OD Measurements Research Articles

Role of planktonic and sessile extracellular metabolic byproducts on Pseudomonas aeruginosa and Escherichia coli intra and interspecies relationships

Bacterial species are found primarily as residents of complex surface-associated communities, known as biofilms. Although these structures prevail in nature, bacteria still exist in planktonic lifestyle and differ from those in morphology, physiology, and metabolism. This study aimed to investigate the influence of physiological states of Pseudomonas aeruginosa and Escherichia coli in cell-to-cell interactions. Filtered supernatants obtained under planktonic and biofilm cultures of each single species were supplemented with tryptic soy broth (TSB) and used as the growth media (conditioned media) to planktonic and sessile growth of both single- and two-species cultures. Planktonic bacterial growth was examined through OD(640) measurement. One-day-old biofilms were evaluated in terms of biofilm biomass (CV), respiratory activity (XTT), and CFU number. Conditioned media obtained either in biofilm or in planktonic mode of life triggered a synergistic effect on planktonic growth, mainly for E. coli single cultures growing in P. aeruginosa supernatants. Biofilms grown in the presence of P. aeruginosa biofilms-derived metabolites presented less mass and activity. These events highlight that, when developed in biofilm, P. aeruginosa release signals or metabolites able to prejudice single and binary biofilm growth of others species and of their own species. However, products released by their planktonic counterparts did not impair biofilm growth or activity. E. coli, living as planktonic or sessile cultures, released signals and metabolites or removed un-beneficial compounds which promoted the growth and activity of all the species. Our findings revealed that inter and intraspecies behaviors depend on the involved bacteria and their adopted mode of life.

Combined effect of lysozyme and nisin at different incubation temperature and mild heat treatment on the probability of time to growth of Bacillus cereus

Stochastic models can be useful to predict the risk of foodborne illness. The presence of Bacillus cereus in liquid egg can pose a serious hazard to the food industry, since a mild heat treatment cannot guarantee its complete inactivation. However, most of the information available in the scientific literature is deterministic, including growth of B. cereus. In this paper, a stochastic approach to evaluate growth of B. cereus cells influenced by different stresses (presence of nisin and lysozyme separately or in combination) was performed, using an individual-based approach of growth through OD measurements. Lag phase duration was derived from the growth curves obtained. From results obtained, histograms of the lag phase were generated and distributions were fitted. Normal and Weibull distributions were ranked as the bestfit distributions in experiments performed at 25 °C. At 16 °C, lag values (obtained in presence of combinations of both antimicrobials) were also fitted by a Gamma distribution. Predictions were compared with growth curves obtained in liquid egg exposed to mild heat, nisin and/or lysozyme to assess their validity.

Abstract 5428: Histone deacetylase (HDAC) enzyme inhibition potentiates taxanes and doxorubicin activity in thyroid cancer cell lines

