Myocardial contrast echocardiography (MCE) allows visualization of radiofrequency (RF) ablation lesions in the left ventricle in an animal model. Aim: To test whether MCE allows visualization of RF and cryo ablation lesions in the human right atrium using three-dimensional echocardiography.18 patients underwent catheter ablation of a supraventricular tachycardia and were included in this prospective single-blind study. Twelve patients were ablated inside Koch's triangle and 6, who served as controls, outside this area. Three-dimensional echocardiography of Koch's triangle was performed before and after the ablation procedure in all patients, using respiration and ECG gated pullback of a 9 MHz ICE transducer, with and without continuous intravenous echocontrast infusion (SonoVue, Bracco). Two independent observers analyzed the data off-line.MCE identified ablation lesions as a low contrast area within the normal atrial myocardial tissue. Craters on the endocardial surface were seen in 10 (83%) patients after ablation. Lesions were identified in 11 out of 12 patients (92%). None of the control patients were recognized as having been ablated. The confidence score of the independent echo reviewer tended to be higher when the number of applications increased.1. MCE allows direct visualization of ablation lesions in the human atrial myocardium. 2. Both RF and cryo energy lesions can be identified using MCE. Catheter ablation is a curative treatment for most patients with arrhythmias. In some patients, the results are still suboptimal because 18 patients were included into this study. The clinical characteristics of the patients are shown in Table Standard electrophysiology (EP) and ablation procedures were undertaken using quadripolar electrode catheters in the high right atrium, to record the His bundle electrogram, in the right ventricle and a decapolar diagnostic catheter was inserted into the coronary sinus (CS). The initial portion of the EP procedure was directed at determining the presence of dual AV nodal physiology or accessory pathways, measuring the conduction properties and refractory periods of the fast and slow AV nodal pathways (if present), and determining the mechanism of the paroxysmal SVT. Mapping was performed and after the target site was identified, ablation was applied. Cryothermy and radiofrequency energy were used alternately during the study period.MCE was performed using SonoVue (Bracco), which is a second generation contrast agent made of microbubbles stabilized by phospholipids and containing sulphur hexafluoride. The mean bubble diameter is 2.5 μm and more than 90% of the bubbles are smaller than 8 μm. The blood level curve shows a distribution half-life of about 1 minute and an elimination half-life of about 6 minutes[The ClearView™ system (CardioVascular Imaging Systems Inc, Fremont, CA) was used with an 8F sheath-based ICE imaging catheter that incorporates a 9 MHz beveled single-element transducer rotating at 1800 rpm (model 9900, EP Technologies, Boston Scientific Corp., San Jose, CA, USA).A custom-designed ECG- and respiratory-gated pullback device and a 3D-ultrasound workstation (EchoScan, TomTec GmbH, Munich, Germany) were used to acquire and process the ICE images using a technique described elsewhere. The pulContinuous variables are expressed as mean ± standard deviation. Correlation analysis between the confidence scores and number of ablation lesions were performed using Pearson's test. The level of significance was set at a p value of 0.05.Of the 18 patients undergoing catheter ablation of supraventricular arrhythmias 12 had AVNRT tachycardia. 6 out of these 12 patients were ablated using cryothermy. All but one patient were successfully ablated (one patient with AVNRT). The number of applications in Koch's triangle was 6 ± 4.9, ranging from 1 to 15 applications. The fluoroscopy and procedure times were 45.7 ± 30.8 min and 196 ± 80.2 min, respectively.MCE identified ablation lesions as a low contrast area within the normal atrial myocardial tissue Figure . LesionsKoch's triangle was successfully reconstructed in 3D in all patients in whom the ablation lesion was previously identified. Lesions could be easily found by 3D echocardiography. An "en face" view of the lesion could be reconstructed in all of these patients Figure .This study demonstrates that MCE allows visualization of ablation lesions in the human right atrial myocardium during continuous venous echocontrast infusion. This potentially opens a new avenue for objective, and easily accessible evaluation of ablation lesions. Since transmural lesion formation is critical for successful ablation, the MCECrater formation and tissue changes as increased echo-density have been detected using ICE immediately after RF ablation,13. HoweHuman use does not seem to be practical in this way and neither was safety assessed. In the present study we used continuous peripheral venous echocontrast infusion and we screened lesions in Koch's triangle in humans. We showed in atrial myocardial tissue that the lesions can be visualized and continuous venous infusion provides sufficient differences in echo contrast intensity to directly visualize ablation lesions after focal ablation. In this study we used cryothermy as well as radiofrequency energy for creating lesions in Koch's triangle. We do not have a sufficient number of patients for volumetric comparison of ablation lesions using this method, but it seems that both types of ablation lesion can be reliably visualized using this technique.This single blind controlled study allowed two independent reviewers to examine the 2D ICE recordings. Although in none of the control patients a lesion in the area of interest was recognized, there was one patient with a single ablation lesion who was not identified by the two independent reviewers. This may suggest that the sensitivity of the technique is still suboptimal. One possible reason is that the concentration of the echocontrast infusion was titrated too low. This is also reflected in the results of the confidence scores. These showed a linear correlation with the number of applications. Furthermore, a correlative study to gross inspection and histopathology would be advantageous. Obviously, this cannot be done in humans.3D reconstruction and a creation of a volumetric data set allow visualization of an "en face" view of the ablation lesion. Without 3D reconstruction determination of the depth and the shape of the ablation lesion was not possible. Therefore the technique could be used to assess whether the ablation lesion was transmural. Furthermore, with 3D reconstruction, the volume of the lesion can be determined. This could potentially be a major asset for clinical electrophysiology since on line 3D echocardiography would allow real-time assessment of ablation lesions. Most importantly, the appropriateness of such lesions could be judged during linear ablations and the continuity of the lines could be checked.In conclusion, MCE is a safe and promising new method to detect ablation lesion in the human atrial tissue.


Primary human tissues are an invaluable widely used tool for discovery of gene expression patterns which characterize disease states. Tissue processing methods remain unstandardized, leading to unanswered concerns of how to best store collected tissues and maintain reproducibility between laboratories. We subdivided uterine myometrial tissue specimens and stored split aliquots using the most common tissue processing methods (fresh, frozen, RNALater) before comparing quantitative RNA expression profiles on the Affymetrix U133 human expression array. Split samples and inclusion of duplicates within each processing group allowed us to undertake a formal genome-wide analysis comparing the magnitude of result variation contributed by sample source (different patients), processing protocol (fresh vs. frozen vs. 24 or 72 hours RNALater), and random background (duplicates). The dataset was randomly permuted to define a baseline pattern of ANOVA test statistic values against which the observed results could be interpreted.14,639 of 22,283 genes were expressed in at least one sample. Patient subjects provided the greatest sources of variation in the mixed model ANOVA, with replicates and processing method the least. The magnitude of variation conferred by processing method (24 hours RNALater vs 72 hours RNALater vs. fresh vs frozen) was similar to the variability seen within replicates. Subset analysis of the test statistic according to gene functional class showed that the frequency of "outlier" ANOVA results within each functional class is overall no greater than expected by chance.Ambient storage of tissues for 24 or 72 hours in RNALater did not contribute any systematic shift in quantitative RNA expression results relative to the alternatives of fresh or frozen tissue. This nontoxic preservative enables decentralized tissue collection for expression array analysis without a requirement for specialized equipment. Many of the hopes for achieving clinical benefits of genomic medicine will hinge on the ability to develop an efficient specimen conduit from clinic to laboratory. Quantitative gene expression studies have created unprecedented tissue collection and handling challenges. In particular, the rapid degeneration of RNA, and possible perturbation of expression following excision place a high premium on prompt stabilization of tissue samples intended for expression analysis. This can be accomplished by sending a dedicated trained technologist outfitted with the necessary specialized equipment, such as liquid nitrogen, into the clinical environment. Alternatively, clinicians can be enabled to process the specimens directly in the course of patient care and send them in some stable form by unrushed and routine means for centralized processing. The latter is greatly preferred when patients are physically dispersed, and becomes essential in a multi-institutional setting.High throughput quantification of RNA expression in solid tissues has become a commonplace modality for genome-wide discovery of mechanisms of disease. Typically, groups of samples classified into comparison groups are used as a training set for expression pattern discovery, followed by validation in a fresh challenge set of annotated cases. The likelihood of success is highly dependent on the accuracy of classification within the training set, and ability to control random variables introduced during tissue processing and analytical measurement of RNA abundance. Efforts to standardize RNA quantification include sharing of information regarding probe design and use , or centFlash freezing, either by immersion in liquid nitrogen or on dry ice, is the most common means of stabilizing tissue samples intended for RNA analysis. Local access to the necessary materials and expense of cold shipping and/or storage limit these collection capabilities in most clinical settings. An additional disadvantage of frozen storage is that homogenization of frozen tissue must be accomplished rapidly to avoid the rapid RNA degeneration that occurs during thawing of a previously frozen sample.Room temperature immersion of fresh tissue samples in aqueous sulfate salt solutions (such as ammonium sulfate) at controlled pH precipitates degenerative RNAses  and otheWe studied the effects of differences between storage conditions on gene expression as measured by expression array. Duplicate uterine myometrial tissue samples from three women were processed under each of 4 fixed storage conditions – fresh, frozen, 24 hours RNA-later and 72 hours RNA-later. The 24 labeled cRNA samples Figure  were hybWe found no systematic bias in measured quantitative level of gene expression by processing method, indicating that short term storage in RNALater is a valid alternative to traditional frozen storage.3 possible ways of permuting 4 pairs of replicate samples within each subject were considered. For each of these, the F statistics were computed for each gene. To control the overall error rate, the distributions of the maximum F statistics over the genes were used. That is, for each gene, the p-value is the proportion of permutations with the maximum F statistics over all genes greater or equal to the observed value for a particular gene. A test declaring as significant any genes with p < 0.05 then guarantees that the chance of any false positives being selected is < 5%. Similar analyses were performed replacing the distribution of the maximum F statistic with the distribution of the F statistics at the 95th percentile and then at the 90th percentile. After closer examination of the 387 genes in the 5% tail, we noted that most were exhibiting expression values below 100 for all 24 samples. In fact, within a storage condition, 2 out of 3 patients exhibited null expression while the third patient showed expression values other than null but less than 100 for at least one of their replicates. Therefore, as an additional analysis, any expression values less than 100 were recoded as 100. Genes that showed expression levels of 100 across all 24 samples, and therefore lacked variability, were then removed from the analysis. This resulted in 7,853 genes for which there was at least one sample with expression level greater than 100 across all 24 samples.Of the 22,283 genes, 14,639 did not have absolutely null expression across all 24 samples. We fit