Μm Pore Filters Research Articles

Potential exposure of adults and children to particles from resuspended nano-enabled consumer sprays

The increasing application of nanotechnology has resulted in a growing number of nano-enabled consumer products, and they could be important contributors to indoor particulate matter, with potential adverse health effects. This study investigated the exposure of adults and children to the released and resuspended manufactured particles from seven nano-enabled consumer sprays. Sedimentation and resuspension of released particles were investigated in a newly constructed 2.8 × 1.6 × 2.4 m3 chamber. The resuspension of deposited particles was investigated as a function of product type, flooring material (e.g., carpet and vinyl), resuspension force (e.g., walking by an adult and motion of a robotic sampler that simulated a child), and measurement height. The concentration of released and resuspended particles in the air was determined using Button Aerosol Samplers (SKC Inc.) with 25-mm 2 μm-pore PTFE filters. Samplers were positioned in the experimenter's breathing zone (e.g., 1.5 m for adults and 0.3 m for a child-simulating robot) and at fixed stations of 0.3 m and 1.1 m heights. Resuspended particle mass concentrations ranged from 28 to 905 μg/m3, and the resuspension rates of deposited spray particles for the same variable combinations varied from 10−4 to 10−1 h−1, depending on product type, flooring material, sampling height, and resuspension force. Particle resuspension rates from carpet were up to 320 % higher than resuspension rates from vinyl flooring, resuspension rates measured at 0.3 m were up to 195 % higher than the rates measured with a 1.1 m stationary sampler, and resuspension rates due to a walking adult were up to 243 % higher than resuspension rates caused to a moving robot that simulated a child. Overall, these data on the resuspension of particles from nano-enabled consumer sprays could help us understand the resulting exposures and support future studies on human exposure reduction.

Ultrafiltration to characterize PM2.5 water-soluble iron and its sources in an urban environment

Soluble forms of transition metals in airborne particles are linked to adverse health impacts. Iron (Fe) is often the most abundant transition metal in ambient PM2.5. In Atlanta, the water-soluble fraction of Fe had a stronger association with adverse cardiovascular outcomes than PM2.5 mass and total Fe. Water solubility is operationally defined. One determining factor is the pore size in the liquid filtration (0.45 μm in this study) used to isolate water-soluble species, implying colloidal particles smaller than the pore size may be included in the water-soluble group. Here, we characterize the properties of PM2.5 water-soluble iron (WS Fe) utilizing ultrafiltration and investigate sources of the various isolated fractions based on one-year of samples collected in urban Atlanta (N = 355). Annual average Fe solubility (WS Fe divided by total Fe) was 12%, with the highest solubility in the warm season (May–Sept.) when aerosol acidity (pH of 1.8–2.7) and oxalate concentrations were highest. Ultrafiltration showed that 84% of WS Fe (extract that passed through 0.45 μm pore filter) was smaller than nominally 4 nm, with 61% of the soluble iron was smaller than nominally 2 nm. A correlation analysis, experiments involving addition of oxalate to sample extract and a pH cycling experiment with selected Fe species were consistent with the oxalate-Fe ligand being mainly found in the smallest ultrafiltrate size (< nominally 2 nm), whereas pH cycling could lead to the formation of colloidal particles with sizes between nominally 2–4 nm. Magnetite, a solid Fe nanoparticle (smaller than 0.1 μm) detected in human organs in previous studies, was undetectable in the PM2.5 WS Fe in urban Atlanta. These data show that most WS Fe is composed of soluble species or very small colloidal particles (roughly <4 nm), making them highly bioavailable.

Microplastics particle size affects cloth filter performance

Can the unit process or operation in the drinking water treatment plant remove Microplastics (MPs)? Is an additional unit operation/process needed? This study aims to determine the cloth filters' ability to remove MPs based on the particle size of MPs. The cloth filter can be used as an additional unit operation after a conventional rapid sand filter or as surface filtration in the ultrafiltration process. The cloth filter media was made by 194 μm, 115 μm, and 57.5 μm polyester. The artificial MPs size varied from 210 to 420 μm and > 420 μm. A 194 μm filter pore could remove 41.5% of MPs which particle size >420 μm, while 115 μm and 57.5 μm filters pore could remove 55.5% and 83.7%, respectively. A 194 μm filter pore could remove 34% of MPs which particle size range of 210–420 μm, while 115 μm and 57.5 μm filters pore could remove 52.85% and 85.3%, respectively. Based on tensile strength and elongation, the 194 μm filters pore were better than both 115 μm and 57.5 μm filter pores. However, quality (tensile strength and elongation) had no impact on MPs removal due to its large pore size. Filters which pore sizes 194 μm and 115 μm showed that the MPs size had a significant effect on removal (p < 0.05) on which also be shown by regression coefficient of 34.7% and 15.8%. However, for 57.5 μm pore filter, the MPs size did not affect MPs removal (p-value>0.05) on which also be shown by a low regression coefficient of 1%.