Abstract Thyroid cancer is the fastest growing cancer in the US. Doxorubicin, the only approved medical therapy, has limited efficacy; therefore alternative treatment options are warranted. We previously showed increased cytotoxicity when HDAC inhibitor was combined with taxanes and carboplatin in lung cancer cell lines. We now studied the combination of HDAC inhibitor and cytotoxic agents in thyroid cancer cell lines. Exponentially growing cells (Cal 62, PTC1, and BCPAP) were treated by continuous exposure for 72 hrs to vehicle or increasing concentrations of doxorubicin, paclitaxel or docetaxel ± fixed concentration of LBH589. Surviving cell fraction was estimated by sulforhodamine B (SRB) colorimetric assay. IC50 concentration was determined by non-linear regression modeling of OD measurements. In a separate experiment, Western blot assay for markers of apoptosis (caspase and PARP cleavage), cell cycle regulation (p21, p27) and tubulin acetylation was performed using cell lysates from cells treated for 48 hrs with IC50 concentration of chemotherapy drugs ± LBH589. Similarly treated cells were analyzed for changes in cell cycle distribution using propidium iodide staining and flow cytometry. Doxorubicin, docetaxel and paclitaxel showed potent nanomolar activity with up to 80% reduction in the IC50 concentrations when combined with LBH589 (paclitaxel: 0% - 70%; docetaxel: 60% - 84%; doxorubicin: 2% - 65%). Flow cytometry showed qualitative evidence for increased cell death with a 1.5 to 6-fold increase in the subG1 apoptotic cell fraction with combined doxorubicin and LBH589 versus doxorubicin alone. The increased cytotoxicity correlated with increased PARP cleavage and increased p21 protein expression; p27 induction by doxorubicin was not augmented by co-treatment with LBH589. HDAC inhibition augments in vitro cytotoxicity of microtubule and DNA-damaging agents in thyroid cancer cell lines. This combination deserves further evaluation in this disease.DrugIC50 Concentrations in Thyroid Cancer Cell Lines (nM) BCPAPCAL 62PTC1 − L+L− L+L− L+L Paclitaxel3.60.641.310.538.6631.365Docetaxel6.9852.1450.710.282.252.25Doxorubicin34.2512.142278.6778.7188.542LBH569 16.84 11.94 22.71Flow Cytometry Analysis Following Propidium Iodide Staining BCPAPCAL 62PTC1TreatmentVDoxDox + LVDoxDox + LVDoxDox + L%Sub G145.5456.362.286.825.6766.775.456.914.21V: Vehicle;Dox: Doxorubicin;L: LBH589Full details of Western blot assay and complete flow analysis to be presented at the meeting.Acknowledgement: Taofeek Owonikoko, Suresh Ramalingam, Fadlo Khuri and Shi-Yong Sun are all supported by the Georgia Cancer Coalition as Distinguished Cancer Scholars. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5428.

BOD Exertion and OD600 Measurements in Presence of Heavy Metal Ions Using Microbes from Dairy Wastewater as a Seed

BOD measurements in presence of cobalt, nickel, copper, zinc, silver and cadmium are reported using wastewater from dairy industry as a seed. Extent of inhibition in BOD is studied for variables like, concen-tration of metal ions (0.2 mM to 1.4 mM), pH (3 to 8) and temperature of incubation (15℃, 20℃, 25℃, 30℃ and 35℃). Results of BOD inhibition are supported by absorbance measurement (OD600) studies of microbial matter preserved in Luriya broth medium. OD measurement results are used to derive minimum inhibitory concentration, i.e., threshold concentration of each metal showing toxicity towards microbes. Sil-ver is found to be the most toxic element.

Using the rate of respiration to monitor events in the infection of Escherichia coli cultures by bacteriophage T4

The growing interest in applications of bacteriophages creates a need for improvements in the production processes. Continuous monitoring of the phage production is an essential aspect of any control strategy and, at present, there is no completely satisfactory option. The approach presented here uses IR-spectrometry to continuously measure the rate of respiration (CO(2) released) of Escherichia coli infected by phage T4 at various multiplicities of infection (MOI). Within the trends in these data, or in other aspects of the rate of respiration, it was possible to reliably and reproducibly identify five features that reflected specific events in the infection process. These included two events in the host cell apparent growth rate and events in the magnitude of the host cell density, in the measurement of OD(600) or in the specific rate of respiration. All of these correlations were within 95% confidence showing that they are suitable for the monitoring and control of E. coli populations infected by phage T4. This method is reliable, cheap, and can be operated in-line and in real time.

Prediction of time to growth of Listeria monocytogenes using Monte Carlo simulation or regression analysis, influenced by sublethal heat and recovery conditions

Stochastic models, including the variability in extent and probability of microbial growth, are useful for estimating the risk of foodborne illness (i.e. Nauta, 2000). Risk assessment typically has to embrace all sources of variability. In this paper, a stochastic approach to evaluate growth of heat damaged Listeria monocytogenes cells influenced by different stresses (pH and presence of eugenol) was performed, using an individual-based approach of growth through OD measurements. Both the lag phase duration and the “work to be done” ( h 0 parameter) were derived from the growth curves obtained. From results obtained histograms of the lag phase were generated and distributions were fitted. Histograms showed a shift to longer lag phases and an increase in variability with high stress levels. Using the distributions fitted, predictions of time to unacceptable growth (10 2 cfu/g) of L. monocytogenes were established by Monte Carlo simulation and they were compared with results from statistical methods. It was evidenced that both methods (Monte Carlo and regression analysis) gave a good indication of the probability of a certain level of growth other than the average. Tornado plots were obtained to establish a sensitivity analysis of the influence of the conditions tested (heat, pH, eugenol) applied to the microorganism and their combinations.