the mixed model ANOVA from their log values and recorded this F statistic. The permutation distribution was used to assess the significance of F statistics calculated for each gene in the dataset. In this approach all 13,824 or (4!)Patient subjects provided the greatest sources of variation in the mixed model ANOVA, with replicates and processing method the least Figure . The magth percentile was 2.58. Corresponding p-values were 0.94, 0.55 and 0.51, respectively. The values of test statistics seen at the 95% level in a randomly permuted dataset  are evenly intermingled throughout the myometrial compartment, lending itself to physical subdivision into equivalent aliquots. This would not be possible with more complex tissues in which differing cell types are distributed asymmetrically within the specimen. Despite the equivalency of subdivided fractions that underwent varying storage treatments, it must be noted that this is a hormonally responsive tissue whose expression patterns would be expected to differ between individual women as a function of monthly changes in circulating sex hormones. We did not control for hormonal factors or indication for hysterectomy (prolapse or fibroids) but selected patients randomly. It comes as no surprise that expression differences between women, irrespective of processing method, emerged as the dominant source of inter-sample variation. This was anticipated in constructing the model, by assigning the subject source of specimens as a random variable which could be measured against the fixed processing effects. It is likely that if a larger number of women had been included in the study, the observed biologic variation attributable to subject would have been even greater. Since our goal was to compare magnitude of variation contributed by subjects to that conferred by processing method, we achieved a balanced design by having comparable degrees of freedom for those two variables.There are several critical procedural elements that must be highlighted for successful preservation of solid tissues in aqueous sulfate salt solutions such as RNALater. These reagents enter the tissue through passive diffusion, a process which follows simple physical principles. The distance between the tissue surface, which is exposed to preservative, and the innermost regions of the fragment should be minimized. We did this by cutting the tissues into 2 mm thick slices, thereby reducing the diffusion distance to 1 mm or less. Clumping of multiple fragments into a mass that excludes preservative may obviate the benefits of fine division. This can be avoided either by gentle agitation or placement in a sufficiently large container that individual pieces are likely to disperse. Results reported here are for tissues stored at room temperature (23–25°C). Storage under cooler conditions (4°C) as recommend by the manufacturer of RNALater were not directly evaluated in this experiment because it was our intent to mimic storage interval and conditions commonly encountered when sending a specimen by express courier to a centralized processing facility. Storage at temperatures substantially higher than 25°C, especially before the preservative has had an opportunity to penetrate the tissue, should be avoided.Split samples of fresh human tissue yield quantitatively similar RNA expression profiles whether processed fresh, frozen, or following 24–72 hour storage in RNALater. Formal statistical analysis shows patient source is the predominant source of variation between samples, with processing method contributing a random level of variation comparable to that seen in split duplicates (replicates). Subset analysis by functional gene category did not identify a specific class of genes which responded differently by processing method.Use of nontoxic ambient environment tissue preservatives makes it practical to engage practicing clinicians directly in decentralized sample collection for high throughput expression analysis in a central location. Tissue handling closely resembles that used by clinicians to prepare specimens for routine pathology analysis. Upon receipt in a centralized facility, the samples can either be immediately homogenized or archived at -60°C.Normal fresh uterine myometrial tissues were collected randomly from three women undergoing hysterectomy for benign uterine disease. For each hysterectomy, a single 4 to 8 gram tissue fragment was subdivided into eight aliquots composed of thin slices measuring no more than 2 mm in thickness. Replicate aliquots were immediately triaged into one of four storage conditions prior to homogenization: 1)immediate homogenization; 2)flash frozen in liquid nitrogen and storage for 48 hours at -80°C; 3)24 hour immersion in RNALater at room temperature with gentle agitation; or 4)72 hours immersion in RNALater at room temperature with gentle agitation.Tissue was solubilized in Trizol reagent (Gibco BRL, Grand Island, NY), and RNA isolated according to the manufacturers instructions. In brief, the aqueous phase was resolved by addition of chloroform, and RNA precipitated from the aqueous phase by addition of isopropyl alcohol. Pelleted RNA was washed with 70% ethanol, dried, and resuspended in water. Quality of total RNA was assessed by running a non-denaturing 1% agarose tris-acetate buffer which confirmed the integrity of 18S and 28S ribosomal bands for all 24 total RNA preparations.® Expression Analysis Technical Manual) and all arrays were scanned under a low PMT (Photo Multiplier Tube) of 570 nm. Global scaling to a target value of 75 was applied to normalize all the arrays so they were comparable . The Affymetrix average-difference expression data and the P/A calls were used in the analysis. Those probe sets determined to have no detectible signal above background mismatch hybridization  were assigned a nominal value of 1 to facilitate future log transformations. Probesets having at least one tissue with detectable expression  and an average difference above either 1 or 100 were selected to define subsets of 14639 permissively or 7853 stringently expressed genes, respectively. Further analysis was performed using the natural log transformed data of these probe subsets. Data files for all specimens processed are deposited online at the Gene Expression Omnibus at the National Center for Biotechnology Information [Double-stranded cDNA was generated from 8 μg total RNA using the Superscript Choice System (Life Technologies) with T7-(dT)24 oligomer. cDNA was purified by phenol/chloroform extraction and ethanol precipitation. Biotin-labeled cRNA was prepared using the Enzo BioArray HighYield RNA Transcript labeling kit (Affymetrix). Unincorporated NTPs were removed from the biotinylated cRNA using an RNeasy kit (Qiagen). 10 μg of quality, fragmented cRNA was hybridized to each Affymetrix HG-U133A arrays containing probe sets representing approximately 22,000 genes. Array hybridization, washing was done according to the manufacturer's protocol was used, regarding storage condition as a fixed factor with four levels and subject as a random factor with three levels. The analysis of variance calculations for sums of squares in the mixed model ANOVA are identical to those for the fixed ANOVA model. Similarly, the degrees of freedom and mean squares are exactly the same. The mixed ANOVA model departs from the fixed ANOVA model only in the expected mean squares and the consequence choice of the appropriate test statistic. The mixed model also included a random storage by subject interaction. Replicate samples enabled us to estimate the replication error in the model. To test for the presence of storage main effects for each gene we divided the mean square for storage by the mean square for the interaction effect between storage and subject . The ANOtm download center [Functional annotation of probesets on the U133A chip, were downloaded from the NetaffxGM and JW conceived and designed the research plan and participated in all aspects of data collection and analysis. DF participated in data analysis and interpretation. DN and DZ performed the statistical analysis. CL and HB performed the RNA isolations, chip hybridizations, and data collation.


Hydrocarbon-degrading biofilms in the treatment of contaminated groundwaters have received increasing attention due to the role played in the so-called "biobarriers". These are bioremediation systems in which a microbial consortium adherent to a solid support is placed across the flow of a contaminated plume, thus promoting biodegradation of the pollutant.Rhodococcus erythropolis and Pseudomonas marginalis were the predominant bacterial species in the microbial consortium.A microbial consortium adherent to pumice granules (biofilm) developed from a toluene-enriched microflora in a mini-scale system, following continuous supply of a mineral medium containing toluene, over a 12-month period. Observation by scanning electron microscopy, together with quantification of the biomass attached to pumice, evidenced the presence of abundant exopolymeric material surrounding the cells in the biofilm. Toluene removal monitored during 12-month operation, reached 99%. Identification of the species, based on comparative 16S ribosomal DNA (rDNA) sequence analysis, revealed that Rhodococcus erythropolis and Pseudomonas marginalis, developed on pumice granules, in a mini-scale apparatus continuously fed with toluene.A structurally complex toluene-degrading biofilm, mainly formed by  More recently, biofilms originating from the indigenous microflora of a contaminated groundwater have received increasing attention due to the possibility to develop arriers" . These aIndeed, the reactive system of a biobarrier is represented by the biofilm developed on the solid support starting from the autochtonous microbial population of the groundwater, where potentially degrading species are present.In this communication, we report the characterization of a toluene-degrading biofilm developed on pumice granules in two packing mini-columns, employed as a laboratory-scale biobarrier, over a 12-month period. That time was long enough for a mature consortium to establish on a solid support [The main objective of the work was to analyse the structure and composition of the biofilm established on pumice stones in a mini-scale apparatus and to ascertain its degradative capability towards toluene, as a starting point for potential applications of a pumice-bound microbial consortium in bioremediation. Pumice, a rock of volcanic origin very abundant in Southern Italy, was chosen as a support material for its high-surface-area-for-unit-volume and relatively low price.Pumice granules packed mini-columns of two different heights were colonized by a microbial consortium, isolated from a gasoline contaminated groundwater sample and enriched in the presence of toluene as the sole carbon and energy source. Afterwards, the mini-columns (henceforth named columns 1 and 2, see Methods for details) were continuously supplied with a mineral medium containing toluene as the target contaminant, and then sacrificed after 6- and 12-month operation, respectively.Biofilms were characterized with three complementary approaches: observation by scanning electron microscopy, quantification of the attached biomass, and identification of the bacterial species, based on comparative 16S ribosomal DNA (rDNA) sequence analysis. Further, the biodegradative capability of the biofilm developed in column 2 was determined by measuring the efficiency of toluene removal, over a 12-month period.Scanning electron micrographs of Fig. Table The discrepancy between data obtained with the two procedures could be attributed to the presence of a massive exopolymeric material in the 12 month-old biofilm, presumably of polysaccharidic nature and unavoidably weighed when the burning method is employed. The electron microscopy analysis suggests that this material is present both in the upper and lower part of the column; its production is linked to the biofilm maturation occurring in the second 6-month period. In fact, an abundant extracellular material covering the cells was evident by scanning electron microscopy of pumice granules sampled from both the upper and lower part of the column , whereas Pseudomonas marginalis (98% of identity) represented only 10% of the entire consortium. On the contrary, in the enriched culture , Pseudomonas marginalis was the predominant species (86%) and Rhodococcus erythropolis was only 10% of the consortium. Apparently, adhesion to the pumice support promoted the growth of Rhodococcus erythropolis, modifying the initial ratio between the two species.Identification of the species based on 16S rDNA comparative analysis revealed that in both biofilms (column 1 and 2) the majority (85%) of the attached cells was represented by Agrobacterium tumefaciens (98% of identity), Hydrogenophaga palleronii (98% of identity), Chryseobacterium scophtalum (98% of identity), Leucobacter komagatae (98% of identity), Brachybacterium articum (96% of identity), Beta proteobacterium (98% of identity), Microbacterium liquefaciens (99% of identities) and Acinetobacter sp.