Identification of Free-Living Amoebas in Tap Water of Buildings with Storage Tanks in Korea.

Free-living amoebas (FLAs) can cause severe disease in humans and animals when they become infected. However, there are no accurate survey reports on the prevalence of FLAs in Korea. In this study, we collected 163 tap water samples from buildings, apartments, and restrooms of highway service areas in 7 Korean provinces with high population density. All these buildings and facilities have water storage tanks in common. The survey was separated into categories of buildings, apartments, and highway service areas. Five hundred milliliters of tap water from each building was collected and filtered with 0.2 μm pore filter paper. The filters were incubated in agar plates with heated E. coli at 25°C. After axenization, genomic DNA was collected from each FLA, and species classification was performed using partial 18S-rDNA PCR-sequencing analysis. We found that 12.9% of tap water from buildings with storage tanks in Korea was contaminated with FLAs. The highway service areas had the highest contamination rate at 33.3%. All of the FLAs, except one, were genetically similar to Vermamoeba vermiformis (Hartmannella vermiformis). The remaining FLA (KFA21) was very similar to Acanthamoeba lugdunensis (KA/E26). Although cases of human infection by V. vermiformis are very rare, we must pay attention to the fact that one-third of tap water supplies in highway service areas have been contaminated.

Tratamiento de lixiviados con una combinación de procesos Fenton/filtración/adsorción

Fenton and adsorption are two of the most widely used physicochemical processes for leachates treatment. Each one separately has shown limited treatment capabilities, reaching COD removals up to 60-70%. The Fenton process oxidizes both organic and inorganic matter; nevertheless, low-density sludges are produced and hard to decant. To remove them, the filtration process could be an efficient alternative to eliminate these solids the remaining substances could be removed by means of the adsorption process. This study presents the results of a Fenton/filtration/adsorption treatment train, under the following conditions: a) Fenton: pH = 4, contact time= 60 minutes, [Fe2+]/[H2O2] = 0.6, [COD]/[H2O2] = 9; b) leachate filtered through 4 µm pore filter paper; c) adsorption was performed in a packed column with macroporous granular activated carbon. The COD removals reached 99.9%, where 90.8% was achieved with the Fenton/filtration process and 9.1% was removed by adsorption. The 95.7% of color was removed in the Fenton/filtration stage and 4.3% by adsorption. Although, the COD, BOD5, color and TSS removals from the adsorption process were lower than the Fenton/filtration process, the additional removal allows to the effluent reach the values required to meet the current Mexican Norms

A metabarcoding survey for seasonal picophytoplankton composition in two coral reefs around Sesoko Island, Okinawa, Japan

Okinawa is the southwesternmost part of Japan and represents the northern limit of coral reefs that are influenced by the Kuroshio warm current. We have been studying two coral reefs off Sesoko Island, in northern Okinawa, to evaluate coral reef environments since 2014. In this study, we applied a metagenomic approach to analyze the seasonal composition of picophytoplankton at these two reef sites (Sesoko Minami and Ishikawabaru). The two sites were selected for their differences: Sesoko Minami faces the open ocean and experiences relatively small land effects, while Ishikawabaru experiences relatively high land effects and is surrounded by barrier reefs. Seawater samples were collected from these two sites and filtered through a 1.6 μm pore filter followed by a 0.2 μm membrane filter. DNA was extracted from the 0.2 μm filters, and the V1-V2 region of the 16S rRNA gene (260 bp) was amplified before sequencing using next-generation sequencing techniques. Cyanobacteria or chloroplast sequences were selected for further metabarcording analyses. Interestingly, almost no differences were indicated between the two sites and more than 95% of the picophytoplankton at both sampling sites was composed of only 5 to 7 OTUs. The seasonal composition of picophytoplankton clearly changed; in summer (Jul 2014, May to Jul 2015, and May to Aug 2016) OTU 1 (Synechococcus sp. NC007513) was most abundant. OTU 4 (Prochlorococcus marinus NC009091) also reached its maximum abundance in summer (May to Aug 2016), while OTU 6 (Bathycoccous prasinos FN563099) increased in winter. OTU 8 (Micromonas commoda RCC299 FJ858267) was present at every sampling period and accounted for 5–20% of the total picophytoplankton contig numbers. Our results indicate that seasonal changes clearly influence fluctuations in picophytoplankton community compositions.