Development of and results from a new coiled tubing inspection system

We have now built systems that will perform NDE inspections of (a) the body and (b) the seam weld of new and used electric-weld coiled carbon steel tubulars. The full-body inspection system provides four functions: OD and ovality measurement, internal and external imperfection detection and assessment and wall thickness measurement, all by noncontact measurements. The magnetising system places a high magnetic field across the seam weld and searches for either flaws or permeability variations there-in. Advanced forms of data processing are used to image the magnetic flux leakage (MFL) (1) from wall anomalies. Systems may be built into coiled tubing (CT) rigs, and can be configured to add fatigue models (2) , so that fatigue loss due to tubing defects can be assessed. Two systems are in operation in CT mills, while one operates for a commercial oil country tubular goods (OCTG) inspection company. Results from inspections using these tools are presented.

Coadministration of Telomerase Genetic Vaccine and a Novel TLR9 Agonist in Nonhuman Primates

The human telomerase reverse transcriptase (hTERT) is an attractive target for human cancer vaccination because its expression is reactivated in most human tumors. We have evaluated the ability of DNA electroporation (DNA-EP) and adenovirus serotype 6 (Ad6) to induce immune responses against hTERT in nonhuman primates (NHPs) (Macaca mulatta). Vaccination was effective in all treated animals, and the adaptive immune response remained detectable and long lasting without side effects. To further enhance the efficacy of the hTERT vaccine, we evaluated the combination of hTERT vaccine and a novel TLR9 agonist, referred to as immunomodulatory oligonucleotide (IMO). Monkeys were dosed weekly with IMO concurrently with the vaccine regimen and showed increases in cytokine secretion and activation of natural killer (NK) cells compared with the group that received vaccine alone. Using a peptide array, a specific profile of B-cell reactive epitopes was identified when hTERT vaccine was combined with IMO. The combination of IMO with hTERT genetic vaccine did not impact vaccine-induced TERT-specific cell-mediated immunity. Our results show that appropriate combination of a DNA-EP/Ad6-based cancer vaccine against hTERT with IMO induces multiple effects on innate and adaptive immune responses in NHPs.

Sequence-specific bacterial growth inhibition by peptide nucleic acid targeted to the mRNA binding site of 16S rRNA

Peptide nucleic acid (PNA) targeted to the functional domains of 23S rRNA can inhibit translation and cell growth. However, effective inhibition of translation and cell growth using 16S rRNA-targeted PNA has still not been achieved. Here, we report that PNA targeted to the functional site of 16S rRNA could inhibit both gene expression in vitro and bacterial growth in pure culture with sequence specificity. We used 10-mer PNAs conjugated with a cell-penetrating peptide, which targeted the mRNA binding site at the 3' end of 16S rRNA. Using 0.6 microM of the peptide-PNAs, cell-free ss-galactosidase production decreased by 50%, whereas peptide-PNAs with one or two mismatches to the target sequence showed much weaker inhibition effects. To determine the growth inhibition and bactericidal effects of the peptide-PNA conjugate, we performed OD measurement and viable cell counting. We observed dose- and sequence-dependent inhibition of cell growth and bactericidal effects. These growth inhibitory effects are observed both in the Gram-negative bacterium of Escherichia coli and the Gram-positive bacteria Bacillus subtilis and Corynebacterium efficiens, although inhibitory concentrations were different for each bacterial species. These results present possibilities for 16S rRNA sequence-based specific bacterial growth inhibition using a peptide-PNA conjugate.