(98% of identity).Less represented species in the three consortia examined (biofilms from column 1 and 2, as well as enriched culture) were identified as vs. the loading rate (LR) applied to column 2 , calculated as the slope of the line obtained plotting the average toluene elimination rate (ER) Toluene oxidation in the presence of nitrate as electron acceptor  may be pRhodococcus erythropolis and Pseudomonas marginalis were the predominant species. Apparently, the two species, once attached to pumice stone, gave rise to a specialised community by the production of exopolymers functioning as biofilm stabilizers [Pseudomonas sp. is the most studied single-species, biofilm-forming bacterium [Rhodococcus sp. have not been investigated so far, though the genus Rhodococcus is present in biofilm reactors [The results obtained in this work demonstrate that a structurally complex toluene-degrading biofilm developed on pumice granules, following their inoculation with a microbial consortium obtained by enrichment of toluene-contaminated water. In the biofilm, bilizers . Pseudomacterium ,7; differeactors  for its reactors .Although the microbiological complexity of the consortium established on pumice deserves further investigation in order to clarify the specific role of each species and its contribution to toluene degradation, the results obtained in this work may be relevant for a final design of a biobarrier in which a cheap support as pumice is used for biofilm formation. In particular, the molecular analysis performed revealed the strong impact of immobilization on the structure of the toluene-degrading community, demonstrating the importance of a molecular approach to characterize microbial biofilms.-1 toluene  as the sole carbon and energy source, sparged with air and incubated at 25°C for two weeks. After this acclimation period, 25% (v/v) of the culture was transferred into fresh MSM medium containing the same toluene concentration and incubated in the same conditions. To allow culture enrichment, the transfer was repeated 10 times, until a biomass (dry weight determination after centrifugation at 3000 g and washing of the collected cells) of 1.2 mg d. w. ml-1 was achieved.A microbial consortium was obtained following enrichment of a gasoline contaminated groundwater sample and subsequently used to inoculate the packing material in the columns. The enrichment was carried out in 500 ml-flasks, adding 100 ml of groundwater to 100 ml of minimal salt medium (MSM), containing high sulphate and nitrate concentrations [-1), respectively previously washed with distilled water, autoclaved (120°C, 20 min) and then soaked in MSM medium containing 20 mg l-1 toluene. The ratio between column and particle diameter was ~50 to reduce wall effects and preferred channelling; the column void volume was 60%. The columns were provided with teflon supports, tubes and connections to prevent abiotic removal of toluene.To study the biofilm development, two glass columns (2.5 cm in diameter and 1 or 3 cm in height), were packed with pumice granules (0.4–0.6 mm in size, average particle density 0.48 g ml-1) for one week. After this period, the two columns, henceforth referred as column 1 (2.5 × 1 cm) and column 2 (2.5 × 3 cm), were continuously fed upwards at a flow rate (Q) of 0.026 ± 0.003 l h-1, with MSM plus toluene for 6 and 12 months, respectively, and then sacrificed. Operations were carried out at 25°C. MSM was continuously sparged with air and mixed with the solution containing toluene just before entering the columns. The set-up for the continuous operation of the two minicolumns is schematically presented in Fig. For each column, the attachment of microbial cells to the support material was carried out by recirculating the enriched culture at a low flow rate (0.008 l h 2 2.5 ×  cm, werei) was changed step-wise every 4 weeks, from an initial concentration of 0.77 ± 0.03 up to 4.42 ± 1.21 mg l-1, allowing two weeks to achieve culture acclimation before conducting the biodegradation assays in the following two weeks. A total of six values of toluene concentration in the inlet were established, being the highest inlet concentration achieved during the sixth month of operation; then, column 1 was sacrificed, and column 2 was fed with 4.42 ± 1.21 mg l-1 toluene during the remaining 6 months, in order to permit biofilm maturation under constant feeding conditions.The inlet concentration of toluene (Si) and in the outlet (Se) were employed to calculate toluene loading rate (LR) and elimination rate (ER) according to the following equations: ER = Q (Si-Se) / V and LR = Si·Q/V, where, Q is the inlet feed flow rate (0.026 l h-1) and V is the total volume (0.015 l) of the column, respectively.In the case of column 2, values of toluene concentration in the inlet (SToluene concentration in the gas and liquid phase was determined by the headspace method on a gas chromatographic system (HP 6890 Series) equipped with flame ionization detector (FID), calculating the area of the chromatographic peaks. Toluene concentration values in the outlet of column 2 were the average of at least 10 determinations, carried out either after culture acclimation during the first 6-month operation, or every week during the following 6-month period, when column 2 was maintained at the highest LR. Neither evidence of abnormal biomass growth nor loss of mechanical properties of the supports were recorded during the entire time course of the experiment.-1 NaCl for two hours at 4°C, processed according to [To perform scanning electron microscopy analysis, pumice granules collected from both columns 1 and 2, together with not colonised granules were fixed with 2.5 % glutaraldehyde in 15 g lThe biomass attached to the support was estimated as volatile solids (VS), after drying at 105°C and then burning at 600°C of the colonised pumice granules, collected from both columns. The difference in mass between the dried and the burned samples was the weight of VS . Biomass was also indirectly determined by total protein assay (Bio-Rad, Richmond, CA, USA) after detachment of the cells from the support by bead-beating in a homogenizer (Fast prep, BIO 101 Thermo Savant) and their lysis (60°C for 90 min with 0.6% w/v SDS), assuming that the average protein content in bacteria is about 50% of cell dry weight . Since cTo identify the microbial species in the biofilm of columns 1 and 2, cells removed from the pumice granules by bead-beating (see above) were plated on TSA (Tryptone Soy Agar). An average of 100 colonies were isolated after serial dilutions from each consortium, and cultured individually in Tryptone Soy Broth (TSB). In parallel, to identify the microbial species present in the enriched culture employed to inoculate the packing material, an average of 100 colonies from the enriched culture were isolated on TSA and cultured individually in TSB.B. subtilis rrnE. These primers are normally utilized as universal primers for eubacteria. After PCR amplification, 1.5 Kbp 16S ribosomal DNA fragments were purified from agarose gels and sequenced. Ribosomal DNA gene sequences of the isolates where compared by BLAST program with those present in the DNA GenBank [Chromosomal DNA was extracted from cells of the isolated strains according to standard methods for bacteria  and used GenBank .DLA:carried out biofilm characterization. VM:carried out the identification of species and helped to draft the manuscript. PP: partecipated in the design of the study. VR: partecipated in the design of the study and setting up of laboratory apparatus. BA: performed toluene analysis. SP: partecipated in the setting up of laboratory apparatus. SR: partecipated in the design of the study and helped to draft the manuscript. DAE: conceived of the study, and partecipated in its design and coordination, drafted the manuscript. All authors read and approved the final manuscript.


Dietary administration of 1% canavanine had been shown to improve survival in female BALB/c mice consuming diets containing 23.4% protein (dry matter basis).In order to determine if this effect also obtains at more moderate dietary protein concentrations, 30 female BALB/c mice were fed a basal diet with 14% protein (15.7% dry matter basis) and another 30 were fed the same diet plus 1% canavanine.Neither mean  nor median  lifespans differed between groups.Although mean antinuclear antibody (ANA) titers did not differ between control and canavanine-treated mice at 833 days of age , one canavanine-treated mouse displayed an outlying ANA value of 50  denoting possible early sign of incipient autoimmune disease in that individual.There may be an interaction between dietary protein level and canavanine with respect to lifespan in mice. L-canavanine is a common non-protein amino acid found naturally in alfalfa sprouts, broad beans, jack beans, and a number of other legume foods and animal feed ingredients  at up toCanavanine has also been reported to induce a condition that mimics systemic lupus erythematosus (SLE) in primates ,12, to i2 with Yates correction = 24.8).In our previous study , eighteeThe death rate and median life span of control mice were consistent with previously reported survival studies of female BALB-C mice fed high protein diets . NecropsKristal and Yu  have sugApparently, canavanine postponed the type of physiological decline that results in earlier death due to the inability of these aging mice to maintain homeostasis in the face of undetectable disease or some other stress.These were serendipitous findings during a project designed to explore the mechanism of diet-induced autoimmunity. Ironically, no positive ANAs or anti SSDNA or anti dsDNA titers were detected in these mice as a result of canavanine treatment.Previous reports of life span extension by restricting the intake of energy , proteinOur earlier results did leave unanswered the question of whether canavanine was extending life or merely offsetting the life-shortening effects of a very high protein diet. Since the objectives of the original investigation were unrelated to lifespan determination and required the harvest of body tissues, full lifespan data were not available.The purpose of the experiment reported here was to test the hypothesis that canavanine will reduce middle age mortality and extend the lifespan of female BALB/c mice consuming a diet containing moderate concentrations of protein (15.7% DMB).Five female 100-day-old 20-gram BALB/cAnHsd mice (first generation offspring of mice obtained from Harlan Sprague-Dawley) were assigned to each of twelve cages and six cages were randomly assigned to each of two treatments: Control = 25 g of ground Agway 1000 lab animal feed or Canavanine = 25 g of a mixture containing 1.56% canavanine sulfate  and 98.At 833 days of age, blood samples were taken from the supraorbital sinus of the 37 surviving mice (19 controls and 18 canavanine-fed) and analyzed for antinuclear antibodies (ANA) by the methods of Naiki, et al .Variations in lifespan and ANA values were evaluated by analyses of variance and median values were contrasted by the Mann Whitney procedure .Neither mean (870.0 vs 873.2, SEM = 34.2) nor median (885 vs 902, W = 923.5) lifespan differed significantly between mice fed canavanine and those fed the same basal diet without canavanine, respectively.  indices of ANA titers at 833 days of age were also similar for canavanine and control treatments, one canavanine-fed mouse displayed an unusually high titer (10X SEM above mean).  displayed mammary tumors, pulmonary adenocarcinomas or both at the time of death  (Table The median longevity of control mice observed in this experiment (in which a 15.7% protein was fed) considerably exceeds that of the previous trial in which the basal diet contained 23.4% protein (902 d vs 449 d). Storer  fed BALBA number of populations consume protein in amounts that far exceed known nutritional requirements . The impact of these protein intakes on human and domestic animal longevity and the possible corrective effects of non-protein amino acids on those effects should interest a variety of medical, veterinary and nutritional workers.For the second time, we have failed to induce significant autoimmune disease signs with dietary canavanine in BALB/c mice. Since this can be done in autoimmune susceptible strains and hybrids and in the "normal" (DBA/2) strain of mice , geneticThese preliminary experiments were not contemporary comparisons, nor were they conducted with both sexes, with the same source of BALB/c mice, the same basal diets or at the same locations. Known autoimmune-susceptible lines were not challenged in those trials. Although the data from those trials did indicate a canavanine-protein concentration interaction, we propose a contemporary, comprehensive, integrated experiment including both autoimmune susceptible and more normal populations. The proposed work will be more conclusive and provide an opportunity to examine the generality of the canavanine longevity effect, suggest mechanisms for its occurrence and find out if positive effects on oxidation event-dependent aging might be offset by autoimmunity in susceptible individuals. Examination of such indices as tissue and urine nitrate/nitrite, 8-OH deoxyguanidine, canavanyl residues in specific and total proteins would all contribute to a better understanding of mechanisms of longevity modulation.This and a previous study suggest that an interaction between dietary protein concentration and canavanine effects on longevity may exist in BALB/c mice. Contemporary trials that vary both protein and canavanine concentrations are needed to test this possibility more conclusively.The author(s) declare that they have no competing interests.