Abstract LB-320: Evaluating changes in circulating tumor cell count in prostate cancer patients during androgen deprivation plus chemotherapy by using AxonDx enumeration device

Abstract Background: Circulating tumor cell (CTC) counts have clinical utility as a prognostic marker in cancer prostate cancer (PCa). Using the FDA-approved CellSearch system, CTC counts &amp;gt5 CTC/7.5 ml convey a negative prognosis with respect to overall survival. The significance of sequential CTC counts during treatment is not fully understood. To determine whether longitudinal CTC counts can be used as a prognostic and/or predictive marker of clinical outcome in PCa patients undergoing androgen deprivation therapy (ADT) plus chemotherapy, we used the AxonDx nCyte non-enrichment-based CTC detection and enumeration system to evaluate CTC counts at baseline, during, and after therapy in 16 patients for up to 11 months. Methods: Whole blood was drawn from PCa patients participating in a clinical trial of ADT plus chemotherapy. CTCs were determined at multiple time points: at PSA evaluation (every 8 wks, ±radiographic reevaluation [every 16 wks]). Blood was collected into vacutainers containing K3-EDTA and processed within 6 hr. PBMC containing nucleated cells harvested from 6 ml of whole blood were fixed, permeabilized and blocked with BSA buffer, and stained using a proprietary antibody cocktail containing DAPI, an anti-cytokeratin mixture, the pan-leukocyte marker CD45, and markers selective for granulocytes and lymphocytes. Stained cells were drawn onto 1.2 µm pore filters, washed, placed on counting slides, and analyzed. CTCs are identified as cytokeratin-positive, nucleus-positive and leukocyte marker-negative. Results: CTCs were obtained from 12 patients for 2-9 months at 2-7 time points; from 2 patients for 11 months at 5-7 time points, and from 2 patients for 1 month at 1 time point. We detected time-dependent changes in CTC counts following treatment that did not necessarily correlate with patient's local prostate specific antigen (PSA) levels but were early indicators of subsequent increase in tumor burden. Conclusions: The AxonDx nCyte system can reliably detect CTCs from PCa patient samples that trend along with other signs of disease progression. CTCs can be used longitudinally for clinical monitoring of patients with PCa prior to signs of progression via tumor markers or scans during ADT plus chemotherapy. To further evaluate sequential CTC counts as a reliable biomarker for PCa, we plan additional studies to confirm our data and compare the AxonDx nCyte system with the CellSearch device in our ongoing clinical trial PCa program. Citation Format: Robert J. Amato, Reynolds Brobey, Yue Wang, Mehdi Dehghani, Zoe Tramel, Kent Murphy, Jeff Smith. Evaluating changes in circulating tumor cell count in prostate cancer patients during androgen deprivation plus chemotherapy by using AxonDx enumeration device [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-320.

Evaluating changes in circulating tumor cell count in prostate cancer patients during androgen deprivation plus chemotherapy by using AxonDx enumeration device.