Impacts of gold nanoparticle exposure on two freshwater species: a phytoplanktonic alga (Scenedesmus subspicatus) and a benthic bivalve (Corbicula fluminea)

For years, nanotechnologies have developed the use of common materials, such as iron or silica, at an extremely small scale because of their new properties (reactivity, conductivity, optical sensitivity). More precisely, gold nanoparticles are used in numerous technologies such as electronics, new paints or research on cancer. But, despite their promising future and expansive utilization, only a few studies deal with their behaviors or impacts on the environment. Thus, we decided to explore the impacts of amine-coated 10nm gold nanoparticle (AuNp) contaminations on two freshwater aquatic models. The green algaScenedesmus subspicatus was submitted to 24 h-direct exposures at four AuNp concentrations (1.6×102, 1.6×103, 1.6×104 and 1.6×105 AuNp/cell) along with a control condition. The process used for the freshwater bivalvesCorbicula fluminea was a trophic exposure during 7 days to three AuNp concentrations (1.6×103, 1.6×104 and 1.6×105 AuNp/cell). These conditions were tested in triplicate with controls. For these experiments, OD measurements (γ= 520nm) were performed to verify AuNp concentrations in the water (stability). Cell numerations of algae were used to determine the growth/mortality effects on this species. Cellular impacts and AuNp distributions in the two species were revealed by transmission electron microscopy (TEM). The bioaccumulation rates were assessed by gold dosagesvia MS-ICP procedures. Molecular impacts were analyzed by quantifications of metallothionein concentrations (metal detoxification protein) and genetic expressions via real-time RT-PCR. Our study focused on the expression of six genes encoding proteins involved in: metal detoxification (metallothionein), the response to oxidative stress (catalase and superoxide-dismutase), the mitochondrial respiratory chain (subunit 1 of the cytochrome-C-oxidase), the concentration of mitochondria (RNA12s) and the response to xenobiotics (glutathione S transferase); using the β-actin as reference of the basal rates of gene expressions. The results showed a marked impact on the algae after a 24h-exposure to amine-coated 10nm gold nanoparticles, leading to 20% of mortality for the lowest contamination condition, while the highest one reached 50%. TEM examinations showed that AuNp were strongly adsorbed by the cell wall of algae, leading to progressive intracellular and wall disturbances. The bivalve contaminations revealed the ability of these particles to be bioaccumulated and to penetrate gills and digestive epithelia. Their lysosomial localization leads to the loss of their coating, which brought on an oxidative stress.

A High-Throughput In Vitro Model Illustrating Potential Microbiological Interactions During Treatment of Pseudomonas Aeruginosa Biofilm Associated Infections

Objective: Amend a real-time, high-throughput method of bacterial growth detection for use as a model of biofilm response to co-administered pharmaceuticals during the treatment of devise associated infections. Background: Biofilms are the root etiology for chronic infections, particularly in regard to infections in patients with implanted medical devices. Calcium channel blockers (CCBs) are used for control of hypertension and angina and are commonly prescribed to elderly patients. We address potential interference of commonly prescribed CCBs with levofloxacin for treatment of Pseudomonas aeruginosa biofilms. Methods: Inoculum of 1–3×106CFU∕mL in the log phase were seeded into each well of a polystyrene plate. Biofilms developed over 6h at 37°C, was washed and medium containing various CCBs plus levofloxacin was added to the biofilm. OD measurements were obtained at 1h intervals over 90h at 37°C. Changes in turbidity were kinetically measured with a vertical photometer with a wide-band filter. Results: Mibefradil and diltiazem appear to be strongly antagonistic toward levofloxacin where both of them decrease antibiofilm effect of levofloxacin and they encourage the selection of resistant mutants from biofilm. Discussion: Implanted medical devices are quite common and are subjected to biofilm infections. Increasing multi-drug resistance underscores the need to conserve current antibiotics by judicious use. This necessitates consideration of evidence regarding antagonistic or synergistic activity of commonly prescribed drugs of different classes toward commonly used antibiotics. The combinations described here show vital and previously unreported effects of some CCBs when co-prescribed with levofloxacin on Pseudomonas aeruginosa biofilm.