Umbilical metastases from a malignant neoplasm, also termed Sister Mary Joseph's nodule, are not commonly reported in the English literature, and they have usually been considered as a sign of a poor prognosis for the patient. The present article reports on the current view point on umbilical metastasis besides discussing the epidemiology, clinical presentation, pathophysiology and treatment.A search of Pubmed was carried out using the term 'umblic*' and 'metastases' or metastasis' revealed no references. Another search was made using the term "Sister Joseph's nodule" or sister Joseph nodule" that revealed 99 references. Of these there were 14 review articles, however when the search was limited to English language it yielded only 20 articles. Articles selected from these form the basis of this report along with cross references.The primary lesions usually arise from gastrointestinal or genitourinary tract malignancies and may be the presenting symptom or sign of a primary tumour in an unknown site.A careful evaluation of all umbilical lesions, including an early biopsy if appropriate, is recommended. Recent studies suggest an aggressive surgical approach combined with chemotherapy for such patients may improve survival. Cutaneous metastases localised to the umbilicus are named "Sister Mary Joseph's nodules". In 1949 Sir Hamilton Bailey initially used this eponym in his book "Physical Signs in Clinical Surgery" to describe umbilical metastases, in honour of Sister Mary Joseph, the superintendent nurse and surgical assistant of Dr. William Mayo at St. Mary's Hospital in Rochester (presently the Mayo Clinic in Minnesota, USA). Sister Mary Joseph was the first to note the link between umbilical nodules and intra abdominal malignancy [The occurrence of cutaneous metastases from malignant neoplasms occurs in from 1% to up to 9% of individuals, as determined at autopsy. Those metastases to the umbilicus are uncommon and represent only 10% of all secondary tumours which have spread to the skin ,6. EpideFrom a review of the literature, umbilical neoplastic nodules can be due to a primary tumour in 38% of cases, due to endometriosis in 32% of individuals, and in 30% are actually secondary tumour deposits from a primary tumour elsewhere -11.Sister Mary Joseph's nodules usually present as a painful lump on the anterior abdominal wall. It has irregular margins and a hard fibrous consistency. The surface may be ulcerated and necrotic, with either blood, serous, purulent, or mucous discharge from it. The size of the nodule usually ranges from 0.5 to 2 cm, although some nodules may reach up to 10 cm in size . High-reA full understanding of the mechanisms whereby the tumour spreads to the umbilicus remains unclear. However, following anatomical criteria, and several hypothesis have been proposed.The umbilical ring is a scar invaginated on the abdominal wall between the transversalis fascia and peritoneum. After birth, the foetal cord structures develop into ligaments or peritoneal folds: 1) median umbilical ligament secondary to the obliterating urachus, 2) medial umbilical ligaments ; 3) ligamentum teres  that continues into 4) the falciform ligament. On the lateral umbilical folds the inferior epigastric vessels and, sometimes, a vestigial vitelline duct connecting the umbilicus to the ileum can be recognised. The umbilical region shows a rich arterial supply that includes the inferior epigastric and deep circumflex iliac branches of the external iliac artery, and the superior epigastric branch of the internal mammary artery.The venous drainage includes several anastomotic branches, coming from cranially the axillary vein, through the internal mammary vein, and caudally, the femoral vein through the superficial epigastric vein. In addition, the umbilicus may be connected with the portal system, through small umbilical veins.The lymphatic system connects the umbilical region to the axillary, inguinal, and para-aortic lymph nodes. The deep lymphatic system passes along the falciform ligament, pierces the diaphragm and enters the anterior mediastinum or courses to the nodes around the iliac arteries -15.All these systems  as described represent possible routes by which metastatic tumour cells could implant into the umbilical region.vice versa.It is reasonable to suggest that direct extension of tumour through the peritoneum is the preferred route for gastrointestinal tumours. Furthermore, the common association between hepatic and umbilical metastases might suggests that the hypothesis that the tumour spreads from the primary tumour to the liver through the portal system and then through the lymphatic and/or venous channels, they spread to the umbilicus. It is still unclear if the umbilical tumour spread precedes the hepatic spread or Renal cell carcinoma typically spreads via extra-renal extension, lymphatic dissemination or venous invasion by the tumour. Intraperitoneal spread may occur as a result of disruption of the renal capsule . The disHaematogenous, lymphatic and venous spread all represent valid mechanisms of tumour spread from gynaecological cancers ,14.Usually the presence of an umbilical metastasis indicates a poor prognosis, is a sign of advanced neoplastic disease, and may not be amenable to surgery. The survival of these patients without treatment has been reported to range from 2 to 11 months from the time of initial diagnosis -19.However, recent studies have suggested that there are several factors which are able to influence the prognosis of such patients. Certain data has shown a better survival (mean 9.7 months) in patients who detect an umbilical metastasis before definitive treatment of the primary tumour. In contrast, when the lesion appears after the primary tumour has been treated then the survival for these patients does not exceed the 7.6 months ,17.Moreover, the aetiology of the primary malignancy determines the prognosis. For example, a better survival rate for patients with primary ovarian carcinoma has been reported previously .Finally, the type of treatment seems able to influence the patient's prognosis. Despite some authors proposing only palliative treatment because of these patients poor prognosis ,17,20, rObviously, the appropriateness of such an aggressive treatment approach is determined by the clinical state of the patient.A careful examination of all umbilical lesions is recommended, especially in those patients with gastrointestinal and genitourinary tract malignancies. All umbilical mass lesions should be biopsied to determinate the pathological nature of the lesion.Clinical experience suggests that, whenever it is possible, an aggressive surgical approach combined with chemotherapy treatment may be considered to offer the patient the best survival probability.The author(s) declare that they have no competing interests.RG contributed to conception, design, literature search and preparation of manuscript.MC contributed to conception, design, literature search and preparation of manuscript.FE contributed to conception, design, literature search and preparation of manuscript.MB contributed to conception, design, literature search and preparation of manuscript.All authors read and approved the manuscript


Inappropriate medication use is a major healthcare issue for the elderly population. This study explored the prevalence of potentially inappropriate prescriptions (PIPs) in long-term care in metropolitan Quebec.A cross sectional chart review of 2,633 long-term care older patients of the Quebec City area was performed. An explicit criteria list for PIPs was developed based on the literature and validated by a modified Delphi method. Medication orders were reviewed to describe prescribing patterns and to determine the prevalence of PIPs. A multivariate analysis was performed to identify predictors of PIPs.Almost all residents (94.0%) were receiving one or more prescribed medication; on average patients had 4.8 prescribed medications. A majority (54.7%) of treated patients had a potentially inappropriate prescription (PIP). Most common PIPs were drug interactions (33.9% of treated patients), followed by potentially inappropriate duration (23.6%), potentially inappropriate medication (14.7%) and potentially inappropriate dosage (9.6%). PIPs were most frequent for medications of the central nervous system (10.8% of prescribed medication). The likelihood of PIP increased significantly as the number of drugs prescribed increased  and with the length of stay (OR: 1.78, CI: 1.43 – 2.20). On the other hand, the risk of receiving a PIP decreased with age.Potentially inappropriate prescribing is a serious problem in the highly medicated long-term care population in metropolitan Quebec. Use of explicit criteria lists may help identify the most critical issues and prioritize interventions to improve quality of care and patient safety. Inappropriate medication use is a major health care issue for the elderly population -3. OlderPrevalence estimates of PIPs are likely to vary with the criteria that are applied. Some authors have based their assessment on the Beers criteria -7,9-12. In Canada, a list of explicit criteria was developed by a panel of experts in 1997 . The CanThe long-term care elderly population is particularly vulnerable to inappropriate medication use; it is composed of frail older patients who typically have functional disabilities and acute and chronic medical histories that require complex medication regimens ,21. AsseA cross-sectional chart review of long-term care patients aged 65 years and over living in the Quebec City area was performed in the period between April 1995 and December 1996. All long-term care facilities located in the Quebec City area were contacted and the majority (29 out of 33) agreed to participate in the study. Within the 29 participating facilities, there were a total of 71 long-term care units. Numbers of beds in these units averaged 41 (10 to 190). Units were visited once during the study period. Data on drugs currently being prescribed the day of the visit was collected using medication charts. Demographic data included age, gender and length of stay. This study was approved by the ethics committees at Université Laval, Hôpital Saint-François d'Assise and Hôpital de l'Enfant-Jésus.For each medication order, the name, dosage, frequency of dosing and nature of prescription (scheduled or given on an as-needed basis) were collected. To capture the fullest possible extent of potentially inappropriate prescribing, it was assumed that all medications prescribed on an as-needed basis were taken. The total daily dose of an as-needed prescription was calculated by multiplying the prescribed unit dose with the indicated daily frequency of administration. Prescriptions for creams, ointments and drops were not included. Each medication was classified using the Anatomical Therapeutic Chemical (ATC) classification system . The maxA list of explicit criteria for PIP in older patients was developed based on a review of the literature ,13,14,22Medication charts were reviewed and compared with the list of explicit criteria. PIPs were categorized as:• Potentially inappropriate medication;• Potentially inappropriate duration;• Potentially inappropriate dosage; and• Potentially inappropriate drug-drug interaction.t tests were used to compare proportions and means, respectively. Association between age and drug utilization was evaluated by analysis of variance. Factors