266 Background: Circulating tumor cell (CTC) counts have clinical utility as a prognostic marker in cancer. For prostate cancer (PCa), CTC counts ≥5 CTC/7.5 ml using the FDA-approved CellSearch system convey a negative prognosis with respect to overall survival, but the significance of changes in CTC count during treatment is not fully understood. To determine whether longitudinal CTC counts can be used as a prognostic and/or predictive marker of clinical outcome in PCa patients undergoing androgen deprivation therapy (ADT) plus chemotherapy, we used the AxonDx nCyte non-enrichment-based CTC detection and enumeration system to evaluate CTC counts at baseline, during, and after therapy in 18 patients for up to 11 months. Methods: Whole blood was drawn from PCa patients on clinical trials for ADT plus chemotherapy at PSA evaluation (±radiographic reevaluation) into vacutainers containing K3-EDTA and processed within 6 hr. PBMC containing nucleated cells harvested from 6 ml of whole blood were fixed, permeabilized and blocked with BSA buffer, and stained using a proprietary antibody cocktail containing DAPI, an anti-cytokeratin mixture, the pan-leukocyte marker CD45, and markers selective for granulocytes and lymphocytes. Stained cells were drawn onto 1.2 µm pore filters, washed, placed on counting slides, and analyzed. CTCs are identified as cytokeratin-positive, nucleus-positive and leukocyte marker-negative. Results: CTCs were obtained from 12 patients for 2-9 months at 2-7 time points; from 2 patients for 11 months at 5-7 time points, and from 4 patients for 1 month at 1 time point. We detected time-dependent changes in CTC counts following treatment that did not necessarily correlate with patient’s local prostate specific antigen (PSA) levels but were early indicators of subsequent increase in tumor burden. Conclusions: The AxonDx nCyte system can reliably detect CTCs from PCa patient samples that trend along with other signs of disease progression. CTCs can be used longitudinally for clinical monitoring of patients with PCa prior to signs of progression via tumor markers or scans during ADT plus chemotherapy, providing a reliable biomarker for PCa.

Mesenchymal Stromal Cells as Active Regulators of Lymphocyte Recruitment to Blood Vascular Endothelial Cells.

Methods are described for analyzing adhesion and migration of isolated lymphocytes on endothelial cell monolayers which have been cocultured with different mesenchymal stromal cells, with or without additional cytokine treatment. The different cells types are grown on opposite sides of 3.0 or 0.4 μm pore filters, depending on whether migration through the whole construct is to be analyzed, or adhesion to the endothelial cells alone. Migration away from the sub-endothelial space and through the stromal layer can also be assessed by culturing mesenchymal stromal cells within a 3-D collagen gel overlaid with endothelial cells. Assays may be "static" or the filter-based constructs can be incorporated into flow chambers so that cell behavior can be directly observed under conditions simulating those in vivo. In general, by choice of method, one can evaluate efficiency of attachment, and ability of cells to migrate across the endothelial monolayer, through the filter and through the stromal cell layer in 2-D or 3-D. Fluorescence microscopic examination of fixed filters can be used, e.g., to ascertain whether lymphocytes are retained by stromal cells. In general, static assays have the higher throughput and greatest ease of use, while the flow-based assays are more physiologically relevant and allow detailed recording of cell behavior in real time.

Giant viruses: update, enigmas, controversies and perspectives

Unlike microbes known in his time, the first virus (that of tobacco mosaic disease) was discovered by Ivanoski in 1892 because it was not retained by Chamberland's porcelain candles. For more than a century afterward, viruses were equated with this simple property that is still extensively used today (using modern 0,2 µm pore filters) as a practical criterion to delineate the "viral fraction" from other microbes in medical or environmental samples. The first documented exception to the simplistic criterion of particle size came with the discovery of Mimivirus, the viral nature of which was eventually recognized in 2003, following ten years during which it was mistaken for an obligate intracellular bacterium. Thirteen more years later, we now realize that non-filtering "giant viruses" are not rare, probably ubiquitous, and come in a large variety of virion shapes, genome sizes, gene contents, and replication strategies. Following a quick description of the 4 giant virus families known today, we discuss the enigmas, controversies and perspectives of conceptual revolutions that are brought about by this new and booming area of virology.

Interaction of rare earth elements and components of the Horonobe deep groundwater