Multi-method approach indicates no presence of sub-lethally injured Listeria monocytogenes cells after mild heat treatment

Application of mild inactivation treatments follows an increasing trend in the food industry and is often combined with sub-optimal intrinsic product conditions to ensure appropriate level of microbial safety. Listeria monocytogenes was subjected to mild heat treatment (20 min at 60 °C) and subsequently exposed to various mild preservation conditions based on increased NaCl concentration and decreased pH. Recovery and resuscitation of L. monocytogenes cells were studied using various methods. Using 12-fold Most Probable Number (MPN) method no difference in the amount of recovered cells under adverse conditions was noted between heat-treated and non-treated L. monocytogenes cells. Time-to-detection method using on-line OD measurements showed that heat-treated L. monocytogenes cells reached detection limit faster in acidified media and NaCl supplemented media in comparison with non-heated control cells. Flow cytometry (FCM) analysis using 5–6-carboxyfluorescein diacetate (cFDA) and propidium iodide (PI) staining showed presence of low numbers of viable cells. Overall, there was no indication of sub-lethal injury in L. monocytogenes cells after mild heat treatment.

Accurate, precise modeling of cell proliferation kinetics from time-lapse imaging and automated image analysis of agar yeast culture arrays

BackgroundGenome-wide mutant strain collections have increased demand for high throughput cellular phenotyping (HTCP). For example, investigators use HTCP to investigate interactions between gene deletion mutations and additional chemical or genetic perturbations by assessing differences in cell proliferation among the collection of 5000 S. cerevisiae gene deletion strains. Such studies have thus far been predominantly qualitative, using agar cell arrays to subjectively score growth differences. Quantitative systems level analysis of gene interactions would be enabled by more precise HTCP methods, such as kinetic analysis of cell proliferation in liquid culture by optical density. However, requirements for processing liquid cultures make them relatively cumbersome and low throughput compared to agar. To improve HTCP performance and advance capabilities for quantifying interactions, YeastXtract software was developed for automated analysis of cell array images.ResultsYeastXtract software was developed for kinetic growth curve analysis of spotted agar cultures. The accuracy and precision for image analysis of agar culture arrays was comparable to OD measurements of liquid cultures. Using YeastXtract, image intensity vs. biomass of spot cultures was linearly correlated over two orders of magnitude. Thus cell proliferation could be measured over about seven generations, including four to five generations of relatively constant exponential phase growth. Spot area normalization reduced the variation in measurements of total growth efficiency. A growth model, based on the logistic function, increased precision and accuracy of maximum specific rate measurements, compared to empirical methods. The logistic function model was also more robust against data sparseness, meaning that less data was required to obtain accurate, precise, quantitative growth phenotypes.ConclusionMicrobial cultures spotted onto agar media are widely used for genotype-phenotype analysis, however quantitative HTCP methods capable of measuring kinetic growth rates have not been available previously. YeastXtract provides objective, automated, quantitative, image analysis of agar cell culture arrays. Fitting the resulting data to a logistic equation-based growth model yields robust, accurate growth rate information. These methods allow the incorporation of imaging and automated image analysis of cell arrays, grown on solid agar media, into HTCP-driven experimental approaches, such as global, quantitative analysis of gene interaction networks.

Sensing of Gram-positive bacteria in Drosophila: GNBP1 is needed to process and present peptidoglycan to PGRP-SA

Genetic evidence indicates that Drosophila defense against Gram-positive bacteria is mediated by two putative pattern recognition receptors acting upstream of Toll, namely Gram-negative binding protein 1 (GNBP1) and peptidoglycan recognition protein SA (PGRP-SA). Until now however, the molecular recognition proceedings for sensing of Gram-positive pathogens were not known. In the present, we report the physical interaction between GNBP1 and PGRP-SA using recombinant proteins. GNBP1 was able to hydrolyze Gram-positive peptidoglycan (PG), while PGRP-SA bound highly purified PG fragments (muropeptides). Interaction between these proteins was enhanced in the presence of PG or muropeptides. PGRP-SA binding depended on the polymerization status of the muropeptides, pointing to constraints in the number of PGRP-SA molecules bound for signaling initiation. We propose a model whereby GNBP1 presents a processed form of PG for sensing by PGRP-SA and that a tripartite interaction between these proteins and PG is essential for downstream signaling.

