strategies to enrich potentially beneficial or deplete detrimental t-18:1 isomers in beef. The possible contribution of seemingly beneficial t-18:1 isomers to global food and nutrition security is highlighted. The review also describes the importance of developing a regulatory framework to govern the consumption of individual t-18:1 isomers to enhance and sustain their contribution to food and nutrition security.

Heterodimer-specific TLR2 stimulation results in divergent functional outcomes in B-cell precursor acute lymphoblastic leukemia.

Reports of spontaneous acute lymphoblastic leukemia (ALL) remissions following severe bacterial infections suggest that bacterial components may trigger elimination of ALL. To date, TLR2, which recognizes a broad range of bacterial pathogens through TLR1 or TLR6 heterodimerization, has not been fully evaluated for direct effects on ALL. Studies investigating TLR2 signaling in other tumor cell types utilizing single ligands have yielded contradictory results, and comparative, heterodimer-specific analyses of TLR2 stimulation are lacking. In this study, we report that two well-characterized heterodimer-specific TLR2 ligands, Pam3 CSK4 (TLR2/1), and Pam2 CSK4 (TLR2/6), induce ALL cell lines and primary ALL samples to upregulate CD40 expression. However, only Pam3 CSK4 triggers Caspase-8-mediated apoptosis and sensitizes cells to vincristine-mediated cytotoxicity. Consistent with this result, stimulation of ALL cells through TLR2/1 or TLR2/6 activates Mal, p38 and the NF-κB and PI3K signaling pathways with divergent kinetics that may underlie their distinct downstream effects. Our results reveal a novel branching in downstream responses to heterodimer-specific TLR2 stimulation in ALL cells and emphasize the need for comparative studies to determine differential biological effects observed in specific tumor cells. Based on our results, TLR2/1 ligand Pam3 CSK4 possesses potential for generating anti-ALL activity through its direct effects on leukemic blasts.

Long-term Coexposure to Hexavalent Chromium and B[a]P Causes Tissue-Specific Differential Biological Effects in Liver and Gastrointestinal Tract of Mice.

Complex mixtures of environmental agents often cause mixture-specific health effects that cannot be accounted for by a single mechanism. To study the biological effects of exposure to a mixture of chromium-VI and benzo[a]pyrene (B[a]P), often found together in the environment, we exposed mice for 60 days to 0, 55, 550, or 5500 ppb Cr(VI) in drinking water followed by 90 days of coexposure to B[a]P at 0, 1.25, 12.5, or 125 mg/kg/day and examined liver and gastrointestinal (GI) tract for exposure effects. In the liver, the mixture caused more significant histopathology than expected from the sum of effects of the individual components, while in the GI tract, Cr(VI) alone caused significant enterocyte hypertrophy and increases in cell proliferation and DNA damage that were also observed in mice coexposed to B[a]P. Expression of genes involved in drug metabolism, tumor suppression, oxidative stress, and inflammation was altered in mixed exposures relative to control and to singly exposed mice. Drug metabolism and oxidative stress genes were upregulated and tumor suppressor and inflammation genes downregulated in the proximal GI tract, whereas most markers were upregulated in the distal GI tract and downregulated in the liver. Oral exposure to Cr(VI) and B[a]P mixtures appears to have tissue-specific differential consequences in liver and GI tract that cannot be predicted from the effects of each individual toxicant. Tissue specificity may be particularly critical in cases of extended exposure to mixtures of these agents, as may happen in the occupational setting or in areas where drinking water contains elevated levels of Cr(VI).

Inhibition of platelet activation and thrombus formation by adenosine and inosine: studies on their relative contribution and molecular modeling.