To better understand the migration behavior of minor actinides in deep groundwater, the interactions between doped rare earth elements (REEs) and components of Horonobe deep groundwater were investigated. Approximately 10 ppb of the REEs, i.e. Y, La, Ce, Pr, Nd, Sm, Eu, Gd, Tb, Dy, Er, Tm, and Yb were doped into a groundwater sample collected from a packed section in a borehole drilled at 140 m depth in the experiment drift of Horonobe Underground Research Laboratory in Hokkaido, Japan. The groundwater sample was sequentially filtered with a 0.2 μm pore filter, and 10 kDa, 3 kDa and 1 kDa nominal molecular weight limit (NMWL) ultrafilters with conditions kept inert. Next, the filtrate solutions were analyzed with inductively coupled plasma mass spectrometry (ICP-MS) to determine the concentrations of the REEs retained in solution at each filtration step, while the used filters were analyzed through neutron activation analysis (NAA) and TOF-SIMS element mapping to determine the amounts and chemical species of the trapped fractions of REEs on each filter. A strong relationship between the ratios of REEs retained in the filtrate solutions and the ionic radii of the associated REEs was observed; i.e. smaller REEs occur in larger proportions dissolved in the solution phase under the conditions of the Horonobe groundwater. The NAA and TOF-SIMS analyses revealed that portions of the REEs were trapped by the 0.2 μm pore filter as REE phosphates, which correspond to the species predicted to be predominant by chemical equilibrium calculations for the conditions of the Horonobe groundwater. Additionally, small portions of colloidal REEs were trapped by the 10 kDa and 3 kDa NMWL ultrafilters. These results suggest that phosphate anions play an important role in the chemical behavior of REEs in saline (seawater-based) groundwater, which may be useful for predicting the migration behavior of trivalent actinides released from radioactive waste repositories in the far future.

Evaluation of Nuclepore Membrane Filteration Technique for Diagnosis of Schistosoma haematobium Infection

Shistosomiasis is a parasitic disease . It is one of the most important diseases of humans in tropical and subtropical parts of the world , rapid characterization of schistosome eggs considered as a key step for clinical management, diagnosis of Schistosoma haematobium is generally based on the detection of eggs in a single urine specimen, Urine filteration is one of the methods recommended by the WHO for the detection of S.haematobium. In 1976, Peters et al adapted nucleopore filter for the diagnosis of S.haematobium eggs . They used filter 13 mm in diameter with 8 µm pores ,In another study done by Peters et al (1976),they proved that under field conditions , filteration of two 5 ml aliquots using 8 µm pore filters was very practical , if eggs of S.haematobium are present (size 150 by 60 µm), they are unable to pass through the filter and can be observed and counted under a microscope fitted with a 10x objective. This study used both the microscopic examination techniques of urine, centrifugation sedimentation technique and Nuclepore membrane filteration technique to detect Schistosoma haematobium eggs in urine for 1000 patient samples, to evaluate each technique alone and the two techniques together . This study showed that nuclepore filteration method more valid than sedimentation centrifugation method, nuclepore filteration detect 77 infected cases with sensitivity of 97.5%, but sedimentation detect 64 infected cases with a sensitivity of 81%, in this study we considered the sum of nuclepore and sedimentation as a gold standard, they detected 79 infected cases to compare them with another methods.