Prostaglandin J2 Alters Pro-survival and Pro-death Gene Expression Patterns and 26 S Proteasome Assembly in Human Neuroblastoma Cells

Many neurodegenerative disorders are characterized by two pathological hallmarks: progressive loss of neurons and occurrence of inclusion bodies containing ubiquitinated proteins. Inflammation may be critical to neurodegeneration associated with ubiquitin-protein aggregates. We previously showed that prostaglandin J2 (PGJ2), one of the endogenous products of inflammation, induces neuronal death and the accumulation of ubiquitinated proteins into distinct aggregates. We now report that temporal microarray analysis of human neuroblastoma SK-N-SH revealed that PGJ2 triggered a "repair" response including increased expression of heat shock, protein folding, stress response, detoxification and cysteine metabolism genes. PGJ2 also decreased expression of cell growth/maintenance genes and increased expression of apoptotic genes. Over time pro-death responses prevailed over pro-survival responses, leading to cellular demise. Furthermore, PGJ2 increased the expression of proteasome and other ubiquitin-proteasome pathway genes. This increase failed to overcome PGJ2 inhibition of 26 S proteasome activity. Ubiquitinated proteins are degraded by the 26 S proteasome, shown here to be the most active proteasomal form in SK-N-SH cells. We demonstrate that PGJ2 impairs 26 S proteasome assembly, which is an ATP-dependent process. PGJ2 perturbs mitochondrial function, which could be critical to the observed 26 S proteasome disassembly, suggesting a cross-talk between mitochondrial and proteasomal impairment. In conclusion neurotoxic products of inflammation, such as PGJ2, may play a role in neurodegenerative disorders associated with the aggregation of ubiquitinated proteins by impairing 26 S proteasome activity and inducing a chain of events that culminates in neuronal cell death. Temporal characterization of these events is relevant to understanding the underlying mechanisms and to identifying potential early biomarkers.

Diamagnetic levitation changes growth and gene expression of Saccharomyces cereviasiae

Background Magnetic flux (MF) is used in magnetic traps to levitate biological specimens by exploiting the diamagnetism of virtually all materials. However, little is known of the effects of MF during magnetic levitation (ML). This report investigates how changes in MF and gravity (GR) affect the eukaryotic organism Saccharomyces cerevisiae during ML. Methods Wild type (WT) yeast was compared to strains with genetic modifications in transcription factors Msn4, Sfp1 or Rap1 as they have been implicated in stress and gravity responses. Yeasts were levitated at various fluxes, and subjected to various gravity levels. Cell growth was determined by OD measurements in WT vs. Msn4 and Sfp1 deletion strains. Gene expression changes were determined by fluorescent content of strains bearing GFP-tagged reporters for YIL52C (Sfp1-depedent) YST-2 (Sfp1/Rap1-dependent) or SSA4 (Msn4-dependent) by flow cytometry. Results MF had no effect on growth of WT (12% reduction, p >0.05 vs. control), but it reduced growth in Msn4 and Sfp1-deletion strains (26% and 15%, p <0.05 vs. control). GR reduced growth in the WT and Msn4-deletion strains (30% and 33%, 0 g vs. 2 g, p < 0.05) but had no effect on Sfp1-deletion strain (0% change). MF increased expression of YIL52C and YST-2 strains (57% and 46%, p > 0.05 vs. control). GR reduced expression of YIL52C and SSA4 strains (41% and 65%, 0 g vs. 2 g, p < 0.05). Conclusions MF and GR had independent effects on yeast cell growth and gene expression in a strain dependent manner during ML. Supported by NASA NRA NAG8-1344, NIH P20 RR017659 grants and N.O. VA.