predicting PIP were identified by logistic regression analyses. Independent variables were age, sex, number of prescribed drugs and length of stay. An initial bivariate analysis allowed calculation of crude odds ratios, identification of variables individually associated with the risk of PIP, and determination of the appropriate scale for each variable. A multivariate analysis with a significance threshold of 0.10 for the inclusion of variables subsequently yielded adjusted odds ratios for the number of prescribed medications, age and length of stay. Data were analyzed for collinearity and overdispersion. Data analyses were performed using SAS version 6.12 (SAS Institute Inc. Cary, NC).Drug prescribed and PIP data were stratified by age and gender. Chi-square and Student p = .0001).The study population included 2,633 individuals, aged 65 years and older, residing in long-term care facilities for a mean duration of 8.5 years. Mean age was 82 ± 8 years and the majority of individuals were women (74.2%). Women were older than men (84 ± 8 years versus 79 ± 8 years, Most residents (94%, n = 2,481) had one or more prescribed medications and 48% (n = 1,266) of the total population had five or more medications. Residents had on average 4.8 prescribed medications. Proportions of patients by number of prescribed medications were similar for men and women, but varied according to age. The oldest patients, aged 85 years and more, received significantly less medications than their youngest counterparts aged between 65 and 74 years; 43.8% of patients aged over 85 years received five medications or more, compared to 59.4% of those aged 65 to 74 years. Of the 12,707 medications prescribed, 86% were scheduled administrations and 82% were prescribed for more than three months.A majority of patients (85.5%, n = 2,251 patients) had a prescription for medications of the central nervous system (CNS). Cardiovascular medications (46.4%, n = 1,221 patients) and medications of the alimentary tract and metabolism (29.3%, n = 772 patients) were the following most frequently prescribed anatomical groups of medications. Most commonly prescribed therapeutic classes included analgesics (48.0%), anxiolytics (41.4%), antipsychotics (35.0%) and loop (high-ceiling) diuretics (18.6%)  had one or more PIPs; 29.5% had one PIP, 12.5% had two PIPs, 7.5% had three PIPs and 5.3% had four or more PIPs.A total of 12,707 drugs were prescribed of which 1807 were given on an as-needed basis. The proportion of PIPs among scheduled and as-needed prescriptions were 9.2% and 11.5%, respectively. If we exclude as-needed prescriptions, 46.4% of all residents had one or more PIPs.The most common type of PIP was drug-drug interaction, affecting 33.9% of patients treated with drugs, followed by potentially inappropriate duration (23.6%), potentially inappropriate medication 14.7%), and potentially inappropriate dosage (9.6%) ; more than half of those PIPs were for the anxiolytic oxazepam Table . A substMultivariate analysis indicated that patients with a length of stay 10 years or over were 1.78 times at greater risk of being prescribed a PIP than those with less than 10 years of stay  . Estimates of PIP prevalence in the literature vary between 4.8%  and 45.61.5%. EstA large number of patients were receiving CNS medication 85%) and the most common PIPs were related to that category of drugs. Thirty-five percent of patients were prescribed antipsychotics and 22.9% had benzodiazepine for potentially inappropriate duration, defined as more than a month . A numbe% and theThe length of stay was positively associated with PIPs, while the prevalence of PIPs decreased with age. Although the association between length of stay and the likelihood of receiving a PIP in nursing homes was studied in the past , to our The results presented here should be viewed in light of potential limitations. As in previous studies , we did This study evaluated prescription patterns rather than the actual consumption of medication. The low prevalence of as-needed medication (14%) and the long-term care setting, in which medication is administered to patients by a health caregiver, suggest that this limitation did not have a significant impact on the results. As-needed prescriptions may have accounted for repeat prescriptions, which may in turn have led to overestimation of the number of drug-drug interactions. However, even after excluding as-needed prescriptions from the analysis, the proportion of residents with a PIP remains high.Predictors of PIPs were assessed using a multivariate analysis. It allowed us to adjust for potential confounding variables. However, we were not able to adjust for facility variables as those were not available.This study is the first to describe and qualify prescribing practices in long-term care facilities in urban Quebec. In particular, it highlights the extent of potentially inappropriate prescribing in elderly long-term care patients, which are among the frailest of society ,21. InapInappropriate prescribing is highly prevalent in the elderly long-term care population in metropolitan Quebec. The use of a explicit criteria list to identify PIPs is a first step towards identifying most critical issues and implementing strategies to improve quality of care and patient safety. Identifying predictors of PIPs may help to target problems and prioritize interventions that are most needed in the rapidly expanding older population.Carol Rancourt and Jean-Pierre Grégoire were employed by Merck Frosst Canada at the time of the preparation of this article.CR, in partial fulfillment for the grade of M.Sc., lead the protocol development, expert panel consultation, data analyses, discussion of results, and manuscript preparation. JM contributed to all steps of this research project and manuscript preparation. LB contributed to protocol development, presentation and discussion of results and manuscript preparation and participated in the expert panel to define the explicit criteria. RV is the principal investigator for the initial research project which generated the drug prescription data used for this study. He contributed to protocol development, presentation and discussion of results, manuscript preparation and participated in the expert panel to define the explicit criteria. DL was a co-investigator for the initial research project, which generated the drug prescription data used for this study, and contributed to protocol development, data analyses and manuscript preparation. All authors read and approved the final manuscript. JPG contributed to protocol development presentation and discussion of results, manuscript preparation and participated in the expert panel to define the explicit criteria.The pre-publication history for this paper can be accessed here:Appendix: List of explicit criteria used to assess the quality of prescribing in long-term care for elderly patients provide as additional fileClick here for file


In this work we explored if the ability of aqueous garlic extract to prevent in vitro Cu2+-induced lipoprotein oxidation in human serum is affected by heating (a) aqueous garlic extracts or (b) garlic cloves. In the first case, aqueous extract of raw garlic and garlic powder were studied. In the second case, aqueous extract of boiled garlic cloves, microwave-treated garlic cloves, and pickled garlic were studied. It was also studied if the above mentioned preparations were able to chelate Cu2+.It has been shown that several extracts and compounds derived from garlic are able to inhibit Cu2+-induced lipoprotein oxidation in human serum was followed by the formation of conjugated dienes at 234 nm and 37°C by 240 min in a phosphate buffer 20 mM, pH 7.4. Blood serum and CuSO4 were added to a final concentration of 0.67% and 0.0125 mM, respectively. The lag time and the area under the curve from the oxidation curves were obtained. The Cu2+-chelating properties of garlic extracts were assessed using an approach based upon restoring the activity of xanthine oxidase inhibited in the presence of 0.050 mM Cu2+. The activity of xanthine oxidase was assessed by monitoring the production of superoxide anion at 560 nm and the formation of uric acid at 295 nm. Data were compared by parametric or non-parametric analysis of variance followed by a post hoc test.Cu2+-induced lipoprotein oxidation. The heating of garlic extracts or garlic cloves was unable to alter significantly the increase in lag time and the decrease in the area under the curve observed with the unheated garlic extracts or raw garlic. In addition, it was found that the garlic extracts were unable to chelate Cu2+.Extracts from garlic powder and raw garlic inhibited in a dose-dependent way Cu2+-induced lipoprotein oxidation in human serum, and (b) this ability is not secondary to Cu2+-chelation.(a) the heating of aqueous extracts of raw garlic or garlic powder or the heating of garlic cloves by boiling, microwave or pickling do not affect garlic's ability to inhibit Cu Allium sativum) has been cultivated since ancient times and used as a spice and condiment for many centuries [2+-induced low density lipoprotein (LDL) oxidation. In addition, Ide et al. [et al. [2+-induced LDL oxidation. Ou et al. [2+-induced LDL oxidation. Huang et al. [2+-induced LDL oxidation may be explained, at least in part, for their ability to chelate Cu2+. Interestingly, the diethyl ether extract of aged garlic extract, which also inhibits Cu2+-induced LDL oxidation, is unable to chelate Cu2+ [2+-chelation.Garlic  heated extract of garlic powder, (b) heated extract of raw garlic, (c) extract of boiled garlic cloves, (d) extract of microwave-treated garlic cloves, and (e) extract of pickled garlic. In addition it was studied if the above mentioned preparations are able to chelate Cu2+.Several studies have been performed to test the effect of heating on several garlic properties. It has been shown that the boiling of garlic cloves by 15 min impairs significantly its ability to inhibit cyclooxygenase activity  and throynthesis . In addiynthesis . Interesynthesis . In a siynthesis . In contr pylori  and the r pylori . Howeverin vitro Cu2+-induced lipoprotein oxidation in human serum, and (b) this protective effect was unrelated to Cu2+-chelation.It was found that (a) the heating of garlic extracts or garlic cloves had no influence on the ability of garlic extracts to prevent 2EDTA, Na2CO3, KH2PO4 , and Na2HPO4 were from JT Baker (Mexico City, Mexico). Copper sulfate was dissolved in distilled water. Xanthine oxidase, xanthine, and nitroblue tetrazolium (NBT) were from Sigma Chemical Co. (St. Louis, MO., USA).Bulbs of garlic were from a local market. Garlic powder was from McCormick (Mexico City, Mexico). Copper sulfate, NaGarlic powder was weighted (0.6 g), dissolved, and stirred with 6 mL of distilled water for 20 min. This solution was centrifuged at 20,124 × g for 5 min at 4°C. The supernatant was recovered and used at the final concentration of 0.05, 0.075, 0.10, and 0.25 mg/mL.The procedure was similar to the previous one except that the mixture was boiled for 20 min before the centrifugation. The supernatant was recovered and used at the final concentration of 0.05 and 0.10 mg/mL.Garlic cloves were peeled off, weighted, chopped, and homogenized with distilled water in a Polytron (Model PT2000, Brinkmann, Switzerland). This homogenate was centrifuged at 1,277 × g for 10 min and the supernatant was centrifuged at 20,124 × g