BackgroundThe inhibitory effect of adenosine on platelet aggregation is abrogated after the addition of adenosine-deaminase. Inosine is a naturally occurring nucleoside degraded from adenosine.ObjectivesThe mechanisms of antiplatelet action of adenosine and inosine in vitro and in vivo, and their differential biological effects by molecular modeling were investigated.ResultsAdenosine (0.5, 1 and 2 mmol/L) inhibited phosphatidylserine exposure from 52±4% in the control group to 44±4 (p<0.05), 29±2 (p<0.01) and 20±3% (p<0.001). P-selectin expression in the presence of adenosine 0.5, 1 and 2 mmol/L was inhibited from 32±4 to 27±2 (p<0.05), 14±3 (p<0.01) and 9±3% (p<0.001), respectively. At the concentrations tested, only inosine to 4 mmol/L had effect on platelet P-selectin expression (p<0.05). Adenosine and inosine inhibited platelet aggregation and ATP release stimulated by ADP and collagen. Adenosine and inosine reduced collagen-induced platelet adhesion and aggregate formation under flow. At the same concentrations adenosine inhibited platelet aggregation, decreased the levels of sCD40L and increased intraplatelet cAMP. In addition, SQ22536 (an adenylate cyclase inhibitor) and ZM241385 (a potent adenosine receptor A2A antagonist) attenuated the effect of adenosine on platelet aggregation induced by ADP and intraplatelet level of cAMP. Adenosine and inosine significantly inhibited thrombosis formation in vivo (62±2% occlusion at 60 min [n = 6, p<0.01] and 72±1.9% occlusion at 60 min, [n = 6, p<0.05], respectively) compared with the control (98±2% occlusion at 60 min, n = 6). A2A is the adenosine receptor present in platelets; it is known that inosine is not an A2A ligand. Docking of adenosine and inosine inside A2A showed that the main difference is the formation by adenosine of an additional hydrogen bond between the NH2 of the adenine group and the residues Asn253 in H6 and Glu169 in EL2 of the A2A receptor.ConclusionTherefore, adenosine and inosine may represent novel agents lowering the risk of arterial thrombosis.

A colloidal singularity reveals the crucial role of colloidal stability for nanomaterials in-vitro toxicity testing: nZVI-microalgae colloidal system as a case study.

Aggregation raises attention in Nanotoxicology due to its methodological implications. Aggregation is a physical symptom of a more general physicochemical condition of colloidal particles, namely, colloidal stability. Colloidal stability is a global indicator of the tendency of a system to reduce its net surface energy, which may be achieved by homo-aggregation or hetero-aggregation, including location at bio-interfaces. However, the role of colloidal stability as a driver of ENM bioactivity has received little consideration thus far. In the present work, which focuses on the toxicity of nanoscaled Fe° nanoparticles (nZVI) towards a model microalga, we demonstrate that colloidal stability is a fundamental driver of ENM bioactivity, comprehensively accounting for otherwise inexplicable differential biological effects. The present work throws light on basic aspects of Nanotoxicology, and reveals a key factor which may reconcile contradictory results on the influence of aggregation in bioactivity of ENMs.

Genotoxicity of charged particles of importance in space flight using murine kidney epithelial cells

Ionizing radiation presents significant challenges for human space flight including an increased cancer risk. High-energy heavy ions in the galactic cosmic radiation can produce qualitative and quantitative differences in biological effects when compared with sparsely ionizing radiations. Mutations are induced by charged particle exposure and are integral to the formation and/or progression of human cancers. Most cancer-associated mutations occur on autosomal chromosomes, and most solid cancers occur in epithelial tissues. Here, a combined in vitro/in vivo approach was used to evaluate cell killing and the induction of mutations at a model autosomal locus, Aprt, in mouse kidney epithelium. For in vitro exposures, Aprt heterozygous kidney cells (clones 1a, 4a or 6a) were used from C57BL/6×DBA/2 mice. Additional experiments were performed using whole body irradiation of mice with the same genotype. Both males and females were irradiated in approximately equal numbers. Irradiations were performed at the NASA Space Radiation Laboratories at Brookhaven National Laboratory. For in vitro studies, cells from primary kidney clones were irradiated and seeded at limiting dilution immediately post-irradiation to determine the toxicity of the treatment. The irradiated kidney cells were also seeded in mutation assays within 1 week post-irradiation to determine the Aprt mutant fraction at the earliest time post-exposure. This work was complemented by studies wherein mice were exposed to the same ions with kidneys harvested several months post-irradiation to determine the residual toxicity and the Aprt mutant fraction. Our previous studies focused on sparsely ionizing 1 GeV protons (LET = 0.24 keV/µm) and densely ionizing 1 GeV/amu Fe ions (LET = 151 keV/µm). Our most recent studies have included work with Si ions (240 MeV/amu for in vitro studies, LET = 78 keV/µm; 263 MeV/amu initial energy for in vivo studies to achieve 78 keV/µm near the midline of the animal) and O ions (250 MeV/amu in vitro studies only, LET = 25 keV/µm). Toxicity for the cultured kidney cells in vitro follows this pattern: Fe > Si > O > protons when the results are expressed per unit dose. D0 values were 92 cGy for Fe ions, 103 cGy for Si ions, 192 cGy for O ions and 340 cGy for protons. With regard to the induction of Aprt mutations, Fe ions were more mutagenic than protons. Si ions were also quite mutagenic with evidence for a linear dose–response for Aprt mutations in kidney cells exposed in vitro or in kidneys harvested from mice irradiated several months earlier. These results are consistent with the linear dose–response data obtained previously for Aprt mutation induction following Fe ion exposure in vitro or in vivo, but the results for Si ions differ from the curvilinear dose–response data we recently published following similar exposures to energetic protons [ 1, 2]. Our most recent studies examined the molecular characteristics of Si ion-induced Aprt mutants following in vitro exposure. A dose of 160 cGy was used to collect 58 Aprt kidney cell mutants. Mutational events were classified as follows based on PCR-based analyses of polymorphic markers along mouse chromosome 8: intragenic events, apparent mitotic recombination, interstitial deletions of Aprt only, multilocus deletions, discontinuous loss of heterozygosity or whole chromosome loss. The results for this group of mutants will be compared against our previous studies on Aprt mutants arising after exposure to sparsely ionizing 1 GeV protons or densely ionizing 1 GeV/amu Fe ions. Additional studies are ongoing to define mutational spectra following Si ion exposure to kidney epithelium in vivo.Clinical Trial Registration number: not applicable.