P67: Use of extracellular RNA from urine for the diagnosis of prostate cancer

Differently sized microparticles, including exosomes (30–100 nm), prostasomes (50–500 nm), oncosomes (50–500 nm) and other microparticles (100–1000 nm) were found in blood and urine. Exosomes from prostate cancer (PCa) patients can potentially contain cancer-specific nucleic acids, and thus can represent a valuable source of diagnostic material. In this study, we have investigated microvesicles and miRNA from urine of healthy donors and PCa patients. To isolate miRNAs from urine and microparticles, novel methods for miRNA isolation were elaborated (Rus. patent application No 2014137763, priority date 17.09.2014). The study population included 14 patients with PCa (63–82 years, T2-3NxMx1) and control group of 20 healthy volunteers with no previous history of prostate disease (48–73 years). Urine was clarified by two serial centrifugations at 400 g , 20 °C, 20 min and at 17000 g , 20 °C, 20 min. Microparticles were precipitated from the resulting supernatant by high-speed centrifugation at 100000 g , 18 °C, 90 min, the pellet was resuspended and pelleted by centrifugation under the same conditions. To isolate exosomes, total microparticles were filtered through 0.1 μm pore filters and reprecipitated. The resulting pellets were resuspended and exosome samples were investigated by transmission electron microscopy (TEM). MiRNAs were isolated by one-step single-phase protocol and purified using “BioSilica” spin- columns (Zaporozhchenko et al., Anal. Biochem, upcoming, doi: 10.1016/j.ab.2015.03.028) and by recently developed method based on precipitation of excess biopolymers, allowing to isolate miRNAs with better efficiency than commercially available kits. The size and quantity assessment of extracellular RNA were performed using capillary electrophoresis system on Agilent 2100 Bioanalyzer. Concentrations of miRNAs (miR19b, miR25, miR205, miR125b, miR126) were measured by qRT-PCR and normalized to miR-16 using dCq method. TEM demonstrated the presence of 20–300 nm microparticles in urine of healthy donors and PCa patients. Approximately 50–70% of all urine microparticles are represented by 30–100 nm exosomes and residual 30–50% by particles larger than 100 nm. (The pool of urine microvesicles consists of 50–70% exosomes and 30–50% particles larger than 100 nm.). The major part of extracellular RNA found both in exosomes and total microparticles fraction of healthy donors and patients with PCa, is 25–200 nt long and can include tRNA (73–93 n.), 5.8 rRNA (∼150 n.), snoRNK (10–20 n.), snRNA (60–300 n.) piRNAs (29–30 n.), miRNAs (20–25 n.), siRNA (21–25 n.). Concentration of extracellular urine RNA in exosomes and total microparticles of healthy donors and patients with PCa amounts to100 pg/ml of urine on average.Receiver Operating Characteristic (ROC) curve analysis of all miRNAs in samples isolated from total urine did not demonstrate any diagnostic significance. In contrast, in training cohort concentration of miR-19b in exosomes and total microparticles fraction provide 95%/75% and 93%/100% sensitivity and specificity, respectively. Thus, microparticles, isolated from urine of PCa patients represent a valuable source of diagnostically significant miRNAs. To investigate miR-19b, a large testing cohort is required. Search for other potential miRNA-markers by microarray profiling of cell-free miRNA isolated from urine of PCa patients is also required to reveal a sold set of markers for confident PCa diagnostics.

CUB domain containing protein 1 (CDCP1) modulates adhesion and motility in colon cancer cells.

BackgroundDeregulated expression of the transmembrane glycoprotein CDCP1 (CUB domain-containing protein-1) has been detected in several cancers including colon, lung, gastric, breast, and pancreatic carcinomas. CDCP1 has been proposed to either positively or negatively regulate tumour metastasis. In this study we assessed the role of CDCP1 in properties of cells that are directly relevant to metastasis, namely adhesion and motility. In addition, association between CDCP1 and the tetraspanin protein CD9 was investigated.MethodsCDCP1 and CD9 protein expression was measured in a series of colon cancer cell lines by flow cytometry and Western blotting. Adhesion of Colo320 and SW480 cells was determined using a Matrigel adhesion assay. The chemotactic motility of SW480 cells in which CDCP1 expression had been reduced by RNA interference was analysed using the xCELLigence system Real-Time Cell Analyzer Dual Plates combined with 8 μm pore filters. Detergent-resistant membrane fractions were generated following density gradient centrifugation and the CDCP1 and CD9 protein composition of these fractions was determined by Western blotting. The potential association of the CDCP1 and CD9 proteins was assessed by co-immunoprecipitation.ResultsEngineered CDCP1 expression in Colo320 cells resulted in a reduction in cell adhesion to Matrigel. Treatment of SW480 cells with CDCP1 siRNA reduced serum-induced chemotaxis. CDCP1 and CD9 cell-surface protein and mRNA levels showed a positive correlation in colon cancer cell lines and the proteins formed a low-level, but detectable complex as judged by co-sedimentation of detergent lysates of HT-29 cells in sucrose gradients as well as by co-immunoprecipitation in SW480 cell lysates.ConclusionsA number of recent studies have assigned a potentially important role for the cell-surface protein CDCP1 in invasion and metastasis of a several types of human cancer cells. In this study, CDCP1 was shown to modulate cell-substratum adhesion and motility in colon cancer cell lines, with some variation depending on the colon cancer cell type. CDCP1 and CD9 were co-expressed at the mRNA and protein level and we obtained evidence for the presence of a molecular complex of these proteins in SW480 colon cancer cells.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2407-14-754) contains supplementary material, which is available to authorized users.