for 5 min at 4°C. The supernatant was recovered and used at the final concentration of 0.125, 0.25, 0.5, and 0.75 mg/mL.The procedure was similar to the previous one except that the homogenate was boiled for 20 min before the second centrifugation step. The amount of water evaporated was replaced at the end of the heating. The supernatant was recovered and used at the final concentration of 0.25 and 0.5 mg/mL.Unpeeled garlic cloves were boiled in water for 10 min. After this time, garlic cloves were peeled off and the aqueous extract was prepared as described before (extract of raw garlic). The supernatant was recovered and used at the final concentration of 0.25 and 0.5 mg/mL.Unpeeled garlic cloves were submitted to microwave heating for 1 min (1100 watts). After this time, garlic cloves were peeled off and the aqueous extract was prepared as described before (extract of raw garlic). When allinase is inactivated by heating, the cascade of thiosulfinate formation is blocked from alliin, and allicin and its derivates can not be formed. It has been shown that as little as 60 seconds of microwave heating can totally destroy allinase enzyme activity whereas microwave heating for 30 seconds inhibits 90% of allinase activity compared with unheated garlic . The supGarlic cloves were peeled off carefully to avoid allinase activation and put in an aqueous solution of vinegar (1:1, v/v) and then heated to the boiling point for 30 min. Garlic cloves were put into jars with same solution and then pasteurized for 5 min at 72°C. The jars were closed immediately and stored at 4°C. The experiments with pickled garlic were performed five weeks after. The aqueous extract was prepared as described before (extract of raw garlic). The supernatant was recovered and used at the final concentration of 0.25 and 0.5 mg/mL.Blood samples were obtained from 2 male and 3 female healthy volunteers aged 24 to 46 years. The experimental protocol is in compliance with the Helsinki Declaration and written informed consent was obtained from all subjects. A fasting blood sample was drawn from the antecubital fossa vein into glass tubes, allowed to clot, and then centrifuged at 2,000 × g for 10 min. The serum removed was aliquoted and frozen at -80°C until assayed. The concentration of glucose, cholesterol and triglycerides in serum was measured by an autoanalyzer .et al. [2+, assessed by measuring changes in absorbance at 234 nm. The formation of conjugated dienes in lipoprotein deficient serum exposed to Cu2+ is absent, indicating that diene formation in lipoprotein fatty acids is primarily responsible for the increase in absorbance [2. Oxidation was initiated by the addition of CuSO4 to a final concentration of 0.0125 mM. The formation of conjugated dienes was followed by monitoring the change in absorbance at 234 nm at 37°C on a Beckman DU-64 spectrophotometer equipped with a six position automatic sample changer (Fullerton, CA, USA). Absorbance readings were taken every 10 min over 240 min. The aqueous garlic extracts were added at the indicated concentrations (ranging from 0.05 to 0.75 mg/mL). Control tubes consisted of identical assays conditions but without the garlic extract. Since the transition from lag phase to propagation phase was continuous, lag time was defined as the intercept of the tangents of the propagation and lag phases and expressed in minutes.A modification of the serum oxidation method described by Regnstrom et al.  was usedsorbance -28. Seru2+ chelating properties of aqueous garlic extracts were assessed using an approach based upon restoring the activity of xanthine oxidase described previously [4 [2+.The Cueviously . This enously [4 . The act2CO3 0.4 M. Superoxide anions were generated in a reaction volume of 1 mL containing in a final concentration: xanthine 0.087 mM, Na2CO3 15 mM, NBT 0.022 mM, and 50 mM phosphate buffer pH 7.4 or garlic extract in a volume of 0.1 mL for control or experimental tube, respectively. In additional tubes with or without garlic extract, CuSO4 was added in a final concentration of 0.050 mM. The reaction was initiated by the addition of 0.025 U of xanthine oxidase, and superoxide anion production was monitored at 560 nm. In the same experiment, xanthine oxidase activity was measured by following the uric acid production at 295 nm [2+-induced lipoprotein oxidation along with (+CuSO4) or without (-CuSO4) 0.050 mM CuSO4. Control tubes contained all the reagents but without garlic extracts and they considered as 100% production of uric acid and superoxide anion. In a separate tube with 0.050 mM CuSO4, 0.060 mM EDTA was added as an additional control in which was expected the restoration of xanthine oxidase since EDTA is a metal-chelating agent.Xanthine oxidase activity was measured by NBT reduction and uric acid production. The following concentrated solutions were prepared in distilled water: xanthine oxidase 168 U/L, xanthine 0.30 mM, NBT 0.15 mM, and NaData are expressed as mean ± SEM of five determinations using different serum samples. The variables used to describe the difference between the oxidation curves were lag time, area under the oxidation curve (AUC), and slope of the propagation phase. These parameters were obtained using the GraphPad Prism software v. 3.02 (San Diego, CA, USA). All parameters were compared using either one way analyses of variance followed by Bonferroni's t test or Kruskall-Wallis analysis of variance followed by Dunn's test. p < 0.05 was considered significant.The concentration, in mmol/L, of glucose, cholesterol, and triglycerides in serum, of the subjects involved in this study was 4.56 ± 0.15, 4.24 ± 0.42, and 1.05 ± 0.11, respectively. These data show that the human beings from whom the blood serum samples were obtained for this study had no alterations in the circulating levels of glucose, cholesterol, and triglycerides.2+-induced lipoprotein oxidation in human serum. The increasing concentrations of garlic extracts displaced the curve to the right, compared to the control curve obtained without garlic extract, indicating that the inhibition of Cu2+-induced lipoprotein oxidation is dose-dependent. Lag time, AUC, and slope were obtained from these oxidation curves. Figure Unheated garlic extracts from raw garlic or from garlic powder inhibited lipoprotein oxidation in a dose-dependent way. Figure 2+-induced lipoprotein oxidation. The decrease in the slope was significant only at the higher concentration for GP and HGP  to know if they were able to chelate Cu2+. The results for each extract at the higher concentration are presented in Fig. 2+-induced a decrease in xanthine oxidase activity measured both by uric acid production at 295 nm which decreased 71% and by NBT reduction at 560 nm which decreased 96%  the extracts were unable to modify uric acid production indicating that they were unable to modify xanthine oxidase activity. In absence of Cu2+, NBT reduction was decreased significantly by RG indicating that this extract quenched superoxide anion. In presence of Cu2+ (+CuSO4) the extracts were unable to restore (a) uric acid production (p > 0.05 vs. CT without Cu2+), with the exception of RG which was able to restore it partially (p < 0.05 vs. CT without Cu2+), and (b) NBT reduction (p > 0.05 vs. CT without Cu2+). In summary, the extracts were unable to restore xanthine oxidase activity indicating that they do not chelate Cu2+. Only RG showed a weak Cu2+-chelating activity.To investigate if the ability of garlic extracts to inhibit Cu96% Fig. . Uric acon Figs. ,4,5,6. Iin vivo enhances endogenous antioxidant system [Garlic has been used for millennia in folk medicine of many cultures to treat cardiovascular diseases and other disorders -8. It hat system  and prevt system ,35. Chrot system  and prott system  or ischet system  and supet system . The heat system . In addi2+-induced lipoprotein oxidation in human serum. Alliin, the main component in extracts from boiled garlic cloves, microwave-treated garlic cloves and pickled garlic, scavenges hydroxyl radicals [2+-induced lipoprotein oxidation in human serum. Interestingly, it has been shown that another garlic compounds such as S-allylcysteine [2+-induced LDL oxidation. The antioxidant properties of S-allylcysteine [in vivo in an experimental model of nephrotoxicity induced by gentamicin.Furthermore, the antioxidant properties of some isolated garlic compounds also have been studied. Allicin, the main component in aqueous extract from raw garlic and garlic powder, scavenges hydroxyl radicals and inhibit lipid peroxidation  and prevradicals , hydrogeradicals , and inhradicals  and LDL radicals . This macysteine ,13, N-accysteine , S-allylcysteine , alliin cysteine , allixincysteine , and S-ecysteine  are ablecysteine , S-allylcysteine , diallylcysteine , and diacysteine  also hav2+-induced lipoprotein oxidation in human serum is preserved in spite of inactivation of allinase by boiling, microwave or pickling or by the heating of garlic extracts and that the compound(s) involved in the inhibition of Cu2+-induced lipoprotein oxidation are heat stable. Our data are in contrast with previous studies in the literature showing that the heating may impair significantly several garlic properties. For example, microwave-treatment for 1 min impaired the anticancer properties of garlic [2 synthesis [Helicobacter pylori [et al. [et al. [et al. [et al. [et al. [2+-induced lipoprotein oxidation is also preserved after the heating of garlic.Our data strongly suggest that the ability of garlic to prevent Cuf garlic  and the ynthesis , and playnthesis , and theynthesis . The hear pylori . Interes [et al.  have fou [et al.  found th [et al.  and Kasu [et al.  strongly [et al.  suggesti2+ suggesting that the ability of these extracts to inhibit Cu2+-induced lipoprotein oxidation is not secondary to Cu2+-chelation. Only RG showed a weak Cu2+-chelation activity, which was more evident at 295 nm. Based on previous data with aged garlic extracts [2+-chelating activity. The discrepancy with our data may simply reflect differences in composition in each garlic extract. This is additionally supported by the fact that the diethyl ether extract from aged garlic extract has no Cu2+-chelating activity [2+-induced lipoprotein oxidation remains to be studied.In addition, it was found that the garlic extracts used in our study were unable to chelate Cuextracts  and someextracts , we expeactivity . The pre2+-induced lipoprotein oxidation in human serum, and (b) this ability is not secondary to Cu2+-chelation.(a) the heating of aqueous extracts of raw garlic or garlic powder or the heating of garlic cloves by boiling, microwave or pickling do not affect garlic's ability to inhibit Cu2+-chelation and studied the effect of boiled garlic, picked garlic and garlic submitted to microwave heating on Cu2+-induced lipoprotein oxidation in human serum. GA developed in our laboratory the method to oxidize lipoproteins by Cu2+ and performed studies with garlic extracts of raw garlic and garlic powder. LBE participated in the studies on Cu2+-chelation. MM participated in the blood obtention and clinical chemistry analyses of blood serum. ONMC participated in the statistical analyses and developed the method to analyze the Cu2+ chelating properties of garlic extracts. All authors read and approved the final manuscript.JPCH conceived, designed and coordinated the study and drafted the manuscript. MGO performed the studies on CuNone declared.