SU-D-137-06: Effect of Dose-Per-Pulse and Pulse Repetition Frequency in X-Ray Beam Irradiation of In-Vitro Cell Cultures

Purpose: It has been reported that flattening‐filter‐free (FFF) beams may have a different biological effect than flattened beams, possibly due to the increased dose‐per‐pulse in FFF beams. The purpose of this work is to determine if in‐vitro cell survival is affected by the higher dose‐per‐pulse and/or repetition rates generated by flattening‐filter‐free beams in comparison to flattened beams. Methods: three cell lines, T98G (p53 mutant human glioblastoma), U87‐MG (p53 wild type human glioblastoma) and V79‐4 (Chinese hamster lung fibroblast) were irradiated to 5 Gy and 10 Gy with Linac X‐ray photon energies of 6 MV, 6 MV FFF and 10 MV FFF, using dose rates of 400, 1400 and 2400 MU/min. The relative variation of the average dose‐per‐pulse among the beams was measured as 1.0: 2.8: 4.6 for 6X: 6XFFF: 10XFFF. The dose delivered to each sample was verified with a parallel‐plate ionization chamber. Gafchromic film measurements were made to confirm the dosimetry methodology. Clonogenic cell survival assays were used to evaluate biological effect of each beam. Three or more biological replicates were performed for each experiment. Results: For T98G cells, surviving fractions at 5 Gy for 6MV at 400 MU/min, 6MVFFF for 400MU/min and 1400MU/min, 10XFFF 400MU/min and 2400MU/min, were 0.59(0.09), 0.53(0.07), 0.55(0.08), 0.58(0.08), and 0.60(0.07) respectively. For U87‐MG also at 5 Gy surviving fractions were 0.27(0.02), 0.29(0.05), 0.29(0.05), 0.32(0.05), and 0.31(0.06). For V79‐4 survival at 5 Gy was 0.59(0.12), 0.56(0.06), 0.50(0.08), 0.51(0.03), and 0.54(0.04). Increased cell killing was observed using 10 Gy doses but no difference in biological effect among beams was observed. Conclusion: No effects due to dose‐per‐pulse or pulse repetition frequency variation were observed on cell killing at either 5 Gy or 10 Gy for any of the cell lines tested.

Antioxidants in Italian Head Lettuce (Lactuca sativa var. capitata L.) Grown in Organic and Conventional Systems under Greenhouse Conditions

Lettuce belongs to the Asteraceae family and it is native of the Mediterranean region. In our research, the effect of cultivation practices (organic versus conventional) on antioxidants in Italian head lettuce (Lactuca sativa var. capitata L.) grown in greenhouse was investigated. Bioactive molecule levels, antioxidant properties and the cytotoxicity of polyphenolic extracts from the edible part of head lettuce in Caco-2 cells were determined. FRAP values are 5.33 ± 0.35 and 6.35 ± 0.08 mmol/kg in organic products and 4.83 ± 0.16 and 1.80 ± 0.04 mmol/kg in conventional products. FRAP values differ significantly (P < 0.001) between organic and conventional lettuces harvested in September. Significant differences are found between organic and conventional extracts in some target compounds such as quercetin, rutin, lutein, β-carotene and vitamin C. No difference in biological effect is observed between organic and conventional products harvested in July and in September, whereas a major cytotoxicity is found for polyphenol extracts derived from organic and conventional lettuces harvested in September. Practical Applications Nowadays, a new image of agriculture is emerging: agricultural production systems are closely related to the needs of consumers and to the search of products that meet food quality and safety. The results of this investigation highlighted the importance of factors such as cultivar, agronomic practices, climatic conditions, degree of ripeness, time of harvest and storage conditions. The variation of these elements represents, in fact, a key tool to obtain healthful and more nutritious food crops by increasing and maximizing the levels of bioactive molecules in foods. Our results suggest a potential to increase the commercial production of the selected specialty leafy greens.