Novel Role of Toll-Like Receptor 3 in Hepatitis C-Associated Glomerulonephritis

Hepatitis C virus (HCV) infection is frequently complicated by glomerulonephritis with immune complexes containing viral RNA. We examined the potential influence of Toll-like receptors (TLRs), specifically TLR3 recognition of viral dsRNA exemplified by polyriboinosinic:polyribocytidylic acid [poly(I:C) RNA]. Normal human kidney stained positive for TLR3 on mesangial cells (MCs), vascular smooth muscle cells, and collecting duct epithelium. Cultured MCs have low TLR3 mRNA levels with predominant intracellular protein localization, which was increased by tumor necrosis factor-alpha, interleukin (IL)-1beta, interferon (IFN)-gamma, and the TLR3 ligand poly(I:C) RNA. Poly(I:C) RNA stimulation of MCs increased mRNA and protein synthesis of IL-6, IL-1beta, M-CSF, IL-8/CXCL8, RANTES/CCL5, MCP-1/CCL2, and ICAM-I; it also increased anti-proliferative and proapoptotic effects, the latter of which was decreased by inhibiting caspase-8. In microdissected glomeruli of normal and non-HCV membranoproliferative glomerulonephritis biopsies, TLR3 mRNA expression was low. In contrast TLR3 mRNA expression was significantly increased in hepatitis C-positive glomerulonephritis and was associated with enhanced mRNA for RANTES/CCL5 and MCP-1/CCL2. We hypothesize that immune complexes containing viral RNA activate mesangial TLR3 during HCV infection, thereby contributing to chemokine/cytokine release and effecting proliferation and apoptosis. Thus, TLR3 expression on renal cells, and especially MCs, may establish a link between viral infections and glomerular diseases.

66. Gene Transfer-Mediated Expression of Murine Factor VIIa Improves Clot Formation in a Mouse Model for Bernard-Soulier Syndrome

Moderate to severe bleeding occurs in Bernard-Soulier syndrome (BSS) patients due to a rare inherited dysfunction of their platelet GPIb|[alpha]|/|[beta]|-IX-V receptor, a four-gene encoded protein complex. Defective receptor disrupts the interaction between platelets and vascular wall and form abnormal macrothrombocytopenia, in addition to causing impaired thrombin generation and defective platelet plug at the tissue injury site. While platelet transfusion is the conventional therapy, treatment with a bolus injection of recombinant human factor VIIa (rhVIIa, NovoSeven|[reg]|) offers an alternative strategy especially when alloimmunization develops against administered platelets. At pharmacological plasma levels, rhVIIa led to cessation of mild and severe bleeding episodes in a number of BSS patients (Ozelo et al., Ann Hematol, 84(12), 2005 and Almeida et al, Br J Haematol, 121(3) 2003). However, the short half-life of the protein and its high expense remain as two major disadvantages of a rhVIIa regimen. We here report a gene therapy-based approach resulting in continuous expression of activated murine factor VII (mVIIa) in the plasma of a mouse model for BSS. Male BSS mice were tail-injected with three doses of AAV8-mVIIa vector ranging from 0.3 to 4.8e12 vg/mouse. The liver specific expression of mVIIa resulted in a dose- dependent increase in plasma mVIIa levels and shortening of prothrombin time (PT) with a plateau at four weeks after injection. At mVIIa levels >500 ng/ml (n=5), there was a significant increase in the rate of initial and progressing clot formation in whole blood measured by a thromboelastogram (ROTEM|[reg]|) compared to untreated age- and gender-matched control mice (n=5); CT (266|[plusmn]|44 vs 387|[plusmn]|59, P<0.01), CFT (125|[plusmn]|19 vs. 222|[plusmn]|76, P<0.03) and a angle (66.4|[plusmn]|3.6 vs. 52.7|[plusmn]|9.5, P<0.02), respectively. Moreover, tail clip assay also showed a dose-dependent decrease in blood loss, which was lower in mice receiving doses of 1.2 (n=12) and 4.8e12 (n=5) vg/mouse; OD measurement of hemoglobin released after lysis of RBC (1.0|[plusmn]|0.5, P<0.005 and 0.4|[plusmn]|0.3, P<5e-5) in the two doses vs. (1.5|[plusmn]|0.1) in untreated controls (n=5), respectively. Higher thrombin- anti thrombin (TAT) levels (49|[plusmn]|36 vs 12|[plusmn]|7, P<0.05) in the highest dose vs. control, respectively, reflected the increased overall thrombin generation upon VIIa treatment. Our data suggest that the increase in both the amount and rate of thrombin generation, resulting from continuous expression of mVIIa, could contribute to a better fibrin clot and partially correct the abnormal hemostasis in BSS mice. Whereas the mechanism of VIIa action is independent of the genetic defect, a gene approach to compensate for the ineffectiveness of receptor components largely depends on the affected gene(s), hence narrowing down the number of already rare recipients. Moreover, our approach could be expanded to include other bleeding diatheses due to more common platelet defects such as Glanzmann thrombasthenia, especially with failure of transfusion therapy.