The lipoprotein lipase (LPL) hydrolyses circulating triacylglycerol-rich lipoproteins. Thereby, LPL acts as a metabolic gate-keeper for fatty acids partitioning between adipose tissue for storage and skeletal muscle primarily for energy use. Transgenic mice that markedly over-express LPL exclusively in muscle, show increases not only in LPL activity, but also in oxidative enzyme activities and in number of mitochondria, together with an impaired glucose tolerance. However, the role of LPL in intracellular nutrient pathways remains uncertain. To examine differences in muscle nutrient uptake and fatty acid oxidative pattern, transgenic rabbits harboring a DNA fragment of the human LPL gene (hLPL) and their wild-type littermates were compared for two muscles of different metabolic type, and for perirenal fat.ex vivo using [1-14C]oleate as substrate, was lower in hLPL rabbits than in wild-type rabbits for the two muscles under study. Both insulin-sensitive glucose transporter GLUT4 and muscle fatty acid binding protein (H-FABP) contents were higher in hLPL rabbits than in wild-type littermates for the pure oxidative semimembranosus proprius muscle, but differences between groups did not reach significance when considering the fast-twitch glycolytic longissimus muscle. Variations in both glucose uptake potential, intra-cytoplasmic binding of fatty acids, and lipid oxidation rate observed in hLPL rabbits compared with their wild-type littermates, were not followed by any modifications in tissue lipid content, body fat, and plasma levels in energy-yielding metabolites.Analyses of skeletal muscles and adipose tissue showed the expression of the hLPL DNA fragment in tissues of the hLPL group only. Unexpectedly, the activity level of LPL in both tissues was similar in the two groups. Nevertheless, mitochondrial fatty acid oxidation rate, measured Expression of intracellular binding proteins for both fatty acids and glucose, and their following oxidation rates in skeletal muscles of hLPL rabbits were not fully consistent with the physiology rules. The modifications observed in muscle metabolic properties might not be directly associated with any LPL-linked pathways, but resulted likely of transgene random insertion into rabbit organism close to any regulatory genes. Our findings enlighten the risks for undesirable phenotypic modifications in micro-injected animals and difficulties of biotechnology in mammals larger than mice. The endothelial cell-associated lipoprotein lipase (LPL) works to break down triacylglycerol-rich dietary fats absorbed after a meal, thus generating free fatty acids transported in the blood. Earlier works suggested that LPL acts as a metabolic gate-keeper for fatty acid partitioning between adipose tissue for storage and muscle primarily for energy use . Then, vAltogether, metabolic studies in muscles of transgenic animals help to understand the biological links between fatty acid uptake, intracellular lipid metabolism, and some metabolic disorders such as diabetes in human beings. However, most data have been established in mice. Potential advantages of rabbit compared with mouse as human disease model ,16 relatsemimembranosus proprius and longissimus muscles of hLPL group, proving the expression of the transgene in tissues of the rabbit organism. On the contrary, no signal was detected in the same tissues of wild-type animals (figure An amplification product corresponding to hLPL fragment was evidenced in adipose tissue, s figure . Surpriss figure .Plasma concentrations in triglycerides, free fatty acids, and glucose, were similar in the two genetic groups table . Moreovesemimembranosus proprius samples of hLPL rabbits compared with wild-type littermates (table longissimus muscle. This was probably related to a high intra-assay variability, due to the low expression of H-FABP in low-fat glycolytic muscles. The content in insulin-sensitive glucose transporter GLUT4 (the first step in glucose utilization by tissues) was 36% higher (P = 0.07) in hLPL rabbits than in wild-type animals for semimembranosus proprius muscle, but it did not vary in other sites under study (table P < 0.05) in semimembranosus proprius and longissimus muscles, respectively (figure Muscle content in H-FABP (responsible for cytoplasmic binding of fatty acids in muscle cells), was 30% higher in y figure , in hLPLDespite a clear evidence for expression of the hLPL transgene in the tissues under study in the hLPL rabbits only, total LPL (human +native) activity in skeletal muscles or perirenal adipose tissue was similar in hLPL rabbits and in their wild-type littermates. This situation contrasts with the moderately enhanced LPL activity in post-heparin plasma reported in another line of hLPL transgenic rabbits , and witsemimembranosus proprius (a muscle composed exclusively of slow-twitch type I fibers), and to a lesser extent in the fast-twitch glycoytic longissimus muscle. However, unlike results in mice, our findings reported a lower muscle fatty acid oxidation rates in hLPL rabbits. Others have observed that over-expression of hLPL gene specifically in skeletal muscle of transgenic mice rather led to a dose-dependent increase in oxidative enzymes and to a proliferation of the oxidative specialized organelles [Surprisingly, despite the lack of difference in LPL activity, many metabolic differences were found between hLPL rabbits and their wild-type littermates in ganelles ,4.According to the well-known fatty acid-glucose cycle in skeletal muscles , decreassemimembranosus proprius muscle of our hLPL rabbits compared with wild-type animals remains largely unknown. Indeed, a preferential involvement of H-FABP in delivering intracellular fatty acids to sites of oxidation has been widely suggested [In vitro and ex vivo findings indeed recently suggest that non-adipose tissue, such as cardiomyocytes, can re-export fatty acids when influx exceeds oxidation rate [Finally, the reason for a higher content of intra-cytoplasmic fatty acid binding proteins (H-FABP) in uggested . Here, fion rate . Howeverion rate ,4 dependAltogether, the various metabolic disruptions in skeletal muscles of hLPL rabbits are in favor to a random integration of the micro-injected hLPL DNA within or close to endogenous genes. In transgenic mice, estimates of the frequency of these insertional mutations range from 7 to 20% . This maDuring the last 15 years, transgenesis has been extended from mice to larger mammals, with the aim of benefiting human health. Transgenic rabbits for LPL gene may offer useful models to test the relationships between uptake of nutrients, intracellular trafficking and subsequent metabolic fate in a species sharing a lipoprotein profile closely similar to that in Human. However, we currently reported alterations of nutrient bindings and oxidative metabolism in skeletal muscles of hLPL rabbits, despite the lack of difference in tissue LPL activity between transgenic rabbits and their wild-type littermates. It is thus suggested that hLPL phenotype emerged from insertional mutation of hLPL DNA within or close to endogenous genes. This study underlined the risk of unpredictable phenotypic properties in micro-injected transgenic rabbits, and thereby the difficulty of animal biotechnology in mammals larger than mice. Nevertheless, transgenic rabbits remain useful tools for understanding the relative importance of the various metabolic pathways involved in the control of tissue lipid content, especially when the genetic map now under progress will be available in the rabbit.The Genetic Committee of the French Ministry of Agriculture approved the experiment. Rabbits were reared and killed in accordance with the French regulations for humane care and use of animals in research. New-Zealand White rabbit does were super-ovulated by injections of porcine-follicle-stimulating hormone, as previously described , and werTwo transgenic littermate male founders were cross-bred with 20 females of a standard New-Zealand White line (A-1067, INRA, France) at the INRA experimental unit (Le Magneraud, Surgères, France), to provide F1 animals for analysis. Pup genotypes were determined at the hLPL locus by PCR from tail tip DNA at the age of 4 weeks, using the primers described above. Control PCR amplification of the actin gene was performed in parallel to ensure DNA quality.n = 6 in each genotype) were selected within litters, and bled in the fed state.After weaning (5 weeks), young rabbits were housed collectively by genotype , under a controlled light/dark cycle (16/8 h). They were offered free access to water and to a standard rabbit pelleted diet . At 10 weeks of age, pairs of hLPL rabbits and wild-type littermates of similar body weight , were used to determine levels of triglycerides (kit PAP 150, BioMérieux, Marcy l'Etoile, France), free fatty acids (kit Wako NEFA-C kit, Richmond, VA, USA) and glucose (kit PAP 1200, BioMérieux, Marcy l'Etoile, France) in rabbit plasma collected at the time of the death.semimembranosus proprius (SMP) as a muscle composed solely of slow-twitch oxidative fibers, and longissimus (LL) muscle representing predominantly fast-twitch glycolytic fibers, were stored at -70°C until RNA analysis and biochemical measurements. About 300 mg of each muscle was homogenized immediately after sacrifice in an appropriated buffer for measurements of ex vivo oxidation rates, as described previously [Portions of perirenal fat, eviously ,29.Total RNA from 600 mg of tissues was extracted by acid guanidium thiocyanate-phenol chloroform method , and wasg for 20 min at 4°C. As previously described [® (Pharmacia, Stockholm, Sweden), into which [3H] triolein has been incorporated, was used as the substrate. Liberated [3H]-free fatty acids were quantified by liquid scintillation.Lipoprotein lipase (LPL) activity was assessed after homogenization of the tissues in a buffer composed of ammonia-HCl (25 mM) pH 8.2, containing EDTA (5 mM), Triton-X-100 (8 g/l), sodium dodecyl sulfate (0.10 g/l), heparin (5000 IU/l) and peptidase inhibitors. Insoluble material was discarded by centrifugation at 20000 × escribed , rat serTissue lipid content was determined after chloroform/methanol extraction . Muscle 14C]oleic acid bound to defatted albumin in a 5:1 molar ratio was used as substrate. Oleate oxidation was measured using L-carnitine and other cofactors, in the absence  or presence  of mitochondrial inhibitors of the respiratory chain (i.e., 75.6 μM antimycin A, and 10 μM rotenone, SIGMA, St-Louis, MO). The difference between total oxidation and peroxisomal oxidation was considered to be mitochondrial oxidation. All assays were performed in triplicates.Oxidation rate of oleic acid was determined in freshly excised muscles as described earlier for rat and bovine muscles , with miThe Kruskal-Wallis non-parametric test was used to analyze differences between groups (SAS Inst, Cary NC, NY, USA). All data are presented as mean ± SEM.FG, JFH and PH conceived of the study, participated in its design and co-ordination. FG, SBJ and JFH carried out biochemical analyses. MD carried out pup genotyping. CV and LMH carried out micro-injection and provide transgenic breeder animals. FG, JFH and MD drafted the manuscript. All authors read and approved the final manuscript.