Natural interferon-beta plus ribavirin therapy led to sustained virological response after seven unsuccessful courses of anti-viral treatment in a chronic hepatitis C patient

We describe a patient infected with hepatitis C virus (HCV) genotype 1 who failed to respond to interferon with or without ribavirin seven times but who then achieved sustained virological response by the combination of interferon-beta plus ribavirin treatment for 48weeks. He did not respond to stronger treatment such as prolonged peginterferon plus ribavirin treatment for 87weeks. It might be rare to treat a patient with interferon 8 times considering the cost and time, since patients failing interferon treatment several times are regarded to be refractory to interferon. It is possible that the difference in biological effects between interferon-alfa and interferon-beta might have resulted in a different outcome in our patient. Interferon-beta plus ribavirin therapy might be one of the treatment options for certain patients chronically infected with HCV who do not respond to standard care treatment.

REVIEWING AND IDENTIFYING AMINO ACIDS OF HUMAN, MURINE, CANINE AND EQUINE TLR4 / MD-2 RECEPTOR COMPLEXES CONFERRING ENDOTOXIC INNATE IMMUNITY ACTIVATION BY LPS/LIPID A, OR ANTAGONISTIC EFFECTS BY ERITORAN, IN CONTRAST TO SPECIES-DEPENDENT MODULATION BY LIPID IVA

There is literature evidence gathered throughout the last two decades reflecting unexpected species differences concerning the immune response to lipid IVa which provides the opportunity to gain more detailed insight by the molecular modeling approach described in this study. Lipid IVa is a tetra-acylated precursor of lipid A in the biosynthesis of lipopolysaccharide (LPS) in Gram-negative bacteria. Lipid A of the prototypic E. coli-type is a hexa-acylated structure that acts as an agonist in all tested mammalian species by innate immunorecognition via the Toll-like receptor 4 (TLR4)/myeloid differentiation factor 2 (MD-2) receptor complex. In contrast, lipid IVa is proinflammatory in mouse cells (agonism) but it remains inactive to human macrophages and even antagonizes the action of potent agonists like E. coli-type lipid A. This particular ambivalent activity profile of lipid IVa has been confirmed in other mammalian species: in equine cells Lipid IVa also acts in a weak agonistic manner, whereas being inactive and antagonizing the lipid A-induced activation of canine TLR4/MD-2. Intriguingly, the respective TLR4 amino acid sequences of the latter species are more identical to the human (67%, 68%) than to the murine (62%, 58%) ortholog. In order to address the unpaired activity-sequence dualism for human, murine, canine and equine species regarding the activity of lipid IVa as compared to LPS and lipid A and, we review the literature and computationally pinpoint the differential biological effects of lipid IVa versus LPS and lipid A to specific amino acid residues. In contrast to lipid IVa the structurally related synthetic compound Eritoran (E5564) acts consistently in an antagonistic manner in these mammalian species and serves as a reference ligand for molecular modeling in this study. The combined evaluation of data sets provided by prior studies and in silico homology mapping of differential residues of TLR4/MD-2 complexes lends detailed insight into the driving forces of the characteristic binding modes of the lipid A domain in LPS and the precursor structure lipid IVa to the receptor complex in individual mammalian species.

Purification and differential biological effects of ginger-derived substances on normal and tumor cell lines

This study describes an optimization of [6]-, [8]- and [10]-gingerol isolation and purification in semi-preparative HPLC scale and their anti-proliferative activity. The gingerols purification was carried out in HPLC system using a Luna-C18 and the best mobile phase evaluated was MeOH/H2O (75:25, v/v). This new methodology for the gingerols isolation was very effective, since considerable amounts (in the range of milligrams) with a good purity degree (∼98%) were achieved in 30min of chromatographic run. [6]-, [8]- and [10]-Gingerol purified by this methodology inhibited the proliferation of MDA-MB-231 tumor cell line with IC50 of 666.2±134.6μM, 135.6±22.6μM and 12.1±0.3μM, respectively. These substances also inhibited human fibroblasts (HF) cell proliferation, however in concentrations starting from 500μM. In conclusion, our results demonstrate an optimization of gingerols isolation and their specific anti-proliferative activities against tumor cells, suggesting their use as important models for drug design in an attempt to develop new compounds with fewer side effects when compared to conventional chemotherapy.