Assessment of open and incomplete bite correction by incisor overlap and optical density of polyvinyl siloxane bite registration

Open bite (OB) is a generalized term, which could incorporate subgroups that react differently to vertical correction. The objectives of the present study were to detect vertical treatment changes in incomplete bite (IB: inter-incisor overlap with no lower incisor contact with teeth or palate) and OB (no inter-incisor overlap) groups compared with a complete bite (CB: inter-incisor overlap with full lower incisor contact with teeth or palate) control group, to evaluate treatment response of the central and lateral incisors, and to study the vertico-sagittal interaction. Dental casts were taken at three time points, pre-treatment, post-treatment, and after one year of retention, from 54 Class II patients (22 males and 32 females with a mean age of 11 years 6 months) divided into three groups: CB (n = 21), IB (n = 18) and OB (n = 15). Measurements included incisor overlap (mm) and optical density (OD/mm2) of occlusal bite registration made of polyvinl siloxane. Both CB and IB groups demonstrated post-retention bite opening. However, bite opening in the CB group was three times greater than that in the IB group (e.g. lower lateral = -1.42 mm, 118 OD/mm2 versus -0.40 mm, 107 OD/mm2). Conversely, the OB group showed a significant (P < 0.001) bite closure (e.g. lower lateral = 1.30 mm, -377 OD/mm2). Overjet changes affected OD measurements, causing diversity in OD and millimetric measurements of the lateral incisors in the IB group. In conclusion, the OB group demonstrated a significant stable vertical correction; a post-treatment non-contact inter-incisor relationship was determined by a vertico-sagittal relapse; and full compensation of an IB was not possible.

THE INFLUENCE OF PRE-CULTURAL CONDITIONS ON THE INDIVIDUAL CELL LAG PHASE AND GENERATION TIME OF LISTERIA MONOCYTOGENES

In this study, we assessed the impact of pre-cultural temperature and pH on the lag phase of individual Listeria monocytogenes cells at 7 degrees C. In a first step, the pre-cultural temperature effect (37, 15, 10, 7, 4 and 2 degrees C) was investigated on a subsequent growth at 7 degrees C and pH 7.4. In a second step, low pre-cultural temperatures (10, 7 and 4 degrees C) were combined with a controlled pH at 7.4 and 5.7 with a subsequent growth at 7 degrees C and different pH values (7.4, 6.0 and 5.5). Growth was monitored by OD measurements at 600 nm. For all temperature-pH combinations, the individual cell lag phase and the subsequent growth rate were determined using a three-phase linear growth model. Around 100 replications were made for each set of conditions. The results were plotted as histograms, and distributions were fitted to these data. It was observed that at low pre-cultural temperatures, a high proportion of L. monocytogenes cells was able to grow with almost no lag phase. Regarding the pH effect, the pH transition from the pre-cultural to a growth media was proportional to the mean values of the lag phases. There was no remarkable effect observed on the generation time. Regarding the statistical distributions, the exponential distribution yielded the best fit in most cases, as the highest probability was observed for very small lag phases and decreased with lag phase increase. To apply the results obtained at an industrial level, a simulation in @RISK to predict the distribution of the lag phase of L. monocytogenes at different transitions of temperature was performed. These findings have important consequences to estimate the possible risks concerned L. monocytogenes for refrigerated food products.