Expression of C1q, an early component of the classical complement pathway, has been shown to be induced in neurons in hippocampal slices, following accumulation of exogenous Aβ42. Microglial activation was also detected by surface marker expression and cytokine production. To determine whether C1q induction was correlated with intraneuronal Aβ and/or microglial activation, D-(-)-2-amino-5-phosphonovaleric acid (APV, an NMDA receptor antagonist) and glycine-arginine-glycine-aspartic acid-serine-proline peptide (RGD, an integrin receptor antagonist), which blocks and enhances Aβ42 uptake, respectively, were assessed for their effect on neuronal C1q synthesis and microglial activation. APV inhibited, and RGD enhanced, microglial activation and neuronal C1q expression. However, addition of Aβ10–20 to slice cultures significantly reduced Aβ42 uptake and microglial activation, but did not alter the Aβ42-induced neuronal C1q expression. Furthermore, Aβ10–20 alone triggered C1q production in neurons, demonstrating that neither neuronal Aβ42 accumulation, nor microglial activation is required for neuronal C1q upregulation. These data are compatible with the hypothesis that multiple receptors are involved in Aβ injury and signaling in neurons. Some lead to neuronal C1q induction, whereas other(s) lead to intraneuronal accumulation of Aβ and/or stimulation of microglia. Alzheimer's disease (AD) is the most common form of dementia in the elderly. Its main pathological features include extracellular amyloid beta (Aβ) deposition in plaques, neurofibrillary tangles (composed of hyperphosphorylated tau protein) in neurons, progressive loss of synapses and cortical/hippocampal neurons, and upregulation of inflammatory components including activated microglia and astrocytes and complement activation . AlthougIn vitro studies have shown both protection and toxicity contributed by microglia in response to Aβ depending on the state of activation of microglia [Microglial cells, when activated, are believed to be responsible for much of the Aβ clearance through receptor-mediated phagocytosis ,3. Upon icroglia ,10. Corricroglia , althougicroglia ,13. Aβ iicroglia . NeverthComplement proteins have been shown to be associated with Aβ plaques in AD brains, specifically those plaques containing the fibrillar form of the Aβ peptide . ComplemInterestingly, C1q and, upon complement activation, C3 also can bind to apoptotic cells and blebs and promote ingestion of those dying cells -26. ElevWe have previously reported that in rat hippocampal slice cultures treated with exogenous Aβ42, C1q expression was detected in pyramidal neurons following the internalization of Aβ peptide. This upregulation of neuronal C1q could be a response to injury from Aβ that would facilitate removal of dying cells. Concurrently, microglial activation was prominent upon Aβ treatment. In the present study, the relationship of Aβ-induced neuronal C1q production to microglia activation and Aβ uptake in slice cultures was investigated.2O and subsequently N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES) was added to make a final concentration of 10 mM HEPES, 500 μM peptide. This solution was immediately diluted in serum-free medium and added to slices. Glycine-arginine-glycine-aspartic acid-serine-proline (RGD) peptide was purchased from Calbiochem (San Diego, CA). D-(-)-2-amino-5-phosphonovaleric acid (APV) was purchased from Sigma (St. Louis, MO). Both compounds were dissolved in sterile Hanks' balanced salt solution (HBSS) without glucose at 0.2 M and 5 mM, respectively, before diluted in serum-free medium. Antibodies used in experiments are listed in Table Aβ 1–42, obtained from Dr. C. Glabe (UC, Irvine), was synthesized as previously described . Aβ 10–22 before addition to the slices. Aβ 1–42 or Aβ 10–20 was added to slice cultures as described previously [Hippocampal slice cultures were prepared according to the method of Stoppini et al  and as deviously . BrieflyAt the end of the treatment period, media was removed, the slices were washed with serum-free media and subjected to trypsinization as previously described  for 15 m2, 1 mM MgCl2, 0.15 M NaCl, 0.3% NP-40) containing protease inhibitors pepstatin (2 μg/ml), leupeptin (10 μg/ml), aprotinin (10 μg/ml), and PMSF (1 mM). Protein concentration was determined by BCA assay (Pierce, Rockford, IL) using BSA provided for the standard curve.Slices were homogenized in ice-cold extraction buffer  according to the manufacturer's instructions. RNA was treated with RNase-free DNase (Fisher, Pittsburgh, PA) to remove genomic DNA contamination. Each RNA sample was extracted from 3 to 5 hippocampal organotypic slices in the same culture insert. The reverse transcription (RT) reaction conditions were 42°C for 50 min, 70°C for 15 min. Tubes were then centrifuged briefly and held at 4°C. Primer sequences and PCR conditions are listed in Table We have previously reported that C1q was detected in cells positive for neuronal markers and that microglial cells were activated in slices following Aβ42 ingestion . Lynch aIt has been shown that an integrin receptor antagonist peptide, GRGDSP (RGD), can enhance Aβ ingestion by neurons in hippocampal slice cultures . TherefoAddition of RGD peptide by itself did not result in neuronal C1q induction or microglial activation (CD45) compared to no treatment control, as assessed by immunostaining (data not shown). While greater ingestion was seen at 30 μM Figure , additioData reported by Giulian et al suggests that residues 13–16, the HHQK domain in human Aβ peptide, mediate Aβ-microglia interaction . To invein vivo [It is known that activated microglia cells can produce pro-inflammatory cytokines, chemokines, and nitric oxide, as well as higher expression of co-stimulatory molecules like CD40 and B7 . Many ofRT-PCR revealed that mRNAs for CD40 and IL-8 were enhanced in Aβ treated slice cultures relative to the control after 3 days Figure  and 6b. Macrophage-colony stimulating factor (MCSF), a proinflammatory mediator for microglial proliferation and activation, has been shown to be expressed by neurons upon Aβ stimulation . The expPreviously, it has been shown that Aβ is taken up by pyramidal neurons in hippocampal slice culture and that the synthesis of complement protein C1q is induced in neurons . Here weSince Aβ10–20 alone can induce a level of C1q expression in neurons comparable to Aβ42, it is hypothesized that amino acids 10–20 in Aβ peptide contain the sequence that is recognized by at least one Aβ receptor. It was reported by Giulian et al. that the HHQK domain (residues 13–16) in Aβ is critical for Aβ-microglia interaction and activation of microglia, as they demonstrated that small peptides containing HHQK suppress microglial activation and Aβ-induced microglial mediated neurotoxicity . We haveNeurons are the major type of cells that accumulate exogenous Aβ in slice cultures. Microglial activation, as assessed by CD45, OX42, and ED1, was increased with enhanced neuronal Aβ42 uptake and inhibited when Aβ42 uptake was blocked by APV or Aβ10–20 in this slice culture system. These data would be consistent with a model in which neurons, upon internalization of Aβ peptide, secrete molecules to modulate microglial activation ,41,42 , and cathepsin D ,37. SeveThe data presented thus far suggest the hypothesis that neurons, upon uptake and accumulation of Aβ, release certain substances that activate microglia. One possible candidate of those neuron-produced substances is MCSF, which has been reported to be induced in neuronal cultures upon Aβ stimulation ,46, and in vivo.In this organotypic slice culture, no significant neuronal damage was observed in 3 day treatment with Aβ at concentrations that have been reported to cause neurotoxicity in cell cultures. One possible explanation is that the peptide has to penetrate the astrocyte layer surrounding the tissue to reach the multiple layers of neurons. Thus, the effective concentration of Aβ on neurons is certainly much lower than the added concentration. Aβ failing to induce neurotoxicity in slices to the same extent as in cell cultures may also indicate the loss of certain protective mechanisms in isolated cells. A distinct advantage of the slice culture model is that the tissue contains all of the cell types present in brain, the cells are all at the same developmental stage, and cells may communicate in similar fashion as Our data demonstrating distinct pathways for the induction of neuronal C1q and the activation of microglial by amyloid peptides suggest the involvement of multiple Aβ receptors on multiple cell types in response to Aβ Figure , model aIn summary, induction of C1q expression in hippocampal neurons by exogenous Aβ42 is dependent upon specific cellular interactions with Aβ peptide that require HHQK region-containing sequence, but does not require intraneuronal accumulation of Aβ or microglial activation. Thus, induction of neuronal C1q synthesis may be an early response to injury to facilitate clearance of damaged cells, while modulating inflammation and perhaps facilitating repair. Microglial activation in slice culture involves the induction of CD45, CD40, CR3, and IL-8, which correlates with intraneuronal accumulation of Aβ, indicating contribution of factors released by neurons upon Aβ exposure. MCSF may be one of those stimulatory factors, though by itself MCSF cannot fully activate microglia.in vivo.Removal of Aβ to prevent deposition and of cellular debris to avoid excitotoxicity would be a beneficial role of microglial activation in AD. However, activated microglia also produce substances that are neurotoxic. Therefore, the goal of modulating the inflammatory response in neurodegenerative diseases like AD is to enhance the phagocytic function of glial cells and inhibit the production of proinflammatory molecules. Being able to distinguish in the slice system C1q expression (which has been shown to facilitate phagocytosis of apoptotic cells in other systems ) from miAβ: amyloid beta; AD: Alzheimer's disease; APV: D-(-)-2-amino-5-phosphonovaleric acid; BSA: bovine serum albumin; GRGDSP (RGD): glycine-arginine-glycine-aspartic acid-serine-proline; HBSS: Hanks' balanced salt solution; HEPES: N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid; MCSF: macrophage colony stimulating factor; NMDA: N-methyl-D-aspartic acid; PMSF: phenylmethylsulfonylfluoride; TAE: triethanolamine.The author(s) declare that they have no competing interests.RF cultured and processed the tissue, performed all experiments (immunohistochemistry, ELISA, PCR and others), analyzed the data, and drafted the manuscript. AJT contributed to the design of the study, guided data interpretation and presentation and edited the manuscript.