The Influence of Nuclear Magnetic Resonance Therapy (NMRT) and Interleukin IL1-β Stimulation on Cal 78 Chondrosarcoma Cells and C28/I2 Chondrocytes

The Influence of Nuclear Magnetic Resonance Therapy (NMRT) and Interleukin IL1-β Stimulation on Cal 78 Chondrosarcoma Cells and C28/I2 Chondrocytes

Influence of Apical Patency and Cleaning of the Apical Foramen on Periapical Extrusion in Retreatment

This study evaluated the influence of apical patency, root filling removal technique and cleaning of the apical foramen, concerning the amount of debris extruded during root canal retreatment. Forty mandibular incisors were randomly assigned to 4 groups - GIM (n=10), GIIM (n=10), GIPT (n=10) and GIIPT (n=10), which were named according to leaving (I) or not (II) apical patency during canal preparation and filling removal technique (manual - M or ProTaper system - PT). After filling material removal, each specimen of each group had the apical foramen cleaned by sizes 15, 20 and 25 instruments, generating 12 subgroups: GIM15, GIM20, GIM25, GIIM15, GIIM20, GIIM25, GIPT15, GIPT20, GIPT25, GIIPT15, GIIPT20 and GIIPT25. Extruded filling debris was collected by a Milipore filtration system, an HV-durapore, 0.45 µm pore filter with a 25 mm diameter. The filters were weighed before and after the collection on an analytical scale (10-5 g), and the difference was calculated. The mean weight of extruded filling debris was analyzed statistically by Kruskal-Wallis and Friedman ANOVA tests (α=0.05). The mean values found in the groups (in mg) were: GIM (0.95 ± 0.94), GIIM (0.47 ± 0.62), GIPT (0.30 ± 0.31) and GIIPT (0.32 ± 0.44). There was no statistically significant difference among any of the groups or subgroups (p>0.05). ProTaper provided the smallest amount of extruded filling material, regardless of presence or absence of apical patency, followed by manual technique, without and with apical patency. Additional amounts of debris were collected during cleaning of the apical foramen, regardless of the instrument, presence/absence of patency or root filling removal technique.

Cortactin Expression Is Tightly Connected to B-Cell Chronic Lymphocytic Leukemia Aggressiveness

AbstractAbstract 4561 Background.Cortactin is an ubiquitous actin-binding protein, encoded by EMS1 gene and localized in chromosome 11q13 region. This protein is expressed in nearly all mammalian tissues and mostly localizes to dynamic actin structures. As a consequence, cortactin is involved in regulating several actin-dependent processes, including lamellipodium protrusion, trafficking of the key invadopodia proteases and intracellular transducer downstream to kinase-mediated cell signalling upon phosphorylation by Src and Syk tyrosine kinase families. Cortactin is over-expressed in several tumors, such as esophageal squamous cell carcinoma, head and neck squamous cell carcinoma and gastric carcinoma, and so tied to tumor aggressiveness by the promotion of cell invasion and metastasis. We previously showed that cortactin is over-expressed also in neoplastic B cells of patients with B-Cell Chronic Lymphocytic Leukemia (B-CLL). In the present study we investigated the regulation of cortactin activity by the Src kinase Lyn in neoplastic B-CLL cells and the biological implications of cortactin overexpression in this leukemia. Methods.Twenty patients and ten normal controls were enrolled. Informed consent was obtained from all patients according to the Declaration of Helsinki. CD19+ lymphocytes were purified from peripheral blood by negative selection using the RosetteSep cells isolation kit (StemCell Technologies). Cortactin localization was performed by Confocal Microscope analysis at basal condition, in presence of the chemotactic stimulus CXCL12 and Src kinase inhibitor PP2. Lyn kinase and cortactin phosphorylation levels were evaluated by western blotting analysis. Migration assay was performed using 3 μm pore filters (Transwell Permeable Supports) in the presence of CXCL12, Src kinase inhibitor PP2 and after cortactin silencing. Results.We found that cortactin localization is regulated by Lyn kinase through its phosphorylation. In CLL cells, cortactin was over-phosphorylated and it did not co-localize with actin, as compared to normal cells. PP2-inhibition of Lyn decreased cortactin phosphorylation and triggered its co-localization with actin. Cortactin over-expression proved to be associated to the increased B-CLL spreading through Lyn activity. In fact, not only cortactin over-expression correlated with leukemic cell increased response to CXCL12 (r = 0.9), but also the inhibition of cortactin activity, through PP2 inhibitor or cortactin silencing, drastically reduced neoplastic cell migration after chemotactic triggering. Conclusions.These results suggest that cortactin is involved in aggressiveness and spreading of B-CLL cells and that Lyn-cortactin axis could represent an alternative target for the development of new therapeutic strategies. Disclosures:No relevant conflicts of interest to declare.

Measurement of airborne nanoparticle surface area using a filter-based gas adsorption method for inhalation toxicology experiments

Measurement of the surface area of airborne nanoparticles as administered to an experimental subject is critical for characterizing exposures during inhalation experiments. A filter-based surface area measurement methodology is described herein that allows for such determinations. Krypton gas adsorption was used to determine total particle surface area. Track-etched polycarbonate 0.4 μm pore filters were chosen as the collection substrate for metal oxide particles due to their highly reproducible surface areas and low background weights. The subject nanomaterials included two different batches of ultrafine TiO2, TiO2 nanorods, and SiO2. The instrument detection limit for surface area was 200 cm2 (0.02 m2). Ninety percent confidence interval estimates of method accuracy were 17.7–23.5% with a point estimate of 20.8%. The filter-based surface area measurement strategy is demonstrated to be a viable sampling and analysis methodology that provides much needed physical characterization information of particles as administered in an animal inhalation chamber.

PMN TRANSMIGRATION THROUGH ENDOTHELIUM IS ENHANCED BY THE COMBINED INJURY OF BURN AND INFECTION

PMNs are important in the host defense against microbes. PMNs also play an important role in tissue damages in injury conditions. We showed that the combined injury of burn (B) and infection (CLP) increased PMN accumulation in the intestine and intestinal microvascular permeability to albumin. Those changes were greater in the combined injury than B or CLP alone. Activated PMNs, increased chemoattractants, or both appeared to be responsible for those changes. In order to evaluate PMN contribution to those changes, we studied PMN transmigration and albumin permeability through the endothelium using endothelial monolayers in vitro in the present study. Materials and Methods Male Sprague-Dawley rats were divided into 4 groups: B group (30% total body surface area), CLP group, B+CLP group, and sham (S) group. One day after the injury, blood was collected and PMNs were isolated. A rat microvascular endothelial cell-line was cultured on 3 μm pore filter inserts in 24-well plates. When confluent endothelial cell monolayers (END) developed, the isolated PMNs and I125 -albumin were placed on END and cultured for an hour. PMNs and I125 -albumin, which passed through END, were counted. PMN productions of reactive oxygen species (ROS) and elastase were also observed. Results Both ROS and elastase productions increased in B+CLP group more than B or CLP groups. Ecadherin decreased in B+CLP group more than B or CLP groups.FigureConclusion PMNs play an important role in the enhancement of PMN transmigration in the combined injury of B + CLP. (Supported by NIH RO1 grants GM53235 and GM56865, and Folk research foundation.)

071 TGF‐ß‐Mediated Paracrine Regulation of Keratinocyte Wound Closure

The importance of stromal‐epithelial interactions in wound healing is well established. These interactions likely involve autocrine and paracrine action of multiple growth factors, including members of the TGF‐ß family. TGF‐ß1, ß2 and ß3 isoforms signal by sequentially binding to the TGF‐ß type II and type I receptors, respectively. We address the role of TGF‐ß signaling in dermal fibroblasts using a conditional fibroblastic TGF‐ß type II receptor knockout mouse model (termed FßKO). We found that the loss of TGF‐ß signaling in the dermal fibroblasts results in accelerated excision‐wound closure compared with similar wounds in wild type mice. The mechanism of the altered rate of re‐epitheliaization in the FßKO mice was examined with regard to keratiocyte motility and proliferation. The migration of keratinocytes through collagen I coated 8 μm pore filters in the presence or absence of fibroblast‐conditioned media was tested. These experiments showed increased keratinocyte migration when incubated with FßKO dermal fibroblast conditioned media compared to media conditioned in wild type fibroblasts. Immuno‐histochemical staining of paraffin embedded intact skin indicated both wild type and FßKO mice had similar low levels of keratinocyte proliferation, based on Ki67 staining. In healing wounds, only the distal wound edges of wild type mice were proliferative. In contrast, the FßKO mice exhibited elevated proliferation across the length of the wound, including the leading edge of epithelial closure. Together our results suggest TGF‐ß signaling by the dermal fibroblasts suppresses re‐epithelialization of excision wounds by regulating keratinocyte motility and proliferation through paracrine mechanisms. Funding: DOD BC99184 and NIH CA85492.